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Sample records for k-ras increases radiosensitivity

  1. Lin28-let7 Modulates Radiosensitivity of Human Cancer Cells With Activation of K-Ras

    SciTech Connect

    Oh, Jee-Sun.; Kim, Jae-Jin; Byun, Ju-Yeon; Kim, In-Ah

    2010-01-15

    Purpose: To evaluate the potential of targeting Lin28-let7 microRNA regulatory network for overcoming the radioresistance of cancer cells having activated K-Ras signaling. Methods and Materials: A549 lung carcinoma cells and ASPC1 pancreatic cancer cells possessing K-RAS mutation were transfected with pre-let7a microRNA or Lin28 siRNA, respectively. Clonogenic assay, quantitative reverse transcription polymerase chain reaction, and Western analysis were performed. The effects of Lin28 on SQ20B cells having wild-type K-RAS, and a normal fibroblast were also assessed. Results: The overexpression of let-7a decreased expression of K-Ras and radiosensitized A549 cells. Inhibition of Lin28, a repressor of let-7, attenuated K-Ras expression and radiosensitized A549 and ASPC1 cells. Neither SQ20B cells expressing wild-type K-RAS nor HDF, the normal human fibroblasts, were radiosensitized by this approach. Conclusions: The Lin28-let7 regulatory network may be a potentially useful therapeutic target for overcoming the radioresistance of human cancers having activated K-Ras signaling.

  2. Comparative Analysis of Radiosensitizers for K-RAS Mutant Rectal Cancers

    PubMed Central

    Kim, Stephen Y.; Hong, Theodore S.; Haigis, Kevin M.

    2013-01-01

    Approximately 40% of rectal cancers harbor activating K-RAS mutations, and these mutations are associated with poor clinical response to chemoradiotherapy. We aimed to identify small molecule inhibitors (SMIs) that synergize with ionizing radiation (IR) (“radiosensitizers”) that could be incorporated into current treatment strategies for locally advanced rectal cancers (LARCs) expressing mutant K-RAS. We first optimized a high-throughput assay for measuring individual and combined effects of SMIs and IR that produces similar results to the gold standard colony formation assay. Using this screening platform and K-RAS mutant rectal cancer cell lines, we tested SMIs targeting diverse signaling pathways for radiosensitizing activity and then evaluated our top hits in follow-up experiments. The two most potent radiosensitizers were the Chk1/2 inhibitor AZD7762 and the PI3K/mTOR inhibitor BEZ235. The chemotherapeutic agent 5-fluorouracil (5-FU), which is used to treat LARC, synergized with AZD7762 and enhanced radiosensitization by AZD7762. This study is the first to compare different SMIs in combination with IR for the treatment of K-RAS mutant rectal cancer, and our findings suggest that Chk1/2 inhibitors should be evaluated in new clinical trials for LARC. PMID:24349411

  3. Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

    PubMed

    Muñoz-Félix, José M; Fuentes-Calvo, Isabel; Cuesta, Cristina; Eleno, Nélida; Crespo, Piero; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-10-01

    The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-β1 (TGF-β1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-β1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-β1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc.

  4. Altered detoxification status and increased resistance to oxidative stress by K-ras transformation.

    PubMed

    Recktenwald, Christian V; Kellner, Roland; Lichtenfels, Rudolf; Seliger, Barbara

    2008-12-15

    Mutated K-ras is frequently found in human malignancies and plays a key role in many signal transduction processes resulting in an altered gene and/or protein expression pattern. Proteins controlled by a constitutive activated mitogen-activated protein kinase pathway are primarily related to alterations in the mitochondrial and nuclear compartments. Therefore, different K-Ras mutants and respective control cells were subjected to two-dimensional gel electrophoresis using basic pH gradients. This approach led to the identification of differentially expressed proteins, such as members of the heterogeneous ribonucleoprotein family, and enzymes involved in cellular detoxification as well as in oxidative stress. Increased expression of these enzymes was paralleled by an elevated tolerance of K-ras mutants against the cytotoxic potential of hydrogen peroxide and formaldehyde as well as an altered redox status based on enhanced intracellular glutathione (GSH) levels indicating an improved detoxification potential of defined K-ras transfectants, whereas down-regulation by RNA interference of candidate proteins reversed the tolerance against these compounds. This hypothesis is supported by an up-regulated expression of a key enzyme of the pentose phosphate pathway resulting in an increased production of NADPH required for anabolic processes as well as the rebuilding of oxidized GSH. Both the enhanced resistance against xenobiotic compounds as well as an altered oxidative pathway might confer growth advantages for tumor cells carrying dominant-positive K-ras mutations such as in lung or pancreatic adenocarcinoma.

  5. Down-regulation of let-7 microRNA increased K-ras expression in lung damage induced by radon.

    PubMed

    Chen, Zhihai; Wang, Dapeng; Gu, Chao; Liu, Xing; Pei, Weiwei; Li, Jianxiang; Cao, Yi; Jiao, Yang; Tong, Jian; Nie, Jihua

    2015-09-01

    Radon has long been recognized as a human carcinogen leading to lung cancer, but the underlying mechanisms remain obscure. Recent studies have shown that the let-7 microRNA and K-ras play an important role in the development of various cancers. However, the exact role between let-7 and K-ras in radon induced lung damage has not been explored so far. In the present study, wistar rats and human bronchial epithelial (HBE) cells were long-term exposed to radon, and then alterations in histological pathology of rat lung tissue, ROS, antioxidant enzymes activities and clonogenic formation in HBE cells, as well as changes in let-7 and K-ras expression were determined to observe the adverse effects induced by radon. The results showed that long-term exposure to radon produced severe lung damage in rats, significantly increased ROS production and clonogenic formation ratios and decreased SOD activities in HBE cells. In addition, an obvious down-regulation of let-7 and up-regulation of K-ras were also revealed both in mRNA and in protein level in lung tissue of rats and HBE cells exposed to radon. Furthermore, a significant down-regulation of K-ras was then confirmed in both let-7b-3p and let-7a-2-3p transfected HBE cells. Taken together, the present results propose an involvement of let-7 microRNA and K-ras in radon induced lung damage both in vivo and in vitro, which may thus be of potential value in early diagnosis and therapy of radon-induced lung tumorgenesis.

  6. Activated K-RAS Increases Polyamine Uptake in Human Colon Cancer Cells Through Modulation of Caveolar Endocytosis

    PubMed Central

    Basu Roy, Upal K.; Rial, Nathaniel S.; Kachel, Karen L.; Gerner, Eugene W.

    2008-01-01

    Endocytic pathways have been implicated in polyamine transport in mammalian cells, but specific mechanisms have not been described. We have shown that expression of a dominant negative (DN) form of the GTPase Dynamin, but not Eps15, diminished polyamine uptake in colon cancer cells indicating a caveolar and nonclathrin uptake mode. Polyamines co-sediment with lipid raft/caveolin-1 rich fractions, of the plasma membrane in a sucrose density gradient. Knock down of caveolin-1 significantly increased polyamine uptake. Conversely, ectopic expression of this protein resulted in diminished polyamine uptake. We also found that presence of an activated K-RAS oncogene significantly increased polyamine uptake by colon cancer cells. This effect is through an increase in caveolin-1 phosphorylation at tyrosine residue 14. Caveolin-1 is a negative regulator of caveolar endocytosis and phosphorylation in a K-RAS dependent manner leads to an increase in caveolar endocytosis. In cells expressing wild type K-RAS, addition of exogenous uPA was sufficient to stimulate caveolar endocytosis of polyamines. This effect was abrogated by the addition of a SRC kinase inhibitor. These data indicate that polyamine transport follows a dynamin-dependent and clathrin-independent endocytic uptake route, and this route is positively regulated by the oncogenic expression of K-RAS in a caveolin-1 dependent manner. PMID:18176934

  7. High-LET Radiation Increases Tumor Progression in a K-Ras-Driven Model of Lung Adenocarcinoma.

    PubMed

    Asselin-Labat, Marie-Liesse; Rampersad, Rishi; Xu, Xia; Ritchie, Matthew E; Michalski, Jacob; Huang, Lingling; Onaitis, Mark W

    2017-09-27

    High-linear energy transfer (LET) radiation encountered by astronauts in space generates clustered DNA damage that is potentially oncogenic. Analysis of the impact of exposure to space radiation on cancer formation is necessary to determine the best ways to prepare astronauts for space travel so they are protected for the duration of the space mission. A mouse model of lung adenocarcinoma driven by oncogenic K-Ras was used to ascertain the effect of low- and high-LET radiation on tumor formation. We observed increased tumor progression and tumor cell proliferation after single dose or fractionated high-LET doses, which was not observed in mice exposed to low-LET radiation. Location of the tumor nodules was not affected by radiation, indicating that the cell of origin of K-Ras-driven tumors was the same in irradiated or nonirradiated mice. Gene expression analysis revealed an upregulation of genes involved in cell proliferation and DNA damage repair. This study provides evidence that exposure to a single dose or fractionated doses of high-LET radiation induces molecular and cellular changes that accelerate lung tumor growth.

  8. Silencing the wild-type and mutant K-ras increases the resistance to 5-flurouracil in HCT-116 as a colorectal cancer cell line.

    PubMed

    Teimoori-Toolabi, Ladan; Hashemi, Saba; Azadmanesh, Kayhan; Eghbalpour, Farnaz; Safavifar, Farnaz; Khorramizadeh, Mohammad Reza

    2015-02-01

    Colon cancer is the second to third common cancer worldwide. Several efforts have been made to reveal the pathways responsible for drug resistance in this type of cancer. We aimed to investigate the effect of silencing both mutant and wild-type Kristen Rous sarcoma (k-ras) on the response of human colorectal tumor 116 (HCT-116) as a colon cancer cell line to the cytotoxic effect of 5-flurouracil (5-FU). One oligonucelotide against mutant k-ras (12th codon, namely 207) and two against wild-type k-ras (namely 535 and 689) were cloned into pSilencer neo2.1. The linearized vectors besides the negative control plasmid were stably transfected into HCT-116. The proliferation rates of these cells in different concentrations of 5-FU and the apoptosis rates of the cells after treatment with lethal doses of 5-FU were studied. Moreover, the cell cycle in these cells was also analyzed by staining the cells with propidium iodide. Stably transfected cells were named HCT207ks, HCT535ks, HCT689ks, and HCT-Sc (transfected with the negative control plasmid). Decreased expression of k-ras in HCT207ks, HCT535ks, and to a lesser extent in HCT689ks was proved by quantitative real-time PCR. Although in HCT207ks the cells were mostly in G0/G1 and G2/M phases, in HCT535ks and HCT689ks, the cells in the S phase were higher in comparison with nontransfected HCT-116. Lethal doses of 5-FU in HCT-116 and HCT-Sc were 2.5-3 and 3-3.5 µmol/l, whereas in HCT207ks, HCT535ks, and HCT689ks, they were 35-40, 37.5-40, and 22.5-25 µmol/l. In conclusion, silencing mutant and wild-type k-ras would increase the resistance of HCT-116 cell line as a model of colorectal cancer to 5-FU. The degree of resistance was related directly to the k-ras mRNA level. Therefore, both mutant and wild-type k-ras may play a role in sensitizing colorectal cancer cells to 5-FU as a common chemotherapeutic drug.

  9. Radiosensitization of EGFR/HER2 positive pancreatic cancer is mediated by inhibition of Akt independent of Ras mutational status

    PubMed Central

    Kimple, Randall J.; Vaseva, Angelina V.; Cox, Adrienne D.; Baerman, Kathryn M.; Calvo, Benjamin F.; Tepper, Joel E.; Shields, Janiel M.; Sartor, Carolyn I.

    2009-01-01

    Purpose Epidermal growth factor receptor family members (e.g., EGFR, HER2, HER3, and HER4) are commonly overexpressed in pancreatic cancer. We investigated the effects of inhibition of EGFR/HER2 signaling on pancreatic cancer to elucidate the role(s) of EGFR/HER2 in radiosensitization and to provide evidence in support of further clinical investigations. Experimental Design Expression of EGFR family members in pancreatic cancer lines was assessed by qRT-PCR. Cell growth inhibition was determined by MTS assay. The effects of inhibition of EGFR family receptors and downstream signaling pathways on in vitro radiosensitivity were evaluated using clonogenic assays. Growth delay was used to evaluate the effects of nelfinavir on in vivo tumor radiosensitivity. Results Lapatinib inhibited cell growth in four pancreatic cancer cell lines, but radiosensitized only wild-type K-ras-expressing T3M4 cells. Akt activation was blocked in a wild-type K-ras cell line, whereas constitutive phosphorylation of Akt and ERK was seen in lines expressing mutant K-ras. Overexpression of constitutively-active K-ras(G12V) abrogated lapatinib-mediated inhibition of both Akt phosphorylation and radiosensitization. Inhibition of MEK/ERK signaling with U0126 had no effect on radiosensitization, whereas inhibition of activated Akt with LY294002 (enhancement ratio 1.2–1.8) or nelfinavir (enhancement ratio 1.2–1.4) radiosensitized cells regardless of K-ras mutation status. Oral nelfinavir administration to mice bearing mutant K-ras-containing Capan-2 xenografts resulted in a greater than additive increase in radiation-mediated tumor growth delay (synergy assessment ratio of 1.5). Conclusions Inhibition of EGFR/HER2 enhances radiosensitivity in wild-type K-ras pancreatic cancer. Nelfinavir, and other PI3K/Akt inhibitors, are effective pancreatic radiosensitizers regardless of K-ras mutation status. PMID:20103665

  10. Deletion of cyclooxygenase-2 inhibits K-ras-induced lung carcinogenesis.

    PubMed

    Pan, Yong; Jiang, Yan; Tan, Lin; Ravoori, Murali K; Gagea, Mihai; Kundra, Vikas; Fischer, Susan M; Yang, Peiying

    2015-11-17

    The purpose of this study was to identify the role COX-2 plays in K-ras-induced lung carcinogenesis. We crossed COX-2-homozygous knockout mice with K-rasLA1 (G12D) expressing mice to obtain COX-2-deficient mice with K-ras expression (K-ras/COX-2(-/-) mice) and COX-2 wild type mice with K-ras expression (K-ras mice). At 3.5 months of age, the K-ras/COX-2(-/-) mice had significantly fewer lung adenocarcinomas and substantially smaller tumors than K-ras mice. K-ras/COX-2(-/-) mice also had significantly fewer bronchioalveolar hyperplasias than K-ras mice. Compared with lung tumors from K-Ras mice, the levels of prostaglandin E2 (PGE2) were significantly lower, whereas levels of the PGE2 metabolite 13,14-dihydro-15-keto-PGE2 were significantly higher, in lung tumors from K-ras/COX-2(-/-) mice. In addition, K-ras/COX-2(-/-) mice had strikingly lower rates of tumor cell proliferation and expressed less MEK and p-Erk1/2 protein than K-ras mice did. In line with this, knocking down COX-2 in mutant K-ras non-small cell lung cancer A549 cells reduced colony formation, PGE2 synthesis and ERK phosphorylation compared to that of vector control cells. Taken together, these findings suggest that COX-2 deletion contributes to the repression of K-ras-induced lung tumorigenesis by reducing tumor cell proliferation, decreasing the production of PGE2, and increasing the production of 13,14-dihydro-15-keto-PGE2, possibly via the MAPK pathway. Thus, COX-2 is likely important in lung tumorigenesis, and COX-2 and its product, PGE2, are potential targets for lung cancer prevention.

  11. Oncogenic K-Ras signals through epidermal growth factor receptor and wild-type H-Ras to promote radiation survival in pancreatic and colorectal carcinoma cells.

    PubMed

    Cengel, Keith A; Voong, K Rahn; Chandrasekaran, Sanjay; Maggiorella, Laurence; Brunner, Thomas B; Stanbridge, Eric; Kao, Gary D; McKenna, W Gillies; Bernhard, Eric J

    2007-04-01

    Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor alpha was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers.

  12. Oncogenic K-Ras Signals through Epidermal Growth Factor Receptor and Wild-Type H-Ras to Promote Radiation Survival in Pancreatic and Colorectal Carcinoma Cells1

    PubMed Central

    Cengel, Keith A.; Voong, K. Rahn; Chandrasekaran, Sanjay; Maggiorella, Laurence; Brunner, Thomas B.; Stanbridge, Eric; Kao, Gary D.; McKenna, W. Gillies; Bernhard, Eric J.

    2007-01-01

    Pancreatic and colorectal carcinomas frequently express oncogenic/mutant K-Ras that contributes to both tumorigenesis and clinically observed resistance to radiation treatment. We have previously shown that farnesyltransferase inhibitors (FTI) radiosensitize many pancreatic and colorectal cancer cell lines that express oncogenic K-ras at doses that inhibit the prenylation and activation of H-Ras but not K-Ras. In the present study, we have examined the mechanism of FTI-mediated radiosensitization in cell lines that express oncogenic K-Ras and found that wild-type H-Ras is a contributor to radiation survival in tumor cells that express oncogenic K-Ras. In these experiments, inhibiting the expression of oncogenic K-Ras, wild-type H-Ras, or epidermal growth factor receptor (EGFR) led to similar levels of radiosensitization as treatment with the FTI tipifarnib. Treatment with the EGFR inhibitor gefitinib led to similar levels of radiosensitization, and the combinations of tipifarnib or gefitinib plus inhibition of K-Ras, H-Ras, or EGFR expression did not provide additional radiosensitization compared with tipifarnib or gefitinib alone. Finally, supplementing culture medium with the EGFR ligand transforming growth factor α was able to reverse the radiosensitizing effect of inhibiting K-ras expression. Taken together, these findings suggest that EGFR-activated H-Ras signaling is initiated by oncogenic K-Ras to promote radiation survival in pancreatic and colorectal cancers. PMID:17460778

  13. K-Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

    EPA Science Inventory

    K-Ras mutant fraction (MF) was measured to examine the default assumption of low dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of ten male A/J mice (7-9 weeks-old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P, and were sacrifi...

  14. K-Ras mutant fraction in A/J mouse lung increases as a function of benzo[a]pyrene dose

    EPA Science Inventory

    K-Ras mutant fraction (MF) was measured to examine the default assumption of low dose linearity in the benzo[a]pyrene (B[a]P) mutational response. Groups of ten male A/J mice (7-9 weeks-old) received a single i.p. injection of 0, 0.05, 0.5, 5, or 50 mg/kg B[a]P, and were sacrifi...

  15. Degradation of Activated K-Ras Orthologue via K-Ras-specific Lysine Residues Is Required for Cytokinesis*

    PubMed Central

    Sumita, Kazutaka; Yoshino, Hirofumi; Sasaki, Mika; Majd, Nazanin; Kahoud, Emily Rose; Takahashi, Hidenori; Takeuchi, Koh; Kuroda, Taruho; Lee, Susan; Charest, Pascale G.; Takeda, Kosuke; Asara, John M.; Firtel, Richard A.; Anastasiou, Dimitrios; Sasaki, Atsuo T.

    2014-01-01

    Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers. PMID:24338482

  16. Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon.

    PubMed

    Calcagno, Shelly R; Li, Shuhua; Colon, Migdalisel; Kreinest, Pamela A; Thompson, E Aubrey; Fields, Alan P; Murray, Nicole R

    2008-06-01

    Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-ras(G12D) allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-ras(G12D) throughout the colon (LSL-K-ras(G12D)/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-ras(G12D) induces increased expression of procarcinogenic protein kinase C beta II (PKC beta II), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-ras(G12D) inhibits expression of procarcinogenic PKC beta II and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice. (c) 2008 Wiley-Liss, Inc.

  17. Oncogenic K-ras promotes early carcinogenesis in the mouse proximal colon

    PubMed Central

    Calcagno, Shelly R.; Li, Shuhua; Colon, Migdalisel; Kreinest, Pamela A.; Thompson, E. Aubrey; Fields, Alan P.; Murray, Nicole R.

    2014-01-01

    Oncogenic K-ras mutations are frequently observed in colon cancers and contribute to transformed growth. Oncogenic K-ras is detected in aberrant crypt foci (ACF), precancerous colonic lesions, demonstrating that acquisition of a K-ras mutation is an early event in colon carcinogenesis. Here, we investigate the role of oncogenic K-ras in neoplastic initiation and progression. Transgenic mice in which an oncogenic K-rasG12D allele is activated in the colonic epithelium by sporadic recombination (K-rasLA2 mice) develop spontaneous ACF that are morphologically indistinguishable from those induced by the colon carcinogen azoxymethane (AOM). Similar neoplastic changes involving the entire colon are induced in transgenic mice constitutively expressing K-rasG12D throughout the colon (LSL-K-rasG12D/Villin-Cre mice). However, the biochemistry and fate of K-ras-induced lesions differ depending upon their location within the colon in these mice. In the proximal colon, K-rasG12D induces increased expression of procarcinogenic protein kinase CβII (PKCβII), activation of the MEK/ERK signaling axis and increased epithelial cell proliferation. In contrast, in the distal colon, K-rasG12D inhibits expression of procarcinogenic PKCβII and induces apoptosis. Treatment of K-rasLA2 mice with AOM leads to neoplastic progression of small ACF to large, dysplastic microadenomas in the proximal, but not the distal colon. Thus, oncogenic K-ras functions differently in the proximal and distal colon of mice, inducing ACF capable of neoplastic progression in the proximal colon, and ACF with little or no potential for progression in the distal colon. Our data indicate that acquisition of a K-ras mutation is an initiating neoplastic event in proximal colon cancer development in mice. PMID:18271008

  18. Identification of Mutant K-Ras-dependent Phenotypes Using a Panel of Isogenic Cell Lines*

    PubMed Central

    Vartanian, Steffan; Bentley, Carolyn; Brauer, Matthew J.; Li, Li; Shirasawa, Senji; Sasazuki, Takehiko; Kim, Jung-Sik; Haverty, Pete; Stawiski, Eric; Modrusan, Zora; Waldman, Todd; Stokoe, David

    2013-01-01

    To assess the consequences of endogenous mutant K-Ras, we analyzed the signaling and biological properties of a small panel of isogenic cell lines. These include the cancer cell lines DLD1, HCT116, and Hec1A, in which either the WT or mutant K-ras allele has been disrupted, and SW48 colorectal cancer cells and human mammary epithelial cells in which a single copy of mutant K-ras was introduced at its endogenous genomic locus. We find that single copy mutant K-Ras causes surprisingly modest activation of downstream signaling to ERK and Akt. In contrast, a negative feedback signaling loop to EGFR and N-Ras occurs in some, but not all, of these cell lines. Mutant K-Ras also had relatively minor effects on cell proliferation and cell migration but more dramatic effects on cell transformation as assessed by growth in soft agar. Surprisingly, knock-out of the wild type K-ras allele consistently increased growth in soft agar, suggesting tumor-suppressive properties of this gene under these conditions. Finally, we examined the effects of single copy mutant K-Ras on global gene expression. Although transcriptional programs triggered by mutant K-Ras were generally quite distinct in the different cell lines, there was a small number of genes that were consistently overexpressed, and these could be used to monitor K-Ras inhibition in a panel of human tumor cell lines. We conclude that there are conserved components of mutant K-Ras signaling and phenotypes but that many depend on cell context and environmental cues. PMID:23188824

  19. Mutant K-RAS Promotes Invasion and Metastasis in Pancreatic Cancer Through GTPase Signaling Pathways

    PubMed Central

    Padavano, Julianna; Henkhaus, Rebecca S; Chen, Hwudaurw; Skovan, Bethany A; Cui, Haiyan; Ignatenko, Natalia A

    2015-01-01

    Pancreatic ductal adenocarcinoma is one of the most aggressive malignancies, characterized by the local invasion into surrounding tissues and early metastasis to distant organs. Oncogenic mutations of the K-RAS gene occur in more than 90% of human pancreatic cancers. The goal of this study was to investigate the functional significance and downstream effectors of mutant K-RAS oncogene in the pancreatic cancer invasion and metastasis. We applied the homologous recombination technique to stably disrupt K-RAS oncogene in the human pancreatic cell line MiaPaCa-2, which carries the mutant K-RASG12C oncogene in both alleles. Using in vitro assays, we found that clones with disrupted mutant K-RAS gene exhibited low RAS activity, reduced growth rates, increased sensitivity to the apoptosis inducing agents, and suppressed motility and invasiveness. In vivo assays showed that clones with decreased RAS activity had reduced tumor formation ability in mouse xenograft model and increased survival rates in the mouse orthotopic pancreatic cancer model. We further examined molecular pathways downstream of mutant K-RAS and identified RhoA GTP activating protein 5, caveolin-1, and RAS-like small GTPase A (RalA) as key effector molecules, which control mutant K-RAS-dependent migration and invasion in MiaPaCa-2 cells. Our study provides rational for targeting RhoA and RalA GTPase signaling pathways for inhibition of pancreatic cancer metastasis. PMID:26512205

  20. Radiosensitization Effect of STI-571 on Pancreatic Cancer Cells In Vitro

    SciTech Connect

    Chung, Hye Won; Wen, Jing; Lim, Jong-Baeck; Bang, Seung Min; Park, Seung Woo; Song, Si Young

    2009-11-01

    Purpose: To examine STI-571-induced radiosensitivity in human pancreatic cancer cells in vitro. Methods and Materials: Three human pancreatic cancer cell lines (Bxpc-3, Capan-1, and MiaPaCa-2) exhibiting different expression levels of c-Kit and platelet-derived growth factor receptor beta (PDGFRbeta) and showing different K-ras mutation types were used. For evaluation of the antitumor activity of STI-571 in combination with radiation, clonogenic survival assays, Western blot analysis, and the annexin V/propidium iodide assay with microscopic evaluation by 4',6-diamidino-2-phenylindole were conducted. Results: Dramatic phosphorylated (p)-c-Kit and p-PDGFRbeta attenuation, a modest dose- and time-dependent growth inhibition, and significant radiosensitization were observed after STI-571 treatment in view of apoptosis, although the levels of growth inhibition and increased radiosensitization were different according to cell lines. The grades of radiosensitivity corresponded to the attenuation levels of p-c-Kit and p-PDGFRbeta by STI-571, particularly to those of p-c-Kit, and the radiosensitivity was partially affected by K-ras mutation in pancreatic cancer cells. Among downstream pathways associated with c-Kit or PDGFRbeta, p-PLCgamma was more closely related to radiosensitivity compared with p-Akt1 or p-extracellular signal-regulated kinase 1. Conclusion: STI-571 enhances radiation response in pancreatic cancer cells. This effect is affected by the attenuation levels of p-c-Kit or p-PDGFRbeta, and K-ras mutation status. Among them, p-c-Kit plays more important roles in the radiosensitivity in pancreatic cancer compared with p-PDGFRbeta or K-ras mutation status.

  1. K-ras oncogene mutation in pterygium.

    PubMed

    Ozturk, B T; Yıldırım, M S; Zamani, A; Bozkurt, B

    2017-03-01

    PurposePterygium is claimed to be a benign proliferation triggered by prolonged exposure to ultraviolet radiation. The frequency of K-ras oncogene mutation, which is among the initial mutations in tumorigenesis, is evaluated in this study.Patients and methodsIn this prospective randomized clinical, trial pterygium tissues and normal conjunctiva tissue specimens are obtained from the superotemporal quadrant of patients who underwent primary pterygium excision with autograft transplantation. DNA extraction from tissues was performed using the QIAamp DNA FFPE tissue kit. A PCR reaction was performed to amplify sequences containing codons 12, 13, and 61 of the K-ras gene in DNA. These PCR products then underwent the 'pyrosequencing' procedure for mutations at these codons.ResultsPterygium and normal conjunctival tissue samples of 25 patients (10 females, 15 males) were evaluated in the study. The mean age of the patients was 54.54±13.13 years. Genetic analysis revealed no K-ras mutations in normal conjunctival tissues, whereas pterygium tissues of the same cases demonstrated mutation at codon 12 in one case and mutations at codon 61 in seven cases, which was statistically significant (P<0.05). The point missense mutations at codon 61 were glutamine to arginine (Glu61Arg CAA>CGA) in four cases and glutamine to leucine (Glu61Leu CAA>CTA) in three cases.ConclusionThe significantly higher frequency of codon 61 mutation of the ras oncogene in primary and bilateral pterygium specimens compared with normal conjunctiva supports the tumoral origin of pterygium, and thus set the stage for research into a targeted therapy for pterygium with better outcomes than surgical excision.

  2. Autophagy Inhibition to Increase Radiosensitization in Breast Cancer

    PubMed Central

    Liang, Diana Hwang; El-Zein, Randa; Dave, Bhuvanesh

    2015-01-01

    Currently, many breast cancer patients with localized breast cancer undergo breast-conserving therapy, consisting of local excision followed by radiation therapy. Following radiation therapy, breast cancer cells are noted to undergo induction of autophagy, development of radioresistance, and enrichment of breast cancer stem cell subpopulations. It is hypothesized that inhibition of the cytoprotective autophagy that arises following radiation therapy increases radiosensitivity and confers longer relapse-free survival by eliminating tumor-initiating breast cancer stem cells. Therefore, we reviewed the controversial role of autophagy in breast cancer tumorigenesis and progression, autophagy induction by radiotherapy, and utilization of autophagy inhibitors to increase radiosensitivity of breast cancer and to target radioresistant breast cancer stem cells. PMID:26613064

  3. Acute sensitivity of the oral mucosa to oncogenic K-ras

    PubMed Central

    van der Weyden, Louise; Alcolea, Maria P; Jones, Philip H; Rust, Alistair G; Arends, Mark J; Adams, David J

    2011-01-01

    Mouse models of cancer represent powerful tools for analysing the role of genetic alterations in carcinogenesis. Using a mouse model that allows tamoxifen-inducible somatic activation (by Cre-mediated recombination) of oncogenic K-rasG12D in a wide range of tissues, we observed hyperplasia of squamous epithelium located in moist or frequently abraded mucosa, with the most dramatic effects in the oral mucosa. This epithelium showed a sequence of squamous hyperplasia followed by squamous papilloma with dysplasia, in which some areas progressed to early invasive squamous cell carcinoma, within 14 days of widespread oncogenic K-ras activation. The marked proliferative response of the oral mucosa to K-rasG12D was most evident in the basal layers of the squamous epithelium of the outer lip with hair follicles and wet mucosal surface, with these cells staining positively for pAKT and cyclin D1, showing Ras/AKT pathway activation and increased proliferation with Ki-67 and EdU positivity. The stromal cells also showed gene activation by recombination and immunopositivity for pERK indicating K-Ras/ERK pathway activation, but without Ki-67 positivity or increase in stromal proliferation. The oral neoplasms showed changes in the expression pattern of cytokeratins (CK6 and CK13), similar to those observed in human oral tumours. Sporadic activation of the K-rasG12D allele (due to background spontaneous recombination in occasional cells) resulted in the development of benign oral squamous papillomas only showing a mild degree of dysplasia with no invasion. In summary, we show that oral mucosa is acutely sensitive to oncogenic K-ras, as widespread expression of activated K-ras in the murine oral mucosal squamous epithelium and underlying stroma can drive the oral squamous papilloma–carcinoma sequence. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:21381032

  4. K-ras mutations, rectal crypt cells proliferation, and meat consumption in patients with left-sided colorectal carcinoma.

    PubMed

    O'Brien, H; Matthew, J A; Gee, J M; Watson, M; Rhodes, M; Speakman, C T; Stebbings, W S; Kennedy, H J; Johnson, I T

    2000-02-01

    It has been suggested that N-nitroso compounds derived from meat may increase the risk of K-ras mutations in the human colon. We sought evidence of associations between red meat consumption, frequency and type of K-ras mutations in resected tumours, and the rate of crypt cell proliferation (CCP) in the normal mucosa of patients with left-sided colorectal carcinoma. Meat consumption was assessed by food frequency questionnaire, and CCP was determined in rectal biopsies obtained prior to surgery. K-ras mutations in the resected tumours were determined using a PCR-based oligonucleotide hybridization assay. Fifteen K-ras mutations were detected in tumours from 43 patients; 13/15 in codon 12, 3/15 in codon 13, and 1/15 in both codons 12 and 13. All mutations were G-->A or G-->T transitions. There was no statistically significant difference between intakes of red meat in patients with a K-ras mutation (92.4 +/- 9.7 g/day) and those without (82.3 +/- 7.7 g/day). Rectal CCP was significantly higher in patients than in healthy controls, but there was no correlation with meat consumption or K-ras mutation. These data do not support the hypothesis that meat consumption is a risk factor for acquisition of K-ras mutations during colorectal carcinogenesis.

  5. Mutated K-ras(Asp12) promotes tumourigenesis in Apc(Min) mice more in the large than the small intestines, with synergistic effects between K-ras and Wnt pathways.

    PubMed

    Luo, Feijun; Brooks, David G; Ye, Hongtao; Hamoudi, Rifat; Poulogiannis, George; Patek, Charles E; Winton, Douglas J; Arends, Mark J

    2009-10-01

    Summary K-ras mutations are found in 40-50% of human colorectal adenomas and carcinomas, but their functional contribution remains incompletely understood. Here, we show that a conditional mutant K-ras mouse model (K-ras(Asp12)/Cre), with transient intestinal Cre activation by beta-Naphthoflavone (beta-NF) treatment, displayed transgene recombination and K-ras(Asp12) expression in the murine intestines, but developed few intestinal adenomas over 2 years. However, when crossed with Apc(Min/+) mice, the K-ras(Asp12)/Cre/Apc(Min/+) offspring showed acceleration of intestinal tumourigenesis with significantly changed average lifespan (P < 0.05) decreased to 18.4 +/- 5.4 weeks from 20.9 +/- 4.7 weeks (control Apc(Min/+) mice). The numbers of adenomas in the small intestine and large intestine were significantly (P < 0.01) increased by 1.5-fold and 5.7-fold, respectively, in K-ras(Asp12)/Cre/Apc(Min/+) mice compared with Apc(Min/+) mice, with the more marked increase in adenoma prevalence in the large intestine. To explore possible mechanisms for K-ras(Asp12) and Apc(Min) co-operation, the Mitogen-activated protein kinase (Mapk), Akt and Wnt signalling pathways, including selected target gene expression levels, were evaluated in normal large intestine and large intestinal tumours. K-ras(Asp12) increased activation of Mapk and Akt signalling pathway targets phospho-extracellular signal-regulated kinase (pErk) and pAkt, and increased relative expression levels of Wnt pathway targets vascular endothelial growth factor (VEGF), gastrin, cyclo-oxygenase 2 (Cox2) and T-cell lymphoma invasion and metastasis 1 (Tiam1) in K-ras(Asp12)/Cre/Apc(Min/+) adenomas compared with that of Apc(Min/+) adenomas, although other Wnt signalling pathway target genes such as Peroxisome proliferator-activated receptor delta (PPARd), matrix metalloproteinase 7 (MMP7), protein phosphatase 1 alpha (PP1A) and c-myc remained unchanged. In conclusion, intestinal expression of K-ras(Asp12) promotes mutant

  6. Co-dependency of PKCδ and K-Ras: inverse association with cytotoxic drug sensitivity in KRAS mutant lung cancer.

    PubMed

    Ohm, A M; Tan, A-C; Heasley, L E; Reyland, M E

    2017-07-27

    Recent studies suggest that the presence of a KRAS mutation may be insufficient for defining a clinically homogenous molecular group, as many KRAS mutant tumors lose reliance on K-Ras for survival. Identifying pathways that support K-Ras dependency may define clinically relevant KRAS subgroups and lead to the identification of new drug targets. We have analyzed a panel of 17 KRAS mutant lung cancer cell lines classified as K-Ras-dependent or -independent for co-dependency on protein kinase C δ (PKCδ). We show that functional dependency on K-Ras and PKCδ co-segregate, and that dependency correlates with a more epithelial-like phenotype. Furthermore, we show that the pro-apoptotic and pro-tumorigenic functions of PKCδ also segregate based on K-Ras dependency, as K-Ras-independent cells are more sensitive to topoisomerase inhibitors, and depletion of PKCδ in this subgroup suppresses apoptosis through increased activation of extracellular signal-regulated kinase (ERK). In contrast, K-Ras-dependent lung cancer cells are largely insensitive to topoisomerase inhibitors, and depletion of PKCδ can increase apoptosis and decrease activation of ERK in this subgroup. We have previously shown that nuclear translocation of PKCδ is necessary and sufficient for pro-apoptotic signaling. Our current studies show that K-Ras-dependent cells are refractive to PKCδ-driven apoptosis. Analysis of this subgroup showed increased PKCδ expression and an increase in the nuclear:cytoplasmic ratio of PKCδ. In addition, targeting PKCδ to the nucleus induces apoptosis in K-Ras-independent, but not K-Ras-dependent non-small-cell lung cancer (NSCLC) cells. Our studies provide tools for identification of the subset of patients with KRAS mutant tumors most amenable to targeting of the K-Ras pathway, and identify PKCδ as a potential target in this tumor population. These subgroups are likely to be of clinical relevance, as high PKCδ expression correlates with increased overall survival and

  7. Co-dependency of PKCδ and K-Ras: Inverse association with cytotoxic drug sensitivity in KRAS mutant lung cancer

    PubMed Central

    Ohm, Angela M.; Tan, Aik-Choon; Heasley, Lynn E.; Reyland, Mary E.

    2017-01-01

    Recent studies suggest that the presence of a KRAS mutation may be insufficient for defining a clinically homogenous molecular group, as many KRAS mutant tumors lose reliance on K-Ras for survival. Identifying pathways that support K-Ras dependency may define clinically relevant KRAS sub-groups and lead to the identification of new drug targets. We have analyzed a panel of 17 KRAS mutant lung cancer cell lines classified as K-Ras dependent or independent, for co-dependency on PKCδ. We show that functional dependency on K-Ras and PKCδ co-segregate, and that dependency correlates with a more epithelial-like phenotype. Furthermore, we show that the pro-apoptotic and pro-tumorigenic functions of PKCδ also segregate based on K-Ras dependency, as K-Ras independent cells are more sensitive to topoisomerase inhibitors, and depletion of PKCδ in this sub-group suppresses apoptosis through increased activation of ERK. In contrast, K-Ras dependent lung cancer cells are largely insensitive to topoisomerase inhibitors, and depletion of PKCδ can increase apoptosis and decrease activation of ERK in this sub-group. We have previously shown that nuclear translocation of PKCδ is necessary and sufficient for pro-apoptotic signaling. Our current studies show that K-Ras dependent cells are refractive to PKCδ driven apoptosis. Analysis of this sub-group showed increased PKCδ expression and an increase in the nuclear:cytoplasmic ratio of PKCδ. In addition, targeting PKCδ to the nucleus induces apoptosis in K-Ras independent, but not K-Ras dependent NSCLC cells. Our studies provide tools for identification of the subset of patients with KRAS mutant tumors most amenable to targeting of the K-Ras pathway, and identify PKCδ as a potential target in this tumor population. These sub-groups are likely to be of clinical relevance, as high PKCδ expression correlates with increased overall survival and a more epithelial tumor phenotype in patients with KRAS mutant lung adenocarcinomas

  8. K-RAS(V12) Induces Autocrine Production of EGFR Ligands and Mediates Radioresistance Through EGFR-Dependent Akt Signaling and Activation of DNA-PKcs

    SciTech Connect

    Minjgee, Minjmaa; Toulany, Mahmoud; Kehlbach, Rainer; Giehl, Klaudia; Rodemann, H. Peter

    2011-12-01

    Purpose: It is known that postirradiation survival of tumor cells presenting mutated K-RAS is mediated through autocrine activation of epidermal growth factor receptor (EGFR). In this study the molecular mechanism of radioresistance of cells overexpressing mutated K-RAS(V12) was investigated. Methods and Materials: Head-and-neck cancer cells (FaDu) presenting wild-type K-RAS were transfected with empty vector or vector expressing mutated K-RAS(V12). The effect of K-RAS(V12) on autocrine production of EGFR ligands, activation of EGFR downstream pathways, DNA damage repair, and postirradiation survival was analyzed. Results: Conditioned medium collected from K-RAS(V12)-transfected cells enhanced activation of the phosphatidylinositol-3-kinase-Akt pathway and increased postirradiation survival of wild-type K-RAS parental cells when compared with controls. These effects were reversed by amphiregulin (AREG)-neutralizing antibody. In addition, secretion of the EGFR ligands AREG and transforming growth factor {alpha} was significantly increased upon overexpression of K-RAS(V12). Expression of mutated K-RAS(V12) resulted in an increase in radiation-induced DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation at S2056. This increase was accompanied by increased repair of DNA double-strand breaks. Abrogation of DNA-PKcs phosphorylation by serum depletion or AREG-neutralizing antibody underscored the role of autocrine production of EGFR ligands, namely, AREG, in regulating DNA-PKcs activation in K-RAS mutated cells. Conclusions: These data indicate that radioresistance of K-RAS mutated tumor cells is at least in part due to constitutive production of EGFR ligands, which mediate enhanced repair of DNA double-strand breaks through the EGFR-phosphatidylinositol-3-kinase-Akt cascade.

  9. Endogenous K-ras signaling in erythroid differentiation.

    PubMed

    Zhang, Jing; Lodish, Harvey F

    2007-08-15

    K-ras is one of the most frequently mutated genes in virtually all types of human cancers. Using mouse fetal liver erythroid progenitors as a model system, we studied the role of endogenous K-ras signaling in erythroid differentiation. When oncogenic K-ras is expressed from its endogenous promoter, it hyperactivates cytokine-dependent signaling pathways and results in a partial block in erythroid differentiation. In erythroid progenitors deficient in K-ras, cytokine-dependent Akt activation is greatly reduced, leading to delays in erythroid differentiation. Thus, both loss- and gain-of-Kras functions affect erythroid differentiation through modulation of cytokine signaling. These results support the notion that in human cancer patients oncogenic Ras signaling might be controlled by antagonizing essential cytokines.

  10. [Mouse models of K-ras-initiated oncogenesis].

    PubMed

    Barrière, C; Marjou, F El; Louvard, D; Robine, S

    2009-12-01

    Activating mutations of the oncogene K-ras are found in one third of all human cancers. Much of our knowledge on K-ras signal transduction and its influence on tumor initiation and progression come from in vitro studies with cell lines. However, mouse models of human cancer allow a much more faithful recapitulation of the human disease, and the in vivo perspective is crucial for our understanding of neoplasia. In recent years, several new murine models for K-ras-induced tumorigenesis have been described. They allow new insights into the specific role that oncogenic K-ras proteins play in different solid tumors, and they permit the molecular dissection of the pathways that are initiated by somatic mutations in subsets of cells. Key advances have been made by the use of tissue-specific and inducible control of expression, which is achieved by the Cre/loxP technology or the tetracycline system. From these sophisticated models, a common picture emerges: the effects of K-ras on tumor initiation depend strongly on the cellular context, and different tissues vary in their susceptibility to K-ras transformation.

  11. The Rac1 splice form Rac1b promotes K-ras-induced lung tumorigenesis

    PubMed Central

    Zhou, Chun; Licciulli, Silvia; Avila, Jacqueline L.; Cho, Miyoung; Troutman, Scott; Jiang, Ping; Kossenkov, Andrew; Showe, Louise C.; Liu, Qin; Vachani, Anil; Albelda, Steven M.; Kissil, Joseph L.

    2012-01-01

    Rac1b, an alternative splice form of Rac1, has been previously shown to be upregulated in colon and breast cancer cells, suggesting an oncogenic role for Rac1b in these cancers. Our analysis of NSCLC tumor and matched normal tissue samples indicates Rac1b is upregulated in a significant fraction of lung tumors in correlation with mutational status of K-ras. To directly assess the oncogenic potential of Rac1b in vivo, we employed a mouse model of lung adenocarcinoma, in which the expression of Rac1b can be conditionally activated specifically in the lung. While expression of Rac1b alone is insufficient to drive tumor initiation, the expression of Rac1b synergizes with an oncogenic allele of K-ras resulting in increased cellular proliferation and accelerated tumor growth. Finally, we show that in contrast to our previous findings demonstrating a requirement for Rac1 in K-ras-driven cell proliferation, Rac1b is not required in this context. Given the partially overlapping spectrum of downstream effectors regulated by Rac1 and Rac1b, our findings further delineate the signaling pathways downstream of Rac1 that are required for K-ras driven tumorigenesis. PMID:22430205

  12. Impact of oncogenic K-RAS on YB-1 phosphorylation induced by ionizing radiation

    PubMed Central

    2011-01-01

    repair of DNA-DSB. Likewise, YB-1 siRNA increased radiation sensitivity. Conclusions IR induces YB-1 phosphorylation. YB-1 phosphorylation induced by oncogenic K-Ras or IR enhances repair of DNA-DSB and postirradiation survival via erbB1 downstream PI3K/Akt and MAPK/ERK signaling pathways. PMID:21392397

  13. Loss of miR-125a expression in a model of K-ras-dependent pulmonary premalignancy

    PubMed Central

    Liclican, Elvira L.; Walser, Tonya C.; Hazra, Saswati; Krysan, Kostyantyn; Park, Stacy J.; Pagano, Paul C.; Gardner, Brian K.; Larsen, Jill E.; Minna, John D.; Dubinett, Steven M.

    2014-01-01

    Understanding the molecular pathogenesis of lung cancer is necessary to identify biomarkers/targets specific to individual airway molecular profiles and to identify options for targeted chemoprevention. Herein, we identify mechanisms by which loss of microRNA (miRNA)-125a-3p (miR-125a) contributes to the malignant potential of human bronchial epithelial cells (HBECs) harboring an activating point mutation of the K-ras proto-oncogene (HBEC K-ras). Among other miRNAs, we identified significant miR-125a loss in HBEC K-ras lines and determined that miR-125a is regulated by the PEA3 transcription factor. PEA3 is upregulated in HBEC K-ras cells, and genetic knockdown of PEA3 restores miR-125a expression. From a panel of inflammatory/angiogenic factors, we identified increased CXCL1 and vascular endothelial growth factor (VEGF) production by HBEC K-ras cells and determined that miR-125a overexpression significantly reduces K-ras-mediated production of these tumorigenic factors. miR-125a overexpression also abrogates increased proliferation of HBEC K-ras cells and suppresses anchorage-independent growth (AIG) of HBEC K-ras/P53 cells, the latter of which is CXCL1-dependent. Finally, pioglitazone increases levels of miR-125a in HBEC K-ras cells via PEA3 downregulation. In addition, pioglitazone and miR-125a overexpression elicit similar phenotypic responses, including suppression of both proliferation and VEGF production. Our findings implicate miR-125a loss in lung carcinogenesis and lay the groundwork for future studies to determine if miR-125a is a possible biomarker for lung carcinogenesis and/or a chemoprevention target. Moreover, our studies illustrate that pharmacologic augmentation of miR-125a in K-ras-mutated pulmonary epithelium effectively abrogates several deleterious downstream events associated with the mutation. PMID:24913817

  14. Elevated K-ras activity with cholestyramine and lovastatin, but not konjac mannan or niacin in lung—importance of mouse strain

    PubMed Central

    Calvert, Richard J.; Tepper, Shirley; Kammouni, Wafa; Anderson, Lucy M.; Kritchevsky, David

    2007-01-01

    Our previous work established that hypocholesterolemic agents altered K-ras intracellular localization in lung. Here, we examined K-ras activity to define further its potential importance in lung carcinogenesis. K-ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For three weeks, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% konjac mannan (KM), or were injected with lovastatin 25 mg/kg three or five times weekly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After three weeks, serum cholesterol was assayed with a commercial kit. Activated K-ras protein from lung was affinity precipitated with a Raf-1 ras binding domain-glutathione-S-transferase fusion protein bound to glutathione-agarose beads, followed by Western blotting, K-ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in 2 of 3 mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-ras activity increased 1.8-fold versus control (p = 0.009). In normal lung with wild-type K-ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-ras activity increased 2.1-fold (p = 0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-ras activity. These data are consistent with the concept that K-ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type. PMID:17005160

  15. Alterations in K-ras, APC and p53-multiple genetic pathway in colorectal cancer among Indians.

    PubMed

    Malhotra, Pooja; Anwar, Mumtaz; Nanda, Neha; Kochhar, Rakesh; Wig, Jai Dev; Vaiphei, Kim; Mahmood, Safrun

    2013-06-01

    The incidence of colorectal cancer (CRC) is increasing rapidly in Asian countries during the past few decades, but no comprehensive analysis has been done to find out the exact cause of this disease. In this study, we investigated the frequencies of mutations and expression pattern of K-ras, APC (adenomatosis polyposis coli) and p53 in tumor, adjoining and distant normal mucosa and to correlate these alterations with patients clinicopathological parameters as well as with the survival. Polymerase chain reaction (PCR)-restriction digestion was used to detect mutations in K-ras and PCR-SSCP (Single Strand Conformation Polymorphism) followed by DNA sequencing was used to detect mutations in APC and p53 genes. Immunohistochemistry was used to detect the expression pattern of K-ras, APC and p53 proteins. The frequencies of mutations of K-ras, APC and p53 in 30 tumor tissues samples were 26.7 %, 46.7 % and 20 %, respectively. Only 3.3 % of tumors contained mutations in all the three genes. The most common combination of mutation was APC and p53 whereas mutation in both p53 and K-ras were extremely rare. There was no association between the mutations and expression pattern of K-ras, APC and p53 (p>0.05). In Indians, the frequency of alterations of K-ras and APC is similar as in Westerns, whereas the frequency of p53 mutation is slightly lower. The lack of multiple mutations in tumor specimens suggests that these genetic alterations might have independent influences on CRC development and there could be multiple alternative genetic pathways to CRC in our present study cohort.

  16. The effect of forced expression of mutated K-RAS gene on gastrointestinal cancer cell lines and the IGF-1R targeting therapy.

    PubMed

    Matsunaga, Yasutaka; Adachi, Yasushi; Sasaki, Yasushi; Koide, Hideyuki; Motoya, Masayo; Nosho, Katsuhiko; Takagi, Hideyasu; Yamamoto, Hiroyuki; Sasaki, Shigeru; Arimura, Yoshiaki; Tokino, Takashi; Carbone, David P; Imai, Kohzoh; Shinomura, Yasuhisa

    2017-02-01

    Mutation in K-RAS (K-RAS-MT) plays important roles in both cancer progression and resistance to anti-epidermal growth factor receptor (EGFR) therapy in gastrointestinal tumors. Insulin-like growth factor-1 receptor (IGF-1R) signaling is required for carcinogenicity and progression of many tumors as well. We have previously shown successful therapy for gastrointestinal cancer cell lines bearing a K-RAS mutation using an anti-IGF-1R monoclonal antibody. In this study, we sought to evaluate effects of forced K-RAS-MT expression on gastrointestinal cancer cell lines representing a possible second resistance mechanism for anti-EGFR therapy and IGF-1R-targeted therapy for these transfectants. We made stable transfectants of K-RAS-MT in two gastrointestinal cancer cell lines, colorectal RKO and pancreatic BxPC-3. We assessed the effect of forced expression of K-RAS-MT on proliferation, apoptosis, migration, and invasion in gastrointestinal cancer cells. Then we assessed anti-tumor effects of dominant negative IGF-1R (IGF-1R/dn) and an IGF-1R inhibitor, picropodophyllin, on the K-RAS-MT transfectants. Overexpression of K-RAS-MT in gastrointestinal cancer cell lines led to more aggressive phenotypes, with increased proliferation, decreased apoptosis, and increased motility and invasion. IGF-1R blockade suppressed cell growth, colony formation, migration, and invasion, and up-regulated chemotherapy-induced apoptosis of gastrointestinal cancer cells, even when K-RAS-MT was over-expressed. IGF-1R blockade inhibited the Akt pathway more than the extracellular signal-regulated kinase (ERK) pathway in the K-RAS-MT transfectants. IGF-1R/dn, moreover, inhibited the growth of murine xenografts expressing K-RAS-MT. Thus, K-RAS-MT might be important for progressive phonotype observed in gastrointestinal cancers. IGF-1R decoy is a candidate molecular therapeutic approach for gastrointestinal cancers even if K-RAS is mutated. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals

  17. K-ras-driven engineered mouse models for pancreatic cancer.

    PubMed

    Yin, Xuyuan; Su, Jingna; Zhou, Xiuxiao; Guo, Jianping; Wei, Wenyi; Wang, Zhiwei

    2015-01-01

    Pancreatic cancer (PC) is one of the most fatal cancers largely due to the lack of early diagnosis and effective therapies. Therefore, further understanding the underlying molecular mechanisms of PC development and progression is pivotal to the development of more effective targeted therapies. Emerging evidence has suggested that using mouse models, especially genetically engineered mouse models, is ideal to explore the mechanisms of pancreatic tumorigenesis. To this end, it has been known that a K-ras mutation on codon 12 (K-ras G12D) plays a critical role in the PC development. Thus, most mouse models of PC have been developed by targeting a conditionally mutated K-ras (G12D) together with concomitant deletion or mutation of other key genes to recapitulate the PC progression in human patients. Here, we summarize a number of K-ras-driven engineered mouse models to provide molecular insights into PC disease development and progression. We hope that these mouse models will help design a novel therapeutic strategy and to further improve the treatment of PC patients.

  18. Discordance of Mutation Statuses of Epidermal Growth Factor Receptor and K-ras between Primary Adenocarcinoma of Lung and Brain Metastasis.

    PubMed

    Rau, Kun-Ming; Chen, Han-Ku; Shiu, Li-Yen; Chao, Tsai-Ling; Lo, Yi-Ping; Wang, Chin-Chou; Lin, Meng-Chih; Huang, Chao-Cheng

    2016-04-07

    Mutations on epidermal growth factor receptor (EGFR) of adenocarcinomas of lung have been found to be associated with increased sensitivity to EGFR tyrosine kinase inhibitors and K-ras mutations may correlate with primary resistance. We aimed to explore the discordant mutation statuses of EGFR and K-ras between primary tumors and matched brain metastases in adenocarcinomas of lung. We used a sensitive Scorpion ARMS method to analyze EGFR mutation, and Sanger sequencing followed by allele-specific real-time polymerase chain reaction to analyze K-ras mutation. Forty-nine paired tissues with both primary adenocarcinoma of lung and matched brain metastasis were collected. Thirteen patients (26.5%) were discordant for the status of EGFR between primary and metastatic sites. K-ras gene could be checked in paired specimens from 33 patients, thirteen patients (39.6%) were discordant for the status of K-ras. In primary lung adenocarcinoma, there were 14 patients of mutant EGFR had mutant K-ras synchronously. This study revealed that the status of EGFR mutation in lung adenocarcinomas is relatively consistent between primary and metastatic sites compared to K-ras mutation. However, there are still a few cases of adenocarcinoma of lung showing discordance for the status of EGFR mutation. Repeated analysis of EGFR mutation is highly recommended if tissue from metastatic or recurrent site is available for the evaluation of target therapy.

  19. K-rasG12V mediated lung tumor models identified three new quantitative trait loci modifying events post-K-ras mutation.

    PubMed

    Saito, Hiromitsu; Suzuki, Noboru

    2014-10-03

    A high incidence of oncogenic K-ras mutations is observed in lung adenocarcinoma of human cases and carcinogen-induced animal models. The process of oncogenic K-ras-mediated lung adenocarcinogenesis can be dissected into two parts: pre- and post-K-ras mutation. Adoption of transgenic lines containing a flox-K-rasG12V transgene eliminates the use of chemical carcinogens and enables us to study directly crucial events post-K-ras mutation without considering the cellular events involved with oncogenic K-ras mutation, e.g., distribution and metabolism of chemical carcinogens, DNA repair, and somatic recombination by host factors. We generated two mouse strains C57BL/6J-Ryr2(tm1Nobs) and A/J-Ryr2(tm1Nobs) in which K-rasG12V can be transcribed from the cytomegalovirus early enhancer/chicken beta actin promoter in virtually any tissue. Upon K-rasG12V induction in lung epithelial cells by an adenovirus expressing the Cre recombinase, the number of tumors in the C57BL/6J-Ryr2(tm1Nobs/+) mouse line was 12.5 times that in the A/J-Ryr2(tm1Nobs/+) mouse line. Quantitative trait locus (QTL) analysis revealed that new three modifier loci, D3Mit19, D3Mit45 and D11Mit20, were involved in the differential susceptibility between the two lines. In addition, we found that differential expression of the wild-type K-ras gene, which was genetically turn out to be anti-oncogenic activity on K-rasG12V, could not account for the different susceptibility in our two K-rasG12V-mediated lung tumor models. Thus, we provide a genetic system that enables us to explore new downstream modifiers post-K-ras mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane

    PubMed Central

    Cho, Kwang-jin; van der Hoeven, Dharini; Zhou, Yong; Maekawa, Masashi; Ma, Xiaoping; Chen, Wei

    2015-01-01

    K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization. PMID:26572827

  1. Inhibition of Acid Sphingomyelinase Depletes Cellular Phosphatidylserine and Mislocalizes K-Ras from the Plasma Membrane.

    PubMed

    Cho, Kwang-Jin; van der Hoeven, Dharini; Zhou, Yong; Maekawa, Masashi; Ma, Xiaoping; Chen, Wei; Fairn, Gregory D; Hancock, John F

    2015-11-16

    K-Ras must localize to the plasma membrane for biological activity; thus, preventing plasma membrane interaction blocks K-Ras signal output. Here we show that inhibition of acid sphingomyelinase (ASM) mislocalizes both the K-Ras isoforms K-Ras4A and K-Ras4B from the plasma membrane to the endomembrane and inhibits their nanoclustering. We found that fendiline, a potent ASM inhibitor, reduces the phosphatidylserine (PtdSer) and cholesterol content of the inner plasma membrane. These lipid changes are causative because supplementation of fendiline-treated cells with exogenous PtdSer rapidly restores K-Ras4A and K-Ras4B plasma membrane binding, nanoclustering, and signal output. Conversely, supplementation with exogenous cholesterol restores K-Ras4A but not K-Ras4B nanoclustering. These experiments reveal different operational pools of PtdSer on the plasma membrane. Inhibition of ASM elevates cellular sphingomyelin and reduces cellular ceramide levels. Concordantly, delivery of recombinant ASM or exogenous ceramide to fendiline-treated cells rapidly relocalizes K-Ras4B and PtdSer to the plasma membrane. K-Ras4B mislocalization is also recapitulated in ASM-deficient Neimann-Pick type A and B fibroblasts. This study identifies sphingomyelin metabolism as an indirect regulator of K-Ras4A and K-Ras4B signaling through the control of PtdSer plasma membrane content. It also demonstrates the critical and selective importance of PtdSer to K-Ras4A and K-Ras4B plasma membrane binding and nanoscale spatial organization.

  2. Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis.

    PubMed

    Ulus-Senguloglu, G; Arlett, C F; Plowman, P N; Parnell, J; Patel, N; Bourton, E C; Parris, C N

    2012-10-23

    The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells. Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.

  3. Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis

    PubMed Central

    Ulus-Senguloglu, G; Arlett, C F; Plowman, P N; Parnell, J; Patel, N; Bourton, E C; Parris, C N

    2012-01-01

    Background: The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients. Methods: Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells. Results: Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis. Conclusion: We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity. PMID:23093295

  4. v-K-ras leads to preferential farnesylation of p21ras in FRTL-5 cells: Multiple interference with the isoprenoid pathway

    PubMed Central

    Laezza, Chiara; Di Marzo, Vincenzo; Bifulco, Maurizio

    1998-01-01

    The isoprenoid pathway in FRTL-5 thyroid cells was found to be deeply altered on transformation with v-K-ras. A dramatic overall reduction of protein prenylation was found in v-K-ras-transformed cells in comparison with the parent FRTL-5 cells, as shown by labeling cells with [3H]mevalonic acid. This phenomenon was accompanied by a relative increase of p21ras farnesylation and by a decrease of the ratio between the amounts of geranylgeraniol and farnesol bound to prenylated proteins. Analysis of protein prenylation in FRTL-5 cells transformed by a temperature-sensitive mutant of the v-K-ras oncogene indicated that these variations represent an early and specific marker of active K-ras. Conversely, FRTL-5 cells transformed with Harvey-ras showed a pattern of [3H]-mevalonate (MVA)-labeled proteins similar to that of nontransformed cells. The K-ras oncogene activation also resulted in an overall decrease of [3H]-MVA incorporation into isopentenyl-tRNA together with an increase of unprocessed [3H]-MVA and no alteration in [3H]-MVA uptake. The effects of v-K-ras on protein prenylation could be mimicked in FRTL-5 cells by lowering the concentration of exogenous [3H]-MVA whereas increasing the [3H]-MVA concentration did not revert the alterations observed in transformed cells. Accordingly, v-K-ras expression was found to: (i) down-regulate mevalonate kinase; (ii) induce farnesyl-pyrophosphate synthase expression; and (iii) augment protein farnesyltransferase but not protein geranylgeranyl-transferase-I activity. Among these events, mevalonate kinase down-regulation appeared to be related strictly to differential protein prenylation. This study represents an example of how expression of the v-K-ras oncogene, through multiple interferences with the isoprenoid metabolic pathway, may result in the preferential farnesylation of the ras oncogene product p21ras. PMID:9811854

  5. K-Ras4B proteins are expressed in the nucleolus: Interaction with nucleolin

    SciTech Connect

    Birchenall-Roberts, Maria C. . E-mail: birchena@mail.ncifcrf.gov; Fu, Tao; Kim, Soo-Gyung; Huang, Ying K.; Dambach, Michael; Resau, James H.; Ruscetti, Francis W.

    2006-09-22

    Kirsten Ras4B (K-Ras4B) is a potent onco-protein that is expressed in the majority of human cell types and is frequently mutated in carcinomas. K-Ras4B, like other members of the Ras family of proteins, is considered to be a cytoplasmic protein that must be localized to the plasma membrane for activation. Here, using confocal microscopy and biochemical analysis, we show that K-Ras4B, but not H-Ras or the closely related K-Ras4A, is also present in the nucleoli of normal and transformed cells. Subcellular fractionation and immunostaining show that K-Ras4B is located not only in the cytoplasm, but also in the nucleolar compartment. Modification of a C-terminal hexa-lysine motif unique to K-Ras4B results in exclusively cytoplasmic forms of the protein. Nucleolin, a pleiotropic regulator of cellular processes, including transcriptional regulation, is also characterized by a nucleolar-like nuclear appearance. We show that K-Ras4B and nucleolin co-localize within the nucleus and that nucleolin physically associates with K-Ras4B. Inhibition of K-Ras4B/nucleolin association blocked nucleolar localization of K-Ras4B. Using siRNA to knockdown the expression of nucleolin eliminated the nucleolar localization of K-Ras4B and significantly repressed the activation of the well-characterized K-Ras4B transcriptional target Ap-1, but stimulated Elk1. These data provide evidence of a nucleolar localization of K-Ras4B and describe a functional association between K-Ras4B and nucleolin.

  6. 4-Hydroxytamoxifen induces autophagic death through K-Ras degradation.

    PubMed

    Kohli, Latika; Kaza, Niroop; Coric, Tatjana; Byer, Stephanie J; Brossier, Nicole M; Klocke, Barbara J; Bjornsti, Mary-Ann; Carroll, Steven L; Roth, Kevin A

    2013-07-15

    Tamoxifen is widely used to treat estrogen receptor-positive breast cancer. Recent findings that tamoxifen and its derivative 4-hydroxytamoxifen (OHT) can exert estrogen receptor-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in estrogen receptor-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNST) where estrogen is not involved. In this study, we gained insights into the estrogen receptor-independent cytotoxic effects of OHT by studying how it kills MPNST cells. Although caspases were activated following OHT treatment, caspase inhibition provided no protection from OHT-induced death. Rather, OHT-induced death in MPNST cells was associated with autophagic induction and attenuated by genetic inhibition of autophagic vacuole formation. Mechanistic investigations revealed that OHT stimulated autophagic degradation of K-Ras, which is critical for survival of MPNST cells. Similarly, we found that OHT induced K-Ras degradation in breast, colon, glioma, and pancreatic cancer cells. Our findings describe a novel mechanism of autophagic death triggered by OHT in tumor cells that may be more broadly useful clinically in cancer treatment.

  7. Blocking anaplerotic entry of glutamine into the TCA cycle sensitizes K-Ras mutant cancer cells to cytotoxic drugs.

    PubMed

    Saqcena, M; Mukhopadhyay, S; Hosny, C; Alhamed, A; Chatterjee, A; Foster, D A

    2015-05-14

    Cancer cells undergo a metabolic transformation that allows for increased anabolic demands, wherein glycolytic and tricarboxylic acid (TCA) cycle intermediates are shunted away for the synthesis of biological molecules required for cell growth and division. One of the key shunts is the exit of citrate from the mitochondria and the TCA cycle for the generation of cytosolic acetyl-coenzyme A that can be used for fatty acid and cholesterol biosynthesis. With the loss of mitochondrial citrate, cancer cells rely on the 'conditionally essential' amino acid glutamine (Q) as an anaplerotic carbon source for TCA cycle intermediates. Although Q deprivation causes G1 cell cycle arrest in non-transformed cells, its impact on the cancer cell cycle is not well characterized. We report here a correlation between bypass of the Q-dependent G1 checkpoint and cancer cells harboring K-Ras mutations. Instead of arresting in G1 in response to Q-deprivation, K-Ras-driven cancer cells arrest in either S- or G2/M-phase. Inhibition of K-Ras effector pathways was able to revert cells to G1 arrest upon Q deprivation. Blocking anaplerotic utilization of Q mimicked Q deprivation--causing S- and G2/M-phase arrest in K-Ras mutant cancer cells. Significantly, Q deprivation or suppression of anaplerotic Q utilization created synthetic lethality to the cell cycle phase-specific cytotoxic drugs, capecitabine and paclitaxel. These data suggest that disabling of the G1 Q checkpoint could represent a novel vulnerability of cancer cells harboring K-Ras and possibly other mutations that disable the Q-dependent checkpoint.

  8. Downregulation of a novel human gene, ROGDI, increases radiosensitivity in cervical cancer cells

    PubMed Central

    Chen, Yi-Fan; Cho, Jonathan J.; Huang, Tsai-Hua; Tseng, Chao-Neng; Huang, Eng-Yen; Cho, Chung-Lung

    2016-01-01

    ABSTRACT ROGDI is a protein that contains a leucine zipper domain and may be involved in cell proliferation. In addition, ROGDI is associated with genome stability by regulating the activity of a DNA damage marker, γ-H2AX. The role of ROGDI in tumor radiosensitization has not been investigated. Previous studies have indicated that radiosensitivity is associated with DNA repair and the cell cycle. In general, the G2/M DNA damage checkpoint is more sensitive to radiation, whereas the G1/S phase transition is more resistant to radiation. Inhibition of cyclin-dependent kinases (CDKs) can lead to a halt of cell cycle progression and a stay at different phases or checkpoints. Our data show that the downregulation of ROGDI led to a decreased expression of CDK 1, 2, cyclin A, B and resulted in a G2/M phase transition block. In addition, the downregulation of ROGDI increased cell accumulation at the G2 phase as detected using flow cytometry and decreased cell survival as revealed by clonogenic assay in HeLa and C33A cells following irradiation. These findings suggest that the downregulation of ROGDI can mediate radiosensitivity by blocking cells at G2/M, the most radiosensitive phase of the cell cycle, as well as exerting deleterious effects in the form of DNA damage, as shown by increased γ-H2AX activation. PMID:27636029

  9. Downregulation of a novel human gene, ROGDI, increases radiosensitivity in cervical cancer cells.

    PubMed

    Chen, Yi-Fan; Cho, Jonathan J; Huang, Tsai-Hua; Tseng, Chao-Neng; Huang, Eng-Yen; Cho, Chung-Lung

    2016-08-12

    ROGDI is a protein that contains a leucine zipper domain and may be involved in cell proliferation. In addition, ROGDI is associated with genome stability by regulating the activity of a DNA damage marker, γ-H2AX. The role of ROGDI in tumor radiosensitization has not been investigated. Previous studies have indicated that radiosensitivity is associated with DNA repair and the cell cycle. In general, the G2/M DNA damage checkpoint is more sensitive to radiation, whereas the G1/S phase transition is more resistant to radiation. Inhibition of cyclin-dependent kinases (CDKs) can lead to a halt of cell cycle progression and a stay at different phases or checkpoints. Our data show that the downregulation of ROGDI led to a decreased expression of CDK 1, 2, cyclin A, B and resulted in a G2/M phase transition block. In addition, the downregulation of ROGDI increased cell accumulation at the G2 phase as detected using flow cytometry and decreased cell survival as revealed by clonogenic assay in HeLa and C33A cells following irradiation. These findings suggest that the downregulation of ROGDI can mediate radiosensitivity by blocking cells at G2/M, the most radiosensitive phase of the cell cycle, as well as exerting deleterious effects in the form of DNA damage, as shown by increased γ-H2AX activation.

  10. Pten Inactivation Accelerates Oncogenic K-ras-Initiated Tumorigenesis in a Mouse Model of Lung Cancer

    PubMed Central

    Iwanaga, Kentaro; Yang, Yanan; Raso, Maria Gabriela; Ma, Lijiang; Hanna, Amy E.; Thilaganathan, Nishan; Moghaddam, Seyed; Evans, Christopher M.; Li, Huaiguang; Cai, Wei-Wen; Sato, Mitsuo; Minna, John D.; Wu, Hong; Creighton, Chad J.; Demayo, Francesco J.; Wistuba, Ignacio I.; Kurie, Jonathan M.

    2009-01-01

    Phosphatase and tensin homologue deleted from chromosome 10 (Pten) is expressed aberrantly in non-small cell lung cancer cells, but the role of Pten in lung neoplasia has not been fully elucidated. In this study, we used a genetic approach to inactivate Pten in the bronchial epithelium of mice. Although, by itself, Pten inactivation had no discernible effect on bronchial epithelial histology, it accelerated lung tumorigenesis initiated by oncogenic K-ras, causing more rapid lethality than that induced by oncogenic K-ras alone (8 weeks versus 24 weeks of median duration of survival, respectively). Lung tumors arose in K-ras mutant, Pten-deficient mice that rapidly obstructed bronchial lumina and replaced alveolar spaces. Relative to K-ras mutant tumors, the K-ras mutant, Pten-deficient tumors exhibited more advanced histologic severity and more prominent inflammation and vascularity. Thus, Pten inactivation cooperated with oncogenic K-ras in promoting lung tumorigenesis. PMID:18281487

  11. IMP-1 displays cross-talk with K-Ras and modulates colon cancer cell survival through the novel proapoptotic protein CYFIP2.

    PubMed

    Mongroo, Perry S; Noubissi, Felicite K; Cuatrecasas, Miriam; Kalabis, Jiri; King, Catrina E; Johnstone, Cameron N; Bowser, Mark J; Castells, Antoni; Spiegelman, Vladimir S; Rustgi, Anil K

    2011-03-15

    Insulin-like growth factor 2 mRNA-binding protein-1 (IMP-1) is an oncofetal protein that binds directly to and stabilizes oncogenic c-Myc and regulates, in turn, its posttranscriptional expression and translation. In contrast to normal adult tissue, IMP-1 is reexpressed and/or overexpressed in human cancers. We show that knockdown of c-Myc in human colon cancer cell lines increases the expression of mature let-7 miRNA family members and downregulates several of its mRNA targets: IMP-1, Cdc34, and K-Ras. We further show that loss of IMP-1 inhibits Cdc34, Lin-28B, and K-Ras, suppresses SW-480 cell proliferation and anchorage-independent growth, and promotes caspase- and lamin-mediated cell death. We also found that IMP-1 binds to the coding region and 3'UTR of K-Ras mRNA. RNA microarray profiling and validation by reverse transcription PCR reveals that the p53-inducible proapoptotic protein CYFIP2 is upregulated in IMP-1 knockdown SW480 cells, a novel finding. We also show that overexpression of IMP-1 increases c-Myc and K-Ras expression and LIM2405 cell proliferation. Furthermore, we show that loss of IMP-1 induces Caspase-3- and PARP-mediated apoptosis, and inhibits K-Ras expression in SW480 cells, which is rescued by CYFIP2 knockdown. Importantly, analysis of 228 patients with colon cancers reveals that IMP-1 is significantly upregulated in differentiated colon tumors (P ≤ 0.0001) and correlates with K-Ras expression (r = 0.35, P ≤ 0.0001) relative to adjacent normal mucosa. These findings indicate that IMP-1, interrelated with c-Myc, acts upstream of K-Ras to promote survival through a novel mechanism that may be important in colon cancer pathogenesis. ©2011 AACR.

  12. Deletion of Pim Kinases Elevates the Cellular Levels of Reactive Oxygen Species and Sensitizes to K-Ras-Induced Cell Killing

    PubMed Central

    Song, Jin H.; An, Ningfei; Chatterjee, Shilpak; Kistner-Griffin, Emily; Mahajan, Sandeep; Mehrotra, Shikhar; Kraft, Andrew S.

