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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

    Kleiman, Laura B; Krebs, Angela M; 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. PMID:26873620

  4. 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...

  5. The Differential Effects of Wild-Type and Mutated K-Ras on MST2 Signaling Are Determined by K-Ras Activation Kinetics

    PubMed Central

    Romano, David; Maccario, Helene; Doherty, Carolanne; Quinn, Niall P.

    2013-01-01

    K-Ras is frequently mutated in human cancers. Mutant (mt) K-Ras can stimulate both oncogenic transformation and apoptosis through activation of extracellular signal-regulated kinase (ERK) and AKT pathways and the MST2 pathway, respectively. The biological outcome is determined by the balance and cross talk between these pathways. In colorectal cancer (CRC), a K-Ras mutation is negatively correlated with MST2 expression, as mt K-Ras can induce apoptosis by activating the MST2 pathway. However, wild-type (wt) K-Ras can prevent the activation of the MST2 pathway upon growth factor stimulation and enable transformation by mt K-Ras in CRC cells that express MST2. Here we have investigated the mechanism by which wt and mt K-Ras differentially regulate the MST2 pathway and MST2-dependent apoptosis. The ability of K-Ras to activate MST2 and MST2-dependent apoptosis is determined by the differential activation kinetics of mt K-Ras and wt K-Ras. Chronic activation of K-Ras by mutation or overexpression of Ras exchange factors results in the activation of MST2 and LATS1, increased MST2-LATS1 complex formation, and apoptosis. In contrast, transient K-Ras activation upon epidermal growth factor (EGF) stimulation prevents the formation of the MST2-LATS1 complex in an AKT-dependent manner. Our data suggest that the close relationship between Ras prosurvival and proapoptotic signaling is coordinated via the differential regulation of the MST2-LATS1 interaction by transient and chronic stimuli. PMID:23459937

  6. 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

  7. Oncogenic K-ras confers SAHA resistance by up-regulating HDAC6 and c-myc expression.

    PubMed

    Wang, Qun; Tan, Rong; Zhu, Xin; Zhang, Yi; Tan, Zhiping; Su, Bing; Li, Yu

    2016-03-01

    Histone deacetylase inhibitors (HDIs) represent a new class of anticancer drugs. Suberoylanilide hydroxamic acid (SAHA), the first HDI approved for the treatment of cutaneous T cell lymphoma (CTCL), is currently being tested in clinical trials for other cancers. However, SAHA has been ineffective against solid tumors in many clinical trials. A better understanding of molecular mechanisms of SAHA resistance may provide the basis for improved patient selection and the enhancement of clinical efficacy. Here we demonstrate that oncogenic K-ras contributes to SAHA resistance by upregulating HDAC6 and c-myc expression. We find that the high levels of HDAC6 expression are associated with activated K-ras mutant in colon cancer patients. And expressions of HDAC6 and c-myc are increased in fibroblasts transformed with activated K-ras. Surprisingly, we find that activated K-ras transformed cells are more resistant to SAHA inhibition on cell growth and anchorage-independent colony formation. We show that a K-ras inhibitor sensitizes K-ras mutated lung cancer cells to SAHA induced growth inhibition. We also find that mutant K-ras induces HDAC6 expression by a MAP kinase dependent pathway. Our study suggests that combined treatment with SAHA and K-ras inhibitors may represent an effective strategy to overcome SAHA resistance. PMID:26848526

  8. Oncogenic K-ras confers SAHA resistance by up-regulating HDAC6 and c-myc expression

    PubMed Central

    Zhang, Yi; Tan, Zhiping; Su, Bing; Li, Yu

    2016-01-01

    Histone deacetylase inhibitors (HDIs) represent a new class of anticancer drugs. Suberoylanilide hydroxamic acid (SAHA), the first HDI approved for the treatment of cutaneous T cell lymphoma (CTCL), is currently being tested in clinical trials for other cancers. However, SAHA has been ineffective against solid tumors in many clinical trials. A better understanding of molecular mechanisms of SAHA resistance may provide the basis for improved patient selection and the enhancement of clinical efficacy. Here we demonstrate that oncogenic K-ras contributes to SAHA resistance by upregulating HDAC6 and c-myc expression. We find that the high levels of HDAC6 expression are associated with activated K-ras mutant in colon cancer patients. And expressions of HDAC6 and c-myc are increased in fibroblasts transformed with activated K-ras. Surprisingly, we find that activated K-ras transformed cells are more resistant to SAHA inhibition on cell growth and anchorage-independent colony formation. We show that a K-ras inhibitor sensitizes K-ras mutated lung cancer cells to SAHA induced growth inhibition. We also find that mutant K-ras induces HDAC6 expression by a MAP kinase dependent pathway. Our study suggests that combined treatment with SAHA and K-ras inhibitors may represent an effective strategy to overcome SAHA resistance. PMID:26848526

  9. Profiling of transcripts and proteins modulated by K-ras oncogene in the lung tissues of K-ras transgenic mice by omics approaches.

    PubMed

    Lee, Sojung; Kang, Jungwoo; Cho, Minchul; Seo, Eunhee; Choi, Heesook; Kim, Eunjin; Kim, Junghee; Kim, Heejong; Kang, Gum Yong; Kim, Kwang Pyo; Park, Young-Ho; Yu, Dae-Yeul; Yum, Young Na; Park, Sue-Nie; Yoon, Do-Young

    2009-01-01

    The mutated K-ras gene is involved in approximately 30% of human cancers. In order to search for K-ras oncogene-induced modulators in lung tissues of K-ras transgenic mice, we performed microarray and proteomics (LC/ESI-MS/MS) analysis. Genes (RAB27b RAS family, IL-1RA, IL-33, chemokine ligand 6, epiregulin, EGF-like domain and cathepsin) related to cancer development (Wnt signaling pathway) and inflammation (chemokine/cytokine signaling pathway, Toll receptor signaling) were up-regulated while genes (troponin, tropomodulin 2, endothelial lipase, FGFR4, integrin alpha8 and adenylate cyclase 8) related to the tumor suppression such as p53 pathway, TGF-beta signaling pathway and cadherin signaling pathway were down-regulated by K-ras oncogene. Proteomics approach revealed that up-regulated proteins in lung adenomas of K-ras mice were classified as follows: proteins related to the metabolism/catabolism (increased from 7 to 22% by K-ras gene), proteins related to translation/transcription and nucleotide (from 4 to 6%), proteins related to signal transduction (from 3 to 5%), proteins related to phosphorylation (from 1 to 2%). ATP synthase, Ras oncogene family, cytochrome c oxidase, flavoprotein, TEF 1, adipoprotein A-1 BP, glutathione oxidase, fatty acid BP 4, diaphorase 1, MAPK4 and transgelin were up-regulated by K-ras oncogene. However, integrin alpha1, Ras-interacting protein (Rain), endothelin-converting enzyme-1d and splicing factor 3b were down-regulated. These studies suggest that genes related to cancer development and inflammation were up-regulated while genes related to the tumor suppression were down-regulated by K-ras, resulting in the tumor growth. Putative biomarkers such as cell cycle related genes (Cdc37), cancer cell adhesion (Glycam 1, integrin alpha8, integrin alphaX and Clec4n), signal transduction (Tlr2, IL-33, and Ccbp2), migration (Ccr1, Ccl6, and diaphorase 1 (Cyb5r3) and cancer development (epiregulin) can be useful for diagnosis and as

  10. ERK2-dependent reactivation of Akt mediates the limited response of tumor cells with constitutive K-RAS activity to PI3K inhibition

    PubMed Central

    Toulany, Mahmoud; Minjgee, Minjmaa; Saki, Mohammad; Holler, Marina; Meier, Friedegund; Eicheler, Wolfgang; Rodemann, H Peter

    2014-01-01

    K-RAS mutated (K-RASmut) non-small cell lung cancer (NSCLC) cells are resistant to EGFR targeting strategies. We investigated the impact of K-RAS activity irrespective of mutational status in the EGFR-independent increase in clonogenic cell survival. An analysis of the K-RAS activity status revealed a constitutively high K-RAS activity in K-RASmut NSCLC cells and also in head and neck squamous cell carcinoma (HNSCC) cells overexpressing wild-type K-RAS (K-RASwt). Similar to K-RAS-mutated cells, increased K-RAS activity in HNSCC cells overexpressing K-RASwt was associated with the stimulated production of the EGFR ligand amphiregulin and resistance to EGFR tyrosine kinase (EGFR-TK) inhibitors such as erlotinib. Expression of mutated K-RAS stimulated Akt phosphorylation and increased plating efficiency. Conversely, knockdown of K-RAS in K-RASmut NSCLC cells and in HNSCC cells presenting overexpression of K-RASwt resulted in sensitization to the anti-clonogenic activity of erlotinib. K-RAS activity results in EGFR-dependent and EGFR-independent Akt activity. The short-term treatment (2 h) of cells with EGFR-TK or PI3K inhibitors (erlotinib and PI-103) resulted in the repression of Akt activation, whereas long-term treatment (24 h) with inhibitors led to the reactivation of Akt and improved clonogenicity. The Akt re-activation was MAPK-ERK2-dependent and associated with a lack of complete response to anti-clonogenic activity of PI-103. A complete response was observed when PI-103 was combined with MEK inhibitor PD98059. Together, clonogenicity inhibition in tumor cells presenting constitutive K-RAS activity independent of K-RAS mutational status can be achieved by targeting of EGFR downstream pathways, i.e., PI3K alone or the combination of PI3K and MAPK inhibitors. PMID:24351425

  11. 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.

  12. 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.

  13. 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. PMID:23526092

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

    PubMed Central

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

    2016-01-01

    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. PMID:27070580

  15. 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

    2016-01-01

    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. PMID:27070580

  16. 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

  17. 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

    2015-01-01

    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

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

    PubMed

    Birchenall-Roberts, Maria C; 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. PMID:16889753

  19. 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.

  20. Deletion of Pim kinases elevates the cellular levels of reactive oxygen species and sensitizes to K-Ras-induced cell killing.

    PubMed

    Song, J H; An, N; Chatterjee, S; Kistner-Griffin, E; Mahajan, S; Mehrotra, S; Kraft, A S

    2015-07-01

    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-Ras(G12V)) and undergo cell death. Transduction of K-Ras(G12V) into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuated ROS production and reversed the cytotoxic effects of K-Ras(G12V) 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-Ras(G12V)-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-Ras(G12V)-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases have an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth. PMID:25241892

  1. TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

    PubMed Central

    Luu, Carrie; Heinrich, Eileen L.; Duldulao, Marjun; Arrington, Amanda K.; Fakih, Marwan; Garcia-Aguilar, Julio; Kim, Joseph

    2013-01-01

    Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRASmut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53−/−, TP53+/−, TP53mut/+, and TP53mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRASmut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (−/− or −/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer. PMID:23936455

  2. Regulation of K-Ras4B Membrane Binding by Calmodulin.

    PubMed

    Sperlich, Benjamin; Kapoor, Shobhna; Waldmann, Herbert; Winter, Roland; Weise, Katrin

    2016-07-12

    K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell. PMID:27410739

  3. A novel combination of K-ras and myc amplification accompanied by point mutational activation of K-ras in a human lung cancer.

    PubMed Central

    Taya, Y; Hosogai, K; Hirohashi, S; Shimosato, Y; Tsuchiya, R; Tsuchida, N; Fushimi, M; Sekiya, T; Nishimura, S

    1984-01-01

    Amplifications of two oncogenes, c-K-ras-2 and c-myc, were found in a human lung giant cell carcinoma (LGCC) Lu-65, which is maintained in nude mice. The extent of c-K-ras-2 and myc amplifications were estimated to be 10- and 8-fold, respectively, by means of the Southern hybridization procedure. In addition, NIH3T3 cells were transformed by transfection of Lu-65 DNA and the transforming gene was identified as c-K-ras-2. c-K-ras-2 genes were cloned from a gene library of Lu-65 and a single point mutation causing a substitution of cysteine for glycine in codon 12 was found by DNA sequencing. It was concluded that the amplification of the c-myc and c-K-ras-2 genes are accompanied by point mutational activation of c-K-ras-2 in the human LGCC Lu-65. This is the first report of multiple gene amplification accompanied by a point mutation of oncogenes in human cancer cells, providing further support for the idea that co-operation of at least two activated cellular oncogenes is required for carcinogenesis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:6098458

  4. CaM interaction and Ser181 phosphorylation as new K-Ras signaling modulators

    PubMed Central

    Alvarez-Moya, Blanca; Barceló, Carles; Tebar, Francesc; Jaumot, Montserrat

    2011-01-01

    The small G-protein Ras was the first oncogene to be identified and has a very important contribution to human cancer development (20–23% prevalence). K-RasB, one of the members of the Ras family, is the one that is most mutated and plays a prominent role in pancreatic, colon and lung cancer development. Ras proteins are membrane bound GTPases that cycle between inactive, GDP-bound and active, GTP-bound, states. Most of the research into K-RasB activity regulation has focused on the analysis of how GTP-exchange factors (GEFs) and GTPase activating proteins (GAPs) are regulated by external and internal signals. In contrast, oncogenic K-RasB has a very low GTPase activity and furthermore is not deactivated by GAPs. Consequently, the consensus was that activity of oncogenic K-RasB was not modulated. In this extra view we recapitulate some recent data showing that calmodulin binding to K-RasB inhibits phosphorylation of K-RasB at Ser181, near to the membrane anchoring domain, modulating signaling of both non-oncogenic and oncogenic K-RasB. This may be relevant to normal cell physiology, but also opens new therapeutic perspectives for the inhibition of oncogenic K-RasB signaling in tumors. PMID:21776410

  5. K-ras gene mutation in gall bladder carcinomas and dysplasia.

    PubMed Central

    Ajiki, T; Fujimori, T; Onoyama, H; Yamamoto, M; Kitazawa, S; Maeda, S; Saitoh, Y

    1996-01-01

    Epithelial dysplasia of gall bladder is an important precancerous lesion of gall bladder carcinogenesis. To investigate the frequency of K-ras gene mutation in gall bladder carcinoma and dysplasia, K-ras codon 12 mutations were investigated by the polymerase chain reaction/restriction enzyme based method following direct sequencing. Mutation was detected in 59% (30 of 51) of gall bladder carcinomas, in 73% (8 of 11) of gall bladder dysplasia in gall stone cases, and in 0% of the normal gall bladder epithelium. There was, however, no correlation between K-ras mutation and clinicopathological factors of gall bladder carcinoma. K-ras gene mutation occurs even in gall bladder dysplasia at an incidence similar to that in carcinomas, suggesting that testing for K-ras gene mutation may prove useful as an adjunct to bile cytological or biopsy analysis. Images Figure 1 Figure 2 Figure 3 PMID:8675098

  6. Analysis of K-Ras Nuclear Expression in Fibroblasts and Mesangial Cells

    PubMed Central

    Fuentes-Calvo, Isabel; Blázquez-Medela, Ana M.; Santos, Eugenio; López-Novoa, José M.; Martínez-Salgado, Carlos

    2010-01-01

    Background Ras GTPases are considered cytoplasmic proteins that must be localized to cell membranes for activation, and there are few evidences of the presence of any Ras isoform in nuclei of eukaryotic cells. Methodology/Principal Findings Using conventional antibodies and inmunocytochemistry, differential centrifugation and western blot, we have observed the putative presence of K-Ras isoform in the nuclei of fibroblasts and mesangial cells. In order to avoid cross-reactions with other Ras isoforms, and using antibodies against K-Ras (R-3400, H3845-M01, sc-30) or pan-Ras (05-516, OP40) in cells that only expressed the K-Ras isoform (fibroblasts obtained from H-ras−/−,N-ras−/− mice) we also detected some nuclear positive expression. To further probe the identity of nuclear K-Ras, we have generated K-Ras knockout (K-ras−/−) embrionary fibroblasts by mating of K-ras+/− heterozygote mice. Using specific antibodies, only H- and N-Ras isoforms were observed in the cytoplasm of K-ras−/− fibroblasts. However, both K-Ras4A and K-Ras4B positive signals were detected by immunocytochemistry and Western blot with two commercial antibodies (sc-522 and sc-521 against each isoforms, respectively) in both cytoplasm and nuclei from K-ras−/− fibroblasts. Conclusions/Significance We show that the presence of K-Ras4B in fibroblast nuclei, already described by other authors, is probably due to a cross-reaction of the antibody with an undetermined nucleolar protein. Although this study also shows the possible nuclear expression of K-Ras isoform in fibroblasts or in mesangial cells, it also reveals the importance of being cautious in these studies about distribution of protein isoforms due to some important limitations imposed by the unspecificity of the antibodies or contaminations in cellular preparations. PMID:20090846

  7. Synergistic effects of sorafenib in combination with gemcitabine or pemetrexed in lung cancer cell lines with K-ras mutations

    PubMed Central

    Wang, Shu; Su, Zeng-feng; Yuan, Yuan

    2016-01-01

    K-ras is currently accepted as the most frequently mutated oncogene in non-small cell lung cancer (NSCLC, including squamous carcinoma, adenocarcinoma, and large cell carcinoma). NSCLC patients with the K-ras mutation appear to be refractory to the majority of systemic therapies. In the present study, the in vitro antitumor effects and correlated molecular mechanisms of sorafenib combined with gemcitabine or pemetrexed were explored in the K-ras mutation-positive NSCLC A549 cell line. Sorafenib was seen to exhibit dose-dependent growth inhibition in the A549 cells, while sorafenib combined with pemetrexed demonstrated a greater synergism compared with sorafenib combined with gemcitabine. Sorafenib arrested the cell cycle at the G1 phase, while gemcitabine and pemetrexed caused arrest at the S phase. The molecular mechanism of this synergism was due to the downstream signalling pathways, which were efficiently suppressed by sorafenib, therefore increasing the incidence of the entry of the chemotherapeutic drugs into the apoptotic pathways. Moreover, sorafenib and pemetrexed demonstrated stronger synergism, demonstrating that inhibiting the Ras/Raf/Mek/Erk and Ras/PI3K/Akt pathways concurrently may achieve improved antitumor effects. PMID:27095937

  8. Bi-transgenic Mice Reveal that K-rasVal12 Augments a p53-independent Apoptosis When Small Intestinal Villus Enterocytes Reenter the Cell Cycle

    PubMed Central

    Coopersmith, Craig M.; Chandrasekaran, Chitra; McNevin, M. Shane; Gordon, Jeffrey I.

    1997-01-01

    Studies in cell culture systems have indicated that oncogenic forms of Ras can affect apoptosis. Activating mutations of Ras occur in ∼30% of all human tumors and 50% of colorectal carcinomas. Since these mutations appear at early or intermediate stages in multistep journeys to neoplasia, an effect on apoptosis may help determine whether initiated cells progress towards a more neoplastic state. We have tested the effects of K-rasVal12 on apoptosis in transgenic mice. A lineage-specific promoter was used to direct expression of human K-rasVal12, with or without wild-type (wt) or mutant SV-40 T antigens (TAg), in postmitotic villus enterocytes, the principal cell type of the small intestinal epithelium. Enterocytes can be induced to reenter the cell cycle by TAgWt. Reentry is dependent upon the ability of TAg to bind pRB and is associated with a p53-independent apoptosis. Analyses of K-rasVal12 × TAgWt bi-transgenic animals indicated that K-rasVal12 can enhance this apoptosis threefold but only in cycling cells; increased apoptosis does not occur when K-rasVal12 is expressed alone or with a TAg containing Glu107,108→ Lys107,108 substitutions that block its ability to bind pRB. Analysis of bi-transgenic K-rasVal12 × TAgWt mice homozygous for wild-type or null p53 alleles established that the enhancement of apoptosis occurs through a p53-independent mechanism, is not attributable to augmented proliferation or to an increase in abortive cell cycle reentry (compared to TAgWt mice), and is not associated with detectable changes in the crypt–villus patterns of expression of apoptotic regulators (Bcl-2, Bcl-xL, Bak, and Bax) or mediators of epithelial cell–matrix interactions and survival (e.g., α5β1 integrin and its ligand, fibronectin). Coexpression of K-rasVal12 and TAgWt produces dysplasia. The K-rasVal12-augmented apoptosis is unrelated to this dysplasia; enhanced apoptosis is also observed in cycling nondysplastic enterocytes that produce K-rasVal12 and a

  9. 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.

  10. IL6 Blockade Reprograms the Lung Tumor Microenvironment to Limit the Development and Progression of K-ras-Mutant Lung Cancer.

    PubMed

    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

    2016-06-01

    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 IL6, 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 IL6 using a monoclonal anti-IL6 antibody in a K-ras-mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL6 blockade significantly inhibited lung cancer promotion, tumor cell-intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL6 inhibition reduced expression of protumor type 2 molecules (arginase 1, Fizz 1, Mgl, and IDO), number of M2-type macrophages and granulocytic myeloid-derived suppressor cells, and protumor T-regulatory/Th17 cell responses. This was accompanied by increased expression of antitumor type 1 molecule (Nos2), and antitumor Th1/CD8 T-cell responses. Our study demonstrates that IL6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also reeducates the lung microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces IL6 as a potential druggable target for prevention and treatment of K-ras-mutant lung tumors. Cancer Res; 76(11); 3189-99. ©2016 AACR. PMID:27197187

  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. Combined Inactivation of MYC and K-Ras Oncogenes Reverses Tumorigenesis in Lung Adenocarcinomas and Lymphomas

    PubMed Central

    Koh, Shan; Komatsubara, Kim; Chen, Joy; Horng, George; Bellovin, David I.; Giuriato, Sylvie; Wang, Craig S.; Whitsett, Jeffrey A.; Felsher, Dean W.

