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Characterization of dihydroartemisinin-resistant colon carcinoma HCT116/R cell line.  


Dihydroartemisinin (DHA) is an important artemisinin derivative and presents profound anti-tumor potential. A DHA-resistant cell line named HCT116/R derived from colon carcinoma cell line HCT116 was established in our previous study. Herein, we found that HCT116/R cells were much more resistant to DHA- or artesunate-induced proliferation inhibition and more tolerant to DHA-induced cell cycle arrest and apoptosis compared with those of the parent HCT116 cells. The protein levels of P-glycoprotein and MDR-associated protein 1 and the accumulation of doxorubicin in cells were similar in both cell lines. Moreover, HCT116/R cells were still sensitive to camptothecin- and doxorubicin-induced cell growth inhibition. To further explore the characterization of HCT116/R cell line, a proteomic study employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed. Eight different expressed proteins between the two cell lines were identified including some heat shock proteins, annexins, etc. This study not only indicates that exposure to DHA may not induce a tumor multi-drug-resistant phenotype but also affords new clues for the further investigation of the anti-cancer mechanisms of DHA and other artemisinin derivatives. PMID:21959972

Lu, Jin-Jian; Chen, Si-Meng; Ding, Jian; Meng, Ling-Hua



Changes in Subcellular Localization of Visfatin in Human Colorectal HCT-116 Carcinoma cell Line After Cytochalasin-B Treatment  

PubMed Central

The aim of the study was to assess the expression and subcellular localization of visfatin in HCT-116 colorectal carcinoma cells after cytokinesis failure using Cytochalasin B (CytB) and the mechanism of apoptosis of cells after CytB. We observed translocation of visfatin’s antigen in cytB treated colorectal carcinoma HCT-116 cells from cytosol to nucleus. Statistical and morphometric analysis revealed significantly higher area-related numerical density visfatin-bound nano-golds in the nuclei of cytB-treated HCT-116 cells compared to cytosol. Reverse relation to visfatin subcellular localization was observed in un-treated HCT-116 cells. The total amount of visfatin protein and visfatin mRNA level in HCT-116 cells was also decreased after CytB treatment. Additionally, CytB significantly decreased cell survival, increased levels of G2/M fractions, induced bi-nuclei formation as well as increased reactive oxygen species (ROS) level in HCT-116 cells. CytB treatment showed cytotoxic effect that stem from oxidative stress and is connected with the changes in the cytoplasmic/nuclear amount of visfatin in HCT-116 cells.

Skonieczna, M.; Buldak, L; Matysiak, N.; Mielanczyk, L; Wyrobiec, G.; Kukla, M.; Michalski, M.; Zwirska-Korczala, K.



Urokinase-type plasminogen activator receptor regulates apoptotic sensitivity of colon cancer HCT116 cell line to TRAIL via JNK-p53 pathway.  


The urokinase-type plasminogen activator receptor (uPAR) serves not only as an anchor for urokinase-type plasminogen activator but also participates in intracellular signal transduction events. In this study, we investigated whether uPAR could modulate TRAIL-induced apoptosis in human colon cancer cells HCT116. Using an antisense strategy, we established a stable HCT116 cell line with down-regulated uPAR. The sensitivity to TRAIL-induced apoptosis was evaluated by FACS analysis. Our results show that the inhibition of uPAR could sensitize HCT116 to TRAIL-induced apoptosis. uPAR inhibition changed the expression of mitochondrial apoptotic pathway proteins, including Bcl-2, Bax, Bid and p53, in a pro-apoptotic manner. We also found that the inhibition of uPAR down-regulated the phosphorylation of FAK, ERK and JNK. The inhibition of p53 by RNA interference rescued cells from enhanced apoptosis, thus indicating that p53 is critical for enhancing TRAIL-induced apoptosis. Furthermore, JNK, but not ERK, inhibition involved in the up-regulation of p53. JNK negatively regulated p53 protein level. Overall, our results show that uPAR inhibition can sensitize colon cancer cells HCT116 to TRAIL-induced apoptosis via active p53 and mitochondrial apoptotic pathways that JNK inhibition is involved. PMID:25113506

Liu, Xiufeng; Qiu, Fan; Liu, Zhipeng; Lan, Yan; Wang, Kai; Zhou, Ping-Kun; Wang, Yao; Hua, Zi-Chun



NCI60 Cancer Cell Line Panel Data and RNAi Analysis Help Identify EAF2 as a Modulator of Simvastatin and Lovastatin Response in HCT-116 Cells  

PubMed Central

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the pharmacological data available for all the NCI60 cell lines to classify simvastatin or lovastatin resistant and sensitive cell lines, respectively. Next, we performed whole-genome single marker case-control association tests for the lovastatin and simvastatin resistant and sensitive cells using their publicly available Affymetrix 125K SNP genomic data. The results were then evaluated using RNAi methodology. After correction of the p-values for multiple testing using False Discovery Rate, our results identified three genes (NRP1, COL13A1, MRPS31) and six genes (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) associated with resistance to simvastatin and lovastatin, respectively. Functional validation using RNAi confirmed that silencing of EAF2 expression modulated the response of HCT-116 colon cancer cells to both statins. In summary, we have successfully utilized the publicly available data on the NCI60 cell lines to perform whole-genome association studies for simvastatin and lovastatin. Our results indicated genes involved in the cellular response to these statins and siRNA studies confirmed the role of the EAF2 in response to these drugs in HCT-116 colon cancer cells. PMID:21483694

Savas, Sevtap; Azorsa, David O.; Jarjanazi, Hamdi; Ibrahim-Zada, Irada; Gonzales, Irma M.; Arora, Shilpi; Henderson, Meredith C.; Choi, Yun Hee; Briollais, Laurent; Ozcelik, Hilmi; Tuzmen, Sukru



Characteristics of apoptosis in HCT116 colon cancer cells induced by deoxycholic acid.  


Hydrophobic bile acids induce apoptosis in both colon cancer cells and hepatocytes. The mechanism by which colon cancer cells respond to bile acids is thought to be different from that of hepatocytes. Therefore, we investigated the characteristics of apoptosis in colon cancer cell line HCT116. Hydrophobic bile acids, i.e., deoxycholic acid (DCA), and chenodeoxycholic acid, induced apoptosis in HCT116 cells. Apoptotic indications were detectable at as early as 30 min and the extent increased in time- and concentration-dependent manners. SDS and a hydrophilic bile acid, cholic acid, did not induce apoptosis even at cytotoxic concentrations. Pretreatment with cycloheximide failed to inhibit apoptosis, suggesting that protein synthesis is not involved in the apoptotic response. Release of cytochrome c from mitochondria and activation of caspase-9 were detectable after 5 and 10 min, respectively, whereas remarkable activation of Bid was not detected. Ursodeoxycholic acid (UDCA) protected HCT116 cells from DCA-induced apoptosis but a preincubation period of > or =5 h was required. Nevertheless, UDCA did not inhibit cytochrome c release from mitochondria. Our results indicate that hydrophobic bile acids induce apoptosis in HCT116 cells by releasing cytochrome c from mitochondria via an undefined but specific mechanism, and that UDCA protects HCT116 cells by acting downstream of cytochrome c release. PMID:16091589

Yui, Satoko; Saeki, Tohru; Kanamoto, Ryuhei; Iwami, Kimikazu



Regulation of p53 stability and function in HCT116 colon cancer cells.  


We have used a lentiviral vector to stably express p53 at a physiological level in p53 knockout HCT116 cells. Cells transduced with wild type p53 responded to genotoxic stress by stabilizing p53 and expressing p53 target genes. The reconstituted cells underwent G(1) arrest or apoptosis appropriately depending on the type of stress, albeit less efficiently than parental wild type cells. Compared with cells expressing exogenous wild type p53, the apoptotic response to 5-fluorouracil (5FU) was >50% reduced in cells expressing S15A or S20A mutant p53, and even more reduced by combined mutation of serines 6, 9, 15, 20, 33, and 37 (N6A). Among a panel of p53 target genes tested by quantitative PCR, the gene showing the largest defect in induction by 5FU was BBC3 (PUMA), which was induced 4-fold by wild type p53 and 2-fold by the N6A mutant. Mutation of N-terminal phosphorylation sites did not prevent p53 stabilization by doxorubicin or 5FU. MDM2 silencing by RNA interference activated p53 target gene expression in normal fibroblasts but not in HCT116 cells, and exogenous p53 could be stabilized in HCT116 knockout cells despite combined mutation of p53 phosphorylation sites and silencing of MDM2 expression. The MDM2 feedback loop is thus defective, and other mechanisms must exist to regulate p53 stability and function in this widely used tumor cell line. PMID:14665630

Kaeser, Matthias D; Pebernard, Stephanie; Iggo, Richard D



Suppression of microRNA-31 increases sensitivity to 5-FU at an early stage, and affects cell migration and invasion in HCT-116 colon cancer cells  

PubMed Central

Background MicroRNAs (miRNAs) are endogenously expressed noncoding RNAs with important biological and pathological functions. Although several studies have shown that microRNA-31 (miR-31) is obviously up-regulated in colorectal cancer (CRC), there is no study on the functional roles of miR-31 in CRC. Methods Anti-miR™ miRNA 31 inhibitor (anti-miR-31) is a sequence-specific and chemically modified oligonucleotide to specifically target and knockdown miR-31 molecule. The effect of anti-miR-31 transfection was investigated by real-time PCR. HCT-116p53+/+ and HCT-116p53-/-colon cancer cells were treated by anti-miR-31 with or without 5-fluorouracil (5-FU), cell proliferation was determined by MTT assay; apoptosis was detected by DAPI staining; cell cycle was evaluated by flow cytometry; colony formation, migration and invasion assays were performed to investigate the effect of suppression of miR-31 on the cell lines. Results Real-time PCR results showed that anti-miR-31 was efficiently introduced into the cells and reduced miR-31 levels to 44.1% in HCT-116p53+/+ and 67.8% in HCT-116p53-/-cell line (p = 0.042 and 0.046). MTT results showed that anti-miR-31 alone had no effect on the proliferation of HCT-116p53+/+ or HCT-116p53-/-. However, when combined with 5-FU, anti-miR-31 inhibited the proliferation of the two cell lines as early as 24 h after exposure to 5-FU (p = 0.038 and 0.044). Suppression of miR-31 caused a reduction of the migratory cells by nearly 50% compared with the negative control in both HCT-116p53+/+ and HCT-116p53-/-(p = 0.040 and 0.001). The invasive ability of the cells were increased by 8-fold in HCT-116p53+/+ and 2-fold in HCT-116p53-/- (p = 0.045 and 0.009). Suppression of miR-31 had no effect on cell cycle and colony formation (p > 0.05). Conclusions Suppression of miR-31 increases sensitivity to 5-FU at an early stage, and affects cell migration and invasion in HCT-116 colon cancer cells. PMID:21062447



Induction of apoptosis by 7-piperazinethylchrysin in HCT-116 human colon cancer cells.  


The antitumor activity of 7-piperazinethylchrysin (7-PEC) was investigated in HCT-116 human colon cancer cells. MTT assay revealed that the IC50 of 7-PEC in HCT-116 cells was 1.5 µM after 72 h of treatment, much lower than that of chrysin (>100 µM). The data showed that 7-PEC was able to inhibit the growth of HCT-116 cells in a concentration- and time-dependent manner. Topical morphological changes of apoptotic body formation after 7-PEC treatment were observed by Hoechst 33258 staining. 7-PEC reduced mitochondrial membrane potential (??m) of cells in a concentration-dependent manner and increased the production of intracellular reactive oxygen species (ROS). After treatment with 7-PEC, a significant increase of Bax protein expression and decrease of Bcl-2 protein expression were observed at the same time. These events paralleled with activation of p53, caspase-3 and -9 and the release of cytochrome c (cyt?c), as well as poly(ADP-ribose) polymerase-1 (PARP1) cleavage and downregulation of p-Akt. However, the apoptosis induced by 7-PEC was blocked by Ac-DEVD-CHO, a caspase-3 inhibitor. These results demonstrate that 7-PEC-induced mitochondrial dysfunction in HCT-116 human colon cancer cells triggers events responsible for caspase-dependent apoptosis pathways, and the elevated ratio of Bax/Bcl-2 is likely involved in this effect. PMID:22948973

Ren, Jie; Cheng, Hong; Xin, Wen Qun; Chen, Xin; Hu, Kun



Cell specific apoptosis by RLX is mediated by NF?B in human colon carcinoma HCT-116 cells  

PubMed Central

Background Resistance to chemotherapy represents a major obstacle in correcting colorectal carcinomas (CRC). Inspite of recent advances in the treatment of metastatic disease, the prognosis of the patients remains poor. RLX, a vasicinone analogue has been reported to possess potent bronchodilator, anti-asthmatic and anti-inflammatory properties. However, its anti-cancer activity is unknown. Results Here, we report for the first time that RLX has anti-cancer property against panel of human cancer cell lines and most potent activity was found against HCT-116 cells with IC50 value of 12 ?M and have further investigated the involvement of NF?B and caspase-3 in RLX action in CRC apoptosis. Following RLX and BEZ-235 treatment in HCT-116, we observed significant down-regulation of NF?B (1 to 0.1 fold) and up-regulation of caspase-3 (1 to 2 fold) protein expressions. Additionally, morphological studies revealed membrane blebbing, cell shrinkage, chromatin condensation and finally apoptosis in HCT-116 cells. Conclusions Overall, these findings indicate that RLX is a potent small molecule which triggers apoptosis, and promising potential candidate to be a chemotherapeutic agent. PMID:25303828



Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells.  


The purine-derived analogs, roscovitine and purvalanol are selective synthetic inhibitors of cyclin-dependent kinases (CDKs) induced cell cycle arrest and lead to apoptotic cell death in various cancer cells. Although a number of studies investigated the molecular mechanism of each CDK inhibitor on apoptotic cell death mechanism with their therapeutic potential, their regulatory role on autophagy is not clarified yet. In this paper, our aim was to investigate molecular mechanism of CDK inhibitors on autophagy and apoptosis in wild type (wt) and Bax deficient HCT 116 cells. Exposure of HCT 116 wt and Bax(-/-) cells to roscovitine or purvalanol for 24h decreased cell viability in dose-dependent manner. However, Bax deficient HCT 116 cells were found more resistant against purvalanol treatment compared to wt cells. We also established that both CDK inhibitors induced apoptosis through activating mitochondria-mediated pathway in caspase-dependent manner regardless of Bax expression in HCT 116 colon cancer cells. Concomitantly, we determined that purvalanol was also effective on autophagy in HCT 116 colon cancer cells. Inhibition of autophagy by 3-MA treatment enhanced the purvalanol induced apoptotic cell death in HCT 116 Bax(-/-) cells. Our results revealed that mechanistic action of each CDK inhibitor on cell death mechanism differs. While purvalanol treatment activated apoptosis and autophagy in HCT 116 cells, roscovitine was only effective on caspase-dependent apoptotic pathway. Another important difference between two CDK inhibitors, although roscovitine treatment overcame Bax-mediated drug resistance in HCT 116 cells, purvalanol did not exert same effect. PMID:25088259

Coker-Gurkan, Ajda; Arisan, Elif Damla; Obakan, Pinar; Guvenir, Esin; Unsal, Narcin Palavan



Corosolic acid induces apoptotic cell death in HCT116 human colon cancer cells through a caspase-dependent pathway.  


Corosolic acid (CA), a pentacyclic triterpene isolated from Lagerstroemia speciosa L. (also known as Banaba), has been shown to exhibit anticancer properties in various cancer cell lines. However, the anticancer activity of CA on human colorectal cancer cells and the underlying mechanisms remain to be elucidated. In this study, we investigated the effects of CA on cell viability and apoptosis in HCT116 human colon cancer cells. CA dose-dependently inhibited the viability of HCT116 cells. The typical hallmarks of apoptosis, such as chromatin condensation, a sub-G1 peak and phosphatidylserine externalization were detected by Hoechst 33342 staining, flow cytometry and Annexin V staining following treatment with CA. Western blot analysis revealed that CA induced a decrease in the levels of procaspase-8, -9 and -3 and the cleavage of poly(ADP-ribose) polymerase (PARP). The apoptotic cell death induced by CA was accompanied by the activation of caspase-8, -9 and -3, which was completely abrogated by the pan-caspase inhibitor, z-VAD?FMK. Furthermore, CA upregulated the levels of pro-apoptotic proteins, such as Bax, Fas and FasL and downregulated the levels of anti-apoptotic proteins, such as Bcl-2 and survivin. Taken together, our data provide insight into the molecular mechanisms of CA-induced apoptosis in colorectal cancer (CRC), rendering this compound a potential anticancer agent for the treatment of CRC. PMID:24481288

Sung, Bokyung; Kang, Yong Jung; Kim, Dong Hwan; Hwang, Seong Yeon; Lee, Yujin; Kim, Minjeong; Yoon, Jeong-Hyun; Kim, Cheol Min; Chung, Hae Young; Kim, Nam Deuk



Knockdown of Slug by RNAi inhibits the proliferation and invasion of HCT116 colorectal cancer cells.  


Colorectal cancer is one of the most common alimentary malignancies. Slug has been shown to be an ideal target for cancer gene therapy by numerous studies due to its strong anti?apoptotic effect. The elevated expression of Slug is a frequent genetic abnormality observed in colorectal cancer. In the present study, a Slug short hairpin RNA (shRNA) expression vector that was able to efficiently inhibit the expression of Slug in HCT116 cells was prepared. Following transfection, the mRNA expression levels were detected by RT?PCR analysis. Western blotting detected a similar inhibition of the Slug protein levels in the cells transfected with the pGCsi?Slug plasmid. Downregulation of Slug resulted in a significant inhibition of cancer cell growth in vitro. Cell invasion and apoptosis were decreased concomitantly with the reduction in Slug protein expression. These results suggested that RNA interference (RNAi) was able to downregulate the Slug protein level in HCT116 cells and significantly inhibit tumor growth in vitro. These findings suggest that RNAi has therapeutic potential for the treatment of colorectal cancer, as well as other types of cancer, by targeting the overexpression of oncogenes, including Slug. PMID:23900394

Qian, Jiang; Liu, Hong; Chen, Wangsheng; Wen, Kunming; Lu, Weidong; Huang, Chun; Fu, Zhongxue



Transcriptional regulation of the legumain gene by p53 in HCT116 cells.  


Legumain (EC is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was found to be highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro and in vivo. In this study, we found a p53-binding site in intron 1 of the human legumain gene using computational analysis. To determine whether transcription of the legumain gene is regulated by p53, HCT116 cells were transfected with p53 siRNA and the effect of knockdown of p53 expression on legumain expression was examined. The results showed that expression levels of both legumain mRNA and protein were decreased in the siRNA-treated cells. Furthermore, enzyme activity of legumain was also increased by doxorubicin and its activity was reduced by knockdown of p53 in HCT116 cells. These results suggest that legumain expression and its enzyme activity are regulated by p53. PMID:23942113

Yamane, Takuya; Murao, Sato; Kato-Ose, Izumi; Kashima, Lisa; Yuguchi, Motoki; Kozuka, Miyuki; Takeuchi, Keisuke; Ogita, Hisakazu; Ohkubo, Iwao; Ariga, Hiroyoshi



Synthetic Genistein Glycosides Inhibiting EGFR Phosphorylation Enhance the Effect of Radiation in HCT 116 Colon Cancer Cells.  


The need to find new EGFR inhibitors for use in combination with radiotherapy in the treatment of solid tumors has drawn our attention to compounds derived from genistein, a natural isoflavonoid. The antiproliferative potential of synthetic genistein derivatives used alone or in combination with ionizing radiation was evaluated in cancer cell lines using clonogenic assay. EGFR phosphorylation was assessed with western blotting. Genistein derivatives inhibited clonogenic growth of HCT 116 cancer cells additively or synergistically when used in combination with ionizing radiation, and decreased EGFR activation. Our preclinical evaluation of genistein-derived EGFR inhibitors suggests that these compounds are much more potent sensitizers of cells to radiation than the parent isoflavonoid, genistein and indicate that these compounds may be useful in the treatment of colon cancer with radiation therapy. PMID:25401399

Gruca, Aleksandra; Krawczyk, Zdzis?aw; Szeja, Wies?aw; Grynkiewicz, Grzegorz; Rusin, Aleksandra



Xanthorrhizol, a natural sesquiterpenoid, induces apoptosis and growth arrest in HCT116 human colon cancer cells.  


Xanthorrhizol is a sesquiterpenoid from the rhizome of Curcuma xanthorrhiza. In our previous studies, xanthorrhizol suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, inhibited cancer cell growth, and exerted an anti-metastatic effect in an animal model. However, the exact mechanisms for its inhibitory effects against cancer cell growth have not yet been fully elucidated. In the present study, we investigated the growth inhibitory effect of xanthorrhizol on cancer cells. Xanthorrhizol dose-dependently exerted antiproliferative effects against HCT116 human colon cancer cells. Xanthorrhizol also arrested cell cycle progression in the G0/G1 and G2/M phase and induced the increase of sub-G1 peaks. Cell cycle arrest was highly correlated with the downregulation of cyclin A, cyclin B1, and cyclin D1; cyclin-dependent kinase 1 (CDK1), CDK2, and CDK4; proliferating cell nuclear antigen; and inductions of p21 and p27, cyclin-dependent kinase inhibitors. The apoptosis by xanthorrhizol was markedly evidenced by induction of DNA fragmentation, release of cytochrome c, activation of caspases, and cleavage of poly-(ADP-ribose) polymerase. In addition, xanthorrhizol increased the expression and promoter activity of pro-apoptotic non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1). These findings provide one plausible mechanism for the growth inhibitory activity of xanthorrhizol against cancer cells. PMID:19926935

Kang, You-Jin; Park, Kwang-Kyun; Chung, Won-Yoon; Hwang, Jae-Kwan; Lee, Sang Kook



Molecular cloning, genomic characterization and over-expression of a novel gene, XRRA1, identified from human colorectal cancer cell HCT116Clone2_XRR and macaque testis  

PubMed Central

Background As part of our investigation into the genetic basis of tumor cell radioresponse, we have isolated several clones with a wide range of responses to X-radiation (XR) from an unirradiated human colorectal tumor cell line, HCT116. Using human cDNA microarrays, we recently identified a novel gene that was down-regulated by two-fold in an XR-resistant cell clone, HCT116Clone2_XRR. We have named this gene as X-ray radiation resistance associated 1 (XRRA1) (GenBank BK000541). Here, we present the first report on the molecular cloning, genomic characterization and over-expression of the XRRA1 gene. Results We found that XRRA1 was expressed predominantly in testis of both human and macaque. cDNA microarray analysis showed three-fold higher expression of XRRA1 in macaque testis relative to other tissues. We further cloned the macaque XRRA1 cDNA (GenBank AB072776) and a human XRRA1 splice variant from HCT116Clone2_XRR (GenBank AY163836). In silico analysis revealed the full-length human XRRA1, mouse, rat and bovine Xrra1 cDNAs. The XRRA1 gene comprises 11 exons and spans 64 kb on chromosome 11q13.3. Human and macaque cDNAs share 96% homology. Human XRRA1 cDNA is 1987 nt long and encodes a protein of 559 aa. XRRA1 protein is highly conserved in human, macaque, mouse, rat, pig, and bovine. GFP-XRRA1 fusion protein was detected in both the nucleus and cytoplasm of HCT116 clones and COS-7 cells. Interestingly, we found evidence that COS-7 cells which over-expressed XRRA1 lacked Ku86 (Ku80, XRCC5), a non-homologous end joining (NHEJ) DNA repair molecule, in the nucleus. RT-PCR analysis showed differential expression of XRRA1 after XR in HCT116 clones manifesting significantly different XR responses. Further, we found that XRRA1 was expressed in most tumor cell types. Surprisingly, mouse Xrra1 was detected in mouse embryonic stem cells R1. Conclusions Both XRRA1 cDNA and protein are highly conserved among mammals, suggesting that XRRA1 may have similar functions. Our results also suggest that the genetic modulation of XRRA1 may affect the XR responses of HCT116 clones and that XRRA1 may have a role in the response of human tumor and normal cells to XR. XRRA1 might be correlated with cancer development and might also be an early expressed gene. PMID:12908878

Mesak, Felix M; Osada, Naoki; Hashimoto, Katsuyuki; Liu, Qing Y; Ng, Cheng E



Dibenzocyclooctadiene lignans, gomisins J and N inhibit the Wnt/{beta}-catenin signaling pathway in HCT116 cells  

SciTech Connect

Graphical abstract: Schematic diagram of the possible molecular mechanism underlying the inhibition of the Wnt/{beta}-catenin signaling pathway and the induction of G0/G1-phase arrest by gomisins J and N, derived from the fruits of S. chinensis, in HCT116 human colon cancer cells. Highlights: Black-Right-Pointing-Pointer Gomisins J and N inhibited Wnt/{beta}-catenin signaling pathway in HCT116 cells. Black-Right-Pointing-Pointer Gomisins J and N disrupted the binding of {beta}-catenin to specific DNA sequences, TBE. Black-Right-Pointing-Pointer Gomisins J and N inhibited the HCT116 cell proliferation through G0/G1 phase arrest. Black-Right-Pointing-Pointer Gomisins J and N inhibited the expression of Cyc D1, a Wnt/{beta}-catenin target gene. -- Abstract: Here, we report that gomisin J and gomisin N, dibenzocyclooctadiene type lignans isolated from Schisandra chinensis, inhibit Wnt/{beta}-catenin signaling in HCT116 cells. Gomisins J and N appear to inhibit Wnt/{beta}-catenin signaling by disrupting the interaction between {beta}-catenin and its specific target DNA sequences (TCF binding elements, TBE) rather than by altering the expression of the {beta}-catenin protein. Gomisins J and N inhibit HCT116 cell proliferation by arresting the cell cycle at the G0/G1 phase. The G0/G1 phase arrest induced by gomisins J and N appears to be caused by a decrease in the expression of Cyclin D1, a representative target gene of the Wnt/{beta}-catenin signaling pathway, as well as Cdk2, Cdk4, and E2F-1. Therefore, gomisins J and N, the novel Wnt/{beta}-catenin inhibitors discovered in this study, may serve as potential agents for the prevention and treatment of human colorectal cancers.

Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye; Lee, Hee Ju [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of)] [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); Kim, Chul Young [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of) [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of); College of Pharmacy, Hanyang University, Ansan 426-791 (Korea, Republic of); Nho, Chu Won, E-mail: [Functional Food Center, Korea Institute of Science and Technology, Gangneung 210-340 (Korea, Republic of)



Stereospecific ligands and their complexes. Part XII. Synthesis, characterization and in vitro antiproliferative activity of platinum(IV) complexes with some O,O?-dialkyl esters of (S,S)-ethylenediamine-N,N?-di-2-propanoic acid against colon cancer (HCT-116) and breast cancer (MDA-MB-231) cell lines  

NASA Astrophysics Data System (ADS)

Synthesis of three new platinum(IV) complexes C1-C3, with bidentate N,N?-ligand precursors, O,O?-dialkyl esters (alkyl = propyl, butyl and pentyl), of (S,S)-ethylenediamine-N,N?-di-2-propanoic acid, H2-S,S-eddp were reported. The reported platinum(IV) complexes characterized by elemental analysis and their structures were discussed on the bases of their infrared, 1H and 13C NMR spectroscopy. In vitro antiproliferative activity was determined on tumor cell lines: human colon carcinoma HCT-116 and human breast carcinoma MDA-MB-231, using MTT test.

Stojkovi?, Danijela Lj.; Jevti?, Verica V.; Radi?, Gordana P.; ?a?i?, Dragana S.; ?ur?i?, Milena G.; Markovi?, Snežana D.; Ðinovi?, Vesna M.; Petrovi?, Vladimir P.; Trifunovi?, Sre?ko R.



Isoreserpine promotes {beta}-catenin degradation via Siah-1 up-regulation in HCT116 colon cancer cells  

SciTech Connect

Aberrant accumulation of intracellular {beta}-catenin in intestinal epithelial cells is a frequent early event during the development of colon cancer. To identify small molecules that decrease the level of intracellular {beta}-catenin, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells to detect compounds that inhibit TOPFlash reporter activity, which was stimulated by Wnt3a-conditioned medium. We found that isoreserpine promoted the degradation of intracellular {beta}-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of {beta}-catenin/T-cell factor (TCF)-dependent genes, such as cyclin D1 and c-myc, resulting in the suppression of HCT116 cell proliferation. Our findings suggest that isoreserpine can potentially be used as a chemotherapeutic agent against colon cancer.

Gwak, Jungsug; Song, Taeyun [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)] [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Song, Jie-Young; Yun, Yeon-Sook [Laboratory of Radiation Cancer Science, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of)] [Laboratory of Radiation Cancer Science, Korea Institute of Radiological and Medical Sciences, Seoul 139-706 (Korea, Republic of); Choi, Il-Whan [Department of Microbiology, Center for Viral Disease Research, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)] [Department of Microbiology, Center for Viral Disease Research, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Jeong, Yongsu [Department of Genetic Engineering, and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701 (Korea, Republic of)] [Department of Genetic Engineering, and Graduate School of Biotechnology, Kyung Hee University, Yongin 446-701 (Korea, Republic of); Shin, Jae-Gook [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of) [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of); Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan 614-735 (Korea, Republic of); Oh, Sangtaek, E-mail: [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)] [PharmacoGenomics Research Center, Inje University, Busan 614-735 (Korea, Republic of)



Preclinical Study of Treatment Response in HCT-116 Cells and Xenografts with 1H-decoupled 31P MRS  

PubMed Central

The topoisomerase I inhibitor, irinotecan, and its active metabolite SN-38 have been shown to induce G2/M cell cycle arrest without significant cell death in human colon carcinoma cells (HCT-116). Subsequent treatment of these G2/M-arrested cells with the cyclin-dependent kinase inhibitor, flavopiridol, induced these cells to undergo apoptosis. The goal of this study was to develop a noninvasive metabolic biomarker for early tumor response and target inhibition of irinotecan followed by flavopiridol treatment in a longitudinal study. A total of eleven mice bearing HCT-116 xenografts were separated into two cohorts where one cohort was administered saline and the other treated with a sequential course of irinotecan followed by flavopiridol. Each mouse xenograft was longitudinally monitored with proton (1H)-decoupled phosphorus (31P) magnetic resonance spectroscopy (MRS) before and after treatment. A statistically significant decrease in phosphocholine (p = 0.0004) and inorganic phosphate (p = 0.0103) levels were observed in HCT-116 xenografts following treatment, which were evidenced within twenty-four hours of treatment completion. Also, a significant growth delay was found in treated xenografts. To discern the underlying mechanism for the treatment response of the xenografts, in vitro HCT-116 cell cultures were investigated with enzymatic assays, cell cycle analysis, and apoptotic assays. Flavopiridol had a direct effect on choline kinase as measured by a 67% reduction in the phosphorylation of choline to phosphocholine. Cells treated with SN-38 alone underwent 83±5% G2/M cell cycle arrest compared to untreated cells. In cells, flavopiridol alone induced 5±1% apoptosis while the sequential treatment (SN-38 then flavopiridol) resulted in 39±10% apoptosis. In vivo 1H-decoupled 31P MRS indirectly measures choline kinase activity. The decrease in phosphocholine may be a potential indicator of early tumor response to the sequential treatment of irinotecan followed by flavopiridol in noninvasive and/or longitudinal studies. PMID:21994185

Darpolor, Moses M.; Kennealey, Peter T.; Carl Le, H; Zakian, Kristen L.; Ackerstaff, Ellen; Rizwan, Asif; Chen, Jin-Hong; Sambol, Elliot B.; Schwartz, Gary K.; Singer, Samuel; Koutcher, Jason A.



Expression profiling of choline and ethanolamine kinases in MCF7, HCT116 and HepG2 cells, and the transcriptional regulation by epigenetic modification.  


The function of choline kinase (CK) and ethanolamine kinase (EK) is to catalyse the phosphorylation of choline and ethanolamine, respectively, in order to yield phosphocholine (PCho) and phosphoethanolamine (PEtn). A high expression level of PCho, due to elevated CK activity, has previously been associated with malignant transformation. In the present study, a quantitative polymerase chain reaction was performed to determine the mRNA expression profiles of ck and ek mRNA variants in MCF7 breast, HCT116 colon and HepG2 liver cancer cells. The ck and ek mRNA expression profiles showed that total ck? was expressed most abundantly in the HepG2 cells. The HCT116 cells exhibited the highest ck? and ek1 mRNA expression levels, whereas the highest ek2? mRNA expression levels were detected in the MCF7 cells. The ck? variant had higher mRNA expression levels, as compared with total ck?, in both the MCF7 and HCT116 cells. Relatively low ek1 mRNA expression levels were detected, as compared with ek2? in the MCF7 cells; however, this was not observed in the HCT116 and HepG2 cells. Notably, the mRNA expression levels of ck?2 were markedly low, as compared with ck?1, in all three cancer cell lines. The effects of epigenetic modification on ck and ek mRNA expression, by treatment of the cells with the histone deacetylase inhibitor trichostatin A (TSA), were also investigated. The results of the present study showed that the mRNA expression levels of ck?, ck? and ek2? were affected by TSA. An increase >8?fold was observed in ek2? mRNA expression upon treatment with TSA, in a concentration? and time?dependent manner. In conclusion, the levels of ck and ek transcript variants in the three cancer cell lines were varied. The effects of TSA treatment on the mRNA expression levels of ck and ek imply that ck and ek mRNA expression may be regulated by epigenetic modification. PMID:25333818

Ling, Chua Siang; Yin, Khoo Boon; Cun, See Too Wei; Ling, Few Ling



Negative regulation of cell motile and invasive activities by lysophosphatidic acid receptor-3 in colon cancer HCT116 cells.  


Lysophosphatidic acid (LPA) mediates a wide range of biological responses with G protein-coupled transmembrane receptors (LPA receptors). So far, at least six types of LPA receptors (LPA receptor-1 (LPA(1)) to LPA(6)) have been identified. Recently, it has been reported that LPA(3) indicates opposite effects on cellular functions of cancer cells. In the present study, to assess a biological role of LPA(3) on cell migration ability of colon cancer cells, we generated LPA receptor-3 (LPAR3) knockdown (HCT-sh3-3) cells from HCT116 and measured cell motile and invasion activities. In motility assay with a cell culture insert, HCT-sh3-3 cells showed significantly high cell motile activity, compared with control cells. For invasion assay, the filter was coated with Matrigel. The invasive activity of HCT-sh3-3 cells was significantly higher than that of control cells. Furthermore, we also examined the effects of LPAR3 knockdown on the interaction between colon cancer cells and endothelial F-2 cells. When F-2 cells were cultured with serum-free DMEM containing a supernatant from HCT-sh3-3 cells, the cell growth rate and migration activity of F-2 cells were significantly stimulated, associating with the elevated expressions of vascular endothelial growth factor (VEGF)-A and VEGF-C genes in HCT-sh3-3 cells. These results suggest that LPA(3) may act as a negative regulator on cell motile and invasive abilities of colon cancer HCT116 cells. PMID:22763559

Fukui, Rie; Tanabe, Eriko; Kitayoshi, Misaho; Yoshikawa, Kyohei; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi



Bone morphogenetic protein-4 is overexpressed in colonic adenocarcinomas and promotes migration and invasion of HCT116 cells  

SciTech Connect

Bone morphogenetic protein (BMP), a member of the TGF-{beta} superfamily, is involved in development, morphogenesis, cell proliferation and apoptosis. Dysregulation of BMP signaling has been suggested in tumorigenesis. In an analysis of human colon normal mucosa and tumors at different stages by immunohistochemistry, we observed that the intensity of BMP-4 staining in late-adenocarcinomas was stronger than that in normal mucosa and adenomas, while there was no difference in the staining of its receptors (BMPR-IA and BMPR-II) at all stages. The up-regulation of BMP-4 was further validated in another panel of tumor tissues by real-time RT-PCR, showing that BMP-4 mRNA levels in primary colonic carcinomas with liver metastasis were significantly higher than that in the matched normal mucosa. In order to understand the functional relevance of BMP-4 expression in colon cancer progression, BMP-4-overexpressing cell clones were generated from HCT116 cells. Overexpression of BMP-4 did not affect the HCT116 cell growth. The cells overexpressing BMP-4 became resistant to serum-starvation-induced apoptosis and exhibited enhanced migration and invasion characteristics. Overexpression of BMP-4 changed cell morphology to invasive spindle phenotype and induced the expression and activity of urokinase plasminogen activator (uPA). These results indicate that BMP-4 confers invasive phenotype during progression of colon cancer.

Deng Haiyun [Division of Surgical Research, Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY 11030 (United States); Makizumi, Ryouji [Division of Surgical Research, Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY 11030 (United States); Ravikumar, T.S. [Division of Surgical Research, Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY 11030 (United States); Dong Huali [Division of Surgical Research, Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY 11030 (United States); Yang Wancai [Department of Oncology, Montefiore Medical Center and Albert Einstein Cancer Center, Bronx, NY 10461 (United States); Yang, W.-L. [Division of Surgical Research, Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY 11030 (United States) and Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY 11030 (United States)]. E-mail:



Bamboo salt has in vitro anticancer activity in HCT-116 cells and exerts anti-metastatic effects in vivo.  


Bamboo salt is a traditional food widely used in Korea. The in vitro anticancer effects of this salt were evaluated in HCT-116 human colon cancer cells using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay. A 1% salt concentration of bamboo salt baked nine times (9×) inhibited the growth of HCT-116 cells by 53%, which was higher than salt baked three times (3×) or once (1×; 44% and 41%, respectively) and much higher than solar sea salt (Korean sea salt) and purified salt (22% and 18%, respectively). To elucidate the inhibitory mechanisms underlying the anticancer effect of the salt samples in cancer cells, expression of genes associated with apoptosis, inflammation, and metastasis was measured with reverse transcription-polymerase chain reaction and Western blotting. Bamboo salt (9×) significantly induced apoptosis in cancer cells (P<.05) by upregulating Bax, caspase-9, and caspase-3, and downregulating Bcl-2. The expression of genes associated with inflammation (NF-?B, iNOS, and COX-2) was significantly downregulated (P<.05) by 9× bamboo salt, demonstrating its anti-inflammatory properties. The 9× bamboo salt also exerted a greater anti-metastatic effect on cancer cells than the other salts as demonstrated by decreased mRNA expression of MMP genes and increased expression of tissue inhibitors of metalloproteinases, which was confirmed by the inhibition of tumor metastasis induced in colon 26-M3.1 cells in BALB/c mice. In contrast, purified and solar salts increased metastasis in the mice. Our results demonstrated that 9× bamboo salt had the most potent in vitro anticancer effect, induced apoptosis, had anti-inflammatory activities, and exerted in vivo anti-metastatic effects. Additionally, the anticancer, anti-inflammatory, and anti-metastatic effects of the 1× and 3× bamboo salts were stronger than those of the purified and solar salts. PMID:23256441

Zhao, Xin; Kim, So-Young; Park, Kun-Young



Carnosol induces apoptosis through generation of ROS and inactivation of STAT3 signaling in human colon cancer HCT116 cells.  


Carnosol, an active constituent of rosemary, has been reported to possess anti-inflammatory and anticancer activities. However, the molecular mechanisms underlying the anticancer effects of carnosol remain poorly understood. In the present study, we found that carnosol significantly reduced the viability of human colon cancer (HCT116) cells in a concentration- and time-dependent manner. Treatment of cells with carnosol induced apoptosis, which was associated with activation of caspase-9 and -3 and the cleavage of poly-(ADP-ribose) polymerase (PARP). Incubation with carnosol elevated the expression of Bax and inhibited the levels of Bcl-2 and Bcl-xl. Carnosol induced expression of p53 and inhibited that of murine-double minute-2 (Mdm2). Moreover, carnosol generated reactive oxygen species (ROS), and pretreatment with N-acetyl cysteine abrogated carnosol-induced cleavage of caspase-3 and PARP. The constitutive phosphorylation, the DNA binding and reporter gene activity of signal transducer and activator of transcription-3 (STAT3) was diminished by treatment with carnosol. To further elucidate the molecular mechanisms of STAT3 inactivation, we found that carnosol attenuated the phosphorylation of Janus-activated kinase-2 (Jak2) and Src kinase. Pharmacological inhibition of Jak2 and Src inhibited STAT3 phosphorylation. Furthermore, carnosol attenuated the expression of STAT3 target gene products, such as survivin, cyclin-D1, -D2, and -D3. Taken together, our study provides the first report that carnosol induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases and inhibition of STAT3 signaling pathway. PMID:24481553

Park, Ki-Woong; Kundu, Juthika; Chae, In-Gyeong; Kim, Do-Hee; Yu, Mi-Hee; Kundu, Joydeb Kumar; Chun, Kyung-Soo



Ursodeoxycholic acid protects colon cancer HCT116 cells from deoxycholic acid-induced apoptosis by inhibiting apoptosome formation.  


We previously demonstrated that ursodeoxycholic acid (UDC) requires prolonged (?5 h) preincubation to exhibit effective protection of colon cancer HCT116 cells from deoxycholic acid (DC)-induced apoptosis. Although UDC diminished DC-mediated caspase-9 activation, cytochrome c release from the mitochondria was not inhibited, indicating that UDC acts on the steps of caspase-9 activation. In the present study, therefore, we investigated the effects of UDC on the factors involved in caspase-9 activation. We found that UDC had no significant effect on the expression of antiapoptotic XIAP. Furthermore, UDC did not affect the expression or release of proapoptotic Smac/DIABLO, or the association of XIAP and Smac/DIABLO. In contrast, association of Apaf-1 and caspase-9 stimulated by 500 ?M DC was inhibited by UDC pretreatment. Although UDC caused remarkable activation of Akt/PKB, phosphatidylinositol-3-kinase (PI3K) inhibitor did not significantly reduce UDC-mediated cytoprotection. Furthermore, phosphorylation of threonine residues on caspase-9 after UDC pretreatment could not be detected. UDC-mediated cytoprotection was independent of the MAPK pathway, and cyclic AMP (cAMP) analogue did not inhibit DC-induced apoptosis. Our results indicate that UDC protects colon cancer cells from apoptosis induced by hydrophobic bile acids, by inhibiting apoptosome formation independently of the survival signals mediated by the PI3K, MAPK, or cAMP pathways. PMID:22497644

Saeki, Tohru; Yui, Satoko; Hirai, Tadashi; Fujii, Takami; Okada, Sawami; Kanamoto, Ryuhei



MLH1-deficient HCT116 colon tumor cells exhibit resistance to the cytostatic and cytotoxic effect of the poly(A) polymerase inhibitor cordycepin (3'-deoxyadenosine) in vitro.  


Cordycepin (3'-deoxyadenosine) is an inhibitor of poly(A) polymerase (PAP), an enzyme crucial to mRNA 3'-end processing, which produces the shortening of poly(A) tails, leading to the destabilization of mRNAs. Cordycepin inhibits proliferation and induces apoptosis in tumor cells, indicating its antitumor activity. Defective 3'-end processing is associated with hypersensitivity to UV treatment. We investigated the effects of cordycepin on proliferation and apoptosis in MLH1-deficient and MLH1-proficient HCT116 colon tumor cells. MLH1 is a DNA mismatch repair (MMR) protein involved in the processing of damaged DNA. Cells with defective MMR show resistance to certain anticancer drugs. The results showed that MLH1-deficient HCT116 cells are 2-fold less sensitive to the cytostatic effect of cordycepin, as compared to MLH1-proficient cells. This reduced sensitivity to cordycepin in MLH1-deficient cells was associated with reduced upregulation of the cell cycle inhibitor p21. MLH1-deficient cells also exhibited reduced susceptibility to apoptosis upon treatment with cordycepin, as demonstrated by the reduced PARP-1 cleavage. Our findings showed that MLH1-deficient HCT116 colon tumor cells are resistant to the cytostatic and cytotoxic effect of cordycepin, indicating a possible involvement of MMR in mRNA polyadenylation. The findings also suggest that cordycepin is not suitable to therapeutically encounter tumor cells lacking MLH1 expression. PMID:22740928

Imesch, Patrick; Goerens, Anouk; Fink, Daniel; Fedier, André



MLH1-deficient HCT116 colon tumor cells exhibit resistance to the cytostatic and cytotoxic effect of the poly(A) polymerase inhibitor cordycepin (3?-deoxyadenosine) in vitro  

PubMed Central

Cordycepin (3?-deoxyadenosine) is an inhibitor of poly(A) polymerase (PAP), an enzyme crucial to mRNA 3?-end processing, which produces the shortening of poly(A) tails, leading to the destabilization of mRNAs. Cordycepin inhibits proliferation and induces apoptosis in tumor cells, indicating its antitumor activity. Defective 3?-end processing is associated with hypersensitivity to UV treatment. We investigated the effects of cordycepin on proliferation and apoptosis in MLH1-deficient and MLH1-proficient HCT116 colon tumor cells. MLH1 is a DNA mismatch repair (MMR) protein involved in the processing of damaged DNA. Cells with defective MMR show resistance to certain anticancer drugs. The results showed that MLH1-deficient HCT116 cells are 2-fold less sensitive to the cytostatic effect of cordycepin, as compared to MLH1-proficient cells. This reduced sensitivity to cordycepin in MLH1-deficient cells was associated with reduced upregulation of the cell cycle inhibitor p21. MLH1-deficient cells also exhibited reduced susceptibility to apoptosis upon treatment with cordycepin, as demonstrated by the reduced PARP-1 cleavage. Our findings showed that MLH1-deficient HCT116 colon tumor cells are resistant to the cytostatic and cytotoxic effect of cordycepin, indicating a possible involvement of MMR in mRNA polyadenylation. The findings also suggest that cordycepin is not suitable to therapeutically encounter tumor cells lacking MLH1 expression. PMID:22740928




Apoptosis induced by glycoprotein (150-kDa) isolated from Solanum nigrum L. is not related to intracellular reactive oxygen species (ROS) in HCT116 cells  

Microsoft Academic Search

This study was carried out to investigate the apoptotic effects of glycoprotein [Solanum nigrum L. (SNL) glycoprotein, 150-kDa] isolated from Solanum nigrum L., which has been used as an antipyretic and anticancer agent in folk medicine. With the purified SNL glycoprotein, we evaluated\\u000a the cytotoxic and apoptotic effects of SNL glycoprotein on HCT-116 cells, DNA fragmentation and nuclear staining assays,

Sei-Jung Lee; Kye-Taek Lim



A 150-kDa glycoprotein isolated from Solanum nigrum L. has cytotoxic and apoptotic effects by inhibiting the effects of protein kinase C alpha, nuclear factor-kappa B and inducible nitric oxide in HCT116 cells  

Microsoft Academic Search

This study was carried out to investigate the anticancer effects of a 150-kDa glycoprotein isolated from Solanum nigrum L. (SNL glycoprotein) on spontaneously and experimentally induced tumor promotion in HCT-116 cells. For spontaneously induced tumor promotion, we evaluated the cytotoxic and apoptotic effects in HCT-116 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT), DNA fragmentation, and H33342 and ethidium bromide staining

Sei-Jung Lee; Phil-Sun Oh; Jeong-Hyeon Ko; Kwang Lim; Kye-Taek Lim



Pro-growth role of the JMJD2C histone demethylase in HCT-116 colon cancer cells and identification of curcuminoids as JMJD2 inhibitors  

PubMed Central

Colon tumors are a major cause of cancer death, yet their molecular intricacies are not fully understood. We demonstrate that the histone demethylases JMJD2A, JMJD2B and JMJD2C are overexpressed in colon cancer cell lines, whereas another related protein, JMJD2D, is not. Interestingly, despite their high homology, the intracellular localization of JMJD2A-C is different in colon and other cancer cells, with JMJD2A being present comparably in the cytoplasm and nucleus, JMJD2B more prevalent in the nucleus and JMJD2C strongly associated with chromatin. This suggests that each of these three proteins performs different, non-redundant functions. Moreover, we show that JMJD2C (also called KDM4C) forms complexes with ?-catenin, an oncoprotein whose overexpression is crucial for the development of most colonic tumors. In addition, JMJD2C downregulation reduced both growth and clonogenic capacity of HCT-116 colon cancer cells. Further, JMJD2C was required for efficient expression of the growth stimulatory proteins FRA1 and cyclin D1 as well as the survival factor BCL2. Lastly, we identified derivatives of curcumin as in vitro inhibitors of JMJD2 enzymes, suggesting that these curcuminoids could be useful for decreasing JMJD2 activity in vivo. In conclusion, our data highlight that overexpression of JMJD2C confers a pro-growth effect on colon cancer cells and, therefore, its inhibition by curcuminoids or other small molecules could be beneficial as an adjuvant therapy for colon cancer patients. PMID:24936217

Kim, Tae-Dong; Fuchs, James R; Schwartz, Eric; Abdelhamid, Dalia; Etter, Jonathan; Berry, William L; Li, Chenglong; Ihnat, Michael A; Li, Pui-Kai; Janknecht, Ralf



Induction of intrinsic apoptosis pathway in colon cancer HCT-116 cells by novel 2-substituted-5,6,7,8-tetrahydronaphthalene derivatives.  