    2014-01-01

    The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-RasG12V) and undergo cell death. Transduction of K-RasG12V into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuates ROS production and reversed the cytotoxic effects of K-RasG12V in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-RasG12V-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-RasG12V-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases play an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth. PMID:25241892

  13. Alcohol and the risk of colon and rectal cancer with mutations in the K-ras gene.

    PubMed

    Bongaerts, Brenda W C; de Goeij, Anton F P M; van den Brandt, Piet A; Weijenberg, Matty P

    2006-04-01

    The first metabolite of alcohol, acetaldehyde, may trigger replication errors and mutations in DNA, which may predispose to developing colorectal cancer (CRC). In a prospective study on colon and rectal cancer, we investigated the following hypotheses: alcohol consumption is associated with an increased risk of mutations in the K-ras oncogene, and beer consumption is associated with an increased risk of G-->A mutations in this gene. Therefore, we studied the associations between consumption of alcohol and alcoholic beverages and the risk of CRC without and with specific K-ras gene mutations. In 1986, 120,852 men and women, aged 55-69 years, completed a questionnaire on risk factors for cancer. The case-cohort approach was used for data processing and analyses. After 7.3 years of follow-up, excluding the first 2.3 years, complete data from 4,076 subcohort members, 428 colon and 150 rectal cancer patients, were available for data analyses. Incidence rate ratios (RRs) and corresponding 95% confidence intervals (95% CIs) were estimated using Cox proportional hazards models. Compared to abstaining, a total alcohol consumption of 30.0 g/day and more was associated with the risk of colon and rectal cancer with and without a K-ras mutation in both men and women. Independent from alcohol intake, liquor consumption when compared to nonliquor consumption was associated with an increased risk of rectal cancer with a wild type K-ras in men (RR: 2.25, 95% CI: 1.0-5.0). Beer consumption was not clearly associated with the risk of colon and rectal tumors harboring G-->A mutations in the K-ras gene in men. This association could not be assessed in women because of sparse beer consumption. In conclusion, alcohol does not seem to be involved in predisposing to CRC through mutations in the K-ras gene, and specifically beer consumption is not associated with colon and rectal tumors harboring a G-->A mutation.

  14. Nicotine does not enhance tumorigenesis in mutant K-ras-driven mouse models of lung cancer.

    PubMed

    Maier, Colleen R; Hollander, M Christine; Hobbs, Evthokia A; Dogan, Irem; Linnoila, R Ilona; Dennis, Phillip A

    2011-11-01

    Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiologic levels of nicotine on lung tumor formation, tumor growth, or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Tumors that develop in this model have mutations in K-ras, which is commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size and did not affect overall survival. Likewise, in a syngeneic model using lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable with those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiologic data showing that NRT does not enhance lung cancer risk in former smokers.

  15. Nicotine does not enhance tumorigenesis in mutant K-Ras-driven mouse models of lung cancer

    PubMed Central

    Maier, Colleen R.; Hollander, M. Christine; Hobbs, Evthokia A.; Dogan, Irem; Dennis, Phillip A.

    2011-01-01

    Smoking is the leading cause of preventable cancer deaths in the United States. Nicotine replacement therapies (NRT) have been developed to aid in smoking cessation, which decreases lung cancer incidence. However, the safety of NRT is controversial because numerous preclinical studies have shown that nicotine enhances tumor cell growth in vitro and in vivo. We modeled NRT in mice to determine the effects of physiological levels of nicotine on lung tumor formation, tumor growth or metastasis. Nicotine administered in drinking water did not enhance lung tumorigenesis after treatment with the tobacco carcinogen, NNK. Tumors that develop in this model have mutations in K-ras, which is a commonly observed in smoking-related, human lung adenocarcinomas. In a transgenic model of mutant K-ras-driven lung cancer, nicotine did not increase tumor number or size, and did not affect overall survival. Likewise, in a syngeneic model of lung cancer cell lines derived from NNK-treated mice, oral nicotine did not enhance tumor growth or metastasis. These data show that nicotine does not enhance lung tumorigenesis when given to achieve levels comparable to those of NRT, suggesting that nicotine has a dose threshold, below which it has no appreciable effect. These studies are consistent with epidemiological data showing that NRT does not enhance lung cancer risk in former smokers. PMID:22027685

  16. The noncoding RNAs SNORD50A and SNORD50B bind K-Ras and are recurrently deleted in human cancer

    PubMed Central

    Siprashvili, Zurab; Webster, Dan E; Johnston, Danielle; Shenoy, Rajani M; Ungewickell, Alexander J; Bhaduri, Aparna; Flockhart, Ross; Zarnegar, Brian J; Che, Yonglu; Meschi, Francesca; Puglisi, Joseph D; Khavari, Paul A

    2017-01-01

    Small nucleolar RNAs (snoRNAs) are conserved noncoding RNAs best studied as ribonucleoprotein (RNP) guides in RNA modification1,2. To explore their role in cancer, we compared 5,473 tumor-normal genome pairs to identify snoRNAs with frequent copy number loss. The SNORD50A-SNORD50B snoRNA locus was deleted in 10–40% of 12 common cancers, where its loss was associated with reduced survival. A human protein microarray screen identified direct SNORD50A and SNORD50B RNA binding to K-Ras. Loss of SNORD50A and SNORD50B increased the amount of GTP-bound, active K-Ras and hyperactivated Ras-ERK1/ERK2 signaling. Loss of these snoRNAs also increased binding by farnesyltransferase to K-Ras and increased K-Ras prenylation, suggesting that KRAS mutation might synergize with SNORD50A and SNORD50B loss in cancer. In agreement with this hypothesis, CRISPR-mediated deletion of SNORD50A and SNORD50B in KRAS-mutant tumor cells enhanced tumorigenesis, and SNORD50A and SNORD50B deletion and oncogenic KRAS mutation co-occurred significantly in multiple human tumor types. SNORD50A and SNORD50B snoRNAs thus directly bind and inhibit K-Ras and are recurrently deleted in human cancer. PMID:26595770

  17. Metastatic colorectal cancer first-line treatment with bevacizumab: the impact of K-ras mutation.

    PubMed

    Rossi, Luigi; Veltri, Enzo; Zullo, Angelo; Zoratto, Federica; Colonna, Maria; Longo, Flavia; Mottolese, Marcella; Giannarelli, Diana; Ruco, Luigi; Marchetti, Paolo; Romiti, Adriana; Barucca, Viola; Giannini, Giuseppe; Bianchi, Loredana; Tomao, Silverio

    2013-01-01

    Bevacizumab plus chemotherapy prolongs progression-free survival (PFS) and overall survival (OS) in metastatic colorectal cancer (mCRC). Although there is strong evidence to suggest that the mutational status of the K-ras oncogene has a role as a predictive factor for activity in patients treated with cetuximab and panitumumab, few data have been obtained in patients treated with bevacizumab. We conducted an additional retrospective analysis to investigate the prognostic value of K-ras mutation relative to mCRC first-line treatment with bevacizumab. A total of 108 patients were retrospectively reviewed. K-ras status was assessed in the overall population by sequencing. Statistical association for PFS and OS was analyzed using the Kaplan-Meier method, and the prognostic role of K-ras was determined using the logrank test. Median PFS was 10 months both for patients with wild-type (WT) K-ras and mutated (MT) K-ras (hazard ratio [HR] 0.94, P=0.75); neither difference in median OS was significant (27 months WT K-ras versus 26 months MT K-ras, HR 0.92; P=0.70). A further analysis was carried out in the two groups according to metastatic sites. No statistically significant difference in terms of PFS and OS was demonstrated between WT K-ras and MT K-ras with liver metastases only and in those with extrahepatic disease. Although further study is required, our results seem to confirm that K-ras mutation does not have a prognostic role in mCRC patients receiving first-line treatment with bevacizumab.

  18. Identification of effective siRNA against K-ras in human pancreatic cancer cell line MiaPaCa-2 by siRNA expression cassette.

    PubMed

    Wang, Wei; Wang, Chun-You; Dong, Ju-Hua; Chen, Xiong; Zhang, Min; Zhao, Gang

    2005-04-07

    We shall construct the small interfering RNA (siRNA) expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities. Three different sites of SECs were constructed by PCR. K1/siRNA, K2/siRNA and K3/siRNA are located at sites 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively. Introduction of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells. K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras but K3/siRNA has no effect, demonstrating that K1/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

  19. Multiple cellular proteins modulate the dynamics of K-ras association with the plasma membrane.

    PubMed

    Bhagatji, Pinkesh; Leventis, Rania; Rich, Rebecca; Lin, Chen-ju; Silvius, John R

    2010-11-17

    Although specific proteins have been identified that regulate the membrane association and facilitate intracellular transport of prenylated Rho- and Rab-family proteins, it is not known whether cellular proteins fulfill similar roles for other prenylated species, such as Ras-family proteins. We used a previously described method to evaluate how several cellular proteins, previously identified as potential binding partners (but not effectors) of K-ras4B, influence the dynamics of K-ras association with the plasma membrane. Overexpression of either PDEδ or PRA1 enhances, whereas knockdown of either protein reduces, the rate of dissociation of K-ras from the plasma membrane. Inhibition of calmodulin likewise reduces the rate of K-ras dissociation from the plasma membrane, in this case in a manner specific for the activated form of K-ras. By contrast, galectin-3 specifically reduces the rate of plasma membrane dissociation of activated K-ras, an effect that is blocked by the K-ras antagonist farnesylthiosalicylic acid (salirasib). Multiple cellular proteins thus control the dynamics of membrane association and intercompartmental movement of K-ras to an important degree even under basal cellular conditions. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. Detection of K-ras oncogene from the human genomic DNA using ultrasonication and a quantum dots-based microfluidic chip.

    PubMed

    Noh, Han Na; Kim, Jong Sung

    2013-09-01

    In clinical diagnostics, single-stranded DNAs (ssDNA) have been prepared from the human genomic DNA for the detection of a specific gene. In this study, the human genomic DNA was degraded via ultrasonication in solution, and K-ras oncogene was detected from the DNA fragments via the fluorescence quenching of quantum dots (QDs) by intercalating dyes after hybridization of the target, to probe DNAs in a microfluidic chip. K-ras is one of the most activated common oncogenes, and many human tumors are known to be due to the mutation of this gene. QDs are nano-sized semiconductors with a wide selection of emission wavelengths and exceptional stability against photo bleaching. In this study, probe DNA-conjugated QDs were immobilized to polystyrene microbeads, and the DNA-microbead-QDs complexes were packed through a microchannel by pillars that trap the beads in the microfluidic chip. The fluorescence of the QDs could be quenched by intercalating dye (TOTO-3) after hybridization of K-ras oncogene to the probe DNA in the channel. The fluorescence intensity decrease of the QDs can be used as an indication of the K-ras oncogene. By introducing an alkaline buffer solution, the DNAs were denatured, and the fluorescence intensity of the QDs again increased, which shows the possibility of reuse of the microfluidic chip for the detection of the K-ras gene.

  1. Gene expression studies demonstrate that the K-ras/Erk MAP kinase signal transduction pathway and other novel pathways contribute to the pathogenesis of cumene-induced lung tumors.

    PubMed

    Wakamatsu, Nobuko; Collins, Jennifer B; Parker, Joel S; Tessema, Mathewos; Clayton, Natasha P; Ton, Thai-Vu T; Hong, Hue-Hua L; Belinsky, Steven; Devereux, Theodora R; Sills, Robert C; Lahousse, Stephanie A

    2008-07-01

    National Toxicology Program (NTP) inhalation studies demonstrated that cumene significantly increased the incidence of alveolar/bronchiolar adenomas and carcinomas in B6C3F1 mice. Cumene or isopropylbenzene is a component of crude oil used primarily in the production of phenol and acetone. The authors performed global gene expression analysis to distinguish patterns of gene regulation between cumene-induced tumors and normal lung tissue and to look for patterns based on the presence or absence of K-ras and p53 mutations in the tumors. Principal component analysis segregated the carcinomas into groups with and without K-ras mutations, but failed to separate the tumors based on p53 mutation status. Expression of genes associated with the Erk MAP kinase signaling pathway was significantly altered in carcinomas with K-ras mutations compared to tumors without K-ras mutations or normal lung. Gene expression analysis also suggested that cumene-induced carcinomas with K-ras mutations have greater malignant potential than those without mutations. In addition, significance analysis of function and expression (SAFE) demonstrated expression changes of genes regulated by histone modification in carcinomas with K-ras mutations. The gene expression analysis suggested the formation of alveolar/bronchiolar carcinomas in cumene-exposed mice typically involves mutation of K-ras, which results in increased Erk MAP kinase signaling and modification of histones.

  2. SIGNAL TRANSDUCTION. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling.

    PubMed

    Zhou, Yong; Wong, Ching-On; Cho, Kwang-jin; van der Hoeven, Dharini; Liang, Hong; Thakur, Dhananiay P; Luo, Jialie; Babic, Milos; Zinsmaier, Konrad E; Zhu, Michael X; Hu, Hongzhen; Venkatachalam, Kartik; Hancock, John F

    2015-08-21

    Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras-dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

  3. Zidovudine, abacavir and lamivudine increase the radiosensitivity of human esophageal squamous cancer cell lines.

    PubMed

    Chen, Xuan; Wang, Cong; Guan, Shanghui; Liu, Yuan; Han, Lihui; Cheng, Yufeng

    2016-07-01

    Telomerase is a type of reverse transcriptase that is overexpressed in almost all human tumor cells, but not in normal tissues, which provides an opportunity for radiosensitization targeting telomerase. Zidovudine, abacavir and lamivudine are reverse transcriptase inhibitors that have been applied in clinical practice for several years. We sought to explore the radiosensitization effect of these three drugs on human esophageal cancer cell lines. Eca109 and Eca9706 cells were treated with zidovudine, abacavir and lamivudine for 48 h before irradiation was administered. Samples were collected 1 h after irradiation. Clonal efficiency assay was used to evaluate the effect of the combination of these drugs with radiation doses of 2, 4, 6 and 8 Gy. DNA damage was measured by comet assay. Telomerase activity (TA) and relative telomere length (TL) were detected and evaluated by real-time PCR. Apoptosis rates were assessed by flow cytometric analysis. The results showed that all the drugs tested sensitized the esophageal squamous cell carcinoma (ESCC) cell lines to radiation through an increase in radiation-induced DNA damage and cell apoptosis, deregulation of TA and decreasing the shortened TL caused by radiation. Each of the drugs investigated (zidovudine, abacavir and lamivudine) could be used for sensitizing human esophageal cancer cell lines to radiation. Consequently, the present study supports the potential of these three drugs as therapeutic agents for the radiosensitization of esophageal squamous cell cancer.

  4. Involvement of oncogenic K-ras on cell migration stimulated by lysophosphatidic acid receptor-2 in pancreatic cancer cells.

    PubMed

    Yoshikawa, Kyohei; Tanabe, Eriko; Shibata, Ayano; Inoue, Serina; Kitayoshi, Misaho; Okimoto, Souta; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2013-02-01

    Lysophosphatidic acid (LPA) mediates a variety of cellular responses with atleast six G protein-coupled transmembrane receptors (LPA receptor-1 (LPA(1)-LPA(6))). The interaction between LPA receptors and other cellular molecules on the biological function is not fully understood. Recently, we have reported that LPA(1) suppressed and LPA(3) stimulated cell migration of pancreatic cancer cells. In the present study, to evaluate the function of LPA(2) on motile and invasive activities of pancreatic cancer cells, we generated Lpar2 knockdown (HPD-sh2) cells from hamster pancreatic cancer cells and measured their cell migration ability. In cell motility and invasive assays with an uncoated Cell Culture Insert, HPD-sh2 cells showed significantly lower intrinsic activity than control (HPD-GFP) cells. Since K-ras mutations were frequently detected in pancreatic cancer, we next investigated whether oncogenic K-ras is involved in cell migration induced by LPA(2) using K-ras knockdown (HPD-K2) cells. The cell motile ability of HPD-K2 cells was significantly lower than that of control cells. To confirm LPA(2) increases cell migration activity, cells were pretreated with dioctylglycerol pyrophosphate (DGPP) which is the antagonist of LPA(1)/LPA(3). The cell motile and invasive abilities of DGPP -treated HPD-GFP cells were markedly higher than those of untreated cells, but DGPP did not stimulate cell migration of HPD-K2 cells. These results suggest that cell migration activity of pancreatic cancer cells stimulated by LPA(2) may be enhanced by oncogenic K-ras. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Dual targeting of PI3K and MEK enhances the radiation response of K-RAS mutated non-small cell lung cancer

    PubMed Central

    Toulany, Mahmoud; Iida, Mari; Keinath, Simone; Iyi, Firdevs F.; Mueck, Katharina; Fehrenbacher, Birgit; Mansour, Wael Y.; Schaller, Martin; Wheeler, Deric L.; Rodemann, H. Peter

    2016-01-01

    Despite the significant contribution of radiotherapy to non-small lung cancer (NSCLC), radioresistance still occurs. One of the major radioresistance mechanisms is the hyperactivation of the PI3K/Akt pathway in which Akt facilitates the repair of DNA double-strand breaks (DSBs) through the stimulation of DNA-PKcs. We investigated if targeting PI3K would be a potential approach for enhancing the radiosensitivity of K-RAS mutated (K-RASmut) NSCLC cell lines A549 and H460. Short-term (1-2 h) pre-treatment of cells with the PI3K inhibitor PI-103 (1 μM) inhibited Akt/DNA-PKcs activity, blocked DSBs repair and induced radiosensitivity, while long-term (24 h) pre-treatment did not. Lack of an effect after 24 h of PI-103 pre-treatment was due to reactivation of K-Ras/MEK/ERK-dependent Akt. However, long-term treatment with the combination of PI-103 and MEK inhibitor PD98059 completely blocked reactivation of Akt and impaired DSBs repair through non-homologous end joining (NHEJ) leading to radiosensitization. The effect of PI3K inhibition on Akt signaling was also tested in A549 mouse xenografts. P-Akt and P-DNA-PKcs were inhibited 30 min post-irradiation in xenografts, which were pretreated by PI-103 30 min before irradiation. However, Akt was reactivated 30 min post-irradiation in tumors, which were pre-treated for 3 h with PI-103 before irradiation. After a 24 h pretreatment with PI-103, a significant reactivation of Akt was achieved 24 h after irradiation. Thus, due to MEK/ERK-dependent reactivation of Akt, targeting PI3K alone is not a suitable approach for radiosensitizing K-RASmut NSCLC cells, indicating that dual targeting of PI3K and MEK is an efficient approach to improve radiotherapy outcome. PMID:27248324

  6. Nitrative and oxidative DNA damage caused by K-ras mutation in mice

    SciTech Connect

    Ohnishi, Shiho; Saito, Hiromitsu; Suzuki, Noboru; Ma, Ning; Hiraku, Yusuke; Murata, Mariko; Kawanishi, Shosuke

    2011-09-23

    Highlights: {yields} Mutated K-ras in transgenic mice caused nitrative DNA damage, 8-nitroguanine. {yields} The mutagenic 8-nitroguanine seemed to be generated by iNOS via Ras-MAPK signal. {yields} Mutated K-ras produces additional mutagenic lesions, as a new oncogenic role. -- Abstract: Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-{kappa}B, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.

  7. Molecular interaction between K-Ras and H-REV107 in the Ras signaling pathway.

    PubMed

    Han, Chang Woo; Jeong, Mi Suk; Jang, Se Bok

    2017-09-16

    Ras proteins are small GTPases that serve as master moderators of a large number of signaling pathways involved in various cellular processes. Activating mutations in Ras are found in about one-third of cancers. H-REV107, a K-Ras binding protein, plays an important role in determining K-Ras function. H-REV107 is a member of the HREV107 family of class II tumor suppressor genes and a growth inhibitory Ras target gene that suppresses cellular growth, differentiation, and apoptosis. Expression of H-REV107 was strongly reduced in about 50% of human carcinoma cell lines. However, the specific molecular mechanism by which H-REV107 inhibits Ras is still unknown. In the present study, we suggest that H-REV107 forms a strong complex with activating oncogenic mutation Q61H K-Ras from various biochemical binding assays and modeled structures. In addition, the interaction sites between K-Ras and H-REV107 were predicted based on homology modeling. Here, we found that some structure-based mutants of the K-Ras disrupted the complex formation with H-REV107. Finally, a novel molecular mechanism describing K-Ras and H-REV107 binding is suggested and insights into new K-Ras effector target drugs are provided. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

    PubMed Central

    Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

    2015-01-01

    K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

  9. IL-6 blockade reprograms the lung tumor microenvironment to limit the development and progression of K-ras mutant lung cancer

    PubMed Central

    Caetano, Mauricio S.; Zhang, Huiyuan; Cumpian, Amber M.; Gong, Lei; Unver, Nese; Ostrin, Edwin J.; Daliri, Soudabeh; Chang, Seon Hee; Ochoa, Cesar E.; Hanash, Samir; Behrens, Carmen; Wistuba, Ignacio I.; Sternberg, Cinthya; Kadara, Humam; Ferreira, Carlos Gil; Watowich, Stephanie S.; Moghaddam, Seyed Javad

    2016-01-01

    Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer. Unfortunately, attempts to target K-ras mutant lung tumors have thus far failed, clearly indicating the need for new approaches in patients with this molecular profile. We have previously shown NF-κB activation, release of IL-6, and activation of its responsive transcription factor STAT3 in K-ras mutant lung tumors, which was further amplified by the tumor enhancing effect of chronic obstructive pulmonary disease (COPD)-type airway inflammation. These findings suggest an essential role for this inflammatory pathway in K-ras mutant lung tumorigenesis and its enhancement by COPD. Therefore, here we blocked IL-6 using a monoclonal anti-IL-6 antibody in a K-ras mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL-6 blockade significantly inhibited lung cancer promotion, tumor cell intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL-6 inhibition reduced expression of pro-tumor type 2 molecules (Arginase 1, Fizz 1, Mgl, and IDO), number of M2 type macrophages and G-MDSCs, and pro-tumor T-regulatory/T helper 17 cell responses. This was accompanied by increased expression of anti-tumor type 1 molecule (Nos2), and anti-tumor T helper 1/CD8 T cell responses. Our study demonstrates that IL-6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also re-educates the lung microenvironment toward an anti-tumor phenotype by altering the relative proportion between pro-tumor and anti-tumor immune cells. This information introduces IL-6 as a potential druggable target for prevention and treatment of K-ras mutant lung tumors. PMID:27197187

  10. [Clinical relevance of the K-ras oncogene in colorectal cancer: experience in a Mexican population].

    PubMed

    Cabrera-Mendoza, F; Gainza-Lagunes, S; Castañeda-Andrade, I; Castro-Zárate, A

    2014-01-01

    Colorectal cancer is frequent in the developed countries, with a cancer-specific mortality rate of 33%. Different biomarkers are associated with overall survival and the prediction of monoclonal treatment effectiveness. The presence of mutations in the K-ras oncogene alters the response to target therapy with cetuximab and could be an independent prognostic factor. To analyze the difference in survival between patients with mutated K-ras and those with K-ras wild-type status. Thirty-one clinical records were retrospectively analyzed of patients presenting with colorectal cancer that underwent K-ras sequencing through real-time polymerase chain reaction within the time frame of 2009 to 2012 at the Hospital de Alta Especialidad de Veracruz of the Instituto para la Salud y Seguridad Social de los Trabajadores del Estado (HAEV-ISSSTE). Survival analysis for patients with and without K-ras mutation was performed using the Kaplan Meier method. Contrast of covariates was performed using logarithmic transformations. No statistically significant difference was found in relation to survival in the patients with mutated K-ras vs. those with K-ras wild-type (P=.416), nor were significant differences found when analyzing the covariants and survival in the patients with mutated K-ras: ECOG scale (P=.221); age (less than, equal to or greater than 65years, P=.441); clinical stage according to the AJCC (P=.057), and primary lesion site (P=.614). No relation was found between the K-ras oncogene mutation and reduced survival, in contrast to what has been established in the international medical literature. Further studies that include both a larger number of patients and those receiving monoclonal treatment, need to be conducted. There were only 5 patients in the present study that received cetuximab, resulting in a misleading analysis. Copyright © 2013 Asociación Mexicana de Gastroenterología. Published by Masson Doyma México S.A. All rights reserved.

  11. Lead identification for the K-Ras protein: virtual screening and combinatorial fragment-based approaches

    PubMed Central

    Pathan, Akbar Ali Khan; Panthi, Bhavana; Khan, Zahid; Koppula, Purushotham Reddy; Alanazi, Mohammed Saud; Sachchidanand; Parine, Narasimha Reddy; Chourasia, Mukesh

    2016-01-01

    Objective Kirsten rat sarcoma (K-Ras) protein is a member of Ras family belonging to the small guanosine triphosphatases superfamily. The members of this family share a conserved structure and biochemical properties, acting as binary molecular switches. The guanosine triphosphate-bound active K-Ras interacts with a range of effectors, resulting in the stimulation of downstream signaling pathways regulating cell proliferation, differentiation, and apoptosis. Efforts to target K-Ras have been unsuccessful until now, placing it among high-value molecules against which developing a therapy would have an enormous impact. K-Ras transduces signals when it binds to guanosine triphosphate by directly binding to downstream effector proteins, but in case of guanosine diphosphate-bound conformation, these interactions get disrupted. Methods In the present study, we targeted the nucleotide-binding site in the “on” and “off” state conformations of the K-Ras protein to find out suitable lead compounds. A structure-based virtual screening approach has been used to screen compounds from different databases, followed by a combinatorial fragment-based approach to design the apposite lead for the K-Ras protein. Results Interestingly, the designed compounds exhibit a binding preference for the “off” state over “on” state conformation of K-Ras protein. Moreover, the designed compounds’ interactions are similar to guanosine diphosphate and, thus, could presumably act as a potential lead for K-Ras. The predicted drug-likeness properties of these compounds suggest that these compounds follow the Lipinski’s rule of five and have tolerable absorption, distribution, metabolism, excretion and toxicity values. Conclusion Thus, through the current study, we propose targeting only “off” state conformations as a promising strategy for the design of reversible inhibitors to pharmacologically inhibit distinct conformations of K-Ras protein. PMID:27217775

  12. Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Su Young; Seo, Si Hyung; Park, Won Sang; Nam, Suk Woo; Song, Sang Yong; Han, Joung Ho; Park, Cheol Keun; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2005-05-01

    Both epidermal growth factor receptor (EGFR) and RAS gene mutations contribute to the development of non-small cell lung cancer (NSCLC). Because RAS is one of the downstream molecules in the EGFR signal transduction, the association between the somatic mutations of EGFR and RAS may be important in the pathogenesis of NSCLC . However, to date, such data are lacking. In this study, we analyzed the hotspot regions of K-RAS gene (codons 12, 13, 59 and 61) and EGFR gene (exons 18, 19 and 21) in 153 NSCLC tissue samples including 69 adenocarcinomas. Overall, we detected 30 EGFR mutations (19.6%) and 6 K-RAS mutations (3.9%) in the 153 NSCLCs. In the 69 adenocarcinomas, 26 EGFR mutations (37.7%) and six K-RAS mutations (8.7%) were detected. Of note, the 26 tumors with EGFR mutations did not harbor any K-RAS mutations, and the six tumors with K-RAS mutations did not harbor any EGFR mutations. Inverse relationship between K-RAS and EGFR mutations in the lung adenocarcinoma was statistically significant (P=0.046, chi2 test). As regards smoking history, EGFR mutation was significantly associated with never-smoking history, whereas K-RAS mutation was significantly associated with smoking history. Our data suggest that mutations of EGFR and K-RAS genes might separately, but not cooperatively, contribute to lung adenocarcinoma pathogenesis, and that EGFR and K-RAS mutants could separately be anti-neoplastic targets in lung adenocarcinomas.

  13. APC, K-ras, and p53 gene mutations in colorectal cancer patients: correlation to clinicopathologic features and postoperative surveillance.

    PubMed

    Hsieh, Jan-Sing; Lin, Shiu-Ru; Chang, Mei-Yin; Chen, Fang-Ming; Lu, Chien-Yu; Huang, Tsung-Jen; Huang, Yu-Sheng; Huang, Che-Jen; Wang, Jaw-Yuan

    2005-04-01

    Current researches have proposed a genetic model for colorectal cancer (CRC), in which the sequential accumulation of mutations in specific cancer-related genes, including adenomatous polyposis coli (APC), K-ras, and p53, drives the transition from normal epithelium through increasing adenomatous dysplasia to colorectal cancer. To identify patients with an increased risk of tumor recurrence or metastasis and evaluate the prognostic values of APC, K-ras, and p53 gene mutations, we investigated the frequency of these three mutated genes in tumors and sera of CRC patients. APC, K-ras, and p53 gene mutations in primary tumor tissues and their paired preoperative serum samples of 118 CRC patients were detected by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, followed by direct DNA sequencing of the PCR-amplified genomic DNA. Subsequently, serum molecular markers were analyzed for their correlation with patients' clinicopathologic features and presence of postoperative recurrence/metastasis. We did not observe any significant difference in the association of APC or K-ras or p53 gene mutations in primary tumors with patients' demographic data (all were P > 0.05). In contrast, both serum APC and p53 molecular markers were closely correlated with lymph node metastasis and TNM stage (both P < 0.05). Moreover, the serum overall molecular markers (at least one of the three markers) were prominently associated with depth of tumor invasion (P = 0.033), lymph node metastasis (P < 0.001), and TNM stage (P < 0.001). In addition, a significantly higher postoperative metastasis/recurrence rate in patients positive for overall molecular markers compared to those negative for these molecular markers were also demonstrated (P < 0.001). APC and K-ras molecular markers were more frequently observed in patients with locoregional metastasis (both P < 0.05), while p53 molecular marker was usually detected in the cases of peritoneal metastasis (P

  14. Gold nanoparticles in combination with megavoltage radiation energy increased radiosensitization and apoptosis in colon cancer HT-29 cells.

    PubMed

    Saberi, Alihossein; Shahbazi-Gahrouei, Daryoush; Abbasian, Mahdi; Fesharaki, Mehrafarin; Baharlouei, Azam; Arab-Bafrani, Zahra

    2017-03-01

    Gold nanoparticles (GNP) act as a radiosensitizer in radiation therapy. However, recent studies have shown contradictory evidence in terms of radiosensitization in the presence of GNP combined with X-ray megavoltage energy (MV) on different cell types. In this study, the effect of GNP on radiosensitization enhancement of HT-29 human colorectal cancer cells at MV X-ray energy was evaluated. The cytotoxicity and radiosensitization of GNP were evaluated in HT-29 human colorectal cancer cells by MTS-assay and multiple MTS-assay, respectively. Cellular uptake was assayed using graphite furnace atomic absorption spectrometry (GFAAS). Apoptosis and cell cycle progression were determined by an Annexin V-FITC/propidium iodide (PI) kit and PI/RNase solution with flow cytometry, respectively. Results showed that the cell viability of the HT-29 cells was not influenced by exposure to different concentrations of GNP (10-100 μM). GNP alone did not affect the cell cycle progression and apoptosis. In contrast, GNP, in combination with radiation (9 MV), induced more apoptosis. The interaction of GNP with MV energy resulted in a significant radiosensitization enhancement compared with irradiation alone. It was concluded that GNP may work as bio-inert material on HT-29 cancer cells and their enhancement of radiosensitization may be due to increase in the absorbed irradiation dose.

  15. Nicotinamide increases thyroid radiosensitivity by stimulating nitric oxide synthase expression and the generation of organic peroxides.

    PubMed

    Agote Robertson, M; Finochietto, P; Gamba, C A; Dagrosa, M A; Viaggi, M E; Franco, M C; Poderoso, J J; Juvenal, G J; Pisarev, M A

    2006-01-01

    Differentiated thyroid cancer and hyperthyroidism are treated with radioiodine. However, when the radioisotope dose exceeds certain limits, the patient must be hospitalized to avoid contact with people that would otherwise be exposed to radiation. It would be desirable to obtain a similar therapeutic effect using lower radioiodine doses. Radiosensitizers can be utilized for this purpose. Nicotinamide (NA) increases thyroid radiosensitivity to 131I in both normal and goitrous glands. NA causes a significant increase in thyroid blood flow, which would increase tissue oxygenation and tissue damage via free radicals. Wistar rats were treated with either nicotinamide (NA), 131I or both. The expression of the three isoforms of nitric oxide synthase (NOS) in the thyroid (Western blot) and the activities of SOD, GPx, catalase and organic peroxides were determined. Treatment with NA or 131I increased the expression of eNOS and the generation of organic peroxides. When administered jointly, they showed a synergistic effect. No changes were observed in the other NOS isoforms or in the activities of catalase, glutathione peroxidase and superoxide dismutase. NA potentiates the effect of 131I by increasing eNOS, which would in turn stimulate NO production, increasing thyroid blood flow and tissue damage via organic peroxides.

  16. Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling

    PubMed Central

    Zhou, Yong; Wong, Ching-On; Cho, Kwang-jin; van der Hoeven, Dharini; Liang, Hong; Thakur, Dhananiay P.; Luo, Jialie; Babic, Milos; Zinsmaier, Konrad E.; Zhu, Michael X.; Hu, Hongzhen; Venkatachalam, Kartik; Hancock, John F.

    2015-01-01

    Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras–dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits. PMID:26293964

  17. Across the universe of K-RAS mutations in non-small-cell-lung cancer.

    PubMed

    Piva, Sheila; Ganzinelli, Monica; Garassino, Marina Chiara; Caiola, Elisa; Farina, Gabriella; Broggini, Massimo; Marabese, Mirko

    2014-01-01

    RAS family proteins are important signaling molecules that regulate cell growth, survival and differentiation by coupling receptor activation to downstream effector pathways. Three distinct genes encode for the three different proteins H-, K-, and N- RAS. These proteins share high sequence homology, particularly at the N-Terminal domain. Among them, K-RAS is one of the most frequently mutated in human cancer. The majority of the mutations present in K-RAS are at codon 12 (from 80 to 100%) followed by codon 13 and 61. In all cases, aminoacid change leads to a constitutively activated protein. K-RAS mutations have a role in tumor development as well as in tumor progression and resistance. Despite the various studies which have been published, the prognostic and predictive role of K-RAS mutations is still under debate. Keeping in mind that the glycine present at position 12 can be substituted by valine, aspartic acid or cysteine, it could be well understood that each different substitution plays a different role in K-RAS-dependent processes. The present article focuses on the molecular and biological characteristics of K-RAS protein, its role in NSCLC tumor development and progression. We also present an overview of the preclinical models both in vitro and in vivo available to determine the role of K-RAS in tumor progression and response to treatment and on the recent results obtained in this field. Finally, we have considered the impact of KRAS mutations in clinical practice, analyzing the different recent trials that have taken into consideration K-RAS.

  18. Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer.

    PubMed

    Palmirotta, Raffaele; Savonarola, Annalisa; Ludovici, Giorgia; De Marchis, Maria Laura; Covello, Renato; Ettorre, Giuseppe Maria; Ialongo, Cristiano; Guadagni, Fiorella

    2011-01-01

    The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine) at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine) at codon 57. In addition, we found in the same patient's sample a silent polymorphism at codon 11 (Ala11Ala) of exon 1.

  19. Expanding the Scope of Electrophiles Capable of Targeting K-Ras Oncogenes.

    PubMed

    McGregor, Lynn M; Jenkins, Meredith L; Kerwin, Caitlin; Burke, John E; Shokat, Kevan M

    2017-06-27

    There is growing interest in reversible and irreversible covalent inhibitors that target noncatalytic amino acids in target proteins. With a goal of targeting oncogenic K-Ras variants (e.g., G12D) by expanding the types of amino acids that can be targeted by covalent inhibitors, we survey a set of electrophiles for their ability to label carboxylates. We functionalized an optimized ligand for the K-Ras switch II pocket with a set of electrophiles previously reported to react with carboxylates and characterized the ability of these compounds to react with model nucleophiles and oncogenic K-Ras proteins. Here, we report that aziridines and stabilized diazo groups preferentially react with free carboxylates over thiols. Although we did not identify a warhead that potently labels K-Ras G12D, we were able to study the interactions of many electrophiles with K-Ras, as most of the electrophiles rapidly label K-Ras G12C. We characterized the resulting complexes by crystallography, hydrogen/deuterium exchange, and differential scanning fluorimetry. Our results both demonstrate the ability of a noncatalytic cysteine to react with a diverse set of electrophiles and emphasize the importance of proper spatial arrangements between a covalent inhibitor and its intended nucleophile. We hope that these results can expand the range of electrophiles and nucleophiles of use in covalent protein modulation.

  20. Slug inhibition increases radiosensitivity of oral squamous cell carcinoma cells by upregulating PUMA.

    PubMed

    Jiang, Fangfang; Zhou, Lijie; Wei, Changbo; Zhao, Wei; Yu, Dongsheng

    2016-08-01

    As a new strategy, radio-gene therapy was widely used for the treatment of cancer patients in recent few years. Slug was involved in the radioresistance of various cancers and has been found to have an anti-apoptotic effect. This study aims to investigate whether the modulation of Slug expression by siRNA affects oral squamous cell carcinoma sensitivity to X-ray irradiation through upregulating PUMA. Two oral squamous cell carcinoma cell lines (HSC3 and HSC6) were transfected with small interfering RNA (siRNA) targeting Slug and subjected to radiotherapy in vitro. After transfection with Slug siRNA, both HSC3 and HSC6 cells showed relatively lower expression of Slug and higher expression of PUMA. The Slug siRNA transfected cells showed decreased survival and proliferation rates, an increased apoptosis rate and enhanced radiosensitivity to X-ray irradiation. Our results revealed that Slug siRNA transfection in combination with radiation increased the expression of PUMA, which contributed to radiosensitivity of oral squamous cell carcinoma cells. Thus, controlling the expression of Slug might contribute to enhance sensitivity of HSC3 and HSC6 cells toward X-ray irradiation in vitro by upregulating PUMA.

  1. A signature motif in LIM proteins mediates binding to checkpoint proteins and increases tumour radiosensitivity

    PubMed Central

    Xu, Xiaojie; Fan, Zhongyi; Liang, Chaoyang; Li, Ling; Wang, Lili; Liang, Yingchun; Wu, Jun; Chang, Shaohong; Yan, Zhifeng; Lv, Zhaohui; Fu, Jing; Liu, Yang; Jin, Shuai; Wang, Tao; Hong, Tian; Dong, Yishan; Ding, Lihua; Cheng, Long; Liu, Rui; Fu, Shenbo; Jiao, Shunchang; Ye, Qinong

    2017-01-01

    Tumour radiotherapy resistance involves the cell cycle pathway. CDC25 phosphatases are key cell cycle regulators. However, how CDC25 activity is precisely controlled remains largely unknown. Here, we show that LIM domain-containing proteins, such as FHL1, increase inhibitory CDC25 phosphorylation by forming a complex with CHK2 and CDC25, and sequester CDC25 in the cytoplasm by forming another complex with 14-3-3 and CDC25, resulting in increased radioresistance in cancer cells. FHL1 expression, induced by ionizing irradiation in a SP1- and MLL1-dependent manner, positively correlates with radioresistance in cancer patients. We identify a cell-penetrating 11 amino-acid motif within LIM domains (eLIM) that is sufficient for binding CHK2 and CDC25, reducing the CHK2–CDC25 and CDC25–14-3-3 interaction and enhancing CDC25 activity and cancer radiosensitivity accompanied by mitotic catastrophe and apoptosis. Our results provide novel insight into molecular mechanisms underlying CDC25 activity regulation. LIM protein inhibition or use of eLIM may be new strategies for improving tumour radiosensitivity. PMID:28094252

  2. Increased Chromosomal Radiosensitivity in Women Carrying BRCA1/BRCA2 Mutations Assessed With the G2 Assay

    SciTech Connect

    Ernestos, Beroukas; Nikolaos, Pandis; Koulis, Giannoukakos; Eleni, Rizou; Konstantinos, Beroukas; Alexandra, Giatromanolaki; Michael, Koukourakis

    2010-03-15

    Purpose: Several in vitro studies suggest that BRCA1 and BRCA2 mutation carriers present increased sensitivity to ionizing radiation. Different assays for the assessment of deoxyribonucleic acid double-strand break repair capacity have been used, but results are rather inconsistent. Given the concerns about the possible risks of breast screening with mammography in mutation carrier women and the potentially damaging effects of radiotherapy, the purpose of this study was to further investigate the radiosensitivity of this population. Methods and Materials: The G2 chromosomal radiosensitivity assay was used to assess chromosomal breaks in lymphocyte cultures after exposure to 1 Gy. A group of familiar breast cancer patients carrying a mutation in the BRCA1 or BRCA2 gene (n = 15) and a group of healthy mutation carriers (n = 5) were investigated and compared with a reference group of healthy women carrying no mutation (n = 21). Results: BRCA1 and BRCA2 mutation carriers had a significantly higher number of mean chromatid breaks per cell (p = 0.006) and a higher maximum number of breaks (p = 0.0001) as compared with their matched controls. Both healthy carriers and carriers with a cancer history were more radiosensitive than controls (p = 0.002 and p = 0.025, respectively). Age was not associated with increased radiosensitivity (p = 0.868). Conclusions: Our results indicate that BRCA1 and BRCA2 mutation carriers show enhanced radiosensitivity, presumably because of the involvement of the BRCA genes in deoxyribonucleic acid repair and cell cycle control mechanisms.

  3. Dietary supplementation of lutein reduces colon carcinogenesis in DMH-treated rats by modulating K-ras, PKB, and β-catenin proteins.

    PubMed

    Reynoso-Camacho, R; González-Jasso, E; Ferriz-Martínez, R; Villalón-Corona, B; Loarca-Piña, G F; Salgado, L M; Ramos-Gomez, M

    2011-01-01

    In colon cancer, disturbances have been detected in genes coding for proteins involved in cellular proliferation, such as K-ras, β-catenin, extracellular signal-regulated kinases (ERKs), and the protein kinase B (PKB). Although carotenoids such as lutein have an important role to prevent and treat some types of cancer, there are very few studies about the effect of lutein against colon cancer and its activity at the molecular level. Therefore, the aim of this study was to evaluate the chemoprotective activity of lutein against colon cancer induced by dimethylhydrazine (DMH). The results showed a significant increase in protein expression for K-ras and β-catenin in tumors of DMH-treated rats. Simultaneously, we detected changes in the phosphorylation state of ERK1/2 and PKB in DMH-treated animals. Lutein given in the diet (0.002%), before (prevention) and after (treatment) DMH administration, diminished the number of tumors by 55% and 32%, respectively. Moreover, lutein significantly decreased in tumors the expression of K-ras (25%) and β-catenin (28%) and the amount of pPKB (32%), during the prevention, and 39%, 26%, and 26% during the treatment stage, respectively. This study demonstrates the chemoprotective effect of lutein against colon cancer by modulating the proliferative activity of K-ras, PKB, and β-catenin proteins.

  4. K-ras mutations and mucin profile in preneoplastic lesions and colon tumors induced in rats by 1,2-dimethylhydrazine.

    PubMed

    Femia, Angelo Pietro; Tarquini, Elena; Salvadori, Maddalena; Ferri, Stefania; Giannini, Augusto; Dolara, Piero; Caderni, Giovanna

    2008-01-01

    K-ras and mucin profile variations, associated with intestinal carcinogenesis, were studied in the preneoplastic lesions, mucin-depleted foci (MDF) and aberrant crypt foci (ACF), and in colonic tumors induced in rats by 1,2-dimethylhydrazine (DMH). The frequency of lesions with K-ras mutations was 23% (3/13), 5.5% (1/18) and 100% (14/14) in MDF, tumors and ACF, respectively. Two of three MDF mutated in K-ras also carried a missense mutation in Apc. We also tested the expression of MUC2, a mucin abundantly expressed in normal colon and M1/MUCA5C, up-regulated in colon carcinogenesis, using immunohistochemistry. MDF and tumors showed a dramatic reduction in the expression of MUC2, whereas ACF showed only a slight reduction. The expression of M1/MUC5AC was almost absent in normal mucosa, but was increased in all the lesions (MDF, tumors and ACF). The expression of the intestinal trefoil factor (ITF), a marker of goblet cell lineage, was reduced in MDF and tumors compared to normal mucosa but not in ACF. In conclusion, although K-ras mutations are present in all ACF, they are less frequent in MDF and tumors; M1/MUC5AC is a marker associated with all preneoplastic events while the reduction of MUC2 and ITF expression is selectively associated with more advanced lesions such as MDF and tumors.

  5. IMP-1 displays crosstalk with K-Ras and modulates colon cancer cell survival through the novel pro-apoptotic protein CYFIP2

    PubMed Central

    Mongroo, Perry S.; Noubissi, Felicite K.; Cuatrecasas, Miriam; Kalabis, Jiri; King, Catrina E.; Johnstone, Cameron N.; Bowser, Mark J.; Castells, Antoni; Spiegelman, Vladimir S.; Rustgi, Anil K.

    2011-01-01

    Insulin-like growth factor 2 mRNA-binding protein-1 (IMP-1) is an oncofetal protein that binds directly to and stabilizes oncogenic c-Myc and regulates in turn its post-transcriptional expression and translation. In contrast to normal adult tissue, IMP-1 is re-expressed and/or overexpressed in human cancers. We demonstrate that knock-down of c-Myc in human colon cancer cell lines increases the expression of mature let-7 miRNA family members and downregulates several of its mRNA targets: IMP-1, Cdc34, and K-Ras. We further demonstrate that loss of IMP-1 inhibits Cdc34, Lin-28B, and K-Ras, and suppresses SW-480 cell proliferation and anchorage-independent growth, and promotes caspase and lamin-mediated cell death. We also found that IMP-1 binds to the coding region and 3′UTR of K-Ras mRNA. RNA microarray profiling and validation by reverse transcription PCR reveals that the p53-inducible pro-apoptotic protein, CYFIP2, is upregulated in IMP-1 knock-down SW480 cells, a novel finding. We also show that overexpression of IMP-1 increases c-Myc and K-Ras expression, and LIM2405 cell proliferation. Furthermore, we show that loss of IMP-1 induces Caspase-3 and Parp–mediated apoptosis, and inhibits K-Ras expression in SW480 cells, which is rescued by CYFIP2 knock-down. Importantly, analysis of 228 patients with colon cancers reveals that IMP-1 is significantly upregulated in differentiated colon tumors (p ≤ 0.0001) and correlates with K-Ras expression (r=0.35, p ≤ 0.0001) relative to adjacent normal mucosa. These findings indicate that IMP-1, interrelated with c-myc, acts upstream of K-Ras to promote survival through a novel mechanism that may be important in colon cancer pathogenesis. PMID:21252116

  6. The higher level of complexity of K-Ras4B activation at the membrane

    PubMed Central

    Jang, Hyunbum; Banerjee, Avik; Chavan, Tanmay S.; Lu, Shaoyong; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2016-01-01

    Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5′-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.—Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane. PMID:26718888

  7. K-Ras(V14I) -induced Noonan syndrome predisposes to tumour development in mice.

    PubMed

    Hernández-Porras, Isabel; Schuhmacher, Alberto J; Garcia-Medina, Raquel; Jiménez, Beatriz; Cañamero, Marta; de Martino, Alba; Guerra, Carmen

    2016-06-01

    The Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant proportion of NS patients may also develop myeloproliferative disorders (MPDs), including juvenile myelomonocytic leukaemia (JMML). Surprisingly, scarce information is available in relation to other tumour types in these patients. We have previously developed and characterized a knock-in mouse model that carries one of the most frequent KRAS-NS-related mutations, the K-Ras(V14I) substitution, which recapitulates most of the alterations described in NS patients, including MPDs. The K-Ras(V14I) mutation is a mild activating K-Ras protein; thus, we have used this model to study tumour susceptibility in comparison with mice expressing the classical K-Ras(G12V) oncogene. Interestingly, our studies have shown that these mice display a generalized tumour predisposition and not just MPDs. In fact, we have observed that the K-Ras(V14I) mutation is capable of cooperating with the p16Ink4a/p19Arf and Trp53 tumour suppressors, as well as with other risk factors such as pancreatitis, thereby leading to a higher cancer incidence. In conclusion, our results illustrate that the K-Ras(V14I) activating protein is able to induce cancer, although at a much lower level than the classical K-Ras(G12V) oncogene, and that it can be significantly modulated by both genetic and non-genetic events. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  8. Mutations in Succinate Dehydrogenase Subunit C Increase Radiosensitivity and Bystander Responses

    NASA Astrophysics Data System (ADS)

    Zhou, Hongning; Hei, Tom K.

    Although radiation-induced bystander effect is well studied in the past decade, the precise mech-anisms are still unclear. It is likely that a combination of pathways involving both primary and secondary signaling processes is involved in producing a bystander effect. There is recent evidence that mitochondria play a critical role in bystander responses. Recently studies found that a mutation in succinate dehydrogenese subunit C (SDHC), an integral membrane protein in complex II of the electron transport chain, resulted in increased superoxide, oxidative stress, apoptosis, tumorigenesis, and genomic instability, indicating that SDHC play a critical role in maintaining mitochondrial function. In the present study, using Chinese hamster fibroblasts (B1 cells) and the mutants (B9 cells) containing a single base substitution that produced a premature stop codon resulting in a 33-amino acid COOH-terminal truncation of the SDHC protein, we found that B9 cells had an increase in intracellular superoxide content, nitric oxide species, and mitochondrial membrane potential when compared with wild type cells. After irradiated with a grade of doses of gamma rays, B9 cells show an increased radiosensitivity, especially at high doses. The HPRT- mutant yield after gamma-ray irradiation in B9 cells was significantly higher than that of B1 cells. A single, 3Gy dose of gamma-rays increased the background mutant level by more than 4 fold. In contrast, the mutant induction was less than 2 fold in B1 cells. In addition, B9 cells produced a higher bystander mutagenesis after alpha particle irradiation than the B1 cells. Furthermore, pretreated with carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), a nitric oxide scavenger, significantly decreased the bystander effect. Our findings demonstrate that a mutation in SDHC increases radiosensitivity in both directly irradiated cells and in neighboring bystander cells, and mito-chondrial function play an essential role in

  9. Enhanced MET translation and signaling sustains K-Ras driven proliferation under anchorage-independent growth conditions

    PubMed Central

    Fujita-Sato, Saori; Galeas, Jacqueline; Truitt, Morgan; Pitt, Cameron; Urisman, Anatoly; Bandyopadhyay, Sourav; Ruggero, Davide; McCormick, Frank

    2015-01-01

    Oncogenic K-Ras mutation occurs frequently in several types of cancers including pancreatic and lung cancers. Tumors with K-Ras mutation are resistant to chemotherapeutic drugs as well as molecular targeting agents. Although numerous approaches are ongoing to find effective ways to treat these tumors, there are still no effective therapies for K-Ras mutant cancer patients. Here we report that K-Ras mutant cancers are more dependent on K-Ras in anchorage independent culture conditions than in monolayer culture conditions. In seeking to determine mechanisms that contribute to the K-Ras dependency in anchorage independent culture conditions, we discovered the involvement of Met in K-Ras-dependent, anchorage independent cell growth. The Met signaling pathway is enhanced and plays an indispensable role in anchorage independent growth even in cells in which Met is not amplified. Indeed, Met expression is elevated under anchorage-independent growth conditions and is regulated by K-Ras in a MAPK/ERK kinase (MEK)-dependent manner. Remarkably, in spite of a global down-regulation of mRNA translation during anchorage independent growth, we find that Met mRNA translation is specifically enhanced under these conditions. Importantly, ectopic expression of an active Met mutant rescues K-Ras ablation-derived growth suppression, indicating that K-Ras mediated Met expression drives “K-Ras addiction” in anchorage independent conditions. Our results indicate that enhanced Met expression and signaling is essential for anchorage independent growth of K-Ras mutant cancer cells and suggests that pharmacological inhibitors of Met could be effective for K-Ras mutant tumor patients. PMID:25977330

  10. Identification of translocation products but not K-RAS mutations in memory B cells from patients with multiple myeloma.

    PubMed

    Rasmussen, Thomas; Haaber, Jacob; Dahl, Inger Marie; Knudsen, Lene M; Kerndrup, Gitte B; Lodahl, Marianne; Johnsen, Hans E; Kuehl, Michael

    2010-10-01

    Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells. The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells. Cells with the phenotype of tumor plasma cells (CD38(++)CD19(-)CD45(-/+)CD56(-/+/++)) or memory B cells (CD38(-)/CD19(+)/CD27(+)) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes (FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K-RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K-RAS mutations were identified. The increased expression of a specific "early" oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack "late" oncogene (K-RAS) mutations but express the "early" oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.

  11. Cancer stem cell drugs target K-ras signaling in a stemness context

    PubMed Central

    Najumudeen, A K; Jaiswal, A; Lectez, B; Oetken-Lindholm, C; Guzmán, C; Siljamäki, E; Posada, I M D; Lacey, E; Aittokallio, T; Abankwa, D

    2016-01-01

    Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC. PMID:26973241

  12. A novel K-ras mutation in colorectal cancer. A case report and literature review.

    PubMed

    Palmirotta, Raffaele; Savonarola, Annalisa; Formica, Vincenzo; Ludovici, Giorgia; Del Monte, Girolamo; Roselli, Mario; Guadagni, Fiorella

    2009-08-01

    Activating mutations in the K-ras oncogene mainly occur in codons 12 and 13 and may be predictive of response to drugs directly linked to the K-ras signaling pathway, such as panitumumab and cetuximab. K-ras analysis was carried out on DNA extracted from paraffin-embedded tumor samples after microdissection. Exons 1 and 2 were amplified by PCR and then sequenced. A never-reported K-ras mutation (CAG>TAG) determining a premature stop signal at codon 22 (Gln22Stop) was found in a patient with metastatic colorectal cancer. BRAF and p53 were not found to be modified and microsatellite instability was not present. The patient, however, was found to be unresponsive to an anti-EGFR MAb treatment. This study is the first report of a novel K-ras truncating mutation in a patient with metastatic colorectal cancer and is also suggestive for the evaluation of alternative pathways to better identify individuals who are likely to benefit from targeted therapies.

  13. Association of K-ras codon 12 transversions with short survival in non-small cell lung cancer.

    PubMed

    Vega, F; Iniesta, P; Caldes, T; Sanchez, A; Lopez, J; Dejuan, C; Diazrubio, E; Torres, A; Balibrea, J; Benito, M

    1996-12-01

    K-ras activating point mutations appear to have a role in human lung cancer, however, the prognostic significance of these abnormalities remains unclear. The aim of our work was to clarify the role of K-ras mutations as prognostic indicators in patients affected by non-small cell lung cancer (NSCLC). We studied 94 resected primary NSCLCs for K-ras mutations by the PCR-RFLP technique, followed by sequencing. K-ras activating mutations were present in 34% of tumors, a higher incidence being detected in adenocarcinomas. Comparing the impact of K-ras mutation types, we found that K-ras transversions were associated with a shorter survival in NSCLC.

  14. Carbon metabolism and the sign of control coefficients in metabolic adaptations underlying K-ras transformation.

    PubMed

    de Atauri, Pedro; Benito, Adrian; Vizán, Pedro; Zanuy, Miriam; Mangues, Ramón; Marín, Silvia; Cascante, Marta

    2011-06-01

    Metabolic adaptations are associated with changes in enzyme activities. These adaptations are characterized by patterns of positive and negative changes in metabolic fluxes and concentrations of intermediate metabolites. Knowledge of the mechanism and parameters governing enzyme kinetics is rarely available. However, the signs-increases or decreases-of many of these changes can be predicted using the signs of metabolic control coefficients. These signs require the only knowledge of the structure of the metabolic network and a limited qualitative knowledge of the regulatory dependences, which is widely available for carbon metabolism. Here, as a case study, we identified control coefficients with fixed signs in order to predict the pattern of changes in key enzyme activities which can explain the observed changes in fluxes and concentrations underlying the metabolic adaptations in oncogenic K-ras transformation in NIH-3T3 cells. The fixed signs of control coefficients indicate that metabolic changes following the oncogenic transformation-increased glycolysis and oxidative branch of the pentose-phosphate pathway, and decreased concentration in sugar-phosphates-could be associated with increases in activity for glucose-6-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase, and decrease for transketolase. These predictions were validated experimentally by measuring specific activities. We conclude that predictions based on fixed signs of control coefficients are a very robust tool for the identification of changes in enzyme activities that can explain observed metabolic adaptations in carbon metabolism.

  15. Characterization of a novel oncogenic K-ras mutation in colon cancer

    SciTech Connect

    Akagi, Kiwamu . E-mail: akagi@cancer-c.pref.saitama.jp; Uchibori, Ryosuke; Yamaguchi, Kensei; Kurosawa, Keiko; Tanaka, Yoichiro; Kozu, Tomoko

    2007-01-19

    Activating mutations of RAS are frequently observed in subsets of human cancers, indicating that RAS activation is involved in tumorigenesis. Here, we identified and characterized a novel G to T transversion mutation of the K-ras gene at the third position of codon 19 (TTG) which substituted phenylalanine for leucine in 3 primary colon carcinomas. Biological and biochemical activity was examined using transformed NIH3T3 cells expressing mutant or wild-type K-ras. Transformants harboring the K-ras mutation at codon 19 showed proliferative capacity under serum-starved conditions, less contact inhibition, anchorage-independent growth, tumorigenicity in nude mice and elevation of active Ras-GTP levels. These results indicated that this novel mutation possesses high oncogenic activity.

  16. Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients

    PubMed Central

    Vincenzi, Bruno; Cremolini, Chiara; Sartore-Bianchi, Andrea; Russo, Antonio; Mannavola, Francesco; Perrone, Giuseppe; Pantano, Francesco; Loupakis, Fotios; Rossini, Daniele; Ongaro, Elena; Bonazzina, Erica; Dell'Aquila, Emanuela; Imperatori, Marco; Zoccoli, Alice; Bronte, Giuseppe; De Maglio, Giovanna; Fontanini, Gabriella; Natoli, Clara; Falcone, Alfredo; Santini, Daniele; Onetti-Muda, Andrea; Siena, Salvatore; Tonini, Giuseppe; Aprile, Giuseppe

    2015-01-01

    Introduction: Activating mutations of K-Ras gene have a well-established role as predictors of resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) patients. Their prognostic value is controversial, and no data regarding the prognostic value of mutation rate, defined as the percentage of mutated alleles/tumor sample, are available. We aimed to evaluate the prognostic value of K-Rasmutation rate in a homogenous cohort of mCRC patients receiving first-line doublet plus bevacizumab. Patients and Methods: This retrospective study enrolled 397 K-Ras mutant mCRC patients from 6 Italian centers, and 263 patients were fully evaluable for our analysis. K-Ras mutation rate was assessed by pyrosequencing. Patients with less than 60% of cancer cells in tumor tissue were excluded. No patients received anti-EGFR containing anticancer therapy, at any time. Median mutation rate was 40% and was adopted as cut-off. The primary and secondary endpoints were PFS and OS respectively. Results: At univariate analysis, K-Ras mutation rate higher than 40% was significantly associated with lower PFS (7.3 vs 9.1 months; P < 0.0001) and OS (21 vs 31 months; P = 0.004). A multivariate model adjusted for age at diagnosis, site of origin of tumor tissue (primary vs metastases), referral center, number of metastatic sites, and first-line chemotherapy backbone, showed that K-Ras mutation rate remained a significant predictor of PFS and OS in the whole population. Discussion: Our data demonstrate an association between K-Ras mutation rate and prognosis in mCRC patients treated with bevacizumab-containing first-line therapy. These data deserve to be verified in an independent validation set. PMID:26384309

  17. Ribozyme-mediated inactivation of mutant K-ras oncogene in a colon cancer cell line

    PubMed Central

    Tokunaga, T; Tsuchida, T; Kijima, H; Okamoto, K; Oshika, Y; Sawa, N; Ohnishi, Y; Yamazaki, H; Miura, S; Ueyama, Y; Nakamura, M

    2000-01-01

    Mutation of c-K-ras oncogene is an important step in progression of colon cancer. We used a hammerhead ribozyme (KrasRz) against mutated K-ras gene transcripts (codon 12, GTT) to inactivate mutant K-ras function in the colon cancer cell line SW480, harbouring a mutant K-ras gene. The β-actin promoter-driven KrasRz sequence (pHβ/KrasRz) was introduced into these cells (SW480/KrasRz), and we evaluated its effects on growth of the colon cancer. The gene expression of angiogenesis-related molecules (vascular endothelial growth factor and thrombospondin) was also estimated in SW480/KrasRz. KrasRz specifically and efficiently cleaved the mutant K-ras mRNA but not wild-type mRNA in vitro. SW480/KrasRz showed decreased growth rate under tissue culture conditions (P< 0.01, Dunnett’s test). The xenotransplantability of SW480/KrasRz (XeSW480/KrasRz) was significantly decreased in nude mice (P< 0.05, Fisher’s exact test). Tumour volume of the xenografts XeSW480/KrasRz was significantly smaller than that of XeSW480/DisKrasRz (P< 0.01, Dunnett’s test). Gene expression of VEGF was suppressed in SW480/KrasRz, while TSP1 gene expression was enhanced. The SW480/KrasRz cells showed apoptosis-related features including nuclear condensation and DNA fragmentation. These results suggested that the hammerhead ribozyme-mediated inactivation of the mutated K-ras mRNA induced growth suppression, apoptosis and alteration of angiogenic factor expression. © 2000 Cancer Research Campaign PMID:10952790

  18. Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients.

    PubMed

    Vincenzi, Bruno; Cremolini, Chiara; Sartore-Bianchi, Andrea; Russo, Antonio; Mannavola, Francesco; Perrone, Giuseppe; Pantano, Francesco; Loupakis, Fotios; Rossini, Daniele; Ongaro, Elena; Bonazzina, Erica; Dell'Aquila, Emanuela; Imperatori, Marco; Zoccoli, Alice; Bronte, Giuseppe; De Maglio, Giovanna; Fontanini, Gabriella; Natoli, Clara; Falcone, Alfredo; Santini, Daniele; Onetti-Muda, Andrea; Siena, Salvatore; Tonini, Giuseppe; Aprile, Giuseppe

    2015-10-13

    Activating mutations of K-Ras gene have a well-established role as predictors of resistance to anti-EGFR monoclonal antibodies in metastatic colorectal cancer (mCRC) patients. Their prognostic value is controversial, and no data regarding the prognostic value of mutation rate, defined as the percentage of mutated alleles/tumor sample, are available. We aimed to evaluate the prognostic value of K-Rasmutation rate in a homogenous cohort of mCRC patients receiving first-line doublet plus bevacizumab. This retrospective study enrolled 397 K-Ras mutant mCRC patients from 6 Italian centers, and 263 patients were fully evaluable for our analysis. K-Ras mutation rate was assessed by pyrosequencing. Patients with less than 60% of cancer cells in tumor tissue were excluded. No patients received anti-EGFR containing anticancer therapy, at any time. Median mutation rate was 40% and was adopted as cut-off. The primary and secondary endpoints were PFS and OS respectively. At univariate analysis, K-Ras mutation rate higher than 40% was significantly associated with lower PFS (7.3 vs 9.1 months; P < 0.0001) and OS (21 vs 31 months; P = 0.004). A multivariate model adjusted for age at diagnosis, site of origin of tumor tissue (primary vs metastases), referral center, number of metastatic sites, and first-line chemotherapy backbone, showed that K-Ras mutation rate remained a significant predictor of PFS and OS in the whole population. Our data demonstrate an association between K-Ras mutation rate and prognosis in mCRC patients treated with bevacizumab-containing first-line therapy. These data deserve to be verified in an independent validation set.

  19. Prevalence of K-Ras mutations in hepatocellular carcinoma: A Turkish Oncology Group pilot study

    PubMed Central

    TURHAL, NAZIM SERDAR; SAVAŞ, BERNA; ÇOŞKUN, ÖZNUR; BAŞ, EMINE; KARABULUT, BÜLENT; NART, DENIZ; KORKMAZ, TANER; YAVUZER, DILEK; DEMIR, GÖKHAN; DOĞUSOY, GÜLEN; ARTAÇ, MEHMET

    2015-01-01

    Hepatocellular carcinoma (HCC) is the fifth most common male-predominant type of cancer worldwide. There is no effective treatment regimen available for advanced-stage disease and chemotherapy is generally ineffective in these patients. The number of studies on the prevalence of K-Ras mutations in HCC patients is currently limited. A total of 58 patients from 6 comprehensive cancer centers in 4 metropolitan cities of Turkey were enrolled in this study. Each center committed to enroll approximately 10 random patients whose formalin-fixed paraffin-embedded tumor tissues were available for K-Ras, exon 2 genotyping. Two methods were applied based on the availability of adequate amounts of tumor DNA. In the first method, the samples were processed using TheraScreen. The genomic DNA was further used to detect the 7 most frequent somatic mutations (35G>A; 35G>C; 35G>T; 34G>A; 34G>C; 34G>T and 38G>A) in codons 12 and 13 in exon 2 of the K-Ras oncogene by quantitative polymerase chain reaction (PCR). In the second method, the genomic DNA was amplified by PCR using primers specific for K-Ras exon 2 with the GML SeqFinder Sequencing System's KRAS kit. The identified DNA sequence alterations were confirmed by sequencing both DNA strands in two independent experiments with forward and reverse primers. A total of 40 samples had adequate tumor tissue for the mutation analysis. A total of 33 (82.5%) of the investigated samples harbored no mutations in exon 2. All the mutations were identified via a direct sequencing technique, whereas none were identified by TheraScreen. In conclusion, in our patients, HCC exhibited a remarkably low (<20%) K-Ras mutation rate. Patients harboring K-Ras wild-type tumors may be good candidates for treatment with epidermal growth factor inhibitors, such as cetuximab. PMID:26807232

  20. Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions

    PubMed Central

    Vatansever, Sezen; Gümüş, Zeynep H.; Erman, Burak

    2016-01-01

    K-Ras is the most frequently mutated oncogene in human cancers, but there are still no drugs that directly target it in the clinic. Recent studies utilizing dynamics information show promising results for selectively targeting mutant K-Ras. However, despite extensive characterization, the mechanisms by which K-Ras residue fluctuations transfer allosteric regulatory information remain unknown. Understanding the direction of information flow can provide new mechanistic insights for K-Ras targeting. Here, we present a novel approach –conditional time-delayed correlations (CTC) – using the motions of all residue pairs of a protein to predict directionality in the allosteric regulation of the protein fluctuations. Analyzing nucleotide-dependent intrinsic K-Ras motions with the new approach yields predictions that agree with the literature, showing that GTP-binding stabilizes K-Ras motions and leads to residue correlations with relatively long characteristic decay times. Furthermore, our study is the first to identify driver-follower relationships in correlated motions of K-Ras residue pairs, revealing the direction of information flow during allosteric modulation of its nucleotide-dependent intrinsic activity: active K-Ras Switch-II region motions drive Switch-I region motions, while α-helix-3L7 motions control both. Our results provide novel insights for strategies that directly target mutant K-Ras. PMID:27845397

  1. Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions

    NASA Astrophysics Data System (ADS)

    Vatansever, Sezen; Gümüş, Zeynep H.; Erman, Burak

    2016-11-01

    K-Ras is the most frequently mutated oncogene in human cancers, but there are still no drugs that directly target it in the clinic. Recent studies utilizing dynamics information show promising results for selectively targeting mutant K-Ras. However, despite extensive characterization, the mechanisms by which K-Ras residue fluctuations transfer allosteric regulatory information remain unknown. Understanding the direction of information flow can provide new mechanistic insights for K-Ras targeting. Here, we present a novel approach –conditional time-delayed correlations (CTC) – using the motions of all residue pairs of a protein to predict directionality in the allosteric regulation of the protein fluctuations. Analyzing nucleotide-dependent intrinsic K-Ras motions with the new approach yields predictions that agree with the literature, showing that GTP-binding stabilizes K-Ras motions and leads to residue correlations with relatively long characteristic decay times. Furthermore, our study is the first to identify driver-follower relationships in correlated motions of K-Ras residue pairs, revealing the direction of information flow during allosteric modulation of its nucleotide-dependent intrinsic activity: active K-Ras Switch-II region motions drive Switch-I region motions, while α-helix-3L7 motions control both. Our results provide novel insights for strategies that directly target mutant K-Ras.

  2. Implication of K-ras and p53 in colorectal cancer carcinogenesis in Tunisian population cohort.

    PubMed

    Ines, Chaar; Donia, Ounissi; Rahma, Boughriba; Ben Ammar, Azza; Sameh, Amara; Khalfallah, Taher; Abdelmajid, Ben Hmida; Sabeh, Mzabi; Saadia, Bouraoui

    2014-07-01

    According to the multistep route of genetic alterations in the colorectal adenoma-carcinoma sequence, the complex K-ras/p53 mutation is one of the first alterations to occur and represent an important genetic event in colorectal cancer (CRC). An evaluation of the mutation spectra in K-ras and p53 gene was effected in 167 Tunisian patients with sporadic CRC to determine whether our populations have similar pattern of genetic alteration as in Maghrebin's population. Mutation patterns of codon 12-13 of K-ras and exon 5-8 of p53 were analyzed by immunohistochemistry and PCR-SSCP and confirmed by sequencing. Mutations in the K-ras gene were detected in 31.13 % and affect the women more than the men (p = 0.008). Immunostaining showed that expression of p21 ras was correlated with the advanced age (p = 0.004), whereas loss of signal was associated with mucinous histotype (p = 0.003). Kaplan-Meier survival curve found that patients with the K-ras mutation had a shorter survival compared with patients without mutation (p = 0.005). Alteration in p53 was seen in 17.4 % of patients and affects three hot spot codons such as 175, 245, and 248. Overexpression of p53 was seen in 34.1 % and correlated with tumor node metastasis (TNM) advanced stage (p = 0.037) and mucinous histotype (p = 0.001). A high concordance between p53 expression and alteration (p<0.005) was shown. Concomitant mutations in K-ras and p53 gene were detected in only 4 % of tumors. K-ras and p53 undergo separate pathways in colorectal tumorogenesis. Interestingly, mutations in the K-ras gene might be considered a valuable prognostic factor correlated to poor outcome. p53 gene alterations were rather low in our set, and methylation pattern of p53 is required to elucidate the molecular basis of this protein in CRC.

  3. The Chk1 inhibitor MK-8776 increases the radiosensitivity of human triple-negative breast cancer by inhibiting autophagy

    PubMed Central

    Zhou, Zhi-rui; Yang, Zhao-zhi; Wang, Shao-jia; Zhang, Li; Luo, Ju-rui; Feng, Yan; Yu, Xiao-li; Chen, Xing-xing; Guo, Xiao-mao

    2017-01-01

    MK-8776 is a recently described inhibitor that is highly selective for checkpoint kinase 1 (Chk1), which can weaken the DNA repair capacity in cancer cells to achieve chemo-sensitization. A number of studies show that MK-8776 enhances the cytotoxicity of hydroxyurea and gemcitabine without increasing normal tissue toxicities. Thus far, there is no evidence that MK-8776 can be used as a radiotherapy sensitization agent. In this study, we investigated the effects of MK-8776 on the radiosensitivity of 3 human triple-negative breast cancer (TNBC) cell lines MDA-MB-231, BT-549 and CAL-51. MK-8776 dose-dependently inhibited the proliferation of MDA-MB-231, BT-549 and CAL-51 cells with IC50 values of 9.4, 17.6 and 2.1 μmol/L, respectively. Compared with irradiation-alone treatment, pretreatment with a low dose of MK-8776 (100–400 nmol/L) significantly increased irradiation-induced γH2A.X foci in the 3 TNBC cell lines, suggesting enhanced DNA damage by MK-8776, inhibited the cell proliferation and increased the radiosensitivity of the 3 TNBC cell lines. Similar results were obtained in MDA-MB-231 xenograft tumors in nude mice that received MK-8776 (15 or 40 mg/kg, ip) 26 d after irradiation. To explore the mechanisms underlying the radio-sensitization by MK-8776, we used TEM and found that irradiation significantly increased the numbers of autophagosomes in the 3 TNBC cell lines. Moreover, irradiation markedly elevated the levels of Atg5, and promoted the transformation of LC3-I to LC3-II in the cells. Pretreatment with the low dose of MK-8776 suppressed these effects. The above results suggest that MK-8776 increases human TNBC radiosensitivity by inhibiting irradiation-induced autophagy and that MK-8776 may be a potential agent in the radiosensitization of human TNBC. PMID:28042876

  4. The anticancer effects of Saccharina japonica on 267B1/K-ras human prostate cancer cells.

    PubMed

    Jo, Mi Jeong; Kim, Hyung Rak; Kim, Gun Do

    2012-11-01

    Saccharina japonica (S. japonica), a brown macro-alga, has been used as a traditional medicine in Korea for thousands of years. In this study, the potential anticancer effects of S. japonica were evaluated on 267B1/K-ras human prostate cancer cells. The exposure of cells to the extract induced inhibition of cell growth by increasing the number of apoptotic cells with cell shrinkage and inhibition of cell cycle progression. The effects of the extract on the cells were assessed by studying the cleavage of caspases and the target proteins of caspases. The increased expression of various cleaved caspases and changed expression of other proteins related in the apoptosis pathway were observed. 4'-6-Diamidino-2-phenylindole (DAPI) and immunofluorescence staining showed the cells undergoing apoptosis. Apoptosis induced changes in the expression of proteins involved in a variety of signaling pathways such as endocellular reticulum (ER) stress, death receptor and mammalian target of rapamycin (mTOR)-FoxO-mediated pathways. The data suggest that the extract (n-hexane sub-fraction) of S. japonica, induces apoptosis and cell cycle arrest in 267B1/K-ras human prostate cancer cells, and has potential as a complementary agent for cancer prevention.