    2008-01-01

    Background Conditional transgenic models have established that tumors require sustained oncogene activation for tumor maintenance, exhibiting the phenomenon known as “oncogene-addiction.” However, most cancers are caused by multiple genetic events making it difficult to determine which oncogenes or combination of oncogenes will be the most effective targets for their treatment. Methodology/Principal Findings To examine how the MYC and K-rasG12D oncogenes cooperate for the initiation and maintenance of tumorigenesis, we generated double conditional transgenic tumor models of lung adenocarcinoma and lymphoma. The ability of MYC and K-rasG12D to cooperate for tumorigenesis and the ability of the inactivation of these oncogenes to result in tumor regression depended upon the specific tissue context. MYC-, K-rasG12D- or MYC/K-rasG12D-induced lymphomas exhibited sustained regression upon the inactivation of either or both oncogenes. However, in marked contrast, MYC-induced lung tumors failed to regress completely upon oncogene inactivation; whereas K-rasG12D-induced lung tumors regressed completely. Importantly, the combined inactivation of both MYC and K-rasG12D resulted more frequently in complete lung tumor regression. To account for the different roles of MYC and K-rasG12D in maintenance of lung tumors, we found that the down-stream mediators of K-rasG12D signaling, Stat3 and Stat5, are dephosphorylated following conditional K-rasG12D but not MYC inactivation. In contrast, Stat3 becomes dephosphorylated in lymphoma cells upon inactivation of MYC and/or K-rasG12D. Interestingly, MYC-induced lung tumors that failed to regress upon MYC inactivation were found to have persistent Stat3 and Stat5 phosphorylation. Conclusions/Significance Taken together, our findings point to the importance of the K-Ras and associated down-stream Stat effector pathways in the initiation and maintenance of lymphomas and lung tumors. We suggest that combined targeting of oncogenic

  13. The brain microenvironment negatively regulates miRNA-768-3p to promote K-ras expression and lung cancer metastasis

    PubMed Central

    Subramani, Arasukumar; Alsidawi, Samer; Jagannathan, Sajjeev; Sumita, Kazutaka; Sasaki, Atsuo T.; Aronow, Bruce; Warnick, Ronald E.; Lawler, Sean; Driscoll, James J.

    2013-01-01

    The brain microenvironment promotes metastasis through mechanisms that remain elusive. Co-culture of lung cancer cells with astrocytes - the most abundant cell type within the metastatic brain niche – lead to downregulation of miRNA-768-3p which drives K-ras expression and key signaling pathways, enhances cell viability and promotes chemotherapeutic resistance. Vector-based forced expression of miRNA-768-3p complementary sequence or a chemically-engineered miRNA-768-3p inhibitor recapitulated the astrocyte effect to increase tumor cell viability. The miRNA-768-3p inhibitor targeted the K-ras 3′-UTR as demonstrated by increased luminescence from a luciferase reporter and strikingly increased the K-ras protein and the downstream effectors ERK1/2 and B-Raf. miRNA-768-3p was reduced in patient brain metastases compared to normal brain tissue and was lower in patient tissue from brain metastases compared to same-patient primary tumour tissue. The brain microenvironment negatively regulates miRNA-768-3p to enhance K-ras and promote metastasis. We propose that therapeutic replacement of the metastasis suppressor miRNA-768-3p holds clinical promise. PMID:23928793

  14. 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. PMID:27174785

  15. k-RAS mutations in non-small cell lung cancer patients treated with TKIs among smokers and non-smokers: a meta-analysis

    PubMed Central

    Lu, Hui-Yu

    2016-01-01

    Aim of the study Recent studies have suggested that k-RAS mutations are related to the response to epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitions (TKIs) in advanced non-small cell lung cancer (NSCLC) treatment. The aim of this meta-analysis was to assess the relationship between smoking history and k-RAS mutations in NSCLC treated with TKIs. Material and methods We searched MEDLINE and Web of Science up to 15 March 2014. The pooled relative risk (RR) was estimated by using fixed effect model or random effect model, according to heterogeneity between studies. We also carried out power analyses. Results We identified 12 studies with 1193 patients, including 196 patients (16.4%) with k-RAS mutations. The pooled k-RAS mutations incidence was 22.8% (174/764) in patients with smoke expose vs. 5.4% (23/429) in those with no smoke exposure. The pooled RR was 2.991 (95% CI: 1.884–4.746; Z = 4.65, p = 0.000). No publication bias was found (Begg's test: z = 1.09, p = 0.274 and Egger's test: t = 1.38, p = 0.201). In subgroup analyses, the pooled RR was 3.336 (95% CI: 1.925–5.779; Z = 4.30, p = 0.000) in the Caucasian subgroup, while in the Asian subgroup the pooled RR was 2.093 (95% CI: 0.909–4.822; Z = 1.73, p = 0.083), but the sample size was underpowered (0.465). Conclusions The current meta-analysis found that smoking was related to increased incidence of k-RAS mutations in non-small cell lung cancer treated with TKIs. This may be further evidence that smoking will lead to a worse prognosis in NSCLC patients treated with TKIs. PMID:27358590

  16. K-Ras Mutations in Non-Small-Cell Lung Cancer: Prognostic and Predictive Value

    PubMed Central

    D'Arcangelo, Manolo; Cappuzzo, Federico

    2012-01-01

    Non-small-cell lung cancer (NSCLC) is a heterogeneous disease due to the presence of different clinically relevant molecular subtypes. Until today, several biological events have been identified in lung adenocarcinoma, including epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) translocations, offering new hopes to patients with metastatic disease. Unfortunately, in approximately 50% of adenocarcinoma and for those harbouring K-RAS mutations, the most frequent mutation in Caucasian lung adenocarcinoma, so far no specific drug demonstrated efficacy. The rat sarcoma (RAS) genes, including H-RAS, K-RAS, and N-RAS, encode a family of proteins regulating cell growth, differentiation, and apoptosis. K-RAS mutations are present in 20–30% of NSCLC and occur most commonly, but not exclusively, in adenocarcinoma histology and life-long smokers. Although in colorectal cancer patients K-RAS mutations represent a validated negative predictive biomarker for treatment with anti-EGFR monoclonal antibodies, their role in selecting specific treatment for NSCLC patients remains undefined. Aim of the present paper is to critically analyze the prognostic and predictive value of K-RAS mutations in NSCLC.

  17. K-ras mutations in beryllium-induced mouse lung tumors

    SciTech Connect

    Belinsky, S.A.; Mitchell, C.E.

    1994-11-01

    Previous studies at ITRI have shown that single, nose-only exposure of F344/N rats to beryllium metal (Be) produced a 64% incidence of lung tumors over the lifetime of the rat. Long tumors induced by Be metal were subsequently analyzed for alterations in the K-ras and p53 genes. Mutation of the K-ras gene was both a rare (2 of 24 tumors) and late event in Be-induced carcinogenesis. In addition, no mutations were detected in exons 5 - 8 of the p53 gene. These results indicated that the mechanisms underlying the development of Be-induced lung cancer in rats did not involve gene dysfunction commonly associated with human non-small-cell lung cancer. The purpose of this study was to determine and compare the prevalence and specificity for mutation of the K-ras gene in lung tumors induced in the A/J mouse by Be to mutations in spontaneous tumors.

  18. 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.

  19. p16 and K-ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma.

    PubMed

    Moskaluk, C A; Hruban, R H; Kern, S E

    1997-06-01

    Pancreatic adenocarcinoma is thought to arise from a noninvasive neoplastic precursor, the pancreatic intraductal lesion (PIL). Mutations of the K-ras gene are known to occur in PILs, but their high prevalence among PILs within the general population probably limit the use of K-ras as a marker of eventual clinical risk. In search of genetic constellations that might indicate the progression of some PILs toward an invasive phenotype, mutations at both the K-ras and p16 genes were sought within PILs of 10 pancreata resected for adenocarcinoma. K-ras mutations were present in most PILs and in nearly all PILs having nuclear atypia. In half of the patients, two or more unique K-ras mutations were identified among distinct PILs, which is evidence for the separate clonal evolution of multiple pancreatic neoplasms within individual patients. p16 alterations (one homozygous deletion and three point mutations) were found in 4 of the 10 carcinomas; these four pancreata harbored p16 alterations in three of nine PILs, of which one was a "histologically early" lesion. Two patients had p16 alterations in PILs matching those of the associated carcinomas. p16 mutations were not found in PILs of pancreata having wild-type p16 in the carcinoma, nor were they found in ducts having normal histology. It is suggested that alterations of the p16 gene affect a subset of PILs that contain mutations of the K-ras gene and that these mutations might identify high-risk precursors of the invasive malignancy. PMID:9187111

  20. Multiple cells-of-origin of mutant K-Ras-induced mouse lung adenocarcinoma.

    PubMed

    Sutherland, Kate D; Song, Ji-Ying; Kwon, Min Chul; Proost, Natalie; Zevenhoven, John; Berns, Anton

    2014-04-01

    Much controversy surrounds the cell-of-origin of mutant K-Ras (K-RasG12D)-induced lung adenocarcinoma. To shed light on this issue, we have used technology that enables us to conditionally target K-RasG12D expression in Surfactant Protein C (SPC)(+) alveolar type 2 cells and in Clara cell antigen 10 (CC10)(+) Clara cells by use of cell-type-restricted recombinant Adeno-Cre viruses. Experiments were performed both in the presence and absence of the tumor suppressor gene p53, enabling us to assess what effect the cell-of-origin and the introduced genetic lesions have on the phenotypic characteristics of the resulting adenocarcinomas. We conclude that both SPC-expressing alveolar type 2 cells and CC10-expressing Clara cells have the ability to initiate malignant transformation following the introduction of these genetic alterations. The lungs of K-Ras(lox-Stop-lox-G12D/+) and K-Ras(lox-Stop-lox-G12D/+);tumor suppressor gene Trp53(F/F) mice infected with Adeno5-SPC-Cre and Adeno5-CC10-Cre viruses displayed differences in their tumor spectrum, indicating distinct cellular routes of tumor initiation. Moreover, using a multicolor Cre reporter line, we demonstrate that the resulting tumors arise from a clonal expansion of switched cells. Taken together, these results indicate that there are multiple cellular paths to K-RasG12D-induced adenocarcinoma and that the initiating cell influences the histopathological phenotype of the tumors that arise. PMID:24586047

  1. 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

  2. Identification of a ternary protein-complex as a therapeutic target for K-Ras-dependent colon cancer

    PubMed Central

    Hou, Songwang; Li, Gang; Yin, Ning; Dong, Lei; Lepp, Adrienne; Chesnik, Marla A.; Mirza, Shama P.; Szabo, Aniko; Tsai, Susan; Basir, Zainab; Wu, Shixiu; Chen, Guan

    2014-01-01

    A cancer phenotype is driven by several proteins and targeting a cluster of functionally interdependent molecules should be more effective for therapeutic intervention. This is specifically important for Ras-dependent cancer, as mutated (MT) Ras is non-druggable and targeting its interaction with effectors may be essential for therapeutic intervention. Here, we report that a protein-complex activated by the Ras effector p38γ MAPK is a novel therapeutic target for K-Ras-dependent colon cancer. Unbiased proteomic screening and immune-precipitation analyses identified p38γ interaction with heat shock protein 90 (Hsp90) and K-Ras in K-Ras MT, but not wild-type (WT), colon cancer cells, indicating a role of this complex in Ras-dependent growth. Further experiments showed that this complex requires p38γ and Hsp90 activity to maintain MT, but not WT, K-Ras protein expression. Additional studies demonstrated that this complex is activated by p38γ-induced Hsp90 phosphorylation at S595, which is important for MT K-Ras stability and for K-Ras dependent growth. Of most important, pharmacologically inhibition of Hsp90 or p38γ activity disrupts the complex, decreases K-Ras expression, and selectively inhibits the growth of K-Ras MT colon cancer in vitro and in vivo. These results demonstrated that the p38γ-activated ternary complex is a novel therapeutic target for K-Ras-dependent colon cancer. PMID:24962213

  3. 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. PMID:24763823

  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. PMID:22941421

  5. 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. PMID:27220342

  6. VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activating-mutations.

    PubMed

    Hervé, Virginie; Rabbe, Nathalie; Guilleminault, Laurent; Paul, Flora; Schlick, Laurène; Azzopardi, Nicolas; Duruisseaux, Michael; Fouquenet, Delphine; Montharu, Jérôme; Redini, Françoise; Paintaud, Gilles; Lemarié, Etienne; Cadranel, Jacques; Wislez, Marie; Heuzé-Vourc'h, Nathalie

    2014-01-01

    K-ras mutations promote angiogenesis in lung cancer and contribute to the drug resistance of cancer cells. It is not clear whether K-ras mutated adenocarcinomas are sensitive to anti-angiogenic therapy with monoclonal antibodies (mAbs) that target vascular endothelial growth factor (VEGF). Anti-angiogenic mAbs are usually delivered systemically, but only a small proportion reaches the lung after intravenous injection. We investigated the relevance of a non-invasive pulmonary route for the delivery of anti-VEGF mAbs in the mouse K-ras(LA1) model. We found that pulmonary delivery of these mAbs significantly reduced the number of tumor lesions and inhibited malignant progression. The antitumor effect involves the VEGFR2-dependent inhibition of blood vessel growth, which impairs tumor proliferation. Pharmacokinetic analysis of aerosolized anti-VEGF showed its low rate of passage into the bloodstream, suggesting that this delivery route is associated with reduced systemic side effects. Our findings highlight the value of the aerosol route for administration of anti-angiogenic mAbs in pulmonary adenocarcinoma with K-ras activating-mutations. PMID:25484066

  7. c-K-ras overexpression is characteristic for metastases derived from a methylcholanthrene-induced fibrosarcoma.

    PubMed

    Algarra, I; Perez, M; Serrano, M J; Garrido, F; Gaforio, J J

    We investigated the relationship between the activation of the c-myc and c-K-ras proto-oncogenes and the acquisition of metastatic potential in a methylcholanthrene-induced BALB/c fibrosarcoma. The murine fibrosarcoma GR9 was originally induced in BALB/c mice following exposure to the carcinogenic chemical 3-methylcholanthrene. To induce spontaneous metastasis, we used two tumor cell clones (B9 and G2) known to differ in their metastatic potential, local tumor growth, H-2 class I expression and sensitivity to natural killer (NK) cells. The metastatic nodes were obtained from the lung, liver and kidney. The results showed: (1) amplification of the c-myc proto-oncogene in original tumor clones as well as in all metastatic nodes; (2) mRNA overexpression without amplification of the K-ras proto-oncogene in the metastatic cells, regardless of their anatomical location; (3) no c-K-ras point mutations at codons 12 and 61, and (4) in general, a statistically significantly reduced in vitro sensitivity of metastatic tumor cells to NK cells as compared with the tumor clones used to induce them (p<0.05). These results therefore suggest that overexpressed c-K-ras mRNA is important during tumor progression, perhaps rendering metastatic tumor cells more resistant to lysis by NK cells. PMID:10729771

  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 hypervariable region of K-Ras4B is responsible for its specific interactions with Calmodulin

    PubMed Central

    Abraham, Sherwin J.; Nolet, Ryan P.; Calvert, Richard J.; Anderson, Lucy M.; Gaponenko, Vadim

    2009-01-01

    K-Ras4B belongs to the family of p21 Ras GTPases, which play an important role in cell proliferation, survival and motility. The p21 Ras proteins such as K-Ras4B, K-Ras4A, H-Ras, and N-Ras, share 85% sequence homology and activate very similar signaling pathways. Only the C-terminal hypervariable regions differ significantly. A growing body of literature demonstrates that each Ras isoform possesses unique functions in normal physiological processes as well as in pathogenesis. One of the central questions in the field of Ras biology is how these very similar proteins achieve such remarkable specificity in protein-protein interactions that regulate signal transduction pathways. Here we explore specific binding of K-Ras4B to calmodulin. Using NMR techniques and isothermal titration calorimetry we demonstrate that the hypervariable region of K-Ras contributes in a major way to the interaction with calmodulin while the catalytic domain of K-Ras4B provides a way to control the interaction by nucleotide binding. The hypervariable region of K-Ras4B binds specifically to the C-terminal domain of Ca2+-loaded calmodulin with micromolar affinity, while the GTP-γ-S loaded catalytic domain of K-Ras4B may interact with the N-terminal domain of calmodulin. PMID:19583261

  10. Partial knockdown of TRF2 increase radiosensitivity of human mesenchymal stem cells.

    PubMed

    Orun, O; Tiber, P Mega; Serakinci, N

    2016-09-01

    Telomere repeat binding factor TRF2 is a member of shelterin complex with an important role in protecting and stabilizing chromosomal ends. In the present study, we investigated the effect of partial knockdown of TRF2 on radiosensitivity of telomerase immortalized human mesenchymal stem cells (hMSC-telo1), which have a higher radioresistance compared to non telomerized counterpart. Partial knockdown of the protein achieved 15-20% reduction in TRF2 protein levels. The study compared the effect of 2.5Gy radiation in two-four days after irradiation for hMSC-telo1 cells and the cells transfected with siTRF2 and null control vector. Radio-response of the cells were examined using senescence associated β-Gal assay (β-Gal), colony forming assay (CFU) and γ-H2AX phosphorylation. TRF2 deficiency substantially increased radiosensitivity of cells compared to controls in both proliferation and senescence assay (2.4 fold increase in β-Gal, 1.6 fold decrease in CFU). In addition, it increased the γ-H2AX foci as revealed by both immunfluorescence and Western blot analysis. Our data suggests that partial knockdown of TRF2 in hMSC-telo1 cells cause increased γ-H2AX foci which led to fail TRF2 to protect telomeres from radiation thus TRF2 deficiency led to a 1,5-2 fold increase in the radiosensitivity of hMSC-telo1 cells through telomere destabilization. PMID:26598048

  11. K-Ras promotes the non-small lung cancer cells survival by cooperating with sirtuin 1 and p27 under ROS stimulation.

    PubMed

    Cheng, Dezhi; Zhao, Liang; Xu, Yunsheng; Ou, Rongying; Li, Gang; Yang, Han; Li, Wenfeng

    2015-09-01

    Cigarette smoking might lead to lung cancer. However, the related signaling pathways at molecular level remained unknown until now. In this study, we studied the signaling processes associated between tobacco exposure and lung cancer. First, we detected and validated pathway-specific gene expression at bronchial epithelium. These proteins reflected the activation of signaling pathways relevant to tobacco exposure, including ATM, BCL2, GPX1, K-Ras, IKBKB, and SIRT1. Tobacco smoking was simulated via reactive oxygen species (ROS) pathway. ROS not only arrested cell cycle at G1/S stage but also increased expressions of Sirt1 and p27. Further studies showed that the expression of p27 was dependent on ERK1/2 activation, and p27 itself could halt cell cycle by inhibiting the activation of CDKs. Moreover, activation of K-Ras, the key regulator of Ras/ERK pathway, was tightly regulated by enzyme activity of Sirt1. Deacetylation of K-Ras by Sirt1 increased the transformation of Ras-GTP to Ras-GDP, promoting the activation of downstream of ERK1/2. In reverse, Ras/ERK pathway could also regulate Sirt1 transcription. In conclusion, inhibition of Sirt1 may be an effective strategy for the prevention of tumor progression in high-risk patients or as a therapeutic strategy in established tumors. PMID:25894374

  12. Epidermal growth factor receptor and K-Ras in non-small cell lung cancer-molecular pathways involved and targeted therapies

    PubMed Central

    de Mello, Ramon Andrade; Marques, Dânia Sofia; Medeiros, Rui; Araújo, António MF

    2011-01-01

    Lung cancer is currently the leading cause of cancer death in Western nations. Non-small cell lung cancer (NSCLC) represents 80% of all lung cancers, and adenocarcinoma is the predominant histological type. Despite the intensive research carried out on this field and therapeutic advances, the overall prognosis of these patients remains unsatisfactory, with a 5-year overall survival rate of less than 15%. Nowadays, pharmacogenetics and pharmacogenomics represent the key to successful treatment. Recent studies suggest the existence of two distinct molecular pathways in the carcinogenesis of lung adenocarcinoma: one associated with smoking and activation of the K-Ras oncogene and the other not associated with smoking and activation of the epidermal growth factor receptor (EGFR). The K-ras mutation is mainly responsible for primary resistance to new molecules which inhibit tyrosine kinase EGFR (erlotinib and gefitinib) and most of the EGFR mutations are responsible for increased tumor sensitivity to these drugs. This article aims to conduct a systematic review of the literature regarding the molecular pathways involving the EGFR, K-Ras and EGFR targeted therapies in NSCLC tumor behavior. PMID:22087435

  13. Activated K-Ras, But Not H-Ras or N-Ras, Regulates Brain Neural Stem Cell Proliferation in a Raf/Rb-Dependent Manner

    PubMed Central

    Bender, R. Hugh F.; Haigis, Kevin M.; Gutmann, David H.

    2016-01-01

    Neural stem cells (NSCs) give rise to all the major cell types in the brain, including neurons, oligodendrocytes, and astrocytes. However, the intracellular signaling pathways that govern brain NSC proliferation and differentiation have been incompletely characterized to date. Since some neurodevelopmental brain disorders (Costello syndrome and Noonan syndrome) are caused by germline activating mutations in the RAS genes, Ras small GTPases are likely critical regulators of brain NSC function. In the mammalian brain, Ras exists as three distinct molecules (H-Ras, K-Ras, and N-Ras), each with different subcellular localizations, downstream signaling effectors, and biological effects. Leveraging a novel series of conditional-activated Ras molecule-expressing genetically engineered mouse strains, we demonstrate that activated K-Ras, but not H-Ras or N-Ras, expression increases brain NSC growth in a Raf-dependent, but Mek-independent, manner. Moreover, we show that activated K-Ras regulation of brain NSC proliferation requires Raf binding and suppression of retinoblastoma (Rb) function. Collectively, these observations establish tissue-specific differences in activated Ras molecule regulation of brain cell growth that operate through a noncanonical mechanism. PMID:25788415

  14. 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. PMID:27277529

  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. Geranylgeranyl Diphosphate Synthase Modulates Fetal Lung Branching Morphogenesis Possibly through Controlling K-Ras Prenylation.

    PubMed

    Jia, Wen-Jun; Jiang, Shan; Tang, Qiao-Li; Shen, Di; Xue, Bin; Ning, Wen; Li, Chao-Jun

    2016-06-01

    G proteins play essential roles in regulating fetal lung development, and any defects in their expression or function (eg, activation or posttranslational modification) can lead to lung developmental malformation. Geranylgeranyl diphosphate synthase (GGPPS) can modulate protein prenylation that is required for protein membrane-anchoring and activation. Here, we report that GGPPS regulates fetal lung branching morphogenesis possibly through controlling K-Ras prenylation during fetal lung development. GGPPS was continuously expressed in lung epithelium throughout whole fetal lung development. Specific deletion of geranylgeranyl diphosphate synthase 1 (Ggps1) in lung epithelium during fetal lung development resulted in neonatal respiratory distress syndrome-like disease. The knockout mice died at postnatal day 1 of respiratory failure, and the lungs showed compensatory pneumonectasis, pulmonary atelectasis, and hyaline membranes. Subsequently, we proved that lung malformations in Ggps1-deficient mice resulted from the failure of fetal lung branching morphogenesis. Further investigation revealed Ggps1 deletion blocked K-Ras geranylgeranylation and extracellular signal-related kinase 1 or 2/mitogen-activated protein kinase signaling, which in turn disturbed fibroblast growth factor 10 regulation on fetal lung branching morphogenesis. Collectively, our data suggest that GGPPS is essential for maintaining fetal lung branching morphogenesis, which is possibly through regulating K-Ras prenylation. PMID:27106761

  17. 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.

  18. The K-Ras 4A isoform promotes apoptosis but does not affect either lifespan or spontaneous tumor incidence in aging mice

    SciTech Connect

    Plowman, Sarah J.; Arends, Mark J.; Brownstein, David G.; Luo Feijun; Devenney, Paul S.; Rose, Lorraine; Ritchie, Ann-Marie; Berry, Rachel L.; Harrison, David J.; Hooper, Martin L.; Patek, Charles E. . E-mail: Charles.Patek@ed.ac.uk

    2006-01-01

    Ras proteins function as molecular switches in signal transduction pathways, and, here, we examined the effects of the K-ras4A and 4B splice variants on cell function by comparing wild-type embryonic stem (ES) cells with K-ras {sup tm{delta}}{sup 4A/tm{delta}}{sup 4A} (exon 4A knock-out) ES cells which express K-ras4B only and K-ras {sup -/-} (exons 1-3 knock-out) ES cells which express neither splice variant, and intestinal epithelium from wild-type and K-ras {sup tm{delta}}{sup 4A/tm{delta}}{sup 4A} mice. RT-qPCR analysis found that K-ras4B expression was reduced in K-ras {sup tm{delta}}{sup 4A/tm{delta}}{sup 4A} ES cells but unaffected in small intestine. K-Ras deficiency did not affect ES cell growth, and K-Ras4A deficiency did not affect intestinal epithelial proliferation. K-ras {sup tm{delta}}{sup 4A/tm{delta}}{sup 4A} and K-ras {sup -/-} ES cells showed a reduced capacity for differentiation following LIF withdrawal, and K-ras {sup -/-} cells were least differentiated. K-Ras4A deficiency inhibited etoposide-induced apoptosis in ES cells and intestinal epithelial cells. However, K-ras {sup tm{delta}}{sup 4A/tm{delta}}{sup 4A} ES cells were more resistant to etoposide-induced apoptosis than K-ras {sup -/-} cells. The results indicate that (1) K-Ras4A promotes apoptosis while K-Ras4B inhibits it, and (2) K-Ras4B, and possibly K-Ras4A, promotes differentiation. The findings raise the possibility that alteration of the K-Ras4A/4B isoform ratio modulates tumorigenesis by differentially affecting stem cell survival and/or differentiation. However, K-Ras4A deficiency did not affect life expectancy or spontaneous overall tumor incidence in aging mice.

  19. 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.

  20. 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

  1. K-ras mutation at codon 12 in stage I pancreatic adenocarcinoma: analysis by laser capture microdissection and direct sequencing.

    PubMed

    Chang, M C; Chang, Y T; Wu, M S; Shun, C T; Tien, Y W; Lin, J T

    2001-05-01

    Pancreatic ductal adenocarcinoma has been reported to carry a rate mutation high in codon 12 of the K-ras oncogene. To avoid the pitfalls of conventional methods of tissue dissection that might affect the sensitivity and specificity of detecting K-ras mutation, laser capture microdissection (LCM) technique was used. Pancreatic adenocarcinoma tissues were obtained from 15 patients who underwent Whipple's procedure. Selected tissues procured by LCM were analyzed by direct sequencing after polymerase chain reaction amplification of K-ras sequences at codon 12. K-ras mutation was noted in nine patients. All mutations showed G to A substitution at codon 12. The mutational pattern (GGT to GAT) is similar in both western and eastern reports. LCM is a feasible method to effectively obtain pure tumor cells from a surgical specimen. It remains to be determined whether this low mutation rate is a result of relatively early stage of disease or different carcinogenesis in different geographic regions. PMID:11432318

  2. Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

    PubMed Central

    2011-01-01

    Background Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied. Methods In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses. Results Under normoxic conditions, a half maximal inhibitory concentration (IC50) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions. Conclusion Our

  3. 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

  4. Inhibition of PARP-1 by olaparib (AZD2281) increases the radiosensitivity of a lung tumor xenograft.

    PubMed

    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

    2011-10-01

    PARP-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, although 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 hemodynamics in non-small cell lung carcinoma (NSCLC). In irradiated Calu-6 and A549 cells, olaparib enhanced the cytotoxic effects of radiation (sensitizer enhancement ratio at 10% survival = 1.5 and 1.3) and DNA double-strand breaks persisted for at least 24 hours 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 radiosensitization was solely due 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 preconstricted 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

  5. 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. PMID:25450481

  6. Tissue-specific hypomethylation of the human c-K-ras gene.

    PubMed Central

    Metter, J; Cho, C

    1989-01-01

    Methylation of the c-K-ras gene was examined in a wide variety of human tissues using the methylation sensitive restriction endonuclease HpaII. All tissues showed hypomethylation in the region of exon zero. Specific hypomethylation of a particular HpaII site in the second intron was observed in gastrointestinal and tracheobronchial epithelial cell DNAs. Specific hypomethylation was also observed in a cluster of HpaII sites within the first intron in sperm, endometrium and placenta DNAs. These regions were predominantly methylated in a wide variety of other tissues, including fetal gut. The possible implications are discussed. Images PMID:2476726

  7. The role of antiangiogenic agents in the treatment of patients with advanced colorectal cancer according to K-RAS status.

    PubMed

    García-Alfonso, Pilar; Grande, Enrique; Polo, Eduardo; Afonso, Ruth; Reina, Juan José; Jorge, Mónica; Campos, Juan Manuel; Martínez, Virginia; Angeles, Cristina; Montagut, Clara

    2014-10-01

    Colorectal cancer (CRC) is the fourth most commonly diagnosed cancer worldwide. Recently, it has been found that about 40 % of patients with CRC have mutations in the K-RAS gene. Several clinical trials have showed that patients with metastatic colorectal cancer (mCRC) who present tumour-promoting mutations in signalling pathways involving the epidermal growth factor receptor (EGFR), which includes activating K-RAS mutations, do not respond to anti-EGFR drugs such as panitumumab and cetuximab. Hence, K-RAS status is now considered an important negative predictive factor for response to anti-EGFR drugs. Moreover, K-RAS status seems to have also a prognostic role in CRC, but this fact is somewhat controversial. Activity of antiangiogenic agents seems not to be influenced by K-RAS gene status. Tumour angiogenesis has attracted interest in attempts to improve the management of mCRC. The vascular endothelial growth factor (VEGF) pathway is fundamental to the regulation of angiogenesis, and research has focused on developing agents that selectively target it. In this way, the anti-VEGF antibody bevacizumab in combination with chemotherapy has provided important clinical benefits in terms of response rate, progression-free survival and overall survival to patients with mCRC. Efficacy data of bevacizumab in K-RAS wild-type patients seem to be comparable with the efficacy data observed with anti-EGFR therapies in a cross-trial comparison. Although there is a lack of prospective and randomized data in this setting, the combination of chemotherapy plus antiangiogenic agents could be considered as an effective alternative for the treatment of mCRC with independence of K-RAS gene status. Here, we review the available data we have in the literature of the use of antiangiogenic strategies in the treatment of mCRC nowadays. PMID:24793846

  8. Increased radiosensitivity of HPV-positive head and neck cancers: Molecular basis and therapeutic perspectives.

    PubMed

    Mirghani, Haïtham; Amen, Furrat; Tao, Yungan; Deutsch, Eric; Levy, Antonin

    2015-12-01

    Human papillomavirus driven head and neck squamous cell carcinoma (HNSCC), particularly oropharyngeal squamous cell carcinoma (OPSCC), are characterized by a significant survival advantage over their HPV-negative counterparts. Although the reasons behind this are still not fully elucidated, it is widely accepted that these tumors have a higher response to ionizing radiation that might explain their favorable outcomes. Potential underlying intrinsic mechanisms include impaired DNA repair abilities, differences in activated repopulation-signaling pathways and cell cycle control mechanisms. The role of the microenvironment is increasingly highlighted, particularly tumor oxygenation and the immune response. Recent studies have shown a distinct pattern of intratumoral immune cell infiltrates, according to HPV status, and have suggested that an increased cytotoxic T-cell based antitumor immune response is involved in improved prognosis of patients with HPV-positive OPSCC. These significant milestones, in the understanding of HPV-induced HNSCC, pave the way to new therapeutic opportunities. This article reviews the current evidence on the biological basis of increased radiosensitivity in HPV-positive HNSCC and discusses potential therapeutic implications. PMID:26476574

  9. Downregulation of high mobility group box 1 modulates telomere homeostasis and increases the radiosensitivity of human breast cancer cells.

    PubMed

    Ke, Shaobo; Zhou, Fuxiang; Yang, Hui; Wei, Yuehua; Gong, Jun; Mei, Zijie; Wu, Lin; Yu, Haijun; Zhou, Yunfeng

    2015-03-01

    The functions of the high mobility group box 1 (HMGB1) in tumor cells include replenishing telomeric DNA and maintaining cell immortality. There is a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Our aim was to elucidate the relationship among HMGB1, telomere homeostasis and radiosensitivity in MCF-7 cells. In this study, we established stably transfected control (MCF-7-NC) and HMGB1 knockdown (MCF-7-shHMGB1) cell lines. The expression of HMGB1 mRNA and the relative telomere length were examined by real-time PCR. Radiosensitivity was detected by clonogenic assay. The protein expressions were determined by western blot analysis. The telomerase activity was detected by PCR-ELISA. Proliferation ability was examined by CCK-8 assay. Cell cycle and apoptosis were examined by flow cytometry. DNA damage foci were detected by immunofluorescence. ShRNA-mediated downregulation of HMGB1 expression increased the radiosensitivity of MCF-7 cells, and reduced the accumulation of hTERT and cyclin D1. Moreover, knockdown of HMGB1 in MCF-7 cells inhibited telomerase activity and cell proliferation, while increasing the extent of apoptosis. Downregulation of HMGB1 modulated telomere homeostasis by changing the level of telomere-binding proteins, such as TPP1 (PTOP), TRF1 and TRF2. This downregulation also inhibited the ATM and ATR signaling pathways. The current data demonstrate that knockdown of HMGB1 breaks telomere homeostasis, enhances radiosensitivity, and suppresses the repair of DNA damage in human breast cancer cells. These results suggested that HMGB1 might be a potential radiotherapy target in human breast cancer. PMID:25501936

  10. 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

  11. The Bisphenol A analogue Bisphenol S binds to K-Ras4B--implications for 'BPA-free' plastics.

    PubMed

    Schöpel, Miriam; Herrmann, Christian; Scherkenbeck, Jürgen; Stoll, Raphael

    2016-02-01

    K-Ras4B is a small GTPase that belongs to the Ras superfamily of guanine nucleotide-binding proteins. GTPases function as molecular switches in cells and are key players in intracellular signalling. Ras has been identified as an oncogene and is mutated in more than 20% of human cancers. Here, we report that Bisphenol S binds into a binding pocket of K-Ras4B previously identified for various low molecular weight compounds. Our results advocate for more comprehensive safety studies on the toxicity of Bisphenol S, as it is frequently used for Bisphenol A-free food containers. PMID:26867649

  12. 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.

  13. The cell of origin and subtype of K-Ras-induced lung tumors are modified by Notch and Sox2

    PubMed Central

    Xu, Xia; Huang, Lingling; Futtner, Christopher; Schwab, Brian; Rampersad, Rishi R.; Lu, Yun; Sporn, Thomas A.; Hogan, Brigid L.M.

    2014-01-01

    Cell type-specific conditional activation of oncogenic K-Ras is a powerful tool for investigating the cell of origin of adenocarcinomas in the mouse lung. Our previous studies showed that K-Ras activation with a CC10(Scgb1a1)-CreER driver leads to adenocarcinoma in a subset of alveolar type II cells and hyperplasia in the bronchioalveolar duct region. However, no tumors develop in the bronchioles, although recombination occurs throughout this region. To explore underlying mechanisms, we simultaneously modulated either Notch signaling or Sox2 levels in the CC10+ cells along with activation of K-Ras. Inhibition of Notch strongly inhibits adenocarcinoma formation but promotes squamous hyperplasia in the alveoli. In contrast, activation of Notch leads to widespread Sox2+, Sox9+, and CC10+ papillary adenocarcinomas throughout the bronchioles. Chromatin immunoprecipitation demonstrates Sox2 binding to NOTCH1 and NOTCH2 regulatory regions. In transgenic mouse models, overexpression of Sox2 leads to a significant reduction of Notch1 and Notch2 transcripts, while a 50% reduction in Sox2 leads to widespread papillary adenocarcinoma in the bronchioles. Taken together, our data demonstrate that the cell of origin of K-Ras-induced tumors in the lung depends on levels of Sox2 expression affecting Notch signaling. In addition, the subtype of tumors arising from type II cells is determined in part by Notch activation or suppression. PMID:25184679

  14. Immunophenotype and K-RAS mutation in mucinous ovarian adenocarcinoma with mural nodule of high-grade sarcoma: case report.

    PubMed

    Desouki, Mohamed M; Fadare, Oluwole; Kanbour, Anisa; Kanbour-Shakir, Amal

    2014-03-01

    Ovarian mucinous tumors with mural nodules are rare. The mural nodules are microscopically divergent neoplasms of varying sizes that may be benign (eg, sarcoma-like and carcinosarcoma-like), or malignant (eg, anaplastic carcinoma and sarcoma). The K-RAS gene mutation in ovarian mucinous neoplasms with mural nodules has not been previously reported. This is a case report of a 25-year-old female diagnosed with ovarian invasive mucinous adenocarcinoma with mural nodule of high-grade sarcoma. The mucinous tumor component demonstrated a K-RAS codon 12/13 mutation (p.G12V, c.35 G>T), whereas the sarcomatous component demonstrated a K-RAS codon 12/13 mutation (p.G12D, c.35 G>A). Although both tumor components revealed a mutation in codon 12 of K-RAS, they were of different nucleotide substitutions, indicating that these 2 tumor components were of different clonal origins. However, the fact that the 2 mutations identified in the tumor components are the most common mutations reported in mucinous tumors of the ovary, raises the possibility that sarcomatous mural nodules simply represent a form of dedifferentiation in mucinous tumors. PMID:24487474

  15. Translation-dependent mechanisms lead to PML upregulation and mediate oncogenic K-RAS-induced cellular senescence

    PubMed Central

    Scaglioni, Pier Paolo; Rabellino, Andrea; Yung, Thomas M; Bernardi, Rosa; Choi, Sooyeon; Konstantinidou, Georgia; Nardella, Caterina; Cheng, Ke; Pandolfi, Pier Paolo

    2012-01-01

    Expression of oncogenic K-RAS in primary cells elicits oncogene-induced cellular senescence (OIS), a form of growth arrest that potently opposes tumourigenesis. This effect has been largely attributed to transcriptional mechanisms that depend on the p53 tumour suppressor protein. The PML tumour suppressor was initially identified as a component of the PML-RARα oncoprotein of acute promyelocytic leukaemia (APL). PML, a critical OIS mediator, is upregulated by oncogenic K-RAS in vivo and in vitro. We demonstrate here that oncogenic K-RAS induces PML protein upregulation by activating the RAS/MEK1/mTOR/eIF4E pathway even in the absence of p53. Under these circumstances, PML mRNA is selectively associated to polysomes. Importantly, we find that the PML 5′ untranslated mRNA region plays a key role in mediating PML protein upregulation and that its presence is essential for an efficient OIS response. These findings demonstrate that upregulation of PML translation plays a central role in oncogenic K-RAS-induced OIS. Thus, selective translation initiation plays a critical role in tumour suppression with important therapeutic implications for the treatment of solid tumours and APL. PMID:22359342

  16. 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.

  17. Detection of K-ras Mutations in Predicting Efficacy of Epidermal Growth Factor Receptor Tyrosine Kinase (EGFR-TK) Inhibitor in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Li, Ze; Liu, Xue-Wei; Chi, Zhao-Cheng; Sun, Bao-Sheng; Cheng, Ying; Cheng, Long-Wei

    2015-01-01

    Epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors are useful in treating different advanced human cancers; however, their clinical efficacy varies. This study detected K-ras mutations to predict the efficacy of EGFR-TK inhibitor cetuximab treatment on Chinese patients with metastatic colorectal cancer (mCRC). A total of 87 patients with metastatic colorectal cancer were treated with cetuximab for 2-16 months, in combination with chemotherapy between August 2008 and July 2012, and tissue samples were used to detect K-ras mutations. The data showed that K-ras mutation occurred in 27/87 (31%). The objective response rates and disease control rate in K-ras wild type and mutant patients were 42% (25/60) versus 11% (3/27) (p<0.05) and 60% (36/60) versus 26% (7/27) (p<0.05), respectively. Patients with the wild-type K-ras had significantly higher median survival times and progression-free survival, than patients with mutated K-ras (21 months versus 17 months, p=0.017; 10 months versus 6 months, p=0.6). These findings suggest that a high frequency of K-ras mutations occurs in Chinese mCRC patients and that K-ras mutation is required to select patients for eligibility for cetuximab therapy. Further prospective studies using a large sample size are needed to confirm these preliminary findings. PMID:25950441

  18. Novel chemical enhancers of heat shock increase thermal radiosensitization through a mitotic catastrophe pathway.

    PubMed

    Sekhar, Konjeti R; Sonar, Vijayakumar N; Muthusamy, Venkatraj; Sasi, Soumya; Laszlo, Andrei; Sawani, Jamil; Horikoshi, Nobuo; Higashikubo, Ryuji; Bristow, Robert G; Borrelli, Michael J; Crooks, Peter A; Lepock, James R; Roti Roti, Joseph L; Freeman, Michael L

    2007-01-15

    Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(+/-)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation. PMID:17234780

  19. The combination of tetraiodothyroacetic acid and cetuximab inhibits cell proliferation in colorectal cancers with different K-ras status.

    PubMed

    Lee, Yee-Shin; Chin, Yu-Tang; Yang, Yu-Chen S H; Wei, Po-Li; Wu, Han-Chung; Shih, Ai; Lu, Yueh-Tong; Pedersen, Jens Z; Incerpi, Sandra; Liu, Leroy F; Lin, Hung-Yun; Davis, Paul J

    2016-07-01

    Thyroid hormone induces cancer cell proliferation through its cell surface receptor integrin αvβ3. Acting via integrin αvβ3, the deaminated T4 analog tetraiodothyroacetic acid (tetrac), and its nanoparticle formulation nano-diamino-tetrac (NDAT) could inhibit cell proliferation and xenograft growth. In this study, we investigated the T4 effects on proliferation in colorectal cancer cell lines based on the proliferation marker expressions at both mRNA and protein levels. The effects of tetrac/NDAT, the monoclonal anti-EGFR antibody cetuximab, and their combinations on colorectal cancer cell proliferation were examined according to the relevant gene expression profiles and cell count analysis. The results showed that T4 significantly enhanced PCNA, Cyclin D1 and c-Myc levels in both K-ras wild type HT-29 and mutant HCT 116 cells. In HCT 116 cells, the combination of NDAT and cetuximab significantly suppressed the mRNA expressions of proliferative genes PCNA, Cyclin D1, c-Myc and RRM2 raised by T4 compared to cetuximab alone. In addition, T4-suppressed mRNA expressions of pro-apoptotic genes p53 and RRM2B could be significantly elevated by the combination of NDAT and cetuximab compared to cetuximab alone. In the K-ras mutant HCT 116 cells, but not in the K-ras wild type COLO 205 cells, the combinations of tetrac/NDAT and cetuximab significantly reduced cell proliferation compared to cetuximab alone. In conclusion, T4 promoted colorectal cancer cell proliferation which could be repressed by tetrac and NDAT. The combinations of tetrac/NDAT and cetuximab potentiated cetuximab actions in K-ras mutant colorectal cancer cells. PMID:26980146

  20. 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

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

    PubMed

    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-Ras(G12D) on tumor burden and gene expression signature using compound mutant mouse lines. Concomitant activation of COX-2 and K-Ras(G12D) 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

  2. 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.

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

    PubMed

    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-Ras4B(WT)-GTP/GDP) catalytic domain, the K-Ras4B(WT)-GTP-GAP complex, and the mutants (K-Ras4B(G12C/G12D/G12V)-GTP/GDP, K-Ras4B(G13D)-GTP/GDP, K-Ras4B(Q61H)-GTP/GDP) and their complexes with GAP. In addition, we simulated 'exchanged' nucleotide states. These comprehensive simulations reveal that in solution K-Ras4B(WT)-GTP exists in two, active and inactive, conformations. Oncogenic mutations differentially elicit an inactive-to-active conformational transition in K-Ras4B-GTP; in K-Ras4B(G12C/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

  4. 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. PMID:27225938

  5. 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.

  6. 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.

  7. 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.

  8. Frequent k- ras -2 mutations and p16(INK4A)methylation in hepatocellular carcinomas in workers exposed to vinyl chloride.

    PubMed

    Weihrauch, M; Benicke, M; Lehnert, G; Wittekind, C; Wrbitzky, R; Tannapfel, A

    2001-04-01

    Vinyl chloride (VC) is a know animal and human carcinogen associated with liver angiosarcomas (LAS) and hepatocellular carcinomas (HCC). In VC-associated LAS mutations of the K- ras -2 gene have been reported; however, no data about the prevalence of such mutations in VC associated HCCs are available. Recent data indicate K- ras -2 mutations induce P16 methylation accompanied by inactivation of the p16 gene. The presence of K- ras -2 mutations was analysed in tissue from 18 patients with VC associated HCCs. As a control group, 20 patients with hepatocellular carcinoma due to hepatitis B (n = 7), hepatitis C (n = 5) and alcoholic liver cirrhosis (n = 8) was used. The specific mutations were determined by direct sequencing of codon 12 and 13 of the K- ras -2 gene in carcinomatous and adjacent non-neoplastic liver tissue after microdissection. The status of p16 was evaluated by methylation-specific PCR (MSP), microsatellite analysis, DNA sequencing and immunohistochemical staining. All patients had a documented chronic quantitated exposure to VC (average 8883 ppmy, average duration: 245 months). K- ras -2 mutations were found in 6 of 18 (33%) examined VC-associated HCCs and in 3 cases of adjacent non-neoplastic liver tissue. There were 3 G --> A point mutations in the tumour tissue. All 3 mutations found in non-neoplastic liver from VC-exposed patients were also G --> A point mutations (codon 12- and codon 13-aspartate mutations). Hypermethylation of the 5' CpG island of the p16 gene was found in 13 of 18 examined carcinomas (72%). Of 6 cancers with K- ras -2 mutations, 5 specimens also showed methylated p16. Within the control group, K- ras -2 mutation were found in 3 of 20 (15%) examined HCC. p16 methylation occurred in 11 out of 20 (55%) patients. K- ras -2 mutations and p16 methylation are frequent events in VC associated HCCs. We observed a K- ras -2 mutation pattern characteristic of chloroethylene oxide, a carcinogenic metabolite of VC. Our results strongly

  9. Long-term follow-up of patients with resected pancreatic cancer following vaccination against mutant K-ras.

    PubMed

    Wedén, Synne; Klemp, Marianne; Gladhaug, Ivar P; Møller, Mona; Eriksen, Jon Amund; Gaudernack, Gustav; Buanes, Trond

    2011-03-01

    K-ras mutations are frequently found in adenocarcinomas of the pancreas and can elicit mutation-specific immune responses. Targeting the immune system against mutant Ras may thus influence the clinical course of the disease. Twenty-three patients who were vaccinated after surgical resection for pancreatic adenocarcinoma (22 pancreaticoduodenectomies, one distal resection), in two previous Phase I/II clinical trials, were followed for more than 10 years with respect to long-term immunological T-cell reactivity and survival. The vaccine was composed of long synthetic mutant ras peptides designed mainly to elicit T-helper responses. Seventeen of 20 evaluable patients (85%) responded immunologically to the vaccine. Median survival for all patients was 27.5 months and 28 months for immune responders. The 5-year survival was 22% and 29%, respectively. Strikingly, 10-year survival was 20% (four patients out of 20 evaluable) versus zero (0/87) in a cohort of nonvaccinated patient treated in the same period. Three patients mounted a memory response up to 9 years after vaccination. The present observation of long-term immune response together with 10-year survival following surgical resection indicates that K-ras vaccination may consolidate the effect of surgery and represent an adjuvant treatment option for the future. PMID:20473937

  10. STAT3 supports experimental K-RasG12D–induced murine myeloproliferative neoplasms dependent on serine phosphorylation

    PubMed Central

    Gough, Daniel J.; Marié, Isabelle J.; Lobry, Camille; Aifantis, Iannis

    2014-01-01

    Juvenile myelomonocytic leukemia, acute myeloid leukemia (AML), and other myeloproliferative neoplasms (MPNs) are genetically heterogeneous but frequently display activating mutations in Ras GTPases and activation of signal transducer and activator of transcription 3 (STAT3). Altered STAT3 activity is observed in up to 50% of AML correlating with poor prognosis. Activated STAT proteins, classically associated with tyrosine phosphorylation, support tumor development as transcription factors, but alternative STAT functions independent of tyrosine phosphorylation have been documented, including roles for serine-phosphorylated STAT3 in mitochondria supporting transformation by oncogenic Ras. We examined requirements for STAT3 in experimental murine K-Ras–dependent hematopoietic neoplasia. We show that STAT3 is phosphorylated on S727 but not Y705 in diseased animals. Moreover, a mouse with a point mutation abrogating STAT3 S727 phosphorylation displayed delayed onset and decreased disease severity with significantly extended survival. Activated K-Ras required STAT3 for cytokine-independent growth of myeloid progenitors in vitro, and mitochondrially restricted STAT3 and STAT3-Y705F, both transcriptionally inert mutants, supported factor-independent growth. STAT3 was dispensable for growth of normal or K-Ras–mutant myeloid progenitors in response to cytokines. However, abrogation of STAT3-S727 phosphorylation impaired factor-independent malignant growth. These data document that serine-phosphorylated mitochondrial STAT3 supports neoplastic hematopoietic cell growth induced by K-Ras. PMID:25150294

  11. Prevention of K-Ras- and Pten-mediated intravaginal tumors by treatment with camptothecin-loaded PLGA nanoparticles

    PubMed Central

    Blum, Jeremy S.; Weller, Caroline E.; Booth, Carmen J.; Babar, Imran A.; Liang, Xianping; Slack, Frank J.

    2014-01-01

    Primary squamous cell carcinoma of the vagina is an uncommon disease that often exhibits few symptoms before reaching an advanced stage. Topical intravaginal therapies for resolving precancerous and cancerous vaginal lesions have the potential to be non-invasive and safer alternatives to existing treatment options. Two factors limit the testing of this approach: lack of a preclinical intravaginal tumor model and absence of safe and effective topical delivery systems. In this study, we present both an inducible genetic model of vaginal squamous cell carcinoma in mice and a novel topical delivery system. Tumors were generated via activation of oncogenic K-Ras and inactivation of tumor suppressor Pten in LSL-K-RasG12D/+ PtenloxP/loxP mice. This was accomplished by exposing the vaginal epithelium to a recombinant adenoviral vector expressing Cre recombinase (AdCre). As early as 3 weeks after AdCre exposure exophytic masses protruding from the vagina were observed; these were confirmed to be squamous cell carcinoma by histology. We utilized this model to investigate an anticancer therapy based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with camptothecin (CPT); our earlier work has shown that PLGA nanoparticles can penetrate the vaginal epithelium and provide sustained CPT release. Particles were lavaged into the vaginal cavity of AdCre-infected mice. None of the mice receiving CPT nanoparticles developed tumors. These results demonstrate a novel topical strategy to resolve precancerous and cancerous lesions in the female reproductive tract. PMID:25419505

  12. MEKs/ERKs inhibitor U0126 increases the radiosensitivity of rhabdomyosarcoma cells in vitro and in vivo by down regulating growth and DNA repair signals

    PubMed Central

    Marampon, Francesco; Gravina, Giovanni Luca; Di Rocco, Agnese; Bonfili, Pierluigi; Di Staso, Mario; Fardella, Caterina; Polidoro, Lorella; Ciccarelli, Carmela; Festuccia, Claudio; Popov, Vladimir M.; Pestell, Richard G.; Tombolini, Vincenzo; Zani, Bianca Maria

    2010-01-01

    Multimodal treatment has improved the outcome of many solid tumors, and in some cases the use of radiosensitizers has significantly contributed to this gain. Activation of the extracellular signaling kinase pathway (MEK/ERK) generally results in stimulation of cell growth and confers a survival advantage playing the major role in human cancer. The potential involvement of this pathway in cellular radiosensitivity remains unclear. We previously reported that the disruption of c-Myc through MEK/ERK inhibition blocks the expression of the transformed phenotype, affects in vitro and in vivo growth, angiogenic signaling, and induces myogenic differentiation in the embryonal rhabdomyosarcoma (ERMS) cell lines (RD). The present study was designed to examine whether the ERK pathway affects intrinsic radiosensitivity of rhabdomyosarcoma cancer cells. Exponentially growing human ERMS, RD, xenograft-derived RD-M1 and TE671 cell lines were used. The specific MEK/ERK inhibitor, U0126, reduced the clonogenic potential of the three cell lines, and was effected by radiation. U0126 inhibited phospho/active ERK1/2 and reduced DNAPKcs suggesting that ERKs and DNA-PKcs cooperate in radioprotection of rhabdomyosarcoma cells. The TE671 cell line-xenotransplanted in mice showed a reduction in tumor mass and increase in the time of tumor progression with U0126 treatment associated with reduced DNAPKcs, an effect enhanced by radiotherapy. Thus, our results show that MEK/ERK inhibition enhances radiosensitivity of rhabdomyosarcoma cells suggesting a rational approach in combination with radiotherapy. PMID:21220498

  13. 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

  14. The HER2-Binding Affibody Molecule (ZHER2∶342)2 Increases Radiosensitivity in SKBR-3 Cells

    PubMed Central

    Ekerljung, Lina; Lennartsson, Johan; Gedda, Lars

    2012-01-01

    We have previously shown that the HER2-specific affibody molecule (ZHER2∶342)2 inhibits proliferation of SKBR-3 cells. Here, we continue to investigate its biological effects in vitro by studying receptor dimerization and clonogenic survival following irradiation. We found that (ZHER2∶342)2 sensitizes the HER2-overexpressing cell line SKBR-3 to ionizing radiation. The survival after exposure to (ZHER2∶342)2 and 8 Gy (S8Gy 0.006) was decreased by a factor four compared to the untreated (S8Gy 0.023). The low HER2-expressing cell line MCF-7 was more radiosensitive than SKBR-3 but did not respond to (ZHER2∶342)2. Treatment by (ZHER2∶342)2 strongly increased the levels of dimerized and phosphorylated HER2 even after 5 minutes of stimulation. The monomeric ZHER2∶342 does not seem to be able to induce receptor phosphorylation and dimerization or sensitize cells to irradiation. PMID:23166716

  15. Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization.

    PubMed

    Bhatia, Shilpa; Baig, Nimrah A; Timofeeva, Olga; Pasquale, Elena B; Hirsch, Kellen; MacDonald, Tobey J; Dritschilo, Anatoly; Lee, Yi Chien; Henkemeyer, Mark; Rood, Brian; Jung, Mira; Wang, Xiao-Jing; Kool, Marcel; Rodriguez, Olga; Albanese, Chris; Karam, Sana D

    2015-04-20

    The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target. PMID:25879388

  16. Knockdown of EphB1 receptor decreases medulloblastoma cell growth and migration and increases cellular radiosensitization

    PubMed Central

    Timofeeva, Olga; Pasquale, Elena B.; Hirsch, Kellen; MacDonald, Tobey J.; Dritschilo, Anatoly; Lee, Yi Chien; Henkemeyer, Mark; Rood, Brian; Jung, Mira; Wang, Xiao-Jing; Kool, Marcel

    2015-01-01

    The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased β1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target. PMID:25879388

  17. Modulation of tumor induction and progression of oncogenic K-ras-positive tumors in the presence of TGF- b1 haploinsufficiency.

    PubMed

    Pandey, Jyotsna; Umphress, Sarah M; Kang, Yang; Angdisen, Jerry; Naumova, Alena; Mercer, Kim L; Jacks, Tyler; Jakowlew, Sonia B

    2007-12-01

    Oncogenic K-ras is one of the most common genetic alterations in human lung adenocarcinomas. In addition, inactivation of clusters of tumor suppressor genes is required to bring about classical characteristics of cancer including angiogenesis as a prelude to invasion and metastasis. Transforming growth factor-beta (TGF-beta) 1 is a tumor suppressor gene that is implicated in lung cancer progression. Although in vitro studies have shown that TGF-beta1 and Ras pathways cooperate during tumorigenesis, the biology of interaction of TGF-beta1 and Ras has not been studied in in vivo tumorigenesis. We hypothesized that inactivation of TGF-beta1 in addition to oncogeneic activation of K-ras would lead to early initiation and faster progression to lung adenocarcinoma and invasion and metastasis. Heterozygous (HT) TGF-beta1 mice were mated with latent activatable (LA) mutated K-ras mice to generate TGF-beta1(+/+), K-ras LA (wild-type (WT)/LA) and TGF-beta1(+/-), K-ras LA (HT/LA) mice. Both HT/LA and WT/LA mice developed spontaneous lung tumors, but HT/LA mice progressed to adenocarcinomas significantly earlier compared with WT/LA mice. In addition, WT/LA adenocarcinomas had significantly higher angiogenic activity compared with HT/LA adenocarcinomas. Thus, while oncogenic K-ras mutation and insensitivity to the growth regulatory effects of TGF-beta1 is essential for initiation and progression of mouse lung tumors to adenocarcinoma, a full gene dosage of TGF-beta1 is required for tumor-induced angiogenesis and invasive potential. This study identifies a number of genes not previously associated with lung cancer that are involved in tumor induction and progression. In addition, we provide evidence that progression to invasive angiogenic lesions requires TGF-beta1 responsiveness in addition to Ras mutation. PMID:17690114

  18. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis; the role of p53 and p21

    PubMed Central

    Escandell, José M.; Kaler, Pawan; Recio, M. Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija

    2008-01-01

    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke3 cells. However, the presence of oncogenic k-Ras significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransfromed intestinal epithelial cells with inducible expression of k-RasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3. PMID:18561895

  19. Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21.

    PubMed

    Escandell, José M; Kaler, Pawan; Recio, M Carmen; Sasazuki, Takehiko; Shirasawa, Senji; Augenlicht, Leonard; Ríos, José-Luis; Klampfer, Lidija

    2008-07-15

    Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3. PMID:18561895

  20. The relationship between microvessel count and the expression of vascular endothelial growth factor, p53, and K-ras in non-small cell lung cancer.

    PubMed Central

    Kang, Y. H.; Kim, K. S.; Yu, Y. K.; Lim, S. C.; Kim, Y. C.; Park, K. O.

    2001-01-01

    Using immunohistochemical staining, we studied the relationship between the microvessel count (MC) and the expression of K-ras, mutant p53 protein, and vascular endothelial growth factor (VEGF) in 61 surgically resected non-small cell lung cancers (NSCLC) (42 squamous cell carcinoma, 14 adenocarcinoma, 2 large cell carcinoma, 2 adenosquamous carcinoma, and 1 mucoepidermoid carcinoma). MC of the tumors with lymph node (LN) metastasis was significantly higher than that of tumors without LN metastasis (66.1+/-23.1 vs. 33.8+/-13.1, p<0.05). VEGF was positive in 54 patients (88.5%). MC was 58.1+/-25.2 (mean+/-S.D.) in a x200 field, and it was significantly higher in VEGF(+) tumors than in VEGF(-) tumors (61.4+/-23.7 vs. 32.9+/-23.8, p<0.05). VEGF expression was higher in K-ras-positive or mutant p53-positive tumors than in negative tumors (p<0.05). MC was significantly higher in K-ras(+) tumors than in K-ras(-) tumors, although it did not differ according to the level of mutant p53 protein expression. Survival did not differ with VEGF, mutant p53, or K-ras expression, or the level of MC. In conclusion, there is a flow of molecular alterations from K-ras and p53, to VEGF expression, leading to angiogenesis and ultimately lymph node metastasis. Correlations between variables in close approximation and the lack of prognostic significance of individual molecular alterations suggest that tumorigenesis and metastasis are multifactorial processes. PMID:11511786

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

    PubMed Central

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

    2016-01-01

    Background 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. Methods 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. Results 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

  2. A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand.

    PubMed

    Sun, Qi; Phan, Jason; Friberg, Anders R; Camper, DeMarco V; Olejniczak, Edward T; Fesik, Stephen W

    2014-09-01

    K-Ras is a well-validated cancer target but is considered to be "undruggable" due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets. PMID:25087006

  3. Cathepsin L suppression increases the radiosensitivity of human glioma U251 cells via G2/M cell cycle arrest and DNA damage

    PubMed Central

    Zhang, Qing-qing; Wang, Wen-juan; Li, Jun; Yang, Neng; Chen, Gang; Wang, Zhong; Liang, Zhong-qin

    2015-01-01

    Aim: Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro. Methods: Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy. Results: Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G2/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2. Conclusion: Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro. PMID:26095040

  4. Aerosol delivery of beclin1 enhanced the anti-tumor effect of radiation in the lungs of K-rasLA1 mice.

    PubMed

    Shin, Ji-Young; Lim, Hwang-Tae; Minai-Tehrani, Arash; Noh, Mi-Suk; Kim, Ji-Eun; Kim, Ji-Hye; Jiang, Hu-Lin; Arote, Rohidas; Kim, Doo-Yeol; Chae, Chanhee; Lee, Kee-Ho; Kim, Mi-Sook; Cho, Myung-Haing

    2012-07-01

    Radiotherapy alone has several limitations for treating lung cancer. Inhalation, a non-invasive approach for direct delivery of therapeutic agents to the lung, may help to enhance the therapeutic efficacy of radiation. Up-regulating beclin1, known as a tumor suppressor gene that plays a major role in autophagy, may sensitize tumors and lead to tumor regression in lungs of K-ras(LA1) lung cancer model mice. To minimize the side-effects of radiotherapy, fractionated exposures (five times, 24-h interval) with low dose (2 Gy) of radiation to the restricted area (thorax, 2 cm) were conducted. After sensitizing the lungs with radiation, beclin1, complexed with a nano-sized biodegradable poly(ester amine), was prepared and delivered into the murine lung via aerosol three times/week for four weeks. In a histopathological analysis, animals treated with beclin1 and radiation showed highly significant tumor regression and low progression to adenocarcinoma. An increase in the number of autophagic vacuoles and secondary lysosomes was detected. Dissociation of beclin1-bcl2 stimulated autophagy activation and showed a synergistic anti-tumor effect by inhibiting the Akt-mTOR pathway, cell proliferation and angiogenesis. The combination of radiation with non-invasive aerosol delivery of beclin1 may provide a prospect for developing novel therapy regimens applicable in clinics. PMID:22843615

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

    PubMed Central

    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

    2015-01-01

    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-RasG12V-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. PMID:25971796

  6. 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

  7. 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.

  8. 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

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

    PubMed

    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

  10. 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.
    <...

  11. Comparison of a PNA clamp PCR and an ARMS/Scorpion PCR assay for the detection of K-ras mutations.

    PubMed

    Nordgård, Oddmund; Oltedal, Satu; Janssen, Emiel A M; Gilje, Bjørnar; Kørner, Hartwig; Tjensvoll, Kjersti; Smaaland, Rune

    2012-03-01

    Point mutations in the K-ras gene have been shown to confer resistance against epidermal growth factor receptor-directed therapy of metastatic colorectal cancer. Accordingly, K-ras mutation testing has become mandatory in hospitals offering such treatment. We compared the performance and reagent costs of 2 sensitive methods for detection of K-ras mutations: a peptide nucleic acid (PNA) clamp polymerase chain reaction (PCR) assay and a commercially available amplification refractory mutation system/Scorpion (ARMS/S) PCR assay. Both methods were applied in parallel to 101 formalin-fixed, paraffin-embedded tumor and metastasis samples from patients with colon cancer. The PNA clamp PCR assay detected K-ras mutations in 35% (35 of 101) of the samples, whereas the ARMS/S PCR assay detected mutations in 27% (27 of 101) of them. There was 92% (93 of 101) concordance between the 2 methods and the κ coefficient for the comparison was 0.82. The 8 discordant cases were exclusively positive by PNA clamp PCR. Finally, the reagent costs of the PNA clamp PCR assay were estimated to be at least 20 times lower than the ARMS/S assay. We concluded that the high performance and low costs associated with the PNA clamp PCR assay encourage its use in the administration of personalized epidermal growth factor receptor-directed therapy. PMID:22306670

  12. 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.

  13. Identification and characterization of a new G-quadruplex forming region within the kRAS promoter as a transcriptional regulator.

    PubMed

    Morgan, Rhianna K; Batra, Harshul; Gaerig, Vanessa C; Hockings, Jennifer; Brooks, Tracy A

    2016-02-01

    kRAS is one of the most prevalent oncogenic aberrations. It is either upregulated or mutationally activated in a multitude of cancers, including pancreatic, lung, and colon cancers. While a significant effort has been made to develop drugs that target kRAS, their clinical activity has been disappointing due to a variety of mechanistic hurdles. The presented works describe a novel mechanism and molecular target to downregulate kRAS expression--a previously undescribed G-quadruplex (G4) secondary structure within the proximal promoter acting as a transcriptional silencer. There are three distinct guanine-rich regions within the core kRAS promoter, including a previously examined region (G4near). Of these regions, the most distal region does not form an inducible and stable structure, whereas the two more proximal regions (termed near and mid) do form strong G4s. G4near is predominantly a tri-stacked structure with a discontinuous guanine run incorporated; G4mid consists of seven distinct runs of continuous guanines and forms numerous competing isoforms, including a stable three-tetrad stacked mixed parallel and antiparallel loop structures with longer loops of up to 10 nucleotides. Comprehensive analysis of the regulation of transcription by higher order structures has revealed that the guanine-rich region in the middle of the core promoter, termed G4mid, is a stronger repressor of promoter activity than G4near. Using the extensive guanine-rich region of the kRAS core promoter, and particularly the G4mid structure, as the primary target, future drug discovery programs will have potential to develop a potent, specifically targeted small molecule to be used in the treatment of pancreatic, ovarian, lung, and colon cancers. PMID:26597160

  14. Ultra-sensitive biosensor for K-ras gene detection using enzyme capped gold nanoparticles conjugates for signal amplification.

    PubMed

    Fang, Xian; Bai, Lijuan; Han, Xiaowei; Wang, Jiao; Shi, Anqi; Zhang, Yuzhong

    2014-09-01

    In this study, an ultra-sensitive hairpin DNA-based electrochemical DNA biosensor for K-ras gene detection is described. Gold nanoparticles (Au-NPs) and horseradish peroxidase (HRP)-streptavidin capped Au-NPs (HAS) conjugates are used for signal amplification. Initially, hairpin DNA dually labeled with thiol at its 5' end and with biotin at its 3' end is immobilized on the surface of Au-NPs modified electrode, and the hairpin DNA is in a "closed" state; hence, the HAS conjugates are shielded from being approached by the biotin due to steric hindrance. However, in the presence of target DNA, the target DNA hybridizes with the loop structure of hairpin DNA and causes conformational change, resulting in biotin forced away from the electrode surface, thereby becoming accessible for the HAS conjugates. Thus, the HAS conjugates are linked to the electrode surface via the specific interaction between biotin and streptavidin. Electrochemical detection was performed in phosphate-buffered saline (PBS) containing tetramethylbenzidine (TMB) and H2O2. Under optimal conditions, the peak current differences (ΔI) are linear with the target DNA in the range from 0.1 fM to 1 nM with a detection limit of 0.035 fM. Furthermore, this biosensor also demonstrates its excellent specificity for single-base mismatched DNA. PMID:24939462

  15. Transcriptional profiling of immortalized and K-ras-transformed mouse fibroblasts upon PKA stimulation by forskolin in low glucose availability.

    PubMed

    Chiaradonna, Ferdinando; Pirola, Yuri; Ricciardiello, Francesca; Palorini, Roberta

    2016-09-01

    Forskolin (FSK) induces activation of protein kinase A (PKA). This activation protects specifically some cancer cells from death induced by glucose starvation. Cell effects upon FSK treatment prompted us to investigate in detail the physiological role of PKA in the activation of pro-survival mechanisms in glucose starvation. In this regard we performed a microarray analysis of normal NIH3T3 and transformed NIH3T3-K-ras mouse fibroblasts cultured at 1 mM glucose and daily treated or not with 10 μM FSK until 72 h of growth, when the samples were collected. The microarray is deposited into Gene Expression Omnibus under Series GSE68266. The microarray data revealed that the activation of PKA regulates the expression of genes involved in metabolic, stress-response and pro-survival processes, like glutamine metabolism, autophagy and unfolded protein response, preventing cancer cell death in glucose starvation. Altogether these findings suggest that PKA activation, by inducing a complex transcriptional program, leads to cancer survival in nutrient stress, a typical feature of developing tumor. These transcriptional data, identifying this important role of PKA, will be useful to identify novel target in cancer therapy. PMID:27486565

  16. Zerumbone increases oxidative stress in a thiol-dependent ROS-independent manner to increase DNA damage and sensitize colorectal cancer cells to radiation.

    PubMed

    Deorukhkar, Amit; Ahuja, Niharika; Mercado, Armando-Lopez; Diagaradjane, Parmeswaran; Raju, Uma; Patel, Nalini; Mohindra, Pranshu; Diep, Nga; Guha, Sushovan; Krishnan, Sunil

    2015-02-01

    Locally advanced rectal cancers are treated with neoadjuvant chemoradiation therapy followed by surgery. In a minority (~20%) of patients, no tumor is present at the time of surgery; these patients with a complete pathologic response (pathCR) to neoadjuvant therapy have better treatment outcomes. Unfortunately, the inherent radioresistance of colorectal cancer (CRC) cells dictates that the majority of patients do not achieve a pathCR. Efforts to improve these odds have fueled the search for novel, relatively less-toxic radiosensitizers with distinct molecular mechanism(s) and broad-spectrum anticancer activities. Here, we use zerumbone, a sesquiterpene from the edible ginger (Zingiber zerumbet Smith), to enhance radiosensitivity of CRC cells. Short exposure to zerumbone (7 h) profoundly sensitized CRC cells, independent of their p53 or k-RAS status. Zerumbone enhanced radiation-induced cell cycle arrest (G2/M), increased radiation-induced apoptosis, but induced little apoptosis by itself. Zerumbone significantly enhanced radiation-induced DNA damage, as evident by delayed resolution of post-irradiation nuclear γH2AX foci, whereas zerumbone treatment alone did not induce γH2AX foci formation. Zerumbone pretreatment inhibited radiation-induced nuclear expression of DNA repair proteins ataxia-telangiectasia mutated (ATM) and DNA-PKcs. Interestingly, zerumbone-mediated radiosensitization did not involve reactive oxygen species (ROS), but was mediated through depletion of cellular glutathione (GSH). Ability of only thiol-based antioxidants to abrogate zerumbone-mediated radiosensitization further corroborated this hypothesis. The α,β-unsaturated carbonyl group in zerumbone was found to be essential for its bioactivity as zerumbone analog α-Humulene that lacks this functional group, could neither radiosensitize CRC cells, nor deplete cellular GSH. Our studies elucidate novel mechanism(s) of zerumbone's ability to enhance CRC radiosensitivity. PMID:25450478

  17. A Crosstalk Between K ras (Kirsten Rat Sarcoma Viral Oncogene Homologue) and Adherence Molecular Complex Leads to Disassociation of Cells-A Possible Contribution Towards Metastasis in Colorectal Cancer.

    PubMed

    Murtaza, Bibi Nazia; Doak, Shareen; Morgan, Claire; Nadeem, Muhammad Shahid; Al-Ghanim, Khalid A; Shakoori, Abdul Rauf

    2016-10-01

    Constitutive activation of mutant K ras (Kirsten rat sarcoma viral oncogene homologue) and disassembly of E-cadherin-catenin complex (E-cadherin, α-catenin, β-catenin, and γ-catenin) play an important role in apoptosis, differentiation, and cell proliferation. In this study, the expression pattern of K ras and E-cadherin-catenin complex has been evaluated in normal and mutant colorectal cancer cell lines with an object to determine its impact on disassociation of cells from one another. We addressed the expression analysis of K ras with reference to its association with adherence molecules in two colorectal cancer cell lines, that is, Caco-2 (wild type K ras served as a control) and DLD1 (heterozygous mutation at codon 13) at message level by qRT-PCR and translational level by western blotting. Compared to the control Caco-2 cell lines, the K ras in DLD1 cell lines showed slightly higher values while α-catenin showed a slight lower (1.3-folds), β-catenin and E-cadherin showed significantly lower expression (4.2-fold decrease). It can be inferred that a possible cross talk exists between K ras and adherent junction mediated signalling. Mutation at codon 13 (G to D) leads to the overexpression of K ras and reduced expression of adherent junction complex resulting in metastasis. J. Cell. Biochem. 117: 2340-2345, 2016. © 2016 Wiley Periodicals, Inc. PMID:26945839

  18. A method for the second-site screening of K-Ras in the presence of a covalently attached first-site ligand

    PubMed Central

    Sun, Qi; Phan, Jason; Friberg, Anders R.; Camper, DeMarco V.; Olejniczak, Edward T.; Fesik, Stephen W.

    2015-01-01

    K-Ras is a well-validated cancer target but is considered to be “undruggable” due to the lack of suitable binding pockets. We previously discovered small molecules that bind weakly to K-Ras but wanted to improve their binding affinities by identifying ligands that bind near our initial hits that we could link together. Here we describe an approach for identifying second site ligands that uses a cysteine residue to covalently attach a compound for tight binding to the first site pocket followed by a fragment screen for binding to a second site. This approach could be very useful for targeting Ras and other challenging drug targets. PMID:25087006

  19. 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. PMID:23939923

  20. 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

  1. Aerosol delivery of eukaryotic translation initiation factor 4E-binding protein 1 effectively suppresses lung tumorigenesis in K-rasLA1 mice.

    PubMed

    Chang, S-H; Kim, J-E; Lee, J-H; Minai-Tehrani, A; Han, K; Chae, C; Cho, Y-H; Yun, J-H; Park, K; Kim, Y-S; Cho, M-H

    2013-06-01

    Conventional radiotherapy or chemotherapy for the long-term survival of patients with lung cancer is still difficult for treatment in metastatic and advanced tumors. Therefore, the safe and effective approaches to the treatment of lung cancer are needed. In this study, the effect of delivered eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) on lung cancer progression was evaluated. Recombinant adeno-associated virus (rAAV)-M3/4E-BP1 was delivered into 6-week-old K-rasLA1 lung cancer model mice through a nose-only inhalation system twice a week for 4 weeks. Long-term repeated delivery of 4E-BP1 effectively reduced tumor progression in the lungs of K-rasLA1 mice. Reduction of eIF4E by overexpression of 4E-BP1 resulted in suppression of cap-dependent protein expression of basic fibroblast growth factor (bFGF or FGF-2) and vascular endothelial growth factor (VEGF). In addition, delivered 4E-BP1 inhibited the proliferation of lung cancer cells in K-rasLA1 mice model. Our results suggest that long-term repeated viral delivery of 4E-BP1 may provide a useful tool for designing lung cancer treatment. PMID:23640516

  2. 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. PMID:12211696

  3. 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.

  4. Rothmund-Thomson syndrome: two case reports show heterogeneous cutaneous abnormalities, an association with genetically programmed ageing changes, and increased chromosomal radiosensitivity.

    PubMed Central

    Kerr, B; Ashcroft, G S; Scott, D; Horan, M A; Ferguson, M W; Donnai, D

    1996-01-01

    Rothmund-Thomson syndrome is a rare, autosomal recessive disorder associated with characteristic cutaneous changes, sparse hair, juvenile cataracts, short stature, skeletal defects, dystrophic teeth and nails, and hypogonadism. Mental retardation is unusual. An increased incidence of certain malignancies has been reported. Clonal or mosaic chromosome abnormalities and abnormalities in DNA repair mechanisms have been reported in some cases. We report two cases of Rothmund-Thomson syndrome, both with intellectual handicap, associated in one with a previously undescribed histological appearance of involved skin, suggesting that the spectrum of abnormalities is even more heterogeneous than previously presumed. Both cases exhibited chromosomal radiosensitivity of lymphocytes which may be an indication of a DNA repair defect. This is the first report of an association between Rothmund-Thomson syndrome and unique, intrinsic, age related skin changes. Images PMID:8950673

  5. 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.

  6. Higher Levels of c-Met Expression and Phosphorylation Identify Cell Lines With Increased Sensitivity to AMG-458, a Novel Selective c-Met Inhibitor With Radiosensitizing Effects

    SciTech Connect

    Li Bo; Torossian, Artour; Sun, Yunguang; Du, Ruihong; Dicker, Adam P.; Lu Bo

    2012-11-15

    Purpose: c-Met is overexpressed in some non-small cell lung cancer (NSCLC) cell lines and tissues. Cell lines with higher levels of c-Met expression and phosphorylation depend on this receptor for survival. We studied the effects of AMG-458 on 2 NSCLC cell lines. Methods and Materials: 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium assays assessed the sensitivities of the cells to AMG-458. Clonogenic survival assays illustrated the radiosensitizing effects of AMG-458. Western blot for cleaved caspase 3 measured apoptosis. Immunoblotting for c-Met, phospho-Met (p-Met), Akt/p-Akt, and Erk/p-Erk was performed to observe downstream signaling. Results: AMG-458 enhanced radiosensitivity in H441 but not in A549. H441 showed constitutive phosphorylation of c-Met. A549 expressed low levels of c-Met, which were phosphorylated only in the presence of exogenous hepatocyte growth factor. The combination of radiation therapy and AMG-458 treatment was found to synergistically increase apoptosis in the H441 cell line but not in A549. Radiation therapy, AMG-458, and combination treatment were found to reduce p-Akt and p-Erk levels in H441 but not in A549. H441 became less sensitive to AMG-458 after small interfering RNA knockdown of c-Met; there was no change in A549. After overexpression of c-Met, A549 became more sensitive, while H441 became less sensitive to AMG-458. Conclusions: AMG-458 was more effective in cells that expressed higher levels of c-Met/p-Met, suggesting that higher levels of c-Met and p-Met in NSCLC tissue may classify a subset of tumors that are more sensitive to molecular therapies against this receptor.

  7. Radiosensitive severe combined immunodeficiency disease.

    PubMed

    Dvorak, Christopher C; Cowan, Morton J

    2010-02-01

    Inherited defects in components of the nonhomologous 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 that are traditionally used for conditioning before allogeneic hematopoietic cell transplantation (HCT). Known causes of radiosensitive SCID include deficiencies of Artemis, DNA ligase IV, DNA-dependent protein kinase catalytic subunit, and Cernunnos-XLF, all of which have been treated with HCT. Because of these patients' sensitivity to certain forms of chemotherapy, the approach to donor selection and the type of conditioning regimen used for a patient with radiosensitive SCID requires careful consideration. Significantly more research needs to be done to determine the long-term outcomes of patients with radiosensitive SCID after HCT and to discover novel nontoxic approaches to HCT that might benefit those patients with intrinsic radiosensitivity and chemosensitivity as well as potentially all patients undergoing an HCT. PMID:20113890

  8. 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

  9. ¹H, ¹³C and ¹⁵N resonance assignment for the human K-Ras at physiological pH.

    PubMed

    Vo, Uybach; Embrey, Kevin J; Breeze, Alexander L; Golovanov, Alexander P

    2013-10-01

    K-Ras, a member of the Ras family of small GTPases, is involved in cell growth, proliferation, differentiation and apoptosis and is frequently mutated in cancer. The activity of Ras is mediated by the inter-conversion between GTP- and GDP- bound states. This conversion is regulated by binding of effector proteins such as guanine nucleotide exchange factors and GTPase activating proteins. Previously, NMR signals from these effector-binding regions of Ras often remained unassigned and largely unobservable due to conformational exchange and polysterism inherent to this protein. In this paper, we report the complete backbone and C(β), as well as partial H(α), H(β) and C(γ), NMR assignment for human K-Ras (residues 1-166) in the GDP-bound form at a physiological pH of 7.4. These data thereby make possible detailed monitoring of the functional cycle of Ras and its interactions with nucleotides and effector proteins through the observation of fingerprint signals from all the functionally important regions of the protein. PMID:22886485

  10. Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

    PubMed Central

    Sloan, S R; Newcomb, E W; Pellicer, A

    1990-01-01

    Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA. Images PMID:2403644

  11. MGMT promoter hypermethylation and K-RAS, PTEN and TP53 mutations in tamoxifen-exposed and non-exposed endometrial cancer cases

    PubMed Central

    Nagy, E; Gajjar, K B; Patel, I I; Taylor, S; Martin-Hirsch, P L; Stringfellow, H F; Martin, F L; Phillips, D H

    2014-01-01

    Background: Tamoxifen has anti-oestrogenic and anti-tumour activity in the breast, but is oestrogenic and carcinogenic in the endometrium. It can induce experimental tumours by both hormonal and DNA-damaging mechanisms, but its carcinogenic mode of action in human endometrium remains unclear. Methods: We investigated whether an epigenetic mechanism, involving promoter hypermethylation of the gene for the DNA repair enzyme MGMT (O6-methylguanine DNA methyltransferase), was associated with K-RAS, TP53 and PTEN mutations in endometrial tumours from women treated with tamoxifen (TAM, n=30) or unexposed to the drug (EC, n=38). Results: There were significant (P<0.05) differences in tumour grade between the TAM and EC groups, with more favourable morphology in the latter. K-RAS mutations, predominantly G>A, occurred in small numbers in both groups. TP53 mutations were of mainly A>G, C>T and indel modifications in both groups, but more frequent in TAM cases. PTEN mutations dominated in EC tumours and were of the type that has large impact on protein function, such as indel or nonsense mutations. These observations alongside the mutational spectrum in PTEN suggest that the malignancies arise from different backgrounds, hence pointing to an effect of tamoxifen. Both groups displayed MGMT promoter hypermethylation. This coincided with mutations more frequently in the TAM (78%) than in the EC (50%) group, even though there were significantly (P<0.05) fewer mutations and methylations in TAM cases. Conclusions: Although the difference in coincidence did not reach significance with the current sample size, the findings suggest that epigenetic processes may play a role in the way tamoxifen induces endometrial cancer. PMID:24853176

  12. 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

  13. Lack of DNA polymerase theta (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation.

    PubMed

    Goff, Julie P; Shields, Donna S; Seki, Mineaki; Choi, Serah; Epperly, Michael W; Dixon, Tracy; Wang, Hong; Bakkenist, Christopher J; Dertinger, Stephen D; Torous, Dorothea K; Wittschieben, John; Wood, Richard D; Greenberger, Joel S

    2009-08-01

    Abstract Mammalian POLQ (pol theta) is a specialized DNA polymerase with an unknown function in vivo. Roles have been proposed in chromosome stability, as a backup enzyme in DNA base excision repair, and in somatic hypermutation of immunoglobulin genes. The purified enzyme can bypass AP sites and thymine glycol. Mice defective in POLQ are viable and have been reported to have elevated spontaneous and radiation-induced frequencies of micronuclei in circulating red blood cells. To examine the potential roles of POLQ in hematopoiesis and in responses to oxidative stress responses, including ionizing radiation, bone marrow cultures and marrow stromal cell lines were established from Polq(+/+) and Polq(-/-) mice. Aging of bone marrow cultures was not altered, but Polq(-/-) cells were more sensitive to gamma radiation than were Polq(+/+) cells. The D(0) was 1.38 +/- 0.06 Gy for Polq(+/+) cells compared to 1.27 +/- 0.16 and 0.98 +/- 0.10 Gy (P = 0.032) for two Polq(-/-) clones. Polq(-/-) cells were moderately more sensitive to bleomycin than Polq(+/+) cells and were not hypersensitive to paraquat or hydrogen peroxide. ATM kinase activation appeared to be normal in gamma-irradiated Polq(-/-) cells. Inhibition of ATM kinase activity increased the radiosensitivity of Polq(+/+) cells slightly but did not affect Polq(-/-) cells. Polq(-/-) mice had more spontaneous and radiation-induced micronucleated reticulocytes than Polq+/+ and (+/-) mice. The sensitivity of POLQ-defective bone marrow stromal cells to ionizing radiation and bleomycin and the increase in micronuclei in red blood cells support a role for this DNA polymerase in cellular tolerance of DNA damage that can lead to double-strand DNA breaks. PMID:19630521

  14. 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.

  15. 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.

  16. [Synthesis and radiosensitizing activity of benzimidazoles].

    PubMed

    Li, M J; Li, S Z; Zhuang, X L; Chen, A; Zhang, H Q; Pang, X C; Hu, B

    1992-01-01

    In search for effective radiosensitizer with low neurotoxicity, benzimidazole compounds were synthesized. Reaction of 2-nitrobenzimidazole with ethyl chloroformate yielded ethyl alpha-(2-nitrobenzimidazolyl-1)-formate(1) or ethyl alpha-(2-hydroxybenzimidazolyl-1)-formate(2), depending upon the solvents used. Reaction of 2-nitrobenzimidazole with 1,2-epoxy-3-chloropropane gave a cyclized compound which was confirmed to be benzimidazo(1,2b)-5'-chloromethyl-oxazolidine(3). In attempt to increase hydrophilicity, 1-substituted 2(3'-pyridyl)-5-nitrobenzimidazoles were prepared by reaction of 2-(3'-pyridyl)-5(6)-nitrobenzimidazole(4) with alkyl epoxides or ethyl chloroacetate. Some of the compounds synthesized were tested for radiosensitizing activity in Ehrlich ascites carcinoma-bearing mice. Preliminary results showed that some compounds have radiosensitizing activity. The radiosensitizing enhancement ratio (SER) of compounds 3, 5 and 6 were found to be 1.50, 1.52 and 1.65 respectively. PMID:1293936

  17. 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.

  18. 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

  19. Novel Ras pathway inhibitor induces apoptosis and growth inhibition of K-ras-mutated cancer cells in vitro and in vivo.

    PubMed

    Jasinski, Piotr; Zwolak, Pawel; Terai, Kaoru; Dudek, Arkadiusz Z

    2008-11-01

    MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind. PMID:19010291

  20. 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

  1. Predictive value of K-ras and PIK3CA in non-small cell lung cancer patients treated with EGFR-TKIs: a systemic review and meta-analysis

    PubMed Central

    Chen, Jie-Ying; Cheng, Ya-Nan; Han, Lei; Wei, Feng; Yu, Wen-Wen; Zhang, Xin-Wei; Cao, Shui; Yu, Jin-Pu

    2015-01-01

    Objective A meta-analysis was performed to augment the insufficient data on the impact of mutative EGFR downstream phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on the clinical efficiency of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment of non-small cell lung cancer (NSCLC) patients. Methods Network databases were explored in April, 2015. Papers that investigated the clinical outcomes of NSCLC patients treated with EGFR-TKIs according to the status of K-ras and/or PIK3CA gene mutation were included. A quantitative meta-analysis was conducted using standard statistical methods. Odds ratios (ORs) for objective response rate (ORR) and hazard ratios (HRs) for progression-free survival (PFS) and overall survival (OS) were calculated. Results Mutation in K-ras significantly predicted poor ORR [OR =0.22; 95% confidence interval (CI), 0.13-0.35], shorter PFS (HR =1.56; 95% CI, 1.27-1.92), and shorter OS (HR =1.59; 95% CI, 1.33-1.91) in NSCLC patients treated with EGFR-TKIs. Mutant PIK3CA significantly predicted shorter OS (HR =1.83; 95% CI, 1.05-3.20), showed poor ORR (OR =0.70; 95% CI, 0.22-2.18), and shorter PFS (HR =1.79; 95% CI, 0.91-3.53) in NSCLC patients treated with EGFR-TKIs. Conclusion K-ras mutation adversely affected the clinical response and survival of NSCLC patients treated with EGFR-TKIs. PIK3CA mutation showed similar trends. In addition to EGFR, adding K-ras and PIK3CA as routine gene biomarkers in clinical genetic analysis is valuable to optimize the effectiveness of EGFR-TKI regimens and identify optimal patients who will benefit from EGFR-TKI treatment. PMID:26175928

  2. 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. PMID:26655363

  3. 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.

  4. 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. PMID:26081808

  5. Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer

    PubMed Central

    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.

    2015-01-01

    The toxicity of pharmacological ascorbate is mediated by the generation of H2O2 via the oxidation of ascorbate. Since 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 pharmacological 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 non-tumorigenic 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, pharmacological 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 pharmacological ascorbate as a radiosensitizer in the treatment of pancreatic cancer. PMID:26081808

  6. 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.

  7. Recent developments in radiosensitization.

    PubMed

    Linam, Justin; Yang, Li-Xi

    2015-05-01

    Radiation therapy is essential for local tumor control for many types of cancer histologies. Technological advancements in recent years have allowed for precise irradiation of target tissues while minimizing the dose to non-target tissues. To enhance radiation damage to cancer cells and further limit the radiation effects on normal tissue, researchers have explored compounds that specifically target cancer cells and make them more sensitive to ionizing radiation. Recent radiosensitization research has focused on promising compounds that alter hypoxia, inhibit topoisomerases, interfere with microtubules, and activate caspases, among other mechanisms. Many such compounds have shown impressive results in pre-clinical trials against a variety of cell types, but their safety, efficacy and practicability in clinical trials remains to be demonstrated. This review seeks to provide an overview of recent research in radiosensitization, detailing some of the more successful compounds, and illustrating avenues for future research. PMID:25964520

  8. Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1.

    PubMed

    Norden, Pieter R; Kim, Dae Joong; Barry, David M; Cleaver, Ondine B; Davis, George E

    2016-01-01

    A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network

  9. Cdc42 and k-Ras Control Endothelial Tubulogenesis through Apical Membrane and Cytoskeletal Polarization: Novel Stimulatory Roles for GTPase Effectors, the Small GTPases, Rac2 and Rap1b, and Inhibitory Influence of Arhgap31 and Rasa1

    PubMed Central

    Norden, Pieter R.; Kim, Dae Joong; Barry, David M.; Cleaver, Ondine B.; Davis, George E.

    2016-01-01

    A critical and understudied property of endothelial cells is their ability to form lumens and tube networks. Although considerable information has been obtained concerning these issues, including the role of Cdc42 and Rac1 and their effectors such as Pak2, Pak4, Par6b, and co-regulators such as integrins, MT1-MMP and Par3; many key questions remain that are necessary to elucidate molecular and signaling requirements for this fundamental process. In this work, we identify new small GTPase regulators of EC tubulogenesis including k-Ras, Rac2 and Rap1b that act in conjunction with Cdc42 as well as the key downstream effectors, IQGAP1, MRCKβ, beta-Pix, GIT1, and Rasip1 (which can assemble into multiprotein complexes with key regulators including α2β1 integrin and MT1-MMP). In addition, we identify the negative regulators, Arhgap31 (by inactivating Cdc42 and Rac) and Rasa1 (by inactivating k-Ras) and the positive regulator, Arhgap29 (by inactivating RhoA) which play a major functional role during the EC tubulogenic process. Human EC siRNA suppression or mouse knockout of Rasip1 leads to identical phenotypes where ECs form extensive cord networks, but cannot generate lumens or tubes. Essential roles for these molecules during EC tubulogenesis include; i) establishment of asymmetric EC cytoskeletal polarization (subapical distribution of acetylated tubulin and basal membrane distribution of F-actin); and ii) directed membrane trafficking of pinocytic vacuoles or other intracellular vesicles along acetylated tubulin tracks to the developing apical membrane surface. Cdc42 co-localizes subapically with acetylated tubulin, while Rac1 and k-Ras strongly label vacuole/ vesicle membranes which accumulate and fuse together in a polarized, perinuclear manner. We observe polarized apical membrane and subapical accumulation of key GTPases and effectors regulating EC lumen formation including Cdc42, Rac1, Rac2, k-Ras, Rap1b, activated c-Raf and Rasip1 to control EC tube network

  10. 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.

  11. Radiosensitization of mouse skin by oxygen and depletion of glutathione

    SciTech Connect

    Stevens, G.; Joiner, M.; Joiner, B.

    1995-09-30

    To determine the oxygen enhancement ratio (OER) and shape of the oxygen sensitization curve of mouse foot skin, the extent to which glutathione (GSH) depletion radiosensitized skin, and the dependence of such sensitization on the ambient oxygen tension. Carbogen caused the greatest radiosensitization of skin, with a reproducible enhancement of 2.2 relative to the anoxic response. The OER of 2.2 is lower than other reports for mouse skin. This may indicate that the extremes of oxygenation were not produced, although there was no direct evidence for this. Depletion of GSH caused minimal radiosensitization when skin was irradiated under anoxic or well-oxygenated conditions. Radiosensitization by GSH depletion was maximal at intermediate oxygen tensions of 10-21% O{sub 2} in the ambient gas. Increasing the extent of GSH depletion led to increasing radiosensitization, with sensitization enhancement ratios of 1.2 and 1.1, respectively, for extensive and intermediated levels of GSH depletion. In mice exposed to 100% O{sub 2}, a significant component of skin radiosensitivity was due to diffusion of oxygen directly through the skin. Pentobarbitone anesthesia radiosensitized skin in mice exposed to 100% O{sub 2} by a factor of 1.2, but did not further sensitize skin in mice exposed to carbogen. Glutathione levels and the local oxygen tension at the time of irradiation were important determinants of mouse foot skin radiosensitivity. The extent to which GSH levels altered the radiosensitivity of skin was critically dependent on the local oxygen tension. These results have significant implications for potential clinical applications of GSH depletion. 53 refs., 7 figs., 2 tabs.

  12. 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

  13. Radiosensitization of E. coli B/r by arylhydrazonopropanedinitriles.

    PubMed

    Crump, P W; Bisby, R H; Cundall, R B; Thomas, E W

    1989-04-01

    Several arylhydrazonopropanedinitriles and an arylhydrazonopropane-diethyl ester (derivatives of well-known uncouplers of oxidative phosphorylation) have been studied with respect to their ability to radiosensitize E. coli B/r under oxic and hypoxic conditions. Of the compounds studied, 2-carboxyphenylhydrazono-propanedinitrile and 2-carboxyphenylhydrazonopropanediethylester were found to be the most efficient radiosensitizers under hypoxia, whilst the former compound was also found to provide radiosensitization under oxic conditions. Increased radiosensitization by 4-carboxyphenylhydrazonopropanedinitrile was observed on decreasing the pH of the irradiation incubation medium. The results are discussed with respect to the physicochemical properties of these compounds and their reactivity with thiols, for which data are presented. PMID:2564869

  14. Benzamide and nicotinamide radiosensitizers

    SciTech Connect

    Lee, W.W.; Brown, J.M.; Grange, E.W.; Martinez, A.P.

    1993-06-01

    A method to destroy hypoxic tumor cells in a warmblooded animal is described, which method comprises: (a) administering to said warmblooded animal a compound of the formula: wherein X is O or S; Z is H, OR, SR or NHR in which R is H, hydrocarbyl (1-6C) including cyclic and unsaturated hydrocarbyl, optionally substituted with 1 or 2 substituents selected from the group consisting of halo, hydroxy, epoxy, alkoxy, alkylthio, amino including morpholino, acyloxy and acylamido and their thio analogs, alkylsulfonyl or alkylphosphonyl, carboxy or alkoxycarbonyl, or carbamyl or alkylcarbamyl, and in which R can optionally be interrupted by a single ether (-O-) linkage; or Z os O(CO)R, NH(CO)R, O(SO)R, or O(POR)R in which R is as above defined, in an amount effective to radiosensitize said hypoxic tumor cells, (b) followed by, after a time period to provide maximum radiosensitization, irradiating said tumor cells with a dose of radiation effective to destroy said cells.

  15. Factors Affecting the Radiosensitivity of Hexaploid Wheat to -Irradiation: Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.).

    PubMed

    Han, Bing; Gu, Jiayu; 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

  16. Interfaces with Tunable Mechanical and Radiosensitizing Properties.

    PubMed

    Berg, Nora G; Pearce, Brady L; Snyder, Patrick J; Rohrbaugh, Nathaniel; Nolan, Michael W; Adhikari, Prajesh; Khan, Saad A; Ivanisevic, Albena

    2016-08-31

    We report the fabrication of a composite containing nanostructured GaOOH and Matrigel with tunable radiosensitizing and stiffness properties. Composite characterization was done with microscopy and rheology. The utility of the interface was tested in vitro using fibroblasts. Cell viability and reactive oxygen species assays quantified the effects of radiation dosages and GaOOH concentrations. Fibroblasts' viability decreased with increasing concentration of GaOOH and composite stiffness. During ionizing radiation experiments the presence of the scintillating GaOOH triggered a different cellular response. Reactive oxygen species data demonstrated that one can reduce the amount of radiation needed to modulate the behavior of cells on interfaces with different stiffness containing a radiosensitizing material. PMID:26882455

  17. Irinotecan and radiosensitization in rectal cancer.

    PubMed

    Illum, Henrik

    2011-04-01

    Neoadjuvant radiation therapy with concurrent 5-fluorouracil-based chemotherapy is currently considered the standard of care for locally advanced rectal cancer. Pathologically complete response is a desirable outcome and has been associated with increased disease-free survival. There is a need to improve on this approach given that only approximately 10% achieve a pathologically complete response. Irinotecan has an established role in the treatment of metastatic rectal cancer. Both in-vitro and in-vivo data have shown promising radiosensitization properties. This study provides an overview of the published clinical trials evaluating the role of irinotecan as a radiosensitizer in the management of locally advanced rectal cancer. Although early-phase clinical trials initially showed promising results, this did not translate into improved outcome in a larger randomized phase II trial. Increased topoisomerase I expression has recently been identified as a possible predictive marker for improved response to irinotecan-based radiosensitization. This finding could help identify a subset of patients more likely to benefit from the addition of irinotecan in future trials. PMID:21160419

  18. 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. PMID:27315162

  19. 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. PMID:27315161

  20. 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

  1. A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation

    PubMed Central

    Fujimori, Hiroaki; Sato, Akira; Kikuhara, Sota; Wang, Junhui; Hirai, Takahisa; Sasaki, Yuka; Murakami, Yasufumi; Okayasu, Ryuichi; Masutani, Mitsuko

    2015-01-01

    A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1β in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1β complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1β foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses. PMID:26667181

  2. A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation.

    PubMed

    Fujimori, Hiroaki; Sato, Akira; Kikuhara, Sota; Wang, Junhui; Hirai, Takahisa; Sasaki, Yuka; Murakami, Yasufumi; Okayasu, Ryuichi; Masutani, Mitsuko

    2015-01-01

    A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1β-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1β in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1β complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1β foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses. PMID:26667181

  3. 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

  4. Radiosensitizing Properties of Bortezomib Depend on Therapeutic Schedule

    SciTech Connect

    Labussiere, Marianne; Pinel, Sophie; Vandamme, Marc; Plenat, Francois; Chastagner, Pascal

    2011-03-01

    Purpose: To investigate the influence of the bortezomib (BTZ) on malignant glioma radiosensitivity in two xenograft models. Methods and Materials: For TCG3 and U87 models, we evaluated the antitumor activity of BTZ, radiotherapy, and BTZ plus radiothearapy according to two therapeutic schedules: a 'nonfractionated' schedule corresponding to a single dose of treatment per week, and a 'fractionated' schedule corresponding to the same weekly dose divided into 5 fractions. Treatments influence on proliferation and apoptosis indexes, cell cycle distribution, and nuclear factor-{kappa}B pathway were explored. Results: The radiosensitizing properties of BTZ observed with the nonfractionated schedule were lost with the fractionated schedule. Bortezomib-mediated radiosensitization was associated with an increased apoptosis response and major changes in cell proliferation, but the nuclear factor-{kappa}B pathway was not involved. Most of the cellular effects induced by BTZ when tumors received a single irradiation were cancelled out if radiotherapy was fractionated. Conclusion: The influence of BTZ on glioma radiosensitivity seems to depend on the treatment fractionation schedule, emphasizing the need to clarify the mechanisms underlying BTZ's radiosensitizing effects before further clinical trials are initiated.

  5. The Search for New Hypoxic Cell Radiosensitizers

    PubMed Central

    Mansfield, Carl M.; Kimler, Bruce F.; Cheng, C.C.; Abrahams, Iris L.; Podrebarac, Eugene G.; Wittek, Philip J.; Reddy, Eashwer K.

    1980-01-01

    A number of newly synthesized compounds whose chemical structure suggested possible or remotely possible ability to radiosensitize hypoxic mammalian cells were studied in an in-vitro system. Those compounds that were not excluded because of insolubility or extreme cytotoxicity were tested for radiosensitizing ability. The correlation between chemical structure and radiosensitizing ability will be used for the rational design of additional compounds with a high probability of being effective hypoxic cell radiosensitizers. It is hoped that this will contribute to attempts to improve the cure rate of patients with malignant tumors through the use of radiation therapy and hypoxic cell radiosensitizers. PMID:7401185

  6. 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.

  7. G9a/RelB regulates self-renewal and function of colon-cancer-initiating cells by silencing Let-7b and activating the K-RAS/β-catenin pathway.

    PubMed

    Cha, Shih-Ting; Tan, Ching-Ting; Chang, Cheng-Chi; Chu, Chia-Yu; Lee, Wei-Jiunn; Lin, Been-Zen; Lin, Ming-Tsan; Kuo, Min-Liang

    2016-09-01

    Epigenetic reprogramming has been associated with the functional plasticity of cancer-initiating cells (CICs); however, the regulatory pathway has yet to be elucidated. A siRNA screen targeting known epigenetic genes revealed that G9a profoundly impairs the chemo-resistance, self-renewal and metastasis of CICs obtained from patients with colorectal cancer (CRC). Patients with elevated G9a were shown to face a high risk of relapse and poor survival rates. From a mechanistic perspective, G9a binds with and stabilizes RelB, thereby recruiting DNA methyltransferase 3 on the Let-7b promoter and repressing its expression. This leads to the activation of the K-RAS/β-catenin pathway and regulates self-renewal and function of CICs. These findings indicate that the G9a/RelB/Let-7b axis acts as a critical regulator in the maintenance of CIC phenotypes and is strongly associated with negative clinical outcomes. Thus, these findings may have diagnostic as well as therapeutic implications for the treatment of chemotherapy-resistant or metastatic CRC. PMID:27525719

  8. Targeted radiosensitization of cells expressing truncated DNA polymerase {beta}.

    PubMed

    Neijenhuis, Sari; Verwijs-Janssen, Manon; van den Broek, Lenie J; Begg, Adrian C; Vens, Conchita

    2010-11-01

    Ionizing radiation (IR) is an effective anticancer treatment, although failures still occur. To improve radiotherapy, tumor-targeted strategies are needed to increase radiosensitivity of tumor cells, without influencing normal tissue radiosensitivity. Base excision repair (BER) and single-strand break repair (SSBR) contribute to the determination of sensitivity to IR. A crucial protein in BER/SSBR is DNA polymerase β (polβ). Aberrant polβ expression is commonly found in human tumors and leads to inhibition of BER. Here, we show that truncated polβ variant (polβ-Δ)-expressing cells depend on homologous recombination (HR) for survival after IR, indicating that a considerable fraction of polβ-Δ-induced lesions are subject to repair by HR. Increased sensitization was found not to result from involvement in DNA-dependent protein kinase-dependent nonhomologous end joining, the other major double-strand break repair pathway. Caffeine and the ATM inhibitor Ku55933 cause polβ-Δ-dependent radiosensitization. Consistent with the observed HR dependence and the known HR-modulating activity of ATM, polβ-Δ-expressing cells showed increased radiosensitization after BRCA2 knockdown that is absent under ATM-inhibited conditions. Our data suggest that treatment with HR modulators is a promising therapeutic strategy for exploiting defects in the BER/SSBR pathway in human tumors. PMID:20978197

  9. 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.

  10. Cyclopentenylcytosine does not enhance cisplatin-induced radiosensitization in human lung tumour cells

    PubMed Central

    RODERMOND, HANS M.; CATE, ROSEMARIE TEN; HAVEMAN, JAAP; VAN KUILENBURG, ANDRÉ; MEDEMA, JAN PAUL; VAN BREE, CHRIS; FRANKEN, NICOLAAS A.P.

    2010-01-01

    The search for agents that enhance the effect of ionizing radiation has been an object of study for decades. In this study, the sensitizing properties of cyclopentenylcytosine (CPEC) on radiation and cisplatin-induced radiosensitization in human squamous lung carcinoma cells were investigated. Human lung tumour SW-1573 cells (SWp, parental; SWg, gemcitabine-resistant) were incubated with CPEC and cisplatin and subsequently irradiated with different doses of γ-rays. Clonogenic survival was determined to measure the effectiveness of the treatments. CPEC (1 or 2 μM) treatment for 4 h decreased the plating efficiency to 75 and 50% in SWp and SWg cells, respectively. In the SWg cells, 0.1 and 1 μM CPEC for 4 h enhanced the cell killing effect of cisplatin. However, an increase was not noted in the SWp cells. Due to the moderate toxicity of 1 μM for 4 h, this CPEC dose was used in the radiosensitization experiments. However, CPEC neither radiosensitized the lung tumour cells nor enhanced the radiosensitizing effect of cisplatin. A 2-h incubation with 4 μM cisplatin also decreased the plating efficiency to 75–80% in the two cell lines. Using this cisplatin dose, radiosensitization was obtained in the two cell lines. Although cisplatin treatment clearly radiosensitized the lung tumour cells, CPEC treatment did not. Cisplatin-induced radiosensitization was also not enhanced by CPEC. PMID:22966339

  11. 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

  12. Heat inactivation of Ku autoantigen: possible role in hyperthermic radiosensitization.

    PubMed

    Burgman, P; Ouyang, H; Peterson, S; Chen, D J; Li, G C

    1997-07-15

    Heat shock prior, during, or immediately after ionizing radiation synergistically increases cell killing, a phenomenon termed hyperthermic radiosensitization. Recently, we have shown a constitutive DNA-binding factor in rodent cells that is inactivated by heat shock to be identical to Ku autoantigen. Ku, consisting of an Mr 70,000 (Ku70) and an Mr 86,000 (Ku80) subunit, is a heterodimeric nuclear protein and is the DNA-binding regulatory component of the mammalian DNA-dependent protein kinase DNA-PK. Recent genetic and biochemical studies indicate the involvement of Ku and DNA-PK in DNA double-strand break repair and V(D)J recombination. On the basis of these findings, we propose that heat-induced loss of the DNA-binding activity of Ku may lead to hyperthermic radiosensitization. To test this hypothesis, we examined and compared the DNA-binding activity of Ku, the DNA-PK kinase activity, and hyperthermic radiosensitization in rodent cells immediately after heat shock and during post-heat shock recovery at 37 degrees C. Our results show that the heat-induced loss of Ku-DNA binding activity correlates well with an increased radiosensitivity of the heat-shocked cells, and furthermore, the loss of synergistic interaction between heat and radiation parallels the recovery of the DNA-binding activity of Ku. On the other hand, the heat-induced decrease of DNA-PK activity did not correlate with hyperthermic radiosensitization. Our data, for the first time, provide evidence for a role of Ku protein in modulating the cellular response to combined treatments of heat shock and ionizing radiation. PMID:9230187

  13. Base excision repair of both uracil and oxidatively damaged bases contribute to thymidine deprivation-induced radiosensitization

    SciTech Connect

    Allen, Bryan G.; Johnson, Monika; Marsh, Anne E.; Dornfeld, Kenneth J. . E-mail: kenneth-dornfeld@uiowa.edu

    2006-08-01

    Purpose: Increased cellular sensitivity to ionizing radiation due to thymidine depletion is the basis of radiosensitization with fluoropyrimidine and methotrexate. The mechanism responsible for cytotoxicity has not been fully elucidated but appears to involve both the introduction of uracil into, and its removal from, DNA. The role of base excision repair of uracil and oxidatively damaged bases in creating the increased radiosensitization during thymidine depletion is examined. Methods and Materials: Isogenic strains of S. cerevisiae differing only at loci involved in DNA repair functions were exposed to aminopterin and sulfanilamide to induce thymidine deprivation. Cultures were irradiated and survival determined by clonogenic survival assay. Results: Strains lacking uracil base excision repair (BER) activities demonstrated less radiosensitization than the parental strain. Mutant strains continued to show partial radiosensitization with aminopterin treatment. Mutants deficient in BER of both uracil and oxidatively damaged bases did not demonstrate radiosensitization. A recombination deficient rad52 mutant strain was markedly sensitive to radiation; addition of aminopterin increased radiosensitivity only slightly. Radiosensitization observed in rad52 mutants was also abolished by deletion of the APN1, NTG1, and NTG2 genes. Conclusion: These data suggest radiosensitization during thymidine depletion is the result of BER activities directed at both uracil and oxidatively damaged bases.