2-Acetyl tetralin (1) reacted with N,N-dimethylformamide dimethylacetal (DMF-DMA) to afford the enaminone 3. The reaction of 3 with piperidine and morpholine afforded the trans enaminone 5a,b, respectively. Compound 3 was treated with primary aromatic amines to give secondary enaminones 6a-e. The enaminone 3 reacted with acetylglycine and hippuric acid to yield pyranones 10a, b, respectively. The reaction of enaminone 3 with 1,4-benzoquinone and 1,4-naphthoquinone gave benzofuranyl tetralin derivatives 14a,b, respectively. Also, when 3 reacted with 5-amino-3-phenyl-1H-pyrazole 15a and 5-amino-1,2,3-triazole 15b, it afforded the new pyrazolo[1,5-a]pyrimidine 17a and 1,2,3-triazolo[1,5-a]pyrimidine 17b, respectively. While the reaction of 3 with pyrimidines 18a, b resulted in the formation of pyrido[2,3-d]pyrimidine derivatives 20a, b, respectively. Investigations of the cytotoxic effect of those compounds against different human cell lines indicated that some compounds showed high selective cytotoxicity against colon cancer HCT-116 cells. Some of these compounds led to DNA damaging and fragmentation that was associated with the induction of apoptosis via mitochondrial pathway. This pathway is initiated by the impairment of mitochondrial transmembrane potential (??m) and in response to that the mitochondria released cytochrome c increased, that in turn activated caspase-9 and caspase-3 and induced apoptosis. Compounds 17b and 20b were promising anti-cancer agents that induced intrinsic apoptosis pathway in colon cancer cells. PMID:24657569

Gamal-Eldeen, Amira M; Hamdy, Nehal A; Abdel-Aziz, Hatem A; El-Hussieny, Enas A; Fakhr, Issa M I



Differential growth inhibitory effects of highly oxygenated guaianolides isolated from the Middle Eastern indigenous plant Achillea falcata in HCT-116 colorectal cancer cells.  


Medicinal plants play a crucial role in traditional medicine and in the maintenance of human health worldwide. Sesquiterpene lactones represent an interesting group of plant-derived compounds that are currently being tested as lead drugs in cancer clinical trials. Achillea falcata is a medicinal plant indigenous to the Middle Eastern region and belongs to the Asteraceae family, which is known to be rich in sesquiterpene lactones. We subjected Achillea falcata extracts to bioassay-guided fractionation against the growth of HCT-116 colorectal cancer cells and identified four secotanapartholides, namely 3-?-methoxy-isosecotanapartholide (1), isosecotanapartholide (2), tanaphallin (3), and 8-hydroxy-3-methoxyisosecotanapartholide (4). Three highly oxygenated guaianolides were isolated for the first time from Achillea falcata, namely rupin A (5), chrysartemin B (6), and 1?, 2?-epoxy-3?,4?,10?-trihydroxyguaian-6?,12-olide (7). These sesquiterpene lactones showed no or minor cytotoxicity while exhibiting promising anticancer effects against HCT-116 cells. Further structure-activity relationship studies related the bioactivity of the tested compounds to their skeleton, their lipophilicity, and to the type of functional groups neighboring the main alkylating center of the molecule. PMID:23860275

Tohme, Rita; Al Aaraj, Lamis; Ghaddar, Tarek; Gali-Muhtasib, Hala; Saliba, Najat A; Darwiche, Nadine



Antheraea pernyi silk fibroin-coated PEI/DNA complexes for targeted gene delivery in HEK 293 and HCT 116 cells.  


Polyethylenimine (PEI) has attracted much attention as a DNA condenser, but its toxicity and non-specific targeting limit its potential. To overcome these limitations, Antheraea pernyi silk fibroin (ASF), a natural protein rich in arginyl-glycyl-aspartic acid (RGD) peptides that contains negative surface charges in a neutral aqueous solution, was used to coat PEI/DNA complexes to form ASF/PEI/DNA ternary complexes. Coating these complexes with ASF caused fewer surface charges and greater size compared with the PEI/DNA complexes alone. In vitro transfection studies revealed that incorporation of ASF led to greater transfection efficiencies in both HEK (human embryonic kidney) 293 and HCT (human colorectal carcinoma) 116 cells, albeit with less electrostatic binding affinity for the cells. Moreover, the transfection efficiency in the HCT 116 cells was higher than that in the HEK 293 cells under the same conditions, which may be due to the target bonding affinity of the RGD peptides in ASF for integrins on the HCT 116 cell surface. This result indicated that the RGD binding affinity in ASF for integrins can enhance the specific targeting affinity to compensate for the reduction in electrostatic binding between ASF-coated PEI carriers and cells. Cell viability measurements showed higher cell viability after transfection of ASF/PEI/DNA ternary complexes than after transfection of PEI/DNA binary complexes alone. Lactate dehydrogenase (LDH) release studies further confirmed the improvement in the targeting effect of ASF/PEI/DNA ternary complexes to cells. These results suggest that ASF-coated PEI is a preferred transfection reagent and useful for improving both the transfection efficiency and cell viability of PEI-based nonviral vectors. PMID:24776757

Liu, Yu; You, Renchuan; Liu, Guiyang; Li, Xiufang; Sheng, Weihua; Yang, Jicheng; Li, Mingzhong



D. candidum has in vitro anticancer effects in HCT-116 cancer cells and exerts in vivo anti-metastatic effects in mice  

PubMed Central

BACKGROUND/OBJECTIVES D. candidum is a traditional Chinese food or medicine widely used in Asia. There has been little research into the anticancer effects of D. candidum, particularly the effects in colon cancer cells. The aim of this study was to investigate the anticancer effects of D. candidum in vitro and in vivo. MATERIALS/METHODS The in vitro anti-cancer effects on HCT-116 colon cancer cells and in vivo anti-metastatic effects of DCME (Dendrobium canidum methanolic extract) were examined using the experimental methods of MTT assay, DAPI staining, flow cytometry analysis, RT-PCR, and Western blot analysis. RESULTS At a concentration of 1.0 mg/mL, DCME inhibited the growth of HCT-116 cells by 84%, which was higher than at concentrations of 0.5 and 0.25 mg/mL. Chromatin condensation and formation of apoptotic bodies were observed in cancer cells cultured with DCME as well. In addition, DCME induced significant apoptosis in cancer cells by upregulation of Bax, caspase 9, and caspase 3, and downregulation of Bcl-2. Expression of genes commonly associated with inflammation, NF-?B, iNOS, and COX-2, was significantly downregulated by DCME. DCME also exerted an anti-metastasis effect on cancer cells as demonstrated by decreased expression of MMP genes and increased expression of TIMPs, which was confirmed by the inhibition of induced tumor metastasis in colon 26-M3.1 cells in BALB/c mice. CONCLUSIONS Our results demonstrated that D. candidum had a potent in vitro anti-cancer effect, induced apoptosis, exhibited anti-inflammatory activities, and exerted in vivo anti-metastatic effects.

Zhao, Xin; Sun, Peng; Qian, Yu



The anti-cancer activity of dihydroartemisinin is associated with induction of iron-dependent endoplasmic reticulum stress in colorectal carcinoma HCT116 cells.  


Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is among the artemisinin derivatives possessing potent anti-malarial and anti-cancer activities. In the present study, we found that DHA displayed significant anti-proliferative activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis. To further elucidate the mechanism of action of DHA, a proteomic study employed two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed. Glucose-regulated protein 78 (GRP78), which is related with endoplasmic reticulum stress (ER stress), was identified to be significantly up-regulated after DHA treatment. Further study demonstrated that DHA enhanced expression of GRP78 as well as growth arrest and DNA-damage-inducible gene 153 (GADD153, another ER stress-associated molecule) at both mRNA and protein levels. DHA treatment also led to accumulation of GADD153 in cell nucleus. Moreover, pretreatment of HCT116 cells with the iron chelator deferoxamine mesylate salt (DFO) abrogated induction of GRP78 and GADD153 upon DHA treatment, indicating iron is required for DHA-induced ER stress. This result is consistent with the fact that the anti-proliferative activity of DHA is also mediated by iron. We thus suggest the unbalance of redox may result in DHA-induced ER stress, which may contribute, at least in part, to its anti-cancer activity. PMID:20607588

Lu, Jin-Jian; Chen, Si-Meng; Zhang, Xiao-Wei; Ding, Jian; Meng, Ling-Hua



UDN glycoprotein regulates activities of manganese-superoxide dismutase, activator protein-1, and nuclear factor-kappaB stimulated by reactive oxygen radicals in lipopolysaccharide-stimulated HCT-116 cells.  


This study was carried out to investigate the anti-inflammatory effects of glycoprotein (UDN glycoprotein, 116-kDa) isolated from Ulmus davidiana Nakai, which has been used to heal inflammatory diseases in Korean herbal medicine. We found that UDN glycoprotein has strong scavenging effect on the production of intracellular superoxide anion (O(2)(-)), hydrogen peroxides (H(2)O(2)), and nitric oxide (NO) without any cytotoxicity, and that the glycoprotein also selectively normalizes the aberrant activation of manganese-superoxide dismutases (Mn-SOD) activity in lipopolysaccaride (LPS)-treated cancerous human colon epithelial cells (HCT-116 cells). The results obtained from electrophoretic mobility shift assay (EMSA) and Western blot analysis showed that UDN glycoprotein blocks the DNA binding activities of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1), and attenuates the activities of NF-kappaB subunits (p50 and p65), and AP-1 subunits (c-Jun and c-Fos), respectively. To further verify the anti-inflammatory effect of UDN glycoprotein, we investigated the activity of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinases-9 (MMP-9) in LPS-treated HCT-116 cells, using Western blot analysis and gelatin zymographic assay. Results in this study indicated that 200mug/ml of UDN glycoprotein has inhibitory effects on the activations of iNOS, COX-2, and MMP-9. Therefore, UDN glycoprotein, a natural antioxidant, is a potential modulator of inflammatory signal pathways in LPS-treated HCT-116 cells. PMID:17459574

Lee, Sei-Jung; Lim, Kye-Taek



GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells  

Microsoft Academic Search

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116

F. Hohla; S. Buchholz; A. V. Schally; S. Seitz; F. G. Rick; L. Szalontay; J. L. Varga; M. Zarandi; G. Halmos; I. Vidaurre; A. Krishan; M. Kurtoglu; S. Chandna; E. Aigner; C. Datz



Stability of XIST repression in relation to genomic imprinting following global genome demethylation in a human cell line.  


DNA methylation is essential in X chromosome inactivation and genomic imprinting, maintaining repression of XIST in the active X chromosome and monoallelic repression of imprinted genes. Disruption of the DNA methyltransferase genes DNMT1 and DNMT3B in the HCT116 cell line (DKO cells) leads to global DNA hypomethylation and biallelic expression of the imprinted gene IGF2 but does not lead to reactivation of XIST expression, suggesting that XIST repression is due to a more stable epigenetic mark than imprinting. To test this hypothesis, we induced acute hypomethylation in HCT116 cells by 5-aza-2'-deoxycytidine (5-aza-CdR) treatment (HCT116-5-aza-CdR) and compared that to DKO cells, evaluating DNA methylation by microarray and monitoring the expression of XIST and imprinted genes IGF2, H19, and PEG10. Whereas imprinted genes showed biallelic expression in HCT116-5-aza-CdR and DKO cells, the XIST locus was hypomethylated and weakly expressed only under acute hypomethylation conditions, indicating the importance of XIST repression in the active X to cell survival. Given that DNMT3A is the only active DNMT in DKO cells, it may be responsible for ensuring the repression of XIST in those cells. Taken together, our data suggest that XIST repression is more tightly controlled than genomic imprinting and, at least in part, is due to DNMT3A. PMID:25387668

de Araújo, E S S; Vasques, L R; Stabellini, R; Krepischi, A C V; Pereira, L V



p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells  

SciTech Connect

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53{sup wt}) or being p(HCT-116 p53{sup -/-}), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53{sup -/-} xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53{sup wt} cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53{sup wt} cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75{sup NTR}, p53 and Bax.

Janssen, Astrid; Schiffmann, Susanne; Birod, Kerstin; Maier, Thorsten J.; Wobst, Ivonne; Geisslinger, Gerd [pharmazentrum frankfurt/ZAFES, Institut fuer Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universitaet Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt (Germany); Groesch, Sabine [pharmazentrum frankfurt/ZAFES, Institut fuer Klinische Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universitaet Frankfurt, Theodor Stern Kai 7, 60590 Frankfur (Germany)], E-mail:



The anticancer effect of saffron in two p53 isogenic colorectal cancer cell lines  

PubMed Central

Background Saffron extract, a natural product, has been shown to induce apoptosis in several tumor cell lines. Nevertheless, the p53-dependency of saffron’s mechanism of action in colon cancer remains unexplored. Material and methods In order to examine saffron’s anti-proliferative and pro-apoptotic effects in colorectal cancer cells, we treated two p53 isogenic HCT116 cell lines (HCT wildtype and HCT p53?/?) with different doses of the drug and analyzed cell proliferation and apoptosis in a time-dependent manner. MTT viability and crystal violet assays were performed in order to determine the effective dose of saffron on both cell lines. The cell cycle progress was examined by Flow cytometric analysis. Apoptosis was assessed using Annexin-PI-staining and Western Blotting for caspase 3 and PARP cleavage. Autophagy was determined by Western Blotting of the light chain 3 (LC3)-II and Beclin 1 proteins. The protein content of phospho-H2AX (?H2AX), a sensor of DNA double strand breaks, was also analyzed by Western Blotting. Results Saffron extract induced a p53-dependent pattern of cell cycle distribution with a full G2/M stop in HCT116 p53 wildtype cells. However, it induced a remarkable delay in S/G2 phase transit with entry into mitosis in HCT116 p53 ?/? cells. The apoptotic Pre-G1 cell fraction as well as Annexin V staining and caspase 3 cleavage showed a more pronounced apoptosis induction in HCT116 p53 wildtype cells. Obviously, the significantly higher DNA-damage, reflected by ?H2AX protein levels in cells lacking p53, was coped by up-regulation of autophagy. The saffron-induced LC3-II protein level was a remarkable indication of the accumulation of autophagosomes, a response to the cellular stress condition of drug treatment. Conclusions This is the first study showing the effect of saffron in HCT116 colorectal cancer cells with different p53 status. Saffron induced DNA-damage and apoptosis in both cell lines. However, autophagy has delayed the induction of apoptosis in HCT116 p53 ?/? cells. Considering the fact that most tumors show a functional p53 inactivation, further research is needed to elucidate the long-term effects of saffron in p53 ?/? tumors. PMID:22640402



Supraadditive effect of 2?,2?-difluorodeoxycytidine (gemcitabine) in combination with oxaliplatin in human cancer cell lines  

Microsoft Academic Search

Purpose: This study assessed the cytotoxic effects of the nucleoside analog gemcitabine in combination with the diaminocyclohexane\\u000a platinum compound oxaliplatin. Methods: Growth inhibition studies were performed using the human CEM leukemia cell line and the colon-cancer cell lines HCT 116\\u000a and Colo 320 DM. Gemcitabine-oxaliplatin combinations were compared with gemcitabine-cisplatin combinations in the same cell\\u000a lines using similar experimental settings.

Sandrine Faivre; Eric Raymond; Jan M. Woynarowski; Esteban Cvitkovic



Altered Growth of Human Colon Cancer Cell Lines Disrupted at Activated Ki-ras  

Microsoft Academic Search

Point mutations that activate the Ki-ras proto-oncogene are present in about 50 percent of human colorectal tumors. To study the functional significance of these mutations, the activated Ki-ras genes in two human colon carcinoma cell lines, DLD-1 and HCT 116, were disrupted by homologous recombination. Compared with parental cells, cells disrupted at the activated Ki-ras gene were morphologically altered, lost

Senji Shirasawa; Masanori Furuse; Nobuhiko Yokoyama; Takehiko Sasazuki



Potent Vinblastine C20? Ureas Displaying Additionally Improved Activity Against a Vinblastine-Resistant Cancer Cell Line  

PubMed Central

A series of disubstituted C20?–urea derivatives of vinblastine were prepared from 20?-aminovinblastine that was made accessible through a unique Fe(III)/NaBH4- mediated alkene functionalization reaction of anhydrovinblastine. Three analogs were examined across a panel of 15 human tumor cell lines, displaying remarkably potent cell growth inhibition activity (avg. IC50 = 200–300 pM), being 10–200-fold more potent than vinblastine (avg. IC50 = 6.1 nM). Significantly, the analogs also display further improved activity against the vinblastine-resistant HCT116/VM46 cell line that bears the clinically relevant overexpression of Pgp, exhibiting IC50 values on par with that of vinblastine against the sensitive HCT116 cell line, 100–200-fold greater than the activity of vinblastine against the resistant HCT116/VM46 cell line, and display a reduced 10–20-fold activity differential between the matched sensitive and resistant cell lines (vs 100-fold for vinblastine). PMID:24223237

Barker, Timothy J.; Duncan, Katharine K.; Otrubova, Katerina; Boger, Dale L.



Synthesis and biological evaluation of bile carboxamide derivatives with pro-apoptotic effect on human colon adenocarcinoma cell lines.  


We previously reported that the cinnamylpiperazinyl group in the side chain of the chenodeoxycholic acid showed apoptosis-inducing activity on multiple myeloma cancer cell line KMS-11. In the present study, we synthesized and tested the pro-apoptotic potency of fifteen new piperazinyl bile carboxamide derived from cholic, ursodeoxycholic, chenodeoxycholic, deoxycholic and lithocholic acids on human colon adenocarcinoma cell lines DLD-1, HCT-116, and HT-29. Cell viability was first measured using XTT assay. The most of the synthetic bile carboxamide derivatives decreased significantly cell viability in a dose-dependent manner. HCT-116 and DLD-1 cell lines were more sensitive than HT-29 to tested compounds. 9c, 9d showed the best in vitro results in term of solubility and dose-response effect on the three colon adenocarcinoma cell lines. Additionally, flow cytometric and Western-blotting analysis showed that 9c induced pro-apoptosis in DLD-1 and HCT-116 whereas 9d did not. We conclude that the benzyl group improved anti-proliferative activity and that the ?-hydroxyl group was found to be more beneficial at the 7-position in steroid skeleton. PMID:25173827

Brossard, Dominique; Lechevrel, Mathilde; El Kihel, Laïla; Quesnelle, Céline; Khalid, Mohamed; Moslemi, Safa; Reimund, Jean-Marie



Chemotherapeutic candidate inducing immunological death of human tumor cell lines.  


The immunological death induction by EY-6 on the human tumor cell lines was screened. Human colon carcinoma (HCT15, HCT116), gastric carcinoma (MKN74, SNU668), and myeloma (KMS20, KMS26, KMS34) cells were died by EY-6 treatment with dose-dependent manner. CRT expression, a typical marker for the immunological death, was increased on the EY-6-treated colorectal and gastric cancer cells. Interestingly, the effects on the myeloma cell lines were complicated showing cell line dependent differential modulation. Cytokine secretion from the EY-6 treated tumor cells were dose and cell-dependent. IFN-? and IL-12 secretion was increased in the treated cells (200% to over 1000% of non-treated control), except HCT116, SNU668 and KMS26 cells which their secretion was declined by EY-6. Data suggest the potential of EY-6 as a new type of immuno-chemotherapeutics inducing tumor-specific cell death. Further studies are planned to confirm the efficacy of EY-6 including in vivo study. PMID:22740792

Oh, Su-Jin; Ryu, Chung-Kyu; Choi, Inhak; Baek, So-Young; Lee, Hyunah



Apoptotic effect of Naphthoquinone derivatives on HCT116 colon cancer cells  

Microsoft Academic Search

Naphthoquinone is found in the core structure of many natural compounds, most notably the K vitamins. Numerous molecules with\\u000a the 1,4-naphthoquinone moiety are known to display distinct biological activities including anti-cancer, anti-inflammatory\\u000a and anti-bacterial activities. Vitamin K2 and doxorubicin, which are used to treat bleeding and lymphoma respectively, belong\\u000a to this class of chemicals. Although the exact mechanism of action

Young-Sam Im; Yongseog Chung; Dae Yeon Won; Soo Han Kwon; Hye-Ryun Kim; Dong Geun Lee; Seung-Ryul Kim; Kyung Do Park; Hak-Kyo Lee; Joong-Kook Choi



Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells.  


Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116, does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G(1) to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance. PMID:23063976

Chiaro, Christopher; Lazarova, Darina L; Bordonaro, Michael



CD8 + T cells from a patient with colon carcinoma, specific for a mutant p21-Ras-derived peptide (GLY 13 ?ASP), are cytotoxic towards a carcinoma cell line harbouring the same mutation  

Microsoft Academic Search

Several T lymphocyte clones (TLC), specific for a p21-Ras-derived peptide expressing a Gly13?Asp mutation and of the CD8+ subtype, were generated from peripheral blood of a colon carcinoma patient. The TLC exerted cytotoxicity against an interferon-? (IFN?)-pretreated colon carcinoma cell line, HCT116, which harbours the Gly13?Asp mutation and shares both HLA-A2 and HLA-B12(44) with the patient. This cytotoxic effect could

Beate Fossum; Anna Catharina Olsen; Erik Thorsby; Gustav Gaudernack



Cell Context Dependent p53 Genome-Wide Binding Patterns and Enrichment at Repeats  

PubMed Central

The p53 ability to elicit stress specific and cell type specific responses is well recognized, but how that specificity is established remains to be defined. Whether upon activation p53 binds to its genomic targets in a cell type and stress type dependent manner is still an open question. Here we show that the p53 binding to the human genome is selective and cell context-dependent. We mapped the genomic binding sites for the endogenous wild type p53 protein in the human cancer cell line HCT116 and compared them to those we previously determined in the normal cell line IMR90. We report distinct p53 genome-wide binding landscapes in two different cell lines, analyzed under the same treatment and experimental conditions, using the same ChIP-seq approach. This is evidence for cell context dependent p53 genomic binding. The observed differences affect the p53 binding sites distribution with respect to major genomic and epigenomic elements (promoter regions, CpG islands and repeats). We correlated the high-confidence p53 ChIP-seq peaks positions with the annotated human repeats (UCSC Human Genome Browser) and observed both common and cell line specific trends. In HCT116, the p53 binding was specifically enriched at LINE repeats, compared to IMR90 cells. The p53 genome-wide binding patterns in HCT116 and IMR90 likely reflect the different epigenetic landscapes in these two cell lines, resulting from cancer-associated changes (accumulated in HCT116) superimposed on tissue specific differences (HCT116 has epithelial, while IMR90 has mesenchymal origin). Our data support the model for p53 binding to the human genome in a highly selective manner, mobilizing distinct sets of genes, contributing to distinct pathways. PMID:25415302

Botcheva, Krassimira; McCorkle, Sean R.



New sesquiterpene lactones from Glechoma hederacea L. and their cytotoxic effects on human cancer cell lines.  


Three new sesquiterpene lactones, 1 ?,10 ?-epoxy-4-hydroxy-glechoma-5-en-olide (1), 1 ?,10 ?-epoxy-4,8-dihydroxy-glechoma-5-en-olide (2), and 1 ?,10 ?;4 ?,5 ?-diepoxy-8-methoxy-glechoman-8 ?,12-olide (3), were isolated from the whole plant of Glechoma hederacea, together with four known sesquiterpene lactones. The structures of the three new sesquiterpene lactones were determined by spectroscopic evidence. Cytotoxic effects of the isolated compounds were examined against MDA-MB-231 (breast), HCT116 (colon), SW620 (colon), and DU145 (prostate) human cancer cell lines. PMID:21243589

Kim, JinPyo; Lee, IkSoo; Ha, DoThi; Seo, JeongJu; Min, ByungSun; Yoo, IckDong; Bae, KiHwan



Effect of S-adenosyl-l-methionine (SAM), an allosteric activator of cystathionine-?-synthase (CBS) on colorectal cancer cell proliferation and bioenergetics in vitro.  


Recent data show that colon cancer cells selectively overexpress cystathionine-?-synthase (CBS), which produces hydrogen sulfide (H2S), to maintain cellular bioenergetics, support tumor growth and stimulate angiogenesis and vasorelaxation in the tumor microenvironment. The purpose of the current study was to investigate the effect of the allosteric CBS activator S-adenosyl-l-methionine (SAM) on the proliferation and bioenergetics of the CBS-expressing colon cancer cell line HCT116. The non-transformed, non-tumorigenic colon epithelial cell line NCM356 was used as control. For assessment of cell proliferation, the xCELLigence system was used. Bioenergetic function was measured by Extracellular Flux Analysis. Experiments using human recombinant CBS or HCT116 homogenates complemented the cell-based studies. SAM markedly enhanced CBS-mediated H2S production in vitro, especially when a combination of cysteine and homocysteine was used as substrates. Addition of SAM (0.1-3mM) to HCT116 cells induced a concentration-dependent increase H2S production. SAM exerted time- and concentration-dependent modulatory effects on cell proliferation. At 0.1-1mM SAM increased HCT116 proliferation between 0 and 12h, while the highest SAM concentration (3mM) inhibited proliferation. Over a longer time period (12-24h), only the lowest concentration of SAM used (0.1mM) stimulated cell proliferation; higher SAM concentrations produced a concentration-dependent inhibition. The short-term stimulatory effects of SAM were attenuated by the CBS inhibitor aminooxyacetic acid (AOAA) or by stable silencing of CBS. In contrast, the inhibitory effects of SAM on cell proliferation was unaffected by CBS inhibition or CBS silencing. In contrast to HCT116 cells, the lower rate of proliferation of the low-CBS expressor NCM356 cells was unaffected by SAM. Short-term (1h) exposure of HCT116 cells to SAM induced a concentration-dependent increase in oxygen consumption and bioenergetic function at 0.1-1mM, while 3mM was inhibitory. Longer-term (72h) exposure of HCT116 cells to all concentrations of SAM tested suppressed mitochondrial oxygen consumption rate, cellular ATP content and cell viability. The stimulatory effect of SAM on bioenergetics was attenuated in cells with stable CBS silencing, while the inhibitory effects were unaffected. In NCM356 cells SAM exerted smaller effects on cellular bioenergetics than in HCT116 cells. We have also observed a downregulation of CBS in response to prolonged exposure of SAM both in HCT116 and NCM356 cells. Taken together, the results demonstrate that H2S production in HCT116 cells is stimulated by the allosteric CBS activator, SAM. At low-to intermediate levels and early time periods the resulting H2S serves as an endogenous cancer cell growth and bioenergetic factor. In contrast, the inhibition of cell proliferation and bioenergetic function by SAM does not appear to relate to adverse autocrine effects of H2S resulting from CBS over-stimulation but, rather to CBS-independent pharmacological effects. PMID:24667534

Módis, Katalin; Coletta, Ciro; Asimakopoulou, Antonia; Szczesny, Bartosz; Chao, Celia; Papapetropoulos, Andreas; Hellmich, Mark R; Szabo, Csaba



Tcf3 and cell cycle factors contribute to butyrate resistance in colorectal cancer cells  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer We investigate mechanisms responsible for butyrate resistance in colon cancer cells. Black-Right-Pointing-Pointer Tcf3 modulates butyrate's effects on Wnt activity and cell growth in resistant cells. Black-Right-Pointing-Pointer Tcf3 modulation of butyrate's effects differ by cell context. Black-Right-Pointing-Pointer Cell cycle factors are overexpressed in the resistant cells. Black-Right-Pointing-Pointer Reversal of altered gene expression can enhance the anti-cancer effects of butyrate. -- Abstract: Butyrate, a fermentation product of dietary fiber, inhibits clonal growth in colorectal cancer (CRC) cells dependent upon the fold induction of Wnt activity. We have developed a CRC cell line (HCT-R) that, unlike its parental cell line, HCT-116, does not respond to butyrate exposure with hyperactivation of Wnt signaling and suppressed clonal growth. PCR array analyses revealed Wnt pathway-related genes, the expression of which differs between butyrate-sensitive HCT-116 CRC cells and their butyrate-resistant HCT-R cell counterparts. We identified overexpression of Tcf3 as being partially responsible for the butyrate-resistant phenotype, as this DNA-binding protein suppresses the hyperinduction of Wnt activity by butyrate. Consequently, Tcf3 knockdown in HCT-R cells restores their sensitivity to the effects of butyrate on Wnt activity and clonal cell growth. Interestingly, the effects of overexpressed Tcf3 differ between HCT-116 and HCT-R cells; thus, in HCT-116 cells Tcf3 suppresses proliferation without rendering the cells resistant to butyrate. In HCT-R cells, however, the overexpression of Tcf3 inhibits Wnt activity, and the cells are still able to proliferate due to the higher expression levels of cell cycle factors, particularly those driving the G{sub 1} to S transition. Knowledge of the molecular mechanisms determining the variable sensitivity of CRC cells to butyrate may assist in developing approaches that prevent or reverse butyrate resistance.

Chiaro, Christopher, E-mail: [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States)] [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States); Lazarova, Darina L., E-mail: [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States); Bordonaro, Michael, E-mail: [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States)] [Department of Basic Sciences, The Commonwealth Medical College, 525 Pine Street, Scranton, PA 18509 (United States)



Essential oil content of the rhizome of Curcuma purpurascens Bl. (Temu Tis) and its antiproliferative effect on selected human carcinoma cell lines.  


Curcuma purpurascens Bl., belonging to the Zingiberaceae family, is known as temu tis in Yogyakarta, Indonesia. In this study, the hydrodistilled dried ground rhizome oil was investigated for its chemical content and antiproliferative activity against selected human carcinoma cell lines (MCF7, Ca Ski, A549, HT29, and HCT116) and a normal human lung fibroblast cell line (MRC5). Results from GC-MS and GC-FID analysis of the rhizome oil of temu tis showed turmerone as the major component, followed by germacrone, ar-turmerone, germacrene-B, and curlone. The rhizome oil of temu tis exhibited strong cytotoxicity against HT29 cells (IC50 value of 4.9 ± 0.4??g/mL), weak cytotoxicity against A549, Ca Ski, and HCT116 cells (with IC50 values of 46.3 ± 0.7, 32.5 ± 1.1, and 35.0 ± 0.3??g/mL, resp.), and no inhibitory effect against MCF7 cells. It exhibited mild cytotoxicity against a noncancerous human lung fibroblast cell line (MRC5), with an IC50 value of 25.2 ± 2.7??g/mL. This is the first report on the chemical composition of this rhizome's oil and its selective antiproliferative effect on HT29. The obtained data provided a basis for further investigation of the mode of cell death. PMID:25177723

Hong, Sok-Lai; Lee, Guan-Serm; Syed Abdul Rahman, Syarifah Nur; Ahmed Hamdi, Omer Abdalla; Awang, Khalijah; Aznam Nugroho, Nurfina; Abd Malek, Sri Nurestri



[Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines].  


Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells. PMID:16568831

Tronov, V A; Kramarenko, I I; Smirnova, T D; Terekhov, S M



Zinc finger nuclease mediated knockout of ADP-dependent glucokinase in cancer cell lines: effects on cell survival and mitochondrial oxidative metabolism.  


Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of ADPGK, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of ADPGK, and were ADPGK-null by immunoblotting. ADPGK knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p?=?0.002) and 4.3±0.8% (p?=?0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when ADPGK was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of ADPGK in HCT116 cells caused few changes in global gene expression, knockout of ADPGK in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the ADPGK knockouts, in some cases markedly so. Collectively, the results demonstrate that ADPGK can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of ADPGK is cell line dependent and appears to be unrelated to priming of glycolysis in these lines. PMID:23799003

Richter, Susan; Morrison, Shona; Connor, Tim; Su, Jiechuang; Print, Cristin G; Ronimus, Ron S; McGee, Sean L; Wilson, William R



NCI in vitro and in silico anticancer screen, cell cycle pertubation and apoptosis-inducing potential of new acylated, benzylidene and isopropylidene derivatives of andrographolide.  


Andrographolide (AGP) is the main bioactive constituent isolated from the traditional medicinal, Andrographis paniculata which contributes towards its various biological activities, including anticancer property. In this study, a series of new AGP derivatives were semi-synthesised and screened against the NCI in vitro 60 cell lines. From the screening results, we had identified SRS07 as the most potent AGP derivative, against breast and colon cancer cell lines. Subsequently, SRS07 was tested for its capability to induce cell cycle arrest and apoptosis in MCF-7 and HCT116 cancer cells. SRS07 effectively induced G1 cell cycle arrest in both cell lines and ultimately apoptosis by inducing DNA fragmentation in HCT116 cells. The apoptotic cell death induced by SRS07 was confirmed via FITC Annexin-V double staining. Western blot analysis of SRS07-treated HCT116 cells revealed that the compound induced apoptosis be activating caspase 8 which in turn cleaved Bid to t-Bid to initiate cell death cascade. Prediction of the possible mode of action of SRS07 by utilising NCI COMPARE analysis failed to reveal a distinct mechanism category. Hence, it is speculated that SRS07 possesses novel mechanism of action. In conclusion, SRS07 demonstrated superior in vitro anticancer profiles and emerged as a potential lead anticancer candidate. PMID:25168151

Wong, Charng Choon; Sagineedu, Sreenivasa Rao; Sumon, Shariful Hasan; Sidik, Shiran Mohamad; Phillips, Roger; Lajis, Nordin H; Stanslas, Johnson



GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells.  


We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo. High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10 muM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (Delta Psi m). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21(Waf1/Cip1) was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p < 0.05) and extended tumor doubling time (p < 0.001). Our observations suggest that GHRH antagonist JMR-132 exerts its antiproliferative effect on experimental colon cancer cells through p21(Waf1/Cip1) mediated S-phase arrest along with apoptosis involving the intrinsic pathway. PMID:19755849

Hohla, Florian; Buchholz, Stefan; Schally, Andrew V; Seitz, Stefan; Rick, Ferenc G; Szalontay, Luca; Varga, Jozsef L; Zarandi, Marta; Halmos, Gabor; Vidaurre, Irving; Krishan, Awtar; Kurtoglu, Metin; Chandna, Sudhir; Aigner, Elmar; Datz, Christian



Synthesis of novel ciprofloxacin analogues and evaluation of their anti-proliferative effect on human cancer cell lines.  


A series of twenty two novel 1-cyclopropyl-6-fluoro-4-oxo-7-(4-substituted piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid analogues have been synthesized, characterized ((1)H NMR, (13)C NMR and LCMS) and evaluated for their inhibitory activity on the proliferation of human caucasian acute lymphoblastic leukemia cells (CCRF-CEM), breast adenocarcinoma cells (MDA-MB-468) and human colon carcinoma cells (HCT-116). Among all the synthesized ciprofloxacin analogues 3t at 50 ?M showed comparable potency to doxorubicin (10 ?M) in all three cell lines and 3j inhibited proliferation of MDA-MB-468 up to 35% selectively over other two cell lines. PMID:24138941

Suresh, Narva; Nagesh, Hunsur Nagendra; Sekhar, Kondapalli Venkata Gowri; Kumar, Anil; Shirazi, Amir N; Parang, Keykavous



Ganoderma lucidum polysaccharides target a Fas/caspase dependent pathway to induce apoptosis in human colon cancer cells.  


Ganoderma lucidum polysaccharides (GLP) extracted from Ganoderma lucidum have been shown to induce cell death in some kinds of cancer cells. This study investigated the cytotoxic and apoptotic effect of GLP on HCT-116 human colon cancer cells and the molecular mechanisms involved. Cell proliferation, cell migration, lactate dehydrogenase (LDH) levels and intracellular free calcium levels ([Ca(2+)]i) were determined by MTT, wound-healing, LDH release and fluorescence assays, respectively. Cell apoptosis was observed by scanning and transmission electron microscopy. For the mechanism studies, caspase-8 activation, and Fas and caspase-3 expression were evaluated. Treatment of HCT-116 cells with various concentrations of GLP (0.625-5 mg/mL) resulted in a significant decrease in cell viability (P< 0.01). This study showed that the antitumor activity of GLP was related to cell migration inhibition, cell morphology changes, intracellular Ca(2+) elevation and LDH release. Also, increase in the levels of caspase-8 activity was involved in GLP-induced apoptosis. Western blotting indicated that Fas and caspase-3 protein expression was up-regulated after exposure to GLP. This investigation demonstrated for the first time that GLP shows prominent anticancer activities against the HCT-116 human colon cancer cell line through triggering intracellular calcium release and the death receptor pathway. PMID:24935584

Liang, Zengenni; Guo, Yu-Tong; Yi, You-Jin; Wang, Ren-Cai; Hu, Qiu-Long; Xiong, Xing-Yao



Correlation between T-cadherin gene expression and aberrant methylation of T-cadherin promoter in human colon carcinoma cells  

Microsoft Academic Search

Previous researches showed T-cadherin (CDH13) expression was downregulated in colon cancer tissues and was associated with\\u000a increase of invasive and metastatic potential. This research was to observe the mechanisms responsible for inactivation of\\u000a T-cadherin gene in colon carcinoma; we investigated the methylation status around the 5? promoter region of T-cadherin gene\\u000a of Hct116 colon cancer cell line by methylation-specific polymerase

Jian-zhen Ren; Ji-rong Huo


Hedgehog-glioma-associated oncogene homolog-1 signaling in colon cancer cells and its role in the celecoxib-mediated anti-cancer effect  

PubMed Central

Hedgehog (Hh) signaling is activated in numerous malignant tumors, but its role in human colorectal cancer remains uncertain. Celecoxib, a selective cyclooxygenase-2 inhibitor, has been shown to exhibit chemoprevention in colorectal cancer, however, the effects of celecoxib on Hh signaling remain unknown. The current study presents an evaluation of Hh signaling in colon cancer cell lines and the effects of celecoxib in vitro. Active Hh signaling was observed in LoVo and HT-29 cells, with particularly high levels in the LoVo cells. However, Hh signaling activity was absent in HCT-116 cells. Quantitative polymerase chain reaction indicated that the expression of Hh receptor patched homolog 1 (PTCH1) was absent in the LoVo cells, but that they exhibited high levels of glioma-associated oncogene homolog-1 (GLI1) expression, while high expression levels of PTCH1 and low expression levels of smoothened (SMO) and GLI1 were observed in the HCT-116 cells. The HCT-116 cells were extremely sensitive to celecoxib, whereas the LoVo cells were resistant to the anticancer effect of the drug. Celecoxib downregulated the expression of GLI1 in the HCT-116 and HT-29 cells, but did not change the expression of GLI1 in the LoVo cells. The results presented in this study indicated that the anticancer effect of celecoxib is selective in colon cancer cells; celecoxib may target cancer cells via the SMO-independent modulation of GLI1 activity, and Hh signaling may be significant in maintaining the malignant state of LoVo cells. These findings may aid in improving our understanding of the carcinogenesis of colon cancer and the development of novel approaches for the targeted therapy of this disease. PMID:25295109




Resveratrol inhibits proliferation, angiogenesis and induces apoptosis in colon cancer cells: calorie restriction is the force to the cytotoxicity.  


The aim of this study was to examine the antitumour activity of resveratrol in human colorectal cancer cell lines (HCT116 and Caco2) and to explore its mechanism of action assuming that it is by calorie-restriction effect. Resveratrol inhibited the proliferation of colon cancer cells with half maximal inhibitory concentration (IC(50)) equal to 50 and 130 ?M for HCT116 and Caco2, respectively. Caco2 cells appeared with significant time-dependent increase in the glycolytic pathway, a behaviour that was absent in HCT116 cells. Resveratrol (100 ?M) significantly decreased the glycolytic enzymes (pyruvate kinase and lactate dehydrogenase) in Caco2 cells, while an increase in citrate synthase activity and a decrease in glucose consumption were observed in both cell lines. Moreover, resveratrol downregulated the expressions of leptin and c-Myc, and decreased the content of vascular endothelial growth factor. The apoptotic markers, caspases 3 and 8, were activated and the Bax/BCl2 ratio was increased. The study suggested a promising anticancer activity of resveratrol, calorie-restriction pathway may be one of the driving forces for this activity. PMID:23536519

Fouad, M A; Agha, A M; Merzabani, M M Al; Shouman, S A



Intrinsic cellular resistance to oxazaphosphorines exhibited by a human colon carcinoma cell line expressing relatively large amounts of a class-3 aldehyde dehydrogenase.  


A cultured human colon carcinoma cell line, viz. colon C, exhibiting intrinsic cellular resistance to mafosfamide mediated by relatively elevated levels of a cytosolic class-3 aldehyde dehydrogenase was identified. Colon C cells were found to be much less sensitive/more resistant (about 10-fold as judged by LC90 values) to mafosfamide than were two other cultured human colon carcinoma cell lines, viz. RCA and HCT 116b, and, as compared to the barely detectable aldehyde dehydrogenase activity (NADP-dependent enzyme-catalyzed oxidation of benzaldehyde to benzoic acid) in RCA and HCT 116b cells, that in colon C cells was about 200-fold greater. The three cell lines were equisensitive to phosphoramide mustard. Aldehyde dehydrogenase activity was confined to the cytosol in colon C cells (as well as in the other two cell lines) and, on the basis of its physical, immunological and catalytic characteristics, the operative enzyme was judged to be a Type-1 ALDH-3 identical to the Type-1 ALDH-3 expressed in methylcholanthrene-treated human breast adenocarcinoma MCF-7/0 cells and very nearly identical to the Type-1 ALDH-3 expressed in human normal stomach mucosa. Class-1 and class-2 aldehyde dehydrogenases were not found in these cells. The relative insensitivity to mafosfamide on the part of colon C cells was not observed when exposure to mafosfamide was in the presence of benzaldehyde or 4-(diethylamino)benzaldehyde, each a relatively good substrate for ALDH-3, whereas it was retained when exposure to mafosfamide was in the presence of acetaldehyde, a relatively poor substrate for this enzyme. Sensitivity to mafosfamide on the part of HCT 116b and RCA cells, and to phosphoramide mustard on the part of all three cell lines, was unaffected when drug exposure was in the presence of any of the three aldehydes. Together with earlier reports from our laboratory, these observations demonstrate that intrinsic, as well as stable and transient acquired, resistance to oxazaphosphorines, such as mafosfamide and cyclophosphamide, can be mediated by relatively increased levels of cytosolic class-3 aldehyde dehydrogenases. PMID:7986206

Rekha, G K; Sreerama, L; Sladek, N E



Proteomic profiling of human colon cancer cells treated with the histone deacetylase inhibitor belinostat.  


The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 muM belinostat were analysed by 2-D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed 45 unique differentially expressed proteins that were identified by LC-MSMS analysis. Among these proteins, of particular interest are the downregulated proteins nucleophosmin and stratifin, and the upregulated proteins nucleolin, gelsolin, heterogeneous nuclear ribonucleoprotein K, annexin 1, and HSP90B that all were related to the proto-oncogene proteins p53, Myc, activator protein 1, and c-fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be discussed. PMID:20717991

Beck, Hans Christian; Petersen, Jørgen; Nielsen, Søren Jensby; Morsczeck, Christian; Morszeck, Christian; Jensen, Peter B; Sehested, Maxwell; Grauslund, Morten



Cytotoxicity of Probiotics from Philippine Commercial Dairy Products on Cancer Cells and the Effect on Expression of cfos and cjun Early Apoptotic-Promoting Genes and Interleukin-1? and Tumor Necrosis Factor-? Proinflammatory Cytokine Genes  

PubMed Central

This study determined cytotoxicity of probiotic Lactobacillus spp. from Philippine dairy products on cancer cells and normal fibroblasts and their effects on expression of early apoptotic-promoting cfos, cjun and proinflammatory cytokine IL-1?, TNF-? genes. Cultures were from Yakult, Bear Brand Probiotic Drink, Nido3+ Powdered Milk. Filter-sterilized supernatants from cultures of Lactobacillus spp. were evaluated for cytotoxicity to colon cancer cells (HT-29 and HCT116), leukemia cells (THP-1), and normal human dermal fibroblasts (HDFn) using PrestoBlue. Bleomycin was the positive control. Absolute quantification of transcript levels was conducted using qRT-PCR. Cytotoxicity index profiles on HDFn, THP-1 of all probiotic supernatants and negative controls suggest nontoxicity to the cells when compared to bleomycin, whereas all probiotic supernatants were found to be cytotoxic to HT-29 and HCT-116 colon cancer cell lines. Expression of cfos, cjun transcripts was significantly upregulated in HT-29 and HCT116 cells treated with probiotic supernatants compared to untreated baseline levels (P < 0.05). Expression of IL-1? and TNF-? by lipopolysaccharide-treated macrophages was significantly downregulated in cells with probiotic supernatants compared to those exposed to MRS medium (P < 0.05). Results provide strong support for the role of Lactobacillus spp. studied in anticancer therapy and in prevention of inflammation that may act as precursor to carcinogenesis.

Shyu, Peter T.; Oyong, Glenn G.; Cabrera, Esperanza C.



Systems analysis of cancer cell heterogeneity in caspase-dependent apoptosis subsequent to mitochondrial outer membrane permeabilization.  


Deregulation of apoptosis is a hallmark of carcinogenesis. We here combine live cell imaging and systems modeling to investigate caspase-dependent apoptosis execution subsequent to mitochondrial outer membrane permeabilization (MOMP) in several cancer cell lines. We demonstrate that, although most cell lines that underwent MOMP also showed robust and fast activation of executioner caspases and apoptosis, the colorectal cancer cell lines LoVo and HCT-116 Smac(-/-), similar to X-linked inhibitor of apoptosis protein (XIAP)-overexpressing HeLa (HeLa XIAP(Adv)) cells, only showed delayed and often no caspase activation, suggesting apoptosis impairment subsequent to MOMP. Employing APOPTO-CELL, a recently established model of apoptosis subsequent to MOMP, this impairment could be understood by studying the systemic interaction of five proteins that are present in the apoptosis pathway subsequent to MOMP. Using APOPTO-CELL as a tool to study detailed molecular mechanisms during apoptosis execution in individual cell lines, we demonstrate that caspase-9 was the most important regulator in DLD-1, HCT-116, and HeLa cells and identified additional cell line-specific co-regulators. Developing and applying a computational workflow for parameter screening, systems modeling identified that apoptosis execution kinetics are more robust against changes in reaction kinetics in HCT-116 and HeLa than in DLD-1 cells. Our systems modeling study is the first to draw attention to the variability in cell specific protein levels and reaction rates and to the emergent effects of such variability on the efficiency of apoptosis execution and on apoptosis impairment subsequent to MOMP. PMID:23038270

Schmid, Jasmin; Dussmann, Heiko; Boukes, Gerhardt J; Flanagan, Lorna; Lindner, Andreas U; O'Connor, Carla L; Rehm, Markus; Prehn, Jochen H M; Huber, Heinrich J



Crocin from Crocus Sativus Possesses Significant Anti-Proliferation Effects on Human Colorectal Cancer Cells  

PubMed Central

Aim To investigate the anti-proliferative effects of Crocus sativus extract and its major constituent, crocin, on three colorectal cancer cell lines (HCT-116, SW-480, and HT-29). The cell growth inhibition effect was compared to that of non-small cell lung cancer (NSCLC) cells. In addition, Crocus sativus' effect on non-cancer cells was evaluated. Methods Using high performance liquid chromatography (HPLC), the purity of crocin and the content of crocin extract were determined. Anti-proliferative effects of Crocus sativus extract and crocin on test cells was evaluated by MTS assay. Results The purity of crocin was found to be 95.9% and the content of crocin in the extract was 22.9%. Significant concentration-related inhibition effects of the extract on all three colorectal cancer cell lines were observed (P < 0.01). The proliferation was reduced most significantly in HCT-116 cells, to 45.5% at 1.0 mg/ml and to 6.8 % at 3.0 mg/ml. Crocin at 1.0 mM, significantly reduced HCT-116, SW-480, and HT-29 cell proliferation to 2.8%, 52%, and 16.8%, respectively (P < 0.01). Since 3.0 mg/ml Crocus sativus extract contained approximately 0.6 mM crocin, the observed effects suggest that crocin is a major responsible constituent in the extract. Significant anti-proliferative effects were also observed in non-small cell lung cancer cells. However, Crocus sativus extract did not significantly affect the growth of non-cancer young adult mouse colon cells. Conclusion Data from this study demonstrated that Crocus sativus extract and its major constituent, crocin, significantly inhibited the growth of colorectal cancer cells while not affecting normal cells. Crocus sativus extract should be investigated further as a viable option in the treatment of colorectal cancer. PMID:18004240

Aung, H.H.; Wang, C.Z.; Ni, M.; Fishbein, A.; Mehendale, S.R.; Xie, J.T.; Shoyama, A.Y.; Yuan, C.S.



150 kDa glycoprotein isolated from Solanum nigrum Linne stimulates caspase-3 activation and reduces inducible nitric oxide production in HCT116 cells  

Microsoft Academic Search

This study was carried out to investigate the apoptotic effects of glycoprotein (SNL glycoprotein, 150-kDa) isolated from Solanum nigrum Linne, which has been used as an antipyretic and anticancer agent in folk medicine. We found that SNL glycoprotein consists of carbohydrate content (69.74%) and protein content (30.26%), which contains more than 50% hydrophobic amino acids such as glycine and proline.