  5. Hyperplastic (serrated) polyps of the colorectum: relationship of CpG island methylator phenotype and K-ras mutation to location and histologic subtype.

    PubMed

    O'Brien, Michael J; Yang, Shi; Clebanoff, Jennifer L; Mulcahy, Elizabeth; Farraye, Francis A; Amorosino, Mark; Swan, Niall

    2004-04-01

    We investigated the frequency of promoter region CpG island methylation (CIM) of hMLH1, MGMT, MINT1, MINT2, and p16 and K-ras mutations in a total of 79 hyperplastic (serrated) polyps (HPs) from 75 patients and correlated the molecular profiles to polyp location in the colorectum, histologic variation, and other factors. Methylation-specific PCR (MS-PCR) was used to assay CIM status. HPs that showed CIM of one or more or two or more of the genes assayed were classified as CpG island methylator phenotype (CIMP) and CIMP-high (CIMP-H), respectively. PCR restriction fragment length polymorphism was used to assay K-ras codon 12 and 13 mutations. Logistic regression indicated a statistically significant trend for increasing odds for CIMP (P = 0.002) and CIMP-H (P < 0.001) according to proximity to the cecum or distance from the rectum. Conversely, K-ras codon 12 mutation was present in 13 of 40 (32.5%) distally located HPs compared with 2 of 39 (5.1%) proximal HPs (P = 0.006). Histologic subtype distribution varied by proximal and distal locations. Frequency of CIMP in serrated polyps with abnormal proliferation (SPAPs), differed significantly from goblet cell serrated polyps (GCSPs) (24 of 26, 92.3% vs. 6 of 13, 46.2%) (P = 0.003) and microvesicular serrated polyps (MVSPs) (26 of 38, 68.4%) (P = 0.03). Frequency of K-ras mutation in GCSPs (7 of 13, 54%) differed from that of MVSPs (6 of 38, 16%) (P = 0.01) and SPAPs (2 of 26, 8%) (P = 0.003). Location in the colorectum and histologic subtype were major determinants of the molecular profile of HPs. The molecular findings of CIMP and K-ras mutations appear to encompass most if not all HPs; CIMP profiles suggest that SPAP is the most advanced morphologic variant. We postulate that MVSP and GCSP may be precursor lesions that, if proximally located or larger, can progress to SPAP. Frequent K-ras mutations and infrequent CIMP distinguish the distal GCSP variant.

  6. Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output.

    PubMed

    Zhou, Yong; Prakash, Priyanka; Liang, Hong; Cho, Kwang-Jin; Gorfe, Alemayehu A; Hancock, John F

    2017-01-12

    K-Ras is targeted to the plasma membrane by a C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain. We used quantitative spatial imaging and atomistic molecular dynamics simulations to examine molecular details of K-Ras plasma membrane binding. We found that the K-Ras anchor binds selected plasma membrane anionic lipids with defined head groups and lipid side chains. The precise amino acid sequence and prenyl group define a combinatorial code for lipid binding that extends beyond simple electrostatics; within this code lysine and arginine residues are non-equivalent and prenyl chain length modifies nascent polybasic domain lipid preferences. The code is realized by distinct dynamic tertiary structures of the anchor on the plasma membrane that govern amino acid side-chain-lipid interactions. An important consequence of this specificity is the ability of such anchors when aggregated to sort subsets of phospholipids into nanoclusters with defined lipid compositions that determine K-Ras signaling output.

  7. Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress

    PubMed Central

    Shaw, Alice T.; Winslow, Monte M.; Magendantz, Margaret; Ouyang, Chensi; Dowdle, James; Subramanian, Aravind; Lewis, Timothy A.; Maglathin, Rebecca L.; Tolliday, Nicola; Jacks, Tyler

    2011-01-01

    Activating K-RAS mutations are the most frequent oncogenic mutations in human cancer. Numerous downstream signaling pathways have been shown to be deregulated by oncogenic K-ras. However, to date there are still no effective targeted therapies for this genetically defined subset of patients. Here we report the results of a small molecule, synthetic lethal screen using mouse embryonic fibroblasts derived from a mouse model harboring a conditional oncogenic K-rasG12D allele. Among the >50,000 compounds screened, we identified a class of drugs with selective activity against oncogenic K-ras–expressing cells. The most potent member of this class, lanperisone, acts by inducing nonapoptotic cell death in a cell cycle- and translation-independent manner. The mechanism of cell killing involves the induction of reactive oxygen species that are inefficiently scavenged in K-ras mutant cells, leading to oxidative stress and cell death. In mice, treatment with lanperisone suppresses the growth of K-ras–driven tumors without overt toxicity. Our findings establish the specific antitumor activity of lanperisone and reveal oxidative stress pathways as potential targets in Ras-mediated malignancies. PMID:21555567

  8. Alterations in the K-ras and p53 genes in rat lung tumors

    SciTech Connect

    Belinsky, S.A.; Swafford, D.S.; Finch, G.L.; Mitchell, C.E.

    1997-06-01

    Activation of the K-ras protooncogene and inactivation of the p53 tumor suppressor gene are events common to many types of human cancers. Molecular epidemiology studies have associated mutational profiles in these genes with specific exposures. The purpose of this paper is to review investigations that have examined the role of the K-ras and p53 genes in lung tumors induced in the F344 rat by mutagenic and nonmutagenic exposures. Mutation profiles within the K-ras and p53 genes, if present in rat lung tumors, would help to define some of the molecular mechanisms underlying cancer induction by various environmental agents. Pulmonary adenocarcinomas or squamous cell carcinomas were induced by tetranitromethane (TNM), 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK), beryllium metal, plutonium-239, X-ray, diesel exhaust, or carbon black. These agents were chosen because the tumors they produced could arise via different types of DNA damage. Mutation of the K-ras gene was determined by approaches that included DNA transfection, direct sequencing, mismatch hybridization, and restriction fragment length polymorphism analysis. The frequency for mutation of the K-ras gene was exposure dependent. The transition mutations formed could have been derived from deamination of cytosine. Alteration in the p53 gene was assessed by immunohistochemical analysis for p53 protein and single-strand conformation polymorphism (SSCP) analysis of exons 4 to 9. None of the 93 adenocarinomas examined was immunoreactive toward the anti-p53 antibody CM1. In contrast, 14 of 71 squamous cell carcinomas exhibited nuclear p53 immunoreactivity with no correlation to type of exposure. However, SSCP analysis only detected mutations in 2 of 14 squamous cell tumors that were immunoreactive, suggesting that protein stabilization did not stem from mutations within the p53 gene. Thus, the p53 gene does not appear to be involved in the genesis of most rat lung tumors. 2 figs., 2 tabs., 48 refs.

  9. The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting.

    PubMed

    Jonckheere, Nicolas; Vasseur, Romain; Van Seuningen, Isabelle

    2017-03-01

    RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives.

  10. [Effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-catenin/p120 protein complex formation and RhoA protein activity].

    PubMed

    Li, Jing-nan; Li, Xiao; Qian, Jia-ming; Lu, Xin-qing; Yang, Hong

    2010-02-01

    To explore the effects of K-ras gene mutation on colon cancer cell line Caco-2 metastasis by regulating E-cadherin/beta-catenin/p120 protein complex formation and RhoA protein activity. K-ras wild-type colon cancer cell line Caco-2 was transiently transfected by phr-GFP vector (control group), transfected by mutant K-ras gene phr-K-ras (Val12) vector (transfection group), transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific MAPK pathway inhibitor PD98059 (MAPK inhibition group), or transfected by mutant K-ras gene phr-K-ras (Val12) vector and treated by specific PI-3K pathway inhibitor LY294002 (PI-3K inhibition group), respectively. Cell migration was tested by Transwell experiment. E-cadherin and beta-catenin protein expression and intracellular location were detected by cell immunofluorescence method. Intracellular p120 protein expression was detected by Western blot. beta-catenin protein level which combined with E-cadherin was detected by immunoprecipitation. RhoA activity was analyzed by Pull-down assay. The Caco-2 cell migration rate was (19.8 +/- 5.6) % in transfection group, which was significantly higher than that in control group [(14.0 +/- 4.2) %] (P = 0.001) and in MAPK inhibition group [(15.8 +/- 1.2) %] (P = 0.044), but was not significantly different from that in PI-3K inhibition group [(17.5 +/- 2.8) %] (P = 0.095). Immunofluorescence method showed that the E-cadherin and beta-catenin stain located in the cell membrane decreased in transfection group. Western blot showed that the total intracellular p120 protein decreased in transfection group and PI-3K inhibition group. Immunoprecipitation data showed that beta-catenin protein level combined with E-cadherin decreased in transfection group and PI-3K group. Pull-down test showed that RhoA protein activity was up-regulated in transfection group. K-ras gene mutation stimulates the migration of colon cancer cell Caco-2, which may be achieved by decreasing the E

  11. K-Ras(G12D)-selective inhibitory peptides generated by random peptide T7 phage display technology.

    PubMed

    Sakamoto, Kotaro; Kamada, Yusuke; Sameshima, Tomoya; Yaguchi, Masahiro; Niida, Ayumu; Sasaki, Shigekazu; Miwa, Masanori; Ohkubo, Shoichi; Sakamoto, Jun-Ichi; Kamaura, Masahiro; Cho, Nobuo; Tani, Akiyoshi

    2017-03-11

    Amino-acid mutations of Gly(12) (e.g. G12D, G12V, G12C) of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras), the most promising drug target in cancer therapy, are major growth drivers in various cancers. Although over 30 years have passed since the discovery of these mutations in most cancer patients, effective mutated K-Ras inhibitors have not been marketed. Here, we report novel and selective inhibitory peptides to K-Ras(G12D). We screened random peptide libraries displayed on T7 phage against purified recombinant K-Ras(G12D), with thorough subtraction of phages bound to wild-type K-Ras, and obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) as a consensus sequence. KRpep-2 showed more than 10-fold binding- and inhibition-selectivity to K-Ras(G12D), both in SPR analysis and GDP/GTP exchange enzyme assay. KD and IC50 values were 51 and 8.9 nM, respectively. After subsequent sequence optimization, we successfully generated KRpep-2d (Ac-RRRRCPLYISYDPVCRRRR-NH2) that inhibited enzyme activity of K-Ras(G12D) with IC50 = 1.6 nM and significantly suppressed ERK-phosphorylation, downstream of K-Ras(G12D), along with A427 cancer cell proliferation at 30 μM peptide concentration. To our knowledge, this is the first report of a K-Ras(G12D)-selective inhibitor, contributing to the development and study of K-Ras(G12D)-targeting drugs.

  12. Cetuximab in the first-line treatment of K-ras wild-type metastatic colorectal cancer: the choice and schedule of fluoropyrimidine matters.

    PubMed

    Ku, Geoffrey Y; Haaland, Benjamin A; de Lima Lopes, Gilberto

    2012-08-01

    Cetuximab, a monoclonal antibody against the epidermal growth factor receptor, inconsistently improves response rates (RR), progression-free survival (PFS) and overall survival (OS) in the first-line treatment of advanced colorectal cancer patients with K-ras wild-type (WT) tumors. We performed a meta-analysis of four trials where K-ras WT Pts received a fluoropyrimidine (infusional vs. bolus 5-fluorouracil (5-FU) vs. capecitabine) and oxaliplatin or irinotecan with and without cetuximab (CRYSTAL, OPUS, COIN and NORDIC VII trials) and two trials, where K-ras WT and mutant patients received cetuximab and a fluoropyrimidine (capecitabine in a German AIO study and infusional 5-FU in the CECOG study) with oxaliplatin versus irinotecan. We sought to determine whether the choice of fluoropyrimidine or of oxaliplatin versus irinotecan affects the response to cetuximab. Meta-analysis was performed in the context of a mixed effects model with a random effect for each study. Only patients treated with infusional 5-FU-based chemotherapy derived benefit from cetuximab. Relative to infusional 5-FU, patients treated with capecitabine/bolus 5-FU-based doublet chemotherapy had a 42 % (95 % CI 21-58 %; p < 0.001) decrease in response probability and a 52 % (95 % CI 20-93 %; p < 0.001) and 33 % (95 % CI 7-65 %; p = 0.012) increase, respectively, in risk of progression and death. The choice of oxaliplatin or irinotecan did not affect benefit from cetuximab. The lack of benefit for cetuximab with capecitabine/bolus 5-FU regimens is unexpected. Cetuximab should only be used with infusional 5-FU regimens in the first-line treatment of K-ras WT colorectal cancer patients. Further study is urgently needed to elucidate the basis of this observation.

  13. EGF RECEPTOR SIGNALING IS ESSENTIAL FOR K-RAS ONCOGENE-DRIVEN PANCREATIC DUCTAL ADENOCARCINOMA

    PubMed Central

    Navas, Carolina; Hernández-Porras, Isabel; Schuhmacher, Alberto J; Sibilia, Maria; Guerra, Carmen; Barbacid, Mariano

    2013-01-01

    Clinical evidence indicates that mutation/activation of EGF receptors (EGFRs) is mutually exclusive with the presence of K-RAS oncogenes in lung and colon tumors. We have validated these observations using genetically engineered mouse models. However, pancreatic ductal adenocarcinomas driven by K-Ras oncogenes are totally dependent on EGFR signaling. Similar results were obtained using human pancreatic tumor cell lines. EGFRs were also essential even in the context of pancreatic injury and absence of p16Ink4a/p19Arf. Only loss of p53 made pancreatic tumors independent of EGFR signaling. Additional inhibition of PI3K and STAT3 effectively prevented proliferation of explants derived from these p53–defective pancreatic tumors. These findings may provide the bases for more rational approaches to treat pancreatic tumors in the clinic. PMID:22975375

  14. Radiosensitivity increases with differentiation status of murine hemopoietic progenitor cells selected using enriched marrow subpopulations and recombinant growth factors

    SciTech Connect

    Baird, M.C.; Hendry, J.H.; Testa, N.G. )

    1990-09-01

    The radiosensitivity of populations of colony-forming cells (CFC) in murine bone marrow was investigated using different recombinant colony-stimulating factors (CSFs; murine IL-3 and granulocyte-macrophage CSF and human granulocyte CSF), or purified murine macrophage CSF. With unfractionated normal bone marrow the CFC increased in radiosensitivity as they progressed through the granulocyte lineage. The D0 values ranged from 129 +/- 12 cGy for CFC stimulated with GM-CSF down to 42 +/- 2 cGy after stimulation with G-CSF. IL-3 stimulated a CFC population which gave the only survival curve with a shoulder (n = 1.9 +/- 0.3). With semipurified populations of primitive or bipotential CFC, D0 values were generally lower with respect to the equivalent values for unpurified bone marrow (range 62 +/- 7 cGy to 135 +/- 7 cGy). Changes in cluster/colony ratio and colony morphology together possibly with products of accessory cells influence the interpretation of the radiosensitivity parameters.

  15. Specific repression of mutant K-RAS by 10-23 DNAzyme: Sensitizing cancer cell to anti-cancer therapies

    SciTech Connect

    Yu, S.-H.; Wang, T.-H.; Au, L.-C.

    2009-01-09

    Point mutations of the Ras family are frequently found in human cancers at a prevalence rate of 30%. The most common mutation K-Ras(G12V), required for tumor proliferation, survival, and metastasis due to its constitutively active GTPase activity, has provided an ideal target for cancer therapy. 10-23 DNAzyme, an oligodeoxyribonucleotide-based ribonuclease consisting of a 15-nucleotide catalytical domain flanked by two target-specific complementary arms, has been shown to effectively cleave the target mRNA at purine-pyrimidine dinucleotide. Taking advantage of this specific property, 10-23 DNAzyme was designed to cleave mRNA of K-Ras(G12V)(GGU {yields} GUU) at the GU dinucleotide while left the wild-type (WT) K-Ras mRNA intact. The K-Ras(G12V)-specific 10-23 DNAzyme was able to reduce K-Ras(G12V) at both mRNA and protein levels in SW480 cell carrying homozygous K-Ras(G12V). No effect was observed on the WT K-Ras in HEK cells. Although K-Ras(G12V)-specific DNAzymes alone did not inhibit proliferation of SW480 or HEK cells, pre-treatment of this DNAzyme sensitized the K-Ras(G12V) mutant cells to anti-cancer agents such as doxorubicin and radiation. These results offer a potential of using allele-specific 10-23 DNAzyme in combination with other cancer therapies to achieve better effectiveness on cancer treatment.

  16. SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling

    PubMed Central

    Siljamäki, Elina

    2016-01-01

    The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner. PMID:27503857

  17. Genetic Interaction between Rb and K-ras in the Control of Differentiation and Tumor Suppression

    PubMed Central

    Takahashi, Chiaki; Contreras, Bernardo; Bronson, Roderick T.; Loda, Massimo; Ewen, Mark E.

    2004-01-01

    Although the retinoblastoma protein (pRb) has been implicated in the processes of cellular differentiation, there is no compelling genetic or in vivo evidence that such activities contribute to pRb-mediated tumor suppression. Motivated by cell culture studies suggesting that Ras is a downstream effector of pRb in the control of differentiation, we have examined the tumor and developmental phenotypes of Rb and K-ras double-knockout mice. We find that heterozygosity for K-ras (i) rescued a unique subset of developmental defects that characterize Rb-deficient embryos by affecting differentiation but not proliferation and (ii) significantly enhanced the degree of differentiation of pituitary adenocarcinomas arising in Rb heterozygotes, leading to their prolonged survival. These observations suggest that Rb and K-ras function together in vivo, in the contexts of both embryonic and tumor development, and that the ability to affect differentiation is a major facet of the tumor suppressor function of pRb. PMID:15542848

  18. Anti-tumor activity of ESX1 on cancer cells harboring oncogenic K-ras mutation

    SciTech Connect

    Nakajima, Junta; Ishikawa, Susumu; Hamada, Jun-Ichi; Yanagihara, Masatomo; Koike, Takao; Hatakeyama, Masanori

    2008-05-23

    Human ESX1 is a 65-kilodalton (kDa) paired-like homeoprotein that is proteolytically processed into N-terminal 45-kDa and C-terminal 20-kDa fragments. The N-terminal ESX1 fragment, which contains the homeodomain, localizes to the nucleus and represses mRNA transcription from the K-ras gene. When we inoculated human colorectal carcinoma HCT116 constitutive expressing N-terminal region of ESX1 (N-ESX1) into nude mice, transfectant cells uniformly showed decreased tumor-forming activity compared with that of the parental cells. Furthermore, pretreatment of HCT116 carcinoma cells with a fusion protein consisting of N-ESX1 and the protein-transduction domain derived from the human immunodeficiency virus type-1 TAT protein gave rise to a dramatic reduction in the tumorigenicity of HCT116 cells in nude mice. Our results provide first in vivo evidence for the molecular targeting therapeutic application of the K-ras repressor ESX1, especially TAT-mediated transduction of N-ESX1, in the treatment of human cancers having oncogenic K-ras mutations.

  19. K-RasV14I recapitulates Noonan syndrome in mice.

    PubMed

    Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R; Guerra, Carmen; Barbacid, Mariano

    2014-11-18

    Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-Ras(V14I), a recurrent KRAS mutation in NS patients. K-Ras(V14I)-mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-Ras(V14I)-mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome.

  20. K-RasV14I recapitulates Noonan syndrome in mice

    PubMed Central

    Hernández-Porras, Isabel; Fabbiano, Salvatore; Schuhmacher, Alberto J.; Aicher, Alexandra; Cañamero, Marta; Cámara, Juan Antonio; Cussó, Lorena; Desco, Manuel; Heeschen, Christopher; Mulero, Francisca; Bustelo, Xosé R.; Guerra, Carmen; Barbacid, Mariano

    2014-01-01

    Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-RasV14I, a recurrent KRAS mutation in NS patients. K-RasV14I–mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-RasV14I–mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome. PMID:25359213

  1. CYP1B1 polymorphisms and k-ras mutations in patients with pancreatic ductal adenocarcinoma.

    PubMed

    Crous-Bou, Marta; De Vivo, Immaculata; Porta, Miquel; Pumarega, José A; López, Tomàs; Alguacil, Joan; Morales, Eva; Malats, Núria; Rifà, Juli; Hunter, David J; Real, Francisco X

    2008-05-01

    The frequency of CYP1B1 polymorphisms in pancreatic cancer has never been reported. There is also no evidence on the relationship between CYP1B1 variants and mutations in ras genes (K-, H- or N-ras) in any human neoplasm. We analyzed the following CYP1B1 polymorphisms in 129 incident cases of pancreatic ductal adenocarcinoma (PDA): the m1 allele (Val to Leu at codon 432) and the m2 allele (Asn to Ser at codon 453). The calculated frequencies for the m1 Val and m2 Asn alleles were 0.45 and 0.68, respectively. CYP1B1 genotypes were out of Hardy-Weinberg equilibrium; this was largely due to K-ras mutated PDA cases. The Val/Val genotype was over five times more frequent in PDA cases with a K-ras mutation than in wild-type cases (OR = 5.25; P = 0.121). In PDA, polymorphisms in CYP1B1 might be related with K-ras activation pathways.

  2. SPRED1 Interferes with K-ras but Not H-ras Membrane Anchorage and Signaling.

    PubMed

    Siljamäki, Elina; Abankwa, Daniel

    2016-10-15

    The Ras/mitogen-activated protein kinase (MAPK) signaling pathway is tightly controlled by negative feedback regulators, such as the tumor suppressor SPRED1. The SPRED1 gene also carries loss-of-function mutations in the RASopathy Legius syndrome. Growth factor stimulation translocates SPRED1 to the plasma membrane, triggering its inhibitory activity. However, it remains unclear whether SPRED1 there acts at the level of Ras or Raf. We show that pharmacological or galectin-1 (Gal-1)-mediated induction of B- and C-Raf-containing dimers translocates SPRED1 to the plasma membrane. This is facilitated in particular by SPRED1 interaction with B-Raf and, via its N terminus, with Gal-1. The physiological significance of these novel interactions is supported by two Legius syndrome-associated mutations that show diminished binding to both Gal-1 and B-Raf. On the plasma membrane, SPRED1 becomes enriched in acidic membrane domains to specifically perturb membrane organization and extracellular signal-regulated kinase (ERK) signaling of active K-ras4B (here, K-ras) but not H-ras. However, SPRED1 also blocks on the nanoscale the positive effects of Gal-1 on H-ras. Therefore, a combinatorial expression of SPRED1 and Gal-1 potentially regulates specific patterns of K-ras- and H-ras-dependent signaling output. More broadly, our results open up the possibility that related SPRED and Sprouty proteins act in a similar Ras and Raf isoform-specific manner.

  3. Changes in epidermal radiosensitivity with time associated with increased colony numbers.

    PubMed

    van den Aardweg, G J; Morris, G M; Bywaters, A; Bakker, E J; Mooi, W J

    2001-05-01

    Epidermal clonogenic cell survival and colony formation following irradiation were investigated and related to radiosensitivity. A rapid in vivo/in vitro assay was developed for the quantification of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. Bromodeoxyuridine (BrdU)-labelled cells in full thickness epidermal sheets were visualized using standard immunohistochemistry. In unirradiated skin, approximately 900 BrdU-positive cells mm(-2) were counted. In a time sequence experiment, BrdU-positive cell numbers increased from an average of 900 cells mm(-2) to approximately 1400 cells mm(-2) after BrdU-labelling for 2-24 h. In irradiated skin, colonies containing >/=16 BrdU-positive cells were seen for the first time at days 14/15 after irradiation. The number of these colonies per cm(2) as a function of skin surface dose yielded a cell survival curve with a D(0)-value (+/-SE) of 3.9+/-0.6 Gy. This relatively high D(0)-value is possibly due to a rapid fall off in depth dose distribution for the iridium-192 source and consequently a substantial contribution of hair follicular epithelium to colony formation. At 14/15 days after irradiation, the ED(50) level of 33.6 Gy for the in vivo response of moist desquamation corresponded with 2.7 colonies cm(-2). Surprisingly, the number of colonies increased with time after irradiation with an estimated doubling time of approximately 4 days, while the D(0)-value remained virtually unchanged. This increase in colony numbers could be due to migration of clonogenic cells, to the recruitment of dormant clonogenic cell survivors by elevated levels of cytokines, or to both. Although frequent biopsying caused increased cytokine levels, which had a systemic effect on unirradiated skin, it had no influence on colony formation in irradiated skin. Smaller colonies, containing 4-8 cells or 9-15 cells, were abundant, particularly after higher doses, which resulted in higher D(0)-values. The majority of

  4. The let-7a microRNA protects from growth of lung carcinoma by suppression of k-Ras and c-Myc in nude mice.

    PubMed

    He, Xiao-Yan; Chen, Jun-xia; Zhang, Zheng; Li, Chun-lei; Peng, Qiong-le; Peng, Hui-min

    2010-07-01

    Down-regulation of let-7 microRNA (miRNA) plays an important role in the pathogenesis of lung cancer. k-Ras and c-Myc, two key oncogenes in lung cancer, have been found to be targeted by let-7 in vitro. However, the in vivo relevance of these findings is unknown. The aim of the present study is to determine the effect of let-7a, a member of let-7 family, on the growth of lung cancer in vivo and to investigate whether let-7-induced suppression of k-Ras and c-Myc is involved in lung cancer. A549-let-7a cell line and A549-control cell line, two stable transfected cell lines over-expressing let-7a and the control miRNA, were established and preserved in our lab. A549, A549-control, and A549-let-7a cells were injected subcutaneously into nude mice, respectively. After 30 days, the mice were killed; the xenografts were excised and weighed. The expression of let-7a in tumor xenografts was assessed by real-time reverse transcription-PCR (RT-PCR). The expression of k-Ras and c-Myc in xenografts were determined by western blot and immunohistochemistry detection. Real-time RT-PCR showed the expression of let-7a was increased significantly in A549-let-7a cells-injected group, compared with A549-control cells-injected group and A549 cells-injected group (P < 0.01). In the xenografts of A549-let-7a cells-injected group, a significant depression in tumor weight (P < 0.05) and significant decrease of k-Ras and c-Myc protein were observed (P < 0.01), compared to A549 cells-injected group and A549-control cells-injected group. Overexpression of let-7a can inhibit the growth of lung cancer transplanted subcutaneously in nude mice by suppression of k-Ras and c-Myc.

  5. Is Increased Low-dose somatic Radiosensitivity Associated with Increased Transgenerational Germline Mutation

    SciTech Connect

    Brenner, David J.

    2008-10-02

    Using single-molecule polymerase chain reaction, the frequency of spontaneous and radiation-induced mutation at an expanded simple tandem repeat (ESTR) locus was studied in DNA samples extracted from sperm and bone marrow of Atm knockout (Atm+/–) heterozygous male mice. The frequency of spontaneous mutation in sperm and bone marrow in Atm+/– males did not significantly differ from that in wild-type BALB/c mice. Acute gamma-ray exposure did not affect ESTR mutation frequency in bone marrow and resulted in similar increases in sperm samples taken from Atm+/– and BALB/c males. Taken together, these results suggest that the Atm haploinsufficiency analyzed in our study does not affect spontaneous and radiation-induced ESTR mutation frequency in mice.

  6. Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

    PubMed

    Gong, Jianhua; Wang, Zhongde; Polejaeva, Irina; Salgia, Ravi; Kao, Chien-Min; Chen, Chin-Tu; Chen, Guangchun; Chen, Liaohai

    2014-01-01

    Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

  7. Activating the Expression of Human K-rasG12D Stimulates Oncogenic Transformation in Transgenic Goat Fetal Fibroblast Cells

    PubMed Central

    Gong, Jianhua; Wang, Zhongde; Polejaeva, Irina; Salgia, Ravi; Kao, Chien-Min; Chen, Chin-Tu; Chen, Guangchun; Chen, Liaohai

    2014-01-01

    Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established. PMID:24594684

  8. Activation of c-myc and c-K-ras oncogenes in primary rat tumors induced by ionizing radiation.

    PubMed Central

    Sawey, M J; Hood, A T; Burns, F J; Garte, S J

    1987-01-01

    An activated K-ras oncogene was detected by transfection in NIH 3T3 cells and by Southern blot analysis in 6 of 12 rat skin tumors induced by ionizing radiation. The DNA from 10 of the 12 tumors also showed c-myc gene amplification and restriction polymorphisms. Evidence for tissue specificity was observed in patterns of oncogene activation, with each of three clear cell carcinomas exhibiting activation of both c-myc and K-ras oncogenes. Images PMID:3547086

  9. Cooperativity of Oncogenic K-Ras and Downregulated p16/INK4A in Human Pancreatic Tumorigenesis

    PubMed Central

    Ling, Jianhua; Zhuang, Zhuonan; Li, Zhongkui; Wang, Huamin; Fleming, Jason B.; Freeman, James W.; Yu, Dihua; Huang, Peng; Chiao, Paul J.

    2014-01-01

    Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC. PMID:25029561

  10. Cooperativity of oncogenic K-ras and downregulated p16/INK4A in human pancreatic tumorigenesis.

    PubMed

    Chang, Zhe; Ju, Huaiqiang; Ling, Jianhua; Zhuang, Zhuonan; Li, Zhongkui; Wang, Huamin; Fleming, Jason B; Freeman, James W; Yu, Dihua; Huang, Peng; Chiao, Paul J

    2014-01-01

    Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGFα signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC.

  11. AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

    PubMed

    Cho, Kwang-Jin; Casteel, Darren E; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A; Kim, Choel; Capon, Robert J; Lacey, Ernest; Cunha, Shane R; Gorfe, Alemayehu A; Hancock, John F

    2016-12-15

    K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

  12. Increased radiosensitivity of colorectal tumors with intra-tumoral injection of low dose of gold nanoparticles

    PubMed Central

    Shi, Minghan; Paquette, Benoit; Thippayamontri, Thititip; Gendron, Louis; Guérin, Brigitte; Sanche, Léon

    2016-01-01

    The potential of gold nanoparticles (GNPs) as radiosensitizers for the treatment of malignant tumors has been limited by the large quantities of GNPs that must be administered and the requirement for low-energy X-ray irradiation to optimize radiosensitization. In this study, we enhance the radiosensitivity of HCT116 human colorectal cells with tiopronin-coated GNPs (Tio-GNPs) combined with a low-energy X-ray (26 keV effective energy) source, similar to the Papillon 50 clinical irradiator used for topical irradiation of rectal tumors. Sensitizer enhancement ratios of 1.48 and 1.69 were measured in vitro, when the HCT116 cells were incubated with 0.1 mg/mL and 0.25 mg/mL of Tio-GNPs, respectively. In nude mice bearing the HCT116 tumor, intra-tumoral (IT) injection of Tio-GNPs allowed a 94 times higher quantity of Tio-GNPs to accumulate than was possible by intravenous injection and facilitated a significant tumor response. The time following irradiation, for tumors growing to four times their initial tumor volume (4Td) was 54 days for the IT injection of 366.3 μg of Tio-GNPs plus 10 Gy, compared to 37 days with radiation alone (P=0.0018). Conversely, no significant improvement was obtained when GNPs were injected intravenously before tumor irradiation (P=0.6547). In conclusion, IT injection of Tio-GNPs combined with low-energy X-rays can significantly reduce the growth of colorectal tumors. PMID:27789945

  13. Early recognition of lung cancer by integrin targeted imaging in K-ras mouse model.

    PubMed

    Ermolayev, Vladimir; Mohajerani, Pouyan; Ale, Angelique; Sarantopoulos, Athanasios; Aichler, Michaela; Kayser, Gian; Walch, Axel; Ntziachristos, Vasilis

    2015-09-01

    Non-small cell lung cancer is characterized by slow progression and high heterogeneity of tumors. Integrins play an important role in lung cancer development and metastasis and were suggested as a tumor marker; however their role in anticancer therapy remains controversial. In this work, we demonstrate the potential of integrin-targeted imaging to recognize early lesions in transgenic mouse model of lung cancer based on spontaneous introduction of mutated human gene bearing K-ras mutation. We conducted ex vivo and fluorescence molecular tomography-X-ray computed tomography (FMT-XCT) in vivo imaging and analysis for specific targeting of early lung lesions and tumors in rodent preclinical model for lung cancer. The lesions and tumors were characterized by histology, immunofluorescence and immunohistochemistry using a panel of cancer markers. Ex vivo, the integrin-targeted fluorescent signal significantly differed between wild type lung tissue and K-ras pulmonary lesions (PL) at all ages studied. The panel of immunofluorescence experiments demonstrated that PL, which only partially show cancer cell features were detected by αvβ3-integrin targeted imaging. Human patient material analysis confirmed the specificity of target localization in different lung cancer types. Most importantly, small tumors in the lungs of 4-week-old animals could be noninvasively detected in vivo on the fluorescence channel of FMT-XCT. Our findings demonstrated αvβ3-integrin targeted fluorescent imaging to specifically detect premalignant pleural lesions in K-ras mice. Integrin targeted imaging may find application areas in preclinical research and clinical practice, such as early lung cancer diagnostics, intraoperative assistance or therapy monitoring.

  14. Conformational SERS Classification of K-Ras Point Mutations for Cancer Diagnostics.

    PubMed

    Morla-Folch, Judit; Gisbert-Quilis, Patricia; Masetti, Matteo; Garcia-Rico, Eduardo; Alvarez-Puebla, Ramon A; Guerrini, Luca

    2017-02-20

    Point mutations in Ras oncogenes are routinely screened for diagnostics and treatment of tumors (especially in colorectal cancer). Here, we develop an optical approach based on direct SERS coupled with chemometrics for the study of the specific conformations that single-point mutations impose on a relatively large fragment of the K-Ras gene (141 nucleobases). Results obtained offer the unambiguous classification of different mutations providing a potentially useful insight for diagnostics and treatment of cancer in a sensitive, fast, direct and inexpensive manner.

  15. [Study on the effect of grape seed proanthocyanidins on increasing the radiosensitivity for X-ray].

    PubMed

    Pan, Xiao-jing; Wang, Min; Wang, Xue-xi; Liu, Bin; Zhang, Hong

    2012-02-01

    To investigate the effect of Grape Seed Proanthocyanidins (GSPs) on enhancing the radiosensitivity of human hepatic carcinoma cell line HepG2, human cervical cancer cell line Hela and human leukemia cell line K562 for X-ray in vitro. The killing effect of GSPs combined with X-ray on cells was evaluated by SRB and clone formation assay. The GSPs had obvious cytotoxicity on three cell lines in a dose-dependent and time-dependent manners. However, inhibition rate of different cell line were quite different, the strongest one was human leukemia K562 cells and the others were weak. The sensitization ranges calculated by univariate analysis were 6.25-12.5 microg/mL for human leukemia K562 cells. Sensitization enhancement ratio was 1.94 using curve fitting method for K562 cells. GSPs can obviously enhance the radiosensitivity of cancer cells in vitro. The mechanism of sensitization effect may be related to the effects of GSPs on oxygen balance and cell cycle.

  16. Toll-like receptor 9 agonist IMO cooperates with cetuximab in K-ras mutant colorectal and pancreatic cancers.

    PubMed

    Rosa, Roberta; Melisi, Davide; Damiano, Vincenzo; Bianco, Roberto; Garofalo, Sonia; Gelardi, Teresa; Agrawal, Sudhir; Di Nicolantonio, Federica; Scarpa, Aldo; Bardelli, Alberto; Tortora, Giampaolo

    2011-10-15

    K-Ras somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong in vivo activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab. In the present study, we investigated, both in vitro and in vivo, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring K-Ras mutations and resistance to EGFR inhibitors. We showed that IMO was able to significantly restore the sensitivity of K-Ras mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen-activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. In vivo, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using K-Ras mutant colorectal cancer cell models obtained through homologous recombination technology. We showed that IMO markedly inhibits growth of K-Ras mutant colon and pancreatic cancers in vitro and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for K-Ras wild-type as well for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancers. ©2011 AACR.

  17. Knockdown of Importin 7 Inhibits Lung Tumorigenesis in K-ras(LA1) Lung Cancer Mice.

    PubMed

    Lee, Ah Young; Kim, Sangwha; Lee, Somin; Jiang, Hu-Lin; Kim, Sang-Bum; Hong, Seong-Ho; Cho, Myung-Haing

    2017-05-01

    Background/Aim: Lung cancer shows the highest estimated deaths in both males and females in the Unites States. Importin 7 is overexpressed in lung adenocarcinoma tissues. In this study, we aimed to demonstrate the anticancer effect of importin 7 down-regulation, especially in lung cancer. Materials and Methods: Glycerol propoxylate triacrylate spermine (GPT-SPE) is a biocompatible carrier used for aerosol gene delivery. Repeated aerosol delivery of GPT-SPE/shImportin 7 complexes was performed to 10-week-old male K-ras(LA1)mice (a murine lung cancer model) twice a week for 4 weeks (8 times) in a nose-only exposure chamber. Results: Aerosol delivery of GPT-SPE/shImportin 7 inhibits lung cancer in K-ras(LA1)mice compared to control and scramble control groups. Moreover, importin 7-down-regulated stable cell-line demonstrates suppression of proliferation through Akt inhibition and apoptosis. Conclusion: Down-regulation of importin 7 significantly suppresses lung cancer in vitro and in vivo. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  18. 3-Methyl pyruvate enhances radiosensitivity through increasing mitochondria-derived reactive oxygen species in tumor cell lines.

    PubMed

    Nishida, Naoya; Yasui, Hironobu; Nagane, Masaki; Yamamori, Tohru; Inanami, Osamu

    2014-05-01

    Considerable interest has recently been focused on the special characteristics of cancer metabolism, and several drugs designed to modulate cancer metabolism have been tested as potential anticancer agents. To date, however, very few studies have been conducted to investigate the combined effects of anticancer drugs and radiotherapy. In this study, to evaluate the role of mitochondria-derived reactive oxygen species (ROS) in the radiation-induced cell death of tumor cells, we have examined the effect of 3-methyl pyruvate (MP). MP is a membrane-permeable pyruvate derivative that is capable of activating mitochondrial energy metabolism in human lung carcinoma A549 cells and murine squamous carcinoma SCCVII cells. Pretreatment with MP significantly enhanced radiation-induced cell death in both cell lines, and also led to increases in the mitochondrial membrane potential, intracellular adenosine triphosphate content, and mitochondria-derived ROS production following the exposure of the cells to X-rays. In A549 cells, MP-induced radiosensitization was completely abolished by vitamin C. In contrast, it was partially abolished in SCCVII cells. These results therefore suggest that the treatment of the cells with MP induced radiosensitization via the production of excess mitochondria-derived ROS in tumor cells.

  19. Increased chemosensitivity and radiosensitivity of human breast cancer cell lines treated with novel functionalized single-walled carbon nanotubes

    PubMed Central

    Jia, Yijun; Weng, Ziyi; Wang, Chuanying; Zhu, Mingjie; Lu, Yunshu; Ding, Longlong; Wang, Yongkun; Cheng, Xianhua; Lin, Qing; Wu, Kejin

    2017-01-01

    Hypoxia is a major cause of treatment resistance in breast cancer. Single-walled carbon nanotubes (SWCNTs) exhibit unique properties that make them promising candidates for breast cancer treatment. In the present study, a new functionalized single-walled carbon nanotube carrying oxygen was synthesized; it was determined whether this material could increase chemosensitivity and radiosensitivity of human breast cancer cell lines, and the underlying mechanisms were investigated. MDA-MB-231 cells growing in folic acid (FA) free medium, MDA-MB-231 cells growing in medium containing FA and ZR-75-1 cells were treated with chemotherapy drugs or radiotherapy with or without tombarthite-modified-FA-chitosan (R-O2-FA-CHI)-SWCNTs under hypoxic conditions, and the cell viability was determined by water-soluble tetrazolium salts-1 assay. The cell surviving fractions were determined by colony forming assay. Cell apoptosis induction was monitored by flow cytometry. Expression of B-cell lymphoma 2 (Bcl-2), survivin, hypoxia-inducible factor 1-α (HIF-1α), multidrug resistance-associated protein 1 (MRP-1), P-glycoprotein (P-gp), RAD51 and Ku80 was monitored by western blotting. The novel synthesized R-O2-FA-CHI-SWCNTs were able to significantly enhance the chemosensitivity and radiosensitivity of human breast cancer cell lines and the material exhibited its expected function by downregulating the expression of Bcl-2, survivin, HIF-1α, P-gp, MRP-1, RAD51 and Ku80. PMID:28123543

  20. Increased chemosensitivity and radiosensitivity of human breast cancer cell lines treated with novel functionalized single-walled carbon nanotubes.

    PubMed

    Jia, Yijun; Weng, Ziyi; Wang, Chuanying; Zhu, Mingjie; Lu, Yunshu; Ding, Longlong; Wang, Yongkun; Cheng, Xianhua; Lin, Qing; Wu, Kejin

    2017-01-01

    Hypoxia is a major cause of treatment resistance in breast cancer. Single-walled carbon nanotubes (SWCNTs) exhibit unique properties that make them promising candidates for breast cancer treatment. In the present study, a new functionalized single-walled carbon nanotube carrying oxygen was synthesized; it was determined whether this material could increase chemosensitivity and radiosensitivity of human breast cancer cell lines, and the underlying mechanisms were investigated. MDA-MB-231 cells growing in folic acid (FA) free medium, MDA-MB-231 cells growing in medium containing FA and ZR-75-1 cells were treated with chemotherapy drugs or radiotherapy with or without tombarthite-modified-FA-chitosan (R-O2-FA-CHI)-SWCNTs under hypoxic conditions, and the cell viability was determined by water-soluble tetrazolium salts-1 assay. The cell surviving fractions were determined by colony forming assay. Cell apoptosis induction was monitored by flow cytometry. Expression of B-cell lymphoma 2 (Bcl-2), survivin, hypoxia-inducible factor 1-α (HIF-1α), multidrug resistance-associated protein 1 (MRP-1), P-glycoprotein (P-gp), RAD51 and Ku80 was monitored by western blotting. The novel synthesized R-O2-FA-CHI-SWCNTs were able to significantly enhance the chemosensitivity and radiosensitivity of human breast cancer cell lines and the material exhibited its expected function by downregulating the expression of Bcl-2, survivin, HIF-1α, P-gp, MRP-1, RAD51 and Ku80.

  1. K-ras genetic mutation and influencing factor analysis for Han and Uygur nationality colorectal cancer patients

    PubMed Central

    Eli, Mayinur; Mollayup, Ablikim; Muattar; Liu, Chao; Zheng, Chao; Bao, Yong-Xing

    2015-01-01

    To investigate the K-ras genetic mutation status in colorectal cancer patients, compare the difference of K-ras genetic mutation rate in Han and Uygur nationality and analyze the influencing factor. 91 cases (52 cases of Han nationality and 39 cases of Uygur nationality) of colorectal biopsy or surgical ablation pathology specimen from the first affiliated hospital of Xinjiang Medical University during January, 2010 to March, 2013 were collected to detect the 12th and 13th code mutation status of K-ras gene exon 2 with pyrosequencing method and compare the difference of K-ras gene mutation rate between Han and Uygur nationality patients. Single factor analysis and multiple factor logistic regression analysis were utilized to analyze the influencing factor for K-ras genetic mutation. 33 cases of patients with K-ras genetic mutation were found from the 91 cases colorectal cancer patients and the total mutation rate was 36.3%. Among them, 24 cases (72.7%) were found with mutation only in the 12th code, 9 cases (27.3%) were found with mutation only in the 13th code and no one case was found with mutation in both the two codes. Mutation rate of the 12th code in the Uygur nationality was significantly higher than that in the Han nationality (P<0.05), but there were no significant difference in the comparison of the total mutation rate and the 13th code mutation rate between the two groups (P>0.05). There were no associativity (P>0.05) between the K-ras genetic mutation and sex, age, smoking history, drinking history, tumor location, macropathology type, differentiation level, staging, invasive depth, lymph nodes transferring and metastasis in colorectal cancer patients (P>0.05). K-ras genetic mutation rate is high in colorectal cancer patients. The mutation rate of 12th code in Uygur nationality is higher than that in Han nationality. There is no significant associativity between K-ras genetic mutation rate and patients’ clinical pathology characteristic. PMID:26309716

  2. K-ras genetic mutation and influencing factor analysis for Han and Uygur nationality colorectal cancer patients.

    PubMed

    Eli, Mayinur; Mollayup, Ablikim; Muattar; Liu, Chao; Zheng, Chao; Bao, Yong-Xing

    2015-01-01

    To investigate the K-ras genetic mutation status in colorectal cancer patients, compare the difference of K-ras genetic mutation rate in Han and Uygur nationality and analyze the influencing factor. 91 cases (52 cases of Han nationality and 39 cases of Uygur nationality) of colorectal biopsy or surgical ablation pathology specimen from the first affiliated hospital of Xinjiang Medical University during January, 2010 to March, 2013 were collected to detect the 12th and 13th code mutation status of K-ras gene exon 2 with pyrosequencing method and compare the difference of K-ras gene mutation rate between Han and Uygur nationality patients. Single factor analysis and multiple factor logistic regression analysis were utilized to analyze the influencing factor for K-ras genetic mutation. 33 cases of patients with K-ras genetic mutation were found from the 91 cases colorectal cancer patients and the total mutation rate was 36.3%. Among them, 24 cases (72.7%) were found with mutation only in the 12th code, 9 cases (27.3%) were found with mutation only in the 13th code and no one case was found with mutation in both the two codes. Mutation rate of the 12th code in the Uygur nationality was significantly higher than that in the Han nationality (P<0.05), but there were no significant difference in the comparison of the total mutation rate and the 13th code mutation rate between the two groups (P>0.05). There were no associativity (P>0.05) between the K-ras genetic mutation and sex, age, smoking history, drinking history, tumor location, macropathology type, differentiation level, staging, invasive depth, lymph nodes transferring and metastasis in colorectal cancer patients (P>0.05). K-ras genetic mutation rate is high in colorectal cancer patients. The mutation rate of 12th code in Uygur nationality is higher than that in Han nationality. There is no significant associativity between K-ras genetic mutation rate and patients' clinical pathology characteristic.

  3. Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

    NASA Astrophysics Data System (ADS)

    Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

    2017-01-01

    Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

  4. Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

    PubMed Central

    Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

    2017-01-01

    Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes. PMID:28067274

  5. Low somatic K-ras mutation frequency in colorectal cancer diagnosed under the age of 45 years.

    PubMed

    Alsop, Kathryn; Mead, Leeanne; Smith, Letitia D; Royce, Simon G; Tesoriero, Andrea A; Young, Joanne P; Haydon, Andrew; Grubb, Garry; Giles, Graham G; Jenkins, Mark A; Hopper, John L; Southey, Melissa C

    2006-07-01

    Somatic mutation of K-ras is known to be a common event in colorectal cancer tumourigenesis however its association with age at onset has not been widely explored. In this study, we have analyzed tumours from a population-based study of colorectal cancer diagnosed before the age of 45 years, in which cases had been previously screened for germ-line mismatch repair gene mutations and for microsatellite instability. We used a micro-dissection and sequencing approach to search for somatic K-ras mutations in codons 12, 13 and 61 in 101 early-onset colorectal cancers. Six (6%) somatic K-ras mutations were detected; five in codon 12 (4 G>T transitions and 1 G>A) and one in codon 13 (G>A transition). All codon 12 mutations were identified in microsatellite stable tumours and the codon 13 mutation was identified in a MSI-high tumour. Four cases with K-ras mutations had no reported family history of colorectal cancer and two had some family history of colorectal cancer. None were known to carry a germ-line mutation in hMSH2, hMLH1, hMSH6 or hPMS2. The role of somatic K-ras mutations in early-onset colorectal cancer carcinogenesis appears to be minor, in contrast to its significant role in colorectal cancer of later age of onset.

  6. Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis.

    PubMed

    Prakash, Priyanka; Zhou, Yong; Liang, Hong; Hancock, John F; Gorfe, Alemayehu A

    2016-03-08

    K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15-20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1-3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy.

  7. The Frequency and Type of K-RAS Mutations in Mexican Patients With Colorectal Cancer: A National Study.

    PubMed

    Cárdenas-Ramos, Susana G; Alcázar-González, Gregorio; Reyes-Cortés, Luisa M; Torres-Grimaldo, Abdiel A; Calderón-Garcidueñas, Ana L; Morales-Casas, José; Flores-Sánchez, Patricia; De León-Escobedo, Raúl; Gómez-Díaz, Antonio; Moreno-Bringas, Carmen; Sánchez-Guillén, Jorge; Ramos-Salazar, Pedro; González-de León, César; Barrera-Saldaña, Hugo A

    2017-06-01

    Current metastatic colorectal cancer (mCRC) therapy uses monoclonal antibodies against the epidermal growth factor receptor. This treatment is only useful in the absence of K-RAS gene mutations; therefore the study of such mutations is part of a personalized treatment. The aim of this work is to determine the frequency and type of the most common K-RAS mutations in Mexican patients with metastatic disease by nucleotide sequencing. We studied 888 patients with mCRC from different regions of Mexico. The presence of mutations in exon 2, codons 12 and 13, of the K-RAS gene was determined by nucleotide sequencing. Patients exhibited K-RAS gene mutations in 35% (310/888) of cases. Mutation frequency of codons 12 and 13 was 71% (221/310) and 29% (89/310), respectively. The most common mutation (45.7%) in codon 12 was c.35G>A (p.G12D), whereas the one in codon 13 was c.38G>A (p.G13D) (78.7%). Given the frequency of K-RAS mutations in Mexicans, making a genetic study before deciding to treat mCRC patients with monoclonal antibodies is indispensable.

  8. Radiosensitizing effects of the prenyltransferase inhibitor AZD3409 against RAS mutated cell lines.

    PubMed

    Cengel, Keith A; Deutsch, Eric; Stephens, Trevor C; Voong, K Ranh; Kao, Gary D; Bernhard, Eric J

    2006-09-01

    Mutations at the H-, N- and K-ras loci are among the most frequent genetic alterations in human cancers. In this study, we have investigated the effect of AZD3409, a novel, peptidomimetic prenyltransferase inhibitor (PTI), on the radiosensitivity of cells with mutated ras alleles. AZD3409, developed by AstraZeneca, inhibits both farnesyl- and geranylgeranyl transferase in cell free systems. AZD3409 inhibits the growth of a variety of human cancer cell lines, including cells that express mutant alleles of either K- or H- ras and was well tolerated when administered orally to healthy volunteers in a phase I clinical trial. We have previously shown that PTI can radiosensitize human and rodent cancer cell lines that express activated RAS. Here we assessed the ability of AZD3409 to radiosensitize human cancer cell lines in vivo and in vitro and the activation state of RAS proteins in treated cells. Once daily oral administration of AZD3409 to nude mice bearing PSN-1 and MiaPaCa-2 human pancreatic cancer xenografts expressing mutant K-ras was well tolerated and resulted in a supra-additive reduction in clonogenic cell survival after irradiation. Similarly, AZD3409 reduced clonogenic survival in cells that express either mutant K- or H- ras in vitro. We next examined the effect of AZD3409 on the processing and activation of K- and H-RAS. AZD3409-mediated radiosensitization, both in vivo and in vitro, correlates with a decrease in H-RAS processing without detectable effect on K-RAS processing. RAS activation assays show that the decreased H-RAS processing is accompanied by decreased H-RAS activation in cell lines with mutations in either K- or H-ras. However, no decrease in K-RAS activation was detected. Thus, radiosensitization of human cancer cells that express mutated K-RAS occurred under conditions where AZD3409 inihibits the activation of farneyslated H-RAS, but did not inhibit K-RAS activation.

  9. The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

    PubMed Central

    Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

    2016-01-01

    Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP–GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer. PMID:26902995

  10. The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B

    NASA Astrophysics Data System (ADS)

    Lu, Shaoyong; Jang, Hyunbum; Nussinov, Ruth; Zhang, Jian

    2016-02-01

    Ras mediates cell proliferation, survival and differentiation. Mutations in K-Ras4B are predominant at residues G12, G13 and Q61. Even though all impair GAP-assisted GTP → GDP hydrolysis, the mutation frequencies of K-Ras4B in human cancers vary. Here we aim to figure out their mechanisms and differential oncogenicity. In total, we performed 6.4 μs molecular dynamics simulations on the wild-type K-Ras4B (K-Ras4BWT-GTP/GDP) catalytic domain, the K-Ras4BWT-GTP-GAP complex, and the mutants (K-Ras4BG12C/G12D/G12V-GTP/GDP, K-Ras4BG13D-GTP/GDP, K-Ras4BQ61H-GTP/GDP) and their complexes with GAP. In addition, we simulated ‘exchanged’ nucleotide states. These comprehensive simulations reveal that in solution K-Ras4BWT-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4BG12C/G12D-GDP they expose the bound nucleotide which facilitates the GDP-to-GTP exchange. These mechanisms may help elucidate the differential mutational statistics in K-Ras4B-driven cancers. Exchanged nucleotide simulations reveal that the conformational transition is more accessible in the GTP-to-GDP than in the GDP-to-GTP exchange. Importantly, GAP not only donates its R789 arginine finger, but stabilizes the catalytically-competent conformation and pre-organizes catalytic residue Q61; mutations disturb the R789/Q61 organization, impairing GAP-mediated GTP hydrolysis. Together, our simulations help provide a mechanistic explanation of key mutational events in one of the most oncogenic proteins in cancer.

  11. PKCδ regulates integrin αVβ3 expression and transformed growth of K-ras dependent lung cancer cells

    PubMed Central

    Symonds, Jennifer M.; Ohm, Angela M.; Tan, Aik-Choon; Reyland, Mary E.

    2016-01-01

    We have previously shown that Protein Kinase C delta (PKCδ) functions as a tumor promoter in non-small cell lung cancer (NSCLC), specifically in the context of K-ras addiction. Here we define a novel PKCδ -> integrin αVβ3->Extracellular signal-Regulated Kinase (ERK) pathway that regulates the transformed growth of K-ras dependent NSCLC cells. To explore how PKCδ regulates tumorigenesis, we performed mRNA expression analysis in four KRAS mutant NSCLC cell lines that stably express scrambled shRNA or a PKCδ targeted shRNA. Analysis of PKCδ-dependent mRNA expression identified 3183 regulated genes, 210 of which were specifically regulated in K-ras dependent cells. Genes that regulate extracellular matrix and focal adhesion pathways were most highly represented in this later group. In particular, expression of the integrin pair, αVβ3, was specifically reduced in K-ras dependent cells with depletion of PKCδ, and correlated with reduced ERK activation and reduced transformed growth as assayed by clonogenic survival. Re-expression of PKCδ restored ITGAV and ITGB3 mRNA expression, ERK activation and transformed growth, and this could be blocked by pretreatment with a αVβ3 function-blocking antibody, demonstrating a requirement for integrin αVβ3 downstream of PKCδ. Similarly, expression of integrin αV restored ERK activation and transformed growth in PKCδ depleted cells, and this could also be inhibited by pretreatment with PD98059. Our studies demonstrate an essential role for αVβ3 and ERK signalingdownstream of PKCδ in regulating the survival of K-ras dependent NSCLC cells, and identify PKCδ as a novel therapeutic target for the subset of NSCLC patients with K-ras dependent tumors. PMID:26918447

  12. Increased radiosensitivity of a subpopulation of T-lymphocyte progenitors from patients with Fanconi's anemia

    SciTech Connect

    Knox, S.J.; Wilson, F.D.; Greenberg, B.R.; Shifrine, M.; Rosenblatt, L.S.; Reeves, J.D.; Misra, H.

    1981-06-01

    In vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi's anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi's anemia patients. Lymphocytes from patients with Fanconi's anemia were significantly more sensitive to in vitro x irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST and colony formation assay. No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi's anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.

  13. Increased radiosensitivity of a subpopulation ot T-lymphocyte progenitors from patients with Fanconi's anemia

    SciTech Connect

    Knox, S.J.; Wilson, F.D.; Greenberg, B.R.; Shifrine, M.; Rosenblatt, L.S.; Reeves, J.D.; Misra, H.

    1981-06-01

    In vitro radiation survival of peripheral blood T lymphocytes was studied in 15 clinically normal adults and 4 patients with Fanconi's anemia. Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and a newly developed whole blood T-lymphocyte colony assay were used to measure lymphocyte blastogenesis and colony formation in response to phytohemagglutinin (PHA) or concanavalin-A (Con-A) stimulation. Lymphocyte colony formation was found to be consistently more sensitive than the LST for detection of low-level radiation effects using both normal cells and lymphocytes from Fanconi's anemia patients. Lymphocytes from patients with Fanconi's anemia were significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals as measured by their ability to divide when stimulated by PHA in the LST (patients, D37 . 198 R; normals, D37 . 309 R, p . 0.057) and colony formation assay (patients, D37 . 53 R; normals, D37 . 109 R, p . 0.016). No significant difference in the radiosensitivity of the Con-A response was observed between the two groups. The PHA-responsive T-lymphocyte subpopulation in Fanconi's anemia patients appears to be intrinsically defective. The nature of this defect, significance in the disease process, and relevancy of these findings to the establishment of radiation protection standards are discussed.

  14. Generation and analysis of mouse intestinal tumors and organoids harboring APC and K-Ras mutations.

    PubMed

    van Es, Johan H; Clevers, Hans

    2015-01-01

    Genetically engineered mouse models of intestinal cancer are experimental systems in which mice are genetically manipulated to develop malignancies in the gastrointestinal tract. These models enable researchers to study the mechanisms of onset, progression, and metastasis of the disease. They also provide a valuable biological system which is suitable for testing (novel) drugs in vivo. Recently, an in vitro culture model has been established in which intestinal epithelial stem cells can grow into three-dimensional, ever-expanding epithelial organoids that retain their original organ identity and genetic stability. This culture system has been applied to diseased epithelia, such as adenoma, adenocarcinoma, and Barrett's epithelium. These organoids can be particularly useful for studying the mechanisms of intestinal tumors and to test (novel) drugs in vitro. Here, we describe our current laboratory protocols to generate and analyze intestinal tumors and organoids harboring APC and K-Ras double mutations.

  15. Simultaneous detection of 19 K-ras mutations by free-solution conjugate electrophoresis of ligase detection reaction products on glass microchips.

    PubMed

    Albrecht, Jennifer Coyne; Kotani, Akira; Lin, Jennifer S; Soper, Steven A; Barron, Annelise E

    2013-02-01

    We demonstrate here the power and flexibility of free-solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild-type DNA. Here, four large drag-tags are used to achieve free-solution electrophoretic separation of 19 LDR products ranging in size from 42 to 66 nt that correspond to mutations in the K-ras oncogene. LDR-FSCE enabled electrophoretic resolution of these 19 LDR-FSCE products by CE in 13.5 min (E = 310 V/cm) and by microchip electrophoresis in 140 s (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free-solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR-FSCE products were separated in less than 70 s with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K-ras mutations on integrated "sample-in/answer-out" devices with amplification, LDR, and detection all on one platform.

  16. Is there a prognostic role of K-ras point mutations in the serum of patients with advanced non-small cell lung cancer?

    PubMed

    Camps, Carlos; Sirera, Rafael; Bremnes, Roy; Blasco, Ana; Sancho, Eva; Bayo, Pilar; Safont, Maria Jose; Sánchez, José Javier; Tarón, Miquel; Rosell, Rafael

    2005-12-01

    The purpose of this study was to investigate the prognostic significance of K-ras mutations in circulating DNA in advanced non-small lung cancer (NSCLC) patients. Serum samples were assessed prior to platinum-based chemotherapy start in 67 patients with advanced NSCLC (stage IIIB or IV), treated between April 1999 and June 2002. Patients were not previously treated with chemotherapy. K-ras oncogene mutations at codon 12 were analyzed by genomic amplification and direct sequencing of the patient's DNA present in serum. Pre-treatment serum was available in all 67 patients. Twenty patients (30%) demonstrated K-ras mutations while 47 patients (70%) had wild-type K-ras. Among K-ras mutations, the amino acid glycine was substituted by cystein in 90% and valine in 10%. When patients were grouped according to K-ras genotype, there was no significant difference for any of the baseline patient characteristics. There was a tendency towards a higher response rate for patients with K-ras mutations versus wild-type K-ras in serum, however not statistically significant (p=0.37). Median progression-free survival was 7.3 months versus 5.5 months in patients with mutations and with wild-type K-ras, respectively (p=0.23). For median overall survival time, the mutation group was comparable to the wild-type K-ras group with 12.5 and 11.4 months, respectively (p=0.28). In conclusion, there were no significant differences between the patients with K-ras mutations and those with wild-type genotype with respect to baseline patient characteristics, response rates, progression-free survival, or overall survival.

  17. Phosphatidylinositol 3-Kinase Mediates Bronchioalveolar Stem Cell Expansion in Mouse Models of Oncogenic K-ras-Induced Lung Cancer

    PubMed Central

    Yang, Yanan; Iwanaga, Kentaro; Raso, Maria Gabriela; Wislez, Marie; Hanna, Amy E.; Wieder, Eric D.; Molldrem, Jeffrey J.; Wistuba, Ignacio I.; Powis, Garth; Demayo, Francesco J.; Kim, Carla F.; Kurie, Jonathan M.

    2008-01-01

    Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death in Western countries. Developing more effective NSCLC therapeutics will require the elucidation of the genetic and biochemical bases for this disease. Bronchioalveolar stem cells (BASCs) are a putative cancer stem cell population in mouse models of oncogenic K-ras-induced lung adenocarcinoma, an histologic subtype of NSCLC. The signals activated by oncogenic K-ras that mediate BASC expansion have not been fully defined. Methodology/Principal Findings We used genetic and pharmacologic approaches to modulate the activity of phosphatidylinositol 3-kinase (PI3K), a key mediator of oncogenic K-ras, in two genetic mouse models of lung adenocarcinoma. Oncogenic K-ras-induced BASC accumulation and tumor growth were blocked by treatment with a small molecule PI3K inhibitor and enhanced by inactivation of phosphatase and tensin homologue deleted from chromosome 10, a negative regulator of PI3K. Conclusions/Significance We conclude that PI3K is a critical regulator of BASC expansion, supporting treatment strategies to target PI3K in NSCLC patients. PMID:18493606

  18. Inhibition of UBE2D3 Expression Attenuates Radiosensitivity of MCF-7 Human Breast Cancer Cells by Increasing hTERT Expression and Activity

    PubMed Central

    Hu, Liu; Li, Fen; Ren, Li; Yu, Haijun; Liu, Yu; Xia, Ling; Lei, Han; Liao, Zhengkai; Zhou, Fuxiang; Xie, Conghua; Zhou, Yunfeng

    2013-01-01

    The known functions of telomerase in tumor cells include replenishing telomeric DNA and maintaining cell immortality. We have previously shown the existence of a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Here we set out to elucidate the molecular mechanisms underlying regulation by telomerase of radiosensitivity in MCF-7 cells. Toward this aim, yeast two-hybrid (Y2H) screening of a human laryngeal squamous cell carcinoma radioresistant (Hep2R) cDNA library was first performed to search for potential hTERT interacting proteins. We identified ubiquitin-conjugating enzyme E2D3 (UBE2D3) as a principle hTERT-interacting protein and validated this association biochemically. ShRNA-mediated inhibition of UBE2D3 expression attenuated MCF-7 radiosensitivity, and induced the accumulation of hTERT and cyclin D1 in these cells. Moreover, down-regulation of UBE2D3 increased hTERT activity and cell proliferation, accelerating G1 to S phase transition in MCF-7 cells. Collectively these findings suggest that UBE2D3 participates in the process of hTERT-mediated radiosensitivity in human breast cancer MCF-7 cells by regulating hTERT and cyclin D1. PMID:23741361

  19. miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras

    SciTech Connect

    Shin, Ki-Hyuk; Bae, Susan D.; Hong, Hannah S.; Kim, Reuben H.; Kang, Mo K.; Park, No-Hee

    2011-01-28

    Research highlights: {yields} MicroRNA-181a (miR-181a) was frequently downregulated in oral squamous cell carcinoma (OSCC). {yields} Overexpression of miR-181a suppressed OSCC growth. {yields} K-ras is a novel target of miR-181a. {yields} Decreased miR-181a expression is attributed to its lower promoter activity in OSCC. -- Abstract: MicroRNAs (miRNAs) are epigenetic regulators of gene expression, and their deregulation plays an important role in human cancer, including oral squamous cell carcinoma (OSCC). Recently, we found that miRNA-181a (miR-181a) was upregulated during replicative senescence of normal human oral keratinocytes. Since senescence is considered as a tumor suppressive mechanism, we thus investigated the expression and biological role of miR-181a in OSCC. We found that miR-181a was frequently downregulated in OSCC. Ectopic expression of miR-181a suppressed proliferation and anchorage independent growth ability of OSCC. Moreover, miR-181a dramatically reduces the growth of OSCC on three dimensional organotypic raft culture. We also identified K-ras as a novel target of miR-181a. miR-181a decreased K-ras protein level as well as the luciferase activity of reporter vectors containing the 3'-untranslated region of K-ras gene. Finally, we defined a minimal regulatory region of miR-181a and found a positive correlation between its promoter activity and the level of miR-181a expression. In conclusion, miR-181a may function as an OSCC suppressor by targeting on K-ras oncogene. Thus, miR-181a should be considered for therapeutic application for OSCC.

  20. Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis

    PubMed Central

    Prakash, Priyanka; Zhou, Yong; Liang, Hong; Hancock, John F.; Gorfe, Alemayehu A.

    2016-01-01

    K-Ras is a membrane-associated GTPase that cycles between active and inactive conformational states to regulate a variety of cell signaling pathways. Somatic mutations in K-Ras are linked to 15–20% of all human tumors. K-Ras attaches to the inner leaflet of the plasma membrane via a farnesylated polybasic domain; however, the structural details of the complex remain poorly understood. Based on extensive (7.5 μs total) atomistic molecular dynamics simulations here we show that oncogenic mutant K-Ras interacts with a negatively charged lipid bilayer membrane in multiple orientations. Of these, two highly populated orientations account for ∼54% of the conformers whose catalytic domain directly interacts with the bilayer. In one of these orientation states, membrane binding involves helices 3 and 4 of the catalytic domain in addition to the farnesyl and polybasic motifs. In the other orientation, β-strands 1–3 and helix 2 on the opposite face of the catalytic domain contribute to membrane binding. Flexibility of the linker region was found to be important for the reorientation. The biological significance of these observations was evaluated by initial experiments in cells overexpressing mutant K-Ras as well as by an analysis of Ras-effector complex structures. The results suggest that only one of the two major orientation states is capable of effector binding. We propose that the different modes of membrane binding may be exploited in structure-based drug design efforts for cancer therapy. PMID:26958889

  1. KLF4 regulates adult lung tumor-initiating cells and represses K-Ras-mediated lung cancer.

    PubMed

    Yu, T; Chen, X; Zhang, W; Liu, J; Avdiushko, R; Napier, D L; Liu, A X; Neltner, J M; Wang, C; Cohen, D; Liu, C

    2016-02-01

    Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. To identify novel factors that contribute to lung cancer pathogenesis, we analyzed a lung cancer database from The Cancer Genome Atlas and found that Krüppel-like Factor 4 (KLF4) expression is significantly lower in patients' lung cancer tissue than in normal lung tissue. In addition, we identified seven missense mutations in the KLF4 gene. KLF4 is a transcription factor that regulates cell proliferation and differentiation as well as the self-renewal of stem cells. To understand the role of KLF4 in the lung, we generated a tamoxifen-induced Klf4 knockout mouse model. We found that KLF4 inhibits lung cancer cell growth and that depletion of Klf4 altered the differentiation pattern in the developing lung. To understand how KLF4 functions during lung tumorigenesis, we generated the K-ras(LSL-G12D/+);Klf4(fl/fl) mouse model, and we used adenovirus-expressed Cre to induce K-ras activation and Klf4 depletion in the lung. Although Klf4 deletion alone or K-ras mutation alone can trigger lung tumor formation, Klf4 deletion combined with K-ras mutation significantly enhanced lung tumor formation. We also found that Klf4 deletion in conjunction with K-ras activation caused lung inflammation. To understand the mechanism whereby KLF4 is regulated during lung tumorigenesis, we analyzed KLF4 promoter methylation and the profiles of epigenetic factors. We found that Class I histone deacetylases (HDACs) are overexpressed in lung cancer and that HDAC inhibitors induced expression of KLF4 and inhibited proliferation of lung cancer cells, suggesting that KLF4 is probably repressed by histone acetylation and that HDACs are valuable drug targets for lung cancer treatment.

  2. Multiple oncogenic changes (K-RAS(V12), p53 knockdown, mutant EGFRs, p16 bypass, telomerase) are not sufficient to confer a full malignant phenotype on human bronchial epithelial cells.

    PubMed

    Sato, Mitsuo; Vaughan, Melville B; Girard, Luc; Peyton, Michael; Lee, Woochang; Shames, David S; Ramirez, Ruben D; Sunaga, Noriaki; Gazdar, Adi F; Shay, Jerry W; Minna, John D

    2006-02-15

    We evaluated the contribution of three genetic alterations (p53 knockdown, K-RAS(V12), and mutant EGFR) to lung tumorigenesis using human bronchial epithelial cells (HBEC) immortalized with telomerase and Cdk4-mediated p16 bypass. RNA interference p53 knockdown or oncogenic K-RAS(V12) resulted in enhanced anchorage-independent growth and increased saturation density of HBECs. The combination of p53 knockdown and K-RAS(V12) further enhanced the tumorigenic phenotype with increased growth in soft agar and an invasive phenotype in three-dimensional organotypic cultures but failed to cause HBECs to form tumors in nude mice. Growth of HBECs was highly dependent on epidermal growth factor (EGF) and completely inhibited by EGF receptor (EGFR) tyrosine kinase inhibitors, which induced G1 arrest. Introduction of EGFR mutations E746-A750 del and L858R progressed HBECs toward malignancy as measured by soft agar growth, including EGF-independent growth, but failed to induce tumor formation. Mutant EGFRs were associated with higher levels of phospho-Akt, phospho-signal transducers and activators of transcription 3 [but not phospho-extracellular signal-regulated kinase (ERK) 1/2], and increased expression of DUSP6/MKP-3 phosphatase (an inhibitor of phospho-ERK1/2). These results indicate that (a) the HBEC model system is a powerful new approach to assess the contribution of individual and combinations of genetic alterations to lung cancer pathogenesis; (b) a combination of four genetic alterations, including human telomerase reverse transcriptase overexpression, bypass of p16/RB and p53 pathways, and mutant K-RAS(V12) or mutant EGFR, is still not sufficient for HBECs to completely transform to cancer; and (c) EGFR tyrosine kinase inhibitors inhibit the growth of preneoplastic HBEC cells, suggesting their potential for chemoprevention.

  3. K-ras, p53 mutations, and microsatellite instability (MSI) in gallbladder cancer.

    PubMed

    Saetta, Angelica A

    2006-06-15

    Despite the considerable progress in understanding the molecular pathology of carcinogenesis, the genetic mechanisms underlying the development and progression of gallbladder cancer (GC) are poorly understood. The survival of GC patients is generally poor. Therefore, it is very useful to define valuable prognostic factors. The most extensively studied oncogenes in gallbladder carcinogenesis are ras, commonly mutated in neoplasms of the gastrointestinal tract. K-ras oncogene is altered in a subset of gallbladder patients and mainly in those having anomalous junction of the pancreaticobiliary tract. Most of the studies of genetic abnormalities in GC have focused on p53 gene. p53 mutation/overexpression and/or LOH is present in more than 50% of gallbladder carcinomas, suggesting an important role in their pathogenesis. However, these results have not any predictive value yet. Moreover, the involvement of an alternative molecular pathway, that of microsatellite instability (MSI), is found in a limited group of GC patients. Additional research is necessary to establish its possible relation to defects of the mismatch repair (MMR) system and its proposed prognostic significance. Further elucidation of the molecular events specific to GC will help to identify novel molecular targets for the diagnosis and clinical management of the patients. Copyright 2006 Wiley-Liss, Inc.

  4. Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.

    PubMed

    Iwata, Seigo; Masuhara, Kaori; Umeki, Nobuhisa; Sako, Yasushi; Maruta, Shinsaku

    2016-01-01

    A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.

  5. The relationship between 1,2-dimethylhydrazine dose and the induction of colon tumours: tumour development in female SWR mice does not require a K-ras mutational event.