  14. Radiosensitizing Effect of TRPV1 Channel Inhibitors in Cancer Cells.

    PubMed

    Nishino, Keisuke; Tanamachi, Keisuke; Nakanishi, Yuto; Ide, Shunta; Kojima, Shuji; Tanuma, Sei-Ichi; Tsukimoto, Mitsutoshi

    2016-07-01

    Radiosensitizers are used in cancer therapy to increase the γ-irradiation susceptibility of cancer cells, including radioresistant hypoxic cancer cells within solid tumors, so that radiotherapy can be applied at doses sufficiently low to minimize damage to adjacent normal tissues. Radiation-induced DNA damage is repaired by multiple repair systems, and therefore these systems are potential targets for radiosensitizers. We recently reported that the transient receptor potential vanilloid type 1 (TRPV1) channel is involved in early responses to DNA damage after γ-irradiation of human lung adenocarcinoma A549 cells. Therefore, we hypothesized that TRPV1 channel inhibitors would have a radiosensitizing effect by blocking repair of radiation-induced cell damage. Here, we show that pretreatment of A549 cells with the TRPV1 channel inhibitors capsazepine, AMG9810, SB366791 and BCTC suppressed the γ-ray-induced activation of early DNA damage responses, i.e., activation of the protein kinase ataxia-telangiectasia mutated (ATM) and accumulation of p53-binding protein 1 (53BP1). Further, the decrease of survival fraction at one week after γ-irradiation (2.0 Gy) was enhanced by pretreatment of cells with these inhibitors. On the other hand, inhibitor pretreatment did not affect cell viability, the number of apoptotic or necrotic cells, or DNA synthesis at 24 h after irradiation. These results suggest that inhibition of DNA repair by TRPV1 channel inhibitors in irradiated A549 cells caused gradual loss of proliferative ability, rather than acute facilitation of apoptosis or necrosis. TRPV1 channel inhibitors could be novel candidates for radiosensitizers to improve the efficacy of radiation therapy, either alone or in combination with other types of radiosensitizers. PMID:27150432

  15. Andrographolide radiosensitizes human ovarian cancer SKOV3 xenografts due to an enhanced apoptosis and autophagy.

    PubMed

    Zhang, Chao; Qiu, Xingsheng

    2015-11-01

    Andrographolide (AND), a diterpenoid lactone isolated from Andrographis paniculata, has been shown to have radiosensitivity in several types of cancer. Whether AND can radiosensitize ovarian cancer remains unknown. The present study investigated the radiosensitizing effects of AND in human ovarian SKOV3 xenografts and examined the molecular mechanisms of AND-mediated radiosensitization. Nude mice bearing human ovarian SKOV3 were treated with AND to investigate the effects of drug administration on tumor growth, radiosensitivity, apoptosis, and autophagy. Subsequent Western blot analysis and monodansylcadaverine (MDC) staining (autophagy analysis) were used to determine the role of AND. Finally, the pathway of apoptosis was characterized by caspase-3 activity assay as well as TUNEL analysis. AND potently sensitized SKOV3 xenografts to radiation. Moreover, apoptosis and autophagy in radiation combined with drug-treated xenografts increased significantly compared with the simple drug or single radiation treatment. This result was associated with an increase in the Bax/Bcl-2 protein ratio and p-p53 expression after exposure to combination treatment. Meanwhile, the level of Beclin 1 and Atg5 and the conversion from LC3-I to LC3-II, three important proteins involved in autophagy, were increased. AND acts as a strong radiosensitizer in human ovarian SKOV3 xenografts in vivo by increasing the Bax/Bcl-2 protein ratio and promoting the activation of caspase-3, leading to enhanced apoptosis as well as autophagy. PMID:26014516

  16. 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.

  17. 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. PMID:26000387

  18. 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

  19. [Cisplatin influence on: the radiosensitivity and recovery of yeast cells].

    PubMed

    Evstratova, E S; Petin, V G

    2013-01-01

    The effect of the simultaneous combined action of ionizing radiation and cisplatin on the radiosensitivity and liquid holding recovery (LHR) of diploid yeast cells was studied. It was shown that regardless of the cisplatin concentration (0; 0.002; 0.01; 0.02 g/ml) the radiosensitivity of cells was increased by 1.3 times. The ability of a cell to the LHR was progressively decreased with the increasing cisplatin concentration up to the complete inhibition. It was shown that the LHR of yeast cells after a combined action of ionizing radiation and chemical agents is mainly inhibited due to formation of a greater proportion of irreversible damage. The constant of recovery, characterizing the probability of recovery per a unit of time, was independent on cisplatine concentration. PMID:25486742

  20. [Cisplatin influence on: the radiosensitivity and recovery of yeast cells].

    PubMed

    2013-01-01

    The effect of the simultaneous combined action of ionizing radiation and cisplatin on the radiosensitivity and liquid holding recovery (LHR) of diploid yeast cells was studied. It was shown that regardless of the cisplatin concentration (0; 0.002; 0.01; 0.02 g/ml) the radiosensitivity of cells was increased by 1.3 times. The ability of a cell to the LHR was progressively decreased with the increasing cisplatin concentration up to the complete inhibition. It was shown that the LHR of yeast cells after a combined action of ionizing radiation and chemical agents is mainly inhibited due to formation of a greater proportion of irreversible damage. The con- stant of recovery, characterizing the probability of recovery per a unit of time, was independent on cisplatine concentration. PMID:25508873

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. Silibinin Preferentially Radiosensitizes Prostate Cancer by Inhibiting DNA Repair Signaling.

    PubMed

    Nambiar, Dhanya K; Rajamani, Paulraj; Deep, Gagan; Jain, Anil K; Agarwal, Rajesh; Singh, Rana P

    2015-12-01

    Radiotherapy, a frequent mode of cancer treatment, is often restricted by dose-related toxicity and development of therapeutic resistance. To develop a novel and selective radiosensitizer, we studied the radiosensitizing effects and associated mechanisms of silibinin in prostate cancer. The radiosensitizing effect of silibinin with ionizing radiation (IR) was assessed on radioresistant prostate cancer cell lines by clonogenic, cell cycle, cell death, and DNA repair assays. Tumor xenograft growth, immunohistochemical (IHC) analysis of tumor tissues, and toxicity-related parameters were measured in vivo. Silibinin (25 μmol/L) enhanced IR (2.5-10 Gy)-caused inhibition (up to 96%, P < 0.001) of colony formation selectively in prostate cancer cells, and prolonged and enhanced IR-caused G2-M arrest, apoptosis, and ROS production. Mechanistically, silibinin inhibited IR-induced DNA repair (ATM and Chk1/2) and EGFR signaling and attenuated the levels of antiapoptotic proteins. Specifically, silibinin suppressed IR-induced nuclear translocation of EGFR and DNA-PK, an important mediator of DSB repair, leading to an increased number of γ-H2AX (ser139) foci suggesting lesser DNA repair. In vivo, silibinin strongly radiosensitized DU145 tumor xenograft inhibition (84%, P < 0.01) with higher apoptotic response (10-fold, P < 0.01) and reduced repair of DNA damage, and rescued the mice from IR-induced toxicity and hematopoietic injury. Overall, silibinin enhanced the radiotherapeutic response via suppressing IR-induced prosurvival signaling and DSB repair by inhibiting nuclear translocation of EGFR and DNA-PK. Because silibinin is already in phase II clinical trial for prostate cancer patients, the present finding has translational relevance for radioresistant prostate cancer. PMID:26516160

  6. 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

  7. 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.

  8. Dependence of Gold Nanoparticle Radiosensitization on Functionalizing Layer Thickness.

    PubMed

    Spaas, Cedric; Dok, Rüveyda; Deschaume, Olivier; De Roo, Bert; Vervaele, Mattias; Seo, Jin Won; Bartic, Carmen; Hoet, Peter; Van den Heuvel, Frank; Nuyts, Sandra; Locquet, Jean-Pierre

    2016-04-01

    Gold nanoparticles functionalized with polyethylene glycol of different chain lengths are used to determine the influence of the capping layer thickness on the radiosensitizing effect of the particles. The size variations in organic coating, built up with polyethylene glycol polymers of molecular weight 1-20 kDa, allow an evaluation of the decrease in dose enhancement percentages caused by the gold nanoparticles at different radial distances from their surface. With localized eradication of malignant cells as a primary focus, radiosensitization is most effective after internalization in the nucleus. For this reason, we performed controlled radiation experiments, with doses up to 20 Gy and particle diameters in a range of 5-30 nm, and studied the relaxation pattern of supercoiled DNA. Subsequent gel electrophoresis of the suspensions was performed to evaluate the molecular damage and consecutively quantify the gold nanoparticle sensitization. In conclusion, on average up to 58.4% of the radiosensitizing efficiency was lost when the radial dimensions of the functionalizing layer were increased from 4.1 to 15.3 nm. These results serve as an experimental supplement for biophysical simulations and demonstrate the influence of an important parameter in the development of nanomaterials for targeted therapies in cancer radiotherapy. PMID:26950059

  9. Cellular and Molecular Mechanisms Underlying Oxygen-Dependent Radiosensitivity

    PubMed Central

    Liu, Chao; Lin, Qun; Yun, Zhong

    2015-01-01

    Molecular oxygen has long been recognized as a powerful radiosensitizer that enhances the cell-killing efficiency of ionizing radiation. Radiosensitization by oxygen occurs at very low concentrations with the half-maximum radiosensitization at approximately 3 mmHg. However, robust hypoxia-induced signal transduction can be induced at <15 mmHg and can elicit a wide range of cellular responses that will affect therapy response as well as malignant progression. Great strides have been made, especially since the 1990s, toward identification and characterization of the oxygen-regulated molecular pathways that affect tumor response to ionizing radiation. In this review, we will discuss the current advances in our understanding of oxygen-dependent molecular modification and cellular signal transduction and their impact on tumor response to therapy. We will specifically address mechanistic distinctions between radiobiological hypoxia (0–3 mmHg) and pathological hypoxia (3–15 mmHg). We also propose a paradigm that hypoxia increases radioresistance by maintaining the cancer stem cell phenotype. PMID:25938770

  10. 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.

  11. 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.

  12. Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells.

    PubMed

    Hirai, Takahisa; Saito, Soichiro; Fujimori, Hiroaki; Matsushita, Keiichiro; Nishio, Teiji; Okayasu, Ryuichi; Masutani, Mitsuko

    2016-09-01

    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. PMID:27425251

  13. On the Path to Seeking Novel Radiosensitizers

    SciTech Connect

    Katz, David; Ito, Emma; Liu Feifei

    2009-03-15

    Radiation therapy is a highly effective cancer treatment modality, and extensive investigations have been undertaken over the years to augment its efficacy in the clinic. This review summarizes the current understanding of the biologic bases underpinning many of the clinically used radiosensitizers. In addition, this review illustrates how the advent of innovative, high-throughput technologies with integration of different disciplines could be harnessed for an expeditious discovery process for novel radiosensitizers, providing an exciting future for such pursuits in radiation biology and oncology.

  14. 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.

  15. 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.

  16. Tumor radiosensitization by monomethyl auristatin E: mechanism of action and targeted delivery

    PubMed Central

    Crisp, Jessica L.; Jones, Karra A.; Hicks, Angel M.; Scanderbeg, Daniel J.; Nguyen, Quyen T.; Sicklick, Jason K.; Lowy, Andrew M.; Tsien, Roger Y.; Advani, Sunil J.

    2015-01-01

    Intrinsic tumor resistance to radiotherapy limits the efficacy of ionizing radiation (IR). Sensitizing cancer cells specifically to IR would improve tumor control and decrease normal tissue toxicity. The development of tumor targeting technologies allows for developing potent radiosensitizing drugs. We hypothesized that the anti-tubulin agent monomethyl auristatin E (MMAE), a component of a clinically approved antibody-directed conjugate, could function as a potent radiosensitizer and be selectively delivered to tumors using an activatable cell penetrating peptide targeting matrix metalloproteinases and RGD binding integrins (ACPP-cRGD-MMAE). We evaluated the ability of MMAE to radiosensitize both established cancer cells and a low passage cultured human pancreatic tumor cell line using clonogenic and DNA damage assays. MMAE sensitized colorectal and pancreatic cancer cells to IR in a schedule and dose dependent manner correlating with mitotic arrest. Radiosensitization was evidenced by decreased clonogenic survival and increased DNA double strand breaks in irradiated cells treated with MMAE. MMAE in combination with IR resulted in increased DNA damage signaling and activation of CHK1. To test a therapeutic strategy of MMAE and IR, PANC-1 or HCT-116 murine tumor xenografts were treated with non-targeted free MMAE or tumor targeted MMAE (ACPP-cRGD-MMAE). While free MMAE in combination with IR resulted in tumor growth delay, tumor targeted ACPP-cRGD-MMAE with IR produced a more robust and significantly prolonged tumor regression in xenograft models. Our studies identify MMAE as a potent radiosensitizer. Importantly, MMAE radiosensitization can be localized to tumors by targeted activatable cell penetrating peptides. PMID:25681274

  17. Radiosensitized treatment of malignant brain tumors

    NASA Astrophysics Data System (ADS)

    Bloznelyte-Plesniene, Laima

    2003-12-01

    Around 12,000 deaths from glioblastoma occurs within the European Community annually. At present, the best available treatment for malignant brain tumors results in a median survival of patients of 15 months despite surgery, radiotherapy, and chemotherapy. The purpose of this paper is to review our results of radiosensitized treatment of malignant brain tumors.

  18. 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

  19. Dependence of 5-fluorouracil-mediated radiosensitization on DNA-directed effects

    SciTech Connect

    Lawrence, T.S.; Davis, M.A.; Maybaum, J. )

    1994-06-15

    Although 5-fluorouracil (FUra) has been demonstrated to be a radiation sensitizer both in the laboratory and the clinic, it is not known whether radiosensitization results primary from FUra's DNA or RNA-directed effects. The authors studied the radiosensitizing effects of FUra [+-] thymidine (dThd) on HT29 human colon cancer cells, which are relatively sensitive to the DNA-directed action of FUra, in comparison to SW620 and HuTu80 human colon cancer cells, which are relatively resistant to FUra's DNA-directed effects. They hypothesized that if FUra were acting chiefly through DNA dependent mechanisms, HT29 cells would (a) show greater radiosensitization than SW620 and HuTu80 cells under the same conditions of exposure; and (b) demonstrate selective reversal of radiation sensitivity (compared to cytotoxicity) in the presence of FUra + dThd, compared to FUra alone. They found that the enhancement ratio produced by a 24 h exposure to 10 [mu]M FUra was significantly greater in HT29 cells compared to SW620 and HuTu80 cells (enhancement ratios of 2.1 [+-] 0.1; 1.1 [+-] 0.1, and 1.3 [+-] 0.1, respectively). Furthermore, in HT29 cells, dThd blocked FUra-mediated radiosensitization to a greater extent than FUra-mediated cytotoxicity. Thus, the hypotheses were confirmed. These findings support the concept that the manipulation of FUra's DNA-dependent actions, for example, through modulators of thymidylate synthase (TS) activity, may increase radiosensitization in clinical trials in the treatment of gastrointestinal cancers. However, since resistance to the DNA-directed effects of fluoropyrimidines can result from mechanisms unrelated to TS inhibition, additional strategies will be required to potentiate fluoropyrimidine-mediated radiosensitization. 15 refs., 2 figs., 1 tab.

  20. ZnFe2O4 nanoparticles for potential application in radiosensitization

    NASA Astrophysics Data System (ADS)

    Hidayatullah, M.; Nurhasanah, I.; Budi, W. S.

    2016-03-01

    Radiosensitizer is a material that can increase the effects of radiation in radiotherapy application. Various materials with high effective atomic number have been developed as a radiosensitizer, such as metal, iron oxide and quantum dot. In this study, ZnFe2O4 nanoparticles are included in iron oxide class were synthesized by precipitation method from the solution of zinc nitrate and ferrite nitrate and followed by calcination at 700° C for 3 hours. The XRD pattern shows that most of the observed peaks can be indexed to the cubic phase of ZnFe2O4 with a lattice parameter of 8.424 Å. SEM image reveals that nanoparticles are the sphere-like shape with size in the range 84-107 nm. The ability of ZnFe2O4 nanoparticles as radiosensitizer was examined by loading those nanoparticles into Escherichia coli cell culture which irradiated with photon energy of 6 MV at a dose of 2 Gy. ZnFe2O4 nanoparticles showed ability to increase the absorbed dose by 0.5 to 1.0 cGy/g. In addition, the presence of 1 g/L ZnFe2O4 nanoparticles resulted in an increase radiation effect by 6.3% higher than if exposed to radiation only. These results indicated that ZnFe2O4 nanoparticles can be used as the radiosensitizer for increasing radiation effect in radiotherapy.

  1. The gangliosides as a possible molecular coupling factor between the proportion of radiosensitive cells in vitro and the metastatic potential in vivo within a human melanoma cell line.

    PubMed Central

    Thomas, C. P.; Buronfosse, A.; Portoukalian, J.; Fertil, B.

    1997-01-01

    With an experimental model of spontaneous lung metastases in immunosuppressed newborn rats, seven clones and variants with different metastatic potential and gangliosides expression were derived from a single parental human melanoma cell line M4Be. The cellular radiosensitivity of M4Be and its seven sublines was estimated using an in vitro colony assay. The total amount of gangliosides in M4Be and its seven sublines was determined by cell extraction and thin-layer chromatography, while the expression of GD3 gangliosides was estimated by flow cytometry with a monoclonal antibody. The radiation-cell survival curves of most clones and variants derived from M4Be showed a zero dose extrapolation clearly lower than 100%, suggesting that two populations of cells of very different radiosensitivity coexist within each of these clones and variants. Although the proportion of radiosensitive cells could be estimated from the shape of the survival curve, its radiosensitivity is too high to be properly evaluated by the colony assay. The eight survival curves differ essentially in the proportion of radiosensitive cells--which varied from 0% to 40% among M4Be and its seven sublines--whereas the cellular radiosensitivity of the radioresistant population was similar among them. The metastatic potential in vivo of M4Be and its seven sublines was not significantly related to the cellular radiosensitivity of their corresponding radioresistant population, but significantly increased with the fraction of radiosensitive cells. This relationship is valid only when the highly metastatic cells are cultured for no more than five passages in vitro as the fraction of radiosensitive cells is rapidly lost during subcultures. The relationship remains valid in vivo as metastatic melanoma-bearing newborn rats whole body irradiated with 20 cGy show no lung metastasis compared with controls. The radiosensitive cell fraction is inversely correlated with both the total ganglioside content (r = 0.84, P

  2. In vitro 3-dimensional tumor model for radiosensitivity of HPV positive OSCC cell lines

    PubMed Central

    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. PMID:26046692

  3. 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.

  4. 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.

  5. RRx-001, A novel dinitroazetidine radiosensitizer.

    PubMed

    Oronsky, Bryan; Scicinski, Jan; Ning, Shoucheng; Peehl, Donna; Oronsky, Arnold; Cabrales, Pedro; Bednarski, Mark; Knox, Susan

    2016-06-01

    The 'holy grail' in radiation oncology is to improve the outcome of radiation therapy (RT) with a radiosensitizer-a systemic chemical/biochemical agent that additively or synergistically sensitizes tumor cells to radiation in the absence of significant toxicity. Similar to the oxygen effect, in which DNA bases modified by reactive oxygen species prevent repair of the cellular radiation damage, these compounds in general magnify free radical formation, leading to the permanent "fixation" of the resultant chemical change in the DNA structure. The purpose of this review is to present the origin story of the radiosensitizer, RRx-001, which emerged from the aerospace industry. The activity of RRx-001 as a chemosensitizer in multiple tumor types and disease states including malaria, hemorrhagic shock and sickle cell anemia, are the subject of future reviews. PMID:26841903

  6. Seeking genetic signature of radiosensitivity - a novel method for data analysis in case of small sample sizes

    PubMed Central

    2014-01-01

    Background The identification of polymorphisms and/or genes responsible for an organism's radiosensitivity increases the knowledge about the cell cycle and the mechanism of the phenomena themselves, possibly providing the researchers with a better understanding of the process of carcinogenesis. Aim The aim of the study was to develop a data analysis strategy capable of discovering the genetic background of radiosensitivity in the case of small sample size studies. Results Among many indirect measures of radiosensitivity known, the level of radiation-induced chromosomal aberrations was used in the study. Mathematical modelling allowed the transformation of the yield-time curve of radiation-induced chromosomal aberrations into the exponential curve with limited number of parameters, while Gaussian mixture models applied to the distributions of these parameters provided the criteria for mouse strain classification. A detailed comparative analysis of genotypes between the obtained subpopulations of mice followed by functional validation provided a set of candidate polymorphisms that might be related to radiosensitivity. Among 1857 candidate relevant SNPs, that cluster in 28 genes, eight SNPs were detected nonsynonymous (nsSNP) on protein function. Two of them, rs48840878 (gene Msh3) and rs5144199 (gene Cc2d2a), were predicted as having increased probability of a deleterious effect. Additionally, rs48840878 is capable of disordering phosphorylation with 14 PKs. In silico analysis of candidate relevant SNP similarity score distribution among 60 CGD mouse strains allowed for the identification of SEA/GnJ and ZALENDE/EiJ mouse strains (95.26% and 86.53% genetic consistency respectively) as the most similar to radiosensitive subpopulation Conclusions A complete step-by-step strategy for seeking the genetic signature of radiosensitivity in the case of small sample size studies conducted on mouse models was proposed. It is shown that the strategy, which is a combination of

  7. 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

  8. 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.

  9. 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

  10. MicroRNA-218 Enhances the Radiosensitivity of Human Cervical Cancer via Promoting Radiation Induced Apoptosis

    PubMed Central

    Yuan, Wang; Xiaoyun, Han; Haifeng, Qiu; Jing, Li; Weixu, Hu; Ruofan, Dong; Jinjin, Yu; Zongji, Shen

    2014-01-01

    We previously reported frequent loss of microRNA-218 (miR-218) in cervical cancer, which was associated with tumor progression and poor prognosis. As microRNAs were found invovled in the regulation of radiosensitivity in various human cancers, we therefore aim to investigate the effects of miR-218 on radiosensitivity of cervical cancer in the present study. The clonogenic survival assay demonstrated that loss of miR-218 could predict radioresistance in the primary cervical cancer cells (R2=0.6516, P<0.001). In vitro, abundant miR-218 increased the radiosensitivity in cervical cancer cells (P<0.001 for HeLa, P=0.009 for SiHa, P=0.016 for C33A and P=0.01 for CaSki). Upregulation of miR-218 significantly enhanced the radiation-induced apoptosis, which was further enhanced by the combination of miR-218 overexpression and radiation In xenograft growth assay, combination of miR-218 overexpression and radiation notably induced cellular apoptosis and suppressed tumor growth. In conclusion, we demonstrated that miR-218 resensitized cervical cancer cells to radiation via promoting cellular apoptosis. Moreover, we proved that miR-218 as a potent predictor of radiosensitivity in cervical cancer, especially for those patients with loss of miR-218. PMID:24843318

  11. 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.

  12. 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

  13. 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

  14. Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers12

    PubMed Central

    Wei, Dongping; Zhang, Qiang; Schreiber, Jason S.; Parsels, Leslie A.; Abulwerdi, Fardokht A.; Kausar, Tasneem; Lawrence, Theodore S.; Sun, Yi; Nikolovska-Coleska, Zaneta; Morgan, Meredith A.

    2015-01-01

    In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells. PMID:25749177

  15. Targeting BRG1 chromatin remodeler via its bromodomain for enhanced tumor cell radiosensitivity in vitro and in vivo.

    PubMed

    Kwon, Su-Jung; Lee, Seul-Ki; Na, Juri; Lee, Shin-Ai; Lee, Han-Sae; Park, Ji-Hye; Chung, June-Key; Youn, Hyewon; Kwon, Jongbum

    2015-02-01

    Radiotherapy treats cancer by inducing DNA double-strand breaks (DSB) in tumor cells using ionizing radiation. However, DNA repair in tumor cells often leads to radioresistance and unsuccessful outcome. Inhibition of DNA repair by targeting repair proteins can increase radiosensitivity of tumor cells. The BRG1 chromatin remodeling enzyme assists DSB repair by stimulating γ-H2AX formation and BRG1 binding to acetylated histones at DSBs via bromodomain (BRD) is critical for this activity. Here, we show that ectopic expression of BRG1-BRD inhibited γ-H2AX and DSB repair after irradiation and increased the radiosensitivity in various human cancer cells, including HT29 colon cancer. Dimerization of BRG1-BRD, increasing its chromatin binding affinity, aggravated the defects in γ-H2AX and DSB repair and further enhanced the radiosensitivity. While little affecting the upstream ATM activation, BRG1-BRD in irradiated HT29 cells inhibited the recruitment of 53BP1 to damaged chromatin, the downstream event of γ-H2AX, and compromised the G2-M checkpoint and increased apoptosis. Importantly, in a xenograft mouse model, BRG1-BRD increased the radiosensitivity of HT29 tumors, which was further enhanced by dimerization. These data suggest that BRG1-BRD radiosensitizes tumor cells by a dominant negative activity against BRG1, which disrupts γ-H2AX and its downstream 53BP1 pathways, leading to inefficient DNA repair, G2-M checkpoint defect, and increased apoptosis. This work therefore identifies BRG1-BRD as a novel tumor radiosensitizer and its action mechanism, providing the first example of chromatin remodeler as a target for improving cancer radiotherapy. PMID:25504753

  16. DNMT (DNA methyltransferase) inhibitors radiosensitize human cancer cells by suppressing DNA repair activity

    PubMed Central

    2012-01-01

    Background Histone modifications and DNA methylation are two major factors in epigenetic phenomenon. Unlike the histone deacetylase inhibitors, which are known to exert radiosensitizing effects, there have only been a few studies thus far concerning the role of DNA methyltransferase (DNMT) inhibitors as radiosensitizers. The principal objective of this study was to evaluate the effects of DNMT inhibitors on the radiosensitivity of human cancer cell lines, and to elucidate the mechanisms relevant to that process. Methods A549 (lung cancer) and U373MG (glioblastoma) cells were exposed to radiation with or without six DNMT inhibitors (5-azacytidine, 5-aza-2'-deoxycytidine, zebularine, hydralazine, epigallocatechin gallate, and psammaplin A) for 18 hours prior to radiation, after which cell survival was evaluated via clonogenic assays. Cell cycle and apoptosis were analyzed via flow cytometry. Expressions of DNMT1, 3A/3B, and cleaved caspase-3 were detected via Western blotting. Expression of γH2AX, a marker of radiation-induced DNA double-strand break, was examined by immunocytochemistry. Results Pretreatment with psammaplin A, 5-aza-2'-deoxycytidine, and zebularine radiosensitized both A549 and U373MG cells. Pretreatment with psammaplin A increased the sub-G1 fraction of A549 cells, as compared to cells exposed to radiation alone. Prolongation of γH2AX expression was observed in the cells treated with DNMT inhibitors prior to radiation as compared with those treated by radiation alone. Conclusions Psammaplin A, 5-aza-2'-deoxycytidine, and zebularine induce radiosensitivity in both A549 and U373MG cell lines, and suggest that this effect might be associated with the inhibition of DNA repair. PMID:22429326

  17. 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.

  18. DNA-Dependent Protein Kinase As Molecular Target for Radiosensitization of Neuroblastoma Cells

    PubMed Central

    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. PMID:26716839

  19. 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

  20. 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.

  1. 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.

  2. 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

    2015-01-01

    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. PMID:26125914

  3. 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. PMID:25028150

  4. 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.

  5. Radiosensitivity of chromosomes in two successive mitotic cycles of human lymphocytes

    SciTech Connect

    Luchnik, N.V.; Poryadkova, N.A.

    1988-11-01

    A culture of human lymphocytes was irradiated with /gamma/-quanta in a dose of 0.5 Gy with different ratios of cells in first (M1) and second (M2) mitotic cycle and the frequency of aberrations induced at stage G2 was analyzed. With increase in interval of time between the start of culturing and irradiation, total yield of aberrations increased in a regular way. However, if the M1:M2 ratio is considered, then it turns out that in M2 chromosomes are /approximately/1.5 times more sensitive than in M1: within the limits of each cycle, radiosensitivity is constant and does not depend on its duration. It was established in accordance with data of other authors that 5-bromodeoxyuridine (5-BdU) increases radiosensitivity materially.

  6. 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. PMID:26608458

  7. 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.

  8. Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

    PubMed

    Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

    2016-01-01

    To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. PMID:25059546

  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. 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

  11. The High Radiosensitizing Efficiency of a Trace of Gadolinium-Based Nanoparticles in Tumors.

    PubMed

    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. Rational design of cancer-targeted BSA protein nanoparticles as radiosensitizer to overcome cancer radioresistance.

    PubMed

    Huang, Yanyu; Luo, Yi; Zheng, Wenjie; Chen, Tianfeng

    2014-01-01

    Radiotherapy displays curative potential for cervical cancer management, but radioresistance occurs during long-term therapy. To overcome this limitation, tumor-targeted nanotechnology has been proposed to enhance the radiosensitivity of solid tumors. Herein, we used biocompatible bovine serum albumin nanoparticles (BSANPs) as carriers of organic selenocompound (PSeD) with folate (FA) as the targeting ligand to fabricate a cancer-targeted nanosystem. The combination of PSeD and BSANPs endowed the nanosystem with higher light absorption and reactive oxygen species (ROS) generation owing to their properties of surface plasmon resonance (SPR) effect, heavy metal effect, high refractive index and nanoparticulate interfacial effect. The combined treatment drastically increased the ROS overproduction, VEGF/VEGFR2 inactivation and inhibition of XRCC-1-mediated repair of DNA damage, thus triggering G2/M phase arrest and apoptosis. Taken together, our findings demonstrate the utility of FA-BSANPs as a promising radiosensitizer to improve cancer radiotherapy. PMID:25314331

  13. 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.

  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. Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization.

    PubMed

    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

  16. Molecularly targeted radiosensitization chances towards gene aberration-due organ confined/regionally advanced prostate cancer radioresistance

    PubMed Central

    ALBERTI, C.

    2015-01-01

    Considering that the prostate cancer radioresistance occurs in a significant percentage – as 20–40% of prostate cancer (PCa) patients undergone external beam radiation therapy developing, within ten years, recurrent and more aggressive tumor – the resort to customized radiosensitizer measures, focusly targeting PCa radioresistance-linked individual molecular aberrations, can increase the successful outcomes of PCa radiotherapy. PMID:26188759

  17. 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.

  18. Metabolic potentiation of the radiosensitization of hypoxic bacterial cells afforded by nitroaromatic compounds.

    PubMed

    Anderson, R F; Patel, K B

    1983-03-01

    Prolonged preirradiation incubation of nitroaromatic radiosensitizers with Escherichia coli cells has been found to increase the degree of radiosensitization of the cells in anoxia. Studies with E. coli strains which differ in their nitroreductase activity indicate that the increase in sensitization arises from the action of metabolites produced by the nitroreductase system of the cell. The metabolites alone appear to decrease the extrapolation number of irradiated hypoxic cells and when combined with the parent compound give a biphasic survival curve. The combination of misonidazole (1 mmole dm-3) and its metabolites (1 mmole dm-3) gave initial and final enhancement ratios of 2.4 and 1.4, respectively. The final enhancement ratio is that expected for 1 mmole dm-3 misonidazole alone, whereas the initial enhancement ratio indicates that the metabolites potentiate the action of misonidazole. The preirradiation incubation effect is removed by dithiothreitol at concentrations which do not affect the radiosensitization level of the nitroaromatic sensitizer. This result indicates that the active metabolite probably depletes a certain amount of the free-thiol compounds inside the cell which assist in the repair of radiation-induced damage. PMID:6344127

  19. 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.

  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. Cellular and molecular mechanisms affecting tumour radiosensitivity : An in vitro study

    NASA Astrophysics Data System (ADS)

    Power, Olive Mary

    less in the radiosensitive cell lines at this dose and was directly related to the increased level of apoptosis observed in these cell lines. It is speculated that the induction of apoptosis occurs after cells have attempted to repair damage before the onset of mitosis, eliminating those cells with irreparable damage. Apoptosis at low doses were not significantly different to constitutive levels, suggesting a limited role of apoptosis in the low dose hypersensitivity response previously observed in some of the cell lines in this study. Radiosensitive cell lines also demonstrated reduced initial DNA damage, measured by CHEF, (1.3-1.7 versus 1.8-2.2 excluding mutant radiosensitive phenotypes) and DSB break repair (20-30% versus 15-20%). While the responses to radiation were expectedly varied it is speculated that radioresistance is associated with increased repair while radiosensitivity is associated with increased apoptosis.

  2. Physical basis and biological mechanisms of gold nanoparticle radiosensitization.

    PubMed

    Butterworth, Karl T; McMahon, Stephen J; Currell, Fred J; Prise, Kevin M

    2012-08-21

    The unique properties of nanomaterials, in particular gold nanoparticles (GNPs) have applications for a wide range of biomedical applications. GNPs have been proposed as novel radiosensitizing agents due to their strong photoelectric absorption coefficient. Experimental evidence supporting the application of GNPs as radiosensitizing agents has been provided from extensive in vitro investigation and a relatively limited number of in vivo studies. Whilst these studies provide experimental evidence for the use of GNPs in combination with ionising radiation, there is an apparent disparity between the observed experimental findings and the level of radiosensitization predicted by mass energy absorption and GNP concentration. This review summarises experimental findings and attempts to highlight potential underlying biological mechanisms of response in GNP radiosensitization. PMID:22767423

  3. 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.

  4. 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

  5. [Radioresistance parameters in head and neck cancers and methods to radiosensitize].

    PubMed

    Biau, J; Chautard, E; Miroir, J; Lapeyre, M

    2015-08-01

    Head and neck cancers have been widely studied concerning their sensitivity to radiation therapy. Several parameters affect tumour response to radiation therapy. Some parameters are linked to the tumour. Large or invasive tumours, localization, such as oral cavity or adenopathy, are factors of radioresistance. Others parameters are linked to the patients themselves. Tobacco intoxication during radiotherapy and a low hemoglobin level contribute to radioresistance. More recently, a positive human papilloma virus (HPV) status has been reported to positively affect radiosensitivity. Finally, other parameters are related to tumour biology. Hypoxia, intrinsic radiosensitivity of tumour cells, tumour differentiation and repopulation (provided by Ki-67 index or EGFR level) are components of radiosensitivity. Currently, concurrent chemoradiotherapy is one of the gold standard treatments to overcome clinical outcome of locally advanced head and neck cancer. This combination increases locoregional control and survival. Taxane-based induction chemotherapy can also be an alternative. Another validated approach is the association of radiotherapy with cetuximab (EGFR targeting) but only one randomized study has been published. Fractionation modifications, especially hyperfractionation, have given positive results on both tumour control and survival. Strategies targeting hypoxia improve locoregional control but have less clinical impact. PMID:26119219

  6. Radiosensitizing effect of zinc oxide and silica nanocomposites on cancer cells.

    PubMed

    Generalov, Roman; Kuan, Woo Boon; Chen, Wei; Kristensen, Solveig; Juzenas, Petras

    2015-05-01

    Nanoparticulates responsive to X-rays offer increased efficacy of radiation therapy. However, successful demonstrations of such nanoparticle use are limited so far due to lack of significant radiosensitizing effects or poor nanoparticle stability in a biological system. Zinc oxide (ZnO) is the most promising biocompatible material for medicinal applications. In this paper, we report preparation and characterization of scintillating ZnO/SiO2 core-shell nanoparticles. The ZnO/SiO2 nanoparticles absorb ultraviolet (UV) radiation (below 360nm) and emit green fluorescence (400-750nm, maximum 550nm). Under X-ray irradiation (200kVp), the nanoparticles scintillate emitting luminescence in the region 350-700nm (maximum 420nm). The synthesized ZnO/SiO2 nanoparticles are stable in a biologically relevant environment (water and cell growth medium). The potential of the ZnO/SiO2 nanoparticles for radiosensitization is demonstrated in human prostate adenocarcinoma cell lines (LNCaP and Du145). The nanoparticles enhance radiation-induced reduction in cell survival about 2-fold for LNCaP and 1.5-fold for Du145 cells. Radiosensitizing effect can be attributed to X-ray-induced radiocatalysis by the nanoparticles. PMID:25829130

  7. Radiosensitizing effect of medroxyprogesterone acetate on endometrial cancer cells in vitro

    SciTech Connect

    Huber, H.; Husslein, P.; Michalica, W.; Wagenbichler, P.

    1984-09-15

    From clinical experience it is known that medroxyprogesterone acetate (MPA) can increase the radiosensitivity of adenocarcinomas of the corpus uteri. This study investigates this phenomenon in vitro. Primary explants of highly differentiated adenocarcinomas were irradiated with or without pretreatment with MPA and compared with an untreated control group and to a group treated with MPA only. Cell culture itself was performed on an agarose medium in order to prevent overgrowth by fibroblasts. Untreated samples formed 43 +/- 5 clones, explants treated with MPA only produced 39 +/- 5 clones, a difference which was not statistically different; samples irradiated without pretreatment produced 16 +/- 8 and samples after combined treatment 9 +/- 3 clones (all values means +/- SD). This numeric reduction of cell growth through preirradiation treatment with MPA was statistically significant. The effect of MPA as a radiosensitizer may be due to its potential to prolong the radiosensitive G2 phase of the cell cycle. This effect of MPA may be useful also in other hormone-dependent tumors.

  8. Dimethoxycurcumin, a metabolically stable analogue of curcumin enhances the radiosensitivity of cancer cells: Possible involvement of ROS and thioredoxin reductase.

    PubMed

    Jayakumar, Sundarraj; Patwardhan, R S; Pal, Debojyoti; Sharma, Deepak; Sandur, Santosh K

    2016-09-01

    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. PMID:27381867

  9. Comment on 'Implications on clinical scenario of gold nanoparticle radiosensitization in regards to photon energy, nanoparticle size, concentration and location'

    NASA Astrophysics Data System (ADS)

    McMahon, Stephen J.; Prise, Kevin M.; Currell, Fred J.

    2012-01-01

    A recent paper by Lechtman et al (2011 Phys. Med. Biol. 56 4631-47) presented Monte Carlo modelling of gold nanoparticle dose modification. In it, they predict that the introduction of gold nanoparticles has the strongest effect with x-rays at kilovoltage energies, and that negligible increases in dose are expected at megavoltage energies. While these results are in agreement with others in the literature (including those produced by our group), the conclusion that '(gold nanoparticle) radiosensitization using a 6 MV photon source is not clinically feasible' appears to conflict with recently published experimental studies which have shown radiosensitization using 6 MV x-ray sources with relatively low gold concentrations. The increasing disparity between theoretical predictions of dose enhancement and experimental results in the field of gold nanoparticle radiosensitization suggests that, while the ability of gold nanoparticles to modify dose within a tumour volume is well understood, the resulting radiosensitization is not simply correlated with this measure. This highlights the need to validate theoretical predictions of this kind against experimental measurements, to ensure that the scenarios and values being modelled are meaningful within a therapeutic context.

  10. Comment on 'implications on clinical scenario of gold nanoparticle radiosensitization in regard to photon energy, nanoparticle size, concentration and location'.

    PubMed

    McMahon, Stephen J; Prise, Kevin M; Currell, Fred J

    2012-01-01

    A recent paper by Lechtman et al (2011 Phys. Med. Biol. 56 4631-47) presented Monte Carlo modelling of gold nanoparticle dose modification. In it, they predict that the introduction of gold nanoparticles has the strongest effect with x-rays at kilovoltage energies, and that negligible increases in dose are expected at megavoltage energies. While these results are in agreement with others in the literature (including those produced by our group), the conclusion that ‘(gold nanoparticle) radiosensitization using a 6 MV photon source is not clinically feasible’ appears to conflict with recently published experimental studies which have shown radiosensitization using 6 MV x-ray sources with relatively low gold concentrations. The increasing disparity between theoretical predictions of dose enhancement and experimental results in the field of gold nanoparticle radiosensitization suggests that, while the ability of gold nanoparticles to modify dose within a tumour volume is well understood, the resulting radiosensitization is not simply correlated with this measure. This highlights the need to validate theoretical predictions of this kind against experimental measurements, to ensure that the scenarios and values being modelled are meaningful within a therapeutic context. PMID:22156112