Sei-Jung Lee; Kye-Taek Lim



Infant intestinal Enterococcus faecalis down-regulates inflammatory responses in human intestinal cell lines  

PubMed Central

AIM: To investigate the ability of Lactic acid bacteria (LAB) to modulate inflammatory reaction in human intestinal cell lines (Caco-2, HT-29 and HCT116). Different strains of LAB isolated from new born infants and fermented milk, together with the strains obtained from culture collections were tested. METHODS: LABs were treated with human intestinal cell lines. ELISA was used to detect IL-8 and TGF-? protein secretion. Cytokines and Toll like receptors (TLRs) gene expression were assessed using RT-PCR. Conditional medium, sonicated bacteria and UV killed bacteria were used to find the effecter molecules on the bacteria. Carbohydrate oxidation and protein digestion were applied to figure out the molecules’ residues. Adhesion assays were further carried out. RESULTS: It was found that Enterococcus faecalis is the main immune modulator among the LABs by downregulation of IL-8 secretion and upregulation of TGF-?. Strikingly, the effect was only observed in four strains of E. faecalis out of the 27 isolated and tested. This implies strain dependent immunomodulation in the host. In addition, E. faecalis may regulate inflammatory responses through TLR3, TLR4, TLR9 and TRAF6. Carbohydrates on the bacterial cell surface are involved in both its adhesion to intestinal cells and regulation of inflammatory responses in the host. CONCLUSION: These data provide a case for the modulation of intestinal mucosal immunity in which specific strains of E. faecalis have uniquely evolved to maintain colonic homeostasis and regulate inflammatory responses. PMID:18286689

Wang, Shugui; Ng, Lydia Hui Mei; Chow, Wai Ling; Lee, Yuan Kun



Inhibition of PCAF histone acetyltransferase, cytotoxicity and cell permeability of 2-acylamino-1-(3- or 4-carboxy-phenyl)benzamides.  


Small molecule HAT inhibitors are useful tools to unravel the role of histone acetyltransferases (HATs) in the cell and they also have relevance in oncology. We synthesized a series of 2-acylamino-1-(3- or 4-carboxyphenyl)benzamides 8–19 bearing C6, C8, C10, C12, C14, and C16 acyl chains at the 2-amino position of 2-aminobenzoic acid. Enzyme inhibition of these compounds was investigated using in vitro PCAF HAT assays. The inhibitory activities of compounds 8–10, 16, and 19 were similar to that of anacardic acid, and 17 was found to be more active than anacardic acid at 100 ?M. Compounds 11–15 showed the low inhibitory activity on PCAF HAT. The cytotoxicity of the synthesized compounds was evaluated by SRB (sulforhodamine B) assay against seven human cancer cell lines: HT-29 (colon), HCT-116 (colon), MDA-231 (breast), A549 (lung), Hep3B (hepatoma), HeLa (cervical) and Caki (kidney) and one normal cell line (HSF). Compound 17 was more active than anacardic acid against human colon cancer (HCT 116, IC(50): 29.17 ?M), human lung cancer (A549, IC??: 32.09 ?M) cell lines. 18 was more active than anacardic acid against human colon cancer (HT-29, IC??: 35.49 ?M and HCT 116, IC??: 27.56 ?M), human lung cancer (A549, IC??: 30.69 ?M), and human cervical cancer (HeLa, IC??: 34.41 ?M) cell lines. The apparent permeability coefficient (P(app), cm/s) values of two compounds (16 and 17) were evaluated as 68.21 and 71.48 × 10?? cm/s by Caco-2 cell permeability assay. PMID:23128090

Park, Woong Jae; Ma, Eunsook



Cell death by the quinoxaline dioxide DCQ in human colon cancer cells is enhanced under hypoxia and is independent of p53 and p21  

Microsoft Academic Search

INTRODUCTION: We have shown that the radio sensitizer DCQ enhances sensitivity of HCT116 human colon cancer cells to hypoxia. However, it is not known whether the p53 or p21 genes influence cellular response to DCQ. In this study, we used HCT116 that are either wildtype for p53 and p21, null for p53 or null for p21 to understand the role

Mona El-Khatib; Fady Geara; Makhluf J Haddadin; Hala Gali-Muhtasib



Induction of apoptosis against cancer cell lines by four ascomycetes (endophytes) from Malaysian rainforest.  


Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with IC(50) values less than 17 ?g/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis. PMID:22397996

Hazalin, Nurul Aqmar Mohamad Nor; Ramasamy, Kalavathy; Lim, Siong Meng; Cole, Anthony L J; Majeed, Abu Bakar Abdul



A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.  


Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA), epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape seed [GS] extract) at low cytotoxic concentrations on CRC cells and test their activity on mTOR and translational regulation. The CRC cell lines HCT116 and SW480 were treated for 24h with combinations of EPA-FFA (0-150 µM), EGCG (0-175 µM) and GS extract (0-15 µM) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 µM, EGCG 175 µM and GS extract 15 µM completely inhibited the mTOR signaling in HCT116 and SW480 cells, reaching an effect stronger than or comparable to that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells, respectively. Moreover, the treatment led to changes of protein translation of ribosomal proteins, c-Myc and cyclin D1. In addition, we found a reduction of clonal capability in both cell lines, with block of cell cycle in G0G1 and induction of apoptosis. Our data suggest that the low cytotoxic combination of EPA-FFA, EGCG and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment. PMID:25123131

D'Angelo, Leonarda; Piazzi, Giulia; Pacilli, Annalisa; Prossomariti, Anna; Fazio, Chiara; Montanaro, Lorenzo; Graziani, Giulia; Fogliano, Vincenzo; Munarini, Alessandra; Bianchi, Francesca; Belluzzi, Andrea; Bazzoli, Franco; Ricciardiello, Luigi



Antiproliferative effects of protopanaxadiol ginsenosides on human colorectal cancer cells  

PubMed Central

Ginsenosides are the main biologically active components of ginseng. In this study, seven types of protopanaxadiol ginsenosides were assessed for their antiproliferative activity on the HCT-116 and HT-29 human colorectal cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The experimental results indicated that the native protopanaxadiol ginsenosides Rb1 and Rb2 inhibited the proliferation of the colorectal cancer cells in a dose-dependent manner. The deglycosylation products F2 and CO (from ginsenosides Rb1 and Rb2, respectively) significantly inhibited the growth of the human colorectal cancer cell lines, whereas product C-K (from Rb1 and Rb2) exerted no antiproliferative effects on the cancer cell lines assessed in this study. HT-29 cells were more sensitive to these ginsenosides compared to HCT-116 cells. In addition, the antiproliferative activity of ginsenosides was found to be correlated with the number and type of sugar residues. The potent growth inhibitory effect of protopanaxadiol ginsenosides on cancer cells may be used in the pharmaceutical industry. PMID:24648985




Withaferin A modulates the Spindle assembly checkpoint by degradation of Mad2-Cdc20 complex in colorectal cancer cell lines.  


Withania somnifera L. Dunal (Ashwagandha) is used over centuries in the ayurvedic medicines in India. Withaferin A, a withanolide, is the major compound present in leaf extract of the plant which shows anticancer activity against leukemia, breast cancer and colorectal cancer. It arrests the ovarian cancer cells in the G2/M phase in dose dependent manner. In the current study we show the effect of Withaferin A on cell cycle regulation of colorectal cancer cell lines HCT116 and SW480 and its effect on cell fate. Treatment of these cells with this compound leads to apoptosis in a dose dependent manner. It causes the G2/M arrest in both the cell lines. We show that Withaferin A (WA) causes mitotic delay by blocking Spindle assembly checkpoint (SAC) function. Apoptosis induced by Withaferin A is associated with proteasomal degradation of Mad2 and Cdc20, an important constituent of the Spindle Checkpoint Complex. Further overexpression of Mad2 partially rescues the deleterious effect of WA by restoring proper anaphase initiation and keeping more number of cells viable. We hypothesize that Withaferin A kills cancer cells by delaying the mitotic exit followed by inducing chromosome instability. PMID:24995417

Das, Tania; Roy, Kumar Singha; Chakrabarti, Tulika; Mukhopadhyay, Sibabrata; Roychoudhury, Susanta



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

PubMed Central

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

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



B7h triggering inhibits the migration of tumor cell lines.  


Vascular endothelial cells (ECs) and several cancer cells express B7h, which is the ligand of the ICOS T cell costimulatory molecule. We have previously shown that B7h triggering via a soluble form of ICOS (ICOS-Fc) inhibits the adhesion of polymorphonuclear and tumor cell lines to HUVECs; thus, we suggested that ICOS-Fc may act as an anti-inflammatory and antitumor agent. Because cancer cell migration and angiogenesis are crucial for metastasis dissemination, the aim of this work was to evaluate the effect of ICOS-Fc on the migration of cancer cells and ECs. ICOS-Fc specifically inhibited the migration of HUVECs, human dermal lymphatic ECs, and the HT29, HCT116, PC-3, HepG2, JR8, and M14 tumor cell lines expressing high levels of B7h, whereas it was ineffective in the RPMI7932, PCF-2, LM, and BHT-101 cell lines expressing low levels of B7h. Furthermore, ICOS-Fc downmodulated hepatocyte growth factor facilitated the epithelial-to-mesenchymal transition in HepG2 cells. Moreover, ICOS-Fc downmodulated the phosphorylation of focal adhesion kinase and the expression of ?-Pix in both HUVECs and tumor cell lines. Finally, treatment with ICOS-Fc inhibited the development of lung metastases upon injection of NOD-SCID-IL2R?null mice with CF-PAC1 cells, as well as C57BL/6 mice with B16-F10 cells. Therefore, the B7h-ICOS interaction may modulate the spread of cancer metastases, which suggests the novel use of ICOS-Fc as an immunomodulatory drug. However, in the B16-F10-metastasized lungs, ICOS-Fc also increased IL-17A/RORc and decreased IL-10/Foxp3 expression, which indicates that it also exerts positive effects on the antitumor immune response. PMID:24729612

Dianzani, Chiara; Minelli, Rosalba; Gigliotti, Casimiro Luca; Occhipinti, Sergio; Giovarelli, Mirella; Conti, Laura; Boggio, Elena; Shivakumar, Yogesh; Baldanzi, Gianluca; Malacarne, Valeria; Orilieri, Elisabetta; Cappellano, Giuseppe; Fantozzi, Roberto; Sblattero, Daniele; Yagi, Junji; Rojo, Josè Maria; Chiocchetti, Annalisa; Dianzani, Umberto



ER stress signaling in ARPE-19 cells after inhibition of protein kinase CK2 by CX-4945.  


Protein kinase CK2 is a critical factor for the survival of cells. It is overexpressed in many cancer cells and provides protection against apoptosis in these cells. Inhibition of CK2 kinase activity in various cancer cells leads to apoptosis, which makes CK2 an attractive target for cancer therapy. Little is, however, known about CK2 inhibition in non-cancerous cells. Using the human retinal pigment epithelial cell line ARPE-19, we analyzed the formation of reactive oxygen species (ROS) and the ER stress signaling pathway after CK2 inhibition with CX-4945. Following CK2 inhibition, we did not find any significant generation of ROS in neither ARPE-19 non-cancer cells nor in HCT116 cancer cells. We found an induction of the ER stress pathway including the activation of eIF2? and ATF4 in both cell types. This activation was sufficient for ARPE-19 cells to cope with the ER stress. Furthermore, in contrast to HCT116 cancer cells, there was no induction of the pro-apoptotic transcription factor CHOP and no induction of apoptosis in the ARPE-19 cells. Overexpression of CHOP, however, induced apoptosis in ARPE-19 cells indicating that this step in the ER stress pathway is abrogated in normal cells compared to cancer cell. PMID:24686080

Intemann, Johanna; Saidu, Nathaniel Edward Bennett; Schwind, Lisa; Montenarh, Mathias



The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells  

PubMed Central

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis post-exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered N-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer. PMID:18413749

Derdak, Zoltan; Mark, Nicholas M.; Beldi, Guido; Robson, Simon C.; Wands, Jack R.; Baffy, Gyorgy



Aspirin upregulates expression of urokinase type plasminogen activator receptor (uPAR) gene in human colon cancer cells through AP1.  


In this study, the effects of acetylsalicylic acid (aspirin) on the expression of uPAR and the mechanism by which it regulates expression of uPAR was examined in two different colon cancer cell lines HCT116 and GEO, respectively. The study shows that under physiological concentration, aspirin upregulates steady-state level expression of uPAR mRNA as well as expression of uPAR protein. Using a transient transfection assay, a region corresponding to -1 to -398 region of uPAR promoter has been identified which shows maximum responsiveness to aspirin treatment and found that this region is sufficient for the aspirin-induced up-regulation of uPAR. A stable integration of a single copy of this region coupled to luciferase reporter gene into the HCT116 genome also behaved similarly. Using gel mobility shift assays, it is found that the distal AP1 region between -171 and -186 is responsible for the aspirin-induced up-regulation of uPAR. Mutation of this region reduced up-regulation. Supershift assays identify that the bound proteins at this region are c-Jun and Fra-1. Real-time PCR analysis showed more than 4-fold increase in the binding of c-Jun and a 1.6-fold increase in the binding of Fra-1 in this region and this up-regulation corresponds to an increased binding of acetylated histone H4 in this region. Since an increase in the expression of uPAR corresponds to an increase in the migration of the cell, a migration assay was performed and result showed a 3-fold increased migration of HCT116 cells through the vitronectin-coated layer. Thus, an AP1 mediated pathway for aspirin induced up-regulation of uPAR has been identified. PMID:16893520

Jamaluddin, Md Saha



KRAS G13D Mutation and Sensitivity to Cetuximab or Panitumumab in a Colorectal Cancer Cell Line Model  

PubMed Central

ABSTRACT BACKGROUND: The treatment of metastatic colorectal cancer (mCRC) includes drugs targeting the epidermal growth factor receptor (EGFR). Mutation in codon 12 or 13 in the Kirsten rat sarcoma viral oncogene homolog (KRAS) gene, downstream of the EGFR, evokes constitutive activation of the RAS/RAF/MAPK signaling pathway and correlates with resistance to anti-EGFR monoclonal antibody (mAb) therapies. However, a retrospective study reported that a proportion of patients with the KRAS G13D mutation may respond to cetuximab. A similar analysis for panitumumab was not as conclusive. We sought to determine the sensitivity of CRC cell lines to cetuximab or panitumumab treatment and to investigate the correlation of the KRAS mutational status of the CRC cell lines to the responsiveness to cetuximab or panitumumab. METHODS: To determine the responsiveness of CRC cell lines to cetuximab or panitumumab, cell lines were treated with an optimized concentration of each mAb, and proliferation assays were conducted. RESULTS: After treatment with cetuximab or panitumumab, at the optimum concentration of 8 ?g/well, the KRAS G13D mutant cell lines HCT-116, LoVo, and T84 showed intermediate sensitivity to both treatments, between the resistant KRAS G12V mutant cell line SW480 and the sensitive KRAS wild-type cell line LIM1215. One of the G13D cell lines was significantly more sensitive to panitumumab than to cetuximab (P = .02). CONCLUSION: The specific KRAS mutation determines the responsiveness to anti-EGFR monoclonal antibody treatment, corresponding to reported clinical observations. PMID:24558511

Kumar, Shalini Sree; Price, Timothy J.; Mohyieldin, Omar; Borg, Matthew; Townsend, Amanda



Chrysin promotes tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induced apoptosis in human cancer cell lines.  


Chrysin exists widely in plants, honey and propolis. The anti-cancer property of chrysin has been demonstrated though the molecular mechanism is not clear. In this study, we found that pre-treatment with chrysin could promote the cell death induced by TRAIL according to the morphological changes and appearance of sub-G1 peak in four human cancer cell lines. In HCT-116 cells, the results of flow cytometry analysis showed that the percentage of sub-G1 reached (38.89 ± 3.78) % when pre-treatment of chrysin was used at 40 ?M, but that was only (2.53 ± 0.10) % in the untreated group and (13.22 ± 0.20) % in TRAIL alone group. The differences between the combination and the untreated or TRAIL alone group were all significant (P<0.05) and dose-dependent effect was obvious. Similar results were obtained in CNE1 cells. In the search of molecular mechanisms, we found that pre-treatment with chrysin could increase TRAIL-induced degradation of caspase 3, caspase 8, PARP proteins. Z-VAD-fmk, which is a pan-caspase inhibitor, could inhibit the apoptosis enhanced by the combination of chrysin and TRAIL. All data indicate that chrysin can enhance the apoptosis induced by TRAIL, and the apoptosis is caspase-dependent and related to the activation of caspase 8. PMID:21195158

Li, Xin; Wang, Jian-Ning; Huang, Jun-Ming; Xiong, Xi-Kun; Chen, Mei-Fen; Ong, Choon-Nam; Shen, Han-Ming; Yang, Xing-Fen



Hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells.  


In this paper, a targeted drug delivery system has been developed based on hyaluronic acid (HA) modified mesoporous silica nanoparticles (MSNs). HA-MSNs possess a specific affinity to CD44 over-expressed on the surface of a specific cancer cell line, HCT-116 (human colon cancer cells). The cellular uptake performance of fluorescently labelled MSNs with and without HA modification has been evaluated by confocal microscopy and fluorescence-activated cell sorter (FACS) analysis. Compared to bare MSNs, HA-MSNs exhibit a higher cellular uptake via HA receptor mediated endocytosis. An anticancer drug, doxorubicin hydrochloride (Dox), has been loaded into MSNs and HA-MSNs as drug delivery vehicles. Dox loaded HA-MSNs show greater cytotoxicity to HCT-116 cells than free Dox and Dox-MSNs due to the enhanced cell internalization behavior of HA-MSNs. It is expected that HA-MSNs have a great potential in targeted delivery of anticancer drugs to CD44 over-expressing tumors. PMID:23076766

Yu, Meihua; Jambhrunkar, Siddharth; Thorn, Peter; Chen, Jiezhong; Gu, Wenyi; Yu, Chengzhong



Targeting colorectal cancer cells with single-walled carbon nanotubes conjugated to anticancer agent SN-38 and EGFR antibody.  


In this study, single-walled carbon nanotubes (SWNTs) conjugated with antibody C225 were used to achieve targeted therapy against EGFR over-expressed colorectal cancer cells. In addition, the control release of the chemotherapeutic drug, 7-Ethyl-10-hydroxy-camptothecin (SN38), was studied. We used three different colorectal cancer cell lines, HCT116, HT29, and SW620, listed in the order of decreasing expression levels of EGFR. Our results showed that SWNT could use C225 to specifically bind to EGFR-expressed cells. The cellular uptakes of SWNT of EGFR over-expressed cells (HCT116 and HT29) were much higher than that of the negative control (SW620). We, next, demonstrated that receptor-mediated endocytosis was the primary cell entry route for SWNT. As a consequence, abundant amount of SN38 was released and EGFR over-expressed cells were killed. The drug control release process was studied by utilizing human carboxylesterase enzyme (hCE) that would break the bond linking SN38 and SWNT-carrier in cytoplasm. The intracellular SN38 release observed by confocal microscopy showed that SN38 actually dissociated from the SWNT-carrier first. SN38's entry to nucleus was then followed while the SWNT-carrier still remained in the cytoplasm. Overall, all these data suggested that SWNT could be a good carrier for targeting controlled release therapy. PMID:23937913

Lee, Pei-Chi; Chiou, Yu-Chi; Wong, Jau-Min; Peng, Cheng-Liang; Shieh, Ming-Jium



p53 is involved in clearance of ionizing radiation-induced RAD51 foci in a human colon cancer cell line  

SciTech Connect

We have investigated p53-related differences in cellular response to DNA damaging agents, focusing on p53s effects on RAD51 protein level and sub-cellular localization post exposure to ionizing radiation. In a human colon cancer cell line, HCT116 and its isogenic p53-/- subcell line we show here p53-independent RAD51 foci formation but interestingly the resolution of RAD51 foci showed clear p53 dependence. In p53 wt cells, but not in p53-/- cells, RAD51 protein level decreased 48 h post irradiation and fluorescence immunostaining showed resolution of RAD51 foci and relocalization of RAD51 to nucleoli at time points corresponding to the decrease in RAD51 protein level. Both cell lines rejoined DNA double strand breaks efficiently with similar kinetics and p53 status did not influence sensitivity to DNA damaging agents. We suggest that p53 has a role in RAD51 clearance post DSB repair and that nucleoli might be sites of RAD51 protein degradation.

Orre, Lukas M. [Cancer Centrum Karolinska Institutet, Department of Oncology and Pathology, Division of Medical Radiation Biology, Stockholm (Sweden)]. E-mail:; Stenerloew, Bo [Division of Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Stockholm (Sweden); Dhar, Sumeer [Division of Clinical Pharmacology, Department of Medical Sciences, University Hospital, Uppsala (Sweden); Larsson, Rolf [Division of Clinical Pharmacology, Department of Medical Sciences, University Hospital, Uppsala (Sweden); Lewensohn, Rolf [Cancer Centrum Karolinska Institutet, Department of Oncology and Pathology, Division of Medical Radiation Biology, Stockholm (Sweden); Lehtioe, Janne [Cancer Centrum Karolinska Institutet, Department of Oncology and Pathology, Division of Medical Radiation Biology, Stockholm (Sweden)



Oyaksungisan, a Traditional Herbal Formula, Inhibits Cell Proliferation by Induction of Autophagy via JNK Activation in Human Colon Cancer Cells  

PubMed Central

Oyaksungisan (OY) is a traditional herbal formula broadly used to treat beriberi, vomiting, diarrhea, and circulatory disturbance in Asian countries from ancient times. The effect of OY on cancer, however, was not reported until now. In this study, we have demonstrated that OY inhibits cell proliferation and induces cell death via modulating the autophagy on human colon cancer cells. In HCT116 cells, OY increased the ratio of LC3-II/LC3-I, a marker of autophagy, and treatment with 3-MA, an inhibitor of autophagy, and considerably reduced the formation of autophagosomes. In addition, OY regulated mitogen-activated protein kinase (MAPK) cascades; especially, JNK activation was closely related with autophagy effect by OY in HCT116 cells. Our results indicate that autophagy induction is responsible for the antiproliferative effect by OY, despite the weak apoptosis induction in HCT116 cells. In conclusion, OY might have a potential to be developed as an herbal anticancer remedy. PMID:23573119

Yim, Nam-Hui; Jung, Young Pil; Kim, Aeyung; Ma, Choong Je; Cho, Won-Kyung; Ma, Jin Yeul



Methylated DNA-binding protein 2 antisense inhibitors suppress tumourigenesis of human cancer cell lines in vitro and in vivo.  


Methylated DNA-binding protein 2 (MBD2) has been proposed to function both as a silencer of methylated genes and as a DNA demethylase. Our previous data indicated that knockdown of MBD2 inhibited tumourigenesis of human cancer lines and MBD2-deficient mice were recently shown to be resistant to intestinal tumourigenesis. MBD2 is an attractive anticancer target since MBD2-deficient mice were previously shown to be viable and fertile and knockdown of MBD2 was reported to have no effect on cellular growth parameters of non-transformed cells. In this paper we test the hypothesis that pharmacological inhibition of MBD2 inhibits cancer growth in vivo using human tumour lines implanted in mice as a model. We develop sequence-specific antisense inhibitors of MBD2 and we show that these agents inhibit anchorage-independent growth of human lung (A549) and colorectal (HCT116) cancer cell lines in vitro and tumourigenic growth of human cancer cell xenografts in vivo. MBD2 antisense oligonucleotide does not inhibit the growth of normal and transformed cell lines and does not alter cell cycle parameters in vitro and does not exhibit overt toxicity in vivo in comparison with a scrambled control oligonucleotide, as determined by measuring body mass, blood cell parameters and liver and kidney enzymes. Our data provide a proof of principle that MBD2 is a new anticancer target and that pharmacological inhibition of MBD2 by agents such as the antisense inhibitors described in this paper is a potential new anticancer therapy, which in contrast to the vast majority of current approaches does not target normal progression of the cell cycle. PMID:14688029

Campbell, Paul M; Bovenzi, Veronica; Szyf, Moshe



Proteomic profiling identifies cyclooxygenase-2-independent global proteomic changes by celecoxib in colorectal cancer cells.  


Celecoxib, a selective inhibitor of the enzyme cyclooxygenase-2 (COX-2), has been shown to be a promising chemoprevention agent. The chemopreventive efficacy of celecoxib is believed to be a consequence of its COX-2-dependent and COX-2-independent effects on a variety of cellular processes including proliferation, apoptosis, angiogenesis, and immunosurveillance. In an attempt to identify proteomic markers modulated by celecoxib that are independent of its inhibitory effect on COX-2, the colorectal cancer cell line HCT-116, a nonexpresser of COX-2, was treated with celecoxib. We used the powerful, state-of-the-art two-dimensional difference gel electrophoresis technology coupled with mass spectrometric sequencing to compare global proteomic profiles of HCT-116 cells before and after treatment with celecoxib. Among the differentially expressed proteins identified following celecoxib treatment were proteins involved in diverse cellular functions including glycolysis, protein biosynthesis, DNA synthesis, mRNA processing, protein folding, phosphorylation, redox regulation, and molecular chaperon activities. Our study presents a comprehensive analysis of large-scale celecoxib-modulated proteomic alterations, at least some of which may be mechanistically related to the COX-2-independent chemopreventive effect of celecoxib. PMID:16985019

Lou, Jianrong; Fatima, Naheed; Xiao, Zhen; Stauffer, Stacy; Smythers, Gary; Greenwald, Peter; Ali, Iqbal Unnisa



DRO1 sensitizes colorectal cancer cells to receptor-mediated apoptosis  

PubMed Central

The molecule DRO1 (down?regulated by oncogenes 1) is a potential tumor suppressor protein that is frequently down?regulated in primary colorectal cancers and colorectal cancer cell lines. Although the mechanism of DRO1 action has yet to be elucidated, previous data have suggested that DRO1 interferes with tumor growth by sensitizing cells to apoptosis. The effect of DRO1 expression on receptor-, mitochondrial- and endoplasmic reticulum?mediated apoptosis in colorectal cancer cell lines was analyzed in this study, following the generation of DLD1/DRO1 and HCT116/DRO1 cell lines. Cells were cultured, and then analyzed using flow cytometry. DRO1 was found to sensitize cells to receptor-mediated apoptosis by promoting the activation of components of the death-inducing signaling complex (DISC). PMID:22866160

Herbst, Andreas; Bayer, Constanze; Wypior, Claudia; Kolligs, Frank T.



Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53- and DNMT1(DNA methyltransferase 1)-independent manner  

PubMed Central

Epigenetic silencing of gene expression is important in cancer. Aberrant DNA CpG island hypermethylation and histone modifications are involved in the aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) is a H3K4 (histone H3 Lys4) demethylase associated with gene repression and is overexpressed in multiple cancer types. LSD1 has also been implicated in targeting p53 and DNMT1 (DNA methyltransferase 1), with data suggesting that the demethylating activity of LSD1 on these proteins is necessary for their stabilization. To examine the role of LSD1 we generated LSD1 heterozygous (LSD1+/?) and homozygous (LSD1?/?) knockouts in the human colorectal cancer cell line HCT116. The deletion of LSD1 led to a reduced cell proliferation both in vitro and in vivo. Surprisingly, the knockout of LSD1 in HCT116 cells did not result in global increases in its histone substrate H3K4me2 (dimethyl-H3K4) or changes in the stability or function of p53 or DNMT1. However, there was a significant difference in gene expression between cells containing LSD1 and those null for LSD1. The results of the present study suggested that LSD1 is critical in the regulation of cell proliferation, but also indicated that LSD1 is not an absolute requirement for the stabilization of either p53 or DNMT1. PMID:23072722

Jin, Lihua; Hanigan, Christin L.; Wu, Yu; Wang, Wei; Park, Ben Ho; Woster, Patrick M.; Casero, Robert A.



The ethanolic extract of bark from Salix aegyptiaca L. inhibits the metastatic potential and epithelial to mesenchymal transition of colon cancer cell lines.  


Willow bark extracts have been used for centuries as a natural pain killer. Recently their potential as anticancer agents has been reported. We have shown the high antioxidant activity, phenolic and flavonoid content in the ethanolic extract of bark (EEB) from Salix aegyptiaca, a species endogenous to the Middle East. We have also reported that incubation with EEB resulted in a reduction in cell proliferation through the induction of apoptosis and cell cycle arrest via the inhibition of phosphatidyl inositol 3-kinase/Protein kinase B and mitogen activated protein kinases signaling pathways as strongly as commercial inhibitors. The current study demonstrates the robust inhibition of anchorage-independent growth, motility, migration, and adhesion of colon cancer cell lines HCT-116 and HT-29 by EEB. These in vitro functional changes were accompanied by a restoration of E-cadherin expression, a reduction in EGFR, SNAI1, SNAI2, and Twist1 and the matrix metalloproteases MMP9 and MMP2. Many of these proteins are involved in the process of epithelial to mesenchymal transition, which is considered as a critical step in the progression of noninvasive tumor cells into malignant, metastatic carcinomas. We therefore propose that EEB from Salix aegyptiaca is a potent nutraceutical causing cancer chemoprevention by inhibiting epithelial to mesenchymal transition and can be consumed for its health promoting effects. PMID:25175673

Enayat, Shabnam; Banerjee, Sreeparna



Role of Bax in quercetin-induced apoptosis in human prostate cancer cells  

PubMed Central

The aim of this study was to investigate the effect of quercetin, a flavonoid, on the apoptotic pathway in a human prostate cell line (LNCaP). We observed that treatment of cells for 24 h with quercetin induced cell death in a dose-dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in quercetin-treated cells. Treatment of LNCaP cells with an apoptosis inducing concentration of quercetin (100 ?M) resulted in a rapid decrease in the inhibitory Ser(473) phosphorylation of Akt leading to inhibition of its kinase activity. Quercetin treatment (100 ?M) also caused a decrease in Ser(136) phosphorylation of Bad, which is a downstream target of Akt. Protein interaction assay revealed that during treatment with quercetin, Bcl-xL dissociated from Bax and then associated with Bad. Our results also show that quercetin decreases the Bcl-xL:Bax ratio and increases translocation and multimerization of Bax to the mitochondrial membrane. The translocation is accompanied by cytochrome c release, and procaspases-3, -8 and -9 cleavage and increased poly (ADP-ribose) polymerase (PARP) cleavage. Similar results were observed in human colon cancer HCT116Bax+/+ cell line, but not HCT116Bax?/? cell line. Interestingly, at similar concentrations (100 ?M), quercetin treatment did not affect the viability or rate of apoptosis in normal human prostate epithelial cell line (PrEC) and rat prostate epithelial cell line (YPEN-1). Our results indicate that the apoptotic processes caused by quercetin are mediated by the dissociation of Bax from Bcl-xL and the activation of caspase families in human prostate cancer cells. PMID:18455702

Lee, Dae-Hee; Szczepanski, Miroslaw; Lee, Yong J.



Differential expression of nanog1 and nanogp8 in colon cancer cells  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Nanog is expressed in a majority of colon cancer cell lines examined. Black-Right-Pointing-Pointer Both nanog1 and nanogp8 are expressed in colon cancer cells with varying ratios. Black-Right-Pointing-Pointer Nanog mediates cell proliferation of colon cancer cells. Black-Right-Pointing-Pointer Nanog predominantly localizes in cytoplasm of colon cancer cells. -- Abstract: Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

Ishiguro, Tatsuya; Sato, Ai; Ohata, Hirokazu; Sakai, Hiroaki [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)] [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Nakagama, Hitoshi, E-mail: [Division of Cancer Development System, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)] [Division of Cancer Development System, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Okamoto, Koji, E-mail: [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)] [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)



Systematic Analyses of the Cytotoxic Effects of Compound 11a, a Putative Synthetic Agonist of Photoreceptor-Specific Nuclear Receptor (PNR), in Cancer Cell Lines  

PubMed Central

Photoreceptor cell-specific receptor (PNR/NR2E3) is an orphan nuclear receptor that plays a critical role in retinal development and photoreceptor maintenance. The disease-causing mutations in PNR have a pleiotropic effect resulting in varying retinal diseases. Recently, PNR has been implicated in control of cellular functions in cancer cells. PNR was reported to be a novel regulator of ER? expression in breast cancer cells, and high PNR expression correlates with favorable response to tamoxifen treatment. Moreover, PNR was shown to increase p53 stability in HeLa cells, implying that PNR may be a therapeutic target in this and other cancers that retain a wild type p53 gene. To facilitate further understanding of PNR functions in cancer, we characterized compound 11a, a synthetic, putative PNR agonist in several cell-based assays. Interestingly, we showed that 11a failed to activate PNR and its cytotoxicity was independent of PNR expression, excluding PNR as a mediator for 11a cytotoxicity. Systematic analyses of the cytotoxic effects of 11a in NCI-60 cell lines revealed a strong positive correlation of cytotoxicity with p53 status, i.e., p53 wild type cell lines were significantly more sensitive to 11a than p53 mutated or null cell lines. Furthermore, using HCT116 p53+/+ and p53-/- isogenic cell lines we revealed that the mechanism of 11a-induced cytotoxicity occurred through G1/S phase cell cycle arrest rather than apoptosis. In conclusion, we observed a correlation of 11a sensitivity with p53 status but not with PNR expression, suggesting that tumors expressing wild type p53 might be responsive to this compound. PMID:24066170

Zhao, Zibo; Wang, Lu; Wen, Zhi; Ayaz-guner, Serife; Wang, Yidan; Ahlquist, Paul; Xu, Wei



Overexpression of COX-2 in human osteosarcoma cells decreases proliferation and increases apoptosis.  


Overexpression of cyclooxygenase-2 (COX-2) is generally considered to promote tumorigenesis. To investigate a potential role of COX-2 in osteosarcoma, we overexpressed COX-2 in human osteosarcoma cells. Saos-2 cells deficient in COX-2 expression were retrovirally transduced or stably transfected with murine COX-2 cDNA. Functional expression of COX-2 was confirmed by Northern and Western analyses and prostaglandin production. Overexpression of COX-2 reduced cell numbers by 50% to 70% compared with controls. Decreased proliferation in COX-2-overexpressing cells was associated with cell cycle prolongation in G(2)-M. Apoptosis, measured by both Annexin V binding assay and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling staining, was increased in cells overexpressing COX-2, and the increase was not reversed by treatment with NS-398, indicating that the effects were not mediated by prostaglandins. Retroviral COX-2 overexpression in two other human osteosarcoma cell lines, U2OS and TE85, also decreased cell viability. However, in the human colon carcinoma HCT-116 cell line, which is deficient in COX-2, retroviral overexpression of COX-2, at similar efficiency as in Saos-2 cells, increased resistance to apoptosis. Reactive oxygen species (ROS), measured by flow cytometry, were increased by COX-2 overexpression in Saos-2 cells but not in HCT-116 cells. Inhibition of peroxidase activity, but not of COX activity, blocked the ROS increase. Antioxidants blocked the increase in ROS and the increase in apoptosis due to COX-2 overexpression in Saos-2 cells. Our results suggest that (a) COX-2 overexpression in osteosarcoma cells may increase resistance to tumorigenesis by increasing ROS to levels that decrease cell viability and (b) the effects of COX-2 overexpression are cell type/tissue dependent. PMID:16818639

Xu, Zheng; Choudhary, Shilpa; Voznesensky, Olga; Mehrotra, Meenal; Woodard, Monica; Hansen, Marc; Herschman, Harvey; Pilbeam, Carol



Knockdown of FAM3B triggers cell apoptosis through p53-dependent pathway.  


FAM3B, also named PANDER, is a cytokine-like protein identified in 2002. Previous studies showed that FAM3B regulates glucose and lipid metabolism through interaction with liver and endocrine pancreas. FAM3B is also expressed by other tissues but its basic function is unclear. In this study, we found that FAM3B was expressed in mouse colon, intestine, liver and lung tissues and multiple types of cell lines, including murine pancreatic ?-cell (Min6), microglia (N9) and muscle cell (C2C12); human colon cancer cells (HCT8, HCT116, HT29), hepatocyte (HL-7702), hepatocellular carcinoma cell (SMMC-7721) and lung carcinoma cell (A549). Inhibition of FAM3B expression by RNA interference induced apoptotic cell death of HCT8, HCT116, A549, N9, C2C12 and Min6 cells and decreased cell viability of HL-7702 and murine primary hepatocytes. Further studies with HCT8 cells showed that knockdown of FAM3B increased the protein levels of membrane-bound Fas and Bax, reduced the expression of Bcl-2, promoted the cleavage of caspases-8, -3, -9 and PARP, and the nuclear translocation of cleaved PARP. These results suggest that FAM3B silencing activates both extrinsic and intrinsic apoptotic pathways. Mechanistic studies showed that neutralizing antibody against Fas or silencing Fas-associated death domain had no effect on, while caspase inhibitors could significantly reverse FAM3B knockdown induced apoptosis, suggesting Fas and death receptor mediated extrinsic apoptotic pathway is not involved in FAM3B silencing induced apoptosis. Further studies showed that p53 was significantly upregulated after FAM3B knockdown. Silencing p53 could almost completely reverse FAM3B knockdown induced upregulation of Bax, downregulation of Bcl-2, cleavage of caspases-8, -9, -3, and apoptotic cell death, suggesting p53-dependent pathway plays critical roles in FAM3B silencing induced apoptosis. Studies with HCT116 cells confirmed that inhibition of FAM3B expression induced apoptosis through p53-dependent pathway. Furthermore, knockdown of FAM3B reduced the protein level of Mdm2 and promoted p53 phosphorylation. Taken together, our studies demonstrated that silencing FAM3B promoted p53 phosphorylation and induced p53 accumulation by decreasing Mdm2 expression, which resulted in apoptotic cell death. PMID:23246487

Mou, Haiwei; Li, Zongmeng; Yao, Pengle; Zhuo, Shu; Luan, Wei; Deng, Bo; Qian, Lihua; Yang, Mengmei; Mei, Hong; Le, Yingying



Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation  

SciTech Connect

Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)] [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)] [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)



news and views nature cell biology volume 10 | number 5 | mAY 2008 501  

E-print Network

changes in cell morphology and motility so that they adopt mesenchymal characteristics. Expression of E-cadherin cell-adhesion protein e-cadherin, highlighting the potential importance of miRnas in eRNAs was sufficient to reduce E-cadherin expression, increase cell motility and thus induce EMT in MDCK3 and HCT116

Miska, Eric


Wild celery (Smyrnium olusatrum L.) oil and isofuranodiene induce apoptosis in human colon carcinoma cells.  


Smyrnium olusatrum (Apiaceae), well known as wild celery, is a biennal celery-scented plant used for many centuries as a vegetable, then abandoned after the introduction of celery. In the present work, the essential oil obtained from inflorescences and the amounts of its main constituents isofuranodiene, curzerene and germacrone were analyzed by GC as well as by HPLC because of their degradation (Cope rearrangement) occurring at high temperatures. The oil and the main constituents were assayed for cytotoxic activity on the human colon cancer cell line (HCT116) by MTT assay. Flower oil and isofuranodiene showed noteworthy activity on tumor cells with IC50 of 10.71 and 15.06 ?g/ml, respectively. Analysis of the cytotoxic activity showed that wild celery oil and isofuranodiene are able to induce apoptosis in colon cancer cells in a time and concentration-dependent manner suggesting a potential role as models for the development of chemopreventive agents. PMID:24924290

Quassinti, Luana; Maggi, Filippo; Barboni, Luciano; Ricciutelli, Massimo; Cortese, Manuela; Papa, Fabrizio; Garulli, Chiara; Kalogris, Cristina; Vittori, Sauro; Bramucci, Massimo



Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models  

PubMed Central

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer, and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in colorectal cancer cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45?85-fold higher than parental cells. The IL-8-transfected clones secreted 19?28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (P < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance suggesting that IL-8 not only provides a proliferative advantage, but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in colorectal cancers. PMID:20648559

Ning, Yan; Manegold, Philipp C; Hong, Young Kwon; Zhang, Wu; Pohl, Alexandra; Lurje, Georg; Winder, Thomas; Yang, Dongyun; LaBonte, Melissa J; Wilson, Peter M; Ladner, Robert D; Lenz, Heinz-Josef



Depletion of Securin Induces Senescence After Irradiation and Enhances Radiosensitivity in Human Cancer Cells Regardless of Functional p53 Expression  

SciTech Connect

Purpose: Radiotherapy is one of the best choices for cancer treatment. However, various tumor cells exhibit resistance to irradiation-induced apoptosis. The development of new strategies to trigger cancer cell death besides apoptosis is necessary. This study investigated the role of securin in radiation-induced apoptosis and senescence in human cancer cells. Methods and Materials: Cell survival was determined using clonogenic assays. Western blot analysis was used to analyze levels of securin, caspase-3, PARP, p53, p21, Rb, gamma-H2AX, and phospho-Chk2. Senescent cells were analyzed using a beta-galactosidase staining assay. A securin-expressed vector (pcDNA-securin) was stably transfected into securin-null HCT116 cells. Securin gene knockdown was performed by small interfering RNA and small hairpin RNA in HCT116 and MDA-MB-231 cells, respectively. Results: Radiation was found to induce apoptosis in securin wild type HCT116 cells but induced senescence in securin-null cells. Restoration of securin reduced senescence and increased cell survival in securin-null HCT116 cells after irradiation. Radiation-induced gamma-H2AX and Chk2 phosphorylation were induced transiently in securin-wild-type cells but exhibited sustained activation in securin-null cells. Securin gene knockdown switches irradiation-induced apoptosis to senescence in both HCT116 p53-null and MDA-MB-231 cells. Conclusions: Our results demonstrated that the level of securin expression plays a determining role in the radiosensitivity and fate of cells. Depletion of securin impairs DNA repair after irradiation, increasing DNA damage and promoting senescence in the residual surviving cells regardless of functional p53 expression. The knockdown of securin may contribute to a novel radiotherapy protocol for the treatment of human cancer cells that are resistant to irradiation.

Chen Wenshu; Yu Yichu [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Lee Yijang [Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan (China); Chen, J.-H. [Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien, Taiwan (China); Hsu, H.-Y. [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Chiu, S.-J., E-mail: [Department of Life Science, Tzu Chi University, Hualien, Taiwan (China); Institute of Radiation Sciences, Tzu Chi Technology College, Hualien, Taiwan (China)



Nuclear Expression of ?-Catenin Promotes RB Stability and Resistance to TNF-Induced Apoptosis in Colon Cancer Cells  

PubMed Central

Tumor necrosis factor (TNF)-? promotes tumor development under chronic inflammation. Because TNF also activates caspase-8, selective inhibition of TNF-induced extrinsic apoptosis would be required for inflammation-associated tumor growth. In a mouse model of inflammation-associated colon carcinogenesis, we found nuclear expression of ?-catenin in tumors of wild-type, but not mutant, mice that were made resistant to TNF-induced apoptosis by a germline mutation blocking caspase cleavage of the retinoblastoma (RB) protein, despite similar frequencies of ?-catenin exon-3 mutations in these two genetic backgrounds. TNF-induced apoptosis was also attenuated in human colon cancer cell lines with genetically activated ?-catenin. However, we found that HCT116 cells, which contain an activated allele of ?-catenin but do not express nuclear ?-catenin, were sensitive to TNF-induced apoptosis. In HCT116 cells, TNF stimulated efficient RB cleavage that preceded chromatin condensation. In contrast, TNF did not induce RB cleavage in colon cancer cells expressing nuclear ?-catenin and these cells could be sensitized to basal and/or TNF-induced apoptosis by the knockdown of ?-catenin or RB. In the apoptosis-resistant colon cancer cells, knockdown of ?-catenin led to a reduction in the RB protein without affecting RB mRNA. Furthermore, ectopic expression of the caspase-resistant, but not the wild-type, RB re-established resistance to TNF-induced caspase activation in colon cancer cells without ?-catenin. Together, these results suggest that nuclear ?-catenin–dependent RB stabilization suppresses TNF-induced apoptosis in caspase-8–positive colon cancer cells. PMID:23339186

Han, Jinbo; Soletti, Rossana C.; Sadarangani, Anil; Sridevi, Priya; Ramirez, Michael E.; Eckmann, Lars; Borges, Helena L.; Wang, Jean Y.J.



Folic acid-mediated inhibition of serum-induced activation of EGFR promoter in colon cancer cells.  


Although accumulating evidence suggests a chemopreventive role for folic acid (FA) in colorectal carcinogenesis, the underlying mechanisms are largely unknown. Previously, we reported that supplemental FA inhibits the expression and activation of epidermal growth factor receptor (EGFR) in colon cancer cell lines. To determine the mechanism(s) by which FA affects EGFR function, we have examined whether and to what extent supplemental FA or its metabolites 5-methyltetrahydrofolate (MTF), dihydrofolate (DF), and tetrahydrofolate (TF) will modulate basal and serum-induced activation of the EGFR promoter in the HCT-116 colon cancer cell line. HCT-116 cells were preincubated with or without (control) FA or one of its metabolites (10 microg/ml) for 48 h, transfected with the EGFR promoter luciferase reporter construct, and incubated for 48 h with FA, DF, TF, or 5-MTF in the absence or presence of 10% FBS. Supplemental FA as well as its metabolites markedly inhibited EGFR promoter activity and its methylation status. Exposure of the cells to 10% FBS caused a marked stimulation of EGFR promoter activity and its expression, both of which were greatly abrogated by supplemental FA and 5-MTF. In contrast, serum-induced activation of c-fos promoter activity was unaffected by 5-MTF. The 5-MTF-induced inhibition of serum-mediated stimulation of EGFR promoter activity and EGFR expression was reversed when methylation was inhibited by 5-aza-2'-deoxycytidine. Our data suggest that FA and its metabolite 5-MTF inhibit EGFR promoter activity in colon cancer cells by enhancing methylation. This could partly be responsible for FA-mediated inhibition of growth-related processes in colorectal neoplasia. PMID:15075253

Nagothu, Kiran K; Rishi, Arun K; Jaszewski, Richard; Kucuk, Omer; Majumdar, Adhip P N



Effect of a nutrient mixture on matrix metalloproteinase-9 dimers in various human cancer cell lines.  


Strong clinical and experimental evidence demonstrates association of elevated levels of matrix metalloproteinase MMP-9 with cancer progression, metastasis and shortened patient survival, as it plays a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. MMP-9 is secreted in both the monomeric and dimeric form. Although there is little research on MMP-9 dimers, some studies have shown the dimer to be associated with more aggressive tumor progression. Our objective was to study the relative secretion patterns of MMP-9 monomer and dimer in a variety of cancer cell lines and the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract on MMP-9 secretion. The cancer cell lines were grown in their respective media, supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0,10, 50, 100, 500 and 1000 µg/ml. Parallel sets of cultures were treated with PMA (100 ng/ml) for induction of MMP-9. Cell MMP-9 secretion was assayed by gelatinase zymography. MMP-9 dimer secretion patterns of cancer cells fell into different categories. We observed no MMP-9 dimer in prostate DU-145 and PC-3, pancreatic MIA-Pa-Ca2, colon HCT-116, bladder T-24, head and neck FaDu, glioblastoma A-172, T-98 and LN-18 and leukemia HL-60, Jurkat, and Raji cell lines. MMP-dimer secretion only with PMA induction was seen in breast MCF-7 and MDA-MB-231, uterine SK-UT-1, lung A-549, tongue SC-25, melanoma A2058, osteosarcoma U-2OS, rhabdomyosarcoma, fibrosarcoma HT-1080, chondrosarcoma SW-1350 and liposarcoma SW-872. Cervical HeLa and DoTc 2 4510, renal 786-0 and HCC SK-Hep-1 cells exhibited MMP-9 dimer without PMA treatment and increased secretion with PMA treatment. Sarcomas had the highest levels of MMP-9 monomer and dimer with and without PMA among these cancer cell lines. Cervical, uterine and male breast cancer cell lines showed the next highest levels of MMP-9, followed by breast cancer cell lines. Melanoma, renal, lung, head and neck and HCC showed lower levels and prostate, glioblastoma, bladder and leukemia cell lines the lowest. NM showed dose-dependent inhibition of MMP-9 monomer and dimer in all cell lines tested. In conclusion, high MMP-9 and dimer secretion levels correlated with the most aggressive cancer cell lines. NM was effective in inhibiting MMP-9 and dimer secretion in all cell lines tested, suggesting its therapeutic potential as an antimetastatic agent. PMID:24378964

Roomi, M W; Kalinovsky, T; Rath, M; Niedzwiecki, A



Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines  

PubMed Central

Background Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-?-tocopherol isoform is found primarily in the US diet, while RRR-?-tocopherol is highest in the plasma. Methods The effectiveness of RRR-?- and RRR-?-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-?- or RRR-?-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. Results Treatment with RRR-?-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-?-tocopherol did not. Further, RRR-?-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-?-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-?-tocopherol to induce cell death. Conclusion This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-?-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-?-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-?-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-?-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents. PMID:16417629

Campbell, Sharon E; Stone, William L; Lee, Steven; Whaley, Sarah; Yang, Hongsong; Qui, Min; Goforth, Paige; Sherman, Devin; McHaffie, Derek; Krishnan, Koyamangalath



Mitogen-activated protein kinase phosphatase-1 inhibition and sustained extracellular signal-regulated kinase 1/2 activation in camptothecin-induced human colon cancer cell death  

PubMed Central

Camptothecins are commonly used chemotherapeutics; in some models, they enhance signaling via the mitogen-activated protein kinase (MAPK) pathway through effects on upstream kinases. To evaluate the impact of camptothecin (CPT) on MAPKs in human colon cancer, we studied HCT116 and CaCo2 colon cancer cells. We found that HCT116 cells highly express mitogen-activated protein kinase phosphatase-1 (MKP1), which selectively inactivates extracellular signal-regulated kinase (ERK), whereas MKP1 levels were undetectable in CaCo2 cells. CPT did not affect ERK activity in CaCo2 cells, but did induce a striking increase in ERK activity in HCT116 cells in association with a corresponding decrease in MKP1. The reduction in MKP1 expression occurred at a posttranscriptional level and was blocked by the proteasome inhibitor MG132, whereas that CPT-induced downregulation of MKP1 was not due to proteasome-mediated degradation. Treatment of HCT116 cells with CPT induced a sustained activation of nuclear ERK, which was required for CPT-induced apoptosis. P38 and JNK activity were unaffected by CPT, suggesting that the effects of CPT are mediated specifically by ERK. These results suggest that targeting dual-specificity MAPK phosphatases in colon cancer cells may be a viable strategy for optimizing camptothecin-based therapeutic protocols. PMID:24005240

Lee, Minyoung; Young Kim, Sun; Kim, JongGuk; Kim, Hak-Su; Kim, Sang-Man; Kim, Eun Ju



Tumor-derived hydrogen sulfide, produced by cystathionine-?-synthase, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer.  