    PubMed

    Jackson, P E; Cooper, D P; O'Connor, P J; Povey, A C

    1999-03-01

    In this study we have investigated the relationship between the dose of 1,2-dimethylhydrazine (DMH) and the yield (and location) of tumours in a mouse strain susceptible to colon tumour induction. Female SWR mice were injected with 6.8 mg/kg DMH i.p. once a week for 1, 5, 10 and 20 weeks and the animals were followed for almost 2 years. Administration of increasing doses of DMH resulted in a dose-dependent decrease in survival time. Colon tumours developed in 26, 76 and 87% of mice given a total dose of 34, 68 and 136 mg/kg DMH, respectively: no tumours were detected in animals treated with a total dose of 6.8 mg/kg. Most colon tumours (79%) were located in the distal colon with the remainder being found in the mid colon and none were detected in either the proximal colon or small intestine. As mutations in the K-ras gene are thought to be key events in the pathogenesis of human and rodent colon tumours, we determined the frequency of codon 12 and 13 K-ras mutations in these tumours by restriction site mutation analysis and/or DNA sequencing. A total of 50 colon tumour samples were analysed for codon 12 mutations and of these 29 were also screened for codon 13 mutations. No mutations were detected in either of these codons. The mutational activation of the K-ras gene is not an essential step in the development of DMH-induced colon tumours in female SWR mice and if similar considerations apply to humans, then the aetiological role of alkylating agents may be underestimated from the prevalence of K-ras GC-->AT transitions in human tumours.

  6. Inverse Relationship between Microsatellite Instability and K-ras and p53 Gene Alterations in Colon Cancer

    PubMed Central

    Samowitz, Wade S.; Holden, Joseph A.; Curtin, Karen; Edwards, Sandra L.; Walker, Adrianne R.; Lin, Heather A.; Robertson, Margaret A.; Nichols, Melanie F.; Gruenthal, Kristin M.; Lynch, Beverly J.; Leppert, Mark F.; Slattery, Martha L.

    2001-01-01

    Some studies have shown an inverse relationship between microsatellite instability in colon cancer and mutations in p53 and K-ras, whereas others have not. We therefore evaluated these features in a population-based sample of 496 individuals with colon cancer. Microsatellite instability was determined by a panel of 10 tetranucleotide repeats, the Bethesda consensus panel of mono- and dinucleotide repeats, and coding mononucleotide repeats in transforming growth factor-beta receptor type II, hMSH3, BAX, hMSH6, and insulin-like growth factor receptor type II. Mutations in codons 12 and 13 in K-ras were evaluated by sequencing. p53 overexpression (as detected by immunohistochemistry) was used as an indicator of p53 mutation; this was evaluated in 275 of the tumors. K-ras mutations were present in 33.2% of tumors, p53 overexpression in 51.5%, and microsatellite instability (as determined by the Bethesda consensus panel) in 12.5%. K-ras mutations were significantly less common in unstable tumors than stable tumors (11.8% versus 36.9%, P < 0.001). p53 overexpression was significantly less common in unstable tumors than stable tumors (20.0% versus 55.7%, P < 0.001). These inverse relationships between microsatellite instability and ras gene mutations and p53 overexpression were shown to be independent of tumor site in logistic regression analyses. All other measures of instability also showed statistically significant inverse relationships independent of tumor site with alterations in ras and p53, and instability results determined by the panel of 10 tetranucleotide repeats were highly significantly related to those determined by the Bethesda consensus panel. Coding mononucleotide repeat mutations were significantly more common in unstable tumors than stable tumors (85.7% versus 1.0%, P < 0.001). We conclude that there is an inverse relationship between microsatellite instability and mutations in p53 and K-ras, and that the molecular profile of colon cancers with

  7. Association between coffee drinking and K-ras mutations in exocrine pancreatic cancer. PANKRAS II Study Group

    PubMed Central

    Porta, M.; Malats, N.; Guarner, L.; Carrato, A.; Rifa, J.; Salas, A.; Corominas, J. M.; Andreu, M.; Real, F. X.

    1999-01-01

    STUDY OBJECTIVE: To analyse the relation between coffee consumption and mutations in the K-ras gene in exocrine pancreatic cancer. DESIGN: Case- case study. Consumption of coffee among cases with the activating mutation in the K-ras gene was compared with that of cases without the mutation. SETTING AND PATIENTS: All cases of pancreatic cancer newly diagnosed at five hospitals in Spain during three years were included in the PANKRAS II Study (n = 185, of whom 121 whose tissue was available for molecular analysis are the object of the present report). Over 88% were personally interviewed in hospital. DNA was amplified from paraffin wax embedded tissues, and mutations in codon 12 of K-ras were detected by the artificial RFLP technique. MAIN RESULTS: Mutations were found in tumours from 94 of 121 patients (77.7%). Mutations were more common among regular coffee drinkers than among non-regular coffee drinkers (82.0% v 55.6%, p = 0.018, n = 107). The odds ratio adjusted by age, sex, smoking and alcohol drinking was 5.41 (95% CI 1.64, 17.78). The weekly intake of coffee was significantly higher among patients with a mutated tumour (mean of 14.5 cups/week v 8.8 among patients with a wild type tumour, p < 0.05). With respect to non- regular coffee drinkers, the odds ratio of a mutated tumour adjusted by age, sex, smoking and alcohol drinking was 3.26 for drinkers of 2-7 cups/week, 5.77 for drinkers of 8-14 cups/week and 9.99 for drinkers of > or = 15 cups/week (p < 0.01, test for trend). CONCLUSIONS: Pancreatic cancer cases without activating mutations in the K-ras gene had drank significantly less coffee than cases with a mutation, with a significant dose response relation: the less they drank, the less likely their tumours were to harbour a mutation. In exocrine pancreatic cancer the K-ras gene may be activated less often among non-regular coffee drinkers than among regular drinkers. Caffeine, other coffee compounds or other factors with which coffee drinking is

  8. Regulation of uptake of 18F-FDG by a follicular human thyroid cancer cell line with mutation-activated K-ras.

    PubMed

    Prante, Olaf; Maschauer, Simone; Fremont, Valerie; Reinfelder, Julia; Stoehr, Robert; Szkudlinski, Mariusz; Weintraub, Bruce; Hartmann, Arndt; Kuwert, Torsten

    2009-08-01

    Dedifferentiation of thyroid carcinoma is accompanied by increased accumulation of the PET tracer (18)F-FDG. The molecular mechanisms responsible for this phenomenon are poorly understood. Therefore, we studied the regulation of (18)F-FDG uptake by the human follicular thyroid carcinoma cell line ML-1 and the as-yet-unknown oncogene expression of that cell line. The data obtained in ML-1 were compared with those of a well-differentiated thyroid cell line of rat origin (FRTL-5). The expression of the thyroid-stimulating hormone (TSH) receptor was investigated by immunocytochemistry, and the expression of the glucose transporters (GLUTs) was determined by Western blotting. Mutation analysis of ML-1 was performed for K-ras codons 12 and 13. The effect of TSH on intracellular cAMP levels was determined by a competitive enzyme immunoassay. Cells were incubated with (18)F-FDG (0.5-1.0 MBq/mL) for 1 h, and tracer uptake was related to protein concentration. The effects of bovine TSH, the cAMP analog (Bu)(2)cAMP, and the phosphatidylinositol-3-kinase (PI3-kinase) inhibitor LY294002 on (18)F-FDG uptake were investigated. The TSH receptor was present in both cell lines. FRTL-5 clearly expressed GLUT-1 and also GLUT-4. In ML-1 only, the expression of GLUT-3 was detected. TSH and (Bu)(2)cAMP had a significant effect on (18)F-FDG uptake or GLUT-1 expression in FRTL-5, but not in ML-1 cells. PI3-kinase inhibition by LY294002 downregulated (18)F-FDG uptake in FRTL-5 by 58% +/- 9% (n = 6) and in ML-1 by 26% +/- 5% (n = 42, both P < 0.05). Mutation analysis of ML-1 cells revealed a Gly12Ser point mutation at codon 12 of the K-ras gene. (18)F-FDG uptake in the thyroid carcinoma cell line ML-1 is no longer regulated by TSH or cAMP or mediated by GLUT-1. However, in this cell line, this variable is still governed to some extent by PI3-kinase located downstream to the constitutively active K-ras in the Ras-PI3-kinase-Akt pathway. These data suggest that increases in (18)F-FDG uptake in

  9. Tumor necrosis factor links chronic obstructive pulmonary disease and K-ras mutant lung cancer through induction of an immunosuppressive pro-tumor microenvironment

    PubMed Central

    da Silva Caetano, Mauricio; Cumpian, Amber M.; Daliri, Soudabeh; Garza Flores, Alejandra; Chang, Seon Hee; Evans, Christopher M.; Yu, Zhentao; Moghaddam, Seyed Javad

    2016-01-01

    ABSTRACT Tumor necrosis factor (TNF) is known as an important regulator of tumor microenvironment and inflammation. TNF levels are markedly elevated in the bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), which is an independent risk factor for lung cancer. We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model (CC-LR mouse). This was associated with a significant increase of neutrophils in BALF, accompanied by a marked increase in TNF level, suggesting a link between COPD, TNF, and lung cancer promotion. Therefore, we first overexpressed TNF in the airway epithelium of CC-LR mice, which promoted lung cancer by ∼2-fold. This was associated with increased numbers of Ki67 and CD31 positive cells in lung tumors of CC-LR/TNF-Tg mice. We also found a robust increase in NF-κB activation, and numbers of neutrophils and myeloid-derived suppressor cells (MDSCs) in lung. Accordingly, we depleted MDSCs in CC-LR/TNF-Tg mice, which lead to significant tumor suppression emphasizing on the role of TNF-induced MDSCs in K-ras induced lung tumorigenesis. Finally, we targeted TNF expression by crossing CC-LR mice with TNF knock-out mice (CC-LR/TNF-KO), which resulted in a significant decrease in lung tumor burden in the absence or presence of COPD-like airway inflammation. Interestingly, there were less MDSCs and lower Ki67 and CD31 expression in the lung of the CC-LR/TNF-KO mice. We conclude that TNF links COPD to lung cancer promotion by induction of an immunosuppressive MDSC response, and subsequent amplification of proliferation and angiogenesis in tumors. PMID:27853654

  10. Tumor necrosis factor links chronic obstructive pulmonary disease and K-ras mutant lung cancer through induction of an immunosuppressive pro-tumor microenvironment.

    PubMed

    Gong, Lei; da Silva Caetano, Mauricio; Cumpian, Amber M; Daliri, Soudabeh; Garza Flores, Alejandra; Chang, Seon Hee; Ochoa, Cesar E; Evans, Christopher M; Yu, Zhentao; Moghaddam, Seyed Javad

    2016-01-01

    Tumor necrosis factor (TNF) is known as an important regulator of tumor microenvironment and inflammation. TNF levels are markedly elevated in the bronchoalveolar lavage fluid (BALF) of patients with chronic obstructive pulmonary disease (COPD), which is an independent risk factor for lung cancer. We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model (CC-LR mouse). This was associated with a significant increase of neutrophils in BALF, accompanied by a marked increase in TNF level, suggesting a link between COPD, TNF, and lung cancer promotion. Therefore, we first overexpressed TNF in the airway epithelium of CC-LR mice, which promoted lung cancer by ∼2-fold. This was associated with increased numbers of Ki67 and CD31 positive cells in lung tumors of CC-LR/TNF-Tg mice. We also found a robust increase in NF-κB activation, and numbers of neutrophils and myeloid-derived suppressor cells (MDSCs) in lung. Accordingly, we depleted MDSCs in CC-LR/TNF-Tg mice, which lead to significant tumor suppression emphasizing on the role of TNF-induced MDSCs in K-ras induced lung tumorigenesis. Finally, we targeted TNF expression by crossing CC-LR mice with TNF knock-out mice (CC-LR/TNF-KO), which resulted in a significant decrease in lung tumor burden in the absence or presence of COPD-like airway inflammation. Interestingly, there were less MDSCs and lower Ki67 and CD31 expression in the lung of the CC-LR/TNF-KO mice. We conclude that TNF links COPD to lung cancer promotion by induction of an immunosuppressive MDSC response, and subsequent amplification of proliferation and angiogenesis in tumors.

  11. Selective 'stencil'-aided pre-PCR cleavage of wild-type sequences as a novel approach to detection of mutant K-RAS.

    PubMed

    Lichtenstein, A V; Serdjuk, O I; Sukhova, T I; Melkonyan, H S; Umansky, S R

    2001-09-01

    The enriched PCR widely used for detection of mutant K-RAS in either tumor tissues or circulating DNA was modified so that abundant wild-type K-RAS alleles are cleaved prior to PCR. We took advantage of an AluI recognition site located immediately upstream of the K-RAS codon 12. The site was reconstituted upon DNA denaturation followed by annealing with a 'stencil', a 16-bp synthetic oligonucleotide complementary to the wild-type sequence. As opposed to normal K-RAS, the mutant allele forms, upon annealing with the stencil, a mismatch at the codon 12 which lies within the AluI enzyme binding site and partially inhibits its activity. The mismatch also lowers the melting temperature of the stencil-mutant K-RAS double helix as compared to stencil-wild-type duplex, so that only the latter is double stranded and selectively digested by AluI at elevated temperatures. The proposed method of stencil-aided mutation analysis (SAMA) based on selective pre-PCR elimination of wild-type sequences can be highly advantageous for detection of mutant K-RAS due to: (i) an enhanced sensitivity because of reduced competition with a great excess of normal K-RAS, and (ii) a decrease in a number of false-positive results from Taq polymerase errors. Application of SAMA for generalized detection of DNA mutations is discussed.

  12. TGFβ1 Inhibition Increases the Radiosensitivity of Breast Cancer Cells In Vitro and Promotes Tumor Control by Radiation In Vivo

    PubMed Central

    Bouquet, Fanny; Pal, Anupama; Pilones, Karsten A.; Demaria, Sandra; Hann, Byron; Akhurst, Rosemary J.; Babb, Jim S.; Lonning, Scott M.; DeWyngaert, J. Keith; Formenti, Silvia C.; Barcellos-Hoff, Mary Helen

    2013-01-01

    Purpose To determine whether inhibition of TGFβ signaling prior to irradiation sensitizes human and murine cancer cells in vitro and in vivo. Experimental Design TGFβ-mediated growth and Smad phosphorylation of MCF7, Hs578T, MDA-MB-231, and T47D human breast cancer cell lines were examined and correlated with clonogenic survival following graded radiation doses with and without pretreatment with LY364947, a small molecule inhibitor of the TGFβ type I receptor kinase. The DNA damage response was assessed in irradiated MDA-MB-231 cells pretreated with LY364947 in vitro and LY2109761, a pharmacokinetically stable inhibitor of TGFβ signaling, in vivo. The in vitro response of a syngeneic murine tumor, 4T1, was tested using a TGFβ neutralizing antibody, 1D11, with single or fractionated radiation doses in vivo. Results Human breast cancer cell lines pretreated with TGFβ small molecule inhibitor were radio-sensitized, irrespective of sensitivity to TGFβ growth inhibition. Consistent with increased clonogenic cell death, radiation-induced phosphorylation of H2AX and p53 was significantly reduced in MDA-MB-231 triple-negative breast cancer cells when pretreated in vitro or in vivo with a TGFfS type I receptor kinase inhibitor. Moreover, TGFβ neutralizing antibodies increased radiation sensitivity, blocked γH2AX foci formation, and significantly increased tumor growth delay in 4T1 murine mammary tumors in response to single and fractionated radiation exposures. Conclusion These results show that TGFβ inhibition prior to radiation attenuated DNA damage responses, increased clonogenic cell death, and promoted tumor growth delay, and thus may be an effective adjunct in cancer radiotherapy. PMID:22028490

  13. K-ras activation occurs frequently in mucinous adenocarcinomas and rarely in other common epithelial tumors of the human ovary.

    PubMed Central

    Enomoto, T.; Weghorst, C. M.; Inoue, M.; Tanizawa, O.; Rice, J. M.

    1991-01-01

    To explore the role of mutational activation of members of the ras family of cellular protooncogenes in the development of human ovarian neoplasms, a series of 37 ovarian tumors from Japanese patients was studied. These included 30 common epithelial tumors (1 mucinous tumor of borderline malignancy, 7 mucinous adenocarcinomas, and 22 nonmucinous carcinomas: 10 serous, 3 clear cell, 8 endometrioid, and 1 undifferentiated), 5 tumors of germ cell origin, and 2 sex cord/stromal cell tumors. Polymerase chain reaction was performed from selected areas of deparaffinized sections of formalin-fixed paraffin-embedded tissue, and the presence of activating point mutations in codons 12, 13, and 61 of the H-, N-, and K-ras genes was probed by dot-blot hybridization analysis with mutation specific oligonucleotides. Mutations in K-ras were also looked for by direct genomic sequencing. The overall frequency of ras gene mutations was 10/37 (27%). Mutations were detected only in K-ras, and were found in most of the mucinous tumors, including the one such tumor of borderline malignancy (6/8; 75%). In one mucinous adenocarcinoma, two mutations were detected in paraffin-embedded material that had not previously been found in high molecular weight DNA isolated from frozen tissue from the same case. K-ras mutations occurred significantly more frequently in mucinous tumors (6/8, 75%) than in serous carcinomas (2/10, 20%; P = 0.031) or in all nonmucinous types of epithelial ovarian tumors combined (3/22, 14%; P = 0.0031). Images Figure 1 Figure 2 PMID:1656759

  14. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling.

    PubMed

    Campbell, Paul M; Groehler, Angela L; Lee, Kwang M; Ouellette, Michel M; Khazak, Vladimir; Der, Channing J

    2007-03-01

    Mutational activation of the K-Ras oncogene is well established as a key genetic step in the development and growth of pancreatic adenocarcinomas. However, the mechanism by which aberrant Ras signaling promotes uncontrolled pancreatic tumor cell growth remains to be fully elucidated. The recent use of primary human cells to study Ras-mediated oncogenesis provides important model cell systems to dissect this mechanism. We have used a model of telomerase-immortalized human pancreatic duct-derived cells (E6/E7/st) to study mechanisms of Ras growth transformation. First, we found that human papillomavirus E6 and E7 oncogenes, which block the function of the p53 and Rb tumor suppressors, respectively, and SV40 small t antigen were required to allow mutant K-Ras(12D) growth transformation. Second, K-Ras(12D) caused growth transformation in vitro, including enhanced growth rate and loss of density dependency for growth, anchorage independence, and invasion through reconstituted basement membrane proteins, and tumorigenic transformation in vivo. Third, we determined that the Raf, phosphatidylinositol 3-kinase (PI3K), and Ral guanine nucleotide exchange factor effector pathways were activated, although extracellular signal-regulated kinase (ERK) activity was not up-regulated persistently. Finally, pharmacologic inhibition of Raf/mitogen-activated protein kinase/ERK and PI3K signaling impaired K-Ras-induced anchorage-independent growth and invasion. In summary, our studies established, characterized, and validated E6/E7/st cells for the study of Ras-induced oncogenesis.

  15. Mutations of APC, K-ras, and p53 are associated with specific chromosomal aberrations in colorectal adenocarcinomas.

    PubMed

    Leslie, Amy; Pratt, Norman R; Gillespie, Karen; Sales, Mark; Kernohan, Neil M; Smith, Gillian; Wolf, C Roland; Carey, Francis A; Steele, Robert J C

    2003-08-01

    It is widely accepted that both large-scale chromosomal abnormalities and mutation of specific genes, such as APC, K-ras, and/or p53, occur in the majority of colorectal adenocarcinomas. Whether or not a relationship exists between these different forms of genetic abnormalities was previously unknown. Using comparative genomic hybridization and mutational analysis of APC, K-ras, and p53 to evaluate 50 colorectal adenocarcinomas, we have shown that mutation of p53 is significantly associated with gain of 20q, 13q, and 8q and loss of 18q (P = 0.000, 0.02, 0.044, and 0.001, respectively). Conversely, APC mutation did not associate with any of the above-mentioned aberrations but did associate significantly with gain of 7p (P = 0.01). Gain of chromosomal arm 12p, although a less common aberration, was significantly associated with K-ras mutation (P = 0.011). The associations we have described should refine the search for candidate genes underlying chromosomal aberrations and assist in the definition of distinct pathways in colorectal tumorigenesis.

  16. Prevalence of G-to-T transversions among K-ras oncogene mutations in human colorectal tumors in Yugoslavia.

    PubMed

    Urosević, N; Krtolica, K; Skaro-Milić, A; Knezević-Usaj, S; Dujić, A

    1993-05-08

    Human colorectal carcinoma tissue sampled from 37 patients, routinely graded into Dukes' stages A, B and C and histologically examined for the level of differentiation, were analyzed for the presence of point mutations in the K-ras oncogene. Seventeen cases out of the 37 analyzed were found to have a mutation in either the 12th or the 13th codon of the K-ras gene, giving an overall frequency of mutation of 46%. The incidence of mutations in Dukes' stages A, B and C was 33, 46 and 58% respectively. Although the frequency of mutation appears to be similar to that reported for the USA population, the spectrum of point mutations in codons 12 and 13 of the K-ras gene in the Yugoslav population appears to differ significantly. G-to-T transversions make up 77% of all mutations present, with the distribution as follows: 18% at the first base and 59% at the second base of codons 12 and 13. G-to-A transitions at the second base is the only other mutation identified, occurring mainly in codon 13 in colorectal tumors of all 3 stages.

  17. Frequency and spectrum of mutations at codons 12 and 13 of the C-K-ras gene in human tumors

    SciTech Connect

    Capella, G.; Cronauer-Mitra, S.; Peinado, M.A.; Perucho, M. )

    1991-06-01

    The frequency of point mutations at codons 12 and 13 of the c-K-ras gene has been determined in a panel of more than 400 human tumors. Mutant c-K-ras genes were detected in about 75% of adenocarcinomas of the pancreas; 40% of adenomas and carcinomas of the colon and rectum; 30% of carcinomas of the bile duct; 25% of carcinomas of the lung, and in lower frequency in other carcinomas, including liver, stomach, and kidney. No mutations were found in carcinomas of the breast, prostate, esophagus, and gall bladder, among others. Comparative analysis of the spectrum of mutations show that while G to A transitions were the most frequent mutations in pancreatic and colo-rectal tumors, G to T transversions were more prevalent in lung carcinomas. The aspartic acid mutation at codon 13 (GGC {r arrow} GAC) was relatively frequent in colo-rectal tumors but rare in pancreatic and lung carcinomas. The differences in the mutation spectrum of the c-K-ras gene in cancers of the gastrointestinal and respiratory tracts are suggestive of differential exposure to genotoxic agents.

  18. K-Ras and cyclooxygenase-2 coactivation augments intraductal papillary mucinous neoplasm and Notch1 mimicking human pancreas lesions

    PubMed Central

    Chiblak, Sara; Steinbauer, Brigitte; Pohl-Arnold, Andrea; Kucher, Dagmar; Abdollahi, Amir; Schwager, Christian; Höft, Birgit; Esposito, Irene; Müller-Decker, Karin

    2016-01-01

    Mutational activation of K-Ras is an initiating event of pancreatic ductal adenocarcinomas (PDAC) that may develop either from pancreatic intraepithelial neoplasia (PanIN) or intraductal papillary mucinous neoplasms (IPMN). Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is causally related to pancreatic carcinogenesis. Here, we deciphered the impact of COX-2, a key modulator of inflammation, in concert with active mutant K-RasG12D on tumor burden and gene expression signature using compound mutant mouse lines. Concomitant activation of COX-2 and K-RasG12D accelerated the progression of pancreatic intraepithelial lesions predominantly with a cystic papillary phenotype resembling human IPMN. Transcriptomes derived from laser capture microdissected preneoplastic lesions of single and compound mutants revealed a signature that was significantly enriched in Notch1 signaling components. In vitro, Notch1 signaling was COX-2-dependent. In line with these findings, human IPMN stratified into intestinal, gastric and pancreatobillary types displayed Notch1 immunosignals with high prevalence, especially in the gastric lesions. In conclusion, a yet unknown link between activated Ras, protumorigenic COX-2 and Notch1 in IPMN onset was unraveled. PMID:27381829

  19. Identification and Characterization of a Small Inhibitory Peptide That Can Target DNA-PKcs Autophosphorylation and Increase Tumor Radiosensitivity

    SciTech Connect

    Sun Xiaonan; Yang Chunying; Liu Hai; Wang Qi; Wu Shixiu; Li Xia; Xie Tian; Brinkman, Kathryn L.; Teh, Bin S.; Butler, E. Brian; Xu Bo; Zheng, Shu

    2012-12-01

    Purpose: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. Methods and Materials: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. Results: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged {gamma}-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. Conclusions: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

  20. Inhibition of poly(ADP-ribose) polymerase-1 by olaparib (AZD2281) increases the radiosensitivity of a lung tumor xenograft

    PubMed Central

    Senra, Joana M.; Telfer, Brian A.; Cherry, Kim E.; McCrudden, Cian M.; Hirst, David G.; O’Connor, Mark J.; Wedge, Stephen R.; Stratford, Ian J.

    2011-01-01

    Poly(ADP-ribose) polymerase-1 is a critical enzyme in the repair of DNA strand breaks. Inhibition of PARP-1 increases the effectiveness of radiation in killing tumor cells. However, while the mechanism(s) are well understood for these radiosensitizing effects in vitro, the underlying mechanism(s) in vivo are less clear. Nicotinamide, a drug structurally related to the first generation PARP-1 inhibitor, 3-aminobenzamide, reduces tumor hypoxia by preventing transient cessations in tumor blood flow, thus improving tumor oxygenation and sensitivity to radiotherapy. Here we investigate whether olaparib, a potent PARP-1 inhibitor, enhances radiotherapy, not only by inhibiting DNA repair but also by changing tumor vascular haemodynamics in non-small cell lung carcinoma. In irradiated Calu-6 and A549 cells, olaparib enhanced the cytotoxic effects of radiation (SER10=1.5 and 1.3) and DNA double strand breaks persisted for at least 24 h after treatment. Combination treatment of Calu-6 xenografts with olaparib and fractionated radiotherapy caused significant tumor regression (p=0.007) relative to radiotherapy alone. To determine whether this radiosensitisation was due solely to effects on DNA repair we used a dorsal window chamber model to establish the drug/radiation effects on vessel dynamics. Olaparib alone, when given as single or multiple daily doses, or in combination with fractionated radiotherapy, increased the perfusion of tumor blood vessels. Furthermore, an ex vivo assay in phenylephrine pre-constricted arteries confirmed olaparib to have higher vasodilatory properties than nicotinamide. This study suggests that olaparib warrants consideration for further development in combination with radiotherapy in clinical oncology settings such as NSCLC. PMID:21825006

  1. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

    PubMed

    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples (n=5) and cervical intraepithelial neoplasia tissue samples (n=5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P<0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group (P<0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group (P<0.05) but higher than the miR-143 mimic group significantly (P<0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control group and the

  2. Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis

    PubMed Central

    Doyen, Jérome; Parks, Scott K.; Marcié, Serge; Pouysségur, Jacques; Chiche, Johanna

    2013-01-01

    The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pHi)-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pHo manipulations and hypoxia (1% O2) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pHi-regulating capacity of fibroblasts through inhibition of Na+/H+ exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pHi-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pHi regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pHi-regulating CAs as an anti-cancer strategy. PMID:23316475

  3. Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage.

    PubMed

    Bayart, Emilie; Pouzoulet, Frédéric; Calmels, Lucie; Dadoun, Jonathan; Allot, Fabien; Plagnard, Johann; Ravanat, Jean-Luc; Bridier, André; Denozière, Marc; Bourhis, Jean; Deutsch, Eric

    2017-01-01

    Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2'-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 μM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy.

  4. Enhancement of IUdR Radiosensitization by Low-Energy Photons Results from Increased and Persistent DNA Damage

    PubMed Central

    Bayart, Emilie; Pouzoulet, Frédéric; Calmels, Lucie; Dadoun, Jonathan; Allot, Fabien; Plagnard, Johann; Ravanat, Jean-Luc; Bridier, André; Denozière, Marc; Bourhis, Jean; Deutsch, Eric

    2017-01-01

    Low-energy X-rays induce Auger cascades by photoelectric absorption in iodine present in the DNA of cells labeled with 5-iodo-2’-deoxyuridine (IUdR). This photoactivation therapy results in enhanced cellular sensitivity to radiation which reaches its maximum with 50 keV photons. Synchrotron core facilities are the only way to generate such monochromatic beams. However, these structures are not adapted for the routine treatment of patients. In this study, we generated two beams emitting photon energy means of 42 and 50 keV respectively, from a conventional 225 kV X-ray source. Viability assays performed after pre-exposure to 10 μM of IUdR for 48h suggest that complex lethal damage is generated after low energy photons irradiation compared to 137Cs irradiation (662KeV). To further decipher the molecular mechanisms leading to IUdR-mediated radiosensitization, we analyzed the content of DNA damage-induced foci in two glioblastoma cell lines and showed that the decrease in survival under these conditions was correlated with an increase in the content of DNA damage-induced foci in cell lines. Moreover, the follow-up of repair kinetics of the induced double-strand breaks showed the maximum delay in cells labeled with IUdR and exposed to X-ray irradiation. Thus, there appears to be a direct relationship between the reduction of radiation survival parameters and the production of DNA damage with impaired repair of these breaks. These results further support the clinical potential use of a halogenated pyrimidine analog combined with low-energy X-ray therapy. PMID:28045991

  5. The anti-malarial atovaquone increases radiosensitivity by alleviating tumour hypoxia

    PubMed Central

    Ashton, Thomas M.; Fokas, Emmanouil; Kunz-Schughart, Leoni A.; Folkes, Lisa K.; Anbalagan, Selvakumar; Huether, Melanie; Kelly, Catherine J.; Pirovano, Giacomo; Buffa, Francesca M.; Hammond, Ester M.; Stratford, Michael; Muschel, Ruth J.; Higgins, Geoff S.; McKenna, William Gillies

    2016-01-01

    Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy. PMID:27453292

  6. Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells.

    PubMed

    Collis, S J; Tighe, A; Scott, S D; Roberts, S A; Hendry, J H; Margison, G P

    2001-04-01

    The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20-50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to gamma-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of approximately 2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the alpha parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation.

  7. Ribozyme minigene-mediated RAD51 down-regulation increases radiosensitivity of human prostate cancer cells

    PubMed Central

    Collis, S. J.; Tighe, A.; Scott, S. D.; Roberts, S. A.; Hendry, J. H.; Margison, G. P.

    2001-01-01

    The strand transferase RAD51 is a component of the homologous recombination repair pathway. To examine the contribution of RAD51 to the genotoxic effects of ionising radiation, we have used a novel ribozyme strategy. A reporter gene vector was constructed so that expression of an inserted synthetic double-stranded ribozyme-encoding oligonucleotide would be under the control of the cytomegalovirus immediate-early gene enhancer/promoter system. The prostate tumour cell line LNCaP was transfected with this vector or a control vector, and a neomycin resistance gene on the vector was used to create geneticin-resistant stable cell lines. Three stable cell lines were shown by western blot analysis to have significant down-regulation of RAD51 to 20–50% of the levels expressed in control cell lines. All three cell lines had a similar increased sensitivity to γ-irradiation by 70 and 40%, respectively, compared to normal and empty vector-transfected cells, corresponding to dose-modifying factors of ∼2.0 and 1.5 in the mid-range of the dose-response curves. The amount of RAD51 protein in transfected cell lines was shown to strongly correlate with the α parameter obtained from fitted survival curves. These results highlight the importance of RAD51 in cellular responses to radiation and are the first to indicate the potential use of RAD51-targeted ribozyme minigenes in tumour radiosensitisation. PMID:11266555

  8. K-Ras mutation detection in liquid biopsy and tumor tissue as prognostic biomarker in patients with pancreatic cancer: a systematic review with meta-analysis.

    PubMed

    Li, Tao; Zheng, Yuanting; Sun, Hong; Zhuang, Rongyuan; Liu, Jing; Liu, Tianshu; Cai, Weimin

    2016-07-01

    K-Ras gene mutations have been found in most pancreatic cancers; however, conflicting data on the prognostic value of K-Ras mutations in pancreatic cancer have been published. We conducted a meta-analysis to assess its prognostic significance. Literature searches of PubMed, EMBASE, Cochrane Library, Web of Science and Google Scholar were performed through December 2015 to identify publications exploring the association of K-Ras mutation with overall survival. Forty eligible studies involving 3427 patients with pancreatic cancer were included in the present meta-analysis. Our analysis showed a hazard ratio (HR) of negative association with survival of 1.61 [95 % confidence interval (CI) 1.36-1.90; p < 0.01] in K-Ras mutant pancreatic cancer patients. In subgroup analyses, K-Ras mutations detected in tumor tissues and in liquid biopsies had HRs of 1.37 (95 % CI 1.20-1.57; p < 0.01) and 3.16 (95 % CI 2.1-4.71; p < 0.01), respectively. In addition, the HR was higher when K-Ras mutations were detected in fresh frozen samples (HR = 2.01, 95 % CI 1.28-3.16, p = 0.002) than in formalin-fixed, paraffin-embedded (FFPE) samples (HR = 1.29, 95 % CI 1.12-1.49, p < 0.01). Though K-Ras alterations are more frequent among non-East Asian individuals than East Asian individuals, there were no significant differences in HRs of survival between the two ethnic subgroups. In conclusion, this meta-analysis suggests that K-Ras mutations are associated with a worse overall survival in pancreatic cancer patients, especially when mutations are detected in liquid biopsies or fresh frozen tumor tissue samples.

  9. Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins.

    PubMed

    Gulyás, Gergö; Radvánszki, Glória; Matuska, Rita; Balla, András; Hunyady, László; Balla, Tamas; Várnai, Péter

    2017-09-22

    Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Posttranslational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C β or with a rapamycin-inducible system in which various PI phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescent energy transfer (BRET)-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P2, but not PtdIns(3,4,5)P3, was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasine, an inhibitor of the Ras-phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [3H]-Leucine in K-Ras-expressing cells, suggesting that Golgi-localized K-Ras is not as signaling competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras mediated signals. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  10. Sphingosine-1-Phosphate Receptor Subtype 3: A Novel Therapeutic Target of K-Ras Mutant Driven Non-Small Cell Lung Carcinoma

    DTIC Science & Technology

    2015-10-01

    mediated lung adenocarcinoma (AdC) progression, and to examine the novel therapeutic utility for K-Ras mutant-driven lung AdC by targeting S1PR3...and adenocarcinomas will be assessed. We have completed the animal treatments proposed in Aims 1 and 2. The collected mouse lung specimens are...currently being analyzed. 15. SUBJECT TERMS: Oncogenic K-Ras mutant, lung adenocarcinoma , sphingosine-1-phosphate receptor subtype 3 16. SECURITY

  11. Growth Regulation via Insulin-Like Growth Factor Binding Protein-4 and -2 in Association with Mutant K-ras in Lung Epithelia

    PubMed Central

    Sato, Hanako; Yazawa, Takuya; Suzuki, Takehisa; Shimoyamada, Hiroaki; Okudela, Koji; Ikeda, Masaichi; Hamada, Kenji; Yamada-Okabe, Hisafumi; Yao, Masayuki; Kubota, Yoshinobu; Takahashi, Takashi; Kamma, Hiroshi; Kitamura, Hitoshi

    2006-01-01

    Gain-of-function point mutations in K-ras affect early events in pulmonary bronchioloalveolar carcinoma. We investigated altered mRNA expression on K-Ras activation in human peripheral lung epithelial cells (HPL1A) using oligonucleotide microarrays. Mutated K-Ras stably expressed in HPL1A accelerated cell growth and induced the expression of insulin-like growth factor (IGF)-binding protein (IGFBP)-4 and IGFBP-2, which modulate cell growth via IGF. Other lung epithelial cell lines (NHBE and HPL1D) revealed the same phenomena as HPL1A by mutated K-ras transgene. Lung cancer cell growth was also accelerated by mutated K-ras gene transduction, whereas IGFBP-4/2 induction was weaker compared with mutated K-Ras-expressing lung epithelial cells. To understand the differences in IGFBP-4/2 inducibility via K-Ras-activated signaling between nonneoplastic lung epithelia and lung carcinoma, we addressed the mechanisms of IGFBP-4/2 transcriptional activation. Our results revealed that Egr-1, which is induced on activation of Ras-mitogen-activated protein kinase signaling, is crucial for transactivation of IGFBP-4/2. Furthermore, IGFBP-4 and IGFBP-2 promoters were often hypermethylated in lung carcinoma, yielding low basal expression/weak induction of IGFBP-4/2. These findings suggest that continuous K-Ras activation accelerates cell growth and evokes a feedback system through IGFBP-4/2 to prevent excessive growth. Moreover, this growth regulation is disrupted in lung cancers because of promoter hypermethylation of IGFBP-4/2 genes. PMID:17071580

  12. Oncogenic K-Ras and Basic Fibroblast Growth Factor Prevent FAS-Mediated Apoptosis in Fibroblasts through Activation of Mitogen-Activated Protein Kinase

    PubMed Central

    Kazama, Hirotaka; Yonehara, Shin

    2000-01-01

    By an expression cloning method using Fas-transgenic Balb3T3 cells, we tried to obtain inhibitory genes against Fas-mediated apoptosis and identified proto-oncogene c-K-ras. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibiting Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, including Raf (mitogen-activated protein kinase kinase kinase [MAPKKK]), phosphatidylinositol 3 (PI-3) kinase, and Ral guanine nucleotide exchange factor (RalGDS), only the constitutively active form of Raf (Raf-CAAX) could inhibit Fas-mediated apoptosis. In addition, the constitutively active form of MAPKK (SDSE-MAPKK) suppressed Fas-mediated apoptosis, and MKP-1, a phosphatase specific for classical MAPK, canceled the protective activity of oncogenic K-Ras (K-RasV12), Raf-CAAX, and SDSE-MAPKK. Furthermore, physiological activation of Ras by basic fibroblast growth factor (bFGF) protected Fas-transgenic Balb3T3 cells from Fas-mediated apoptosis. bFGF protection was also dependent on the activation of the MAPK pathway through Ras. All the results indicate that the activation of MAPK through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis. PMID:10662780

  13. Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer

    SciTech Connect

    Naumov, Inna; Kazanov, Dina; Lisiansky, Victoria; Starr, Alex; Aroch, Ilan; Shapira, Shiran; Kraus, Sarah; Arber, Nadir

    2012-01-15

    Background: Functional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras. Results: A recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our 'gene therapy' approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce {approx} 50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by {approx} 35% tumor progression in vivo in already established tumors. Conclusions: Selective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

  14. Assessment of epidermal growth factor receptor mutation/copy number and K-ras mutation in esophageal cancer.

    PubMed

    Guo, Kang; Wang, Wu-Ping; Jiang, Tao; Wang, Ju-Zheng; Chen, Zhao; Li, Yong; Zhou, Yong-An; Li, Xiao-Fei; Lu, Qiang; Zhang, Lan-Jun

    2016-07-01

    The molecular status of epidermal growth factor receptor (EGFR) in esophageal cancer has not been well elucidated. The purpose of the study was to investigate the prevalence of EGFR and K-ras mutation, and EGFR gene copy number status as well as its association with clinicopathologic characteristics, and also to identify the prognostic value of EGFR gene copy number in esophageal cancer. EGFR mutation in exon 19/exon 21 and K-ras mutation in codon 12/codon 13 were detected by real-time PCR method, while EGFR gene copy number status was analyzed by fluorescent in situ hybridization (FISH). EGFR gene amplification and high polysomy were defined as high EGFR gene copy number status (FISH-positive), and all else were defined as low EGFR gene copy number status (FISH-negative). The relationship between EGFR gene copy number status and clinicpathologic characteristics was analyzed. Kaplan-Meier method and Cox proportional hazards regression model were employed to evaluate the effects of EGFR gene copy number status on the patients' survival. A total of 57 esophageal squamous cell carcinoma (ESCC) patients and 9 esophageal adenocarcinoma (EADC) patients were enrolled in the study. EGFR mutation was identified in one patient who was diagnosed as ESCC with stage IIIC disease. K-ras mutation was identified in one patient who was diagnosed as EADC. In all, 34 of 66 (51.5%) samples were detected as FISH-positive, which includes 30 ESCC and 4 EADC tumor samples. The correlation analysis showed that FISH-positive was significantly associated with the tumor stage (P=0.019) and lymph node metastasis (P=0.005) in esophageal cancer patients, and FISH-positive was also significantly associated with the tumor stage (P=0.007) and lymph node metastasis (P=0.008) in ESCC patients. Cox regression analysis showed that high EGFR gene copy number was not a significant predictor of a poor outcome for esophageal cancer patients (P=0.251) or for ESCC patients (P=0.092), but esophageal cancer

  15. [Correlation analysis between abundance of K-ras mutation in plasma free DNA and its correlation with clinical outcome and prognosis in patients with metastatic colorectal cancer].

    PubMed

    Bai, Yan-qing; Liu, Xiao-jing; Wang, Yan; Ge, Fei-jiao; Zhao, Chuan-hua; Fu, Ya-li; Lin, Li; Xu, Jian-ming

    2013-09-01

    To detect K-ras gene mutations in plasma free DNA by peptide nucleic acid clamp PCR assay (PNA-PCR) and nested primer PCR, and to analyze the correlation between K-ras mutations and prognosis in patients with metastatic colorectal cancer (mCRC). Peripheral blood was collected and free DNA was extracted from plasma in 106 patients with mCRC. Nested primer PCR and PNA-PCR were used to detect K-ras gene mutation in the plasma free DNA. The patients were divided into three groups by K-ras status: wild-type group (wild-type determined by both methods), low mutation group (mutation by PNA-PCR method, wild-type by nested primer PCR method) and high mutation group (mutation by two methods). The correlation between K-ras mutations and prognosis was analyzed. The mutation rate of K-ras in tumor tissues of the 106 patients was 40.6%. The Mutation rate of K-ras in plasma free DNA detected by PNA-PCR was 31.1%, significantly higher than that of 15.1% detected by nested primer PCR (P = 0.006). The consistent rate of the K-ras status in plasma free DNA detected by PNA-PCR and that in tumor tissue detected by traditional method was up to 83.0%. The median overall survival (OS) of patients of the wild type, low mutation and high mutation groups was 23.5 months, 17.3 months and 13.9 months, respectively (P = 0.002). The median progression-free survival (PFS) of the K-ras wild-type, low mutation and high mutation groups with first-line chemotherapy was 6.8 months, 6.1 months and 3.2 months, respectively (P = 0.002), and the median OS of them were 23.0 months, 15.5 months and 13.9 months, respectively (P = 0.036). The overall response rate (ORR) was improved in the K-ras wide-type patients who received cetuximab combined with chemotherapy as first-line therapy (75.0% vs. 23.4%, P = 0.058). Cetuximab combined with in second-line therapy chemotherapy led to a significant improvement in disease control rate (DCR) ( 100% vs. 35.7%, P < 0.001) as compared with those of chemotherapy alone

  16. K-ras cancer gene mutations in lung tumors from female Swiss (CD-1) mice exposed transplacentally to 3'-azido-3'-deoxythymidine.

    PubMed

    Koujitani, Takatoshi; Ton, Tai-Vu T; Lahousse, Stephanie A; Hong, Hue-Hua L; Wakamatsu, Nobuko; Sills, Robert C

    2008-12-01

    A transplacental carcinogenicity study was conducted by exposing pregnant Swiss (CD-1) mice to 0, 50, 100, 200, or 300 mg 3'-azido-3'-deoxythymidine (AZT)/kg body weight (BW) daily for the duration of gestation (18-19 days) [National Toxicology Program,2006]. The incidence of alveolar/bronchiolar adenomas and carcinomas in the 200 and 300 mg/kg groups was significantly higher (P = 0.027 and 0.007, respectively) in male offspring, but not in females (P = 0.338 and 0.315, respectively). The purpose of the present study was to evaluate K-ras mutation status in lung tumors from the female offspring in AZT exposed groups and to determine whether at the molecular level there were signature K-ras mutations in lung tumors that were different from spontaneous tumors. K-ras mutation was detected by cycle sequencing of polymerase chain reaction (PCR)-amplified DNA, isolated from formalin-fixed, paraffin-embedded lung tumors. K-ras mutations were detected in 17 of 28 (61%) lung tumors from the female offspring in AZT exposed groups. No K-ras mutations were detected in the 8 tumors examined from the female control group. The predominant mutations were Codon 12 G-->T transversions in the 50, 100, and 300 mg/kg groups, and Codon 12 G-->C transversions in the 200 and 300 mg/kg groups. K-ras Codon 12 G-->T transversions (TGT mutations) may be induced by oxidative DNA damage and 8-oxoguanine (8-oxoG), while K-ras Codon 12 G-->C transversions (CGT mutations) may be due to further oxidative lesions of guanine and 8-oxoG.

  17. Response to first-line chemotherapy in patients with non-small-cell lung cancer according to epidermal growth factor receptor and K-RAS mutation status.

    PubMed

    Dong, Xiaopeng; Zhao, Xiaogang; Hao, Yingtao; Wei, Yucheng; Yin, Qiuwei; Du, Jiajun

    2013-11-01

    Epidermal growth factor receptor (EGFR)-targeted therapy has shown a favorable efficacy in patients with non-small-cell lung cancer (NSCLC). Conversely, K-RAS mutations were reported to have an adverse effect on the survival of patients with NSCLC. These studies suggest that the tumor biology of patients with EGFR or K-RAS mutations is different from that of patients with wild-type mutations. Therefore, we hypothesized that the response to cytotoxic chemotherapy may differ among patients with and without EGFR or K-RAS mutations. A total of 229 patients with advanced NSCLC who received platinum doublet chemotherapy were included in this retrospective study, and their clinical outcomes were analyzed according to EGFR and K-RAS mutation status. EGFR and K-RAS mutations were found in 52.4% and 27.9% of patients, respectively. Progression-free survival (PFS) was significantly higher in patients with EGFR mutations than in patients with wild-type EGFR (P = .008), and multivariate analysis showed that EGFR mutation was an independent factor to chemotherapy (P = .01). Among the patients with EGFR mutations, the disease control rate for docetaxel was higher than for gemcitabine-based therapy (P = .031). In addition, docetaxel or vinorelbine showed a longer PFS than gemcitabine-based chemotherapy in patients with EGFR mutations (P = .033 and P = .028). However, no similar differences were found according to the K-RAS mutations. EGFR, but not K-RAS mutation, is associated with improved survival time to platinum-based chemotherapy. In patients with EGFR mutations, PFS for docetaxel and gemcitabine was higher than for vinorelbine-based chemotherapies. The predictive meaning of EGFR mutation for chemotherapy should be further investigated. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Membrane-associated Ras dimers are isoform-specific: K-Ras dimers differ from H-Ras dimers.

    PubMed

    Jang, Hyunbum; Muratcioglu, Serena; Gursoy, Attila; Keskin, Ozlem; Nussinov, Ruth

    2016-06-15

    Are the dimer structures of active Ras isoforms similar? This question is significant since Ras can activate its effectors as a monomer; however, as a dimer, it promotes Raf's activation and MAPK (mitogen-activated protein kinase) cell signalling. In the present study, we model possible catalytic domain dimer interfaces of membrane-anchored GTP-bound K-Ras4B and H-Ras, and compare their conformations. The active helical dimers formed by the allosteric lobe are isoform-specific: K-Ras4B-GTP favours the α3 and α4 interface; H-Ras-GTP favours α4 and α5. Both isoforms also populate a stable β-sheet dimer interface formed by the effector lobe; a less stable β-sandwich interface is sustained by salt bridges of the β-sheet side chains. Raf's high-affinity β-sheet interaction is promoted by the active helical interface. Collectively, Ras isoforms' dimer conformations are not uniform; instead, the isoform-specific dimers reflect the favoured interactions of the HVRs (hypervariable regions) with cell membrane microdomains, biasing the effector-binding site orientations, thus isoform binding selectivity.

  19. Genetic Alterations in K-ras and p53 Cancer Genes in Lung Neoplasms From B6C3F1 Mice Exposed to Cumene

    PubMed Central

    Hong, Hue-Hua L.; Ton, Thai-Vu. T.; Kim, Yongbaek; Wakamatsu, Nobuko; Clayton, Natasha P.; Chan, Po-Chuen; Sills, Robert C.; Lahousse, Stephanie A.

    2009-01-01

    The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the controls. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87 % cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56 % cumene-induced neoplasms and mutations were detected in 52 % neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions and codon 133 C to T transitions. No p53 mutation and one of 7 (14 %) K-ras mutation was detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13 %) and on chromosome 6 near the K-ras gene (12 %). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene. PMID:18648094

  20. A subset of lung adenocarcinomas and atypical adenomatous hyperplasia-associated foci are genotypically related: an EGFR, HER2, and K-ras mutational analysis.

    PubMed

    Sartori, Giuliana; Cavazza, Alberto; Bertolini, Federica; Longo, Lucia; Marchioni, Alessandro; Costantini, Matteo; Barbieri, Fausto; Migaldi, Mario; Rossi, Giulio

    2008-02-01

    Atypical adenomatous hyperplasia (AAH) is considered the preinvasive lesion of pulmonary adenocarcinoma, and mutations of EGFR, HER2, and K-ras are involved in the early stage of lung adenocarcinoma carcinogenesis, also predicting clinical response to anti-EGFR small molecule inhibitors. We analyzed 18 cases of primary lung adenocarcinoma with concomitant AAH foci from 13 patients for mutations of EGFR (exons 18-21), HER2 (exons 19-20), and K-ras (exon 2) by direct sequencing polymerase chain reaction. Among mutated cases, concordant mutations of EGFR or K-ras in adenocarcinoma and related AAH were observed in 5 (63%) of 8 cases. In particular, 3 of 4 adenocarcinomas with EGFR mutations (all L858R point mutations in women, never or former smokers) had a concomitant and identical mutation in AAH, and 2 of 4 adenocarcinomas with K-ras mutations (both at codon 12 in women, a never and a current smoker) showed the same mutation in concomitant AAH. All cases were wild-type for HER2. Mutations of EGFR and K-ras genes represent an early event in lung adenocarcinomagenesis, and AAH convincingly seems to be a precursor lesion in a subset of cases of adenocarcinoma.

  1. Genetic alterations in K-ras and p53 cancer genes in lung neoplasms from B6C3F1 mice exposed to cumene.

    PubMed

    Hong, Hue-Hua L; Ton, Thai-Vu T; Kim, Yongbaek; Wakamatsu, Nobuko; Clayton, Natasha P; Chan, Po-Chuen; Sills, Robert C; Lahousse, Stephanie A

    2008-07-01

    The incidences of alveolar/bronchiolar adenomas and carcinomas in cumene-treated B6C3F1 mice were significantly greater than those of the control animals. We evaluated these lung neoplasms for point mutations in the K-ras and p53 genes that are often mutated in humans. K-ras and p53 mutations were detected by cycle sequencing of PCR-amplified DNA isolated from paraffin-embedded neoplasms. K-ras mutations were detected in 87% of cumene-induced lung neoplasms, and the predominant mutations were exon 1 codon 12 G to T transversions and exon 2 codon 61 A to G transitions. P53 protein expression was detected by immunohistochemistry in 56% of cumene-induced neoplasms, and mutations were detected in 52% of neoplasms. The predominant mutations were exon 5, codon 155 G to A transitions, and codon 133 C to T transitions. No p53 mutations and one of seven (14%) K-ras mutations were detected in spontaneous neoplasms. Cumene-induced lung carcinomas showed loss of heterozygosity (LOH) on chromosome 4 near the p16 gene (13%) and on chromosome 6 near the K-ras gene (12%). No LOH was observed in spontaneous carcinomas or normal lung tissues examined. The pattern of mutations identified in the lung tumors suggests that DNA damage and genomic instability may be contributing factors to the mutation profile and development of lung cancer in mice exposed to cumene.

  2. Computational Modeling Reveals that Signaling Lipids Modulate the Orientation of K-Ras4A at the Membrane Reflecting Protein Topology.

    PubMed

    Li, Zhen-Lu; Buck, Matthias

    2017-04-04

    The structural, dynamical, and functional characterization of the small GTPase K-Ras has become a research area of intense focus due to its high occurrence in human cancers. Ras proteins are only fully functional when they interact with the plasma membrane. Here we present all-atom molecular dynamics simulations (totaling 5.8 μs) to investigate the K-Ras4A protein at membranes that contain anionic lipids (phosphatidyl serine or phosphatidylinositol bisphosphate). We find that similarly to the homologous and highly studied K-Ras4B, K-Ras4A prefers a few distinct orientations at the membrane. Remarkably, the protein surface charge and certain lipids can strongly modulate the orientation preference. In a novel analysis, we reveal that the electrostatic interaction (attraction but also repulsion) between the protein's charged residues and anionic lipids determines the K-Ras4A orientation, but that this is also influenced by the topology of the protein, reflecting the geometry of its surfaces.

  3. Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways.

    PubMed

    Zhang, Jing; Liu, Yangang; Beard, Caroline; Tuveson, David A; Jaenisch, Rudolf; Jacks, Tyler E; Lodish, Harvey F

    2007-06-15

    When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-ras(G12D)) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-ras(G12D) leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-ras(G12D) fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-ras(G12D) display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.

  4. Resveratrol-Induced Apoptosis and Increased Radiosensitivity in CD133-Positive Cells Derived From Atypical Teratoid/Rhabdoid Tumor

    SciTech Connect

    Kao, C.-L.; Huang, P.-I; Tsai, P.-H.; Tsai, M.-L.; Lo, J.-F.; Lee, Y.-Y.; Chen, Y.-J.; Chen, Y.-W.; Chiou, S.-H.

    2009-05-01

    Purpose: CD133 has recently been proposed as a marker for cancer stem-like cells (CSC) in brain tumors. The aim of the present study was to investigate the possible role of resveratrol (RV) in radiosensitivity of CD133-positive/-negative cells derived from atypical teratoid/rhabdoid tumors (AT/RT-CD133{sup +/-}). Materials and Methods: AT/RT-CD133{sup +/-} were isolated and characterized by flow cytometry and quantitative real-time reverse transcription-polymerase chain reaction, and then treated with RV at different doses. Migratory ability, colony formation, apoptotic activity, and xenotransplantation were assessed for RV alone, ionizing radiation (IR) alone, and IR with RV conditions. Results: AT/RT-CD133{sup +} displayed enhanced self-renewal and highly coexpressed 'stem cell' genes and drug-resistant genes, in addition to showing significant resistance to chemotherapeutic agents and radiotherapy as compared with CD133{sup -} cells. After treatment with 200 {mu}M RV, the in vitro proliferation rates and in vivo tumor restoration abilities of ATRT-CD133{sup +} were dramatically inhibited. Importantly, treatment with 150 {mu}M RV can effectively inhibit the expression of drug-resistant genes in AT/RT-CD133{sup +}, and further facilitate to the differentiation of CD133{sup +} into CD133{sup -}. In addition, treatment with 150 {mu}M RV could significantly enhance the radiosensitivity and IR-mediated apoptosis in RV-treated ATRT-CD133{sup +/-}. Kaplan-Meier survival analysis indicated that the mean survival rate of mice with ATRT-CD133{sup +} that were treated with IR could be significantly improved when IR was combined with 150 {mu}M RV treatment. Conclusions: AT/RT-CD133{sup +} exhibit CSC properties and are refractory to IR treatment. Our results suggest that RV treatment plays crucial roles in antiproliferative, proapoptotic, and radiosensitizing effects on treated-CD133{sup +/-}; RV may therefore improve the clinical treatment of AT/RT.

  5. c-Raf, but not B-Raf, is essential for development of K-Ras oncogene driven non-small cell lung carcinoma

    PubMed Central

    Blasco, Rafael B.; Francoz, Sarah; Santamaría, David; Cañamero, Marta; Dubus, Pierre; Charron, Jean; Baccarini, Manuela; Barbacid, Mariano

    2013-01-01

    SUMMARY We have interrogated the role of individual members of the Raf/Mek/Erk cascade in the onset of K-Ras oncogene-driven non-small cell lung carcinoma (NSCLC). Ablation of Erk1 or Erk2 in K-Ras oncogene expressing lung cells had no significant effect due to compensatory activities. Yet, elimination of both Erk kinases completely blocked tumor development. Similar results were obtained with Mek kinases. Ablation of B-Raf had no significant effect on tumor development. However, c-Raf expression was absolutely essential for the onset of NSCLC. Interestingly, concomitant elimination of c-Raf and B-Raf in adult mice had no deleterious consequences for normal homeostasis. These results indicate that c-Raf plays a unique role in mediating K-Ras signaling and makes it a suitable target for therapeutic intervention. PMID:21514245

  6. Assessment of epidermal growth factor receptor and K-ras mutation status in cytological stained smears of non-small cell lung cancer patients: correlation with clinical outcomes.

    PubMed

    Lozano, Maria D; Zulueta, Javier J; Echeveste, Jose I; Gúrpide, Alfonso; Seijo, Luis M; Martín-Algarra, Salvador; Del Barrio, Anabel; Pio, Ruben; Idoate, Miguel Angel; Labiano, Tania; Perez-Gracia, Jose Luis

    2011-01-01

    Epidermal growth factor receptor (EGFR) and K-ras mutations guide treatment selection in non-small cell lung cancer (NSCLC) patients. Although mutation status is routinely assessed in biopsies, cytological specimens are frequently the only samples available. We determined EGFR and K-ras mutations in cytological samples. DNA was extracted from 150 consecutive samples, including 120 Papanicolau smears (80%), 10 cell blocks (7%), nine fresh samples (6%), six ThinPrep® tests (4%), and five body cavity fluids (3.3%). Papanicolau smears were analyzed when they had >50% malignant cells. Polymerase chain reaction and direct sequencing of exons 18-21 of EGFR and exon 2 of K-ras were performed. EGFR mutations were simultaneously determined in biopsies and cytological samples from 20 patients. Activity of EGFR tyrosine kinase inhibitors (TKIs) was assessed. The cytological diagnosis was adenocarcinoma in 110 samples (73%) and nonadenocarcinoma in 40 (27%) samples. EGFR mutations were identified in 26 samples (17%) and K-ras mutations were identified in 18 (12%) samples. EGFR and K-ras mutations were mutually exclusive. In EGFR-mutated cases, DNA was obtained from stained smears in 24 cases (92%), pleural fluid in one case (4%), and cell block in one case (4%). The response rate to EGFR TKIs in patients harboring mutations was 75%. The mutation status was identical in patients who had both biopsies and cytological samples analyzed. Assessment of EGFR and K-ras mutations in cytological samples is feasible and comparable with biopsy results, making individualized treatment selection possible for NSCLC patients from whom tumor biopsies are not available.

  7. K-Ras gene mutation status as a prognostic and predictive factor in patients with colorectal cancer undergoing irinotecan- or oxaliplatin-based chemotherapy

    PubMed Central

    Stec, Rafał; Bodnar, Lubomir; Charkiewicz, Radosław; Korniluk, Jan; Rokita, Marta; Smoter, Marta; Ciechowicz, Marzena; Chyczewski, Lech; Nikliński, Jacek; Kozłowski, Wojciech; Szczylik, Cezary

    2012-01-01

    Background: CRC caused more than 600,000 estimated deaths in 2008. Dysregulated signaling through the RAS/RAF/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway due to mutations in K-Ras and B-Raf are common events in CRC. Methods: Incidence of mutations in codons 12 and 13 of K-Ras and exons 11 and 15 of B-Raf were analyzed in amplified PCR products from primary tumors of 273 patients with CRC, and their prognostic and predictive significance was assessed. The prognostic role of clinical and pathological factors was also examined. Results: K-Ras mutations were present in 89 patients (32.6%), of whom 76 (85.4%) had mutations in codon 12 and 10 (11.2%) had mutations in codon 13. B-Raf gene mutations were present in 17 patients (6.9%), of whom 6 (35.3%) had mutations in exon 15. Multivariate analysis revealed a predictive significance for K-Ras mutations with respect to time to progression in patients treated with irinotecan and oxaliplatin as first-line chemotherapy. There was no predictive significance for B-Raf gene mutation status in these patients. The following risk factors were found to affect overall survival (OS) rates: primary tumor location, lymph node involvement grade, carcinoembryonic antigen (CEA) level before treatment, and performance status according to WHO criteria. Conclusions: Based on the results of this study, K-Ras mutation status may be a suitable indicator of patient eligibility and a prognostic indicator for responsiveness to anti-EGFR therapy alone, or in combination with chemotherapy. Also, K-Ras mutation status may predict time to progression in patients treated with irinotecan and oxaliplatin. PMID:22909976

  8. Clinical outcome of patients with non-small cell lung cancer receiving front-line chemotherapy according to EGFR and K-RAS mutation status.

    PubMed

    Kalikaki, Aristea; Koutsopoulos, Anastasios; Hatzidaki, Dora; Trypaki, Maria; Kontopodis, Emmanouel; Stathopoulos, Efstathios; Mavroudis, Dimitris; Georgoulias, Vassilis; Voutsina, Alexandra

    2010-07-01

    Somatic mutations in EGFR and K-RAS may predict for sensitivity and resistance to EGFR tyrosine kinase inhibitors (TKIs). Whether EGFR and K-RAS mutations could also predict clinical outcome of non-small cell lung cancer (NSCLC) patients following front-line chemotherapy has not yet been established. One hundred and sixty-two chemotherapy-naïve patients with locally advanced/metastatic NSCLC who received front-line chemotherapy were included in this retrospective study and their clinical outcome data was analyzed according to EGFR and K-RAS mutation status of their tumors. Classical activating EGFR and K-RAS mutations were found in 8.2 and 22.6% of patients respectively and were not associated with patients' clinicopathological characteristics. Patients with classical EGFR mutations had a higher probability of response to front-line chemotherapy as compared to those with wild type EGFR (p=0.023). Multivariate analysis showed that the presence of activating EGFR mutations was an independent factor associated with response to front-line chemotherapy (HR=4.85; 95% CI: 1.13-20.83, p=0.034). K-RAS mutation status was not associated with response to front-line chemotherapy. The presence of activating EGFR but not of K-RAS mutations was associated with a significantly higher overall survival compared to patients without mutations treated with platinum-based front-line chemotherapy (p=0.043). The data indicate that EGFR mutation status could be predictive for response to cytotoxic front-line chemotherapy in patients with NSCLC. Additional prospective studies are needed in order to validate this observation and to define whether these patients should be preferentially treated with front-line TKIs or chemotherapy. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

  9. GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

    PubMed Central

    Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

    2015-01-01

    K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

  10. Assessment of Epidermal Growth Factor Receptor and K-Ras Mutation Status in Cytological Stained Smears of Non-Small Cell Lung Cancer Patients: Correlation with Clinical Outcomes

    PubMed Central

    Zulueta, Javier J.; Echeveste, Jose I.; Gúrpide, Alfonso; Seijo, Luis M.; Martín-Algarra, Salvador; del Barrio, Anabel; Pio, Ruben; Idoate, Miguel Angel; Labiano, Tania; Perez-Gracia, Jose Luis

    2011-01-01

    Objective. Epidermal growth factor receptor (EGFR) and K-ras mutations guide treatment selection in non-small cell lung cancer (NSCLC) patients. Although mutation status is routinely assessed in biopsies, cytological specimens are frequently the only samples available. We determined EGFR and K-ras mutations in cytological samples. Methods. DNA was extracted from 150 consecutive samples, including 120 Papanicolau smears (80%), 10 cell blocks (7%), nine fresh samples (6%), six ThinPrep® tests (4%), and five body cavity fluids (3.3%). Papanicolau smears were analyzed when they had >50% malignant cells. Polymerase chain reaction and direct sequencing of exons 18–21 of EGFR and exon 2 of K-ras were performed. EGFR mutations were simultaneously determined in biopsies and cytological samples from 20 patients. Activity of EGFR tyrosine kinase inhibitors (TKIs) was assessed. Results. The cytological diagnosis was adenocarcinoma in 110 samples (73%) and nonadenocarcinoma in 40 (27%) samples. EGFR mutations were identified in 26 samples (17%) and K-ras mutations were identified in 18 (12%) samples. EGFR and K-ras mutations were mutually exclusive. In EGFR-mutated cases, DNA was obtained from stained smears in 24 cases (92%), pleural fluid in one case (4%), and cell block in one case (4%). The response rate to EGFR TKIs in patients harboring mutations was 75%. The mutation status was identical in patients who had both biopsies and cytological samples analyzed. Conclusion. Assessment of EGFR and K-ras mutations in cytological samples is feasible and comparable with biopsy results, making individualized treatment selection possible for NSCLC patients from whom tumor biopsies are not available. PMID:21572125

  11. The impact of the genetic background in the Noonan syndrome phenotype induced by K-RasV14I

    PubMed Central

    Hernández-Porras, Isabel; Jiménez-Catalán, Beatriz; Schuhmacher, Alberto J; Guerra, Carmen

    2015-01-01

    Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. A significant fraction of NS-patients also develop myeloproliferative disorders. The penetrance of these defects varies considerably among patients. In this study, we have examined the effect of 2 genetic backgrounds (C57BL/6J.OlaHsd and 129S2/SvPasCrl) on the phenotypes displayed by a mouse model of NS induced by germline expression of the mutated K-RasV14I allele, one of the most frequent NS-KRAS mutations. Our results suggest the presence of genetic modifiers associated to the genetic background that are essential for heart development and function at early stages of postnatal life as well as in the severity of the haematopoietic alterations. PMID:26458870

  12. Biweekly cetuximab and first-line chemotherapy in chinese patients with k-ras wild-type colorectal cancers.

    PubMed

    Chan, Wing-Lok; Lee, Victor Ho Fun; Siu, Wai Kwan Steven; Ho, Pui Ying Patty; Liu, Rico King Yin; Leung, To Wai

    2014-07-01

    The efficacy and safety of using combination chemotherapy with cetuximab as first-line treatment in patients with K-ras wild-type colorectal cancers has been well established. In general, weekly cetuximab was given with biweekly chemotherapy FOLFOX-4 or FOLFIRI, synchronizing them would be appealed to both patients and health care professionals. This Phase II, prospective study investigated the efficacy and safety of using biweekly cetuximab 500 mg/m(2) with chemotherapy FOLFOX-4 or FOLFIRI as first-line treatment for Chinese patients with K-ras wild-type metastatic colorectal cancer. The study endpoints included overall objective response (OR), progression-free survival (PFS), overall survival (OS) and safety. Total 15 Chinese patients (male: 10 [67%]; median age: 60 [range 41-80]) were enrolled. Patients received median 12 cycles (range 2-12) of chemotherapy + cetuximab (FOLFOX-4 + cetuximab: 9 [60%]; FOLFIRI + cetuximab: 6 [40%]). Six patients (40%) with non-progressive disease after 12 cycles of chemotherapy + cetuximab carried on maintenance cetuximab. Median duration of follow-up (FU) was 23.7 months. The OR was 40% (complete response: 0%; partial response: 40%) for a disease control rate of 87%. Median PFS and OS were 7.8 months and 17.9 months respectively. For maintenance cetuximab phase, median PFS since the start of maintenance cetuximab was 6.8 months and median OS was 17.0 months. The only grade 3-4 toxicities were neutropenia (26.7%) in chemotherapy phase and acneiform rashes (16.7%) in maintenance phase. Biweekly cetuximab with combination chemotherapy was effective and safe as weekly dose. Further studies are warranted for the role of maintenance cetuximab.

  13. Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage.

    PubMed

    García-Beccaria, María; Martínez, Paula; Méndez-Pertuz, Marinela; Martínez, Sonia; Blanco-Aparicio, Carmen; Cañamero, Marta; Mulero, Francisca; Ambrogio, Chiara; Flores, Juana M; Megias, Diego; Barbacid, Mariano; Pastor, Joaquín; Blasco, Maria A

    2015-07-01

    Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.

  14. Binding hotspots on K-Ras: consensus ligand binding sites and other reactive regions from probe-based molecular dynamics analysis

    PubMed Central

    Prakash, Priyanka; Hancock, John F.; Gorfe, Alemayehu A.

    2015-01-01

    We have used probe-based molecular dynamics (pMD) simulations to search for interaction hotspots on the surface of the therapeutically highly relevant oncogenic K-Ras G12D. Combining the probe-based query with an ensemble-based pocket identification scheme and an analysis of existing Ras-ligand complexes, we show that (i) pMD is a robust and cost-effective strategy for binding site identification, (ii) all four of the previously reported ligand binding sites are suitable for structure-based ligand design, and (iii) in some cases probe binding and expanded sampling of configurational space enable pocket expansion and increase the likelihood of site identification. Furthermore, by comparing the distribution of hotspots in non-pocket-like regions with known protein- and membrane-interacting interfaces, we propose that pMD has the potential to predict surface patches responsible for protein-biomolecule interactions. These observations have important implications for future drug design efforts and will facilitate the search for potential interfaces responsible for the proposed transient oligomerization or interaction of Ras with other biomolecules in the cellular milieu. PMID:25740554

  15. Binding hotspots on K-ras: consensus ligand binding sites and other reactive regions from probe-based molecular dynamics analysis.

    PubMed

    Prakash, Priyanka; Hancock, John F; Gorfe, Alemayehu A

    2015-05-01

    We have used probe-based molecular dynamics (pMD) simulations to search for interaction hotspots on the surface of the therapeutically highly relevant oncogenic K-Ras G12D. Combining the probe-based query with an ensemble-based pocket identification scheme and an analysis of existing Ras-ligand complexes, we show that (i) pMD is a robust and cost-effective strategy for binding site identification, (ii) all four of the previously reported ligand binding sites are suitable for structure-based ligand design, and (iii) in some cases probe binding and expanded sampling of configurational space enable pocket expansion and increase the likelihood of site identification. Furthermore, by comparing the distribution of hotspots in nonpocket-like regions with known protein- and membrane-interacting interfaces, we propose that pMD has the potential to predict surface patches responsible for protein-biomolecule interactions. These observations have important implications for future drug design efforts and will facilitate the search for potential interfaces responsible for the proposed transient oligomerization or interaction of Ras with other biomolecules in the cellular milieu.

  16. Chromosomes, cancer and radiosensitivity

    SciTech Connect

    Samouhos, E.

    1983-08-01

    Some specific chromosomal abnormalities are associated with certain cancers. The earliest description of such a specific association is the one of the Philadelphia chromosome and myelogenous leukemia (1960). Other congenital karyotype abnormalities are associated with specific cancers. Examples of these are Down's syndrome with leukemia and Klinefelter's syndrome with male breast cancer. Genetic diseases of increased chromosome breakage, or of defective chromosome repair, are associated with greatly increased cancer incidence. Three such diseases have been recognized: 1) Fanconi's anemia, associated with leukemias and lymphomas, 2) Bloom's syndrome, associated with acute leukemias and lymphosarcoma, and 3) ataxia telangiectasia, associated with Hodgkin's disease, leukemia, and lymphosarcomas. Ten percent of individuals with ataxia telangiectasia will develop one of these neoplasms. Individuals with certain of these syndromes display an unusually high radiosensitivity. Radiation therapy for cancers has been fatal in patients who received as low as 3000 rad. This remarkable radiosensitivity has been quantitated in cell cultures from such cases. Evidence suggests that the apparent sensitivity may reflect subnormal ability to repair radiation damage. The rapid proliferation of information in this field stems from the interdigitation of many disciplines and specialties, including cytogenetics, cell biology, molecular biology, epidemiology, radiobiology, and several others. This paper is intended for clinicians; it presents a structured analytic scheme for correlating and classifying this multidisciplinary information as it becomes available.

  17. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

    EPA Science Inventory

    Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

    Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
    <...

  18. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS

    EPA Science Inventory

    Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis

    Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
    <...

  19. Radiosensitive Severe Combined Immunodeficiency Disease

    PubMed Central

    Dvorak, Christopher C.; Cowan, Morton J.

    2009-01-01

    Synopsis Inherited defects in components of the non-homologous end joining DNA repair mechanism produce a T-B-NK+ severe combined immunodeficiency disease (SCID) characterized by heightened sensitivity to ionizing radiation. Patients with the radiosensitive form of SCID may also have increased short- and long-term sensitivity to the alkylator-based chemotherapy regimens traditionally utilized for conditioning prior to allogeneic hematopoietic cell transplantation (HCT). Known etiologies of radiosensitive SCID include deficiencies of Artemis, DNA Ligase IV, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and Cernunnos-XLF, all of which have been treated with HCT. Because of their sensitivity to certain forms of chemotherapy, the approach to donor selection and type of conditioning regimen utilized for a radiosensitive SCID patient requires careful consideration. Significantly more research needs to be done in order to determine the long-term outcomes of radiosensitive SCID patients following HCT, as well as to discover novel non-toxic approaches to HCT that might benefit those with intrinsic radio- and chemo-sensitivity, as well as potentially all patients undergoing an HCT. PMID:20113890

  20. The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

    SciTech Connect

    Kim, Han-Soo; Kim, Ju Won; Gang, Jingu; Wen, Jing; Koh, Sang Seok; Koh, Jong Sung; Chung, Hyun-Ho; Song, Si Young . E-mail: gisong@yumc.yonsei.ac.kr

    2006-09-15

    LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain the differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.

  1. A murine model of K-ras and β-catenin induced renal tumors express high levels of E2F1 and resemble human Wilms Tumor

    PubMed Central

    Yi, Yajun; Polosukhina, Dina; Love, Harold D.; Hembd, Austin; Pickup, Michael; Moses, Harold L.; Lovvorn, Harold N.; Zent, Roy; Clark, Peter E.

    2016-01-01

    Background Wilms tumor (WT) is the most common renal neoplasm of childhood. We previously showed that restricted activation of the WNT/β-catenin pathway in renal epithelium late in kidney development is sufficient to induce small primitive neoplasms with features of epithelial WT. Metastatic disease progression required simultaneous addition of an activating mutation of the oncogene K-RAS. Here, we sought to define the molecular pathways activated in this process and their relationship to human renal malignancies. Methods Affymetrix expression microarray data from murine kidneys with activation of K-ras, Ctnnb1 (β-catenin), or both restricted to renal epithelium were analyzed and compared to publically available expression data from normal and neoplastic human renal tissue. Target genes were verified by immunoblot and immunohistochemistry. Results Mouse kidney tumors with activation of K-ras and Ctnnb1 and human renal malignancies have similar mRNA expression signatures and are associated with activation of networks centered on β-catenin and TP53. Up-regulation of WNT/β-catenin targets (MYC, Survivin, FOXA2, Axin2, Cyclin D1) was confirmed by immunoblotting. K-RAS/β-catenin murine kidney tumors were more similar to human WT than other renal malignancies and demonstrated activation of a TP53 dependent network of genes including the transcription factor E2F1. Up-regulation of E2F1 was confirmed in both murine and human WT samples. Conclusions Simultaneous activation of K-RAS and β-catenin in embryonic renal epithelium leads to neoplasms similar to human WT associated with activation of TP53 and up-regulation of E2F1. Further studies to evaluate the role of TP53 and E2F1 in human WT are warranted. PMID:25934441

  2. The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status.

    PubMed

    Ii, Masanori; Li, Hua; Adachi, Yasushi; Yamamoto, Hiroyuki; Ohashi, Hirokazu; Taniguchi, Hiroaki; Arimura, Yoshiaki; Carbone, David P; Imai, Kohzoh; Shinomura, Yasuhisa

    2011-08-01

    Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal cancers. We have previously shown successful targeting therapy for colorectal, pancreatic, gastric, and esophageal carcinomas using recombinant adenoviruses expressing dominant negative IGF-IR. Mutation in k-ras is one of key factors in gastrointestinal cancers. In this study, we sought to evaluate the effect of a new monoclonal antibody for IGF-IR, figitumumab (CP-751,871), on the progression of human gastrointestinal carcinomas with/without k-ras mutation. We assessed the effect of figitumumab on signal transduction, proliferation, and survival in six gastrointestinal cancer cell lines with/without k-ras mutation, including colorectal and pancreatic adenocarcinoma, esophageal squamous cell carcinoma, and hepatoma. Combination effects of figitumumab and chemotherapy were also studied. Then figitumumab was evaluated in the treatment of xenografts in nude mice. Figitumumab blocked autophosphorylation of IGF-IR and its downstream signals. The antibody suppressed proliferation and tumorigenicity in all cell lines. Figitumumab inhibited survival by itself and up-regulated chemotherapy (5-FU and gemcitabine) induced apoptosis. Moreover, the combination of this agent and chemotherapy was effective against tumors in mice. The effect of figitumumab was not influenced by the mutation status of k-ras. Figitumumab reduced expression of IGF-IR but not insulin receptor in these xenografted tumors. The drug did not affect murine body weight or blood concentrations of glucose, insulin, IGF binding protein 3, and growth hormone. IGF-IR might be a good molecular therapeutic target and figitumumab may thus have therapeutic value in human gastrointestinal malignancies even in the presence of k-ras mutations. ©2011 AACR.

  3. K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells

    PubMed Central

    Van Meter, Margaret E. M.; Díaz-Flores, Ernesto; Archard, Joehleen A.; Passegué, Emmanuelle; Irish, Jonathan M.; Kotecha, Nikesh; Nolan, Garry P.

    2007-01-01

    Defining how cancer-associated mutations perturb signaling networks in stem/progenitor populations that are integral to tumor formation and maintenance is a fundamental problem with biologic and clinical implications. Point mutations in RAS genes contribute to many cancers, including myeloid malignancies. We investigated the effects of an oncogenic KrasG12D allele on phosphorylated signaling molecules in primary c-kit+ lin−/low hematopoietic stem/progenitor cells. Comparison of wild-type and KrasG12D c-kit+ lin−/low cells shows that K-RasG12D expression causes hyperproliferation in vivo and results in abnormal levels of phosphorylated STAT5, ERK, and S6 under basal and stimulated conditions. Whereas KrasG12D cells demonstrate hyperactive signaling after exposure to granulocyte-macrophage colony-stimulating factor, we unexpectedly observe a paradoxical attenuation of ERK and S6 phosphorylation in response to stem cell factor. These studies provide direct biochemical evidence that cancer stem/progenitor cells remodel signaling networks in response to oncogenic stress and demonstrate that multi-parameter flow cytometry can be used to monitor the effects of targeted therapeutics in vivo. This strategy has broad implications for defining the architecture of signaling networks in primary cancer cells and for implementing stem cell–targeted interventions. PMID:17192389

  4. Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon.

    PubMed

    Pekow, Joel; Meckel, Katherine; Dougherty, Urszula; Butun, Fatma; Mustafi, Reba; Lim, John; Crofton, Charis; Chen, Xindi; Joseph, Loren; Bissonnette, Marc

    2015-02-01

    The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.

  5. Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon

    PubMed Central

    Meckel, Katherine; Dougherty, Urszula; Butun, Fatma; Mustafi, Reba; Lim, John; Crofton, Charis; Chen, Xindi; Joseph, Loren; Bissonnette, Marc

    2014-01-01

    The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition. PMID:25477374

  6. Daily rhythms of radiosensitivity of animals and several determining causes

    NASA Technical Reports Server (NTRS)

    Druzhinin, Y. P.; Malyutina, T. S.; Seraya, V. M.; Rodina, G. P.; Vatsek, A.; Rakova, A.

    1974-01-01

    Daily rhythms of radiosensitivity in rats and mice were determined by survival rates after acute total radiation at the same dosage at different times of the day. Radiosensitivity differed in animals of different species and varieties. Inbred mice exhibited one or two increases in radiosensitivity during the dark, active period of the day. These effects were attributed to periodic changes in the state of stem hematopoietic cells.

  7. Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-kappaB activation in mouse embryonic stem cells.

    PubMed

    Tan, Mingjia; Zhu, Yueming; Kovacev, Jordan; Zhao, Yongchao; Pan, Zhen-Qiang; Spitz, Douglas R; Sun, Yi

    2010-09-15

    SAG (sensitive to apoptosis gene; also known as RBX2 or ROC2) is a dual-function protein with antioxidant activity when acting alone or E3 ligase activity when complexed with other components of SCF (Skp1, cullins, F-box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene-trapping strategy significantly sensitized mouse embryonic stem (ES) cells to radiation, with a sensitizing enhancement rate of 1.5-1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24h after irradiation as well as enhancement of radiation-induced apoptosis. Furthermore, Sag elimination abrogated IkappaBalpha degradation leading to inhibition of NF-kappaB activation. Further detailed analysis revealed that IkappaBalpha is a direct substrate of SAG-SCF(beta-TrCP) E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NF-kappaB. Copyright 2010 Elsevier Inc. All rights reserved.

  8. Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-κB activation in mouse embryonic stem cells

    PubMed Central

    Tan, Mingjia; Zhu, Yueming; Kovacev, Jordan; Zhao, Yongchao; Pan, Zhen-Qiang; Spitz, Douglas R.; Sun, Yi

    2010-01-01

    SAG (Sensitive to Apoptosis Gene) (also known as RBX2 or ROC2) is a dual function protein with antioxidant activity while acting alone or E3 ligase activity when complexed with other components of SCF (Skp1-cullins-F box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene trapping strategy significantly sensitized mouse embryonic stem cells to radiation with a SER (sensitizing enhancement rate) of 1.5–1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24 h following irradiation as well as enhancement of radiation-induced apoptosis. Furthermore Sag elimination abrogated IκBα degradation leading to inhibition of NFκB activation. Further detailed analysis revealed that I IκBα is a direct substrate of SAG-SCFβTrCP E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NFκB. PMID:20638939

  9. Inhibition of DNA-PKcs enhances radiosensitivity and increases the levels of ATM and ATR in NSCLC cells exposed to carbon ion irradiation

    PubMed Central

    YANG, LINA; LIU, YUANYUAN; SUN, CHAO; YANG, XINRUI; YANG, ZHEN; RAN, JUNTAO; ZHANG, QIUNING; ZHANG, HONG; WANG, XINYU; WANG, XIAOHU

    2015-01-01

    Non-small cell lung cancer (NSCLC) exhibits radioresistance to conventional rays, due to its DNA damage repair systems. NSCLC may potentially be sensitized to radiation treatment by reducing those factors that continuously enhance the repair of damaged DNA. In the present study, normal lung fibroblast MRC-5 and lung cancer A549 cells were treated with NU7026 and CGK733, which are inhibitors of the DNA-dependent protein kinase catalytic subunit (PKcs) and ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively, followed by exposure to X-rays and carbon ion irradiation. The cytotoxic activity, cell survival rate, DNA damage repair ability, cell cycle arrest and apoptosis rate of the treated cells were analyzed with MTT assay, colony formation assay, immunofluorescence and flow cytometry, respectively. The transcription and translation levels of the ATM, ATR and DNA-PKcs genes were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The results indicated that the radiosensitivity and DNA repair ability of A549 cells were reduced, and the percentages of apoptotic cells and those arrested at the G2/M phase of the cell cycle were significantly increased, following ionizing radiation with inhibitor-pretreatment. The expression levels of ATM, ATR, DNA-PKcs and phosphorylated histone H2AX, a biomarker for DNA double-strand breaks, were all upregulated at the transcriptional or translational level in A549 cells treated with carbon ion irradiation, compared with the control and X-rays-treated cells. In addition, the treatment with 5–50 µM NU7026 or CGK733 did not produce any obvious cytotoxicity in MRC-5 cells, and the effect of the DNA-PKcs-inhibitor on enhancing the radiosensitivity of A549 cells was stronger than that observed for the ATM and ATR-inhibitor. These findings demonstrated a minor role for ATM and ATR in radiation-induced cell death, since the upregulation of

  10. Inhibition of DNA-PKcs enhances radiosensitivity and increases the levels of ATM and ATR in NSCLC cells exposed to carbon ion irradiation.

    PubMed

    Yang, Lina; Liu, Yuanyuan; Sun, Chao; Yang, Xinrui; Yang, Zhen; Ran, Juntao; Zhang, Qiuning; Zhang, Hong; Wang, Xinyu; Wang, Xiaohu

    2015-11-01

    Non-small cell lung cancer (NSCLC) exhibits radioresistance to conventional rays, due to its DNA damage repair systems. NSCLC may potentially be sensitized to radiation treatment by reducing those factors that continuously enhance the repair of damaged DNA. In the present study, normal lung fibroblast MRC-5 and lung cancer A549 cells were treated with NU7026 and CGK733, which are inhibitors of the DNA-dependent protein kinase catalytic subunit (PKcs) and ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR), respectively, followed by exposure to X-rays and carbon ion irradiation. The cytotoxic activity, cell survival rate, DNA damage repair ability, cell cycle arrest and apoptosis rate of the treated cells were analyzed with MTT assay, colony formation assay, immunofluorescence and flow cytometry, respectively. The transcription and translation levels of the ATM, ATR and DNA-PKcs genes were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The results indicated that the radiosensitivity and DNA repair ability of A549 cells were reduced, and the percentages of apoptotic cells and those arrested at the G2/M phase of the cell cycle were significantly increased, following ionizing radiation with inhibitor-pretreatment. The expression levels of ATM, ATR, DNA-PKcs and phosphorylated histone H2AX, a biomarker for DNA double-strand breaks, were all upregulated at the transcriptional or translational level in A549 cells treated with carbon ion irradiation, compared with the control and X-rays-treated cells. In addition, the treatment with 5-50 µM NU7026 or CGK733 did not produce any obvious cytotoxicity in MRC-5 cells, and the effect of the DNA-PKcs-inhibitor on enhancing the radiosensitivity of A549 cells was stronger than that observed for the ATM and ATR-inhibitor. These findings demonstrated a minor role for ATM and ATR in radiation-induced cell death, since the upregulation of

  11. Analysis of p53, K-ras gene mutation & Helicobacter pylori infection in patients with gastric cancer & peptic ulcer disease at a tertiary care hospital in north India.

    PubMed

    Saxena, Ashish; Shukla, Sanket Kumar; Prasad, Kashi Nath; Ghoshal, Uday Chand

    2012-10-01

    Mutations in the oncogene and tumour suppressor genes play an important role in carcinogenesis. We investigated the association of p53 and K-ras gene mutation and Helicobacter pylori infection in patients with gastric cancer (GC) and peptic ulcer disease (PUD) attending a tertiary care hospital in north India. In total, 348 adult patients [62 GC, 45 PUD and 241 non-ulcer dyspepsia (NUD)] who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology and PCR. Mutation in the exon 5-8 of p53 gene was analyzed by PCR-single stranded conformational polymorphism (SSCP) and confirmed by sequence analysis. K-ras gene codon 12 mutation was analyzed by PCR-based restriction fragment length polymorphism. Overall p53 gene mutation was found in 4.6 per cent of the study population, and its distribution in GC, PUD and NUD was 21, 4.4 and 0.4 per cent, respectively. p53 gene mutation was significantly higher in patients with GC than PUD (P<0.05) and NUD (P<0.001). No difference in p53 gene mutation was observed between H. pylori infected and non-infected individuals. K-ras gene mutation was absent in all the patients. Our results show that p53 gene mutation may be associated with gastric carcinogenesis independent to H. pylori infection and absence of K-ras gene mutation questions its role in the pathogenesis of GC and PUD in Indian patients.

  12. Analysis of p53, K-ras gene mutation & Helicobacter pylori infection in patients with gastric cancer & peptic ulcer disease at a tertiary care hospital in north India

    PubMed Central

    Saxena, Ashish; Shukla, Sanket Kumar; Prasad, Kashi Nath; Ghoshal, Uday Chand

    2012-01-01

    Background & objectives: Mutations in the oncogene and tumour suppressor genes play an important role in carcinogenesis. We investigated the association of p53 and K-ras gene mutation and Helicobacter pylori infection in patients with gastric cancer (GC) and peptic ulcer disease (PUD) attending a tertiary care hospital in north India. Methods: In total, 348 adult patients [62 GC, 45 PUD and 241 non-ulcer dyspepsia (NUD)] who underwent an upper gastrointestinal endoscopy were enrolled. H. pylori infection was diagnosed by rapid urease test, culture, histopathology and PCR. Mutation in the exon 5-8 of p53 gene was analyzed by PCR-single stranded conformational polymorphism (SSCP) and confirmed by sequence analysis. K-ras gene codon 12 mutation was analyzed by PCR-based restriction fragment length polymorphism. Results: Overall p53 gene mutation was found in 4.6 per cent of the study population, and its distribution in GC, PUD and NUD was 21, 4.4 and 0.4 per cent, respectively. p53 gene mutation was significantly higher in patients with GC than PUD (P<0.05) and NUD (P<0.001). No difference in p53 gene mutation was observed between H. pylori infected and non-infected individuals. K-ras gene mutation was absent in all the patients. Interpretation & conclusions: Our results show that p53 gene mutation may be associated with gastric carcinogenesis independent to H. pylori infection and absence of K-ras gene mutation questions its role in the pathogenesis of GC and PUD in Indian patients. PMID:23168708

  13. Identification of predictive markers of response to the MEK1/2 inhibitor selumetinib (AZD6244) in K-ras-mutated colorectal cancer.

    PubMed

    Tentler, John J; Nallapareddy, Sujatha; Tan, Aik Choon; Spreafico, Anna; Pitts, Todd M; Morelli, M Pia; Selby, Heather M; Kachaeva, Maria I; Flanigan, Sara A; Kulikowski, Gillian N; Leong, Stephen; Arcaroli, John J; Messersmith, Wells A; Eckhardt, S Gail

    2010-12-01

    Mutant K-ras activity leads to the activation of the RAS/RAF/MEK/ERK pathway in approximately 44% of colorectal cancer (CRC) tumors. Accordingly, several inhibitors of the MEK pathway are under clinical evaluation in several malignancies including CRC. The aim of this study was to develop and characterize predictive biomarkers of response to the MEK1/2 inhibitor AZD6244 in CRC in order to maximize the clinical utility of this agent. Twenty-seven human CRC cell lines were exposed to AZD6244 and classified according to the IC(50) value as sensitive (≤ 0.1 μmol/L) or resistant (>1 μmol/L). All cell lines were subjected to immunoblotting for effector proteins, K-ras/BRAF mutation status, and baseline gene array analysis. Further testing was done in cell line xenografts and K-ras mutant CRC human explants models to develop a predictive genomic classifier for AZD6244. The most sensitive and resistant cell lines were subjected to differential gene array and pathway analyses. Members of the Wnt signaling pathway were highly overexpressed in cell lines resistant to AZD6244 and seem to be functionally involved in mediating resistance by shRNA knockdown studies. Baseline gene array data from CRC cell lines and xenografts were used to develop a k-top scoring pair (k-TSP) classifier, which predicted with 71% accuracy which of a test set of patient-derived K-ras mutant CRC explants would respond to AZD6244, providing the basis for a patient-selective clinical trial. These results also indicate that resistance to AZD6244 may be mediated, in part, by the upregulation of the Wnt pathway, suggesting potential rational combination partners for AZD6244 in CRC.

  14. K-Ras resides on the Arf6-mediated CIE system and its active type interacted with Arf6T27N.

    PubMed

    Xie, Chuan-gao; Wei, Shu-mei; Cai, Jian-ting

    2012-02-01

    Ras is known as an oncogene transferring signals from the plasma membrane. Recent studies have demonstrated that plasma membrane was not the unique platform for Ras signaling. Ras could also be endocytosed and transported to different endomembrane compartments, evoking different signal pathways there. It is of great significance to exploit the unique intracellular trafficking features of different Ras isoforms to develop new anti-Ras drugs. ADP-ribosylation factor 6 (Arf6) was known to mediate one of the clathrin-independent endocytosis (CIE) pathways. The role of Arf6 in K-Ras dynamic remains largely unknown. In this study, we showed that K-RasG12V co-localized with Arf6 at the plasma membrane, and entered the tubular endosomes or protrusions induced by cytochalasin D or aluminum fluoride in the same way as H-RasG12V does. A subcellular fractionation experiment demonstrated that Arf6 siRNA treatment reduced the plasma membrane presence of both endogenous Ras isoforms and inhibited the phosphorylation of Erk triggered by EGF. When co-expressed with Arf6Q67L, both isoforms were sequestered into the large phosphatidylinositol 4,5-biphosphate [PI(4,5)P2]-enriched vacuoles. However, when co-expressed with Arf6T27N, K-RasG12V co-localized with Arf6T27N at the tubular endosomes significantly than H-RasG12V. Immunoprecipitation and GST fusion protein pull-down studies found out for the first time that K-RasG12V interacted with Arf6T27N. Swapping mutation study showed that the above difference was due to different C-termini. Our study indicated that Arf6 was involved in the dynamic regulation of both Ras isoforms. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Targeting the K-Ras/PDEδ protein-protein interaction: the solution for Ras-driven cancers or just another therapeutic mirage?

    PubMed

    Frett, Brendan; Wang, Yuanxiang; Li, Hong-Yu

    2013-10-01

    The holy grail, finally? After years of unsuccessful attempts at drugging the Ras oncogene, a recent paper by Zimmerman et al. has revealed the possibility of inhibiting Ras signaling on a clinically relevant level by blocking the K-Ras/PDEδ protein-protein interaction. The results, reported in Nature, are highlighted herein with future implications and directions to evaluate the full clinical potential of this research. Copyright © 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Activation and increase of radio-sensitive CD11b+ recruited Kupffer cells/macrophages in diet-induced steatohepatitis in FGF5 deficient mice

    PubMed Central

    Nakashima, Hiroyuki; Nakashima, Masahiro; Kinoshita, Manabu; Ikarashi, Masami; Miyazaki, Hiromi; Hanaka, Hiromi; Imaki, Junko; Seki, Shuhji

    2016-01-01

    We have recently reported that Kupffer cells consist of two subsets, radio-resistant resident CD68+ Kupffer cells and radio-sensitive recruited CD11b+ Kupffer cells/macrophages (Mφs). Non-alcoholic steatohepatitis (NASH) is characterized not only by hepatic steatosis but also chronic inflammation and fibrosis. In the present study, we investigated the immunological mechanism of diet-induced steatohepatitis in fibroblast growth factor 5 (FGF5) deficient mice. After consumption of a high fat diet (HFD) for 8 weeks, FGF5 null mice developed severe steatohepatitis and fibrosis resembling human NASH. F4/80+ Mφs which were both CD11b and CD68 positive accumulated in the liver. The production of TNF and FasL indicated that they are the pivotal effectors in this hepatitis. The weak phagocytic activity and lack of CRIg mRNA suggested that they were recruited Mφs. Intermittent exposure to 1 Gy irradiation markedly decreased these Mφs and dramatically inhibited liver inflammation without attenuating steatosis. However, depletion of the resident subset by clodronate liposome (c-lipo) treatment increased the Mφs and tended to exacerbate disease progression. Recruited CD11b+ CD68+ Kupffer cells/Mφs may play an essential role in steatohepatitis and fibrosis in FGF5 null mice fed with a HFD. Recruitment and activation of bone marrow derived Mφs is the key factor to develop steatohepatitis from simple steatosis. PMID:27708340

  17. K-ras oncogene DNA sequences in pink salmon in streams impacted by the Exxon Valdez oil spill: no evidence of oil-induced heritable mutations.

    PubMed

    Cronin, Matthew A; Wickliffe, Jeffrey K; Dunina, Yelena; Baker, Robert J

    2002-08-01

    It was hypothesized in previous studies that the Exxon Valdez oil spill in Prince William Sound, Alaska, induced heritable mutations and resulted in mortality of pink salmon (Oncorhynchus gorbuscha) embryos. In one of these studies, laboratory exposure of pink salmon embryos to crude oil resulted in apparent mutation-induction in exon 1 and exon 2 of the K-ras oncogene, but no fish from the area impacted by the oil spill were analyzed. We assessed K-ras exon 1 and exon 2 DNA sequences in pink salmon from five streams that were oiled and five streams that were not oiled by the Exxon Valdez oil spill in Prince William Sound, and two streams with natural oil seeps and one stream without seeps on the Alaska Peninsula. Of the 79 fish analyzed for exon 1 and the 89 fish analyzed for exon 2, none had the nucleotide substitutions representing the mutations induced in the laboratory study. Other variable nucleotides occurred in similar proportions in oiled and non-oiled streams and probably represent natural allelic variation. These data do not support the hypothesis that heritable mutations in the K-ras gene were induced by the Exxon Valdez oil spill or oil seeps.

  18. Oncogenic K-ras expression is associated with derangement of the cAMP/PKA pathway and forskolin-reversible alterations of mitochondrial dynamics and respiration.

    PubMed

    Palorini, R; De Rasmo, D; Gaviraghi, M; Sala Danna, L; Signorile, A; Cirulli, C; Chiaradonna, F; Alberghina, L; Papa, S

    2013-01-17

    The Warburg effect in cancer cells has been proposed to involve several mechanisms, including adaptation to hypoxia, oncogenes activation or loss of oncosuppressors and impaired mitochondrial function. In previous papers, it has been shown that K-ras transformed mouse cells are much more sensitive as compared with normal cells to glucose withdrawal (undergoing apoptosis) and present a high glycolytic rate and a strong reduction of mitochondrial complex I. Recent observations suggest that transformed cells have a derangement in the cyclic adenosine monophosphate/cAMP-dependent protein kinase (cAMP/PKA) pathway, which is known to regulate several mitochondrial functions. Herein, the derangement of the cAMP/PKA pathway and its impact on transformation-linked changes of mitochondrial functions is investigated. Exogenous stimulation of PKA activity, achieved by forskolin treatment, protected K-ras-transformed cells from apoptosis induced by glucose deprivation, enhanced complex I activity, intracellular adenosine triphosphate (ATP) levels, mitochondrial fusion and decreased intracellular reactive oxygen species (ROS) levels. Several of these effects were almost completely prevented by inhibiting the PKA activity. Short-time treatment with compounds favoring mitochondrial fusion strongly decreased the cellular ROS levels especially in transformed cells. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial complex I decrease, reduction of ATP formation, prevalence of mitochondrial fission over fusion, and thereby opening new approaches for development of anticancer drugs.

  19. High frequency of codon 61 K-ras A-->T transversions in lung and Harderian gland neoplasms of B6C3F1 mice exposed to chloroprene (2-chloro-1,3-butadiene) for 2 years, and comparisons with the structurally related chemicals isoprene and 1,3-butadiene.

    PubMed

    Sills, R C; Hong, H L; Melnick, R L; Boorman, G A; Devereux, T R

    1999-04-01

    Chloroprene is the 2-chloro analog of 1,3-butadiene, a potent carcinogen in laboratory animals. Following 2 years of inhalation exposure to 12.8, 32 or 80 p.p.m. chloroprene, increased incidences of lung and Harderian gland (HG) neoplasms were observed in B6C3F1 mice at all exposure concentrations. The present study was designed to characterize genetic alterations in the K- and H-ras proto-oncogenes in chloroprene-induced lung and HG neoplasms. K-ras mutations were detected in 80% of chloroprene-induced lung neoplasms (37/46) compared with only 30% in spontaneous lung neoplasms (25/82). Both K- and H-ras codon 61 A-->T transversions were identified in 100% of HG neoplasms (27/27) compared with a frequency of 56% (15/27) in spontaneous HG neoplasms. The predominant mutation in chloroprene-induced lung and HG neoplasms was an A-->T transversion at K-ras codon 61. This mutation has not been detected in spontaneous lung tumors of B6C3F1 mice and was identified in only 7% of spontaneous HG neoplasms. In lung neoplasms, greater percentages (80 and 71%) of A-->T transversions were observed at the lower exposures (12.8 and 32 p.p.m.), respectively, compared with 18% at the high exposure. In HG neoplasms, the percentage of A-->T transversions was the same at all exposure concentrations. The chloroprene-induced ras mutation spectra was similar to that seen with isoprene, where the predominant base change was an A-->T transversion at K-ras codon 61. This differed from 1,3-butadiene, where K-ras codon 13 G-->C transitions and H-ras codon 61 A-->G transitions were the predominant mutations. The major finding of K-ras A-->T transversions in lung and Harderian gland neoplasms suggests that this mutation may be important for tumor induction by this class of carcinogens.

  20. Extracting the normal lung dose-response curve from clinical DVH data: a possible role for low dose hyper-radiosensitivity, increased radioresistance

    NASA Astrophysics Data System (ADS)

    Gordon, J. J.; Snyder, K.; Zhong, H.; Barton, K.; Sun, Z.; Chetty, I. J.; Matuszak, M.; Ten Haken, R. K.

    2015-09-01

    In conventionally fractionated radiation therapy for lung cancer, radiation pneumonitis’ (RP) dependence on the normal lung dose-volume histogram (DVH) is not well understood. Complication models alternatively make RP a function of a summary statistic, such as mean lung dose (MLD). This work searches over damage profiles, which quantify sub-volume damage as a function of dose. Profiles that achieve best RP predictive accuracy on a clinical dataset are hypothesized to approximate DVH dependence. Step function damage rate profiles R(D) are generated, having discrete steps at several dose points. A range of profiles is sampled by varying the step heights and dose point locations. Normal lung damage is the integral of R(D) with the cumulative DVH. Each profile is used in conjunction with a damage cutoff to predict grade 2 plus (G2+) RP for DVHs from a University of Michigan clinical trial dataset consisting of 89 CFRT patients, of which 17 were diagnosed with G2+ RP. Optimal profiles achieve a modest increase in predictive accuracy—erroneous RP predictions are reduced from 11 (using MLD) to 8. A novel result is that optimal profiles have a similar distinctive shape: enhanced damage contribution from low doses (<20 Gy), a flat contribution from doses in the range ~20-40 Gy, then a further enhanced contribution from doses above 40 Gy. These features resemble the hyper-radiosensitivity / increased radioresistance (HRS/IRR) observed in some cell survival curves, which can be modeled using Joiner’s induced repair model. A novel search strategy is employed, which has the potential to estimate RP dependence on the normal lung DVH. When applied to a clinical dataset, identified profiles share a characteristic shape, which resembles HRS/IRR. This suggests that normal lung may have enhanced sensitivity to low doses, and that this sensitivity can affect RP risk.

  1. Extracting the normal lung dose-response curve from clinical DVH data: a possible role for low dose hyper-radiosensitivity, increased radioresistance.

    PubMed

    Gordon, J J; Snyder, K; Zhong, H; Barton, K; Sun, Z; Chetty, I J; Matuszak, M; Ten Haken, R K

    2015-09-07

    In conventionally fractionated radiation therapy for lung cancer, radiation pneumonitis' (RP) dependence on the normal lung dose-volume histogram (DVH) is not well understood. Complication models alternatively make RP a function of a summary statistic, such as mean lung dose (MLD). This work searches over damage profiles, which quantify sub-volume damage as a function of dose. Profiles that achieve best RP predictive accuracy on a clinical dataset are hypothesized to approximate DVH dependence.Step function damage rate profiles R(D) are generated, having discrete steps at several dose points. A range of profiles is sampled by varying the step heights and dose point locations. Normal lung damage is the integral of R(D) with the cumulative DVH. Each profile is used in conjunction with a damage cutoff to predict grade 2 plus (G2+) RP for DVHs from a University of Michigan clinical trial dataset consisting of 89 CFRT patients, of which 17 were diagnosed with G2+ RP.Optimal profiles achieve a modest increase in predictive accuracy--erroneous RP predictions are reduced from 11 (using MLD) to 8. A novel result is that optimal profiles have a similar distinctive shape: enhanced damage contribution from low doses (<20 Gy), a flat contribution from doses in the range ~20-40 Gy, then a further enhanced contribution from doses above 40 Gy. These features resemble the hyper-radiosensitivity / increased radioresistance (HRS/IRR) observed in some cell survival curves, which can be modeled using Joiner's induced repair model.A novel search strategy is employed, which has the potential to estimate RP dependence on the normal lung DVH. When applied to a clinical dataset, identified profiles share a characteristic shape, which resembles HRS/IRR. This suggests that normal lung may have enhanced sensitivity to low doses, and that this sensitivity can affect RP risk.

  2. Differential Expression of IL-17, 22 and 23 in the Progression of Colorectal Cancer in Patients with K-ras Mutation: Ras Signal Inhibition and Crosstalk with GM-CSF and IFN-γ

    PubMed Central

    Petanidis, Savvas; Anestakis, Doxakis; Argyraki, Maria; Hadzopoulou-Cladaras, Margarita; Salifoglou, Athanasios

    2013-01-01

    Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future. PMID:24040001

  3. Transition in Survival From Low-Dose Hyper-Radiosensitivity to Increased Radioresistance Is Independent of Activation of ATM SER1981 Activity

    SciTech Connect

    Krueger, Sarah A.; Collis, Spencer J.; Joiner, Michael C.; Wilson, George D.; Marples, Brian

    2007-11-15

    Purpose: The molecular basis of low-dose hyper-radiosensitivity (HRS) is only partially understood. The aim of this study was to define the roles of ataxia telangiectasia mutated (ATM) activity and the downstream ATM-dependent G{sub 2}-phase cell cycle checkpoint in overcoming HRS and triggering radiation resistance. Methods and Materials: Survival was measured using a high-resolution clonogenic assay. ATM Ser1981 activation was measured by Western blotting. The role of ATM was determined in survival experiments after molecular (siRNA) and chemical (0.4 mM caffeine) inhibition and chemical (20 {mu}g/mL chloroquine, 15 {mu}M genistein) activation 4-6 h before irradiation. Checkpoint responsiveness was assessed in eight cell lines of differing HRS status using flow cytometry to quantify the progression of irradiated (0-2 Gy) G{sub 2}-phase cells entering mitosis, using histone H3 phosphorylation analysis. Results: The dose-response pattern of ATM activation was concordant with the transition from HRS to radioresistance. However, ATM activation did not play a primary role in initiating increased radioresistance. Rather, a relationship was discovered between the function of the downstream ATM-dependent early G{sub 2}-phase checkpoint and the prevalence and overcoming of HRS. Four cell lines that exhibited HRS failed to show low-dose (<0.3-Gy) checkpoint function. In contrast, four HRS-negative cell lines exhibited immediate cell cycle arrest for the entire 0-2-Gy dose range. Conclusion: Overcoming HRS is reliant on the function of the early G{sub 2}-phase checkpoint. These data suggest that clinical exploitation of HRS could be achieved by combining radiotherapy with chemotherapeutic agents that modulate this cell cycle checkpoint.

  4. Inhibition of LDH-A by oxamate induces G2/M arrest, apoptosis and increases radiosensitivity in nasopharyngeal carcinoma cells.

    PubMed

    Zhai, Xiaoming; Yang, Yang; Wan, Jianmei; Zhu, Ran; Wu, Yiwei

    2013-12-01

    An elevated rate of glucose consumption and the dependency on aerobic glycolysis for ATP generation have long been observed in cancer cells, a phenomenon known as the Warburg effect. the altered energy metabolism in cancer cells provides an attractive opportunity for developing novel cancer therapeutic strategies. Lactate dehydrogenase (LDH), which catalyzes the transformation of pyruvate to lactate, plays a vital role in the process of glycolysis. It has been reported that the level of LDH-A expression is increased both in head and neck cancer cells and in the blood serum of nasopharyngeal carcinoma (NPC) patients, and is associated with poor prognosis. However, the effect of LDH-A inhibition on NPC cells remains unknown. Here, in the present study, we found that oxamate, a classical inhibitor of LDH-A, suppressed cell proliferation in a dose- and time-dependent manner both in CNE-1 and CNE-2 cells, two NPC cancer cell lines. LDH inhibition by oxamate induced G2/M cell cycle arrest via downregulation of the CDK1/cyclin B1 pathway and promoted apoptosis through enhancement of mitochondrial ROS generation. N-acetylcysteine, a specific scavenger of ROS, significantly blocked the growth inhibition effect induced by oxamate. We also identified that oxamate increased sensitivity to ionizing radiation in the two NPC cancer cell lines. Furthermore, we verified similar results in tumor xenograft models. collectively, these results suggest that LDH-A may serve as a promising therapeutic target for NPC treatment.

  5. High dietary iron increases oxidative stress and radiosensitivity in the rat retina and vasculature after exposure to fractionated gamma radiation

    PubMed Central

    Theriot, Corey A; Westby, Christian M; Morgan, Jennifer L L; Zwart, Sara R; Zanello, Susana B

    2016-01-01

    Radiation exposure in combination with other space environmental factors including microgravity, nutritional status, and deconditioning is a concern for long-duration space exploration missions. Astronauts experience altered iron homeostasis due to adaptations to microgravity and an iron-rich food system. Iron intake reaches three to six times the recommended daily allowance due to the use of fortified foods on the International Space Station. Iron is associated with certain optic neuropathies and can potentiate oxidative stress. This study examined the response of eye and vascular tissue to gamma radiation exposure (3 Gy fractionated at 37.5 cGy per day every other day for 8 fractions) in rats fed an adequate-iron diet or a high-iron diet. Twelve-week-old Sprague-Dawley rats were assigned to one of four experimental groups: adequate-iron diet/no radiation (CON), high-iron diet/no radiation (IRON), adequate-iron diet/radiation (RAD), and high-iron diet/radiation (IRON+RAD). Animals were maintained on the corresponding iron diet for 2 weeks before radiation exposure. As previously published, the high-iron diet resulted in elevated blood and liver iron levels. Dietary iron overload altered the radiation response observed in serum analytes, as evidenced by a significant increase in catalase levels and smaller decrease in glutathione peroxidase and total antioxidant capacity levels. 8-OHdG immunostaining, showed increased intensity in the retina after radiation exposure. Gene expression profiles of retinal and aortic vascular samples suggested an interaction between the response to radiation and high dietary iron. This study suggests that the combination of gamma radiation and high dietary iron has deleterious effects on retinal and vascular health and physiology. PMID:28725729

  6. Phenotype of bone metastases of non-small cell lung cancer: epidermal growth factor receptor expression and K-RAS mutational status.

    PubMed

    Badalian, Gayane; Barbai, Tamás; Rásó, Erzsébet; Derecskei, Katalin; Szendrôi, Miklós; Tímár, József

    2007-01-01

    Bone metastasis is a frequent complication of lung cancer progression, however, studies on bone metastatic tissues are scanty. Here we have collected a small cohort of 11 non-small cell lung cancer cases where primary tumors and corresponding bone metastases were available for pathological analysis. We have tested two molecular markers: EGFR protein expression and K-RAS mutation at codon 12 using immunohistochemistry and RFLPPCR, respectively. We have shown that using improved protocols, EGFR protein (both the extracellular as well as the cytoplasmic domain) is readily detectable in decalcified bone tissue. We found that the EGFR expression status is highly similar in bone metastases compared to the primary tumors, although the expression levels may change. Individual comparison of corresponding primary and metastatic NSCLC tissues indicated that downregulation of EGFR was a rare event (2/11) compared to upregulation (4/11) in bone metastases. On the other hand, our data indicate that the K-RAS mutational status of the primary tumor does not predict the status of the bone metastatic tissue of NSCLC, since we have observed both emergence of mutant clones in metastases from wild-type (wt) primary tumors and loss of mutant clones in metastases from mutant primaries in addition to the maintained mutant status. Our data support that at least two progression models occur in NSCLC, the samegene as well as the clonal selection one. It is noteworthy that in NSCLC cases with wt- or mutant KRAS, downregulation of EGFR expression was a rare event although upregulation in bone metastases was observed more frequently in wt K-RAS cases.

  7. p53, erbB-2 and K-ras gene alterations are rare in spontaneous and plutonium-239-induced canine lung neoplasia

    SciTech Connect

    Tierney, L.A.; Hahn, F.F.; Lechner, J.F.

    1996-02-01

    Inhalation of high-linear energy transfer radiation in the form of radon progeny is a suspected cause of human lung cancer. To gain insight into the types of genetic derangements caused by this type of radiation, lung tumors from beagle dogs exposed to {sup 239}PuO{sub 2} and those arising in animals with no known carcinogen exposure were examined for evidence of aberrations in genes known to be altered in lung tumors. Altered expression of the p53 tumor suppressor gene and proto-oncogene erbB-2 proteins (p185{sup erbB2}) was evaluated by immunohistochemical analysis of 117 tumors representing different histological types in exposed (n = 80) and unexposed (n = 37) animals. Twenty-eight tumors were analyzed for K-ras proto-oncogene mutations by polymerase chain reaction amplification and direct sequencing. Fourteen percent (16/116) of all lung neoplasms showed elevated nuclear accumulation of p53 protein. Regardless of exposure history, adenosquamous and squamous cell cancers comprised 94% of all tumors with p53 abnormalities. Eighteen percent (21/117) of all tumors had evidence of erbB-2 protein overexpression. K-ras mutations were not detected in codons 12, 13 or 61 of tumors from unexposed (n = 9) or plutonium-exposed dogs (n = 19). These data indicate that p53 and K-ras gene abnormalities as a result of missense mutation are infrequent events in spontaneous and {sup 239}PuO{sub 2}-induced lung neoplasia in this colony of beagle dogs. Alternative mechanisms of gene alteration may be involved in canine pulmonary carcinogenesis. 45 refs., 3 figs., 2 tabs.

  8. A recombinantly tailored β-defensin that displays intensive macropinocytosis-mediated uptake exerting potent efficacy against K-Ras mutant pancreatic cancer

    PubMed Central

    Du, Yue; Shang, Bo-yang; Sheng, Wei-jin; Zhang, Sheng-hua; Li, Yi; Miao, Qing-fang; Zhen, Yong-su

    2016-01-01

    K-Ras mutant pancreatic cancer cells display intensive macropinocytosis, indicating that this process may be exploited in the design of anticancer targeted therapies. In this study, we constructed a macropinocytosis-oriented recombinantly tailored defensin (DF-HSA) which consists of human β-defensin-2 (DF) and human serum albumin (HSA). The macropinocytosis intensity and cytotoxicity of DF-HSA were investigated in K-Ras mutant MIA PaCa-2 cells and wild-type BxPC-3 cells. As found, the DF-HSA uptake in MIA PaCa-2 cells was much higher than that in wild-type BxPC-3 cells. Correspondingly, the cytotoxicity of DF-HSA to MIA PaCa-2 cells was more potent than that to BxPC-3 cells. In addition, the cytotoxicity of DF-HSA was much stronger than that of β-defensin HBD2. DF-HSA suppressed cancer cell proliferation and induced mitochondrial pathway apoptosis. Notably, DF-HSA significantly inhibited the growth of human pancreatic carcinoma MIA PaCa-2 xenograft in athymic mice at well tolerated dose. By in vivo imaging, DF-HSA displayed a prominent accumulation in the tumor. The study indicates that the recombinantly tailored β-defensin can intensively enter into the K-Ras mutant pancreatic cancer cells through macropinocytosis-mediated process and exert potent therapeutic efficacy against the pancreatic carcinoma xenograft. The novel format of β-defensin may play an active role in macropinocytosis-mediated targeting therapy. PMID:27517152

  9. Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.

    PubMed

    Ward, R; Hawkins, N; O'Grady, R; Sheehan, C; O'Connor, T; Impey, H; Roberts, N; Fuery, C; Todd, A

    1998-08-01

    The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

  10. On the radiosensitivity of man in space

    NASA Astrophysics Data System (ADS)

    Esposito, R. D.; Durante, M.; Gialanella, G.; Grossi, G.; Pugliese, M.; Scampoli, P.; Jones, T. D.

    Astronauts' radiation exposure limits are based on experimental and epidemiological data obtained on Earth. It is assumed that radiation sensitivity remains the same in the extraterrestrial space. However, human radiosensitivity is dependent upon the response of the hematopoietic tissue to the radiation insult. It is well known that the immune system is affected by microgravity. We have developed a mathematical model of radiation-induced myelopoiesis which includes the effect of microgravity on bone marrow kinetics. It is assumed that cellular radiosensitivity is not modified by the space environment, but repopulation rates of stem and stromal cells are reduced as a function of time in weightlessness. A realistic model of the space radiation environment, including the HZE component, is used to simulate the radiation damage. A dedicated computer code was written and applied to solar particle events and to the mission to Mars. The results suggest that altered myelopoiesis and lymphopoiesis in microgravity might increase human radiosensitivity in space.

  11. Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer.

    PubMed

    Du, Juan; Cieslak, John A; Welsh, Jessemae L; Sibenaller, Zita A; Allen, Bryan G; Wagner, Brett A; Kalen, Amanda L; Doskey, Claire M; Strother, Robert K; Button, Anna M; Mott, Sarah L; Smith, Brian; Tsai, Susan; Mezhir, James; Goswami, Prabhat C; Spitz, Douglas R; Buettner, Garry R; Cullen, Joseph J

    2015-08-15

    The toxicity of pharmacologic ascorbate is mediated by the generation of H2O2 via the oxidation of ascorbate. Because pancreatic cancer cells are sensitive to H2O2 generated by ascorbate, they would also be expected to become sensitized to agents that increase oxidative damage such as ionizing radiation. The current study demonstrates that pharmacologic ascorbate enhances the cytotoxic effects of ionizing radiation as seen by decreased cell viability and clonogenic survival in all pancreatic cancer cell lines examined, but not in nontumorigenic pancreatic ductal epithelial cells. Ascorbate radiosensitization was associated with an increase in oxidative stress-induced DNA damage, which was reversed by catalase. In mice with established heterotopic and orthotopic pancreatic tumor xenografts, pharmacologic ascorbate combined with ionizing radiation decreased tumor growth and increased survival, without damaging the gastrointestinal tract or increasing systemic changes in parameters indicative of oxidative stress. Our results demonstrate the potential clinical utility of pharmacologic ascorbate as a radiosensitizer in the treatment of pancreatic cancer.

  12. Sulforaphane, quercetin and catechins complement each other in elimination of advanced pancreatic cancer by miR-let-7 induction and K-ras inhibition

    PubMed Central

    APPARI, MAHESH; BABU, KAMESH R.; KACZOROWSKI, ADAM; GROSS, WOLFGANG; HERR, INGRID

    2014-01-01

    Pancreatic ductal adenocarcinoma (PDA) has the worst prognosis of all malignancies, and current therapeutic options do not target cancer stem cells (CSCs), which may be the reason for the extreme aggressiveness. The dietary agents sulforaphane and quercetin enriched e.g., in broccoli, and the main and best studied green tea catechin EGCG hold promise as anti-CSC agents in PDA. We examined the efficacy of additional catechins and the combination of these bioactive agents to stem cell features and miRNA signaling. Two established and one primary PDA cell line and non-malignant pancreatic ductal cells were used. Whereas each agent strongly inhibited colony formation, the catechins ECG and CG were more effective than EGCG. A mixture of green tea catechins (GTCs) significantly inhibited viability, migration, expression of MMP-2 and -9, ALDH1 activity, colony and spheroid formation and induced apoptosis, but the combination of GTCs with sulforaphane or quercetin was superior. Following treatment with bioactive agents, the expression of miR-let7-a was specifically induced in cancer cells but not in normal cells and it was associated with K-ras inhibition. These data demonstrate that sulforaphane, quercetin and GTC complement each other in inhibition of PDA progression by induction of miR-let7-a and inhibition of K-ras. PMID:25017900

  13. K-Ras, H-Ras, N-Ras and B-Raf mutation and expression analysis in Wilms tumors: association with tumor growth.

    PubMed

    Dalpa, Efterpi; Gourvas, Victor; Soulitzis, Nikolaos; Spandidos, Demetrios A

    2017-01-01

    Nephroblastoma (Wilms tumor) is a kidney neoplasia, predominately occurring at very young age, resulting from the malignant transformation of renal stem cells. The Ras proto-oncogenes and B-Raf are members of an intracellular cascade pathway, which regulates cell growth and differentiation, and ultimately cancer development. Our objective was to determine the mutation rate and to measure the mRNA levels of the three Ras genes and of B-Raf in formalin-fixed paraffin-embedded tissue samples from 32 patients with nephroblastoma and 10 controls. No mutations were detected in the four studied genes among our Wilms tumors cases, while Ras and B-Raf expression was higher in malignant samples versus controls. Statistical analysis revealed a positive correlation of K-Ras (p < 0.001) and B-Raf (p = 0.006) with tumor size, a negative correlation of K-Ras (p = 0.041) and H-Ras (p = 0.033) with the percentage of tissue necrosis, and an association of N-Ras (p = 0.047) and B-Raf (p = 0.044) with tissue histology. From the above, we deduce that although Ras and B-Raf mutations are rare events in Wilms tumors, their expression pattern suggests that they play an important role in the development and progression of this malignancy.

  14. Early increase of radiation-induced γH2AX foci in a human Ku70/80 knockdown cell line characterized by an enhanced radiosensitivity.

    PubMed

    Vandersickel, Veerle; Depuydt, Julie; Van Bockstaele, Bram; Perletti, Gianpaolo; Philippe, Jan; Thierens, Hubert; Vral, Anne

    2010-01-01

    A better understanding of the underlying mechanisms of DNA repair after exposure to ionizing radiation represents a research priority aimed at improving the outcome of clinical radiotherapy. Because of the close association with DNA double strand break (DSB) repair, phosphorylation of the histone H2AX protein (γH2AX), quantified by immunodetection, has recently been used as a method to study DSB induction and repair at low and clinically relevant radiation doses. However, the lack of consistency in literature points to the need to further validate the role of H2AX phosphorylation in DSB repair and the use of this technique to determine intrinsic radiosensitivity. In the present study we used human mammary epithelial MCF10A cells, characterized by a radiosensitive phenotype due to reduced levels of the Ku70 and Ku80 repair proteins, and investigated whether this repair-deficient cell line displays differences in the phosphorylation pattern of H2AX protein compared to repair-proficient MCF10A cells. This was established by measuring formation and disappearance of γH2AX foci after irradiating synchronized cell populations with (60)Co γ-rays. Our results show statistically significant differences in the number of γH2AX foci between the repair-deficient and -proficient cell line, with a higher amount of γH2AX foci present at early times post-irradiation in the Ku-deficient cell line. However, the disappearance of those differences at later post-irradiation times questions the use of this assay to determine intrinsic radiosensitivity, especially in a clinical setting.

  15. Azidothymidine Enhances Fluorodeoxyuridine-Mediated Radiosensitization

    SciTech Connect

    Chen, C.-M.; Johnson, Monika; Smith, Brian J.; Dornfeld, Ken

    2010-03-01

    Purpose: To examine the role of DNA repair and altered thymidine analogues in altering the response to radiation during thymidine deprivation. Methods and Materials: Mismatch repair-deficient and -proficient cell lines HEC59 and HC-2.4 were treated with fluorodeoxyuridine (FUdR), azidothymidine (AZT), and irradiation either alone or in combination, and outcomes of clonogenic survival and cell-cycle distributions were determined. Results: Survival outcomes for all treatments were similar for both cell lines, suggesting that hMSH2 does not significantly influence thymidine deprivation toxicity or radiosensitization. The chain-terminating thymidine analogue AZT increased the toxicity of FUdR and increased DNA fragmentation. The combination of FUdR and AZT afforded greater radiosensitization than either drug alone. Drug enhancement ratios, the degree of excess radiation-induced cell death in drug-treated cultures compared with radiation alone for HEC59, were 1.2, 1.4, and 1.8 for AZT, FUdR, and the combination, respectively. Enhancement ratios for HC-2.4 were 1.3, 1.5, and 1.8 for AZT, FUdR, and the combination, respectively. Conclusion: Azidothymidine, a chain-terminating thymidine analogue, can enhance the radiosensitizing affects of thymidine deprivation. Deoxyribonucleic acid strand breaks may play an important role in the mechanism of thymidine deprivation-induced radiosensitization.

  16. Comparative study of mutations in SNP loci of K-RAS, hMLH1 and hMSH2 genes in neoplastic intestinal polyps and colorectal cancer

    PubMed Central

    Yan, Zhi-Hui; Cui, Li-Hong; Wang, Xiao-Hui; Li, Chao; He, Xing

    2014-01-01

    AIM: To clarify the molecular mechanism involved in pathogenesis of colorectal cancer as well as clinical significance of genetic analysis of histological samples. METHODS: A total of 480 blood and tissue specimens were collected in our hospital from January 2011 to October 2012. In the observation group, there were 120 blood specimens and 120 intestinal tract tissue specimens collected from patients with neoplastic intestinal polyps. In the control group I there were 80 blood specimens and 80 intestinal tract tissue specimens collected from patients with colorectal cancer. In the control group II there were 40 blood specimens and 40 intestinal tract tissue specimens collected from healthy individuals. The gene segments were amplified using PCR and DNA gel electrophoresis along with DNA sequence analysis were employed for the detection of the following single nucleotide polymorphisms (SNPs): K-RAS codons 12 and 13; hMLH1 (human mutS homolog 1) gene missense mutation at Va1384Asp; hMSH2 (human mutS homolog 2) gene missense mutation at 2783C/A. RESULTS: The mutation rate of the SNP at Va1384Asp locus of the hMLH1 gene from blood and tissue specimens in the observation group showed no statistical difference from those in the control group I. The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene were significantly lower in the observation group than in the control group I (χ2 = 15.476, 29.670, 10.811, 16.618, 33.538, 7.898, P < 0.05). The mutation rate of SNP at Va1384Asp locus of the hMLH1 gene was significantly higher in the observation group when compared to the control group II (χ2 = 10.486, 4.876, P < 0.05). The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene did not show any statistical difference from those in the control group II. CONCLUSION: There may be important clinical significance and relevance between neoplastic intestinal polyps and colorectal cancer in terms

  17. Change in radiosensitivity of rats during hypokinetic stress

    NASA Technical Reports Server (NTRS)

    Chernov, I. P.

    1980-01-01

    The laws governing stress modification of radiation sickness in relation to hypokinetic stress were investigated. It was found that gamma irradiation (800 rad) of rats on the third day of exposure to hypokinesia increased the radiosensitivity of the animals which was determined by the survival rate and the dynamics of body weight and the weight of some internal organs. The same radiation dose was given on the 20th day of hypokinesia and on the third day of recovery from the 20 day hypokinesia decreased the radiosensitivity of rats. It is concluded that the variations in the radiosensitivity observed may be due to a stress effect of hypokinesia.

  18. Phase II study of selumetinib (AZD6244, ARRY-142886) plus irinotecan as second-line therapy in patients with K-RAS mutated colorectal cancer.

    PubMed

    Hochster, H S; Uboha, N; Messersmith, W; Gold, P J; ONeil, B H; Cohen, D; Denlinger, C; Cohen, S; Leichman, C G; Leichman, L; Lenz, H-J

    2015-01-01

    More than half of colorectal tumors harbor activating mutations in RAS/RAF proteins. Selumetinib (AZD6244, ARRY-142886) is a small molecule kinase inhibitor targeting MEK kinase, downstream of RAS. We examined the efficacy and safety of selumetinib with irinotecan in second-line therapy. Patients with K-RAS mutated colorectal cancer, progressing on first-line oxaliplatin-based chemotherapy with bevacizumab, were eligible for this multicenter open-label phase I/II trial. In part A, a dose was determined using a standard "3 + 3" design; in part B, efficacy was determined. The primary endpoint was RECIST response rate. Historical data for irinotecan were used as reference. Secondary endpoints included progression-free survival and overall survival. Thirty-two patients entered the study, and 31 were treated. All had K-RAS exon 2 mutated tumors. In phase I, the recommended oral dose of selumetinib was 75 mg twice per day with intravenous (IV) irinotecan, 180 mg/m² every 2 weeks. Three patients (9.7 %) had partial response . Sixteen patients (51.6 %) had stable disease for ≥4 weeks, including three >1 year. The most common grade 3 adverse events included diarrhea, neutropenia, fatigue, anemia, nausea, and dehydration. The study was terminated before a pre-planned accrual of 45 subjects. Despite termination before full accrual, the point estimates of RR and median PFS show promising results, suggesting that further investigations of MEK inhibition in the treatment of metastatic colorectal cancer are warranted. Studies combining MEK inhibitors with cytotoxics or other targeted agents may lead to improved clinical activity based on the emerging preclinical data.

  19. Radiosensitization of DNA by Cisplatin Adducts Results from an Increase in the Rate Constant for the Reaction with Hydrated Electrons and Formation of PtI

    PubMed Central

    Behmand, B.; Marignier, J.-L.; Mostafavi, M.; Wagner, J. R.; Hunting, D. J.; Sanche, L.

    2015-01-01

    Pulse radiolysis measurements of the decay of hydrated electrons in solutions containing different concentrations of the oligonucleotide GTG with and without a cisplatin adduct show that the presence of a cisplatin moiety accelerates the reaction between hydrated electrons and the oligonucleotide. The rate constant of the reaction is found to be 2.23 × 1010 mol−1 L s−1, which indicates that it is diffusion controlled. In addition, we show for the first time the formation of a PtI intermediate as a result of the reaction of hydrated electrons with GTG-cisplatin. A putative reaction mechanism is proposed, which may form the basis of the radiosensitization of cancer cells in concomitant chemoradiation therapy with cisplatin. PMID:26098937

  20. Radiosensitizers, radioprotectors, and radiation mitigators.

    PubMed

    Raviraj, Jayam; Bokkasam, Vijay Kumar; Kumar, Venkata Suneel; Reddy, Uday Shankar; Suman, Venkata

    2014-01-01

    Radiotherapy is regarded as one of the most important therapeutic modality for the treatment of malignant lesions. This field is undergoing rapid advancements in the recent times. With the use of radiosensitizers and radioprotective agents, the course of radiotherapy has improved the sensitization of tumor cells and protection of normal cells, respectively. The aim of this paper was to critically review and analyze the available compounds used as radiosensitizers, radioprotectors, and radiation mitigators. For reviewing, the author used the electronic search for the keywords 'Radiosensitizers', 'Radioprotectors', 'Radiation mitigators' on PubMed for inclusion of previously published articles and further search of reference papers on individual radiosensitizing and radioprotecting agents was done. Radiosensitizers are agents that sensitize the tumor cells to radiation. These compounds apparently promote fixation of the free radicals produced by radiation damage at the molecular level. The mechanism of action is similar to the oxygen effect, in which biochemical reactions in the damaged molecules prevent repair of the cellular radiation damage. Free radicals such as OH + are captured by the electron affinity of the radiosensitizers, rendering the molecules incapable of repair. Radioprotectors are compounds that are designed to reduce the damage in normal tissues caused by radiation. These compounds are often antioxidants and must be present before or at the time of radiation for effectiveness. Other agents, termed mitigators, may be used to minimize toxicity even after radiation has been delivered. This article tries to discuss the various aspects of radiosensitizers, radioprotectors, and radiation mitigators including the newer agents.

  1. Coamplification at lower denaturation temperature polymerase chain reaction enables selective identification of K-Ras mutations in formalin-fixed, paraffin-embedded tumor tissues without tumor-cell enrichment.

    PubMed

    Yu, Shaorong; Xie, Li; Hou, Zhibo; Qian, Xiaoping; Yu, Lixia; Wei, Jia; Ding, Yitao; Liu, Baorui

    2011-09-01

    Conventional polymerase chain reaction-based Sanger sequencing is the standard assay for the detection of K-Ras mutations. However, this method is deficient in identifying small numbers of mutation-bearing cells, and tumor-cell enrichment methods such as microdissection or macrodissection are labor intensive and not always achievable. We applied the recently described coamplification at lower denaturation temperature polymerase chain reaction, which amplifies minority alleles selectively, to detect K-Ras mutations directly in 29 formalin-fixed, paraffin-embedded pancreatic specimens and compared the results with those of conventional polymerase chain reaction. To avoid a false-negative result from the coamplification at lower denaturation temperature polymerase chain reaction assay, we applied a more sensitive peptide nucleic acid polymerase chain reaction method as the gold standard. Dilution experiments indicated an approximately 5-fold improvement in sensitivity with coamplification at lower denaturation temperature polymerase chain reaction-based Sanger sequencing. Conventional polymerase chain reaction detected K-Ras mutations in 11 formalin-fixed, paraffin-embedded pancreatic specimens (37.9%), whereas coamplification at lower denaturation temperature polymerase chain reaction could identify all of those mutations as well as mutations in 10 additional samples, for a total of 21 (72.4%, P = .002) of 29. Unlike peptide nucleic acid polymerase chain reaction, coamplification at lower denaturation temperature polymerase chain reaction identified all K-Ras mutations in specimens in which tumor cells accounted for at least 20% of the total. Adoption of coamplification at lower denaturation temperature polymerase chain reaction is straightforward and requires no additional reagents or instruments. The technique is a good strategy to detect K-Ras mutations selectively in formalin-fixed, paraffin-embedded tissues without tumor-cell enrichment.

  2. Caffeic Acid Phenethyl Ester Increases Radiosensitivity of Estrogen Receptor-Positive and -Negative Breast Cancer Cells by Prolonging Radiation-Induced DNA Damage

    PubMed Central

    Khoram, Nastaran Masoudi; Bigdeli, Bahareh; Nikoofar, Alireza

    2016-01-01

    Purpose Breast cancer is an important cause of death among women. The development of radioresistance in breast cancer leads to recurrence after radiotherapy. Caffeic acid phenethyl ester (CAPE), a polyphenolic compound of honeybee propolis, is known to have anticancer properties. In this study, we examined whether CAPE enhanced the radiation sensitivity of MDA-MB-231 (estrogen receptor-negative) and T47D (estrogen receptor-positive) cell lines. Methods The cytotoxic effect of CAPE on MDA-MB-231 and T47D breast cancer cells was evaluated by performing an 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. To assess clonogenic ability, MDA-MB-231 and T47D cells were treated with CAPE (1 µM) for 72 hours before irradiation, and then, a colony assay was performed. A comet assay was used to determine the number of DNA strand breaks at four different times. Results CAPE decreased the viability of both cell lines in a dose- and time-dependent manner. In the clonogenic assay, pretreatment of cells with CAPE before irradiation significantly reduced the surviving fraction of MDA-MB-231 cells at doses of 6 and 8 Gy. A reduction in the surviving fraction of T47D cells was observed relative to MDA-MB-231 at lower doses of radiation. Additionally, CAPE maintained radiation-induced DNA damage in T47D cells for a longer period than in MDA-MB-231 cells. Conclusion Our results indicate that CAPE impairs DNA damage repair immediately after irradiation. The induction of radiosensitivity by CAPE in radioresistant breast cancer cells may be caused by prolonged DNA damage. PMID:27066092

  3. Non-covalent interactions of the carcinogen (+)-anti-BPDE with exon 1 of the human K-ras proto-oncogene

    NASA Astrophysics Data System (ADS)

    Rodriguez, Jorge H.; Deligkaris, Christos

    2013-03-01

    Investigating the complementary, but different, effects of physical (non-covalent) and chemical (covalent) mutagen-DNA and carcinogen-DNA interactions is important for understanding possible mechanisms of development and prevention of mutagenesis and carcinogenesis. A highly mutagenic and carcinogenic metabolite of the polycyclic aromatic hydrocarbon benzo[ α]pyrene, namely (+)-anti-BPDE, is known to undergo both physical and chemical complexation with DNA. The major covalent adduct, a promutagenic, is known to be an external (+)-trans-anti-BPDE-N2-dGuanosine configuration whose origins are not fully understood. Thus, it is desirable to study the mechanisms of external non-covalent BPDE-DNA binding and their possible relationships to external covalent trans adduct formation. We present a detailed codon-by-codon computational study of the non-covalent interactions of (+)-anti-BPDE with DNA which explains and correctly predicts preferential (+)-anti-BPDE binding at minor groove guanosines. Due to its relevance to carcinogenesis, the interaction of (+)-anti-BPDE with exon 1 of the human K-ras gene has been studied in detail. Present address: Department of Physics, Drury University

  4. A vertically-stacked, polymer, microfluidic point mutation analyzer: Rapid, high accuracy detection of low-abundance K-ras mutations

    PubMed Central

    Han, Kyudong; Lee, Tae Yoon; Nikitopoulos, Dimitris E.; Soper, Steven A.; Murphy, Michael C.

    2011-01-01

    Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well-trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user-base for these types of molecular assays, a vertically-stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employed a primary PCR coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers and ExoSAP-IT® purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in ~1 h, an 80% reduction in time compared to conventional bench-top instrumentation. Purifying the post-PCR products with the ExoSAP-IT® enzyme led to optimized LDR performance minimizing false positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25 μL sample, equivalent to DNA from 42 mutant cells. PMID:21771577

  5. [Efficacy and safety of panitumumab for K-ras wild-type unresectable or recurrent colorectal cancer - a study focusing on first-line treatment].

    PubMed

    Mitomo, Shingo; Suto, Takayuki; Umemura, Akira; Ishida, Kaoru; Kanno, Kiminori; Takeda, Daiki; Fujita, Tomonori; Otsuka, Koki; Nitta, Hiroyuki; Uesugi, Noriyuki; Sugai, Tamotsu; Wakabayashi, Go

    2014-06-01

    Panitumumab was approved in June 2010 for use in the treatment of unresectable advanced/recurrent colorectal cancer. Here, we report outcomes and adverse events of panitumumab combination therapy or single-agent chemotherapy for K-ras wild-type unresectable or recurrent colorectal cancers. Our study focused on first-line treatments. The study involved 18 patients who started receiving panitumumab in October 2010. Nine patients received panitumumab as a first-line treatment; 4, as a second-line treatment; and 5, as a third-line or subsequent treatment. The overall response rate was 27.8%. Among the patients who received panitumumab as a first-line treatment, the response rate was 55.6%. Grade 1 and 2 skin disorders were common adverse events. Grade 2 interstitial pneumonia was observed in 1 patient(5.6%). Grade 3 or higher events comprised peripheral neuropathy in 1 patient(5.6%)and neutropenia in another patient(5.6%). The treatment was beneficial, and metastatic foci were resected in 3 patients. In this study, the only adverse events of Grade 3 or higher were 1 case each of peripheral neuropathy and neutropenia. Accordingly, adequate control seemed possible. The specific line of treatment that panitumumab should belong to remains controversial. However, active initiation as first-line treatment should be considered for cases in which resection of metastatic foci can be expected from tumor reductions due to panitumumab.

  6. Differential radiosensitivity among B cell subpopulations

    SciTech Connect

    Riggs, J.E.; Lussier, A.M.; Lee, S.K.; Appel, M.C.; Woodland, R.T.

    1988-09-15

    We have previously shown that low doses of ionizing radiation selectively impair a functionally defined B cell subpopulation. Normal mice, after exposure to 200 rad of ionizing radiation, have normal or near normal splenic plaque-forming cell responses to thymus-independent type 1 Ag, but reduced responses to thymus-independent type 2 Ag. Here, we confirm and extend the original findings by using hapten-specific serum RIA to demonstrate this differential radiosensitivity is systemic. We also examined splenocytes stained with a panel of lymphocyte surface Ag by FACS analysis to determine if these functional changes are accompanied by a physical alteration of the B cell pool of irradiated mice. Single-parameter FACS analyses demonstrate a diminution in both B cell number and the heterogeneity of membrane Ag expression within the surviving B cell pool after irradiation. In contrast, T cells are relatively radioresistant as the relative percentage of T cells in the irradiated splenocyte pool increases, whereas the heterogeneity of membrane Ag expression remains constant. Multiparameter FACS analyses indicate that B cells with the sIgM much greater than sIgD phenotype are more radiosensitive than B cells of the sIgM much less than sIgD phenotype. In addition, immunohistochemical analysis of splenic sections stained with anti-IgM or anti-IgD reveal the enhanced radiosensitivity of marginal zone B cells.

  7. Preventive effects of butyric acid, nicotinamide, calcium glucarate alone or in combination during the 7, 12-dimethylbenz (a) anthracene induced mouse skin tumorigenesis via modulation of K-Ras-PI3K-AKTpathway and associated micro RNAs.

    PubMed

    Tiwari, Prakash; Sahay, Satya; Pandey, Manuraj; Qadri, Syed S Y H; Gupta, Krishna P

    2016-02-01

    Skin cancer is among the most common cancers worldwide and identifiable molecular changes for early and late stage of skin tumorigenesis can suggest the better targets for its control. In this study, we investigated the status of K-Ras-PI3K-AKTpathway followed by NF-κB, cyclin D1, MMP-9 and regulatory micro RNA during 7, 12-dimethylbenz[a]anthracene (DMBA) induced mouse skin tumorigenesis and its prevention by butyric acid (BA), nicotinamide (NA) and calcium glucarate (CAG), individually or in combination with respect to time. DMBA upregulated the K-Ras, PI3K, Akt, NF-κB, cyclin D1 and MMP-9, but downregulated the PTEN in a time dependent manner. DMBA also reduced the levels of micoRNA let-7a but induced the levels of miR-21 and miR-20a as a function of time. BA, NA and CAG were found to prevent DMBA induced changes, but they were most effective when used together in a combination. Reduced let-7a and miR-211 were correlated with the overexpression of K-Ras and MMP-9. Overexpression of miR-21 and miR-20a was correlated with the down regulation of PTEN and overexpression of Cyclin D1. Collectively, the enhanced chemopreventive potential of natural compound in combination via regulation of K-Ras-PI3K-AKTpathway along with regulatory micro RNAs provide a newer and effective mean for cancer management.

  8. MicroRNA let-7a inhibits the proliferation and invasion of nonsmall cell lung cancer cell line 95D by regulating K-Ras and HMGA2 gene expression.

    PubMed

    Wang, Yan-Ying; Ren, Tao; Cai, Ying-Yun; He, Xiao-Ye

    2013-03-01

    MicroRNAs are closely linked to tumor metastasis and let-7a may play a role in inhibiting the proliferation, invasion, and metastasis of lung cancer. In vitro, we aim to observe the impact of let-7a on the proliferation and invasion of the nonsmall cell lung cancer cell line 95D by constructing a lentiviral vector that expresses let-7a. Cell proliferation assays and Transwell experiments were used to compare the proliferation and invasion of the 95D cell group with let-7a overexpressed or inhibited. Real-time polymerase chain reaction and immunoblotting analysis were used to compare the expression of K-RAS and HMGA2 at mRNA and the protein level in the above groups. The results showed the cells in the let-7a overexpressed group were significantly less proliferative and invasive than those in the let-7a inhibited group (p < 0.05). K-RAS and HMGA2 mRNA levels were significantly higher in the let-7a overexpressed group than those in the let-7a inhibited group (p < 0.05). However, the protein levels of K-RAS and HMGA2 were significantly lower in the let-7a overexpressed group than those in the let-7a inhibited group (p < 0.05). We suppose that let-7a inhibits the proliferation and invasion of the cell line 95D by regulating the translation of K-RAS and HMGA2 mRNA, not the transcription of the mRNA itself.

  9. Effects of low-level chronic irradiation on radiosensitivity of mammals: modeling and experimental studies

    NASA Astrophysics Data System (ADS)

    Smirnova, O. A.; Yonezawa, M.

    Effects of low dose rate chronic irradiation on radiosensitivity of mammals mice are studied by experimental and modeling methods Own and reference experiments show that priming chronic low-level short-term and long-term exposures to radiation induce respectively elevated radiosensitivity and lowered radiosensitivity radioresistance in mice The manifestation of these radiosensitization and radioprotection effects are respectively increased and decreased mortality of preirradiated specimens after challenge acute irradiation in comparison with those for previously unexposed ones Taking into account that the reason of the animal death in the experiments was the hematopoietic syndrome the biophysical models of the critical body system hematopoiesis are used to simulate the dynamics of the major hematopoietic lines in mice exposed to challenge acute irradiation following the chronic one Juxtaposition of the modeling results obtained and the relevant experimental data shows that the radiosensitization effect of chronic low-level short-term less than 1 month preirradiation on mice is due to increased radiosensitivity of lymphopoietic granulocytopoietic and erythropoietic systems accompanied by increased or close to the normal level radiosensitivity of thrombocytopoietic system which are induced by the above-indicated exposure In turn the radioprotection effect of chronic low-level long-term more than 1 month preirradiation on mice is caused by decreased radiosensitivity radioresistance of the granulocytopoietic system which

  10. Combined inhibition of Wee1 and PARP1/2 for radiosensitization in pancreatic cancer

    PubMed Central

    Karnak, David; Engelke, Carl G.; Parsels, Leslie A.; Kausar, Tasneem; Wei, Dongping; Robertson, Jordan R.; Marsh, Katherine B.; Davis, Mary A.; Zhao, Lili; Maybaum, Jonathan; Lawrence, Theodore S.; Morgan, Meredith A.

    2014-01-01

    Purpose While the addition of radiation to chemotherapy improves survival in patients with locally advanced pancreatic cancer, more effective therapies are urgently needed. Thus, we investigated the radiosensitizing efficacy of the novel drug combination of Wee1 and PARP1/2 [poly (ADP-ribose) polymerase 1/2] inhibitors (AZD1775 and olaparib, respectively) in pancreatic cancer. Experimental Design Radiosensitization of AsPC-1 or MiaPaCa-2 human pancreatic cancer cells was assessed by clonogenic survival and tumor growth assays. Mechanistically, the effects of AZD1775, olaparib, and radiation on cell cycle, DNA damage (γH2AX) and HRR (homologous recombination repair) were determined. Results Treatment of AsPC-1 and MiaPaCa-2 cells with either AZD1775 or olaparib caused modest radiosensitization while treatment with the combination significantly increased radiosensitization. Radiosensitization by the combination of AZD1775 and olaparib was associated with G2 checkpoint abrogation and persistent DNA damage. In addition, AZD1775 inhibited HRR activity and prevented radiation-induced Rad51 focus formation. Finally, in vivo, in MiaPaCa-2-derived xenografts, olaparib did not radiosensitize, while AZD1775 produced moderate, yet significant, radiosensitization (P<0.05). Importantly, the combination of AZD1775 and olaparib produced highly significant radiosensitization (P<0.0001) evidenced by a 13-day delay in tumor volume doubling (vs radiation alone) and complete eradication of 20% of tumors. Conclusions Taken together, these results demonstrate the efficacy of combined inhibition of Wee1 and PARP inhibitors for radiosensitizing pancreatic cancers and support the model that Wee1 inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization through inhibition of HRR and abrogation of the G2 checkpoint, ultimately resulting in unrepaired, lethal DNA damage and radiosensitization. PMID:25117293

  11. Hypoxic radiosensitizers: substituted styryl derivatives.

    PubMed

    Nudelman, A; Falb, E; Odesa, Y; Shmueli-Broide, N

    1994-10-01

    A number of novel styryl epoxides, N-substituted-styryl-ethanolamines, N-mono and N,N'-bis-(2-hydroxyethyl)-cinnamamides--analogues to the known radiosensitizers RSU-1069, pimonidazole and etanidazole--display selective hypoxic radiosensitizing activity. The styryl group, especially when substituted by electron withdrawing groups, was found to be bioisosteric to the nitroimidazolyl functionality. The most active derivative 2-(2'-nitrophenyl)ethen-1-yl-oxirane 8a displayed a sensitizer enhancement ratio (SER) of 5 relative to misonidazole.