The physiological functions of hydrogen sulfide (H2S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H2S-producing enzyme cystathionine-?-synthase (CBS) in colon cancer, resulting in an increased rate of H2S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H2S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H2S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-?-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H2S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H2S as a tumor growth factor and anticancer drug target. PMID:23836652

Szabo, Csaba; Coletta, Ciro; Chao, Celia; Módis, Katalin; Szczesny, Bartosz; Papapetropoulos, Andreas; Hellmich, Mark R



Tumor-derived hydrogen sulfide, produced by cystathionine-?-synthase, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer  

PubMed Central

The physiological functions of hydrogen sulfide (H2S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H2S-producing enzyme cystathionine-?-synthase (CBS) in colon cancer, resulting in an increased rate of H2S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H2S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H2S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-?-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H2S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H2S as a tumor growth factor and anticancer drug target. PMID:23836652

Szabo, Csaba; Coletta, Ciro; Chao, Celia; Modis, Katalin; Szczesny, Bartosz; Papapetropoulos, Andreas; Hellmich, Mark R.



High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Specific enzyme for HMGB1 phosphorylation and its secretion is proposed. Black-Right-Pointing-Pointer Inhibition of PKC-{zeta} leads to significant reduction of the secreted HMGB1. Black-Right-Pointing-Pointer Phosphorylation of specific site of HMGB1 redirects its secretion in cancer cells. Black-Right-Pointing-Pointer Activation of PKC-{zeta} in cancers explains the enhanced HMGB1 secretion. -- Abstract: High mobility group box-1 (HMGB1), a nuclear protein, is overexpressed and secreted in cancer cells. Phosphorylation on two different nuclear localization signal regions are known to be important for the nuclear-to-cytoplasmic transport and secretion of HMGB1. However, little is known about the biochemical mechanism of HMGB1 modifications and its subsequent secretion from cancer cells. To identify the specific enzyme and important sites for HMGB1 phosphorylation, we screened the protein kinase C (PKC) family in a colon cancer cell line (HCT116) for HMGB1 binding by pull-down experiments using a 3XFLAG-HMGB1 construct. Strong interactions between atypical PKCs (PKC-{zeta}, {lambda}, and {iota}) and cytoplasmic HMGB1 were observed in HCT116 cells. We further identified the most critical PKC isotype that regulates HMGB1 secretion is PKC-{zeta} by using PKC inhibitors and siRNA experiments. The serine residues at S39, S53 and S181 of HMGB1 were related to enhancing HMGB1 secretion. We also demonstrated overexpression and activation of PKC-{zeta} in colon cancer tissues. Our findings suggest that PKC-{zeta} is involved in the phosphorylation of HMGB1, and the phosphorylation of specific serine residues in the nuclear localization signal regions is related to enhanced HMGB1 secretion in colon cancer cells.

Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of) [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Yun Gi [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 (Korea, Republic of)] [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 (Korea, Republic of); Shin, Jeon-Soo [Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of)] [Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Kim, Hoguen, E-mail: [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of) [Department of Pathology, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of); Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul (Korea, Republic of)



Resveratrol Inhibits Invasion and Metastasis of Colorectal Cancer Cells via MALAT1 Mediated Wnt/?-Catenin Signal Pathway  

PubMed Central

Resveratrol, extracted from Chinese herbal medicine Polygonum cuspidatum, is known to inhibit invasion and metastasis of human colorectal cancer (CRC), in which long non-coding Metastasis Associated Lung Adenocarcinoma Transcript 1 (RNA-MALAT1) also plays an important role. Using MALAT1 lentiviral shRNA and over-expression constructs in CRC derived cell lines, LoVo and HCT116, we demonstrated that the anti-tumor effects of resveratrol on CRC are through inhibiting Wnt/?-catenin signaling, thus the expression of its target genes such as c-Myc, MMP-7, as well as the expression of MALAT1. In detail, resveratrol down-regulates MALAT1, resulting in decreased nuclear localization of ?-catenin thus attenuated Wnt/?-catenin signaling, which leads to the inhibition of CRC invasion and metastasis. This finding of ours surely provides important pre-clinical evidence supporting future use of resveratrol in prevention and treatment of CRC. PMID:24244343

Fu, Xiaoling; Zhang, Long; Sui, Hua; Zhou, Lihong; Sun, Jian; Cai, Jianfeng; Qin, Jianmin; Ren, Jianlin; Li, Qi



Celecoxib Induced Tumor Cell Radiosensitization by Inhibiting Radiation Induced Nuclear EGFR Transport and DNA-Repair: A COX-2 Independent Mechanism  

SciTech Connect

Purpose: The purpose of the study was to elucidate the molecular mechanisms mediating radiosensitization of human tumor cells by the selective cyclooxygenase (COX)-2 inhibitor celecoxib. Methods and Materials: Experiments were performed using bronchial carcinoma cells A549, transformed fibroblasts HH4dd, the FaDu head-and-neck tumor cells, the colon carcinoma cells HCT116, and normal fibroblasts HSF7. Effects of celecoxib treatment were assessed by clonogenic cell survival, Western analysis, and quantification of residual DNA damage by {gamma}H{sub 2}AX foci assay. Results: Celecoxib treatment resulted in a pronounced radiosensitization of A549, HCT116, and HSF7 cells, whereas FaDu and HH4dd cells were not radiosensitized. The observed radiosensitization could neither be correlated with basal COX-2 expression pattern nor with basal production of prostaglandin E2, but was depended on the ability of celecoxib to inhibit basal and radiation-induced nuclear transport of epidermal growth factor receptor (EGFR). The nuclear EGFR transport was strongly inhibited in A549-, HSF7-, and COX-2-deficient HCT116 cells, which were radiosensitized, but not in FaDu and HH4dd cells, which resisted celecoxib-induced radiosensitization. Celecoxib inhibited radiation-induced DNA-PK activation in A549, HSF7, and HCT116 cells, but not in FaDu and HH4dd cells. Consequentially, celecoxib increased residual {gamma}H2AX foci after irradiation, demonstrating that inhibition of DNA repair has occurred in responsive A549, HCT116, and HSF7 cells only. Conclusions: Celecoxib enhanced radiosensitivity by inhibition of EGFR-mediated mechanisms of radioresistance, a signaling that was independent of COX-2 activity. This novel observation may have therapeutic implications such that COX-2 inhibitors may improve therapeutic efficacy of radiation even in patients whose tumor radioresistance is not dependent on COX-2.

Dittmann, Klaus H. [Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen (Germany)], E-mail:; Mayer, Claus; Ohneseit, Petra A. [Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen (Germany); Raju, Uma [Department of Experimental Radiation Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX (United States); Andratschke, Nickolaus H. [Klinik und Poliklinik fuer Strahlentherapie und Radiologische Onkologie, Klinikum Rechts Der Isar, Technische Universitaet Muenchen, Munich (Germany); Milas, Luka [Department of Experimental Radiation Oncology, University of Texas, M.D. Anderson Cancer Center, Houston, TX (United States); Rodemann, H. Peter [Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Tuebingen (Germany)



Dihydrocapsaicin (DHC), a saturated structural analog of capsaicin, induces autophagy in human cancer cells in a catalase-regulated manner.  


Although capsaicin, a pungent component of red pepper, is known to induce apoptosis in several types of cancer cells, the mechanisms underlying capsaicin-induced cytotoxicity are unclear. Here, we showed that dihydrocapsaicin (DHC), an analog of capsaicin, is a potential inducer of autophagy. DHC was more cytotoxic than capsaicin in HCT116, MCF-7 and WI38 cell lines. Capsaicin and DHC did not affect the sub-G(1) apoptotic peak, but induced G(0)/G(1) arrest in HCT116 and MCF-7 cells. DHC caused the artificial autophagosome marker GFP-LC3 to redistribute and upregulated expression of autophagy-related proteins. Blocking of autophagy by 3-methyladenine (3MA) as well as siRNA Atg5 induced a high level of caspase-3 activation. Although pretreatment with zVAD completely inhibited caspase-3 activation by 3MA, it did not prevent cell death. DHC-induced autophagy was enhanced by zVAD pretreatment, as shown by increased accumulation of LC3-II protein. DHC attenuated basal ROS levels through catalase induction; this effect was enhanced by antioxidants, which increased both LC3-II expression and caspase-3 activation. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) abrogated DHC-induced expression of LC3-II, overexpression of the catalase gene increased expression of LC3-II protein, and knockdown decreased it. Additionally, DHC-induced autophagy was independent of p53 status. Collectively, DHC activates autophagy in a p53-independent manner and that may contribute to cytotoxicity of DHC. PMID:18818525

Oh, Seon Hee; Kim, Young Soon; Lim, Sung Chul; Hou, Yi Feng; Chang, In Youb; You, Ho Jin



Overexpression of Arginine Transporter CAT-1 Is Associated with Accumulation of L-Arginine and Cell Growth in Human Colorectal Cancer Tissue  

PubMed Central

We previously showed that L-arginine (Arg) accumulates in colorectal cancer tissues. The aim of this study was to investigate the mechanism by which Arg accumulates and determine its biological significance. The concentration of Arg and Citrulline (Cit) in sera and tumor tissues from colorectal cancer (CRC) patients was analyzed by high-performance liquid chromatography (HPLC). The expression of Arg transporters was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis of tissue microarray. We also transfected the colon cancer cell line HCT-116 with siRNA specific for the Arg transporter CAT-1 and measured the induction of apoptosis by flow cytometry and cell proliferation by MTT assay. Consistent with our previous results, serum Arg and Cit concentrations in colorectal cancer patients were significantly lower than those in normal volunteers, while Arg and Cit concentrations in colorectal cancer tissues were significantly higher than in matched adjacent normal colon tissues. Quantitative RT-PCR showed that the CAT-1 gene was highly overexpressed in 70.5% of colorectal cancer tissue samples relative to adjacent normal colon tissues in all 122 patients with colorectal cancer. Immunohistochemical analysis of tissue microarray confirmed that the expression of CAT-1 was higher in all 25 colorectal cancer tissues tested. CAT-1 siRNA significantly induced apoptosis of HCT-116 cells and subsequently inhibited cell growth by 20–50%. Our findings indicate that accumulation of L-Arg and Cit and cell growth in colorectal cancer tissues is associated with over-expression of the Arg transporter gene CAT-1. Our results may be useful for the development of molecular diagnostic tools and targeted therapy for colorectal cancer. PMID:24040099

Wang, Junchen; Yang, Chunzhang; Mao, Huiming; Fu, Xuelian; Wu, Yanling; Cai, Jingping; Han, Junyi; Xu, Zengguang; Zhuang, Zhengping; Liu, Zhongmin; Hu, Hai; Chen, Bingguan



In Vitro and In Vivo Enhancement of Chemoradiation Using the Oral PARP Inhibitor ABT-888 in Colorectal Cancer Cells  

SciTech Connect

Purpose: Poly(ADP-ribose) polymerase plays a critical role in the recognition and repair of DNA single-strand breaks and double-strand breaks (DSBs). ABT-888 is an orally available inhibitor of this enzyme. This study seeks to evaluate the use of ABT-888 combined with chemotherapy and radiation therapy (RT) in colorectal carcinoma models. Methods and Materials: RT clonogenic assays were performed on HCT116 and HT29 cells treated with 5-fluorouracil, irinotecan, or oxaliplatin with or without ABT. The surviving fraction at 2 Gy and dose-modifying factor at 10% survival were analyzed. Synergism was assessed by isobologram analysis for combination therapies. ?H2AX and neutral comet assays were performed to assess the effect of therapy on DSB formation/repair. In vivo assessments were made by use of HCT116 cells in a xenograft mouse model. Tumor growth delay was measured at a volume of 500 mm{sup 3}. Results: Both lines were radiosensitized by ABT alone, and ABT further increased chemotherapy dose-modifying factors to the 1.6 to 1.8 range. All combinations were synergistic (combination indices <0.9). ABT treatment significantly increased DSB after RT (?H2AX, 69% vs 43%; P=.017) and delayed repair. We found tumor growth delays of 7.22 days for RT; 11.90 days for RT and ABT; 13.5 days for oxaliplatin, RT, and ABT; 14.17 days for 5-fluorouracil, RT, and ABT; and 23.81 days for irinotecan, RT, and ABT. Conclusion: ABT-888 radiosensitizes at similar or higher levels compared with classic chemotherapies and acts synergistically with these chemotherapies to enhance RT effects. In vivo confirmation of these results indicates a potential role for combining its use with existing chemoradiation regimens.

Shelton, Joseph W., E-mail: [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Waxweiler, Timothy V.; Landry, Jerome; Gao, Huiying; Xu, Yanbo; Wang, Lanfang [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)] [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); El-Rayes, Bassel [Department of Hematology and Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)] [Department of Hematology and Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States); Shu, Hui-Kuo G. [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)] [Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, Georgia (United States)



Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation  

SciTech Connect

Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the significant functional impact of Mesenchymal Stem Cell-secreted PAI-1 on colon cancer cells.

Hogan, Niamh M. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland)] [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Joyce, Myles R. [Department of Colorectal Surgery, University College Hospital, Galway (Ireland)] [Department of Colorectal Surgery, University College Hospital, Galway (Ireland); Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy [Regenerative Medicine Institute, National University of Ireland, Galway (Ireland)] [Regenerative Medicine Institute, National University of Ireland, Galway (Ireland); Kerin, Michael J. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland)] [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Dwyer, Roisin M., E-mail: [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland)



Evaluation of Cancer Stem Cell Markers CD133, CD44, CD24: Association with AKT Isoforms and Radiation Resistance in Colon Cancer Cells  

PubMed Central

The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms) expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1) expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133high/CD44high) were more resistant to gamma radiation than the ten percent with lowest expression (CD133low/CD44low). The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2) did not differ. Our results demonstrate that CD133high/CD44high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting. PMID:24760019

Sahlberg, Sara Haggblad; Spiegelberg, Diana; Glimelius, Bengt; Stenerlow, Bo; Nestor, Marika



Aberrant, ectopic expression of VEGF and VEGF receptors 1 and 2 in malignant colonic epithelial cells. Implications for these cells growth via an autocrine mechanism  

SciTech Connect

Highlights: •Malignant colonic epithelial cells express VEGF and its receptors. •Cultured colon cancer cells secrete VEGF into the medium. •Inhibition of VEGF receptor significantly decreases colon cancer cell proliferation. •VEGF is critical for colon cancer cell growth. -- Abstract: Vascular endothelial growth factor A (referred to as VEGF) is implicated in colon cancer growth. Currently, the main accepted mechanism by which VEGF promotes colon cancer growth is via the stimulation of angiogenesis, which was originally postulated by late Judah Folkman. However, the cellular source of VEGF in colon cancer tissue; and, the expression of VEGF and its receptors VEGF-R1 and VEGF-R2 in colon cancer cells are not fully known and are subjects of controversy. Material and methods: We examined and quantified expression of VEGF, VEGF-R1 and VEGF-R2 in three different human colonic tissue arrays containing sections of adenocarcinoma (n = 43) and normal mucosa (n = 41). In human colon cancer cell lines HCT116 and HT29 and normal colon cell lines NCM356 and NCM460, we examined expression of VEGF, VEGF-R1 and VEGF-R2 mRNA and protein, VEGF production and secretion into the culture medium; and, the effect of a potent, selective inhibitor of VEGF receptors, AL-993, on cell proliferation. Results: Human colorectal cancer specimens had strong expression of VEGF in cancer cells and also expressed VEGF-R1 and VEGF-R2.In vitro studies showed that human colon cancer cell lines, HCT116 and HT29, but not normal colonic cell lines, express VEGF, VEGF-R1 and VEGF-R2 and secrete VEGF into the medium up to a concentration 2000 pg/ml within 48 h. Furthermore, we showed that inhibition of VEGF receptors using a specific VEGF-R inhibitor significantly reduced proliferation (by >50%) of cultured colon cancer cell lines. Conclusions: Our findings support the contention that VEGF generated by colon cancer cells stimulates their growth directly through an autocrine mechanism that is independent of its primary function in the induction of angiogenesis.

Ahluwalia, Amrita [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States)] [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Jones, Michael K. [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States) [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Department of Medicine, University of California, Irvine, CA (United States); Szabo, Sandor [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States) [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Department of Pathology, University of California, Irvine, CA (United States); Tarnawski, Andrzej S., E-mail: [Veterans Affairs Long Beach Healthcare System, Long Beach, CA (United States); Department of Medicine, University of California, Irvine, CA (United States)



Cytotoxic flavonoids and isoflavonoids from Erythrina sigmoidea towards multi-factorial drug resistant cancer cells.  


Introduction Continuous efforts from scientists of diverse fields are necessary not only to better understand the mechanism by which multidrug resistant (MDR) cancer cells occur, but also to boost the discovery of new cytotoxic compounds. This work was designed to assess the cytotoxicity and the mechanism of action of flavonoids abyssinone IV (1), atalantoflavone (3) and neocyclomorusin (6) and isoflavonoids sigmoidin I (2), sophorapterocarpan A (4), bidwillon A (5) and 6?-hydroxyphaseollidin (7) isolated from Erythrina sigmoidea against nine drug sensitive and multidrug resistant (MDR) cancer cell lines. Methods The resazurin reduction assay was used to evaluate the cytotoxicity of the studied compounds whilst caspase-Glo assay was used to detect the activation of caspases enzymes by 1, 2, 4 and 7. Cell cycle, mitochondrial membrane potential and levels of reactive oxygen species were all analyzed via flow cytometry. Results The pterocarpan isoflavonoid 7 displayed the best antiproliferative activity with the IC50 values below 10 ?M obtained on the nine tested cancer cell lines. The IC50 values below 50 ?M were also recorded with compounds 1, 2 and 4 against the nine cancer cell lines whilst 3, 5 and 6 showed selective activities. The IC50 values varied from 14.43 ?M (against MDA-MB-231-pcDNA cells) to 20.65 ?M [towards HCT116 (p53 (+/+)) cells] for compound 1, from 4.24 ?M (towards CCRF-CEM cells) to 30.98 ?M (towards MDA-MB-231-BCRP cells) for 2, from 3.73 ?M (towards CCRF-CEM cells) to 14.81 ?M (against U87MG.?EGFR cells) for 4, from 3.36 ?M (towards CCRF-CEM cells) to 6.44 ?M (against HepG2 cells) for 7, and from 0.20 ?M (against CCRF-CEM cells) and 195.12 ?M (against CEM/ADR5000 cells) for the positive control drug, doxorubicin. Compared to their corresponding sensitive cell lines, collateral sensitivity was observed with HCT116 (p53 (-/-)) to 1, 2, 4, 5, and 7 and with U87MG.?EGFR to 1 to 6. Compound 7 induced apoptosis in CCRF-CEM cells mediated by the activation of caspases 3/7, 8 and 9 and breakdown of MMP and increase in ROS production, whereas the apoptotic process induced by 1, 2 and 4 was mediated by the loss of MMP as well as increase in ROS production. Conclusions Compounds from Erythrina sigmoidea and mostly 6?-hydroxyphaseollidin are potential antiproliferative natural products that deserve more investigations to develop novel anticancer drugs against sensitive and otherwise drug-resistant phenotypes. PMID:25034000

Kuete, Victor; Sandjo, Louis P; Djeussi, Doriane E; Zeino, Maen; Kwamou, Guy M N; Ngadjui, Bonaventure; Efferth, Thomas



Optimized pregelatinized starch technique for cell block preparation in cell cultures.  


The aim of the present study was to optimize the pregelatinized starch technique for cell block preparation and apply this approach in cultured cells of all types of growing forms, suspension and adherent. In order to evenly mix the starch powder and the cell suspension, we crafted a special plastic dropper. To prove the effectiveness of this optimized technique we used different cell lines, NCI-H69, NCI-H345, HCT-116, SKBR3 and MDA-MB-231. The morphology features, immunocytochemistry (ICC) and fluorescent/chromogenic in-situ hybridization (FISH/CISH) on the cell block sections were evaluated. The morphology features, the ICC and ISH results of cell block sections prepared by the new method were satisfactory comparing with the results obtained in biopsies, the gold standard test for this kind of analysis. The most attractive advantage of our optimized pregelatinized starch technique is that this new method is based on cell suspensions instead of cell sediment, so with our technique every section will contain cells due to the even distribution of the starch powder and the cells forming a homogeneous cell block. To the authors' knowledge, this is the first description on cell block preparation based on cell suspension. PMID:23797005

Zhu, Ya-Zhen; Cui, Feng-Yun; Yang, Yu; Peng, Hui; Li, Wei-Ping; Huang, Zhen-Dong; Zhu, Hong-Guang; He, Qing-Lian; Zheng, Guang-Juan



Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53  

Microsoft Academic Search

Ginsenosides are the main bioactive components in American ginseng, a commonly used herb. In this study, we showed that the ginsenoside Rh2 exhibited significantly more potent cell death activity than the ginsenoside Rg3 in HCT116 and SW480 colorectal cancer cells. Cell death induced by Rh2 is mediated in part by the caspase-dependent apoptosis and in part by the caspase-independent paraptosis,

Binghui Li; Jiong Zhao; Chong-Zhi Wang; Jennifer Searle; Tong-Chuan He; Chun-Su Yuan; Wei Du



Tumor Associated Macrophages Protect Colon Cancer Cells from TRAILInduced Apoptosis through IL1beta- Dependent Stabilization of Snail in Tumor Cells  

Microsoft Academic Search

BackgroundWe recently reported that colon tumor cells stimulate macrophages to release IL-1?, which in turn inactivates GSK3? and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.Principal FindingsHere we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1? by neutralizing IL-1? antibody, or silencing

Pawan Kaler; Vincent Galea; Leonard Augenlicht; Lidija Klampfer; Dong-Yan Jin



PRIMA-1 induces autophagy in cancer cells carrying mutant or wild type p53.  


PRIMA-1 is a chemical compound identified as a growth suppressor of tumor cells expressing mutant p53. We previously found that in the MDA-MB-231 cell line expressing high level of the mutant p53-R280K protein, PRIMA-1 induced p53 ubiquitination and degradation associated to cell death. In this study, we investigated the ability of PRIMA-1 to induce autophagy in cancer cells. In MDA-MB-231 and HCT116 cells, expressing mutant or wild type p53, respectively, autophagy occurred following exposure to PRIMA-1, as shown by acridine orange staining, anti-LC3 immunofluorescence and immunoblots, as well as by electron microscopy. Autophagy was triggered also in the derivative cell lines knocked-down for p53, although to a different extent than in the parental cells expressing mutant or wild type p53. In particular, while wild type p53 limited PRIMA-1 induced autophagy, mutant p53 conversely promoted autophagy, thus sustaining cell viability following PRIMA-1 treatment. Therefore, the autophagic potential of PRIMA-1, besides being cell context dependent, could be modulated in a different way by the presence of wild type or mutant p53. Furthermore, since both cell lines lacking p53 were more sensitive to the cytotoxic effect of PRIMA-1 than the parental ones, our findings suggest that a deregulated autophagy may favor cell death induced by this drug. PMID:23545415

Russo, Debora; Ottaggio, Laura; Foggetti, Giorgia; Masini, Matilde; Masiello, Pellegrino; Fronza, Gilberto; Menichini, Paola



1,3-Bis(2-chloroethyl)-1-nitrosourea enhances the inhibitory effect of Resveratrol on 5-fluorouracil sensitive/resistant colon cancer cells  

PubMed Central

AIM: To study the mechanism of 5-fluorouracil (5-FU) resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment. METHODS: We established and characterized a 5-FU resistance (5-FU-R) cell line derived from continuous exposure (25 ?mol/L) to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells. The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting, respectively, after treatment with Resveratrol (Res) and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU). Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis, respectively. The extent of DNA damage was measured by the Comet assay. We measured the visible changes in the DNA damage/repair cascade by Western blotting. RESULTS: The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner. Combined application of BCNU and Res caused more apoptosis in 5-FU sensitive cells in comparison to individual treatment. In addition, the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells. We established a 5-FU resistance cell line (5-FU-R) from 5-FU-sensitive HCT-116 (mismatch repair deficient) cells that was not resistant to other chemotherapeutic agents (e.g., BCNU, Res) except 5-FU. The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay. There was no significant cell death noted in 5-FU-R cells in comparison to 5-FU sensitive cells after 5-FU treatment. This resistant cell line overexpressed anti-apoptotic [e.g., AKT, nuclear factor ?B, FLICE-like inhibitory protein), DNA repair (e.g., DNA polymerase beta (POL-?), DNA polymerase eta (POLH), protein Flap endonuclease 1 (FEN1), DNA damage-binding protein 2 (DDB2)] and 5-FU-resistance proteins (thymidylate synthase) but under expressed pro-apoptotic proteins (e.g., DAB2, CK1) in comparison to the parental cells. Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells. BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells. Fifty percent cell death were noted in parental cells when 18 ?mol/L of Res was associated with fixed concentration (20 ?mol/L) of BCNU, but a much lower concentration of Res (8 ?mol/L) was needed to achieve the same effect in 5-FU resistant cells. Interestingly, increased levels of adenomatous polyposis coli and decreased levels POL-?, POLH, FEN1 and DDB2 were noted after the same combined treatment in resistant cells. CONCLUSION: BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance. PMID:24259968

Das, Dipon; Preet, Ranjan; Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Kundu, Chanakya Nath



CD133+CXCR4+ colon cancer cells exhibit metastatic potential and predict poor prognosis of patients  

PubMed Central

Background Colorectal cancer (CRC), which frequently metastasizes to the liver, is one of the three leading causes of cancer-related deaths worldwide. Growing evidence suggests that a subset of cells exists among cancer stem cells. This distinct subpopulation is thought to contribute to liver metastasis; however, it has not been fully explored in CRC yet. Methods Flow cytometry analysis was performed to detect distinct subsets with CD133 and CXCR4 markers in human primary and metastatic CRC tissues. The 'stemness' and metastatic capacities of different subpopulations derived from the colon cancer cell line HCT116 were compared in vitro and in vivo. The roles of epithelial-mesenchymal transition (EMT) and stromal-cell derived factor-1 (SDF-1) in the metastatic process were also investigated. A survival curve was used to explore the correlation between the content of CD133+CXCR4+ cancer cells and patient survival. Results In human specimens, the content of CD133+CXCR4+ cells was higher in liver metastases than in primary colorectal tumors. Clonogenic and tumorigenic cells were restricted to CD133+ cells in the HCT116 cell line, with CXCR4 expression having no impact on the 'stemness' properties. We found that CD133+CXCR4+ cancer cells had a high metastatic capacity in vitro and in vivo. Compared with CD133+CXCR4- cells, CD133+CXCR4+ cancer cells experienced EMT, which contributed partly to their metastatic phenotype. We then determined that SDF-1/CXCL12 treatment could further induce EMT in CD133+CXCR4+ cancer cells and enhance their invasive behavior, while this could not be observed in CD133+CXCR4- cancer cells. Blocking SDF-1/CXCR4 interaction with a CXCR4 antagonist, AMD3100 (1,10-[1,4-phenylenebis(methylene)]bis-1,4,8,11 -tetraazacyclotetradecane octahydrochloride), inhibited metastatic tumor growth in a mouse hepatic metastasis model. Finally, a high percentage of CD133+CXCR4+ cells in human primary CRC was associated with a reduced two-year survival rate. Conclusions Strategies targeting the SDF-1/CXCR4 interaction may have important clinical applications in the suppression of colon cancer metastasis. Further investigations on how high expression of CXCR4 and EMT occur in this identified cancer stem cell subset are warranted to provide insights into our understanding of tumor biology. PMID:22871210



Down-regulation of GPR137 expression inhibits proliferation of colon cancer cells.  


G protein-coupled receptors (GPRs) are highly related to oncogenesis and cancer metastasis. G protein-coupled receptor 137 (GPR137) was initially reported as a novel orphan GPR about 10 years ago. Some orphan GPRs have been implicated in human cancers. The aim of this study is to investigate the role of GPR137 in human colon cancer. Expression levels of GRP137 were analyzed in different colon cancer cell lines by quantitative polymerase chain reaction and western blot analysis. Lentivirus-mediated short hairpin RNA was specifically designed to knock down GPR137 expression in colon cancer cells. Cell viability was measured by methylthiazoletetrazolium and colony formation assays. In addition, cell cycle characteristic was investigated by flow cytometry. GRP137 expression was observed in all seven colon cancer cell lines at different levels. The mRNA and protein levels of GPR137 were down-regulated in both HCT116 and RKO cells after lentivirus infection. Lentivirus-mediated silencing of GPR137 reduced the proliferation rate and colonies numbers. Knockdown of GPR137 in both cell lines led to cell cycle arrest in the G0/G1 phase. These results indicated that GPR137 plays an important role in colon cancer cell proliferation. A better understanding of GPR137's effects on signal transduction pathways in colon cancer cells may provide insights into the novel gene therapy of colon cancer. PMID:25301753

Zhang, Kai; Shen, Zhen; Liang, Xianjun; Liu, Tongjun; Wang, Tiejun; Jiang, Yang



Cellular distribution of tumour suppressor protein p53 and high-risk human papillomavirus (HPV)-18 E6 fusion protein in wild-type p53 cell lines.  


Human papillomaviruses (HPVs) are very important pathogens that can be classified as high- and low-risk types based on the lesions they cause. Mucosal high-risk HPV E6 can target and degrade the tumour suppressor p53, hence it is recognized as the major cause of cervical cancer, however, due to a lack of reliable anti-E6 antibodies, the distribution of high-risk HPV E6 protein remains elusive. The present study, therefore, used a mammalian green fluorescent protein (GFP) expression system to express GFP-18 E6 fusion proteins in wild-type p53 cells, SMMC-7721 and HCT116, in order to trace the location and expression of HPV E6 and p53 protein. Following transfection, expression of GFP-18 E6 was found to be located in the nucleus, and endogenous wild-type p53 was also located there with GFP-18 E6. PMID:18831896

Sun, L; Zhang, G; Li, Z; Lei, T; Huang, C; Song, T; Si, L



Antiproliferative activity and induction of apoptosis in human colon cancer cells treated in vitro with constituents of a product derived from Pistacia lentiscus L. var. chia  

Microsoft Academic Search

In this report, we demonstrate that a 50% ethanol extract of the plant-derived product, Chios mastic gum (CMG), contains compounds which inhibit proliferation and induce death of HCT116 human colon cancer cells in vitro. CMG-treatment induces cell arrest at G1, detachment of the cells from the substrate, activation of pro-caspases-8, -9 and -3, and causes several morphological changes typical of

K. V. Balan; J. Prince; Z. Han; K. Dimas; M. Cladaras; J. H. Wyche; N. M. Sitaras; P. Pantazis



Biology of SNU Cell Lines  

PubMed Central

SNU (Seoul National University) cell lines have been established from Korean cancer patients since 1982. Of these 109 cell lines have been characterized and reported, i.e., 17 colorectal carcinoma, 12 hepatocellular carcinoma, 11 gastric carcinoma, 12 uterine cervical carcinoma, 17 B-lymphoblastoid cell lines derived from cancer patients, 5 ovarian carcinoma, 3 malignant mixed Mllerian tumor, 6 laryngeal squamous cell carcinoma, 7 renal cell carcinoma, 9 brain tumor, 6 biliary tract, and 4 pancreatic carcinoma cell lines. These SNU cell lines have been distributed to biomedical researchers domestic and worldwide through the KCLB (Korean Cell Line Bank), and have proven to be of value in various scientific research fields. The characteristics of these cell lines have been reported in over 180 international journals by our laboratory and by many other researchers from 1987. In this paper, the cellular and molecular characteristics of SNU human cancer cell lines are summarized according to their genetic and epigenetic alterations and functional analysis. PMID:19956504

Ku, Ja-Lok



Dual-Color Imaging of Nuclear-Cytoplasmic Dynamics, Viability, and Proliferation of Cancer Cells in the Portal Vein Area  

Microsoft Academic Search

We used dual-color in vivo cellular imaging to visualize trafficking, nuclear-cytoplasmic dynamics, and the viability of cancer cells after their injection into the portal vein of mice. For these studies, we used dual-color fluorescent cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and retroviral red fluorescent protein (RFP) in the cytoplasm. Human HCT-116-GFP-RFP

Kazuhiko Tsuji; Kensuke Yamauchi; Meng Yang; Ping Jiang; Michael Bouvet; Hitoshi Endo; Yoshikatsu Kanai; Koji Yamashita; Abdool R. Moossa


Using real-time impedance-based assays to monitor the effects of fibroblast-derived media on the adhesion, proliferation, migration and invasion of colon cancer cells  

PubMed Central

Increasing our knowledge of the mechanisms regulating cell proliferation, migration and invasion are central to understanding tumour progression and metastasis. The local tumour microenvironment contributes to the transformed phenotype in cancer by providing specific environmental cues that alter the cells behaviour and promotes metastasis. Fibroblasts have a strong association with cancer and in recent times there has been some emphasis in designing novel therapeutic strategies that alter fibroblast behaviour in the tumour microenvironment. Fibroblasts produce growth factors, chemokines and many of the proteins laid down in the ECM (extracellular matrix) that promote angiogenesis, inflammation and tumour progression. In this study, we use a label-free RTCA (real-time cell analysis) platform (xCELLigence) to investigate how media derived from human fibroblasts alters cancer cell behaviour. We used a series of complimentary and novel experimental approaches to show HCT116 cells adhere, proliferate and migrate significantly faster in the presence of media from human fibroblasts. As well as this, we used the xCELLigence CIM-plates system to show that HCT116 cells invade matrigel layers aggressively when migrating towards media derived from human fibroblasts. These data strongly suggest that fibroblasts have the ability to increase the migratory and invasive properties of HCT116 cells. This is the first study that provides real-time data on fibroblast-mediated migration and invasion kinetics of colon cancer cells. PMID:24935351

Dowling, Catriona M.; Herranz Ors, Carmen; Kiely, Patrick A.



Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT  

PubMed Central

Objective Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment. Methods Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU. Results Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (?1-integrin, ICAM-1), transforming growth factor-? signaling molecules (TGF-?3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-?B, MMP-13), TGF-?3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment. Conclusion Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-? and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.

Buhrmann, Constanze; Kraehe, Patricia; Lueders, Cora; Shayan, Parviz; Goel, Ajay; Shakibaei, Mehdi



Astrocyte Elevated Gene-1 Mediates Glycolysis and Tumorigenesis in Colorectal Carcinoma Cells via AMPK Signaling  

PubMed Central

To investigate the role of AEG-1 in glycolysis and tumorigenesis, we construct myc-AEG-1 expression vector and demonstrate a novel mechanism that AEG-1 may increase the activity of AMPK by Thr172 phosphorylation. The higher expression levels of AEG-1 in colorectal carcinoma cells were found but showed significant difference in different cell lines. To study the role of AEG-1 in colorectal cells, myc-AEG-1 vector was constructed and transfected into NCM460 colonic epithelial cells. We observed consistent increasing of glucose consumption and lactate production, typical features of anaerobic glycolysis, suggesting that AEG-1 may promote anaerobic glycolysis. Moreover, we noted that AMPK phosphorylation at Thr172 as well as pPFK2 (Ser466) was increased in NCM460 cells overexpressing AEG-1. Compound C may block AMPK and PFK2 phosphorylation in both control and AEG-1-overexpressed cells and decrease the glucose consumption and lactate production. The present findings indicated that reduced AEG-1 protein levels by RNAi may decrease the glucose consumption and lactate production in HCT116 colorectal carcinoma cells. The present identified AEG-1/AMPK/PFK2 glycolysis cascade may be essential to cell proliferation and tumor growth. The present results may provide us with a mechanistic insight into novel targets controlled by AEG-1, and the components in the AEG-1/AMPK/PFK2 glycolysis process may be targeted for the clinical treatment of cancer. PMID:24829520

Song, Hong-tao; Qin, Yu; Yao, Guo-dong; Tian, Zhen-nan; Fu, Song-bin; Geng, Jing-shu



Growth inhibition of colon cancer cells by compounds affecting AMPK activity  

PubMed Central

AIM: To determine if other molecules reported to modulate AMP-dependent protein kinase (AMPK) activity would have effects resembling those of metformin and phenformin on colon cancer cell proliferation and metabolism. METHODS: Studies were performed with four human colon cancer cell lines, Caco-2, HCT116, HT29 and SW1116. The compounds that were studied included A-769662, 5-aminoimidazole-4-carboxamide-1-ribofuranoside, butyrate, (-)-epigallocatechin gallate (EGCG), KU-55933, quercetin, resveratrol and salicylates. The parameters that were measured were cell proliferation and viability, glucose uptake, lactate production and acidification of the incubation medium. RESULTS: Investigations with several molecules that have been reported to be associated with AMPK activation (A-769662, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside, EGCG, KU-55933, quercetin, resveratrol and salicylates) or AMPK inhibition (compound C) failed to reveal increased medium acidification and increased glucose uptake in colon cancer cells as previously established with metformin and phenformin. The only exception was 5-aminosalicylic acid with which there were apparently lower glucose levels in the medium after incubation for 72 h. Further study in the absence of cells revealed that the effect was an artifact due to inhibition of the enzyme-linked glucose assay. The compounds were studied at concentrations that inhibited cell proliferation. CONCLUSION: It was concluded that treatment with several agents that can affect AMPK activity resulted in the inhibition of the proliferation of colon cancer cells under conditions in which glucose metabolism is not enhanced, in contrast to the effect of biguanides. PMID:25024815

Lea, Michael A; Pourat, Jacob; Patel, Rupali; desBordes, Charles



Dihydroceramide delays cell cycle G1/S transition via activation of ER stress and induction of autophagy.  


Dihydroceramides, the precursors of ceramides in the de novo sphingolipid synthesis, have been recently implicated in active signalling. We previously demonstrated that dihydroceramide accumulation, in response to treatment with the dihydroceramide desaturase inhibitor XM462, induced autophagy with no sign of cell death in the gastric carcinoma HCG27 cell line. Here we show that XM462 treatment induces a transient early increase in dihydroceramides that are successively metabolized into other sphingolipids. Dihydroceramides accumulation is associated with cyclin D1 expression modulation, delayed G1/S transition of cell cycle and increased autophagy. Moreover, XM462 treatment induces ER stress via the activation of the translation inhibitor eIF2? and the pro-survival transcriptional factor Xbp1. Exogenous addition of a short chain dihydroceramide analog reproduces the effects of endogenous accumulation of dihydroceramides, causing cell cycle delay of the G1/S transition, autophagy enhancement, eIF2? activation and Xbp1 splicing. Blocking autophagy with 3-methyladenine abrogates the effect of XM462 on cell cycle and reduces cell survival to XM462 treatment. Furthermore, the XM462-induced survival response is able to reduce etoposide toxicity in HCG27 and HCT116 cancer cells. Our data suggest a role of dihydroceramide in regulating cell proliferation and survival. PMID:22960157

Gagliostro, Vincenzo; Casas, Josefina; Caretti, Anna; Abad, Jose L; Tagliavacca, Luigina; Ghidoni, Riccardo; Fabrias, Gemma; Signorelli, Paola



Nicotinamide Phosphoribosyl Transferase (Nampt) Is a Target of MicroRNA-26b in Colorectal Cancer Cells  

PubMed Central

A number of cancers show increased expression of Nicotinamide phosphoribosyl transferase (Nampt). However, the mechanism through which Nampt is upregulated is unclear. In our study, we found that the Nampt-specific chemical inhibitor FK866 significantly inhibited cell survival and reduced nicotinamide adenine dinucleotide (NAD) levels in LoVo and SW480 cell lines. Bioinformatics analyses suggested that miR-26b targets Nampt mRNA. We identified Nampt as a new target of miR-26b and demonstrated that miR-26b inhibits Nampt expression at the protein and mRNA levels by binding to the Nampt 3?-UTR. Moreover, we found that miR-26b was down regulated in cancer tissues relative to that in adjacent normal tissues in 18 colorectal cancer patients. A statistically significant inverse correlation between miR-26b and Nampt expression was observed in samples from colorectal cancer patients and in 5 colorectal cell lines (HT-29, SW480, SW1116, LoVo, and HCT116). In addition, over expression of miR-26b strongly inhibited LoVo cell survival and invasion, an effect partially abrogated by the addition of NAD. In conclusion, this study demonstrated that the NAD-salvaging biosynthesis pathway involving Nampt might play a role in colorectal cancer cell survival. MiR-26b may serve as a tumor suppressor by targeting Nampt. PMID:23922874

Huang, Gang



Toxins VapC and PasB from Prokaryotic TA Modules Remain Active in Mammalian Cancer Cells  

PubMed Central

Among the great number of addictive modules which have been discovered, only a few have been characterized. However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine. In our study, we tested two different toxins derived from class II addictive modules, pasAB from plasmid pTF-FC2 (Thiobacillus ferrooxidans) and vapBC 2829Rv (Mycobacterium tuberculosis), in terms of their usefulness as growth inhibitors of human cancer cell lines, namely KYSE 30, MCF-7 and HCT 116. Transfection of the pasB and vapC genes into the cells was conducted with the use of two different expression systems. Cellular effects, such as apoptosis, necrosis and changes in the cell cycle, were tested by applying flow cytometry with immunofluorescence staining. Our findings demonstrated that toxins VapC and PasB demonstrate proapoptotic activity in the human cancer cells, regardless of the expression system used. As for the toxin PasB, observed changes were more subtle than for the VapC. The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line. PMID:25271785

Wieteska, Lukasz; Skulimowski, Aleksander; Cybula, Magdalena; Szemraj, Janusz



Tax1 enhances cancer cell proliferation via Ras-Raf-MEK-ERK signaling pathway.  


Erbin is an ErbB2 binding protein, which belongs to the LAP (leucine-rich repeat (LRR) and PDZ domain) protein family. We previously reported that Tax1, a protein of the human T-cell leukemia virus type I (HTLV-I), associated with Erbin by using Erbin PDZ domain as a bait to screen a human T lymphocyte cDNA library by a yeast two hybrid strategy. In the present study, we demonstrated that Tax1 enhances cancer cell proliferation via Ras-Raf-MEK-ERK signaling pathway by using molecular section strategy. The pull-down assay showed that the four amino acid domain, that is, Tax1 350-353, might specifically interact with Erbin, but not any other Tax1 deletion mutants. The coimmunoprecipitation assay confirmed that Tax1 350-353 domain bound with Erbin in vivo. Functional study demonstrated that overexpression of Tax1 in cancer cell lines of liver cancer SMMC-7721, colon cancer HCT-116, and breast cancer MCF-7 facilitated the cell proliferation. And the transfection of Tax1 353 in MCF-7 cells with endogenous Erbin expression markedly increased phosphorylation of Ras, Raf, MEK1/2, ERK1/2, PI3K, and IkappaBalpha, suggesting that Tax1-enhanced cell proliferation tracks Ras-Raf-MEK-ERK signaling pathway. PMID:19472191

Song, Chunjiao; Wang, Weimin; Li, Meng; Liu, Yanxin; Zheng, Dexian



Synthetic lethal targeting of PTEN-deficient cancer cells using selective disruption of polynucleotide kinase/phosphatase  

PubMed Central

A recent screen of 6961 siRNAs to discover possible synthetic lethal partners of the DNA repair protein polynucleotide kinase/phosphatase (PNKP) led to the identification of the potent tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Here we have confirmed the PNKP/PTEN synthetic lethal partnership in a variety of different cell lines including the PC3 prostate cancer cell line, which is naturally deficient in PTEN. We provide evidence that co-depletion of PTEN and PNKP induces apoptosis. In HCT116 colon cancer cells the loss of PTEN is accompanied by an increased background level of DNA double strand breaks, which accumulate in the presence of an inhibitor of PNKP DNA 3?-phosphatase activity. Complementation of PC3 cells with several well-characterized mutated PTEN cDNAs indicated that the critical function of PTEN required to prevent toxicity induced by an inhibitor of PNKP is most likely associated with its cytoplasmic lipid phosphatase activity. Finally, we show that modest inhibition of PNKP in a PTEN knockout background enhances cellular radiosensitivity, suggesting that such a “synthetic sickness” approach involving the combination of PNKP inhibition with radiotherapy may be applicable to PTEN-deficient tumors. PMID:23883586

Mereniuk, Todd R.; El Gendy, Mohamed A.M.; Mendes-Pereira, Ana M.; Lord, Christopher J.; Ghosh, Sunita; Foley, Edan; Ashworth, Alan; Weinfeld, Michael



Thyroid cell lines in research on goitrogenesis.  


Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

Gerber, H; Peter, H J; Asmis, L; Studer, H



Down-Regulation of miR-126 Is Associated with Colorectal Cancer Cells Proliferation, Migration and Invasion by Targeting IRS-1 via the AKT and ERK1/2 Signaling Pathways  

PubMed Central

Background Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126. Methods The effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3?-untranslated region (3?-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay. Results We found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3?-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein. Conclusions MiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways. PMID:24312276

Zhou, Yu; Feng, Xiao; Liu, Ya-ling; Ye, Shi-cai; Wang, Hao; Tan, Wen-kai; Tian, Ting; Qiu, Yu-mei; Luo, He-sheng



The mechanisms responsible for the radiosensitizing effects of sorafenib on colon cancer cells.  