  12. Synthesis and radiosensitization properties of hydrogen peroxide and sodium hyaluronate complex

    SciTech Connect

    Rosli, Nur Ratasha Alia Md.; Mohamed, Faizal; Heng, Cheong Kai; Rahman, Irman Abdul; Ahmad, Ainee Fatimah; Mohamad, Hur Munawar Kabir

    2014-09-03

    Cancer cells which are large in size are resistant towards radiation therapy due to the presence of large amount of anti-oxidative enzymes and hypoxic cancer cells. Thus radiosensitizer agents have been developed to enhance the therapeutic effect of radiotherapy by increasing the sensitivity of these cancer cells towards radiation. This study is conducted to investigate the radiosensitization properties of radiosensitizer complex containing hydrogen peroxide and sodium hyaluronate. Combination with sodium hyaluronate may decrease reactivity of hydrogen peroxide but maintain the oxygen concentration needed for radiosensitizing effect. HepG2 cancer cells are cultured as the mean of test subject. Cancer cell samples which are targeted and not targeted with these radiosensitizers are irradiated with 2Gy single fractionated dose. Results obtained shows that the cancer cells which are not targeted with radiosensitizers has a cell viability of 98.80±0.37% after a time interval of 48 hours and has even repopulated over 100% after a 72 hour time interval. This shows that the cancer cells are resistant towards radiation. However, when the cancer cells are targeted with radiosensitizers prior to irradiation, there is a reduction of cell viability by 25.50±10.81% and 10.30±5.10% at time intervals of 48 and 72 hours respectively. This indicates that through the use of these radiosensitizers, cancer cells are more sensitive towards radiation.

  13. Differential radiosensitivity among B cell subpopulations

    SciTech Connect

    Riggs, J.E.

    1988-01-01

    The selective radiosensitivity of sIgM >> sIgD marginal zone B cells is associated with the selective loss of B cell function. The simultaneous restoration of impaired function and recovery of these cells with time supports this premise. B cell recovery, delayed one week after irradiation, is in progress at two weeks, and virtually complete by three weeks. XID mice reveal similar recovery kinetics although there are fewer recovering cells and these bear reduced levels of Ia. This observation represents additional evidence that xid B cells are distinct from those of normal mice. The simultaneous loss, and concurrent recovery, of sIgM >> sIgD B cells and TI-2 responsiveness in irradiated mice suggests the existence of a unique B cell subpopulation possessing both phenotypes. Additional support for this hypothesis is provided by demonstrating that splenocytes, depleted of IgD{sup +} cells adoptively reconstitute this response in XID mice. The peritoneal B cell pool, which, compared to the spleen, consist of increased numbers of sIgM >> sIgD B cells, is shown to be a source of radiosensitive B cells that are TI-2 responsive. These observations represent additional evidence for an association between sIgM >> sIgD B cells and TI-2 responsiveness.

  14. Radiosensitization in prostate cancer: mechanisms and targets

    PubMed Central

    2013-01-01

    Prostate cancer is the second most commonly diagnosed cancer in American men over the age of 45 years and is the third most common cause of cancer related deaths in American men. In 2012 it is estimated that 241,740 men will be diagnosed with prostate cancer and 28,170 men will succumb to prostate cancer. Currently, radiation therapy is one of the most common definitive treatment options for localized prostate cancer. However, significant number of patients undergoing radiation therapy will develop locally persistent/recurrent tumours. The varying response rates to radiation may be due to 1) tumor microenvironment, 2) tumor stage/grade, 3) modality used to deliver radiation, and 4) dose of radiation. Higher doses of radiation has not always proved to be effective and have been associated with increased morbidity. Compounds designed to enhance the killing effects of radiation, radiosensitizers, have been extensively investigated over the past decade. The development of radiosensitizing agents could improve survival, improve quality of life and reduce costs, thus benefiting both patients and healthcare systems. Herin, we shall review the role and mechanisms of various agents that can sensitize tumours, specifically prostate cancer. PMID:23351141

  15. Roadmap to clinical use of gold nanoparticles for radiosensitization

    PubMed Central

    Schuemann, J.; Berbeco, R.; Chithrani, B. D.; Cho, S.; Kumar, R.; McMahon, S.; Sridhar, S.; Krishnan, S.

    2015-01-01

    The past decade has seen a dramatic increase in interest in the use of Gold Nanoparticles (GNPs) as radiation sensitizers for radiotherapy. This interest was initially driven by their strong absorption of ionizing radiation and the resulting ability to increase dose deposited within target volumes even at relatively low concentrations. These early observations are supported by extensive experimental validation, showing GNPs’ efficacy at sensitizing tumors in both in vitro and in vivo systems to a range of types of ionizing radiation, including kilovoltage and megavoltage X-rays as well as charged particles. Despite this experimental validation, there has been limited translation of GNP-mediated radiosensitization to a clinical setting. One of the key challenges in this area is the wide range of experimental systems that have been investigated, spanning a range of particle sizes, shapes and preparations. As a result, mechanisms of uptake and radiosensitization have remained difficult to clearly identify. This has proven a significant impediment to the identification of optimal GNP formulations which strike a balance among their radiosensitizing properties, their specificity to the tumors, their biocompatibility, and their imageability in vivo. This white paper reviews the current state of knowledge in each of the areas concerning the use of GNPs as radiosensitizers, and outlines the steps which will be required to advance GNP-enhanced radiation therapy from their current pre-clinical setting to clinical trials and eventual routine usage. PMID:26700713

  16. Differentiation and radiosensitivity of hemopoietic stem cells of mice during hypokinesia

    NASA Technical Reports Server (NTRS)

    Shvets, V. N.

    1980-01-01

    The potential for differentiation and radiosensitivity of the stem hemopoietic cells (KOE) under conditions of initial and later hypokinesia is examined. It is established that in the initial period of hypokinesia (3 days) when a stress reaction prevails, changes occur in the erythroid differentiation and radiosensitivity of KOE. This effect is associated with redistribution of T-lymphocytes that increase in number in the bone marrow of mice during hypokinesia. At later periods of hypokinesia (30 days) when changes in the organism are related to hypokinesia proper, differentiation and radiosensitivity of KOE were normalized.

  17. Anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize.

    PubMed

    Adams, Stephen R; Yang, Howard C; Savariar, Elamprakash N; Aguilera, Joe; Crisp, Jessica L; Jones, Karra A; Whitney, Michael A; Lippman, Scott M; Cohen, Ezra E W; Tsien, Roger Y; Advani, Sunil J

    2016-10-04

    Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery.

  18. Radiosensitizing effect of 5-aminolevulinic acid-induced protoporphyrin IX in glioma cells in vitro.

    PubMed

    Yamamoto, Junkoh; Ogura, Shun-Ichiro; Tanaka, Tohru; Kitagawa, Takehiro; Nakano, Yoshiteru; Saito, Takeshi; Takahashi, Mayu; Akiba, Daisuke; Nishizawa, Shigeru

    2012-06-01

    5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy and fluorescence-guided resection of malignant gliomas due to its high cellular uptake in tumours. Porphyrin compounds act not only as photosensitizers but also as radiosensitizers. In the present study, the possible use of 5-ALA as a radiosensitizer for malignant gliomas was examined in vitro. Rat glioma cell lines (9L, C6) were pre-treated with 5-ALA and exposed to ionizing irradiation. The radiosensitizing effect of 5-ALA was evaluated by colony-forming assay. Intracellular reactive oxygen species (ROS) produced by 5-ALA and irradiation were evaluated by confocal laser scanning microscopy. Pre-treatment with 5-ALA enhanced the radiosensitivity of 9L cells to single-dose ionizing irradiation compared with controls (D0 value, 4.35 ± 0.20 and 4.84 ± 0.23 Gy, respectively, P ≤ 0.05). Exposure to multi-dose ionizing irradiation revealed high radiosensitivity in both 9L and C6 cells pre-treated with 5-ALA compared to controls. Production of intracellular ROS increased in 9L cells pre-treated with 5-ALA after ionizing irradiation compared to control cells. Thus, 5-ALA functions as a specific radiosensitizer for malignant gliomas. Intracellular 5-ALA-induced PpIX plays an important role in the production of ROS and the radiosensitizing effect under ionizing irradiation conditions.

  19. Anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize

    PubMed Central

    Adams, Stephen R.; Yang, Howard C.; Savariar, Elamprakash N.; Aguilera, Joe; Crisp, Jessica L.; Jones, Karra A.; Whitney, Michael A.; Lippman, Scott M.; Cohen, Ezra E. W.; Tsien, Roger Y.; Advani, Sunil J.

    2016-01-01

    Tumour resistance to radiotherapy remains a barrier to improving cancer patient outcomes. To overcome radioresistance, certain drugs have been found to sensitize cells to ionizing radiation (IR). In theory, more potent radiosensitizing drugs should increase tumour kill and improve patient outcomes. In practice, clinical utility of potent radiosensitizing drugs is curtailed by off-target side effects. Here we report potent anti-tubulin drugs conjugated to anti-ErbB antibodies selectively radiosensitize to tumours based on surface receptor expression. While two classes of potent anti-tubulins, auristatins and maytansinoids, indiscriminately radiosensitize tumour cells, conjugating these potent anti-tubulins to anti-ErbB antibodies restrict their radiosensitizing capacity. Of translational significance, we report that a clinically used maytansinoid ADC, ado-trastuzumab emtansine (T-DM1), with IR prolongs tumour control in target expressing HER2+ tumours but not target negative tumours. In contrast to ErbB signal inhibition, our findings establish an alternative therapeutic paradigm for ErbB-based radiosensitization using antibodies to restrict radiosensitizer delivery. PMID:27698471

  20. Radiosensitization by the histone deacetylase inhibitor vorinostat under hypoxia and with capecitabine in experimental colorectal carcinoma

    PubMed Central

    2012-01-01

    Background The histone deacetylase inhibitor vorinostat is a candidate radiosensitizer in locally advanced rectal cancer (LARC). Radiosensitivity is critically influenced by hypoxia; hence, it is important to evaluate the efficacy of potential radiosensitizers under variable tissue oxygenation. Since fluoropyrimidine-based chemoradiotherapy (CRT) is the only clinically validated regimen in LARC, efficacy in combination with this established regimen should be assessed in preclinical models before a candidate drug enters clinical trials. Methods Radiosensitization by vorinostat under hypoxia was studied in four colorectal carcinoma cell lines and in one colorectal carcinoma xenograft model by analysis of clonogenic survival and tumor growth delay, respectively. Radiosensitizing effects of vorinostat in combination with capecitabine were assessed by evaluation of tumor growth delay in two colorectal carcinoma xenografts models. Results Under hypoxia, radiosensitization by vorinostat was demonstrated in vitro in terms of decreased clonogenicity and in vivo as inhibition of tumor growth. Adding vorinostat to capecitabine-based CRT increased radiosensitivity of xenografts in terms of inhibited tumor growth. Conclusions Vorinostat sensitized colorectal carcinoma cells to radiation under hypoxia in vitro and in vivo and improved therapeutic efficacy in combination with capecitabine-based CRT in vivo. The results encourage implementation of vorinostat into CRT in LARC trials. PMID:23017053

  1. Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells.

    PubMed

    Mamo, Tewodros; Mladek, Ann C; Shogren, Kris L; Gustafson, Carl; Gupta, Shiv K; Riester, Scott M; Maran, Avudaiappan; Galindo, Mario; van Wijnen, Andre J; Sarkaria, Jann N; Yaszemski, Michael J

    2017-04-29

    Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Factors Affecting the Radiosensitivity of Hexaploid Wheat to γ-Irradiation: Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)

    PubMed Central

    Zhao, Linshu; Guo, Huijun; Xie, Yongdun; Zhao, Shirong; Song, Xiyun; Han, Longzhi; Liu, Luxiang

    2016-01-01

    Understanding the radiosensitivity of plants, an important factor in crop mutation breeding programs, requires a thorough investigation of the factors that contribute to this trait. In this study, we used the highly radiosensitive wheat (Triticum aestivum L.) variety HY1 and J411, a γ-irradiation-insensitive control, which were screened from a natural population, to examine the factors affecting radiosensitivity, including free radical content and total antioxidant capacity, as well as the expression of TaKu70 and TaKu80 (DNA repair-related genes) as measured by real-time PCR. We also investigated the alternative splicing of this gene in the wild-type wheat ecotype by sequence analysis. Free radical contents and total antioxidant capacity significantly increased upon exposure of HY1 wheat to γ-irradiation in a dose-dependent manner. By contrast, in J411, the free radical contents exhibited a similar trend, but the total antioxidant capacity exhibited a downward trend upon increasing γ-irradiation. Additionally, we detected dose-dependent increases in TaKu70 and TaKu80 expression levels in γ-irradiated HY1, while in J411, TaKu70 expression levels increased, followed by a decline. We also detected alternative splicing of TaKu70 mRNA, namely, intron retention, in HY1 but not in J411. Our findings indicate that γ-irradiation induces oxidative stress and DNA damage in hexaploid wheat, resulting in growth retardation of seedlings, and they suggest that TaKu70 may play a causal role in radiosensitivity in HY1. Further studies are required to exploit these factors to improve radiosensitivity in other wheat varieties. PMID:27551965

  3. Disturbance of redox status enhances radiosensitivity of hepatocellular carcinoma

    PubMed Central

    Sun, Chao; Wang, Zhen-hua; Liu, Xiong-xiong; Yang, Li-na; Wang, Yali; Liu, Yang; Mao, Ai-hong; Liu, Yuan-yuan; Zhou, Xin; Di, Cui-xia; Gan, Lu; Zhang, Hong

    2015-01-01

    Aims: High constitutive expression of Nrf2 has been found in many types of cancers, and this high level of Nrf2 also favors resistance to drugs and radiation. Here we investigate how isoliquiritigenin (ISL), a natural antioxidant, inhibits the Nrf2-dependent antioxidant pathway and enhances the radiosensitivity of HepG2 cells and HepG2 xenografts. Results: Treatment of HepG2 cells with ISL for 6 h selectively enhanced transcription and expression of Keap1. Keap1 effectively induced ubiquitination and degradation of Nrf2, and inhibited translocation of Nrf2 to the nucleus. Consequently, expression of Nrf2 downstream genes was reduced, and the Nrf2-dependent antioxidant system was suppressed. Endogenous ROS was higher than before ISL treatment, causing redox imbalance and oxidative stress in HepG2 cells. Moreover, pretreatment with ISL for 6 h followed by X-ray irradiation significantly increased γ-H2AX foci and cell apoptosis, and reduced clonogenic potential compared with cells irradiated with X-rays alone. In addition, HepG2 xenografts, ISL, and X-ray co-treatments induced greater apoptosis and tumor growth inhibition, when compared with X-ray treatments alone. Additionally, HepG2 xenografts, in which Nrf2 was expressed at very low levels due to ectopic expression of Keap1, showed that ISL-mediated radiosensitization was Keap1 dependent. Innovation and Conclusions: ISL inhibited the Nrf2-antioxidant pathway by increasing the levels of Keap1 and ultimately inducing oxidative stress via disturbance of the redox status. The antioxidant ISL possessed pro-oxidative properties, and enhanced the radiosensitivity of liver cancer cells, both in vivo and in vitro. Taken together, these results demonstrated the effectiveness of using ISL to decrease radioresistance, suggesting that ISL could be developed as an adjuvant radiosensitization drug. Disturbance of redox status could be a potential target for radiosensitization. PMID:26101703

  4. Panitumumab and irinotecan every 3 weeks is an active and convenient regimen for second-line treatment of patients with wild-type K-RAS metastatic colorectal cancer.

    PubMed

    Carrato, A; Gómez, A; Escudero, P; Chaves, M; Rivera, F; Marcuello, E; González, E; Grávalos, C; Constenla, M; Manzano, J Luis; Losa, F; Maurel, J; Dueñas, R; Massuti, B; Gallego, J; Aparicio, J; Antón, A; Aranda, E

    2013-09-01

    To evaluate the efficacy and safety profile of the combination of panitumumab and irinotecan every 3 weeks in a phase II trial as second-line treatment in patients with advanced wild-type (WT) K-RAS colorectal cancer (CRC). Fifty-three patients received 9 mg/kg of panitumumab followed by 350 mg/m(2) of irinotecan every 21 days until disease progression, unacceptable toxicity or consent withdrawal. Median age of patients included was 67 years. All patients had previously received 5-fluorouracil, 84 % oxaliplatin and 8 % irinotecan as first-line treatment. Patients received a median of five infusions of panitumumab and irinotecan. On an intention-to-treat analysis, 12 patients (23 %) achieved partial responses and 22 patients (41 %) achieved disease stabilization. Median progression-free survival and overall survival were 4.5 and 15.1 months, respectively. The most frequent treatment-related severe toxicities per patient were diarrhoea (35.8 %), followed by skin rash (32.1 %), asthenia (18.9 %) and neutropenia (13.2 %). A significant association between clinical response and incidence and grade of skin toxicity was observed (p = 0.0032). This study shows that the administration of panitumumab plus irinotecan every 3 weeks is safe, active and feasible as second-line treatment in patients with advanced WT K-RAS CRC.

  5. Radiosensitization by porphyrins in 60Co-irradiated cells of different natures

    NASA Astrophysics Data System (ADS)

    Luksiene, Zivile; Atkocius, Vydmantas

    1994-03-01

    Efficiency of cell radiosensitization by porphyrins is a function of the irradiation dose, the nature of the cell and physico-chemical properties of the sensitizer. The maximal radio- sensitization for E. ascites tumor cells was reached at 2 Gy, for Saccharomyces cerevisiae- 25Gy. The radiosensitization by different porphyrins in E.ascites tumor cells increases their cytotoxicity by a factor of 3, in Saccharomyces cerevisae --inhibition of growth 0.5 - 2.5 time.

  6. Suppression of autophagy augments the radiosensitizing effects of STAT3 inhibition on human glioma cells

    SciTech Connect

    Yuan, Xiaopeng; Du, Jie; Hua, Song; Zhang, Haowen; Gu, Cheng; Wang, Jie; Yang, Lei; Huang, Jianfeng; Yu, Jiahua Liu, Fenju

    2015-01-15

    Radiotherapy is an essential component of the standard therapy for newly diagnosed glioblastoma. To increase the radiosensitivity of glioma cells is a feasible solution to improve the therapeutic effects. It has been suggested that inhibition of signal transducer and activator of transcription 3 (STAT3) can radiosensitize glioma cells, probably via the activation of mitochondrial apoptotic pathway. In this study, human malignant glioma cells, U251 and A172, were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The radiosensitizing effects of STAT3 inhibition were confirmed in glioma cells. Intriguingly, combination of ionizing radiation exposure and STAT3 inhibition triggered a pronounced increase of autophagy flux. To explore the role of autophagy, glioma cells were treated with 3-methyladenine or siRNA for autophagy-related gene 5, and it was demonstrated that inhibition of autophagy further strengthened the radiosensitizing effects of STAT3 inhibition. Accordingly, more apoptotic cells were induced by the dual inhibition of autophagy and STAT3 signaling. In conclusion, our data revealed a protective role of autophagy in the radiosensitizing effects of STAT3 inhibition, and inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells. - Highlights: • Inactivation of STAT3 signaling radiosensitizes malignant glioma cells. • STAT3 inhibition triggers a significant increase of autophagy flux induced by ionizing radiation in glioma cells. • Suppression of autophagy further strengthens the radiosensitizing effects of STAT3 inhibition in glioma cells. • Dual inhibition of autophagy and STAT3 induce massive apoptotic cells upon exposure to ionizing radiation.

  7. miR-424 acts as a tumor radiosensitizer by targeting aprataxin in cervical cancer

    PubMed Central

    Jin, Hua; Zou, Hua; Xia, Wei; Dai, Nan; Dai, Xiao-Yan; Wang, Dong; Xu, Cheng-Xiong; Qing, Yi

    2016-01-01

    Previous studies have shown that some dysregulated miRNAs are involved in radioresistance of tumor cells. Here, we identified significantly decreased miR-424 expression in radioresistant cervical cancer cells and specimens from cervical cancer patients with radioresistance compared to their radiosensitive parental cells and specimens from radiosensitive patients, respectively. Ectopic expression of miR-424 significantly increased radiation-induced DNA damage, cell apoptosis and G2/M cell cycle arrest in radioresistant cervical cancer cells. Notably, miR-424 agomiR treatment can sensitize radioresistant cervical cancer cells to radiation in a xenograft model. Furthermore, we demonstrated that miR-424 regulated radiosensitivity by directly targeting aprataxin. Taken together, these findings suggest that miR-424 acts as a radiosensitizing miRNA and reveal a new therapeutic strategy for radioresistant cervical cancers. PMID:27769049

  8. Glyoxylic compounds as radiosensitizers of hypoxic cells

    SciTech Connect

    Cornago, M.P.; Lopez Zumel, M.C.; Alvarez, M.V.; Izquierdo, M.C. )

    1990-06-01

    The radiosensitizing effect of five glyoxal derivatives on the survival of TC-SV40 cells has been measured, under aerobic and hypoxic conditions. A toxicity study was previously performed in order to use nontoxic concentrations. The OER for the TC-SV40 cells was 2.74. None of the glyoxylic compounds showed radiosensitizing activity under aerobic conditions while in hypoxia their radiosensitizing factors decreased in the order phenylglyoxylic acid (1.68 at 8 x 10(-3) mole dm-3) greater than phenylglyoxal (1.55 at 5 x 10(-6) mole dm-3) greater than 2-2' furil (1.48 at 5 x 10(-5) mole dm-3) greater than glyoxylic acid (1.39 at 1 x 10(-3) mole dm-3) greater than glyoxal (1.30 at 5 x 10(-5) mole dm-3). The dose-modifying factors were also determined at two equimolar concentrations 5 x 10(-5) and 5 x 10(-6) mole dm-3. A concentration effect was noticed for all the compounds although their relative radiosensitizing activity kept, independently of the concentration, the same order noted above. Glyoxals with aromatic or heterocyclic rings exert a greater radiosensitization than the others. The acidic compounds have less radiosensitizing activity than their aldehydic counterparts. Interaction of these glyoxals with NPSH cellular groups was tested and the low degree of inhibition shows that this mechanism would contribute very little, if any, to the radiosensitization effect.

  9. Ganetespib radiosensitization for liver cancer therapy

    PubMed Central

    Chettiar, Sivarajan T.; Malek, Reem; Annadanam, Anvesh; Nugent, Katriana M.; Kato, Yoshinori; Wang, Hailun; Cades, Jessica A.; Taparra, Kekoa; Belcaid, Zineb; Ballew, Matthew; Manmiller, Sarah; Proia, David; Lim, Michael; Anders, Robert A.; Herman, Joseph M.; Tran, Phuoc T.

    2016-01-01

    ABSTRACT Therapies for liver cancer particularly those including radiation are still inadequate. Inhibiting the stress response machinery is an appealing anti-cancer and radiosensitizing therapeutic strategy. Heat-shock-protein-90 (HSP90) is a molecular chaperone that is a prominent effector of the stress response machinery and is overexpressed in liver cancer cells. HSP90 client proteins include critical components of pathways implicated in liver cancer cell survival and radioresistance. The effects of a novel non-geldanamycin HSP90 inhibitor, ganetespib, combined with radiation were examined on 3 liver cancer cell lines, Hep3b, HepG2 and HUH7, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γH2AX foci kinetics and client protein expression in pathways important for liver cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined ganetespib-radiation treatment on tumor cell proliferation in a HepG2 hind-flank tumor graft model. Nanomolar levels of ganetespib alone exhibited liver cancer cell anti-cancer activity in vitro as shown by decreased clonogenic survival that was associated with increased apoptotic cell death, prominent G2-M arrest and marked changes in PI3K/AKT/mTOR and RAS/MAPK client protein activity. Ganetespib caused a supra-additive radiosensitization in all liver cancer cell lines at low nanomolar doses with enhancement ratios between 1.33–1.78. These results were confirmed in vivo, where the ganetespib-radiation combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in HepG2 tumor grafts. Our data suggest that combined ganetespib-radiation therapy exhibits promising activity against liver cancer cells, which should be investigated in clinical studies. PMID:26980196

  10. Ganetespib radiosensitization for liver cancer therapy.

    PubMed

    Chettiar, Sivarajan T; Malek, Reem; Annadanam, Anvesh; Nugent, Katriana M; Kato, Yoshinori; Wang, Hailun; Cades, Jessica A; Taparra, Kekoa; Belcaid, Zineb; Ballew, Matthew; Manmiller, Sarah; Proia, David; Lim, Michael; Anders, Robert A; Herman, Joseph M; Tran, Phuoc T

    2016-04-02

    Therapies for liver cancer particularly those including radiation are still inadequate. Inhibiting the stress response machinery is an appealing anti-cancer and radiosensitizing therapeutic strategy. Heat-shock-protein-90 (HSP90) is a molecular chaperone that is a prominent effector of the stress response machinery and is overexpressed in liver cancer cells. HSP90 client proteins include critical components of pathways implicated in liver cancer cell survival and radioresistance. The effects of a novel non-geldanamycin HSP90 inhibitor, ganetespib, combined with radiation were examined on 3 liver cancer cell lines, Hep3b, HepG2 and HUH7, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γH2AX foci kinetics and client protein expression in pathways important for liver cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined ganetespib-radiation treatment on tumor cell proliferation in a HepG2 hind-flank tumor graft model. Nanomolar levels of ganetespib alone exhibited liver cancer cell anti-cancer activity in vitro as shown by decreased clonogenic survival that was associated with increased apoptotic cell death, prominent G2-M arrest and marked changes in PI3K/AKT/mTOR and RAS/MAPK client protein activity. Ganetespib caused a supra-additive radiosensitization in all liver cancer cell lines at low nanomolar doses with enhancement ratios between 1.33-1.78. These results were confirmed in vivo, where the ganetespib-radiation combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in HepG2 tumor grafts. Our data suggest that combined ganetespib-radiation therapy exhibits promising activity against liver cancer cells, which should be investigated in clinical studies.

  11. Competitive but Not Allosteric mTOR Kinase Inhibition Enhances Tumor Cell Radiosensitivity1

    PubMed Central

    Hayman, Thomas J; Kramp, Tamalee; Kahn, Jenna; Jamal, Muhammad; Camphausen, Kevin; Tofilon, Philip J

    2013-01-01

    The mechanistic target of rapamycin (mTOR) is a critical kinase in the regulation of gene translation and has been suggested as a potential target for radiosensitization. The goal of this study was to compare the radiosensitizing activities of the allosteric mTOR inhibitor rapamycin with that of the competitive mTOR inhibitor PP242. On the basis of immunoblot analyses, whereas rapamycin only partially inhibited mTOR complex 1 (mTORC1) activity and had no effect on mTOR complex 2 (mTORC2), PP242 inhibited the activity of both mTOR-containing complexes. Irradiation alone had no effect on mTORC1 or mTORC2 activity. Clonogenic survival was used to define the effects of the mTOR inhibitors on in vitro radiosensitivity. In the two tumor cell lines evaluated, PP242 treatment 1 hour before irradiation increased radiosensitivity, whereas rapamycin had no effect. Addition of PP242 after irradiation also enhanced the radiosensitivity of both tumor lines. To investigate the mechanism of radiosensitization, the induction and repair of DNA double-strand breaks were evaluated according γH2AX foci. PP242 exposure did not influence the initial level of γH2AX foci after irradiation but did significantly delay the dispersal of radiation-induced γH2AX foci. In contrast to the tumor cell lines, the radiosensitivity of a normal human fibroblast cell line was not influenced by PP242. Finally, PP242 administration to mice bearing U251 xenografts enhanced radiation-induced tumor growth delay. These results indicate that in a preclinical tumor model PP242 enhances tumor cell radiosensitivity both in vitro and in vivo and suggest that this effect involves an inhibition of DNA repair. PMID:23730416

  12. Retraction: "Activated K-Ras and INK4a/Arf Deficiency Promote Aggressiveness of Pancreatic Cancer by Induction of EMT Consistent With Cancer Stem Cell Phenotype" by Wang et al.

    PubMed

    2016-10-01

    The above article, published online on November 23, 2012 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 4B and C to be inappropriately manipulated and re-labeled. Literature Cited Wang Z, Ali S, Banerjee S, Bao B, Li Y, Azmi AS, Korc M, Sarkar FH. 2013. Activated K-Ras and INK4a/Arf deficiency promote aggressiveness of pancreatic cancer by induction of EMT consistent with cancer stem cell phenotype. J Cell Physiol 228:556-562; doi: 10.1002/jcp.24162.

  13. Retraction: "Inactivation of Ink4a/Arf Leads to Deregulated Expression of miRNAs in K-Ras Transgenic Mouse Model of Pancreatic Cancer" by Ali et al.

    PubMed

    2016-10-01

    The above article, published online on June 21, 2012 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Gary S. Stein, and Wiley Periodicals, Inc. The retraction has been agreed following an investigation from Wayne State University involving the first author and the corresponding author that found Figure 5A to be inappropriately manipulated. Literature Cited Ali S, Banerjee S, Logna F, Bao B, Philip PA, Korc M, Sarkar FH. 2012. Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer. J Cell Physiol 227:3373-3380; doi: 10.1002/jcp.24036.

  14. Effects of glutathione depletion by buthionine sulfoximine on radiosensitization by oxygen and misonidazole in vitro

    SciTech Connect

    Shrieve, D.C.; Denekamp, J.; Minchinton, A.I.

    1985-06-01

    Buthionine sulfoximine (BSO) has been used to deplete glutathione (GSH) in V79-379A cells in vitro, and the effect on the efficiency of oxygen and misonidazole (MISO) as radiosensitizers has been determined. Treatment with 50 or 500 ..mu..M BSO caused a rapid decline in GSH content to less than 5% of control values after 10 hr of exposure. Removal of BSO resulted in a rapid regeneration of GSH after 50 ..mu..M BSO, but little regeneration was observed over the subsequent 10-hr period after 500 ..mu..M. Cells irradiated in monolayer on glass had an oxygen enhancement ratio (OER) of 3.1. After 10-14 hr pretreatment with 50 ..mu..M BSO, washed cells were radiosensitized by GSH depletion at all oxygen tensions tested. The OER was reduced to 2.6, due to greater radiosensitization of hypoxic cells than aerated ones by GSH depletion. In similar experiments performed with MISO, an enhancement ratio of 2.0 could be achieved with 0.2 mM MISO in anoxic BSO-pretreated cells, compared to 2.7 mM MISO in non-BSO-treated cells. These apparent increases in radiosensitizer efficiency in GSH-depleted cells could be explained on the basis of radiosensitization of hypoxic cells by GSH depletion alone. These results are consistent with hypoxic cell radiosensitization by GSH depletion and by MISO or oxygen acting by separate mechanisms.

  15. Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51

    PubMed Central

    Liu, Zhaojian; Wang, Yu; Zhao, Minnan; Hao, Chunyan; Feng, Shuai; Guo, Haiyang; Xu, Bing; Yang, Qifeng; Gong, Yaoqin; Shao, Changshun

    2011-01-01

    Background Esophageal squamous cell carcinomas (ESCC) have poor prognosis. While combined modality of chemotherapy and radiotherapy increases survival, most patients die within five years. Development of agents that confer cancer cell-specific chemo- and radiosensitivity may improve the therapy of ESCC. We here reported the discovery of berberine as a potent radiosensitizing agent on ESCC cells. Principal Findings Berberine at low concentrations (<15 µM) substantially radiosensitized ESCC cells. X-ray induced DNA double-strand breaks (DSBs) persist longer in ESCC cells pretreated with berberine. Berberine pretreatment led to a significant downregulation of RAD51, a key player in homologous recombination repair, in ESCC cells, but not in non-malignant human cells. Downregulation of RAD51 by RNA interference similarly radiosensitized the cancer cells, and, conversely, introduction of exogenous RAD51 was able to significantly counteract the radiosensitizing effect of berberine, thus establishing RAD51 as a key determinant in radiation sensitivity. We also observed that RAD51 was commonly overexpressed in human ESCC tissues, suggesting that it is necessary to downregulate RAD51 to achieve high radio- or chemotherapeutic efficacy of ESCC in clinic, because overexpression of RAD51 is known to confer radio- and chemoresistance. Conclusions/Significance Berberine can effectively downregulate RAD51 in conferring radiosensitivity on esophageal cancer cells. Its clinical application as an adjuvant in chemotherapy and radiotherapy of esophageal cancers should be explored. PMID:21858113

  16. Circadian rhythmometry of mammalian radiosensitivity

    NASA Technical Reports Server (NTRS)

    Haus, E.; Halberg, F.; Loken, M. K.; Kim, Y. S.

    1974-01-01

    In the case of human bone marrow, the largest number of mitoses is seen in the evening in diurnally active men, mitotic activity being at a minimum in the morning. The opposite pattern is observed for nocturnal animals such as rats and mice on a regimen of light during the daytime alternating with darkness during the night hours. The entirety of these rhythms plays an important role in the organism's responses to environmental stimuli, including its resistance to potentially harmful agents. Conditions under which circadian rhythms can be observed and validated by inferential statistical means are discussed while emphasizing how artifacts of the laboratory environment can be shown to obscure circadian periodic variations in radiosensitivity.

  17. Circadian rhythmometry of mammalian radiosensitivity

    NASA Technical Reports Server (NTRS)

    Haus, E.; Halberg, F.; Loken, M. K.; Kim, Y. S.

    1974-01-01

    In the case of human bone marrow, the largest number of mitoses is seen in the evening in diurnally active men, mitotic activity being at a minimum in the morning. The opposite pattern is observed for nocturnal animals such as rats and mice on a regimen of light during the daytime alternating with darkness during the night hours. The entirety of these rhythms plays an important role in the organism's responses to environmental stimuli, including its resistance to potentially harmful agents. Conditions under which circadian rhythms can be observed and validated by inferential statistical means are discussed while emphasizing how artifacts of the laboratory environment can be shown to obscure circadian periodic variations in radiosensitivity.

  18. Radiosensitization of non-small cell lung cancer by kaempferol.

    PubMed

    Kuo, Wei-Ting; Tsai, Yuan-Chung; Wu, His-Chin; Ho, Yung-Jen; Chen, Yueh-Sheng; Yao, Chen-Han; Yao, Chun-Hsu

    2015-11-01

    The aim of the present study was to determine whether kaempferol has a radiosensitization potential for lung cancer in vitro and in vivo. The in vitro radio-sensitization activity of kaempferol was elucidated in A-549 lung cancer cells by using an MTT (3-(4 5-dimethylthiazol-2-yl)-25-diphenyl-tetrazolium bromide) assay, cell cycle analysis and clonogenic assay. The in vivo activity was evaluated in the BALB/c nude mouse xenograft model of A-549 cells by hematoxylin and eosin staining and immunohistochemistry, and the tumor volume was recorded. Protein levels of the apoptotic pathway were detected by western blot analysis. Treatment with kaempferol inhibited the growth of A-549 cells through activation of apoptotic pathway. However, the same doses did not affect HFL1 normal lung cell growth. Kaempferol induced G2/M cell cycle arrest and the enhancement of radiation-induced death and clonogenic survival inhibition. The in vivo data showed that kaempferol increased tumor cell apoptosis and killing of radiation. In conclusion, the findings demonstrated that kaempferol increased tumor cell killing by radiation in vitro and in vivo through inhibition of the AKT/PI3K and ERK pathways and activation of the mitochondria apoptosis pathway. The results of the present study provided solid evidence that kaempferol is a safe and potential radiosensitizer.

  19. Radiosensitization: enhancing the radiation inactivation of foodborne bacteria

    NASA Astrophysics Data System (ADS)

    Borsa, J.; Lacroix, M.; Ouattara, B.; Chiasson, F.

    2004-09-01

    Irradiation of meat products to kill pathogens can be limited by radiation-induced detriment of sensory quality. Since such detriment is directly related to dose, one approach to reduce it is by devising means to lower the dose of radiation required for processing. Increasing the radiation sensitivity of the target microorganisms would lower the dose required for a given level of microbial kill. In this work, the radiation sensitivities of inoculated Escherichia coli and Salmonella typhi in ground beef were examined under a variety of conditions. Results showed that specific manipulations of treatment conditions significantly increased the radiation sensitivity of the test organisms, ranging from a few percent to several-fold reduction in D10. In particular, radiation sensitization could be effected by certain additives, including carvacrol, thymol and trans-cinnamaldehyde, and also by certain compositions of modified atmosphere in the package headspace. A combination of additives and modified atmosphere effected a greater radiosensitization effect than could be achieved by either factor applied alone. Radiosensitization could be demonstrated with irradiation of either fresh or frozen ground meat. The radiosensitization phenomenon may be of practical utility in enhancing the technical effectiveness and feasibility of irradiation of a variety of meat and other food products.

  20. Modification of radiosensitivity by the so-called tissue recovery stimulator. I. Radiosensitizing effects of solcoseryl.

    PubMed

    Kumar, A; Kimura, H; Aoyama, T; Sugahara, T

    1992-12-01

    The effect of solcoseryl on the growth, radiosensitization and ability of V79 cells to recover from X-ray-induced damage has been observed. Solcoseryl at 0.8 mg/ml was the optimal concentration for the stimulation of cell growth. Increased sensitivity to X-irradiation was found in the shoulder region of V79 cells treated before and after irradiation with solcoseryl (0.8 mg/ml). The Dq and extrapolation number (n) decreased. Solcoseryl treatment apparently does not reduce split dose recovery or inhibit the repair of potentially lethal damage. Flow cytofluorometry studies of the cell cycle distribution and mitotic index show that solcoseryl inhibits the expression of radiation-induced cell arrest in the G2 phase of the cell cycle. Although this action increases radiation sensitization, additional mechanisms probably exist.

  1. Low-Dose Radiation Cataract and Genetic Determinants of Radiosensitivity

    SciTech Connect

    Kleiman, Norman Jay

    2013-11-30

    The lens of the eye is one of the most radiosensitive tissues in the body. Ocular ionizing radiation exposure results in characteristic, dose related, progressive lens changes leading to cataract formation. While initial, early stages of lens opacification may not cause visual disability, the severity of such changes progressively increases with dose until vision is impaired and cataract extraction surgery may be required. Because of the transparency of the eye, radiation induced lens changes can easily be followed non-invasively over time. Thus, the lens provides a unique model system in which to study the effects of low dose ionizing radiation exposure in a complex, highly organized tissue. Despite this observation, considerable uncertainties remain surrounding the relationship between dose and risk of developing radiation cataract. For example, a growing number of human epidemiological findings suggest significant risk among various groups of occupationally and accidentally exposed individuals and confidence intervals that include zero dose. Nevertheless, questions remain concerning the relationship between lens opacities, visual disability, clinical cataract, threshold dose and/or the role of genetics in determining radiosensitivity. Experimentally, the response of the rodent eye to radiation is quite similar to that in humans and thus animal studies are well suited to examine the relationship between radiation exposure, genetic determinants of radiosensitivity and cataractogenesis. The current work has expanded our knowledge of the low-dose effects of X-irradiation or high-LET heavy ion exposure on timing and progression of radiation cataract and has provided new information on the genetic, molecular, biochemical and cell biological features which contribute to this pathology. Furthermore, findings have indicated that single and/or multiple haploinsufficiency for various genes involved in DNA repair and cell cycle checkpoint control, such as Atm, Brca1 or Rad9

  2. Molecular Parameters of Hyperthermia for Radiosensitization

    PubMed Central

    Pandita, Tej K.; Pandita, Shruti; Bhaumik, Sukesh R.

    2011-01-01

    Hyperthermia is a potent sensitizer of cell killing by ionizing radiation (IR), however, the precise mechanism of heat-induced cell death is not yet clear. Radiosensitization can be attributed to the fact that heat is a pleiotropic damaging agent, affecting multiple cell components to varying degrees by altering protein structures, thus influencing the DNA damage response. Hyperthermia alone induces several steps associated with IR signaling in cells. For example, hyperthermia enhances ATM kinase activity and increases cellular ATM autophosphorylation. This prior activation of ATM or other components of the IR-induced signaling pathway by heat interferes with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cell killing post irradiation. Hyperthermia also induces heat shock protein 70 (HSP70) synthesis and enhances telomerase activity. HSP70 expression is associated with radioresistance. Inactivation of HSP70 and telomerase increases residual DNA DSBs post IR exposure, which correlates with increased cell killing, supporting the role of HSP70 and telomerase in IR-induced DNA damage repair. Thus, hyperthermia influences several molecular parameters involved in sensitizing tumor cells to radiation and can enhance the potential of targeted radiotherapy. PMID:19883367

  3. Radiosensitization effect of zidovudine on human malignant glioma cells

    SciTech Connect

    Zhou Fuxiang; Liao Zhengkai; Dai Jing; Xiong Jie; Xie CongHua; Luo Zhiguo; Liu Shiquan; Zhou Yunfeng . E-mail: yfzhouwhu@163.com

    2007-03-09

    Telomeres are shortened with each cell division and play an important role in maintaining chromosomal integrity and function. Telomerase, responsible for telomere synthesis, is activated in 90% of human tumor cells but seldom in normal somatic cells. Zidovudine (AZT) is a reverse transcriptase inhibitor. In this study, we have investigated the effects of {gamma}-radiation in combination with AZT on telomerase activity (TA), telomere length, DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and the changes in radiosensitivity of human malignant glioma cell line U251. The results showed that the TA was suppressed by AZT but enhanced by irradiation, resulting in a deceleration of restored rate of shortened telomere, decreased repair rate of DNA strand breaks, and increased radiosensitivity of U251 cells. Our results suggested that telomerase activity and telomere length may serve as markers for estimating the efficacy of cancer radiotherapy and reverse transcriptase inhibitors, such as AZT, may be used clinically as a new radiosensitizer in cancer radiotherapy.

  4. Preclinical evaluation of radiosensitizing activity of Pluronic block copolymers

    PubMed Central

    Perera, Reshani H.; Patel, Ravi; Wu, Hanping; Gangolli, Mihika; Traughber, Bryan; Oleinick, Nancy; Exner, Agata A.

    2014-01-01

    Purpose Pluronic block copolymers are non-ionic surfactants with demonstrated sensitizing activity in chemotherapy and hyperthermia in various tumor cell lines. In the current study we investigated the potential activity of Pluronic as a radiosensitizing agent. Materials and methods As a possible mechanism, the effect of Pluronic on Hsp70 and Hsp90 was examined. Gli36 human glioma cells were treated with radiation alone as well as with a combination treatment of Pluronic and radiation. Results Clonogenic cell survival assays show that Pluronic has an elevated effect on radiosensitization (50% high, p < 0.01), even with radiation doses as low as 2 Gy. The Hsp90 level was reduced 24 h after the combined treatment in both in vitro and in vivo. Similarly, Hsp70 levels were also decreased 24 h post treatment. When Gli36 cells were exposed to Pluronic before and during irradiation, DNA DSB: double-strand breaks repair was reduced, and elevated apoptosis was also seen in tumor xenografts. Conclusion Data suggest the potential use of L10 as a radiosensitizer. While the mechanism of sensitization requires additional investigation, the presented results indicate that the effect may be due, in part, to a decrease in Hsp90 and 70 levels and increased DNA damage. PMID:23631609

  5. Individual Radiosensitivity Measured With Lymphocytes May Predict the Risk of Acute Reaction After Radiotherapy

    SciTech Connect

    Borgmann, Kerstin; Hoeller, Ulrike; Nowack, Sven; Bernhard, Michael; Roeper, Barbara; Brackrock, Sophie; Petersen, Cordula; Szymczak, Silke; Ziegler, Andreas; Feyer, Petra; Alberti, Winfried; Dikomey, Ekkehard

    2008-05-01

    Purpose: We tested whether the chromosomal radiosensitivity of in vitro irradiated lymphocytes could be used to predict the risk of acute reactions after radiotherapy. Methods and Materials: Two prospective studies were performed: study A with 51 patients included different tumor sites and study B included 87 breast cancer patients. Acute reaction was assessed using the Radiation Therapy Oncology Group score. In both studies, patients were treated with curative radiotherapy, and the mean tumor dose applied was 55 Gy (40-65) {+-} boost with 11 Gy (6-31) in study A and 50.4 Gy {+-} boost with 10 Gy in study B. Individual radiosensitivity was determined with lymphocytes irradiated in vitro with X-ray doses of either 3 or 6 Gy and scoring the number of chromosomal deletions. Results: Acute reactions displayed a typical spectrum with 57% in study A and 53% in study B showing an acute reaction of Grade 2-3. Individual radiosensitivity in both studies was characterized by a substantial variation and the fraction of patients with Grade 2-3 reaction was found to increase with increasing individual radiosensitivity measured at 6 Gy (study A, p = 0.238; study B, p = 0.023). For study B, this fraction increased with breast volume, and the impact of individual radiosensitivity on acute reaction was especially pronounced (p = 0.00025) for lower breast volume. No such clear association with acute reaction was observed when individual radiosensitivity was assessed at 3 Gy. Conclusion: Individual radiosensitivity determined at 6 Gy seems to be a good predictor for risk of acute effects after curative radiotherapy.

  6. DNA-PKcs-Dependent Modulation of Cellular Radiosensitivity by a Selective Cyclooxygenase-2 Inhibitor

    SciTech Connect

    Kodym, Elisabeth; Kodym, Reinhard; Chen, Benjamin P.; Chen, David J.; Morotomi-Yano, Keiko; Choy, Hak; Saha, Debabrata

    2007-09-01

    Purpose: Inhibition of cyclooxygenase-2 has been shown to increase radiosensitivity. Recently, the suppression of radiation-induced DNA-dependant protein kinase (DNA-PK) activity by the selective cyclooxygenase-2 inhibitor celecoxib was reported. Given the importance of DNA-PK for repair of radiation-induced DNA double-strand breaks by nonhomologous end-joining and the clinical use of the substance, we investigated the relevance of the DNA-PK catalytic subunit (DNA-PKcs) for the modulation of cellular radiosensitivity by celecoxib. Methods and Materials: We used a syngeneic model of Chinese hamster ovarian cell lines: AA8, possessing a wild-type DNK-PKcs; V3, lacking a functional DNA-PKcs; and V3/WT11, V3 stably transfected with the DNA-PKcs. The cells were treated with celecoxib (50 {mu}M) for 24 h before irradiation. The modulation of radiosensitivity was determined using the colony formation assay. Results: Treatment with celecoxib increased the cellular radiosensitivity in the DNA-PKcs-deficient cell line V3 with a dose-enhancement ratio of 1.3 for a surviving fraction of 0.5. In contrast, clonogenic survival was increased in DNA-PKcs wild-type-expressing AA8 cells and in V3 cells transfected with DNA-PKcs (V3/WT11). The decrease in radiosensitivity was comparable to the radiosensitization in V3 cells, with a dose-enhancement ratio of 0.76 (AA8) and 0.80 (V3/WT11) for a survival of 0.5. Conclusions: We have demonstrated a DNA-PKcs-dependent differential modulation of cellular radiosensitivity by celecoxib. These effects might be attributed to alterations in signaling cascades downstream of DNA-PK toward cell survival. These findings offer an explanation for the poor outcomes in some recently published clinical trials.

  7. Enhanced radiosensitization by the cationic liposome-encapsulated thymidine analogue BrdU through the increased intracellular BrdU-uptake on human melanoma as compared to anionic or nonionic liposomal or free BrdU.

    PubMed

    Kato, Shinya; Kimura, Masatsugu; Miwa, Nobuhiko

    2014-11-01

    The synthetic thymidine analogue, 5-bromo-2'-deoxy-uridine (BrdU) was encapsulated in cationic liposome composed of dipalmitoylphosphatidylcholine, cholesterol and stearylamine (molar ratio = 1/1/0.2; diameter = 120 nm), and the radiosensitization of cationic liposomal BrdU was assessed on human melanoma cells HMV-II, with comparing to anionic or nonionic liposomal BrdU and free-BrdU. HMV-II cells were pretreated by cationic liposomal BrdU or free-BrdU and then exposed to X-ray, followed by WST-8 assay to examine cell proliferation. The radiation-induced change of nuclei was defined with Hoechst33342 staining. The rates of thymidine replacement by BrdU and DNA double-strand breaks on HMV-II cells were determined with an anti-BrdU antibody and anti-53BP1 antibody, respectively. On 7th day after X-ray irradiation at 3 Gy, cell proliferation was suppressed more markedly in the administration of cationic liposomal BrdU than free-BrdU at equivalent BrdU doses. Giant polykaryocytes were observed in cationic liposomal BrdU-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with BrdU doses of 0.1-0.8 μM in the order: cationic liposomal BrdU > anionic liposomal BrdU > nonionic liposomal BrdU (see symbol) free-BrdU. Similarly, the cationic liposomal BrdU augmented the rate of thymidine-moiety replacement by BrdU and DNA double-strand breaks more appreciably than free-BrdU. Therefore, the cationic liposome-encapsulation of BrdU would be one of favorable drug deliveries for facilitating the X-ray therapy against cancer.

  8. Radiosensitizing effect of the histone acetyltransferase inhibitor anacardic acid on various mammalian cell lines

    PubMed Central

    CATE, ROSEMARIE TEN; KRAWCZYK, PRZEMEK; STAP, JAN; ATEN, JACOB A.; FRANKEN, NICOLAAS A.P.

    2010-01-01

    Agents that enhance the effectiveness of ionizing radiation have been investigated over many decades. A relatively new group of potential radiosensitizers consists of agents that inhibit histone acetyltransferases (HATs). This study evaluated the radiosensitizing properties of the HAT inhibitor anacardic acid (AA), used at a low-toxic concentration of 100 μM in V79, SW1573 and U2OS cells. Radiation survival curves were analyzed according to the linear quadratic model. Significant radiosensitization by AA was only obtained in U2OS cells. AA significantly increased the value of the linear parameter α, but not of the quadratic parameter β, indicating fixation of potentially lethal damage and an intact repair function of sublethal damage. The increase of the α value was also observed in SW1573 cells, but was not accompanied by a significant radiosensitization. A likely explanation for the enhancement of the α value may be an increase in the amount of lethal lesions due to the compacted chromatin structure. Despite the conflicting results of the radiosensitizing effect of AA in the three cell lines tested, the ability of AA to increase the α value suggests potential advantages for clinical application. PMID:22966377

  9. Inhibiting CD146 by its Monoclonal Antibody AA98 Improves Radiosensitivity of Cervical Cancer Cells

    PubMed Central

    Cheng, Huawen

    2016-01-01

    Background Cervical cancer is one of the major causes of cancer death of females worldwide. Radiotherapy is considered effective for cervical cancer treatment, but the low radiosensitivity found in some cases severely affects therapeutic outcomes. This study aimed to reveal the role of CD146, an important adhesion molecule facilitating tumor angiogenesis, in regulating radiosensitivity of cervical cancer cells. Material/Methods CD146 protein expression was compared in normal cells, cervical cancer cells with lower radiosensitivity, and cervical cancer cells with higher sensitivity from cervical squamous cell carcinoma patients. Anti-CD146 monoclonal antibody AA98 was used to inhibit CD146 in human cervical cancer SiHa cells with relatively low radiosensitivity, and then the cell survival and apoptosis changes after radiation were detected by colony formation assay and flow cytometry. Results CD146 protein was significantly up-regulated in cervical cancer cells (P<0.001), especially in cancer cells with lower radiosensitivity. The SiHa cells treated with AA98 showed more obvious inhibition in cell survival (P<0.05) and promotion in cell apoptosis (P<0.01) after radiation, compared to the untreated cells. More dramatic changes in apoptotic factors Caspase 3 and Bcl-XL were also detected in AA98-treated cells. Conclusions These results indicate that inhibiting CD146 improves the effect of radiation in suppressing SiHa cells. This study shows the potential of CD146 as a target for increasing radiosensitivity of cervical cancer cells, which might allow improvement in treatment outcome in cervical cancer. Further studies are necessary for understanding the detailed mechanism of CD146 in regulating radiosensitivity. PMID:27647179

  10. Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells

    SciTech Connect

    Hirai, Takahisa; Saito, Soichiro; Fujimori, Hiroaki; Matsushita, Keiichiro; Nishio, Teiji; Okayasu, Ryuichi; Masutani, Mitsuko

    2016-09-09

    The poly(ADP-ribose) polymerase (PARP)-1 regulates DNA damage responses and promotes base excision repair. PARP inhibitors have been shown to enhance the cytotoxicity of ionizing radiation in various cancer cells and animal models. We have demonstrated that the PARP inhibitor (PARPi) AZD2281 is also an effective radiosensitizer for carbon-ion radiation; thus, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. Institutes for biological experiments using proton beam are limited worldwide. This study was performed as a cooperative research at heavy ion medical accelerator in Chiba (HIMAC) in National Institute of Radiological Sciences. HIMAC can generate various ion beams; this enabled us to compare the radiosensitization effect of the PARPi on cells subjected to proton and carbon-ion beams from the same beam line. After physical optimization of proton beam irradiation, the radiosensitization effect of the PARPi was assessed in the human lung cancer cell line, A549, and the pancreatic cancer cell line, MIA PaCa-2. The effect of the PARPi, AZD2281, on radiosensitization to Bragg peak was more significant than that to entrance region. The PARPi increased the number of phosphorylated H2AX (γ-H2AX) foci and enhanced G2/M arrest after proton beam irradiation. This result supports our hypothesis that a PARPi could be applied to a wide therapeutic range of LET radiation by blocking the DNA repair response. - Highlights: • Effective radiosensitizers for particle radiation therapy have not been reported. • PARP inhibitor treatment radiosensitized after proton beam irradiation. • The sensitization at Bragg peak was greater than that at entrance region. • DSB induction and G2/M arrest is involved in the sensitization mechanism.

  11. Cisplatin-mediated radiosensitization of non-small cell lung cancer cells is stimulated by ATM inhibition.

    PubMed

    Toulany, Mahmoud; Mihatsch, Julia; Holler, Marina; Chaachouay, Hassan; Rodemann, H Peter

    2014-05-01

    Cisplatin activates ataxia-telangiectasia-mutated (ATM), a protein with roles in DNA repair, cell cycle progression and autophagy. We investigated the radiosensitizing effect of cisplatin with respect to its effect on ATM pathway activation. Non-small cell lung cancer cells (NSCLC) cell lines (A549, H460) and human fibroblast (ATM-deficient AT5, ATM-proficient 1BR3) cells were used. The effects of cisplatin combined with irradiation on ATM pathway activity, clonogenicity, DNA double-strand break (DNA-DSB) repair and cell cycle progression were analyzed with Western blotting, colony formation and γ-H2AX foci assays as well as FACS analysis, respectively. Cisplatin radiosensitized H460 cells, but not A549 cells. Radiosensitization of H460 cells was not due to impaired DNA-DSB repair, increased apoptosis or cell cycle dysregulation. The lack of radiosensitization demonstrated for A549 cells was associated with cisplatin-mediated stimulation of ATM (S1981) and AMPKα (T172) phosphorylation and autophagy. However, in both cell lines inhibition of ATM and autophagy by KU-55933 and chloroquine diphosphate (CQ) respectively resulted in a significant radiosensitization. Combined treatment with the AMPK inhibitor compound-C led to radiosensitization of A549 but not of H460 cells. As compared to the treatment with KU-55933 alone, radiosensitivity of A549 cells was markedly stimulated by the combination of KU-55933 and cisplatin. However, the combination of CQ and cisplatin did not modulate the pattern of radiation sensitivity of A549 or H460 cells. In accordance with the results that cisplatin via stimulation of ATM activity can abrogate its radiosensitizing effect, ATM deficient cells were significantly sensitized to ionizing radiation by cisplatin. The results obtained indicate that ATM targeting can potentiate cisplatin-induced radiosensitization. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. Enhancement of radiosensitivity of melanoma cells by pegylated gold nanoparticles under irradiation of megavoltage electrons.

    PubMed

    Mousavi, Mehdi; Nedaei, Hassan Ali; Khoei, Samideh; Eynali, Samira; Khoshgard, Karim; Robatjazi, Mostafa; Iraji Rad, Rasoul

    2017-02-01

    Gold nanoparticles (GNP) have significant potential as radiosensitizer agents due to their distinctive properties. Several studies have shown that the surface modification of nanoparticles with methyl polyethylene glycol (mPEG) can increase their biocompatibility. However, the present study investigated the radiosensitization effects of mPEG-coated GNP (mPEG-GNP) in B16F10 murine melanoma cells under irradiation of 6 MeV Electron beam. The synthesized GNP were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy, and zeta potential. Enhancement of radiosensitization was evaluated by the clonogenic assay at different radiation doses of megavoltage electron beams. It was observed that mPEG-GNP with a hydrodynamic size of approximately 50 nm are almost spherical and cellular uptake occurred at all concentrations. Both proliferation efficiency and survival fraction decreased with increasing mPEG-GNP concentration. Furthermore, significant GNP sensitization occurred with a maximum dose enhancement factor of 1.22 at a concentration of 30 μM. Pegylated-GNP are taken up by B16F10 cancer cells and cause radiosensitization in the presence of 6 MeV electrons. The radiosensitization effects of GNP may probably be due to biological processes. Therefore, the underlying biological mechanisms beyond the physical dose enhancement need to be further clarified.

  13. Clofarabine Acts as Radiosensitizer In Vitro and In Vivo by Interfering With DNA Damage Response

    SciTech Connect

    Cariveau, Mickael J.; Stackhouse, Murray; Cui Xiaoli; Tiwari, Kamal; Waud, William; Secrist, John A.; Xu Bo

    2008-01-01

    Purpose: Combination treatment with radiotherapy and chemotherapy has emerged as the dominant form of cancer adjuvant regimens in recent years. Clofarabine, a newly approved drug for pediatric leukemia, is a second-generation purine nucleoside analogue that can block DNA synthesis and inhibit DNA repair. Therefore, we hypothesized that clofarabine could work synergistically with radiotherapy to increase the tumor cell response. Methods and Materials: The effects of clofarabine on radiosensitivity have been established in several tumor cell lines in vitro and in vivo using colony-forming assays and tumor xenografts. The effect of clofarabine on the DNA damage response was also studied in vitro by measuring {gamma}-H2AX focus formation. Results: Clonogenic survival was significantly reduced in irradiated cells treated with clofarabine, demonstrating the strong radiosensitizing effect of clofarabine. Furthermore, clofarabine displayed a radiosensitizing effect that was greater than gemcitabine or 5-fluorouracil. We also found that low doses of clofarabine can prolong the presence of radiation-induced {gamma}-H2AX nuclear focus formation, and high doses of clofarabine can induce DNA double-strand breaks, suggesting that clofarabine can interfere with DNA damage response pathways. In addition, clofarabine-induced radiosensitization was also established in vivo using a colorectal cancer model, DLD-1, in athymic nude mice. When combined with fractionated radiotherapy, a moderate dose of clofarabine led to a significant increase in tumor growth inhibition. Conclusion: Clofarabine acts as a powerful radiosensitizer both in vitro and in vivo by interfering with the DNA damage response.

  14. Lanthanum fluoride nanoparticles for radiosensitization of tumors

    NASA Astrophysics Data System (ADS)

    Kudinov, Konstantin; Bekah, Devesh; Cooper, Daniel; Shastry, Sathvik; Hill, Colin; Bradforth, Stephen; Nadeau, Jay

    2016-03-01

    Dense inorganic nanoparticles have recently been identified as promising radiosensitizers. In addition to dose enhancement through increased attenuation of ionizing radiation relative to biological tissue, scintillating nanoparticles can transfer energy to coupled photosensitizers to amplify production of reactive oxygen species, as well as provide UVvisible emission for optical imaging. Lanthanum fluoride is a transparent material that is easily prepared as nanocrystals, and which can provide radioluminescence at a number of wavelengths through simple substitution of lanthanum ions with other luminescent lanthanides. We have prepared lanthanum fluoride nanoparticles doped with cerium, terbium, or both, that have good spectral overlap with chlorine6 or Rose Bengal photosensitizer molecules. We have also developed a strategy for stable conjugation of the photosensitizers to the nanoparticle surface, allowing for high energy transfer efficiencies on a per molecule basis. Additionally, we have succeeded in making our conjugates colloidally stable under physiological conditions. Here we present our latest results, using nanoparticles and nanoparticle-photosensitizer conjugates to demonstrate radiation dose enhancement in B16 melanoma cells. The effects of nanoparticle treatment prior to 250 kVp x-ray irradiation were investigated through clonogenic survival assays and cell cycle analysis. Using a custom apparatus, we have also observed scintillation of the nanoparticles and conjugates under the same conditions that the cell samples are irradiated.

  15. The Hsp70 inhibiting peptide aptamer A17 potentiates radiosensitization of tumor cells by Hsp90 inhibition.

    PubMed

    Schilling, Daniela; Garrido, Carmen; Combs, Stephanie E; Multhoff, Gabriele

    2017-04-01

    The inhibition of heat shock protein 90 (Hsp90) is a promising strategy to increase the radiosensitivity of tumor cells. However, Hsp90 inhibition induces the expression of Hsp70 which is a prominent cytoprotective protein. Therefore, dual targeting of Hsp70 and Hsp90 might be beneficial to increase the radiosensitivity of tumor cells. Hsp70 inhibiting peptide aptamers have been shown to increase the sensitivity of tumor cells to apoptosis induced by different anticancer drugs. Herein, we studied the radiosensitizing activity of the Hsp70 inhibiting peptide aptamer A17 in combination with the Hsp90 inhibitor NVP-AUY922. Whereas A17 significantly increased apoptosis induction by NVP-AUY922 it did not significantly affect the radiosensitivity of human lung and breast cancer cells. However, Hsp70 inhibition by the aptamer A17 potentiated the radiosensitizing effects of the Hsp90 inhibitor NVP-AUY922. Mechanistically we speculate that an increased number of DNA double strand breaks and an enhanced G2/M arrest might be responsible for the increased radiosensitization in A17 expressing tumor cells. Therefore, the simultaneous inhibition of Hsp90 and Hsp70 combined with radiotherapy might provide a promising anti-cancer strategy.

  16. Radiosensitization of DNA in presence of Pt(II)-based compounds

    NASA Astrophysics Data System (ADS)

    Śmiałek, Małgorzata A.; Ptasińska, Sylwia; Gow, Jason; Pieve, Chiara Da; Mason, Nigel J.

    2014-04-01

    X-ray irradiation of plasmid DNA in presence of platinum (II)-based compounds was carried out in order to assess the radiosensitization capabilities of these drugs. In present investigations pBR322 plasmid DNA was used to monitor the effectiveness of chosen compounds in inducing strand breaks. Samples were incubated in the presence of potential radiosensitisers: platinum (II) bromide and cis-diamminedibromoplatinum (II). The results were examined against a common cancer chemotherapy drug cis-diamminedichloroplatinum (II). It was found that platinum (II) bromide can greatly increase the levels of single- and double-strand break formation observed in the irradiated samples with respect to the samples containing platinum as a radiosensitizer only, possessing very little chemotherapeutic activity. The suggested drugs exhibit much higher level of radiosensitivity than widely used cisplatin and thus may be good candidates for cancer treatment.

  17. Effect of anemia on tumor radiosensitivity under normo and hyperbaric conditions

    SciTech Connect

    Rojas, A.; Stewart, F.A.; Smith, K.A.; Soranson, J.A.; Randhawa, V.S.; Stratford, M.R.; Denekamp, J.

    1987-11-01

    The effect of chronic anemia on tumor radiosensitivity in a murine tumor has been investigated. Anemia was induced by bilateral kidney irradiation given several months before tumor implantation. Anemic, anemic transfused, and normal non-anemic age-matched tumor bearing animals were irradiated with X rays (2 F/24 hr) either in air, air plus misonidazole, or under hyperbaric oxygen. The most resistant response was that of tumors grown in normal mice treated in air. Anemia produced an increase in radiosensitivity which was further enhanced by red blood cell replacement. The most sensitive overall response was seen in the anemic-transfused group treated with HBO.

  18. Prostate cancer radiosensitization through PARP-1 hyperactivation

    PubMed Central

    Dong, Ying; Bey, Erik A.; Li, Long-Shan; Kabbani, Wareef; Yan, Jingsheng; Xie, Xian-Jin; Hsieh, Jer-Tsong; Gao, Jinming; Boothman, David A.

    2010-01-01

    The clinical experimental agent, β-lapachone (Arq 501), can act as a potent radiosensitizer in vitro through an unknown mechanism. In this study, we analyzed the mechanism to determine whether β-lapachone may warrant clinical evaluation as a radiosensitizer. β-lapachone killed prostate cancer cells by NAD(P)H:quinone oxidoreductase 1 (NQO1) metabolic bioactivation, triggering a massive induction of reactive oxygen species (ROS), irreversible DNA single strand breaks (SSBs), PARP-1 hyperactivation, NAD+/ATP depletion, and μ-calpain-induced programmed necrosis. In combination with ionizing radiation (IR), β-lapachone radiosensitized NQO1+ prostate cancer cells, under conditions where nontoxic doses of either agent alone achieved threshold levels of SSBs required for hyperactivation of PARP-1. Combination therapy significantly elevated SSBs, γ-H2AX foci formation, and poly(ADP-ribosylation) of PARP-1, which were associated with ATP loss and induction of μ-calpain-induced programmed cell death. Radiosensitization by β-lapachone was blocked by the NQO1 inhibitor, dicoumarol, or the PARP-1 inhibitor, DPQ. In a mouse xenograft model of prostate cancer, β-lapachone synergized with IR to promote antitumor efficacy. NQO1 levels were elevated in ~60% of human prostate tumors evaluated relative to adjacent normal tissue, where β-lapachone might be efficacious alone or in combination with radiation. Our findings offer a rationale for clinical assessment of β-lapachone (Arq501) as a radiosensitizer in prostate cancers that overexpress NQO1, offering a potentially synergistic targeting strategy to exploit PARP-1 hyperactivation. PMID:20940411

  19. Radiosensitization by the novel DNA intercalating agent vosaroxin.

    PubMed

    Gordon, Ira K; Graves, Christian; Kil, Whoon J; Kramp, Tamalee; Tofilon, Philip; Camphausen, Kevin

    2012-02-27

    Vosaroxin is a first in class naphthyridine analog structurally related to quinolone antibacterials, that intercalates DNA and inhibits topoisomerase II. Vosaroxin is not a P-glycoprotein receptor substrate and its activity is independent of p53, thus evading common drug resistance mechanisms. To evaluate vosaroxin as a clinically applicable radiation sensitizer, we investigated its effects on tumor cell radiosensitivity in vitro and in vivo. Vosaroxin's effect on post-irradiation sensitivity of U251, DU145, and MiaPaca-2 cells was assessed by clonogenic assay. Subsequent mechanistic and in vivo studies were performed with U251 cells. Cell cycle distribution and G2 checkpoint integrity was analyzed by flow cytometry. DNA damage and repair was evaluated by a high throughput gamma-H2AX assay. Apoptosis was assessed by flow cytometry. Mitotic catastrophe was assessed by microscopic evidence of fragmented nuclei by immunofluorescence. In vivo radiosensitization was measured by subcutaneous tumor growth delay. 50-100 nmol/L treatment with vosaroxin resulted in radiosensitization of all 3 cell lines tested with a dose enhancement factor of 1.20 to 1.51 measured at a surviving fraction of 0.1. The maximal dose enhancement was seen in U251 cells treated with 75 nmol/L vosaroxin (DEF 1.51). Vosaroxin exposure did not change cell cycle distribution prior to irradiation nor alter G2 checkpoint integrity after irradiation. No difference was seen in the apoptotic fraction regardless of drug or radiation treatment. The number of cells in mitotic catastrophe was significantly greater in irradiated cells treated with vosaroxin than cells receiving radiation only at 72 hr (p = 0.009). Vosaroxin alone did not significantly increase mitotic catastrophe over control (p = 0.53). Cells treated with vosaroxin and radiation maintained significantly higher gamma-H2AX levels than cells treated with vehicle control (p = 0.014), vosaroxin (p = 0.042), or radiation alone (p = 0.039) after 24

  20. G2 checkpoint control and G2 chromosomal radiosensitivity in cancer survivors and their families

    PubMed Central

    Cadwell, Kevin K.; Curwen, Gillian B.; Tawn, E. Janet; Winther, Jeanette F.; Boice, John D.

    2011-01-01

    Significant inter-individual variation in G2 chromosomal radiosensitivity, measured as radiation-induced chromatid-type aberrations in the subsequent metaphase, has been reported in peripheral blood lymphocytes of both healthy individuals and a range of cancer patients. One possible explanation for this variation is that it is driven, at least in part, by the efficiency of G2–M checkpoint control. The hypothesis tested in the current analysis is that increased G2 chromosomal radiosensitivity is facilitated by a less efficient G2–M checkpoint. The study groups comprised 23 childhood and adolescent cancer survivors, their 23 partners and 38 of their offspring (Group 1) and 29 childhood and young adult cancer survivors (Group 2). Following exposure to 0.5 Gy of 300 kV X-rays, lymphocyte cultures were assessed for both G2 checkpoint delay and G2 chromosomal radiosensitivity. In Group 1, the extent of G2 checkpoint delay was measured by mitotic inhibition. No statistically significant differences in G2 checkpoint delay were observed between the cancer survivors (P = 0.660) or offspring (P = 0.171) and the partner control group nor was there any significant relationship between G2 checkpoint delay and G2 chromosomal radiosensitivity in the cancer survivors (P = 0.751), the partners (P = 0.634), the offspring (P = 0.824) or Group 1 taken as a whole (P = 0.379). For Group 2, G2 checkpoint delay was assessed with an assay utilising premature chromosome condensation to distinguish cell cycle stage. No significant relationship between G2 checkpoint delay and G2 chromosomal radiosensitivity was found (P = 0.284). Thus, this study does not support a relationship between G2–M checkpoint efficiency and variation in G2 chromosomal radiosensitivity. PMID:21044988

  1. G2 checkpoint control and G2 chromosomal radiosensitivity in cancer survivors and their families.

    PubMed

    Cadwell, Kevin K; Curwen, Gillian B; Tawn, E Janet; Winther, Jeanette F; Boice, John D

    2011-03-01

    Significant inter-individual variation in G(2) chromosomal radiosensitivity, measured as radiation-induced chromatid-type aberrations in the subsequent metaphase, has been reported in peripheral blood lymphocytes of both healthy individuals and a range of cancer patients. One possible explanation for this variation is that it is driven, at least in part, by the efficiency of G(2)-M checkpoint control. The hypothesis tested in the current analysis is that increased G(2) chromosomal radiosensitivity is facilitated by a less efficient G(2)-M checkpoint. The study groups comprised 23 childhood and adolescent cancer survivors, their 23 partners and 38 of their offspring (Group 1) and 29 childhood and young adult cancer survivors (Group 2). Following exposure to 0.5 Gy of 300 kV X-rays, lymphocyte cultures were assessed for both G(2) checkpoint delay and G(2) chromosomal radiosensitivity. In Group 1, the extent of G(2) checkpoint delay was measured by mitotic inhibition. No statistically significant differences in G(2) checkpoint delay were observed between the cancer survivors (P = 0.660) or offspring (P = 0.171) and the partner control group nor was there any significant relationship between G(2) checkpoint delay and G(2) chromosomal radiosensitivity in the cancer survivors (P = 0.751), the partners (P = 0.634), the offspring (P = 0.824) or Group 1 taken as a whole (P = 0.379). For Group 2, G(2) checkpoint delay was assessed with an assay utilising premature chromosome condensation to distinguish cell cycle stage. No significant relationship between G(2) checkpoint delay and G(2) chromosomal radiosensitivity was found (P = 0.284). Thus, this study does not support a relationship between G(2)-M checkpoint efficiency and variation in G(2) chromosomal radiosensitivity.

  2. Macrophages enhance the radiosensitizing activity of lipid A: A novel role for immune cells in tumor cell radioresponse

    SciTech Connect

    Ridder, Mark de . E-mail: Mark.De.Ridder@vub.ac.be; Verovski, Valeri N.; Darville, Martine I.; Berge, Dirk L. van den; Monsaert, Christinne; Eizirik, Decio L.; Storme, Guy A.