Colorectal cancer is one of the most common malignancies in the world, and is generally treated more effectively by chemoradiotherapy rather than radiotherapy or chemotherapy alone. Targeted radiosensitizers are often used in order to enhance the radiosensitivity of tumor cells. The aim of the present study was to identify the mechanism of radiosensitization by sorafenib in colorectal cancer. Three human colorectal adenocarcinoma cell lines (HCT116, HT29 and SW480) were treated with sorafenib alone or radiation followed by sorafenib. In vitro tests were performed using colony forming assays, FACS analysis, immunohistochemistry, tumor cell motility assays, invasion assays and endothelial tube formation assays. Sorafenib enhanced the anti-proliferative effects of radiation, reducing colony formation, increasing G2/M arrest and enhancing radiation-induced apoptosis by reactive oxygen species. Sorafenib also inhibited the repair of radiation-induced DNA damage by blocking the activation of DNA-dependent protein kinase. Combination treatment significantly inhibited tumor cell migration, tumor cell invasion and vascular endothelial growth factor-mediated angiogenesis in vitro. Taken together, our results provide a scientific rationale for the use of sorafenib with radiotherapy in colon cancer and suggest a clinical utility for this approach. PMID:25242034

Kim, Eun Ho; Kim, Mi-Sook; Jung, Won-Gyun



Survivin-2B promotes autophagy by accumulating IKK alpha in the nucleus of selenite-treated NB4 cells  

PubMed Central

Survivin-2B, a known splice variant of survivin, has been reported to promote cell death in some cancer cells, although it keeps prosurvival function in others, and the mechanisms are unclear. In this report, we discovered that selenite, an antitumor agent, switched protective autophagy to apoptosis in NB4 cells. In this process, the level of survivin-2B was decreased and the interaction between IKK alpha and survivin-2B in the nucleus was attenuated, which further led to the decrease of nuclear IKK alpha. As a result, P73, a known transcript factor of UVRAG, was downregulated. Therefore, the expression of UVRAG, one of the initiators of autophagy, was inhibited. The regulatory status of survivin-2B was also proved in NB4 cells after different chemicals' exposure and in other tumor cell lines (Jurkat, HCT116). Finally, experiments in vivo confirmed that the alterations of survivin-2B, IKK alpha, P73 and UVRAG were the same as that in vitro. Taken together, survivin-2B promoted autophagy and further regulated cell death by accumulating and stabilizing IKK alpha in the nucleus. PMID:24556686

Shi, K; An, J; Shan, L; Jiang, Q; Li, F; Ci, Y; Wu, P; Duan, J; Hui, K; Yang, Y; Xu, C



NPRL-Z-1, as a New Topoisomerase II Poison, Induces Cell Apoptosis and ROS Generation in Human Renal Carcinoma Cells  

PubMed Central

NPRL-Z-1 is a 4?-[(4?-benzamido)-amino]-4?-O-demethyl-epipodophyllotoxin derivative. Previous reports have shown that NPRL-Z-1 possesses anticancer activity. Here NPRL-Z-1 displayed cytotoxic effects against four human cancer cell lines (HCT 116, A549, ACHN, and A498) and exhibited potent activity in A498 human renal carcinoma cells, with an IC50 value of 2.38 µM via the MTT assay. We also found that NPRL-Z-1 induced cell cycle arrest in G1-phase and detected DNA double-strand breaks in A498 cells. NPRL-Z-1 induced ataxia telangiectasia-mutated (ATM) protein kinase phosphorylation at serine 1981, leading to the activation of DNA damage signaling pathways, including Chk2, histone H2AX, and p53/p21. By ICE assay, the data suggested that NPRL-Z-1 acted on and stabilized the topoisomerase II (TOP2)–DNA complex, leading to TOP2cc formation. NPRL-Z-1-induced DNA damage signaling and apoptotic death was also reversed by TOP2? or TOP2? knockdown. In addition, NPRL-Z-1 inhibited the Akt signaling pathway and induced reactive oxygen species (ROS) generation. These results demonstrated that NPRL-Z-1 appeared to be a novel TOP2 poison and ROS generator. Thus, NPRL-Z-1 may present a significant potential anticancer candidate against renal carcinoma. PMID:25372714

Wu, Szu-Ying; Pan, Shiow-Lin; Xiao, Zhi-Yan; Hsu, Jui-Ling; Chen, Mei-Chuan; Lee, Kuo-Hsiung; Teng, Che-Ming



Oncostatin M Mediates STAT3-Dependent Intestinal Epithelial Restitution via Increased Cell Proliferation, Decreased Apoptosis and Upregulation of SERPIN Family Members  

PubMed Central

Objective Oncostatin M (OSM) is produced by activated T cells, monocytes, and dendritic cells and signals through two distinct receptor complexes consisting of gp130 and LIFR (I) or OSMR-? and gp130 (II), respectively. Aim of this study was to analyze the role of OSM in intestinal epithelial cells (IEC) and intestinal inflammation. Methods OSM expression and OSM receptor distribution was analyzed by PCR and immunohistochemistry experiments, signal transduction by immunoblotting. Gene expression studies were performed by microarray analysis and RT-PCR. Apoptosis was measured by caspases-3/7 activity. IEC migration and proliferation was studied in wounding and water soluble tetrazolium assays. Results The IEC lines Caco-2, DLD-1, SW480, HCT116 and HT-29 express mRNA for the OSM receptor subunits gp130 and OSMR-?, while only HCT116, HT-29 and DLD-1 cells express LIFR mRNA. OSM binding to its receptor complex activates STAT1, STAT3, ERK-1/2, SAPK/JNK-1/2, and Akt. Microarray analysis revealed 79 genes that were significantly up-regulated (adj.-p?0.05) by OSM in IEC. Most up-regulated genes belong to the functional categories “immunity and defense” (p?=?2.1×10?7), “apoptosis” (p?=?3.7×10?4) and “JAK/STAT cascade” (p?=?3.4×10?6). Members of the SERPIN gene family were among the most strongly up-regulated genes. OSM significantly increased STAT3- and MEK1-dependent IEC cell proliferation (p<0.05) and wound healing (p?=?3.9×10?5). OSM protein expression was increased in colonic biopsies of patients with active inflammatory bowel disease (IBD). Conclusions OSM promotes STAT3-dependent intestinal epithelial cell proliferation and wound healing in vitro. Considering the increased OSM expression in colonic biopsy specimens of patients with active IBD, OSM upregulation may modulate a barrier-protective host response in intestinal inflammation. Further in vivo studies are warranted to elucidate the exact role of OSM in intestinal inflammation and the potential of OSM as a drug target in IBD. PMID:24710357

Beigel, Florian; Friedrich, Matthias; Probst, Corina; Sotlar, Karl; Goke, Burkhard; Diegelmann, Julia; Brand, Stephan



Isolation and antiproliferative activity of Lotus corniculatus lectin towards human tumour cell lines.  


The objective of the study was to investigate the anti cancer activity of a lectin isolated from Lotus corniculatus seeds. A tetrameric 70kDa galactose specific lectin was purified using two step simple purification protocol which involved affinity chromatography on AF-BlueHC650M and gel filtration on Sephadex G-100. The lectin was adsorbed on AF-BlueHC650M and desorbed using 1M NaCl in the starting buffer. Gel filtration on Sephadex G-100 yielded a major peak absorbance that gave two bands of 15kDa and 20kDa in SDS PAGE. Hemagglutination activity was completely preserved, when the temperature was in the range of 20-60°C. However, drastic reduction in activity occurred at temperatures above 60°C. Full hemagglutination activity was retained at ambient pH 4-12. Thereafter no activity was observed above pH 13. Hemaglutination of the lectin was inhibited by d-galactose. The lectin showed a strong antiproliferative activity towards human leukemic (THP-1) cancer cells followed by lung cancer (HOP62) cells and HCT116 with an IC50 of 39?g/ml and 50?g/ml and 60?g/ml respectively. Flow cytometry analysis showed an increase in the percentage of cells in sub G0G1 phase confirming that Lotus corniculatus lectin induced apoptosis. Morphological observations showed that Lotus corniculatus lectin (LCL) treated THP-1 cells displayed apparent apoptosis characteristics such as nuclear fragmentation, appearance of membrane enclosed apoptotic bodies and DNA fragmentation. Lotus corniculatus lectin (LCL) effectively inhibits the cell migration in a dose dependent manner as indicated by the wound healing assay. PMID:24055517

Rafiq, Shaista; Majeed, Rabiya; Qazi, Asif Khurshid; Ganai, Bashir Ahmad; Wani, Ishfak; Rakhshanda, Syed; Qurishi, Yasrib; Sharma, P R; Hamid, Abid; Masood, Akbar; Hamid, Rabia



Relative inhibition of lipid peroxidation, cyclooxygenase enzymes, and human tumor cell proliferation by natural food colors.  


The most abundant water soluble natural food colors are betacyanins and anthocyanins. Similarly, lycopene, bixin, beta-carotene, and chlorophyll are water insoluble colors. Pure betanin, bixin, lycopene, chlorophyll, beta-carotene, and cyanidin-3-O-glucoside were isolated from Beta vulgaris, Bixa orellana,Lycopersicum esculentum, Spinacia oleracea, Daucus carrota, and Prunus cerasus, respectively. These natural pigments, alone and in combination, were evaluated for their relative potencies against cyclooxygenase enzymes and tumor cell growth inhibition by using MCF-7 (breast), HCT-116 (colon), AGS (stomach), CNS (central nervous system), and NCI-H460 (lung) tumor cell lines. Among the colors tested, betanin, cyanidin-3-O-glucoside, lycopene, and beta-carotene inhibited lipid peroxidation. However, all pigments tested gave COX-1 and COX-2 inhibition and showed a dose-dependent growth inhibition against breast, colon, stomach, central nervous system, and lung tumor cells, respectively. The mixtures of these pigments were also evaluated for their synergistic effects and chemical interactions at various concentrations. The mixture of anthocyanin and betanin negated their efficacy in the cell growth inhibitory assay and did not enhance the COX enzyme inhibitory activity. This is the first report of a comparative evaluation and the impact on biological activities of these pigments alone and in combination. PMID:16277432

Reddy, Muntha K; Alexander-Lindo, Ruby L; Nair, Muraleedharan G



Upregulated SLC1A5 promotes cell growth and survival in colorectal cancer  

PubMed Central

Glutamine metabolism is essential for tumorigenesis of colorectal cancer, cancer cells remodel their glutamine metabolic pathways to fuel rapid proliferation. SLC1A5 is an important transporter of glutamine various cancer cells. In this study, we investigated SLC1A5 protein expression in colorectal cancer and evaluated its clinical significance and functional importance. Immunohistochemical analysis was performed on tissue microarrays containing 90 pairs of cancer and adjacent normal tissues from colorectal cancer patients, we found that SLC1A5 expression increased significantly in colorectal cancer compared with normal mucosa tissues (P < 0.001). We further validated SLC1A5 overexpression in 12 pairs of fresh cancer and adjacent normal mucosa tissues from colorectal cancer patients by Western blot (P < 0.05). SLC1A5 expression levels were strongly associated with T stage of tumor (P < 0.05), and the tubular adenocarcinoma subtype (P < 0.001). Moreover, downregulation of SLC1A5 by synthetic siRNA could suppress proliferation and induce apoptosis in colorectal cancer cell lines HT29 and HCT116. In conclusion, our results provide for the first time the differential expression in human colorectal cancer and normal tissues, and a functional link between SLC1A5 expression and growth and survival of colorectal cancer, making it an attractive target in colorectal cancer treatment. PMID:25337245

Huang, Fang; Zhao, Yingchao; Zhao, Junzhang; Wu, Shuang; Jiang, Yao; Ma, Hong; Zhang, Tao



Asymmetric triplex metallohelices with high and selective activity against cancer cells  

NASA Astrophysics Data System (ADS)

Small cationic amphiphilic ?-helical peptides are emerging as agents for the treatment of cancer and infection, but they are costly and display unfavourable pharmacokinetics. Helical coordination complexes may offer a three-dimensional scaffold for the synthesis of mimetic architectures. However, the high symmetry and modest functionality of current systems offer little scope to tailor the structure to interact with specific biomolecular targets, or to create libraries for phenotypic screens. Here, we report the highly stereoselective asymmetric self-assembly of very stable, functionalized metallohelices. Their anti-parallel head-to-head-to-tail ‘triplex’ strand arrangement creates an amphipathic functional topology akin to that of the active sub-units of, for example, host-defence peptides and p53. The metallohelices display high, structure-dependent toxicity to the human colon carcinoma cell-line HCT116 p53++, causing dramatic changes in the cell cycle without DNA damage. They have lower toxicity to human breast adenocarcinoma cells (MDA-MB-468) and, most remarkably, they show no significant toxicity to the bacteria methicillin-resistant Staphylococcus aureus and Escherichia coli.

Faulkner, Alan D.; Kaner, Rebecca A.; Abdallah, Qasem M. A.; Clarkson, Guy; Fox, David J.; Gurnani, Pratik; Howson, Suzanne E.; Phillips, Roger M.; Roper, David I.; Simpson, Daniel H.; Scott, Peter



Rapid purification of cell encapsulated hydrogel beads from oil phase to aqueous phase in a microfluidic device.  


In this paper, we demonstrate a new type of microfluidic chip that can realize continuous-flow purification of hydrogel beads from a carrier oil into aqueous solution by using a laminar-like oil/water interface. The microfluidic chip is composed by two functional components: (1) a flow-focusing bead generation module that can control size and shape of beads, (2) a bead extraction module capable of purifying hydrogel beads from oil into aqueous solution. This module is featured with large branch channels on one side and small ones on the opposite side. Water is continuously infused into the bead extraction module through the large branch channels, resulting in a laminar-like oil/water interface between the branch junctions. Simulation and experimental data show that the efficiency of oil depletion is determined by the relative flow rates between infused water and carrier oil. By using such a microfluidic device, viable cells (HCT116, colon cancer cell line) can be encapsulated in the hydrogel beads and purified into a cell culture media. Significantly improved cell viability was achieved compared to that observed by conventional bead purification approaches. This facile microfluidic chip could be a promising candidate for sample treatment in lab-on-a-chip applications. PMID:22012540

Deng, Yuliang; Zhang, Nangang; Zhao, Libo; Yu, Xiaolei; Ji, Xinghu; Liu, Wei; Guo, Shishang; Liu, Kan; Zhao, Xing-Zhong



Surface-Exposed Histone-Like Protein A Modulates Adherence of Streptococcus gallolyticus to Colon Adenocarcinoma Cells ?  

PubMed Central

Streptococcus gallolyticus (formerly known as Streptococcus bovis biotype I) is a low-grade opportunistic pathogen which is considered to be associated with colon cancer. It is thought that colon polyps or tumors are the main portal of entry for this bacterium and that heparan sulfate proteoglycans (HSPGs) at the colon tumor cell surface are involved in bacterial adherence during the first stages of infection. In this study, we have shown that the histone-like protein A (HlpA) of S. gallolyticus is a genuine anchorless bacterial surface protein that binds to lipoteichoic acid (LTA) of the gram-positive cell wall in a growth phase-dependent manner. In addition, HlpA was shown to be one of the major heparin-binding proteins of S. gallolyticus able to bind to the HSPG-expressing colon tumor cell lines HCT116 and HT-29. Strikingly, although wild-type levels of HlpA appeared to contribute to adherence, coating of additional HlpA at the bacterial surface resulted in reduced binding to colon tumor cells. This may be explained by the fact that heparan sulfate and LTA compete for the same binding site in HlpA. Altogether, this study implies that HlpA serves as a fine-tuning factor for bacterial adherence. PMID:19752027

Boleij, Annemarie; Schaeps, Renee M. J.; de Kleijn, Stan; Hermans, Peter W.; Glaser, Philippe; Pancholi, Vijay; Swinkels, Dorine W.; Tjalsma, Harold



Molecular size fractions of bay leaf (Laurus nobilis) exhibit differentiated regulation of colorectal cancer cell growth in vitro.  


Numerous in vitro studies using solvent or aqueous extracts of raw dietary plant material have demonstrated modulation of colon cancer cell growth and apoptosis and effects on immune and nonimmune pathways of inflammation. We have developed a generic, 3-staged food-compatible process involving heating for conversion of dietary plants into food ingredients and report results on potential colon cancer-regulating properties of processed forms of Bay leaf (Laurus nobilis). In vitro studies demonstrated inhibition of cancer cell growth by processed Bay leaf products in HT-29, HCT-116, Caco-2, and SW-480 human cancer cell lines, which were accompanied by variable levels of elevated apoptosis. Bay leaf also exerted moderate inhibition of cycloxygenase 2 and 5 lipoxygenase enzymatic activity. In addition, these extracts significantly downregulated interferon-? production in T helper Type 1-stimulated whole blood from healthy donors. Furthermore, size fractionation of the extracts revealed that antiproliferative and proapoptotic activities were associated with low mass (primarily polyphenolics and essential oils) and high mass (primarily proteins including polyphenol oxidase) chemical classes, respectively. Bay leaf exerted in vitro bioactivity that might be relevant to protecting against early events in sporadic colorectal cancer, with potential for further optimization of bioactivity by size-based fractionation. PMID:23859043

Bennett, Louise; Abeywardena, Mahinda; Burnard, Sharon; Forsyth, Santina; Head, Richard; King, Kerryn; Patten, Glen; Watkins, Peter; Williams, Roderick; Zabaras, Dimitrios; Lockett, Trevor



Persistent infection of SARS coronavirus in colonic cells in vitro.  


Severe acute respiratory syndrome coronavirus (SARS-CoV) can produce gastrointestinal symptoms. The intestinal tract is the only extrapulmonary site where viable viruses have been detected. This study examined seven established human intestinal cell lines, DLD-1, HCT-116, HT-29, LoVo, LS-180, SW-480 and SW-620, for their permissiveness to SARS-CoV infection. The results showed that only LoVo cells were permissive to SARS-CoV infection as evident by positive findings from indirect immunofluorescence staining for intracellular viral antigens, in situ hybridization for intracellular viral RNA, and electron microscopy for intracellular viral particles. In contrast to Vero cells, SARS-CoV did not produce cytopathic effects on LoVo cells. However, LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3 x 10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV. Our results on the expression of angiotensin-converting enzyme 2 (ACE2), a recently identified cellular receptor for SARS-CoV, in these cell lines indicated that it might not be the sole determinant for cells to be susceptible to SARS-CoV infection. PMID:15258961

Chan, Paul K S; To, Ka-Fai; Lo, Anthony W I; Cheung, Jo L K; Chu, Ida; Au, Florence W L; Tong, Joanna H M; Tam, John S; Sung, Joseph J Y; Ng, Ho-Keung



Expression of the MAP kinase phosphatase DUSP4 is associated with microsatellite instability in colorectal cancer (CRC) and causes increased cell proliferation.  


DUSP4 (MKP-2), a member of the mitogen-activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen-activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer (CRC) with high frequent microsatellite instability (MSI-H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI-H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT-qPCR. DUSP4 was overexpressed in all 19 MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p < 0.001). Consistently, MSI-H CRC cell lines showed 6.8-fold higher DUSP4 mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYC and CDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore, DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC. PMID:22965873

Gröschl, Benedikt; Bettstetter, Marcus; Giedl, Christian; Woenckhaus, Matthias; Edmonston, Tina; Hofstädter, Ferdinand; Dietmaier, Wolfgang



Induction of apoptosis in human pancreatic MiaPaCa-2 cells through the loss of mitochondrial membrane potential (??m) by Gentiana kurroo root extract and LC-ESI-MS analysis of its principal constituents.  


The objective of the current study was to evaluate the methanolic root extract of Gentiana kurroo for antioxidant and antiproliferative activities as well as to study the effect of the extract on the induction of apoptosis in human pancreatic cancer cell line (MiaPaCa-2). The extract exerted significant antioxidant activity as verified by DPPH, hydroxyl radical, lipid peroxidation and protective oxidative DNA damage assays. The results were comparable to standard antioxidants like ?-tocopherol, catechin and BHT used in such experiments. Antioxidant potential of G. kurroo may be attributed to the presence of high phenolic and flavonoid content (73±1.02 and 46±2.05 mg/g extract respectively). The anti-proliferative property of Gentiana kurroo root extract was determined by sulphorhodamine B (SRB) assay against Human colon cancer cell line (HCT-116), Lung carcinoma cell line (A-549), Pancreatic cancer cell line (MiaPaCa-2), Lung cancer cell line (HOP-62) and acute monocytic leukaemia cell line (THP-1). G. kurroo root extract inhibited cancer cell growth depending upon the cell line used and in a dose dependent manner. The extract induced potent apoptotic effects in MiaPaCa-2 cells. The population of apoptotic cells increased from 11.4% in case of control to 49.6% at 100 ?g/ml of G. kurroo root extract. The extract also induced a remarkable decrease in mitochondrial membrane potential (??m) leading to apoptosis of cancer cells used. The main chemical constituents identified by the liquid chromatography-tandem mass spectrometry (LC-ESI-MSMS) were found to be iridoid glucosides (iridoids and secoiridoids), xanthones and flavonoids. Iridoid glucosides are the bitter principles of Gentiana species. Loganic acid, Sweroside, Swertiamarin, Gentiopicroside, Gentisin, Isogentisin, Gentioside, Norswertianolin, Swertianolin, 4?-O-?-D-glucosyl-6'-O-(4-O-?-D-glucosylcaffeoyl)-linearoside and Swertisin were the principal compounds present in the methanol root extract of G. kurroo. PMID:23453831

Wani, Bilal A; Ramamoorthy, D; Rather, Manzoor A; Arumugam, N; Qazi, Asif Khurshid; Majeed, Rabiya; Hamid, Abid; Ganie, Showkat A; Ganai, Bashir A; Anand, Rajneesh; Gupta, Ajai P



Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F?F?-ATP synthase  

PubMed Central

We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53+/+ cells simultaneously show increased O? consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F?F?-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F?F?-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria. PMID:23966169

Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssiere, Jean-Luc; Rincheval, Vincent



Colon cancer cell apoptosis is induced by combined exposure to the n-3 fatty acid docosahexaenoic acid and butyrate through promoter methylation  

PubMed Central

DNA methylation and histone acetylation contribute to the transcriptional regulation of genes involved in apoptosis. We have demonstrated that docosahexaenoic acid (DHA, 22:6 n-3) and butyrate enhance colonocyte apoptosis. To determine if DHA and/or butyrate elevate apoptosis through epigenetic mechanisms thereby restoring the transcription of apoptosis-related genes, we examined global methylation; gene-specific promoter methylation of 24 apoptosis-related genes; transcription levels of Cideb, Dapk1, and Tnfrsf25; and global histone acetylation in the HCT-116 colon cancer cell line. Cells were treated with combinations of (50 ?M) DHA or linoleic acid (18:2 n-6), (5 mM) butyrate or an inhibitor of DNA methyltransferases, and 5-aza-2?-deoxycytidine (5-Aza-dC, 2 ?M). Among highly methylated genes, the combination of DHA and butyrate significantly reduced methylation of the proapoptotic Bcl2l11, Cideb, Dapk1, Ltbr, and Tnfrsf25 genes compared to untreated control cells. DHA treatment reduced the methylation of Cideb, Dapk1, and Tnfrsf25. These data suggest that the induction of apoptosis by DHA and butyrate is mediated, in part, through changes in the methylation state of apoptosis-related genes. PMID:24495951

Cho, Youngmi; Turner, Nancy D; Davidson, Laurie A; Chapkin, Robert S; Carroll, Raymond J; Lupton, Joanne R



Downregulation of MSP58 inhibits growth of human colorectal cancer cells via regulation of the cyclin D1-cyclin-dependent kinase 4-p21 pathway.  


We have investigated the expression and role of the 58-kDa microspherule protein (MSP58) in colorectal carcinoma (CRC). By immuhistochemistry and immunofluorescence, we observed MSP58 in the nucleus and cytoplasm of CRC cells, and found MSP58 to be present in CRC specimens more often than in adjacent non-tumor tissues (92.5 vs 36.3%, P < 0.01). The average staining score in adjacent non-tumor tissues was significantly lower than in CRC tissues (2.05 +/- 1.13 vs 5.23 +/- 1.38, P < 0.01). Moreover, MSP58 mRNA and protein appeared to be upregulated in six fresh CRC samples compared to their adjacent non-cancerous tissues. MSP58 expression was also detected in the human CRC-derived cell lines LoVo, CoLo205, HCT116, HT-29, SW620, and SW480. Downregulation of MSP58 inhibited in vitro growth and attenuated tumor growth in animal models by induction of cell cycle arrest, and was associated with reduced levels of cyclin D1, cyclin-dependent kinase 4, phosphorylation-Rb (p-Rb), p21, and Retino blastoma (Rb) proteins. These results indicated that MSP58 might play an important role in the carcinogenesis of CRC via regulation of the cyclin D1-cyclin-dependent kinase 4-p21 pathway. PMID:19549253

Shi, Hai; Chen, Sunxiao; Jin, Haifeng; Xu, Chunsheng; Dong, Guanglong; Zhao, Qingchuan; Wang, Weizhong; Zhang, Hongwei; Lin, Wei; Zhang, Jing; Davidovic, Laetitia; Yao, Libo; Fan, Daiming



Molluscan cells in culture: primary cell cultures and cell lines  

PubMed Central

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

Yoshino, T. P.; Bickham, U.; Bayne, C. J.



Phenethyl isothiocyanate inhibits hypoxia-induced accumulation of HIF-1? and VEGF expression in human glioma cells.  


Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, inhibits angiogenesis but the molecular mechanisms that underlie this effect are not known. In this study, under hypoxic conditions (1% O2), we examined the effect of PEITC on the intracellular level of the hypoxia inducible factor (HIF-1?) and extracellular level of the vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that PEITC suppressed the HIF-1? accumulation during hypoxia in human glioma U87, human prostate cancer DU145, colon cancer HCT116, liver cancer HepG2, and breast cancer SkBr3 cells. PEITC treatment also significantly reduced the hypoxia-induced secretion of VEGF. Suppression of HIF-1? accumulation during treatment with PEITC in hypoxia was related to PI3K and MAPK pathways. Taken together, these results suggest that PEITC inhibits the HIF-1? expression through inhibiting the PI3K and MAPK signalling pathway and provide a new insight into a potential mechanism of the anticancer properties of PEITC. PMID:23870899

Gupta, Brinda; Chiang, Linda; Chae, KyungMin; Lee, Dae-Hee



Silencing of CCDC6 reduces the expression of 14-3-3? in colorectal carcinoma cells.  


Coiled-coil domain containing 6 (CCDC6) is frequently rearranged in papillary thyroid carcinomas participating in the formation of RET/PTC1 oncogene. Other rearrangements involving CCDC6 have also been identified demonstrating its high susceptibility to chromosomal recombination. Malignancies bearing CCDC6 fusion genes are developed in a background where CCDC6 is either lost or deregulated. Our aim was to identify interacting proteins which are affected by the silencing of CCDC6 expression and could possibly link CCDC6 deregulation to cancer causality. Therefore, a proteomic approach was adopted using a human cancer cell-line (HCT116) where CCDC6 expression was silenced by lentiviral shRNA constructs. 14-3-3? down-regulation in the absence of CCDC6 was revealed and verified by western blot analysis and confocal microscopy. Only the levels and not the topology of CCDC6 were altered. The down-regulation of 14-3-3? in the absence of CCDC6 demonstrated their direct association and supports the notion that CCDC6 contributes to cancer development, possibly through malignant pathways involving 14-3-3?. PMID:22399611

Thanasopoulou, Angeliki; Xanthopoulou, Alexandra G; Anagnostopoulos, Athanasios K; Konstantakou, Eumorphia G; Margaritis, Lukas H; Papassideri, Isidora S; Stravopodis, Dimitrios J; Tsangaris, George T; Anastasiadou, Ema



Cell line: 2004-2014.  


2014 marks Cell's 40th anniversary, and over the year we have looked back at how discoveries of the last four decades have molded our understanding of biology. The final decade of the Cell Line features a selection of the exceptional scientific work-both landmark papers and essential reviews. Select entries can be read as an "Annotated Classic," which includes the original paper and accompanying reflections of a leading scientist, considering the work from our current vantage point. Our last installment includes a harbinger of the interplay between microbiota and mammalian hosts in 2004, revolutionary papers in 2006 and 2007 unlocking cellular reprogramming, the discovery of beige adipocytes in 2012, and the first example of CRISPR-based genome editing in a nonhuman primate in 2014. In addition to landmark publications, there were innovative developments at the journal in this decade, with the complete redesign of the print journal and the creation of Leading Edge in late 2005 and the restructuring of the online display of the article in 2010. Keeping pace with the changing nature of biological research, over the decade Cell added new article types, introduced guidelines for the organization of supplementary material, and expanded the journal's web-based content to bring editors' and authors' excitement and perspective on individual papers to the readership. An interactive version of the timeline, with links to the papers, full author lists, and Annotated Classics, is available at PMID:25416957



High expression of microRNA-625-3p is associated with poor response to first-line oxaliplatin based treatment of metastatic colorectal cancer.  


The backbone of current cytotoxic treatment of metastatic colorectal cancer (mCRC) consists of a fluoropyrimidine together with either oxaliplatin (XELOX/FOLFOX) or irinotecan (XELIRI/FOLFIRI). With an overall objective response rate of approximately 50% for either treatment combination, a major unsolved problem is that no predictors of response to these treatments are available. To address this issue, we profiled 742 microRNAs in laser-capture microdissected cancer cells from responding and non-responding patients receiving XELOX/FOLFOX as first-line treatment for mCRC, and identified, among others, high expression of miR-625-3p, miR-181b and miR-27b to be associated with poor clinical response. In a validation cohort of 94 mCRC patients treated first-line with XELOX, high expression of miR-625-3p was confirmed to be associated with poor response (OR = 6.25, 95%CI [1.8; 21.0]). Independent analyses showed that miR-625-3p was not dysregulated between normal and cancer samples, nor was its expression associated with recurrence of stage II or III disease, indicating that miR-625-3p solely is a response marker. Finally, we also found that these miRNAs were up-regulated in oxaliplatin resistant HCT116/oxPt (miR-625-3p, miR-181b and miR-27b) and LoVo/oxPt (miR-181b) colon cancer cell lines as compared with their isogenic parental cells. Altogether, our results suggest an association between miR-625-3p and response to first-line oxaliplatin based chemotherapy of mCRC. PMID:23506979

Rasmussen, Mads H; Jensen, Niels F; Tarpgaard, Line S; Qvortrup, Camilla; Rømer, Maria U; Stenvang, Jan; Hansen, Tine P; Christensen, Lise L; Lindebjerg, Jan; Hansen, Flemming; Jensen, Benny V; Hansen, Torben F; Pfeiffer, Per; Brünner, Nils; Ørntoft, Torben F; Andersen, Claus L



The BH3 mimetic ABT-263 synergizes with the MEK1/2 inhibitor selumetinib/AZD6244 to promote BIM-dependent tumour cell death and inhibit acquired resistance.  


Tumour cells typically exhibit a G(1) cell cycle arrest in response to the MEK1/2 [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitor selumetinib, but do not die, and thus they acquire resistance. In the present study we examined the effect of combining selumetinib with the BH3 [BCL2 (B-cell lymphoma 2) homology domain 3]-mimetic BCL2 inhibitor ABT-263. Although either drug alone caused little tumour cell death, the two agents combined to cause substantial caspase-dependent cell death and inhibit long-term clonogenic survival of colorectal cancer and melanoma cell lines with BRAF(V600E) or RAS mutations. This cell death absolutely required BAX (BCL2-associated X protein) and was inhibited by RNAi (RNA interference)-mediated knockdown of BIM (BCL2-interacting mediator of cell death) in the BRAF(V600E)-positive COLO205 cell line. When colorectal cancer cell lines were treated with selumetinib plus ABT-263 we observed a striking reduction in the incidence of cells emerging with acquired resistance to selumetinib. Similar results were observed when we combined ABT-263 with the BRAF(V600E)-selective inhibitor PLX4720, but only in cells expressing BRAF(V600E). Finally, cancer cells in which acquired resistance to selumetinib arises through BRAF(V600E) amplification remained sensitive to ABT-263, whereas selumetinib-resistant HCT116 cells (KRAS(G13D) amplification) were cross-resistant to ABT-263. Thus the combination of a BCL2 inhibitor and an ERK1/2 pathway inhibitor is synthetic lethal in ERK1/2-addicted tumour cells, delays the onset of acquired resistance and in some cases overcomes acquired resistance to selumetinib. PMID:23234544

Sale, Matthew J; Cook, Simon J



PPAR? deficiency disrupts hypoxia-mediated tumorigenic potential of colon cancer cells.  


Peroxisome proliferator-activated receptor (PPAR) ? is highly expressed in colon epithelial cells and closely linked to colon carcinogenesis. However, the role of PPAR? in colon cancer cells in a hypoxic tumor microenvironment is not fully understood. We found that expression of the tumor-promoting cytokines, IL-8 and VEGF, induced by hypoxia (<1% O2 ) and deferoxamine (a hypoxia mimetic) was significantly attenuated in PPAR?-deficient HCT116 colon cancer cells. Consequently, PPAR?-knockout colon cancer cells exposed to hypoxia and deferoxamine failed to stimulate endothelial cell vascularization and macrophage migration/proliferation, whereas wild-type cells were able to induce angiogenesis and macrophage activation in response to hypoxic stress. Hypoxic stress induced transcriptional activation of PPAR?, but not its protein expression, in HCT116 cells. Exogenous expression of p300 potentiated deferoxamine-induced PPAR? transactivation, while siRNA knockdown of p300 abolished hypoxia- and deferoxamine-induced PPAR? transactivation. PPAR? associated with p300 upon hypoxic stress as demonstrated by coimmunoprecipitation studies. PI3K inhibitors or siRNA knockdown of Akt suppressed the PPAR? transactivation induced by hypoxia and deferoxamine in HCT116 cells, leading to decreased expression of IL-8 and VEGF. Collectively, these results reveal that PPAR? is required for hypoxic stress-mediated cytokine expression in colon cancer cells, resulting in promotion of angiogenesis, macrophage recruitment, and macrophage proliferation in the tumor microenvironment. p300 and the PI3K/Akt pathway play a role in the regulation of PPAR? transactivation induced by hypoxic stress. Our results demonstrate the positive crosstalk between PPAR? in tumor cells and the hypoxic tumor microenvironment and provide potential therapeutic targets for colon cancer. © 2014 Wiley Periodicals, Inc. PMID:24610641

Jeong, Eunshil; Koo, Jung Eun; Yeon, Sang Hyeon; Kwak, Mi-Kyoung; Hwang, Daniel H; Lee, Joo Young



Inhibition of phosphatidylinositol 3-kinase promotes tumor cell resistance to chemotherapeutic agents via a mechanism involving delay in cell cycle progression  

SciTech Connect

Approaches to overcome chemoresistance in cancer cells have involved targeting specific signaling pathways such as the phosphatidylinositol 3-kinase (PI3K) pathway, a stress response pathway known to be involved in the regulation of cell survival, apoptosis and growth. The present study determined the effect of PI3K inhibition on the clonogenic survival of human cancer cells following exposure to various chemotherapeutic agents. Treatment with the PI3K inhibitors LY294002 or Compound 15e resulted in increased survival of MDA-MB-231 breast carcinoma cells after exposure to doxorubicin, etoposide, 5-fluorouracil, and vincristine. Increased survival following PI3K inhibition was also observed in DU-145 prostate, HCT-116 colon and A-549 lung carcinoma cell lines exposed to doxorubicin. Increased cell survival mediated by LY294002 was correlated with a decrease in cell proliferation, which was linked to an increase in the proportion of cells in the G{sub 1} phase of the cell cycle. Inhibition of PI3K signaling also resulted in higher levels of the cyclin-dependent kinase inhibitors p21{sup Waf1/Cip1} and p27{sup Kip1}; and knockdown of p27{sup kip1} with siRNA attenuated resistance to doxorubicin in cells treated with LY294002. Incubation in the presence of LY294002 after exposure to doxorubicin resulted in decreased cell survival. These findings provide evidence that PI3K inhibition leads to chemoresistance in human cancer cells by causing a delay in cell cycle; however, the timing of PI3K inhibition (either before or after exposure to anti-cancer agents) may be a critical determinant of chemosensitivity.

McDonald, Gail T.; Sullivan, Richard; Pare, Genevieve C.; Graham, Charles H., E-mail:



Dual drug delivery of 5-fluorouracil (5-FU) and methotrexate (MTX) through random copolymeric nanomicelles of PLGA and polyethylenimine demonstrating enhanced cell uptake and cytotoxicity.  


We now report the synthesis of a random copolymer of poly-lactic-co-glycolic acid (PLGA) grafted branched polyethylenimine (BPEI) and the use of it as a multi drug delivery system (DDS). The methotrexate (MTX) was conjugated to BPEI through DCC/NHS chemistry. The copolymer-drug conjugate (PBP-MTX) was characterised by FT-IR and (1)H NMR spectroscopy. The PBP-MTX was converted into nanomicelles with entrapped 5-fluorouracil (5-FU) through nanoprecipitation technique. The size, shape, morphology and surface charge of the nanomicelles were confirmed using different techniques. The thermal behaviour and distribution of both conjugated and entrapped drug through the polymeric matrix were assessed by differential scanning calorimetry (DSC) and powder X-ray diffraction analysis (PXRD). In vitro drug release pattern of the nanomicelles was examined to ascertain the release pattern of two drugs namely 5-FU and MTX. The cellular uptake studies demonstrated higher uptake of the nanomicelles in colon cancer cell line HCT 116. Further the cytotoxicity evaluation of nanomicelles illustrated promising action which confirms the use of the system as a potential DDS to colon cancer. PMID:25108479

Ashwanikumar, N; Kumar, Nisha Asok; Nair, S Asha; Kumar, G S Vinod



Combined effects of storage and processing on the bioactive compounds and pro-apoptotic properties of color-fleshed potatoes in human colon cancer cells.  


Potatoes can be stored for up to 1 year before being processed and consumed. The objective of this study was to determine the extent to which fresh and stored color-fleshed potatoes retain their anticancer properties after baking and chipping compared with unprocessed potatoes. We utilized white-, yellow-, and purple-fleshed potato clones and tested their phenolic and anthocyanin content, antioxidant activity, metabolite profile, and antiproliferative and pro-apoptotic properties. When compared with unprocessed samples, baking or chipping led to significant losses in the phenolic and anthocyanin content and antioxidant activity of the potatoes. However, with storage, total phenolic and anthocyanin content and antioxidant activity increased in baked samples while in the chipped samples they remained constant. Ethanolic extracts of baked and chipped samples suppressed proliferation and elevated apoptosis (p < 0.05) in HCT-116 (p53 wild-type; ras mutated) and HT-29 (p53 mutated; ras wild-type) human colon cancer cell lines. Antiproliferative and pro-apoptotic properties of baked potatoes were similar to that of fresh potatoes, while chipping caused a significant suppression. Phenolic content and antioxidant activity of purple-fleshed potatoes, after baking, were comparable with those of anthocyanin-rich berries. Hence, purple-fleshed potatoes can be a healthier choice for consumers as they possess greater levels of bioactive compounds and anticancer properties even after processing as compared with their white- and yellow-fleshed counterparts. PMID:23039105

Madiwale, Gaurav P; Reddivari, Lavanya; Stone, Martha; Holm, David G; Vanamala, Jairam



Chemotherapeutic agents in low noncytotoxic concentrations increase immunogenicity of human colon cancer cells  

Microsoft Academic Search

Background  We have recently reported that chemotherapeutic agents in ultra low noncytotoxic concentrations may block the ability of tumor\\u000a cells to suppress functional activation of dendritic cells (DCs).\\u000a \\u000a \\u000a \\u000a \\u000a Methods  HCT-116 human colon cancer cells were treated with 0.5 nM paclitaxel (PAC) or 2 nM doxorubicin (DOX) with the aim of defining\\u000a the immunogenic changes induced by ultra low noncytotoxic concentrations of antineoplastic

Ramon Kaneno; Galina V. Shurin; Felipe M. Kaneno; Hiam Naiditch; Jianhua Luo; Michael R. Shurin



Bim contributes to phenethyl isothiocyanate-induced apoptosis in breast cancer cells.  


Phenethyl isothiocyanate (PEITC) is a highly promising cancer chemopreventive constituent of cruciferous vegetables (e.g., watercress) with in vivo efficacy in experimental rodent cancer models. Research thus far implicates apoptosis induction in cancer chemopreventive response to PEITC, but the mechanism of proapoptotic effect is not fully understood. The present study demonstrates that p53 upregulated modulator of apoptosis (PUMA)-independent apoptosis by PEITC is mediated by B-cell lymphoma 2 interacting mediator of cell death (Bim). Exposure of a cell line (BRI-JM04) derived from spontaneously developing mammary tumor of a MMTV-neu transgenic mouse to pharmacological concentrations of PEITC resulted in decreased cell viability coupled with apoptosis induction, characterized by release of histone-associated DNA fragments into the cytosol and cleavage of poly-(ADP-ribose)-polymerase and procaspase-3. The PEITC-induced apoptosis in BRI-JM04 cells was associated with up-regulation of Bak, PUMA, and Bim (long and short forms of Bim), increased S65 phosphorylation of BimEL (extra-long form), and down-regulation of Bcl-xL and Bcl-2. On the other hand, a non-tumorigenic human mammary epithelial cell line (MCF-10A) was significantly more resistant to PEITC-induced apoptosis compared with BRI-JM04 despite induction of Bax and PUMA due to concomitant overexpression of anti-apoptotic proteins, including Bcl-xL, Bcl-2, and Mcl-1. Wild-type HCT-116 cells and its isogenic PUMA knockout variant exhibited comparable sensitivity to PEITC-induced apoptosis. On the other hand, small interfering RNA knockdown of Bim protein imparted partial but statistically significant protection against PEITC-induced apoptosis in BRI-JM04, MCF-7, and MDA-MB-231 cells. In conclusion, the present study provides novel insight into the mechanism of PEITC-induced apoptosis involving Bim. PMID:21739479

Hahm, Eun-Ryeong; Singh, Shivendra V



The Sensitivity of Cancer Cells to Pheophorbide a-Based Photodynamic Therapy Is Enhanced by NRF2 Silencing  

PubMed Central

Photodynamic therapy (PDT) has emerged as an effective treatment for various solid tumors. The transcription factor NRF2 is known to protect against oxidative and electrophilic stress; however, its constitutive activity in cancer confers resistance to anti-cancer drugs. In the present study, we investigated NRF2 signaling as a potential molecular determinant of pheophorbide a (Pba)-based PDT by using NRF2-knockdown breast carcinoma MDA-MB-231 cells. Cells with stable NRF2 knockdown showed enhanced cytotoxicity and apoptotic/necrotic cell death following PDT along with increased levels of singlet oxygen and reactive oxygen species (ROS). A confocal microscopic visualization of fluorogenic Pba demonstrated that NRF2-knockdown cells accumulate more Pba than control cells. A subsequent analysis of the expression of membrane drug transporters showed that the basal expression of BCRP is NRF2-dependent. Among measured drug transporters, the basal expression of breast cancer resistance protein (BCRP; ABCG2) was only diminished by NRF2-knockdown. Furthermore, after incubation with the BCRP specific inhibitor, differential cellular Pba accumulation and ROS in two cell lines were abolished. In addition, NRF2-knockdown cells express low level of peroxiredoxin 3 compared to the control, which implies that diminished mitochondrial ROS defense system can be contributing to PDT sensitization. The role of the NRF2-BCRP pathway in Pba-PDT response was further confirmed in colon carcinoma HT29 cells. Specifically, NRF2 knockdown resulted in enhanced cell death and increased singlet oxygen and ROS levels following PDT through the diminished expression of BCRP. Similarly, PDT-induced ROS generation was substantially increased by treatment with NRF2 shRNA in breast carcinoma MCF-7 cells, colon carcinoma HCT116 cells, renal carcinoma A498 cells, and glioblastoma A172 cells. Taken together, these results indicate that the manipulation of NRF2 can enhance Pba-PDT sensitivity in multiple cancer cells. PMID:25226504

Choi, Bo-hyun; Ryoo, In-geun; Kang, Han Chang; Kwak, Mi-Kyoung



Effects of the kava chalcone flavokawain A differ in bladder cancer cells with wild-type versus mutant p53.  


Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G(1) arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G(2)-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G(2)-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G(2)-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G(2)-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer. PMID:19138991

Tang, Yaxiong; Simoneau, Anne R; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin



Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53  

PubMed Central

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2 and then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G2-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation and then led to a G2-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G2-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G2-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer. PMID:19138991

Tang, Yaxiong; Simoneau, Anne R.; Xie, Jun; Shahandeh, Babbak; Zi, Xiaolin



Therapeutic potential of nitric oxide-modified drugs in colon cancer cells.  


We have examined the influence of the nitric oxide (NO)-modified anti-inflammatory drug (S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid (VGX-1027) named GIT-27NO or the NO-modified antiviral drug saquinavir (Saq) named Saq-NO on two colon cancer cell lines, mouse CT26CL25 and human HCT116. The effects of the drugs on cell viability, apoptosis, proliferation, and metastatic potential were analyzed. The release of NO and oxygen and nitrogen species was also determined. The efficacy of the drugs was evaluated in vivo in BALB/c mice injected with CT26CL25 cells. Both agents suppressed the growth of colon cancer cells in vitro and reduced tumor volume in syngeneic BALB/c mice. However, their mechanisms of action were different because GIT-27NO released larger amounts of nitrite than Saq-NO in cell cultures and its antitumor action depended on the intracellular NO release inside the cells. On the contrary, Saq-NO released barely detectable amounts of NO and its antitumor action was NO-independent. In fact, cotreatment with an NO-peroxynitrite scavenger revealed that GIT-27NO but not Saq-NO acts through peroxynitrite-mediated cell destruction. At the cellular level, GIT-27NO prevalently induced proapoptotic signals followed by caspase-dependent apoptosis. In contrast, Saq-NO blocked cell proliferation, changed the adhesive, migratory, and invasive properties of the cells, and decreased metastatic potential in vivo. In conclusion, differences in NO release and oxidative stress generation between GIT-27NO and Saq-NO resulted in different mechanisms that caused cell death. PMID:22798453

Mojic, Marija; Mijatovic, Sanja; Maksimovic-Ivanic, Danijela; Miljkovic, Djordje; Stosic-Grujicic, Stanislava; Stankovic, Marija; Mangano, Katia; Travali, Salvatore; Donia, Marco; Fagone, Paolo; Zocca, Mai-Britt; Al-Abed, Yousef; McCubrey, James A; Nicoletti, Ferdinando



The iroquois homeobox gene 5 is regulated by 1,25-dihydroxyvitamin D3 in human prostate cancer and regulates apoptosis and the cell cycle in LNCaP prostate cancer cells.  


1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the most active metabolite of vitamin D3, has significant antitumor activity in a broad range of preclinical models of cancer. In this study, we show that the Iroquois homeobox gene 5 (Irx5) is down-regulated by 1,25(OH)2D3 in human prostate cancer samples from patients randomly assigned to receive weekly high-dose 1,25(OH)2D3 or placebo before radical prostatectomy. Down-regulation of Irx5 by 1,25(OH)2D3 was also shown in the human androgen-sensitive prostate cancer cell line LNCaP and in estrogen-sensitive MCF-7 breast cancer cells. Knockdown of Irx5 by RNA interference showed a significant reduction in LNCaP cell viability, which was accompanied by an increase in p21 protein expression, G2-M arrest, and an increase in apoptosis. The induced apoptosis was partially mediated by p53, and p53 protein expression was increased as a result of Irx5 knockdown. Cell survival was similarly reduced by Irx5 knockdown in the colon cancer cell line HCT 116 and in MCF-7 breast cancer cells, each being derived from clinical tumor types that seem to be inhibited by 1,25(OH)2D3. Overexpression of Irx5 led to a reduction of p21 and p53 expression. This is the first report that Irx5 is regulated by 1,25(OH)2D3 in humans and the first report to show that Irx5 is involved in the regulation of both the cell cycle and apoptosis in human prostate cancer cells. Irx5 may be a promising new therapeutic target in cancer treatment. PMID:18519790

Myrthue, Anne; Rademacher, Brooks L S; Pittsenbarger, Janet; Kutyba-Brooks, Bozena; Gantner, Marin; Qian, David Z; Beer, Tomasz M



MicroRNA-26a regulates glucose metabolism by direct targeting PDHX in colorectal cancer cells  

PubMed Central

Background Reprogramming energy metabolism has been an emerging hallmark of cancer cells. MicroRNAs play important roles in glucose metabolism. Methods The targets of microRNA-26a (miR-26a) were predicted by bioinformatics tools. The efficacy of miR-26a binding the 3?-untranslated region (UTR) of pyruvate dehydrogenase protein X component (PDHX) mRNA was evaluated using a dual-luciferase reporter assay. The PDHX expression at the mRNA and protein level in several colon cancer cell lines was quantified with real-time PCR and Western blot analysis respectively. The effects of miR-26a on glucose metabolism were determined by detecting the content of glucose consumption, production of lactate, pyruvate, and acetyl-coenzyme A. Results The expression of miR-26a is inversely associated with the level of its targeting protein PDHX in several colon cancer cell lines with different malignancy potentials. MiR-26a inhibits PDHX expression by direct targeting the 3?-UTR of PDHX mRNA. The glucose consumption and lactate concentration were both greatly increased in colon cancer cells than the normal colon mucosal epithelia under physiological conditions. The overexpression of miR-26a in HCT116 cells efficiently improved the accumulation of pyruvate and decreased the production of acetyl coenzyme A. Meanwhile the inhibition of miR-26a expression induced inverse biological effects. Conclusions MiR-26a regulates glucose metabolism of colorectal cancer cells by direct targeting the PDHX, which inhibits the conversion of pyruvate to acetyl coenzyme A in the citric acid cycle. PMID:24935220



ABT-737 Induces Expression of the Death Receptor 5 and Sensitizes Human Cancer Cells to TRAIL-induced Apoptosis*S?  

PubMed Central

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-?B site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-?B activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies. PMID:18599488

Song, Jin H.; Kandasamy, Karthikeyan; Kraft, Andrew S.



Gene expression profiles modulated by the human carcinogen aristolochic acid I in human cancer cells and their dependence on TP53  

SciTech Connect

Aristolochic acid (AA) is the causative agent of urothelial tumours associated with aristolochic acid nephropathy. These tumours contain TP53 mutations and over-express TP53. We compared transcriptional and translational responses of two isogenic HCT116 cell lines, one expressing TP53 (p53-WT) and the other with this gene knocked out (p53-null), to treatment with aristolochic acid I (AAI) (50-100 {mu}M) for 6-48 h. Modulation of 118 genes was observed in p53-WT cells and 123 genes in p53-null cells. Some genes, including INSIG1, EGR1, CAV1, LCN2 and CCNG1, were differentially expressed in the two cell lines. CDKN1A was selectively up-regulated in p53-WT cells, leading to accumulation of TP53 and CDKN1A. Apoptotic signalling, measured by caspase-3 and -7 activity, was TP53-dependent. Both cell types accumulated in S phase, suggesting that AAI-DNA adducts interfere with DNA replication, independently of TP53 status. The oncogene MYC, frequently over-expressed in urothelial tumours, was up-regulated by AAI, whereas FOS was down-regulated. Observed modulation of genes involved in endocytosis, e.g. RAB5A, may be relevant to the known inhibition of receptor-mediated endocytosis, an early sign of AA-mediated proximal tubule injury. AAI-DNA adduct formation was significantly greater in p53-WT cells than in p53-null cells. Collectively, phenotypic anchoring of the AAI-induced expression profiles to DNA adduct formation, cell-cycle parameters, TP53 expression and apoptosis identified several genes linked to these biological outcomes, some of which are TP53-dependent. These results strengthen the importance of TP53 in AA-induced cancer, and indicate that other alterations, e.g. to MYC oncogenic pathways, may also contribute.