    2004-10-01

    Purpose: This study examines whether activated macrophages may radiosensitize tumor cells through the release of proinflammatory mediators. Methods and materials: RAW 264.7 macrophages were activated by lipid A, and the conditioned medium (CM) was analyzed for the secretion of cytokines and the production of nitric oxide (NO) through inducible nitric oxide synthase (iNOS). EMT-6 tumor cells were exposed to CM and analyzed for hypoxic cell radiosensitivity. The role of nuclear factor (NF)-{kappa}B in the transcriptional activation of iNOS was examined by luciferase reporter gene assay. Results: Clinical immunomodulator lipid A, at a plasma-relevant concentration of 3 {mu}g/mL, stimulated RAW 264.7 macrophages to release NO, tumor necrosis factor (TNF)-{alpha}, and other cytokines. This in turn activated iNOS-mediated NO production in EMT-6 tumor cells and drastically enhanced their radiosensitivity. Radiosensitization was abrogated by the iNOS inhibitor aminoguanidine but not by a neutralizing anti-TNF-{alpha} antibody. The mechanism of iNOS induction was linked to NF-{kappa}B but not to JAK/STAT signaling. Interferon-{gamma} further increased the NO production by macrophages to a level that caused radiosensitization of EMT-6 cells through the bystanding effect of diffused NO. Conclusions: We demonstrate for the first time that activated macrophages may radiosensitize tumor cells through the induction of NO synthesis, which occurs in both tumor and immune cells.

  3. Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks

    PubMed Central

    Martin, N T; Nakamura, K; Paila, U; Woo, J; Brown, C; Wright, J A; Teraoka, S N; Haghayegh, S; McCurdy, D; Schneider, M; Hu, H; Quinlan, A R; Gatti, R A; Concannon, P

    2014-01-01

    The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis. PMID:24651433

  4. Solution structure of an oncogenic DNA duplex, the K-ras gene and the sequence containing a central C.A or A.G mismatch as a function of pH: nuclear magnetic resonance and molecular dynamics studies.

    PubMed

    Boulard, Y; Cognet, J A; Gabarro-Arpa, J; Le Bret, M; Carbonnaux, C; Fazakerley, G V

    1995-02-10

    The DNA duplex 5' d(GCCACCAGCTC)-d(GAGCTGGTGGC) corresponds to the sequence 29 to 39 of the K-ras gene, which contains a hot spot for mutations. This has been studied by one and two-dimensional nuclear magnetic resonance, energy minimization and molecular dynamics. The results show that it adopts a globally B-DNA type structure. We have introduced, at the central base-pair, the mismatches C.A and A.G. The mismatch position is that of the first base of the Gly12 codon, the hot spot. For the C.A mismatch we observe a structural change as a function of pH with an apparent pKa of 7.2. At low pH, the mismatch pair adopts a structure close to a classic wobble conformation with the cytidine residue displaced into the major groove. It is stabilised by two hydrogen bonds in which the adenosine residue is protonated and the cytidine residue has a significant C3'-endo population. At high pH, the mispair structure is in equilibrium between wobble and reverse wobble conformations. Similar studies are reported on the A.G mismatch, which also undergoes a transition as a function of pH. 31P spectra have been recorded on all systems and as a function of pH. No evidence for BII phosphodiester backbone conformations was found. The NMR results are well corroborated by molecular dynamics calculations performed with or without distance constraints. The dynamics at the mismatch sites have been examined. Although the overall structures are close to B-DNA, helical parameters fluctuate differently at these sites. Different hydrogen bonding alternatives in dynamic equilibrium that can involve three-centred hydrogen bonds are observed.

  5. Analysis of chromosomal radiosensitivity of healthy BRCA2 mutation carriers and non-carriers in BRCA families with the G2 micronucleus assay.

    PubMed

    Baert, Annelot; Depuydt, Julie; Van Maerken, Tom; Poppe, Bruce; Malfait, Fransiska; Van Damme, Tim; De Nobele, Sylvia; Perletti, Gianpaolo; De Leeneer, Kim; Claes, Kathleen B M; Vral, Anne

    2017-03-01

    Breast cancer risk drastically increases in individuals with a heterozygous germline BRCA1 or BRCA2 mutation, while it is estimated to equal the population risk for relatives without the familial mutation (non-carriers). The aim of the present study was to use a G2 phase-specific micronucleus assay to investigate whether lymphocytes of healthy BRCA2 mutation carriers are characterized by increased radiosensitivity compared to controls without a family history of breast/ovarian cancer and how this relates to healthy non-carrier relatives. BRCA2 is active in homologous recombination, a DNA damage repair pathway, specifically active in the late S/G2 phase of the cell cycle. We found a significantly increased radiosensitivity in a cohort of healthy BRCA2 mutation carriers compared to individuals without a familial history of breast cancer (P=0.046; Mann-Whitney U test). At the individual level, 50% of healthy BRCA2 mutation carriers showed a radiosensitive phenotype (radiosensitivity score of 1 or 2), whereas 83% of the controls showed no radiosensitivity (P=0.038; one-tailed Fisher's exact test). An odds ratio of 5 (95% CI, 1.07-23.47) indicated an association between the BRCA2 mutation and radiosensitivity in healthy mutation carriers. These results indicate the need for the gentle use of ionizing radiation for either diagnostic or therapeutic use in BRCA2 mutation carriers. We detected no increased radiosensitivity in the non-carrier relatives.

  6. Analysis of chromosomal radiosensitivity of healthy BRCA2 mutation carriers and non-carriers in BRCA families with the G2 micronucleus assay

    PubMed Central

    Baert, Annelot; Depuydt, Julie; Van Maerken, Tom; Poppe, Bruce; Malfait, Fransiska; Van Damme, Tim; De Nobele, Sylvia; Perletti, Gianpaolo; De Leeneer, Kim; Claes, Kathleen B.M.; Vral, Anne

    2017-01-01

    Breast cancer risk drastically increases in individuals with a heterozygous germline BRCA1 or BRCA2 mutation, while it is estimated to equal the population risk for relatives without the familial mutation (non-carriers). The aim of the present study was to use a G2 phase-specific micronucleus assay to investigate whether lymphocytes of healthy BRCA2 mutation carriers are characterized by increased radiosensitivity compared to controls without a family history of breast/ovarian cancer and how this relates to healthy non-carrier relatives. BRCA2 is active in homologous recombination, a DNA damage repair pathway, specifically active in the late S/G2 phase of the cell cycle. We found a significantly increased radiosensitivity in a cohort of healthy BRCA2 mutation carriers compared to individuals without a familial history of breast cancer (P=0.046; Mann-Whitney U test). At the individual level, 50% of healthy BRCA2 mutation carriers showed a radiosensitive phenotype (radiosensitivity score of 1 or 2), whereas 83% of the controls showed no radiosensitivity (P=0.038; one-tailed Fishers exact test). An odds ratio of 5 (95% CI, 1.07–23.47) indicated an association between the BRCA2 mutation and radiosensitivity in healthy mutation carriers. These results indicate the need for the gentle use of ionizing radiation for either diagnostic or therapeutic use in BRCA2 mutation carriers. We detected no increased radiosensitivity in the non-carrier relatives. PMID:28184943

  7. In vitro 3-dimensional tumor model for radiosensitivity of HPV positive OSCC cell lines.

    PubMed

    Zhang, Mei; Rose, Barbara; Lee, C Soon; Hong, Angela M

    2015-01-01

    The incidence of oropharyngeal squamous cell carcinoma (OSCC) is increasing due to the rising prevalence of human papillomavirus (HPV) positive OSCC. HPV positive OSCC is associated with better outcomes than HPV negative OSCC. Our aim was to explore the possibility that this favorable prognosis is due to the enhanced radiosensitivity of HPV positive OSCC. HPV positive OSCC cell lines were generated from the primary OSCCs of 2 patients, and corresponding HPV positive cell lines generated from nodal metastases following xenografting in nude mice. Monolayer and 3 dimensional (3D) culture techniques were used to compare the radiosensitivity of HPV positive lines with that of 2 HPV negative OSCC lines. Clonogenic and protein assays were used to measure survival post radiation. Radiation induced cell cycle changes were studied using flow cytometry. In both monolayer and 3D culture, HPV positive cells exhibited a heterogeneous appearance whereas HPV negative cells tended to be homogeneous. After irradiation, HPV positive cells had a lower survival in clonogenic assays and lower total protein levels in 3D cultures than HPV negative cells. Irradiated HPV positive cells showed a high proportion of cells in G1/S phase, increased apoptosis, an increased proliferation rate, and an inability to form 3D tumor clumps. In conclusion, HPV positive OSCC cells are more radiosensitive than HPV negative OSCC cells in vitro, supporting a more radiosensitive nature of HPV positive OSCC.

  8. Atm deletion with dual recombinase technology preferentially radiosensitizes tumor endothelium.

    PubMed

    Moding, Everett J; Lee, Chang-Lung; Castle, Katherine D; Oh, Patrick; Mao, Lan; Zha, Shan; Min, Hooney D; Ma, Yan; Das, Shiva; Kirsch, David G

    2014-08-01

    Cells isolated from patients with ataxia telangiectasia are exquisitely sensitive to ionizing radiation. Kinase inhibitors of ATM, the gene mutated in ataxia telangiectasia, can sensitize tumor cells to radiation therapy, but concern that inhibiting ATM in normal tissues will also increase normal tissue toxicity from radiation has limited their clinical application. Endothelial cell damage can contribute to the development of long-term side effects after radiation therapy, but the role of endothelial cell death in tumor response to radiation therapy remains controversial. Here, we developed dual recombinase technology using both FlpO and Cre recombinases to generate primary sarcomas in mice with endothelial cell-specific deletion of Atm to determine whether loss of Atm in endothelial cells sensitizes tumors and normal tissues to radiation. Although deletion of Atm in proliferating tumor endothelial cells enhanced the response of sarcomas to radiation, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. Blocking cell cycle progression reversed the effect of Atm loss on tumor endothelial cell radiosensitivity. These results indicate that endothelial cells must progress through the cell cycle in order to be radiosensitized by Atm deletion.

  9. SU11657 Enhances Radiosensitivity of Human Meningioma Cells

    SciTech Connect

    Milker-Zabel, Stefanie Bois, Angelika Zabel-du; Ranai, Gholamreza; Trinh, Thuy; Unterberg, Andreas; Debus, Juergen; Lipson, Kenneth E.; Abdollahi, Amir; Huber, Peter E.

    2008-03-15

    Purpose: To analyze the effect of the multireceptor tyrosine kinase inhibitor SU11657 (primarily vascular endothelial growth factor, platelet-derived growth factor) in combination with irradiation in freshly isolated primary human meningioma cells. Methods and Materials: Tumor specimens were obtained from meningioma patients undergoing surgery at the Department of Neurosurgery, University of Heidelberg, Germany. For the present study only cells up to passage 6 were used. Benign and atypical meningioma cells and human umbilical vein endothelial cells (HUVEC) were treated with SU11657 alone and in combination with 6-MV photons (0-10 Gy). Clonogenic survival and cell proliferation were determined alone and in coculture assays to determine direct and paracrine effects. Results: Radiation and SU11657 alone reduced cell proliferation in atypical and benign meningioma cells as well as in HUVEC in a dose-dependent manner. SU11657 alone also reduced clonogenic survival of benign and atypical meningioma cells. SU11657 increased radiosensitivity of human meningioma cells in clonogenic survival and cell number/proliferation assays. The anticlonogenic and antiproliferative effects alone and the radiosensitization effects of SU11657 were more pronounced in atypical meningioma cells compared with benign meningioma cells. Conclusion: Small-molecule tyrosine kinase inhibitors like SU11657 are capable of amplifying the growth inhibitory effects of irradiation in meningioma cells. These data provide a rationale for further clinical evaluation of this combination concept, especially in atypical and malignant meningioma patients.

  10. Numb/Notch signaling pathway modulation enhances human pancreatic cancer cell radiosensitivity.

    PubMed

    Bi, Yi-Liang; Min, Min; Shen, Wei; Liu, Yan

    2016-11-01

    The present study aims to evaluate whether repression of the Numb/Notch signaling pathway affects the radiosensitivity of human pancreatic cancer cell lines. Different doses of X-rays (0, 2, 3, 4, and 5 Gy) were applied to the PANC-1, SW1990, and MIA PaCa-2 human pancreatic cancer cell lines, and the Numb/Notch pathway inhibitor DAPT was added at different doses (0, 1, 3, and 5 μmol/l). MTT assay, colony formation assay, flow cytometry, scratch assay, and Transwell experiments were performed, and qRT-PCR and Western blot were conducted for the detection of Numb expression. Tumorigenicity assay in nude mice was carried out to verify the influence of blocker of the Numb/Notch signaling pathway on the radiosensitivity of xenograft tumors. The MTT assay, colony formation assay and flow cytometry experiments revealed that proliferation decreased as radiation dose increased. The viability of PANC-1 cells at 5 Gy, SW 1990 cells at 4 Gy and 5 Gy, and MIA PaCa-2 cells at 2-5 Gy was significantly lower than that of non-irradiated cells (all P < 0.05). The migration and invasion assays indicated that the PANC-1 cell line was least radiosensitive, while the MIA PaCa-2 cell line was the most radiosensitive. Numb expression significantly increased with increasing radiation dose, whereas the expression of Hes1, Notch1, and Hes5 significantly decreased compared to non-irradiated cells (P < 0.05). Compared to untreated control cells, DAPT dose dependently increased Numb expression and inhibited Notch1, Hes1, and Hes5 expressions at 2 Gy (P < 0.05). Subcutaneous tumorigenicity assay in nude mice demonstrated that DAPT increased the radiosensitivity of PANC-1, SW 1990, and MIA PaCa-2 cells. These findings suggest that Numb/Notch signaling in pancreatic cancer cells is associated with X-ray radiation and that inhibition of the Numb/Notch signaling pathway can enhance radiosensitivity, suggesting that inhibition of the Numb/Notch signaling pathway may serve as a potential

  11. Characterization of in vitro radiosensitization in mammalian cells using biomathematical modelling: implications for hypofractionated radiotherapy with a combined modality approach.

    PubMed

    Seo, Yuji; Tamari, Keisuke; Yoshioka, Yasuo; Isohashi, Fumiaki; Suzuki, Osamu; Hayashi, Kazuhiko; Takahashi, Yutaka; Baek, SungJae; Otani, Keisuke; Ogawa, Kazuhiko

    2016-06-01

    It is unclear whether radiosensitization is beneficial when radiotherapy is administered at a high dose per fraction. The aim of this study was to assess the impact of radiation dose on the effectiveness of a broad range of radiosensitizers. We analyzed 653 pairs of clonogenic survival curves in 285 published articles, in which modifications of radiosensitivity were studied using the colony-forming assay. The modifications of radiosensitivity were arbitrarily classified into 20 classes. The survival curves were fitted to two biomathematical models: the linear-quadratic model and the repair-misrepair (RMR) model. We found that radiosensitization was predominantly characterized by an increase of the α value (α-sensitization) without an increase of the β value (β-sensitization). A subset analysis revealed that all 20 classes showed significant α-sensitization. In contrast, only oxygen/hypoxic sensitizers (oxygen) and poly(adenosine diphosphate-ribose) polymerase inhibition (PARPi) exhibited β-sensitization. An analysis using the RMR model revealed two major sources of radiosensitization: an increased residual DNA lesion through repair inhibition and a shift from linear repairs to quadratic misrepairs, leading to enhanced lethal chromosomal aberrations. Oxygen and PARPi were found to show β-sensitization, which was favourable for eliciting a comparable degree of sensitization in the higher dose range. Reduced fidelity of the repair was suggested to be a possible mechanism of β-sensitization. Further study targeting β-sensitization is needed to develop a novel combined modality therapy with high-dose-per-fraction radiotherapy. Radiosensitization can be classified into two groups, α- and β-sensitizations. These two phenomena may stem from distinct underlying mechanisms.

  12. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  13. Short Hairpin RNA Suppression of Thymidylate Synthase Produces DNA Mismatches and Results in Excellent Radiosensitization

    SciTech Connect

    Flanagan, Sheryl A.; Cooper, Kristin S.; Mannava, Sudha; Nikiforov, Mikhail A.; Shewach, Donna S.

    2012-12-01

    Purpose: To determine the effect of short hairpin ribonucleic acid (shRNA)-mediated suppression of thymidylate synthase (TS) on cytotoxicity and radiosensitization and the mechanism by which these events occur. Methods and Materials: shRNA suppression of TS was compared with 5-fluoro-2 Prime -deoxyuridine (FdUrd) inactivation of TS with or without ionizing radiation in HCT116 and HT29 colon cancer cells. Cytotoxicity and radiosensitization were measured by clonogenic assay. Cell cycle effects were measured by flow cytometry. The effects of FdUrd or shRNA suppression of TS on dNTP deoxynucleotide triphosphate imbalances and consequent nucleotide misincorporations into deoxyribonucleic acid (DNA) were analyzed by high-pressure liquid chromatography and as pSP189 plasmid mutations, respectively. Results: TS shRNA produced profound ({>=}90%) and prolonged ({>=}8 days) suppression of TS in HCT116 and HT29 cells, whereas FdUrd increased TS expression. TS shRNA also produced more specific and prolonged effects on dNTPs deoxynucleotide triphosphates compared with FdUrd. TS shRNA suppression allowed accumulation of cells in S-phase, although its effects were not as long-lasting as those of FdUrd. Both treatments resulted in phosphorylation of Chk1. TS shRNA alone was less cytotoxic than FdUrd but was equally effective as FdUrd in eliciting radiosensitization (radiation enhancement ratio: TS shRNA, 1.5-1.7; FdUrd, 1.4-1.6). TS shRNA and FdUrd produced a similar increase in the number and type of pSP189 mutations. Conclusions: TS shRNA produced less cytotoxicity than FdUrd but was equally effective at radiosensitizing tumor cells. Thus, the inhibitory effect of FdUrd on TS alone is sufficient to elicit radiosensitization with FdUrd, but it only partially explains FdUrd-mediated cytotoxicity and cell cycle inhibition. The increase in DNA mismatches after TS shRNA or FdUrd supports a causal and sufficient role for the depletion of dTTP thymidine triphosphate and consequent DNA

  14. Differential Radiosensitizing Effect of Valproic Acid in Differentiation Versus Self-Renewal Promoting Culture Conditions

    SciTech Connect

    Debeb, Bisrat G.; Xu Wei; Mok, Henry; Li Li; Robertson, Fredika; Ueno, Naoto T.; Reuben, Jim; Lucci, Anthony; Cristofanilli, Massimo; Woodward, Wendy A.

    2010-03-01

    Purpose: It has been shown that valproic acid (VA) enhances the proliferation and self-renewal of normal hematopoietic stem cells and that breast cancer stem/progenitor cells can be resistant to radiation. From these data, we hypothesized that VA would fail to radiosensitize breast cancer stem/progenitor cells grown to three-dimensional (3D) mammospheres. Methods and Materials: We used the MCF7 breast cancer cell line grown under stem cell-promoting culture conditions (3D mammosphere) and standard nonstem cell monolayer culture conditions (two-dimensional) to examine the effect of pretreatment with VA on radiation sensitivity in clonogenic survival assays and on the expression of embryonic stem cell transcription factors. Results: 3D-cultured MCF-7 cells expressed higher levels of Oct4, Nanog, and Sox2. The 3D passage enriched self-renewal and increased radioresistance in the 3D mammosphere formation assays. VA radiosensitized adherent cells but radioprotected 3D cells in single-fraction clonogenic assays. Moreover, fractionated radiation sensitized VA-treated adherent MCF7 cells but did not have a significant effect on VA-treated single cells grown to mammospheres. Conclusion: We have concluded that VA might preferentially radiosensitize differentiated cells compared with those expressing stem cell surrogates and that stem cell-promoting culture is a useful tool for in vitro evaluation of novel cancer therapeutic agents and radiosensitizers.

  15. Chromosomal radiosensitivity of human immunodeficiency virus positive/negative cervical cancer patients in South Africa

    PubMed Central

    HERD, OLIVIA; FRANCIES, FLAVIA; KOTZEN, JEFFREY; SMITH, TRUDY; NXUMALO, ZWIDE; MULLER, XANTHENE; SLABBERT, JACOBUS; VRAL, ANNE; BAEYENS, ANS

    2016-01-01

    Cervical cancer is the second most common cancer amongst South African women and is the leading cause of cancer-associated mortality in this region. Several international studies on radiation-induced DNA damage in lymphocytes of cervical cancer patients have remained inconclusive. Despite the high incidence of cervical cancer in South Africa, and the extensive use of radiotherapy to treat it, the chromosomal radiosensitivity of South African cervical cancer patients has not been studied to date. Since a high number of these patients are human immunodeficiency virus (HIV)-positive, the effect of HIV infection on chromosomal radiosensitivity was also investigated. Blood samples from 35 cervical cancer patients (20 HIV-negative and 15 HIV-positive) and 20 healthy controls were exposed to X-rays at doses of 6 MV of 2 and 4 Gy in vitro. Chromosomal radiosensitivity was assessed using the micronucleus (MN) assay. MN scores were obtained using the Metafer 4 platform, an automated microscopic system. Three scoring methods of the MNScore module of Metafer were applied and compared. Cervical cancer patients had higher MN values than healthy controls, with HIV-positive patients having the highest MN values. Differences between groups were significant when using a scoring method that corrects for false positive and false negative MN. The present study suggested increased chromosomal radiosensitivity in HIV-positive South African cervical cancer patients. PMID:26549042

  16. Optimal energy for cell radiosensitivity enhancement by gold nanoparticles using synchrotron-based monoenergetic photon beams.

    PubMed

    Rahman, Wan Nordiana; Corde, Stéphanie; Yagi, Naoto; Abdul Aziz, Siti Aishah; Annabell, Nathan; Geso, Moshi

    2014-01-01

    Gold nanoparticles have been shown to enhance radiation doses delivered to biological targets due to the high absorption coefficient of gold atoms, stemming from their high atomic number (Z) and physical density. These properties significantly increase the likelihood of photoelectric effects and Compton scattering interactions. Gold nanoparticles are a novel radiosensitizing agent that can potentially be used to increase the effectiveness of current radiation therapy techniques and improve the diagnosis and treatment of cancer. However, the optimum radiosensitization effect of gold nanoparticles is strongly dependent on photon energy, which theoretically is predicted to occur in the kilovoltage range of energy. In this research, synchrotron-generated monoenergetic X-rays in the 30-100 keV range were used to investigate the energy dependence of radiosensitization by gold nanoparticles and also to determine the photon energy that produces optimum effects. This investigation was conducted using cells in culture to measure dose enhancement. Bovine aortic endothelial cells with and without gold nanoparticles were irradiated with X-rays at energies of 30, 40, 50, 60, 70, 81, and 100 keV. Trypan blue exclusion assays were performed after irradiation to determine cell viability. Cell radiosensitivity enhancement was indicated by the dose enhancement factor which was found to be maximum at 40 keV with a value of 3.47. The dose enhancement factor obtained at other energy levels followed the same direction as the theoretical calculations based on the ratio of the mass energy absorption coefficients of gold and water. This experimental evidence shows that the radiosensitization effect of gold nanoparticles varies with photon energy as predicted from theoretical calculations. However, prediction based on theoretical assumptions is sometimes difficult due to the complexity of biological systems, so further study at the cellular level is required to fully characterize the effects

  17. Celecoxib Enhances the Radiosensitizing Effect of 7-Hydroxystaurosporine (UCN-01) in Human Lung Cancer Cell Lines

    SciTech Connect

    Kim, Young-Mee; Jeong, In-Hye; Pyo, Hongryull

    2012-07-01

    Purpose: 7-Hydroxystaurosporine (UCN-01), a Chk1-specific inhibitor, showed promising in vitro and in vivo chemo- or radiosensitizing activity. However, there have been concerns about its limited therapeutic efficacy and risk of side effects. A method of enhancing the treatment efficacy of UCN-01 while not increasing its side effects on normal tissue may therefore be required to apply this drug in clinical settings. Celecoxib is a cyclooxygenase-2 (COX-2)-specific inhibitor that downregulates ataxia telangiectasia and rad3-related (ATR) protein, an upstream kinase of Chk1. In this study, we investigated whether the addition of celecoxib can potentiate the radiosensitizing effect of UCN-01. Methods and Materials: The cooperative radiosensitizing effects and the underlying molecular mechanisms of UCN-01 plus celecoxib were determined by clonogenic assay, tumor growth delay assay, flow cytometry, and Western blotting. Synergism of the three agents combined (UCN-01 plus celecoxib plus radiation) were evaluated using median drug effect analysis and drug-independent action model analysis. Results: The combination of UCN-01 and celecoxib could induce synergistic cytotoxicity and radiosensitizing effects in in vitro and in vivo systems. The combination of both drugs also cooperatively inhibited IR-induced G{sub 2}/M arrest, and increased the G{sub 2} to mitotic transition. Conclusions: Combined treatment with UCN-01 and celecoxib can exert synergistically enhanced radiosensitizing effects via cooperative inhibition of the ionizing radiation-activated G{sub 2} checkpoint. We propose that this combination strategy may be useful in clinical applications of UCN-01 for radiotherapy of cancer patients.

  18. Optimal energy for cell radiosensitivity enhancement by gold nanoparticles using synchrotron-based monoenergetic photon beams

    PubMed Central

    Rahman, Wan Nordiana; Corde, Stéphanie; Yagi, Naoto; Abdul Aziz, Siti Aishah; Annabell, Nathan; Geso, Moshi

    2014-01-01

    Gold nanoparticles have been shown to enhance radiation doses delivered to biological targets due to the high absorption coefficient of gold atoms, stemming from their high atomic number (Z) and physical density. These properties significantly increase the likelihood of photoelectric effects and Compton scattering interactions. Gold nanoparticles are a novel radiosensitizing agent that can potentially be used to increase the effectiveness of current radiation therapy techniques and improve the diagnosis and treatment of cancer. However, the optimum radiosensitization effect of gold nanoparticles is strongly dependent on photon energy, which theoretically is predicted to occur in the kilovoltage range of energy. In this research, synchrotron-generated monoenergetic X-rays in the 30–100 keV range were used to investigate the energy dependence of radiosensitization by gold nanoparticles and also to determine the photon energy that produces optimum effects. This investigation was conducted using cells in culture to measure dose enhancement. Bovine aortic endothelial cells with and without gold nanoparticles were irradiated with X-rays at energies of 30, 40, 50, 60, 70, 81, and 100 keV. Trypan blue exclusion assays were performed after irradiation to determine cell viability. Cell radiosensitivity enhancement was indicated by the dose enhancement factor which was found to be maximum at 40 keV with a value of 3.47. The dose enhancement factor obtained at other energy levels followed the same direction as the theoretical calculations based on the ratio of the mass energy absorption coefficients of gold and water. This experimental evidence shows that the radiosensitization effect of gold nanoparticles varies with photon energy as predicted from theoretical calculations. However, prediction based on theoretical assumptions is sometimes difficult due to the complexity of biological systems, so further study at the cellular level is required to fully characterize the

  19. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase

    SciTech Connect

    Jayakumar, Sundarraj; Patwardhan, R.S.; Pal, Debojyoti; Sharma, Deepak; Sandur, Santosh K.

    2016-09-09

    Dimethoxycurcumin (DIMC), a structural analogue of curcumin, has been shown to have more stability, bioavailability, and effectiveness than its parent molecule curcumin. In this paper the radiosensitizing effect of DIMC has been investigated in A549 lung cancer cells. As compared to its parent molecule curcumin, DIMC showed a very potent radiosensitizing effect as seen by clonogenic survival assay. DIMC in combination with radiation significantly increased the apoptosis and mitotic death in A549 cells. This combinatorial treatment also lead to effective elimination of cancer stem cells. Further, there was a significant increase in cellular ROS, decrease in GSH to GSSG ratio and also significant slowdown in DNA repair when DIMC was combined with radiation. In silico docking studies and in vitro studies showed inhibition of thioredoxin reductase enzyme by DIMC. Overexpression of thioredoxin lead to the abrogation of radiosensitizing effect of DIMC underscoring the role of thioredoxin reductase in radiosensitization. Our results clearly demonstrate that DIMC can synergistically enhance the cancer cell killing when combined with radiation by targeting thioredoxin system. - Highlights: • DIMC enhances radiosensitivity of cancer cells by inducing cell death. • DIMC with radiation disrupted the cellular redox and targeted cancer stem cells. • DNA repair is hampered when cells are treated with DIMC. • DIMC inhibited thioredoxin reductase in cancer cells.

  20. The dominant negative mutant Artemis enhances tumor cell radiosensitivity.

    PubMed

    Liu, Hai; Sun, XiaoNan; Zhang, Shuo; Ge, WeiTing; Zhu, YongLiang; Zhang, JiaWei; Zheng, Shu

    2011-10-01

    Tumor radioresistance often leads to treatment failure during radiotherapy. New strategies like developing radiosensitizer are clinically important. Intervention with DNA double-strand break repair is an effective way to modulate tumor cell radiosensitivity. This study focused on the mutant Artemis fragment-enhanced radiosensitivity of human cervical cancer cells. We constructed two pEGFP-C1-based eukaryotic expression vectors encoding full-length and the mutant Artemis fragment (D37N-413aa), respectively. HeLa cells were stably transfected with these plasmids or vector. Cell survival was measured by the clonogenic assay. The γH2AX foci assay was used to monitor DNA repair after irradiation. Co-immunoprecipitation and Western blot analysis were performed to study protein interaction and phosphorylation of Artemis. Expression of the mutant Artemis fragment (D37N-413aa) delayed DNA DSB rejoining after irradiation, thereby enhanced radiosensitivity of HeLa cell. Further experiments indicate that this mutant Artemis fragment bind to DNA-PKcs and ATM, inhibited phosphorylation of endogenous Artemis, the key molecule for DNA repair and cell radiosensitivity. The dominant negative mutant Artemis fragment (D37N-413aa) enhanced tumor cell radiosensitivity through blocking activity of endogenous Artemis and DNA repair. It is the first time to modulate tumor cell radiosensitivity via targeting Artemis. This novel mechanism of radiosensitivity strongly suggests the potential role of Artemis in cancer therapy. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  1. Gefitinib radiosensitizes non-small cell lung cancer cells through inhibition of ataxia telangiectasia mutated

    PubMed Central

    2010-01-01

    Purpose Inhibitors of epidermal growth factor receptor (EGFR) have shown dramatic results in a subset of patients with non-small cell lung cancer (NSCLC), and have also been shown to enhance the effect of ionizing radiation (IR). We investigated how gefitinib, an orally given EGFR inhibitor for NSCLC patients, can radiosensitize NSCLC cells. Experimental Design and Results In clonogenic survival assays performed in three NSCLC cell lines, gefitinib radiosensitized NCI-H460 and VMRC-LCD but not A549 cells. Gefitinib pretreatment induced multinucleated cells after IR exposure in NCI-H460 and VMRC-LCD, but not in A549 cells. Gefitinib also inhibited activation of ataxia telangiectasia mutated (ATM) after IR-exposure in NCI-H460 and VMRC-LCD, but not in A549 cells. An ATM specific inhibitor increased IR-induced multinucleated cells in both NCI-H460 and A549 cells. Gefitinib pretreatment inhibited the gradual decrease of γH2AX foci relative to time after IR exposure in NCI-H460 but not in A549 cells. Suppression of COX-2 in A549 cells induced multinucleated cells and caused radiosensitization after gefitinib+IR treatment. In contrast, COX-2 overexpression in NCI-H460 cells attenuated the induction of multinucleation and radiosensitization after the same treatment. Conclusions Our results suggest that gefitinib radiosensitizes NSCLC cells by inhibiting ATM activity and therefore inducing mitotic cell death, and that COX-2 overexpression in NSCLC cells inhibits this action of gefitinib. PMID:20731837

  2. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells.

    PubMed

    Dolman, M Emmy M; van der Ploeg, Ida; Koster, Jan; Bate-Eya, Laurel Tabe; Versteeg, Rogier; Caron, Huib N; Molenaar, Jan J

    2015-01-01

    Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

  3. Prostate-Specific Natural Health Products (Dietary Supplements) Radiosensitize Normal Prostate Cells

    SciTech Connect

    Hasan, Yasmin; Schoenherr, Diane; Martinez, Alvaro A.; Wilson, George D.; Marples, Brian

    2010-03-01

    Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines. Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity was assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing gammaH2AX. Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2mug/mL], Trinovin [10mug/mL], and Prostate Rx [50 mug/mL]). However, both Trinovin (10mug/mL) and Prostate Rx (6mug/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair. Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells.

  4. Taxonomic and developmental aspects of radiosensitivity

    SciTech Connect

    Harrison, F.L.; Anderson, S.L.

    1996-11-01

    Considerable information is available on the effects of radioactivity on adult and early life stages of organisms. The preponderance of data is on mortality after a single irradiation with relatively high doses. Unfortunately, because experiments were carried out under different conditions and for different time periods, the validity of comparing the results from different laxonomic groups is questionable. In general, the conclusions are that there is a relationship (1) between radioresistance to high doses of acute radiation and taxonomy of the organism, primitive forms being more radioresistant than complex vertebrates and (2) between radiosensitivity and developmental stage, early life stages being more sensitive than later stages. The first conclusion may be related to the capability of the organism to repopulate cells and to differentiate and redifferentiate them; the second to the rate of cellular division and to the degree of differentiation. In question, however, is the relevance of the responses from high levels of acute radiation to that of the responses to long-term exposure to low levels of radiation, which are ecologically of more interest. Data from studies of the effects of acute and chronic exposure on development of gametes and zygotes indicate that, for some fishes and invertebrates, responses at the cellular and molecular levels show effect levels comparable to those observed in some mammals. Acute doses between 0,05 and 0.5Cy and dose rates between 0.02 to 0.2mCy/h appear to define critical ranges in which detrimental effects on fertility are first observed in a variety of radiosensitive organisms. To better understand inherent radiosensitivity, we need more information on the ability of cells to repopulate and differentiate and to prevent or repair damage to biological critical molecules, such as DNA, because these factors may alter significantly organisms` responses to radiation.

  5. On the mechanism of salivary gland radiosensitivity

    SciTech Connect

    Konings, Antonius W.T. . E-mail: a.w.t.konings@med.rug.nl; Coppes, Rob P.; Vissink, Arjan

    2005-07-15

    Purpose: To contribute to the understanding of the enigmatic radiosensitivity of the salivary glands by analysis of appropriate literature, especially with respect to mechanisms of action of early radiation damage, and to supply information on the possibilities of amelioration of radiation damage to the salivary glands after radiotherapy of head-and-neck cancer. Methods and Materials: Selected published data on the mechanism of salivary gland radiosensitivity and radioprotection were studied and analyzed. Results: From a classical point of view, the salivary glands should not respond as rapidly to radiation as they appear to do. Next to the suggestion of massive apoptosis, the leakage of granules and subsequent lysis of acinar cells was suggested to be responsible for the acute radiation-induced function loss of the salivary glands. The main problem with these hypotheses is that recently performed assays show no cell loss during the first days after irradiation, while saliva flow is dramatically diminished. The water secretion is selectively hampered during the first days after single-dose irradiation. Literature is discussed that shows that the compromised cells suffer selective radiation damage to the plasma membrane, disturbing signal transduction primarily affecting watery secretion. Although the cellular composition of the submandibular gland and the parotid gland are different, the damage response is very alike. The acute radiation-induced function loss in both salivary glands can be ameliorated by prophylactic treatment with specific receptor agonists. Conclusions: The most probable mechanism of action, explaining the enigmatic high radiosensitivity for early effects, is selective radiation damage to the plasma membrane of the secretory cells, disturbing muscarinic receptor stimulated watery secretion. Later damage is mainly due to classical mitotic cell death of progenitor cells, leading to a hampered replacement capacity of the gland for secretory cells

  6. Doxorubicin-mediated radiosensitivity in multicellular spheroids from a lung cancer cell line is enhanced by composite micelle encapsulation

    PubMed Central

    Xu, Wen-Hong; Han, Min; Dong, Qi; Fu, Zhi-Xuan; Diao, Yuan-Yuan; Liu, Hai; Xu, Jing; Jiang, Hong-Liang; Zhang, Su-Zhan; Zheng, Shu; Gao, Jian-Qing; Wei, Qi-Chun

    2012-01-01

    Background The purpose of this study is to evaluate the efficacy of composite doxorubicinloaded micelles for enhancing doxorubicin radiosensitivity in multicellular spheroids from a non-small cell lung cancer cell line. Methods A novel composite doxorubicin-loaded micelle consisting of polyethylene glycolpolycaprolactone/Pluronic P105 was developed, and carrier-mediated doxorubicin accumulation and release from multicellular spheroids was evaluated. We used confocal laser scanning microscopy and flow cytometry to study the accumulation and efflux of doxorubicin from A549 multicellular spheroids. Doxorubicin radiosensitization and the combined effects of irradiation and doxorubicin on cell migration and proliferation were compared for the different doxorubicin delivery systems. Results Confocal laser scanning microscopy and quantitative flow cytometry studies both verified that, for equivalent doxorubicin concentrations, composite doxorubicin-loaded micelles significantly enhanced cellular doxorubicin accumulation and inhibited doxorubicin release. Colony-forming assays demonstrated that composite doxorubicin-loaded micelles are radiosensitive, as shown by significantly reduced survival of cells treated by radiation + composite micelles compared with those treated with radiation + free doxorubicin or radiation alone. The multicellular spheroid migration area and growth ability verified higher radiosensitivity for the composite micelles loaded with doxorubicin than for free doxorubicin. Conclusion Our composite doxorubicin-loaded micelle was demonstrated to have radiosensitization. Doxorubicin loading in the composite micelles significantly increased its cellular uptake, improved drug retention, and enhanced its antitumor effect relative to free doxorubicin, thereby providing a novel approach for treatment of cancer. PMID:22679376

  7. TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport.

    PubMed

    Zhang, Yushuo; Chen, Fei; Tai, Guomei; Wang, Jiaojiao; Shang, Jun; Zhang, Bing; Wang, Ping; Huang, Baoxing; Du, Jie; Yu, Jiahua; Zhang, Haowen; Liu, Fenju

    2017-03-24

    The up-regulation of thioredoxin reductase-1 (TrxR1) is detected in more than half of gliomas, which is significantly associated with increased malignancy grade and recurrence rate. The biological functions of NADPH-dependent TrxR1 are mainly associated with reduced thioredoxin-1 (Trx1) which plays critical roles in cellular redox signaling and tumour radio-resistance. Our previous work has proved that TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown could notably radiosensitize glioma cells. However, whether TrxR1-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear. In the present study, TrxR1 was stably over-expressed in U-87MG and T98G glioma cells. Both in vitro and in vivo data demonstrated that the radiosensitivity of glioma cells was considerably diminished by TrxR1 overexpression. TIGAR abrogation was able to radiosensitize TrxR1-overexpressing gliomas by inhibiting IR-induced Trx1 nuclear transport. Post-radiotherapy, TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts, which suggested that TIGAR abrogation might be a promising strategy for radiosensitizing TrxR1-overexpressing glial tumours.

  8. Radiosensitization by SAHA in Experimental Colorectal Carcinoma Models-In Vivo Effects and Relevance of Histone Acetylation Status

    SciTech Connect

    Folkvord, Sigurd; Ree, Anne Hansen; Furre, Torbjorn; Halvorsen, Thomas; Flatmark, Kjersti

    2009-06-01

    Purpose: Histone deacetylase inhibitors are being evaluated as antitumor agents in ongoing clinical trials, and promising preclinical results, combined with favorable toxicity profiles, have rendered the drugs as interesting candidates for combination with other treatment modalities, such as radiotherapy. The aim of the present study was to evaluate the radiosensitizing properties of suberoylanilide hydroxamic acid (SAHA) and the possible requirement of histone hyperacetylation at radiation exposure. Methods and materials: Radiosensitization by SAHA was assessed in a colorectal carcinoma cell line and in two colorectal xenograft models by analysis of clonogenic survival and tumor growth delay, respectively. Histone acetylation status at radiation exposure was evaluated by Western blot. Results: In vitro, radiosensitization was demonstrated when cells were preincubated with SAHA, and, in the xenografts, tumor growth was delayed when the mice were treated with fractionated radiation combined with daily SAHA injections compared with radiation alone. Surprisingly, the SAHA-dependent growth delay was still present when radiation was delivered at restored baseline acetylation levels compared with maximal histone hyperacetylation. Conclusion: SAHA was an effective radiosensitizer in experimental colorectal carcinoma models, suggesting that histone deacetylase inhibition might constitute a valuable supplement to current multimodal treatment strategies in rectal cancer. The presence of histone hyperacetylation at radiation was not required to obtain an increased radiation response, questioning the validity of using histone hyperacetylation as a molecular marker for radiosensitivity.

  9. TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport

    PubMed Central

    Zhang, Yushuo; Chen, Fei; Tai, Guomei; Wang, Jiaojiao; Shang, Jun; Zhang, Bing; Wang, Ping; Huang, Baoxing; Du, Jie; Yu, Jiahua; Zhang, Haowen; Liu, Fenju

    2017-01-01

    The up-regulation of thioredoxin reductase-1 (TrxR1) is detected in more than half of gliomas, which is significantly associated with increased malignancy grade and recurrence rate. The biological functions of NADPH-dependent TrxR1 are mainly associated with reduced thioredoxin-1 (Trx1) which plays critical roles in cellular redox signaling and tumour radio-resistance. Our previous work has proved that TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown could notably radiosensitize glioma cells. However, whether TrxR1-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear. In the present study, TrxR1 was stably over-expressed in U-87MG and T98G glioma cells. Both in vitro and in vivo data demonstrated that the radiosensitivity of glioma cells was considerably diminished by TrxR1 overexpression. TIGAR abrogation was able to radiosensitize TrxR1-overexpressing gliomas by inhibiting IR-induced Trx1 nuclear transport. Post-radiotherapy, TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts, which suggested that TIGAR abrogation might be a promising strategy for radiosensitizing TrxR1-overexpressing glial tumours. PMID:28338004

  10. Chromosomal radiosensitivity during the G2 cell-cycle period of skin fibroblasts from individuals with familial cancer

    SciTech Connect

    Parshad, R.; Sanford, K.K.; Jones, G.M.

    1985-08-01

    The authors reported previously that human cells after neoplastic transformation in culture had acquired an increased susceptibility to chromatid damage induced by x-irradiation during the G2 phase of the cell cycle. Evidence suggested that this results from deficient DNA repair during G2 phase. Cells derived from human tumors also showed enhanced G2-phase chromosomal radiosensitivity. Furthermore, skin fibroblasts from individuals with genetic diseases predisposing to a high risk of cancer, including ataxia-telangiectasia, Bloom syndrome, Fanconi anemia, and xeroderma pigmentosum exhibited enhanced G2-phase chromosomal radiosensitivity. The present study shows that apparently normal skin fibroblasts from individuals with familial cancer--i.e., from families with a history of neoplastic disease--also exhibit enhanced G2-phase chromosomal radiosensitivity. This radiosensitivity appears, therefore, to be associated with both a genetic predisposition to cancer and a malignant neoplastic state. Furthermore, enhanced G2-phase chromosomal radiosensitivity may provide the basis for an assay to detect genetic susceptibility to cancer.

  11. Inhibition of PARP1-dependent end-joining contributes to Olaparib-mediated radiosensitization in tumor cells.

    PubMed

    Kötter, Annika; Cornils, Kerstin; Borgmann, Kerstin; Dahm-Daphi, Jochen; Petersen, Cordula; Dikomey, Ekkehard; Mansour, Wael Y

    2014-12-01

    Poly-ADP-ribose-polymerase inhibitors (PARPi) are considered to be optimal tools for specifically enhancing radiosensitivity. This effect has been shown to be replication-dependent and more profound in HR-deficient tumors. Here, we present a new mode of PARPi-mediated radiosensitization which was observed in four out of six HR-proficient tumor cell lines (responders) investigated, but not in normal cells. This effect is replication-independent, as the radiosensitization remained unaffected following the inhibition of replication using aphidicolin. We showed that responders are radiosensitized by Olaparib because their DSB-repair is switched to PARP1-dependent end-joining (PARP1-EJ), as evident by (i) the significant increase in the number of residual γH2AX foci following irradiation with 3Gy and treatment with Olaparib, (ii) the enhanced enrichment of PARP1 at the chromatin after 3Gy and (iii) the inhibition of end-joining activity measured by a specific reporter substrate upon Olaparib treatment. This is the first study which directly demonstrates the switch to PARP1-EJ in tumor cells and its contribution to the response to Olaparib as a radiosensitizer, findings which could widen the scope of application of PARPi in tumor therapy. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Relationship between genetic polymorphisms of DNA ligase 1 and non-small cell lung cancer susceptibility and radiosensitivity.

    PubMed

    Tian, H; He, X; Yin, L; Guo, W J; Xia, Y Y; Jiang, Z X

    2015-06-26

    The aim of this study was to examine the relationship between genetic polymorphisms in DNA ligase 1 (LIG1) and non-small cell lung cancer (NSCLC) susceptibility and radiosensitivity in a Chinese population. This was a case-control study that included 352 NSCLC patients and 448 healthy controls. Polymerase chain reaction-restriction fragment length polymorphism analysis was conducted to detect HaeIII polymorphisms in exon 6 of the LIG1 gene in this popula-tion. This information was used to observe the effects of radiation in pa-tients with different genotypes in order to determine the genotypes as-sociated with radiosensitivity. The CC genotype and C allele frequency were significantly higher in the NSCLC group than in the control group (P = 0.012 and P = 0.023, respectively). The relative risk of experienc-ing NSCLC was 2.55 [95% confidence interval (CI), 1.12-3.98] for CC homozygous patients and 0.87 (95%CI, 0.46-1.88) for AA homozygous patients. Analysis of LIG1 genetic polymorphisms and radiosensitiv-ity of NSCLC patients showed that AA homozygous patients were sig-nificantly more radiosensitive than the control group (AA vs AC, P = 0.014; AA vs CC, P < 0.001; AC vs CC, P = 0.023). Therefore, the LIG1 CC genotype was associated with susceptibility to NSCLC, and the AA genotype demonstrated increased radiosensitivity compared to the AC and CC genotypes.

  13. Potential radiosensitizing agents. 5. 2-Substituted benzimidazole derivatives

    SciTech Connect

    Gupta, R.P.; Larroquette, C.A.; Agrawal, K.C.

    1982-11-01

    A series of 2-substituted benzimidazoles and their derivatives have been synthesized and tested for their ability to selectively sensitize hypoxic Chinese hamster cells (V-79) toward the lethal effect of ionizing radiation. These compounds were prepared by reacting the 2-substituted benzimidazoles with 1,2-epoxy-3-methoxypropane in the presence of potassium carbonate. Reaction of the 2-nitro and 2-methylfonyl analogue with the epoxide also yielded a cyclized material, which was confirmed to be a benzimidazo(2,1-b)oxazole. In an attempt to increase the electron affinity, 5- or 6-nitro-2-substituted-benzimidazoles were also synthesized and then reacted with the epoxide to yield the corresponding 1-substituted derivatives. The results of the biological tests for the radiosensitizing activity of these agents against Chinese hamster cells (V-79) in culture indicated that the 2-nitro-substituted analogues were the most effective sensitizers in this series.

  14. Enhanced cellular radiosensitivity induced by cofilin-1 over-expression is associated with reduced DNA repair capacity

    PubMed Central

    Leu, Jyh-Der; Chiu, Yu-Wen; Lo, Chia-Chien; Chiang, Pei-Hsun; Chiu, Su-Jun; Tsai, Cheng-Han; Hwang, Jeng-Jong; Chen, Ran-Chou; Gorbunova, Vera; Lee, Yi-Jang

    2013-01-01

    Purpose A previous report has indicated that over-expression of cofilin-1 (CFL-1), a member of the actin depolymerizing factor (ADF)/cofilin protein family, enhances cellular radiosensitivity. This study explores, the involvement of various DNA damage responses and repair systems in the enhanced cellular radiosensitivity as well as assessing the role of CFL-1 phosphorylation in radiosensitivity. Materials and Methods Human non-small lung cancer H1299 cells harboring a tet-on gene expression system were used to induce exogenous expression of wild-type CFL-1. Colony formation assays were used to determine cell survival after γ-ray exposure. DNA damage levels were determined by comet assay. DNA repair capacity was assessed by fluorescence-based DNA repair analysis and antibody detection of various repair proteins. The effects of CFL-1 phosphorylation on radiation responses were explored using two mutant CFL-1 proteins, S3D and S3A. Finally, endogenous CFL-1 phosphorylation levels were investigated using latrunculin A (LA), cytochalasin B (CB) and Y27632. Results When phosphorylatable CFL-1 was expressed, radiosensitivity was enhanced after exposure to γ-rays and this was accompanied by DNA damage. Phosphorylated histone H2AX (γ-H2AX) and p53-binding protein-1 (53BP1) foci, as well as Chk1/2 phosphorylation, were apparently suppressed, although ataxia telangiectasia mutated (ATM) kinase activation was apparently unaffected. In addition, two radiation induced double strand break (DSB) repair, systems, namely homologous recombination repair (HRR) and non-homologous end joining (NHEJ), were suppressed. Moreover, over-expression of CFL-1 S3D and CFL-1 S3A both enhanced radiosensitivity. However, enhanced radiosensitivity and reduced γ-H2AX expression were only detected in cells treated with LA which increased endogenous phospho-CFL-1, and not in cells treated with Y27632, which dephosphorylates CFL-1. Conclusion CFL-1 over-expression enhances radiosensitivity and this

  15. Replication-Dependent Radiosensitization of Human Glioma Cells by Inhibition of Poly(ADP-Ribose) Polymerase: Mechanisms and Therapeutic Potential

    SciTech Connect

    Dungey, Fiona A.; Loeser, Dana A.; Chalmers, Anthony J.

    2008-11-15

    Purpose: Current treatments for glioblastoma multiforme are inadequate and limited by the radiation sensitivity of normal brain. Because glioblastoma multiforme are rapidly proliferating tumors within nondividing normal tissue, the therapeutic ratio might be enhanced by combining radiotherapy with a replication-specific radiosensitizer. KU-0059436 (AZD2281) is a potent and nontoxic inhibitor of poly(ADP-ribose) polymerase-1 (PARP-1) undergoing a Phase II clinical trial as a single agent. Methods and Materials: Based on previous observations that the radiosensitizing effects of PARP inhibition are more pronounced in dividing cells, we investigated the mechanisms underlying radiosensitization of human glioma cells by KU-0059436, evaluating the replication dependence of this effect and its therapeutic potential. Results: KU-0059436 increased the radiosensitivity of four human glioma cell lines (T98G, U373-MG, UVW, and U87-MG). Radiosensitization was enhanced in populations synchronized in S phase and abrogated by concomitant exposure to aphidicolin. Sensitization was further enhanced when the inhibitor was combined with a fractionated radiation schedule. KU-0059436 delayed repair of radiation-induced DNA breaks and was associated with a replication-dependent increase in {gamma}H2AX and Rad51 foci. Conclusion: The results of our study have shown that KU-0059436 increases radiosensitivity in a replication-dependent manner that is enhanced by fractionation. A mechanism is proposed whereby PARP inhibition increases the incidence of collapsed replication forks after ionizing radiation, generating persistent DNA double-strand breaks. These observations indicate that KU-0059436 is likely to enhance the therapeutic ratio achieved by radiotherapy in the treatment of glioblastoma multiforme. A Phase I clinical trial is in development.

  16. Actual questions raised by nanoparticle radiosensitization

    NASA Astrophysics Data System (ADS)

    Brun, Emilie; Sicard-Roselli, Cécile

    2016-11-01

    Radiosensitization by metallic nanoparticles (NP) has been explored for more than a decade with promising results in vitro and in cellulo reported in a vast number of publications. Yet, few clinical trials are on-going. This could be related to the lack of selectivity of NP leading to massive quantities to be injected to observe an effect but also to the higher degree of complexity than first thought leading to an absence of consensus probably caused by the lack of standardization in pre-clinical studies. Given the wide panel of NP used, in terms of core nature, size, coating, not to mention of cell lines and irradiation modalities, cross-comparison of data is not a walk in the park. But only a thorough examination could help identifying the key parameters and the possible mechanisms involved. This step is crucial as it should provide guidance for designing the most efficient combination NP/radiation and rationally establishing clinical protocols. In this review, we will combine and confront cellular radiosensitization results with in vitro and numerical experiments in order to give the more recent vision of this complex phenomenon. We decided to address a few hot topics such as the influence of the incident radiation energy, the localization of NP or the so-called ;biological; effect. We will highlight that among the barriers to break down, some are not restricted to the ;nano; community: an incontestable support could be offered by the ;radiation; community in the broadest sense.

  17. Evaluation of nitrobenzimidazoles as hypoxic cell radiosensitizers

    SciTech Connect

    Wright, J.; Frank, L.R.; Bush, D.; Harrison, G.H.

    1983-07-01

    Radiobiological and pharmacokinetic assays were performed to determine the potential of 2-nitrobenzimidazole (NBI) as a hypoxic cell radiosensitizing agent. As judged by comparing survival curve slopes of Serratia marcescens irradiated under aerated and hypoxic conditions, the NBI enhancement ratio (ER) at 2 mM concentration was 2.4 +/- 0.2, compared with an oxygen enhancement ratio of 3.3 +/- 0.3. 2,5-Dinitrobenzimidazole (DNBI) was investigated in vitro; its ER was 3.0 +/- 0.3 at 4 mM concentration. Very poor tissue penetration of DNBI precluded further testing in vivo. Acute toxic signs appeared in C3H/HeJ mice following ip injection of NBI at 100 mg/kg. These would be partly attributable to the stress caused by the high pH of the injection vehicle. The LD50 was estimated to be 125-150 mg/kg. Mammary adenocarcinoma tumors grown in the flanks of these mice exhibited maximum NBI levels at 5 min postinjection (ip). Peak tumor radiosensitization occurred in the interval between 5 and 10 min postinjection. The ER for tumor regrowth delay was 2.1 +/- 0.3 following 50 mg/kg injected into mice 5 min before irradiation. Functional evaluation up to 40 days after treatment revealed no evidence of neurological deficit.

  18. Karenitecin (bnp1350) and flavopridol as radiosensitizers in malignant glioma

    PubMed Central

    Rajesh, Deepika; Robins, H. Ian; Howard, Steven P.

    2016-01-01

    The poor prognosis of malignant glioma patients highlights the need to develop low toxicity, tumor specific agents with the ability to synergize with proven efficacious treatment modalities, e.g., ionizing irradiation. This paper investigates the potential of BNP1350 (karenitecin), a topoisomerase I-targeting anticancer agent, and flavopridol a cyclin-dependent kinase inhibitor as radiosensitizers at clinically relevant doses in glioblastoma cell lines. A clonogenic survival and apoptosis assays were performed to test the effect of karenitecin (0.1 nM to 10 nM), flavopridol, (50 nM to 500 nM), radiation (1 Gy to 5.5 Gy) and a combination of radiation and karenitecin or radiation and flavopridol on the glioma cell lines T986 and M059K. Cells were stained for cyclins B and D using antibodies followed by flow cytometry. Propidium Iodide staining was used to reveal the various phases of the cell cycle; cyclin staining in the G0/G1 and G2/M phase of the cell cycle was estimated as the Mean Fluorescence Intensity (MFI) after subtracting the MFI recorded by the isotype controls. Results demonstrated that in irradiated cells, pretreatment with karenitecin induced apoptosis, a transient arrest in the G2/M phase of the cell cycle and increased the expression of cyclin B1. Flavopridol treatment also induced apoptosis and a transient block in the G2/M phase of the cell cycle. The combined effects of karenitecin and flavopridol displayed synergistic effects. The unique radiosensitizing activity of orally administrable karenitecin and flavopridol is consistent with continued investigation of these compounds preclinically, as well as in the clinical setting. PMID:28111642

  19. Gemcitabine radiosensitization after high-dose samarium for osteoblastic osteosarcoma.

    PubMed

    Anderson, Peter M; Wiseman, Gregory A; Erlandson, Linda; Rodriguez, Vilmarie; Trotz, Barbara; Dubansky, Stephen A; Albritton, Karen

    2005-10-01

    Osteoblastic metastases and osteosarcoma can avidly concentrate bone-seeking radiopharmaceuticals. We sought to increase effectiveness of high-dose (153)Samarium ethylenediaminetetramethylenephosphonate (153Sm-EDTMP, Quadramet) on osteosarcomas using a radiosensitizer, gemcitabine. Fourteen patients with osteoblastic lesions were treated with 30 mCi/kg 153Sm-EDTMP. Gemcitabine was administered 1 day after samarium infusion. Residual total body radioactivity was within the safe range of <3.6 mCi on day +14 (1.1 +/- 0.4 mCi; range, 0.67-1.8 mCi). All patients received autologous stem cell reinfusion 2 weeks after 153Sm to correct expected grade 4 hematopoietic toxicity. Peripheral blood progenitor cells were infused in 11 patients; three patients had marrow infused. Blood count recovery was uneventful after peripheral blood progenitor cells in 11 of 11 patients. Toxicity from a single infusion of gemcitabine (1,500 mg/m2) in combination with 153Sm-EDTMP was minimal (pancytopenia). However, toxicity from a daily gemcitabine regimen (250 mg/m2/d x 4-5 days) was excessive (grade 3 mucositis) in one of two patients. There were no reported episodes of hemorrhagic cystitis (hematuria) or nephrotoxicity. At the 6- to 8-week follow-up, there were six partial remissions, two mixed responses, and six patients with progressive disease. In the 12 patients followed >1 year, there have been no durable responses. Thus, although high-dose 153Sm-EDTMP + gemcitabine has moderate palliative activity (improved pain; radiologic responses) in this poor-risk population, additional measures of local and systemic control are required for durable control of relapsed osteosarcoma with osteoblastic lesions. The strategy of radioactive drug binding to a target followed by a radiosensitizer may provide synergy and improved response rate.

  20. Radiosensitization by Inhibiting STAT1 in Renal Cell Carcinoma

    SciTech Connect

    Hui Zhouguang; Tretiakova, Maria; Zhang Zhongfa; Li Yan; Wang Xiaozhen; Zhu, Julie Xiaohong; Gao Yuanhong; Mai Weiyuan; Furge, Kyle; Qian Chaonan; Amato, Robert; Butler, E. Brian

    2009-01-01

    Purpose: Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. Methods and Materials: The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. Results: STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10{sup -8} for clear cell; and p = 3.6 x 10{sup -4} for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. Conclusion: This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

  1. Radiosensitization of TPGS-emulsified docetaxel-loaded poly(lactic-co-glycolic acid) nanoparticles in CNE-1 and A549 cells.

    PubMed

    Shi, Wei; Yuan, Yin; Chu, Min; Zhao, Shuang; Song, Qingle; Mu, Xiaoqian; Xu, Shuangbing; Zhang, Zhiping; Yang, Kunyu

    2016-03-01

    Docetaxel is among the most effective radiosensitizers. It is widely used as radiosensitizer in many tumors, including head and neck carcinoma. Nevertheless, poor solubility and severe hypersensitivity limit its clinical use and its therapeutic effect remains to be improved. In this study, docetaxel-loaded polymeric nanoparticles were prepared by nanoprecipitation method to be new radiosensitizer with lower side effects and higher efficacy. The physiochemical characteristics of the nanoparticles were studied. Two human tumor cell lines which are resistant to radiotherapy were used in this research. We have compared the radioenhancement efficacy of docetaxel-loaded nanoparticles with docetaxel in A549 and CNE-1 cells. Compared with docetaxel, radiosensitization of docetaxel-loaded nanoparticles was improved significantly (sensitization enhancement ratio in A549 increased 1.24-fold to 1.68-fold when the radiation was applied 2 h after the drug, p < 0.01, sensitization enhancement ratio in CNE-1 increased 1.32-fold to 1.61-fold, p < 0.05). We explored the mechanisms for the radiosensitization efficiency and the difference between docetaxel and docetaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The improved radiosensitization efficacy was associated with enhanced G2/M arrest, promoted apoptosis and the role of D-alpha-tocopheryl polyethylene glycol 1000 succinate which will enhance the cell uptake and inhibit the multiple drug resistance. Moreover, the radiosensitization efficacy of docetaxel-loaded nanoparticles was more prominent than docetaxel. In conclusion, tocopheryl polyethylene glycol 1000 succinate-emulsified docetaxel-loaded PLGA nanoparticles were more efficacious and fewer adverse effects were observed than with the commercial docetaxel formulation. Thus, PLGA nanoparticles hold promise as a radiosensitizing agent.

  2. Comparison of organic and inorganic germanium compounds in cellular radiosensitivity and preparation of germanium nanoparticles as a radiosensitizer.

    PubMed

    Lin, Ming-Hsing; Hsu, Tzu-Sheng; Yang, Pei-Ming; Tsai, Meng-Yen; Perng, Tsong-Pyng; Lin, Lih-Yuan

    2009-03-01

    The aim of this work is to compare the radiosensitizing effect between organic and inorganic germanium compounds and to investigate whether nanometer-sized germanium particles can act as radiosensitizers. Bis (2-carboxyethylgermanium) sesquioxide (Ge-132), germanium oxide (GeO(2)) and germanium nanoparticles were used in this study. Cell viability was determined by clonogenic survival assay. Cellular DNA damage was evaluated by alkaline comet assay, confocal microscopy and the cellular level of phospho-histone H2AX (gamma-H2AX). Nanometer-sized germanium particles were fabricated. They have a similar radiosensitizing effect as that of GeO(2). Conversely, Ge-132 did not enhance the radiosensitivity of cells. Comet assay was employed to evaluate the level of DNA damage and confirmed that inorganic germanium compounds enhanced cellular radiosensitivity. Notably, the comet assay indicated that the nanoparticle itself caused a higher level of DNA damage. The possibility that germanium nanoparticles per se caused DNA damage was ruled out when the cellular level of gamma-H2AX was examined. We demonstrated that inorganic but not organic germanium compounds exerted radiosensitizing effect in cells. Nanometer-sized germanium particles were fabricated and were able to enhance the radiosensitivity of cells. Confounding effect may occur when comet assay is used to estimate the level of DNA damage in the presence of germanium nanoparticles.

  3. Ionizing Radiation Promotes the Acquisition of a Senescence-Associated Secretory Phenotype and Impairs Angiogenic Capacity in Cerebromicrovascular Endothelial Cells: Role of Increased DNA Damage and Decreased DNA Repair Capacity in Microvascular Radiosensitivity

    PubMed Central

    Ungvari, Zoltan; Podlutsky, Andrej; Sosnowska, Danuta

    2013-01-01

    Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2–8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16INK4a), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier). PMID:23689827

  4. Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity.

    PubMed

    Ungvari, Zoltan; Podlutsky, Andrej; Sosnowska, Danuta; Tucsek, Zsuzsanna; Toth, Peter; Deak, Ferenc; Gautam, Tripti; Csiszar, Anna; Sonntag, William E

    2013-12-01

    Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2-8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16 (INK4a) ), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).

  5. Gold nanoparticles and electroporation impose both separate and synergistic radiosensitizing effects in HT-29 tumor cells: an in vitro study

    PubMed Central

    Rezaee, Zohre; Yadollahpour, Ali; Bayati, Vahid; Negad Dehbashi, Fereshteh

    2017-01-01

    Background and objective Radiation therapy (RT) is the gold standard treatment for more than half of known tumors. Despite recent improvements in RT efficiency, the side effects of ionizing radiation (IR) in normal tissues are a dose-limiting factor that restricts higher doses in tumor treatment. One approach to enhance the efficiency of RT is the application of radiosensitizers to selectively increase the dose at the tumor site. Gold nanoparticles (GNPs) and electroporation (EP) have shown good potential as radiosensitizers for RT. This study aims to investigate the sensitizing effects of EP, GNPs, and combined GNPs-EP on the dose enhancement factor (DEF) for 6 MV photon energy. Methods Radiosensitizing effects of EP, GNPs, and combinations of GNPs-EP were comparatively investigated in vitro for intestinal colon cancer (HT-29) and Chinese hamster ovary (CHO) cell lines by MTT assay and colony formation assay at 6 MV photon energy in six groups: IR (control group), GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR. Results Treatment of both cell lines with EP, GNPs, and combined GNPs-EP significantly enhanced the response of cells to irradiation. However, the HT-29 showed higher DEF values for all groups. In addition, the DEF value for HT-29 cells for GNPs+IR, GNPs (24 h)+IR, EP+IR, GNPs+EP+IR, and GNPs (24 h)+EP+IR was, respectively, 1.17, 1.47, 1.36, 2.61, and 2.89, indicating synergistic radiosensitizing effect for the GNPs (24 h)+EP+IR group. Furthermore, the synergistic effect was observed just for HT-29 tumor cell lines. Conclusion Combined GNPs-EP protocols induced synergistic radiosensitizing effect in HT-29 cells, and the effect is also tumor specific. This combined therapy can be beneficially used for the treatment of intrinsically less radiosensitive tumors. PMID:28260889

  6. Celecoxib enhances the radiosensitivity of HCT116 cells in a COX-2 independent manner by up-regulating BCCIP

    PubMed Central

    Xu, Xiao-Ting; Hu, Wen-Tao; Zhou, Ju-Ying; Tu, Yu

    2017-01-01

    It has been reported that celecoxib, a cyclooxygenase-2 (COX-2)-selective nonsteroidal anti-inflammatory drug (NSAID), regulates the radiosensitivity of several cancer cells. BCCIP (BRCA2 and CDKN1A interacting protein) plays a critical role in maintaining the critical functions of p53 in tumor suppression and response to therapy. However, whether the effect of celecoxib on the radiosensitivity of colorectal cancer (CRC) cells is dependent on BCCIP is largely unclear. In this study, we found that celecoxib enhanced the radiosensitivity of HeLa (a human cervical carcinoma cell line), A549 (a human lung carcinoma cell line), and HCT116 cells (a human CRC cells line). Among these cells, COX-2 expression was undetected in HCT116 cells. Treatment with celecoxib significantly increased BCCIP expression in COX-2 negative HCT116 cells. Knockdown of BCCIP obviously abrogated the enhanced radiosensitivity of HCT116 cells induced by celecoxib. A combination of celecoxib and irradiation treatment induced much more γ-H2AX foci formation, higher levels of radiation injury-related proteins phosphorylation, G2/M arrest, apoptosis, and p53 and p21 expression, and lower levels of Cyclin B1 in HCT116 cells than those in cells treated with irradiation alone. However, these changes were undetected in BCCIP-silenced HCT116 cells. Therefore, these data suggest that BCCIP gene may be a radiosensitivity-related gene in CRC. Celecoxib affects the functions of p53 and inhibits the recovery from the irradiation-induced injury by up-regulating the expression of BCCIP, and subsequently regulates the expressions of genes such as p21 and Cyclin B1 to enhance the radiosensitivity of HCT116 cells in a COX-2 independent manner. PMID:28386336

  7. Radiosensitivity of cultured insect cells: II. Diptera

    SciTech Connect

    Koval, T.M.

    1983-10-01

    The radiosensitivity of five dipteran cell lines representing three mosquito genera and one fruit fly genus were examined. These lines are: (1) ATC-10, Aedes aegypti; (2) RU-TAE-14, Toxorhynchites amboinensis; (3) RU-ASE-2A, Anopheles stephensi; (4) WR69-DM-1, Drosophila melanogaster; and (5) WR69-DM-2, Drosophila melanogaster. Population doubling times for these lines range from approximately 16 to 48 hr. Diploid chromosome numbers are six for the mosquito cells and eight for the fruit fly cells D/sub 0/ values are 5.1 and 6.5 Gy for the Drosophila cell lines and 3.6, 6.2, and 10.2 Gy for the mosquito cell lines. The results of this study demonstrate that dipteran insect cells are a few times more resistant to radiation than mammalian cells, but not nearly as radioresistant as lepidopteran cells.

  8. CHK2 is involved in the p53-independent radiosensitizing effects of valproic acid

    PubMed Central

    Choo, Dong Wan; Goh, Sung Ho; Cho, Young Woo; Baek, Hye Jung; Park, Eun Jung; Motoyama, Noboru; Kim, Tae Hyun; Kim, Joo Young; Kim, Sang Soo

    2017-01-01

    Radiotherapy is an effective treatment for the majority of types of localized solid cancer. However, the risk of side effects to the surrounding normal tissues limits radiotherapeutic approaches. Whilst the mechanism of action of valproic acid, an inhibitor of histone deacetylase, remains unknown, the inhibitor is a potential antineoplastic radiosensitizer. The present study demonstrated the in vitro radiosensitizing effects of valproic acid on the human breast cancer MCF7 cell line, and revealed that valproic acid increased the level of DNA breakage, apoptosis and senescence. In addition, western blot analyses revealed that valproic acid induced tumor suppressor protein (p)53 and p21 expression, and activated checkpoint kinase 2 (CHK2) in MCF7 cells and primary mouse embryonic fibroblasts. Notably, treatment with valproic acid also induced increases in the level of p21 protein levels and CHK2 activity in p53-null colon cancer HCT116 cells. Furthermore, the present study demonstrated that valproic acid-induced radiosensitization was largely dependent on the activity of CHK2. The results of the present study reveal that valproic acid may exhibit clinical utility with respect to increasing the anticancer efficacy of radiotherapy by affecting the level of p53. PMID:28454438

  9. HAP1 gene expression is associated with radiosensitivity in breast cancer cells

    SciTech Connect

    Wu, Jing; Zhang, Jun-ying; Yin, Li; Wu, Jian-zhong; Guo, Wen-jie; Wu, Jian-feng; Chen, Meng; Xia, You-you; Tang, Jin-hai; Ma, Yong-chao; He, Xia

    2015-01-02

    Highlights: • Overexpression of HAP1 gene promotes apoptosis in MCF-7 cells after irradiation. • HAP1 reduces tumor volume in nude mice xenograft models after irradiation. • HAP1 increases radiosensitivity of breast cancer cells in vitro and vivo. - Abstract: Objectives: The purpose of this study was to investigate the relationship between huntingtin-associated protein1 (HAP1) gene and radiation therapy of breast cancer cells. Methods: HAP1 gene was transfected into breast cancer MCF-7 cells, which was confirmed by quantitative reverse transcription-polymerase chain reaction analysis (qRT-PCR) and Western blot in vitro. The changes of cell radiosensitivity were assessed by colony formation assay. Apoptosis were examined by flow cytometry. The expressions of two radiation-induced genes were evaluated by Western blot. Tumor growth was investigated in nude mice xenograft models in vivo. Results: Our data showed that HAP1 gene expression was significantly increased in HAP1-transfected MCF-7 cells in comparison with the parental cells or negative control cells. The survival rate in MCF-7/HAP1 cells was significantly decreased after irradiation (0, 2, 4, 6, 8 Gy), compared to cells in MCF-7 and MCF-7/Pb groups in vitro. HAP1 gene increased apoptosis in MCF-7 cells after irradiation. Additionally, the tumor volume and weight in MCF-7/HAP1 + RT group were observably lower than in MCF-7/HAP1 group and MCF-7/Pb + RT group. Conclusion: The present study indicated that HAP1 gene expression was related to the radiosensitivity of breast cancer cells and may play an important role in the regulation of cellular radiosensitivity.

  10. Analogues of RSU-1069: radiosensitization and toxicity in vitro and in vivo.

    PubMed

    Ahmed, I; Jenkins, T C; Walling, J M; Stratford, I J; Sheldon, P W; Adams, G E; Fielden, E M

    1986-07-01

    A series of nitroimidazoles containing aziridine and alkyl-substituted aziridine functions has been synthesized. The 2-nitroimidazole compounds examined all show greater radiosensitizing efficiency in vitro than misonidazole. The 4- and 5-nitroimidazole analogues are also more efficient than equivalent compounds which do not contain the alkylating aziridine moiety. All the compounds show increased toxicity towards hypoxic cells relative to aerobic cells, but this toxicity is reduced by alkyl-substitution of the aziridine ring. In vivo toxicity can also be reduced by modification of the aziridine function, but such alterations appear to have less effect upon the high radiosensitizing efficiency of these compounds in vivo. As a consequence of this study, compounds of potentially improved therapeutic utility compared to RSU-1069, the first reported member of this class, have been identified.

  11. The High Radiosensitizing Efficiency of a Trace of Gadolinium-Based Nanoparticles in Tumors

    PubMed Central

    Dufort, Sandrine; Le Duc, Géraldine; Salomé, Murielle; Bentivegna, Valerie; Sancey, Lucie; Bräuer-Krisch, Elke; Requardt, Herwig; Lux, François; Coll, Jean-Luc; Perriat, Pascal; Roux, Stéphane; Tillement, Olivier

    2016-01-01

    We recently developed the synthesis of ultrasmall gadolinium-based nanoparticles (GBN), (hydrodynamic diameter <5 nm) characterized by a safe behavior after intravenous injection (renal clearance, preferential accumulation in tumors). Owing to the presence of gadolinium ions, GBN can be used as contrast agents for magnetic resonance imaging (MRI) and as radiosensitizers. The attempt to determine the most opportune delay between the intravenous injection of GBN and the irradiation showed that a very low content of radiosensitizing nanoparticles in the tumor area is sufficient (0.1 μg/g of particles, i.e. 15 ppb of gadolinium) for an important increase of the therapeutic effect of irradiation. Such a promising and unexpected result is assigned to a suited distribution of GBN within the tumor, as revealed by the X-ray fluorescence (XRF) maps. PMID:27411781

  12. The High Radiosensitizing Efficiency of a Trace of Gadolinium-Based Nanoparticles in Tumors

    NASA Astrophysics Data System (ADS)

    Dufort, Sandrine; Le Duc, Géraldine; Salomé, Murielle; Bentivegna, Valerie; Sancey, Lucie; Bräuer-Krisch, Elke; Requardt, Herwig; Lux, François; Coll, Jean-Luc; Perriat, Pascal; Roux, Stéphane; Tillement, Olivier

    2016-07-01

    We recently developed the synthesis of ultrasmall gadolinium-based nanoparticles (GBN), (hydrodynamic diameter <5 nm) characterized by a safe behavior after intravenous injection (renal clearance, preferential accumulation in tumors). Owing to the presence of gadolinium ions, GBN can be used as contrast agents for magnetic resonance imaging (MRI) and as radiosensitizers. The attempt to determine the most opportune delay between the intravenous injection of GBN and the irradiation showed that a very low content of radiosensitizing nanoparticles in the tumor area is sufficient (0.1 μg/g of particles, i.e. 15 ppb of gadolinium) for an important increase of the therapeutic effect of irradiation. Such a promising and unexpected result is assigned to a suited distribution of GBN within the tumor, as revealed by the X-ray fluorescence (XRF) maps.

  13. Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization.

    PubMed

    Maier, Patrick; Hartmann, Linda; Wenz, Frederik; Herskind, Carsten

    2016-01-14

    During the last few decades, improvements in the planning and application of radiotherapy in combination with surgery and chemotherapy resulted in increased survival rates of tumor patients. However, the success of radiotherapy is impaired by two reasons: firstly, the radioresistance of tumor cells and, secondly, the radiation-induced damage of normal tissue cells located in the field of ionizing radiation. These limitations demand the development of drugs for either radiosensitization of tumor cells or radioprotection of normal tissue cells. In order to identify potential targets, a detailed understanding of the cellular pathways involved in radiation response is an absolute requirement. This review describes the most important pathways of radioresponse and several key target proteins for radiosensitization.

  14. Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization

    PubMed Central

    Maier, Patrick; Hartmann, Linda; Wenz, Frederik; Herskind, Carsten

    2016-01-01

    During the last few decades, improvements in the planning and application of radiotherapy in combination with surgery and chemotherapy resulted in increased survival rates of tumor patients. However, the success of radiotherapy is impaired by two reasons: firstly, the radioresistance of tumor cells and, secondly, the radiation-induced damage of normal tissue cells located in the field of ionizing radiation. These limitations demand the development of drugs for either radiosensitization of tumor cells or radioprotection of normal tissue cells. In order to identify potential targets, a detailed understanding of the cellular pathways involved in radiation response is an absolute requirement. This review describes the most important pathways of radioresponse and several key target proteins for radiosensitization. PMID:26784176

  15. Metalloporphyrins and their uses as radiosensitizers for radiation therapy

    DOEpatents

    Miura, Michiko; Slatkin, Daniel N.

    2004-07-06

    The present invention covers radiosensitizers containing as an active ingredient halogenated derivatives of boronated porphyrins containing multiple carborane cages having the structure ##STR1## which selectively accumulate in neoplastic tissue within the irradiation volume and thus can be used in cancer therapies including, but not limited to, boron neutron--capture therapy and photodynamic therapy. The present invention also covers methods for using these radiosensitizers in tumor imaging and cancer treatment.

  16. Inhibitors of Histone Deacetylases for Radiosensitization of Prostate Cancer

    DTIC Science & Technology

    2005-02-01

    AD Award Number: W81XWH-04-1-0170 TITLE: Inhibitors of Histone Deacetylases for Radiosensitization of Prostate Cancer PRINCIPAL INVESTIGATOR: Mira 0...of Histone Deacetylases for Radiosensitization W81XWH-04-1-0170 of Prostate Cancer 6. AUTHOR(S) Mira 0. Jung, Ph.D. 7. PERFORMING ORGANIZA TION NAME(S...cellular radiation sensitivity. 14. SUBJECT TERMS 15. NUMBER OF PA GES Radiation sensitivity, histone deacetylase , cytotoxicity 13 16. PRICE CODE 17

  17. Cell-Specific Radiosensitization by Gold Nanoparticles at Megavoltage Radiation Energies

    SciTech Connect

    Jain, Suneil; Coulter, Jonathan A.; Hounsell, Alan R.; Butterworth, Karl T.; McMahon, Stephen J.; Hyland, Wendy B.; Muir, Mark F.; Dickson, Glenn R.; Prise, Kevin M.; Currell, Fred J.; O'Sullivan, Joe M.; Hirst, David G.

    2011-02-01

    Purpose: Gold nanoparticles (GNPs) have been shown to cause sensitization with kilovoltage (kV) radiation. Differences in the absorption coefficient between gold and soft tissue, as a function of photon energy, predict that maximum enhancement should occur in the kilovoltage (kV) range, with almost no enhancement at megavoltage (MV) energies. Recent studies have shown that GNPs are not biologically inert, causing oxidative stress and even cell death, suggesting a possible biological mechanism for sensitization. The purpose of this study was to assess GNP radiosensitization at clinically relevant MV X-ray energies. Methods and Materials: Cellular uptake, intracellular localization, and cytotoxicity of GNPs were assessed in normal L132, prostate cancer DU145, and breast cancer MDA-MB-231 cells. Radiosensitization was measured by clonogenic survival at kV and MV photon energies and MV electron energies. Intracellular DNA double-strand break (DSB) induction and DNA repair were determined and GNP chemosensitization was assessed using the radiomimetic agent bleomycin. Results: GNP uptake occurred in all cell lines and was greatest in MDA-MB-231 cells with nanoparticles accumulating in cytoplasmic lysosomes. In MDA-MB-231 cells, radiation sensitizer enhancement ratios (SERs) of 1.41, 1.29, and 1.16 were achieved using 160 kVp, 6 MV, and 15 MV X-ray energies, respectively. No significant effect was observed in L132 or DU145 cells at kV or MV energies (SER 0.97-1.08). GNP exposure did not increase radiation-induced DSB formation or inhibit DNA repair; however, GNP chemosensitization was observed in MDA-MB-231 cells treated with bleomycin (SER 1.38). Conclusions: We have demonstrated radiosensitization in MDA-MB-231 cells at MV X-ray energies. The sensitization was cell-specific with comparable effects at kV and MV energies, no increase in DSB formation, and GNP chemopotentiation with bleomycin, suggesting a possible biological mechanism of radiosensitization.

  18. Nimotuzumab promotes radiosensitivity of EGFR-overexpression esophageal squamous cell carcinoma cells by upregulating IGFBP-3

    PubMed Central

    2012-01-01

    Background Epidermal growth factor receptor (EGFR) is suggested to predict the radiosensitivity and/or prognosis of human esophageal squamous cell carcinoma (ESCC). The objective of this study was to investigate the efficacy of Nimotuzumab (an anti-EGFR monoclonal antibody) on ESCC radiotherapy (RT) and underlying mechanisms. Methods Nimotuzumab was administrated to 2 ESCC cell lines KYSE30 and TE-1 treated with RT. Cell growth, colony formation and apoptosis were used to measure anti-proliferation effects. The method of RNA interference was used to investigate the role of insulin-like growth factor binding protein-3 (IGFBP-3) in ESCC cells radiosensitivity treated with Nimotuzumab. In vivo effect of Nimotuzumab on ESCC radiotherapy was done using a mouse xenograft model. Results Nimotuzumab enhanced radiation response of KYSE30 cells (with high EGFR expression) in vitro, as evidenced by increased radiation-inhibited cell growth and colony formation and radiation-mediated apoptosis. Mechanism study revealed that Nimotuzumab inhibited phosphorylated EGFR (p-EGFR) induced by EGF in KYSE30 cells. In addition, knockdown of IGFBP-3 by short hairpin RNA significantly reduced KYSE30 cells radiosensitivity (P<0.05), and even after the administration of Nimotuzumab, the RT response of IGFBP-3 silenced KYSE30 cells was not enhanced (P>0.05). In KYSE30 cell xenografts, Nimotuzumab combined with radiation led to significant tumor growth delay, compared with that of radiation alone (P=0.029), and also with IGFBP-3 up-regulation in tumor tissue. Conclusions Nimotuzumab could enhance the RT effect of ESCC cells with a functional active EGFR pathway. In particular, the increased ESCC radiosensitivity by Nimotuzumab might be dependent on the up-regulation of IGFBP-3 through EGFR-dependent pathway. PMID:23232108

  19. Nimotuzumab promotes radiosensitivity of EGFR-overexpression esophageal squamous cell carcinoma cells by upregulating IGFBP-3.

    PubMed

    Zhao, Lei; He, Li-Ru; Xi, Mian; Cai, Mu-Yan; Shen, Jing-Xian; Li, Qiao-Qiao; Liao, Yi-Ji; Qian, Dong; Feng, Zi-Zhen; Zeng, Yi-Xin; Xie, Dan; Liu, Meng-Zhong

    2012-12-11

    Epidermal growth factor receptor (EGFR) is suggested to predict the radiosensitivity and/or prognosis of human esophageal squamous cell carcinoma (ESCC). The objective of this study was to investigate the efficacy of Nimotuzumab (an anti-EGFR monoclonal antibody) on ESCC radiotherapy (RT) and underlying mechanisms. Nimotuzumab was administrated to 2 ESCC cell lines KYSE30 and TE-1 treated with RT. Cell growth, colony formation and apoptosis were used to measure anti-proliferation effects. The method of RNA interference was used to investigate the role of insulin-like growth factor binding protein-3 (IGFBP-3) in ESCC cells radiosensitivity treated with Nimotuzumab. In vivo effect of Nimotuzumab on ESCC radiotherapy was done using a mouse xenograft model. Nimotuzumab enhanced radiation response of KYSE30 cells (with high EGFR expression) in vitro, as evidenced by increased radiation-inhibited cell growth and colony formation and radiation-mediated apoptosis. Mechanism study revealed that Nimotuzumab inhibited phosphorylated EGFR (p-EGFR) induced by EGF in KYSE30 cells. In addition, knockdown of IGFBP-3 by short hairpin RNA significantly reduced KYSE30 cells radiosensitivity (P<0.05), and even after the administration of Nimotuzumab, the RT response of IGFBP-3 silenced KYSE30 cells was not enhanced (P>0.05). In KYSE30 cell xenografts, Nimotuzumab combined with radiation led to significant tumor growth delay, compared with that of radiation alone (P=0.029), and also with IGFBP-3 up-regulation in tumor tissue. Nimotuzumab could enhance the RT effect of ESCC cells with a functional active EGFR pathway. In particular, the increased ESCC radiosensitivity by Nimotuzumab might be dependent on the up-regulation of IGFBP-3 through EGFR-dependent pathway.

  20. The toxic effects, GSH depletion and radiosensitivity by BSO on retinoblastoma

    SciTech Connect

    Xianjin Yi; Li Ding; Yizun Jin; Chuo Ni; Wenji Wang )

    1994-05-15

    Retinoblastoma is the most common intraocular malignant tumor in children. Previous investigations have reported that buthionine sulfoximine (BSO) can deplete intracellular glutathione (GSH) by specific inhibition and increase cellular radiosensitivity. The toxic effects, GSH depletion and radiosensitivity effects of BSO on retinoblastoma cells are reported in this paper. GSH content of retinoblastoma cell lines Y-79, So-Rb50 and retinoblastoma xenograft is 2.7 [+-] 1.3 X 1.0[sup [minus]12] mmol/cell, 1.4 [+-] 0.2 X 1.0[sup [minus]12] mmol/cell, and 2.8 [+-] 1.2 [mu]mol/g, respectively. The ID[sub 50] of BSO on Y-79 and So-Rb50 in air for 3 h exposure is 2.5 mM and 0.2 mM, respectively. GSH depletion by 0.1 mM BSO for 24 h on Y-79 cells and 0.01 mM BSO for 24 h on So-Rb50 cells is 16.35%, and 4.7% of control. GSH depletion in tumor and other organ tissues in retinoblastoma-bearing nude mice after BSO administration is differential. GSH depletion after BSO exposure in Y-79 cells in vitro decreases the Do value of retinoblastoma cells. The SER of 0.01 mM and 0.05 mM BSO for 24 h under hypoxic conditions is 1.21 and 1.36, respectively. Based on these observations, the authors conclude that BSO toxicity on retinoblastoma cells depends on the characteristics of the cell line and that BSO can increase hypoxic retinoblastoma cells' radiosensitivity in vitro. Further study of BSO radiosensitization on retinoblastoma in vivo using nude mouse xenografts is needed. 25 refs., 3 figs., 3 tabs.

  1. Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma.

    PubMed

    Sedlar, Ales; Kranjc, Simona; Dolinsek, Tanja; Cemazar, Maja; Coer, Andrej; Sersa, Gregor

    2013-01-29

    Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer. Solid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD(50) assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated. Single intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect. Single or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation.

  2. Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma

    PubMed Central

    2013-01-01

    Background Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer. Methods Solid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD50 assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated. Results Single intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect. Conclusions Single or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation. PMID:23360213

  3. Enhancement of radiosensitivity in human esophageal carcinoma cells by fenofibrate and its potential mechanism.

    PubMed

    Li, Xiao-Qing; Zhou, Jun-Dong; Zou, Shi-Tao; Yu, Jian; Meng, Xin-Jun; Wu, Jin-Chang

    2015-01-01

    Fenofibrate is a specific agonist of PPARα, and is characterized by relatively low systemic toxicity. Recent studies have revealed that fenofibrate suppresses the growth of several cancer lines in vitro, but the exact relation between fenofibrate and irradiation has not been explored. The purpose of this study was to investigate the radiosensitivity enhancement effects of fenofibrate combined with radiation on the human esophageal carcinoma cell lines Eca-109 and TE1, and the potential mechanism underlying these effects. The Eca-109 and TE1 cell lines were tested by the CCK-8 assay for cell proliferation. The multitarget click model was used to delineate the survival curve and radiosensitivity was determined after cells were treated with fenofibrate and/or x-ray radiation. Flow cytometry was used to examine the effect of fenofibrate and radiation on the cell cycle. The expression of vascular endothelial growth factor (VEGF) protein was detected by Western blot analysis. When given alone, fenofibrate had a time- and concentration-dependent cytotoxic effect on cells. The dose-enhancement ratio for combined fenofibrate and radiation increased markedly compared with fenofibrate alone. Further, the ratio of cells in the G2/M phase after fenofibrate and radiation was higher than that after fenofibrate or irradiation alone. The expression of VEGF protein was suppressed after treatment with fenofibrate alone or fenofibrate plus radiation. Fenofibrate can enhance the radiosensitivity of human esophageal carcinoma cells by increasing G2/M phase arrest. Modulation of VEGF expression could contribute in vivo to a favorable interaction.

  4. Fundamental mechanisms of DNA radiosensitization: damage induced by low-energy electrons in brominated oligonucleotide trimers.

    PubMed

    Park, Yeunsoo; Polska, Katarzyna; Rak, Janusz; Wagner, J Richard; Sanche, Léon

    2012-08-16

    The replacement of nucleobases with brominated analogs enhances DNA radiosensitivity. We examine the chemistry of low-energy electrons (LEEs) in this sensitization process by experiments with thin films of the oligonucleotide trimers TBrXT, where BrX = 5-BrU (5-bromouracil), 5-BrC (5-bromocytosine), 8-BrA (8-bromoadenine), or 8-BrG (8-bromoguanine). The products induced from irradiation of thin (∼ 2.5 nm) oligonucleotide films, with 10 eV electrons, under ultrahigh vacuum (UHV) are analyzed by HPLC-UV. The number of damaged brominated trimers ranges from about 12 to 15 × 10(-3) molecules per incident electron, whereas under the identical conditions, these numbers drop to 4-7 × 10(-3) for the same, but nonbrominated oligonucleotides. The results of HPLC analysis show that the main degradation pathway of trinucleotides containing brominated bases involve debromination (i.e., loss of the bromine atom and its replacement with a hydrogen atom). The electron-induced sum of products upon bromination increases by factors of 2.1 for the pyrimidines and 3.2 for the purines. Thus, substitution of any native nucleobase with a brominated one in simple models of DNA increases LEE-induced damage to DNA and hence its radiosensitivity. Furthermore, besides the brominated pyrimidines that have already been tested in clinical trials, brominated purines not only appear to be promising sensitizers for radiotherapy, but could provide a higher degree of radiosensitization.

  5. Salinomycin radiosensitizes human nasopharyngeal carcinoma cell line CNE-2 to radiation.

    PubMed

    Zhang, Yongqin; Zuo, Yun; Guan, Zhifeng; Lu, Weidong; Xu, Zheng; Zhang, Hao; Yang, Yan; Yang, Meilin; Zhu, Hongcheng; Chen, Xiaochen

    2016-01-01

    Nasopharyngeal carcinoma (NPC) is primarily treated by chemoradiation. However, how to promote radiation sensitivity in NPC remains a challenge. Salinomycin is potentially useful for the treatment of cancer. This study aimed to explore the radiosensitivity of salinomycin on human nasopharyngeal carcinoma cell line CNE-2. CNE-2 were treated with salinomycin or irradiation, alone or in combination. The cytotoxicity effects of salinomycin were measured using CCK-8 assay. Clonogenic survival assay was used to evaluate the effects of salinomycin on the radiosensitivity of CNE-2. The changes of cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of Caspase3/Bax/Bal-2 was detected by Western blotting. DNA damage was detected via γ-H2AX foci counting. The results showed that salinomycin induced apoptosis and G2/M arrest, increased Bax and cleaved Caspase3, decreased Bcl-2 expression, and increased the formation of γ-H2AX nuclear foci. These data suggest that salinomycin may be a radiosensitizer for NPC radiotherapy.

  6. Enhancement of 5- Fluorouracil-induced In Vitro and In Vivo Radiosensitization with MEK Inhibition

    PubMed Central

    Urick, Mary Ellen; Chung, Eun Joo; Shield, William P.; Gerber, Naamit; White, Ayla; Sowers, Anastasia; Thetford, Angela; Camphausen, Kevin; Mitchell, James; Citrin, Deborah E.

    2011-01-01

    Purpose Gastrointestinal cancers frequently exhibit mutational activation of the Ras/MAPK pathway, which is implicated in resistance to ionizing radiation (IR) and chemotherapy. Concurrent radiotherapy and 5-fluorouracil (5-FU) based chemotherapy is commonly used for treatment of gastrointestinal malignancies. We previously reported radiosensitization with selumetinib, an inhibitor of MEK1/2. The purpose of the current study was to evaluate if selumetinib could enhance radiosensitivity induced by 5-FU. Experimental Design Clonogenic survival assays were performed with the HT29 (colorectal), HCT116 (colorectal) and MiaPaca-2 (pancreatic) cell lines using pre-IR treatment with selumetinib, 5-FU and 5-FU+selumetinib. Cell proliferation was determined using a tetrazolium conversion assay. Mitotic catastrophe and DNA repair were analyzed using immunocytochemistry. Flow cytometry was used to analyze cell cycle and apoptosis. Growth delay was used to determine effects of 5-FU+selumetinib on in vivo tumor radiosensitivity. Results Pre-IR treatment with 5-FU+selumetinib significantly decreased clonogenic survival compared to either agent alone. Dose modifying factors at a surviving fraction of 0.1 for 5-FU+selumetinib was 1.78, 1.52, and 1.3 for HT29, HCT116, and MiaPaca-2, respectively. Cell proliferation was decreased by treatment with selumetinib+5-FU as compared to single agent treatment regardless of treatment sequencing. Enhancement of 5-FU cytotoxicity and 5-FU mediated radiosensitization with selumetinib treatment was accompanied by an increase in mitotic catastrophe and apoptosis, and reductions in Stat3 phosphorylation and survivin expression. In vivo, an additive growth delay was observed with 5-FU+selumetinib+5Gy versus 5-FU+5Gy and selumetinib alone. Conclusion These data suggest that selumetinib can be used with 5-FU to augment radiation response. PMID:21690569

  7. Comparison of microwave and magnetic nanoparticle hyperthermia radiosensitization in murine breast tumors

    NASA Astrophysics Data System (ADS)

    Giustini, Andrew J.; Petryk, Alicia A.; Hoopes, Paul J.

    2011-03-01

    Hyperthermia has been shown to be an effective radiosensitizer. Its utility as a clinical modality has been limited by a minimally selective tumor sensitivity and the inability to be delivered in a tumor-specific manner. Recent in vivo studies (rodent and human) have shown that cancer cell-specific cytotoxicity can be effectively and safely delivered via iron oxide magnetic nanoparticles (mNP) and an appropriately matched noninvasive alternating magnetic field (AMF). To explore the tumor radiosensitization potential of mNP hyperthermia we used a syngeneic mouse breast cancer model, dextran-coated 110 nm hydrodynamic diameter mNP and a 169 kHz / 450 Oe (35.8 kA/m) AMF. Intradermally implanted (flank) tumors (150 +/- 40 mm3) were treated by injection of 0.04 ml mNP (7.5 mg Fe) / cm3 into the tumor and an AMF (35.8 kA/m and 169 kHz) exposure necessary to achieve a CEM (cumulative equivalent minute) thermal dose of 60 (CEM 60). Tumors were treated with mNP hyperthermia (CEM 60), radiation alone (15 Gy, single dose) and in combination. Compared to the radiation and heat alone treatments, the combined treatment resulted in a greater than two-fold increase in tumor regrowth tripling time (tumor treatment efficacy). None of the treatments resulted in significant normal tissue toxicity or morbidity. Studies were also conducted to compare the radiosensitization effect of mNP hyperthermia with that of microwave-induced hyperthermia. The effects of incubation of nanoparticles within tumors (to allow nanoparticles to be endocytosed) before application of AMF and radiation were determined. This preliminary information suggests cancer cell specific hyperthermia (i.e. antibody-directed or anatomically-directed mNP) is capable of providing significantly greater radiosensitization / therapeutic ratio enhancement than other forms of hyperthermia delivery.

  8. Radiosensitivity of human cell lines to small doses. Are there some clinical implications?

    SciTech Connect

    Malaise, E.P.; Joiner, M.C.; Lambin, P.

    1994-04-01

    The concept of intrinsic radiosensitivity is now strongly associated with the linear-quadratic (LQ) model which is currently the best and the most reliable method to fit the first three decades of a survival curve for both human fibroblast and human tumor cell lines. This approach has led to the major conclusions that it is the initial part, and not the distal part, of the survival curve which truly characterizes intrinsic cellular radiosensitivity and there is a correlation between the parameters describing mainly the initial part of the survival curve ({alpha}, SF{sub 2} {bar D}) and the clinical radioresponsiveness. More accurate analysis with flow cytometry or a dynamic microscopic image processing scanner (DMIPS) has allowed further study of the survival curve which has shown two sorts of substructure. On one hand, the overall survival curve of exponentially growing cells is described by two or more sets of {alpha}, {beta} parameters (heterogeneity in radiosensitivity due to the cell cycle). On the other hand, hypersensitivity at very low doses (<0.5 Gy) followed by an increase of the radioresistance of the whole population at higher doses has also been observed. This phenomenon is not described by the conventional LQ model and has been interpreted as an induced radioresistance which seems to be negatively correlated with intrinsic radiosensitivity. In clinical radiotherapy, there are two sorts of response of normal tissues: (1) the early and late damage and (2) the carcinogenesis. Concerning the first point, the clinically detectable radiation damage appears at doses usually around 20 Gy (in 2-Gy fractions) with the exception of the hemopoietic and the lymphatic tissues. Therefore, the small doses delivered at the edges or in the penumbrae of treatment fields in routine radiotherapy cannot create detectable damage, despite a potentially much higher effect per unit dose, because the total doses are still very small. 17 refs.

  9. Histone deacetylase enzyme silencing using shRNAs enhances radiosensitivity of SW579 thyroid cancer cells

    PubMed Central

    Wang, Ye; Jin, Tao; Dai, Xueming; Yan, Dongwang; Peng, Zhihai

    2016-01-01

    The aim of the present study was to screen the enzymes that are associated with the radiosensitivity of SW579 thyroid cancer cells, and investigate whether radiation, combined with specific RNA interference on the screened enzymes, enhances radiosensitivity of SW579 thyroid cancer cells. Quantitative polymerase chain reaction (qPCR) was used to analyze epigenetic enzyme expression changes before and after radiotherapy, and four enzymes, histone deacetylase 1 (HDAC1), HDAC2, HDAC4 and HDAC6 were screened. Western blot analysis was performed to analyze the change in HDAC1, HDAC2, HDAC4 and HDAC6 protein expression following radiotherapy. Short hairpin RNA (ShRNA)-HDAC1, shRNA-HDAC2, shRNA-HDAC4 and shRNA-HDAC6 plasmids were constructed and SW579 cells were transfected with corresponding shRNA-HDACs. Reverse transcription-qPCR was used to detect whether downregulation of HDAC mRNAs had been effective. In addition, shRNA and shRNA negative control (NC) pools were established and transfected into the SW579 cells. The samples were divided into four groups; control, trichostatin A, shRNA pool and shRNA NC pool, to analyze the effective enhancement of specific shRNA on radiosensitivity in thyroid cancer cells. The morphological changes were observed in the SW579 cells, and the number of tumor cells decreased markedly in the shRNA pool group compared with that of the other three groups. Therefore, it was concluded that HDACs present a potential target for increasing the sensitivity of thyroid cancer cells to radiotherapy, and shRNA-HDAC interference combined with radiotherapy promotes the radiosensitivity of tumors. PMID:27600599

  10. Radiosensitizing Effect of P2X7 Receptor Antagonist on Melanoma in vitro and in vivo.

    PubMed

    Tanamachi, Keisuke; Nishino, Keisuke; Mori, Natsuki; Suzuki, Toshihiro; Tanuma, Sei-Ichi; Abe, Ryo; Tsukimoto, Mitsutoshi

    2017-03-24

    Melanoma is highly malignant, and generally exhibits radioresistance, responding poorly to radiation therapy. We previously reported that activation of P2X7, P2Y6, and P2Y12 receptors is involved in the DNA damage response after γ-irradiation of human lung adenocarcinoma A549 cells. However, it is not clear whether these receptors are also involved in the case of melanoma cells, although P2X7 receptor is highly expressed in various cancers, including melanoma. Here, we show that P2X7 receptor antagonist enhances radiation-induced cytotoxicity in B16 melanoma cells in vitro and in vivo. We confirmed that these cells express P2X7 receptor mRNA and exhibit P2X7 receptor-mediated activities, such as ATP-induced pore formation and cytotoxicity. We further examined the radiosensitizing effect of P2X7 receptor antagonist Brilliant Blue G (BBG) in vitro by colony formation assay of B16 cells. γ-Irradiation dose-dependently reduced cell survival, and pretreatment with BBG enhanced the radiation-induced cytotoxicity. BBG pretreatment also decreased the number of DNA repair foci in nuclei, supporting involvement of P2X7 receptor in the DNA damage response. Finally, we investigated the radiosensitizing effect of BBG on B16 melanoma cells inoculated into the hind footpad of C57BL/6 mice. Neither 1 Gy γ-irradiation alone nor BBG alone suppressed the increase of tumor volume, but the combination of irradiation and BBG significantly suppressed tumor growth. Our results suggest that P2X7 receptor antagonist BBG has a radiosensitizing effect in melanoma in vitro and in vivo. BBG, which is used as a food coloring agent, appears to be a promising candidate as a radiosensitizer.

  11. Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy.

    PubMed

    Yuk, Jae-Min; Shin, Dong-Min; Song, Kyoung-Sub; Lim, Kyu; Kim, Ki-Hye; Lee, Sang-Hee; Kim, Jin-Man; Lee, Ji-Sook; Paik, Tae-Hyun; Kim, Jun-Sang; Jo, Eun-Kyeong

    2010-01-01

    The cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guerin (BCG/CWS) is an effective antitumor immunotherapy agent. Here, we demonstrate that BCG/CWS has a radiosensitizing effect on colon cancer cells through the induction of autophagic cell death. Exposure of HCT116 colon cancer cells to BCG/CWS before ionizing radiation (IR) resulted in increased cell death in a caspase-independent manner. Treatment with BCG/CWS plus IR resulted in the induction of autophagy in colon cancer cells. Either the autophagy inhibitor 3-methyladenine or knockdown of beclin 1 or Atg7 significantly reduced tumor cell death induced by BCG/CWS plus IR, whereas the caspase inhibitor z-VAD-fmk failed to do so. BCG/CWS plus IR-mediated autophagy and cell death was mediated predominantly by the generation of reactive oxygen species (ROS). The c-Jun NH(2)-terminal kinase pathway functioned upstream of ROS generation in the induction of autophagy and cell death in HCT116 cells after co-treatment with BCG/CWS and IR. Furthermore, toll-like receptor (TLR) 2, and in part, TLR4, were responsible for BCG/CWS-induced radiosensitization. In vivo studies revealed that BCG/CWS-mediated radiosensitization of HCT116 xenograft growth is accompanied predominantly by autophagy. Our data suggest that BCG/CWS in combination with IR is a promising therapeutic strategy for enhancing radiation therapy in colon cancer cells through the induction of autophagy.

  12. Radiosensitization by the ATR Inhibitor AZD6738 through Generation of Acentric Micronuclei

    PubMed Central

    Dillon, Magnus T.; Barker, Holly E.; Pedersen, Malin; Hafsi, Hind; Bhide, Shreerang A.; Newbold, Kate L.; Nutting, Christopher M.

    2017-01-01

    AZD6738 is an orally active ATR inhibitor (ATRi) currently in phase I clinical trials. We found in vitro growth inhibitory activity of this ATRi in a panel of human cancer cell lines. We demonstrated radiosensitization by AZD6738 to single radiation fractions in multiple cancer cell lines independent of both p53 and BRCA2 status by the clonogenic assay. Radiosensitization by AZD6738 to clinically relevant doses of fractionated radiation was demonstrated in vitro using a 3D tumor spheroid model and, in vivo, AZD6738 radiosensitized by abrogating the radiation-induced G2 cell-cycle checkpoint and inhibiting homologous recombination. Mitosis with damaged DNA resulted in mitotic catastrophe as measured by micronucleus formation by live-cell fluorescent-ubiquitination cell-cycle imaging of cell-cycle progression and nuclear morphology. Induction of micronuclei was significantly more prominent for AZD6738 compared with inhibition of the downstream kinase CHK1 alone at isoeffective doses. Micronuclei were characterized as acentric chromosomal fragments, which displayed characteristics of increased DNA damage and cell-cycle dyssynchrony when compared with the primary nucleus. PMID:28062704

  13. N-acetylglucosaminyltransferase V modulates radiosensitivity and migration of small cell lung cancer through epithelial-mesenchymal transition.

    PubMed

    Huang, Chunyue; Huang, Miaojuan; Chen, Wenxia; Zhu, Weiliang; Meng, Hui; Guo, Linlang; Wei, Ting; Zhang, Jian

    2015-11-01

    N-acetylglucosaminyltransferase V (Gnt-V) has been linked to the migration of various human cancers. Recently we have found that inhibition of Gnt-V increases the radiosensitivity of cancer cells. However, the mechanisms by which Gnt-V mediates radiosensitivity and migration, especially in small cell lung cancer (SCLC) remain unknown. In our study, two SCLC cell lines (H1688 and H146) were used to investigate whether Gnt-V modulated the radiosensitivity and migration of SCLC cells through the epithelial-mesenchymal transition (EMT). The results showed that the expression of Gnt-V correlated with the N stage in patients with SCLC. Overexpression of Gnt-V led to a further increase in the relative viable cell number and survival fraction with a decrease in apoptosis rate and Bax/Bcl-2 ratio, when the cells were treated with irradiation. By contrast, knockdown of Gnt-V with irradiation resulted in a further decrease in the relative viable cell number and survival fraction but an increase in apoptosis rate and Bax/Bcl-2 ratio. Cells expressing high levels of Gnt-V increased migration whereas low levels of Gnt-V suppressed cell migration. Besides, the transient knockdown of ZEB2 led to an increase in radiosensitivity and an inhibition in the migration of SCLC cells. Furthermore, Gnt-V was negatively correlated with E-cadherin expression but positively correlated with N-cadherin, vimentin and ZEB2 expression. Finally, an in vivo study revealed that upregulation of Gnt-V caused tumour growth more quickly, as well as the expression of EMT-related markers (N-cadherin, vimentin and ZEB2). Taken together, the study suggested that an elevation of Gnt-V could lead to the radiosensitivity and migration of SCLC cells by inducing EMT, thereby highlighting Gnt-V as a potential therapeutic target for the prevention of EMT-associated tumour radioresistance and migration.

  14. Fulvestrant radiosensitizes human estrogen receptor-positive breast cancer cells

    SciTech Connect

    Wang, Jing; Yang, Qifeng; Haffty, Bruce G.; Li, Xiaoyan; Moran, Meena S.

    2013-02-08

    Highlights: ► Fulvestrant radiosensitizes MCF-7 cells. ► Fulvestrant increases G1 arrest and decreases S phase in MCF-7 cells. ► Fulvestrant down-regulates DNA-PKcs and RAD51 in MCF-7 cells. -- Abstract: The optimal sequencing for hormonal therapy and radiation are yet to be determined. We utilized fulvestrant, which is showing promise as an alternative to other agents in the clinical setting of hormonal therapy, to assess the cellular effects of concomitant anti-estrogen therapy (fulvestrant) with radiation (F + RT). This study was conducted to assess the effects of fulvestrant alone vs. F + RT on hormone-receptor positive breast cancer to determine if any positive or negative combined effects exist. The effects of F + RT on human breast cancer cells were assessed using MCF-7 clonogenic and tetrazolium salt colorimetric (MTT) assays. The assays were irradiated with a dose of 0, 2, 4, 6 Gy ± fulvestrant. The effects of F + RT vs. single adjuvant treatment alone on cell-cycle distribution were assessed using flow cytometry; relative expression of repair proteins (Ku70, Ku80, DNA-PKcs, Rad51) was assessed using Western Blot analysis. Cell growth for radiation alone vs. F + RT was 0.885 ± 0.013 vs. 0.622 ± 0.029 @2 Gy, 0.599 ± 0.045 vs. 0.475 ± 0.054 @4 Gy, and 0.472 ± 0.021 vs. 0.380 ± 0.018 @6 Gy RT (p = 0.003). While irradiation alone induced G2/M cell cycle arrest, the combination of F + RT induced cell redistribution in the G1 phase and produced a significant decrease in the proportion of cells in G2 phase arrest and in the S phase in breast cancer cells (p < 0.01). Furthermore, levels of repair proteins DNA-PKcs and Rad51 were significantly decreased in the cells treated with F + RT compared with irradiation alone. F + RT leads to a decrease in the surviving fraction, increased cell cycle arrest, down regulating of nonhomologous repair protein DNA-PKcs and homologous recombination repair protein RAD51. Thus, our findings suggest that F + RT

  15. Oxidized silicon nanoparticles for radiosensitization of cancer and tissue cells.

    PubMed

    Klein, Stefanie; Dell'Arciprete, Maria L; Wegmann, Marc; Distel, Luitpold V R; Neuhuber, Winfried; Gonzalez, Mónica C; Kryschi, Carola

    2013-05-03

    The applicability of ultrasmall uncapped and aminosilanized oxidized silicon nanoparticles (SiNPs and NH2-SiNPs) as radiosensitizer was studied by internalizing these nanoparticles into human breast cancer (MCF-7) and mouse fibroblast cells (3T3) that were exposed to X-rays at a single dose of 3 Gy. While SiNPs did not increase the production of reactive oxygen species (ROS) in X-ray treated cells, the NH2-SiNPs significantly enhanced the ROS formation. This is due to the amino functionality as providing positive surface charges in aqueous environment. The NH2-SiNPs were observed to penetrate into the mitochondrial membrane, wherein these nanoparticles provoked oxidative stress. The NH2-SiNPs induced mitochondrial ROS production was confirmed by the determination of an increased malondialdehyde level as representing a gauge for the extent of membrane lipid peroxidation. X-ray exposure of NH2-SiNPs incubated MCF-7 and 3T3 cells increased the ROS concentration for 180%, and 120%, respectively. Complementary cytotoxicity studies demonstrate that these silicon nanoparticles are more cytotoxic for MCF-7 than for 3T3 cells.

  16. Radiosensitivity of Human Fibroblasts is Associated With Amino Acid Substitution Variants in Susceptible Genes And Correlates With The Number of Risk Alleles

    SciTech Connect

    Alsbeih, Ghazi . E-mail: galsbeih@kfshrc.edu.sa; El-Sebaie, Medhat; Al-Harbi, Najla; Al-Buhairi, Muneera; Al-Hadyan, Khaled; Al-Rajhi, Nasser

    2007-05-01

    Purpose: Genetic predictive markers of radiosensitivity are being sought for stratifying radiotherapy for cancer patients and risk assessment of radiation exposure. We hypothesized that single nucleotide polymorphisms in susceptible genes are associated with, and the number of risk alleles has incremental effect on, individual radiosensitivity. Methods and Materials: Six amino acid substitution variants (ATM 1853 Asp/Asn G>A, p53 72 Arg/Pro G>C, p21 31 Ser/Arg C>A, XRCC1 399 Arg/Gln G>A, XRCC3 241 Thr/Met C>T, and TGF{beta}1 10 Leu/Pro T>C) were genotyped by direct sequencing in 54 fibroblast strains of different radiosensitivity. Results: The clonogenic survival fraction at 2 Gy range was 0.15-0.50 (mean, 0.34, standard deviation, 0.08). The mean survival fraction at 2 Gy divided the cell strains into radiosensitive (26 cases) and normal (28 controls). A significant association was observed between the survival fraction at 2 Gy and ATM 1853 Asn, XRCC3 241 Met, and TGF{beta}1 10 Leu alleles (p = 0.05, p = 0.02, and p = 0.02, respectively). The p53 72 Arg allele showed a borderline association (p = 0.07). The number of risk alleles increased with increasing radiosensitivity, and the group comparison showed a statistically significant difference between the radiosensitive and control groups (p {<=}0.001). Conclusion: The results of our study have shown that single nucleotide polymorphisms in susceptible genes influence cellular radiation response and that the number of risk alleles has a combined effect on radiosensitivity. Individuals with multiple risk alleles could be more susceptible to radiation effects than those with fewer risk alleles. These results may have implications in predicting normal tissue reactions to radiotherapy and risk assessment of radiation exposure.

  17. Silver nanoparticles outperform gold nanoparticles in radiosensitizing U251 cells in vitro and in an intracranial mouse model of glioma.

    PubMed

    Liu, Peidang; Jin, Haizhen; Guo, Zhirui; Ma, Jun; Zhao, Jing; Li, Dongdong; Wu, Hao; Gu, Ning

    Radiotherapy performs an important function in the treatment of cancer, but resistance of tumor cells to radiation still remains a serious concern. More research on more effective radiosensitizers is urgently needed to overcome such resistance and thereby improve the treatment outcome. The goal of this study was to evaluate and compare the radiosensitizing efficacies of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) on glioma at clinically relevant megavoltage energies. Both AuNPs and AgNPs potentiated the in vitro and in vivo antiglioma effects of radiation. AgNPs showed more powerful radiosensitizing ability than AuNPs at the same mass and molar concentrations, leading to a higher rate of apoptotic cell death. Furthermore, the combination of AgNPs with radiation significantly increased the levels of autophagy as compared with AuNPs plus radiation. These findings suggest the potential application of AgNPs as a highly effective nano-radiosensitizer for the treatment of glioma.

  18. Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

    PubMed

    Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

    2005-11-01

    Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

  19. Silver nanoparticles outperform gold nanoparticles in radiosensitizing U251 cells in vitro and in an intracranial mouse model of glioma

    PubMed Central

    Liu, Peidang; Jin, Haizhen; Guo, Zhirui; Ma, Jun; Zhao, Jing; Li, Dongdong; Wu, Hao; Gu, Ning

    2016-01-01

    Radiotherapy performs an important function in the treatment of cancer, but resistance of tumor cells to radiation still remains a serious concern. More research on more effective radiosensitizers is urgently needed to overcome such resistance and thereby improve the treatment outcome. The goal of this study was to evaluate and compare the radiosensitizing efficacies of gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) on glioma at clinically relevant megavoltage energies. Both AuNPs and AgNPs potentiated the in vitro and in vivo antiglioma effects of radiation. AgNPs showed more powerful radiosensitizing ability than AuNPs at the same mass and molar concentrations, leading to a higher rate of apoptotic cell death. Furthermore, the combination of AgNPs with radiation significantly increased the levels of autophagy as compared with AuNPs plus radiation. These findings suggest the potential application of AgNPs as a highly effective nano-radiosensitizer for the treatment of glioma. PMID:27757033

  20. Nuclear 3D organization and radiosensitivity

    NASA Astrophysics Data System (ADS)

    Eidelman, Y. A.; Slanina, S. V.; Aleshchenko, A. V.; Sen'ko, O. V.; Kononkova, A. D.; Andreev, S. G.

    2017-01-01

    Current mechanisms of radiation-induced chromosomal aberration (CA) formation suggest misrepair of chromosomal lesions being in spatial proximity. In this case CAs have to depend on pattern of chromosomal contacts and on chromosome spatial organization in a cell nucleus. We were interested in whether variation of nucleus 3D organization results in difference of radiation induced CA formation frequency. Experimental data available do not provide information sufficient for definite conclusions. To have more deep insight in this issue we developed the biophysical modeling technique taking into account different levels of chromosome/nuclear organization and radiation damage of DNA and chromosomes. Computer experiments on gamma irradiation were carried out for two types of cells with different 3D organization of nuclei, preferentially peripheral and internal. CA frequencies were found to depend on spatial positioning of chromosomes within a nucleus which determines a pattern of interchromosomal contacts. For individual chromosomes this effect can be more pronounced than for genome averaged. Since significant part of aberrations, for example dicentrics, results in cell death, the proposed technique is capable of evaluating radiosensitivity of cells, both normal and cancer, with the incorporation of 3D genome information. This predictive technology allows to reduce uncertainties of prognosis of biological effects of radiation compared to phenomenological methods and may have variety of biomedical applications, in particular, in cancer radiation therapy.

  1. N-methylformamide: cytotoxic, radiosensitizer, or chemosensitizer

    SciTech Connect

    Clagett-Carr, K.; Sarosy, G.; Plowman, J.; Hoth, D.F.; Leyland-Jones, B.

    1988-05-01

    N-methylformamide (NMF), a polar solvent, is currently being evaluated by the National Cancer Institute (NCI) as an antineoplastic agent because of its activity against colon, mammary, and lung tumor xenografts. Results from preclinical studies suggest that it has radiosensitizing, chemosensitizing, and differentiating activity. Its mechanism of action remains unknown, but may involve cellular depletion of glutathione, cell membrane changes, or modulation of proto-oncogene expression. Preclinical toxicology studies conducted in mice, rats, and beagle dogs showed reversible hepatotoxicity to be dose-limiting. Clinically, NMF is administered both orally and by intravenous (IV) injection. The bioavailability with oral administration is 90% to 95%. The highest reported plasma concentration of NMF is approximately 4 mmol/L in a patient who received a dose of 2,000 mg/m2 of IV NMF. Biphasic elimination with IV NMF is seen on both the daily for five days and weekly for 3 weeks schedule. Approximately 5% to 7% of the total administered IV dose is excreted in the urine. In phase I studies, dose-limiting toxicities included reversible hepatotoxicity, a generalized malaise syndrome, and nausea and vomiting. One partial response has been reported in the 111 patients treated on phase II trials in colorectal, head and neck, and renal carcinomas. Suggestions for the future development of this drug are presented. 82 references.

  2. Celecoxib Induced Tumor Cell Radiosensitization by Inhibiting Radiation Induced Nuclear EGFR Transport and DNA-Repair: A COX-2 Independent Mechanism

    SciTech Connect

    Dittmann, Klaus H. Mayer, Claus; Ohneseit, Petra A.; Raju, Uma; Andratschke, Nickolaus H.; Milas, Luka; Rodemann, H. Peter

    2008-01-01

    Purpose: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. Methods and Materials: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by {gamma}H{sub 2}AX foci assay. Results: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observed radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual {gamma}H2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. Conclusions: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.

  3. Clonal cell populations unresponsive to radiosensitization induced by telomerase inhibition

    SciTech Connect

    Ju, Yeun-Jin; Shin, Hyun-Jin; Park, Jeong-Eun; Juhn, Kyoung-Mi; Woo, Seon Rang; Kim, Hee-Young; Han, Young-Hoon; Hwang, Sang-Gu; Hong, Sung-Hee; Kang, Chang-Mo; Yoo, Young-Do; Park, Won-Bong; Cho, Myung-Haing; Park, Gil Hong; Lee, Kee-Ho

    2010-11-12

    Research highlights: {yields} In our present manuscript, we have clearly showed an interesting but problematic obstacle of a radiosensitization strategy based on telomerase inhibition by showing that: Clonal population unresponsive to this radiosensitization occasionally arise. {yields} The telomere length of unsensitized clones was reduced, as was that of most sensitized clones. {yields} The unsensitized clones did not show chromosome end fusion which was noted in all sensitized clones. {yields} P53 status is not associated with the occurrence of unsensitized clone. {yields} Telomere end capping in unsensitized clone is operative even under telomerase deficiency. -- Abstract: A combination of a radiotherapeutic regimen with telomerase inhibition is valuable when tumor cells are to be sensitized to radiation. Here, we describe cell clones unresponsive to radiosensitization after telomere shortening. After extensive division of individual transformed clones of mTERC{sup -/-} cells, about 22% of clones were unresponsive to radiosensitization even though telomerase action was inhibited. The telomere lengths of unsensitized mTERC{sup -/-} clones were reduced, as were those of most sensitized clones. However, the unsensitized clones did not exhibit chromosomal end-to-end fusion to the extent noted in all sensitized clones. Thus, a defense mechanism preventing telomere erosion is operative even when telomeres become shorter under conditions of telomerase deficiency, and results in unresponsiveness to the radiosensitization generally mediated by telomere shortening.

  4. Testicular orphan nuclear receptor 4 is associated with the radio-sensitivity of prostate cancer.

    PubMed

    Yu, Shicheng; Wang, Mingchao; Ding, Xianfan; Xia, Liqun; Chen, Bide; Chen, Yicheng; Zhang, Zhigen; Niu, Yuanjie; Li, Gonghui; Chang, Chawnshang

    2015-10-01

    It is well known that a significant number of prostate cancers (PCa) showed different extents of radio-resistance and the tumor may recur after treatment. Recent studies demonstrated that Testicular orphan nuclear receptor 4 (TR4) could play a critical role in anti-oxidative stress responses and might modulate the DNA damage repair. The objective of this study is to investigate the role of TR4 in the radiotherapy for PCa. The TR4 expression in tissue samples from PCa patients treated with brachytherapy was measured by immunohistochemistry (IHC). Cell survival test and colony formation assay were applied to test the radio-sensitivity of PCa cells with modulated TR4 gene expression upon irradiation. PCa patients with biochemical recurrence (BCR) after brachytherapy tend to have higher TR4 expression (80%, n = 30) as compared to those without BCR (36.67%, n = 30). Survival analysis demonstrated a significant higher BCR occurrence in patients with high level of TR4 expression (HR = 3.474, 95%CI 1.678-7.192, P = 0.0008). Multivariate analysis showed that the TR4 staining score on IHC was the only significant variable for predicting the PCa patients' clinical outcomes after radiotherapy (OR = 9.919, 95% CI 2.516-39.101, P = 0.001). Using cell survival test and colony forming assay, we found that the addition of functional TR4 in PC3 cells lead to elevated radio-resistance. In contrast, knocking-down TR4 in LNCaP cells resulted in increased radio-sensitivity. The γH2AX foci kinetic analysis suggested that knocking down TR4 might delay the PCa cell's DNA damage repair which would enhance the radio-sensitivity. TR4 could mediate the PCa cells' radio-sensitivity and might become a prognostic indicator for PCa patients received radiotherapy. This study provides a novel approach to manipulate radio-sensitivity of PCa cells, and may bring a promoted therapeutic outcome of radiotherapy to battle PCa in future. © 2015 Wiley Periodicals, Inc.

  5. Radiosensitivity of fibroblasts obtained from a cafe-au-lait spot and normal-appearing skin of a patient with neurofibromatosis (NF-6)

    SciTech Connect

    Hannan, M.A.; Smith, B.P.; Sigut, D.; Sackey, K. )

    1990-07-15

    Fibroblast cells derived from a cafe-au-lait spot and normal-appearing skin of a neurofibromatosis (NF-6) patient were studied for radiosensitivity in comparison with two normal cell lines used as controls. No difference in radiosensitivity was observed between the patient's cell lines and the controls using acute gamma-irradiation. However, a markedly increased radiosensitivity of the fibroblasts obtained from the patient's skin of normal appearance was demonstrated after chronic gamma-irradiation. The cells from the cafe-au-lait spot showed intermediate sensitivity to chronic irradiation as compared with the control cell lines and the fibroblasts derived from the normal skin of the patient. These results showed the usefulness of chronic irradiation in detecting increased cellular radiosensitivity which may result from a unique DNA repair defect in an NF patient. We suggest that enhanced genetic changes in radiosensitive NF patients may lead to formation of cafe-au-lait lesions and certain tumors. Such a transformation may be associated with production of radiotolerant cells.

  6. Targeted Radiosensitization by the Chk1 Inhibitor SAR-020106

    SciTech Connect

    Borst, Gerben R.; McLaughlin, Martin; Kyula, Joan N.; Neijenhuis, Sari; Khan, Aadil; Good, James; Zaidi, Shane; Powell, Ned G.; Meier, Pascal; Collins, Ian; Garrett, Michelle D.; Verheij, Marcel; Harrington, Kevin J.

    2013-03-15

    Purpose: To explore the activity of a potent Chk1 inhibitor (SAR-020106) in combination with radiation. Methods and Materials: Colony and mechanistic in vitro assays and a xenograft in vivo model. Results: SAR-020106 suppressed-radiation-induced G{sub 2}/M arrest and reduced clonogenic survival only in p53-deficient tumor cells. SAR-020106 promoted mitotic entry following irradiation in all cell lines, but p53-deficient cells were likely to undergo apoptosis or become aneuploid, while p53 wild-type cells underwent a postmitotic G{sub 1} arrest followed by subsequent normal cell cycle re-entry. Following combined treatment with SAR-020106 and radiation, homologous-recombination-mediated DNA damage repair was inhibited in all cell lines. A significant increase in the number of pan-γH2AX-staining apoptotic cells was observed only in p53-deficient cell lines. Efficacy was confirmed in vivo in a clinically relevant human head-and-neck cell carcinoma xenograft model. Conclusion: The Chk1 inhibitor SAR-020106 is a potent radiosensitizer in tumor cell lines defective in p53 signaling.

  7. Valproic acid promotes radiosensitization in meningioma stem-like cells.

    PubMed

    Chiou, Hsin-Ying Clair; Lai, Wen-Kuo; Huang, Li-Chun; Huang, Shih-Ming; Chueh, Sheau-Huei; Ma, Hsin-I; Hueng, Dueng-Yuan

    2015-04-30

    Although meningioma stem-like cells have been isolated and characterized, their therapeutic targeting remains a challenge. Meningioma sphere cells (MgSCs) with cancer stem cells properties show chemo- and radioresistance in comparison with meningioma adherent cells (MgACs). We tested the effect of valproic acid (VPA), a commonly used anti-epileptic drug, which passes the blood brain barrier, on cultured MgSCs. VPA reduced the viability of MgSCs and MgACs. In MgSCs, treatment with VPA increased radio-sensitivity, expression of p-cdc2, p-H2AX and cleaved caspase-3 and PARP. Anchorage-independent growth (AIG) was reduced by VPA. AIG was further reduced by combined treatment with irradiation. Expression of a stem cell marker, Oct4, was reduced by VPA. Oct4 was further decreased by combined treatment with irradiation. These results suggest that VPA may be a potential treatment for meningioma through targeting meningioma stem-like cells.

  8. Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

    NASA Technical Reports Server (NTRS)

    Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

    2000-01-01

    The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

  9. Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants

    NASA Technical Reports Server (NTRS)

    Asaad, N. A.; Zeng, Z. C.; Guan, J.; Thacker, J.; Iliakis, G.

    2000-01-01

    The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

  10. Homologous recombination as a potential target for caffeine radiosensitization in mammalian cells: reduced caffeine radiosensitization in XRCC2 and XRCC3 mutants.

    PubMed

    Asaad, N A; Zeng, Z C; Guan, J; Thacker, J; Iliakis, G

    2000-11-23

    The radiosensitizing effect of caffeine has been associated with the disruption of multiple DNA damage-responsive cell cycle checkpoints, but several lines of evidence also implicate inhibition of DNA repair. The role of DNA repair inhibition in caffeine radiosensitization remains uncharacterized, and it is unknown which repair process, or lesion, is affected. We show that a radiosensitive cell line, mutant for the RAD51 homolog XRCC2 and defective in homologous recombination repair (HRR), displays significantly diminished caffeine radiosensitization that can be restored by expression of XRCC2. Despite the reduced radiosensitization, caffeine effectively abrogates checkpoints in S and G2 phases in XRCC2 mutant cells indicating that checkpoint abrogation is not sufficient for radiosensitization. Another radiosensitive line, mutant for XRCC3 and defective in HRR, similarly shows reduced caffeine radiosensitization. On the other hand, a radiosensitive mutant (irs-20) of DNA-PKcs with a defect in non-homologous end-joining (NHEJ) is radiosensitized by caffeine to an extent comparable to wild-type cells. In addition, rejoining of radiation-induced DNA DSBs, that mainly reflects NHEJ, remains unaffected by caffeine in XRCC2 and XRCC3 mutants, or their wild-type counterparts. These observations suggest that caffeine targets steps in HRR but not in NHEJ and that abrogation of checkpoint response is not sufficient to explain radiosensitization. Indeed, immortalized fibroblasts from AT patients show caffeine radiosensitization despite the checkpoint defects associated with ATM mutation. We propose that caffeine radiosensitization is mediated by inhibition of stages in DNA DSB repair requiring HRR and that checkpoint disruption contributes by allowing these DSBs to transit into irreparable states. Thus, checkpoints may contribute to genomic stability by promoting error-free HRR.

  11. Coculture with astrocytes reduces the radiosensitivity of glioblastoma stem-like cells and identifies additional targets for radiosensitization.

    PubMed

    Rath, Barbara H; Wahba, Amy; Camphausen, Kevin; Tofilon, Philip J

    2015-11-01

    Toward developing a model system for investigating the role of the microenvironment in the radioresistance of glioblastoma (GBM), human glioblastoma stem-like cells (GSCs) were grown in coculture with human astrocytes. Using a trans-well assay, survival analyses showed that astrocytes significantly decreased the radiosensitivity of GSCs compared to standard culture conditions. In addition, when irradiated in coculture, the initial level of radiation-induced γH2AX foci in GSCs was reduced and foci dispersal was enhanced suggesting that the presence of astrocytes influenced the induction and repair of DNA double-strand breaks. These data indicate that astrocytes can decrease the radiosensitivity of GSCs in vitro via a paracrine-based mechanism and further support a role for the microenvironment as a determinant of GBM radioresponse. Chemokine profiling of coculture media identified a number of bioactive molecules not present under standard culture conditions. The gene expression profiles of GSCs grown in coculture were significantly different as compared to GSCs grown alone. These analyses were consistent with an astrocyte-mediated modification in GSC phenotype and, moreover, suggested a number of potential targets for GSC radiosensitization that were unique to coculture conditions. Along these lines, STAT3 was activated in GSCs grown with astrocytes; the JAK/STAT3 inhibitor WP1066 enhanced the radiosensitivity of GSCs under coculture conditions and when grown as orthotopic xenografts. Further, this coculture system may also provide an approach for identifying additional targets for GBM radiosensitization.

  12. Development of a Novel Enzyme-Targeting Radiosensitizer (New KORTUC) Using a Gelatin-Based Hydrogel Instead of a Sodium Hyaluronate

    PubMed Central

    Morita-Tokuhiro, Shiho; Ogawa, Yasuhiro; Yokota, Norikazu; Tsuzuki, Akira; Oda, Hideki; Ishida, Naoya; Aoyama, Nobutaka; Nishioka, Akihito

    2016-01-01

    We recently developed Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas (KORTUC) as a strategy to increase intratumoral oxygen concentrations and degrade antioxidant enzymes such as peroxidase and catalase. We then developed KORTUC II, which uses sodium hyaluronate containing hydrogen peroxide as a radiosensitizer. KORTUC II requires twice-weekly administration to sustain its effects, but decreasing the frequency of radiosensitizer injections to once-weekly would reduce the burden on the patients and the physicians. The goal of this study was thus to develop a new formulation of KORTUC (New KORTUC) that only requires once-weekly administration. We performed experimental studies using a mouse tumor model and biodegradable hydrogel. C3H/He mice were allocated to control, KORTUC, or hydrogel groups. At 72 h after injection, each tumor was irradiated with a 6 MeV electron beam to a total dose of 30 Gy. During a 62-day observation period, changes in tumor volume and survival rates were assessed in each group. Tumor growth rate was slowest in the hydrogel groups. These data suggest that hydrogel could represent a useful adjunct as a long-acting radiosensitizer in place of sodium hyaluronate. New KORTUC, which contains hydrogen peroxide and hydrogel, exerted a radiosensitizing effect that persisted beyond 72 h following injection of the agent. Use of this new formulation allows radiosensitizer injections to be performed once-weekly with good effect. PMID:26751477

  13. Platinum complexes with one radiosensitizing ligand (PtCl2(NH3) (sensitizer)): radiosensitization and toxicity studies in vitro

    SciTech Connect

    Skov, K.A.; Farrell, N.P.; Adomat, H.

    1987-11-01

    Complexes of general formula (PtCl2(NH3)L) with one radiosensitizing ligand per platinum are compared with ligand L alone, complexes with two radiosensitizers per platinum (PtCl2L2), and their analogs with NH3 ligands, with respect to radiosensitizing properties and toxicity in CHO cells. Radiosensitizing ligands, L, were misonidazole, metronidazole, 4(5)-nitroimidazole, and 2-amino-5-nitrothiazole, and the ammine analogs were cis- and trans-DDP (diamminedichloroplatinum(II)) and the monoammine, K(PtCl3(NH3)). Results are related to a previous study on plasmid DNA binding by these series. The toxicity of the mono series (PtCl2(NH3)L), attributable to DNA binding, is much higher than the corresponding bis complexes, (PtCl2L2). For L = misonidazole, toxicity is similar to the monoammine, but higher in hypoxic than in aerobic cells. trans-(PtCl2(NH3)-(misonidazole)) is more toxic than the cis isomer. Except for L = 4(5)-nitroimidazole, the complexes (PtCl2(NH3)L) are more toxic than L in air and hypoxia. Hypoxic radiosensitization by the mono complexes is comparable to the monoammine and is not better than free sensitizers, again except for L = 4(5)-nitroimidazole. Significantly lower sensitization is observed in oxic cells. The bis complexes (PtCl2L2), which do not bind to DNA as well as the mono complexes, are less effective radiosensitizers and less toxic than the (PtCl2(NH3)L) series.

  14. Partition coefficient as a guide to the development of radiosensitizers which are less toxic than misonidazole

    SciTech Connect

    Brown, J.M.; Workman, P.

    1980-04-01

    Ten 2-nitroimidazole radiosensitizers of electron affinity equal to that of misonidazole, but differing in their octanol:water partition coefficient over a 100-fold range, were chosen to examine the effect of lipophilicity on the pharmacokinetics of these drugs in BALB/c mice bearing EMT6 tumors. Plasma, tumor, and brain concentrations were assayed, using high performance liquid chromatography (HPLC), as a function of time after a single ip injection of each drug. Peak concentrations in the tumor declined with decreasing lipophilicity. This was due to declining peak plasma concentrations resulting from slower drug absorption and could be overcome by iv injection. The tumor plasma ratio, once sufficient time had elapsed for it to reach its equilibrium value, was independent of partition coefficient (P) over the range 0.026 to 1.5 but showed a 50% reduction in this ratio for the most hydrophilic compound studied (P = 0.014). (This compound was also the one drug in the series which was significantly poorer than misonidazole in its radiosensitization as a function of drug concentration). The brain/plasma ratio, on the other hand, showed a marked dependence on lipophilicity. For misonidazole and more lipophilic compounds, the brain/plasma ratio was 1.0, but as the lipophilicity decreased below that of misonidazole, the compounds showed an increasing difficulty in penetration into the brain, and brain/plasma levels of less than 0.1 were found for the most hydrophilic drugs. These low brain/plasma ratios correlated with an increased acute LD/sub 50/ of the drugs. Bilateral nephrectomy was used to increase the apparent plasma half-life of SR-2508 from 0.8 to 15 hr. This change, however, did not affect the tumor/brain ratio of approximately 10 for this drug. The significance of these pharmacokinetic data is discussed in terms of the development of a radiosensitizer superior to misonidazole for clinical use.

  15. In vivo and in vitro radiosensitivities ofnewly established mouse ascites tumors

    SciTech Connect

    Okamoto, M.; Tsuboi, A.; Tsuchiya, T.

    1981-03-01

    The response of two newly established mouse mammary tumors to x irradiation in vitro and in vivo was studied by colony-forming assay in soft agar. Cells irradiated in vivo were more resistant than those irradiated in vitro. The D/sub 0/ values for in vitro irradiation were 112 rad at both exponential and stationary phases, while those for in vivo irradiation were 303 rad at exponential phase and 556 rad at stationary phase. This increase in D/sub 0/ value, which is greater than the OER, suggests that radiosensitivity in vivo cannot be explained only by hypoxia.

  16. Can Drugs Enhance Hypofractionated Radiotherapy? A Novel Method of Modeling Radiosensitization Using In Vitro Data

    SciTech Connect

    Ohri, Nitin; Dicker, Adam P.; Lawrence, Yaacov Richard

    2012-05-01

    Purpose: Hypofractionated radiotherapy (hRT) is being explored for a number of malignancies. The potential benefit of giving concurrent chemotherapy with hRT is not known. We sought to predict the effects of combined modality treatments by using mathematical models derived from laboratory data. Methods and Materials: Data from 26 published clonogenic survival assays for cancer cell lines with and without the use of radiosensitizing chemotherapy were collected. The first three data points of the RT arm of each assay were used to derive parameters for the linear quadratic (LQ) model, the multitarget (MT) model, and the generalized linear quadratic (gLQ) model. For each assay and model, the difference between the predicted and observed surviving fractions at the highest tested RT dose was calculated. The gLQ model was fitted to all the data from each RT cell survival assay, and the biologically equivalent doses in 2-Gy fractions (EQD2s) of clinically relevant hRT regimens were calculated. The increase in cell kill conferred by the addition of chemotherapy was used to estimate the EQD2 of hRT along with a radiosensitizing agent. For comparison, this was repeated using conventionally fractionated RT regimens. Results: At a mean RT dose of 8.0 Gy, the average errors for the LQ, MT, and gLQ models were 1.63, 0.83, and 0.56 log units, respectively, favoring the gLQ model (p < 0.05). Radiosensitizing chemotherapy increased the EQD2 of hRT schedules by an average of 28% to 82%, depending on disease site. This increase was similar to the gains predicted for the addition of chemotherapy to conventionally fractionated RT. Conclusions: Based on published in vitro assays, the gLQ equation is superior to the LQ and MT models in predicting cell kill at high doses of RT. Modeling exercises demonstrate that significant increases in biologically equivalent dose may be achieved with the addition of radiosensitizing agents to hRT. Clinical study of this approach is warranted.

  17. Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells

    PubMed Central

    Chiang, Ming-Fu; Chen, Hsin-Hong; Chi, Chih-Wen; Sze, Chun-I; Hsu, Ming-Ling; Shieh, Hui-Ru; Lin, Chin-Ping; Tsai, Jo-Ting

    2015-01-01

    WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further

  18. Modulation of Sonic hedgehog signaling and WW domain containing oxidoreductase WOX1 expression enhances radiosensitivity of human glioblastoma cells.

    PubMed

    Chiang, Ming-Fu; Chen, Hsin-Hong; Chi, Chih-Wen; Sze, Chun-I; Hsu, Ming-Ling; Shieh, Hui-Ru; Lin, Chin-Ping; Tsai, Jo-Ting; Chen, Yu-Jen

    2015-03-01

    WW domain containing oxidoreductase, designated WWOX, FOR or WOX1, is a known pro-apoptotic factor when ectopically expressed in various types of cancer cells, including glioblastoma multiforme (GBM). The activation of sonic hedgehog (Shh) signaling, especially paracrine Shh secretion in response to radiation, is associated with impairing the effective irradiation of cancer cells. Here, we examined the role of Shh signaling and WOX1 overexpression in the radiosensitivity of human GBM cells. Our results showed that ionizing irradiation (IR) increased the cytoplasmic Shh and nuclear Gli-1 content in GBM U373MG and U87MG cells. GBM cells with exogenous Shh treatment exhibited similar results. Pretreatment with Shh peptides protected U373MG and U87MG cells against IR in a dose-dependent manner. Cyclopamine, a Hedgehog/Smoothened (SMO) inhibitor, reversed the protective effect of Shh in U87MG cells. Cyclopamine increased Shh plus IR-induced H2AX, a marker of DNA double-strand breaks, in these cells. To verify the role of Shh signaling in the radiosensitivity of GBM cells, we tested the effect of the Gli family zinc finger 1 (Gli-1) inhibitor zerumbone and found that it could sensitize GBM cells to IR. We next examined the role of WOX1 in radiosensitivity. Overexpression of WOX1 enhanced the radiosensitivity of U87MG (possessing wild type p53 or WTp53) but not U373MG (harboring mutant p53 or MTp53) cells. Pretreatment with Shh peptides protected both WOX1-overexpressed U373MG and U87MG cells against IR and increased the cytoplasmic Shh and nuclear Gli-1 content. Zerumbone enhanced the radiosensitivity of WOX1-overexpressed U373MG and U87MG cells. In conclusion, overexpression of WOX1 preferentially sensitized human GBM cells possessing wild type p53 to radiation therapy. Blocking of Shh signaling may enhance radiosensitivity independently of the expression of p53 and WOX1. The crosstalk between Shh signaling and WOX1 expression in human glioblastoma warrants further

  19. Combined radiation-protective and radiation-sensitizing agents. II. Radiosensitivity of hypoxic or aerobic Chinese hamster fibroblasts in the presence of cysteamine and misonidazole: implications for the oxygen effect (with Appendix on calculation of dose-modifying factors. [/sup 60/Co

    SciTech Connect

    Koch, C.J.; Howell, R.L.

    1981-08-01

    Experiments have been done to test whether a hypoxic cell radiosensitizing agent (misonidazole) can be combined with a radioprotecting agent (cysteamine) to equalize partially the radiation response of hypoxic and aerobic mammalian cells in tissue culture. The results indicate that cysteamine will protect against the radiosensitization of a hypoxic cell sensitizing drug (2.5 mM misonidazole) at much lower concentration than it will protect against the radiosensitization of oxygen (350 ..mu..M). Thus the addition of a radiation-protective drug tends to cancel the drug benefit of the radiosensitizer and therefore increases the differential response of hypoxic and aerobic cells rather than equalizing this response. The data suggest that even in situations where tumor tissue absorbs far less radioprotective drug than normal tissue (e.g., WR 2721), one might expect difficulties with the simultaneous administration of radiosensitizing and radioprotecting drugs.

  20. Analysis of individual differences in radiosensitivity using genome editing.

    PubMed

    Matsuura, S; Royba, E; Akutsu, S N; Yanagihara, H; Ochiai, H; Kudo, Y; Tashiro, S; Miyamoto, T

    2016-06-01

    Current standards for radiological protection of the public have been uniformly established. However, individual differences in radiosensitivity are suggested to exist in human populations, which could be caused by nucleotide variants of DNA repair genes. In order to verify if such genetic variants are responsible for individual differences in radiosensitivity, they could be introduced into cultured human cells for evaluation. This strategy would make it possible to analyse the effect of candidate nucleotide variants on individual radiosensitivity, independent of the diverse genetic background. However, efficient gene targeting in cultured human cells is difficult due to the low frequency of homologous recombination (HR) repair. The development of artificial nucleases has enabled efficient HR-mediated genome editing to be performed in cultured human cells. A novel genome editing strategy, 'transcription activator-like effector nuclease (TALEN)-mediated two-step single base pair editing', has been developed, and this was used to introduce a nucleotide variant associated with a chromosomal instability syndrome bi-allelically into cultured human cells to demonstrate that it is the causative mutation. It is proposed that this editing technique will be useful to investigate individual radiosensitivity.

  1. Histone Deacetylation Critically Determines T-cell Subset Radiosensitivity1

    PubMed Central

    Pugh, Jason L.; Sukhina, Alona S.; Seed, Thomas M.; Manley, Nancy R.; Sempowski, Gregory A.; van den Brink, Marcel R.M.; Smithey, Megan J.; Nikolich-Zugich, Janko

    2014-01-01

    Lymphocytes are sensitive to ionizing radiation and naïve lymphocytes are more radiosensitive than their memory counterparts. Less is known about radiosensitivity of memory cell subsets. We examined the radiosensitivity of naïve (TN), effector memory (TEM), and central memory (TCM) T cell subsets in C57BL/6 mice, and found TEM to be more resistant to radiation-induced apoptosis than either TN or TCM. Surprisingly, we found no correlation between the extent of radiation-induced apoptosis in T cell subsets and : (i) levels of pro- and anti-apoptotic Bcl-2 family members; or (ii) the H2-AX content and maximal γH2-AX fold change. Rather, TEM cell survival correlated with higher levels of immediate γH2-AX marking, immediate break binding and genome-wide open chromatin structure. T cells were able to mark DNA damage seemingly instantly (30 s), even if kept on ice. Relaxing chromatin with the histone deacetylase inhibitor valproic acid following radiation or etoposide treatment, improved the survival of TCM and TN cells up to levels seen in the resistant TEM cells, but did not improve survival from caspase-mediated apoptosis. We conclude that an open genome-wide chromatin state is the key determinant of efficient immediate repair of DNA damage in T cells, explaining the observed T cell subset radiosensitivity differences. PMID:24990082

  2. Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule

    PubMed Central

    Djuzenova, Cholpon S.; Fiedler, Vanessa; Katzer, Astrid; Michel, Konstanze; Deckert, Stefanie; Zimmermann, Heiko; Sukhorukov, Vladimir L.; Flentje, Michael

    2016-01-01

    Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and even diminished the radiosensitizing effect of NVP-AUY922. This result can be explained by the activation of PI3K and ERK pathways along with G1-arrest at the time of irradiation. In sharp contrast, PI-103 not only exerted a radiosensitizing effect but also strongly enhanced the radiosensitization by NVP-AUY922 when both inhibitors were added 3 h before irradiation and kept in culture for 24 h. Possible reasons for the observed radiosensitization under this drug-irradiation schedule may be a down-regulation of PI3K and ERK pathways during or directly after irradiation, increased residual DNA damage and strong G2/M arrest 24 h thereafter. We conclude that duration of drug treatment before irradiation plays a key role in the concomitant targeting of PI3K/mTOR and Hsp90 in tumor cells. PMID:27224913

  3. Dual PI3K- and mTOR-inhibitor PI-103 can either enhance or reduce the radiosensitizing effect of the Hsp90 inhibitor NVP-AUY922 in tumor cells: The role of drug-irradiation schedule.

    PubMed

    Djuzenova, Cholpon S; Fiedler, Vanessa; Katzer, Astrid; Michel, Konstanze; Deckert, Stefanie; Zimmermann, Heiko; Sukhorukov, Vladimir L; Flentje, Michael

    2016-06-21

    Inhibition of Hsp90 can increase the radiosensitivity of tumor cells. However, inhibition of Hsp90 alone induces the anti-apoptotic Hsp70 and thereby decreases radiosensitivity. Therefore, preventing Hsp70 induction can be a promising strategy for radiosensitization. PI-103, an inhibitor of PI3K and mTOR, has previously been shown to suppress the up-regulation of Hsp70. Here, we explore the impact of combining PI-103 with the Hsp90 inhibitor NVP-AUY922 in irradiated glioblastoma and colon carcinoma cells. We analyzed the cellular response to drug-irradiation treatments by colony-forming assay, expression of several marker proteins, cell cycle progression and induction/repair of DNA damage. Although PI-103, given 24 h prior to irradiation, slightly suppressed the NVP-AUY922-mediated up-regulation of Hsp70, it did not cause radiosensitization and