Simoes, Maria L.; Hockley, Sarah L. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Schwerdtle, Tanja [Institute of Food Chemistry and Food Toxicology, Technical University of Berlin, TIB 4/3-1, Gustav-Meyer-Allee 25, D-13355 Berlin (Germany); Gamboa da Costa, Goncalo [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Schmeiser, Heinz H. [Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120-Heidelberg (Germany); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom); Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG (United Kingdom)], E-mail:



Targeting FLIP and Mcl-1 using a combination of aspirin and sorafenib sensitizes colon cancer cells to TRAIL.  


The multikinase inhibitor sorafenib is highly effective against certain types of cancer in the clinic and prevents colon cancer cell proliferation in vitro. Non-steroidal anti-inflammatory drugs, such as acetylsalicylic acid (aspirin), have shown activity against colon cancer cells. The aims of this study were to determine whether the combination of aspirin with sorafenib has enhanced anti-proliferative effects and increases recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL)-induced apoptosis in the human SW948, Lovo, Colo205, Colo320, Caco-2 and HCT116 colon cancer cell lines. In four cell lines, aspirin strongly stimulated the anti-proliferative effects of sorafenib (?four-fold enhancement) by inducing cell cycle arrest. Furthermore, combining low doses of aspirin (? 5?mm) and sorafenib (? 2.5?µm) greatly sensitized TRAIL-sensitive and TRAIL-resistant colon cancer cells to rhTRAIL, much more potently than either drug combined with rhTRAIL. The increase in rhTRAIL sensitivity was due to inhibition of FLIP and Mcl-1 protein expression following aspirin and sorafenib co-treatment, as confirmed by knock-down studies. Next, the clinical relevance of targeting FLIP and Mcl-1 in colon cancer was examined. Using immunohistochemistry, we found that Mcl-1 expression was significantly increased in colon adenoma and carcinoma patient material compared to healthy colonic epithelium, similar to the enhanced FLIP expression we recently observed in colon cancer. These results underscore the potential of combining low doses of aspirin with sorafenib to inhibit proliferation and target the anti-apoptotic proteins FLIP and Mcl-1 in colon cancer cells. PMID:23132258

Pennarun, Bodvael; Kleibeuker, Jan H; Boersma-van Ek, Wytske; Kruyt, Frank A E; Hollema, Harry; de Vries, Elisabeth G E; de Jong, Steven



Synthesis of a novel legumain-cleavable colchicine prodrug with cell-specific toxicity.  


Conventional chemotherapy has undesirable toxic side-effects to healthy tissues due to low cell selectivity of cytotoxic drugs. One approach to increase the specificity of a cytotoxic drug is to make a less toxic prodrug which becomes activated at the tumour site. The cysteine protease legumain have remarkable restricted substrate specificity and is the only known mammalian asparaginyl (Asn) endopeptidase. Over-expression of legumain is reported in cancers and unstable atherosclerotic plaques, and utilizing legumain is a promising approach to activate prodrugs. In this study we have synthesized the legumain-cleavable peptide sequence N-Boc-Ala-Ala-Asn-Val-OH. The peptide was subsequently conjugated to deacetyl colchicine during three steps to produce Suc-Ala-Ala-Asn-Val-colchicine (prodrug) with >90% chemical purity. Several cell lines with different expressions and activities of legumain were used to evaluate the general toxicity, specificity and efficacy of the microtubule inhibitor colchicine, valyl colchicine and the legumain-cleavable colchicine prodrug. The prodrug was more toxic to the colorectal cancer HCT116 cells (expressing both the 36kDa active and 56kDa proform of legumain) than SW620 cells (only expressing the 56kDa prolegumain) indicating a relationship between toxicity of the prodrug and activity of legumain in the cells. Also, in monoclonal legumain over-expressing HEK293 cells the prodrug toxicity was higher compared to native HEK293 cells. Furthermore, co-administration of the prodrug either with the potent legumain inhibitor cystatin E/M or the endocytosis inhibitor Dyngo-4a inhibited cell death, indicating that the prodrug toxicity was dependent on both asparaginyl endopeptidase activity and endocytosis. This colchicine prodrug adds to a legumain-activated prodrug strategy approach and could possibly be of use both in targeted anticancer and anti-inflammatory therapy. PMID:24842619

Smith, Robert Løvsletten; Åstrand, Ove Alexander Høgmoen; Nguyen, Luan Minh; Elvestrand, Tina; Hagelin, Gunnar; Solberg, Rigmor; Johansen, Harald Thidemann; Rongved, Pål



GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer.  


Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 ?M) combined with 5-FU (1.25 ?M), irinotecan (1.25 ?M) or cisplatin (1.25 ?M) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G 1 fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40-55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56-85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase. PMID:23095641

Rick, Ferenc G; Seitz, Stephan; Schally, Andrew V; Szalontay, Luca; Krishan, Awtar; Datz, Christian; Stadlmayr, Andreas; Buchholz, Stefan; Block, Norman L; Hohla, Florian



Renal Cell Carcinoma (ccRCC) Human Cell Line

A renal cell carcinoma (RCC) cell line designated UOK171 has been developed from the resected tumor of a patient diagnosed with stage IV high nuclear grade clear cell type renal cell carcinoma (ccRCC). The UOK171 cell line was immortalized spontaneously by mincing the resected tumor into pieces followed by propagation of the cells over more than twenty generations. One of the most prominent characteristics of this cell line is its intact, nonmutated von Hippel-Lindau (VHL) tumor suppressor gene.


MUTYH, an adenine DNA glycosylase, mediates p53 tumor suppression via PARP-dependent cell death  

PubMed Central

p53-regulated caspase-independent cell death has been implicated in suppression of tumorigenesis, however, the regulating mechanisms are poorly understood. We previously reported that 8-oxoguanine (8-oxoG) accumulation in nuclear DNA (nDNA) and mitochondrial DNA triggers two distinct caspase-independent cell death through buildup of single-strand DNA breaks by MutY homolog (MUTYH), an adenine DNA glycosylase. One pathway depends on poly-ADP-ribose polymerase (PARP) and the other depends on calpains. Deficiency of MUTYH causes MUTYH-associated familial adenomatous polyposis. MUTYH thereby suppresses tumorigenesis not only by avoiding mutagenesis, but also by inducing cell death. Here, we identified the functional p53-binding site in the human MUTYH gene and demonstrated that MUTYH is transcriptionally regulated by p53, especially in the p53/DNA mismatch repair enzyme, MLH1-proficient colorectal cancer-derived HCT116+Chr3 cells. MUTYH-small interfering RNA, an inhibitor for p53 or PARP suppressed cell death without an additive effect, thus revealing that MUTYH is a potential mediator of p53 tumor suppression, which is known to be upregulated by MLH1. Moreover, we found that the p53-proficient, mismatch repair protein, MLH1-proficient colorectal cancer cell line express substantial levels of MUTYH in nuclei but not in mitochondria, suggesting that 8-oxoG accumulation in nDNA triggers MLH1/PARP-dependent cell death. These results provide new insights on the molecular mechanism of tumorigenesis and potential new strategies for cancer therapies. PMID:25310643

Oka, S; Leon, J; Tsuchimoto, D; Sakumi, K; Nakabeppu, Y



Up-regulation of the interferon-related genes in BRCA2 knockout epithelial cells.  


BRCA2 mutations are significantly associated with early-onset breast cancer, and the tumour-suppressing function of BRCA2 has been attributed to its involvement in homologous recombination (HR)-mediated DNA repair. In order to identify additional functions of BRCA2, we generated BRCA2-knockout HCT116 human colorectal carcinoma cells. Using genome-wide microarray analyses, we have discovered a link between the loss of BRCA2 and the up-regulation of a subset of interferon (IFN)-related genes, including APOBEC3F and APOBEC3G. The over-expression of IFN-related genes was confirmed in different human BRCA2(-/-) and mouse Brca2(-/-) tumour cell lines, and was independent of senescence and apoptosis. In isogenic wild-type BRCA2 cells, we observed over-expression of IFN-related genes after treatment with DNA-damaging agents, and following ionizing radiation. Cells with endogenous DNA damage because of defective BRCA1 or RAD51 also exhibited over-expression of IFN-related genes. Transcriptional activity of the IFN-stimulated response element (ISRE) was increased in BRCA2 knockout cells, and the expression of BRCA2 greatly decreased IFN?-stimulated ISRE reporter activity, suggesting that BRCA2 directly represses the expression of IFN-related genes through the ISRE. Finally, the colony-forming capacity of BRCA2 knockout cells was significantly reduced in the presence of either IFN? or IFN?, suggesting that IFNs may have potential as therapeutic agents in cancer cells with BRCA2 mutations. The GEO Accession No. for microarray analysis is GSE54830. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:25043256

Xu, Hong; Xian, Jian; Vire, Emmanuelle; McKinney, Steven; Wei, Vivien; Wong, Jason; Tong, Rebecca; Kouzarides, Tony; Caldas, Carlos; Aparicio, Samuel



Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species  

PubMed Central

Background Metals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action. Methods Multiple approaches were employed, including cell viability, cell cycle analysis, multiple measurements of apoptosis, and mitochondrial function. In addition we measured cellular metal contents and employed EPR to record redox cycling of TPEN–metal complexes. Mouse xenografts were also performed to test the efficacy of TPEN in vivo. Results We show that metal chelation using TPEN (5?M) selectively induces cell death in HCT116 colon cancer cells without affecting the viability of non-cancerous colon or intestinal cells. Cell death was associated with increased levels of reactive oxygen species (ROS) and was inhibited by antioxidants and by prior chelation of copper. Interestingly, HCT116 cells accumulate copper to 7-folds higher levels than normal colon cells, and the TPEN-copper complex engages in redox cycling to generate hydroxyl radicals. Consistently, TPEN exhibits robust anti-tumor activity in vivo in colon cancer mouse xenografts. Conclusion Our data show that TPEN induces cell death by chelating copper to produce TPEN-copper complexes that engage in redox cycling to selectively eliminate colon cancer cells. PMID:25047035



PTEN Deletion in Prostate Cancer Cells Does Not Associate With Loss of RAD51 Function: Implications for Radiotherapy and Chemotherapy  

PubMed Central

Purpose PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous-recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported. Experimental Design Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN+/+; DU145-PTEN+/?; PC3-PTEN?/?) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression. Results PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi amongst prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA-damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H2O2 and methyl-methanesulfonate; but not to cisplatin, camptothecin, or paclitaxel. Conclusions These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials. PMID:22114138

Fraser, Michael; Zhao, Helen; Luoto, Kaisa R.; Lundin, Cecilia; Coackley, Carla; Chan, Norman; Joshua, Anthony M.; Bismar, Tarek A.; Evans, Andrew; Helleday, Thomas; Bristow, Robert G.



Thyroid cancer cell lines: an overview.  


Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution. PMID:23162534

Saiselet, Manuel; Floor, Sébastien; Tarabichi, Maxime; Dom, Geneviève; Hébrant, Aline; van Staveren, Wilma C G; Maenhaut, Carine



Thyroid cancer cell lines: an overview  

PubMed Central

Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution. PMID:23162534

Saiselet, Manuel; Floor, Sebastien; Tarabichi, Maxime; Dom, Genevieve; Hebrant, Aline; van Staveren, Wilma C. G.; Maenhaut, Carine



Cell-host, LINE and environment  

PubMed Central

Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship. PMID:23734298

Del Re, Brunella; Giorgi, Gianfranco



Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration  

SciTech Connect

Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes.

Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium)] [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium); De Ridder, Mark, E-mail: [Department of Radiotherapy, Universitair Ziekenhuis Brussel, Vrije Universiteit Brussel, Brussels (Belgium)



Gold Nanorods Targeted to Delta Opioid Receptor: Plasmon-Resonant Contrast and Photothermal Agents  

Microsoft Academic Search

Moleculariy targeted gold nanorods were investigated for applications in both diagnostic imaging and disease treatment with cellular resolution. The nanorods were tested in two genetically engineered cell lines derived from the human colon carcinoma HCT- 116, a model for studying ligand-receptor interactions. One of these lines was modified to express delta opioid receptor (i^OR) and green fluorescent protein, whereas the

Kvar C. Black; Nathaniel D. Kirkpatrick; Timothy S. Troutman; Liping Xu; Josef Vaguer; Robert J. Gillies; Jennifer K. Barton; Urs Utzinger; Marek Romanowski


Lactobacillus gasseri SF1183 Affects Intestinal Epithelial Cell Survival and Growth  

PubMed Central

It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host’s intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis. PMID:23894414

Baccigalupi, Loredana; Calabro, Viola; Crescenzi, Elvira; Ricca, Ezio; Pollice, Alessandra



77 FR 5489 - Identification of Human Cell Lines Project  

Federal Register 2010, 2011, 2012, 2013

...120104006-2006-01] Identification of Human Cell Lines Project AGENCY: National Institute...repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All data and...



Oncogenic K-RAS is required to maintain changes in cytoskeletal organization, adhesion, and motility in colon cancer cells.  


RAS oncogenes are thought to play a role at multiple stages of tumorigenesis. The role and mechanisms by which RAS oncogenes maintain the transformed state of human cancer cells are poorly understood. Here, we have studied the role of oncogenic K-RAS in maintaining cytoskeletal disruption, cell adhesion and motility in metastatic colon carcinoma cells. Targeted deletion of K-RAS(G13D) from HCT116 colon carcinoma cells restored their ability to assemble stress fibers and focal adhesions/complexes, accompanied by increased cell-matrix adhesion and reduced motility. We further show that oncogenic K-Ras induces high Rho activity, but uncouples Rho from stress fiber formation. This uncoupling required the maintenance of high levels of the activator protein-1 family member, Fra-1, via a mitogen-activated protein/extracellular signal-regulated kinase-dependent pathway. We also show that PI3-kinase signaling is required for the motility of HCT116 cells downstream of oncogenic K-Ras. Our findings suggest that mutated K-RAS oncogenes are essential for maintenance of the transformed and invasive phenotype of human colon cancer cells. PMID:15735008

Pollock, Claire B; Shirasawa, Senji; Sasazuki, Takehiko; Kolch, Walter; Dhillon, Amardeep S



Differential SELEX in Human Glioma Cell Lines  

PubMed Central

The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers. PMID:19956692

Cerchia, Laura; Esposito, Carla Lucia; Jacobs, Andreas H.; Tavitian, Bertrand; de Franciscis, Vittorio



Requirement for store-operated calcium entry in sodium butyrate-induced apoptosis in human colon cancer cells.  


The SOCE (store-operated Ca2+ entry) pathway plays a key role in both normal cells and cancerous cells. However, its molecular mechanism remains a long-lasting puzzle of Ca2+ signalling. In this paper, we provide evidence that butyric acid, a dietary fibre-derived short-chain fatty acid, induces apoptosis of colon cancer cells via SOCE signalling networks. We found that sodium butyrate (NaB) induces Ca2+ release from endoplasmic reticulum, which in turn causes extracellular Ca2+ influx in HCT-116 cells. The Ca2+ release and influx are important, because the addition of chelators, EGTA or BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] respectively blocked NaB-induced apoptosis. Furthermore, down-regulation of STIM1 (stromal interaction molecule 1) by RNA interference or pharmacological blockade of the SOCC (store-operated Ca2+ channel) by 2-APB (2-aminoethoxydiphenyl borate) or SKF-96365 inhibited NaB-induced extracellular Ca2+ influx and apoptosis in HCT-116 cells. Thus we conclude that NaB triggers colon cancer cell apoptosis in an SOCE-dependent manner. This finding provides new insights into how butyric acid suppresses colon carcinogenesis. PMID:21699495

Sun, Suxia; Li, Wenjun; Zhang, He; Zha, Longying; Xue, Yong; Wu, Xianbo; Zou, Fei



G protein-coupled lysophosphatidic acid receptors stimulate proliferation of colon cancer cells through the ?-catenin pathway  

PubMed Central

Recent studies suggest that lysophosphatidic acid (LPA) and its G protein-coupled receptors (GPCRs) LPA1, LPA2, or LPA3 may play a role in the development of several types of cancers, including colorectal cancer. However, the specific receptor subtype(s) and their signal-transduction pathways responsible for LPA-induced cancer cell proliferation have not been fully elucidated. We show by specific RNA interference (RNAi) that LPA2 and LPA3 but not LPA1 are targets for LPA-induced proliferation of HCT116 and LS174T colon cancer cells. We determined that LPA-induced colon cancer cell proliferation requires the ?-catenin signaling pathway, because knockdown of ?-catenin by RNAi abolished LPA-induced proliferation of HCT116 cells. Moreover, LPA activates the main signaling events in the ?-catenin pathway: phosphorylation of glycogen synthase kinase 3? (GSK3?), nuclear translocation of ?-catenin, transcriptional activation of T cell factor (Tcf)/lymphoid-enhancer factor (Lef), and expression of target genes. Inhibition of conventional protein kinase C (cPKC) blocked the effects, suggesting its involvement in LPA-induced activation of the ?-catenin pathway. Thus, LPA2 and LPA3 signal the proliferation of colon cancer cells through cPKC-mediated activation of the ?-catenin pathway. These results link LPA and its GPCRs to cancer through a major oncogenic signaling pathway. PMID:15837931

Yang, Ming; Zhong, Wendy W.; Srivastava, Neelam; Slavin, Anthony; Yang, Jianxin; Hoey, Timothy; An, Songzhu



Impact of Phytolacca americana extracts on gene expression of colon cancer cells.  


Native Americans have used Phytolacca americana to treat breast ailments, gastrointestinal disorders, rashes, and inflammation. Some anti-cancer and anti-viral research has been reported on this perennial herb, but none has been published concerning the effects of its extracts on cancer cell genes. In this study, changes in gene expression at the transcription level were evaluated in HCT-116 colon cancer cells after exposure to P. americana ethanol extract and its water fraction using the Human Cancer Pathway Finder PCR Array. Of the genes significantly affected in HCT-116 cells exposed to the ethanol extract at 3200?µg/ml, changes in expression of MYC, PLAU, and TEK may benefit the treatment of colon cancer. Exposing the cells to 1600?µg/ml of the water fraction resulted in several gene changes that may also be beneficial in the treatment of colon cancer: NME4, TEK, and THBS1. A few genes on this array that are known to play a specific role in colon cancer had activities changed in a way that may be detrimental in the treatment of colon cancer. Further studies should be performed to understand how these changes would impact colon cancer treatment. PMID:23553997

Maness, L; Goktepe, I; Chen, H; Ahmedna, M; Sang, S



TAT-RasGAP317-326-mediated tumor cell death sensitization can occur independently of Bax and Bak.  


The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP317-326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP317-326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP317-326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP317-326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP317-326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway. PMID:24362790

Annibaldi, Alessandro; Heulot, Mathieu; Martinou, Jean-Claude; Widmann, Christian




PubMed Central

A clonal cell line derived from a mouse neoplasm is described which shares many properties with smooth muscle. The cells have electrically excitable membranes capable of generating overshooting action potentials, and they contract both spontaneously and with electrical stimulation. They respond to the iontophoretic application of acetylcholine with a depolarizing response, and to norepinephrine with a hyperpolarizing response. Electron microscopy reveals that the cells have a morphology similar in many, but not all, respects to that of smooth muscle cells in vivo. The cells secrete soluble collagen-like molecules in addition to several proteins of undefined function. Finally, there is an increase in the specific activities of creatine phosphokinase and myokinase associated with increased cell density and the cessation of cell division. PMID:4363958

Schubert, David; Harris, A. John; Devine, Carrick E.; Heinemann, Stephen



Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines  

SciTech Connect

Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany) [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany)] [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany)] [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany)] [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)] [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)



Characterization of a new megakaryocytic cell line: the Dami cell  

Microsoft Academic Search

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryo- blastic leukemia. The Dami cells grow primarily in suspen- sion with a doubling time of 24 to 30 hours. By light and electron microscopy. the Dami cells range in size from 1 2 to 120 ?tm in diameter and have lobulated nuclei

SM Greenberg; DS Rosenthal; TA Greeley; R Tantravahi; RI Handin



Radiation sensitivity of Merkel cell carcinoma cell lines  

SciTech Connect

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others] [Queensland Institute of Medical Research (Australia); and others



The Clinical Relevance of Cancer Cell Lines  

PubMed Central

Although advances in genomics during the last decade have opened new avenues for translational research and allowed the direct evaluation of clinical samples, there is still a need for reliable preclinical models to test therapeutic strategies. Human cancer-derived cell lines are the most widely used models to study the biology of cancer and to test hypotheses to improve the efficacy of cancer treatment. Since the development of the first cancer cell line, the clinical relevance of these models has been continuously questioned. Based upon recent studies that have fueled the debate, we review the major events in the development of the in vitro models and the emergence of new technologies that have revealed important issues and limitations concerning human cancer cell lines as models. All cancer cell lines do not have equal value as tumor models. Some have been successful, whereas others have failed. However, the success stories should not obscure the growing body of data that motivates us to develop new in vitro preclinical models that would substantially increase the success rate of new in vitro–assessed cancer treatments. PMID:23434901



Nitric oxide activation of Keap1/Nrf2 signaling in human colon carcinoma cells  

PubMed Central

The transcription factor NF-E2-related nuclear factor 2 (Nrf2) regulates expression of genes that protect cells from oxidative damage. Here, we characterized nitric oxide (•NO)-induced Nrf2–Kelch-like ECH-associated protein 1 (Keap1) signaling and its role in counteracting •NO-induced apoptosis of human colon cancer HCT116 cells. Nrf2 was localized in the cytoplasm in control cells; •NO triggered its rapid nuclear accumulation, transcriptional activation, and up-regulation of HO-1, NQO1, and GCL, but not GST A4 and P1 subunits. Nrf2 accumulation in the nucleus was also associated with enhanced transcription and posttranscriptional modifications. (S)-nitrosation of Keap1 may contribute to nuclear accumulation of Nrf2 by facilitating its dissociation from Keap1, thus initiating •NO-mediated Nrf2–Keap1 signaling. •NO-mediated induction of ARE-dependent genes occurred well before apoptosis, as judged by caspase 3 activation. Collectively, these results show that the Nrf2–Keap1 signaling pathway mediates protective cellular responses to mitigate •NO-induced damage and may contribute to the relative resistance of HCT116 to •NO-induced cytotoxicity. PMID:19706542

Li, Chun-Qi; Kim, Min Young; Godoy, Luiz C.; Thiantanawat, Apinya; Trudel, Laura J.; Wogan, Gerald N.



The anti-proliferative effect of L-carnosine correlates with a decreased expression of hypoxia inducible factor 1 alpha in human colon cancer cells.  


In recent years considerable attention has been given to the use of natural substances as anticancer drugs. The natural antioxidant dipeptide L-carnosine belongs to this class of molecules because it has been proved to have a significant anticancer activity both in vitro and in vivo. Previous studies have shown that L-carnosine inhibits the proliferation of human colorectal carcinoma cells by affecting the ATP and Reactive Oxygen Species (ROS) production. In the present study we identified the Hypoxia-Inducible Factor 1? (HIF-1?) as a possible target of L-carnosine in HCT-116 cell line. HIF-1? protein is over-expressed in multiple types of human cancer and is the major cause of resistance to drugs and radiation in solid tumours. Of particular interest are experimental data supporting the concept that generation of ROS provides a redox signal for HIF-1? induction, and it is known that some antioxidants are able to suppress tumorigenesis by inhibiting HIF-1?. In the current study we found that L-carnosine reduces the HIF-1? protein level affecting its stability and decreases the HIF-1 transcriptional activity. In addition, we demonstrated that L-carnosine is involved in ubiquitin-proteasome system promoting HIF-1? degradation. Finally, we compared the antioxidant activity of L-carnosine with that of two synthetic anti-oxidant bis-diaminotriazoles (namely 1 and 2, respectively). Despite these three compounds have the same ability in reducing intracellular ROS, 1 and 2 are more potent scavengers and have no effect on HIF-1? expression and cancer cell proliferation. These findings suggest that an analysis of L-carnosine antioxidant pathway will clarify the mechanism underlying the anti-proliferative effects of this dipeptide on colon cancer cells. However, although the molecular mechanism by which L-carnosine down regulates or inhibits the HIF-1? activity has not been yet elucidated, this ability may be promising in treating hypoxia-related diseases. PMID:24804733

Iovine, Barbara; Oliviero, Giorgia; Garofalo, Mariangela; Orefice, Maria; Nocella, Francesca; Borbone, Nicola; Piccialli, Vincenzo; Centore, Roberto; Mazzone, Massimiliano; Piccialli, Gennaro; Bevilacqua, Maria Assunta



Activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase pathway is involved in the casticin-induced apoptosis of colon cancer cells  

PubMed Central

Casticin is one of the main components of the fruits of Vitex rotundifolia L. Studies have shown that casticin inhibits the growth of various cancer cells, including colon cancer. In the present study, the anti-carcinogenic effects of casticin on human colon cancer and the underlying mechanisms were investigated. The results revealed that casticin significantly induced apoptosis of HT-29, HCT-116, SW480 and Caco-2 cells, induced the accumulation of reactive oxygen species (ROS) and increased the protein levels of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase (JNK) and B-cell lymphoma 2-interacting mediator of cell death (Bim) in HT-29 cells. Pretreatment with N-acetylcysteine, an antioxidant chemical compound, inhibited the activation of ASK1, JNK and Bim, as well as the apoptosis induced by casticin. Small interfering RNA targeting ASK1 significantly attenuated the induction of JNK and Bim activation and apoptotic cell death by casticin treatment. SP600125, a specific JNK inhibitor, attenuated Bim activation and apoptosis, but did not alter ASK1 phosphorylation levels. In addition, casticin treatment resulted in apoptosis by the same mechanism in HCT-116, SW480 and Caco-2 cells. These results suggest that casticin significantly induced apoptosis by the activation of the ASK1-JNK-Bim signaling cascade and the accumulation of ROS in colon cancer cells. PMID:25289048




Macrophage Cell Lines Use CD81 in Cell Growth Regulation  

PubMed Central

CD81 is an integral membrane protein belonging to the tetraspanin superfamily. It has two extracellular domains that interact with cell surface proteins and two intracellular tails that contribute to cellular processes. Although there are considerable data about how CD81 affects T- and B-cell function, not much is known about how it impacts macrophages. To address this, we established four cell lines from mouse bone marrow in the presence of macrophage colony stimulating factor and transfection with SV40 large T antigen. Two were CD81?/? (ASD1 and ASD2) and two were CD81+/? (2ASD1.10 and 2BSD1.10). Cells were Mac-2-, PU.1-, and c-fms-positive and all the cell lines were phagocytic indicating that they were macrophage-like. In mixtures of the two cell types in tissue culture, CD81?/? cells out competed CD81+/? cells with CD81-bearing cells being undetectable after 50 cell culture passages. Although cell divisions during log-phase growth were not significantly different between CD81+/? macrophage cells and CD81?/? macrophage cells, we found that CD81?/? macrophage cells reached a higher density at confluency than CD81+/? macrophage cells. CD81 transcript levels increased as cultures became confluent, but transcript levels of other tetraspanin-related molecules remained relatively constant. Transfection of CD81 into ASD1 (CD81?/?) cells reduced the density of confluent cultures of transformants compared to cells transfected with vector alone. These data suggest that CD81 potentially plays a role in macrophage cell line growth regulation. PMID:19184252

Mordica, Whitney J.; Woods, Keith M.; Clem, Rollie J.; Passarelli, A. Lorena; Chapes, Stephen K.



In vitro antitumor effects of the cold-water extracts of Mediterranean species of genus Pleurotus (higher Basidiomycetes) on human colon cancer cells.  


The aim of this study was to evaluate whether the cold-water extracts of Pleurotus eryngii var. ferulae (CWE-Pef) and Pleurotus nebrodensis (CWE-Pn), 2 of the most prized wild and cultivated edible mushrooms, can affect the tumor phenotype of human colon cancer HCT116 cells. Our results showed that treatment with CWE-Pef and CWE-Pn resulted in a significant inhibition of the viability of HCT116 cells and promoted apoptosis, as also demonstrated by the increase of Bax-to-Bcl-2 messenger RNA ratio. Moreover, we observed that both extracts were able to inhibit cell migration and to affect homotypic and heterotypic cell-cell adhesion. It also was found that treatment with CWE-Pef and CWE-Pn negatively modulated the phosphorylation of the protein tyrosine as well as the phosphorylation levels of extracellular signal-regulated kinase 1/2. In conclusion, the in vitro antitumor effects of CWE-Pef and CWE-Pn indicate that they can be considered as possible sources for new alternative therapeutic agents for cancer treatment. PMID:24940904

Fontana, Simona; Flugy, Anna; Schillaci, Odessa; Cannizzaro, Alessandra; Gargano, Maria Letizia; Saitta, Alessandro; De Leo, Giacomo; Venturella, Giuseppe; Alessandro, Riccardo



Investigating citrullinated proteins in tumour cell lines  

PubMed Central

Background The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB). Methods Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines. Results 2-D WB and mass spectrometry identified citrullinated ?-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines. Conclusions The citrullination of these proteins suggests a new mechanism in the tumorigenic process. PMID:24099319



Targeted Delivery of Doxorubicin Using a Colorectal Cancer-Specific ssDNA Aptamer.  


Targeted drug delivery is particularly important in cancer treatment because many antitumor drugs are nonspecific and highly toxic to both cancerous and normal cells. The L33 aptamer is a single-stranded DNA (ssDNA) sequence that has the ability to recognize human colorectal cancer (CRC) cell line HCT116 specifically. In this study, we demonstrated that the L33 aptamer can selectively internalize into target HCT116 cells via receptor-mediated endocytosis. Based on this finding, we developed an aptamer-based drug delivery system using L33 as the carrier of the antitumor drug doxorubicin (Dox). The L33-Dox complex exhibited specific and high affinity (Kd ?=?14.3?±?2.2 nM) binding to HCT116 cells. The results of cytotoxicity assays revealed that the L33-Dox complex was capable of selectively delivering the drug to the target HCT116 cells and lowered the toxicity for nontarget CL187 cells. These findings indicate that the aptamer-based targeted drug delivery system has the potential to be used in clinical settings and may overcome drug resistance to a certain extent because high drug dosages can be directed toward target cells. Anat Rec, 297:2280-2288, 2014. © 2014 Wiley Periodicals, Inc. PMID:25044297

Li, Wanming; Chen, Hang; Yu, Min; Fang, Jin



DNA binding properties of novel cytotoxic gold(III) complexes of terpyridine ligands: the impact of steric and electrostatic effects  

Microsoft Academic Search

Four gold(III) complexes of terpyridine derivatives 1–4 have been synthesized and characterized by spectroscopic methods. In vitro data demonstrated that all of them showed higher cytotoxicity than cisplatin against the human non-small-cell lung cancer cell line (A-549), the human stomach carcinoma cell line (SGC-7901), the human cervix carcinoma cell line (HELA), the human colon carcinoma cell line (HCT-116), the human

Pengfei Shi; Qin Jiang; Yongmei Zhao; Yangmiao Zhang; Jun Lin; Liping Lin; Jian Ding; Zijian Guo



Effective inhibition of colon cancer cell growth with MgAl-layered double hydroxide (LDH) loaded 5-FU and PI3K/mTOR dual inhibitor BEZ-235 through apoptotic pathways  

PubMed Central

Colon cancer is the third most common cancer and the third largest cause of cancer-related death. Fluorouracil (5-FU) is the front-line chemotherapeutic agent for colon cancer. However, its response rate is less than 60%, even in combination with other chemotherapeutic agents. The side effects of 5-FU also limit its application. Nanoparticles have been used to deliver 5-FU, to increase its effectiveness and reduce side effects. Another common approach for colon cancer treatment is targeted therapy against the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. A recently-invented inhibitor of this pathway, BEZ-235, has been tested in several clinical trials and has shown effectiveness and low side effects. Thus, it is a very promising drug for colon cancer treatment. The combination of these two drugs, especially nanoparticle-packed 5-FU and BEZ-235, has not been studied. In the present study, we demonstrated that nanoparticles of layered double hydroxide (LDH) loaded with 5-FU were more effective than a free drug at inhibiting colon cancer cell growth, and that a combination treatment with BEZ-235 further increased the sensitivity of colon cancer cells to the treatment of LDH-packed 5-FU (LDH-5-FU). BEZ-235 alone can decrease colon cancer HCT-116 cell viability to 46% of the control, and the addition of LDH-5-FU produced a greater effect, reducing cell survival to 8% of the control. Our data indicate that the combination therapy of nanodelivered 5-FU with a PI3K/Akt inhibitor, BEZ-235, may promise a more effective approach for colon cancer treatment. PMID:25075187

Chen, Jiezhong; Shao, Renfu; Li, Li; Xu, Zhi Ping; Gu, Wenyi



Synergism from the combination of ulinastatin and curcumin offers greater inhibition against colorectal cancer liver metastases via modulating matrix metalloproteinase-9 and E-cadherin expression  

PubMed Central

Liver metastasis is a major cause of mortality in colorectal cancer (CRC). The current study was to investigate the ability of ulinastatin (UTI) and curcumin (CUR) to inhibit CRC liver metastases via modulating matrix metalloproteinase-9 (MMP-9) and E-cadherin expression. Human CRC HCT-116 cells were treated with compounds individually and in combination in order to understand the effect on cell migration and invasion. The HCT-116 cell line was established to stably express luciferase and green fluorescent protein (GFP) by lentiviral transduction (HCT-116-Luc-GFP). We identified an anti-metastasis effect of UTI and CUR on a CRC liver metastasis mouse model. Tumor development and therapeutic responses were dynamically tracked by bioluminescence imaging. Expression of MMP-9 and E-cadherin in metastatic tumors was detected by immunohistochemical assay. Results of wound healing and cell invasion assays suggest that treatment with UTI, CUR, and UTI plus CUR, respectively, significantly inhibit HCT-116 cell migration and invasion. Furthermore, results of CRC hepatic metastasis on a nude mouse model showed that treatment with UTI, CUR alone, and a combination notably inhibited hepatic metastases from CRC and prolonged survival of tumor-bearing mice, especially in the UTI plus CUR group. These results suggest that the combination of UTI and CUR together may offer greater inhibition against metastasis of CRC. PMID:24570592

Shen, Fei; Cai, Wen-Song; Li, Jiang-Lin; Feng, Zhe; Liu, Qi-cai; Xiao, Huan-qing; Cao, Jie; Xu, Bo



Differential expression of CD133 based on microsatellite instability status in human colorectal cancer.  


The association between the types of genomic instability and cancer stem cell (CSC) has not been elucidated. We aimed to investigate the expressions of CSC markers with respect to microsatellite instability (MSI) status in human colorectal cancer (CRC). Immunostainings for CD133, CD44, and CD166, and K-ras mutation analysis were performed on 50 MSI-high (MSI-H), and 50 microsatellite stable (MSS) CRC tissues. In 11 MSS and MSI-H CRC cell lines, CD133 expression and DNA methylation statuses of the CD133 promoter were determined. The proportion of CD133 positive cells and the ability of colosphere formation were compared between HCT116 cells and HCT116 + Chr3 cells (hMLH1-restored HCT116 cells). Immunohistochemistry for CSC markers revealed that high CD133 expression was more frequent in MSS cancers than in MSI-H (P < 0.001, 74.0% vs. 28.0%, respectively), and related with short disease-free survival. Neither CD44 nor CD166 expression differed significantly with respect to MSI status. K-ras mutation showed no association with expressions of CD133, CD44, or CD166. CD133 expression was relatively high in the MSS cell lines compared to those in MSI-H, and showed a reverse correlation with DNA methylation of the CD133 promoter. hMLH1-restored HCT116 cells increased proportions of CD133 positive cells and colosphere forming ability, compared to those in HCT116 cells. In conclusion, high levels of CD133 expression were observed more frequently in MSS CRC than in MSI-H, suggesting that differential expression of colon CSC markers may be linked to tumor characteristics dependent on MSI status. PMID:23065858

Park, Jae Jun; Kwon, Ji-hee; Oh, Sun-Hee; Choi, Junjeong; Moon, Chang Mo; Ahn, Joong Bae; Hong, Sung Pil; Cheon, Jae Hee; Kim, Tae Il; Kim, Hoguen; Kim, Won Ho



Effects of Oplopanax horridus on Human Colorectal Cancer Cells  

PubMed Central

Aim In this study, we investigated the inhibitive effects of Oplopanax horridus extract (OhE) and its fractions (OhF1, OhF2, OhF3, OhF4 and OhF5) on the growth of human colorectal cancer cells and the possible mechanisms. Materials and Methods The anti-proliferative effects were evaluated by MTS cell proliferation assay. Apoptotic effects and cell cycle distribution were analyzed by flow cytometry after staining with Annexin V/PI or PI/RNase. Results After treatment for 48 hr, OhE, OhF4 and OhF5 (10–100 ?g/ml) inhibited proliferation of HCT-116, SW-480 and HT-29 cell lines. And cell growth decreased most with the treatment of OhF4. On the other hand, OhF1, OhF2 and OhF3 were not observed to have obvious suppressive effects on these cell lines at concentrations of 10–100 ?g/ml. OhE, OhF4 and OhF5 (1–10 ?g/ml) noticeably induced apoptosis time- and concentration-dependently compared to the control at the same time point. Treatement with OhE, OhF4 or OhF5 (1–10 ?g/ml) for 24 hr distinctly induced the G2/M phase arrest of the cell cycle in a dose-dependent manner. The trend of increasing cyclin A and cyclin B1 were similar to the increase of G2/M phase cells in all treated groups. Conclusion These results showed that OhE had potential anti-proliferation effects on human colorectal cancer cells, and the active components were enriched in the fractions OhF4 and OhF5. The anticancer mechanism of OhE, OhF4 and OhF5 might be attributed to the induction of apoptotic cells and the regulation of cell cycle transition. PMID:20332432




Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines  

Microsoft Academic Search

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four

O. Felthaus; T. Ettl; M. Gosau; O. Driemel; G. Brockhoff; A. Reck; K. Zeitler; M. Hautmann; T. E. Reichert; G. Schmalz; C. Morsczeck



Evaluation of anti-cancer activity of Acanthester planci extracts obtained by different methods of extraction.  


Acanthaster planci, the crown-of-thorns starfish, naturally endowed with the numerous toxic spines around the dorsal area of its body. Scientific investigations demonstrated several toxico-pharmacological efficacies of A. planci such as, myonecrotic activity, hemorrhagic activity, hemolytic activity, mouse lethality, phospholipase A2 (PLA2) activity, capillary permeability-increasing activity, edema-forming activity, anticoagulant activity and histamine-releasing activity from mast cells. The present study was performed to evaluate the cytotoxic activity of A. planci extracts obtained by different methods of extraction on MCF-7 and HCT-116, human breast and colon cancer cell lines, respectively. Results of the cell proliferation assay showed that PBS extract exhibited very potent cytotoxic activity against both MCF-7 and HCT-116 cell lines with IC(50) of 13.48 ?g/mL and 28.78 ?g/mL, respectively, while the extracts prepared by Bligh and Dyer method showed moderate cytotoxicity effect against MCF-7 and HCT-116 cell lines, for chloroform extract, IC(50) = 121.37 ?g/mL (MCF-7) and 77.65 ?g/mL (HCT-116), and for methanol extract, IC(50) = 46.11 ?g/mL (MCF-7) and 59.29 ?g/mL (HCT-116). However, the extracts prepared by sequential extraction procedure from dried starfish found to be ineffective. This study paves the way for further investigation on the peptide composition in the PBS extract of the starfish to discover potential chemotherapeutic agents. PMID:23009983

Mutee, Ahmed Faisal; Salhimi, Salizawati Muhamad; Ghazali, Farid Che; Aisha, Abdalrahim Fa; Lim, Chung Pin; Ibrahim, Kamarruddin; Asmawi, Mohd Zaini



UDP glucuronosyltransferase 1A expression levels determine the response of colorectal cancer cells to the heat shock protein 90 inhibitor ganetespib.  


HSP90 inhibition represents a promising route to cancer therapy, taking advantage of cancer cell-inherent proteotoxic stress. The HSP90-inhibitor ganetespib showed benefit in advanced clinical trials. This raises the need to identify the molecular determinants of treatment response. We tested the efficacy of ganetespib on a series of colorectal cancer (CRC)-derived cell lines and correlated their sensitivities with comprehensive gene expression analysis. Notably, the drug concentration required for 50% growth inhibition (IC50) varied up to 70-fold (from 36 to 2500?nM) between different cell lines. Correlating cell line-specific IC50s with the corresponding gene expression patterns revealed a strong association between ganetespib resistance (IC50>500?nM) and high expression of the UDP glucuronosyltransferase 1A (UGT1A) gene cluster. Moreover, CRC tumor samples showed a comparable distribution of UGT1A expression levels. The members of the UGT1A gene family are known as drug-conjugating liver enzymes involved in drug excretion, but their function in tumor cells is hardly understood. Chemically unrelated HSP90 inhibitors, for example, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), did not show correlation of drug sensitivities with UGT1A levels, whereas the ganetespib-related compound NVP-AUY922 did. When the most ganetespib-resistant cell line, HT29, was treated with ganetespib, the levels of HSP90 clients were unaffected. However, HT29 cells became sensitized to the drug, and HSP90 client proteins were destabilized by ganetespib upon siRNA-mediated UGT1A knockdown. Conversely, the most ganetespib-sensitive cell lines HCT116 and SW480 became more tolerant toward ganetespib upon UGT1A overexpression. Mechanistically, ganetespib was rapidly glucuronidated and excreted in resistant but not in sensitive CRC lines. We conclude that CRC cell-expressed UGT1A inactivates ganetespib and other resorcinolic Hsp90 inhibitors by glucuronidation, which renders the drugs unable to inhibit Hsp90 and thereby abrogates their biological activity. UGT1A levels in tumor tissues may be a suitable predictive biomarker to stratify CRC patients for ganetespib treatment. PMID:25210794

Landmann, H; Proia, D A; He, S; Ogawa, L S; Kramer, F; Beißbarth, T; Grade, M; Gaedcke, J; Ghadimi, M; Moll, U; Dobbelstein, M



Immunoreactive growth hormone production by human lymphocyte cell lines  

Microsoft Academic Search

1.Two human lymphocyte cell lines, a T-cell line and a B-cell line, were shown to produce and secrete immunoreactive growth hormone (irGH). The irGH molecules secreted by the two cell lines appeared to be de novo synthesized and their molecular size was similar to that of pituitary GH as well as irGH secreted by peripheral blood lymphocytes.2.Affinity-purified irGH molecules had

Ting-Lin Kao; Scott C. Supowit; E. Aubrey Thompson; Walter J. Meyer



Stem cell patterns in cell lines derived from head and neck squamous cell carcinoma.  


The initiation, growth, recurrence and metastasis of solid tumours, including squamous cell carcinoma of the head and neck region, have been related to the behaviour of a small subpopulation of 'tumour-initiating' cells. Cells with stem cell characteristics have also been identified in cell lines derived from cancers and the aim of the present work was to extend examination of such cells. Established cell lines were examined for their patterns of colony morphologies and staining, the presence of a Hoechst dye-excluding 'side population', expression of the putative stem cell markers CD44, CD133 and CD29, and their ability to grow as 'cancer spheroids'. Two cell lines, CaLH2 and CaLH3, recently generated from HNSCC tumour biopsies, were similarly examined. All cell lines showed a holoclone/meroclone/paraclone series of colony morphologies and cell sorting indicated that CD44 marker expression was related to clonogenicity. FACS analysis after exposure to Hoechst dye indicated that the CA1, H357 and UK1 cell lines contain a dye-excluding 'side population', a property associated with stem-like subpopulations. When held in suspension, all cell lines formed spheroids that could be re-passaged. These observations indicate that cell lines derived from HNSCC contain cells with stem cell properties and that such cell lines may provide experimental systems relevant to the behaviour of stem cells present in the tumours of origin and to their responses to therapy. PMID:17944752

Harper, Lisa J; Piper, Kim; Common, John; Fortune, Farida; Mackenzie, Ian C



Establishment and characterization of four human pancreatic carcinoma cell lines  

Microsoft Academic Search

We characterized four pancreatic carcinoma cell lines (designated SNU-213, SNU-324, SNU-410, and SNU-494) established from histopathologically varied primary or liver metastatic tumor samples of Korean patients. Three cell lines grew as adherent monolayers and one as adherent and floating cell clumps. All lines had: (1) relatively high viability; (2) an absence of mycoplasma or bacterial contamination; (3) genetic heterogeneity as

Ja-Lok Ku; Kyong-Ah Yoon; Woo-Ho Kim; Jin Jang; Kyung-Suk Suh; Sun-Whe Kim; Yong-Hyun Park; Jae-Gahb Park



Syngeneic mouse mammary carcinoma cell lines: Two closely related cell lines with divergent metastatic behavior  

Microsoft Academic Search

Two cell lines, Met-1fvb2 and DB-7fvb2, with different metastatic potential, were derived from mammary carcinomas in FVB\\/N-Tg(MMTV-PyVmT) and FVB\\/N-Tg(MMTV-PyVmTY315F\\/Y322F) mice, transplanted into syngeneic FVB\\/N hosts and characterized. The lines maintain a stable morphological and biological phenotype after multiple rounds of in vitro culture and in vivo transplantation. The Met-1fvb2 line derived from a FVB\\/N-Tg(MMTV-PyVmT) tumor exhibits invasive growth and 100%

Alexander D. Borowsky; Ruria Namba; Lawrence J. T. Young; Kent W. Hunter; J. Graeme Hodgson; Clifford G. Tepper; Erik T. McGoldrick; William J. Muller; Robert D. Cardiff; Jeffrey P. Gregg



Combination Effect of Epigenetic Regulation and Ionizing Radiation in Colorectal Cancer Cells  

PubMed Central

Exposure of cells to ionizing radiation (IR) induces, not only, activation of multiple signaling pathways that play critical roles in cell fate determination, but also alteration of molecular pathways involved in cell death or survival. Recently, DNA methylation has been established as a critical epigenetic process involved in the regulation of gene expression in cancer cells, suggesting that DNA methylation inhibition may be an effective cancer treatment strategy. Because alterations of gene expression by DNA methylation have been considered to influence radioresponsiveness, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2?-deoxycytidine (5-aza-dC), on radiosensitivity. In addition, we investigated the underlying cellular mechanisms of combination treatments of ionizing irradiation (IR) and 5-aza-dC in human colon cancer cells. Colon cancer cell lines were initially tested for radiation sensitivity by IR in vitro and were treated with two different doses of 5-aza-dC. Survival of these cell lines was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and clonogenic assays. The effects of 5-aza-dC along with irradiation on cell growth, cell cycle distribution, apoptosis, and apoptosis-related gene expression were examined. Combination irradiation treatment with 5-aza-dC significantly decreased growth activity compared with irradiation treatment alone or with 5-aza-dC treatment alone. The percentage of HCT116 cells in the sub-G1 phase and their apoptotic rate was increased when cells were treated with irradiation in combination with 5-aza-dC compared with either treatment alone. These observations were strongly supported by increased caspase activity, increased comet tails using comet assays, and increased protein levels of apoptosis-associated molecules (caspase 3/9, cleaved PARP). Our data demonstrated that 5-aza-dC enhanced radiosensitivity in colon cancer cells, and the combination effects of 5-aza-dC with radiation showed greater cellular effects than that of single treatment, suggesting that the combination of 5-aza-dC and radiation has the potential to become a clinical strategy for the treatment of cancer. PMID:25136811

Kim, Joong-Gook; Bae, Jin-Han; Kim, Jin-Ah; Heo, Kyu; Yang, Kwangmo; Yi, Joo Mi



SARS-associated coronavirus replication in cell lines.  


Given the potential for laboratory-associated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infections, we must know which cell lines are susceptible to the virus. We investigated 21 cell lines routinely used for virus isolation or research. After infection with SARS-CoV, cells were observed for cytopathic effects, and quantitative real-time polymerase chain reaction was used to measure ongoing viral replication. An indirect immunofluorescence assay was also used as a confirmatory test. The study identified 10 new cell lines capable of supporting the replication of SARS-CoV and confirmed the susceptibility of 4 cell lines previously reported. This study shows that SARS-CoV can be isolated in several cell lines commonly used for diagnostic or research purposes. It also shows that SARS-CoV can achieve high titers in several cell lines, sometimes in the absence of specific cytopathic effects. PMID:16494729

Kaye, Matthew



Lentiviral gene transduction in human and mouse NK cell lines.  


Natural killer (NK) cells play a vital role in the control of cancer and microbial infections. A major hinderance in studying NK cells is the resistance of these cells to gene transfer. Considering over-expression and gene knockdown studies are crucial tools to study the biology of cells, technologies suitable for transferring genes into NK cells are invaluable. Among various technologies available for gene transfer, lentiviral-mediated transduction has been successful in introducing genes into NK cells. We have standardized methods of lentiviral infection in human and mouse NK cell lines. We obtain transduction efficiencies of 15% in the NK-92 cell line and 30-40% in LNK, YT, and DERL7 cell lines. This method allows efficient and stable introduction of genes and shRNAs into NK cell lines. PMID:20033643

Savan, Ram; Chan, Tim; Young, Howard A



Biological behaviors and proteomics analysis of hybrid cell line EAhy926 and its parent cell line A549  

Microsoft Academic Search

BACKGROUND: It is well established that cancer cells can fuse with endothelial cells to form hybrid cells spontaneously, which facilitates cancer cells traversing the endothelial barrier to form metastases. However, up to now, little is known about the biologic characteristics of hybrid cells. Therefore, we investigate the malignant biologic behaviors and proteins expression of the hybrid cell line EAhy926 with

Ze Jun Lu; Ya Qiong Ren; Guo Ping Wang; Qi Song; Mei Li; Sa Sa Jiang; Tao Ning; Yong Song Guan; Jin Yang; Feng Luo



Differentiation of Embryonic Stem Cell Lines Generated from Adult Somatic Cells by Nuclear Transfer  

Microsoft Academic Search

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and

Teruhiko Wakayama; Viviane Tabar; Ivan Rodriguez; Anthony C. F. Perry; Lorenz Studer; Peter Mombaerts



Fucoidan Induces Cancer Cell Apoptosis by Modulating the Endoplasmic Reticulum Stress Cascades  

PubMed Central

Background Cancer metastasis is the main cause leading to disease recurrence and high mortality in cancer patients. Therefore, inhibiting metastasis process or killing metastatic cancer cells by inducing apoptosis is of clinical importance in improving cancer patient survival. Previous studies revealed that fucoidan, a fucose-rich polysaccharide isolated from marine brown alga, is a promising natural product with significant anti-cancer activity. However, little is known about the role of endoplasmic reticulum (ER) stress in fucoidan-induced cell apoptosis. Principal Findings We reported that fucoidan treatment inhibits cell growth and induces apoptosis in cancer cells. Fucoidan treatments resulted in down-regulation of the glucose regulated protein 78 (GRP78) in the metastatic MDA-MB-231 breast cancer cells, and of the ER protein 29 (ERp29) in the metastatic HCT116 colon cancer cells. However, fucoidan treatment promoted ER Ca2+-dependent calmodulin-dependent kinase II (CaMKII) phosphorylation, Bcl-associated X protein (Bax) and caspase 12 expression in MDA-MB-231 cells, but not in HCT116 cells. In both types of cancer cells, fucoidan activated the phosphorylation of eukaryotic initiation factor 2 alpha (p-eIF2?)\\CCAAT/enhancer binding protein homologous protein (CHOP) pro-apoptotic cascade and inhibited the phosphorylation of inositol-requiring kinase 1 (p-IRE-1)\\X-box binding proteins 1 splicing (XBP-1s) pro-survival cascade. Furthermore, CHOP knockdown prevented DNA damage and cell death induced by fucoidan. Conclusion/Significance Fucoidan exerts its anti-tumor function by modulating ER stress cascades. Contribution of ER stress to the fucoidan-induced cell apoptosis augments our understanding of the molecular mechanisms underlying its anti-tumour activity and provides evidence for the therapeutic application of fucoidan in cancer. PMID:25232957

Zhang, Yu; Zhang, Daohai



Expression of tie receptor tyrosine kinase in leukemia cell lines.  


The tie receptor tyrosine kinase mRNA was originally identified as an amplified product in reverse transcription-polymerase chain reaction analysis of human K562 leukemia cell RNA. In situ hybridization analysis revealed that the corresponding mouse gene is expressed predominantly in endothelial cells. We have explored tie mRNA and protein expression in tumor cell lines. The 4.4 kb tie mRNA was expressed at high levels in five of five human megakaryoblastic leukemia cell lines studied and in two IL-3-dependent mouse myeloid leukemia cell lines, but not in 42 other leukemia cell lines representing various hematopoietic lineages. Increased expression of tie mRNA and protein was observed upon treatment of the megakaryoblastic leukemia cells with the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA), known to enhance megakaryoblastic markers. Among several cell lines from solid tumors, two fibrosarcomas, one rhabdomyosarcoma and one melanoma cell line were positive for tie mRNA. These results suggest that among hematopoietic lineages tie is predominantly expressed in cells with megakaryoblastic properties and that the tie tyrosine kinase is a receptor for a regulatory factor specific for megakaryoblasts, endothelial cells, and occasional tumor cell lines derived from mesenchymal tissues. PMID:8412320

Armstrong, E; Korhonen, J; Silvennoinen, O; Cleveland, J L; Lieberman, M A; Alitalo, R



Novel Roles for P53 in the Genesis and Targeting of Tetraploid Cancer Cells  

PubMed Central

Tetraploid (4N) cells are considered important in cancer because they can display increased tumorigenicity, resistance to conventional therapies, and are believed to be precursors to whole chromosome aneuploidy. It is therefore important to determine how tetraploid cancer cells arise, and how to target them. P53 is a tumor suppressor protein and key regulator of tetraploidy. As part of the “tetraploidy checkpoint”, p53 inhibits tetraploid cell proliferation by promoting a G1-arrest in incipient tetraploid cells (referred to as a tetraploid G1 arrest). Nutlin-3a is a preclinical drug that stabilizes p53 by blocking the interaction between p53 and MDM2. In the current study, Nutlin-3a promoted a p53-dependent tetraploid G1 arrest in two diploid clones of the HCT116 colon cancer cell line. Both clones underwent endoreduplication after Nutlin removal, giving rise to stable tetraploid clones that showed increased resistance to ionizing radiation (IR) and cisplatin (CP)-induced apoptosis compared to their diploid precursors. These findings demonstrate that transient p53 activation by Nutlin can promote tetraploid cell formation from diploid precursors, and the resulting tetraploid cells are therapy (IR/CP) resistant. Importantly, the tetraploid clones selected after Nutlin treatment expressed approximately twice as much P53 and MDM2 mRNA as diploid precursors, expressed approximately twice as many p53-MDM2 protein complexes (by co-immunoprecipitation), and were more susceptible to p53-dependent apoptosis and growth arrest induced by Nutlin. Based on these findings, we propose that p53 plays novel roles in both the formation and targeting of tetraploid cells. Specifically, we propose that 1) transient p53 activation can promote a tetraploid-G1 arrest and, as a result, may inadvertently promote formation of therapy-resistant tetraploid cells, and 2) therapy-resistant tetraploid cells, by virtue of having higher P53 gene copy number and expressing twice as many p53-MDM2 complexes, are more sensitive to apoptosis and/or growth arrest by anti-cancer MDM2 antagonists (e.g. Nutlin). PMID:25380055

Davaadelger, Batzaya; Shen, Hong; Maki, Carl G.



Thromboplastic and fibrinolytic activities of cultured human cancer cell lines.  

PubMed Central

Thromboplastic and fibrinolytic activities of 14 lines of cultured human cancer cells were estimated by modified Astrup's methods. High tissue thromboplastic activity was found in one line of urinary-bladder cancer, 2 lines of gastric cancer and one line of lung cancer, but no activity was found in 6 lines of lung cancer. High fibrinolytic activity was noted in one line of gastric cancer and 2 lines of lung cancer, but no activity was seen in 6 lines of lung cancer and one line of gastric cancer. No plasmin activity was found. The tumour cell lines could be classified into 3 groups on the basis of the 2 activities. Cancer cell lines could also be classified into 2 groups: with high or low release of thromboplastin into culture media. Fibrinolytic activity was found in the culture media of all cell lines with high fibrinolytic activity. Fibrinolytic activity, but not thromboplastic activity, seemed to be influenced by the constituents of culture media. No definite correlation was found between the 2 activities and the histological types of the parent tumours of the cultured cells. Images Fig. 1 Fig. 2 Fig. 3 PMID:758928

Kinjo, M.; Oka, K.; Naito, S.; Kohga, S.; Tanaka, K.; Oboshi, S.; Hayata, Y.; Yasumoto, K.



Integrated bioprocess for the production and isolation of urokinase from animal cell culture using supermacroporous cryogel matrices.  


An integrated cell cultivation and protein product separation process was developed using a new type of supermacroporous polyacrylamide gel, called cryogel (pAAm-cryogel) support matrix. Human fibrosarcoma HT1080 and human colon cancer HCT116 cell lines were used to secrete urokinase (an enzyme of immense therapeutic utility) into the culture medium. The secreted protein was isolated from the circulating medium using a chromatographic capture column. A pAAm cryogel support with covalently coupled gelatin (gelatin-pAAm cryogel) was used for the cultivation of anchorage dependent cells in the continuous cell culture mode in 5% carbon dioxide atmosphere. The cells were attached to the matrix within 4-6 h of inoculation and grew as a tissue sheet inside the cryogel matrix. Continuous urokinase secretion into the circulating medium was monitored as a parameter of growth and viability of cells inside the bioreactor. No morphological changes were observed in the cells eluted from the gelatin-cryogel support and re-cultured in T-flask. The gelatin-pAAm cryogel bioreactor was further connected to a pAAm cryogel column carrying Cu(II)-iminodiacetic acid (Cu(II)-IDA)-ligands (Cu(II)-IDA-pAAm cryogel), which had been optimized for the capture of urokinase from the conditioned medium of the cell lines. Thus an automated system was built, which integrated the features of a hollow fiber reactor with a chromatographic protein separation system. The urokinase was continuously captured by the Cu(II)-IDA-pAAm cryogel column and periodically recovered through elution cycles. The urokinase activity increased from 250 PU/mg in the culture fluid to 2,310 PU/mg after recovery from the capture column which gave about ninefold purification of the enzyme. Increased productivity was achieved by operating integrated bioreactor system continuously for 32 days under product inhibition free conditions during which no backpressure or culture contamination was observed. A total 152,600 Plough units of urokinase activity was recovered from 500 mL culture medium using 38 capture columns over a period of 32 days. PMID:16435398

Kumar, Ashok; Bansal, Vibha; Nandakumar, Kutty Selva; Galaev, Igor Yu; Roychoudhury, Pradip K; Holmdahl, Rikard; Mattiasson, Bo



Human cancer cell lines: Experimental models for cancer cells in situ? For cancer stem cells?  


Established human cancer cell lines are routinely used as experimental models for human cancers. Their validity for such use is analyzed and discussed, with particular focus on thyroid tumors. Although cell lines retain some properties of the cells of origin, from the points of view of their genetics, epigenetics and gene expression, they show clear differences in these properties compared to in vivo tumors. This can be explained by a prior selection of initiating cells and a Darwinian evolution in vitro. The properties of the cell lines are compared to those of the postulated cancer stem cells and their use as models in this regard are discussed. Furthermore, other proper and possible uses of the cell lines are discussed. PMID:19167460

van Staveren, W C G; Solís, D Y Weiss; Hébrant, A; Detours, V; Dumont, J E; Maenhaut, C



Authentication of the R06E Fruit Bat Cell Line  

PubMed Central

Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery. PMID:22754654

Jordan, Ingo; Munster, Vincent J.; Sandig, Volker



Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls  

PubMed Central

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.



GREG cells, a dysferlin-deficient myogenic mouse cell line  

SciTech Connect

The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S. [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Morree, Antoine de [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands)] [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands); Pekkurnaz, Gulcin [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Nagaraju, Kanneboyina [Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010 (United States)] [Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010 (United States); Zimmerberg, Joshua, E-mail: [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)



Selective suicide gene therapy of colon cancer exploiting the urokinase plasminogen activator receptor promoter.  


Colon cancer is the third and fourth most prevalent cancer among Iranian men and women, respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application of specific promoters for therapeutic genes should decrease the adverse effects of this modality. The combined aims of this study were to design a specific suicide gene therapy construct for colon cancer and study its effect in distinct representatives of transformed and nontransformed cells. The KRAS oncogene signaling pathway is one of the most important signaling pathways activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR; PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene (thymidine kinase [TK]) and a reporter gene (beta-galactosidase, beta-gal [LacZ]). This promoter is a natural combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors AP1 (FOS/JUN), SP1, SP3, and AP2alpha, and nuclear factor kappa B (NFkappaB). The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to express beta-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma [HCT116] cell lines), while it could not express beta-gal in nontransformed human umbilical vein endothelial cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK decreased the viability of these cells in the presence of ganciclovir 20 and 40 microg/mL (and higher), respectively. Although M30 CytoDEATH antibody could not detect a significant rate of apoptosis induced by ganciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in comparison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive control. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells. This study showed that the uPAR promoter can be considered as a suitable candidate for specific suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is involved in their carcinogenesis process. PMID:20199127

Teimoori-Toolabi, Ladan; Azadmanesh, Kayhan; Amanzadeh, Amir; Zeinali, Sirous



Differential control of growth, apoptotic activity and gene expression in human colon cancer cells by extracts derived from medicinal herbs, Rhazya stricta and Zingiber officinale and their combination  

PubMed Central

AIM: To investigate the effects of extracts from Rhazya stricta (R. stricta) and Zingiber officinale (Z. officinale) on human colorectal cancer cells. METHODS: Human colorectal cancer cells (HCT116) were subjected to increasing doses of crude alkaloid extracts from R. stricta (CAERS) and crude flavonoid extracts from Z. officinale (CFEZO). Cells were then harvested after 24, 48 or 72 h and cell viability was examined by trypan blue exclusion dye test; clonogenicity and soft agar colony-forming assays were also carried out. Nuclear stain (Hoechst 33342), acridine orange/ethidium bromide double staining, agarose gel electrophoresis and comet assays were performed to assess pro-apoptotic potentiality of the extracts. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), using gene-specific primers and Western blot analyses were performed to assess the impact of CAERS and CFEZO on the expression levels of key regulatory proteins in HCT116 cells. RESULTS: Treatment with a combination of CAERS and CFEZO synergistically suppressed the proliferation, colony formation and anchorage-independent growth of HCT116 cells. Calculated IC50, after 24, 48 and 72 h, were 70, 90 and 130 ?g/mL for CAERS, 65, 85 and 120 ?g/mL for CFEZO and 20, 25 and 45 ?g/mL for both agents, respectively. CAERS- and CFEZO-treated cells exhibited morphologic and biochemical features of apoptotic cell death. The induction of apoptosis was associated with the release of mitochondrial cytochrome c, an increase in the Bax/Bcl-2 ratio, activation of caspases 3 and 9 and cleavage of poly ADP-ribose polymerase. CAERS and CFEZO treatments downregulated expression levels of anti-apoptotic proteins including Bcl-2, Bcl-X, Mcl-1, survivin and XIAP, and upregulated expression levels of proapoptotic proteins such as Bad and Noxa. CAERS and CFEZO treatments elevated expression levels of the oncosuppressor proteins, p53, p21 and p27, and reduced levels of the oncoproteins, cyclin D1, cyclin/cyclin-dependent kinase-4 and c-Myc. CONCLUSION: These data suggest that a combination of CAERS and CFEZO is a promising treatment for the prevention of colon cancer. PMID:25386076

Elkady, Ayman I; Hussein, Rania Abd El Hamid; Abu-Zinadah, Osama A



Replicative Capacity of MERS Coronavirus in Livestock Cell Lines.  


Replicative capacity of Middle East respiratory syndrome coronavirus (MERS-CoV) was assessed in cell lines derived from livestock and peridomestic small mammals on the Arabian Peninsula. Only cell lines originating from goats and camels showed efficient replication of MERS-CoV. These results provide direction in the search for the intermediate host of MERS-CoV. PMID:24457147

Eckerle, Isabella; Corman, Victor M; Müller, Marcel A; Lenk, Matthias; Ulrich, Rainer G; Drosten, Christian



Replicative Capacity of MERS Coronavirus in Livestock Cell Lines  

PubMed Central

Replicative capacity of Middle East respiratory syndrome coronavirus (MERS-CoV) was assessed in cell lines derived from livestock and peridomestic small mammals on the Arabian Peninsula. Only cell lines originating from goats and camels showed efficient replication of MERS-CoV. These results provide direction in the search for the intermediate host of MERS-CoV. PMID:24457147

Eckerle, Isabella; Corman, Victor M.; Muller, Marcel A.; Lenk, Matthias; Ulrich, Rainer G.



Development of nuclear receptor transfected Caco-2 cell lines  

E-print Network

DEVELOPMENT OF NUCLEAR RECEPTOR TRANSFECTED CACO-2 CELL LINES Timo Korjamo University of Kuopio Finland 27th October 2006 ? Background ? Cell lines ? Gene expression ? Functional experiments ? Conclusions Intestinal absorption ? Small intestine...B6 Transfection with human PXR Caco/hPXR CYP2B6, MDR1, CYP3A4, CYP2C9, MRP2 Transfection with murine CAR Caco/mCAR Wild type cellsCaco/WT Some target genesModificationCell line Initial characterisation: T. Korjamo, P. Honkakoski, M. R. Toppinen...

Korjamo, Timo



Respiratory epithelial cell lines exposed to anoxia produced inflammatory mediator  

PubMed Central

Human epithelial cell lines were utilized to examine the effects of anoxia on cellular growth and metabolism. Three normal human epithelial cells lines (A549, NHBE, and BEAS-2B) as well as a cystic fibrosis cell line (IB3-1) and its mutation corrected cell line (C38) were grown in the presence and absence of oxygen for varying periods of time. Interleukin-8 (IL-8) levels were measured by enzyme-linked immunosorbent assay technique. Cellular metabolism and proliferation were assayed by determining mitochondrial oxidative burst activity by tetrazolium compound reduction. The viability of cells was indirectly measured by lactate dehydrogenase release. A549, NHBE, and BEAS-2B cells cultured in the absence of oxygen showed a progressive decrease in metabolic activity and cell proliferation after one to three days. There was a concomitant increase in IL-8 production. Cell lines from cystic fibrosis (CF) patients did not show a similar detrimental effect of anoxia. However, the IL-8 level was significantly increased only in IB3-1 cells exposed to anoxia after two days. Anoxia appears to affect certain airway epithelial cell lines uniquely with decreased cellular proliferation and a concomitant increased production of a cytokine with neutrophilic chemotactic activity. The increased ability of the CF cell line to respond to anoxia with increased secretion of inflammatory cytokines may contribute to the inflammatory damage seen in CF bronchial airway. This study indicates the need to use different cell lines in in vitro studies investigating the role of epithelial cells in airway inflammation and the effects of environmental influences. PMID:23301190

Shahriary, Cyrus M.; Nussbaum, Eliezer



Cisplatin resistance induced by decreased apoptotic activity in non-small-cell lung cancer cell lines.  


We have investigated defective steps in apoptosis that might account for the development of resistance. For this purpose, A549 and Calu1 NSCLC (non-small-cell lung cancer) cell lines were treated with cisplatin to obtain resistant sub-lines. Gene expression profiles and the phosphorylation status of the BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) protein were determined for each cell line. Cell death and cytochrome c release were analysed after treating cell lines with their appropriate cisplatin doses. Gene expression of BAD, Bid, caspases 4 and 6 were clearly decreased in the resistant cell lines, and the differential phosphorylation status of BAD also seemed to play a role in the development of cisplatin resistance. Since this is a new cisplatin-resistant Calu1 cell line, it is noteworthy that DNA fragmentation, apoptotic cell ratio and cytochrome c levels were most decreased in the CR-Calu1 cell line. PMID:22397496

Cetintas, Vildan B; Kucukaslan, Ali S; Kosova, Buket; Tetik, Asl?; Selvi, Nur; Cok, Gursel; Gunduz, Cumhur; Eroglu, Zuhal



Induction of apoptosis in colon cancer cells by a novel topoisomerase I inhibitor TopIn  

SciTech Connect

Highlights: {yields} TopIn activates p53-dependent transcription in colon cancer cells. {yields} TopIn induces apoptosis in colon cancer cells. {yields} TopIn selectively inhibits topoisomerase I activity. {yields} TopIn does not affect the activity of BCRP and MDR-1. -- Abstract: The tumor suppressor p53 plays an important role in cellular emergency mechanisms through regulating the genes involved in cell cycle arrest and apoptosis. To identify small molecules that can activate p53-responsive transcription, we performed chemical screening using genetically engineered HCT116 reporter cells. We found that TopIn (7-phenyl-6H-[1,2,5]oxadiazolo[3,4-e]indole 3-oxide) efficiently activated p53-mediated transcriptional activity and induced phosphorylation of p53 at Ser15, thereby stabilizing the p53 protein. Furthermore, TopIn upregulated the expression of p21{sup WAF1/CIP1}, a downstream target of p53, and suppressed cellular proliferation in various colon cancer cells. Additionally, TopIn induced DNA fragmentation, caspase-3/7 activation and poly ADP ribose polymerase cleavage, typical biochemical markers of apoptosis, in p53 wild-type and mutated colon cancer cells. Finally, we found that TopIn inhibited topoisomerase I activity, but not topoisomerase II, in vitro and induced the formation of the topoisomerase I-DNA complex in HCT116 colon cancer cells. Unlike camptothecin (CPT) and its derivative SN38, TopIn did not affect the activity of the ATP-binding cassette transporter breast cancer resistance protein (BCRP) or multidrug-resistant protein-1 (MDR-1). These results suggest that TopIn may present a promising new topoisomerase I-targeting anti-tumor therapeutics.

Bae, Soo Kyung [College of Pharmacy, The Catholic University of Korea, Bucheon 420-743 (Korea, Republic of)] [College of Pharmacy, The Catholic University of Korea, Bucheon 420-743 (Korea, Republic of); Gwak, Jungsug [Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702 (Korea, Republic of)] [Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702 (Korea, Republic of); Song, Im-Sook [PharmcoGenomics Research Center, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)] [PharmcoGenomics Research Center, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Park, Hyung-Soon [Probiond Co., Ltd., Seoul 143-834 (Korea, Republic of)] [Probiond Co., Ltd., Seoul 143-834 (Korea, Republic of); Oh, Sangtaek, E-mail: [Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702 (Korea, Republic of)] [Department of Advanced Fermentation Fusion Science and Technology, Kookmin University, Seoul 136-702 (Korea, Republic of)



UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma

This technology describes the UOK 268 cell line, a spontaneously immortalized renal tumor cell line that may be of great interest to industry for studying HLRCC, drug screening, and searching for tumor markers related to diagnosis, prognosis, and drug resistance.


GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer  

Microsoft Academic Search

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines.

F. G. Rick; S. Seitz; A. V. Schally; L. Szalontay; A. Krishan; C. Datz; A. Stadlmayr; S. Buchholz; N. L. Block; F. Hohla



Apoptosis induced by selenium in human glioma cell lines  

Microsoft Academic Search

Several studies have shown that selenium can inhibit tumorigenesis in tissues. However, little is known about the mechanism\\u000a and the effect of selenium on DNA, especially in brain tumor cells. In this study we examined the biological effect of selenium\\u000a on human glioma cell lines (A172 and T98G). Selenium exhibited an antiproliferative effect on these cell lines (and induced\\u000a the

Zongjian Zhu; Mieko Kimura; Yoshinori Itokawa; Tomokazu Aoki; Jun A. Takahashi; Shouji Nakatsu; Yoshifumi Oda; Haruhiko Kikuchi



An established spleen cell line from Bairdiella chrysura  

Microsoft Academic Search

Summary  A cell line, SP-2, was established from spleen tissue ofBairdiella chrysura (the silver perch). The line is susceptible to lymphocystis virus and the amphibian LT-1 virus but refractory to six additional\\u000a viruses. The modal chromosome number of primary silver perch cells is 48, but SP-2 cells are heteroploid. For growth, Leibovitz\\u000a L-15 medium supplemented with fetal bovine serum and sodium

R. D. Ellender; J. H. Wharton; B. L. Middlebrooks



A kidney epithelial cell line from a bolivian squirrel monkey  

Microsoft Academic Search

Summary  Squirrel monkeys are the most commonly used New World primates in biomedical research, but in vitro studies are restricted\\u000a by the limited number of cell lines available from this species. We report here the development and characterization of a\\u000a continuous, kidney epithelial cell line (SQMK-FP cells) derived from a newborn squirrel monkey. Karyotype was consistent with\\u000a Bolivian squirrel monkey (submetacentric

Jonathan G. Scammell; J. Allan Tucker; Judy A. King; Charleen M. Moore; James L. Wright; Cathy M. Tuck-Muller



Radiation-induced adaptive response in fish cell lines.  


There is considerable interest at present in low-dose radiation effects in non-human species. In this study gamma radiation-induced adaptive response, a low-dose radiation effect, was examined in three fish cell lines, (CHSE-214 (Chinook salmon), RTG-2 (rainbow trout) and ZEB-2J (zebrafish)). Cell survival after exposure to direct radiation with or without a 0.1 Gy priming dose, was determined using the colony forming assay for each cell line. Additionally, the occurrence of a bystander effect was examined by measuring the effect of irradiated cell culture medium from the fish cell lines on unexposed reporter cells. A non-linear dose response was observed for all cell lines. ZEB-2J cells were very sensitive to low doses and a hyper-radiosensitive (HRS) response was observed for doses <0.5 Gy. A typical protective adaptive response was not detected in any of the three fish cell lines tested. Rather, it was found that pre-exposure of these cells to 0.1 Gy radiation sensitized the cells to subsequent high doses. In CHSE-214 cells, increased sensitivity to subsequent high doses of radiation was observed when the priming and challenge doses were separated by 4 h; however, this sensitizing effect was no longer present when the interval between doses was greater than 8 h. Additionally, a "protective" bystander response was observed in these cell lines; exposure to irradiated medium from fish cells caused increased cloning efficiency in unirradiated reporter cells. The data confirm previous conclusions for mammalian cells that the adaptive response and bystander effect are inversely correlated and contrary to expectations probably have different underlying mechanisms. PMID:18054128

Ryan, Lorna A; Seymour, Colin B; O'Neill-Mehlenbacher, Alicia; Mothersill, Carmel E



Changes in gene expression profile in two multidrug resistant cell lines derived from a same drug sensitive cell line.  


Resistance to chemotherapy is one of the most relevant aspects of treatment failure in cancer. Cell lines are used as models to study resistance. We analyzed the transcriptional profile of two multidrug resistant (MDR) cell lines (Lucena 1 and FEPS) derived from the same drug-sensitive cell K562. Microarray data identified 130 differentially expressed genes (DEG) between K562 vs. Lucena 1, 1932 between K562 vs. FEPS, and 1211 between Lucena 1 versus FEPS. The NOTCH pathway was affected in FEPS with overexpression of NOTCH2 and HEY1. The highly overexpressed gene in MDR cell lines was ABCB1, and both presented the ABCB1 promoter unmethylated. PMID:24996974

Moreira, Miguel Angelo Martins; Bagni, Carolina; de Pinho, Marcos Barcelos; Mac-Cormick, Thaís Messias; dos Santos Mota, Mateus; Pinto-Silva, Flávio Eduardo; Daflon-Yunes, Nathalia; Rumjanek, Vivian Mary



Development of NK cell expansion methods using feeder cells from human myelogenous leukemia cell line  

PubMed Central

Background Natural killer (NK) cells constantly survey surrounding tissues and remove newly generated cancer cells, independent of cancer antigen recognition. Although there have been a number of attempts to apply NK cells for cancer therapy, clinical application has been somewhat limited because of the difficulty in preparing a sufficient number of NK cells. Therefore, ex vivo NK cell expansion is one of the important steps for developing NK cell therapeutics. Methods CD3+ depleted lymphocytes were cocultured with IL-2 and with feeder cells (peripheral blood mononuclear cells [PBMCs], K562, and Jurkat) for 15 days. Expanded NK cells were tested for cytotoxicity against cancer cell lines. Results We compared feeder activities of three different cells-PBMC, K562, and Jurkat. K562 expanded NK cells by almost 20 fold and also showed powerful cytotoxic activity against cancer cells. K562-NK cells remarkably expressed the NK cell activation receptors, NKG2D, and DNAM-1. K562-NK cells exhibited more than two-fold production of cytotoxic granules compared with Jurkat-NK cells, producing more perforin and granzyme B than naïve NK cells. Conclusion Our findings suggest that K562 are more efficient feeder cells than Jurkat or PBMCs. K562 feeder cells expanded NK cells by almost 20 fold and showed powerful cytotoxic activity against cancer cells. We herein propose an intriguing approach for a design of NK cell expansion. PMID:25325034

Bae, Duk Seong



Reduced Warburg Effect in Cancer Cells Undergoing Autophagy: Steady- State 1H-MRS and Real-Time Hyperpolarized 13C-MRS Studies  

PubMed Central

Autophagy is a highly regulated, energy dependent cellular process where proteins, organelles and cytoplasm are sequestered in autophagosomes and digested to sustain cellular homeostasis. We hypothesized that during autophagy induced in cancer cells by i) starvation through serum and amino acid deprivation or ii) treatment with PI-103, a class I PI3K/mTOR inhibitor, glycolytic metabolism would be affected, reducing flux to lactate, and that this effect may be reversible. We probed metabolism during autophagy in colorectal HT29 and HCT116 Bax knock-out cells using hyperpolarized 13C-magnetic resonance spectroscopy (MRS) and steady-state 1H-MRS. 24 hr PI103-treatment or starvation caused significant reduction in the apparent forward rate constant (kPL) for pyruvate to lactate exchange compared with controls in HT29 (100 ?M PI-103: 82%, p?=?0.05) and HCT116 Bax-ko cells (10 ?M PI-103: 53%, p?=?0.05; 20 ?M PI-103: 42%, p<0.0001; starvation: 52%, p<0.001), associated with reduced lactate excretion and intracellular lactate in all cases, and unchanged lactate dehydrogenase (LDH) activity and increased NAD+/NADH ratio following PI103 treatment or decreased LDH activity and unchanged NAD+/NADH ratio following starvation. After 48 hr recovery from PI103 treatment, kPL remained below control levels in HT29 cells (74%, p?=?0.02), and increased above treated values, but remained below 24 hr vehicle-treated control levels in HCT116 Bax-ko cells (65%, p?=?0.004) both were accompanied by sustained reduction in lactate excretion, recovery of NAD+/NADH ratio and intracellular lactate. Following recovery from starvation, kPL was significantly higher than 24 hr vehicle-treated controls (140%, p?=?0.05), associated with increased LDH activity and total cellular NAD(H). Changes in kPL and cellular and excreted lactate provided measureable indicators of the major metabolic processes accompanying starvation- and drug-induced autophagy. The changes are reversible, returning towards and exceeding control values on cellular recovery, which potentially identifies resistance. kPL (hyperpolarized 13C-MRS) and lactate (1H-MRS) provide useful biomarkers for the autophagic process, enabling non-invasive monitoring of the Warburg effect. PMID:24667972

Wong Te Fong, Anne-Christine; Hill, Deborah K.; Orton, Matthew R.; Parkes, Harry G.; Koh, Dow-Mu; Robinson, Simon P.; Leach, Martin O.; Eykyn, Thomas R.; Chung, Yuen-Li



miR-18a Inhibits CDC42 and Plays a Tumour Suppressor Role in Colorectal Cancer Cells  

PubMed Central

The miR-17-92 cluster of microRNAs is elevated in colorectal cancer, and has a causative role in cancer development. Of the six miR-17-92 cluster members, miR-19a and b in particular are key promoters of cancer development and cell proliferation, while preliminary evidence suggests that miR-18a may act in opposition to other cluster members to decrease cell proliferation. It was hypothesised that miR-18a may have a homeostatic function in helping to contain the oncogenic effect of the entire miR-17-92 cluster, and that elevated miR-17-92 cluster activity without a corresponding increase in miR-18a may promote colorectal tumour progression. In colorectal cancer samples and corresponding normal colorectal mucosa, miR-18a displayed lower overall expression than other miR-17-92 cluster members. miR-18a was shown to have an opposing role to other miR-17-92 cluster members, in particular the key oncogenic miRNAs, miR-19a and b. Transfection of HCT116 and LIM1215 colorectal cancer cell lines with miR-18a mimics decreased proliferation, while a miR-18a inhibitor increased proliferation. miR-18a was also responsible for decreasing cell migration, altering cell morphology, inducing G1/S phase cell cycle arrest, increasing apoptosis, and enhancing the action of a pro-apoptotic agent. CDC42, a mediator of the PI3K pathway, was identified as a novel miR-18a target. Overexpression of miR-18a reduced CDC42 expression, and a luciferase assay confirmed that miR-18a directly targets the 3?UTR of CDC42. miR-18a mimics had a similar effect on proliferation as a small molecule inhibitor of CDC42. Inhibition of CDC42 expression is likely to be a key mechanism by which miR-18a impairs cancer cell growth, with a target protector experiment revealing miR-18a influences proliferation via direct inhibition of CDC42. Inhibition of CCND1 by miR-18a may also assist in this growth-suppression effect. The homeostatic function of miR-18a within the miR-17-92 cluster in colorectal cancer cells may be achieved through suppression of CDC42 and the PI3K pathway. PMID:25379703

Humphreys, Karen J.; McKinnon, Ross A.; Michael, Michael Z.



Characterization of three new serous epithelial ovarian cancer cell lines  

PubMed Central

Background Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors (TOV-1946, and TOV-2223G), as well as the matched ascites for one case (OV-1946). Methods In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice. Results While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic TP53 mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in BRAF, KRAS or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease. Conclusion This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient. PMID:18507860

Ouellet, Veronique; Zietarska, Magdalena; Portelance, Lise; Lafontaine, Julie; Madore, Jason; Puiffe, Marie-Line; Arcand, Suzanna L; Shen, Zhen; Hebert, Josee; Tonin, Patricia N; Provencher, Diane M; Mes-Masson, Anne-Marie



Nuclear factor-kappaB sensitizes to benzyl isothiocyanate-induced antiproliferation in p53-deficient colorectal cancer cells.  


Benzyl isothiocyanate (BITC), a dietary isothiocyanate derived from cruciferous vegetables, inhibits the proliferation of colorectal cancer cells, most of which overexpress ?-catenin as a result of mutations in the genes for adenomatous polyposis coli or mutations in ?-catenin itself. Because nuclear factor-?B (NF-?B) is a plausible target of BITC signaling in inflammatory cell models, we hypothesized that it is also involved in BITC-inhibited proliferation of colorectal cancer cells. siRNA-mediated knockdown of the NF-?B p65 subunit significantly decreased the BITC sensitivity of human colorectal cancer HT-29 cells with mutated p53 tumor suppressor protein. Treating HT-29 cells with BITC induced the phosphorylation of I?B kinase, I?B-? and p65, the degradation of I?B-?, the translocation of p65 to the nucleus and the upregulation of NF-?B transcriptional activity. BITC also decreased ?-catenin binding to a positive cis element of the cyclin D1 promoter and thus inhibited ?-catenin-dependent cyclin D1 transcription, possibly through a direct interaction between p65 and ?-catenin. siRNA-mediated knockdown of p65 confirmed that p65 negatively affects cyclin D1 expression. On the other hand, when human colorectal cancer HCT-116 cells with wild-type p53 were treated with BITC, translocation of p65 to the nucleus was inhibited rather than enhanced. p53 knockout increased the BITC sensitivity of HCT-116 cells in a p65-dependent manner, suggesting that p53 negatively regulates p65-dependent effects. Together, these results identify BITC as a novel type of antiproliferative agent that regulates the NF-?B pathway in p53-deficient colorectal cancer cells. PMID:25412312

Abe, N; Hou, D-X; Munemasa, S; Murata, Y; Nakamura, Y



Curcumin Glucuronides: Assessing the Proliferative Activity against Human Cell Lines  

PubMed Central

A gram scale synthesis of the glucuronide metabolites of curcumin were completed in four steps. The newly synthesized curcumin glucuronide compounds 2 and 3 along with curcumin 1 were tested and their anti-proliferative effects against KBM-5, Jurkat cell, U266, and A549 cell lines were reported. Biological data revealed that as much as 1 ?M curcumin 1 exhibited anticancer activity and almost 100% cell kill was noted at 10 ?M on two out of four cell lines; while curcumin mono-glucuronide 2 as well as diglucuronide 3 displayed no suppression of cell proliferation. PMID:24280069

Pal, Ashutosh; Sung, Bokyung; Prasad, Basvoju A. Bhanu; Schuber, Paul T.; Prasad, Sahdeo; Aggarwal, Bharat B.; Bornmann, William G.



Development and characterization of a largemouth bass cell line.  


Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014. PMID:25229492

Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R



Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines  

PubMed Central

Background To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n?=?20) or healthy subjects (n?=?25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery. PMID:22586443

Coram, Marc A.; Reddy, Anupama; Deng, Glenn; Telli, Melinda L.; Advani, Ranjana H.; Carlson, Robert W.; Mollick, Joseph A.; Sheth, Shruti; Kurian, Allison W.; Ford, James M.; Stockdale, Frank E.; Quake, Stephen R.; Pease, R. Fabian; Mindrinos, Michael N.; Bhanot, Gyan; Dairkee, Shanaz H.; Davis, Ronald W.; Jeffrey, Stefanie S.



A Potential Rhodium Cancer Therapy: Studies of a Cytotoxic Organorhodium (I) Complex that Binds DNA  

PubMed Central

Described is a novel organorhodium(I) complex that is cytotoxic to the colon cancer cell line HCT116 and alters cell migration, DNA replication, and DNA condensation. Most importantly, the mechanism observed is not seen for the parent organorhodium dimer complex [{RhCl(COD)}2], RhCl3, or the free ligand/proligands (COD and 1-nbutyl-3-methylimidazolium chloride). Thus, the activity of this organorhodium complex is attributable to its unique structure. PMID:23541673

McConnell, Jeanette R.; Rananaware, Dimple P; Ramsey, Deborah M.; Buys, Kai N.; Cole, Marcus L.; McAlpine, Shelli R.



Effects of ethanol on an intestinal epithelial cell line  

SciTech Connect

The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

Nano, J.L.; Cefai, D.; Rampal, P. (Laboratoire de Gastroenterologie et de Nutrition, U.E.R. de Medecine, Nice (France))



Phenotypes and Karyotypes of Human Malignant Mesothelioma Cell Lines  

PubMed Central

Background Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma. Methods Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs. Results Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30–72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5–17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions. Conclusion These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma. PMID:23516439

Relan, Vandana; Morrison, Leanne; Parsonson, Kylie; Clarke, Belinda E.; Duhig, Edwina E.; Windsor, Morgan N.; Matar, Kevin S.; Naidoo, Rishendran; Passmore, Linda; McCaul, Elizabeth; Courtney, Deborah; Yang, Ian A.; Fong, Kwun M.; Bowman, Rayleen V.




EPA Science Inventory

THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE) Methylation of Arsenite by Some Mammalian Cell Lines. Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic. Aim 1: Determine if there is diffe...


A human liposarcoma cell line producing hyaluronic acid.  


A human liposarcoma cell line COLO 222, derived from a primary tumor in a 62-year-old male, elaborates hyaluronic acid. COLO 222 is characterized on the basis of histochemical, ultramorphological, and cytogenetic properties, along with isozyme phenotype and cell products. A chromosome mode of 53 predominates and unique Giemsa-banded marker chromosomes are identified. An autochthonous lymphoid cell line, COLO 143v, was established after the addition of exogenous Epstein-Barr virus. Cytogenetic analysis of Colo 143v is consistent with a normal male karyotype. COLO 143v possesses B-cell characteristics. This autochthonous system had been used for immunological studies and cytotoxicity assays. PMID:6244080

Morgan, R T; Quinn, L A; Moore, G E; Semple, T U; Woods, L K



Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines.  


Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines. PMID:23716874

Gabrani, Reema; Jain, Ramya; Sharma, Anjali; Sarethy, Indira P; Dang, Shweta; Gupta, S



Screening Services – NCI-60 DTP Human Tumor Cell Line Screen

The In Vitro Cell Line Screening Project (IVCLSP) is a dedicated service providing direct support to the DTP anticancer drug discovery program. The in vitro cell line screen was implemented in fully operational form in April of 1990. It required approximately five years (1985 - 1990) to develop, and persistence in the effort reflected dissatisfaction with the performance of prior in vivo primary screens. This project is designed to screen up to 3,000 compounds per year for potential anticancer activity.


Calmodulin modulates Akt activity in human breast cancer cell lines  

Microsoft Academic Search

Growth factor-induced activation of Akt occurs in the majority of human breast cancer cell lines resulting in a variety of\\u000a cellular outcomes, including suppression of apoptosis and enhanced survival. We demonstrate that epidermal growth factor (EGF)-initiated\\u000a activation of Akt is mediated by the ubiquitous calcium sensing molecule, calmodulin, in the majority of human breast cancer\\u000a cell lines. Specifically, in estrogen

Christine M. Coticchia; Chetana M. Revankar; Tushar B. Deb; Robert B. Dickson; Michael D. Johnson



Antibodies to major histocompatibility antigens produced by hybrid cell lines  

Microsoft Academic Search

FUSION between myeloma cells and spleen cells from immunised donors has been shown to be a successful method of deriving homogeneous anti-SRBC (anti-sheep red blood cell) and anti-TNP antibodies1,2. One of the most powerful features of this approach is that, by cloning, one may easily derive cell lines synthesising monoclonal antibodies despite using non-purified immunogens. The multiple components of a

G. Galfre; S. C. Howe; C. Milstein; G. W. BUTCHER; J. C. HOWARD



Gastric cancer cell lines induced by trichostatin A  

PubMed Central

AIM: To explore the effect of trichostatin A (TSA) on apoptosis and acetylated histone H3 levels in gastric cancer cell lines BGC-823 and SGC-7901. METHODS: The effect of TSA on growth inhibition and apoptosis was examined by MTT, fluorescence microscopy and PI single-labeled flow cytometry. The acetylated histone H3 level was detected by Western blot. RESULTS: TSA induced apoptosis in gastric cancer cell lines BGC-823 and SGC-7901 was in a dose and time-dependent manner. Apoptotic cells varied significantly between TSA treated groups (37.5 ng/mL 72 h for BGC-823 cell line and 75 ng/mL 72 h for SGC-7901 cell line) and control group (0.85 ± 0.14 vs 1.14 ± 0.07, P = 0.02; 0.94 ± 0.07 vs 1.15 ± 0.06, P = 0.02). Morphologic changes of apoptosis, including nuclear chromatin condensation and fluorescence strength, were observed under fluorescence microscopy. TSA treatment in BGC-823 and SGC-7901 cell lines obviously induced cell apoptosis, which was demonstrated by the increased percentage of sub-G1 phase cells, the reduction of G1-phase cells and the increase of apoptosis rates in flow cytometric analysis. The result of Western blot showed that the expression of acetylated histone H3 increased in BGC-823 and SGC-7901 TSA treatment groups as compared with the control group. CONCLUSION: TSA can induce cell apoptosis in BGC-823 and SGC-7901 cell lines. The expression of acetylated histone H3 might be correlated with apoptosis. PMID:18720545

Zou, Xiao-Ming; Li, Yun-Long; Wang, Hao; Cui, Wu; Li, Xiao-Lin; Fu, Song-Bin; Jiang, Hong-Chi



Efficacy of ribavirin against malignant glioma cell lines  

PubMed Central

Ribavirin (1-?-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0–1,000 ?M) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant glioma cell growth. Therefore, the results of the current study indicate that ribavirin may have potential as a therapeutic agent in the treatment of malignant gliomas. PMID:25364409




Utility of L-norephedrine in the semisynthesis of novel thiourea and thiazolidine derivatives as a new class of anticancer agents.  


The natural alkaloid 1-norephedrine 1 was utlized in the synthesis of some novel thiourea derivatives 2, 5 and thiazolidinones 4a,b and 6, 7. Structures of the synthesized compounds were confirmed by analytical and spectral data. The synthesized compounds were evaluated in vitro for anticancer activity against the human breast (MCF-7), human liver (HEPG2) and human colon (HCT116) cancer cell lines. Thiazolidinone derivative 7 was the most active against all the cell lines with values IC50 = 2.60, 2.80 and 2.60 microg/mL compared with doxorubicin (IC50 = 5.40, 2.97 and 5.26 microg/mL). Thiazolidinone derivative 6 exhibited higher activity with IC50 value (3.20 microg/mL) against HCT116 when compared with doxorubicin with IC50 value (5.26 microg/mL) as positive control. PMID:25272887

Ghorab, Mostafa M; Alqasoumi, Saleh I; Abdel-Kader, Maged S; Alsaid, Mansour S



DNA-PK/Ku complex binds to latency-associated nuclear antigen and negatively regulates Kaposi's sarcoma-associated herpesvirus latent replication  

SciTech Connect

During latent infection, latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) plays important roles in episomal persistence and replication. Several host factors are associated with KSHV latent replication. Here, we show that the catalytic subunit of DNA protein kinase (DNA-PKcs), Ku70, and Ku86 bind the N-terminal region of LANA. LANA was phosphorylated by DNA-PK and overexpression of Ku70, but not Ku86, impaired transient replication. The efficiency of transient replication was significantly increased in the HCT116 (Ku86 +/-) cell line, compared to the HCT116 (Ku86 +/+) cell line, suggesting that the DNA-PK/Ku complex negatively regulates KSHV latent replication.

Cha, Seho [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)] [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Lim, Chunghun [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of)] [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Lee, Jae Young [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)] [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of); Song, Yoon-Jae [Department of Life Science, Kyungwon University, Seongnam-Si, Kyeonggi-Do 461-701 (Korea, Republic of)] [Department of Life Science, Kyungwon University, Seongnam-Si, Kyeonggi-Do 461-701 (Korea, Republic of); Park, Junsoo [Division of Biological Science and Technology, Yonsei University, Wonju 220-100 (Korea, Republic of)] [Division of Biological Science and Technology, Yonsei University, Wonju 220-100 (Korea, Republic of); Choe, Joonho [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of)] [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701 (Korea, Republic of); Seo, Taegun, E-mail: [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)] [Department of Life Science, Dongguk Univ-Seoul, Seoul 100-715 (Korea, Republic of)



Optimal first-line and second-line treatments for metastatic renal cell carcinoma: current evidence  

PubMed Central

Since 2005, an abundance of targeted agents has been approved for the treatment of metastatic renal cell carcinoma (mRCC), without any specification as to what may be the most optimal first-line and second-line sequence. Hence, our objective was to critically examine the evidence supporting the use of first-line and second-line agents in the management of mRCC. Our review suggests that in first line, sunitinib and pazopanib represent treatment options for patients with favorable or intermediate-risk features and clear cell histology. Unfortunately, the Phase III trial cannot conclusively prove the noninferiority of pazopanib relative to sunitinib. Hence, the use of sunitinib as first-line standard of care remains justified. Pazopanib represents an option for specific patients in whom sunitinib might not be tolerated. In patients with poor-risk features, temsirolimus represents the only option supported with level 1 evidence. Less optimal alternatives include sunitinib and bevacizumab combined with interferon, based on the minimal inclusion of poor-risk patients in pivotal Phase III studies of these two molecules. In patients with non-clear cell mRCC, the use of temsirolimus is supported by Phase III data, unlike for any other molecule. In second line, the options consist of everolimus and axitinib. However, the axitinib data are substantially more robust given the inclusion of more patients considered as true second-line, and validly justify the choice of axitinib over everolimus. Nonetheless, the Phase III trial of everolimus may be considered as level 1 evidence for use as third-line or subsequent lines of therapy. PMID:25378943

Sun, Maxine; Larcher, Alessandro; Karakiewicz, Pierre I



Rabeprazole exhibits antiproliferative effects on human gastric cancer cell lines  

PubMed Central

Intracellular proton extrusion in gastric cancer cells has been reported to promote cancer cell survival under acidic conditions via hydrogen/potassium adenosine triphosphatase (H+/K+-ATPase). Rabeprazole is a frequently used second-generation proton pump inhibitor (PPI) that irreversibly inactivates gastric H+/K+-ATPase. Therefore, we hypothesized that rabeprazole could reduce the viability of gastric cancer cells. In the present study, four human gastric cancer cell lines and one non-cancer gastric cell line were cultured. Cell viability, the ?- and ?-subunits of H+/K+-ATPase and cellular apoptosis were analyzed by dye exclusion assay, reverse transcription-polymerase chain reaction and annexin V-fluorescein isothiocyanate/propidium iodide staining, respectively. The expression level of total extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and phosphorylated-ERK protein was detected by western blot analysis. Gastric cancer cell lines were more tolerant of the acidic culture media than non-cancer cells. Administration of rabeprazole led to a marked decrease in the viability of MKN-28 cells. Exposure to rabeprazole induced significant apoptosis in AGS cells. Rabeprazole completely inhibited the phosphorylation of ERK 1/2 in the MKN-28 cells, whereas the same effect was not observed in either the KATO III or MKN-45 cells. The ERK 1/2 inhibitor, PD98059, attenuated the viability of the AGS cells. A similar antiproliferative effect was observed in the rabeprazole treatment group. In addition, PD98059 and rabeprazole were able to efficaciously inhibit the phosphorylation of ERK 1/2 in the gastric cancer cells. Therefore, it was concluded that rabeprazole can attenuate the cell viability of human gastric cancer cells through inactivation of the ERK1/2 signaling pathway. The results of the present study demonstrate that rabeprazole inhibits the viability of gastric cancer cells in vitro and may serve as a novel antineoplastic agent.




Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells  

Microsoft Academic Search

Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal

Junying Yu; Maxim A. Vodyanik; Kim Smuga-Otto; Jessica Antosiewicz-Bourget; Jennifer L. Frane; Shulan Tian; Jeff Nie; Gudrun A. Jonsdottir; Victor Ruotti; Ron Stewart; Igor I. Slukvin; James A. Thomson



Novel cell lines established from pediatric brain tumors  

PubMed Central

The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat “fingerprints” matching that of the patient’s tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors. PMID:22120608

Xu, Jingying; Erdreich-Epstein, Anat; Gonzalez-Gomez, Ignacio; Melendez, Elizabeth Y.; Smbatyan, Goar; Moats, Rex A.; Rosol, Michael; Biegel, Jaclyn A.



Solid Oxide Fuel Cell Systems PVL Line  

SciTech Connect

In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to test fuel cell components at a scale and under conditions that can be accurately extrapolated to full system performance. This requires specially designed equipment that replicates the pressure (up to 6.5 bara), temperature (about 910 C), anode and cathode gas compositions, flows and power generation density of the full scale design. The SBTS fuel cell anode gas is produced through the reaction of pipeline natural gas with a mixture of steam, CO2, and O2 in a catalytic partial oxidation (CPOX) reactor. Production of the fuel cell anode gas in this manner provides the capability to test a fuel cell with varying anode gas compositions ranging from traditional reformed natural gas to a coal-syngas surrogate fuel. Stark State College and RRFCS have a history of collaboration. This is based upon SSCAs commitment to provide students with skills for advanced energy industries, and RRFCS need for a workforce that is skilled in high temperature fuel cell development and testing. A key to this approach is the access of students to unique SOFC test and evaluation equipment. This equipment is designed and developed by RRFCS, with the participation of SSC interns. In the near-term, the equipment will be used by RRFCS for technology development. When this stage is completed, and RRFCS has moved to commercial products, SSC will utilize this equipment for workforce training. The RRFCS fuel cell design is based upon a unique ceramic substrate architecture in which a porous, flat substrate (tube) provides the support structure for a network of solid oxide fuel cells that are electrically connected in series. These tubes are grouped into a {approx}350-tube repeat configuration, called a stack/block. Stack/block testing, performed at system conditions, provides data that can be confidently scaled to full scale performance. This is the basis for the specially designed and developed test equipment that is required for advancing and accelerating the RRFCS SOFC power system development program. All contract DE-EE0003229 objectives were achieved and deliverables completed during the peri

Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems



The interaction of normal lymphocytes and cells from lymphoid cell lines  

PubMed Central

Human peripheral blood lymphocytes activated by contact with X-irradiated cells from lymphoblastoid cell lines (LCL cells) acquire cytotoxic capacity directed against LCL cells as demonstrated by release of 51Cr from the labelled target. In a large series of cross-over experiments it was possible to demonstrate an element of specificity in the cytotoxic phase in the sense that lymphocytes activated by irradiated cells from line `A' tended to kill target cells of line `A' more efficiently than those of an unrelated line. This `line-directed' specificity was not absolute and in some experiments could not be demonstrated at all. Several factors could be identified which tend to obscure line-directed specificity. Both specific and non-specific cytotoxicity as observed in this in vitro system are probably relevant to the immunological defence of the intact organism against proliferating aberrant lymphoid cells. PMID:4854907

Steel, C. M.; Hardy, D. A.; Ling, N. R.; Lauder, I. J.



Stable, near-haploid mammalian cell line (Dipodomys ordii)  

Microsoft Academic Search

An established SV40-transformed cell line of Dipodomys ordii was cloned for selective loss of chromosomal material. A clone is described which has a modal chromosome number of 50 (in the normal diploid 2n = 72), and has about 66% of the DNA content of normal diploid cells. Karyotype anlysis shows that, although some chromosome rearrangement has taken place, 23 chromosomes

C. J. Bostock; S. Christie; F. T. Hatch; J. A. Mazrimas



Genome-Wide Analysis in Human Colorectal Cancer Cells Reveals Ischemia-Mediated Expression of Motility Genes via DNA Hypomethylation  

PubMed Central

DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumours, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes. PMID:25079072

Skowronki, Karolina; Andrews, Joseph; Rodenhiser, David I.; Coomber, Brenda L.



76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project  

Federal Register 2010, 2011, 2012, 2013

...Comment Request; Identification of Human Cell Lines Project AGENCY: National Institute...repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All data and...



A systematic RNAi synthetic interaction screen reveals a link between p53 and snoRNP assembly  

Microsoft Academic Search

TP53(tumour protein 53) is one of the most frequently mutated genes in human cancer and its role during cellular transformation has been studied extensively. However, the homeostatic functions of p53 are less well understood. Here, we explore the molecular dependency network of TP53 through an RNAi-mediated synthetic interaction screen employing two HCT116 isogenic cell lines and a genome-scale endoribonuclease-prepared short

Dragomir B. Krastev; Mikolaj Slabicki; Maciej Paszkowski-Rogacz; Nina C. Hubner; Magno Junqueira; Andrej Shevchenko; Matthias Mann; Karla M. Neugebauer; Frank Buchholz



Berberine lowers blood glucose in type 2 diabetes mellitus patients through increasing insulin receptor expression  

Microsoft Academic Search

Our previous work demonstrated that berberine (BBR) increases insulin receptor (InsR) expression and improves glucose utility both in vitro and in animal models. Here, we study the InsR–up-regulating and glucose-lowering activities of BBR in humans. Our results showed that BBR increased InsR messenger RNA and protein expression in a variety of human cell lines, including CEM, HCT-116, SW1990, HT1080, 293T,

Hao Zhang; Jing Wei; Rong Xue; Jin-Dan Wu; Wei Zhao; Zi-Zheng Wang; Shu-Kui Wang; Zheng-Xian Zhou; Dan-Qing Song; Yue-Ming Wang; Huai-Ning Pan; Wei-Jia Kong; Jian-Dong Jiang



Highly potent anti-proliferative effects of a gallium(III) complex with 7-chloroquinoline thiosemicarbazone as a ligand: Synthesis, cytotoxic and antimalarial evaluation.  


A gallium(III) complex with 7-chloroquinoline thiosemicarbazone was synthesized and characterized. The complex proved to be thirty-one times more potent on colon cancer cell line, HCT-116, with considerably less cytotoxicity on non-cancerous colon fibroblast, CCD-18Co, when compared to etoposide. Its anti-malarial potential on 3D7 isolate of Plasmodium falciparum was better than lumefantrine. PMID:25147149

Kumar, Kewal; Schniper, Sarah; González-Sarrías, Antonio; Holder, Alvin A; Sanders, Natalie; Sullivan, David; Jarrett, William L; Davis, Krystyn; Bai, Fengwei; Seeram, Navindra P; Kumar, Vipan



Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines  

PubMed Central

Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study. PMID:20011515

Crameri, Gary; Todd, Shawn; Grimley, Samantha; McEachern, Jennifer A.; Marsh, Glenn A.; Smith, Craig; Tachedjian, Mary; De Jong, Carol; Virtue, Elena R.; Yu, Meng; Bulach, Dieter; Liu, Jun-Ping; Michalski, Wojtek P.; Middleton, Deborah; Field, Hume E.; Wang, Lin-Fa



Polo-like kinase 1 inhibitors, mitotic stress and the tumor suppressor p53  

PubMed Central

Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small-molecule inhibitors targeting this kinase have been developed and intensively investigated. Recently, it has been reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with inactive p53, leading to the hypothesis that inactive p53 is a predictive marker for the response of Plk1 inhibition. In our previous study based on different cancer cell lines, we showed that cancer cells with wild type p53 were more sensitive to Plk1 inhibition by inducing more apoptosis, compared with cancer cells depleted of p53. In the present work, we further demonstrate that in the presence of mitotic stress induced by different agents, Plk1 inhibitors strongly induced apoptosis in HCT116 p53+/+ cells, whereas HCT116 p53?/? cells arrested in mitosis with less apoptosis. Depletion of p53 in HCT116 p53+/+ or U2OS cells reduced the induction of apoptosis. Moreover, the surviving HCT116 p53?/? cells showed DNA damage and a strong capability of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition. PMID:23574746

Sanhaji, Mourad; Louwen, Frank; Zimmer, Brigitte; Kreis, Nina-Naomi; Roth, Susanne; Yuan, Juping



90-kDa Heat Shock Protein Inhibition Abrogates the Topoisomerase I Poison-Induced G2/M Checkpoint in p53-Null Tumor Cells by Depleting Chk1 and Wee1S?  

PubMed Central

The G2/M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G2/M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time- and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G2/M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G2/M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1. PMID:18820127

Tse, Archie N.; Sheikh, Tahir N.; Alan, Ho; Chou, Ting-Chao; Schwartz, Gary K.



Generation and establishment of murine adherent cell lines.  


We describe a method to derive cell lines and clones from cells of the murine midgestation aorta-gonads-mesonephros (AGM) microenvironment. We start from subdissected AGM regions in "explant" or "single cell suspension" type cultures from embryos transgenic for tsA58, a temperature-sensitive mutant of the SV40 T antigen gene. The number of cells in such cultures initially expand, but in most cases, this expansion phase is followed by a stable or even decline in cell number. After this so-called crisis phase, cell proliferation is noticeable in more than 90% of the cultures. Stromal cell clones can be isolated from these cultures, some of which have been cultured for more than 50 population doublings, and functionally characterized using various methods These stromal cell clones are valuable tools for the study of the regulation of hematopoietic stem and progenitor cells in the midgestation mouse embryo. PMID:23179840

Istvanffy, Rouzanna; Oostendorp, Robert A J



A better cell line for making hybridomas secreting specific antibodies  

Microsoft Academic Search

FUSION of myeloma cells which grow in tissue culture with spleen cells from an immunised mouse provides a general method for obtaining cell lines (hybridomas) which make antibody of the desired specificity1-3. Hybrids derived from these myelomas make the immunoglobulin (Ig) heavy and light chains of the myeloma parent as well as the antigen-specific heavy and light chains of the

Marc Shulman; C. D. Wilde; Georges Köhler



2,4-Dihydroxychalcone derivatives as novel potent cell division cycle 25B phosphatase inhibitors and protein tyrosine phosphatase 1B inhibitors.  


Eleven 2,4-dihydroxychalcone compounds were synthesized and identified as reversible and competitive cell division cycle 25 (CDC25) B and protein tyrosine phosphatase (PTP) 1B inhibitors with inhibition values in the micromolar range. The results showed that nine compounds significantly inhibited CDC25B phosphatase, whereas seven compounds inhibited the activity against PTP1B in vitro. Compound 8 had the greatest inhibition activity against CDC25B and PTP1B in vitro, with percentage inhibition values of 97.5% and 96.3% at a dose of 20 microg/mL, respectively. Cytotoxic activity assays revealed that compound 8 was the most potent against HCT116, HeLa, and A549 cells. Furthermore, compound 8 exhibited potent antitumor activity in a colo205 xenograft model. PMID:24791588

Xie, Chao; Sun, Yuan; Pan, Cheng-Yan; Tang, Li-Ming; Guan, Li-Ping



Non-targeted radiation effects in vertebrate cell lines  

NASA Astrophysics Data System (ADS)

Radiation effects, such as bystander effects, hyper radiosensitivity/induced radioresistance (HRS/IRR) and adaptive response that are not related to direct DNA damage are now accepted. However the inter-relationship between them and the possible impact on the scientific basis for radiation protection are highly controversial. This thesis attempts to elucidate the mechanisms of some of these well known but little understood effects. Each paper examines some aspect of bystander effects, adaptive responses and HRS/IRR in an effort to understand how they vary with cell type, dose and time of exposure to single or multiple doses. All the effects involve non-linear dose effect curves and are mainly evident following low doses. Overall findings of the thesis include (1) A clear difference was observed between radioresistant, tumorigenic cell lines with mutant p53 gene expression, and radiosensitive, more normal, cell lines with wild type p53. In general death inducing bystander responses are induced in normal cell populations exposed to low doses of radiation while survival inducing IRR and adaptive responses are seen in the radioresistant tumorigenic cell lines. (2) A cohort of fish cell lines which demonstrated survival promoting bystander effects, also did not show a protective adaptive responses. (3) Adaptive responses traditionally occur when a large challenge dose is given 4--6hrs following low (10--100mGy) priming doses but this thesis shows that for the epithelial cell lines tested, the size of the priming dose (range 0.1--2Gy) does not appear to alter the size of the recovery response. Additionally increased survival could be detected in some cases when the challenge dose was given within one hour of the priming dose. The overall conclusion is that cell lines induce either a bystander response or a protective/adaptive response depending on genetic background and other factors. Care is needed in the interpretation of data generated from only one or two cell lines and in the extrapolation of mechanistic ideas based on one or two cell lines to other cell types or to the in vivo situation.

Ryan, Lorna


Adaptation of an insect cell line ( Agallia constricta ) in a mammalian cell culture medium  

Microsoft Academic Search

Summary  An established insect cell line (AC20) from the leafhopperAgallia constricta has been adapted to a mammalian cell culture medium based on the formulation of two commercially available media. The cell\\u000a population doubling time of the adapted line in this medium is approximately 45 hr at 30C.

Arthur H. Mc Intosh; K. Maramorosch; C. Rechtoris



Female Sex Bias in Human Embryonic Stem Cell Lines  

PubMed Central

The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat



Failure of cell cleavage induces senescence in tetraploid primary cells.  


Tetraploidy can arise from various mitotic or cleavage defects in mammalian cells, and inheritance of multiple centrosomes induces aneuploidy when tetraploid cells continue to cycle. Arrest of the tetraploid cell cycle is therefore potentially a critical cellular control. We report here that primary rat embryo fibroblasts (REF52) and human foreskin fibroblasts become senescent in tetraploid G1 after drug- or small interfering RNA (siRNA)-induced failure of cell cleavage. In contrast, T-antigen-transformed REF52 and p53+/+ HCT116 tumor cells rapidly become aneuploid by continuing to cycle after cleavage failure. Tetraploid primary cells quickly become quiescent, as determined by loss of the Ki-67 proliferation marker and of the fluorescent ubiquitination-based cell cycle indicator/late cell cycle marker geminin. Arrest is not due to DNA damage, as the ?-H2AX DNA damage marker remains at control levels after tetraploidy induction. Arrested tetraploid cells finally become senescent, as determined by SA-?-galactosidase activity. Tetraploid arrest is dependent on p16INK4a expression, as siRNA suppression of p16INK4a bypasses tetraploid arrest, permitting primary cells to become aneuploid. We conclude that tetraploid primary cells can become senescent without DNA damage and that induction of senescence is critical to tetraploidy arrest. PMID:25143403

Panopoulos, Andreas; Pacios-Bras, Cristina; Choi, Justin; Yenjerla, Mythili; Sussman, Mark A; Fotedar, Rati; Margolis, Robert L



Ginsenoside compound K, not Rb1, possesses potential chemopreventive activities in human colorectal cancer.  


Ginsenoside compound K (C-K) is an intestinal microbiota metabolite of ginsenoside Rb1, a major constituent in American ginseng. However, previous ginseng anti-cancer observations were largely focused on ginseng parent compounds but not metabolites, and anti-colorectal cancer studies on C-K were limited. This study investigated the anti-proliferative effects of C-K when compared to those of Rb1, and the related mechanisms of action, in HCT-116 and SW-480 colorectal cancer cells. The effects of Rb1 and C-K on the proliferation of HCT-116 and SW-480 human colorectal cancer cells were compared using an MTS assay. Cell cycle and cell apoptosis were assayed using flow cytometry. Enzymatic activities of caspases were determined by colorimetric assay, and interactions of C-K and caspases were explored by docking analysis. C-K showed significant anti-proliferative effects in HCT-116 and SW-480 cells at concentrations of 30-50 µM. At the same concentrations, Rb1 did not show any effects, while C-K arrested the cells in the G1 phase, and significantly induced cell apoptosis. Compared to HCT-116 (p53 wild-type), the p53 mutant cell line SW-480 was more sensitive to C-K as assessed by cell cycle regulation and apoptosis induction. C-K activated expression of caspases 8 and 9, consistent with docking analysis. The docking data suggested that C-K forms hydrogen bonds with Lys253, Thr904 and Gly362 in caspase 8, and with Thr62, Ser63 and Arg207 in caspase 9. C-K, but not its parent ginsenoside Rb1, showed significant anti-proliferative and pro-apoptotic effects in human colorectal cancer cells. These results suggest that C-K could be a potentially effective anti-colorectal cancer agent. PMID:22426808

Wang, Chong-Zhi; Du, Guang-Jian; Zhang, Zhiyu; Wen, Xiao-Dong; Calway, Tyler; Zhen, Zhong; Musch, Mark W; Bissonnette, Marc; Chang, Eugene B; Yuan, Chun-Su



Ginsenoside compound K, not Rb1, possesses potential chemopreventive activities in human colorectal cancer  

PubMed Central

Ginsenoside compound K (C-K) is an intestinal microbiota metabolite of ginsenoside Rb1, a major constituent in American ginseng. However, previous ginseng anticancer observations were largely focused on ginseng parent compounds but not metabolites, and anti-colorectal cancer studies on C-K were limited. This study investigated the antiproliferative effects of C-K when compared to those of Rb1, and the related mechanisms of action, in HCT-116 and SW-480 colorectal cancer cells. The effects of Rb1 and C-K on the proliferation of HCT-116 and SW-480 human colorectal cancer cells were compared using an MTS assay. Cell cycle and cell apoptosis were assayed using flow cytometry. Enzymatic activities of caspases were determined by colorimetric assay, and interactions of C-K and caspases were explored by docking analysis. C-K showed significant antiproliferative effects in HCT-116 and SW-480 cells at concentrations of 30–50 ?M. At the same concentrations, Rb1 did not show any effects, while C-K arrested the cells in the G1 phase, and significantly induced cell apoptosis. Compared to HCT-116 (p53 wild type), the p53 mutant cell line SW-480 was more sensitive to C-K as assessed by cell cycle regulation and apoptosis induction. C-K activated expression of caspases 8 and 9, consistent with docking analysis. The docking data suggested that C-K forms hydrogen bonds with Lys-253, Thr-904 and Gly-362 in caspase 8, and with Thr-62, Ser-63 and Arg-207 in caspase 9. C-K, but not its parent ginsenoside Rb1, showed significant antiproliferative and pro-apoptotic effects in human colorectal cancer cells. These results suggest that C-K could be a potentially effective anti-colorectal cancer agent. PMID:22426808




Human neoplastic cells in tissue culture: two established cell lines derived from giant cell tumor and fibrosarcoma.  


The establishment and cultivation of two human neoplastic cell lines is described. The cell line B-5GT was derived from bone giant cell tumor and B-6FS from poorly differentiated fibrosarcoma. In comparison to the normal skin fibroblasts both cell lines have a potential for "indefinite" multiplication in vitro and they exhibit growth properties which are associated with malignant transformation. The parameters investigated included cell morphology, chromosome characteristics, terminal cell density, growth pattern, residual DNA synthesis and growth in soft agar. Both cell lines exhibited human karyotype with aneuploidy and differed in their karyotype from each other. PMID:1004657

Thurzo, V; Popovic, M; Matoska, J; Blasko, M; Grófová, M; Lizonová, A; Steno, M



Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines  

PubMed Central

Background The treatment of oral squamous cell carcinomas (OSCC) and human osteosarcoma (HOS) includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang) on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20%) were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50) for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ?80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis. PMID:20840769



Cystatin SN neutralizes the inhibitory effect of cystatin C on cathepsin B activity.  


Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3. PMID:24357805

Kim, J-T; Lee, S-J; Kang, M A; Park, J E; Kim, B-Y; Yoon, D-Y; Yang, Y; Lee, C-H; Yeom, Y I; Choe, Y-K; Lee, H G



Inhibitory effect of cinnamoyl compounds against human malignant cell line.  


In the present study, anti-proliferative effects of dietary polyphenolic compounds have been observed and demonstrated the strong anticancer efficacy of curcumin (CMN), an active constituent of dietary spice (turmeric) using human leukemia cancer cell line. CMN inhibited the proliferation of K562 leukemic cells by induction of apoptosis. The current study demonstrated synergy with combination of drug therapy, and suggested that combination of ferulic acid and cisplatin synergistically inhibited cellular proliferation. Cytotoxic synergy was observed independent of the sequence of addition of two drugs to cultured cells. The synergized growth inhibitory effect with cisplatin was probably associated with G2-M arrest in cell cycle progression. These findings suggested that among the cinnamoyl compounds, CMN was most potent and FER appeared to be a better modulating agent on human malignant cell line. PMID:16538860

Indap, M A; Radhika, S; Motiwale, Leena; Rao, K V K



Development of immortalized mouse aortic endothelial cell lines  

PubMed Central

Background The understanding of endothelial cell biology has been facilitated by the availability of primary endothelial cell cultures from a variety of sites and species; however, the isolation and maintenance of primary mouse aortic endothelial cells (MAECs) remain a formidable challenge. Culturing MAECs is difficult as they are prone to phenotypic drift during culture. Therefore, there is a need to have a dependable in vitro culture system, wherein the primary endothelial cells retain their properties and phenotypes. Methods Here, we developed an effective method to prepare immortalized MAEC (iMAEC) lines. Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen. Immortalized cells were then incubated with DiI-acetylated-low density lipoprotein and sorted via flow cytometry to isolate iMAECs. Results iMAECs expressed common markers of endothelial cells, including PECAM1, eNOS, VE-cadherin, and von Willebrand Factor. iMAECs aligned in the direction of imposed laminar shear and retained the ability to form tubes. Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-. Conclusion In summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology. PMID:24690145



Expression of the somatostatin gene in human astrocytoma cell lines.  


Somatostatin (somatotropin release-inhibiting hormone; SRIH) has been demonstrated in neurons of the central nervous system (CNS) as well as in endocrine cells of the pancreas and gastrointestinal tract and can suppress various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production. Since astrocytes possess antigen-presenting activity and can secrete a wide array of immunoregulatory and inflammatory cytokines, we studied SRIH gene expression in both astrocyte cell lines and mitogen-stimulated peripheral blood mononuclear leukocytes from healthy donors. We now report by means of a complementary DNA-based reverse transcription PCR that differential levels of SRIH mRNA were expressed in 9 of 11 human astrocytoma cell lines tested but were undetectable in activated peripheral blood mononuclear leukocytes as well as in a variety of human lymphocyte and monocyte cell lines. The synthesis and secretion of SRIH protein by astrocytoma cells that expressed SRIH transcripts were confirmed by specific radioimmunoassay of cell culture fluids. These findings support the notion that SRIH gene expression occurs in human astrocytoma cells but not in mature lymphoid cells of the immune system. PMID:8991628

Mercure, L; Tannenbaum, G S; Schipper, H M; Phaneuf, D; Wainberg, M A



Characterization of a Breast Cancer Cell Line Derived from a Germ-Line BRCA1 Mutation Carrier1  

Microsoft Academic Search

A tumor cell line, HCC1937, was established from a primary breast carcinoma from a 24-year-old patient with a germ-line \\/\\/AT t \\/ mutation. A corresponding B-lymphoblastoid cell line was established from the patient's peripheral blood lymphocytes. BRCA1 analysis revealed that the tumor cell line is homozygous for the BRCA1 5382insC mutation, whereas the patient's lymphocyte DNA is heterozygous for the

Gail E. Tomlinson; Victor A. Stastny; Arvind K. Virmani; Monique A. Spillman; Vijay Tonk; Joanne L. Blum; Nancy R. Schneider; Ignacio I. Wistuba; Jerry W. Shay; John D. Minna; Adi F. Gazdar


Subcutaneous preconditioning increases invasion and metastatic dissemination in mouse colorectal cancer models  

PubMed Central

Mouse colorectal cancer (CRC) models generated by orthotopic microinjection of human CRC cell lines reproduce the pattern of lymphatic, haematological and transcoelomic spread but generate low metastatic efficiency. Our aim was to develop a new strategy that could increase the metastatic efficiency of these models. We used subcutaneous implantation of the human CRC cell lines HCT116 or SW48 prior to their orthotopic microinjection in the cecum of nude mice (SC+ORT). This subcutaneous preconditioning significantly enhanced metastatic dissemination. In the HCT116 model it increased the number and size of metastatic foci in lymph nodes, lung, liver and peritoneum, whereas, in the SW48 model, it induced a shift from non-metastatic to metastatic. In both models the number of apoptotic bodies in the primary tumour in the SC+ORT group was significantly reduced compared with that in the direct orthotopic injection (ORT) group. Moreover, in HCT116 tumours the number of keratin-positive tumour buddings and single epithelial cells increased at the invasion front in SC+ORT mice. In the SW48 tumour model, we observed a trend towards a higher number of tumour buds and single cells in the SC+ORT group but this did not reach statistical significance. At a molecular level, the enhanced metastatic efficiency observed in the HCT116 SC+ORT model was associated with an increase in AKT activation, VEGF-A overexpression and downregulation of ?1 integrin in primary tumour tissue, whereas, in SW48 SC+ORT mice, the level of expression of these proteins remained unchanged. In summary, subcutaneous preconditioning increased the metastatic dissemination of both orthotopic CRC models by increasing tumour cell survival and invasion at the tumour invasion front. This approach could be useful to simultaneously study the mechanisms of metastases and to evaluate anti-metastatic drugs against CRC. PMID:24487410

Alamo, Patricia; Gallardo, Alberto; Pavón, Miguel A.; Casanova, Isolda; Trias, Manuel; Mangues, Maria A.; Vázquez, Esther; Villaverde, Antonio; Mangues, Ramon; Céspedes, Maria V.



VR09 Cell Line: An EBV-Positive Lymphoblastoid Cell Line with In Vivo Characteristics of Diffuse Large B Cell Lymphoma of Activated B-Cell Type  

PubMed Central

Background small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro. Design and Method we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features. Results VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2?/? ?-chain?/? mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/? CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, ?- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53. Conclusion This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features. PMID:23285191

Nichele, Ilaria; Zamo, Alberto; Bertolaso, Anna; Bifari, Francesco; Tinelli, Martina; Franchini, Marta; Stradoni, Roberta; Aprili, Fiorenza; Pizzolo, Giovanni; Krampera, Mauro



The Natural Compound Cantharidin Induces Cancer Cell Death through Inhibition of Heat Shock Protein 70 (HSP70) and Bcl-2-associated Athanogene Domain 3 (BAG3) Expression by Blocking Heat Shock Factor 1 (HSF1) Binding to Promoters*  

PubMed Central

Heat shock factor 1 (HSF1) enhances the survival of cancer cells under various stresses. The knock-out of HSF1 impairs cancer formation and progression, suggesting that HSF1 is a promising therapeutic target. To identify inhibitors of HSF1 activity, we performed cell-based screening with a library of marketed and experimental drugs and identified cantharidin as an HSF1 inhibitor. Cantharidin is a potent antitumor agent from traditional Chinese medicine. Cantharidin inhibited heat shock-induced luciferase activity with an IC50 of 4.2 ?m. In contrast, cantharidin did not inhibit NF-?B luciferase reporter activity, demonstrating that cantharidin is not a general transcription inhibitor. When the HCT-116 colorectal cancer cells were exposed to heat shock in the presence of cantharidin, the induction of HSF1 downstream target proteins, such as HSP70 and BAG3 (Bcl-2-associated athanogene domain 3), was suppressed. HSP70 and its co-chaperone BAG3 have been reported to protect cells from apoptosis by stabilizing anti-apoptotic Bcl-2 family proteins. As expected, treating HCT-116 cancer cells with cantharidin significantly decreased the amounts of BCL-2, BCL-xL, and MCL-1 protein and induced apoptotic cell death. Chromatin immunoprecipitation analysis showed that cantharidin inhibited the binding of HSF1 to the HSP70 promoter and subsequently blocked HSF1-dependent p-TEFb recruitment. Therefore, the p-TEFb-dependent phosphorylation of the C-terminal domain of RNA polymerase II was blocked, arresting transcription at the elongation step. Protein phosphatase 2A inhibition with PP2CA siRNA or okadaic acid did not block HSF1 activity, suggesting that cantharidin inhibits HSF1 in a protein phosphatase 2A-independent manner. We show for the first time that cantharidin inhibits HSF1 transcriptional activity. PMID:23983126

Kim, Joo Ae; Kim, Youngmi; Kwon, Byoung-Mog; Han, Dong Cho



A CNS catecholaminergic cell line expresses voltage-gated currents.  


CATH.a is a central nervous system (CNS) catecholaminergic cell line derived from a transgenic mouse carrying the SV40 T antigen oncogene under the transcriptional control of regulatory elements from the rat tyrosine hydroxylase gene (Suri et al., 1993). CATH.a cells express several differentiated neuronal characteristics including medium and light chain neurofilament proteins, synaptophysin, tyrosine hydroxylase, and dopamine beta-hydroxylase; they synthesize dopamine and norepinephrine. Conversely, they do not express glial-specific fibrillary acidic protein. To establish definitively that CATH.a cells are of neuronal origin, we characterized the repertoire of voltage-gated inward currents expressed by CATH.a cells. Such inward currents are necessary for neuronal excitability. We report that all CATH.a cells possess a tetrodotoxin-sensitive sodium current (peak amplitude = 590 +/- 319 pA) and 68% possess a high voltage-activated calcium current (peak amplitude = 175 +/- 67 pA). Pharmacological analyses suggest that individual cells express varying levels of L- and N-type calcium current, but no P-type current. In addition, in 55% of the cells with a calcium current, about a half of this current is resistant to selective antagonists for L- and N-type currents, suggesting that another calcium current exists in these CATH.a cells which is not L-, N-, or P-type. The heterogeneous pattern of current detected persisted in several CATH. a subclones, suggesting that factors other than genetic variability influence current expression. The demonstration that CATH.a cells express these currents indicates that they have excitable membrane properties characteristic of neurons. Although many peripheral nervous system (PNS) cell lines exist, very few CNS cell lines with differentiated neuronal properties exist. Since the CATH.a cells can be grown continuously in large amounts, they may be useful for purifying, characterizing, and/or cloning various neuronal-specific molecules and thereby may add to our understanding of CNS catecholaminergic neurons. PMID:8661508

Lazaroff, M; Dunlap, K; Chikaraishi, D M



Molecular cytogenetic analysis of breast cancer cell lines  

PubMed Central

The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at © 2000 Cancer Research Campaign PMID:11044355

Davidson, J M; Gorringe, K L; Chin, S-F; Orsetti, B; Besret, C; Courtay-Cahen, C; Roberts, I; Theillet, C; Caldas, C; Edwards, P A W



Macrophage cell lines use CD81 in cell growth regulation  

Microsoft Academic Search

CD81 is an integral membrane protein belonging to the tetraspanin superfamily. It has two extracellular domains that interact\\u000a with cell surface proteins and two intracellular tails that contribute to cellular processes. Although there are considerable\\u000a data about how CD81 affects T- and B-cell function, not much is known about how it impacts macrophages. To address this, we\\u000a established four cell

Whitney J. Mordica; Keith M. Woods; Rollie J. Clem; A. Lorena Passarelli; Stephen K. Chapes



Validating classical line profile analyses using microbeam diffraction from individual dislocation cell walls and cell interiors  

SciTech Connect

Dislocation structures in deformed metals produce broad asymmetric diffraction line profiles. During analysis, these profiles are generally separated into two nearly symmetric subprofiles corresponding to diffraction by dislocation cell walls and cell interiors. These subprofiles are then interpreted using complex models of dislocation-based line broadening. Until now, it has not been possible to test the many assumptions that are made in such an analysis. Here, depth-resolved microbeam diffraction was used to measure diffraction line profiles from numerous individual dislocation cell walls and cell interiors in a heavily deformed Cu single crystal. Summing these profiles directly constructed the cell-interior and cell-wall subprofiles that have been approximated in the line profile analysis literature for the past 30 years. Direct comparison between the reconstructed subprofiles and the macroscopic asymmetric line profile from the same sample allows the first direct tests of many of the assumptions that have been used for interpreting these X-ray measurements.

Levine, Lyle E. [National Institute of Standards and Technology (NIST); Geantil, P. [University of Southern California; Larson, Ben C [ORNL; Tischler, Jonathan Zachary [ORNL; Kassner, Michael E. [University of Southern California; Liu, Wenjun [Argonne National Laboratory (ANL)



Cell membrane fatty acid composition differs between normal and malignant cell lines.  


Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska



The Sponge-Derived Fijianolide Polyketide Class: Further Evaluation of Their Structural and Cytotoxicity Properties  

PubMed Central

The sponge derived polyketide macrolides fijianolides A (1) and B (2) (a.k.a. isolaulimalide and laulimalide) have taxol-like microtubule-stabilizing activity and the latter exhibits potent cytotoxicity. Insight on the biogeographical and phenotypic variations of Cacospongia mycofijiensis is presented that will enable future study of the biosynthetic pathway that produces the fijianolides. In addition to fijianolides A and B, six new fijianolides, D–I (7–12), were isolated, each with modifications to the C-20 side chain of the macrolide ring. Compounds 7–12 exhibited a range of in vitro activities against HCT-116 and MDA-MB-435 cell lines. Fijianolides 8 and 10 were shown to disrupt interphase and mitotic division but were less potent than 2. An in vivo evaluation of 2 using tumor-bearing SCID mice demonstrated significant inhibition of growth in HCT-116 tumors over 28 days. PMID:17622130

Johnson, Tyler A.; Tenney, Karen; Cichewicz, Robert H.; Morinaka, Brandon I.; White, Kimberly N.; Amagata, Taro; Subramanian, Balanehru; Media, Joseph; Mooberry, Susan L.; Valeriote, Frederick A.; Crews, Phillip



A CNS Catecholaminergic Cell Line Expresses Voltage-gated Currents  

Microsoft Academic Search

.   CATH.a is a central nervous system (CNS) catecholaminergic cell line derived from a transgenic mouse carrying the SV40 T antigen\\u000a oncogene under the transcriptional control of regulatory elements from the rat tyrosine hydroxylase gene (Suri et al., 1993).\\u000a CATH.a cells express several differentiated neuronal characteristics including medium and light chain neurofilament proteins,\\u000a synaptophysin, tyrosine hydroxylase, and dopamine ?-hydroxylase; they

M. Lazaroff; K. Dunlap; D. M. Chikaraishi



Factors Affecting Antigen Uptake by Human Intestinal Epithelial Cell Lines  

Microsoft Academic Search

We assessed the role of size, solubility, and prophagocytic cytokines interferon-? (IFN-?), and granulocyte-macrophage colony stimulatory factor (GM-CSF) in antigen uptake and kinetics by intestinal epithelial cells using keyhole limpet hemocyanin and ovalbumin. Both fluoresceinated keyhole limpet hemocyanin (3000–7500 kDa) and fluoresceinated ovalbumin (45 kDa) were internalized by human colonic epithelial cell lines, with kinetics similar to those of fluoresceinated

AgnesLaiping So; Gillian Small; Kirk Sperber; Kai Becker; Erwin Oei; Max Tyorkin; Lloyd Mayer



Differential effect of artemisinin against cancer cell lines.  


The present study aims at defining the differential cytotoxicity effect of artemisinin toward P815 (murin mastocytoma) and BSR (kidney adenocarcinoma of hamster) cell lines. Cytotoxicity was measured by the growth inhibition using MTT assay. These in vitro cytotoxicity studies were complemented by the determination of apoptotic DNA fragmentation and Annexin V- streptavidin-FITC assay. Furthermore, we examined the in vitro synergism between artemisinin and the chemotherapeutic drug, vincristin. The in vivo study was investigated using the DBA2/P815 (H2d) mouse model. While artemisinin acted on both tumor cell lines, P815 was much more sensitive to this drug than BSR cells, as revealed by the respective IC50 values (12 µM for P815 and 52 µM for BSR cells). On another hand, and interestingly, apoptosis was induced in P815 but not induced in BSR. These data, reveal an interesting differential cytotoxic effect, suggesting the existence of different molecular interactions between artemisinin and the studied cell lines. In vivo, our results clearly showed that the oral administration of artemisinin inhibited solid tumor development. Our study demonstrates that artemisinin caused differential cytotoxic effects depending not only on the concentration and time of exposure but also on the target cells. PMID:24955301

Tilaoui, Mounir; Mouse, Hassan Ait; Jaafari, Abdeslam; Zyad, Abdelmajid



Zebrafish kidney stromal cell lines support multilineage hematopoiesis  

PubMed Central

Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish. PMID:19433857

Stachura, David L.; Reyes, Jason R.; Bartunek, Petr; Paw, Barry H.; Zon, Leonard I.



Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property  

Microsoft Academic Search

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal

R Salingcarnboriboon; H Yoshitake; K Tsuji; M Obinata; T Amagasa; A Nifuji; M Noda



Promotion of cell proliferation and inhibition of ADCC by cancerous immunoglobulin expressed in cancer cell lines  

Microsoft Academic Search

To explore the significance of cancerous immunoglobulin (Ig) in cancer cell growth, HeLa cervical cancer cells were stably transfected with small interfering RNA (siRNA) that specifically, efficiently and consistently silences the expression of heavy chain genes of all immunoglobulin isotypes. This stable cell line was used to examine cell viability, colony formation and tumor growth in athymic nude mice. The

Ming Li; Hui Zheng; Zhi Duan; Haidan Liu; Duosha Hu; Ann Bode; Zigang Dong; Ya Cao



Cadmium and mercury toxicity in a human fetal hepatic cell line (WRL-68 cells)  

Microsoft Academic Search

The toxic effects of cadmium (Cd) and mercury (Hg), as chloride salts, were studied using an hepatic human fetal cell line (WRL-68 cells). From viability curves and the proliferative capacity of the cell in the presence of the metal, three different cell treatments were chosen, (1) 0.5 ?M of the metal chloride for 24 h (acute low dose treatment), (2)

L. Bucio; V. Souza; A. Albores; A. Sierra; E. Chávez; A. Cárabez; M. C. Gutiérrez-Ruiz




EPA Science Inventory

The need to deploy IN VITRO models to test neurotoxic scribes the use of by industry and government regulatory agencies. his research describes the neuroblastoma cell lines to address the relationship between esterase inhibition and neurotoxic outcome following exposure to organo...


Differential Sensitivity in the Survival of Oligodendrocyte Cell Lines to  

E-print Network

Differential Sensitivity in the Survival of Oligodendrocyte Cell Lines to Overexpression of Myelin in oligodendrocyte survival by overexpression studies in vitro and in vivo. The classic and sr proteolipids are targeted to different cellular com- partments in the oligodendrocyte, suggesting different cellular

Bongarzone, Ernesto R.


Characterization of a Selenocystine-Resistant Carrot Cell Line 1  

PubMed Central

A selenocystine-resistant carrot cell line, C-1, was isolated from a haploid carrot (Daucus carota) cell culture, HA. The C-1 variant takes up cystine, but not cysteine, more slowly than does HA. The selenocystine resistance is maintained in culture in the absence of selection and is expressed in regenerated plants. Results based on chromatographic separation of sulfur metabolites from cells fed with [35S]cystine suggest a block either in the uptake or reduction of cystine in the variant. Both lines can grow on cystine as sole sulfur source. Growth of the HA line on cystine suppressed the development of sulfate uptake capacity (Furner, Sung 1982 Proc Natl Acad Sci USA 79: 1149-1153), while cystine-grown C-1 cells have high levels of sulfate uptake capacity. We suggest that the C-1 line, grown on cystine, accumulates an insufficient quantity of some sulfur metabolite, which is involved in the control of sulfate uptake, to suppress the uptake. C-1 grown on cystine is more sensitive than HA to growth inhibition by the sulfate analog selenate. PMID:16662864

Furner, Ian J.; Sung, Zinmay R.



Spontaneous malignant transformation in two epithelial cell lines of rat liver cells.  


The cellular morphology, chromosomal structure, and tumorigenicity of two lines (B and J-13) of rat epithelial cells were examined serially during in vitro cultivation. The cells for such cultures were derived from the hepatic tissues of two 7-day-old male rats of the Donryu strain. The cultured cells were first inoculated into newborn syngenetic rats on the 641st day in vitro (80th subcultures) for line B, and on the 446th day (58 subcultures) for line J-13. The inoculated cells produced tumors with hemorrhagic ascites in rats after long latent periods, viz, 215-599 days in line B and 170-369 days in line J-13. All the tumors were undifferentiated hepatocarcinomas. The pleomorphism in shape and size of the cultured cells gradually became obvious with time of cultivation and was more pronounced in recultured tumor cells. Chromosomes of the culured cells were a normal diploid pattern until about the 200th day in vitro, but thereafter the modal chromsome number shifted to hypodiploid or hypotriploid via hypodiploid stages. The chromosome constitution of recultured tumor cells resembled that of inoculated cells in number distribution, but had changed to a more complicated karyotype. In experiments with line B, the same marker chromosome was detected in all tumor cells analyzed as had been present in inoculated cells. PMID:188308

Masuji, H; Sato, J



Human small cell lung cancer cell lines express functional atrial natriuretic peptide receptors.  


Small cell lung cancer cell (SCLC) lines, NCI-H82, NCI-H660, and NCI-H1284, and HeLa cells were analyzed for the presence of atrial natriuretic peptide (ANP) receptors. In these SCLC cell lines and HeLa cells, ANP A receptor mRNA was identified by Southern blot analyses of polymerase chain reaction products and RNase protection assays using poly(A)(+)-selected RNA. Saturable binding assays revealed that HeLa cells had 2000 to 5000 high affinity atrial natriuretic peptide receptors per cell with a dissociation constant of 140 pM. In the SCLC cell lines, the binding was saturable but too low to accurately estimate the number of binding sites. After addition of human ANP, radioimmunoassays revealed accumulation of cyclic GMP in SCLC cells as well as HeLa cells in a dose-dependent fashion. The half-maximal stimulation concentration of cyclic GMP accumulation in HeLa and these SCLC cell lines was approximately 2 nM. Tetrazolyl blue assays and tritiated thymidine incorporation did not show any remarkable growth inhibition or growth stimulation of SCLC cell lines after addition of human ANP up to 3.3 microM, more than 1000-fold greater than the half-maximal stimulation concentration of cyclic GMP accumulation. Our results indicate that human SCLC cells express functional ANP receptors but ANP addition produced no detectable change in their growth pattern. PMID:8391389

Ohsaki, Y; Yang, H K; Le, P T; Jensen, R T; Johnson, B E



Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines  

PubMed Central

Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 ?g/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p?cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the anticancer potential of açaí may help in the development of chemopreventive drugs and may have therapeutic effects in the treatment of breast cancer. PMID:24886139



The HSP90 inhibitor ganetespib has chemosensitizer and radiosensitizer activity in colorectal cancer.  


The integration of targeted agents to standard cytotoxic regimens has improved outcomes for patients with colorectal cancer (CRC) over recent years; however this malignancy remains the second leading cause of cancer mortality in industrialized countries. Small molecule inhibitors of heat shock protein 90 (HSP90) are one of the most actively pursued classes of compounds for the development of new cancer therapies. Here we evaluated the activity of ganetespib, a second-generation HSP90 inhibitor, in models of CRC. Ganetespib reduced cell viability in a panel of CRC cell lines in vitro with low nanomolar potency. Mechanistically, drug treatment exerted concomitant effects on multiple oncogenic signaling pathways, cell cycle regulation, and DNA damage repair capacity to promote apoptosis. Combinations of ganetespib and low-dose ionizing radiation enhanced the radiosensitivity of HCT 116 cells and resulted in superior cytotoxic activity over either treatment alone. In vivo, the single-agent activity of ganetespib was relatively modest, suppressing HCT 116 xenograft tumor growth by approximately half. However, ganetespib significantly potentiated the antitumor efficacy of the 5-Fluorouracil (5-FU) prodrug capecitabine in HCT 116 xenografts, causing tumor regressions in a model that is intrinsically resistant to fluoropyrimidine therapy. This demonstration of combinatorial benefit afforded by an HSP90 inhibitor to a standard CRC adjuvant regimen provides an attractive new framework for the potential application of ganetespib as an investigational agent in this disease. PMID:24682747

He, Suqin; Smith, Donald L; Sequeira, Manuel; Sang, Jim; Bates, Richard C; Proia, David A



BRITER: A BMP Responsive Osteoblast Reporter Cell Line  

PubMed Central

Background BMP signaling pathway is critical for vertebrate development and tissue homeostasis. High-throughput molecular genetic screening may reveal novel players regulating BMP signaling response while chemical genetic screening of BMP signaling modifiers may have clinical significance. It is therefore important to generate a cell-based tool to execute such screens. Methodology/Principal Findings We have established a BMP responsive reporter cell line by stably integrating a BMP responsive dual luciferase reporter construct in the immortalized calvarial osteoblast cells isolated from tamoxifen inducible Bmp2; Bmp4 double conditional knockout mouse strain. This cell line, named BRITER (BMP Responsive Immortalized Reporter cell line), responds robustly, promptly and specifically to exogenously added BMP2 protein. The sensitivity to added BMP may be further increased by depleting the endogenous BMP2 and BMP4 proteins. Conclusion As the dynamic range of the assay (for BMP responsiveness) is very high for BRITER and as it responds specifically and promptly to exogenously added BMP2 protein, BRITER may be used effectively for chemical or molecular genetic screening for BMP signaling modifiers. Identification of novel molecular players capable of influencing BMP signaling pathway may have clinical significance. PMID:22611465

Bandyopadhyay, Amitabha



Mitochondrial DNA sequence analysis of two mouse hepatocarcinoma cell lines  

PubMed Central

AIM: To study genetic difference of mitochondrial DNA (mtDNA) between two hepatocarcinoma cell lines (Hca-F and Hca-P) with diverse metastatic characteristics and the relationship between mtDNA changes in cancer cells and their oncogenic phenotype. METHODS: Mitochondrial DNA D-loop, tRNAMet+Glu+Ile and ND3 gene fragments from the hepatocarcinoma cell lines with 1100, 1126 and 534 bp in length respectively were analysed by PCR amplification and restriction fragment length polymorphism techniques. The D-loop 3’ end sequence of the hepatocarcinoma cell lines was determined by sequencing. RESULTS: No amplification fragment length polymorphism and restriction fragment length polymorphism were observed in tRNAMet+Glu+Ile, ND3 and D-loop of mitochondrial DNA of the hepatocarcinoma cells. Sequence differences between Hca-F and Hca-P were found in mtDNA D-loop. CONCLUSION: Deletion mutations of mitochondrial DNA restriction fragment may not play a significant role in carcinogenesis. Genetic difference of mtDNA D-loop between Hca-F and Hca-P, which may reflect the environmental and genetic influences during tumor progression, could be linked to their tumorigenic phenotypes. PMID:15633228

Dai, Ji-Gang; Lei, Xia; Min, Jia-Xin; Zhang, Guo-Qiang; Wei, Hong




PubMed Central

Macrophage migration inhibitory factor (MIF) was found in media of human and mouse lymphocyte and fibroblast cell lines that were continuously growing. Its release was dependent on activation of the cells to enter the mitotic cycle, particularly on cells in S phase. The greatest quantity of MIF was detected in supernatants of lymphocytes collected during S phase after the cells were synchronized in G1 and in supernatants of growing fibroblasts. When the latter were contact inhibited little or no MIF was found in media. MIF was also released into media of cells proliferating in homologous serum in the absence of fetal calf serum and into media lacking any protein. The MIF produced by lymphocyte lines eluted from Sephadex G-100 in the same fashion as MIF produced by the interaction of sensitized guinea pig cells and antigen. The results indicated that MIF is not a specific mediator of delayed hypersensitivity and cellular immunity and that MIF released by sensitized lymphocytes incubated with antigen merely reflects that fraction of cells activated by antigen to enter the mitotic cycle. PMID:5060291

Tubergen, David G.; Feldman, Joseph D.; Pollock, E. M.; Lerner, Richard A.



Transient recombinant protein expression in a human amniocyte cell line: the CAP-T® cell system.  


The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult-to-express proteins. For this reason, we evaluated the more recently established novel CAP-T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP-T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes. PMID:22488157

Fischer, Simon; Charara, Nadine; Gerber, Andrea; Wölfel, Jens; Schiedner, Gudrun; Voedisch, Bernd; Geisse, Sabine



Cytotoxicity evaluation of silica nanoparticles using fish cell lines.  


Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO2 NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish