Science.gov

Sample records for cell lines hct-116

  1. [Alpha-1 Antitrypsin Affects U0126-Induced Cytotoxicity in Colon Cancer Cell Line (HCT116)].

    PubMed

    Ljujic, M; Mijatovic, S; Bulatovic, M Z; Mojic, M; Maksimovic-Ivanic, D; Radojkovic, D; Topic, A

    2016-01-01

    Alpha-1-antitrypsin (AAT), an acute phase protein, is the principal circulatory anti-protease. This multifunctional protein is encoded by the SERPINA1 gene. Although AAT was recognised as a potential tumour marker, its role in cancer biology remains unknown. Given that it has been demonstrated that AAT has an anti-apoptotic property against non-malignant cells, we aimed to investigate whether AAT affects apoptosis in a colon cancer cell line (HCT116). The presence of AAT in the HCT116 cell culture antagonized cytotoxicity of blockers of MEK1/2, PI3K/Akt pathways as well as NF-κB. The dominantly recovered cell viability was observed in the co-treatment with MEK1/2 inhibitor U0126. In addition, it was revealed that AAT almost completely abolished U0126-induced apoptosis through maintenance of the autophagy process. Our study revealed for the first time that the observed cyto-protection triggered by AAT was accompanied by sustained autophagy which opposed apoptosis. These results may contribute to understanding of the role of AAT in cancer development and evaluation of efficacy of cancer therapy. PMID:27028823

  2. In Vitro Cytotoxic Activity of Origanum vulgare L. on HCT-116 and MDA-MB-231 Cell Lines

    PubMed Central

    Grbović, Filip; Stanković, Milan S.; Ćurčić, Milena; Đorđević, Nataša; Šeklić, Dragana; Topuzović, Marina; Marković, Snežana

    2013-01-01

    In the present investigation, we examined the cytotoxic effect of methanolic extract from Origanum vulgare on HCT-116 and MDA-MB-231 cell line in vitro. In order to determine the cytotoxic effects we used an MTT viability assay. The results showed that cell growth is significantly lower in extract treated cells compared to untreated control. The effect of inhibition of cell growth was higher in the treatment of HCT-116 cell line than in MDA-MB-231. Based on the results it is determined that O. vulgare is a significant source of biologically active substances that have cytotoxic and antiproliferative activity in vitro. PMID:27137381

  3. Expression of γ-synuclein in colorectal cancer tissues and its role on colorectal cancer cell line HCT116

    PubMed Central

    Ye, Qing; Feng, Bo; Peng, Yuan-Fei; Chen, Xue-Hua; Cai, Qu; Yu, Bei-Qin; Li, Liang-Hui; Qiu, Ming-Yuan; Liu, Bing-Ya; Zheng, Min-Hua

    2009-01-01

    AIM: To investigate the expression pattern of γ-synuclein in colorectal cancer (CRC) tissues, and to study the effects of γ-synuclein on CRC cell line HCT116 biological features in vitro. METHODS: The expression pattern of γ-synuclein was determined in 54 CRC tissues and 30 tumor-matched nonneoplastic adjacent tissues (NNAT) 5 cm away from the tumor via real-time quantitative reverse transcription PCR (RT-PCR) and immunohistochemistry. The relationship between γ-synuclein protein expression and clinicopathological factors of CRC tissues was analyzed. Three small interfering RNA (siRNA) targeting γ-synuclein mRNA plasmids were constructed and transfected into the CRC cell line HCT116. The stable cell lines were selected with G-418 for 28 d, and the biological features of these cells were examined by cell growth curve, soft agar assay, and cell migration and invasion assays in vitro. RESULTS: The expression of γ-synuclein mRNA and protein was much higher in CRC tissue samples than in NNAT samples (P = 0.02, P = 0.036). There was a significant correlation between the γ-synuclein protein expression and clinical stage and lymph node involvement of CRC (P = 0.02, P = 0.033). In functional analysis we found that down-regulation of γ-synuclein expression in HCT116 cells could inhibit the growth, colony formation rate, and migration and invasion ability of HCT116 cells. CONCLUSION: Increased expression of γ-synuclein in CRC tissues and the biological effects of reduced γ-synuclein expression on HCT116 cells suggest that γ-synuclein may play a positive role in the progression of CRC. PMID:19859996

  4. In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line.

    PubMed

    Wang, Jigang; Zhang, Jianbin; Zhang, Chong-Jing; Wong, Yin Kwan; Lim, Teck Kwang; Hua, Zi-Chun; Liu, Bin; Tannenbaum, Steven R; Shen, Han-Ming; Lin, Qingsong

    2016-01-01

    To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQ(TM) quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway Analysis(TM) (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death. PMID:26915414

  5. In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line

    PubMed Central

    Wang, Jigang; Zhang, Jianbin; Zhang, Chong-Jing; Wong, Yin Kwan; Lim, Teck Kwang; Hua, Zi-Chun; Liu, Bin; Tannenbaum, Steven R.; Shen, Han-Ming; Lin, Qingsong

    2016-01-01

    To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQTM quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway AnalysisTM (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death. PMID:26915414

  6. The mycotoxin zearalenone enhances cell proliferation, colony formation and promotes cell migration in the human colon carcinoma cell line HCT116.

    PubMed

    Abassi, Haila; Ayed-Boussema, Imen; Shirley, Sarah; Abid, Salwa; Bacha, Hassen; Micheau, Olivier

    2016-07-01

    Zearalenone (ZEN) and Aflatoxin B1 (AFB1) are fungal secondary metabolites produced by Fusarium and Aspergillus genera, respectively. These mycotoxins are found world-wide as corn and wheat contaminants. AFB1 is probably the most toxic and carcinogenic mycotoxin. It has been demonstrated to be mutagenic, genotoxic, and hepatocarcinogenic. ZEN is a non-steroidal estrogenic mycotoxin that displays hepatotoxicity, immunotoxicity and genotoxicity. Its mutagenic and carcinogenic properties have so far remained controversial and questionable. Using the colon carcinoma cell line HCT116, we will show here that ZEN, at low concentrations, enhances cell proliferation, increases colony formation and fastens cell migration after wound healing. The highest effect of ZEN was observed at a concentration 10 times lower as compared to AFB1. Our findings suggest thus that this mycotoxin exhibits carcinogenesis-like properties in HCT116 cells. PMID:27084041

  7. Metabolomics study on the antitumor effect of marine natural compound flexibilide in HCT-116 colon cancer cell line.

    PubMed

    Gao, Dan; Wang, Yini; Xie, Weiyi; Yang, Ti; Jiang, Yuyang; Guo, Yuewei; Guan, Jin; Liu, Hongxia

    2016-03-01

    A marine natural compound flexibilide isolated from the soft coral Sinularia flexibilis has been found to have antitumor activity. However, its pharmacological mechanism on tumor cells has not been studied. Herein, an ultra-performance liquid chromatography coupled to quadrupole time of-flight mass spectrometry (UPLC/Q-TOF MS) based metabolomics approach was established to investigate the antitumor effect of flexibilide on HCT-116 cells and its action mechanism. Q-TOF MS and MS/MS were used to identify significantly different metabolites. Comparing flexibilide-treated HCT-116 cells group with control group (dimethyl sulfoxide), 19 distinct metabolites involved in sphingolipid metabolism, alanine, aspartate and glutamate metabolism, d-glutamine and d-glutamate metabolism, glycerophospholipid metabolism, pyrimidine metabolism and others were discovered and identified. The significant decrease of phosphatidylcholine (PC) and phosphocholine levels and increase of lysophosphatidylcholine (LysoPC) levels in flexibilide treated cells suggested down-regulation of PC biosynthesis pathway. The decrease of sphingolipids reflected the lesions of cell membrane, and the up-regulation of sphingosine-1-phosphate indicated that TRAF2 and caspase-8 were likely to be activated by flexibilide and further caused cell apoptosis. Furthermore, TCA cycle was deemed to be down-regulated after flexibilide treatment, which might lead to an unsustainable of mitochondrial transmembrane potential MMP). The further measured descreased MMP with the increasing concentration of flexibilide treatment indiciated the dysfunction of mitochondrial which might finally lead to apoptosis. The UPLC/Q-TOF MS based metabolomics approach provides new insights into the mechanistic studies of flexibilide on tumor cells, which benefit its further improvement and application. PMID:26859520

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

    PubMed

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

    2015-02-01

    Colon cancer is the second to third common cancer worldwide. Several efforts have been made to reveal the pathways responsible for drug resistance in this type of cancer. We aimed to investigate the effect of silencing both mutant and wild-type Kristen Rous sarcoma (k-ras) on the response of human colorectal tumor 116 (HCT-116) as a colon cancer cell line to the cytotoxic effect of 5-flurouracil (5-FU). One oligonucelotide against mutant k-ras (12th codon, namely 207) and two against wild-type k-ras (namely 535 and 689) were cloned into pSilencer neo2.1. The linearized vectors besides the negative control plasmid were stably transfected into HCT-116. The proliferation rates of these cells in different concentrations of 5-FU and the apoptosis rates of the cells after treatment with lethal doses of 5-FU were studied. Moreover, the cell cycle in these cells was also analyzed by staining the cells with propidium iodide. Stably transfected cells were named HCT207ks, HCT535ks, HCT689ks, and HCT-Sc (transfected with the negative control plasmid). Decreased expression of k-ras in HCT207ks, HCT535ks, and to a lesser extent in HCT689ks was proved by quantitative real-time PCR. Although in HCT207ks the cells were mostly in G0/G1 and G2/M phases, in HCT535ks and HCT689ks, the cells in the S phase were higher in comparison with nontransfected HCT-116. Lethal doses of 5-FU in HCT-116 and HCT-Sc were 2.5-3 and 3-3.5 µmol/l, whereas in HCT207ks, HCT535ks, and HCT689ks, they were 35-40, 37.5-40, and 22.5-25 µmol/l. In conclusion, silencing mutant and wild-type k-ras would increase the resistance of HCT-116 cell line as a model of colorectal cancer to 5-FU. The degree of resistance was related directly to the k-ras mRNA level. Therefore, both mutant and wild-type k-ras may play a role in sensitizing colorectal cancer cells to 5-FU as a common chemotherapeutic drug. PMID:25325304

  9. 15-Lipoxygenase-1 expression suppresses the invasive properties of colorectal carcinoma cell lines HCT-116 and HT-29.

    PubMed

    Cimen, Ismail; Tunçay, Seda; Banerjee, Sreeparna

    2009-12-01

    Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. 15-Lipoxygenase-1 (15-LO-1), a member of the inflammatory eicosanoid pathway, oxidatively metabolizes linoleic acid and its expression is repressed in CRC. In this study, we investigated the hypothesis that the lack of 15-LO-1 expression in CRC cells might contribute to tumorigenesis. Therefore we introduced 15-LO-1 into HCT-116 and HT-29 cells that do not have detectable levels of 15-LO-1. Our data indicate that expression of 15-LO-1 significantly decreased cell proliferation and increased apoptosis. In addition, we observed a reduction in adhesion to fibronectin, anchorage-independent growth on soft agar, cellular motility and ability to heal a scratch wound, and migratory and invasive capacity across Matrigel. 15-LO-1 expression also reduced the expression of metastasis associated protein-1, a part of the nucleosome remodeling and histone deacetylase silencing complex. We propose that 15-LO-1 expression in CRC might contribute to the inhibition of metastatic capacity in vitro and can be exploited for therapeutic purposes. PMID:19775287

  10. Proliferative and Inhibitory Activity of Siberian ginseng (Eleutherococcus senticosus) Extract on Cancer Cell Lines; A-549, XWLC-05, HCT-116, CNE and Beas-2b.

    PubMed

    Cichello, Simon Angelo; Yao, Qian; Dowell, Ashley; Leury, Brian; He, Xiao-Qiong

    2015-01-01

    Siberian ginseng (Eleutherococcus senticosus) is used primarily as an adaptogen herb and also for its immune stimulant properties in Western herbal medicine. Another closely related species used in East Asian medicine systems i.e. Kampo, TCM (Manchuria, Korea, Japan and Ainu of Hokkaido) and also called Siberian ginseng (Acanthopanax senticosus) also displays immune-stimulant and anti-cancer properties. These may affect tumour growth and also provide an anti-fatigue effect for cancer patients, in particular for those suffering from lung cancer. There is some evidence that a carbohydrate in Siberian ginseng may possess not only immune stimulatory but also anti-tumour effects and also display other various anti-cancer properties. Our study aimed to determine the inhibitory and also proliferative effects of a methanol plant extract of Siberan ginseng (E. senticosus) on various cancer and normal cell lines including: A-549 (small cell lung cancer), XWLC-05 (Yunnan lung cancer cell line), CNE (human nasopharyngeal carcinoma cell line), HCT-116 (human colon cancer) and Beas-2b (human lung epithelial). These cell lines were treated with an extract from E. senticosus that was evaporated and re- constituted in DMSO. Treatment of A-549 (small cell lung cancer) cells with E. senticosus methanolic extract showed a concentration-dependent inhibitory trend from 12.5 - 50μg/mL, and then a plateau, whereas at 12.5 and 25 μg/mL, there is a slight growth suppression in QBC-939 cells, but then a steady suppression from 50, 100 and 200μg/mL. Further, in XWLC-05 (Yunnan lung cancer cell line), E. senticosus methanolic extract displayed an inhibitory effect which plateaued with increasing dosage. Next, in CNE (human nasopharyngeal carcinoma cell line) there was a dose dependent proliferative response, whereas in Beas-2 (human lung epithelial cell line), an inhibitory effect. Finally in colon cancer cell line (HCT-116) we observed an initially weak inhibitory effect and then plateau. PMID:26107240

  11. Effects of ghrelin, leptin and melatonin on the levels of reactive oxygen species, antioxidant enzyme activity and viability of the HCT 116 human colorectal carcinoma cell line.

    PubMed

    Bułdak, Rafał Jakub; Pilc-Gumuła, Katarzyna; Bułdak, Łukasz; Witkowska, Daria; Kukla, Michał; Polaniak, Renata; Zwirska-Korczala, Krystyna

    2015-08-01

    Obesity is associated with an increased risk of certain types of cancer, including colon cancer. Adipose tissue is an endocrine organ that produces biologically active substances, such as leptin and ghrelin. Recent research has suggested that adipose-derived hormones may be associated with mechanisms linked to tumorigenesis and cancer progression. Furthermore, previous studies have demonstrated that pineal gland-derived melatonin possesses important oncostatic and antioxidant properties. The present study aimed to determine the effects of the adipokines ghrelin and leptin, and the melatonin on intracellular levels of reactive oxygen species (ROS) and the activity of selected antioxidant enzymes, such as superoxide dismutase, catalase (CAT) and glutathione peroxidase. The effects of these compounds were also determined on the viability of HCT 116 human colorectal carcinoma cells in vitro. The pro-oxidant and growth inhibitory effects of melatonin resulted in an accumulation of ROS and decreased antioxidant capacity in melatonin-treated cells. Ghrelin administration alone caused a significant decrease in the levels of ROS, due to an increased activity of CAT in the HCT 116 cells. In addition, the present study observed increased lipid peroxidation following melatonin treatment, and decreased levels of malondialdehyde following ghrelin or leptin treatment. In conclusion, ghrelin, leptin and melatonin have various influences on the antioxidant capacity of HCT 116 cells. Compared with the adipokines, treatment with melatonin increased ROS levels and decreased cellular viability. PMID:25873273

  12. Natural product-based design, synthesis and biological evaluation of Albiziabioside A derivatives that selectively induce HCT116 cell death.

    PubMed

    Wei, Gaofei; Cui, Shanshan; Luan, Weijing; Wang, Shuai; Hou, Zhuang; Liu, Yongxiang; Liu, Yang; Cheng, Maosheng

    2016-05-01

    A series of Albiziabioside A coupled substituents of cinnamoyl derivatives were designed and synthesized. The synthesized compounds were screened for anticancer activity against a panel of six human cancer cell lines using a MTT assay. Synthetic derivatives showed excellent selectivity, as they were toxic against only HCT116 cell line. Some compounds exhibited better anti-cancer activity against HCT116 compared to positive controls, such as 5-fluorouracil and Albiziabioside A. Compound 8n was the most active derivative. Importantly, it was also found that the anti-proliferative activity of 8n could be attributed to the induction of cell cycle arrest and apoptosis in HCT116 cells. PMID:26922223

  13. Knockdown of biglycan expression by RNA interference inhibits the proliferation and invasion of, and induces apoptosis in, the HCT116 colon cancer cell line.

    PubMed

    Xing, Xiaojing; Gu, Xiaohu; Ma, Tianfei

    2015-11-01

    Biglycan is an important component of the extracellular matrix, and it is also a member of small leucine-rich proteoglycan family. Previous studies indicated that the expression of biglycan was increased in a variety of tumor tissues, including colon cancer. However, the mechanisms underlying its effects in colon cancer remain to be fully elucidated. In the present study, the effects of biglycan knockdown on colon cancer cell proliferation, migration, invasion and apoptosis were investigated. The mRNA expression levels of biglycan in the HCT116 colon cancer cell line were downregulated using RNA interference, and the stably transfected cell line was obtained through G418 screening for subsequent experiments. The results revealed that downregulation of the expression of biglycan suppressed cell proliferation and caused a cell cycle arrest at the G0/G1 phase. The results of the western blot analysis also revealed that the expression levels of cell cycle‑associated proteins, including cyclin A and cyclin D1, were markedly decreased following silencing of biglycan, whereas the expression levels of p21 and p27 were markedly increased compared with that of the short hairpin RNA control group. Furthermore, the decreased expression of biglycan inhibited colon cancer cell migration and invasion, and induced apoptosis. A complete inhibition of the p38 signaling pathway with SB203580 effectively reversed the increase in apoptotic cell numbers induced by biglycan downregulation. Taken together, the results of the present study indicated that biglycan exerts an important role in cell proliferation, migration, invasion and apoptosis in colon cancer, and that biglycan regulates the p38 MAPK signaling pathway by exerting an antiapoptotic effect. Therefore, biglycan may represent a putative target for colon cancer gene therapy. PMID:26459740

  14. Transforming Growth Factor Beta Receptor 2 (TGFBR2) Changes Sialylation in the Microsatellite Unstable (MSI) Colorectal Cancer Cell Line HCT116

    PubMed Central

    Lee, Jennifer; Ballikaya, Seda; Schönig, Kai; Ball, Claudia R.; Glimm, Hanno; Kopitz, Juergen; Gebert, Johannes

    2013-01-01

    Aberrant glycosylation is a common feature of many malignancies including colorectal cancers (CRCs). About 15% of CRC show the microsatellite instability (MSI) phenotype that is associated with a high frequency of biallelic frameshift mutations in the A10 coding mononucleotide microsatellite of the transforming growth factor beta receptor 2 (TGFBR2) gene. If and how impaired TGFBR2 signaling in MSI CRC cells affects cell surface glycan pattern is largely unexplored. Here, we used the TGFBR2-deficient MSI colon carcinoma cell line HCT116 as a model system. Stable clones conferring doxycycline (dox)-inducible expression of a single copy wildtype TGFBR2 transgene were generated by recombinase-mediated cassette exchange (RMCE). In two independent clones, dox-inducible expression of wildtype TGFBR2 protein and reconstitution of its signaling function was shown. Metabolic labeling experiments using the tritiated sialic acid precursor N-acetyl-D-mannosamine (ManNAc) revealed a significant decline (∼30%) of its incorporation into newly synthesized sialoglycoproteins in a TGFBR2-dependent manner. In particular, we detected a significant decrease of sialylated ß1-integrin upon reconstituted TGFBR2 signaling which did not influence ß1-integrin protein turnover. Notably, TGFBR2 reconstitution did not affect the transcript levels of any of the known human sialyltransferases when examined by real-time RT- PCR analysis. These results suggest that reconstituted TGFBR2 signaling in an isogenic MSI cell line model system can modulate sialylation of cell surface proteins like ß1-integrin. Moreover, our model system will be suitable to uncover the underlying molecular mechanisms of altered MSI tumor glycobiology. PMID:23468914

  15. Butyrate-induced apoptosis in HCT116 colorectal cancer cells includes induction of a cell stress response.

    PubMed

    Fung, Kim Y C; Brierley, Gemma V; Henderson, Steve; Hoffmann, Peter; McColl, Shaun R; Lockett, Trevor; Head, Richard; Cosgrove, Leah

    2011-04-01

    Short chain fatty acids (SCFA), principally butyrate, propionate, and acetate, are produced in the gut through the fermentation of dietary fiber by the colonic microbiotica. Butyrate in particular is the preferred energy source for the cells in the colonic mucosa and has been demonstrated to induce apoptosis in colorectal cancer cell lines. We have used proteomics, specifically 2D-DIGE and mass spectrometry, to identify proteins involved in butyrate-induced apoptosis in HCT116 cells and also to identify proteins involved in the development of butyrate insensitivity in its derivative, the HCT116-BR cells. The HCT116-BR cell line was characterized as being less responsive to the apoptotic effects of butyrate in comparison to its parent cell line. Our analysis has revealed that butyrate likely induces a cellular stress response in HCT116 cells characterized by p38 MAPK activation and an endoplasmic reticulum (ER) stress response, resulting in caspase 3/7 activation and cell death. Adaptive cellular responses to stress-induced apoptosis in HCT116-BR cells may be responsible for the development of resistance to apoptosis in this cell line. We also report for the first time additional cellular processes altered by butyrate, such as heme biosynthesis and dysregulated expression of nuclear lamina proteins, which may be involved in the apoptotic response observed in these cell lines. PMID:21235278

  16. Antiproliferative and Apoptotic Activity of Chamaecyparis obtusa Leaf Extract against the HCT116 Human Colorectal Cancer Cell Line and Investigation of the Bioactive Compound by Gas Chromatography-Mass Spectrometry-Based Metabolomics.

    PubMed

    Kim, Hye-Youn; Lee, Seul-Gi; Oh, Taek-Joo; Lim, Sa Rang; Kim, So-Hyun; Lee, Hong Jin; Kim, Young-Suk; Choi, Hyung-Kyoon

    2015-01-01

    Chamaecyparis obtusa (CO) belongs to the Cupressaceae family, and it is found widely distributed in Japan and Korea. In this study, the anti-proliferative activities of the methanol and water extracts of CO leaves against a human colorectal cancer cell line (HCT116) were investigated. The methanol extract of CO leaves, at a concentration of 1.25 µg/mL, exhibited anti-proliferative activity against HCT116 cells, while displaying no cytotoxicity against Chang liver cells. Comparative global metabolite profiling was performed using gas chromatography-mass spectrometry coupled with multivariate statistical analysis, and it was revealed that anthricin was the major compound contributing to the anti-proliferative activity. The activation of c-Jun N-terminal kinases played a key role in the apoptotic effect of the methanol extract of CO leaves in HCT116 human colon cancer cells. These results suggest that the methanol extract and anthricin derived from CO leaves might be useful in the development of medicines with anti-colorectal cancer activity. PMID:26445036

  17. Evodiamine Induces Apoptosis and Inhibits Migration of HCT-116 Human Colorectal Cancer Cells

    PubMed Central

    Zhao, Lv-Cui; Li, Jing; Liao, Ke; Luo, Nian; Shi, Qing-Qiang; Feng, Zi-Qiang; Chen, Di-Long

    2015-01-01

    Evodiamine (EVO) exhibits strong anti-cancer effects. However, the effect of EVO on the human colorectal cancer cell line HCT-116 has not been explored in detail, and its underlying molecular mechanisms remain unknown. In the present study, cell viability was assessed by Cell Counting Kit-8 (CCK-8). Cell cycle and apoptosis were measured by flow cytometry, and morphological changes in the nucleus were examined by fluorescence microscopy and Hoechst staining. Cell motility was detected by Transwell assay. ELISA was used to assess the protein levels of autocrine motility factor (AMF) in the cell supernatant, and protein expression was determined by Western blotting. Our results showed that EVO inhibited the proliferation of HCT-116 cells, caused accumulation of cells in S and G2/M phases, and reduced the levels of the secreted form of AMF. The protein levels of tumor suppressor protein (p53), Bcl-2 Associated X protein (Bax), B cell CLL/lymphoma-2 (Bcl-2), phosphoglucose isomerase (PGI), phosphorylated signal transducers and activators of transcription 3 (p-STAT3) and matrix metalloproteinase 3 (MMP3) were altered in cells treated with EVO. Taken together, our results suggest that EVO modulates the activity of the p53 signaling pathway to induce apoptosis and downregulate MMP3 expression by inactivating the JAK2/STAT3 pathway through the downregulation of PGI to inhibit migration of HCT-116 human colorectal cancer cells. PMID:26580615

  18. Cellular Uptake of Decitabine by Equilibrative Nucleoside Transporters in HCT116 Cells.

    PubMed

    Ueda, Kumiko; Hosokawa, Mika; Iwakawa, Seigo

    2015-01-01

    DNA hypermethylation, an epigenetic change that silences gene expression without altering nucleotide sequences, plays a critical role in the formation and progression of colorectal cancers as well as in the acquisition of drug resistance. Decitabine (DAC), a DNA methyltransferase 1 inhibitor of nucleoside analogues, has been shown to restore gene expression silenced by hypermethylation. In the present study, the mechanisms underlying both uridine and DAC uptake were examined in the human colon cancer cell line HCT116. Real-time polymerase chain reaction analysis revealed that ENT1 mRNA was the most abundant among the nucleoside transporters examined in HCT116 cells. The ENT1 protein was detected in the membrane fraction, as determined by Western blotting. The uptake of uridine or DAC was time- and concentration-dependent, but also Na(+)-independent. The uptake of these agents was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), an inhibitor of equilibrative nucleoside transporters (ENTs), and was also decreased in cells treated with ENT1 small interfering RNA. The uptake of both uridine and DAC was inhibited by uridine, cytidine, adenosine, or inosine, while that of DAC was also inhibited by thymidine. The expression of MAGEA1 mRNA, the DNA of which was methylated in HCT116 cells, was increased by DAC treatment, and this increment was attenuated by concomitant treatment with NBMPR. The IC50 value of DAC was also increased in the presence of NBMPR. These results suggest that DAC is mainly taken up by ENT1 and that this uptake is one of the key determinants of the activity of DAC in HCT116 cells. PMID:26235575

  19. Anticoagulant properties and cytotoxic effect against HCT116 human colon cell line of sulfated glycosaminoglycans isolated from the Norway lobster (Nephrops norvegicus) shell.

    PubMed

    Sayari, Nadhem; Balti, Rafik; Ben Mansour, Mohamed; Ben Amor, Ikram; Graiet, Imen; Gargouri, Jalel; Bougatef, Ali

    2016-05-01

    Sulfated glycosaminoglycans (SGNL) were extracted for the first time from Norway lobster (Nephrops norvegicus) shell. The monosaccharide composition analysed by GC/MS revealed the presence of galacturonic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine. The analysis of SGNL with acetate cellulose electrophoresis in Zn-acetate revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS). SGNL were evaluated for their anticoagulant activities using activated partial thromboplastin time (aPTT), thrombin time (TT) and prothrombine time (PT) tests. After 21h incubation, HCT116 cell proliferation was inhibited (p<0.05) between 39.7 and 54.8% at 1.5-7.5mg/mL of SGNL. SGNL don't show hemolytic activity towards bovine erythrocytes and no cytotoxicity against the normal lymphocytes. The antiproliferative efficacy of these lobster glycosaminoglycans were probably related with the higher sulfate content. SGNL demonstrated promising antiproliferative and anticoagulant potential, which may be used as a novel, effective and promising antithrombotic agent. PMID:27133072

  20. A novel microtubule depolymerizing colchicine analogue triggers apoptosis and autophagy in HCT-116 colon cancer cells.

    PubMed

    Kumar, Ashok; Singh, Baljinder; Sharma, Parduman R; Bharate, Sandip B; Saxena, Ajit K; Mondhe, D M

    2016-03-01

    Colchicine is a tubulin-binding natural product isolated from Colchicum autumnale. Here we report the in vitro anticancer activity of C-ring modified semi-synthetic derivative of colchicine; N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(4-phenyl-piperidin-1-yl)-5,6,7,9 tetrahydrobenzo[a]heptalen-7-yl]acetamide (4h) on colon cancer HCT-116 cell line. The compound 4h was screened for anti-proliferative activity against different human cancer cell lines and was found to exhibit higher cytotoxicity against colon cancer cell lines HCT-116 and Colo-205 with IC50 of 1 and 0.8 μM respectively. Cytotoxicity of the compound to the normal fR2 breast epithelial cells and normal HEK293 human embryonic kidney cells was evaluated in concentration and time-dependent manner to estimate its selectivity for cancer cells which showed much better selectivity than that of colchicine. Compound 4h induced cell death in HCT-116 cells by activating apoptosis and autophagy pathways. Autophagy inhibitor 3-MA blocked the production of LC3-II and reduced the cytotoxicity in response to 4h, but did not affect apoptosis, suggesting thereby that these two were independent events. Reactive oxygen species scavenger ascorbic acid pretreatment not only decreased the reactive oxygen species level but also reversed 4h induced cytotoxicity. Treatment with compound 4h depolymerized microtubules and the majority of cells arrested at the G2/M transition. Together, these data suggest that 4h has better selectivity and is a microtubule depolymerizer, which activates dual cell-death machineries, and thus, it could be a potential novel therapeutic agent in cancer therapy. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26919061

  1. Cardamonin induces autophagy and an antiproliferative effect through JNK activation in human colorectal carcinoma HCT116 cells.

    PubMed

    Kim, Young-Joo; Kang, Ki Sung; Choi, Kyung-Chul; Ko, Hyeonseok

    2015-06-15

    Cardamonin (2',4'-dihydroxy-6'-methoxychalcone) is derived from Alpinia katsumadai Hayata (Zingiberaceae), a plant that has been used in Traditional Chinese Medicine for thousands of years. Several anticancer agents have been reported to induce autophagy, which either protects cells or further sensitizes cells to drug treatment. However, the possible autophagic and antiproliferative effects of cardamonin on the human colorectal carcinoma HCT116 cell line are unclear. In the present study, experiments were conducted to determine the effects of cardamonin on cell proliferation, cell cycle distribution, and stimulation of autophagy in cultures of the HCT116 cell line. The results showed that cardamonin inhibited cell proliferation, induced G2/M phase cell cycle arrest, and enhanced autophagy in HCT116 cells. We found evidence that cardamonin-induced autophagic and antiproliferative effects are regulated by the tumor protein p53. We also found that the enhanced activation of c-Jun N-terminal kinase (JNK) by cardamonin was partially regulated by p53 and was critical for cardamonin-induced autophagic and antiproliferative effects in HCT116 cells. These findings suggest that cardamonin or other anticancer agents that increase p53/JNK-dependent stimulation of autophagy could be used to effectively treat patients with colorectal carcinoma. PMID:25959811

  2. Pristimerin inhibits proliferation, migration and invasion, and induces apoptosis in HCT-116 colorectal cancer cells.

    PubMed

    Yousef, Bashir A; Hassan, Hozeifa M; Guerram, Mounia; Hamdi, Aida M; Wang, Bin; Zhang, Lu-Yong; Jiang, Zhen-Zhou

    2016-04-01

    Colorectal cancer (CRC) is one of the world's most common cancers with a high mortality rate mainly due to metastasis. Our previous study showed that pristimerin had potent antitumor activities against human CRC cells. In the present study, we further evaluated pristimerin anti-tumor and anti-metastatic properties. MTT assay, Hoechst staining, Annexin V/PI double staining, reactive oxygen species (ROS) measurements were used to assess pristimerin cytotoxicity and apoptotic-inducing effects on HCT-116 cells. Wound healing assay and Transwell assay were used to estimate pristimerin anti-migration and anti-invasion activities on CRC cells. Meanwhile, HCT-116 xenograft model applied for investigating in vivo antitumor activities. Our results showed that pristimerin mediated in vitro HCT-116 cell death, through generation of intracellular ROS and apoptosis induction. Tumor volumes and weights measurements, pathological analysis and Tunnel assay proved that pristimerin inhibited in vivo HCT-116 xenografts growth. Pristimerin was also able to limit CRC invasion and metastasis. It caused downregulation of PI3K/AKT/mTOR pathway and its subsequent downstream p70S6K and E4-BP1 proteins. Collectively, pristimerin exerted both in vitro and in vivo cytotoxic and anti-metastatic effects on HCT-116 cells, suggesting that pristimerin has potential as a new anticancer drug for treatment of colon cancer. PMID:27044819

  3. Synthesis of bile acid derivatives and in vitro cytotoxic activity with pro-apoptotic process on multiple myeloma (KMS-11), glioblastoma multiforme (GBM), and colonic carcinoma (HCT-116) human cell lines.

    PubMed

    Brossard, Dominique; El Kihel, Laïla; Clément, Monique; Sebbahi, Walae; Khalid, Mohamed; Roussakis, Christos; Rault, Sylvain

    2010-07-01

    The novelty of this work derives from the use of nitrogenous heterocycles as building block in the synthesis of conjugate bile acid derivatives. New piperazinyl bile acid derivatives were synthesized and tested in vitro against various human cancer cells (GBM, KMS-11, HCT-116). The best pro-apoptotic activity was obtained with N-[4N-cinnamylpiperazin-1-yl)-3alpha,7beta-dihydroxy-5beta-cholan-24-amide (7b) and N-[4N-cinnamyllpiperazin-1-yl)- 3alpha,7alpha-dihydroxy-5beta-cholan-24-amide (7c) on these human cancer cell lines (IC(50): 8.5-31.4microM). This activity was associated with nuclear and DNA fragmentation, demonstrating that 7b induces cell death by an apoptotic process as 7c. This study shows the possibility of hydrid heterocycle-steroids as new anticancer agents with improved bioactivity and easy to synthesize. PMID:20381215

  4. Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells.

    PubMed

    Huang, Wen-Shih; Yang, Jen-Tsung; Lu, Chien-Chang; Chang, Shun-Fu; Chen, Cheng-Nan; Su, Yu-Ping; Lee, Ko-Chao

    2015-01-01

    A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC). Hence, resistin may play a role in CRC development. Fulvic acid (FA), a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative) and SW-48 (p53-positive) CRC cells and human umbilical vein endothelial cells (HUVECs) were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin. PMID:26690142

  5. Fulvic Acid Attenuates Resistin-Induced Adhesion of HCT-116 Colorectal Cancer Cells to Endothelial Cells

    PubMed Central

    Huang, Wen-Shih; Yang, Jen-Tsung; Lu, Chien-Chang; Chang, Shun-Fu; Chen, Cheng-Nan; Su, Yu-Ping; Lee, Ko-Chao

    2015-01-01

    A high level of serum resistin has recently been found in patients with a number of cancers, including colorectal cancer (CRC). Hence, resistin may play a role in CRC development. Fulvic acid (FA), a class of humic substances, possesses pharmacological properties. However, the effect of FA on cancer pathophysiology remains unclear. The aim of this study was to investigate the effect of resistin on the endothelial adhesion of CRC and to determine whether FA elicits an antagonistic mechanism to neutralize this resistin effect. Human HCT-116 (p53-negative) and SW-48 (p53-positive) CRC cells and human umbilical vein endothelial cells (HUVECs) were used in the experiments. Treatment of both HCT-116 and SW-48 cells with resistin increases the adhesion of both cells to HUVECs. This result indicated that p53 may not regulate this resistin effect. A mechanistic study in HCT-116 cells further showed that this resistin effect occurs via the activation of NF-κB and the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Co-treating cells with both FA and resistin revealed that FA significantly attenuated the resistin-increased NF-κB activation and ICAM-1/VCAM-1 expression and the consequent adhesion of HCT-116 cells to HUVECs. These results demonstrate the role of resistin in promoting HCT-116 cell adhesion to HUVECs and indicate that FA might be a potential candidate for the inhibition of the endothelial adhesion of CRC in response to resistin. PMID:26690142

  6. Impact of antioxidants on the ability of phenolic phytochemicals to kill HCT116 colon cancer cells.

    PubMed

    Murphy, A; Testa, K; Berkelhammer, J; Hopkins, S; Loo, G

    2014-03-01

    Certain phenolic phytochemicals can kill cancer cells. Possible interference from antioxidants is a concern, and this issue has not been studied appreciably. Therefore, the effect of ascorbate and N-acetylcysteine on the ability of epigallocatechin gallate (EGCG) and curcumin to kill HCT116 colon cancer cells was examined. EGCG and curcumin each caused DNA damage in the cells. The DNA-damaging ability of EGCG, but not curcumin, was hindered by either ascorbate or NAC, which was also shown in HT29 and SW480 colon cancer cells. Also, iron chelators (deferoxamine and 2,2'-dipyridyl) inhibited the ability of EGCG, but not curcumin, to cause damage to the DNA in HCT116 cells. Interestingly, curcumin, but not EGCG, increased the expression of growth arrest and DNA damage-inducible gene 153 and also heme oxygenase-1, and this stress gene upregulation by curcumin was antioxidant-insensitive. With prolonged incubation of HCT116 cells with either EGCG or curcumin, cell shrinkage, membrane blebbing, apoptotic bodies, and chromatin condensation/fragmentation were observed. These morphological changes were not apparent in EGCG-treated cells that had been pretreated with either ascorbate or NAC. However, the ascorbate and NAC pretreatments did not prevent the occurrence of the morphological changes in curcumin-treated cells. Thus, these findings suggest that ascorbate and NAC interfere with the ability of EGCG, but not curcumin, to kill HCT116 cells. This basic knowledge may help to better plan and optimize strategies for chemoprevention or chemotherapy. PMID:24256565

  7. Cell specific apoptosis by RLX is mediated by NFκB in human colon carcinoma HCT-116 cells

    PubMed Central

    2014-01-01

    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 μM and have further investigated the involvement of NFκB and caspase-3 in RLX action in CRC apoptosis. Following RLX and BEZ-235 treatment in HCT-116, we observed significant down-regulation of NFκB (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

  8. Herbal Formulation C168 Attenuates Proliferation and Induces Apoptosis in HCT 116 Human Colorectal Carcinoma Cells: Role of Oxidative Stress and DNA Damage

    PubMed Central

    Leong, Lek Mun; Chan, Kok Meng; Hamid, Asmah; Latip, Jalifah; Rajab, Nor Fadilah

    2016-01-01

    The use of herbal formulations has gained scientific interest, particularly in cancer treatment. In this study, the herbal formulation of interest, denoted as C168, is a mixture of eight genera of plants. This study aims to investigate the antiproliferative effect of C168 methanol extract (CME) on various cancer cells and its underlying mechanism of action on the most responsive cell line, namely, HCT 116 cells. CME exerted antiproliferative activities on HCT 116 colorectal carcinoma cells and HepG2 hepatocellular carcinoma cells but not on CCD-841-CoN normal colon epithelial cells, Jurkat E6.1 lymphoblastic leukemic cells, and V79-4 Chinese hamster lung fibroblasts. Further investigation on HCT 116 cells showed that CME induced G2/M cell-cycle arrest and apoptosis. Treatment of CME induced oxidative stress in HCT 116 cells by increasing the superoxide anion level and decreasing the intracellular glutathione. CME also increased tail moment value and H2AX phosphorylation in HCT 116 cells, suggesting DNA damage as an early signal of CME induced apoptosis. Loss of mitochondrial membrane potential in CME-treated cells also indicated the involvement of mitochondria in CME induced apoptosis. This study indicated the selectivity of CME toward colon cancer cells with the involvement of oxidative damage as its possible mechanism of action. PMID:26884792

  9. Herbal Formulation C168 Attenuates Proliferation and Induces Apoptosis in HCT 116 Human Colorectal Carcinoma Cells: Role of Oxidative Stress and DNA Damage.

    PubMed

    Leong, Lek Mun; Chan, Kok Meng; Hamid, Asmah; Latip, Jalifah; Rajab, Nor Fadilah

    2016-01-01

    The use of herbal formulations has gained scientific interest, particularly in cancer treatment. In this study, the herbal formulation of interest, denoted as C168, is a mixture of eight genera of plants. This study aims to investigate the antiproliferative effect of C168 methanol extract (CME) on various cancer cells and its underlying mechanism of action on the most responsive cell line, namely, HCT 116 cells. CME exerted antiproliferative activities on HCT 116 colorectal carcinoma cells and HepG2 hepatocellular carcinoma cells but not on CCD-841-CoN normal colon epithelial cells, Jurkat E6.1 lymphoblastic leukemic cells, and V79-4 Chinese hamster lung fibroblasts. Further investigation on HCT 116 cells showed that CME induced G2/M cell-cycle arrest and apoptosis. Treatment of CME induced oxidative stress in HCT 116 cells by increasing the superoxide anion level and decreasing the intracellular glutathione. CME also increased tail moment value and H2AX phosphorylation in HCT 116 cells, suggesting DNA damage as an early signal of CME induced apoptosis. Loss of mitochondrial membrane potential in CME-treated cells also indicated the involvement of mitochondria in CME induced apoptosis. This study indicated the selectivity of CME toward colon cancer cells with the involvement of oxidative damage as its possible mechanism of action. PMID:26884792

  10. Dichlorvos-induced toxicity in HCT116 cells: involvement of oxidative stress and apoptosis.

    PubMed

    Ben Salem, Intidhar; Boussabbeh, Manel; Bacha, Hassen; Abid, Salwa

    2015-03-01

    Organophosphorous (OP) pesticides are widely used in the agriculture and home. Among those pesticides, Dichlorvos (DDVP) is a worldwide used insecticide for pest control. DDVP is commonly used as an insecticide for maintenance and growth of agricultural products, to control the internal and external parasites of farm animals, and to eradicate insects threatening the household, public health, and stored products. Although substantial information is available regarding the environmental and ecological impact of DDVP, not much is known in regard to its toxicity in the mammalian system. Therefore a study was conducted for the assessment of cytotoxic and genotoxic effects of DDVP in human colon carcinoma (HCT116) cell line. We demonstrated that DDVP significantly decreased cell viability as assessed by the MTT assay. The increase in cell death was accompanied by a reduction in the mitochondrial membrane potential. Besides, pretreatment with Z-VAD-FMK, a general caspases inhibitor, decreased significantly the DDVP-induced cell death. We also shown that DDVP induced reactive oxygen species (ROS) generation followed by lipid peroxidation as evidenced by an increase in the MDA levels. Our results also indicate that DDVP induced a concentration-dependent increase in DNA damage as evident by the comet assay. These data indicate that DDVP produces cytotoxicity and DNA damage in mammalian cells and should be used with caution. PMID:25868818

  11. p21{sup WAF1/CIP1} deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

    SciTech Connect

    Kim, Ae Jeong; Jee, Hye Jin; Song, Naree; Kim, Minjee; Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan ; Jeong, Seon-Young; Department of Medical Genetics, Ajou University School of Medicine ; Yun, Jeanho; Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer p21{sup -/-} HCT116 cells exhibited an increase in mitochondrial mass. Black-Right-Pointing-Pointer The expression levels of PGC-1{alpha} and AMPK were upregulated in p21{sup -/-} HCT116 cells. Black-Right-Pointing-Pointer The proliferation of p21{sup -/-} HCT116 cells in galactose medium was significantly impaired. Black-Right-Pointing-Pointer p21 may play a role in maintaining proper mitochondrial mass and respiratory function. -- Abstract: p21{sup WAF1/CIP1} is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21{sup -/-} HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53{sup -/-} cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1{alpha} and TFAM and AMPK activity were also elevated in p21{sup -/-} cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1{alpha} axis. However, the increase in mitochondrial biogenesis in p21{sup -/-} cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21{sup -/-} cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.

  12. DZNep inhibits the proliferation of colon cancer HCT116 cells by inducing senescence and apoptosis.

    PubMed

    Sha, Mingquan; Mao, Genxiang; Wang, Guofu; Chen, Yufeng; Wu, Xiaojian; Wang, Zhen

    2015-05-01

    EZH2 is over-expressed in human colon cancer and is closely associated with tumor proliferation, metastasis and poor prognosis. Targeting and inhibiting EZH2 may be an effective therapeutic strategy for colon cancer. 3-Deazaneplanocin A (DZNep), as an EZH2 inhibitor, can suppress cancer cell growth. However, the anti-cancer role of DZNep in colon cancer cells has been rarely studied. In this study, we demonstrate that DZNep can inhibit the growth and survival of colon cancer HCT116 cells by inducing cellular senescence and apoptosis. The study provides a novel view of anti-cancer mechanisms of DZNep in human colon cancer cells. PMID:26579445

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

    PubMed

    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

    2014-04-01

    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

  14. Balsalazide Potentiates Parthenolide-Mediated Inhibition of Nuclear Factor-κB Signaling in HCT116 Human Colorectal Cancer Cells

    PubMed Central

    Kim, Hyun-Young; Kim, Se-Lim; Park, Young-Ran; Liu, Yu-Chuan; Seo, Seung Young; Kim, Seong Hun; Kim, In Hee; Lee, Seung Ok; Lee, Soo Teik

    2015-01-01

    Background/Aims Balsalazide is an anti-inflammatory drug used in the treatment of inflammatory bowel disease. Balsalazide can reduce inflammatory responses via several mechanisms, including inhibition of nuclear factor-κB (NF-κB) activity. Parthenolide (PT) inhibits NF-κB and exerts promising anticancer effects by promoting apoptosis. The present investigated the antitumor effects of balsalazide, combined with PT, on NF-κB in a representative human colorectal carcinoma cell line, HCT116. Methods We counted cells and conducted annexin-V assays and cell cycle analysis to measure apoptotic cell death. Western blotting was used investigate the levels of proteins involved in apoptosis. Results PT and balsalazide produced synergistic anti-proliferative effects and induced apoptotic cell death. The combination of balsalazide and PT markedly suppressed nuclear translocation of the NF-κB p65 subunit and the phosphorylation of inhibitor of NF-κB. Moreover, PT and balsalazide dramatically enhanced NF-κB p65 phosphorylation. Apoptosis, through the mitochondrial pathway, was confirmed by detecting effects on Bcl-2 family members, cytochrome c release, and activation of caspase-3 and -8. Conclusions Combination treatment with PT and balsalazide may offer an effective strategy for the induction of apoptosis in HCT116 cells. PMID:26130998

  15. Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells.

    PubMed

    Ko, Hyeonseok; Kim, Young-Joo; Amor, Evangeline C; Lee, Jong Wha; Kim, Han-Cheon; Kim, Hee Ju; Yang, Hyun Ok

    2011-09-01

    Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone; DMC) is a naturally occurring chalcone, and it is the major compound isolated from the leaves of Syzygium samarangense (Blume) Merr. & L.M. Perry (Myrtaceae). Experiments were conducted to determine the effects of DMC on cell proliferation, cell-cycle distribution, and programmed cell death in cultures of human colorectal carcinoma HCT116 and LOVO cells. Results showed that DMC inhibited HCT116 and LOVO cell proliferation and induced G(2) /M cell cycle arrest, which was associated with the conversion of microtubule associated protein light chain 3 (LC3)-I-LC3-II, an autophagosome marker, and the incorporation of monodansylcadaverine (MDC), a marker for the acidic compartment of autolysosomes or acidic vesicular organelles. The treatment of HCT116 and LOVO cells using a combination of DMC with an autophagy inhibitor, such as 3-methyladenine (3-MA), beclin 1 siRNA, or atg5 siRNA, suppressed the effect of DMC-mediated anti-proliferation. These results imply that DMC can suppress colorectal carcinoma HCT116 and LOVO cell proliferation through a G(2) /M phase cell-cycle delay, and can induce autophagy, the hallmark of Type II programmed cell death (PCD). Taken together, our results suggest that DMC may be an effective chemotherapeutic agent for HCT116 and LOVO colorectal carcinoma cells. PMID:21538483

  16. Carnosic acid inhibits STAT3 signaling and induces apoptosis through generation of ROS in human colon cancer HCT116 cells.

    PubMed

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

    2016-06-01

    Carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., has been reported to possess anticancer activity. However, the molecular mechanisms underlying the anticancer effects of CA remain poorly understood. Our study revealed that CA treatment significantly reduced the viability of human colon cancer HCT116, SW480, and HT-29 cells. Treatment with CA induced apoptosis, which was associated with the induction of p53 and Bax, inhibition of Mdm2, Bcl-2, and Bcl-xl expression, activation of caspase-9, and -3, and the cleavage of PARP in HCT116 cells. CA inhibited the constitutive phosphorylation, the DNA binding and the reporter gene activity of STAT3 in HCT116 cells by blocking the phosphorylation of upstream JAK2 and Src kinases. Moreover, CA attenuated the expression of STAT3 target gene products, such as survivin, cyclin D1, D2, and D3. In STAT3-overexpressed HCT116 cells, CA inhibited cell viability and the expression of cyclin D1 and survivin. Furthermore, CA treatment induced the generation of ROS in these colon cancer cells. Pretreatment of cells with ROS scavenger N-acetyl cysteine abrogated the inhibitory effect of CA on the JAK2-STAT3/Src-STAT3 signaling and rescued cells from CA-induced apoptosis by blocking the induction of p53 and the cleavage of caspase-3 and PARP in HCT116 cells. However, L-buthionine-sulfoximine, a pharmacological inhibitor of GSH synthesis, increased CA-induced ROS production, thereby potentiating apoptotic effect of CA. In conclusion, our study provides the first report that CA induced apoptosis in HCT116 cells via generation of ROS, induction of p53, activation of caspases, and inhibition of STAT3 signaling pathway. © 2015 Wiley Periodicals, Inc. PMID:26152521

  17. Columbianadin Inhibits Cell Proliferation by Inducing Apoptosis and Necroptosis in HCT116 Colon Cancer Cells.

    PubMed

    Kang, Ji In; Hong, Ji-Young; Choi, Jae Sue; Lee, Sang Kook

    2016-05-01

    Columbianadin (CBN), a natural coumarin from Angelica decursiva (Umbelliferae), is known to have various biological activities including anti-inflammatory and anti-cancer effects. In this study, the anti-proliferative mechanism of actions mediated by CBN was investigated in HCT-116 human colon cancer cells. CBN effectively suppressed the growth of colon cancer cells. Low concentration (up to 25 µM) of CBN induced apoptosis, and high concentration (50 µM) of CBN induced necroptosis. The induction of apoptosis by CBN was correlated with the modulation of caspase-9, caspase-3, Bax, Bcl-2, Bim and Bid, and the induction of necroptosis was related with RIP-3, and caspase-8. In addition, CBN induced the accumulation of ROS and imbalance in the intracellular antioxidant enzymes such as SOD-1, SOD-2, catalase and GPx-1. These findings demonstrate that CBN has the potential to be a candidate in the development of anti-cancer agent derived from natural products. PMID:27098859

  18. Columbianadin Inhibits Cell Proliferation by Inducing Apoptosis and Necroptosis in HCT116 Colon Cancer Cells

    PubMed Central

    Kang, Ji In; Hong, Ji-Young; Choi, Jae Sue; Lee, Sang Kook

    2016-01-01

    Columbianadin (CBN), a natural coumarin from Angelica decursiva (Umbelliferae), is known to have various biological activities including anti-inflammatory and anti-cancer effects. In this study, the anti-proliferative mechanism of actions mediated by CBN was investigated in HCT-116 human colon cancer cells. CBN effectively suppressed the growth of colon cancer cells. Low concentration (up to 25 μM) of CBN induced apoptosis, and high concentration (50 μM) of CBN induced necroptosis. The induction of apoptosis by CBN was correlated with the modulation of caspase-9, caspase-3, Bax, Bcl-2, Bim and Bid, and the induction of necroptosis was related with RIP-3, and caspase-8. In addition, CBN induced the accumulation of ROS and imbalance in the intracellular antioxidant enzymes such as SOD-1, SOD-2, catalase and GPx-1. These findings demonstrate that CBN has the potential to be a candidate in the development of anti-cancer agent derived from natural products. PMID:27098859

  19. Anticancer potential of an ethanol extract of Asiasari radix against HCT-116 human colon cancer cells in vitro.

    PubMed

    Oh, Se-Mi; Kim, Jinhee; Lee, Jun; Yi, Jin-Mu; Oh, Dal-Seok; Bang, Ok-Sun; Kim, No Soo

    2013-01-01

    Radix of Asiasarum heterotropoides var. mandshuricum F. Maekawa (A. radix) has been prescribed for treating pain, allergies and inflammatory disorders in traditional oriental medicine. However, only limited information on the anticancer effects of A. radix is currently available. The aim of this study was to determine the anticancer effect of the ethanol extract of A. radix (EEAR) on HCT-116 human colon cancer cells and to investigate its underlying mechanisms of action. EEAR significantly induced G2/M cell cycle arrest and apoptosis in HCT-116 cells. EEAR-induced apoptosis was observed in parallel with activation of caspases and an increased ratio of Bax (pro-apoptotic)/Bcl2 (anti-apoptotic). Western blot analyses revealed that EEAR elevated the expression of p53 and p21(Waf/Cip1) and decreased the expression of the regulator proteins of G2/M phase progression, such as cdc2 and cyclin B. The upregulation of p53 by EEAR was due to the increased levels of p53 mRNA without a similar increase in proteasome-mediated p53 degradation. EEAR-induced apoptosis in HCT-116 cells was dependent on p53 expression, as determined by siRNA-mediated p53 knockdown. Taken together, these results suggest that EEAR inhibits the growth of the HCT-116 cells through induction of G2/M cell cycle arrest and apoptosis, which are mediated by p53 expression. PMID:23255939

  20. 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)

    Stojković, Danijela Lj.; Jevtić, Verica V.; Radić, Gordana P.; Đačić, Dragana S.; Ćurčić, Milena G.; Marković, Snežana D.; Ðinović, Vesna M.; Petrović, Vladimir P.; Trifunović, Srećko R.

    2014-03-01

    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.

  1. Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the growth of HT29 and HCT116 colon cancer cells*

    PubMed Central

    Ng, Pek Leng; Rajab, Nor Fadilah; Then, Sue Mian; Mohd Yusof, Yasmin Anum; Wan Ngah, Wan Zurinah; Pin, Kar Yong; Looi, Mee Lee

    2014-01-01

    Objective: The combination effect of Piper betle (PB) and 5-fluorouracil (5-FU) in enhancing the cytotoxic potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. Methods: HT29 and HCT116 cells were subjected to 5-FU or PB treatment. 5-FU and PB were then combined and their effects on both cell lines were observed after 24 h of treatment. PB-5-FU interaction was elucidated by isobologram analysis. Apoptosis features of the treated cells were revealed by annexin V/PI stain. High-performance liquid chromatography (HPLC) was performed to exclude any possible chemical interaction between the compounds. Results: In the presence of PB extract, the cytotoxicity of 5-FU was observed at a lower dose (IC50 12.5 μmol/L) and a shorter time (24 h) in both cell lines. Both cell lines treated with 5-FU or PB alone induced a greater apoptosis effect compared with the combination treatment. Isobologram analysis indicated that PB and 5-FU interacted synergistically and antagonistically in inhibiting the growth of HT29 and HCT116 cells, respectively. Conclusions: In the presence of PB, a lower dosage of 5-FU is required to achieve the maximum drug effect in inhibiting the growth of HT29 cells. However, PB did not significantly reduce 5-FU dosage in HCT116 cells. Our result showed that this interaction may not solely contribute to the apoptosis pathway. PMID:25091987

  2. 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

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

    2003-01-01

    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

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

    SciTech Connect

    Kang, Kyungsu; Lee, Kyung-Mi; Yoo, Ji-Hye; Lee, Hee Ju; Kim, Chul Young; College of Pharmacy, Hanyang University, Ansan 426-791 ; Nho, Chu Won

    2012-11-16

    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.

  4. Deoxyelephantopin from Elephantopus scaber Inhibits HCT116 Human Colorectal Carcinoma Cell Growth through Apoptosis and Cell Cycle Arrest.

    PubMed

    Chan, Chim Kei; Chan, Gomathi; Awang, Khalijah; Abdul Kadir, Habsah

    2016-01-01

    Deoxyelephantopin (DET), one of the major sesquiterpene lactones derived from Elephantopus scaber was reported to possess numerous pharmacological functions. This study aimed to assess the apoptosis inducing effects and cell cycle arrest by DET followed by elucidation of the mechanisms underlying cell death in HCT116 cells. The anticancer activity of DET was evaluated by a MTT assay. Morphological and biochemical changes were detected by Hoescht 33342/PI and Annexin V/PI staining. The results revealed that DET and isodeoxyelephantopin (isoDET) could be isolated from the ethyl acetate fraction of E. scaber leaves via a bioassay-guided approach. DET induced significant dose- and time-dependent growth inhibition of HCT116 cells. Characteristics of apoptosis including nuclear morphological changes and externalization of phosphatidylserine were observed. DET also significantly resulted in the activation of caspase-3 and PARP cleavage. Additionally, DET induced cell cycle arrest at the S phase along with dose-dependent upregulation of p21 and phosphorylated p53 protein expression. DET dose-dependently downregulated cyclin D1, A2, B1, E2, CDK4 and CDK2 protein expression. In conclusion, our data showed that DET induced apoptosis and cell cycle arrest in HCT116 colorectal carcinoma, suggesting that DET has potential as an anticancer agent for colorectal carcinoma. PMID:27007366

  5. Molecular mechanism of diallyl disulfide in cell cycle arrest and apoptosis in HCT-116 colon cancer cells.

    PubMed

    Song, Ju-Dong; Lee, Sang Kwon; Kim, Kang Mi; Park, Si Eun; Park, Sung-Joo; Kim, Koan Hoi; Ahn, Soon Cheol; Park, Young Chul

    2009-01-01

    Diallyl disulfide (DADS) is the most prevalent oil-soluble sulfur compound in garlic and inhibits cell proliferation in many cancer cell lines. Here we examined DADS cytotoxicity in a redox-mediated process, involving reactive oxygen species (ROS) production. In the present study, p53-independent cell cycle arrest at G2/M phase was observed with DADS treatment, along with time-dependent increase of cyclin B1. In addition, apoptosis was also observed upon 24-h DADS treatment accompanied by activation of p53. In HCT-116 cells, DADS application induced a dose-dependent increase and time-dependent changes in ROS production. Scavenging of DADS-induced ROS by N-acetyl cysteine or reduced glutathione inhibited cell cycle arrest, apoptosis and p53 activation by DADS. These results suggest that ROS trigger the DADS-induced cell cycle arrest and apoptosis and that ROS are involved in stress-induced signaling upstream of p53 activation. Transfection of p53 small interfering RNA prevents the accumulation of cleaved poly(ADP-ribose) polymerase and sub-G1 cell population by 65% and 35%, respectively. Moreover, DADS-induced apoptosis was also prevented by treatment with oligomycin, which is known to prevent p53-dependent apoptosis by reducing ROS levels in mitochondria. These results suggest that mitochondrial ROS may serve as second messengers in DADS-induced apoptosis, which requires activation of p53. PMID:19202565

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

    SciTech Connect

    Gwak, Jungsug; Song, Taeyun; Song, Jie-Young; Yun, Yeon-Sook; Choi, Il-Whan; Jeong, Yongsu; Shin, Jae-Gook; Department of Clinical Pharmacology, Inje University Busan Paik Hospital, Busan 614-735 ; Oh, Sangtaek

    2009-09-25

    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.

  7. Selected novel 5'-amino-2'-hydroxy-1, 3-diaryl-2-propen-1-ones arrest cell cycle of HCT-116 in G0/G1 phase.

    PubMed

    Simon, Lalitha; Srinivasan, K K; Kumar, Nitesh; Reddy, Neetinkumar D; Biswas, Subhankar; Rao, C Mallikarjuna; Moorkoth, Sudheer

    2016-01-01

    A series of 5'-amino-2'-hydroxy-1,3-diaryl-2-propen-1-ones (AC1-AC15) were synthesized by Claisen-Schmidt condensation of 5'-acetamido-2'-hydroxy acetophenone with various substituted aromatic aldehydes. The synthesized compounds were characterized by FTIR, (1)H NMR and mass spectrometry and evaluated for their selective cytotoxicity using MTT assay on two cancer cell lines namely breast cancer cell line (MCF-7), colon cancer cell line (HCT-116) and one normal kidney epithelial cell line (Vero). Among the tested compounds, AC-10 showed maximum cytotoxic effect on MCF-7 cell line with IC50 value 74.7 ± 3.5 µM. On HCT-116 cells, AC-13 exhibited maximum cytotoxicity with IC50 value 42.1 ± 4.0 µM followed by AC-14 and AC-10 with IC50 values 62 ± 2.3 µM and 95.4 ± 1.7 µM respectively. All tested compounds were found to be safe on Vero cell line with IC50 value more than 200 µM. Based on their highest efficacy on HCT-116, AC-10, AC-13 and AC-14 were selected for mechanistic study on this cell line by evaluating changes nucleomorphological characteristics using acridine orange-ethidium bromide (AOEB) dual stain and by analyzing cell cycle with flow cytometry using propidium iodide stain. In AOEB staining, all three tested compounds showed significant (p < 0.05) increase in percentage apoptotic nuclei compared to control cells, with highest increase in apoptotic nuclei by AC-13 treatment (31 %). Flow cytometric studies showed cell cycle arrest by AC-10 and AC-14 treatment in G0/G1 phase and by AC-13 in G0/G1 and G2/M phase. The study reflected the potential of AC-10, AC-13 and AC-14 to be the lead molecules for further optimization. PMID:27152112

  8. Selected novel 5'-amino-2'-hydroxy-1, 3-diaryl-2-propen-1-ones arrest cell cycle of HCT-116 in G0/G1 phase

    PubMed Central

    Simon, Lalitha; Srinivasan, K. K.; Kumar, Nitesh; Reddy, Neetinkumar D.; Biswas, Subhankar; Rao, C. Mallikarjuna; Moorkoth, Sudheer

    2016-01-01

    A series of 5'-amino-2'-hydroxy-1,3-diaryl-2-propen-1-ones (AC1-AC15) were synthesized by Claisen-Schmidt condensation of 5'-acetamido-2'-hydroxy acetophenone with various substituted aromatic aldehydes. The synthesized compounds were characterized by FTIR, 1H NMR and mass spectrometry and evaluated for their selective cytotoxicity using MTT assay on two cancer cell lines namely breast cancer cell line (MCF-7), colon cancer cell line (HCT-116) and one normal kidney epithelial cell line (Vero). Among the tested compounds, AC-10 showed maximum cytotoxic effect on MCF-7 cell line with IC50 value 74.7 ± 3.5 µM. On HCT-116 cells, AC-13 exhibited maximum cytotoxicity with IC50 value 42.1 ± 4.0 µM followed by AC-14 and AC-10 with IC50 values 62 ± 2.3 µM and 95.4 ± 1.7 µM respectively. All tested compounds were found to be safe on Vero cell line with IC50 value more than 200 µM. Based on their highest efficacy on HCT-116, AC-10, AC-13 and AC-14 were selected for mechanistic study on this cell line by evaluating changes nucleomorphological characteristics using acridine orange-ethidium bromide (AOEB) dual stain and by analyzing cell cycle with flow cytometry using propidium iodide stain. In AOEB staining, all three tested compounds showed significant (p < 0.05) increase in percentage apoptotic nuclei compared to control cells, with highest increase in apoptotic nuclei by AC-13 treatment (31 %). Flow cytometric studies showed cell cycle arrest by AC-10 and AC-14 treatment in G0/G1 phase and by AC-13 in G0/G1 and G2/M phase. The study reflected the potential of AC-10, AC-13 and AC-14 to be the lead molecules for further optimization. PMID:27152112

  9. Flavokawain C Inhibits Cell Cycle and Promotes Apoptosis, Associated with Endoplasmic Reticulum Stress and Regulation of MAPKs and Akt Signaling Pathways in HCT 116 Human Colon Carcinoma Cells

    PubMed Central

    Phang, Chung-Weng; Karsani, Saiful Anuar; Sethi, Gautam; Abd Malek, Sri Nurestri

    2016-01-01

    Flavokawain C (FKC) is a naturally occurring chalcone which can be found in Kava (Piper methysticum Forst) root. The present study evaluated the effect of FKC on the growth of various human cancer cell lines and the underlying associated mechanisms. FKC showed higher cytotoxic activity against HCT 116 cells in a time- and dose-dependent manner in comparison to other cell lines (MCF-7, HT-29, A549 and CaSki), with minimal toxicity on normal human colon cells. The apoptosis-inducing capability of FKC on HCT 116 cells was evidenced by cell shrinkage, chromatin condensation, DNA fragmentation and increased phosphatidylserine externalization. FKC was found to disrupt mitochondrial membrane potential, resulting in the release of Smac/DIABLO, AIF and cytochrome c into the cytoplasm. Our results also revealed that FKC induced intrinsic and extrinsic apoptosis via upregulation of the levels of pro-apoptotic proteins (Bak) and death receptors (DR5), while downregulation of the levels of anti-apoptotic proteins (XIAP, cIAP-1, c-FlipL, Bcl-xL and survivin), resulting in the activation of caspase-3, -8 and -9 and cleavage of poly(ADP-ribose) polymerase (PARP). FKC was also found to cause endoplasmic reticulum (ER) stress, as suggested by the elevation of GADD153 protein after FKC treatment. After the cells were exposed to FKC (60μM) over 18hrs, there was a substantial increase in the phosphorylation of ERK 1/2. The expression of phosphorylated Akt was also reduced. FKC also caused cell cycle arrest in the S phase in HCT 116 cells in a time- and dose-dependent manner and with accumulation of cells in the sub-G1 phase. This was accompanied by the downregulation of cyclin-dependent kinases (CDK2 and CDK4), consistent with the upregulation of CDK inhibitors (p21Cip1 and p27Kip1), and hypophosphorylation of Rb. PMID:26859847

  10. Antiproliferative activity of O4-benzo[c]phenanthridine alkaloids against HCT-116 and HL-60 tumor cells.

    PubMed

    Hatae, Noriyuki; Fujita, Erina; Shigenobu, Saori; Shimoyama, Sayumi; Ishihara, Yuhsuke; Kurata, Yuhki; Choshi, Tominari; Nishiyama, Takashi; Okada, Chiaki; Hibino, Satoshi

    2015-07-15

    The O4-benzo[c]phenanthridine alkaloids exhibit potent antiproliferative activity against cancer cells, which is derived from their ability to inhibit of topoisomerase I and II. It has been reported that in the alkaloids a cationic quaternary ammonium atom, which results in resonance effects between ring A and B, is necessary for increased antiproliferative activity. These findings indicate the role of their substituents at ring A on inhibition of tumor cell proliferation. In the present study, we systematically assessed the cytotoxic activities of naturally occurring alkaloids and their derivatives containing various ring A substituents against two tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. Among the cationic iminium alkaloids, which displayed more potent activity than the corresponding neutral derivatives, and the 7,8-oxygenated benzo[c]phenanthridine alkaloids, chelerythrine and NK109, exhibited stronger antiproliferative activity than the 8,9- and 9,10-oxygenated alkaloids. The activity of cationic iminium alkaloids could be correlated with the bond lengths of their ring A substituents and the electrostatic potentials of their ammonium molecules by DFT calculation. PMID:26026362

  11. Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak.

    TOXLINE Toxicology Bibliographic Information

    Wang C; Youle RJ

    2012-06-28

    The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax (Bcl2-associated X protein) and Bak (Bcl2-antagonist/killer 1). Although Bax and Bak seem to have redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for fluorouracil-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax, but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant, but they are counteracted by distinct anti-apoptotic Bcl-2 family proteins in different species.

  12. Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1's inhibitory effect on Bak.

    PubMed

    Wang, C; Youle, R J

    2012-06-28

    The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax (Bcl2-associated X protein) and Bak (Bcl2-antagonist/killer 1). Although Bax and Bak seem to have redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for fluorouracil-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax, but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant, but they are counteracted by distinct anti-apoptotic Bcl-2 family proteins in different species. PMID:22056880

  13. Predominant requirement of Bax for apoptosis in HCT116 cells is determined by Mcl-1s inhibitory effect on Bak

    PubMed Central

    Wang, Chunxin; Youle, Richard J.

    2011-01-01

    The intrinsic mitochondrial apoptotic pathway acts through two core pro-apoptotic proteins Bax and Bak. While Bax and Bak seem to play redundant roles in apoptosis, accumulating evidence also suggests that they might not be interchangeable under certain conditions, at least in some human cell lines. Here we report the generation of Bak knockout as well as BaxBak double knockout HCT116 human colon carcinoma cells. We show that Bak is dispensable for apoptosis induced by a variety of stimuli including ABT-737 but not for 5-FU-induced apoptosis. In addition, Bax deficiency only provides partial protection against camptothecin and cisplatin-induced apoptosis and no protection against killing by Puma or ABT-737 plus Noxa overexpression. Moreover, Bak is activated normally in response to many chemotherapeutic drugs in the presence of Bax but remains kept in check by Mcl-1 in the absence of Bax. Our data suggest that Bax and Bak are functionally redundant but they are counteracted by distinct anti-apopotic Bcl-2 family proteins in different species. PMID:22056880

  14. Eugenia jambolana (Java Plum) Fruit Extract Exhibits Anti-Cancer Activity against Early Stage Human HCT-116 Colon Cancer Cells and Colon Cancer Stem Cells

    PubMed Central

    Charepalli, Venkata; Reddivari, Lavanya; Vadde, Ramakrishna; Walia, Suresh; Radhakrishnan, Sridhar; Vanamala, Jairam K. P

    2016-01-01

    The World Health Organization predicts over a 70% increase in cancer incidents in developing nations over the next decade. Although these nations have limited access to novel therapeutics, they do have access to foods that contain chemopreventive bioactive compounds such as anthocyanins, and as such, consumption of these foods can be encouraged to combat cancer. We and others have previously characterized the anti-colon cancer properties of dietary anthocyanins from different sources. Eugenia jambolana (Java plum) is a tropical medicinal fruit rich in anthocyanins, however, its anti-colon cancer properties are not well characterized. Furthermore, recent evidence suggests that colon cancer stem cells (colon CSCs) promote resistance to chemotherapy, relapse of tumors and contribute to poor prognosis. The objectives of this study were to 1) characterize the anthocyanin profile of Java plum using HPLC-MS; and 2) determine the anti-proliferative (cell counting and MTT) and pro-apoptotic (TUNEL and caspase 3/7 glo assay) properties of Java plum fruit extract (JPE) using HCT-116 colon cancer cell line and colon CSCs (positive for CD 44, CD 133 and ALDH1b1 markers). HPLC-MS analysis showed that JPE contains a variety of anthocyanins including glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin. JPE anthocyanins suppressed (p < 0.05) proliferation in HCT-116 cells and elevated (p < 0.05) apoptosis in both HCT-116 cells and colon CSCs. JPE also suppressed the stemness in colon CSCs as evaluated using colony formation assay. These results warrant further assessment of the anti-cancer activity of JPE, and its molecular mechanisms using pre-clinical models of colon cancer. PMID:26927179

  15. Eugenia jambolana (Java Plum) Fruit Extract Exhibits Anti-Cancer Activity against Early Stage Human HCT-116 Colon Cancer Cells and Colon Cancer Stem Cells.

    PubMed

    Charepalli, Venkata; Reddivari, Lavanya; Vadde, Ramakrishna; Walia, Suresh; Radhakrishnan, Sridhar; Vanamala, Jairam K P

    2016-01-01

    The World Health Organization predicts over a 70% increase in cancer incidents in developing nations over the next decade. Although these nations have limited access to novel therapeutics, they do have access to foods that contain chemopreventive bioactive compounds such as anthocyanins, and as such, consumption of these foods can be encouraged to combat cancer. We and others have previously characterized the anti-colon cancer properties of dietary anthocyanins from different sources. Eugenia jambolana (Java plum) is a tropical medicinal fruit rich in anthocyanins, however, its anti-colon cancer properties are not well characterized. Furthermore, recent evidence suggests that colon cancer stem cells (colon CSCs) promote resistance to chemotherapy, relapse of tumors and contribute to poor prognosis. The objectives of this study were to 1) characterize the anthocyanin profile of Java plum using HPLC-MS; and 2) determine the anti-proliferative (cell counting and MTT) and pro-apoptotic (TUNEL and caspase 3/7 glo assay) properties of Java plum fruit extract (JPE) using HCT-116 colon cancer cell line and colon CSCs (positive for CD 44, CD 133 and ALDH1b1 markers). HPLC-MS analysis showed that JPE contains a variety of anthocyanins including glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin. JPE anthocyanins suppressed (p < 0.05) proliferation in HCT-116 cells and elevated (p < 0.05) apoptosis in both HCT-116 cells and colon CSCs. JPE also suppressed the stemness in colon CSCs as evaluated using colony formation assay. These results warrant further assessment of the anti-cancer activity of JPE, and its molecular mechanisms using pre-clinical models of colon cancer. PMID:26927179

  16. Apigenin-induced apoptosis is enhanced by inhibition of autophagy formation in HCT116 human colon cancer cells.

    PubMed

    Lee, Yujin; Sung, Bokyung; Kang, Yong Jung; Kim, Dong Hwan; Jang, Jung-Yoon; Hwang, Seong Yeon; Kim, Minjung; Lim, Hyun Sook; Yoon, Jeong-Hyun; Chung, Hae Young; Kim, Nam Deuk

    2014-05-01

    Apigenin (4',5,7-trihydroxyflavone) is a natural flavonoid, shown to have chemopreventive and/or anticancer properties in a variety of human cancer cells. The involvement of autophagy in apigenin-induced apoptotic cell death of HCT116 human colon cancer cells was investigated. Apigenin induced suppression of cell growth in a concentration-dependent manner in HCT116 cells. Flow cytometric analyses indicated that apigenin resulted in G2/M phase arrest. This flavone also suppressed the expression of both cyclin B1 and its activating partners, Cdc2 and Cdc25c, whereas the expression of cell cycle inhibitors, such as p53 and p53-dependent p21(CIP1/WAF1), was increased after apigenin treatment. Apigenin induced poly (ADP-ribose) polymerase (PARP) cleavage and decreased the levels of procaspase-8, -9 and -3. In addition, the apigenin-treated cells exhibited autophagy, as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles by flow cytometry. Furthermore, the results of the western blot analysis revealed that the levels of LC3-II, the processed form of LC3-I, was increased by apigenin. Treatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly enhanced the apoptosis induced by apigenin, which was accompanied by an increase in the levels of PARP cleavage. These results indicate that apigenin has apoptosis- and autophagy-inducing effects in HCT116 colon cancer cells. Autophagy plays a cytoprotective role in apigenin-induced apoptosis, and the combination of apigenin and an autophagy inhibitor may be a promising strategy for colon cancer control. PMID:24626522

  17. Iron depletion in HCT116 cells diminishes the upregulatory effect of phenethyl isothiocyanate on heme oxygenase-1.

    PubMed

    Bolloskis, Michael P; Carvalho, Fabiana P; Loo, George

    2016-04-15

    Some of the health-promoting properties of cruciferous vegetables are thought to be partly attributed to isothiocyanates. These phytochemicals can upregulate the expression of certain cytoprotective stress genes, but it is unknown if a particular nutrient is involved. Herein, the objective was to ascertain if adequate iron is needed for enabling HCT116 cells to optimally express heme oxygenase-1 (HO-1) when induced by phenethyl isothiocyanate (PEITC). PEITC increased HO-1 expression and also nuclear translocation of Nrf2, which is a transcription factor known to activate the HO-1 gene. However, in HCT116 cells that were made iron-deficient by depleting intracellular iron with deferoxamine (DFO), PEITC was less able to increase HO-1 expression and nuclear translocation of Nrf2. These suppressive effects of DFO were overcome by replenishing the iron-deficient cells with the missing iron. To elucidate these findings, it was found that PEITC-induced HO-1 upregulation can be inhibited with thiol antioxidants (glutathione and N-acetylcysteine). Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin) and a superoxide scavenger (Tiron) each inhibited PEITC-induced HO-1 upregulation. In doing so, diphenyleneiodonium was the most potent and also inhibited nuclear translocation of redox-sensitive Nrf2. Collectively, the results imply that the HO-1 upregulation by PEITC involves an iron-dependent, oxidant signaling pathway. Therefore, it is concluded that ample iron is required to enable PEITC to fully upregulate HO-1 expression in HCT116 cells. As such, it is conceivable that iron-deficient individuals may not reap the full health benefits of eating PEITC-containing cruciferous vegetables that via HO-1 may help protect against multiple chronic diseases. PMID:26945724

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

    SciTech Connect

    Deng Haiyun; Makizumi, Ryouji; Ravikumar, T.S.; Dong Huali; Yang Wancai; Yang, W.-L. . E-mail: wlyang@nshs.edu

    2007-03-10

    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.

  19. Phytochemical investigation of Gynura bicolor leaves and cytotoxicity evaluation of the chemical constituents against HCT 116 cells.

    PubMed

    Teoh, Wuen Yew; Tan, Hooi Poay; Ling, Sui Kiong; Abdul Wahab, Norhanom; Sim, Kae Shin

    2016-02-01

    Gynura bicolor (Compositae) is a popular vegetable in Asia and believed to confer a wide range of benefits including anti-cancer. Our previous findings showed that the ethyl acetate extract of G. bicolor possessed cytotoxicity and induced apoptotic and necrotic cell death in human colon carcinoma cells (HCT 116). A combination of column chromatography had been used to purify chemical constituents from the ethyl acetate and water extract of G. bicolor leaves. Eight chemical constituents 5-p-trans-coumaroylquinic acid (I), 4-hydroxybenzoic acid (II), rutin (III), kampferol-3-O-rutinoside (IV), 3,5-dicaffeoylquinic acid (V), kampferol-3-O-glucoside (VI), guanosine (VII) and chlorogenic acid (VIII) were isolated from G. bicolor grown in Malaysia. To our best knowledge, all chemical constituents were isolated for the first time from G. bicolor leaves except rutin (III). 3,5-dicaffeoylquinic acid (V), guanosine (VII) and chlorogenic acid (VIII) demonstrated selective cytotoxicity (selective index>3) against HCT 116 cancer cells compared to CCD-18Co human normal colon cells. PMID:25738869

  20. Crocin and Quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress.

    PubMed

    Ben Salem, Intidhar; Prola, Alexandre; Boussabbeh, Manel; Guilbert, Arnaud; Bacha, Hassen; Abid-Essefi, Salwa; Lemaire, Christophe

    2015-11-01

    Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity. PMID:26134454

  1. 150 kDa glycoprotein isolated from Solanum nigrum Linne stimulates caspase-3 activation and reduces inducible nitric oxide production in HCT-116 cells.

    PubMed

    Lee, Sei-Jung; Lim, Kye-Taek

    2006-10-01

    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. SNL glycoprotein showed remarkable cytotoxic and apoptotic effects at 40 microg/ml of SNL glycoprotein for 4 h in HCT-116 cells. In the activity of the apoptotic related proteins [caspase-3 and poly(ADP-ribose)polymerase (PARP)], the results showed that SNL glycoprotein (40 microg/ml) has a stimulatory effect on caspase-3 activation and PARP cleavage in HCT-116 cells. Moreover, SNL glycoprotein blocked nuclear factor-kappa B (NF-kappaB) activation and reduced inducible nitric oxide (iNO) production. Interestingly, pyrrolidine dithiocarbamate (PDTC, for NF-kappaB inhibitor) and N omega-Nitro-L-arginine methylester hydrochloride (L-NAME, for NO inhibitor) effectively stimulated the caspase-3 activation in HCT-116 cells. The results in this experiment indicated that SNL glycoprotein induces apoptosis through the NF-kappaB activation and inducible nitric oxide (iNO) production in HCT-116 cells. Here, we speculate that SNL glycoprotein is one of the chemotherapeutic agents and of the modulators for apoptotic signals in HCT-116 cells. PMID:16527444

  2. Methylselenol, a selenium metabolite, plays common and different roles in cancerous colon HCT116 cell and noncancerous NCM460 colon cell proliferation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methylselenol has been hypothesized to be a critical selenium (Se) metabolite for anticancer activity in vivo. To determine differential chemopreventive effects of methylselenol on colon cancer cells versus colon noncancerous cells, colon-cancer-derived HCT-116 cells and noncancerous colonic NCM460 ...

  3. Cochlioquinone derivatives with apoptosis-inducing effects on HCT116 colon cancer cells from the phytopathogenic fungus Bipolaris luttrellii L439.

    PubMed

    Qi, Qiu-Yue; Huang, Li; He, Lu-Wei; Han, Jun-Jie; Chen, Quan; Cai, Lei; Liu, Hong-Wei

    2014-12-01

    A new cochlioquinone derivative, cochlioquinone F (1), as well as three known compounds, anhydrocochlioquinone A (2), isocochlioquinone A (3), and isocochlioquinone C (4), were isolated from the PDB (potato dextrose broth) culture of the phytopathogenic fungus Bipolaris luttrellii. The structure of 1 was elucidated on the basis of NMR techniques. The apoptosis-inducing effects of compounds 1-4 were evaluated against HCT116 cancer cells. Compound 2 exhibited the strongest activity in inducing apoptosis on HCT116 cells within the range of 10-30 μM. In addition, the caspase activation, the release of cytochrome c from mitochondria, and the downregulation of Bcl-2 protein in HCT116 cells treated with compound 2 were detected. PMID:25491333

  4. Acquired resistance to decitabine and cross-resistance to gemcitabine during the long-term treatment of human HCT116 colorectal cancer cells with decitabine

    PubMed Central

    HOSOKAWA, MIKA; SAITO, MAI; NAKANO, AIKO; IWASHITA, SAKURA; ISHIZAKA, AYANO; UEDA, KUMIKO; IWAKAWA, SEIGO

    2015-01-01

    The aim of the present study was to determine the effects of long-term exposure of decitabine (DAC) to HCT116 colorectal cancer (CRC) cells on the acquisition of resistance to DAC as well as cross-resistance to anticancer drugs used for CRC or other epigenetic modifiers. In the present study, DAC-resistant HCT116 CRC cells were established through long-term treatment with increasing concentrations of DAC (10 to 540 nM); and the cross-resistance to other drugs was subsequently examined. DAC-resistant HCT116 cells were obtained following a 104-day treatment with DAC, including DAC-free intervals. The results demonstrated that the IC50 value of DAC was increased ~100-fold in DAC-resistant HCT116 cells. Messenger (m)RNA expression of secreted frizzed-related protein 1 (SFRP1), which is regulated by DNA methylation, was not detected in DAC-resistant cells; however, SFRP1 mRNA was present in HCT116 cells treated with DAC for 52 days. DNA methyltransferase 1 (DNMT1) protein levels were slightly decreased until day 81 and then returned to control levels in DAC-resistant cells. Further experiments using DAC-resistant HCT116 cells revealed that these cells exhibited cross-resistance to gemcitabine (Gem); however, cross-resistance was not observed for other DNMT inhibitors (azacitidine and zebularine), histone deacetylase inhibitors (trichostatin A, vorinostat and valproic acid) or anticancer drugs for CRC (5-fluorouracil, irinotecan and oxaliplatin). Furthermore, the protein expression levels of cytidine deaminase (CDA) were increased, while those of deoxycytidine kinase (dCK) were decreased in DAC-resistant HCT116 cells; by contrast, the mRNA expression levels for these proteins were not significantly altered. In conclusion, the results of the present study indicated that the long-term treatment of HCT116 cells with DAC led to the acquisition of resistance to both DAC and Gem. In addition, these results may be partly attributed to changes in CDA and/or dCK, which are involved in metabolic pathways common to these two drugs. PMID:26622566

  5. Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116 Cells and Theoretical Rationalization.

    PubMed

    Ali, Firoj; Saha, Sukdeb; Maity, Arunava; Taye, Nandaraj; Si, Mrinal Kanti; Suresh, E; Ganguly, Bishwajit; Chattopadhyay, Samit; Das, Amitava

    2015-10-15

    A new rhodamine-based reagent (L1), trapped inside the micellar structure of biologically benign Triton-X 100, could be used for specific recognition of Cr(III) in aqueous buffer medium having physiological pH. This visible light excitable reagent on selective binding to Cr(III) resulted in a strong fluorescence turn-on response with a maximum at ∼583 nm and tail of that luminescence band extended until 650 nm, an optical response that is desired for avoiding the cellular autofluorescence. Interference studies confirm that other metal ions do not interfere with the detection process of Cr(III) in aqueous buffer medium having pH 7.2. To examine the nature of binding of Cr(III) to L1, various spectroscopic studies are performed with the model reagent L2, which tend to support Cr(III)-η(2)-olefin π-interactions involving two olefin bonds in molecular probe L1. Computational studies are also performed with another model reagent LM to examine the possibility of such Cr(III)-η(2)-olefin π-interactions. Presumably, polar functional groups of the model reagent LM upon coordination to the Cr(III) center effectively reduce the formal charge on the metal ion and this is further substantiated by results of the theoretical studies. This assembly is found to be cell membrane permeable and shows insignificant toxicity toward live colon cancer cells (Hct116). Confocal laser scanning microscopic studies further revealed that the reagent L1 could be used as an imaging reagent for detection of cellular uptake of Cr(III) in pure aqueous buffer medium by Hct116 cells. Examples of a specific reagent for paramagnetic Cr(III) with luminescence ON response are scanty in the contemporary literature. This ligand design helped us in achieving the turn on response by utilizing the conversion from spirolactam to an acyclic xanthene form on coordination to Cr(III). PMID:26390369

  6. Generation of ROS by CAY10598 leads to inactivation of STAT3 signaling and induction of apoptosis in human colon cancer HCT116 cells.

    PubMed

    Chae, I G; Kim, D-H; Kundu, J; Jeong, C-H; Kundu, J K; Chun, K-S

    2014-11-01

    Prostaglandin E2 (PGE2) has been reported to play critical roles in cell fate decision by interacting with four types of prostanoid receptors such as EP1, EP2, EP3 and EP4. The present study was aimed at investigating the effect of the EP4-specific agonist CAY10598 in human colon cancer HCT116 cells. Our study revealed that treatment with CAY10598 significantly reduced the cell viability and induced apoptosis in HCT116 cells, as evidenced by the induction of p53 and Bax, release of cytochrome c, cleavage of caspase-9, -7, and -3, and PARP, and the inhibition of Bcl-2, Bcl-xL and survivin expression. Moreover, treatment with CAY10598 diminished the phosphorylation of JAK2, leading to the attenuation of STAT3 activation in HCT116 cells. CAY10598-induced apoptosis in cells which were transiently transfected with EP4 siRNA or treated with an EP4 antagonist prior to incubation with the compound remained unaffected, suggesting an EP4-independent mechanism of apoptosis induction by CAY10598. We found that treatment with CAY10598 generated reactive oxygen species (ROS) and pretreatment of cells with N-acetyl cysteine rescued cells from apoptosis by abrogating the inhibitory effect of CAY10598 on the activation of JAK2/STAT3 signaling. In conclusion, CAY10598 induced apoptosis in HCT116 cells in an EP4-independent manner, but through the generation of ROS and inactivation of JAK2/STAT3 signaling. PMID:25096910

  7. Monoterpene indole alkaloid hydrazone derivatives with apoptosis inducing activity in human HCT116 colon and HepG2 liver carcinoma cells.

    PubMed

    Paterna, Angela; Borralho, Pedro M; Gomes, Sofia E; Mulhovo, Silva; Rodrigues, Cecília M P; Ferreira, Maria-José U

    2015-09-01

    The derivatization of dregamine (1) and tabernaemontanine (2), two epimeric monoterpene indole alkaloids isolated from the methanol extract of the roots of Tabernaemontana elegans, with several hydrazines and hydroxylamine gave rise to ten new derivatives (3-12). Their structures were assigned by spectroscopic methods, including 2D NMR experiments. The compounds were tested for their ability to induce apoptosis in HCT116 colon and HepG2 liver cancer cells. Firstly, the cytotoxicity of all compounds (1-12) was evaluated in both cell lines by the MTS assay. The most active compounds (6, 9, 10) along with 1 and 2 were further investigated for their apoptosis induction capability by Guava ViaCount flow cytometry assays, nuclear morphology evaluation by Hoechst staining, and caspase-3/7 activity assays. Compounds 9 and 10 showed promising apoptosis induction profile, displaying higher activities than 5-fluorouracil, the mainstay in colon cancer treatment. PMID:26169128

  8. A novel fluorescence probe for estimation of cysteine/histidine in human blood plasma and recognition of endogenous cysteine in live Hct116 cells.

    PubMed

    Reddy G, Upendar; Agarwalla, Hridesh; Taye, Nandaraj; Ghorai, Suvankar; Chattopadhyay, Samit; Das, Amitava

    2014-09-01

    A new Cu(II)-complex is used as a "Turn-On" luminescence probe for specific detection of endogenous Cys in live Hct116 cells and Cys present in human blood plasma without any interference from other amino acids, especially GSH and Hcy. Difference in the mechanistic pathway for Cys and His recognition is discussed. PMID:25027189

  9. Activation of ER stress and apoptosis by α- and β-zearalenol in HCT116 cells, protective role of Quercetin.

    PubMed

    Ben Salem, Intidhar; Prola, Alexandre; Boussabbeh, Manel; Guilbert, Arnaud; Bacha, Hassen; Lemaire, Christophe; Abid-Essefi, Salwa

    2016-03-01

    Zearalenone (ZEN) and its metabolites are found in many food products and are known to induce many toxic effects. The major ZEN metabolites are α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). The mechanisms by which they mediate their cytotoxic effects are not well known and seem to differ depending on the type of cells. We investigated the possible underlying mechanism in α-ZOL and β-ZOL-induced toxicity in HCT116 cells. We showed that cell treatment with α-ZOL/β-ZOL generated endoplasmic reticulum (ER) stress and activated the Unfolded Protein Response (UPR) as evidenced by XBP1 mRNA splicing and up-regulation of GADD34, GRP78, ATF4 and CHOP. Apoptosis was triggered by ZEN metabolites-induced ER stress, and executed through a mitochondria-dependent pathway, characterized by a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O2•(-) and caspase 3 activation. Cellular deficiency of the pro-apoptotic proteins Bax and Bak protected cells against α/β-ZOL-induced toxicity. However, treatment with α-ZOL or β-ZOL combined with Quercetin (QUER), a common dietary flavonoid with well-known antioxidant activity, significantly reduced damage induced by α and β-ZOL in all tested markers. We concluded that QUER protects against the cellular toxicity of α and β-ZOL.×. PMID:26584763

  10. Kaempferol induces apoptosis in human HCT116 colon cancer cells via the Ataxia-Telangiectasia Mutated-p53 pathway with the involvement of p53 Upregulated Modulator of Apoptosis.

    PubMed

    Li, Wei; Du, Bingna; Wang, Tianyi; Wang, Siling; Zhang, Jinghai

    2009-01-27

    Dietary flavonols have been found to possess preventive and therapeutic potential against several kinds of cancers. This study is conducted to investigate the anti-proliferation effects of kaempferol, a major component of food flavonols, against colon cancer cells. In the human HCT116 colon cancer cell line, kaempferol induced p53-dependent growth inhibition and apoptosis. Furthermore, kaempferol was found to induce cytochrome c release from mitochondria and activate caspase-3 cleavage. The Bcl-2 family proteins including PUMA were involved in this process. Kaempferol also induced ATM and H2AX phosphorylation in HCT116 cells, inhibition of ATM by a chemical inhibitor resulted in abrogation of the downstream apoptotic cascades. These findings suggest kaempferol could be a potent candidate for colorectal cancer management. PMID:19028473

  11. A novel quinazolinone chalcone derivative induces mitochondrial dependent apoptosis and inhibits PI3K/Akt/mTOR signaling pathway in human colon cancer HCT-116 cells.

    PubMed

    Wani, Zahoor Ahmad; Guru, Santosh Kumar; Rao, A V Subba; Sharma, Sonia; Mahajan, Girish; Behl, Akanksha; Kumar, Ashok; Sharma, P R; Kamal, Ahmed; Bhushan, Shashi; Mondhe, Dilip M

    2016-01-01

    We have synthesized a novel quinazolinone chalcone derivative (QC) and first time reported its in-vitro and in-vivo anticancer potential. It inhibited the cell proliferation of different cancer cell lines like PC-3, Panc-1, Mia-Paca-2, A549, MCF-7 and HCT-116. It induces apoptosis as measured by several biological endpoints such as apoptotic body formation, evident by Hoechst and scanning electron microscopy, enhanced annexinV-FITC binding of the cells, increased sub-G0 cell fraction, loss of mitochondrial membrane potential (Δψm), reduction of Bcl-2/Bax ratio, activation of caspase-9, caspase-3 and PARP-1 (poly-ADP Ribose polymerase) cleavage in HCT-116 cells. In spite of apoptosis, QC significantly hammers the downstream and upstream signaling cascade of PI3K/Akt/mTOR pathway and cell cycle regulator Skp-2, p21 and p27. Interestingly, QC induces the S and G2/M phase of HCT-116 cells at experimental doses. QC inhibits the tumor growth of Ehrlich ascites carcinoma (EAC), Ehrlich tumor (ET, solid) and sarcoma-180(solid) mice models. Furthermore, it was found to be non-toxic as no animal mortality (0/7) occurred during experimental doses. The present study provides an insight of anticancer potential of QC, which may be useful in managing and treating cancer. PMID:26615871

  12. Crataegus azarolus Leaves Induce Antiproliferative Activity, Cell Cycle Arrest, and Apoptosis in Human HT-29 and HCT-116 Colorectal Cancer Cells.

    PubMed

    Mustapha, Nadia; Pinon, Aline; Limami, Youness; Simon, Alain; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

    2016-05-01

    Limited success has been achieved in extending the survival of patients with metastatic colorectal cancer (CRC). There is a strong need for novel agents in the treatment and prevention of CRC. Therefore, in the present study we evaluated the antiproliferative and pro-apoptotic potential of Crataegus azarolus ethyl acetate extract in HCT-116 and HT-29 human colorectal cancer cell lines. Moreover, we attempted to investigate the signaling pathways that should be involved in its cytotoxic effect. The Crataegus azarolus ethyl acetate extract-induced growth inhibitory effect was associated with DNA fragmentation, sub-G1 peak, loss of mitochondrial potential, and poly (ADP-ribose) polymerase (PARP) cleavage. In addition, ethyl acetate extract of Crataegus azarolus induced the cleavage of caspase-8. It has no effect on steady-state levels of total Bcl-2 protein. Whereas Bax levels decreased significantly in a dose-dependent manner in both tested cell lines. Taken together, these findings confirm the involvement of the extrinsic pathway of apoptosis. The apoptotic cell death induced by ethyl acetate extract of Crataegus azarolus was accompanied by an enhancement of the p21 expression but not through p53 activation in human colorectal cancer cells. The above-mentioned data provide insight into the molecular mechanisms of Crataegus azarolus ethyl acetate extract-induced apoptosis in CRC. Therefore, this compound should be a potential anticancer agent for the treatment of CRC. J. Cell. Biochem. 117: 1262-1272, 2016. © 2015 Wiley Periodicals, Inc. PMID:26495895

  13. Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation.

    PubMed

    Zeng, Huawei; Claycombe, Kate J; Reindl, Katie M

    2015-10-01

    Consumption of a high-fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk, while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer-preventive effects. To distinguish these opposing effects of DCA and butyrate (two major metabolites in colon lumen), we examined the effects of physiologically relevant doses of butyrate (0.5-2 mmol/l) and DCA (0.05-0.3 mmol/l) on colon cell proliferation. We hypothesize that butyrate and DCA each modulates the cell cycle and apoptosis via common and distinct cellular signaling targets. In this study, we demonstrated that both butyrate and DCA inhibited cell proliferation by up to 89% and 92% and increased cell apoptosis rate by up to 3.1- and 4.5-fold, respectively. Cell cycle analyses revealed that butyrate led to an increase in G1 and G2 fractions with a concomitant drop in the S-phase fraction, but DCA induced an increase in only G1 fraction with a concomitant drop in the S-phase fraction when compared with the untreated cells. The examination of early cellular signaling revealed that DCA but not butyrate increased intracellular reactive oxygen species, genomic DNA breakage, the activation of ERK1/2, caspase-3 and PARP. In contrast, DCA decreased activated Rb protein level, and butyrate but not DCA increased p21 expression. Collectively, although both butyrate and DCA inhibit colonic cell proliferation, butyrate increases tumor suppressor gene expression, whereas DCA decreases tumor suppressor activation in cell cycle and apoptosis pathways. PMID:26026836

  14. Novel structurally similar chromene derivatives with opposing effects on p53 and apoptosis mechanisms in colorectal HCT116 cancer cells.

    PubMed

    Lima, Cristovao F; Costa, Marta; Proena, M F; Pereira-Wilson, Cristina

    2015-05-25

    In the present work, novel chromene derivatives fused with the imidazo[1,2-a]pyridine nucleus were tested for their anticancer potential in the human colorectal cancer HCT116 cells. Compounds 2a and 2c showed significant growth inhibitory activity with GI50 of 15 ?M and 11 ?M, respectively. Compound 2c, the most potent, has a carbamate group in position 8 of the pyridine ring, and showed significant cell cycle arrest and induction of cell death by apoptosis, even at 5 ?M. Besides different potencies, chromene analogs 2a and 2c showed different mechanisms of action. Whereas the carbamate-free chromene 2a induced cell cycle arrest at G1/G0 phase, compound 2c showed to arrest cell cycle at both S and G2 phases. Chromene derivative 2a at concentrations higher than its GI50 remarkably induced caspases-dependent apoptosis in a p53-independent manner. On the other hand, compound 2c increased significantly p53 levels and induced apoptosis in a p53- and caspases-dependent manner, even at concentrations lower than its GI50. Both compounds increased the Bax/Bcl-2 ratio, induced mitochondria depolarization and activated MAP kinases. In conclusion, two novel and structurally similar chromene derivatives showed cytotoxicity to HCT16 cells through opposing effects on p53 levels and apoptosis mechanisms, which may be relevant for further development of drugs acting on distinct molecular targets useful in the treatment of cancers with different genetic profiles and for personalized medicine. PMID:25746954

  15. Tumor Suppressor DLEC1 can Stimulate the Proliferation of Cancer Cells When AP-2ɑ2 is Down-Regulated in HCT116

    PubMed Central

    Qiu, Guo-Hua; Xie, Xiaojin; Deng, Linhong; Hooi, Shing Chuan

    2015-01-01

    Background: The molecular mechanisms of tumor suppressor gene DLEC1 are largely unknown. Objectives: In this study, we established DLEC1 over-expression stable clones to study the cellular function of DLEC1 in the colorectal cancer cell line, HCT116. Materials and Methods: Stable clones with DLEC1 over-expression were first established by the transfection of DLEC1 expression construct pcDNA31DLEC1 in HCT116. On G418 selection, positive stable clones were screened for DLEC1 expression level by conventional reverse transcription-polymerase chain reaction (RT-PCR), and verified by real-time RT-PCR and Western blotting. Subsequently, these stable clones were subjected to colony formation and cell cycle analyses and identification of factors involved in G1 arrest. Lastly, three stable clones, DLEC1-7 (highest DLEC1 expression), DLEC1-3 (lowest expression) and pcDNA31 vector control, were employed to analyze cell proliferation and cell cycle after AP-2α2 knockdown by siRNAs. Results: The DLEC1 over-expression was found to reduce the number of colonies in colony formation and to induce G1 arrest in seven clones, and apoptosis in one clone in the cell cycle analysis. Furthermore, regardless of the different cell cycle defects in all eight stable clones, the expression level of transcriptional factor AP-2α2 was found to be elevated. More interestingly, we found that when AP-2α2 was knocked down, DLEC1 over-expression neither suppressed cancer cell growth nor induced G1 arrest, yet, instead promoted cell growth and decreased cells in the G1 fraction. This promotion of cell proliferation and release of G1 cells also seemed to be proportional to DLEC1 expression levels in DLEC1 stable clones. Conclusions: DLEC1 suppresses tumor cell growth the presence of AP-2α2 and stimulates cell proliferation in the down-regulation of AP-2α2 in DLEC1 over-expression stable clones of HTC116. PMID:26834787

  16. Effect of the orthoquinone moiety in 9,10-phenanthrenequinone on its ability to induce apoptosis in HCT-116 and HL-60 cells.

    PubMed

    Hatae, Noriyuki; Nakamura, Jun; Okujima, Tetsuo; Ishikura, Minoru; Abe, Takumi; Hibino, Satoshi; Choshi, Tominari; Okada, Chiaki; Yamada, Hiroko; Uno, Hidemitsu; Toyota, Eiko

    2013-08-15

    9,10-Phenanthrenequinone (9,10-PQ) is one of the most abundant quinones among diesel exhaust particulates. Recent data have suggested that quinones induce apoptosis in immune, epithelial and tumor cells, leading to respirator illness; however, the mechanisms by which quinones induce apoptosis and the structure required for this remain unknown. We studied the antitumor activity of 9,10-PQ analogs against two human tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. The loss of the cis-orthoquinone unit in 9,10-PQ abrogated its ability to induce apoptosis in the two tumor cell lines, and the LC50 values of these analogs were indicated over 10 μM. An analog of 9,10-PQ in which the biaryl unit had been deleted displayed a reduced ability to induce tumor cell apoptosis, while the analogs 1,10-phenanthroline-5,6-dione (9) and pyrene-4,5-dione (10), which also had modified biaryl units, exhibited increased tumor cell apoptotic activity. The cis-orthoquinone unit in 9,10-PQ was identified as essential for its ability to induce apoptosis in tumor cells, and its biaryl unit is also considered to influence orthoquinone-mediated apoptotic activity. PMID:23816373

  17. Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of β-catenin in HCT116 cells

    SciTech Connect

    Lee, Kyung-Mi; Yun, Ji Ho; Lee, Dong Hwa; Park, Young Gyun; Son, Kun Ho; Nho, Chu Won; Kim, Yeong Shik

    2015-04-17

    We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of β-catenin in nucleus and inhibits the binding of β-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for β-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/β-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. - Highlights: • CME inhibits cell proliferation in HCT116 cells. • CME increases cell cycle arrest at G0/G1 phase and apoptosis. • CME attenuates cyclin D1 and regulates cell cycle regulatory proteins. • CME inhibits β-catenin translocation to nucleus.

  18. Loss of Nek11 Prevents G2/M Arrest and Promotes Cell Death in HCT116 Colorectal Cancer Cells Exposed to Therapeutic DNA Damaging Agents.

    PubMed

    Sabir, Sarah R; Sahota, Navdeep K; Jones, George D D; Fry, Andrew M

    2015-01-01

    The Nek11 kinase is a potential mediator of the DNA damage response whose expression is upregulated in early stage colorectal cancers (CRCs). Here, using RNAi-mediated depletion, we examined the role of Nek11 in HCT116 WT and p53-null CRC cells exposed to ionizing radiation (IR) or the chemotherapeutic drug, irinotecan. We demonstrate that depletion of Nek11 prevents the G2/M arrest induced by these genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage. Interestingly, Nek11 depletion also led to long-term loss of cell viability that was independent of p53 and exacerbated following IR exposure. CRC cells express four splice variants of Nek11 (L/S/C/D). These are predominantly cytoplasmic, but undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain. In HCT116 cells, Nek11S in particular has an important role in the DNA damage response. These data provide strong evidence that Nek11 contributes to the response of CRC cells to genotoxic agents and is essential for survival either with or without exposure to DNA damage. PMID:26501353

  19. Loss of Nek11 Prevents G2/M Arrest and Promotes Cell Death in HCT116 Colorectal Cancer Cells Exposed to Therapeutic DNA Damaging Agents

    PubMed Central

    Sabir, Sarah R.; Sahota, Navdeep K.; Jones, George D. D.; Fry, Andrew M.

    2015-01-01

    The Nek11 kinase is a potential mediator of the DNA damage response whose expression is upregulated in early stage colorectal cancers (CRCs). Here, using RNAi-mediated depletion, we examined the role of Nek11 in HCT116 WT and p53-null CRC cells exposed to ionizing radiation (IR) or the chemotherapeutic drug, irinotecan. We demonstrate that depletion of Nek11 prevents the G2/M arrest induced by these genotoxic agents and promotes p53-dependent apoptosis both in the presence and absence of DNA damage. Interestingly, Nek11 depletion also led to long-term loss of cell viability that was independent of p53 and exacerbated following IR exposure. CRC cells express four splice variants of Nek11 (L/S/C/D). These are predominantly cytoplasmic, but undergo nucleocytoplasmic shuttling mediated through adjacent nuclear import and export signals in the C-terminal non-catalytic domain. In HCT116 cells, Nek11S in particular has an important role in the DNA damage response. These data provide strong evidence that Nek11 contributes to the response of CRC cells to genotoxic agents and is essential for survival either with or without exposure to DNA damage. PMID:26501353

  20. p53-dependent p21-mediated growth arrest pre-empts and protects HCT116 cells from PUMA-mediated apoptosis induced by EGCG

    PubMed Central

    Thakur, Vijay S; Amin, A.R.M. Ruhul; Paul, Rajib K; Gupta, Kalpana; Hastak, Kedar; Agarwal, Mukesh K; Jackson, Mark W; Wald, David N; Mukhtar, Hasan; Agarwal, Munna L

    2010-01-01

    The tumor suppressor protein p53 plays a key role in regulation of negative cellular growth in response to EGCG. To further explore the role of p53 signaling and elucidate the molecular mechanism, we employed colon cancer HCT116 cell line and its derivatives in which a specific transcriptional target of p53 is knocked down by homologous recombination. Cells expressing p53 and p21 accumulate in G1 upon treatment with EGCG. In contrast, same cells lacking p21 traverse through the cell cycle and eventually undergo apoptosis as revealed by TUNEL staining. Treatment with EGCG leads to induction of p53, p21 and PUMA in p21 wild-type, and p53 and PUMA in p21?/? cells. Ablation of p53 by RNAi protects p21?/? cells, thus indicating a p53-dependent apoptosis by EGCG. Furthermore, analysis of cells lacking PUMA or Bax with or without p21 but with p53 reveals that all the cells expressing p53 and p21 survived after EGCG treatment. More interestingly, cells lacking both PUMA and p21 survived ECGC treatment whereas those lacking p21 and Bax did not. Taken together, our results present a novel concept wherein p21-dependent growth arrest pre-empts and protects cells from otherwise, in its absence, apoptosis which is mediated by activation of pro-apoptotic protein PUMA. Furthermore, we find that p53-dependent activation of PUMA in response to EGCG directly leads to apoptosis with out requiring Bax as is the case in response to agents that induce DNA damage. p21, thus can be used as a molecular switch for therapeutic intervention of colon cancer. PMID:20444544

  1. Quilamine HQ1-44, an iron chelator vectorized toward tumor cells by the polyamine transport system, inhibits HCT116 tumor growth without adverse effect.

    PubMed

    Renaud, Stéphanie; Corcé, Vincent; Cannie, Isabelle; Ropert, Martine; Lepage, Sylvie; Loréal, Olivier; Deniaud, David; Gaboriau, François

    2015-08-01

    Tumor cell growth requires large iron quantities and the deprivation of this metal induced by synthetic metal chelators is therefore an attractive method for limiting the cancer cell proliferation. The antiproliferative effect of the Quilamine HQ1-44, a new iron chelator vectorized toward tumor cells by a polyamine chain, is related to its high selectivity for the Polyamine Transport System (PTS), allowing its preferential uptake by tumoral cells. The difference in PTS activation between healthy cells and tumor cells enables tumor cells to be targeted, whereas the strong dependence of these cells on iron ensures a secondary targeting. Here, we demonstrated in vitro that HQ1-44 inhibits DNA synthesis and cell proliferation of HCT116 cells by modulating the intracellular metabolism of both iron and polyamines. Moreover, in vivo, in xenografted athymic nude mice, we found that HQ1-44 was as effective as cis-platin in reducing HCT116 tumor growth, without its side effects. Furthermore, as suggested by in vitro data, the depletion in exogenous or endogenous polyamines, known to activate the PTS, dramatically enhanced the antitumor efficiency of HQ1-44. These data support the need for further studies to assess the value of HQ1-44 as an adjuvant treatment in cancer. PMID:26070250

  2. Bufalin Inhibits HCT116 Colon Cancer Cells and Its Orthotopic Xenograft Tumor in Mice Model through Genes Related to Apoptotic and PTEN/AKT Pathways

    PubMed Central

    Wang, Jie; Chen, Chao; Wang, Shiying; Zhang, Yong; Yin, Peihao; Gao, Zhongxiang; Xu, Jie; Feng, Dianxu; Zuo, Qinsong; Zhao, Ronghua; Chen, Teng

    2015-01-01

    Aims. To investigate the anticolorectal cancer (CRC) effects of Bufalin, a bioactive polyhydroxysteroid from Venenum Bufonis, using HCT116 human CRC cell and an established orthotopic xenograft model in mice, and to explore the mechanisms of action. Material and Methods. Cultured HCT116 cells or BALB/c mice with orthotopic tumor were treated by Bufalin (positive control: 5-FU). Cell proliferation, apoptosis, and cycling were determined by MTT, Annexin V/PI staining, and flow cytometry, respectively. In mice, tumor inhibition rate and animal survival were calculated. The expressions of PTEN/phosphate-PTEN, AKT/phosphate-AKT, Bad, Bcl-xl, Bax, or Caspase-3 in cells and/or tumors were determined by Western blot or immunohistochemical staining. Results. Bufalin significantly inhibited cell proliferation and induced cell apoptosis and cycle arrest in a dose/time-dependent manner. In the animal model, Bufalin treatment resulted in significant inhibition of tumor growth and prolonged survival. In the Bufalin-treated cultured cells and/or xenograft tumors, the expressions of PTEN, Bad, Bax, and Caspase-3 were significantly increased, while p-AKT and Bcl-xL significantly decreased. Conclusions. Our results indicate that Bufalin inhibit cell proliferation and orthotopic tumor growth by inducing cell apoptosis through the intrinsic apoptotic pathway, which is of pivotal significance in the identification of an anticancer drug that may synergize with Bufalin. PMID:26770191

  3. Tunable Biodegradable Nanocomposite Hydrogel for Improved Cisplatin Efficacy on HCT-116 Colorectal Cancer Cells and Decreased Toxicity in Rats.

    PubMed

    Abdel-Bar, Hend Mohamed; Osman, Rihab; Abdel-Reheem, Amal Youssef; Mortada, Nahed; Awad, Gehanne A S

    2016-02-01

    This work describes the development of a modified nanocomposite thermosensitive hydrogel for controlled cisplatin release and improved cytotoxicity with decreased side effects. The system was characterized in terms of physical properties, morphological architecture and in vitro cisplatin release. Cytotoxicity was tested against human colorectal carcinoma HCT-116. In vivo studies were conducted to evaluate the acute toxicity in terms of rats' survival rate and body weight loss. Nephro and hepatotoxicities were evaluated followed by histopathological alterations of various tissue organs. Nanocomposite thermosensitive hydrogel containing nanosized carrier conferred density and stiffness allowing a zero order drug release for 14 days. Enhanced cytotoxicity with 2-fold decrease in cisplatin IC50 was accomplished. A linear in vivo-in vitro correlation was proved for the system degradation. Higher animal survival rate and lower tissue toxicities proved the decreased toxicity of cisplatin nanocomposite compared to its solution. PMID:26709447

  4. Evaluation of anti-HER2 scFv-conjugated PLGA-PEG nanoparticles on 3D tumor spheroids of BT474 and HCT116 cancer cells

    NASA Astrophysics Data System (ADS)

    Thuy Duong Le, Thi; Pham, Thu Hong; Nghia Nguyen, Trong; Giang Ngo, Thi Hong; Nhung Hoang, Thi My; Huan Le, Quang

    2016-06-01

    Three-dimensional culture cells (spheroids) are one of the multicellular culture models that can be applied to anticancer chemotherapeutic development. Multicellular spheroids more closely mimic in vivo tumor-like patterns of physiologic environment and morphology. In previous research, we designed docetaxel-loaded pegylated poly(D, L-lactide-co-glycolide) nanoparticles conjugated with anti-HER2 single chain antibodies (scFv-Doc-PLGA-PEG) and evaluated them in 2D cell culture. In this study, we continuously evaluate the cellular uptake and cytotoxic effect of scFv-Doc-PLGA-PEG on a 3D tumor spheroid model of BT474 (HER2-overexpressing) and HCT116 (HER2-underexpressing) cancer cells. The results showed that the nanoparticle formulation conjugated with scFv had a significant internalization effect on the spheroids of HER2-overexpressing cancer cells as compared to the spheroids of HER2-underexpressing cancer cells. Therefore, cytotoxic effects of targeted nanoparticles decreased the size and increased necrotic score of HER2-overexpressing tumor spheroids. Thus, these scFv-Doc-PLGA-PEG nanoparticles have potential for active targeting for HER2-overexpressing cancer therapy. In addition, BT474 and HCT116 spheroids can be used as a tumor model for evaluation of targeting therapies.

  5. 5-Methoxyflavanone induces cell cycle arrest at the G2/M phase, apoptosis and autophagy in HCT116 human colon cancer cells

    SciTech Connect

    Shin, Soon Young; Hyun, Jiye; Yu, Jae-Ran; Lim, Yoongho; Lee, Young Han

    2011-08-01

    Natural flavonoids have diverse pharmacological activities, including anti-oxidative, anti-inflammatory, and anti-cancer activities. In this study, we investigated the molecular mechanism underlying the action of 5-methoxyflavanone (5-MF) which has a strong bioavailability and metabolic stability. Our results show that 5-MF inhibited the growth and clonogenicity of HCT116 human colon cancer cells, and that it activated DNA damage responses, as revealed by the accumulation of p53 and the phosphorylation of DNA damage-sensitive proteins, including ataxia-telangiectasia mutated (ATM) at Ser1981, checkpoint kinase 2 (Chk2) at Thr68, and histone H2AX at Ser139. 5-MF-induced DNA damage was confirmed in a comet tail assay. We also found that 5-MF increased the cleavage of caspase-2 and -7, leading to the induction of apoptosis. Pretreatment with the ATM inhibitor KU55933 enhanced 5-MF-induced {gamma}-H2AX formation and caspase-7 cleavage. HCT116 cells lacking p53 (p53{sup -/-}) or p21 (p21{sup -/-}) exhibited increased sensitivity to 5-MF compared to wild-type cells. 5-MF further induced autophagy via an ERK signaling pathway. Blockage of autophagy with the MEK inhibitor U0126 potentiated 5-MF-induced {gamma}-H2AX formation and caspase-2 activation. These results suggest that a caspase-2 cascade mediates 5-MF-induced anti-tumor activity, while an ATM/Chk2/p53/p21 checkpoint pathway and ERK-mediated autophagy act as a survival program to block caspase-2-mediated apoptosis induced by 5-MF. - Graphical abstract: Display Omitted Highlights: > 5-MF inhibits the proliferation of HCT116 colon cancer cells. > 5-MF inhibits cell cycle progression and induces apoptosis. > Inhibition of autophagy triggers 5-MF-induced apoptosis. > Inhibition of ERK signaling blocks 5-MF-induced autophagy but activates apoptosis. > Treatment with 5-MF in combination with an ERK inhibitor may be a potential therapeutic strategy in human colon cancer.

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

    PubMed

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

    2013-01-01

    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-β-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 1β, 2β-epoxy-3β,4α,10α-trihydroxyguaian-6α,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

  7. Apoptotic effects of extract from Cnidium monnieri (L.) Cusson by adenosine monosphosphate-activated protein kinase-independent pathway in HCT116 colon cancer cells.

    PubMed

    Lim, Eun Gyeong; Kim, Guen Tae; Lee, Se Hee; Kim, Sang-Yong; Kim, Young Min

    2016-06-01

    Colon cancer, a common malignancy, can occur due to poor eating habits and increasing age. Consequently, careful regulation of eating habits may serve as a possible method for preventing the occurrence or progression of colon cancer. Extracts of the fruit of Cnidium monnieri (L.) Cusson are well‑known as an effective herbal medicine for the treatment of pain in female genitalia and carbuncle. However, there have been no studies on the apoptotic effects of Cnidium monnieri (L.) Cusson (CME). Adenosine monophosphate‑activated protein kinase (AMPK), the major regulator of energy metabolism, is activated by metabolic stress, including hypoxia and glucose deprivation. Activation of AMPK inhibits cell proliferation and induces apoptosis through the inhibition of phosphorylated (p)‑Akt and control of B‑cell lymphoma 2 (Bcl‑2) family members. The pro‑apoptotic proteins Bcl‑2‑associated X protein (Bax) and Bcl‑2‑homologous antagonist killer (Bak), are activated by their translocation to mitochondria from the cytosol. Translocation of Bax/Bak induces outer membrane permeabilization and is likely to lead to apoptosis through cytochrome C release and caspase activity. In the present study, the apoptotic effects and influence on mitochondria‑mediated apoptotic proteins of CME in HCT116 cells were assessed. We hypothesized that CME may have an effect on the inhibition of p‑Akt in an AMPK‑independent pathway. The present study demonstrated that CME induced the release of LDH and apoptosis through its inhibition of p‑Akt to control Bcl‑2 and activate Bax and Bak. Co‑treatment with CME and AMPK inhibitors showed that CME‑induced apoptosis does not occurr through a AMPK‑dependent pathway. Therefore, the present study determined, for the first time, that CME induced apoptosis as a result of causing metabolic stresses due to directly regulation of the de‑phosphorylation of Akt, whereas it did not control the AMPK-dependent pathway in HCT116 colon cancer cells. PMID:27082059

  8. Combination of lactate calcium salt with 5-indanesulfonamide and α-cyano-4-hydroxycinnamic acid to enhance the antitumor effect on HCT116 cells via intracellular acidification

    PubMed Central

    JEONG, KEUN-YEONG; MANDER, POONAM; SIM, JAE JUN; KIM, HWAN MOOK

    2016-01-01

    Maintenance of a neutral intracellular pH (pHi) is favorable for the survival of tumors, and maintenance of highly acidic extracellular pH (pHe) facilitates tumor invasiveness. The aim of the present study was to investigate the antitumor effects of lactate calcium salt (CaLa), 5-indanesulfonamide (IS) and α-cyano-4-hydroxycinnamic acid (CA) via pH regulation in colon cancer cells. HCT116 cells were treated with CaLa, IS, CA and combinations of the three. Subsequently, the concentration of intracellular lactate was determined. pHi and pHe were measured using cell lysates and culture media. Colony formation assay, cell viability assay and western blot analysis were additionally performed to analyze the consequences of the pH changes. CaLa, IS, CA and combination treatments induced an increase in the concentration of intracellular lactate. Lactate influx into the tumor microenvironment produced an acidic pHi in colon cancer cells. Consequently, colony formation and cell viability were significantly decreased, as well as poly(adenosine diphosphate-ribose) polymerase degradation. The tumor microenvironment may be exploited therapeutically by disrupting the mechanism that regulates pHi, leading to cell apoptosis. The present study indicated that treatment with CaLa, IS and CA induced intracellular acidification via lactate influx, causing apoptosis of colon cancer cells. Additionally, the findings suggested that the combination of CaLa with IS and CA may enhance antitumor activity, and may provide a potential therapeutic approach for the treatment of colon cancer. PMID:26998091

  9. miR-106b fine tunes ATG16L1 expression and autophagic activity in intestinal epithelial HCT116 cells

    PubMed Central

    Zhai, Zili; Wu, Feng; Chuang, Alice Y.; Kwon, John H.

    2014-01-01

    Background miRNAs regulate gene expression at the post-transcriptional level. ATG16L1, an essential component for autophagy and a risk gene for Crohn's disease, contains two binding sites in the 3'UTR for miR-17 family, including miRs-20a, −93, −106a, and −106b. The purpose of this study was to assess the effects of these miRNAs on ATG16L1 expression and autophagic activity in HCT116 cells. Methods The functional binding sites in the ATG16L1 3'UTR were evaluated by transfection of pMIR-GLO vectors bearing the WT or mutant 3'UTR into cells for luciferase reporter assay. The miRNA regulation of ATG16L1 expression was determined by qRT-PCR and Western blot. The miRNA regulation of autophagic activity was evaluated by examining LC3II formation using Western blot and confocal imaging. Results Both miR-106a and miR-106b mimics inhibited starvation-induced autophagy. miR-106b mimic reduced ATG16L1 protein expression. Luciferase reporter assays showed that mutating the binding sequence at the position 1036-1042 abrogated miR-106b regulation of ATG16L1 3'UTR luciferase activity. In addition, miR-106a and miR-106b overexpression inhibited the expression of several other autophagy genes, including ATG12. Conclusions miR-106b targets ATG16L1 and modulates autophagy, partially through the binding site at the 3’ end of ATG16L1 3'UTR. miR-106a regulates autophagy, possibly irrelevant to ATG16L1 regulation. Both miR-106a and miR-106b regulate multiple autophagy genes so that may play an integral role in fine-tuning autophagy. PMID:23899543

  10. Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant

    PubMed Central

    Lan, Yu-Hsuan; Chiang, Jo-Hua; Huang, Wen-Wen; Lu, Chi-Cheng; Chung, Jing-Gung; Wu, Tian-Shung; Jhan, Jia-Hua; Lin, Kuei-Li; Pai, Shu-Jen; Chiu, Yu-Jen; Tsuzuki, Minoru; Yang, Jai-Sing

    2012-01-01

    Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and ?-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer. PMID:22474491

  11. Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant.

    PubMed

    Lan, Yu-Hsuan; Chiang, Jo-Hua; Huang, Wen-Wen; Lu, Chi-Cheng; Chung, Jing-Gung; Wu, Tian-Shung; Jhan, Jia-Hua; Lin, Kuei-Li; Pai, Shu-Jen; Chiu, Yu-Jen; Tsuzuki, Minoru; Yang, Jai-Sing

    2012-01-01

    Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and ?-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer. PMID:22474491

  12. Characterization of sphere-forming HCT116 clones by whole RNA sequencing

    PubMed Central

    Chung, Eunkyung; Oh, Inkyung

    2016-01-01

    Purpose To determine CD133+ cells defined as cancer stem cells (CSCs) in colon cancer, we examined whether CD133+ clones in HCT116 demonstrate known features of CSCs like sphere-forming ability, chemodrug-resistance, and metastatic potential. Methods Magnetic cell isolation and cell separation demonstrated that <1% of HCT116 cells expressed CD133, with the remaining cells being CD133- clones. In colon cancer cells, radioresistance is also considered a CSC characteristic. We performed clonogenic assay using 0.4 Gy γ-irradiation. Results Interestingly, there were no differences between HCT116 parental and HCT116 CD133+ clones when the cells comprised 0.5% of the total cells, and CD133- clone demonstrated radiosensitive changes compared with parental and CD133+ clones. Comparing gene expression profiles between sphere-forming and nonforming culture conditions of HCT116 subclones by whole RNA sequencing failed to obtain specific genes expressed in CD133+ clones. Conclusion Despite no differences of gene expression profiles in monolayer attached culture conditions of each clone, sphere-forming conditions of whole HCT116 subclones, parental, CD133+, and CD133- increased 1,761 coding genes and downregulated 1,384 genes related to CSCs self-renewal and survival. Thus, spheroid cultures of HCT116 cells could be useful to expand colorectal CSCs rather than clonal expansion depending on CD133 expressions. PMID:27073788

  13. Comparison of gene expression in HCT116 treatment derivatives generated by two different 5-fluorouracil exposure protocols

    PubMed Central

    De Angelis, Paula M; Kravik, Katherine L; Tunheim, Siv H; Haug, Terje; Reichelt, Wenche H

    2004-01-01

    Background Established colorectal cancer cell lines subjected to different 5-fluorouracil (5-FU) treatment protocols are often used as in vitro model systems for investigations of downstream cellular responses to 5-FU and to generate 5-FU-resistant derivatives for the investigation of biological mechanisms involved in drug resistance. We subjected HCT116 colon cancer cells to two different 5-FU treatment protocols in an attempt to generate resistant derivatives: one that simulated the clinical bolus regimens using clinically-achievable 5-FU levels, the other that utilized serial passage in the presence of increasing 5-FU concentrations (continuous exposure). HCT116 Bolus3, ContinB, and ContinD, corresponding to independently-derived cell lines generated either by bolus exposure or continuous exposure, respectively, were characterized for growth- and apoptosis-associated phenotypes, and gene expression using 8.5 K oligonucleotide microarrays. Comparative gene expression analyses were done in order to determine if transcriptional profiles for the respective treatment derivatives were similar or substantially different, and to identify the signaling and regulatory pathways involved in mediating the downstream response to 5-FU exposure and possibly involved in development of resistance. Results HCT116 ContinB and ContinD cells were respectively 27-fold and >100-fold more resistant to 5-FU and had reduced apoptotic fractions in response to transient 5-FU challenge compared to the parental cell line, whereas HCT116 Bolus3 cells were not resistant to 5-FU after 3 cycles of bolus 5-FU treatment and had the same apoptotic response to transient 5-FU challenge as the parental cell line. However, gene expression levels and expression level changes for all detected genes in Bolus3 cells were similar to those seen in both the ContinB (strongest correlation) and ContinD derivatives, as demonstrated by correlation and cluster analyses. Regulatory pathways having to do with 5-FU metabolism, apoptosis, and DNA repair were among those that were affected by 5-FU treatment. Conclusion All HCT116 derivative cell lines demonstrated similar transcriptional profiles, despite the facts that they were generated by two different 5-FU exposure protocols and that the bolus exposure derivative had not become resistant to 5-FU. Selection pressures on HCT116 cells as a result of 5-FU challenge are thus similar for both treatment protocols. PMID:15109396

  14. Dietary peptides from the non-digestible fraction of Phaseolus vulgaris L. decrease angiotensin II-dependent proliferation in HCT116 human colorectal cancer cells through the blockade of the renin-angiotensin system.

    PubMed

    Luna-Vital, Diego A; Liang, Katie; González de Mejía, Elvira; Loarca-Piña, Guadalupe

    2016-05-18

    This study aimed to determine the ability of peptides present in the non-digestible fraction (NDF) of common beans to decrease angiotensin II (AngII) through the blockade of RAS and its effect on the proliferation of HCT116 human colorectal cancer cells. Pure synthesized peptides GLTSK and GEGSGA and the peptide fractions (PF) of cultivars Azufrado Higuera and Bayo Madero were used. The cells were pretreated with pure peptides, PF or AGT at their IC50 or IC25 values, in comparison with the simultaneous treatment of peptides and AGT. For western blot and microscopy analysis, 100 μM and 0.5 mg mL(-1) were used for pure peptides and PF treatments, respectively. According to the ELISA tests, GLTSK and GEGSGA decreased (p < 0.05) the conversion rate of AGT to angiotensin I (AngI) by 38 and 28%, respectively. All the peptides tested reduced (p < 0.05) the conversion rate of AngI to AngII from 38 to 50%. When the cells were pretreated with both pure peptides and PF before exposure to AGT, the effectiveness inhibiting cell proliferation was higher than the simultaneous treatment suggesting their preventive effects. GLTSK and GEGSGA interacted with the catalytic site of renin, the angiotensin-I converting enzyme, and the AngII receptor, mainly through hydrogen bonds, polar, hydrophobic and cation-π interactions according to molecular docking. Through confocal microscopy, it was determined that GLTSK and GEGSGA caused the decrease (p < 0.05) of AngII-dependent STAT3 nuclear activation in HCT116 cells by 66 and 23%, respectively. The results suggest that peptides present in the common bean NDF could potentially ameliorate the effects of RAS overexpression in colorectal cancer. PMID:27156533

  15. Integrated in silico and experimental methods revealed that Arctigenin inhibited angiogenesis and HCT116 cell migration and invasion through regulating the H1F4A and Wnt/β-catenin pathway.

    PubMed

    Zhang, Shouyue; Li, Jie; Song, Sicheng; Li, Jing; Tong, Rongsheng; Zang, Zhihe; Jiang, Qinglin; Cai, Lulu

    2015-11-01

    Arctigenin (ARG) has been previously reported to exert diverse biological activities including anti-proliferation, anti-inflammatory, and antiviral, etc. In the current study, the anti-metastasis and anti-angiogenesis activities of ARG were investigated. To further understand how ARG played these bioactivities, proteomic approaches were used to profile the proteome changes in response to ARG treatment using 2DE-MS/MS. Using these approaches, a total of 50 differentially expressed proteins were identified and clustered. Bioinformatics analysis suggested that multiple signalling pathways were involved. Moreover, ARG induced anti-metastatic and anti-angiogenesis activities were mainly accompanied by a deactivation of the Wnt/β-catenin pathway in HCT116 cells. PMID:26267229

  16. Deoxycholic Acid and Selenium Metabolite Methylselenol Exert Common and Distinct Effects on Cell Cycle, Apoptosis, and MAP Kinase Pathway in HCT116 Human Colon Cancer Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bile acid deoxycholic acid (DCA) is a known tumor promoter in colon tumor development. The cell growth inhibition induced by DCA may cause compensatory hyperproliferation of colonic epithelial cells and provide selection for subpopulations of cells resistant to DCA’s inhibitory effect. These survivi...

  17. A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells

    PubMed Central

    Silva, Victoria C; Plooster, Melissa; Leung, Jessica C; Cassimeris, Lynne

    2015-01-01

    Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIPL, a protein that blocks caspase 8 activation. cFLIPL levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIPL in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells. PMID:25602147

  18. Deoxycholic acid and selenium metabolite methylselenol exert common and distinct effects on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cell growth inhibition induced by bile acid deoxycholic acid (DCA) may cause compensatory hyperproliferation of colonic epithelial cells, and consequently increase colon cancer risk. On the other hand, there is increasing evidence for the efficacy of certain forms of selenium (Se) as anticancer ...

  19. Prolonged sulforaphane treatment activates survival signaling in nontumorigenic NCM460 colon cells but apoptotic signaling in tumorigenic HCT116 colon cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sulforaphane (SFN) is a naturally occurring member of the isothiocyanate family of chemopreventive agents and the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon c...

  20. Differential effects of deoxycholic acid versus selenium metabolite methylselenol on cell cycle, apoptosis, and MAP kinase pathway in HCT116 human colon cancer cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: A typical part of the Western diet is a high fat intake that leads to increased levels of fecal bile acids, and these bile acids, primarily deoxycholic acid (DCA) in humans, have been believed to be tumor promoters of colon cancer. The cell growth inhibition induced by bile acid deoxyc...

  1. Combination of Albendazole and 2-Methoxyestradiol significantly improves the survival of HCT-116 tumor-bearing nude mice

    PubMed Central

    2013-01-01

    Background Albendazole (ABZ) is a microtubule-targeting anthelmintic with a remarkable activity against a variety of human cancer cells. In this study, we examined if the antitumor activity of ABZ could be enhanced by its combination with other microtubule-binding agents. Methods The interactions between ABZ and microtubule-binding agents, paclitaxel, vinblastine, colchicine, and 2-methoxyestradiol were characterized using median effect analysis method in HCT-116 colorectal cancer cells and DU145 prostate cancer cell line. The mechanism underlying the synergistic interaction related to tubulin polymerization and apoptosis was then investigated. Finally, the effect of the combination therapy on the survival of HCT-116 tumor-bearing nude mice was evaluated. Results Among the tested drugs, a synergistic anti-proliferative effect was observed with the combination of low concentrations of ABZ plus colchicine and ABZ plus 2-methoxyestradiol (2ME). Exploring the mechanism of the interaction between ABZ and 2ME revealed that the combination therapy synergistically activated the extrinsic pathway of apoptosis. Consistent with in vitro results, the combination of low concentration of ABZ with 2ME prolonged the survival of mice-bearing HCT-116 tumors. High concentration of ABZ in combination with 2ME, however, proved to be less effective than ABZ alone. Conclusions The combination of low doses of ABZ and 2ME has shown promising results in our pre-clinical model. Additionally, the finding that the combination of two microtubule-binding agents that share the same binding site can act synergistically may lead to the development of new therapeutic strategies in cancer treatment. PMID:23432760

  2. Pharmacokinetic, biodistribution and therapeutic efficacy of 5-fluorouracil-loaded pH-sensitive PEGylated liposomal nanoparticles in HCT-116 tumor bearing mouse

    PubMed Central

    Udofot, Ofonime; Affram, Kevin; Smith, Taylor; Tshabe, Bulumko; Krishnan, Sunil; Sachdeva, Mandip; Agyare, Edward

    2016-01-01

    The objective of the study was to investigate the pharmacokinetics and efficacy of 5-FU entrapped pH-sensitive liposomal nanoparticles with surface-modified anti-epidermal growth factor receptor (EGFR) antibody (pHLNps-5-FU) delivery system. Cytotoxicity of 5-FU and pHLNps-5-FU was determined in vitro against HCT-116 cells. The biodistribution and pharmacokinetic parameters of the administered 5-FU and pHLNps-5-FU as well as efficacy of 5-FU and pHLNps-5-FU were determined in HCT-116 subcutaneous mouse model. Mean size of pHLNp-5-FU was 164.3 ± 8.4 nm with entrapment efficiency (E.E) of 54.17%. While cytotoxicity of 5-FU and pHLNps-5-FU showed a strong dose-dependent, pHLNps-5-FU proved to be more effective (2–3 fold high) than that of 5-FU against HCT-116 cells. Pharmacokinetic study showed a prolonged plasma circulation of pHLNps-5-FU and a more significant body exposure while accumulation of pHLNps-5-FU in tumor was significantly higher than that of free 5-FU. Further, the efficacy of pHLNps-5-FU, was greater than free 5-FU at equivalent 5-FU dose. The study suggests that pHLNps may be an effective drug delivery system to enhance the anticancer activity of 5-FU against colorectal tumor growth. PMID:27200415

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

    SciTech Connect

    Janssen, Astrid; Schiffmann, Susanne; Birod, Kerstin; Maier, Thorsten J.; Wobst, Ivonne; Geisslinger, Gerd

    2008-01-25

    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.

  4. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

    PubMed

    Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2014-04-01

    Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. PMID:24550143

  5. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

    PubMed Central

    Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M.; Lee, Ki Won; Dong, Zigang

    2014-01-01

    Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the protein data bank against curcumin. Cyclin dependent kinase 2 (CDK2), a major cell cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of Rb, a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell cycle arrest, we investigated the anti-proliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine if CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantial relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells. PMID:24550143

  6. Oridonin inhibits the proliferation of human colon cancer cells by upregulating BMP7 to activate p38 MAPK.

    PubMed

    Ren, Chun-Mei; Li, Yang; Chen, Qian-Zhao; Zeng, Yu-Hua; Shao, Ying; Wu, Qiu-Xiang; Yuan, Shuang-Xue; Yang, Jun-Qin; Yu, Yu; Wu, Ke; He, Bai-Cheng; Sun, Wen-Juan

    2016-05-01

    Oridonin (ORI), a diterpenoid purified from Rabdosia rubescens, has been reported as a promising chemotherapy drug for colon cancer treatment; yet, the precise mechanisms underlying this anticancer activity remain unclear. In the present study, we investigated the anticancer effect of ORI in HCT116 cells, and dissected the possible molecular mechanisms underlying this activity. With crystal violet staining, flow cytometry and western blot assay, we found that ORI effectively inhibited the proliferation and induced the apoptosis of HCT116 cells. Further analysis of the results indicated that BMP7 was greatly upregulated by ORI in the HCT116 cells, but its endogenous expression in FHC cells was apparently lower than that in the colon cancer cell lines. Exogenous expression of BMP7 inhibited the proliferation of the HCT116 cells, and substantially potentiated the anticancer effect of ORI. However, the specific antibody of BMP7 nearly abolished this anticancer activity of ORI in the HCT116 cells. Meanwhile, ORI exerted no significant effect on the level of phosphorylated Smad1/5/8 or total p38 MAPK, but greatly increased the level of phosphorylated p38 MAPK in the HCT116 cells. A p38 MAPK-specific inhibitor partly reversed the antiproliferative effect of BMP7 in the HCT116 cells, but prominently promoted the effect of the BMP7 antibody on proliferation. Exogenous expression of BMP7 increased the ORI-induced phosphorylation of p38 MAPK, while the BMP7 antibody almost abolished the ORI-elevated p38 MAPK phosphorylation. Our findings suggest that ORI may be an efficacious drug for colon cancer treatment. This anticancer activity of ORI may be mediated by upregulating BMP7 at least to increase the activation of p38 MAPK. PMID:26986967

  7. PKM2 Subcellular Localization Is Involved in Oxaliplatin Resistance Acquisition in HT29 Human Colorectal Cancer Cell Lines

    PubMed Central

    Ginés, Alba; Bystrup, Sara; Ruiz de Porras, Vicenç; Guardia, Cristina; Musulén, Eva; Martínez-Cardús, Anna; Manzano, José Luis; Layos, Laura; Abad, Albert; Martínez-Balibrea, Eva

    2015-01-01

    Chemoresistance is the main cause of treatment failure in advanced colorectal cancer (CRC). However, molecular mechanisms underlying this phenomenon remain to be elucidated. In a previous work we identified low levels of PKM2 as a putative oxaliplatin-resistance marker in HT29 CRC cell lines and also in patients. In order to assess how PKM2 influences oxaliplatin response in CRC cells, we silenced PKM2 using specific siRNAs in HT29, SW480 and HCT116 cells. MTT test demonstrated that PKM2 silencing induced resistance in HT29 and SW480 cells and sensitivity in HCT116 cells. Same experiments in isogenic HCT116 p53 null cells and double silencing of p53 and PKM2 in HT29 cells failed to show an influence of p53. By using trypan blue stain and FITC-Annexin V/PI tests we detected that PKM2 knockdown was associated with an increase in cell viability but not with a decrease in apoptosis activation in HT29 cells. Fluorescence microscopy revealed PKM2 nuclear translocation in response to oxaliplatin in HCT116 and HT29 cells but not in OXA-resistant HTOXAR3 cells. Finally, by using a qPCR Array we demonstrated that oxaliplatin and PKM2 silencing altered cell death gene expression patterns including those of BMF, which was significantly increased in HT29 cells in response to oxaliplatin, in a dose and time-dependent manner, but not in siPKM2-HT29 and HTOXAR3 cells. BMF gene silencing in HT29 cells lead to a decrease in oxaliplatin-induced cell death. In conclusion, our data report new non-glycolytic roles of PKM2 in response to genotoxic damage and proposes BMF as a possible target gene of PKM2 to be involved in oxaliplatin response and resistance in CRC cells. PMID:25955657

  8. Trisubstituted and tetrasubstituted pyrazolines as a novel class of cell-growth inhibitors in tumor cells with wild type p53.

    PubMed

    Abdel-Halim, Mohammad; Keeton, Adam B; Gurpinar, Evrim; Gary, Bernard D; Vogel, Simon M; Engel, Matthias; Piazza, Gary A; Boeckler, Frank M; Hartmann, Rolf W; Abadi, Ashraf H

    2013-12-01

    Derivatives with scaffolds of 1,3,5-tri-substituted pyrazoline and 1,3,4,5-tetra-substituted pyrazoline were synthesized and tested for their inhibitory effects versus the p53(+/+) HCT116 and p53(-/-) H1299 human tumor cell lines. Several compounds were active against the two cell lines displaying IC50 values in the low micromolar range with a clearly more pronounced effect on the p53(+/+) HCT116 cells. The compound class shows excellent developability due to the modular synthesis, allowing independent optimization of all three to four key substituents to improve the properties of the molecules. PMID:24139845

  9. Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway

    PubMed Central

    Su, Li-ya; Shi, Ying-xu; Yan, Mei-rong; Xi, Yaguang; Su, Xiu-lan

    2015-01-01

    Aim: We have reported novel anticancer bioactive peptides (ACBPs) that show tumor-suppressive activities in human gastric cancer, leukemia, nasopharyngeal cancer, and gallbladder cancer. In this study, we investigated the effects of ACBPs on human colorectal cancer and the underlying mechanisms. Methods: Cell growth and apoptosis of human colorectal tumor cell line HCT116 were measured using cell proliferation assay and flow cytometry, respectively. The expression levels of PARP, p53 and Mcl1A were assessed with Western blotting and immunohistochemistry. For evaluation of the in vivo antitumor activity of ACBPs, HCT116 xenograft nude mice were treated with ACBPs (35 μg/mL, ip) for 10 days. Results: Treatment of HCT116 cells with ACBPs (35 μg/mL) for 4–6 days significantly inhibited the cell growth. Furthermore, treatment of HCT116 cells with ACBPs (35 μg/mL) for 6–12 h significantly enhanced UV-induced apoptosis, increased the expression of PARP and p53, and decreased the expression of Mcl-1. Administration of ACBPs did not change the body weight of HCT116 xenograft nude mice, but decreased the tumor growth by approximately 43%, and increased the expression of PARP and p53, and decreased the expression of Mcl-1 in xenograft mouse tumor tissues. Conclusion: Administration of ACBPs inhibits human colorectal tumor cell growth and induces apoptosis in vitro and in vivo through modulating the PARP-p53-Mcl-1 signaling pathway. PMID:26592508

  10. Alteration of Drug Sensitivity in Human Colon Cancer Cells after Exposure to Heat: Implications for Liver Metastasis Therapy using RFA and Chemotherapy

    PubMed Central

    Makizumi, Ryouji; Yang, Weng-Lang; Owen, Randall P.; Sharma, Rohit R.; Ravikumar, T. S.

    2008-01-01

    Radiofrequency ablation (RFA) is gaining popularity for treating colorectal liver metastases by inducing image guided tumor hyperthermia. In order to reduce tumor recurrence, adjuvant therapies have been administered post-RFA. We hypothesized that tumor cells escaping RFA cytotoxicity by being in the sublethal zones of tumor might develop differential behavior toward cytotoxic drugs. Here, we used cultured human colorectal cancer cells to evaluate the interaction between heat treatment and chemotherapeutic agents. Human colon cancer cell lines HT29 and HCT116 were subjected to temperatures of 42° to 50°C for 15 min, in combination with 5-fluorouracil, oxaliplatin, or irinotecan at different sequences. Cytotoxicity was determined by MTT assay. The cell cycle progression was analyzed by flow cytometry with propidium iodide staining. The expression of several genes associated with drug sensitivity was quantitated by real-time RT-PCR before and after heat treatment. Either heat treatment at 45°C by simultaneous or pre-treatment with three different chemotherapeutic agents didn't affect the cytotoxicity of the combined treatment to HT29 and HCT116 cells, except for irinotecan treatment in HCT116 cells. However, when pre-exposure to 45°C, HCT116 cells, but not HT29 cells, developed resistance to these three drugs. In an analysis of cell cycle profile after the drug followed heat treatment, a longer delay in cell cycle progression in HCT116 cells was observed in comparison to HT29 cells. Furthermore, HCT116 and HT29 cells exhibited different expression profiles of several drug-related genes in response to heat treatment at 45°C. An observation of a differential response to the drug and heat treatment sequences between two human colon cancer cell lines suggests that tumor heterogeneity and selection of chemotherapeutic agents need to be under consideration in the clinical setting. PMID:19079666

  11. Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumption of a high fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer preventive effects. To distinguish these opposing effects of DCA and...

  12. Differential Modulation of Nods Signaling Pathways by Fatty Acids in Human Colonic Epithelial HCT116 cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nucleotide-binding oligomerization domain containing proteins (Nods) are intracellular pattern recognition receptors (PRRs) recognizing conserved moieties of bacterial peptidoglycan through their leucine-rich repeats (LRR) domain. The agonists for Nods activate proinflammtory signaling pathways incl...

  13. Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumption of a high fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer preventive effects. To distinguish these opposing effects of D...

  14. Annona squamosa Linn: cytotoxic activity found in leaf extract against human tumor cell lines.

    PubMed

    Wang, De-Shen; Rizwani, Ghazala H; Guo, Huiqin; Ahmed, Mansoor; Ahmed, Maryam; Hassan, Syed Zeeshan; Hassan, Amir; Chen, Zhe-Sheng; Xu, Rui-Hua

    2014-09-01

    Cancer is a common cause of death in human populations. Surgery, chemotherapy and radiotherapy still remain the corner stone of treatment. However, herbal medicines are gaining popularity on account of their lesser harmful side effects on non-targeted human cells and biological environment. Annona squamosa Linn is a common delicious edible fruit and its leaf have been used for the treatment in various types of diseases. The objective of present study is to determine the anticancer potential of the organic and aqueous extracts of leaf of Annona squamosa L. MTT (3-(4, 5-dimethylthiazole-2yl)-2, 5-biphenyl tetrazolium bromide) assay against hepatocellular carcinoma cell line BEL-7404, lung cancer line H460, human epidermoid carcinoma cell line KB-3-1, prostatic cancer cell line DU145, breast carcinoma cell line MDA-MB-435, and colon cancer cell line HCT-116 Human primary embryonic kidney cell line HEK293 as control were used for the study. The crude extract (Zcd) and Ethyl acetate extract (ZE) were found significant anticancer activity only on human epidermoid carcinoma cell line KB-3-1 and colon cancer cell line HCT-116. PMID:25176251

  15. In vitro anti-proliferative activities of Aloe perryi flowers extract on human liver, colon, breast, lung, prostate and epithelial cancer cell lines.

    PubMed

    Al-Oqail, Mai Mohammad; El-Shaibany, Amina; Al-Jassas, Ebtesam; Al-Sheddi, Ebtesam Saad; Al-Massarani, Shaza Mohamed; Farshori, Nida Nayyar

    2016-03-01

    Natural products, especially plant extracts have offered vast opportunities in the field of drug development due to its chemical diversity. The genus Aloe has for long been used for medicinal purposes in different parts of the world. The present study was designed to investigate the phytochemicals and anti-cancer potential of Aloe perryi flowers. The phytochemical analysis revealed the presence of carbohydrates, glycosides, phytosterols, phenols, flavonoids and proteins. While alkaloids and saponins were absent. The percentage inhibition of various extracts (viz. petroleum ether, chloroform, ethyl acetate, butanol and aqueous) of A. perryi flowers on seven human cancer cell lines (HepG2, HCT-116, MCF-7, A549, PC-3, HEp-2 and HeLa) has been evaluated using MTT assay. All the extracts significantly inhibit the proliferation of cancer cells in a concentration-dependent manner. The petroleum ether extract was most active, where the inhibition was recorded as 92.6%, 93.9%, 92%, 90.9%, 88.9%, 82% and 85.7% for HepG2, HCT-116, MCF-7, A-549, PC-3, HEp-2 and HeLa cells, respectively. The results also revealed that HCT-116 cells were more sensitive among all the cell lines studied. PMID:27113311

  16. New benzimidazole acridine derivative induces human colon cancer cell apoptosis in vitro via the ROS-JNK signaling pathway

    PubMed Central

    Chen, Kang; Chu, Bi-zhu; Liu, Feng; Li, Bin; Gao, Chun-mei; Li, Lu-lu; Sun, Qin-sheng; Shen, Zhi-fa; Jiang, Yu-yang

    2015-01-01

    Aim: To investigate the mechanisms underlying anticancer action of the benzimidazole acridine derivative N-{(1H-benzo[d]imidazol-2-yl)methyl}-2-butylacridin-9-amine(8m) against human colon cancer cells in vitro. Methods: Human colon cancer cell lines SW480 and HCT116 were incubated in the presence of 8m, and then the cell proliferation and apoptosis were measured. The expression of apoptotic/signaling genes and proteins was detected using RT-PCR and Western blotting. ROS generation and mitochondrial membrane depolarization were visualized with fluorescence microscopy. Results: 8m dose-dependently suppressed the proliferation of SW480 and HCT116 cells with IC50 values of 6.77 and 3.33 μmol/L, respectively. 8m induced apoptosis of HCT116 cells, accompanied by down-regulation of Bcl-2, up-regulation of death receptor-5 (DR5), truncation of Bid, cleavage of PARP, and activation of caspases (including caspase-8 and caspase-9 as well as the downstream caspases-3 and caspase-7). Moreover, 8m selectively activated JNK and p38 without affecting ERK in HCT116 cells. Knockout of JNK1, but not p38, attenuated 8m-induced apoptosis. In addition, 8m induced ROS production and mitochondrial membrane depolarization in HCT116 cells. Pretreatment with the antioxidants N-acetyl cysteine or glutathione attenuated 8m-induced apoptosis and JNK activation in HCT116 cells. Conclusion: The new benzimidazole acridine derivative, 8m exerts anticancer activity against human colon cancer cells in vitro by inducing both intrinsic and extrinsic apoptosis pathways via the ROS-JNK1 pathway. PMID:26235743

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

    PubMed Central

    Botcheva, Krassimira; McCorkle, Sean R.

    2014-01-01

    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

  18. CELLFOOD™ induces apoptosis in human mesothelioma and colorectal cancer cells by modulating p53, c-myc and pAkt signaling pathways

    PubMed Central

    2014-01-01

    Background CELLFOOD™ (CF) is a nutraceutical non-addictive, non-invasive, and completely non-toxic unique proprietary colloidal-ionic formula. Little is known about its effect on cancer cells in solid tumors. The aim of this study was to evaluate the effect that CF has on different cancer cell lines and the mechanism by which the nutraceutical works. Methods The effect of CF on HFF (normal fibroblasts), Met5A (mesothelium), MSTO-211H, NCI-2452, Ist-Mes1, MPP89, Ist-Mes2 (mesothelioma), M14 (melanoma), H1650, H1975 (lung cancer), SKRB3 (breast cancer), and HCT-116 (colorectal cancer) cell growth was tested by cell proliferation and clonogenic assay. Among all of them, MSTO-211 and HCT-116 were analyzed for cell cycle by flow cytometry and western blot. Results All human cancer lines were suppressed on cell growth upon 1:200 CF treatment for 24 and 48 hours. Death was not observed in HFF and Met5A cell lines. Cell cycle analysis showed an increased sub-G1 with reduction of G1 in MSTO-211 and a cell cycle arrest of in G1 in HCT116. Activation of caspase-3 and cleavage of PARP confirmed an apoptotic death for both cell lines. Increased expression levels of p53, p21, and p27, downregulation of c-myc and Bcl-2, and inhibition of Akt activation were also found in CF-treated MSTO-211 and HCT-116 cells. Conclusions These findings ascertained an interaction between p53, c-myc, p21, p27, Bcl-2, PI3K/Akt pathway, and CF-induced apoptosis in MSTO-211H and HCT-116 cells, suggesting that CF acts as an important regulator of cell growth in human cancer cell lines. CF could be a useful nutraceutical intervention for prevention in colon cancer and mesothelioma. PMID:24598211

  19. Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

    PubMed

    Hassen, Samar; Ali, Akhtar A; Kilaparty, Surya P; Al-Anbaky, Qudes A; Majeed, Waqar; Boman, Bruce M; Fields, Jeremy Z; Ali, Nawab

    2016-01-01

    The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an indicator of apoptosis, showed that MLH1 translocation only occurred in MMR proficient (SW480) cells upon induction of apoptosis further suggested a MSH2 dependent role of MLH1 in apoptosis. These data suggest a role of MLH1 in mediation of apoptosis in a MSH2-dependent manner. Taken together, our data supported an interdependence of mismatch repair proteins, particularly MLH1 and MSH2, in the mediation of apoptosis in human colorectal carcinoma cell lines. PMID:26728996

  20. miR-1915 inhibits Bcl-2 to modulate multidrug resistance by increasing drug-sensitivity in human colorectal carcinoma cells.

    PubMed

    Xu, Ke; Liang, Xin; Cui, Daling; Wu, Yixin; Shi, Weibin; Liu, Jianwen

    2013-01-01

    Colorectal carcinoma is a frequent cause of cancer-related death in the world for men and women. microRNAs are endogenous small noncoding RNAs that regulate gene expression negatively at post-transcriptional level. Here, we investigated the possible role of microRNAs in the development of multidrug resistance (MDR) in colorectal carcinoma cells. We analyzed microRNA (miRNA) expression levels between multidrug resistant colorectal carcinoma cell line HCT116/L-OHP and its parent cell line HCT116 using a miRNA microarray. miR-1915 had the lowest expression of miRNA in HCT116/L-OHP cells compared to its parental cells. Overexpression of Bcl-2 is generally associated with tumor drug resistance, meanwhile Bcl-2 is a predicted target of miR-1915. We found that elevated levels of miR-1915 in the mimics-transfected HCT116/L-OHP cells reduced Bcl-2 protein level and the luciferase activity of a Bcl-2 3'-untranslated region-based reporter, and also sensitized these cells to some anticancer drugs. Taken together, our findings suggest that miR-1915 could play a role in the development of MDR in colorectal carcinoma cells at least in part by modulation of apoptosis via targeting Bcl-2. PMID:22121083

  1. The novel anthraquinone derivative IMP1338 induces death of human cancer cells by p53-independent S and G2/M cell cycle arrest.

    PubMed

    Choi, Hyun Kyung; Ryu, Hwani; Son, A-Rang; Seo, Bitna; Hwang, Sang-Gu; Song, Jie-Young; Ahn, Jiyeon

    2016-04-01

    To identify novel small molecules that induce selective cancer cell death, we screened a chemical library containing 1040 compounds in HT29 colon cancer and CCD18-Co normal colon cells, using a phenotypic cell-based viability assay system with the Cell Counting Kit-8 (CCK-8). We discovered a novel anthraquinone derivative, N-(4-[{(9,10-dioxo-9,10-dihydro-1-anthracenyl)sulfonyl}amino]phenyl)-N-methylacetamide (IMP1338), which was cytotoxic against the human colon cancer cells tested. The MTT cell viability assay showed that treatment with IMP1338 selectively inhibited HCT116, HCT116 p53(-/-), HT29, and A549 cancer cell proliferation compared to that of Beas2B normal epithelial cells. To elucidate the cellular mechanism underlying the cytotoxicity of IMP1338, we examined the effect of IMP1338 on the cell cycle distribution and death of cancer cells. IMP1338 treatment significantly arrested the cell cycle at S and G2/M phases by DNA damage and led to apoptotic cell death, which was determined using FACS analysis with Annexin V/PI double staining. Furthermore, IMP1338 increased caspase-3 cleavage in wild-type p53, p53 knockout HCT116, and HT29 cells as determined using immunoblotting. In addition, IMP1338 markedly induced the phosphorylation of histone H2AX and Chk1 in both cell lines while the combination of 5-fluorouracil (5-FU) and radiation inhibited the viability of HCT116, HCT116 p53(-/-), and HT29 cells compared to 5-FU or radiation alone. Our findings indicated that IMP1338 induced p53-independent cell death through S and G2/M phase arrest as well as DNA damage. These results provide a basis for future investigations assessing the promising anticancer properties of IMP1338. PMID:27044842

  2. Sulforaphane inhibits hypoxia-induced HIF-1α and VEGF expression and migration of human colon cancer cells.

    PubMed

    Kim, Dong Hwan; Sung, Bokyung; Kang, Yong Jung; Hwang, Seong Yeon; Kim, Min Jeong; Yoon, Jeong-Hyun; Im, Eunok; Kim, Nam Deuk

    2015-12-01

    The effects of sulforaphane (a natural product commonly found in broccoli) was investigated on hypoxia inducible factor-1α (HIF-1α) expression in HCT116 human colon cancer cells and AGS human gastric cancer cells. We found that hypoxia-induced HIF-1α protein expression in HCT116 and AGS cells, while treatment with sulforaphane markedly and concentration-dependently inhibited HIF-1α expression in both cell lines. Treatment with sulforaphane inhibited hypoxia-induced vascular endothelial growth factor (VEGF) expression in HCT116 cells. Treatment with sulforaphane modulated the effect of hypoxia on HIF-1α stability. However, degradation of HIF-1α by sulforaphane was not mediated through the 26S proteasome pathway. We also found that the inhibition of HIF-1α by sulforaphane was not mediated through AKT and extracellular signal-regulated kinase phosphorylation under hypoxic conditions. Finally, hypoxia-induced HCT116 cell migration was inhibited by sulforaphane. These data suggest that sulforaphane may inhibit human colon cancer progression and cancer cell angiogenesis by inhibiting HIF-1α and VEGF expression. Taken together, these results indicate that sulforaphane is a new and potent chemopreventive drug candidate for treating patients with human colon cancer. PMID:26498863

  3. Synergistic anticancer activity of curcumin and catechin: an in vitro study using human cancer cell lines.

    PubMed

    Manikandan, R; Beulaja, M; Arulvasu, C; Sellamuthu, S; Dinesh, D; Prabhu, D; Babu, G; Vaseeharan, B; Prabhu, N M

    2012-02-01

    The most practical approach to reduce morbidity and mortality of cancer is to delay the process of carcinogenesis by usage of anticancer agents. This necessitates that safer compounds are to be critically examined for anticancer activity especially, those derived from natural sources. A spice commonly found in India and the surrounding regions, is turmeric, derived from the rhizome of Curcuma longa and the major active component is a phytochemical termed curcumin. Green tea is one of the most popular beverages used worldwide, produced from the leaves of evergreen plant Camellia sinensis and the major active ingredients are polyphenolic compounds known as catechins. In this study, synergistic anticancer activity of curcumin and catechin was evaluated in human colon adenocarcinoma HCT 15, HCT 116, and human larynx carcinoma Hep G-2 cell lines. Although, both curcumin or catechin inhibited the growth of above cell lines, interestingly, in combination of both these compounds highest level of growth control was observed. The anticancer activity shown is due to cytotoxicity, nuclear fragmentation as well as condensation, and DNA fragmentation associated with the appearance of apoptosis. These results suggest that curcumin and catechin in combination can inhibit the proliferation of HCT 15, HCT 116, as well as Hep G-2 cells efficiently through induction of apoptosis. PMID:21780253

  4. Anti-proliferative effect of Melissa officinalis on human colon cancer cell line.

    PubMed

    Encalada, Manuel Alejandro; Hoyos, Kelly Melissa; Rehecho, Sheyla; Berasategi, Izaskun; de Ciriano, Mikel García-Íñiguez; Ansorena, Diana; Astiasarán, Iciar; Navarro-Blasco, Iñigo; Cavero, Rita Yolanda; Calvo, María Isabel

    2011-11-01

    Melissa officinalis L. (Lamiaceae) is consumed as a traditional herbal tea in the Mediterranean region. The cytotoxic effect of the 50% ethanolic and aqueous extract, determined by the MTT and NR assays, was evaluated in vitro on Human Colon Cancer Cell Line (HCT-116), using Triton 10% as positive control. The 50% ethanolic extract showed significant differences after 72 h of treatment, reducing cell proliferation to values close to 40%, even the lowest dose tested (5 μg/ml). In the MTT assay, the same extract caused the lowest cell viability with 13% at a concentration of 1,000 μg/ml after 72 h of treatment, being a value lower than Triton 10%. The antioxidant activity was also confirmed evaluating the capacity of the extracts to scavenge ABTS and DPPH radicals, and IC(50) values were highly correlated with the total phenolic and flavonoid content. Bioassay guided fractionation led to the isolation of an anti-proliferative compound, rosmarinic acid. Its structural elucidation was performed by HPLC/DAD/ESI/MS analysis. High dose of rosmarinic acid (1,000 μg/ml) was clearly cytotoxic against HCT-116 cells, with a significant decrease in cell number since the earliest time point (24 h). PMID:21964875

  5. Crude Extracts of Marine-derived and Soil Fungi of the Genus Neosartorya Exhibit Selective Anticancer Activity by Inducing Cell Death in Colon, Breast and Skin Cancer Cell Lines

    PubMed Central

    Ramos, Alice Abreu; Castro-Carvalho, Bruno; Prata-Sena, Maria; Dethoup, Tida; Buttachon, Suradet; Kijjoa, Anake; Rocha, Eduardo

    2016-01-01

    Background: The crude ethyl acetate extracts of marine-derived fungi Neosartorya tsunodae KUFC 9213 (E1) and N. laciniosa KUFC 7896 (E2), and soil fungus N. fischeri KUFC 6344 (E3) were evaluated for their in vitro anticancer activities on a panel of seven human cancer cell lines. Materials and Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed, after 48 h treatments with different concentrations of extracts, to determine their concentration of the extract or Dox that inhibits cell viability by 50% for each cell line. The effects of the crude extracts on DNA damage, clonogenic potential and their ability to induce cell death were also assessed. Results: E1 was found to the void of anti-proliferative effects. E2 was shown to decrease the clonogenic potential in human colorectal carcinoma cell line (HCT116), human malignant melanoma cell line (A375), human breast adenocarcinoma cell line (MCF7), and human caucasian colon adenocarcinoma Grade II cell line (HT29) cells, whereas E3 showed such effect only in HCT116 and MCF7 cells. Both extracts were found to increase DNA damage in some cell lines. E2 was found to induce cell death in HT29, HCT116, MCF7, and A375 cells while extract E3 increased cell death in MCF7 and HCT116 cell lines. Conclusion: The results reveal that E2 and E3 possess anticancer activities in human colon carcinoma, breast adenocarcinoma, and melanoma cells, validating the interest for an identification of molecular targets involved in the anticancer activity. SUMMARY The crude ethyl acetate extract of N. tsunodae (E1) did not decrease cell viability in any of the tested cell linesThe crude ethyl acetate extracts of N. laciniosa (E2) and N. fischeri (E3) decreased cell proliferation in some human cancer cell lines tested at both short- and long-termN. laciniosa (E2) induced a significant increase in the number of cell death, in part, due to the induction of DNA damageN. fischeri (E3) induce cell death but in some cell lines without induction of DNA damage detected by comet assayCrude ethyl extracts of N. laciniosa (E2) and N. fischeri (E3) exert an anticancer activity in human colon carcinoma, breast adenocarcinoma, and malignant melanoma cells. Abbreviations Used: A375: Human malignant melanoma cell line; A549: Human non-small lung cancer cell line; DAPI: 4,6-diamidino-2-phenylindole; DMEM: Dulbecco's Modified Eagle Medium; DMSO: Dimethylsulfoxide; Dox: Doxorubicin; E1: Neosartorya tsunodae KUFC 9213; E2: Neosartorya laciniosa KUFC 7896; E3: Neosartorya fischeri KUFC 6344; FBS: Fetal bovine serum; HCT116: Human colorectal carcinoma cell line; HEPES: (N-[2-hydroxyethyl] piperazine-N’-[2-ethane-sulfonic acid]); HepG2: Human hepatocellular carcinoma cell line; HT29: Human caucasian colon adenocarcinoma Grade II cell line; IC50: Concentration of the extract or Dox that inhibits cell viability by 50%; MCF7: Human breast adenocarcinoma cell line; MEM: Minimum Essential Medium Eagle; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCI-H460: Human non-small lung cancer cell line; PBS: Phosphate buffered saline; PE: Plating efficiency; RPMI: Roswell park memorial institute medium; SF: Surviving fraction; U-251: Human malignant glioblastoma cell line. PMID:26941530

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

    SciTech Connect

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

    2012-11-09

    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.

  7. Piceatannol, a natural analog of resveratrol, inhibits progression through the S phase of the cell cycle in colorectal cancer cell lines.

    PubMed

    Wolter, Freya; Clausnitzer, Antje; Akoglu, Bora; Stein, Jürgen

    2002-02-01

    Piceatannol, a naturally occurring analog of resveratrol, was previously identified as the active ingredient in herbal preparations in folk medicine and as an inhibitor of p72(Syk). We studied the effects of piceatannol on growth, proliferation, differentiation and cell cycle distribution profile of the human colon carcinoma cell line Caco-2. Growth of Caco-2 and HCT-116 cells was analyzed by crystal violet assay, which demonstrated dose- and time-dependent decreases in cell numbers. Treatment of Caco-2 cells with piceatannol reduced proliferation rate. No effect on differentiation was observed. Determination of cell cycle distribution by flow cytometry revealed an accumulation of cells in the S phase. Immunoblotting demonstrated that cyclin-dependent kinases (cdk) 2 and 6, as well as cdc2 were expressed at steady-state levels, whereas cyclin D1, cyclin B1 and cdk 4 were downregulated. The abundance of p27(Kip1) was also reduced, whereas the protein level of cyclin E was enhanced. Cyclin A levels were enhanced only at concentrations up to 100 micromol/L. These changes also were observed in studies with HCT-116 cells. On the basis of our findings, piceatannol can be considered to be a promising chemopreventive or anticancer agent. PMID:11823594

  8. Anomalous dystroglycan in carcinoma cell lines.

    PubMed

    Losasso, C; Di Tommaso, F; Sgambato, A; Ardito, R; Cittadini, A; Giardina, B; Petrucci, T C; Brancaccio, A

    2000-11-10

    Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non-tumorigenic mammary human cell lines, the expected beta-dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous approximately 31 kDa beta-dystroglycan band. alpha-Dystroglycan was udetectable in most of the cell lines in which beta-dystroglycan was found as a approximately 31 kDa species. An anomalous approximately 31 kDa beta-dystroglycan band was also observed in N-methyl-N-nitrosurea-induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low- and high-ionic strength, we demonstrated that both the 43 kDa and approximately 31 kDa beta-dystroglycan bands harbour their transmembrane segment. PMID:11078877

  9. Relative biological effectiveness of light ions in human tumoural cell lines: role of protein p53

    NASA Technical Reports Server (NTRS)

    Baggio, L.; Cavinato, M.; Cherubini, R.; Conzato, M.; Cucinotta, F.; Favaretto, S.; Gerardi, S.; Lora, S.; Stoppa, P.; Williams, J. R.

    2002-01-01

    Protons and alpha particles of high linear energy transfer (LET) have shown an increased relative biological effectiveness (RBE) with respect to X/gamma rays for several cellular and molecular endpoints in different in vitro cell systems. To contribute to understanding the biochemical mechanisms involved in the increased effectiveness of high LET radiation, an extensive study has been designed. The present work reports the preliminary result of this study on two human tumoural cell lines, DLD1 and HCT116, (with different p53 status), which indicate that for these cell lines, p53 does not appear to take a part in the response to radiation induced DNA damage, suggesting an alternative p53-independent pathway and a cell biochemical mechanism dependent on the cell type.

  10. Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

    SciTech Connect

    Gestl, Erin E.; Anne Boettger, S.

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated with p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53 gene expression levels compared to cell lines without p53 sequestration. Our data reveal the characteristic cytoplasmic sequestration of p53 by the heat shock protein mortalin in human colorectal adenocarcinoma cell lines, as is the case for other cancers, such as glioblastomas and hepatocellular carcinomas.

  11. Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression.

    PubMed

    Mirzayans, Razmik; Andrais, Bonnie; Scott, April; Wang, Ying W; Weiss, Robert H; Murray, David

    2015-01-01

    Phosphorylation of H2AX on Ser139 (γH2AX) after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called γH2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous γH2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s) for this response remains elusive. Here we show that cancer cells (e.g., HCT116; MCF7) that constitutively express detectable levels of p21WAF1 (p21) exhibit low numbers of γH2AX foci (<3/nucleus), whereas p21 knockout cells (HCT116p21-/-) and constitutively low p21-expressing cells (e.g., MDA-MB-231) exhibit high numbers of foci (e.g., >50/nucleus), and that these foci are not associated with apoptosis. The majority (>95%) of cells within HCT116p21-/- and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between γH2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i) p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous γH2AX foci coupled with phosphorylation and nuclear accumulation of p53; and (ii) p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of γH2AX. PMID:26006237

  12. Essential Oil Content of the Rhizome of Curcuma purpurascens Bl. (Temu Tis) and Its Antiproliferative Effect on Selected Human Carcinoma Cell Lines

    PubMed Central

    Hong, Sok-Lai; Lee, Guan-Serm; Ahmed Hamdi, Omer Abdalla; Awang, Khalijah; Aznam Nugroho, Nurfina

    2014-01-01

    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 μg/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 μg/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 μg/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

  13. FRET-Based Probe for Monitoring pH Changes in Lipid-Dense Region of Hct116 Cells.

    PubMed

    Reddy G, Upendar; A, Anila H; Ali, Firoj; Taye, Nandaraj; Chattopadhyay, Samit; Das, Amitava

    2015-11-20

    A rhodamine conjugate (L) with a pseudo Stokes shift of 165 nm is used for probing changes in solution pH under physiological conditions. This reagent is found to be nontoxic, and the luminescence response could be used for imaging changes in endogenous pH induced by dexamethanose (DMT) in the endoplasmic reticulum. PMID:26555683

  14. Icotinib hydrochloride enhances the effect of radiotherapy by affecting DNA repair in colorectal cancer cells.

    PubMed

    Ma, Hong; Bi, Jianping; Liu, Tao; Ke, Yang; Zhang, Sheng; Zhang, Tao

    2015-03-01

    The aim of the present study was to explore the efficacy and mechanism of the radiosensitisation of icotinib hydrochloride (IH), a novel oral epidermal growth factor receptor-tyrosine kinase activity inhibitor, by evaluating the changes in tumour cell double-strand breaks (DSBs) repair, cell cycle and apoptosis following a combination of IH and radiotherapy (RT) in human colorectal adenocarcinoma cell lines. The HT29 and HCT116 human CRC cell lines were treated with IH and/or radiation. Effects on cell viability and cell cycle progression were measured by MTT, a clonogenic survival assay, and flow cytometry. Immunofluorescent staining and western blot analysis were applied to detect the expression of γ-H2AX and 53BP1 in the different treatment groups. Finally, the in vivo effect on the growth of CRC xenografts was assessed in athymic nude mice. IH inhibited the proliferation and enhanced the radiosensitivity in HT29 and HCT116 CRC cells lines. IH combined with radiation increased cell cycle arrest in the G2/M phase compared to the other treatments in the HT29 cell line (P<0.05). Similarly, cell cycle arrest occurred in the HCT116 cell line, although this increase did not result in significant differences in the RT group (P>0.05). IH combined with radiation significantly inhibited the expression of γ-H2AX and 53BP1 based on results of immunofluorescent staining and western blot analysis. In vivo, IH plus radiation significantly inhibited the tumour growth compared to either agent independently. In conclusion, IH significantly increased the radiosensitivity of HT29 and HCT116 cells in vitro and in vivo. Radiation combined with EGFR blockade inhibited tumour proliferation, increased apoptosis, prolonged G2/M arrest and significantly enhanced DNA injury in colorectal cancer. These data support the clinical trials of biologically targeted and conventional therapies in the treatment of cancer. PMID:25572529

  15. Oncolytic reovirus preferentially induces apoptosis in KRAS mutant colorectal cancer cells, and synergizes with irinotecan

    PubMed Central

    Maitra, Radhashree; Seetharam, Raviraja; Tesfa, Lydia; Augustine, Titto A.; Klampfer, Lidija; Coffey, Matthew C.; Mariadason, John M.; Goel, Sanjay

    2014-01-01

    Reovirus is a double stranded RNA virus, with an intrinsic preference for replication in KRAS mutant cells. As 45% of human colorectal cancers (CRC) harbor KRAS mutations, we sought to investigate its efficacy in KRAS mutant CRC cells, and examine its impact in combination with the topoisimerase-1 inhibitor, irinotecan. Reovirus efficacy was examined in the KRAS mutant HCT116, and the isogenic KRAS WT Hke3 cell line, and in the non-malignant rat intestinal epithelial cell line. Apoptosis was determined by flow cytometry and TUNEL staining. Combination treatment with reovirus and irintoecan was investigated in 15 CRC cell lines, including the HCT116 p21 isogenic cell lines. Reovirus preferentially induced apoptosis in KRAS mutant HCT116 cells compared to its isogenic KRAS WT derivative, and in KRAS mutant IEC cells. Reovirus showed a greater degree of caspase 3 activation with PARP 1 cleavage, and preferential inhibition of p21 protein expression in KRAS mutant cells. Reovirus synergistically induced growth inhibition when combined with irinotecan. This synergy was lost upon p21 gene knock out. Reovirus preferentially induces apoptosis in KRAS mutant colon cancer cells. Reovirus and irinotecan combination therapy is synergistic, p21 mediated, and represents a novel potential treatment for patients with CRC. PMID:24798549

  16. Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression.

    PubMed

    Welman, Arkadiusz; Cawthorne, Christopher; Barraclough, Jane; Smith, Nigel; Griffiths, Gareth J; Cowen, Rachel L; Williams, Judith C; Stratford, Ian J; Dive, Caroline

    2005-04-15

    Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S-M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline-regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49-124xHCT116; 178-621xSW480; 261-787xHT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c-Src or dominant negative K-Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double-stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_welman.doc. PMID:15669025

  17. Simultaneous inhibition of ATR and PARP sensitizes colon cancer cell lines to irinotecan

    PubMed Central

    Abu-Sanad, Atlal; Wang, Yunzhe; Hasheminasab, Fatemeh; Panasci, Justin; Noë, Alycia; Rosca, Lorena; Davidson, David; Amrein, Lilian; Sharif-Askari, Bahram; Aloyz, Raquel; Panasci, Lawrence

    2015-01-01

    Enhanced DNA damage repair is one mechanism involved in colon cancer drug resistance. Thus, targeting molecular components of repair pathways with specific small molecule inhibitors may improve the efficacy of chemotherapy. ABT-888 and VE-821, inhibitors of poly-ADP-ribose-polymerase (PARP) and the serine/threonine-kinase Ataxia telangiectasia related (ATR), respectively, were used to treat colon cancer cell lines in combination with the topoisomerase-I inhibitor irinotecan (SN38). Our findings show that each of these DNA repair inhibitors utilized alone at nontoxic single agent concentrations resulted in sensitization to SN38 producing a 1.4–3 fold reduction in the 50% inhibitory concentration (IC50) of SN38 in three colon cancer cell lines. When combined together, nontoxic concentrations of ABT-888 and VE-821 produced a 4.5–27 fold reduction in the IC50 of SN38 with the HCT-116 colon cancer cells demonstrating the highest sensitization as compared to LoVo and HT-29 colon cancer cells. Furthermore, the combination of all three agents was associated with maximal G2 −M arrest and enhanced DNA-damage (γH2AX) in all three colon cancer cell lines. The mechanism of this enhanced sensitization was associated with: (a) maximal suppression of SN38 induced PARP activity in the presence of both inhibitors and (b) ABT-888 producing partial abrogation of the VE-821 enhancement of SN38 induced DNA-PK phosphorylation, resulting in more unrepaired DNA damage; these alterations were only present in the HCT-116 cells which have reduced levels of ATM. This novel combination of DNA repair inhibitors may be useful to enhance the activity of DNA damaging chemotherapies such as irinotecan and help produce sensitization to this drug in colon cancer. PMID:26257651

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

    PubMed

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

    2014-09-01

    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

  19. In vitro anticancer activity of extracts of Mentha Spp. against human cancer cells.

    PubMed

    Sharma, Vikas; Hussain, Shabir; Gupta, Moni; Saxena, Ajit Kumar

    2014-10-01

    In vitro anticancer potential of methanolic and aqueous extracts of whole plants of Mentha arvensis, M. longifolia, M. spicata and M. viridis at concentration of 100 ?g/ml was evaluated against eight human cancer cell lines--A-549, COLO-205, HCT-116, MCF-7, NCI-H322, PC-3, THP-1 and U-87MG from six different origins (breast, colon, glioblastoma, lung, leukemia and prostate) using sulphorhodamine blue (SRB) assay. Methanolic extracts of above-mentioned Mentha Spp. displayed anti-proliferative effect in the range of 70-97% against four human cancer cell lines, namely COLO-205, MCF-7, NCI-H322 and THP-1; however, aqueous extracts were found to be active against HCT-116 and PC-3. The results indicate that Mentha Spp. contain certain constituents with cytotoxic properties which may find use in developing anticancer agents. PMID:25630112

  20. RFPL4A increases the G1 population and decreases sensitivity to chemotherapy in human colorectal cancer cells.

    PubMed

    Naito, Atsushi; Yamamoto, Hirofumi; Kagawa, Yoshinori; Naito, Yoko; Okuzaki, Daisuke; Otani, Keisuke; Iwamoto, Yoriko; Maeda, Sakae; Kikuta, Junichi; Nishikawa, Keizo; Uemura, Mamoru; Nishimura, Junichi; Hata, Taishi; Takemasa, Ichiro; Mizushima, Tsunekazu; Ishii, Hideshi; Doki, Yuichiro; Mori, Masaki; Ishii, Masaru

    2015-03-01

    Cell cycle-arrested cancer cells are resistant to conventional chemotherapy that acts on the mitotic phases of the cell cycle, although the molecular mechanisms involved in halting cell cycle progression remain unclear. Here, we demonstrated that RFPL4A, an uncharacterized ubiquitin ligase, induced G1 retention and thus conferred decreased sensitivity to chemotherapy in the human colorectal cancer cell line, HCT116. Long term time lapse observations in HCT116 cells bearing a "fluorescence ubiquitin-based cell cycle indicator" identified a characteristic population that is viable but remains in the G1 phase for an extended period of time (up to 56 h). Microarray analyses showed that expression of RFPL4A was significantly up-regulated in these G1-arrested cells, not only in HCT116 cells but also in other cancer cell lines, and overexpression of RFPL4A increased the G1 population and decreased sensitivity to chemotherapy. However, knockdown of RFPL4A expression caused the cells to resume mitosis and induced their susceptibility to anti-cancer drugs in vitro and in vivo. These results indicate that RFPL4A is a novel factor that increases the G1 population and decreases sensitivity to chemotherapy and thus may be a promising therapeutic target for refractory tumor conditions. PMID:25605732

  1. RFPL4A Increases the G1 Population and Decreases Sensitivity to Chemotherapy in Human Colorectal Cancer Cells*

    PubMed Central

    Naito, Atsushi; Yamamoto, Hirofumi; Kagawa, Yoshinori; Naito, Yoko; Okuzaki, Daisuke; Otani, Keisuke; Iwamoto, Yoriko; Maeda, Sakae; Kikuta, Junichi; Nishikawa, Keizo; Uemura, Mamoru; Nishimura, Junichi; Hata, Taishi; Takemasa, Ichiro; Mizushima, Tsunekazu; Ishii, Hideshi; Doki, Yuichiro; Mori, Masaki; Ishii, Masaru

    2015-01-01

    Cell cycle-arrested cancer cells are resistant to conventional chemotherapy that acts on the mitotic phases of the cell cycle, although the molecular mechanisms involved in halting cell cycle progression remain unclear. Here, we demonstrated that RFPL4A, an uncharacterized ubiquitin ligase, induced G1 retention and thus conferred decreased sensitivity to chemotherapy in the human colorectal cancer cell line, HCT116. Long term time lapse observations in HCT116 cells bearing a “fluorescence ubiquitin-based cell cycle indicator” identified a characteristic population that is viable but remains in the G1 phase for an extended period of time (up to 56 h). Microarray analyses showed that expression of RFPL4A was significantly up-regulated in these G1-arrested cells, not only in HCT116 cells but also in other cancer cell lines, and overexpression of RFPL4A increased the G1 population and decreased sensitivity to chemotherapy. However, knockdown of RFPL4A expression caused the cells to resume mitosis and induced their susceptibility to anti-cancer drugs in vitro and in vivo. These results indicate that RFPL4A is a novel factor that increases the G1 population and decreases sensitivity to chemotherapy and thus may be a promising therapeutic target for refractory tumor conditions. PMID:25605732

  2. Novel seleno- and thio-urea derivatives with potent in vitro activities against several cancer cell lines.

    PubMed

    Alcolea, Verónica; Plano, Daniel; Karelia, Deepkamal N; Palop, Juan Antonio; Amin, Shantu; Sanmartín, Carmen; Sharma, Arun K

    2016-05-01

    A series of novel selenourea derivatives and corresponding thiourea analogs were synthesized and tested against a panel of six human cancer cell lines: melanoma (1205Lu), lung carcinoma (A549), prostatic carcinoma (DU145), colorectal carcinoma (HCT116), pancreatic epithelioid carcinoma (PANC-1) and pancreatic adenocarcinoma (BxPC3). In general, we found that the selenium-containing derivatives were more potent than their isosteric sulfur analogs. Four selenourea derivatives (1e, 1f, 1g and 1i) showed IC50 values below 10 μM in all of tested cell lines at 72 h. On the basis of its potent activity, compound 1g was selected for further biological evaluation in different colon cancer cell lines. Our results indicated that compound 1g induced apoptosis by caspase activation, along with inhibition of anti-apoptotic proteins. PMID:26922233

  3. Ochratoxin A and T-2 Toxin Induce Clonogenicity and Cell Migration in Human Colon Carcinoma and Fetal Lung Fibroblast Cell Lines.

    PubMed

    Abassi, Haila; Ayed-Boussema, Imen; Shirley, Sarah; Abid, Salwa; Bacha, Hassen

    2016-03-01

    T-2 toxin and Ochratoxin A (OTA) are toxic secondary metabolites produced by various fungi, and together they contaminate feedstuffs worldwide. T-2 toxin and OTA may exert carcinogenic action in rodent. Despite the various in vivo experiments, carcinogenicity of these two mycotoxins has not yet been proven for human. In this current study, we proposed to investigate, in Human colon carcinoma cells and fetal lung fibroblast-like cells transfected with MYC, the effect of T-2 toxin and OTA on cell clonogenicity and cell migration. Results of the present investigation showed that T2-toxin as well as OTA has an important clonogenic effect in all cell lines, suggesting that these mycotoxins could promote the transcription of c-myc gene. Furthermore, T-2 toxin and OTA enhanced the migration effect of HCT116 cells at very low concentrations, proposing that these mycotoxins may exhibit carcinogenesis-like properties in the studied cells. PMID:26849850

  4. Role of miR-100 in the radioresistance of colorectal cancer cells.

    PubMed

    Yang, Xiao-Dong; Xu, Xiao-Hui; Zhang, Shu-Yu; Wu, Yong; Xing, Chun-Gen; Ru, Gan; Xu, Hong-Tao; Cao, Jian-Ping

    2015-01-01

    The prognosis of radioresistant colorectal cancer (CRC) is generally poor. Abnormal expression of microRNAs (miRNAs) is involved in the radiosensitivity of various tumor cells as these RNAs regulate biological signaling pathways. However, radioresistance-associated miRNAs in CRC have not yet been identified. In this study, we filtered out HCT116 and CCL-244 from seven CRC cell lines that showed the highest difference in radiosensitivity in a clonogenic assay. MiRNA sequencing identified 33 differentially expressed miRNAs (13 up-regulated and 20 down-regulated) in CCL-244 and 37 in HCT116 (20 up-regulated and 17 down-regulated) cells. MiR-100 was significantly down-regulated in CCL-244 cells after X-ray irradiation but not in HCT116 cells. Quantitative real-time PCR showed that the expression of miR-100 in CRC tissues was significantly lower than that in normal tissues. Thus, miR-100 seems to be involved in the radioresistance of CCL-244 cells. MiR-100 up-regulation sensitized CCL-244 cells to X-ray irradiation, which probably led to apoptosis and DNA double-strand breaks in these. In conclusion, to our knowledge, this is the first study to show that miR-100 may play an important role in regulating the radiosensitivity of CRC, and it may act as a new clinical target for CRC radiotherapy. PMID:25973296

  5. Role of miR-100 in the radioresistance of colorectal cancer cells

    PubMed Central

    Yang, Xiao-Dong; Xu, Xiao-Hui; Zhang, Shu-Yu; Wu, Yong; Xing, Chun-Gen; Ru, Gan; Xu, Hong-Tao; Cao, Jian-Ping

    2015-01-01

    The prognosis of radioresistant colorectal cancer (CRC) is generally poor. Abnormal expression of microRNAs (miRNAs) is involved in the radiosensitivity of various tumor cells as these RNAs regulate biological signaling pathways. However, radioresistance-associated miRNAs in CRC have not yet been identified. In this study, we filtered out HCT116 and CCL-244 from seven CRC cell lines that showed the highest difference in radiosensitivity in a clonogenic assay. MiRNA sequencing identified 33 differentially expressed miRNAs (13 up-regulated and 20 down-regulated) in CCL-244 and 37 in HCT116 (20 up-regulated and 17 down-regulated) cells. MiR-100 was significantly down-regulated in CCL-244 cells after X-ray irradiation but not in HCT116 cells. Quantitative real-time PCR showed that the expression of miR-100 in CRC tissues was significantly lower than that in normal tissues. Thus, miR-100 seems to be involved in the radioresistance of CCL-244 cells. MiR-100 up-regulation sensitized CCL-244 cells to X-ray irradiation, which probably led to apoptosis and DNA double-strand breaks in these. In conclusion, to our knowledge, this is the first study to show that miR-100 may play an important role in regulating the radiosensitivity of CRC, and it may act as a new clinical target for CRC radiotherapy. PMID:25973296

  6. Cell diameter measurements obtained with a handheld cell counter could be used as a surrogate marker of G2/M arrest and apoptosis in colon cancer cell lines exposed to SN-38

    SciTech Connect

    Tahara, Makiko; Inoue, Takeshi; Fujii, Hirofumi; Kotake, Kenjiro; Sugano, Kokichi

    2013-05-17

    Highlights: •Chemo-sensitivity to SN-38 was assayed by the automated cell counter. •Colon cancer cell line, HCT116 cells were more sensitive to SN-38 than HT29 cells. •Increase of cell size reflects G2/M arrest. •Appearance of small particles indicates cell apoptosis. -- Abstract: In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancer cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the {sup 3}H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.

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

    PubMed

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

    2010-08-01

    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

  8. Omega-3 fatty acids induce Ca2+ mobilization responses in human colon epithelial cell lines endogenously expressing FFA4

    PubMed Central

    Kim, Jung-min; Lee, Kyoung-pil; Park, Soo-jin; Kang, Saeromi; Huang, Jin; Lee, Jung-min; Sato, Koichi; Chung, Hae-young; Okajima, Fumikazu; Im, Dong-soon

    2015-01-01

    Aim: Free fatty acid receptor 4 (FFA4; formerly known as GPR120) is the G protein-coupled receptor (GPCR) for omega-3 polyunsaturated fatty acids. FFA4 has been found to express in the small intestines and colons of mice and humans. In this study we investigate the effects of omega-3 polyunsaturated fatty acids on FFA4 in human colon epithelial cells in vitro. Methods: HCT116 and HT-29 human colon epithelial cell lines endogenously expressing FFA4 were used. Intracellular Ca2+ concentration ([Ca2+]i) was measured in fura 2-AM-loaded cells with fluorescence spectrophotometry. RT-PCR and immunohistochemistry were used to detect FFA4. Results: Ten to 100 μmol/L of omega-3 polyunsaturated fatty acids α-linolenic acid (αLA) or eicosapentaenoic acid (EPA) induced dose-dependent [Ca2+]i increase in HCT116 and HT-29 cells, whereas docosahexaenoic acid (DHA) had no effect. In addition, the omega-6 fatty acids linoleic acid and γ-linoleic acid also dose-dependently increase [Ca2+]i, but the mono-unsaturated fatty acid oleic acid and saturated fatty acids such as stearic acid and palmitic acid had no effect. In HCT116 and HT-29 cells, the αLA-induced [Ca2+]i increase was partially inhibited by pretreatment with EGTA, phospholipase C inhibitor edelfosine, cADPR inhibitors 8-bro-cADPR or DAB, and abolished by pretreatment with Ca2+ATPase inhibitor thapsigargin, but was not affected by Gi/o protein inhibitor PTX or IP3R inhibitor 2-APB. Conclusion: Omega-3 and omega-6 long-chain polyunsaturated fatty acids (C18-20) induce Ca2+ mobilization responses in human colonic epithelial cells in vitro through activation of FFA4 and PTX-insensitive Gi/o protein, followed by Ca2+ release from thapsigargin-sensitive Ca2+ stores and Ca2+ influx across the plasma membrane. PMID:26005911

  9. DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

    PubMed Central

    2009-01-01

    Background Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes. Methods HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity® Pathway Analysis. Results Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed. Conclusion This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer. PMID:19948057

  10. Lipid alterations in human colon epithelial cells induced to differentiation and/or apoptosis by butyrate and polyunsaturated fatty acids.

    PubMed

    Hofmanová, Jiřina; Ciganek, Miroslav; Slavík, Josef; Kozubík, Alois; Stixová, Lenka; Vaculová, Alena; Dušek, Ladislav; Machala, Miroslav

    2012-06-01

    The present study highlights the important association between lipid alterations and differentiation/apoptotic responses in human colon differentiating (FHC) and nondifferentiating (HCT-116) cell lines after their treatment with short-chain fatty acid sodium butyrate (NaBt), polyunsaturated fatty acids (PUFAs), and/or their combination. Our data from GC/MS and LC/MS/MS showed an effective incorporation and metabolization of the supplemented arachidonic acid (AA) or docosahexaenoic acid (DHA), resulting in an enhanced content of the respective PUFA in individual phospholipid (PL) classes and an altered composition of the whole cellular fatty acid spectrum in both FHC and HCT-116 cells. We provide novel evidence that NaBt combined with PUFAs additionally modulated AA and DHA cellular levels and caused their shift from triacylglycerol to PL fractions. NaBt increased, while AA, DHA and their combination with NaBt decreased endogenous fatty acid synthesis in FHC but not in HCT-116 cells. Fatty acid treatment also altered membrane lipid structure, augmented cytoplasmic lipid droplet accumulation, reactive oxygen species (ROS) production and dissipation of the mitochondrial membrane potential. All these parameters were significantly enhanced by combined NaBt/PUFA treatment, but only in FHC cells was this accompanied by highly increased apoptosis and suppressed differentiation. Moreover, the most significant changes of ROS production, differentiation and apoptosis among the parameters studied, the highest effects of combined NaBt/PUFA treatment and a lower sensitivity of HCT-116 cells were confirmed using two-way ANOVA. Our results demonstrate an important role of fatty acid-induced lipid alterations in the different apoptotic/differentiation response of colon cells with various carcinogenic potential. PMID:21775115

  11. Definitive Molecular Cytogenetic Characterization of 15 Colorectal Cancer Cell Lines

    PubMed Central

    Knutsen, Turid; Padilla-Nash, Hesed M.; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J.; Ried, Thomas

    2009-01-01

    In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. We here present the results of a comprehensive investigation of 15 established colorectal cancer cell lines utilizing spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) are described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines, isochromosomes were the most common recurrent abnormalities, and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities result predominantly in copy number changes rather than specific chromosome or gene fusions, suggests this may be the major mechanism leading to carcinogenesis in colorectal cancer. PMID:19927377

  12. Withaferin-A Inhibits Colon Cancer Cell Growth by Blocking STAT3 Transcriptional Activity

    PubMed Central

    Choi, Bu Young; Kim, Bong-Woo

    2015-01-01

    Background: Withania somnifera (known as Ashwagandha) is a medicinal plant used in the ayurvedic medicines in India. Withaferin-A, a withanolide derived from the leaf extract of W. somnifera, has been reported to exhibit anti-tumor activity against various cancer cells, such as leukemia, breast cancer and colon cancer cells. Methods: We investigated the anti-cancer effects of withaferin-A on the proliferation and migration of human colorectal cancer (HCT116) cells. And we evaluated the effects of withaferin-A on the transcriptional activity of STAT3 and the growth of HCT116 cells in xenograft mouse tumor model. Results: In the present study, we found that withaferin-A inhibited the proliferation and migration of HCT116 cells in a concentration-dependent manner. Treatment of HCT116 cells with withaferin-A attenuated interleukin-6-induced activation of STAT3, which has been implicated in the development and progression of colon cancer. To examine the effect of withaferin-A on HCT116 cells proliferation in vivo, we generated HCT116 cells xenograft tumors in Balb/c nude mice and treated the tumor bearing mice with or without withaferin-A intraperitoneally. Treatment with withaferin-A exhibited significant decrease in the volume and weight of tumors as compared to untreated controls. Conclusions: The present study suggests that withaferin-A holds the potential to be developed as a small molecule inhibitor of STAT3 for the treatment of HCT116. PMID:26473157

  13. Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives

    PubMed Central

    Brito, Hugo; Martins, Ana Cláudia; Lavrado, João; Mendes, Eduarda; Francisco, Ana Paula; Santos, Sofia A.; Ohnmacht, Stephan A.; Kim, Nam-Soon; Rodrigues, Cecília M. P.; Moreira, Rui; Neidle, Stephen; Borralho, Pedro M.; Paulo, Alexandra

    2015-01-01

    Background A guanine-rich strand within the promoter of the KRAS gene can fold into an intra-molecular G-quadruplex structure (G4), which has an important role in the regulation of KRAS transcription. We have previously identified indolo[3,2-b]quinolines with a 7-carboxylate group and three alkylamine side chains (IQ3A) as effective G4 stabilizers and promising selective anticancer leads. Herein we investigated the anticancer mechanism of action of these compounds, which we hypothesized due to stabilization of the G4 sequence in the KRAS promoter and subsequent down-regulation of gene expression. Methodology/Principal Findings IQ3A compounds showed greater stabilization of G4 compared to duplex DNA structures and reduced KRAS promoter activity in a dual luciferase reporter assay. Moreover, IQ3A compounds showed high anti-proliferative activity in HCT116 and SW620 colon cancer cells (IC50 < 2.69 μM), without eliciting cell death in non-malignant HEK293T human embryonic kidney, and human colon fibroblasts CCD18co. IQ3A compounds significantly reduced KRAS mRNA and protein steady-state levels at IC50 concentrations, and increased p53 protein steady-state levels and cell death by apoptosis in HCT116 cells (mut KRAS, wt p53). Furthermore, KRAS silencing in HCT116 p53 wild-type (p53(+/+)) and null (p53(-/-)) isogenic cell lines induced a higher level of cell death, and a higher IQ3A-induced cell death in HCT116 p53(+/+) compared to HCT116 p53(-/-). Conclusions Herein we provide evidence that G4 ligands such as IQ3A compounds can target G4 motifs present in KRAS promoter, down-regulate the expression of the mutant KRAS gene through inhibition of transcription and translation, and induce cell death by apoptosis in colon cancer cell lines. Thus, targeting KRAS at the genomic level with G4 ligands may be a new anticancer therapy strategy for colon cancer. PMID:26024321

  14. 5-ASA Affects Cell Cycle Progression in Colorectal Cells by Reversibly Activating a Replication Checkpoint

    PubMed Central

    LUCIANI, M. GLORIA; CAMPREGHER, CHRISTOPH; FORTUNE, JOHN M.; KUNKEL, THOMAS A.; GASCHE, CHRISTOPH

    2007-01-01

    Background & Aims Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. Methods CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116p53−/−, HCT116+chr3, and LoVo were treated with 5-ASA for 2–96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. Results We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. Conclusions Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis. PMID:17241873

  15. Cytotoxicity of Elaoephorbia drupifera and other Cameroonian medicinal plants against drug sensitive and multidrug resistant cancer cells

    PubMed Central

    2013-01-01

    Background Multidrug resistance (MDR) is a major hurdle for cancer treatment worldwide and accounts for chemotherapy failure in over 90% of patients with metastatic cancer. Evidence of the cytotoxicity of Cameroonian plants against cancer cell lines including MDR phenotypes is been intensively and progressively provided. The present work was therefore designed to evaluate the cytotoxicity of the methanol extracts of twenty-two Cameroonian medicinal plants against sensitive and MDR cancer cell lines. Methods The methanol maceration was used to obtain the crude plant extracts whilst the cytotoxicity of the studied extracts was determined using a resazurin reduction assay. Results A preliminary assay on leukemia CCRF-CEM cells at 40 μg/mL shows that six of the twenty plant extract were able to enhance less than 50% of the growth proliferation of CCRF-CEM cells. These include Crinum zeylanicum (32.22%), Entada abyssinica (34.67%), Elaoephorbia drupifera (35.05%), Dioscorea bulbifera (45.88%), Eremomastax speciosa (46.07%) and Polistigma thonningii (45.11%). Among these six plants, E. drupifera showed the best activity with IC50 values below or around 30 μg/mL against the nine tested cancer cell lines. The lowest IC50 value of 8.40 μg/mL was recorded with the extract of E. drupifera against MDA-MB231 breast cancer cell line. The IC50 values below 10 μg/mL were recorded with the extracts of E. drupifera against MDA-MB231 breast cancer cells, C. zeylanicum against HCT116 p53+/+ and HCT116p53-/- colon cancer cells and E. abyssinica against HCT116 p53+/+ cells. Conclusion The results of the present study provide evidence of the cytotoxic potential of some Cameroonian medicinal plants and a baseline information for the potential use of Elaoephorbia drupifera in the treatment of sensitive and drug-resistant cancer cell lines. PMID:24088184

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

    PubMed Central

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

    2008-01-01

    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

  17. Inhibitory activities of Perilla frutescens britton leaf extract against the growth, migration, and adhesion of human cancer cells

    PubMed Central

    Kwak, Youngeun

    2015-01-01

    BACKGROUND/OBJECTIVES Perilla frutescens Britton leaves are a commonly consumed vegetable in different Asian countries including Korea. Cancer is a major cause of human death worldwide. The aim of the current study was to investigate the inhibitory effects of ethanol extract of perilla leaf (PLE) against important characteristics of cancer cells, including unrestricted growth, resisted apoptosis, and activated metastasis, using human cancer cells. MATERIALS/METHODS Two human cancer cell lines were used in this study, HCT116 colorectal carcinoma cells and H1299 non-small cell lung carcinoma cells. Assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were performed for measurement of cell growth. Soft agar and wound healing assays were performed to determine colony formation and cell migration, respectively. Nuclear staining and cell cycle analysis were performed for assessment of apoptosis. Fibronectin-coated plates were used to determine cell adhesion. RESULTS Treatment of HCT116 and H1299 cells with PLE resulted in dose-dependent inhibition of growth by 52-92% (at the concentrations of 87.5, 175, and 350 µg/ml) and completely abolished the colony formation in soft agar (at the concentration of 350 µg/ml). Treatment with PLE at the 350 µg/ml concentration resulted in change of the nucleus morphology and significantly increased sub-G1 cell population in both cells, indicating its apoptosis-inducing activity. PLE at the concentration range of 87.5 to 350 µg/ml was also effective in inhibiting the migration of H1299 cells (by 52-58%) and adhesion of both HCT116 and H1299 cells (by 25-46%). CONCLUSIONS These results indicate that PLE exerts anti-cancer activities against colon and lung cancers in vitro. Further studies are needed in order to determine whether similar effects are reproduced in vivo. PMID:25671062

  18. Inhibitor or promoter? The performance of polysaccharides from Ganoderma lucidum on human tumor cells with different p53 statuses.

    PubMed

    Zhang, Jue; Chen, Jun-Ming; Wang, Xiao-Xia; Xia, Yong-Mei; Cui, Steve W; Li, Jian; Ding, Zhong-Yang

    2016-04-20

    Polysaccharides from Ganoderma lucidum (GLPs) have been taken as effective supplements by both healthy people and cancer patients for many years. However, this short survey indicates that instead of inhibiting cancer cell growth, both submerge-cultured intracellular GLP and fruiting body GLP can stimulate the growth of human carcinoma cell lines lacking functional p53, such as HCT-116 p53(-/-), Saos-2, H1299, HL-60, MDA-MB-157. Conversely, the two GLPs inhibit all other assayed cells with functional p53. These results could be an alert since mutational inactivation of the tumor suppressor protein p53 is the most frequent genetic alteration found in human tumors. PMID:26999513

  19. HSP90 inhibition downregulates thymidylate synthase and sensitizes colorectal cancer cell lines to the effect of 5FU-based chemotherapy.

    PubMed

    Nagaraju, Ganji Purnachandra; Alese, Olatunji B; Landry, Jerome; Diaz, Roberto; El-Rayes, Bassel F

    2014-10-30

    Cell cycle progression and DNA synthesis are essential steps in cancer cell growth. Thymidylate synthase (TS) is a therapeutic target for 5FU. We tested the hypothesis that HSP90 transcriptional and functional inhibition can inhibit cell cycle progression, downregulate TS levels and sensitize colorectal cancer (CRC) cell lines to the effects of 5FU. Treatment with ganetespib (50 nM) for 24 hours inhibited cyclin D1 and pRb at the transcriptional and translational levels and induced p21, leading to G0/G1 cell cycle arrest in both CRC cell lines (HCT-116 and HT-29). This was associated with downregulation of E2F1 and its target gene TS. In addition, ganetespib inhibited PI3K/Akt and ERK signalling pathways. Similar effects were observed with HSP90 knockdown in both cell lines. Ganetespib sensitized CRC cell lines to the effects of oxaliplatin and 5FU. Similar effects were also observed in tumors from animals treated with ganetespib, oxaliplatin and 5FU. In this study, we present in vitro and animal data supporting that the targeting of HSP90 decreases CRC cell survival and proliferation. Ganetespib sensitizes CRC cell lines to the effects of 5FU-based chemotherapy. Combining HSP90 inhibitors with chemotherapy is a rational approach for future drug development in CRC. PMID:25296971

  20. Role of MCP-1 in alcohol-induced aggressiveness of colorectal cancer cells.

    PubMed

    Xu, Mei; Wang, Siying; Qi, Yuanlin; Chen, Li; Frank, Jacqueline A; Yang, Xiuwei H; Zhang, Zhuo; Shi, Xianglin; Luo, Jia

    2016-05-01

    Epidemiological studies demonstrate that alcohol consumption is associated with an increased risk of colorectal cancer (CRC). In addition to promoting carcinogenesis, alcohol may also accelerate the progression of existing CRC. We hypothesized that alcohol may enhance the aggressiveness of CRC. In this study, we investigated the effect of alcohol on the migration/invasion and metastasis of CRC. Alcohol increased the migration/invasion of colorectal cancer cells (DLD1, HCT116, HT29, and SW480) in a concentration-dependent manner. Among these colon cancer cell lines, HCT116 cells were most responsive while HT29 cells were the least responsive to ethanol-stimulated cell migration/invasion. These in vitro results were supported by animal studies which demonstrated that ethanol enhanced the metastasis of colorectal cancer cells to the liver and lung. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays an important role in regulating tumor microenvironment and metastasis. Alcohol increased the expression of MCP-1 and its receptor CCR2 at both protein and mRNA levels. The pattern of alcohol-induced alterations in MCP-1 expression was consistent with its effect on migration/invasion; HCT116 cells displayed the highest up-regulation of MCP-1/CCR2 in response to alcohol exposure. An antagonist of CCR2 blocked alcohol-stimulated migration. Alcohol caused an initial cytosolic accumulation of β-catenin and its subsequent nuclear translocation by inhibiting GSK3β activity. Alcohol stimulated the activity of MCP-1 gene promoter in a β-catenin-dependent manner. Furthermore, knock-down of MCP-1/CCR2 or β-catenin was sufficient to inhibit alcohol-induced cell migration/invasion. Together, these results suggested that alcohol may promote the metastasis of CRC through modulating GSK3β/β-catenin/MCP-1 pathway. © 2015 Wiley Periodicals, Inc. PMID:26014148

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

    PubMed

    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

    2014-10-01

    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 M), EGCG (0-175 M) and GS extract (0-15 M) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 M, EGCG 175 M and GS extract 15 M 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

  2. Reduced host cell reactivation of oxidative DNA damage in human cells deficient in the mismatch repair gene hMSH2.

    PubMed

    Pitsikas, Photini; Lee, David; Rainbow, Andrew J

    2007-05-01

    Germ line mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 account for approximately 98% of hereditary non-polyposis colorectal cancers. In addition, there is increasing evidence for an involvement of MMR gene expression in the response of cells to UV-induced skin cancer. The link between MMR and skin cancer suggests an involvement of MMR gene expression in the response of skin cells to UV-induced DNA damage. In this report, we have used two reporter gene assays to examine the role of hMSH2 and hMLH1 in the repair of oxidative DNA damage induced by UVA light and DNA damage caused by methylene blue plus visible light (MB+VL). UVA and MB+VL produce 8-hydroxyguanines in DNA that are repaired by base excision repair (BER). AdHCMVlacZ is a replication-deficient recombinant adenovirus that expresses the beta-galactosidase (beta-gal) reporter gene under the control of the human cytomegalovirus (CMV) immediate-early promoter. We show a reduced host cell reactivation for beta-gal expression of UVA-treated and MB+VL-treated AdHCMVlacZ in hMSH2-deficient LoVo human colon adenocarcinoma cells compared to their hMSH2-proficient counterpart SW480 cells, but not in hMLH1-deficient HCT116 human colon adenocarcinoma cells compared to hMLH1-proficient HCT116-chr3 cells. We have also reported previously that enhanced expression of the undamaged AdHCMVlacZ reporter gene is induced by the pre-treatment of cells with lower levels of the DNA-damaging agent and to higher expression levels in transcription-coupled repair (TCR)-deficient compared to TCR-proficient cells. Here we show that pre-treatment of cells with UVA or MB+VL enhanced expression of the undamaged reporter gene to a higher level in LoVo compared to SW480 cells but there was little or no difference in HCT116 compared to HCT116-chr3 cells. These results suggest a substantial involvement of hMSH2 but little or no involvement of hMLH1 in the repair of UVA- and MB+VL-induced oxidative DNA damage by BER. PMID:17351251

  3. MicroRNA-101 down-regulates sphingosine kinase 1 in colorectal cancer cells.

    PubMed

    Chen, Min-Bin; Yang, Lan; Lu, Pei-Hua; Fu, Xing-Li; Zhang, Yan; Zhu, Ya-Qun; Tian, Ye

    2015-08-01

    MicroRNAs (miRs) dysregulation is a general feature of colorectal cancer (CRC) and other solid tumors, and is associated cancer progression. In the current study, we demonstrate that microRNA-101 (miR-101) inhibits CRC cells probably through down-regulating sphingosine kinase 1 (SphK1). Our results showed that exogenously expressing miR-101 inhibited CRC cell (HT-29 and HCT-116 lines) growth in vitro. At the molecular level, miR-101 dramatically down-regulated SphK1 mRNA and protein expression, causing pro-apoptotic ceramide production in above CRC cells. On the other hand, inhibition of miR-101 through expressing antagomiR-101 increased SphK1 expression to down-regulate ceramide level in HT-29 cells. miR-101 expression increased the in vitro anti-CRC activity of conventional chemo-agents: paclitaxel and doxorubicin. CRC cells with SphK1-shRNA knockdown showed similar phenotypes as the miR-101-expressed CRC cells, presenting with elevated level of ceramide and high sensitivity to paclitaxel or doxorubicin. In vivo, HCT-116 xenograft growth in severe combined immuno-deficient (SCID) mice was dramatically inhibited by over-expressing miR-101. Further, miR-101 enhanced paclitaxel-induced anti-HCT-116 activity in vivo. Together, these results indicate that miR-101 exerts its anti-CRC activities probably through down-regulating SphK1. PMID:26071354

  4. IND-2, a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells.

    PubMed

    Karthikeyan, Chandrabose; Lee, Crystal; Moore, Joshua; Mittal, Roopali; Suswam, Esther A; Abbott, Kodye L; Pondugula, Satyanarayana R; Manne, Upender; Narayanan, Narayanan K; Trivedi, Piyush; Tiwari, Amit K

    2015-02-01

    Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1″,2″:1,5]pyrazolo[3,4-b]quinoline, exhibited more than ten-fold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and sub-micromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and five-fold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer. PMID:25537531

  5. IND2, a pyrimido[1”,2”:1,5]pyrazolo[3,4-b]quinoline derivative, circumvents multi-drug resistance and causes apoptosis in colon cancer cells

    PubMed Central

    Karthikeyan, Chandrabose; Lee, Crystal; Moore, Joshua; Mittal, Roopali; Suswam, Esther A.; Abbott, Kodye L; Pondugula, Satyanarayana R.; Manne, Upender; Narayanan, Narayanan K.; Trivedi, Piyush; Tiwari, Amit K.

    2014-01-01

    Naturally occurring condensed quinolines have anticancer properties. In efforts to find active analogues, we designed and synthesized eight polycyclic heterocycles with a pyrimido[1”,2”:1,5]pyrazolo[3,4-b]quinoline framework (IND series). The compounds were evaluated for activity against colon (HCT-116 and S1-MI-80), prostate (PC3 and DU-145), breast (MCF-7 and MDAMB-231), ovarian (ov2008 and A2780), and hepatocellular (HepG2) cancer cells and against non-cancerous Madin Darby canine kidney (MDCK), mouse embryonic fibroblast (NIH/3T3), and human embryonic kidney cells (HEK293). IND-2, a 4-chloro-2-methyl pyrimido[1”,2”:1,5]pyrazolo[3,4-b]quinoline, exhibited more than tenfold selectivity and potent cytotoxic activity against colon cancer cells relative to the other cancer and non-cancer cells. With five additional colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo), IND-2 had similar cytotoxicity and selectivity, and submicromolar concentrations caused changes in the morphology of HCT-116 and HCT-15 cells. IND-2 did not activate the transactivating function of the pregnane X receptor (PXR), indicating that it does not induce PXR-regulated ABCB1 or ABCG2 transporters. Indeed, IND-2 was not a substrate of ABCB1 or ABCG2, and it induced cytotoxicity in HEK293 cells overexpressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. IND-2 was cytotoxic to resistant colon carcinoma S1-MI-80 cells, approximately three- and fivefold more than SN-38 and topotecan, respectively. In HCT-116 colon cancer cells, IND-2 produced concentration-dependent changes in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation. These findings suggest that, by increasing apoptosis, IND-2 has potential therapeutic efficacy for colorectal cancer. PMID:25537531

  6. Comparative study of genotoxic, antigenotoxic and cytotoxic activities of monoterpenes camphor, eucalyptol and thujone in bacteria and mammalian cells.

    PubMed

    Nikolić, Biljana; Vasilijević, Bojana; Mitić-Ćulafić, Dragana; Vuković-Gačić, Branka; Knežević-Vukćević, Jelena

    2015-12-01

    Genotoxic/antigenotoxic, mutagenic/antimutagenic and cytotoxic effects of monoterpenes camphor, eucalyptol and thujone were determined in bacteria and mammalian cells using alkaline comet assay, Escherichia coli K12 reversion test and MTT assay, respectively. When applied in low doses (up to 200 μM in bacterial assay and 50 μM in comet assay) monoterpenes protected repair proficient E. coli and Vero cells against UV-induced mutagenesis and 4NQO-induced DNA strand breaks, respectively. Antimutagenic response was not detected in nucleotide excision repair (NER) deficient bacteria. When monoterpenes were applied in higher doses, a weak mutagenic effect was found in mismatch repair (MMR) and NER deficient E. coli strains, while induction of DNA strand breaks was evident in human fetal lung fibroblasts MRC-5, colorectal carcinoma HT-29 and HCT 116 cells, as well as in Vero cells. Moreover, the involvement of NER, MMR and RecBCD pathways in repair of DNA lesions induced by monoterpenes was demonstrated in E. coli. Camphor, eucalyptol and thujone were cytotoxic to MRC-5, HT-29 and HCT 116 cells. The most susceptible cell line was HCT 116, with IC50 values of 4.5 mM for camphor, 4 mM for eucalyptol and 1 mM for thujone. Observed effects of monoterpenes are consistent with hormesis response, characterized by a low dose beneficial effect and a high dose adverse effect of a stressor agent, and provide a basis for further study of both chemopreventive and chemotherapeutic potential of camphor, eucalyptol and thujone. PMID:26482939

  7. Camel milk lactoferrin reduces the proliferation of colorectal cancer cells and exerts antioxidant and DNA damage inhibitory activities.

    PubMed

    Habib, Hosam M; Ibrahim, Wissam H; Schneider-Stock, Regine; Hassan, Hassan M

    2013-11-01

    Lactoferrin (Lf), the main iron-binding protein of milk, has biological activities. We have evaluated the potential of camel milk lactoferrin for its ability to inhibit the proliferation of the colon cancer cell line, HCT-116, in vitro, DNA damage and its antioxidant activities for the first time. The antioxidant capacity of Lf was evaluated by different assays, including ferric-reducing/antioxidant power assay (FRAP), free radical-scavenging activity (DPPH), nitric oxide (NO) radical-scavenging assay, total antioxidant activity and DNA damage, compared with vitamin C and rutin. PMID:23768340

  8. The flavonoid quercetin transiently inhibits the activity of taxol and nocodazole through interference with the cell cycle

    PubMed Central

    Samuel, Temesgen; Fadlalla, Khalda; Turner, Timothy; Yehualaeshet, Teshome E.

    2010-01-01

    Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose dependent manner, 12.5-50μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 hours. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that while long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M. PMID:21058190

  9. Physalin B not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in human colon cancer cells in vitro

    PubMed Central

    Ma, Yi-ming; Han, Wei; Li, Jia; Hu, Li-hong; Zhou, Yu-bo

    2015-01-01

    Aim: To investigate the effects of physalin B insolated from Physalis divericata on human colon cancer cells in vitro and its anticancer mechanisms. Methods: Human HCT116 colon cancer cell line was tested. Cell viability and apoptosis were detected, and relevant proteins were measured using Western blot analyses. Autophagosomes were observed in stable GFP-LC3 HCT116 cells. Localization of autophagosomes and lysosomes was evaluated in GFP-LC3/RFP-LAMP1-co-transfected cells. Microtubules and F-actin microfilaments were observed with confocal microscope. Mitochondrial ROS (mito-ROS) was detected with flow cytometry in the cells stained with MitoSox dye. Results: Physalin B inhibited the viability of HCT116 cells with an IC50 value of 1.35 μmol/L. Treatment of the cells with physalin B (2.5–10 μmol/L) induced apoptosis and the cleavage of PARP and caspase-3. Meanwhile, physalin B treatment induced autophagosome formation, and accumulation of LC3-II and p62, but decreased Beclin 1 protein level. Marked changes of microtubules and F-actin microfilaments were observed in physalin B-treated cells, which led to the blockage of co-localization of autophagosomes and lysosomes. Physalin B treatment dose-dependently increased the phosphorylation of p38, ERK and JNK in the cells, whereas the p38 inhibitor SB202190, ERK inhibitor U0126 or JNK inhibitor SP600125 could partially reduce physalin B-induced PARP cleavage and p62 accumulation. Moreover, physalin B treatment dose-dependently increased mito-ROS production in the cells, whereas the ROS scavenger NAC could reverse physalin B-induced effects, including incomplete autophagic response, accumulation of ubiquitinated proteins, changes of microtubules and F-actin, activation of p38, ERK and JNK, as well as cell death and apoptosis. Conclusion: Physalin B induces mito-ROS, which not only inhibits the ubiquitin-proteasome pathway but also induces incomplete autophagic response in HCT116 cells in vitro. PMID:25832431

  10. Anti-proliferative activity of 2,6-dichloro-9- or 7-(ethoxycarbonylmethyl)-9H- or 7H-purines against several human solid tumour cell lines.

    PubMed

    Morales, Ftima; Ramrez, Alberto; Conejo-Garca, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaqun M

    2014-04-01

    As leads we took several benzo-fused seven- and six-membered scaffolds linked to the pyrimidine or purine moieties with notable anti-proliferative activity against human breast, colon and melanoma cancerous cell lines. We then decided to maintain the double-ringed nitrogenous bases and change the other components to the ethyl acetate moiety. This way six purine and two 5-fluorouracil derivatives were obtained and evaluated against the MCF-7, HCT-116, A-375 and G-361 cancer cell lines. Two QSARs are obtained between the anti-proliferative IC?? values for compounds 26-33 and the clog P against the melanoma cell lines A-375 and G-361. Our results show that two of the analogues [ethyl 2-(2,6-dichloro-9H- or 7H-purine-9- or 7-yl)acetates (30 and 33, respectively)] are potent cytotoxic agents against all the tumour cell lines assayed, showing single-digit micromolar IC?? values. This exemplifies the potential of our previously reported purine compounds to qualify as lead structures for medicinal chemistry campaigns, affording simplified analogues easy to synthesize and with a noteworthy bioactivity. The selective activity of 30 and 33 against the melanoma cell line A-375, via apoptosis, supposes a great advantage for a future therapeutic use. PMID:24583351

  11. Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

    PubMed

    Choudhary, Swati; Pardo, Alessa; Rosinke, Reinhard; Batra, Janendra K; Barth, Stefan; Verma, Rama S

    2016-01-01

    Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system. PMID:26428235

  12. Epigenetic regulation of E-cadherin expression by the histone demethylase UTX in colon cancer cells.

    PubMed

    Zha, Lin; Cao, Qiang; Cui, Xin; Li, Fenfen; Liang, Houjie; Xue, Bingzhong; Shi, Hang

    2016-03-01

    Decreased epithelial cadherin (E-cadherin) gene expression, a hallmark of epithelial-mesenchymal transition (EMT), is essential for triggering metastatic advantage of the colon cancer. Genetic mechanisms underlying the regulation of E-cadherin expression in EMT have been extensively investigated; however, much is unknown about the epigenetic mechanism underlying this process. Here, we identified ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), a histone demethylase involved in demethylating di- or tri-methylated histone 3 lysine 27 (H3K27me2/3), as a positive regulator for the expression of E-cadherin in the colon cancer cell line HCT-116. We showed that inactivation of UTX down-regulated E-cadherin gene expression, while overexpression of UTX did the opposite. Notably, overexpression of UTX inhibited migration and invasion of HCT-116 cells. Moreover, UTX demethylated H3K27me3, a histone transcriptional repressive mark, leading to decreased H3K27me3 at the E-cadherin promoter. Further, UTX interacted with the histone acetyltransferase (HAT) protein CBP and recruited it to the E-cadherin promoter, resulting in increased H3K27 acetylation (H3K27ac), a histone transcriptional active mark. UTX positively regulates E-cadherin expression through coordinated regulation of H3K27 demethylation and acetylation, switching the transcriptional repressive state to the transcriptional active state at the E-cadherin promoter. We conclude that UTX may play a role in regulation of E-cadherin gene expression in HCT-116 cells and that UTX may serve as a therapeutic target against the metastasis in the treatment of colon cancer. PMID:26819089

  13. Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of In Vivo Tumor Engraftment

    PubMed Central

    Ragan, Seamus; Makhanov, Mikhail; Chenchik, Alex; Ruefli-Brasse, Astrid; Quon, Kim; Kassner, Paul D.

    2013-01-01

    Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term “clonal dominance”. We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780cis) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells. PMID:23840661

  14. Expression analysis of secreted and cell surface genes of five transformed human cell lines and derivative xenograft tumors

    PubMed Central

    Stull, Robert A; Tavassoli, Roya; Kennedy, Scot; Osborn, Steve; Harte, Rachel; Lu, Yan; Napier, Cheryl; Abo, Arie; Chin, Daniel J

    2005-01-01

    Background Since the early stages of tumorigenesis involve adhesion, escape from immune surveillance, vascularization and angiogenesis, we devised a strategy to study the expression profiles of all publicly known and putative secreted and cell surface genes. We designed a custom oligonucleotide microarray containing probes for 3531 secreted and cell surface genes to study 5 diverse human transformed cell lines and their derivative xenograft tumors. The origins of these human cell lines were lung (A549), breast (MDA MB-231), colon (HCT-116), ovarian (SK-OV-3) and prostate (PC3) carcinomas. Results Three different analyses were performed: (1) A PCA-based linear discriminant analysis identified a 54 gene profile characteristic of all tumors, (2) Application of MANOVA (Pcorr < .05) to tumor data revealed a larger set of 149 differentially expressed genes. (3) After MANOVA was performed on data from individual tumors, a comparison of differential genes amongst all tumor types revealed 12 common differential genes. Seven of the 12 genes were identified by all three analytical methods. These included late angiogenic, morphogenic and extracellular matrix genes such as ANGPTL4, COL1A1, GP2, GPR57, LAMB3, PCDHB9 and PTGER3. The differential expression of ANGPTL4 and COL1A1 and other genes was confirmed by quantitative PCR. Conclusion Overall, a comparison of the three analyses revealed an expression pattern indicative of late angiogenic processes. These results show that a xenograft model using multiple cell lines of diverse tissue origin can identify common tumorigenic cell surface or secreted molecules that may be important biomarker and therapeutic discoveries. PMID:15836779

  15. Effect of glycosylation patterns of Chinese eggplant anthocyanins and other derivatives on antioxidant effectiveness in human colon cell lines.

    PubMed

    Jing, Pu; Qian, Bingjun; Zhao, Shujuan; Qi, Xin; Ye, Ludan; Mónica Giusti, M; Wang, Xingya

    2015-04-01

    In this study, we compared the scavenging ROS of anthocyanins from Chinese eggplant var. Niu Jiao Qie and other delphinidin derivatives with different glycosylation patterns in HT-29 and HCT-116 cell lines. The eggplant anthocyanins were isolated and identified using LC-MSn and (1)H/(13)C NMR as delphinidin-3-[(4"-trans-p-coumaroyl)-rhamnosyl (1 → 6)glucoside]-5-glucoside, also known as nasunin. Delphinidin derivatives with glycosylation only on C3 (delphinidin-3-glucoside, 3-sambubioside, or 3-rutinoside) exhibited greater effects on ROS reduction as compared to delphinidin derivatives that have glycosylation on C3 and C5 (delphinidin-3,5-diglucoside>delphinidin-3-rutinoside-5-glucoside). Nasunin has glycosylation on C3 and C5 and an acyl group (p-coumaric acid), demonstrated the least effect on ROS reduction. Meanwhile, their ROS reduction activities were consistent with glutathione reductase protein expression levels in HT-29. Although not potent in ROS reduction, nasunin and its deacylated derivatives protected cells from DNA damage in a dose-dependent manner. Taken together, our results suggest that the anthocyanins isolated from Chinese eggplant var. Niu Jiao Qie and other delphinidin have antioxidant activities in colon cancer cells and also protect cells from DNA damage. PMID:25442541

  16. Long non-coding RNA ZFAS1 interacts with CDK1 and is involved in p53-dependent cell cycle control and apoptosis in colorectal cancer

    PubMed Central

    Thorenoor, Nithyananda; Faltejskova-Vychytilova, Petra; Hombach, Sonja; Mlcochova, Jitka; Kretz, Markus; Svoboda, Marek; Slaby, Ondrej

    2016-01-01

    We determined expression of 83 long non-coding RNAs (lncRNAs) and identified ZFAS1 to be significantly up-regulated in colorectal cancer (CRC) tissue. In cohort of 119 CRC patients we observed that 111 cases displayed at least two-times higher expression of ZFAS1 in CRC compared to paired normal colorectal tissue (P < 0.0001). By use of CRC cell lines (HCT116+/+, HCT116−/− and DLD-1) we showed, that ZFAS1 silencing decreases proliferation through G1-arrest of cell cycle, and also tumorigenicity of CRC cells. We identified Cyclin-dependent kinase 1 (CDK1) as interacting partner of ZFAS1 by pull-down experiment and RNA immunoprecipitation. Further, we have predicted by bioinformatics approach ZFAS1 to sponge miR-590-3p, which was proved to target CDK1. Levels of CDK1 were not affected by ZFAS1 silencing, but cyclin B1 was decreased in both cell lines. We observed significant increase in p53 levels and PARP cleavage in CRC cell lines after ZFAS1 silencing indicating increase in apoptosis. Our data suggest that ZFAS1 may function as oncogene in CRC by two main actions: (i) via destabilization of p53 and through (ii) interaction with CDK1/cyclin B1 complex leading to cell cycle progression and inhibition of apoptosis. However, molecular mechanisms behind these interactions have to be further clarified. PMID:26506418

  17. Gallotannin is a DNA damaging compound that induces senescence independently of p53 and p21 in human colon cancer cells.

    PubMed

    Al-Halabi, Racha; Abou Merhi, Raghida; Chakilam, Saritha; El-Baba, Chirine; Hamade, Eva; Di Fazio, Pietro; Ocker, Matthias; Schneider-Stock, Regine; Gali-Muhtasib, Hala

    2015-10-01

    The plant secondary metabolite gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti-carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Here, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53(+/+) /p21(+/+) and null for p53(+/+) /p21(-/-) or p53(-/-) /p21(+/+) , we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N-acetyl cysteine, dithiothreitol, SOD, and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT-induced cell cycle arrest in S-phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re-entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p-H2AX staining. Our findings indicate that GT is an interesting anti colon cancer agent which warrants further study. PMID:24798519

  18. Evaluation of copper-64-labeled somatostatin agonists and antagonist in sstr2-transfected cell lines that are positive and negative for p53: implications for cancer therapy

    PubMed Central

    Nguyen, Kim; Parry, Jesse J.; Rogers, Buck E.; Anderson, Carolyn J.

    2011-01-01

    Objectives Radiolabeled somatostatin analogs have become important agents for molecular imaging and targeted radiotherapy of somatostatin receptor-positive tumors. Here we determine the effect of the tumor suppressor protein, p53, on trafficking 64Cu to tumor cell nuclei from DOTA vs.CB-TE2A-conjugated agonist Y3-TATE and the antagonist 64Cu-CB-TE2A-sst2-ANT in cell lines that are positive or negative for p53. Methods Receptor binding, internalization, cAMP and nuclear localization studies were performed with the SSTr2 agonists, 64Cu-CB-TE2A-Y3-TATE and 64Cu-DOTA-Y3-TATE vs. antagonist, 64Cu-CB-TE2A-sst2-ANT, in SSTr2-transfected p53 +/+ and ?/? HCT116 colorectal carcinoma cells. Results The antagonist, 64Cu-CB-TE2A-sst2-ANT, bound 8-9-fold more SSTr2 binding sites than did the 64Cu-labeled agonists. 64Cu-CB-TE2A-Y3-TATE was more efficiently internalized than 64Cu-DOTA-Y3-TATE, while 64Cu-CB-TE2A-sst2-ANT showed lower, yet significant levels of internalization. CB-TE2A-Y3-TATE acted as a full agonist, inhibiting cAMP production, whereas CB-TE2A-sst2-ANT showed no inhibition of cAMP production.The 64Cu from agonists 64Cu-DOTA-Y3-TATE and 64Cu-CB-TE2A-Y3-TATE showed greater nuclear localization at 24 h in p53 +/+ vs. ?/? cells; however, there was no difference in the levels of 64Cu from the antagonist based on p53 status. Surprisingly, the DOTA and CB-TE2A-conjugated agonists showed similar nuclear localization in the p53 +/+ and ?/? cells, suggesting no difference in 64Cu release from these chelators in the HCT116 cell lines. Conclusion Based on thesein vitro data, the agonist 64Cu-CB-TE2A-Y3-TATE demonstrated the most promise as an agent for targeted radiotherapy in p53 positive, SSTr2-positive tumors. PMID:22056254

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

    SciTech Connect

    Orre, Lukas M. . E-mail: Lukas.Orre@ki.se; Stenerloew, Bo; Dhar, Sumeer; Larsson, Rolf; Lewensohn, Rolf; Lehtioe, Janne

    2006-04-21

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    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.

  1. Potential anti-inflammatory effects of the hydrophilic fraction of pomegranate (Punica granatum L.) seed oil on breast cancer cell lines.

    PubMed

    Costantini, Susan; Rusolo, Fabiola; De Vito, Valentina; Moccia, Stefania; Picariello, Gianluca; Capone, Francesca; Guerriero, Eliana; Castello, Giuseppe; Volpe, Maria Grazia

    2014-01-01

    In this work, we characterized conjugated linolenic acids (e.g., punicic acid) as the major components of the hydrophilic fraction (80% aqueous methanol extract) from pomegranate (Punica granatum L.) seed oil (PSO) and evaluated their anti-inflammatory potential on some human colon (HT29 and HCT116), liver (HepG2 and Huh7), breast (MCF-7 and MDA-MB-231) and prostate (DU145) cancer lines. Our results demonstrated that punicic acid and its congeners induce a significant decrease of cell viability for two breast cell lines with a related increase of the cell cycle G0/G1 phase respect to untreated cells. Moreover, the evaluation of a great panel of cytokines expressed by MCF-7 and MDA-MB-231 cells showed that the levels of VEGF and nine pro-inflammatory cytokines (IL-2, IL-6, IL-12, IL-17, IP-10, MIP-1α, MIP-1β, MCP-1 and TNF-α) decreased in a dose dependent way with increasing amounts of the hydrophilic extracts of PSO, supporting the evidence of an anti-inflammatory effect. Taken together, the data herein suggest a potential synergistic cytotoxic, anti-inflammatory and anti-oxidant role of the polar compounds from PSO. PMID:24962397

  2. Anti-Proliferative Effect of Naringenin through p38-Dependent Downregulation of Cyclin D1 in Human Colorectal Cancer Cells

    PubMed Central

    Song, Hun Min; Park, Gwang Hun; Eo, Hyun Ji; Lee, Jin Wook; Kim, Mi Kyoung; Lee, Jeong Rak; Lee, Man Hyo; Koo, Jin Suk; Jeong, Jin Boo

    2015-01-01

    Naringenin (NAR) as one of the flavonoids observed in grapefruit has been reported to exhibit an anti-cancer activity. However, more detailed mechanism by which NAR exerts anti-cancer properties still remains unanswered. Thus, in this study, we have shown that NAR down-regulates the level of cyclin D1 in human colorectal cancer cell lines, HCT116 and SW480. NAR inhibited the cell proliferation in HCT116 and SW480 cells and decreased the level of cyclin D1 protein. Inhibition of proteasomal degradation by MG132 blocked NAR-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with NAR. In addition, NAR increased the phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine blocked cyclin D1 downregulation by NAR. p38 inactivation attenuated cyclin D1 downregulation by NAR. From these results, we suggest that NAR-mediated cyclin D1 downregulation may result from proteasomal degradation through p38 activation. The current study provides new mechanistic link between NAR, cyclin D1 downregulation and cell growth in human colorectal cancer cells. PMID:26157550

  3. IWR-1 inhibits epithelial-mesenchymal transition of colorectal cancer cells through suppressing Wnt/?-catenin signaling as well as survivin expression

    PubMed Central

    Lee, Sang Chul; Kim, Ok-Hee; Lee, Sang Kuon; Kim, Say-June

    2015-01-01

    Aberrant activation of Wnt/?-catenin signaling is frequently observed in patients with colorectal cancer (CRC) and is considered a major determinant of CRC pathogenesis. CRC pathogenesis is particularly accompanied by epithelial-mesenchymal transition (EMT) and survivin expression. Here, we investigated the potential and mechanism of a novel Wnt/?-catenin inhibitor IWR-1 to suppress tumor metastasis in relation with EMT and survivin expression. We first determined the EMT reversal effects of IWR-1 in in vitro (HCT116 and HT29 cells) and ex vivo (specimens of CRC patients) CRC models. It was shown that IWR-1 inhibited cell proliferation and EMT even in the presence of TNF-?-induced cancer cell stimulation. IWR-1 also significantly suppressed cell migration, invasion, and matrix metalloproteinase activities of CRC cell lines. Furthermore, we showed the evidence that IWR-1 provides EMT reversal effects by directly suppressing survivin expression by the followings: 1) IWR-1 could not completely inhibit EMT in survivin-overexpressing HCT116 cells, 2) EMT reversal effects of IWR-1 were more pronounced in survivin-suppressed cells, and 3) Survivin promoter assay directly identified the survivin promoter region responsible for inhibition of survivin transcription by IWR-1. Taken altogether, our results demonstrate that IWR-1 has the potential to suppress tumor metastasis by inhibiting Wnt/?-catenin pathway as well as survivin expression. Therefore, IWR-1 could be considered for future clinical use as a therapeutic agent to treat CRC. PMID:26450645

  4. Overexpression of Livin promotes migration and invasion of colorectal cancer cells by induction of epithelial–mesenchymal transition via NF-κB activation

    PubMed Central

    Ge, Yang; Cao, Xiankui; Wang, Dalu; Sun, Wei; Sun, Hongli; Han, Bing; Cui, Junpeng; Liu, Baolin

    2016-01-01

    Livin is a novel member of the inhibitors of apoptosis protein family and has been implicated in the development and progression of colorectal cancer (CRC). However, the underlying mechanisms of Livin in CRC remain not fully understood. In this study, we investigated the effects of Livin expression on the proliferation and metastasis of CRC cells and also addressed its related molecular mechanism to metastasis. The expression of Livin in CRC cells (HCT116, SW480, and HT-29 cell lines) was determined by Western blot analysis. Our results show that the overexpression of Livin significantly promotes the proliferation, migration, and invasion of SW480 cells. Concurrently, the inhibition of Livin reduces the proliferation, migration, and invasion of HCT116 cells. In addition, Livin overexpression promotes the epithelial–mesenchymal transition, as evidenced by a decrease in epithelial E-cadherin expression and an increase in mesenchymal markers, including vimentin, Slug, and Snail. Furthermore, adding the NF-κB inhibitor, BAY 11-7028, or transfecting with small interfering RNA against p65 notably restores the expression level of E-cadherin and attenuates the invasive ability of Livin-overexpressing cells. Taken together, these results indicate that Livin potentiates migration and invasion of CRC cells partially through the induction of epithelial–mesenchymal transition via NF-κB activation. Livin may be a potential therapeutic target for CRC. PMID:27013894

  5. Synthesis of 4-piperidone Based Curcuminoids with Anti-inflammatory and Anti-Proliferation Potential in Human Cancer Cell Lines.

    PubMed

    Anthwal, Amit; Singh, Kundan; Rawat, M S M; Tyagi, Amit K; Haque, Ashanul; Ali, Imran; Rawat, Diwan S

    2016-01-01

    A series of 4-piperidone based curcuminoids were synthesized and anticancer potential of these compounds was evaluated against human myeloid leukemia (KBM5) and colon cancer (HCT116) cell lines. Their anti-inflammatory potential was determined through the down-regulation of tumor necrosis factor (TNF)-α-induced nuclear factor (NF)-κB. All compounds, except one, were found to exhibit better cytotoxicity than curcumin at 5 μM. Furthermore, many compounds have shown good potential to inhibit the TNF-α-induced NF-κB activation. Docking study of the compounds with NF-κB revealed that the binding affinity of the compounds ranged from ‒9.0 to ‒6.5 kcal/mol with 0-8 H-bonds. It was also observed that amido-ether based mono-carbonyl compounds bound around the same region of NF-κB where polynucleotides are known to bind to exhibit their activity. PMID:26567619

  6. Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter.

    PubMed

    Di Padova, Monica; Bruno, Tiziana; De Nicola, Francesca; Iezzi, Simona; D'Angelo, Carmen; Gallo, Rita; Nicosia, Daniela; Corbi, Nicoletta; Biroccio, Annamaria; Floridi, Aristide; Passananti, Claudio; Fanciulli, Maurizio

    2003-09-19

    Che-1 is a recently identified human RNA polymerase II binding protein involved in the regulation of gene transcription and cell proliferation. We previously demonstrated that Che-1 inhibits the Rb growth-suppressing function by interfering with Rb-mediated HDAC1 recruitment on E2F target gene promoters. By hybridization of cancer profile arrays, we found that Che-1 expression is strongly down-regulated in several tumors, including colon and kidney carcinomas, compared with the relative normal tissues. Consistent with these data, Che-1 overexpression inhibits proliferation of HCT116 and LoVo human colon carcinoma cell lines by activation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 in a p53-independent manner and by promoting growth arrest at the G1 phase of the cell cycle. Che-1 activates p21WAF1/Cip1 by displacing histone deacetylase (HDAC)1 from the Sp1 binding sites of the p21WAF1/Cip1 gene promoter and accumulating acetylated histone H3 on these sites. Accordingly, Che-1-specific RNA interference negatively affects p21WAF1/Cip1 transactivation and increases cell proliferation in HCT116 cells. Taken together, our results indicate that Che-1 can be considered a general HDAC1 competitor and its down-regulation is involved in colon carcinoma cell proliferation. PMID:12847090

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed

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

    2013-01-01

    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

  9. Capsaicin-mediated tNOX (ENOX2) up-regulation enhances cell proliferation and migration in vitro and in vivo.

    PubMed

    Liu, Nei-Chi; Hsieh, Pei-Fang; Hsieh, Ming-Kun; Zeng, Zih-Ming; Cheng, Hsiao-Ling; Liao, Jiunn-Wang; Chueh, Pin Ju

    2012-03-14

    Cancer chemoprevention is employed to block or reverse the progression of malignancies. To date, several thousands of agents have been found to possess chemopreventative activity, one of which is capsaicin, a component of chili peppers that exhibits antigrowth activity against various cancer cell lines. However, the role of capsaicin in tumorigenesis remains controversial because both cancer prevention and promotion have been proposed. Here, we made the unexpected discovery that treatment with low concentrations of capsaicin up-regulates tNOX (tumor-associated NADH oxidase) expression in HCT116 human colon carcinoma cells in association with enhanced cell proliferation and migration, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX-knockdown in HCT116 cells by RNA interference reversed capsaicin-induced cell proliferation and migration in vitro and decreased tumor growth in vivo. Collectively, these findings provide a basis for explaining the tumor-promoting effect of capsaicin and might imply that caution should be taken when using capsaicin as a chemopreventive agent. PMID:22353011

  10. RSPO2 enriches LGR5+ spheroid colon cancer stem cells and promotes its metastasis by epithelial-mesenchymal transition

    PubMed Central

    Zhang, Shi; Han, Xiaoyan; Wei, Bo; Fang, Jiafeng; Wei, Hongbo

    2016-01-01

    Colon cancer stem cells (CCSCs) account for the tumorigenicity of colon cancer and promote its progression and metastasis. RSPO2, the agonist of canonical Wnt/beta-catenin pathway and serves as the growth factor of intestinal stem cells (ISCs), is considered playing an important role in CCSCs. However, the specific function of RSPO2 in CCSCs remains unclear. In this study, we demonstrated that RSPO2 was highly expressed in CCSCs-enriched HCT116 spheroid cells. Elevates the concentration of RSPO2 in medium in favor of enriching the LGR5+ cells and increasing the LGR5 expression in HCT116 spheroid cells, meanwhile silencing of RSPO2 by small interfering RNA inhibits LGR5 expression in HCT116 spheroid cells. In addition, RSPO2 promotes spheres formation but has little effect on the proliferation of HCT116 spheroid cells in vitro. Moreover, RSPO2 also promotes the invasion of HCT116 spheroid cells through enhancing Epithelial-mesenchymal transition (EMT). These findings suggests that RSPO2 is a potential growth factor for CCSCs, helps enriching the CCSCs by serum-free DMEM/F12 medium (SFM) culture and plays a vital role in the metastasis of colon cancer. PMID:27158331

  11. Piceatannol promotes apoptosis via up-regulation of microRNA-129 expression in colorectal cancer cell lines

    SciTech Connect

    Zhang, Haogang; Jia, Ruichun; Wang, Chunjing; Hu, Tianming; Wang, Fujing

    2014-09-26

    Highlights: • Piceatannol induces apoptosis in cultured CRC cells. • Piceatannol promotes expression of miR-129. • miR-129 mediates proapoptotic effects of piceatannol. - Abstract: Piceatannol, a naturally occurring analog of resveratrol, has been confirmed as an antitumor agent by inhibiting proliferation, migration, and metastasis in diverse cancer. However, the effect and mechanisms of piceatannol on colorectal cancer (CRC) have not been well understood. This study aimed to test whether piceatannol could inhibit growth of CRC cells and reveal its underlying molecular mechanism. MTT assay was used to detect the cell viability in HCT116 and HT29 cells. Flow cytometry analysis was employed to measure apoptosis of CRC cells. Bcl-2, Bax and caspase-3 levels were analyzed by Western blot and miR-129 levels were determined by real-time RT-PCR. Our study showed that piceatannol inhibited HCT116 and HT29 cells growth in a concentration- and time-dependent manner. Piceatannol induced apoptosis by promoting expression of miR-129, and then inhibiting expression of Bcl-2, an known target for miR-129. Moreover, knock down of miR-129 could reverse the reduction of cell viability induced by piceatannol in HCT116 and HT29 cells. Taken together, our study unraveled the ability of piceatannol to suppress colorectal cancer growth and elucidated the participation of miR-129 in the anti-cancer action of piceatannol. Our findings suggest that piceatannol can be considered to be a promising anticancer agent for CRC.

  12. Epibrassinolide alters PI3K/MAPK signaling axis via activating Foxo3a-induced mitochondria-mediated apoptosis in colon cancer cells.

    PubMed

    Coskun, Deniz; Obakan, Pinar; Arisan, Elif Damla; Çoker-Gürkan, Ajda; Palavan-Ünsal, Narçin

    2015-10-15

    Epibrassinolide (EBR), a steroid-derived plant growth regulator, has been recently suggested as an apoptotic inducer in different cancer cells. In this experimental study, we investigated the potential apoptotic effect of EBR on stress-related and survival signaling molecules in colon carcinoma cells. EBR decreased cell viability and colony formation in HCT 116 and HT-29 colon carcinoma cells. The inactivation of PI3K/AKT by EBR treatment led to upregulation of Foxo3a, which in turn induced apoptosis in HCT 116 and HT-29 cells. In addition, the upstream non-receptor protein tyrosine kinase Src was found elevated allowing to the upregulation of p38, stress-activated protein kinase/Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 and their target genes c-jun, c-fos and c-myc in a time-dependent manner in HCT 116 cells within 48h. The alterations in PA metabolism caused intracellular PA pool decrease. The upregulation of pro-apoptotic Bak, Bax, Puma and Bim were accompanied with the decrease in Mcl-1 in HCT 116 and Bcl-xL expression profiles in HT-29 following 48h EBR treatment. We suggest that the upregulation of Bim expression levels might be related with one of the PI3K/AKT target transcription factor Foxo3a, which was dephosphorylated by EBR treatment in HCT 116 and HT-29 cells. PMID:26318418

  13. Active specific immunotherapy for metastatic colorectal carcinoma: phase I study of an allogeneic cell vaccine plus low-dose interleukin-1 alpha.

    PubMed

    Woodlock, T J; Sahasrabudhe, D M; Marquis, D M; Greene, D; Pandya, K J; McCune, C S

    1999-05-01

    A vaccine consisting of four allogeneic colon carcinoma cell lines (DLD-1, HCT116, WiDr, and T84) mixed with the adjuvant DETOX (Mycobacterium phlei cell wall and Salmonella minnesota lipid A) was administered to 25 patients with low-volume metastatic colorectal carcinoma. The first eight patients received vaccine only, given intradermally on three occasions at 3-week intervals. Subsequent patients also received subcutaneous interleukin-1 alpha (IL-1 alpha), 0.3-0.5 microgram/m2 per day for 8 days after each vaccination in an outpatient setting. Vaccine alone caused local erythema, induration, and pruritus. IL-1 caused fevers, chills, and rigors that started in 4 h and lasted 1-2 h. One patient developed a brief loss of consciousness with a rigor that resolved without sequelae. One episode of mild hypotension occurred. Fatigue occurred by day 8 of IL-1. A substantial increase in the number of patients with positive skin tests to DLD-1 and HCT116 occurred after vaccine treatment both without and with IL-1 alpha. An allogeneic cell vaccine plus subcutaneous IL-1 was administered safely to outpatients with some evidence of in vivo effect observed. PMID:10335485

  14. The Effect of Sulfated (1→3)-α-L-Fucan from the Brown Alga Saccharina cichorioides Miyabe on Resveratrol-Induced Apoptosis in Colon Carcinoma Cells

    PubMed Central

    Vishchuk, Olesia S.; Ermakova, Svetlana P.; Zvyagintseva, Tatyana N.

    2013-01-01

    Accumulating data clearly indicate that the induction of apoptosis by nontoxic natural compounds is a potent defense against the development and progression of many malignancies, including colon cancer. Resveratrol and the fucoidans have been shown to possess potent anti-tumor activity in vitro and in vivo. The aim of the present study was to examine whether the combination of a fucoidan from the brown alga Saccharina cichorioides Miyabe and resveratrol would be an effective preventive and/or therapeutic strategy against colon cancer. Based on NMR spectroscopy and MALDI-TOF analysis, the fucoidan isolated and purified from Saccharina cichorioides Miyabe was (1→3)-α-L-fucan with sulfate groups at C2 and C4 of the α-L-fucopyranose residues. The fucoidan enhanced the antiproliferative activity of resveratrol at nontoxic doses and facilitated resveratrol-induced apoptosis in the HCT 116 human colon cancer cell line. Apoptosis was realized by the activation of initiator caspase-9 and effector caspase-7 and -3, followed by the cleavage of PARP. Furthermore, significant inhibition of HCT 116 colony formation was associated with the sensitization of cells to resveratrol by the fucoidan. Taken together, these results demonstrate that the combination of the algal fucoidan with resveratrol may provide a potential therapy against human colon cancer. PMID:23337253

  15. Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells

    PubMed Central

    Akhtar Ali, Muhammad; Younis, Shady; Wallerman, Ola; Gupta, Rajesh; Andersson, Leif; Sjöblom, Tobias

    2015-01-01

    The transcription factor ZBED6 (zinc finger, BED-type containing 6) is a repressor of IGF2 whose action impacts development, cell proliferation, and growth in placental mammals. In human colorectal cancers, IGF2 overexpression is mutually exclusive with somatic mutations in PI3K signaling components, providing genetic evidence for a role in the PI3K pathway. To understand the role of ZBED6 in tumorigenesis, we engineered and validated somatic cell ZBED6 knock-outs in the human colorectal cancer cell lines RKO and HCT116. Ablation of ZBED6 affected the cell cycle and led to increased growth rate in RKO cells but reduced growth in HCT116 cells. This striking difference was reflected in the transcriptome analyses, which revealed enrichment of cell-cycle–related processes among differentially expressed genes in both cell lines, but the direction of change often differed between the cell lines. ChIP sequencing analyses displayed enrichment of ZBED6 binding at genes up-regulated in ZBED6-knockout clones, consistent with the view that ZBED6 modulates gene expression primarily by repressing transcription. Ten differentially expressed genes were identified as putative direct gene targets, and their down-regulation by ZBED6 was validated experimentally. Eight of these genes were linked to the Wnt, Hippo, TGF-β, EGF receptor, or PI3K pathways, all involved in colorectal cancer development. The results of this study show that the effect of ZBED6 on tumor development depends on the genetic background and the transcriptional state of its target genes. PMID:26056301

  16. Highly skewed distribution of miRNAs and proteins between colorectal cancer cells and their exosomes following Cetuximab treatment: biomolecular, genetic and translational implications

    PubMed Central

    Barbagallo, Cristina; Passanisi, Roberta; Alhamdani, Mohamed S.; Destri, Giovanni Li; Cappellani, Alessandro; Barbagallo, Davide; Scalia, Marina; Valadi, Hadi

    2014-01-01

    Exchange of molecules via exosomes is a means of eukaryotic intercellular communication, especially within tumour microenvironments. However, no data are available on alterations of exosomal molecular cargo by environmental cues (eg, pharmacological treatments). To approach this issue, we compared the abundance of 754 miRNAs and 741 cancer-related proteins in exosomes secreted by Caco-2 (Cetuximab-responsive) and HCT- 116 (Cetuximab-resistant) CRC cells, before and after Cetuximab treatment, with that in their source cells. Cetuximab significantly altered the cargo of Caco-2 exosomes: it increased abundance of miRNAs and proteins activating proliferation and inflammation and reduced miRNAs and proteins related to immune suppression. These alterations did not precisely mirror those in source cells, suggesting a Cetuximab-linked effect. Analogous alterations were detected in HCT-116. Transfection of exosomes from Cetuximab-treated Caco-2 into HCT-116 significantly increased HCT-116 viability; conversely, no viability alteration was detected in Caco-2 transfected with exosomes from Cetuximab-treated HCT-116. Analysis of networks, comprising targets of differentially expressed (DE) exosomal miRNAs and DE exosomal proteins, demonstrates a significant involvement of processes related to proliferation, inflammation, immune response, apoptosis. Our data extend existing knowledge on molecular mechanisms of eukaryotic intercellular communication, especially in oncological processes. Their translation to clinical settings may add new weapons to existing therapeutic repertoires against cancer. PMID:25594007

  17. Differential expression of proteins in response to ceramide-mediated stress signal in colon cancer cells by 2-D gel electrophoresis and MALDI-TOF-MS.

    PubMed

    Fillet, M; Cren-Oliv, C; Renert, A-F; Piette, J; Vandermoere, F; Rolando, Ch; Merville, M-P

    2005-01-01

    Comparative cancer cell proteome analysis is a strategy to study the implication of ceramides in the transmission of stress signals. To better understand the mechanisms by which ceramide regulate some physiological or pathological events and the response to the pharmacological treatment of cancer, we performed a differential analysis of the proteome of HCT-116 (human colon carcinoma) cells in response to these substances. We first established the first 2-dimensional map of the HCT-116 proteome. Then, HCT116 cell proteome treated or not with C6-ceramide have been compared using two-dimensional electrophoresis, matrix-assisted laser desorption/ionization-mass spectrometry and bioinformatic (genomic databases). 2-DE gel analysis revealed more than fourty proteins that were differentially expressed in control cells and cells treated with ceramide. Among them, we confirmed the differential expression of proteins involved in apoptosis and cell adhesion. PMID:15952734

  18. Divalent metal-ion transporter 1 is decreased in intestinal epithelial cells and contributes to the anemia in inflammatory bowel disease

    PubMed Central

    Wu, Wei; Song, Yang; He, Chong; Liu, Changqin; Wu, Ruijin; Fang, Leilei; Cong, Yingzi; Miao, Yinglei; Liu, Zhanju

    2015-01-01

    Divalent metal-ion transporter 1 (DMT1) has been found to play an important role in the iron metabolism and hemogenesis. However, little is known about the potential role of DMT1 in the pathogenesis of anemia from patients with inflammatory bowel disease (IBD). Herein, we investigated expression of DMT1 in the intestinal mucosa by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry, and found that DMT1 was significantly decreased in the inflamed mucosa of active IBD patients compared with that in those patients at remission stage and healthy controls. To further study the mechanism, we cultured HCT 116 cell line in vitro. Expression of DMT1 in HCT116 was demonstrated to be markedly decreased under stimulation with TNF for 24 and 48 h, while JNK inhibitor (JNK-IN-7) could significantly reverse the decrease. Interestingly, anti-TNF therapy successfully improved anemia in clinical responsive Crohn’s disease patients, and DMT1 was found to be markedly up-regulated in intestinal mucosa. Taken together, our studies demonstrate that decreased expression of DMT1 in intestinal mucosa leads to compromised absorption and transportation of iron and that blockade of TNF could rescue anemia and promote DMT1 expression in gut mucosa. This work provides a therapeutic approach in the management of anemia in IBD. PMID:26572590

  19. Reducing Compounds Equivocally Influence Oxidation during Digestion of a High-Fat Beef Product, which Promotes Cytotoxicity in Colorectal Carcinoma Cell Lines.

    PubMed

    Van Hecke, Thomas; Wouters, An; Rombouts, Caroline; Izzati, Tazkiyah; Berardo, Alberto; Vossen, Els; Claeys, Erik; Van Camp, John; Raes, Katleen; Vanhaecke, Lynn; Peeters, Marc; De Vos, Winnok H; De Smet, Stefaan

    2016-02-24

    We studied the formation of malondialdehyde, 4-hydroxy-nonenal, and hexanal (lipid oxidation products, LOP) during in vitro digestion of a cooked low-fat and high-fat beef product in response to the addition of reducing compounds. We also investigated whether higher LOP in the digests resulted in a higher cyto- and genotoxicity in Caco-2, HT-29 and HCT-116 cell lines. High-fat compared to low-fat beef digests contained approximately 10-fold higher LOP concentrations (all P < 0.001), and induced higher cytotoxicity (P < 0.001). During digestion of the high-fat product, phenolic acids (gallic, ferulic, chlorogenic, and caffeic acid) displayed either pro-oxidant or antioxidant behavior at lower and higher doses respectively, whereas ascorbic acid was pro-oxidant at all doses, and the lipophilic reducing compounds (α-tocopherol, quercetin, and silibinin) all exerted a clear antioxidant effect. During digestion of the low-fat product, the hydrophilic compounds and quercetin were antioxidant. Decreases or increases in LOP concentrations amounted to 100% change versus controls. PMID:26836477

  20. Assessing chemical constituents of Mimosa caesalpiniifolia stem bark: possible bioactive components accountable for the cytotoxic effect of M. caesalpiniifolia on human tumour cell lines.

    PubMed

    Monção, Nayana Bruna Nery; Araújo, Bruno Quirino; Silva, Jurandy do Nascimento; Lima, Daisy Jereissati Barbosa; Ferreira, Paulo Michel Pinheiro; Airoldi, Flavia Pereira da Silva; Pessoa, Cláudia; Citó, Antonia Maria das Graças Lopes

    2015-01-01

    Mimosa caesalpiniifolia is a native plant of the Brazilian northeast, and few studies have investigated its chemical composition and biological significance. This work describes the identification of the first chemical constituents in the ethanolic extract and fractions of M. caesalpiniifolia stem bark based on NMR, GC-qMS and HRMS analyses, as well as an assessment of their cytotoxic activity. GC-qMS analysis showed fatty acid derivatives, triterpenes and steroid substances and confirmed the identity of the chemical compounds isolated from the hexane fraction. Metabolite biodiversity in M. caesalpiniifolia stem bark revealed the differentiated accumulation of pentacyclic triterpenic acids, with a high content of betulinic acid and minor amounts of 3-oxo and 3β-acetoxy derivatives. Bioactive analysis based on total phenolic and flavonoid content showed a high amount of these compounds in the ethanolic extract, and ESI-(-)-LTQ-Orbitrap-MS identified caffeoyl hexose at high intensity, as well as the presence of phenolic acids and flavonoids. Furthermore, the evaluation of the ethanolic extract and fractions, including betulinic acid, against colon (HCT-116), ovarian (OVCAR-8) and glioblastoma (SF-295) tumour cell lines showed that the crude extract, hexane and dichloromethane fractions possessed moderate to high inhibitory activity, which may be related to the abundance of betulinic acid. The phytochemical and biological study of M. caesalpiniifolia stem bark thus revealed a new alternative source of antitumour compounds, possibly made effective by the presence of betulinic acid and by chemical co-synergism with other compounds. PMID:25751783

  1. Differential expression of nanog1 and nanogp8 in colon cancer cells

    SciTech Connect

    Ishiguro, Tatsuya; Sato, Ai; Ohata, Hirokazu; Sakai, Hiroaki; Nakagama, Hitoshi; Okamoto, Koji

    2012-02-10

    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.

  2. Vitamin K2-derived compounds induce growth inhibition in radioresistant cancer cells.

    PubMed

    Amalia, Helfi; Sasaki, Ryohei; Suzuki, Yoko; Demizu, Yusuke; Bito, Toshinori; Nishimura, Hideki; Okamoto, Yoshiaki; Yoshida, Kenji; Miyawaki, Daisuke; Kawabe, Tetsuya; Mizushina, Yoshiyuki; Sugimura, Kazuro

    2010-01-01

    A strategy to overcome radioresistance in cancer treatment has been expected. To evaluate the strategy, appropriate experimental models are needed. Radioresistant tumour models were originally established from human colon cancer cells, and we evaluated their molecular basis. Next, the growth inhibitory effects of newly synthesized vitamin K2 (VK2)-related compounds were tested. Here, we showed that these novel compounds have growth inhibitory effects not only on cancer cells of various origins, but also on radioresistant cells, through the generation of reactive oxygen species (ROS). Human colon, lung, and breast cancer cell lines were used for testing the growth inhibitory activities of several chemical compounds. Radioresistant tumour models were established by fractionated radiation exposure. Irradiated cells were selected by a single cell cloning method, and their sensitivity to ionizing radiation was evaluated by a colony-forming assay. The VK2 derivatives (named MQ-1, MQ-2, and MQ-3) were chemically synthesized. To evaluate the generation of ROS, flow cytometer analyses were performed. A radioresistant tumour model was established from the HCT116 human colon cancer cell line. The radioresistant cells from HCT116 also showed resistance to cisplatin. In the radioresistant cells, NF-κB was highly activated. MQ-1, MQ-2, and MQ-3 showed greater growth inhibitory activities than VK2 not only in various cancer cells but also in radioresistant cells through the generation of ROS. In conclusion, a radioresistant tumour model was originally established from colon cancer cell lines through NF-κB activation, and it could be a useful tool for evaluating anti-tumour agents. Newly synthesized VK2 derivatives (MQ-1, MQ-2 and MQ-3) seemed to be potential anti-tumour agents in various cancers and radioresistant cancers. The efficacy of those compounds was related to the generation of ROS. These findings together might pave the way for the treatment of radioresistant or recurrent cancers. PMID:21063145

  3. LPA Induces Colon Cancer Cell Proliferation through a Cooperation between the ROCK and STAT-3 Pathways.

    PubMed

    Leve, Fernanda; Peres-Moreira, Rubem J; Binato, Renata; Abdelhay, Eliana; Morgado-Díaz, José A

    2015-01-01

    Lysophosphatidic acid (LPA) plays a critical role in the proliferation and migration of colon cancer cells; however, the downstream signaling events underlying these processes remain poorly characterized. The aim of this study was to investigate the signaling pathways triggered by LPA to regulate the mechanisms involved in the progression of colorectal cancer (CRC). We have used three cell line models of CRC, and initially analyzed the expression profile of LPA receptors (LPAR). Then, we treated the cells with LPA and events related to their tumorigenic potential, such as migration, invasion, anchorage-independent growth, proliferation as well as apoptosis and cell cycle were evaluated. We used the Chip array technique to analyze the global gene expression profiling that occurs after LPA treatment, and we identified cell signaling pathways related to the cell cycle. The inhibition of these pathways verified the conclusions of the transcriptomic analysis. We found that the cell lines expressed LPAR1, -2 and -3 in a differential manner and that 10 μM LPA did not affect cell migration, invasion and anchorage-independent growth, but it did induce proliferation and cell cycle progression in HCT-116 cells. Although LPA in this concentration did not induce transcriptional activity of β-catenin, it promoted the activation of Rho and STAT-3. Moreover, ROCK and STAT-3 inhibitors prevented LPA-induced proliferation, but ROCK inhibition did not prevent STAT-3 activation. Finally, we observed that LPA regulates the expression of genes related to the cell cycle and that the combined inhibition of ROCK and STAT-3 prevented cell cycle progression and increased the LPA-induced expression of cyclins E1, A2 and B1 to a greater degree than either inhibitor alone. Overall, these results demonstrate that LPA increases the proliferative potential of colon adenocarcinoma HCT-116 cells through a mechanism involving cooperation between the Rho-ROCK and STAT3 pathways involved in cell cycle control. PMID:26418031

  4. LPA Induces Colon Cancer Cell Proliferation through a Cooperation between the ROCK and STAT-3 Pathways

    PubMed Central

    Leve, Fernanda; Peres-Moreira, Rubem J.; Binato, Renata; Abdelhay, Eliana; Morgado-Díaz, José A.

    2015-01-01

    Lysophosphatidic acid (LPA) plays a critical role in the proliferation and migration of colon cancer cells; however, the downstream signaling events underlying these processes remain poorly characterized. The aim of this study was to investigate the signaling pathways triggered by LPA to regulate the mechanisms involved in the progression of colorectal cancer (CRC). We have used three cell line models of CRC, and initially analyzed the expression profile of LPA receptors (LPAR). Then, we treated the cells with LPA and events related to their tumorigenic potential, such as migration, invasion, anchorage-independent growth, proliferation as well as apoptosis and cell cycle were evaluated. We used the Chip array technique to analyze the global gene expression profiling that occurs after LPA treatment, and we identified cell signaling pathways related to the cell cycle. The inhibition of these pathways verified the conclusions of the transcriptomic analysis. We found that the cell lines expressed LPAR1, -2 and -3 in a differential manner and that 10 μM LPA did not affect cell migration, invasion and anchorage-independent growth, but it did induce proliferation and cell cycle progression in HCT-116 cells. Although LPA in this concentration did not induce transcriptional activity of β-catenin, it promoted the activation of Rho and STAT-3. Moreover, ROCK and STAT-3 inhibitors prevented LPA-induced proliferation, but ROCK inhibition did not prevent STAT-3 activation. Finally, we observed that LPA regulates the expression of genes related to the cell cycle and that the combined inhibition of ROCK and STAT-3 prevented cell cycle progression and increased the LPA-induced expression of cyclins E1, A2 and B1 to a greater degree than either inhibitor alone. Overall, these results demonstrate that LPA increases the proliferative potential of colon adenocarcinoma HCT-116 cells through a mechanism involving cooperation between the Rho-ROCK and STAT3 pathways involved in cell cycle control. PMID:26418031

  5. The Apoptotic Effect of Plant Based Nanosilver in Colon Cancer Cells is a p53 Dependent Process Involving ROS and JNK Cascade.

    PubMed

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

    2015-04-01

    Here, we report the p53 dependent mitochondria-mediated apoptotic mechanism of plant derived silver-nanoparticle (PD-AgNPs) in colorectal cancer cells (CRCs). PD-AgNPs was synthesized by reduction of AgNO3 with leaf extract of a medicinal plant periwinkle and characterized. Uptake of PD-AgNPs (ξ - 2.52 ± 4.31 mV) in HCT116 cells was 3 fold higher in comparison to synthetic AgNPs (ξ +2.293 ± 5.1 mV). A dose dependent increase in ROS production, activated JNK and decreased mitochondrial membrane potential (MMP) were noted in HCT116 but not in HCT116 p53(-/-) cells after PD-AgNP exposure. PD-AgNP-mediated apoptosis in CRCs is a p53 dependent process involving ROS and JNK cascade. PMID:25359126

  6. Development of an Optimized Protocol for NMR Metabolomics Studies of Human Colon Cancer Cell Lines and First Insight from Testing of the Protocol Using DNA G-Quadruplex Ligands as Novel Anti-Cancer Drugs

    PubMed Central

    Lauri, Ilaria; Savorani, Francesco; Iaccarino, Nunzia; Zizza, Pasquale; Pavone, Luigi Michele; Novellino, Ettore; Engelsen, Søren Balling; Randazzo, Antonio

    2016-01-01

    The study of cell lines by nuclear magnetic resonance (NMR) spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116) by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug). The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested. PMID:26784246

  7. Development of an Optimized Protocol for NMR Metabolomics Studies of Human Colon Cancer Cell Lines and First Insight from Testing of the Protocol Using DNA G-Quadruplex Ligands as Novel Anti-Cancer Drugs.

    PubMed

    Lauri, Ilaria; Savorani, Francesco; Iaccarino, Nunzia; Zizza, Pasquale; Pavone, Luigi Michele; Novellino, Ettore; Engelsen, Søren Balling; Randazzo, Antonio

    2016-01-01

    The study of cell lines by nuclear magnetic resonance (NMR) spectroscopy metabolomics represents a powerful tool to understand how the local metabolism and biochemical pathways are influenced by external or internal stimuli. In particular, the use of adherent mammalian cells is emerging in the metabolomics field in order to understand the molecular mechanism of disease progression or, for example, the cellular response to drug treatments. Hereto metabolomics investigations for this kind of cells have generally been limited to mass spectrometry studies. This study proposes an optimized protocol for the analysis of the endo-metabolome of human colon cancer cells (HCT116) by NMR. The protocol includes experimental conditions such as washing, quenching and extraction. In order to test the proposed protocol, it was applied to an exploratory study of cancer cells with and without treatment by anti-cancer drugs, such as DNA G-quadruplex binders and Adriamycin (a traditional anti-cancer drug). The exploratory NMR metabolomics analysis resulted in NMR assignment of all endo-metabolites that could be detected and provided preliminary insights about the biological behavior of the drugs tested. PMID:26784246

  8. Anticancer Activity of Organogallium(III) Complexes in Colon Cancer Cells.

    PubMed

    Kaluđerović, Milena R; Mojić, Marija; Gómez-Ruiz, Santiago; Mijatović, Sanja; Maksimović-Ivanić, Danijela

    2016-01-01

    In vitro antitumor activity of various organogallium(III) complexes (1-8) has been tested against CT26CL25, HCT116, SW480 colon cancer cell lines. CV and MTT assays were used to assess on the antiproliferative effect of investigated organogallium(III) complexes. From the investigated complexes, the most active was found to be tetranuclear compound 8 against CT26CL25 cells. Flow cytometric analysis of the CT26CL25 cells upon the treatment with 8 was performed in order to determine the role of apoptosis, caspase activation, autophagy and proliferation rate on the cell death caused with this compound. Results indicate cytotoxic potential of the tetranuclear complex 8 by inducing caspase independent apoptosis and blocking most of the cells before first division. PMID:26443026

  9. Thymoquinone hydrazone derivatives cause cell cycle arrest in p53-competent colorectal cancer cells

    PubMed Central

    WIRRIES, ANDR; BREYER, SANDRA; QUINT, KARL; SCHOBERT, RAINER; OCKER, MATTHIAS

    2010-01-01

    Thymoquinone (TQ), the major compound of black seed oil, has been shown to induce pro-apoptotic signaling pathways in various human cancer models. Although TQ is commonly used in traditional medicine, its use in humans is limited due to its chemical properties and poor membrane penetration capacity. We therefore attached saturated and unsaturated fatty acid residues to TQ and evaluated the effect on cell proliferation, apoptosis and underlying signaling pathways in HCT116 and HCT116p53?/? colon cancer and HepG2 hepatoma cells in vitro. Treatment with thymoquinone-4-?-linolenoylhydrazone (TQ-H-10) or thymoquinone-4-palmitoylhydrazone (TQ-H-11) induced a cytostatic effect, particularly in p53-competent HCT116 cells, mediated by an up-regulation of p21cip1/waf1 and a down-regulation of cyclin E, and associated with an S/G2 arrest of the cell cycle. Cells lacking p53 (HCT116p53?/?) or HepG2 liver cancer cells showed only a minor response to TQ-H-10. These findings demonstrate that derivatives of TQ inhibit cell proliferation dependent on p53 status by activating the cell cycle inhibitor p21cip1/waf1 at lower concentrations than unmodified TQ. Structural modifications can therefore contribute to the further clinical development of TQ. PMID:22993551

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

    SciTech Connect

    Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry; Mazella, Jean

    2011-10-14

    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.

  11. Hydrogen sulfide protects colon cancer cells from chemopreventative agent β-phenylethyl isothiocyanate induced apoptosis

    PubMed Central

    Rose, Peter; Moore, Philip K; Ming, Shen Han; Nam, Ong Choon; Armstrong, Jeffrey S; Whiteman, Matt

    2005-01-01

    AIM: Hydrogen sulfide (H2S) is a prominent gaseous constituent of the gastrointestinal (GI) tract with known cytotoxic properties. Endogenous concentrations of H2S are reported to range between 0.2-3.4 mmol/L in the GI tract of mice and humans. Considering such high levels we speculate that, at non-toxic concentrations, H2S may interact with chemical agents and alter the response of colonic epithelium cells to such compounds. The GI tract is a major site for the absorption of phytochemical constituents such as isothiocyanates, flavonoids, and carotenoids, with each group having a role in the prevention of human diseases such as colon cancer. The chemopreventative properties of the phytochemical agent β-phenyethyl isothiocyanate (PEITC) are well recognized. However, little is currently known about the physiological or biochemical factors present in the GI tract that may influence the biological properties of ITCs. The current study was undertaken to determine the effects of H2S on PEITC mediated apoptosis in colon cancer cells. METHODS: Induction of apoptosis by PEITC in human colon cancer HCT116 cells was assessed using classic apoptotic markers namely SubG1 population analysis, caspase-3 like activity and nuclear fragmentation and condensation coupled with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide] viability assay and LDH leakage. RESULTS: PEITC significantly induced apoptosis in HCT116 cells as assessed by SubG1 population formation, nuclear condensation, LDH leakage and caspase-3 activity after 24 h, these data being significant from control groups (P < 0.01). In contrast, co-treatment of cells with physiological concentrations of H2S (0.1-1 mmol/L) prevented PEITC mediated apoptosis as assessed using the parameters described. CONCLUSION: PEITC effectively induced cell death in the human adenocarcinoma cell line HCT116 in vitro through classic apoptotic mechanisms. However, in the presence of H2S, apoptosis was abolished. These data suggest that H2S may play a significant role in the response of colonic epithelial cells to beneficial as well as toxic agents present within the GI tract. PMID:15996021

  12. Notoginseng enhances anti-cancer effect of 5-fluorouracil on human colorectal cancer cells

    PubMed Central

    Wang, Chong-Zhi; Luo, Xiaoji; Zhang, Bin; Song, Wen-Xin; Ni, Ming; Mehendale, Sangeeta; Xie, Jing-Tian; Aung, Han H.; He, Tong-Chuan

    2009-01-01

    Purpose Panax notoginseng is a commonly used Chinese herb. Although a few studies have found that notoginseng shows anti-tumor effects, the effect of this herb on colorectal cancer cells has not been investigated. 5-Fluorouracil (5-FU) is a chemotherapeutic agent for the treatment of colorectal cancer that interferes with the growth of cancer cells. However, this compound has serious side effects at high doses. In this study, using HCT-116 human colorectal cancer cell line, we investigated the possible synergistic anti-cancer effects between notoginseng flower extract (NGF) and 5-FU on colon cancer cells. Methods The anti-proliferation activity of these modes of treatment was evaluated by MTS cell proliferation assay. Apoptotic effects were analyzed by using Hoechst 33258 staining and Annexin-V/PI staining assays. The anti-proliferation effects of four major single compounds from NGF, ginsenosides Rb1, Rb3, Rc and Rg3 were also analyzed. Results Both 5-FU and NGF inhibited proliferation of HCT-116 cells. With increasing doses of 5-FU, the anti-proliferation effect was slowly increased. The combined usage of 5-FU 5 μM and NGF 0.25 mg/ml, significantly increased the anti-proliferation effect (59.4 ± 3.3%) compared with using the two medicines separately (5-FU 5 μM, 31.1 ± 0.4%; NGF 0.25 mg/ml, 25.3 ± 3.6%). Apoptotic analysis showed that at this concentration, 5-FU did not exert an apoptotic effect, while apoptotic cells induced by NGF were observed, suggesting that the anti-proliferation target(s) of NGF may be different from that of 5-FU, which is known to inhibit thymidilate synthase. Conclusions This study demonstrates that NGF can enhance the anti-proliferation effect of 5-FU on HCT-116 human colorectal cancer cells and may decrease the dosage of 5-FU needed for colorectal cancer treatment. PMID:17009031

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

    PubMed

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

    2014-09-01

    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 μg/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

  14. Lgr5 Methylation in Cancer Stem Cell Differentiation and Prognosis-Prediction in Colorectal Cancer

    PubMed Central

    Zhou, Jieqiong; Liang, Yan; Chen, Kequan; Wang, Xinying; Wang, Zhongqiu; Wang, Zhiqing; Chang, Cassie; Han, Weihua; Gong, Wei; Qin, Haitao; Jiang, Bo; Xiong, Huabao; Peng, Liang

    2015-01-01

    Objective Leucine-rich-repeat-containing G-protein-coupled receptor 5 (lgr5) is a candidate marker for colorectal cancer stem cells (CSC). In the current study, we investigated the methylation status within thelgr5 promoter and evaluated its relationship with CSC differentiation, prognosis for colorectal cancer, and its clinicopathological features. Methods The methylation status within Lgr5 promoter was detected with a methylation-specific PCR in six colorectal cancer cell lines as well as 169 primary colorectal tumor tissues. Differentiation of CSC was examined with immunofluorescence and immunocytochemistry. Down-regulation of lgr5 was achieved with gene-specific siRNA. The associations between lgr5 methylation and the clinicopathological features as well as survival of patients were analyzed with statistical methods. Results The lgr5 promoter was methylated to different degrees for the six colorectal cell lines examined, with complete methylation observed in HCT116 cells in which the lgr5 expression was partially recovered following DAC treatment. The stem-cell sphere formation from HCT116 cells was accompanied by increasing methylation within the lgr5 promoter and decreasing expression of lgr5. Knocking down lgr5 by siRNA also led to stem-cell spheres formation. Among primary colorectal tumors, 40% (67/169) were positive for lgr5 methylation, while none of the normal colon tissues were positive for lgr5 methylation. Furthermore, lgr5 methylation significantly associated with higher tumor grade, and negative distant metastasis (p < 0.05), as well as better prognosis (p = 0.001) in patients with colorectal cancer. Conclusions Our data suggests that lgr5 methylation, through the regulation of lgr5 expression and colorectal CSC differentiation, may constitute a novel prognostic marker for colorectal cancer patients. PMID:26599100

  15. DAPK loss in colon cancer tumor buds: implications for migration capacity of disseminating tumor cells

    PubMed Central

    Karamitopoulou, Eva; Dawson, Heather; Koelzer, Viktor Hendrik; Agaimy, Abbas; Garreis, Fabian; Söder, Stephan; Laqua, William; Lugli, Alessandro; Hartmann, Arndt; Rau, Tilman T.; Schneider-Stock, Regine

    2015-01-01

    Defining new therapeutic strategies to overcome therapy resistance due to tumor heterogeneity in colon cancer is challenging. One option is to explore the molecular profile of aggressive disseminating tumor cells. The cytoskeleton-associated Death-associated protein kinase (DAPK) is involved in the cross talk between tumor and immune cells at the invasion front of colorectal cancer. Here dedifferentiated tumor cells histologically defined as tumor budding are associated with a high risk of metastasis and poor prognosis. Analyzing samples from 144 colorectal cancer patients we investigated immunhistochemical DAPK expression in different tumor regions such as center, invasion front, and buds. Functional consequences for tumor aggressiveness were studied in a panel of colon tumor cell lines using different migration, wound healing, and invasion assays. DAPK levels were experimentally modified by siRNA transfection and overexpression as well as inhibitor treatments. We found that DAPK expression was reduced towards the invasion front and was nearly absent in tumor buds. Applying the ECIS system with HCT116 and HCT116 stable lentiviral DAPK knock down cells (HCTshDAPK) we identified an important role for DAPK in decreasing the migratory capacity whereas proliferation was not affected. Furthermore, the migration pattern differed with HCTshDAPK cells showing a cluster-like migration of tumor cell groups. DAPK inhibitor treatment revealed that the migration rate was independent of DAPK's catalytic activity. Modulation of DAPK expression level in SW480 and DLD1 colorectal cancer cells significantly influenced wound closure rate. DAPK seems to be a major player that influences the migratory capability of disseminating tumor cells and possibly affects the dynamic interface between pro- and anti-survival factors at the invasion front of colorectal cancer. This interesting and new finding requires further evaluation. PMID:26405175

  16. PES1 regulates sensitivity of colorectal cancer cells to anticancer drugs

    SciTech Connect

    Xie, Wei; Qu, Like; Meng, Lin; Liu, Caiyun; Wu, Jian; Shou, Chengchao

    2013-02-15

    Highlights: ► PES1 was overexpressed in diverse cancer cell lines. ► PES1-ablation enhances DNA damage response by decreasing DNA repair. ► PES1-ablation increases the sensitivity of HCT116 cells to chemotherapeutic agents. ► PES1-ablation is associated with diminished nuclear entry of RAD51. -- Abstract: PES1 (also known as Pescadillo), a nucleolar protein, was involved in biogenesis of ribosomal RNA. Up-regulation of PES1 has been documented in some human cancers, indicating that PES1 may play some crucial roles in tumorigenesis. In our previous study, it was found that silencing of PES1 resulted in decreased proliferation of colorectal cancer cells. We also noticed that depletion of PES1 altered expression profiles of diverse genes. In the present study, we validated the expression changes of a subset of genotoxic stress-related genes in PES1-silenced HCT116 cells by quantitative RT-PCR. The steady and etoposide-induced phosphorylated H2AX (γ-H2AX) were higher in PES1-silenced cells than in control cells. Besides, etoposide-induced γ-H2AX persisted longer in PES1-silenced cells after removing the etoposide. Next, results of comet assay revealed decreased DNA repair after PES1-ablation. PES1-ablated cells were more sensitive to chemotherapeutic agents, which could be reversed by reconstitution with exogenous PES1. Furthermore, deletion of PES1 diminished steady and DNA damage-induced levels of nuclear RAD51. Our results uncover a potential role of PES1 in chemoresistance by regulating DNA damage response in colorectal cancer cells.

  17. Characterization of the Loss of SUMO Pathway Function on Cancer Cells and Tumor Proliferation

    PubMed Central

    He, Xingyue; Riceberg, Jessica; Pulukuri, Sai M.; Grossman, Steve; Shinde, Vaishali; Shah, Pooja; Brownell, James E.; Dick, Larry; Newcomb, John; Bence, Neil

    2015-01-01

    SUMOylation is a post-translational ubiquitin-like protein modification pathway that regulates important cellular processes including chromosome structure, kinetochore function, chromosome segregation, nuclear and sub-nuclear organization, transcription and DNA damage repair. There is increasing evidence that the SUMO pathway is dysregulated in cancer, raising the possibility that modulation of this pathway may have therapeutic potential. To investigate the importance of the SUMO pathway in the context of cancer cell proliferation and tumor growth, we applied lentivirus-based short hairpin RNAs (shRNA) to knockdown SUMO pathway genes in human cancer cells. shRNAs for SAE2 and UBC9 reduced SUMO conjugation activity and inhibited proliferation of human cancer cells. To expand upon these observations, we generated doxycycline inducible conditional shRNA cell lines for SAE2 to achieve acute and reversible SAE2 knockdown. Conditional SAE2 knockdown in U2OS and HCT116 cells slowed cell growth in vitro, and SAE2 knockdown induced multiple terminal outcomes including apoptosis, endoreduplication and senescence. Multinucleated cells became senescent and stained positive for the senescence marker, SA-β Gal, and displayed elevated levels of p53 and p21. In an attempt to explain these phenotypes, we confirmed that loss of SUMO pathway activity leads to a loss of SUMOylated Topoisomerase IIα and the appearance of chromatin bridges which can impair proper cytokinesis and lead to multinucleation. Furthermore, knockdown of SAE2 induces disruption of PML nuclear bodies which may further promote apoptosis or senescence. In an in vivo HCT116 xenograft tumor model, conditional SAE2 knockdown strongly impaired tumor growth. These data demonstrate that the SUMO pathway is required for cancer cell proliferation in vitro and tumor growth in vivo, implicating the SUMO pathway as a potential cancer therapeutic target. PMID:25860128

  18. Increased SPHK2 Transcription of Human Colon Cancer Cells in Serum-Depleted Culture: The Involvement of CREB Transcription Factor.

    PubMed

    Mizutani, Naoki; Omori, Yukari; Tanaka, Koji; Ito, Hiromi; Takagi, Akira; Kojima, Tetsuhito; Nakatochi, Masahiro; Ogiso, Hideo; Kawamoto, Yoshiyuki; Nakamura, Mitsuhiro; Suzuki, Motoshi; Kyogashima, Mamoru; Tamiya-Koizumi, Keiko; Nozawa, Yoshinori; Murate, Takashi

    2015-10-01

    Sphingosine kinases (SPHK) are important to determine cells' fate by producing sphingosine 1-phosphate. Reportedly, exogenous SPHK2 overexpression induces cell cycle arrest or cell death. However, the regulatory mechanism of SPHK2 expression has not been fully elucidated. Here, we analyzed this issue using human colon cancer cell lines under various stress conditions. Serum depletion (FCS(-)) but not hypoxia and glucose depletion increased mRNA, protein and enzyme activity of SPHK2 but not SPHK1. In HCT116 cells mostly used, SPHK2 activity was predominant over SPHK1, and serum depletion increased both nuclear and cytoplasmic SPHK2 activity. Based on previous reports analyzing cellular response after serum depletion, the temporal changes of intracellular signaling molecules and candidate transcription factors for SPHK2 were examined using serum-depleted HCT116 cells, and performed transfection experiments with siRNA or cDNA of candidate transcription factors. Results showed that the rapid and transient JNK activation followed by CREB activation was the major regulator of increased SPHK2 transcription in FCS(-) culture. EMSA and ChIP assay confirmed the direct binding of activated CREB to the CREB binding site of 5' SPHK2 promoter region. Colon cancer cells examined continued to grow in FCS(-) culture, although mildly, while hypoxia and glucose depletion suppressed cell proliferation or induced cell death, suggesting the different role of SPHK2 in different stress conditions. Because of the unique relationship observed after serum depletion, we examined effects of siRNA for SPHK2, and found the role of SPHK2 as a growth or survival factor but not a cell proliferation inhibitor in FCS(-) culture. PMID:25808826

  19. Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate

    PubMed Central

    Saldanha, Sabita N.; Kala, Rishabh; Tollefsbol, Trygve O.

    2014-01-01

    Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in colorectal cancer cells. PMID:24518414

  20. Combination of Tolfenamic acid and curcumin induces colon cancer cell growth inhibition through modulating specific transcription factors and reactive oxygen species

    PubMed Central

    Sankpal, Umesh T.; Nagaraju, Ganji Purnachandra; Gottipolu, Sriharika R.; Hurtado, Myrna; Jordan, Christopher G.; Simecka, Jerry W.; Shoji, Mamoru; El-Rayes, Bassel; Basha, Riyaz

    2016-01-01

    Curcumin (Cur) has been extensively studied in several types of malignancies including colorectal cancer (CRC); however its clinical application is greatly affected by low bioavailability. Several strategies to improve the therapeutic response of Cur are being pursued, including its combination with small molecules and drugs. We investigated the therapeutic efficacy of Cur in combination with the small molecule tolfenamic acid (TA) in CRC cell lines. TA has been shown to inhibit the growth of human cancer cells in vitro and in vivo, via targeting the transcription factor specificity protein1 (Sp1) and suppressing survivin expression. CRC cell lines HCT116 and HT29 were treated with TA and/or Cur and cell viability was measured 24–72 hours post-treatment. While both agents caused a steady reduction in cell viability, following a clear dose/time-dependent response, the combination of TA+Cur showed higher growth inhibition when compared to either single agent. Effects on apoptosis were determined using flow cytometry (JC-1 staining to measure mitochondrial membrane potential), Western blot analysis (c-PARP expression) and caspase 3/7 activity. Reactive oxygen species (ROS) levels were measured by flow cytometry and the translocation of NF-kB into the nucleus was determined using immunofluorescence. Results showed that apoptotic markers and ROS activity were significantly upregulated following combination treatment, when compared to the individual agents. This was accompanied by decreased expression of Sp1, survivin and NF-kB translocation. The combination of TA+Cur was more effective in HCT116 cells than HT29 cells. These results demonstrate that TA may enhance the anti-proliferative efficacy of Cur in CRC cells. PMID:26672603

  1. Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells

    PubMed Central

    Lai, Ming-Chih; Chang, Chiao-May; Sun, H. Sunny

    2016-01-01

    Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h) dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia. PMID:27078027

  2. Identification of PRKDC (Protein Kinase, DNA-Activated, Catalytic Polypeptide) as an essential gene for colorectal cancer (CRCs) cells.

    PubMed

    Sun, Shangfeng; Cheng, Shuguang; Zhu, Yunxiao; Zhang, Peng; Liu, Ning; Xu, Tong; Sun, Chao; Lv, Yanfeng

    2016-06-10

    Oncogene and non-oncogene addictions describe the phenomenon that tumor cells become reliant on certain genes for maintenance of malignancy. Reversal of these mutations profoundly affects tumor growth and survival, providing a fundamental rationale for development of targeted cancer therapy. However, inadequate knowledge on cancer signaling networks and lack of potential drug targets limited its clinical application. A screen was conducted using a custom small interfering RNA (siRNA) library in colorectal cancer (CRC). Transient knockdown followed by cell proliferation assays were performed to validate the essentiality of PRKDC (Protein Kinase, DNA-Activated, Catalytic Polypeptide) in CRC. Western blot analysis was performed to examine the mechanism by which PRKDC confers selective survival advantage in CRC cells. Inducible knockdown and overexpression cell lines were introduced into nude mice to assess PRKDC dependency of CRC cells in vivo. PRKDC expression level in patient samples and overall survival of patients with low or high PRKDC expression were analyzed. Transient knockdown of PRKDC reduced cell proliferation/survival in HCT116 and DLD1, but not FHC cells. PRKDC down-regulation induced apoptosis partially through inhibiting AKT activation, and sensitized HCT116 cells to chemotherapeutic agents interfering with DNA replication. Inducible knockdown of PRKDC inhibited tumor growth in vivo. PRKDC was up-regulated in cancerous tissues compared with normal tissues. Patients with high PRKDC expression showed poorer overall survival. PRKDC is an essential gene required for CRC cell proliferation/survival, which may represent as a potential prognostic biomarker and an ideal therapeutic target for CRC. PMID:26992638

  3. Methylselenol, a selenium metabolite, plays a critical role in inhibiting colon cancer cell growth in vitro and in vivo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methylselenol is hypothesized to be a critical selenium (Se) metabolite for anticancer activity. In this study, submicromolar methylselenol was generated by incubating methionase with seleno-L methionine, and both colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to...

  4. Expression of DNA damage checkpoint 53BP1 is correlated with prognosis, cell proliferation and apoptosis in colorectal cancer

    PubMed Central

    Bi, Jianping; Huang, Ai; Liu, Tao; Zhang, Tao; Ma, Hong

    2015-01-01

    53BP1, an important mediator of DNA damage checkpoint, plays an essential role in maintaining the cell genome stability, and the aberrant expression of 53BP1 was found to contribute to tumor occurrence and development. In this study, we explored the clinical significance of 53BP1 expression in colorectal cancer and investigated the effects of 53BP1 expression on tumor cell proliferation and apoptosis and its possible mechanisms. Immunohistochemical analysis was performed to detect the expression of 53BP1 in 95 cases of tumor tissues. After establishment of shRNA-mediated knockdown stable HCT-116 cell lines, cell proliferation, apoptosis and cell cycle distribution were detected by MTT and flow cytometry, and expression of up-and down-steam related proteins as γ-H2AX, CHK2 and P53 were tested by Western blot. 53BP1 intensity was found to be associated with tumor location (P < 0.05), and the low expression of 53BP1 revealed decreased survival time compared with high expression in subgroups as male, tumor size > 5 cm, tumor located at right side, T stage as T3-T4, N0, clinical stage as I-II (P < 0.05). In vitro, shRNA-mediated loss of 53BP1 obviously inhibited HCT-116 tumor cell apoptosis, promoted cell proliferation and increased accumulation of cells in S phase. Meanwhile, the expression of γ-H2AX, CHK2 and P53 was significantly reduced (P < 0.05). Our findings suggest 53BP1 may serve as a candidate biomarker for predicting prognosis and disease development in colorectal cancer. PMID:26261485

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

    SciTech Connect

    Chen Wenshu; Yu Yichu; Lee Yijang; Chen, J.-H.; Hsu, H.-Y.; Chiu, S.-J.

    2010-06-01

    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.

  6. Anticolorectal cancer effects and pharmacokinetic application of 2, 2-Bis [4-(4-amino-3-hydroxyphenoxy) phenyl] adamantane

    PubMed Central

    Yang, Po-Sheng; Wang, Jane-Jen; Tsai, Tung-Hu; Wang, Yea-Hwey; Jan, Woan-Ching; Cheng, Shih-Ping; Chi, Chin-Wen; Hsu, Yi-Chiung

    2015-01-01

    2, 2-Bis (4-(4-amino-3-hydroxyphenoxy) phenyl) adamantane (DPA) induced growth inhibition in human cancer cells using the national cancer institute (NCI) anticancer drug screen. In our previous study, we demonstrated that DPA exerted growth inhibitory activities in the three human colon cancer cell lines (Colo 205, HT-29, and HCT-15). To identify the detailed mechanism, we examined the functional importance of p21 and p53 in DPA-induced anticancer effect. We used three isogenic colon cancer cell lines, HCT-116, HCT-116 p53-/-, and HCT-116 p21-/-, to evaluate the roles of p21 and p53 in the in vitro anticancer effects of DPA. DPA dose-dependently inhibited cell growth, cell migration and increased cell cycle at the G0/G1 phase in HCT116 cells but not in p21-/- and p53-/- isogenic HCT-116 cells. Additionally, Western blot showed that DPA treatment induced the p21, p53, and cyclin-E protein expressions in HCT-116 cells. The p21 associated cell cycle regulatory protein such as cyclin D, CDK4, and pRb were decreased after DPA treatment in HCT-116 cells. DPA decreased cell migration in HCT-116 and HCT-116 p53-/- but not in HCT-116 p21-/- cells. We observed the up-regulation of E-cadherin, p-p38, and p-Erk in DPA-treated HCT-116 group but not in HCT-116 p21-/- and HCT-116 p53-/- groups. We assumed that p21 was required for DPA-induced anti-colon cancer effect through the Erk and p38 pathway leading to cell cycle arrest and inhibition of cell motility. Mean (± SE) pharmacokinetic parameters of the DPA were as follows: AUC = 64.44 ± 8.41, Cmax = 1.56 ± 0.48 and t1/2 = 113.92 ± 58.19. The pharmacokinetic data suggest DPA can be applied to further clinical study. This is the first pharmacokinetic study of DPA, and indicated that anti-proliferation and the cell mobility inhibition effects of DPA in HCT116 WT cells may result from the induction of p21 through activation of ERK and p38 pathway. PMID:26628962

  7. CYP2S1 depletion enhances colorectal cell proliferation is associated with PGE2-mediated activation of β-catenin signaling

    SciTech Connect

    Yang, Chao; Li, Changyuan; Li, Minle; Tong, Xuemei; Hu, Xiaowen; Yang, Xuhan; Yan, Xiaomei; He, Lin; Wan, Chunling

    2015-02-15

    Colorectal epithelial cancer is one of the most common cancers in the world and its 5-year survival rate is still relatively low. Cytochrome P450 (CYP) enzymes in epithelial cells lining the alimentary tract play an important role in the oxidative metabolism of a wide range of xenobiotics, including (pro-)carcinogens and endogenous compounds. Although CYP2S1, a member of CYP family, strongly expressed in many extrahepatic tissues, the role of CYP2S1 in cancer remains unclear. To investigate whether CYP2S1 involves in colorectal carcinogenesis, cell proliferation was analyzed in HCT116 cells depleted of CYP2S1 using small hairpin interfering RNA. Our data show that CYP2S1 knockdown promotes cell proliferation through increasing the level of endogenous prostaglandin E2(PGE2). PGE2, in turn, reduces phosphorylation of β-catenin and activates β-catenin signaling, which contributes to the cell proliferation. Furthermore, CYP2S1 knockdown increase tumor growth in xenograft mouse model. In brief, these results demonstrate that CYP2S1 regulates colorectal cancer growth through associated with PGE2-mediated activation of β-catenin signaling. - Highlights: • Knockdown of CYP2S1 expression improve HCT116 cell proliferation in vitro and in vivo. • Elevate PGE2 production in CYP2S1 knockdown cell is associated with its proliferation. • Elevate PGE2 level in CYP2S1 knockdown cells enhance β-catenin accumulation. • β-catenin activate TCF/LEF and target gene expression thus promote cell proliferation.

  8. Lack of functional p53 renders DENSpm-induced autophagy and apoptosis in time dependent manner in colon cancer cells.

    PubMed

    oker-Grkan, Ajda; Arisan, Elif Damla; Obakan, P?nar; Palavan-Unsal, Narin

    2015-01-01

    Polyamines (PAs), such as putrescine, spermidine and spermine, are alkyl-amines that are essential for cell growth, proliferation, differentiation and cancer progression in eukaryotic cells. A designed PA analogue; DENSpm, induces cell cycle arrest, inhibits proliferation and induces apoptosis in melanoma, breast, prostate, lung and colon cancer cells. Although the mechanism by which DENSpm induces apoptosis has been examined, the effect of DENSpm on autophagy has not been investigated yet. Therefore, in this study, our objective was to determine the role of p53 in the DENSpm-induced autophagy/apoptotic regulation in a time-dependent manner in colon cancer cells. Exposure of HCT 116 colon cancer cells to DENSpm decreased cell viability in a dose- and time-dependent manner. However, the p53 mutant, SW480, and deficient HCT 116 p53(-/-) cells were more resistant to DENSpm treatment compared to HCT 116 p53(+/+) cells. The resistant profile caused by p53 defect also caused a cell type-specific response to PA pool depletion and SSAT overexpression. In addition to PA depletion, DENSpm induced apoptosis by activating the mitochondria-mediated pathway in a caspase-dependent manner regardless of p53 expression in colon cancer cells. Concomitantly, we determined that DENSpm also affected autophagy in HCT 116 p53(+/+), SW480 and HCT 116 p53(-/-) colon cancer cells for different periods of exposure to DENSpm. Therefore, this study revealed that effect of DENSpm on cell death differs due to p53 protein expression profile. In addition, DENSpm-induced autophagy may be critical in drug resistance in colon cancer cells. PMID:25311224

  9. LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner

    PubMed Central

    Kramer, Holly B.; Lai, Chun-Fui; Patel, Hetal; Periyasamy, Manikandan; Lin, Meng-Lay; Feller, Stephan M.; Fuller-Pace, Frances V.; Meek, David W.; Ali, Simak; Buluwela, Laki

    2016-01-01

    Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53. PMID:26400164

  10. LRH-1 drives colon cancer cell growth by repressing the expression of the CDKN1A gene in a p53-dependent manner.

    PubMed

    Kramer, Holly B; Lai, Chun-Fui; Patel, Hetal; Periyasamy, Manikandan; Lin, Meng-Lay; Feller, Stephan M; Fuller-Pace, Frances V; Meek, David W; Ali, Simak; Buluwela, Laki

    2016-01-29

    Liver receptor homologue 1 (LRH-1) is an orphan nuclear receptor that has been implicated in the progression of breast, pancreatic and colorectal cancer (CRC). To determine mechanisms underlying growth promotion by LRH-1 in CRC, we undertook global expression profiling following siRNA-mediated LRH-1 knockdown in HCT116 cells, which require LRH-1 for growth and in HT29 cells, in which LRH-1 does not regulate growth. Interestingly, expression of the cell cycle inhibitor p21 (CDKN1A) was regulated by LRH-1 in HCT116 cells. p21 regulation was not observed in HT29 cells, where p53 is mutated. p53 dependence for the regulation of p21 by LRH-1 was confirmed by p53 knockdown with siRNA, while LRH-1-regulation of p21 was not evident in HCT116 cells where p53 had been deleted. We demonstrate that LRH-1-mediated p21 regulation in HCT116 cells does not involve altered p53 protein or phosphorylation, and we show that LRH-1 inhibits p53 recruitment to the p21 promoter, likely through a mechanism involving chromatin remodelling. Our study suggests an important role for LRH-1 in the growth of CRC cells that retain wild-type p53. PMID:26400164

  11. GEN-27, a Newly Synthetic Isoflavonoid, Inhibits the Proliferation of Colon Cancer Cells in Inflammation Microenvironment by Suppressing NF-κB Pathway

    PubMed Central

    Wang, Yajing; Lu, Ping; Zhang, Weifeng; Du, Qianming; Tang, Jingjing; Wang, Hong; Lu, Jinrong; Hu, Rong

    2016-01-01

    Nonresolving inflammation is one of the consistent features of the tumor microenvironment in the intestine and plays a critical role in the initiation and development of colon cancer. Here we reported the inhibitory effects of GEN-27, a new derivative of genistein, on the inflammation-related colon cancer cell proliferation and delineated the mechanism of its action. The results indicated that GEN-27 inhibited the proliferation of human colon tumor HCT116 cells stimulated by culture supernatants of LPS-induced human monocytes THP-1 cells and significantly decreased LPS-induced secretion of proinflammatory cytokines interleukin-6 and interleukin-1β in THP-1 cells. The HCT116 cell proliferation elicited by THP-1-conditioned medium could be blocked by the interleukin-1 receptor antagonist (IL-1RA). Further mechanistic study revealed that GEN-27 remarkably inhibited the nuclear translocation of NF-κB and phosphorylation of IκB and IKKα/β in both HCT116 and THP-1 cells. In addition, GEN-27 markedly suppressed the HCT116 cell proliferation stimulated by IL-1β treatment, which was dependent on the inhibition of NF-κB/p65 nuclear localization, as verified by p65 overexpression and BAY 11-7082, an NF-κB inhibitor. Taken together, our findings established that GEN-27 modulated NF-κB signaling pathway involved in inflammation-induced cancer cells proliferation and therefore could be a potential chemopreventive agent against inflammation-associated colon cancer. PMID:27057094

  12. Sp1 and Sp3 Are the Transcription Activators of Human ek1 Promoter in TSA-Treated Human Colon Carcinoma Cells

    PubMed Central

    Kuan, Chee Sian; See Too, Wei Cun; Few, Ling Ling

    2016-01-01

    Background Ethanolamine kinase (EK) catalyzes the phosphorylation of ethanolamine, the first step in the CDP-ethanolamine pathway for the biosynthesis of phosphatidylethanolamine (PE). Human EK exists as EK1, EK2α and EK2β isoforms, encoded by two separate genes, named ek1 and ek2. EK activity is stimulated by carcinogens and oncogenes, suggesting the involvement of EK in carcinogenesis. Currently, little is known about EK transcriptional regulation by endogenous or exogenous signals, and the ek gene promoter has never been studied. Methodology/Principal Findings In this report, we mapped the important regulatory regions in the human ek1 promoter. 5’ deletion analysis and site-directed mutagenesis identified a Sp site at position (-40/-31) that was essential for the basal transcription of this gene. Treatment of HCT116 cells with trichostatin A (TSA), a histone deacetylase inhibitor, significantly upregulated the ek1 promoter activity through the Sp(-40/-31) site and increased the endogenous expression of ek1. Chromatin immunoprecipitation assay revealed that TSA increased the binding of Sp1, Sp3 and RNA polymerase II to the ek1 promoter in HCT116 cells. The effect of TSA on ek1 promoter activity was cell-line specific as TSA treatment did not affect ek1 promoter activity in HepG2 cells. Conclusion/Significance In conclusion, we showed that Sp1 and Sp3 are not only essential for the basal transcription of the ek1 gene, their accessibility to the target site on the ek1 promoter is regulated by histone protein modification in a cell line dependent manner. PMID:26807725

  13. Polyphenol-rich extract of Salvia chinensis exhibits anticancer activity in different cancer cell lines, and induces cell cycle arrest at the G0/G1-phase, apoptosis and loss of mitochondrial membrane potential in pancreatic cancer cells

    PubMed Central

    ZHAO, QUAN; HUO, XUE-CHEN; SUN, FU-DONG; DONG, RUI-QIAN

    2015-01-01

    Pancreatic cancer (PC) is one of the most aggressive types of human malignancy, which has an overall 5-year survival rate of <2%. PC is the fourth most common cause of cancer-associated mortality in the western world. At present, there is almost no effective treatment available for the treatment of PC. The aim of the present study was to evaluate the anticancer potential of a polyphenol enriched extract obtained from Salvia chinensis, a Chinese medicinal plant. An MTT assay was used to evaluate the cell viability of five cancer cell lines and one normal cell line. In addition, the effects of the extract on apoptotic induction, cell cycle phase distribution, DNA damage and loss of mitochondrial membrane potential (ΛΨm) were evaluated in MiapaCa-2 human PC cells. The effects of the extract on cell cycle phase distribution and ΛΨm were assessed by flow cytometry, using propidium iodide and rhodamine-123 DNA-binding fluorescent dyes, respectively. Fluorescence microscopy, using 4′,6-diamidino-2-phenylindole as a staining agent, was performed in order to detect the morphological changes of the MiapaCa-2 cancer cells and the presence of apoptotic bodies following treatment with the extract. The results of the present study demonstrated that the polyphenol-rich extract from S. chinensis induced potent cytotoxicity in the MCF-7 human breast cancer cells, A549 human lung cancer cells, HCT-116 and COLO 205 human colon cancer cells, and MiapaCa-2 human PC cells. The COLO 205 and MCF-7 cancer cell lines were the most susceptible to treatment with the extract, which exhibited increased rate of growth inhibition. Fluorescence microscopy revealed characteristic morphological features of apoptosis and detected the appearance of apoptotic bodies following treatment with the extract in the PC cells. Flow cytometric analysis demonstrated that the extract induced G0/G1 cell cycle arrest in a dose-dependent manner. In addition, treatment with the extract induced a significant and concentration-dependent reduction in the ΛΨm of the PC cells. PMID:26165362

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

    SciTech Connect

    Lee, Hanna; Park, Minhee; Shin, Nara; Kim, Gamin; Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul ; Kim, Yun Gi; Shin, Jeon-Soo; Kim, Hoguen; Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemoon-Ku, Seoul

    2012-07-27

    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.

  15. Construction of interference vector targeting Ep-CAM gene and its effects on colorectal cancer cell proliferation

    PubMed Central

    Qi, Yanmei; Zhou, Fengqiang; Zhang, Lu; Liu, Lei; Xu, Hong; Guo, Huiguang

    2015-01-01

    Background Prior study indicates that abnormal protein expression and functional changes in the development and progression of colorectal cancer is related to gene expression. The aim of this study was to construct an interference plasmid targeting the Ep-CAM gene and to investigate its effects on the proliferation of colorectal cancer cells. Methods In this study, HT-29 and HCT-116 colorectal cancer cell lines were selected as cell models. The double-stranded micro (mi)RNA oligo was inserted into the pcDNATM6.2-GW/EmGFPmiR vector, which is an expression of miRNA. Lipofectamine™ 2000 was used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg) and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-Ep-CAM-1), respectively. Meanwhile, the nontransferred HT-29 and HCT-116 acts as the blank control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression. The cell proliferation in each group was detected by using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results The results indicated that the Ep-CAM messenger (m)RNA expression in the interference group was lower significantly compared with that of the empty plasmid group and control group (P<0.01). Western blot analysis results showed that Ep-CAM protein expression was significantly lower in interference group compared with that of the empty plasmid group and the control group (P<0.01). MTT assay results demonstrated that the proliferation ability of cells in the interference group was significantly inhibited compared with the two other groups (P<0.05). Conclusion Silencing of Ep-CAM can significantly inhibit the proliferation of colorectal cancer cells. PMID:26028961

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

    PubMed Central

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

    2013-01-01

    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

  17. Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery

    PubMed Central

    De Angelis, Paula M; Svendsrud, Debbie H; Kravik, Katherine L; Stokke, Trond

    2006-01-01

    Background Treatment of cells with the anti-cancer drug 5-fluorouracil (5-FU) causes DNA damage, which in turn affects cell proliferation and survival. Two stable wild-type TP53 5-FU-resistant cell lines, ContinB and ContinD, generated from the HCT116 colon cancer cell line, demonstrate moderate and strong resistance to 5-FU, respectively, markedly-reduced levels of 5-FU-induced apoptosis, and alterations in expression levels of a number of key cell cycle- and apoptosis-regulatory genes as a result of resistance development. The aim of the present study was to determine potential differential responses to 8 and 24-hour 5-FU treatment in these resistant cell lines. We assessed levels of 5-FU uptake into DNA, cell cycle effects and apoptosis induction throughout treatment and recovery periods for each cell line, and alterations in expression levels of DNA damage response-, cell cycle- and apoptosis-regulatory genes in response to short-term drug exposure. Results 5-FU treatment for 24 hours resulted in S phase arrests, p53 accumulation, up-regulation of p53-target genes on DNA damage response (ATF3, GADD34, GADD45A, PCNA), cell cycle-regulatory (CDKN1A), and apoptosis-regulatory pathways (FAS), and apoptosis induction in the parental and resistant cell lines. Levels of 5-FU incorporation into DNA were similar for the cell lines. The pattern of cell cycle progression during recovery demonstrated consistently that the 5-FU-resistant cell lines had the smallest S phase fractions and the largest G2(/M) fractions. The strongly 5-FU-resistant ContinD cell line had the smallest S phase arrests, the lowest CDKN1A levels, and the lowest levels of 5-FU-induced apoptosis throughout the treatment and recovery periods, and the fastest recovery of exponential growth (10 days) compared to the other two cell lines. The moderately 5-FU-resistant ContinB cell line had comparatively lower apoptotic levels than the parental cells during treatment and recovery periods and a recovery time of 22 days. Mitotic activity ceased in response to drug treatment for all cell lines, consistent with down-regulation of mitosis-regulatory genes. Differential expression in response to 5-FU treatment was demonstrated for genes involved in regulation of nucleotide binding/metabolism (ATAD2, GNL2, GNL3, MATR3), amino acid metabolism (AHCY, GSS, IVD, OAT), cytoskeleton organization (KRT7, KRT8, KRT19, MAST1), transport (MTCH1, NCBP1, SNAPAP, VPS52), and oxygen metabolism (COX5A, COX7C). Conclusion Our gene expression data suggest that altered regulation of nucleotide metabolism, amino acid metabolism, cytoskeleton organization, transport, and oxygen metabolism may underlie the differential resistance to 5-FU seen in these cell lines. The contributory roles to 5-FU resistance of some of the affected genes on these pathways will be assessed in future studies. PMID:16709241

  18. Silibinin Suppresses Growth of Human Colorectal Carcinoma SW480 Cells in Culture and Xenograft through Down-regulation of β-Catenin-Dependent Signaling1

    PubMed Central

    Kaur, Manjinder; Velmurugan, Balaiya; Tyagi, Alpna; Agarwal, Chapla; Singh, Rana P; Agarwal, Rajesh

    2010-01-01

    Mutations in APC/β-catenin resulting in an aberrant activation of Wnt/β-catenin pathway are common in colorectal cancer (CRC), suggesting that targeting the β-catenin pathway with chemopreventive/anticancer agents could be a potential translational approach to control CRC. Using human CRC cell lines harboring mutant (SW480) versus wildtype (HCT116) APC gene and alteration in β-catenin pathway, herein we performed both in vitro and in vivo studies to examine for the first time whether silibinin targets β-catenin pathway in its efficacy against CRC. Silibinin treatment inhibited cell growth, induced cell death, and decreased nuclear and cytoplasmic levels of β-catenin in SW480 but not in HCT116 cells, suggesting its selective effect on the β-catenin pathway and associated biologic responses. Other studies, therefore, were performed only in SW480 cells where silibinin significantly decreased β-catenin-dependent T-cell factor-4 (TCF-4) transcriptional activity and protein expression of β-catenin target genes such as c-Myc and cyclin D1. Silibinin also decreased cyclin-dependent kinase 8 (CDK8), a CRC oncoprotein that positively regulates β-catenin activity, and cyclin C expression. In a SW480 tumor xenograft study, 100- and 200-mg/kg doses of silibinin feeding for 6 weeks inhibited tumor growth by 26% to 46% (P < .001). Analyses of xenografts showed that similar to cell culture findings, silibinin decreases proliferation and expression of β-catenin, cyclin D1, c-Myc, and CDK8 but induces apoptosis in vivo. Together, these findings suggest that silibinin inhibits the growth of SW480 tumors carrying the mutant APC gene by down-regulating CDK8 and β-catenin signaling and, therefore, could be an effective agent against CRC. PMID:20454513

  19. Bisleuconothine A, a bisindole alkaloid, inhibits colorectal cancer cell in vitro and in vivo targeting Wnt signaling.

    PubMed

    Kong, Ling-Mei; Feng, Tao; Wang, Yuan-Yuan; Li, Xing-Yao; Ye, Zhen-Nan; An, Tao; Qing, Chen; Luo, Xiao-Dong; Li, Yan

    2016-03-01

    Wnt signaling pathway is aberrantly activated in a variety of cancers, especially in colorectal cancer and small molecule antagonists of Wnt/β-catenin signaling are attractive candidates for developing effective therapeutics. In the present study, we identified Bisleuconothine A, a bisindole alkaloid with an eburnane-aspidosperma type skeleton, as a novel and selective Wnt signaling inhibitor by using a cell-based luciferase assay system. Our study found that Bisleuconothine A down-regulated the endogenous Wnt target gene expression through promoting phosphorylation of β-catenin and the subsequent inhibition of its nuclear translocation in HCT116 and SW480 colorectal cancer cells. In vitro, Bisleuconothine A inhibited cell proliferation through induction of apoptosis by increasing the cleavage of caspases in HCT116 and SW480 colorectal cancer cells. Moreover, in vivo, Bisleuconothine A dramatically suppressed tumor growth in HCT116 Xenograft. And further analysis showed that Bisleuconothine A suppressed the Wnt target gene expression in HCT116 Xenograft, which was associated with up-regulation of β-catenin phosphorylation and subsequent Wnt signaling inhibition. Taken together, our study indicated that bisindole alkaloids could be included as a new chemotype of small-molecule Wnt signaling inhibitors, and have great potential to be further developed for anti-tumor agents. PMID:26862734

  20. Silencing Egr1 Attenuates Radiation-Induced Apoptosis in Normal Tissues while Killing Cancer Cells and Delaying Tumor Growth.

    PubMed

    Zhao, Diana Yi; Jacobs, Keith M; Hallahan, Dennis E; Thotala, Dinesh

    2015-10-01

    Normal tissue toxicity reduces the therapeutic index of radiotherapy and decreases the quality of life for cancer survivors. Apoptosis is a key element of the radiation response in normal tissues like the hippocampus and small intestine, resulting in neurocognitive disorders and intestinal malabsorption. The Early Growth Response 1 (Egr1) transcription factor mediates radiation-induced apoptosis by activating the transcription of proapoptosis genes in response to ionizing radiation (IR). Therefore, we hypothesized that the genetic abrogation of Egr1 and the pharmacologic inhibition of its transcriptional activity could attenuate radiation-induced apoptosis in normal tissues. We demonstrated that Egr1-null mice had less apoptosis in the hippocampus and intestine following irradiation as compared with their wild-type littermates. A similar result was achieved using Mithramycin A (MMA) to prevent binding of Egr1 to target promoters in the mouse intestine. Abolishing Egr1 expression using shRNA dampened apoptosis and enhanced the clonogenic survival of irradiated HT22 hippocampal neuronal cells and IEC6 intestinal epithelial cells. Mechanistically, these events involved an abrogation of p53 induction by IR and an increase in the ratio of Bcl-2/Bax expression. In contrast, targeted silencing of Egr1 in two cancer cell lines (GL261 glioma cells and HCT116 colorectal cancer cells) was not radioprotective, since it reduced their growth while also sensitizing them to radiation-induced death. Further, Egr1 depletion delayed the growth of heterotopically implanted GL261 and HCT116 tumors. These results support the potential of silencing Egr1 in order to minimize the normal tissue complications associated with radiotherapy while enhancing tumor control. PMID:26206332

  1. Synthetic Development of New 3-(4-Arylmethylamino)butyl-5-arylidene-rhodanines under Microwave Irradiation and Their Effects on Tumor Cell Lines and against Protein Kinases.

    PubMed

    Dago, Camille Déliko; Ambeu, Christelle N'ta; Coulibaly, Wacothon-Karime; Békro, Yves-Alain; Mamyrbékova, Janat; Defontaine, Audrey; Baratte, Blandine; Bach, Stéphane; Ruchaud, Sandrine; Guével, Rémy Le; Ravache, Myriam; Corlu, Anne; Bazureau, Jean-Pierre

    2015-01-01

    A new route to 3-(4-arylmethylamino)butyl-5-arylidene-2-thioxo-1,3-thiazolidine-4-one 9 was developed in six steps from commercial 1,4-diaminobutane 1 as starting material. The key step of this multi-step synthesis involved a solution phase "one-pot two-steps" approach assisted by microwave dielectric from N-(arylmethyl)butane-1,4-diamine hydrochloride 6a-f (as source of the first point diversity) and commercial bis-(carboxymethyl)-trithiocarbonate reagent 7 for construction of the rhodanine platform. This platform was immediately functionalized by Knoevenagel condensation under microwave irradiation with a series of aromatic aldehydes 3 as second point of diversity. These new compounds were prepared in moderate to good yields and the fourteen synthetic products 9a-n have been obtained with a Z-geometry about their exocyclic double bond. These new 5-arylidene rhodanines derivatives 9a-n were tested for their kinase inhibitory potencies against four protein kinases: Human cyclin-dependent kinase 5-p25, HsCDK5-p25; porcine Glycogen Synthase Kinase-3, GSK-3α/β; porcine Casein Kinase 1, SsCK1 and human HsHaspin. They have also been evaluated for their in vitro inhibition of cell proliferation (HuH7 D12, Caco 2, MDA-MB 231, HCT 116, PC3, NCI-H727, HaCat and fibroblasts). Among of all these compounds, 9j presented selective micromolar inhibition activity on SsCK1 and 9i exhibited antitumor activities in the HuH7 D12, MDA-MBD231 cell lines. PMID:26184130

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

    SciTech Connect

    Dittmann, Klaus H. Mayer, Claus; Ohneseit, Petra A.; Raju, Uma; Andratschke, Nickolaus H.; Milas, Luka; Rodemann, H. Peter

    2008-01-01

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

  3. Differential Effects of Hepatocyte Nuclear Factor 4? Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells.

    PubMed

    Vuong, Linh M; Chellappa, Karthikeyani; Dhahbi, Joseph M; Deans, Jonathan R; Fang, Bin; Bolotin, Eugene; Titova, Nina V; Hoverter, Nate P; Spindler, Stephen R; Waterman, Marian L; Sladek, Frances M

    2015-10-01

    The nuclear receptor hepatocyte nuclear factor 4? (HNF4?) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4? isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4?2) or P2-driven (HNF4?8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4?2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4?8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/?-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4? and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4? and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4? isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/?-catenin/TCF4 and AP-1 pathways. PMID:26240283

  4. Selenium compounds activate ATM-dependent DNA damage responses via the mismatch repair protein hMLH1 in colorectal cancer cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epidemiological and animal studies indicate that selenium supplementation suppresses risk of colorectal and other cancers. The majority of colorectal cancers are characterized by a defective DNA mismatch repair (MMR) process. Here, we have employed the MMR-deficient HCT 116 colorectal cancer cells ...

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

    PubMed Central

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

    2013-01-01

    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

  6. Effects of folylpolyglutamate synthase modulation on global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells.

    PubMed

    Kim, Sung-Eun; Hinoue, Toshinori; Kim, Michael S; Sohn, Kyoung-Jin; Cho, Robert C; Weisenberger, Daniel J; Laird, Peter W; Kim, Young-In

    2016-03-01

    Folylpolyglutamate synthase (FPGS) plays a critical role in intracellular folate homeostasis. FPGS-induced polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence FPGS modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression and aberrant DNA methylation is mechanistically linked cancer development. We investigated whether FPGS modulation would affect global and gene-specific promoter DNA methylation with consequent functional effects on gene expression profiles in HCT116 colon and MDA-MB-435 breast cancer cells. Although FPGS modulation altered global DNA methylation and DNA methyltransferases (DNMT) activity, the effects of FPGS modulation on global DNA methylation and DNMT activity could not be solely explained by intracellular folate concentrations and content of long-chain folylpolyglutamates, and it may be cell-specific. FPGS modulation influenced differential gene expression and promoter cytosine-guanine dinucleotide sequences (CpG) DNA methylation involved in cellular development, cell cycle, cell death and molecular transport. Some of the altered gene expression was associated with promoter CpG DNA methylation changes. In both the FPGS-overexpressed HCT116 and MDA-MB-435 cell lines, we identified several differentially expressed genes involved in folate biosynthesis and one-carbon metabolism, which might in part have contributed to the observed increased efficacy of 5-fluorouracil in response to FPGS overexpression. Our data suggest that FPGS modulation affects global and promoter CpG DNA methylation and expression of several genes involved in important biological pathways. The potential role of FPGS modulation in DNA methylation and its associated downstream functional effects warrants further studies. PMID:26895662

  7. Insulin, CCAAT/Enhancer-Binding Proteins and Lactate Regulate the Human 11β-Hydroxysteroid Dehydrogenase Type 2 Gene Expression in Colon Cancer Cell Lines

    PubMed Central

    Alikhani-Koupaei, Rasoul; Ignatova, Irena D.; Guettinger, Andreas; Frey, Felix J.; Frey, Brigitte M.

    2014-01-01

    11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon. PMID:25133511

  8. Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

    PubMed

    Göder, Anja; Nagel, Georg; Kraus, Alexander; Dörsam, Bastian; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-08-01

    Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy. PMID:25998848

  9. CLO: The cell line ontology

    PubMed Central

    2014-01-01

    Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

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

    SciTech Connect

    Shelton, Joseph W.; Waxweiler, Timothy V.; Landry, Jerome; Gao, Huiying; Xu, Yanbo; Wang, Lanfang; El-Rayes, Bassel; Shu, Hui-Kuo G.

    2013-07-01

    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. γH2AX 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 (γH2AX, 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.

  11. Colon Cancer Cell Separation by Dielectrophoresis

    NASA Astrophysics Data System (ADS)

    Yang, Fang; Yang, Xiaoming; Jiang, H.; Wood, P.; Hrushesky, W.; Wang, Guiren

    2009-11-01

    Separation of cancer cells from the other biological cells can be useful for clinical cancer diagnosis and cancer treatment. In this presentation, conventional dielectrophoresis (c-DEP) is used in a microfluidic chip to manipulate and collect colorectal cancer HCT116 cell, which is doped with Human Embryonic Kidney 293 cells (HEK 293). It is noticed that, the HCT116 cell are deflected to a side channel from a main channel clearly by apply electric field at particular AC frequency band. This motion caused by negative DEP can be used to separate the cancer cell from others. In this manuscript, chip design, flow condition, the DEP spectrum of the cancer cell are reported respectively, and the separation and collection efficiency are investigated as well. The sorter is microfabricated using plastic laminate technology. -/abstract- This work has been financially supported by the NSF RII funding (EP

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

    SciTech Connect

    Hogan, Niamh M.; Joyce, Myles R.; Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy; Kerin, Michael J.; Dwyer, Roisin M.

    2013-06-14

    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.

  13. Interaction of celecoxib with different anti-cancer drugs is antagonistic in breast but not in other cancer cells

    SciTech Connect

    El-Awady, Raafat A.; Saleh, Ekram M.; Ezz, Marwa; Elsayed, Abeer M.

    2011-09-15

    Celecoxib, an inhibitor of cyclooxygenase-2, is being investigated for enhancement of chemotherapy efficacy in cancer clinical trials. This study investigates the ability of cyclooxygenase-2 inhibitors to sensitize cells from different origins to several chemotherapeutic agents. The effect of the drug's mechanism of action and sequence of administration are also investigated. The sensitivity, cell cycle, apoptosis and DNA damage of five different cancer cell lines (HeLa, HCT116, HepG2, MCF7 and U251) to 5-FU, cisplatin, doxorubicin and etoposide {+-} celecoxib following different incubation schedules were analyzed. We found antagonism between celecoxib and the four drugs in the breast cancer cells MCF7 following all incubation schedules and between celecoxib and doxorubicin in all cell lines except for two combinations in HCT116 cells. Celecoxib with the other three drugs in the remaining four cell lines resulted in variable interactions. Mechanistic investigations revealed that celecoxib exerts different molecular effects in different cells. In some lines, it abrogates the drug-induced G2/M arrest enhancing pre-mature entry into mitosis with damaged DNA thus increasing apoptosis and resulting in synergism. In other cells, it enhances drug-induced G2/M arrest allowing time to repair drug-induced DNA damage before entry into mitosis and decreasing cell death resulting in antagonism. In some synergistic combinations, celecoxib-induced abrogation of G2/M arrest was not associated with apoptosis but permanent arrest in G1 phase. These results, if confirmed in-vivo, indicate that celecoxib is not a suitable chemosensitizer for breast cancer or with doxorubicin for other cancers. Moreover, combination of celecoxib with other drugs should be tailored to the tumor type, drug and administration schedule. - Graphical abstract: Display Omitted Highlights: > Celecoxib may enhance effects of anticancer drugs. > Its combination with four drugs was tested in five cancer cell lines. > It antagonized the effects of the four drugs in the breast cancer cell line MCF7. > Doxorubicin's cytotoxic effects were antagonized by celecoxib in four cell lines. > Cell cycle, apoptosis and DNA damage explain the different interactive effects.

  14. 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

    Ahluwalia, Amrita; Jones, Michael K.; Department of Medicine, University of California, Irvine, CA ; Szabo, Sandor; Department of Pathology, University of California, Irvine, CA ; Tarnawski, Andrzej S.

    2013-08-09

    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.

  15. Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells.

    PubMed

    Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq; Povirk, Lawrence F; Hendrickson, Eric A; Gewirtz, David A

    2016-03-01

    Radiotherapy continues to be a primary modality in the treatment of cancer. In addition to promoting apoptosis, radiation-induced DNA damage can promote autophagy and senescence, both of which can theoretically function to prolong tumor survival. In this work, we tested the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiosensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair-proficient HCT116 colon carcinoma cells and a repair-deficient ligase IV(-/-) isogenic cell line. Exposure to radiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines, however, inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Exposure to radiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the ligase IV(-/-) cells, however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors, olaparib and niraparib, increased the extent of persistent DNA damage induced by radiation exposure as well as the extent of both autophagy and senescence. Neither cell line underwent significant apoptosis by radiation exposure alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiosensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence. PMID:26934368

  16. The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death.

    PubMed

    Lyu, Qing; Tou, Fangfang; Su, Hong; Wu, Xiaoyong; Chen, Xinyi; Zheng, Zhi

    2015-06-19

    Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary, our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. PMID:25935480

  17. Nuclear cathepsin L activity is required for cell cycle progression of colorectal carcinoma cells.

    PubMed

    Tamhane, Tripti; Lllukkumbura, Rukshala; Lu, Shiying; Maelandsmo, Gunhild M; Haugen, Mads H; Brix, Klaudia

    2016-03-01

    Prominent tasks of cysteine cathepsins involve endo-lysosomal proteolysis and turnover of extracellular matrix constituents or plasma membrane proteins for maintenance of intestinal homeostasis. Here we report on enhanced levels and altered subcellular localization of distinct cysteine cathepsins in adenocarcinoma tissue in comparison to adjacent normal colon. Immunofluorescence and immunoblotting investigations revealed the presence of cathepsin L in the nuclear compartment in addition to its expected endo-lysosomal localization in colorectal carcinoma cells. Cathepsin L was represented as the full-length protein in the nuclei of HCT116 cells from which stefin B, a potent cathepsin L inhibitor, was absent. Fluorescence activated cell sorting analyses with synchronized cell cultures revealed deceleration of cell cycle progression of HCT116 cells upon inhibition of cathepsin L activity, while expression of cathepsin L-enhanced green fluorescent protein chimeras accelerated S-phase entry. We conclude that the activity of cathepsin L is high in the nucleus of colorectal carcinoma cells because of lacking stefin B inhibitory activity. Furthermore, we hypothesize that nuclear cathepsin L accelerates cell cycle progression of HCT116 cells thereby supporting the notion that cysteine cathepsins may play significant roles in carcinogenesis due to deregulated trafficking. PMID:26343556

  18. Identification of a distinct population of CD133+CXCR4+ cancer stem cells in ovarian cancer

    PubMed Central

    Cioffi, Michele; D’Alterio, Crescenzo; Camerlingo, Rosalba; Tirino, Virginia; Consales, Claudia; Riccio, Anna; Ieranò, Caterina; Cecere, Sabrina Chiara; Losito, Nunzia Simona; Greggi, Stefano; Pignata, Sandro; Pirozzi, Giuseppe; Scala, Stefania

    2015-01-01

    CD133 and CXCR4 were evaluated in the NCI-60 cell lines to identify cancer stem cell rich populations. Screening revealed that, ovarian OVCAR-3, -4 and -5 and colon cancer HT-29, HCT-116 and SW620 over expressed both proteins. We aimed to isolate cells with stem cell features sorting the cells expressing CXCR4+CD133+ within ovarian cancer cell lines. The sorted population CD133+CXCR4+ demonstrated the highest efficiency in sphere formation in OVCAR-3, OVCAR-4 and OVCAR-5 cells. Moreover OCT4, SOX2, KLF4 and NANOG were highly expressed in CD133+CXCR4+ sorted OVCAR-5 cells. Most strikingly CXCR4+CD133+ sorted OVCAR-5 and -4 cells formed the highest number of tumors when inoculated in nude mice compared to CD133−CXCR4−, CD133+CXCR4−, CD133−CXCR4+ cells. CXCR4+CD133+ OVCAR-5 cells were resistant to cisplatin, overexpressed the ABCG2 surface drug transporter and migrated toward the CXCR4 ligand, CXCL12. Moreover, when human ovarian cancer cells were isolated from 37 primary ovarian cancer, an extremely variable level of CXCR4 and CD133 expression was detected. Thus, in human ovarian cancer cells CXCR4 and CD133 expression identified a discrete population with stem cell properties that regulated tumor development and chemo resistance. This cell population represents a potential therapeutic target. PMID:26020117

  19. Treatment with cucurbitacin B alone and in combination with gefitinib induces cell cycle inhibition and apoptosis via EGFR and JAK/STAT pathway in human colorectal cancer cell lines.

    PubMed

    Yar Saglam, A S; Alp, E; Elmazoglu, Z; Menevse, S

    2016-05-01

    The epidermal growth factor receptor (EGFR) associated with signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), plays an important role in colorectal cancers (CRCs). Gefitinib (Gef) is an orally active inhibitor targeting the adenosine tri phosphate-binding domain of EGFR, and cucurbitacin B (CuB) is a selective inhibitor of JAK/STAT signaling with potent antitumor activity via suppression of STAT3 phosphorylation, but the underlying mechanism is not clear. We aimed to investigate the apoptotic and antiproliferative effects of CuB as a single agent and in combination with Gef on both HT-29 and HCT-116 cell lines. Cell proliferation, cell cycle distribution, and apoptosis were evaluated using viability assay, fluorescent microscopy, cytotoxicity assay, proliferation, DNA fragmentation, and cleaved caspase 3 levels. Real-time polymerase chain reaction and Western blot analyses were performed to determine the expression of relevant genes and proteins including antiapoptotic, proapoptotic, and cell cycle regulation. EGFR, phosphorylated EGFR (pEGFR), STAT3, and pSTAT3 proteins were evalutaed with Western blot analysis. Our results showed that, compared to CuB alone, CuB plus Gef treatment caused a significant growth and cell cycle inhibition and induced apoptosis in both cell lines. Also CuB plus Gef treatment decreased DNA synthesis rate more effectively than CuB alone. Treatment with CuB alone and in combination with Gef decreased the expression levels of B-Cell CLL/Lymphoma 2 (Bcl-2), BCL2-like 1 (BCL2L1), cyclin D1, pSTAT3, and pEGFR and increased the expression levels of Bcl-2-like protein 4, Bcl-2 homologous antagonist/killer, Bcl-2-associated death promoter, Bcl-2-like protein 11, and p27kip1 levels. Our results suggest that treatment with CuB alone and more likely in combination with Gef may be a considerable alternative therapeutic approach for CRC, at least in vitro. PMID:26183715

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

    PubMed

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

    2014-11-01

    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

  1. Combinatorial inhibition of Plk1 and PKC? in cancer cells with different p53 status

    PubMed Central

    Grigat, Juline; Spnkuch, Birgit

    2014-01-01

    PKC? and Plk1 are fascinating targets in cancer therapy. Therefore, we combined Enzastaurin targeting PKC? and SBE13 targeting Plk1 to test synergistic effects in cells with different p53 status. We analyzed cell proliferation and apoptosis induction, and did Western blot and FACScan analyses to examine the combined PKC? and Plk1 inhibition. p53-wild-type cells are more resistant to the combinatorial treatment than p53-deficient cells, which displayed a synergistic reduction of cell proliferation after the combination. HeLa, MCF-7 and HCT116p53wt and HCT116p53-/- cells differed in their cell cycle distribution after combinatorial treatment in dependence on a functional p53-dependent G1/S checkpoint (p53-deficient cells showed an enrichment in S and G2/M, p53-wild-type cells in G0/G1 phase). hTERT-RPE1 cells did not show the synergistic effects of cancer cells. Thus, we demonstrate for the first time that Plk1 inhibition using SBE13 enhances the effects of Enzastaurin in cancer cells. HCT116p53wt and HCT116p53-/- cells confirmed the p53-dependence of different effects after Plk1 and PKC? inhibition observed in HeLa and MCF-7 cells. Obviously, p53 protects cells from the cytotoxicity of Enzastaurin in combination with SBE13. For that reason this combination can be useful to treat p53-deficient cancers, without displaying toxicity to normal cells, which all have functional p53. PMID:24810255

  2. Autophagonizer, a novel synthetic small molecule, induces autophagic cell death

    SciTech Connect

    Choi, In-Kwon; Cho, Yoon Sun; Jung, Hye Jin; Kwon, Ho Jeong

    2010-03-19

    Autophagy is an apoptosis-independent mechanism of cell death that protects the cell from environmental imbalances and infection by pathogens. We identified a novel small molecule, 2-(3-Benzyl-4-oxo-3,4,5,6,7,8-hexahydro-benzo[4,5]thieno[2,3-d] pyrimidin-2-ylsulfanylmethyl)-oxazole-4-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (referred as autophagonizer), using high-content cell-based screening and the autophagosome marker EGFP-LC3. Autophagonizer inhibited growth and induced cell death in the human tumor cell lines MCF7, HeLa, HCT116, A549, AGS, and HT1080 via a caspase-independent pathway. Conversion of cytosolic LC3-I to autophagosome-associated LC3-II was greatly enhanced by autophagonizer treatment. Transmission electron microscopy and acridine orange staining revealed increased autophagy in the cytoplasm of autophagonizer-treated cells. In conclusion, autophagonizer is a novel autophagy inducer with unique structure, which induces autophagic cell death in the human tumor cell lines.

  3. The CDX1-microRNA-215 axis regulates colorectal cancer stem cell differentiation.

    PubMed

    Jones, Matthew F; Hara, Toshifumi; Francis, Princy; Li, Xiao Ling; Bilke, Sven; Zhu, Yuelin; Pineda, Marbin; Subramanian, Murugan; Bodmer, Walter F; Lal, Ashish

    2015-03-31

    The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIP-PCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC. PMID:25775580

  4. The CDX1microRNA-215 axis regulates colorectal cancer stem cell differentiation

    PubMed Central

    Jones, Matthew F.; Hara, Toshifumi; Francis, Princy; Li, Xiao Ling; Bilke, Sven; Zhu, Yuelin; Pineda, Marbin; Subramanian, Murugan; Bodmer, Walter F.; Lal, Ashish

    2015-01-01

    The transcription factor caudal-type homeobox 1 (CDX1) is a key regulator of differentiation in the normal colon and in colorectal cancer (CRC). CDX1 activates the expression of enterocyte genes, but it is not clear how the concomitant silencing of stem cell genes is achieved. MicroRNAs (miRNAs) are important mediators of gene repression and have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in CRC, remain poorly understood. Here, we identified microRNA-215 (miR-215) as a direct transcriptional target of CDX1 by using high-throughput small RNA sequencing to profile miRNA expression in two pairs of CRC cell lines: CDX1-low HCT116 and HCT116 with stable CDX1 overexpression, and CDX1-high LS174T and LS174T with stable CDX1 knockdown. Validation of candidate miRNAs identified by RNA-seq in a larger cell-line panel revealed miR-215 to be most significantly correlated with CDX1 expression. Quantitative ChIPPCR and promoter luciferase assays confirmed that CDX1 directly activates miR-215 transcription. miR-215 expression is depleted in FACS-enriched cancer stem cells compared with unsorted samples. Overexpression of miR-215 in poorly differentiated cell lines causes a decrease in clonogenicity, whereas miR-215 knockdown increases clonogenicity and impairs differentiation in CDX1-high cell lines. We identified the genome-wide targets of miR-215 and found that miR-215 mediates the repression of cell cycle and stemness genes downstream of CDX1. In particular, the miR-215 target gene BMI1 has been shown to promote stemness and self-renewal and to vary inversely with CDX1. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in CRC. PMID:25775580

  5. Isostrychnopentamine, an indolomonoterpenic alkaloid from Strychnos usambarensis, with potential anti-tumor activity against apoptosis-resistant cancer cells.

    PubMed

    Saidou Balde, El-Hadj; Mégalizzi, Véronique; Cao, Martine; Angenot, Luc; Kiss, Robert; Van Damme, Marc; Frederich, Michel

    2010-04-01

    Isostrychnopentamine (ISP) is an indolomonoter-penic alkaloid that is present in the leaves of Strychnos usambarensis, an East African small tree. We have reported previously pro-apoptotic effects induced in vitro by ISP in the human HCT-116 colon cancer cell line, a model that displays relative sensitivity to apoptosis. In the present study, we observed that the in vitro growth inhibitory activities of ISP are similar in cancer cells that display sensitivity versus resistance to apoptosis. We made use of the U373 glioblastoma and the A549 non-small cell lung cancer (NSCLC) cell lines as models relatively resistant to apoptosis, and the human PC-3 prostate cancer cell line as a model relatively sensitive to apoptosis. While ISP induced transient decreases in [ATP]i and apoptosis in human U373 GBM cells, it did not provoke such features in A549 NSCLC cells. It thus seems that ISP-induced anti-cancer activity can lead to pro-apoptotic effects as a consequence, while apoptosis seems not to be the main cause by which ISP induces cancer cell death. ISP is a compound that merits further investigations in order to: i) identify the mechanism(s) of action by which it kills cancer cells, and ii) hemisynthesize novel ISP derivatives aiming to overcome, at least partly, the resistance of metastatic cancers to apoptosis. PMID:20198341

  6. Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron Tagging with Short Homology Donors.

    PubMed

    Natsume, Toyoaki; Kiyomitsu, Tomomi; Saga, Yumiko; Kanemaki, Masato T

    2016-04-01

    Studying the role of essential proteins is dependent upon a method for rapid inactivation, in order to study the immediate phenotypic consequences. Auxin-inducible degron (AID) technology allows rapid depletion of proteins in animal cells and fungi, but its application to human cells has been limited by the difficulties of tagging endogenous proteins. We have developed a simple and scalable CRISPR/Cas-based method to tag endogenous proteins in human HCT116 and mouse embryonic stem (ES) cells by using donor constructs that harbor synthetic short homology arms. Using a combination of AID tagging with CRISPR/Cas, we have generated conditional alleles of essential nuclear and cytoplasmic proteins in HCT116 cells, which can then be depleted very rapidly after the addition of auxin to the culture medium. This approach should greatly facilitate the functional analysis of essential proteins, particularly those of previously unknown function. PMID:27052166

  7. The Impact of ATRA on Shaping Human Myeloid Cell Responses to Epithelial Cell-Derived Stimuli and on T-Lymphocyte Polarization

    PubMed Central

    Gogolak, Péter; Blottière, Hervé M.; Rajnavölgyi, Éva

    2015-01-01

    Vitamin A plays an essential role in the maintenance of gut homeostasis but its interplay with chemokines has not been explored so far. Using an in vitro model system we studied the effects of human colonic epithelial cells (Caco2, HT-29, and HCT116) derived inflammatory stimuli on monocyte-derived dendritic cells and macrophages. Unstimulated Caco2 and HT-29 cells secreted CCL19, CCL21, and CCL22 chemokines, which could attract dendritic cells and macrophages and induced CCR7 receptor up-regulation by retinoic-acid resulting in dendritic cell migration. The chemokines Mk, CXCL16, and CXCL7 were secreted by all the 3 cell lines tested, and upon stimulation by IL-1β or TNF-α this effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1β. In the presence of ATRA the supernatants of these cells induced CD103 expression on monocyte-derived dendritic cells and when conditioned by ATRA and cocultured with CD4+ T-lymphocytes they reduced the proportion of Th17 T-cells. However, in the macrophage-T-cell cocultures the number of these effector T-cells was increased. Thus cytokine-activated colonic epithelial cells trigger the secretion of distinct combinations of chemokines depending on the proinflammatory stimulus and are controlled by retinoic acid, which also governs dendritic cell and macrophage responses. PMID:25944986

  8. Proteasome inhibitors MG-132 and bortezomib induce AKR1C1, AKR1C3, AKR1B1, and AKR1B10 in human colon cancer cell lines SW-480 and HT-29.

    PubMed

    Ebert, Bettina; Kisiela, Michael; Wsól, Vladimir; Maser, Edmund

    2011-05-30

    Aldo-keto reductases (AKRs) play central roles in the reductive metabolism of endogenous signaling molecules and in the detoxification of xenobiotics. AKRC1-1C3, AKR1B1 and AKR1B10 have been shown to be regulated via nuclear factor-erythroid 2 related factor 2 (Nrf2), a transcription factor that is activated upon oxidative stress. Proteasome inhibitors bortezomib and MG-132 produce mild oxidative stress that activates Nrf2-mediated gene expression that in turn may have cytoprotective effects. Bortezomib is clinically approved to treat haematological malignancies and it has also proven activity in solid tumors such as colon cancer. The present study investigated the effect of bortezomib and MG-132 on the expression of AKR1C1-1C4, AKR1B1, and AKR1B10 in colon cancer cell lines HT-29 and SW-480. Human cancer cell lines derived from different organs (lung, colon, pancreas, skin, liver, ovary) were initially assayed for the expression of the AKRs, showing a very unequal distribution. Even among the colon cell lines HT-29, Caco-2, HCT116 and SW-480, the AKRs were expressed quite non-uniformly. HT-29 cells expressed all AKRs on the mRNA level including liver-specific AKR1C4, but AKR1B1 was almost undetectable. In SW-480 cells, treatment with bortezomib (50 nM, 48 h) dramatically increased mRNA levels of AKR1B10 (32-fold), AKR1B1 (5.5-fold), and, to a lesser extent, AKR1C1 and AKR1C3. Drug-efflux transporter MRP2 (ABCC2) and Cox-2 were induced as well. AKR1C2 mRNA was down-regulated in SW-480 but induced in HT-29 cells. MG-132 increased mRNA amounts of AKR1C1, 1C3, 1B1, and 1B10 in a concentration-dependent manner. AKR1B10 and AKR1B1 protein expression was inducible by bortezomib in HT-29 cells, but not detectable in SW-480 cells. In conclusion, treatment with proteasome inhibitors increased the expression of several AKRs as well as of MRP2. It remains to be investigated whether this enzyme induction may contribute to enhanced cell survival and thereby supporting the phenomenon of multidrug resistance upon cancer chemotherapy. PMID:21215737

  9. Role of specific endocytic pathways in electrotransfection of cells

    PubMed Central

    Chang, Chun-Chi; Wu, Mina; Yuan, Fan

    2014-01-01

    Electrotransfection is a technique utilized for gene delivery in both preclinical and clinical studies. However, its mechanisms are not fully understood. The goal of this study was to investigate specific pathways of endocytosis involved in electrotransfection. In the study, three different human cell lines (HEK293, HCT116, and HT29) were either treated with ice cold medium postelectrotransfection or endocytic inhibitors prior to electrotransfection. The inhibitors were pharmacological agents (chlorpromazine, genistein, and amiloride) or different small interfering RNA (siRNA) molecules that could knockdown expression of clathrin heavy chain (CLTC), caveolin-1, and Rab34, respectively. The reduction in gene expressions was confirmed with western blot analysis at 48-72h post-siRNA treatment. It was observed that treatments with either ice cold medium, chlorpromazine, or genistein resulted in significant reductions in electrotransfection efficiency (eTE) in all three cell lines, compared to the matched controls, but amiloride treatment had insignificant effects on eTE. For cells treated with siRNA, only CLTC knockdown resulted in eTE reduction for all three cell lines. Together, these data demonstrated that the clathrin-mediated endocytosis played an important role in electrotransfection. PMID:26052524

  10. c-Met Targeting Enhances the Effect of Irradiation and Chemical Agents against Malignant Colon Cells Harboring a KRAS Mutation

    PubMed Central

    Han, Weihua; Zheng, Yongxiang; Xu, Huan; Zhang, Chuanling; He, Qiuchen; Zhang, Lihe; Li, Zhongxin; Zhou, Demin

    2014-01-01

    Although EGFR-targeted therapy has been beneficial to colorectal cancer patients, several studies have showed this clinical benefit was restricted to patients with wild-type KRAS exon 2 colorectal cancer. Therefore, it is crucial to explore efficient treatment strategies in patients with KRAS mutations. c-Met is an emerging target for the development of therapeutics against colorectal cancer. In this study, we first used the SW620 cell line, which has an activating KRAS mutation, to generate a stable cell line with conditional regulation of c-Met, which is an essential gene for growth and an oncogene. Using this approach, we evaluated the benefits of combined c-Met-targeted therapy with irradiation or chemical agents. In this cell line, we observed that the proliferation and migration of SW620 cells were reduced by the induction of c-Met shRNA. Furthermore, c-Met knockdown enhanced the anti-proliferative effects of 5-FU and Taxol but not cisplatin, irinotecan or sorafenib. These enhancements were also observed in another colon cancer cells line HCT-116, which also has a KRAS mutation. The response of SW620 cells to irradiation was also enhanced by c-Met knockdown. This method and obtained data might have important implications for exploring the combinatory effects of targeted therapies with conventional medications. Moreover, the data suggested that the combination of c-Met-targeted therapy with chemotherapy or irradiation might be an effective strategy against colorectal cancer harboring a KRAS mutation. PMID:25427200

  11. Metformin and soybean-derived bioactive molecules attenuate the expansion of stem cell-like epithelial subpopulation and confer apoptotic sensitivity in human colon cancer cells.

    PubMed

    Montales, Maria Theresa E; Simmen, Rosalia C M; Ferreira, Ederlan S; Neves, Valdir A; Simmen, Frank A

    2015-11-01

    Colorectal cancer (CRC) is a disease whose genesis may include metabolic dysregulation. Cancer stem cells are attractive targets for therapeutic interventions since their aberrant expansion may underlie tumor initiation, progression, and recurrence. To investigate the actions of metabolic regulators on cancer stem cell-like cells (CSC) in CRC, we determined the effects of soybean-derived bioactive molecules and the anti-diabetes drug metformin (MET), alone and together, on the growth, survival, and frequency of CSC in human HCT116 cells. Effects of MET (60 μM) and soybean components genistein (Gen, 2 μM), lunasin (Lun, 2 μM), β-conglycinin (β-con, 3 μM), and glycinin (Gly, 3 μM) on HCT116 cell proliferation, apoptosis, and mRNA/protein expression and on the frequency of the CSC CD133(+)CD44(+) subpopulation by colonosphere assay and fluorescence-activated cell sorting/flow cytometry were evaluated. MET, Gen, and Lun, individually and together, inhibited HCT116 viability and colonosphere formation and, conversely, enhanced HCT116 apoptosis. Reductions in frequency of the CSC CD133(+)CD44(+) subpopulation with MET, Gen, and Lun were found to be associated with increased PTEN and reduced FASN expression. In cells under a hyperinsulinemic state mimicking metabolic dysregulation and without and with added PTEN-specific inhibitor SF1670, colonosphere formation and frequency of the CD133(+)CD44(+) subpopulation were decreased by MET, Lun and Gen, alone and when combined. Moreover, MET + Lun + Gen co-treatment increased the pro-apoptotic and CD133(+)CD44(+)-inhibitory efficacy of 5-fluorouracil under hyperinsulinemic conditions. Results identify molecular networks shared by MET and bioavailable soy food components, which potentially may be harnessed to increase drug efficacy in diabetic and non-diabetic patients with CRC. PMID:26506839

  12. Prominin-1 (CD133, AC133) and dipeptidyl-peptidase IV (CD26) are indicators of infinitive growth in colon cancer cells

    PubMed Central

    Grunt, Thomas W; Hebar, Alexandra; Laffer, Sylvia; Wagner, Renate; Peter, Barbara; Herrmann, Harald; Graf, Alexandra; Bilban, Martin; Posch, Martin; Hoermann, Gregor; Mayerhofer, Matthias; Eisenwort, Gregor; Zielinski, Christoph C; Selzer, Edgar; Valent, Peter

    2015-01-01

    Advanced colorectal cancer is characterized by uncontrolled growth and resistance against anti-cancer agents, including ErbB inhibitors. Recent data suggest that cancer stem cells (CSC) are particularly resistant. These cells may reside within a CD133+ fraction of the malignant cells. Using HCT116 cells we explored the role of CD133 and other CSC markers in drug resistance in colon cancer cells. CD133+ cells outnumbered CD133- cells over time in long-term culture. Both populations displayed the KRAS mutation 38G > A and an almost identical target profile, including EGFR/ErbB1, ErbB2, and ErbB4. Microarray analyses and flow cytometry identified CD26 as additional CSC marker co-expressed on CD133+ cells. However, knock-down of CD133 or CD26 did not affect short-term growth of HCT116 cells, and both cell-populations were equally resistant to various targeted drugs except irreversible ErbB inhibitors, which blocked growth and ERK1/2 phosphorylation in CD133- cells more efficiently than in CD133+ cells. Moreover, the MEK inhibitor AS703026 was found to overcome resistance against ErbB blockers in CD133+ cells. Together, CD133 and CD26 are markers of long-term growth and resistance to ErbB blockers in HCT116 cells, which may be mediated by constitutive ERK activity. PMID:25973297

  13. Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo

    PubMed Central

    Min, Soo Jin; Lim, Ji Ye; Kim, Haeng Ran; Kim, Se-Jae; Kim, Yuri

    2015-01-01

    A rare subpopulation of cancer cells, termed cancer stem cells (CSCs), may be responsible for tumor relapse and resistance to conventional chemotherapy. The development of a non-toxic, natural treatment for the elimination of CSCs is considered a strategy for cancer treatment with minimal side effects. In the present study, the potential for Sasa quelpaertensis leaf extract (SQE) and its two bioactive compounds, tricin and p-coumaric acid, to exert anti-CSC effects by suppressing cancer stemness characteristics were evaluated in colon cancer cells. CD133+CD44+ cells were isolated from HT29 and HCT116 cell lines using flow-activated cell sorting (FACs). SQE treatment was found to significantly suppress the self-renewal capacity of both cell lines. SQE treatment was also associated with the down-regulation of β-catenin and phosphorylated GSK3β, while significantly enhancing cell differentiation by up-regulating CK20 expression and blocking the expression of several stem cell markers, including DLK1, Notch1, and Sox-2. In vivo, SQE supplementation suppressed tumor growth in a xenograft model by down-regulating stem cell markers and β-catenin as well as HIF-1α signaling. Compared with two bioactive compounds of SQE, SQE exhibited the most effective anti-CSC properties. Taken together, these results provide evidence that SQE inhibits colon cancer by regulating the characteristics of CSCs. PMID:25941936

  14. Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo.

    PubMed

    Min, Soo Jin; Lim, Ji Ye; Kim, Haeng Ran; Kim, Se-Jae; Kim, Yuri

    2015-01-01

    A rare subpopulation of cancer cells, termed cancer stem cells (CSCs), may be responsible for tumor relapse and resistance to conventional chemotherapy. The development of a non-toxic, natural treatment for the elimination of CSCs is considered a strategy for cancer treatment with minimal side effects. In the present study, the potential for Sasa quelpaertensis leaf extract (SQE) and its two bioactive compounds, tricin and p-coumaric acid, to exert anti-CSC effects by suppressing cancer stemness characteristics were evaluated in colon cancer cells. CD133+CD44+ cells were isolated from HT29 and HCT116 cell lines using flow-activated cell sorting (FACs). SQE treatment was found to significantly suppress the self-renewal capacity of both cell lines. SQE treatment was also associated with the down-regulation of ?-catenin and phosphorylated GSK3?, while significantly enhancing cell differentiation by up-regulating CK20 expression and blocking the expression of several stem cell markers, including DLK1, Notch1, and Sox-2. In vivo, SQE supplementation suppressed tumor growth in a xenograft model by down-regulating stem cell markers and ?-catenin as well as HIF-1? signaling. Compared with two bioactive compounds of SQE, SQE exhibited the most effective anti-CSC properties. Taken together, these results provide evidence that SQE inhibits colon cancer by regulating the characteristics of CSCs. PMID:25941936

  15. Cytotoxicity of 5-fluorouracil-loaded pH-sensitive liposomal nanoparticles in colorectal cancer cell lines

    PubMed Central

    Udofot, Ofonime; Affram, Kevin; Israel, Bridg'ette; Agyare, Edward

    2015-01-01

    5-Fluorouracil (5-FU) is widely used in cancer therapy, either alone or in combination with other anti-cancer drugs. However, poor membrane permeability and a short half-life (5-20 min) due to rapid metabolism in the body necessitate the continuous administration of high doses of 5-FU to maintain the minimum therapeutic serum concentration. This is associated with significant side effects and a possibility of severe toxic effects. This study aimed to formulate 5-FU-loaded pH-sensitive liposomal nanoparticles (pHLNps-5-FU) and evaluate 5-FU release characteristics and anti-cancer effect of pHLNps-5-FU. Particle size and zeta potential were determined using a particle size analyzer. The release patterns of pHLNps-5-FU formulations were evaluated at 37°C at pH 3, 5, 6.5, and 7.4, while drug release kinetics of 5-FU from a pHLNp3–5-FU formulation were determined at pH 3 and 7.4 at different time points (37°C). Cell viability and clonogenic studies were conducted to evaluate the effectiveness of pHLNps-5-FU against HCT-116 and HT-29 cell lines while cellular uptake of rhodamine-labeled pHLNps-5-FU was determined by flow cytometry and confocal imaging. The average sizes of the pHLNp1–5-FU, pHLNp2–5-FU and pHLNp3–5-FU liposomes were 200nm ± 9.8nm, 181.9 nm ± 9.1 nm, and 164.3 nm ± 8.4 nm respectively. In vitro drug release of 5-FU from different pHLNps-5-FU formulations was the highest at pH 3.8. Both cell lines treated with pHLNps-5-FU exhibited reduced viability, two- or three-fold lower than that of 5-FU-treated cells. Flow cytometry and confocal imaging confirmed high uptake of rhodamine-labeled pHLNps-5-FU in both cell lines. The drug release profile of the chosen pHLNp3-5-FU formulation was optimal at pH 3 and had the poorest release profile at pH 7.4. The release profile of pHLNp3-5-FU showed that 5-FU release was two-fold higher at pH 3 than that at pH 7.4. This study demonstrates that pHLNp3-5-FU may be a potential candidate for the treatment of colorectal cancer. PMID:26691592

  16. Momordica charantia Extract Induces Apoptosis in Human Cancer Cells through Caspase- and Mitochondria-Dependent Pathways

    PubMed Central

    Li, Chia-Jung; Tsang, Shih-Fang; Tsai, Chun-Hao; Tsai, Hsin-Yi; Chyuan, Jong-Ho; Hsu, Hsue-Yin

    2012-01-01

    Plants are an invaluable source of potential new anti-cancer drugs. Momordica charantia is one of these plants with both edible and medical value and reported to exhibit anticancer activity. To explore the potential effectiveness of Momordica charantia, methanol extract of Momordica charantia (MCME) was used to evaluate the cytotoxic activity on four human cancer cell lines, Hone-1 nasopharyngeal carcinoma cells, AGS gastric adenocarcinoma cells, HCT-116 colorectal carcinoma cells, and CL1-0 lung adenocarcinoma cells, in this study. MCME showed cytotoxic activity towards all cancer cells tested, with the approximate IC50 ranging from 0.25 to 0.35 mg/mL at 24 h. MCME induced cell death was found to be time-dependent in these cells. Apoptosis was demonstrated by DAPI staining and DNA fragmentation analysis using agarose gel electrophoresis. MCME activated caspase-3 and enhanced the cleavage of downstream DFF45 and PARP, subsequently leading to DNA fragmentation and nuclear condensation. The apoptogenic protein, Bax, was increased, whereas Bcl-2 was decreased after treating for 24 h in all cancer cells, indicating the involvement of mitochondrial pathway in MCME-induced cell death. These findings indicate that MCME has cytotoxic effects on human cancer cells and exhibits promising anti-cancer activity by triggering apoptosis through the regulation of caspases and mitochondria. PMID:23091557

  17. An efficient method to produce clonal colonies of cancer cells using laser enabled analysis and processing (LEAP)

    NASA Astrophysics Data System (ADS)

    Zordan, Michael; Fatig, Ray; Reece, Lisa; Davisson, V. Jo; Leary, James

    2008-02-01

    Many in vitro studies require a pure clonal population of cells that derive from a single cell. Traditionally this task has been performed using the inefficient manual process of ultimate limiting dilution. We have developed a novel clonal dilution technique using the Laser Enabled Analysis and Processing (LEAP TM) instrument (Cyntellect Inc. San Diego, CA). The LEAP instrument performs automated fluorescence imaging and real time image analysis to identify and measure fluorescence and morphological parameters of cells. The LEAP instrument also features a laser that can be used to manipulate targeted cells. To perform clonal dilution, cells are seeded at a low density (~10 cells/well) into each well of a 384 well plate and viably stained. The LEAP instrument will then image each well and automatically target all of the cells that are present. Then one cell will be chosen to keep (at random or based on a variety of metrics) and the others will be eliminated by laser ablation. We have successfully used this technique to produce single cell clones of HCT116 cells, a heterogeneous colorectal cancer model, in 84 percent of wells (originally containing 5 +/- 2.1 cells/well). This is a marked improvement over the traditional technique of ultimate limiting dilution which produces a clone in only 33 percent of wells. The ability to efficiently produce clonal colonies has great utility in the isolation of subpopulations of cancer cells and purification of transformed cell lines.

  18. UDP-Glucuronosyltransferase 1A Determinates Intracellular Accumulation and Anti-Cancer Effect of β-Lapachone in Human Colon Cancer Cells

    PubMed Central

    Liu, Huiying; Li, Qingran; Cheng, Xuefang; Wang, Hong; Wang, Guangji; Hao, Haiping

    2015-01-01

    β-lapachone (β-lap), an NAD(P)H:quinone oxidoreductase 1 (NQO1) targeting antitumor drug candidate in phase II clinical trials, is metabolically eliminated via NQO1 mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation. This study intends to explore the inner link between the cellular glucuronidation and pharmacokinetics of β-lap and its apoptotic effect in human colon cancer cells. HT29 cells S9 fractions exhibited high glucuronidation activity towards β-lap, which can be inhibited by UGT1A9 competitive inhibitor propofol. UGT1A siRNA treated HT29 cells S9 fractions displayed an apparent low glucuronidation activity. Intracellular accumulation of β-lap in HCT116 cells was much higher than that in HT29 cells, correlated with the absence of UGT1A in HCT116 cells. The cytotoxic and apoptotic effect of β-lap in HT29 cells were much lower than that in HCT116 cells; moreover, β-lap triggered activation of SIRT1-FOXO1 apoptotic pathway was observed in HCT116 cells but not in HT29 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol significantly decreased β-lap’s cytotoxic and apoptotic effects, due to the repression of glucuronidation and the resultant intracellular accumulation. In conclusion, UGT1A is an important determinant, via switching NQO1-triggered redox cycle to metabolic elimination, in the intracellular accumulation of β-lap and thereafter its cytotoxicity in human colon cancer cells. Together with our previous works, we propose that UGTs determined cellular pharmacokinetics is an important determinant in the apoptotic effects of NQO1 targeting substrates serving as chemotherapeutic drugs. PMID:25692465

  19. Expression of Nucleophosmin/NPM1 correlates with migration and invasiveness of colon cancer cells

    PubMed Central

    2012-01-01

    Background We aimed to examine the expression level of Nucleophosmin (NPM1) protein in colon cancer tissues and to investigate the potential role of NPM1 in the regulation of cell migration and invasiveness. Methods Immunohistochemical assay was performed to examine the expression pattern of NPM1 in 31 groups of colonic carcinoma samples, including colon tumors, adjacent normal tissues, and matched metastatic lymph nodes from the same patients. Small interfering RNA technique and exogenous expression of wild type NPM1 methods were used to further verify the function of NPM1. Results High-expression of NPM1 correlates with lymph node metastasis (P = 0.0003) and poor survival rate of human colon cancer patients (P = 0.017). SiRNA-mediated reduction of NPM1 was also shown to inhibit the migration and invasiveness of metastatic colon cancer HCT116 cell line. In addition, the exogenous expression of NPM1 in HT29 cells, a NPM1 low expression and low invasive colon cancer cell line, enhanced cell migration and invasiveness along with increased cell proliferation. Conclusions The current study uncovered the critical role of NPM1 in the regulation of colon cancer cells migration and invasion, and NPM1 may serve as a potential marker for the prognosis of colon cancer patients. PMID:22631075

  20. Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

    PubMed

    Wu, Ya C; Wang, Xiao J; Yu, Le; Chan, Francis K L; Cheng, Alfred S L; Yu, Jun; Sung, Joseph J Y; Wu, William K K; Cho, Chi H

    2012-01-01

    Hydrogen sulfide (H(2)S) is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2)S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC) and a panel of colon cancer cell lines (HT-29, SW1116, HCT116) were exposed to H(2)S at concentrations similar to those found in the human colon. H(2)S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2)S was accompanied by G(1)-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip). Moreover, exposure to H(2)S led to features characteristic of autophagy, including increased formation of LC3B(+) autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2)S. Further mechanistic investigation revealed that H(2)S stimulated the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR) and S6 kinase. Inhibition of AMPK significantly reversed H(2)S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2)S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway. PMID:22679478

  1. Resveratrol-induced transcriptional up-regulation of ASMase (SMPD1) of human leukemia and cancer cells.

    PubMed

    Mizutani, Naoki; Omori, Yukari; Kawamoto, Yoshiyuki; Sobue, Sayaka; Ichihara, Masatoshi; Suzuki, Motoshi; Kyogashima, Mamoru; Nakamura, Mitsuhiro; Tamiya-Koizumi, Keiko; Nozawa, Yoshinori; Murate, Takashi

    2016-02-19

    Resveratrol (RSV) is a plant-derived phytoalexin present in plants, whose pleiotropic effects for health benefits have been previously reported. Its anti-cancer activity is among the current topics for novel cancer treatment. Here, effects of RSV on cell proliferation and the sphingolipid metabolism of K562, a human leukemia cell line, were analyzed. Some experiments were also performed in HCT116, a human colon cancer cell line. RSV inhibited cell proliferation of both cell lines. Increased cellular ceramide and decreased sphingomyelin and S1P by RSV were observed in RSV-treated K562 cells. Further analysis revealed that acid sphingomyelinase mRNA and enzyme activity levels were increased by RSV. Desipramine, a functional ASMase inhibitor, prevented RSV-induced ceramide increase. RSV increased ATF3, EGR1, EGR3 proteins and phosphorylated c-Jun and FOXO3. However, co-transfection using these transcription factor expression vectors and ASMase promoter reporter vector revealed positive effects of EGR1 and EGR3 but not others. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay demonstrated the direct binding of EGR1/3 transcription factors with ASMase 5'-promoter. These results indicate that increased EGR1/3 and ASMase expression play an important role in cellular ceramide increase by RSV treatment. PMID:26809095

  2. Thyroid cell lines in research on goitrogenesis.

    PubMed

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

    1991-12-01

    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

  3. Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo

    PubMed Central

    Lai, Chin-Yu; Tsai, An-Chi; Chen, Mei-Chuan; Chang, Li-Hsun; Sun, Hui-Lung; Chang, Ya-Ling; Chen, Chien-Chih

    2012-01-01

    Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (−/−) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity. PMID:22912688

  4. Selective resistance of tetraploid cancer cells against DNA damage-induced apoptosis.

    PubMed

    Castedo, Maria; Coquelle, Arnaud; Vitale, Ilio; Vivet, Sonia; Mouhamad, Shahul; Viaud, Sophie; Zitvogel, Laurence; Kroemer, Guido

    2006-12-01

    Aneuploidy and chromosomal instability, which are frequent in cancer, can result from the asymmetric division of tetraploid precursors. Genomic instability may favor the generation of more aggressive tumor cells with a reduced propensity for undergoing apoptosis. To assess the impact of tetraploidization on apoptosis regulation, we generated a series of stable tetraploid HCT116 and RKO colon carcinoma cell lines. When comparing diploid parental cells with tetraploid clones, we found that such cells were equally sensitive to a series of cytotoxic agents (staurosporine [STS], hydroxyurea, etoposide), as well as to the lysis by natural killer cells. In strict contrast, tetraploid cells were found to be relatively resistant against a series of DNA-damaging agents, namely cisplatin, oxaliplatin, camptothecin, and gamma- and UVC-irradiation. This increased resistance correlated with a reduced manifestation of apoptotic parameters (such as the dissipation of the mitochondrial transmembrane potential and the degradation of nuclear DNA) in tetraploid as compared to diploid cells subjected to DNA damage. Moreover, tetraploid cells manifested an enhanced baseline level of p53 activation. Inhibition of p53 abolished the difference in the susceptibility of diploid and tetraploid cancer cells to DNA damage-induced apoptosis. These data point to an intrinsic resistance of tetraploid cells against radiotherapy and DNA-targeted chemotherapy that may be linked to the status of the p53 system. PMID:17384245

  5. Stool-fermented Plantago ovata husk induces apoptosis in colorectal cancer cells independently of molecular phenotype.

    PubMed

    Sohn, Vanessa R; Giros, Anna; Xicola, Rosa M; Fluvià, Lourdes; Grzybowski, Mike; Anguera, Anna; Llor, Xavier

    2012-06-01

    Several studies have suggested that the partially fermentable fibre Plantago ovata husk (PO) may have a protective effect on colorectal cancer (CRC). We studied the potentially pro-apoptotic effect of PO and the implicated mechanisms in CRC cells with different molecular phenotypes (Caco-2, HCT116, LoVo, HT-29, SW480) after PO anaerobic fermentation with colonic bacteria as it occurs in the human colon. The fermentation products of PO induced apoptosis in all primary tumour and metastatic cell lines, independent of p53, adenomatous polyposis coli, β-catenin or cyclo-oxygenase-2 status. Apoptosis was caspase-dependent and both intrinsic and extrinsic pathways were implicated. The intrinsic pathway was activated through a shift in the balance towards a pro-apoptotic environment with an up-regulation of B-cell lymphoma protein 2 homologous antagonist killer (BAK) and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) seen in HCT116 and LoVo cells. This resulted in mitochondrial membrane depolarisation, increased expression of caspase activators second mitochondria-derived activator of caspases (Smac)/Diablo, death effector apoptosis-inducing factor, apoptosome member apoptotic protease activating factor 1 and down-regulation of inhibitors of apoptosis Survivin and X-linked inhibitor of apoptosis in most cells. The extrinsic pathway was activated presumably through the up-regulation of death receptor (DR5). Some important differences were seen between primary tumour and metastatic CRC cells. Thus, metastatic PO-treated LoVo cells had a remarkable up-regulation of TNF-α ligand along with death-inducing signalling complex components receptor interacting protein and TNF-α receptor 1-associated death domain protein. The extrinsic pathway modulator FCICE-inhibitory protein (FLIP), an inhibitor of both spontaneous death ligand-independent and death receptor-mediated apoptosis, was significantly down-regulated after PO treatment in all primary tumour cells, but not in metastatic LoVo. These findings suggest that PO could potentially be a useful chemotherapy adjuvant. PMID:22018732

  6. Carvacrol inhibits proliferation and induces apoptosis in human colon cancer cells.

    PubMed

    Fan, Kai; Li, Xiaolei; Cao, Yonggang; Qi, Hanping; Li, Lei; Zhang, Qianhui; Sun, Hongli

    2015-09-01

    Colon cancer is one of the most common malignancies worldwide and has a high mortality rate. Carvacrol is a major component of oregano and thyme essential oils and shows antitumor properties. Here, we investigated the effects of carvacrol on the proliferation and apoptosis of two human colon cancer cell lines, HCT116 and LoVo, and studied the molecular mechanisms of its antitumor properties. We found that carvacrol inhibited the proliferation and migration of the two colon cancer cell lines in a concentration-dependent manner. Cell invasion was suppressed after carvacrol treatment by decreasing the expression of matrix metalloprotease-2 (MMP-2) and MMP-9. Carvacrol treatment also caused cell cycle arrest in the G2/M phase and decreased cyclin B1 expression. Finally, carvacrol induced cell apoptosis in a dose-dependent manner. At the molecular level, carvacrol downregulated the expression of Bcl-2 and induced the phosphorylation of the extracellular-regulated protein kinase and protein kinase B (p-Akt). In parallel, carvacrol upregulated the expression of Bax and c-Jun N-terminal kinase. These results indicate that carvacrol might induce apoptosis in colon cancer cells through the mitochondrial apoptotic pathway and the MAPK and PI3K/Akt signaling pathways. Together, our results suggest that carvacrol may have therapeutic potential for the prevention and treatment of colon cancer. PMID:26214321

  7. Molecular mechanisms for inhibition of colon cancer cells by combined epigenetic-modulating epigallocatechin gallate and sodium butyrate

    SciTech Connect

    Saldanha, Sabita N.; Kala, Rishabh; Tollefsbol, Trygve O.

    2014-05-15

    Bioactive compounds are considered safe and have been shown to alter genetic and epigenetic profiles of tumor cells. However, many of these changes have been reported at molecular concentrations higher than physiologically achievable levels. We investigated the role of the combinatorial effects of epigallocatechin gallate (EGCG), a predominant polyphenol in green tea, and sodium butyrate (NaB), a dietary microbial fermentation product of fiber, in the regulation of survivin, which is an overexpressed anti-apoptotic protein in colon cancer cells. For the first time, our study showed that the combination treatment induced apoptosis and cell cycle arrest in RKO, HCT-116 and HT-29 colorectal cancer cells. This was found to be regulated by the decrease in HDAC1, DNMT1, survivin and HDAC activity in all three cell lines. A G2/M arrest was observed for RKO and HCT-116 cells, and G1 arrest for HT-29 colorectal cancer cells for combinatorial treatment. Further experimentation of the molecular mechanisms in RKO colorectal cancer (CRC) cells revealed a p53-dependent induction of p21 and an increase in nuclear factor kappa B (NF-κB)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (γ-H2AX) protein levels and induction of histone H3 hyperacetylation was also observed with the combination treatment. Further, we observed a decrease in global CpG methylation. Taken together, these findings suggest that at low and physiologically achievable concentrations, combinatorial EGCG and NaB are effective in promoting apoptosis, inducing cell cycle arrest and DNA-damage in CRC cells. - Highlights: • EGCG and NaB as a combination inhibits colorectal cancer cell proliferation. • The combination treatment induces DNA damage, G2/M and G1 arrest and apoptosis. • Survivin is effectively down-regulated by the combination treatment. • p21 and p53 expressions are induced by the combination treatment. • Epigenetic proteins DNMT1 and HDAC1 are effectively down-regulated by the treatment.

  8. The silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway

    PubMed Central

    Zhang, Ling; Zhu, Shengtao; Shi, Xuesen; Sha, Weihong

    2015-01-01

    Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66Shc protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66Shc in the progress of colon cancer still unknown. In this study, we found that p66Shc highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66Shc in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66Shc siRNA. Furthermore, after HCT8 cells treated with p66Shc siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell. PMID:26464652

  9. Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice

    NASA Astrophysics Data System (ADS)

    Hoffman, Robert M.

    2006-02-01

    We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

  10. The synergistic apoptotic interaction of panaxadiol and epigallocatechin gallate in human colorectal cancer cells.

    PubMed

    Du, Guang-Jian; Wang, Chong-Zhi; Qi, Lian-Wen; Zhang, Zhi-Yu; Calway, Tyler; He, Tong-Chuan; Du, Wei; Yuan, Chun-Su

    2013-02-01

    Panaxadiol (PD) is a purified sapogenin of ginseng saponins, which exhibits anticancer activity. Epigallocatechin gallate (EGCG), a major catechin in green tea, is a strong botanical antioxidant. In this study, we investigated the possible synergistic anticancer effects of PD and EGCG on human colorectal cancer cells and explored the potential role of apoptosis in the synergistic activities. Effects of selected compounds on HCT-116 and SW-480 human colorectal cancer cells were evaluated by a modified trichrome stain cell proliferation analysis. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or annexin V/PI. Cell growth was suppressed after treatment with PD (10 and 20?m) for 48?h. When PD (10 and 20?m) was combined with EGCG (10, 20, and 30?m), significantly enhanced antiproliferative effects were observed in both cell lines. Combining 20?m of PD with 20 and 30?m of EGCG significantly decreased S-phase fractions of cells. In the apoptotic assay, the combination of PD and EGCG significantly increased the percentage of apoptotic cells compared with PD alone (p?cells. PMID:22566066

  11. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    PubMed

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin. PMID:26596838

  12. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    PubMed Central

    Tamhane, Tripti; Wolters, Brit K.; Illukkumbura, Rukshala; Maelandsmo, Gunhild M.; Haugen, Mads H.; Brix, Klaudia

    2015-01-01

    The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015) [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP) in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed. PMID:26594658

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

    SciTech Connect

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

    2008-05-23

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

  14. Downregulation of CDC27 inhibits the proliferation of colorectal cancer cells via the accumulation of p21Cip1/Waf1

    PubMed Central

    Qiu, L; Wu, J; Pan, C; Tan, X; Lin, J; Liu, R; Chen, S; Geng, R; Huang, W

    2016-01-01

    Dysregulated cell cycle progression has a critical role in tumorigenesis. Cell division cycle 27 (CDC27) is a core subunit of the anaphase-promoting complex/cyclosome, although the specific role of CDC27 in cancer remains unknown. In our study, we explored the biological and clinical significance of CDC27 in colorectal cancer (CRC) growth and progression and investigated the underlying molecular mechanisms. Results showed that CDC27 expression is significantly correlated with tumor progression and poor patient survival. Functional assays demonstrated that overexpression of CDC27 promoted proliferation in DLD1 cells, whereas knockdown of CDC27 in HCT116 cells inhibited proliferation both in vitro and in vivo. Further mechanistic investigation showed that CDC27 downregulation resulted in G1/S phase transition arrest via the significant accumulation of p21 in HCT116 cells, and the upregulation of CDC27 promoted G1/S phase transition via the attenuation of p21 in DLD1 cells. Furthermore, we also demonstrated that CDC27 regulated inhibitor of DNA binding 1 (ID1) protein expression in DLD1 and HCT116 cells, and rescue assays revealed that CDC27 regulated p21 expression through modulating ID1 expression. Taken together, our results indicate that CDC27 contributes to CRC cell proliferation via the modulation of ID1-mediated p21 regulation, which offers a novel approach to the inhibition of tumor growth. Indeed, these findings provide new perspectives for the future study of CDC27 as a target for CRC treatment. PMID:26821069

  15. 15-LOX-1 suppression of hypoxia-induced metastatic phenotype and HIF-1α expression in human colon cancer cells

    PubMed Central

    Wu, Yuanqing; Mao, Fei; Zuo, Xiangsheng; Moussalli, Micheline J; Elias, Elias; Xu, Weiguo; Shureiqi, Imad

    2014-01-01

    The expression of 15-lipoxygenase-1 (15-LOX-1) is downregulated in colon cancer and other major cancers, and 15-LOX-1 reexpression in cancer cells suppresses colonic tumorigenesis. Various lines of evidence indicate that 15-LOX-1 expression suppresses premetastatic stages of colonic tumorigenesis; nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia, a common feature of the cancer microenvironment, promotes prometastatic mechanisms such as the upregulation of hypoxia-inducible factor (HIF)-1α, a transcriptional master regulator that enhances cancer cell metastatic potential, angiogenesis, and tumor cell invasion and migration. We have, therefore, tested whether restoring 15-LOX-1 in colon cancer cells affects cancer cells' hypoxia response that promotes metastasis. We found that 15-LOX-1 reexpression in HCT116, HT29LMM, and LoVo colon cancer cells inhibited survival, vascular endothelial growth factor (VEGF) expression, angiogenesis, cancer cell migration and invasion, and HIF-1α protein expression and stability under hypoxia. These findings demonstrate that 15-LOX-1 expression loss in cancer cells promotes metastasis and that therapeutically targeting ubiquitous 15-LOX-1 loss in cancer cells has the potential to suppress metastasis. PMID:24634093

  16. Enhancement of CD3AK cell proliferation and killing ability by α-Thujone.

    PubMed

    Zhou, Yu; Liu, Jun-quan; Zhou, Zhong-hai; Lv, Xiao-ting; Chen, Yong-qiang; Sun, Lei-qing; Chen, Fu-xing

    2016-01-01

    Thujone is a monoterpene ketone natural substance found mainly in wormwood and sage. Previous studies have shown that Thujone has various pharmacological effects, such as anti-tumor, analgesic, and insecticide. The effect of α-Thujone to human immune cells is still unknown. Our study focuses on investigating the effects and mechanism of α-Thujone to CD3AK (anti- CD3 antibody induced activated killer) cells proliferation and cytotoxicity to colon cancer cell lines. With cell proliferation and FCM assay, it is found that α-Thujone could significantly enhance CD3AK cell proliferation and expression of CD107a in a dose-dependent manner. The cytotoxicity to colon cancer cells detected by CCK-8 assay is also improved. The expressions of TNF-α and FasL detected with ELISA assay were not significantly changed. Mechanically, the study shows that α-Thujone could enhance the expression of p-ERK1/2 and p-Akt. In addition, α-Thujone has no cytotoxicity to HCT116 and SW620 cells proliferation. In a word, α-Thujone enhances CD3AK cell proliferation and cytotoxicity via the improvement of expression of CD107a, p-Akt and p-ERK1/2. PMID:26655741

  17. The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production

    SciTech Connect

    Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie

    2015-08-07

    In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our in vitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. In vivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation in vitro and in vivo, and ceramide production might be the key mechanism responsible for its actions. - Highlights: • AR-42 is anti-proliferative against primary/established colon cancer cells. • AR-42 induces significant apoptotic death in primary/established colon cancer cells. • Ceramide production mediates AR-42-induced cytotoxicity in colon cancer cells. • AR-42 oral administration potently inhibits SW-620 xenograft growth in SCID mice.

  18. Synthesis and biological evaluation of novel 2,3-dihydrochromeno[3,4-d]imidazol-4(1H)-one derivatives as potent anticancer cell proliferation and migration agents.

    PubMed

    Han, Xuan; Luo, Jiang; Wu, Feng; Hou, XueYan; Yan, Guoyi; Zhou, Meng; Zhang, Mengqi; Pu, Chunlan; Li, Rui

    2016-05-23

    In this study, a series of novel molecules containing chromeno [3,4-d] imidazol-4(1H)-one was synthesized and their biological activities were evaluated. Among them, compound 35 showed a dramatic anticancer activity against HCT116 and MCF-7, and the flow cytometry assays demonstrated that it could arrest G0/G1 cell-cycle and induce apoptosis of SW620 cells in a dose-dependent manner. Besides, it also blocked MCF-7 cancer cell migration. Moreover, it inhibited tumor growth in HCT116 subcutaneously implanted xenografted mice. Taken together, compound 35 may be a promising candidate for anti-cancer agent as well as metastatic one. PMID:26994691

  19. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity.

    PubMed

    Tolba, Mai F; Abdel-Rahman, Sherif Z

    2015-01-01

    Although colorectal cancer (CRC) treatment with 5-fluorouracil (5-FU) is the first line of therapy for this debilitating disease, treatment effectiveness is often hampered by the development of drug resistance and toxicity at high doses. ER-β can play an important role in CRC development and possibly in its response to therapy. Pterostilbene (PT) possesses antioxidant and anticancer effects that are mediated by ER-β. In the current study, we test the hypothesis that PT sensitizes colon cancer cells to 5-FU and we examine the underlying mechanism(s) by which PT exerts its cytotoxic effects in CRC cells. Our data indicate that PT exhibited a more potent cytotoxic effect in Caco-2 compared to HCT-116 cells. PT/5-FU co-treatment was more effective in Caco-2 cells. Our data indicate that ER-β is expressed at higher levels in Caco-2 cells and its levels are further boosted with PT treatment. PT significantly suppressed Akt and ERK phosphorylations, and enhanced FOXO-1 and p27(kip1) levels in Caco-2 cells. PT also induced a significant increase in Caco-2 cells at pre-G phase coupled with increased Bax/Bcl-2 ratio and PARP cleavage. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing ER-β protein. PMID:26472352

  20. Pterostilbine, an active component of blueberries, sensitizes colon cancer cells to 5-fluorouracil cytotoxicity

    PubMed Central

    Tolba, Mai F.; Abdel-Rahman, Sherif Z.

    2015-01-01

    Although colorectal cancer (CRC) treatment with 5-fluorouracil (5-FU) is the first line of therapy for this debilitating disease, treatment effectiveness is often hampered by the development of drug resistance and toxicity at high doses. ER-β can play an important role in CRC development and possibly in its response to therapy. Pterostilbene (PT) possesses antioxidant and anticancer effects that are mediated by ER-β. In the current study, we test the hypothesis that PT sensitizes colon cancer cells to 5-FU and we examine the underlying mechanism(s) by which PT exerts its cytotoxic effects in CRC cells. Our data indicate that PT exhibited a more potent cytotoxic effect in Caco-2 compared to HCT-116 cells. PT/5-FU co-treatment was more effective in Caco-2 cells. Our data indicate that ER-β is expressed at higher levels in Caco-2 cells and its levels are further boosted with PT treatment. PT significantly suppressed Akt and ERK phosphorylations, and enhanced FOXO-1 and p27kip1 levels in Caco-2 cells. PT also induced a significant increase in Caco-2 cells at pre-G phase coupled with increased Bax/Bcl-2 ratio and PARP cleavage. These results provide a rationale for novel combination treatment strategies, especially for patients with 5-FU-resistant tumors expressing ER-β protein. PMID:26472352

  1. Synthesis of Tolmetin Hydrazide-Hydrazones and Discovery of a Potent Apoptosis Inducer in Colon Cancer Cells.

    PubMed

    Küçükgüzel, Ş Güniz; Koç, Derya; Çıkla-Süzgün, Pelin; Özsavcı, Derya; Bingöl-Özakpınar, Özlem; Mega-Tiber, Pınar; Orun, Oya; Erzincan, Pınar; Sağ-Erdem, Safiye; Şahin, Fikrettin

    2015-10-01

    Tolmetin hydrazide and a novel series of tolmetin hydrazide-hydrazones 4a-l were synthesized in this study. The structures of the new compounds were determined by spectral (FT-IR, (1)H NMR) methods. N'-[(2,6-Dichlorophenyl)methylidene]-2-[1-methyl-5-(4-methylbenzoyl)-1H-pyrrol-2-yl]acetohydrazide (4g) was evaluated in vitro using the MTT colorimetric method against the colon cancer cell lines HCT-116 (ATCC, CCL-247) and HT-29 (ATCC, HTB-38) to determine growth inhibition and cell viability at different doses. Compound 4g exhibited anti-cancer activity with an IC50 value of 76 μM against colon cancer line HT-29 (ATCC, HTB-38) and did not display cytotoxicity toward control NIH3T3 mouse embryonic fibroblast cells compared to tolmetin. In addition, this compound was evaluated for caspase-3, caspase-8, caspase-9, and annexin-V activation in the apoptotic pathway, which plays a key role in the treatment of cancer. We demonstrated that the anti-cancer activity of this compound was due to the activation of caspase-8 and caspase-9 involved in the apoptotic pathway. In addition, in this study, we investigated the catalytical effect of COX on the HT-29 cancer line, the apoptotic mechanism, and the moleculer binding of tolmetin and compound 4g on the COX enzyme active site. PMID:26287512

  2. JNK confers 5-fluorouracil resistance in p53-deficient and mutant p53-expressing colon cancer cells by inducing survival autophagy

    PubMed Central

    Sui, Xinbing; Kong, Na; Wang, Xian; Fang, Yong; Hu, Xiaotong; Xu, Yinghua; Chen, Wei; Wang, Kaifeng; Li, Da; Jin, Wei; Lou, Fang; Zheng, Yu; Hu, Hong; Gong, Liu; Zhou, Xiaoyun; Pan, Hongming; Han, Weidong

    2014-01-01

    Deficiency or mutation in the p53 tumor suppressor gene commonly occurs in human cancer and can contribute to disease progression and chemotherapy resistance. Currently, although the pro-survival or pro-death effect of autophagy remains a controversial issue, increasing data seem to support the idea that autophagy facilitates cancer cell resistance to chemotherapy treatment. Here we report that 5-FU treatment causes aberrant autophagosome accumulation in HCT116 p53−/− and HT-29 cancer cells. Specific inhibition of autophagy by 3-MA, CQ or small interfering RNA treatment targeting Atg5 or Beclin 1 can potentiate the re-sensitization of these resistant cancer cells to 5-FU. In further analysis, we show that JNK activation and phosphorylation of Bcl-2 are key determinants in 5-FU-induced autophagy. Inhibition of JNK by the compound SP600125 or JNK siRNA suppressed autophagy and phosphorylation of c-Jun and Bcl-2 but increased 5-FU-induced apoptosis in both HCT116 p53−/− and HT29 cells. Taken together, our results suggest that JNK activation confers 5-FU resistance in HCT116 p53−/− and HT29 cells by promoting autophagy as a pro-survival effect, likely via inducing Bcl-2 phosphorylation. These results provide a promising strategy to improve the efficacy of 5-FU-based chemotherapy for colorectal cancer patients harboring a p53 gene mutation. PMID:24733045

  3. Identifying clinically relevant drug resistance genes in drug-induced resistant cancer cell lines and post- chemotherapy tissues

    PubMed Central

    Tong, Mengsha; Zheng, Weicheng; Lu, Xingrong; Ao, Lu; Li, Xiangyu; Guan, Qingzhou; Cai, Hao; Li, Mengyao; Yan, Haidan; Guo, You; Chi, Pan; Guo, Zheng

    2015-01-01

    Until recently, few molecular signatures of drug resistance identified in drug-induced resistant cancer cell models can be translated into clinical practice. Here, we defined differentially expressed genes (DEGs) between pre-chemotherapy colorectal cancer (CRC) tissue samples of non-responders and responders for 5-fluorouracil and oxaliplatin-based therapy as clinically relevant drug resistance genes (CRG5-FU/L-OHP). Taking CRG5-FU/L-OHP as reference, we evaluated the clinical relevance of several types of genes derived from HCT116 CRC cells with resistance to 5-fluorouracil and oxaliplatin, respectively. The results revealed that DEGs between parental and resistant cells, when both were treated with the corresponding drug for a certain time, were significantly consistent with the CRG5-FU/L-OHP as well as the DEGs between the post-chemotherapy CRC specimens of responders and non-responders. This study suggests a novel strategy to extract clinically relevant drug resistance genes from both drug-induced resistant cell models and post-chemotherapy cancer tissue specimens. PMID:26515599

  4. Patulin induces colorectal cancer cells apoptosis through EGR-1 dependent ATF3 up-regulation

    PubMed Central

    Kwon, Osong; Soung, Nak Kyun; Thimmegowda, N.R.; Jeong, Sook Jung; Jang, Jae Hyuk; Moon, Dong-Oh; Chung, Jong Kyeong; Lee, Kyung Sang; Kwon, Yong Tae; Erikson, Raymond Leo; Ahn, Jong Seog; Kim, Bo Yeon

    2013-01-01

    Patulin is a fungal mycotoxin of Aspergilus and Penicillium that is commonly found in rotting fruits and exerts its potential toxic effect mainly by reactive oxygen species (ROS) generation. However, the effect of patulin on cancer cells as well as its intracellular mechanism has been controversial and not clearly defined yet. In this study, patulin was found to induce G1/S accumulation and cell growth arrest accompanied by caspase-3 activation, PARP cleavage and ATF3 expression in human colon cancer cell line HCT116. Ser/Thr phosphorylation of a transcription factor, EGR-1, was increased while its expression did not change upon patulin treatment to the cells. Knockdown of ATF3 and EGR-1 using their respective siRNAs showed EGR-1 dependent ATF3 expression. Moreover, treatment of the cells with antioxidants N-acetylcysteine (NAC) and glutathione (GSH) revealed that patulin induced ATF3 expression and apoptosis were dependent on ROS generation. ATF3 expression was also increased by patulin in other colorectal cancer cell types, Caco2 and SW620. Collectively, our data present a new anti-cancer molecular mechanism of patulin, suggesting EGR-1 and ATF3 as critical targets for the development of anti-cancer chemotherapeutics. In this regard, patulin could be a candidate for the treatment of colorectal cancers. PMID:22230687

  5. Patulin induces colorectal cancer cells apoptosis through EGR-1 dependent ATF3 up-regulation.

    PubMed

    Kwon, Osong; Soung, Nak Kyun; Thimmegowda, N R; Jeong, Sook Jung; Jang, Jae Hyuk; Moon, Dong-Oh; Chung, Jong Kyeong; Lee, Kyung Sang; Kwon, Yong Tae; Erikson, Raymond Leo; Ahn, Jong Seog; Kim, Bo Yeon

    2012-04-01

    Patulin is a fungal mycotoxin of Aspergilus and Penicillium that is commonly found in rotting fruits and exerts its potential toxic effect mainly by reactive oxygen species (ROS) generation. However, the effect of patulin on cancer cells as well as its intracellular mechanism has been controversial and not clearly defined yet. In this study, patulin was found to induce G1/S accumulation and cell growth arrest accompanied by caspase-3 activation, PARP cleavage and ATF3 expression in human colon cancer cell line HCT116. Ser/Thr phosphorylation of a transcription factor, EGR-1, was increased while its expression did not change upon patulin treatment to the cells. Knockdown of ATF3 and EGR-1 using their respective siRNAs showed EGR-1 dependent ATF3 expression. Moreover, treatment of the cells with antioxidants N-acetylcysteine (NAC) and glutathione (GSH) revealed that patulin induced ATF3 expression and apoptosis were dependent on ROS generation. ATF3 expression was also increased by patulin in other colorectal cancer cell types, Caco2 and SW620. Collectively, our data present a new anti-cancer molecular mechanism of patulin, suggesting EGR-1 and ATF3 as critical targets for the development of anti-cancer chemotherapeutics. In this regard, patulin could be a candidate for the treatment of colorectal cancers. PMID:22230687

  6. Tussilagone suppresses colon cancer cell proliferation by promoting the degradation of β-catenin

    SciTech Connect

    Li, Hua; Lee, Hwa Jin; Ahn, Yeon Hwa; Kwon, Hye Jin; Jang, Chang-Young; Kim, Woo-Young; Ryu, Jae-Ha

    2014-01-03

    Highlights: •Tussilagone (TSL) was purified from plant as an inhibitor of Wnt/β-catenin pathway. •TSL suppressed the β-catenin/T-cell factor transcriptional activity. •The proteasomal degradation of β-catenin was induced by TSL. •TSL suppressed the Wnt/β-catenin target genes, cyclin D1 and c-myc. •TSL inhibit the proliferation of colon cancer cells. -- Abstract: Abnormal activation of the Wnt/β-catenin signaling pathway frequently induces colon cancer progression. In the present study, we identified tussilagone (TSL), a compound isolated from the flower buds of Tussilago farfara, as an inhibitor on β-catenin dependent Wnt pathway. TSL suppressed β-catenin/T-cell factor transcriptional activity and down-regulated β-catenin level both in cytoplasm and nuclei of HEK293 reporter cells when they were stimulated by Wnt3a or activated by an inhibitor of glycogen synthase kinase-3β. Since the mRNA level was not changed by TSL, proteasomal degradation might be responsible for the decreased level of β-catenin. In SW480 and HCT116 colon cancer cell lines, TSL suppressed the β-catenin activity and also decreased the expression of cyclin D1 and c-myc, representative target genes of the Wnt/β-catenin signaling pathway, and consequently inhibited the proliferation of colon cancer cells. Taken together, TSL might be a potential chemotherapeutic agent for the prevention and treatment of human colon cancer.

  7. The Chinese Herb Isolate Yuanhuacine (YHL-14) Induces G2/M Arrest in Human Cancer Cells by Up-regulating p21 Protein Expression through an p53 Protein-independent Cascade*

    PubMed Central

    Zhang, Ruowen; Wang, Yulei; Li, Jingxia; Jin, Honglei; Song, Shaojiang; Huang, Chuanshu

    2014-01-01

    Yuanhuacine (YHL-14), the major component of daphnane diterpene ester isolated from the flower buds of Daphne genkwa, has been reported to have activity against cell proliferation in various cancer cell lines. Nevertheless, the potential mechanism has not been explored yet. Here we demonstrate that YHL-14 inhibits bladder and colon cancer cell growth through up-regulation of p21 expression in an Sp1-dependent manner. We found that YHL-14 treatment resulted in up-regulation of p21 expression and a significant G2/M phase arrest in T24T and HCT116 cells without affecting p53 protein expression and activation. Further studies indicate that p21 induction by YHL-14 occurs at the transcriptional level via up-regulation of Sp1 protein expression. Moreover, our results show that p38 is essential for YHL-14-mediated Sp1 protein stabilization, G2/M growth arrest induction, and anchorage-independent growth inhibition of cancer cells. Taken together, our studies demonstrate a novel mechanism of YHL-14 against cancer cell growth in bladder and colon cancer cell lines, which provides valuable information for the design and synthesis of other new conformation-constrained derivatives on the basis of the structure of YHL-14 for cancer therapy. PMID:24451377

  8. Short-chain ceramides depress integrin cell surface expression and function in colorectal cancer cells.

    PubMed

    Morad, Samy A F; Bridges, Lance C; Almeida Larrea, Alex D; Mayen, Anthony L; MacDougall, Matthew R; Davis, Traci S; Kester, Mark; Cabot, Myles C

    2016-07-01

    Colorectal cancer (CRC) is highly metastatic, significantly so to liver, a characteristic that embodies one of the most challenging aspects of treatment. The integrin family of cell-cell and cell-matrix adhesion receptors plays a central role in migration and invasion, functions that underlie metastatic potential. In the present work we sought to determine the impact of ceramide, which plays a key modulatory role in cancer suppression, on integrin cell surface expression and function in CRC cells in order to reveal possible ceramide-centric effects on tumor cell motility. Human CRC cells LoVo, HT-29, and HCT-116 were employed, which represent lines established from primary and metastatic sites. A cell-permeable, short-chain analog, C6-ceramide, was used as ceramide mimic. Exposure of cells to C6-ceramide (24 h) promoted a dose-dependent (2.5-10 µM) decrease in the expression of cell surface β1 and β4 integrin subunits in all cell lines; at 10 µM C6-ceramide, the decreases ranged from 30 to 50% of the control. Expression of cell surface αVβ6 integrin, which is associated with advanced invasion in CRC, was also suppressed by C6-ceramide. Decreases in integrin expression translated to diminished cellular adhesion, 50% of the control at 5 µM C6-ceramide, and markedly reduced cellular migration, approximately 30-40% of the control in all cell lines. Physicochemical examination revealed potent efficacy of nano-formulated C6-ceramide, but inferior activity of dihydro-C6-ceramide and L-C6-ceramide, compared to the unsaturated counterpart and the natural d-enantiomer, respectively. These studies demonstrate novel actions of ceramides that may have application in suppression of tumor metastasis, in addition to their known tumor suppressor effects. PMID:27045476

  9. Generation of a human antibody that inhibits TSPAN8-mediated invasion of metastatic colorectal cancer cells.

    PubMed

    Kim, Taek-Keun; Park, Chang Sik; Jeoung, Mee Hyun; Lee, Woo Ran; Go, Nam Kyung; Choi, Jong Rip; Lee, Tae Sup; Shim, Hyunbo; Lee, Sukmook

    2015-12-25

    Tetraspanin 8 (TSPAN8) is a tumor-associated antigen implicated in tumor progression and metastasis. However, the validation of TSPAN8 as a potential therapeutic target in metastatic colorectal cancer (mCRC) has not yet been studied. In this study, through several in vitro methodologies, we identified a large extracellular loop of TSPAN8 (TSPAN8-LEL) as a key domain for regulating mCRC invasion. Using phage display technology, we developed a novel anti-TSPAN8-LEL human antibody with subnanomolar affinity that specifically recognizes amino acids 140-205 of TSPAN8-LEL in a conformation-dependent manner. Finally, we demonstrated that the antibody specifically reduces invasion in the HCT116 and LoVo mCRC cell lines more potently than in the HCT-8 and SW480 non-mCRC cell lines. Our data suggest that TSPAN8-LEL may play an important role in mCRC cell invasion, and that the antibody we have developed could be a useful tool for inhibiting the invasion of TSPAN8-expressing mCRCs. PMID:26562525

  10. Synergistic inhibitory effect of cetuximab and tectochrysin on human colon cancer cell growth via inhibition of EGFR signal.

    PubMed

    Park, Mi Hee; Hong, Ji Eun; Hwang, Chul Ju; Choi, Mingi; Choi, Jeong Soon; An, Young Jin; Son, Dong Ju; Hong, Jin Tae

    2016-05-01

    The purpose of this study was to evaluate the enhancing potency of tectochrysin, a flavonoid isolated from Alpinia oxyphylla Miquel by combining cetuximab, an anti-EGFR monoclonal antibody, on human colon cancer cell growth through further inhibition of EGFR pathway. HCT116 and SW480 colon cancer cells were treated with cetuximab (30 μg/mL, 1/10 of IC50), tectochrysin (5 μg/mL, 1/3 of IC50), or the combination of both agents. The growth inhibitory effect was examined using the MTT assay while apoptotic cell death was performed using TUNEL staining assays. The DNA binding activity of NF-kappa B and AP-1 was investigated by electrophoretic mobility shift assay. Protein expression was determined by Western blot. Cell proliferation was significantly inhibited by the combination of cetuximab and tectochrysin than treatment with cetuximab or tectochrysin alone (combination index: 0.572 and 0.533, respectively). Combination treatment of cells with cetuximab and tectochrysin significantly reduced the expressions of p-EGFR and COX-2 in both cell lines. Combination treatment also significantly inhibited activities of NF-kB and AP-1 compared to the single agent treatment. Our results indicate that combined therapy with lower concentration of cetuximab and tectochrysin could significantly enhance the cancer cell growth inhibitory effect through the inhibition of EGFR signaling. PMID:27025376

  11. 5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway.

    PubMed

    Das, Dipon; Preet, Ranjan; Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Siddharth, Sumit; Tamir, Tigist; Jain, Vaibhav; Bharatam, Prasad V; Wyatt, Michael D; Kundu, Chanakya Nath

    2014-12-01

    Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant. PMID:25460919

  12. 5-Fluorouracil mediated anti-cancer activity in colon cancer cells is through the induction of Adenomatous Polyposis Coli: Implication of the long-patch base excision repair pathway

    PubMed Central

    Das, Dipon; Preet, Ranjan; Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Siddharth, Sumit; Tamir, Tigist; Jain, Vaibhav; Bharatam, Prasad V.; Wyatt, Michael D.; Kundu, Chanakya Nath

    2016-01-01

    Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC’s DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain’s interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant. PMID:25460919

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

    PubMed

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

    2014-12-01

    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

  14. Derricin and Derricidin Inhibit Wnt/β-Catenin Signaling and Suppress Colon Cancer Cell Growth In Vitro

    PubMed Central

    Fonseca, Barbara F.; Predes, Danilo; Cerqueira, Debora M.; Reis, Alice H.; Amado, Nathalia G.; Cayres, Marina C. L.; Kuster, Ricardo M.; Oliveira, Felipe L.; Mendes, Fabio A.; Abreu, Jose G.

    2015-01-01

    Overactivation of the Wnt/β-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/β-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/β-catenin pathway. Both chalcones are able to affect the cell distribution of β-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/β-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/β-catenin pathway and colon cancer cell growth in vitro. PMID:25775405

  15. The novel HDAC inhibitor AR-42-induced anti-colon cancer cell activity is associated with ceramide production.

    PubMed

    Xu, Weihong; Xu, Bin; Yao, Yiting; Yu, Xiaoling; Shen, Jie

    2015-08-01

    In the current study, we investigated the potential activity of AR-42, a novel histone deacetylase (HDAC) inhibitor, against colon cancer cells. Our invitro results showed that AR-42 induced ceramide production, exerted potent anti-proliferative and pro-apoptotic activities in established (SW-620 and HCT-116 lines) and primary human colon cancer cells. Exogenously-added sphingosine 1-phosphate (S1P) suppressed AR-42-induced activity, yet a cell-permeable ceramide (C4) facilitated AR-42-induced cytotoxicity against colon cancer cells. In addition, AR-42-induced ceramide production and anti-colon cancer cell activity were inhibited by the ceramide synthase inhibitor fumonisin B1, but were exacerbated by PDMP, which is a ceramide glucosylation inhibitor. Invivo, oral administration of a single dose of AR-42 dramatically inhibited SW-620 xenograft growth in severe combined immunodeficient (SCID) mice, without inducing overt toxicities. Together, these results show that AR-42 dramatically inhibits colon cancer cell proliferation invitro and invivo, and ceramide production might be the key mechanism responsible for its actions. PMID:26026677

  16. Anti-proliferative effect of horehound leaf and wild cherry bark extracts on human colorectal cancer cells.

    PubMed

    Yamaguchi, Kiyoshi; Liggett, Jason L; Kim, Nam-Cheol; Baek, Seung Joon

    2006-01-01

    Marubium vulgare (horehound) and Prunus serotina (wild cherry) have been traditionally used for the treatment of inflammatory-related symptoms such as cold, fever, and sore throat. In this report, we show that extracts of anti-inflammatory horehound leaves and wild cherry bark exhibit anti-proliferative activity in human colorectal cancer cells. Both horehound and wild cherry extracts cause suppression of cell growth as well as induction of apoptosis. We found that horehound extract up-regulates pro-apoptotic non-steroidal anti-inflammatory drug-activated gene (NAG-1) through transactivation of the NAG-1 promoter. In contrast, wild cherry extract decreased cyclin D1 expression and increased NAG-1 expression in HCT-116 and SW480 cell lines. Treatment with wild cherry extract resulted in the suppression of beta-catenin/T cell factor transcription, as assessed by TOP/FOP reporter constructs, suggesting that suppressed beta-catenin signaling by wild cherry extract leads to the reduction of cyclin D1 expression. Our data suggest the mechanisms by which these extracts suppress cell growth and induce apoptosis involve enhanced NAG-1 expression and/or down-regulation of beta-catenin signaling, followed by reduced cyclin D1 expression in human colorectal cancer cells. These findings may provide mechanisms for traditional anti-inflammatory products as cancer chemopreventive agents. PMID:16328068

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

    PubMed Central

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

    2014-01-01

    NPRL-Z-1 is a 4β-[(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 TOP2α or TOP2β 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

  18. NPRL-Z-1, as a new topoisomerase II poison, induces cell apoptosis and ROS generation in human renal carcinoma cells.

    PubMed

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

    2014-01-01

    NPRL-Z-1 is a 4β-[(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 TOP2α or TOP2β 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

  19. Development of a Potent, Specific CDK8 Kinase Inhibitor Which Phenocopies CDK8/19 Knockout Cells.

    PubMed

    Koehler, Michael F T; Bergeron, Philippe; Blackwood, Elizabeth M; Bowman, Krista; Clark, Kevin R; Firestein, Ron; Kiefer, James R; Maskos, Klaus; McCleland, Mark L; Orren, Linda; Salphati, Laurent; Schmidt, Steve; Schneider, Elisabeth V; Wu, Jiansheng; Beresini, Maureen H

    2016-03-10

    Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a series of 6-aza-benzothiophene containing compounds were developed into potent, selective CDK8 inhibitors. When cocrystallized with CDK8 and cyclin C, these compounds exhibit an unusual binding mode, making a single hydrogen bond to the hinge residue A100, a second to K252, and a key cation-π interaction with R356. Structure-based drug design resulted in tool compounds 13 and 32, which are highly potent, kinase selective, permeable compounds with a free fraction >2% and no measurable efflux. Despite these attractive properties, these compounds exhibit weak antiproliferative activity in the HCT-116 colon cancer cell line. Further examination of the activity of 32 in this cell line revealed that the compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner identical to a CDK8 knockout clone, illustrating the complex effects of inhibition of CDK8 kinase activity in proliferation in these cells. PMID:26985305

  20. The different radiation response and radiation-induced bystander effects in colorectal carcinoma cells differing in p53 status.

    PubMed

    Widel, Maria; Lalik, Anna; Krzywon, Aleksandra; Poleszczuk, Jan; Fujarewicz, Krzysztof; Rzeszowska-Wolny, Joanna

    2015-08-01

    Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system. The viability of exposed to X-rays (0-8 Gy) and bystander cells of both lines showed a roughly comparable decline with increasing dose. The frequency of micronuclei was also comparable at lower doses but at higher increased considerably, especially in bystander TP53-/- cells. Moreover, the TP53-/- cells showed a significantly elevated frequency of apoptosis, while TP53+/+ counterparts expressed high level of senescence. The cross-matched experiments where irradiated cells of one line were co-cultured with non-irradiated cells of opposite line show that both cell lines were also able to induce bystander effects in their counterparts, however different endpoints revealed with different strength. Potential mediators of bystander effects, IL-6 and IL-8, were also generated differently in both lines. The knockout cells secreted IL-6 at lower doses whereas wild type cells only at higher doses. Secretion of IL-8 by TP53-/- control cells was many times lower than that by TP53+/+ but increased significantly after irradiation. Transcription of the NFκBIA was induced in irradiated TP53+/+ mainly, but in bystanders a higher level was observed in TP53-/- cells, suggesting that TP53 is required for induction of NFκB pathway after irradiation but another mechanism of activation must operate in bystander cells. PMID:26099456

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

    PubMed

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

    2005-11-16

    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

  2. Selenite-induced autophagy antagonizes apoptosis in colorectal cancer cells in vitro and in vivo.

    PubMed

    Yang, Yang; Luo, Hui; Hui, Kaiyuan; Ci, Yali; Shi, Kejian; Chen, Ge; Shi, Lei; Xu, Caimin

    2016-03-01

    In the present study, we aimed to investigate the relationship between autophagy and apoptosis in selenite?treated colorectal cancer (CRC) cells. The effects of selenite on HCT116 and SW480 cell apoptosis were investigated with an AnnexinV/propidium iodide (PI) double staining kit by flow cytometry. The punctate of LC3 protein following treatment with selenite was observed by a laser scanning confocal microscope and by transmission electron microscopy. Using western blot assays, we detected the apoptotic and autophagic markers in both CRC cells and mouse xenograft tumor models. We found that sodium selenite induced autophagy in the two CRC cell lines. Consistent with the invitro results, we observed that the expression of autophagy marker LC3 was increased. Finally, we discovered that modulation of reactive oxygen species by MnTMPyP inhibited autophagy, while H2O2 activated autophagy. These results help to elucidate the anticancer effect of selenium, providing further evidence to exploit novel anticancer drugs targeting selenium. PMID:26676801

  3. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

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

    2013-01-01

    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

  4. RASSF10 suppresses colorectal cancer growth by activating P53 signaling and sensitizes colorectal cancer cell to docetaxel

    PubMed Central

    Guo, Jing; Yang, Yage; Yang, Yunsheng; Linghu, Enqiang; Zhan, Qimin; Brock, Malcolm V.; Herman, James G.; Zhang, Bingyong; Guo, Mingzhou

    2015-01-01

    RASSF10 has previously been reported to be frequently methylated in a number of malignancies. To understand the importance of RASSF10 inactivation in colorectal carcinogenesis, eight colorectal cancer cell lines, 89 cases of primary colorectal cancer and 5 cases of normal colorectal mucosa were examined. Methylation specific PCR, western blot, siRNA, gene expression array and xenograft mice were employed. The expression of RASSF10 was regulated by promoter regional methylation in colorectal cancer cells. RASSF10 was methylated in 60.7% (54/89) of primary colorectal cancers and was positively associated with tumor stage (p < 0.05) and metastasis (p < 0.05). Restoration of RASSF10 led to inhibition of colorectal cancer cell proliferation in vitro and in vivo and increased apoptosis. Gene expression arrays discovered RASSF10 inhibition of MDM2 expression as a mediator of these effects, which was confirmed with RT-PCR and western blot. RASSF10 was shown to activate P53 signaling in RKO and HCT116 cells after UV exposure, and sensitized these cells to docetaxel. In conclusion, our study demonstrates RASSF10 is frequently methylated in human colorectal cancer leading to loss of expression. RASSF10 normally suppresses human colorectal cancer growth by activating P53 signaling in colorectal cancer, and restored expression sensitizes colorectal cancer to docetaxel. PMID:25638156

  5. Cytotoxicity of the Sesquiterpene Lactones Neoambrosin and Damsin from Ambrosia maritima Against Multidrug-Resistant Cancer Cells

    PubMed Central

    Saeed, Mohamed; Jacob, Stefan; Sandjo, Louis P.; Sugimoto, Yoshikazu; Khalid, Hassan E.; Opatz, Till; Thines, Eckhard; Efferth, Thomas

    2015-01-01

    Multidrug resistance is a prevailing phenomenon leading to chemotherapy treatment failure in cancer patients. In the current study two known cytotoxic pseudoguaianolide sesquiterpene lactones; neoambrosin (1) and damsin (2) that circumvent MDR were identified. The two cytotoxic compounds were isolated using column chromatography, characterized using 1D and 2D NMR, MS, and compared with literature values. The isolated compounds were investigated for their cytotoxic potential using resazurin assays and thereafter confirmed with immunoblotting and in silico studies. MDR cells overexpressing ABC transporters (P-glycoprotein, BCRP, ABCB5) did not confer cross-resistance toward (1) and (2), indicating that these compounds are not appropriate substrates for any of the three ABC transporters analyzed. Resistance mechanisms investigated also included; the loss of the functions of the TP53 and the mutated EGFR. The HCT116 p53-/- cells were sensitive to 1 but resistant to 2. It was interesting to note that resistant cells transfected with oncogenic ΔEGFR exhibited hypersensitivity CS toward (1) and (2) (degrees of resistances were 0.18 and 0.15 for (1) and (2), respectively). Immunoblotting and in silico analyses revealed that 1 and 2 silenced c-Src kinase activity. It was hypothesized that inhibition of c-Src kinase activity may explain CS in EGFR-transfected cells. In conclusion, the significant cytotoxicity of 1 and 2 against different drug-resistant tumor cell lines indicate that they may be promising candidates to treat refractory tumors. PMID:26617519

  6. Effects of omega-3 and omega-6 fatty acids on IGF-I receptor signalling in colorectal cancer cells.

    PubMed

    Seti, Hila; Leikin-Frenkel, Alicia; Werner, Haim

    2009-07-01

    The insulin-like growth factor (IGF) system plays a critical role in normal growth and development as well as in malignant states. Most of the biological activities of the IGFs are mediated by the IGF-IR, which is over-expressed in most tumours and cancer cell lines. Fatty acids have critical roles in both systemic physiological processes (e.g. metabolism) and cellular events (e.g. proliferation, apoptosis, signal transduction, and gene expression). Alpha-linolenic acid (ALA) and linoleic acid (LA) are essential fatty acids of the omega-3 and omega-6 families, respectively. The aim of this study was to investigate the potential interactions between fatty acids and the IGF signal transduction pathways, and to evaluate the impact of this interplay on colon cancer cells survival and proliferation. Results of Western blot analyses revealed that ALA and LA enhanced the ligand-induced IGF-IR phosphorylation and, in addition, increased receptor phosphorylation in an IGF-I independent manner. Furthermore, fatty acid treatment led to phosphorylation of downstream signalling molecules, including Akt and Erk. In addition, FACS analysis and apoptosis measurements indicated that ALA and LA have a potential mitogenic effect on HCT116 cells, as reflected by the number of cells in S phase and by a reduction of PARP cleavage, implying a reduction in apoptotic activity. In summary, our results provide evidence that omega-3 and omega-6 fatty acids modulate IGF-I action in colon cancer cells. PMID:19480565

  7. Depletion of the thiol oxidoreductase ERp57 in tumor cells inhibits proliferation and increases sensitivity to ionizing radiation and chemotherapeutics.

    PubMed

    Hussmann, Melanie; Janke, Kirsten; Kranz, Philip; Neumann, Fabian; Mersch, Evgenija; Baumann, Melanie; Goepelt, Kirsten; Brockmeier, Ulf; Metzen, Eric

    2015-11-17

    Rapidly growing tumor cells must synthesize proteins at a high rate and therefore depend on an efficient folding and quality control system for nascent secretory proteins in the endoplasmic reticulum (ER). The ER resident thiol oxidoreductase ERp57 plays an important role in disulfide bond formation. Lentiviral, doxycycline-inducible ERp57 knockdown was combined with irradiation and treatment with chemotherapeutic agents. The knockdown of ERp57 significantly enhanced the apoptotic response to anticancer treatment in HCT116 colon cancer cells via a p53-dependent mechanism. Instead of a direct interaction with p53, depletion of ERp57 induced cell death via a selective activation of the PERK branch of the Unfolded Protein Response (UPR). In contrast, apoptosis was reduced in MDA-MB-231 breast cancer cells harboring mutant p53. Nevertheless, we observed a strong reduction of proliferation in response to ERp57 knockdown in both cell lines regardless of the p53 status. Depletion of ERp57 reduced the phosphorylation activity of the mTOR-complex1 (mTORC1) as demonstrated by reduction of p70S6K phosphorylation. Our data demonstrate that ERp57 is a promising target for anticancer therapy due to synergistic p53-dependent induction of apoptosis and p53-independent inhibition of proliferation. PMID:26513173

  8. Stevia rebaudiana ethanolic extract exerts better antioxidant properties and antiproliferative effects in tumour cells than its diterpene glycoside stevioside.

    PubMed

    López, Víctor; Pérez, Sergio; Vinuesa, Arturo; Zorzetto, Christian; Abian, Olga

    2016-04-20

    Steviol glycosides are currently being used as natural sweeteners by the food industry and Stevia rebaudiana has long been used as a sweet plant in South America for patients suffering from diabetes. In this study, a Stevia rebaudiana ethanolic extract (SREE) was prepared, analysed and tested for antioxidant activity in terms of free radical scavenging properties and antiproliferative effects in cervix (HeLa), pancreatic (MiaPaCa-2) and colonic (HCT116) cancer cells. The antiproliferative mechanism was confirmed by testing the effects on cyclin D1-CDK4. Bioassays were also performed for the diterpene glycoside stevioside. Our results demonstrate that the extract acts as an antioxidant being able to scavenge free radicals, but this activity was not due to stevioside. The extract also induced cell death in the three cell lines, being more active against cervix cancer cells (HeLa); however, the concentration of stevioside needed to produce antiproliferative effects was higher than the amount of steviol glycosides found in a lower dose of extract inducing cell death. In addition, the extract clearly inhibited CDK4 whereas stevioside did not, concluding that the antiproliferative activity of stevia may be due to inhibition of cyclin-dependent kinases performed by other compounds of the extract. PMID:27071804

  9. Depletion of the thiol oxidoreductase ERp57 in tumor cells inhibits proliferation and increases sensitivity to ionizing radiation and chemotherapeutics

    PubMed Central

    Kranz, Philip; Neumann, Fabian; Mersch, Evgenija; Baumann, Melanie; Goepelt, Kirsten; Brockmeier, Ulf; Metzen, Eric

    2015-01-01

    Rapidly growing tumor cells must synthesize proteins at a high rate and therefore depend on an efficient folding and quality control system for nascent secretory proteins in the endoplasmic reticulum (ER). The ER resident thiol oxidoreductase ERp57 plays an important role in disulfide bond formation. Lentiviral, doxycycline-inducible ERp57 knockdown was combined with irradiation and treatment with chemotherapeutic agents. The knockdown of ERp57 significantly enhanced the apoptotic response to anticancer treatment in HCT116 colon cancer cells via a p53-dependent mechanism. Instead of a direct interaction with p53, depletion of ERp57 induced cell death via a selective activation of the PERK branch of the Unfolded Protein Response (UPR). In contrast, apoptosis was reduced in MDA-MB-231 breast cancer cells harboring mutant p53. Nevertheless, we observed a strong reduction of proliferation in response to ERp57 knockdown in both cell lines regardless of the p53 status. Depletion of ERp57 reduced the phosphorylation activity of the mTOR-complex1 (mTORC1) as demonstrated by reduction of p70S6K phosphorylation. Our data demonstrate that ERp57 is a promising target for anticancer therapy due to synergistic p53-dependent induction of apoptosis and p53-independent inhibition of proliferation. PMID:26513173

  10. Asymmetric triplex metallohelices with high and selective activity against cancer cells

    NASA Astrophysics Data System (ADS)

    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

    2014-09-01

    Small cationic amphiphilic α-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.

  11. ATAD2 Overexpression Identifies Colorectal Cancer Patients with Poor Prognosis and Drives Proliferation of Cancer Cells

    PubMed Central

    Luo, Yang; Ye, Guang-Yao; Qin, Shao-Lan; Yu, Min-Hao; Mu, Yi-Fei; Zhong, Ming

    2015-01-01

    ATPase family AAA domain-containing 2 (ATAD2) has been identified as a critical modulator involved in cell proliferation and invasion. The purpose of this study was to explore the expression of ATAD2 in CRC tissues as well as its relationship with degree of malignancy. Data containing three independent investigations from Oncomine database demonstrated that ATAD2 is overexpressed in CRC compared with normal tissue, and similar result was also found in 32 pairs of CRC tissues by qPCR. The protein expression of ATAD2 was examined in six CRC cell lines and 300 CRC specimens. The results showed that high expression of ATAD2 was significantly correlated with tumor size (P < 0.001), serum CEA (P = 0.012), lymph node metastasis (P = 0.018), liver metastasis (P = 0.025), and clinical stage (P = 0.004). Kaplan-Meier method suggested that higher ATAD2 protein expression significantly associated with the overall survival (OS) of CRC patients (P < 0.001) and was an independent predictor of poor OS. Functional studies showed that suppression of ATAD2 expression with siRNA could significantly inhibit the growth in SW480 and HCT116 cells. These results indicated that ATAD2 could serve as a prognostic marker and a therapeutic target for CRC. PMID:26697062

  12. Identification and Functional Analysis of Epigenetically Silenced MicroRNAs in Colorectal Cancer Cells

    PubMed Central

    Yan, Hongli; Choi, Ae-jin; Lee, Byron H.; Ting, Angela H.

    2011-01-01

    Abnormal microRNA (miRNA) expression has been linked to the development and progression of several human cancers, and such dysregulation can result from aberrant DNA methylation. While a small number of miRNAs is known to be regulated by DNA methylation, we postulated that such epigenetic regulation is more prevalent. By combining MBD-isolated Genome Sequencing (MiGS) to evaluate genome-wide DNA methylation patterns and microarray analysis to determine miRNA expression levels, we systematically searched for candidate miRNAs regulated by DNA methylation in colorectal cancer cell lines. We found 64 miRNAs to be robustly methylated in HCT116 cells; eighteen of them were located in imprinting regions or already reported to be regulated by DNA methylation. For the remaining 46 miRNAs, expression levels of 18 were consistent with their DNA methylation status. Finally, 8 miRNAs were up-regulated by 5-aza-2′-deoxycytidine treatment and identified to be novel miRNAs regulated by DNA methylation. Moreover, we demonstrated the functional relevance of these epigenetically silenced miRNAs by ectopically expressing select candidates, which resulted in inhibition of growth and migration of cancer cells. In addition to reporting these findings, our study also provides a reliable, systematic strategy to identify DNA methylation-regulated miRNAs by combining DNA methylation profiles and expression data. PMID:21698188

  13. Arginine deprivation induces endoplasmic reticulum stress in human solid cancer cells.

    PubMed

    Bobak, Yaroslav; Kurlishchuk, Yuliya; Vynnytska-Myronovska, Bozhena; Grydzuk, Olesia; Shuvayeva, Galyna; Redowicz, Maria J; Kunz-Schughart, Leoni A; Stasyk, Oleh

    2016-01-01

    Deprivation for the single amino acid arginine is a rapidly developing metabolic anticancer therapy, which allows growth control in a number of highly malignant tumors. Here we report that one of the responses of human solid cancer cells to arginine starvation is the induction of prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Systematic study of two colorectal carcinoma HCT-116 and HT29, glioblastoma U251 MG and ovarian carcinoma SKOV3 cell lines revealed, however, that the ER stress triggered by the absence of arginine does not result in massive apoptosis despite a profound upregulation of the proapoptotic gene CHOP. Instead, Akt- and MAPK-dependent pathways were activated which may counteract proapoptotic signaling. Treatment with DMSO as a disaggregating agent or with cycloheximide to block protein synthesis reduced ER stress evoked by arginine deprivation. On the other hand, ER stress and apoptosis induction in arginine-starved cells could be critically augmented by the arginine analog of plant origin canavanine, but not by the classic ER stress inducer tunicamycin. Our data suggest that canavanine treatment applied under the lack of arginine may enhance the efficacy of arginine deprivation-based anticancer therapy. PMID:26546743

  14. Orthotopic Microinjection of Human Colon Cancer Cells in Nude Mice Induces Tumor Foci in All Clinically Relevant Metastatic Sites

    PubMed Central

    Céspedes, María Virtudes; Espina, Carolina; García-Cabezas, Miguel Angel; Trias, Manuel; Boluda, Alicia; Gómez del Pulgar, María Teresa; Sancho, Francesc Josep; Nistal, Manuel; Lacal, Juan Carlos; Mangues, Ramon

    2007-01-01

    Despite metastasis as an important cause of death in colorectal cancer patients, current animal models of this disease are scarcely metastatic. We evaluated whether direct orthotopic cell microinjection, between the mucosa and the muscularis layers of the cecal wall of nude mice, drives tumor foci to the most relevant metastatic sites observed in humans and/or improves its yield as compared with previous methods. We injected eight animals each tested human colorectal cancer cell line (HCT-116, SW-620, and DLD-1), using a especially designed micropipette under binocular guidance, and evaluated the take rate, local growth, pattern and rate of dissemination, and survival time. Take rates were in the 75 to 88% range. Tumors showed varying degrees of mesenteric and retroperitoneal lymphatic foci (57 to 100%), hematogenous dissemination to liver (29 to 67%) and lung (29 to 100%), and peritoneal carcinomatosis (29 to 100%). Tumor staging closely correlated with animal survival. Therefore, the orthotopic cell microinjection procedure induces tumor foci in the most clinically relevant metastatic sites: colon-draining lymphatics, liver, lung, and peritoneum. The replication of the clinical pattern of dissemination makes it a good model for advanced colorectal cancer. Moreover, this procedure also enhances the rates of hematogenous and lymphatic dissemination at relevant sites, as compared with previously described methods that only partially reproduce this pattern. PMID:17322390

  15. γ-Glutamyl hydrolase modulation significantly influences global and gene-specific DNA methylation and gene expression in human colon and breast cancer cells.

    PubMed

    Kim, Sung-Eun; Hinoue, Toshinori; Kim, Michael S; Sohn, Kyoung-Jin; Cho, Robert C; Cole, Peter D; Weisenberger, Daniel J; Laird, Peter W; Kim, Young-In

    2015-01-01

    γ-Glutamyl hydrolase (GGH) plays an important role in folate homeostasis by catalyzing hydrolysis of polyglutamylated folate into monoglutamates. Polyglutamylated folates are better substrates for several enzymes involved in the generation of S-adenosylmethionine, the primary methyl group donor, and hence, GGH modulation may affect DNA methylation. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, and in chromatin modifications, and aberrant or dysregulation of DNA methylation has been mechanistically linked to the development of human diseases including cancer. Using a recently developed in vitro model of GGH modulation in HCT116 colon and MDA-MB-435 breast cancer cells, we investigated whether GGH modulation would affect global and gene-specific DNA methylation and whether these alterations were associated with significant gene expression changes. In both cell lines, GGH overexpression decreased global DNA methylation and DNA methyltransferase (DNMT) activity, while GGH inhibition increased global DNA methylation and DNMT activity. Epigenomic and gene expression analyses revealed that GGH modulation influenced CpG promoter DNA methylation and gene expression involved in important biological pathways including cell cycle, cellular development, and cellular growth and proliferation. Some of the observed altered gene expression appeared to be regulated by changes in CpG promoter DNA methylation. Our data suggest that the GGH modulation-induced changes in total intracellular folate concentrations and content of long-chain folylpolyglutamates are associated with functionally significant DNA methylation alterations in several important biological pathways. PMID:25502219

  16. Refractory lining for electrochemical cell

    SciTech Connect

    Blander, M.; Cook, G.M.

    1987-08-18

    This patent describes an apparatus for processing a melt of molten iron in contact with a molten slag containing iron oxide, the apparatus consists of melt containing means including an electrically conductive refractory lining disposed for contact with an iron oxide containing melt, an anode in the melt containing means electrically separated from the refractory lining, and means for establishing a voltage between the refractory lining as cathode and the anode to reduce iron oxide to iron at the surface of the refractory lining in contact with the iron oxide containing melt, the refractory lining including a metal oxide selected from the group consisting of Mg chromites and MgO.

  17. Synthesis and Characterization of AICAR and DOX Conjugated Multifunctional Nanoparticles as a Platform for Synergistic Inhibition of Cancer Cell Growth.

    PubMed

    Daglioglu, Cenk; Okutucu, Burcu

    2016-04-20

    The success of cancer treatment depends on the response to chemotherapeutic agents. However, malignancies often acquire resistance to drugs if they are used frequently. Combination therapy involving both a chemotherapeutic agent and molecularly targeted therapy may have the ability to retain and enhance therapeutic efficacy. Here, we addressed this issue by examining the efficacy of a novel therapeutic strategy that combines AICAR and DOX within a multifunctional platform. In this context, we reported the bottom-up synthesis of Fe3O4@SiO2(FITC)-FA/AICAR/DOX multifunctional nanoparticles aiming to neutralize survivin (BIRC5) to potentiate the efficacy of DOX against chemoresistance. The structure of nanoparticles was characterized by dynamic light scattering (DLS), zeta-potential measurement, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), and electron microscopy (SEM and STEM with EDX) techniques. Cellular uptake and cytotoxicity experiments demonstrated preferentially targeted delivery of nanoparticles and an efficient reduction of cancer cell viability in five different tumor-derived cell lines (A549, HCT-116, HeLa, Jurkat, and MIA PaCa-2). These results indicate that the multifunctional nanoparticle system possesses high inhibitory drug association and sustained cytotoxic effect with good biocompatibility. This novel approach which combines AICAR and DOX within a single platform might be promising as an antitumor treatment for cancer. PMID:26996194

  18. New ex-ovo colorectal-cancer models from different SdFFF-sorted tumor-initiating cells.

    PubMed

    Mélin, Carole; Perraud, Aurélie; Christou, Niki; Bibes, Romain; Cardot, Philippe; Jauberteau, Marie-Odile; Battu, Serge; Mathonnet, Muriel

    2015-11-01

    Despite effective treatments, relapse of colorectal cancer (CRC) is frequent, in part caused by the existence of tumor-initiating cells (TICs). Different subtypes of TICs, quiescent and activated, coexist in tumors, defining the tumor aggressiveness and therapeutic response. These subtypes have been sorted by hyperlayer sedimentation field-flow fractionation (SdFFF) from WiDr and HCT116 cell lines. On the basis of a new strategy, including TIC SdFFF sorting, 3D Matrigel amplification, and grafting of corresponding TIC colonies on the chick chorioallantoic membrane (CAM), specific tumor matrices could be obtained. If tumors had similar architectural structure with vascularization by the host system, they had different proliferative indices in agreement with their initial quiescent or activated state. Protein analysis also revealed that tumors obtained from a population enriched for "activated" TICs lost "stemness" properties and became invasive. In contrast, tumors obtained from a population enriched for "quiescent" TICs kept their stemness properties and seemed to be less proliferative and invasive. Then, it was possible to produce different kinds of tumor which could be used as selective supports to study carcinogenesis and therapy sensitivity. PMID:26427501

  19. Molecular mechanisms underlying the antitumor activity of (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide in human colorectal cancer cells in vitro and in vivo

    PubMed Central

    Chen, Chun-Han; Lee, Chia-Hwa; Liou, Jing-Ping; Teng, Che-Ming; Pan, Shiow-Lin

    2015-01-01

    Upregulation of class I histone deacetylases (HDAC) correlates with poor prognosis in colorectal cancer (CRC) patients. Previous study revealed that (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide (Compound 11) is a potent and selective class I HDAC inhibitor, exhibited significant anti-proliferative activity in various human cancer cell lines. In current study, we demonstrated that compound 11 exhibited significant anti-proliferative and cytotoxic activity in CRC cells. Notably, compound 11 was less potent than SAHA in inhibiting HDAC6 as evident from the lower expression of acetyl-α-tubulin, suggesting higher selectivity for class I HDACs. Mechanistically, compound 11 induced cell-cycle arrest at the G2/M phase, activated both intrinsic- and extrinsic-apoptotic pathways, altered the expression of Bcl-2 family proteins and exerted a potent inhibitory effect on survival signals (p-Akt, p-ERK) in CRC cells. Moreover, we provide evidence that compound 11 suppressed motility, decreased mesenchymal markers (N-cadherin and vimentin) and increased epithelial marker (E-cadherin) through down-regulation of Akt. The anti-tumor activity and underlying molecular mechanisms of compound 11 were further confirmed using the HCT116 xenograft model in vivo. Our findings provide evidence of the significant anti-tumor activity of compound 11 in a preclinical model, supporting its potential as a novel therapeutic agent for CRC. PMID:26462017

  20. Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in human cells via a p53-dependent mechanism.

    PubMed

    Wohak, Laura E; Krais, Annette M; Kucab, Jill E; Stertmann, Julia; Øvrebø, Steinar; Seidel, Albrecht; Phillips, David H; Arlt, Volker M

    2016-02-01

    The tumour suppressor gene TP53 is mutated in more than 50 % of human tumours, making it one of the most important cancer genes. We have investigated the role of TP53 in cytochrome P450 (CYP)-mediated metabolic activation of three polycyclic aromatic hydrocarbons (PAHs) in a panel of isogenic colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-), TP53(-/-), TP53(R248W/+) or TP53(R248W/-) were treated with benzo[a]pyrene (BaP), dibenz[a,h]anthracene and dibenzo[a,l]pyrene, and the formation of DNA adducts was measured by (32)P-postlabelling analysis. Each PAH formed significantly higher DNA adduct levels in TP53(+/+) cells than in the other cell lines. There were also significantly lower levels of PAH metabolites in the culture media of these other cell lines. Bypass of the need for metabolic activation by treating cells with the corresponding reactive PAH-diol-epoxide metabolites resulted in similar adduct levels in all cell lines, which confirms that the influence of p53 is on the metabolism of the parent PAHs. Western blotting showed that CYP1A1 protein expression was induced to much greater extent in TP53(+/+) cells than in the other cell lines. CYP1A1 is inducible via the aryl hydrocarbon receptor (AHR), but we did not find that expression of AHR was dependent on p53; rather, we found that BaP-induced CYP1A1 expression was regulated through p53 binding to a p53 response element in the CYP1A1 promoter region, thereby enhancing its transcription. This study demonstrates a new pathway for CYP1A1 induction by environmental PAHs and reveals an emerging role for p53 in xenobiotic metabolism. PMID:25398514

  1. Human phosphatase CDC14A is recruited to the cell leading edge to regulate cell migration and adhesion.

    PubMed

    Chen, Nan-Peng; Uddin, Borhan; Voit, Renate; Schiebel, Elmar

    2016-01-26

    Cell adhesion and migration are highly dynamic biological processes that play important roles in organ development and cancer metastasis. Their tight regulation by small GTPases and protein phosphorylation make interrogation of these key processes of great importance. We now show that the conserved dual-specificity phosphatase human cell-division cycle 14A (hCDC14A) associates with the actin cytoskeleton of human cells. To understand hCDC14A function at this location, we manipulated native loci to ablate hCDC14A phosphatase activity (hCDC14A(PD)) in untransformed hTERT-RPE1 and colorectal cancer (HCT116) cell lines and expressed the phosphatase in HeLa FRT T-Rex cells. Ectopic expression of hCDC14A induced stress fiber formation, whereas stress fibers were diminished in hCDC14A(PD) cells. hCDC14A(PD) cells displayed faster cell migration and less adhesion than wild-type controls. hCDC14A colocalized with the hCDC14A substrate kidney- and brain-expressed protein (KIBRA) at the cell leading edge and overexpression of KIBRA was able to reverse the phenotypes of hCDC14A(PD) cells. Finally, we show that ablation of hCDC14A activity increased the aggressive nature of cells in an in vitro tumor formation assay. Consistently, hCDC14A is down-regulated in many tumor tissues and reduced hCDC14A expression is correlated with poorer survival of patients with cancer, to suggest that hCDC14A may directly contribute to the metastatic potential of tumors. Thus, we have uncovered an unanticipated role for hCDC14A in cell migration and adhesion that is clearly distinct from the mitotic and cytokinesis functions of Cdc14/Flp1 in budding and fission yeast. PMID:26747605

  2. Polyploidy Formation in Doxorubicin-Treated Cancer Cells Can Favor Escape from Senescence1

    PubMed Central

    Mosieniak, Grazyna; Sliwinska, Malgorzata A.; Alster, Olga; Strzeszewska, Anna; Sunderland, Piotr; Piechota, Malgorzata; Was, Halina; Sikora, Ewa

    2015-01-01

    Cancer cells can undergo stress-induced premature senescence, which is considered to be a desirable outcome of anticancer treatment. However, the escape from senescence and cancer cell repopulation give rise to some doubts concerning the effectiveness of the senescence-induced anticancer therapy. Similarly, it is postulated that polyploidization of cancer cells is connected with disease relapse. We postulate that cancer cell polyploidization associated with senescence is the culprit of atypical cell divisions leading to cancer cell regrowth. Accordingly, we aimed to dissociate between these two phenomena. We induced senescence in HCT 116 cells by pulse treatment with doxorubicin and observed transiently increased ploidy, abnormal nuclear morphology, and various distributions of some proteins (e.g., p21, Ki-67, SA-β-galactosidase) in the subnuclei. Doxorubicin-treated HCT 116 cells displayed an increased production of reactive oxygen species (ROS) possibly caused by an increased amount of mitochondria, which are characterized by low membrane potential. A decrease in the level of ROS by Trolox partially protected the cells from polyploidization but not from senescence. Interestingly, a decreased level of ROS prevented the cells from escaping senescence. We also show that MCF7 cells senesce, but this is not accompanied by the increase of ploidy upon doxorubicin treatment. Moreover, they were stably growth arrested, thus proving that polyploidy but not senescence per se enables to regain the ability to proliferate. Our preliminary results indicate that the different propensity of the HCT 116 and MCF7 cells to increase ploidy upon cell senescence could be caused by a different level of the mTOR and/or Pim-1 kinases. PMID:26696370

  3. Tumor cell alpha-N-acetylgalactosaminidase activity and its involvement in GcMAF-related macrophage activation.

    PubMed

    Mohamad, Saharuddin B; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

    2002-05-01

    Alpha-N-acetyl galactosaminidase (alpha-NaGalase) has been reported to accumulate in serum of cancer patients and be responsible for deglycosylation of Gc protein, which is a precursor of GcMAF-mediated macrophage activation cascade, finally leading to immunosuppression in advanced cancer patients. We studied the biochemical characterization of alpha-NaGalase from several human tumor cell lines. We also examined its effect on the potency of GcMAF to activate mouse peritoneal macrophage to produce superoxide in GcMAF-mediated macrophage activation cascade. The specific activity of alpha-NaGalases from human colon tumor cell line HCT116, human hepatoma cell line HepG2, and normal human liver cells (Chang liver cell line) were evaluated using two types of substrates; GalNAc-alpha-PNP (exo-type substrate) and Gal-beta-GalNAc-alpha-PNP (endo-type substrate). Tumor-derived alpha-NaGalase having higher activity than normal alpha-NaGalase, had higher substrate specificity to the exo-type substrate than to the endo-type substrate, and still maintained its activity at pH 7. GcMAF enhance superoxide production in mouse macrophage, and pre-treatment of GcMAF with tumor cell lysate reduce the activity. We conclude that tumor-derived alpha-NaGalase is different in biochemical characterization compared to normal alpha-NaGalase from normal Chang liver cells. In addition, tumor cell-derived alpha-NaGalase decreases the potency of GcMAF on macrophage activation. PMID:12062184

  4. 5-Hydroxy-7-Methoxyflavone Triggers Mitochondrial-Associated Cell Death via Reactive Oxygen Species Signaling in Human Colon Carcinoma Cells

    PubMed Central

    Paul, Souren; Jakhar, Rekha; Han, Jaehong; Kang, Sun Chul

    2016-01-01

    Plant-derived compounds are an important source of clinically useful anti-cancer agents. Chrysin, a biologically active flavone found in many plants, has limited usage for cancer chemotherapeutics due to its poor oral bioavailability. 5-Hydroxy-7-methoxyflavone (HMF), an active natural chrysin derivative found in various plant sources, is known to modulate several biological activities. However, the mechanism underlying HMF-induced apoptotic cell death in human colorectal carcinoma cells in vitro is still unknown. Herein, HMF was shown to be capable of inducing cytotoxicity in HCT-116 cells and induced cell death in a dose-dependent manner. Treatment of HCT-116 cells with HMF caused DNA damage and triggered mitochondrial membrane perturbation accompanied by Cyt c release, down-regulation of Bcl-2, activation of BID and Bax, and caspase-3-mediated apoptosis. These results show that ROS generation by HMF was the crucial mediator behind ER stress induction, resulting in intracellular Ca2+ release, JNK phosphorylation, and activation of the mitochondrial apoptosis pathway. Furthermore, time course study also reveals that HMF treatment leads to increase in mitochondrial and cytosolic ROS generation and decrease in antioxidant enzymes expression. Temporal upregulation of IRE1-α expression and JNK phosphorylation was noticed after HMF treatment. These results were further confirmed by pre-treatment with the ROS scavenger N-acetyl-l-cysteine (NAC), which completely reversed the effects of HMF treatment by preventing lipid peroxidation, followed by abolishment of JNK phosphorylation and attenuation of apoptogenic marker proteins. These results emphasize that ROS generation by HMF treatment regulates the mitochondrial-mediated apoptotic signaling pathway in HCT-116 cells, demonstrating HMF as a promising pro-oxidant therapeutic candidate for targeting colorectal cancer. PMID:27116119

  5. 5-Hydroxy-7-Methoxyflavone Triggers Mitochondrial-Associated Cell Death via Reactive Oxygen Species Signaling in Human Colon Carcinoma Cells.

    PubMed

    Bhardwaj, Monika; Kim, Na-Hyung; Paul, Souren; Jakhar, Rekha; Han, Jaehong; Kang, Sun Chul

    2016-01-01

    Plant-derived compounds are an important source of clinically useful anti-cancer agents. Chrysin, a biologically active flavone found in many plants, has limited usage for cancer chemotherapeutics due to its poor oral bioavailability. 5-Hydroxy-7-methoxyflavone (HMF), an active natural chrysin derivative found in various plant sources, is known to modulate several biological activities. However, the mechanism underlying HMF-induced apoptotic cell death in human colorectal carcinoma cells in vitro is still unknown. Herein, HMF was shown to be capable of inducing cytotoxicity in HCT-116 cells and induced cell death in a dose-dependent manner. Treatment of HCT-116 cells with HMF caused DNA damage and triggered mitochondrial membrane perturbation accompanied by Cyt c release, down-regulation of Bcl-2, activation of BID and Bax, and caspase-3-mediated apoptosis. These results show that ROS generation by HMF was the crucial mediator behind ER stress induction, resulting in intracellular Ca2+ release, JNK phosphorylation, and activation of the mitochondrial apoptosis pathway. Furthermore, time course study also reveals that HMF treatment leads to increase in mitochondrial and cytosolic ROS generation and decrease in antioxidant enzymes expression. Temporal upregulation of IRE1-α expression and JNK phosphorylation was noticed after HMF treatment. These results were further confirmed by pre-treatment with the ROS scavenger N-acetyl-l-cysteine (NAC), which completely reversed the effects of HMF treatment by preventing lipid peroxidation, followed by abolishment of JNK phosphorylation and attenuation of apoptogenic marker proteins. These results emphasize that ROS generation by HMF treatment regulates the mitochondrial-mediated apoptotic signaling pathway in HCT-116 cells, demonstrating HMF as a promising pro-oxidant therapeutic candidate for targeting colorectal cancer. PMID:27116119

  6. Genistein induces G2/M cell cycle arrest and apoptosis via ATM/p53-dependent pathway in human colon cancer cells

    PubMed Central

    ZHANG, ZHIYU; WANG, CHONG-ZHI; DU, GUANG-JIAN; QI, LIAN-WEN; CALWAY, TYLER; HE, TONG-CHUAN; DU, WEI; YUAN, CHUN-SU

    2013-01-01

    Soybean isoflavones have been used as a potential preventive agent in anticancer research for many years. Genistein is one of the most active flavonoids in soybeans. Accumulating evidence suggests that genistein alters a variety of biological processes in estrogen-related malignancies, such as breast and prostate cancers. However, the molecular mechanism of genistein in the prevention of human colon cancer remains unclear. Here we attempted to elucidate the anticarcinogenic mechanism of genistein in human colon cancer cells. First we evaluated the growth inhibitory effect of genistein and two other isoflavones, daidzein and biochanin A, on HCT-116 and SW-480 human colon cancer cells. In addition, flow cytometry was performed to observe the morphological changes in HCT-116/SW-480 cells undergoing apoptosis or cell cycle arrest, which had been visualized using Annexin V-FITC and/or propidium iodide staining. Real-time PCR and western blot analyses were also employed to study the changes in expression of several important genes associated with cell cycle regulation. Our data showed that genistein, daidzein and biochanin A exhibited growth inhibitory effects on HCT-116/SW-480 colon cancer cells and promoted apoptosis. Genistein showed a significantly greater effect than the other two compounds, in a time- and dose-dependent manner. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of ATM/p53, p21waf1/cip1 and GADD45α as well as downregulation of cdc2 and cdc25A demonstrated by q-PCR and immunoblotting assay. Interestingly, genistein induced G2/M cell cycle arrest in a p53-dependent manner. These findings exemplify that isoflavones, especially genistein, could promote colon cancer cell growth inhibition and facilitate apoptosis and cell cycle arrest in the G2/M phase. The ATM/p53-p21 cross-regulatory network may play a crucial role in mediating the anticarcinogenic activities of genistein in colon cancer. PMID:23686257

  7. The pro-apoptotic and anti-invasive effects of hypericin-mediated photodynamic therapy are enhanced by hyperforin or aristoforin in HT-29 colon adenocarcinoma cells.

    PubMed

    Šemeláková, Martina; Mikeš, Jaromír; Jendželovský, Rastislav; Fedoročko, Peter

    2012-12-01

    Photodynamic therapy is a rapidly-developing anti-cancer approach for the treatment of various types of malignant as well as non-malignant diseases. In this study, hypericin-mediated photodynamic therapy (HY-PDT) in sub-optimal dose was combined with hyperforin (HP) or its stable derivative aristoforin (AR) in an effort to improve efficacy on the cellular level. The logic of this combination is based on the fact that both bioactive compounds naturally occur in plants of Hypericum sp. At relatively low concentrations up to 5 μM, hyperforin and aristoforin were able to stimulate onset of apoptosis in HT-29 colon adenocarcinoma cells exposed to HY-PDT, inhibit cell cycle progression, suppress expression of matrixmetalloproteinases-2/-9 together with cell adhesivity, thereby affecting the clonogenic potential of the cells. As the action of aristoforin was more pronounced, in line with our assumption, these changes were also linked in this case with hypericin accumulation and increased ROS generation leading to dissipation of mitochondrial membrane potential in a significant portion of the cells, as well as activation of caspase-3. Comparison of HT-29 cells to another colon adenocarcinoma-derived cell line HCT-116 demonstrated significant differences in sensitivity of different cell lines to PDT, however, accumulated effect of HY-PDT with HP/AR proved similar in both tested cell lines. The presented data may help to elucidate the mechanisms of action for different bioactive constituents of St. John's wort, which are increasingly recognized as being able to regulate a variety of pathobiological processes, thus possessing potential therapeutic properties. PMID:23099482

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

    SciTech Connect

    McDonald, Gail T.; Sullivan, Richard; Pare, Genevieve C.; Graham, Charles H.

    2010-11-15

    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.

  9. Nitrogen permease regulator-like 2 enhances sensitivity to oxaliplatin in colon cancer cells.

    PubMed

    Liu, Ming-Na; Liu, Ai-Yun; Du, Ya-Ju; Pei, Feng-Hua; Wang, Xin-Hong; Chen, Jing; Liu, Dan; Liu, Bing-Rong

    2015-07-01

    Colorectal cancer (CRC) is the third most common cancer worldwide. Chemotherapeutic compounds used for the treatment of CRC include oxaliplatin (L-OHP). While L-OHP improves CRC survival, certain patients are resistant. The nitrogen permease regulator like-2 (NPRL2) gene is a candidate tumor suppressor gene that resides in a 120-kb homozygous deletion region on chromosome 3p21.3. In the present study, it was demonstrated that NPRL2 overexpression increases the sensitivity of HCT116 cells to L-OHP. The IC50 of L-OHP was decreased in cells transduced with NPRL2 compared with negative control (NC) cells and the effect of NPRL2 on L-OHP sensitivity was time dependent. Following NPRL2 transduction in HCT116 cells, the cell cycle was arrested in the G1 phase and a partial decrease in the S phase population was observed. Flow cytometric analysis revealed that NPRL2 transduction and L-OHP treatment increased apoptosis compared with NC cells. The mechanism through which NPRL2 overexpression enhances L-OHP sensitivity involves downregulation of the functions of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin network. Furthermore, L-OHP upregulated caspase-3 and caspase-9 to promote apoptosis in NPRL2-overexpressing cells compared with cells that were transduced with NPRL2 or treated with L-OHP and NC cells (P<0.01). NPRL2 overexpression led to the downregulation of CD24, which could significantly reduce tumor invasiveness and decrease the metastatic capacity of HCT116 cells. These mechanisms are likely active in other types of cancer and may be exploited for the development of novel cancer therapies. PMID:25777765

  10. Biomimetic macroporous hydrogel scaffolds in a high-throughput screening format for cell-based assays.

    PubMed

    Dainiak, Maria B; Savina, Irina N; Musolino, Isabella; Kumar, Ashok; Mattiasson, Bo; Galaev, Igor Yu

    2008-01-01

    Macroporous hydrogels (MHs) hold great promise as scaffolds in tissue engineering and cell-based assays. In this study, the possibility of combination of three-dimensional (3D) cell culture with a miniaturized screening format was demonstrated on human colon cancer HCT116, human acute myeloid leukemia KG-1 cells, and embryonic fibroblasts cultured on MHs (12.5 mm x 7.1 mm I.D.) in a 96-minicolumn plate format. MHs were prepared by cryogelation technique and functionalized by coating with type I collagen and by copolymerization with agmatine-based mimetic of cell adhesive peptide RGD (abRGDm). Cancer cells formed multicellular aggregates while fibroblasts formed adhesions on abRGDm-containing and collagen-MHs but not on plain MHs, as was demonstrated by scanning electron microscopy. HCT116 and KG-1 cells grown as aggregates were more resistant to the treatment with cis-diaminedichloroplatinum (II) (cisplatin) and cytosine 1-beta-D-arabinofuranoside (Ara-C), respectively, during the first 18-24 h of incubation, than single cells grown on unmodified MH. HCT116 cells grown as 2D cultures in conventional 96-well tissue culture plates were 1.5- to 3.5-fold more sensitive to the treatment with 70 microM cisplatin than cells in 3D cultures in functionalized MHs. Further development of the described experimental system including matching of a specific cell type with appropriate extracellular matrix (ECM) components and 3D cocultures on ECM-modified MHs may provide a realistic in vitro experimental model for high-throughput toxicity tests. PMID:19194952

  11. 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.

    PubMed

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

    2014-10-01

    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

  12. miRNA-144 suppresses proliferation and migration of colorectal cancer cells through GSPT1.

    PubMed

    Xiao, Ruilin; Li, Cui; Chai, Baofeng

    2015-08-01

    MicroRNAs play a key role in carcinogenesis or tumor progression, which negatively and posttranscriptionally regulate gene expression and function as oncogenes or tumor suppressors, as well as regulators of cell cycle, proliferation, apoptosis, migration and other processes. A number of miRNAs are reported be related to the occurrence and development of colorectal cancer (CRC). However, these studies were not involved in the effect of miRNA 144 of CRC, whose function remains unclear. In this study, we demonstrated that the expression level of miRNA 144 was markedly down-regulated in colorectal cancer HCT116 cells compared with normal control FHC cells. Meanwhile, we found that GSPT1 was over-expressed in human colorectal cancer HCT116 cells. Subsequently, GSPT1 was identified as a target of miRNA 144 through bioinformatics and luciferase reporter assays. Besides, we also confirmed that miRNA 144 can inhibit the proliferation and migration of colorectal cancer HCT116 cells . Next, we observed RNA-mediated knockdown of GSPT1 can also inhibit the proliferation and migration of colorectal cancer cells. Thus, we concluded that miRNA 144 inhibits cell proliferation and migration through GSPT1 in CRC. In addition, further mechanic investigations revealed that miRNA-144 suppressed the expression of GSPT1 to regulate the expression of c-myc, survivin and Bcl2L15 which are involved in cell proliferation, and that metastasis related factor MMP28 was also down-regulated by miRNA144. Our findings suggested that microRNA 144 might be an important element to control the status of colorectal cancer, which has provided a new insight into the mechanism of proliferation and migration and a new target in therapy against colorectal cancer. PMID:26349975

  13. GDNF increases cell motility in human colon cancer through VEGF-VEGFR1 interaction.

    PubMed

    Huang, Ssu-Ming; Chen, Tzu-Sheng; Chiu, Chien-Ming; Chang, Leang-Kai; Liao, Kuan-Fu; Tan, Hsiao-Ming; Yeh, Wei-Lan; Chang, Gary Ro-Lin; Wang, Min-Ying; Lu, Dah-Yuu

    2014-02-01

    Glial cell line-derived neurotrophic factor (GDNF), a potent neurotrophic factor, has been shown to affect cancer cell metastasis and invasion. However, the molecular mechanisms underlying GDNF-induced colon cancer cell migration remain unclear. GDNF is found to be positively correlated with malignancy in human colon cancer patients. The migratory activities of two human colon cancer cell lines, HCT116 and SW480, were found to be enhanced in the presence of human GDNF. The expression of vascular endothelial growth factor (VEGF) was also increased in response to GDNF stimulation, along with VEGF mRNA expression and transcriptional activity. The enhancement of GDNF-induced cancer cell migration was antagonized by a VEGF-neutralizing antibody. Our results also showed that the expression of VEGF receptor 1 (VEGFR1) was increased in response to GDNF stimulation, whereas GDNF-induced cancer cell migration was reduced by a VEGFR inhibitor. The GDNF-induced VEGF expression was regulated by the p38 and PI3K/Akt signaling pathways. Treatment with GDNF increased nuclear hypoxia-inducible factor 1 α (HIF1α) accumulation and its transcriptional activity in a time-dependent manner. Moreover, GDNF increased hypoxia responsive element (HRE)-containing VEGF promoter transcriptional activity but not that of the HRE-deletion VEGF promoter construct. Inhibition of HIF1α by a pharmacological inhibitor or dominant-negative mutant reduced the GDNF-induced migratory activity in human colon cancer cells. These results indicate that GDNF enhances the migration of colon cancer cells by increasing VEGF-VEGFR interaction, which is mainly regulated by the p38, PI3K/Akt, and HIF1α signaling pathways. PMID:24165321

  14. Tumor suppression by resistant maltodextrin, Fibersol-2

    PubMed Central

    So, Eui Young; Ouchi, Mutsuko; Cuesta-Sancho, Sara; Olson, Susan Losee; Reif, Dirk; Shimomura, Kazuhiro; Ouchi, Toru

    2015-01-01

    Resistant maltodextrin Fibersol-2 is a soluble and fermentable dietary fiber that is Generally Recognized As Safe (GRAS) in the United States. We tested whether Fibersol-2 contains anti-tumor activity. Human colorectal cancer cell line, HCT116, and its isogenic cells were treated with FIbersol-2. Tumor growth and tumorigenesis were studied in vitro and in vivo. Apoptotic pathway and generation of reactive oxygen species (ROS) were investigated. We discovered that Fibersol-2 significantly inhibits tumor growth of HCT116 cells by inducing apoptosis. Fibersol-2 strongly induces mitochondrial ROS and Bax-dependent cleavage of caspase 3 and 9, which is shown by isogenic HCT116 variants. Fibersol-2 induces phosphorylation of Akt, mTOR in parental HCT116 cells, but not in HCT116 deficient for Bax or p53. It prevents growth of tumor xenograft without any apparent signs of toxicity in vivo. These results identify Fibersol-2 as a mechanism-based dietary supplement agent that could prevent colorectal cancer development. PMID:25692338

  15. Acquired resistance to 5-fluorouracil via HSP90/Src-mediated increase in thymidylate synthase expression in colon cancer.

    PubMed

    Ahn, Ji-Young; Lee, Ji-Sun; Min, Hye-Young; Lee, Ho-Young

    2015-10-20

    5-fluorouracil (5-FU), one of the first-line chemotherapeutic agents for the treatment of gastrointestinal malignancies, has shown limited efficacy. The expression of thymidylate synthase (TYMS) has been reported to be associated with the resistance to 5-FU. Here, we demonstrate that the enhanced HSP90 function and subsequent activation of Src induce expression of TYMS and acquired resistance to 5-FU in colon cancer. We show that the persistent 5-FU treatment granted 5-FU-sensitive HCT116 colon cancer cells morphologic, molecular, and behavioral characteristic of the epithelial-mesenchymal transition (EMT), contributing to emergence of acquired resistance to 5-FU. HCT116/R, a HCT116 colon cancer cell subline carrying acquired resistance to 5-FU, showed increased expression and activation of HSP90's client proteins and transcriptional up-regulation of TYMS. Forced overexpression of HSP90 or constitutive active Src in HCT116 cells increased TYMS expression. Conversely, pharmacological blockade of HSP90 or Src in HCT116/R cells effectively suppressed the changes involved in 5-FU resistance in vitro and xenograft tumor growth, hematogenous spread, and metastatic tumor development in vivo. This study suggests a novel function of HSP90-Src pathway in regulation of TYMS expression and acquisition of 5-FU resistance. Thus, therapeutics targeting this pathway may be an effective clinical strategy to overcome 5-FU resistance in colon cancer. PMID:26416450

  16. Acquired resistance to 5-fluorouracil via HSP90/Src-mediated increase in thymidylate synthase expression in colon cancer

    PubMed Central

    Min, Hye-Young; Lee, Ho-Young

    2015-01-01

    5-fluorouracil (5-FU), one of the first-line chemotherapeutic agents for the treatment of gastrointestinal malignancies, has shown limited efficacy. The expression of thymidylate synthase (TYMS) has been reported to be associated with the resistance to 5-FU. Here, we demonstrate that the enhanced HSP90 function and subsequent activation of Src induce expression of TYMS and acquired resistance to 5-FU in colon cancer. We show that the persistent 5-FU treatment granted 5-FU-sensitive HCT116 colon cancer cells morphologic, molecular, and behavioral characteristic of the epithelial-mesenchymal transition (EMT), contributing to emergence of acquired resistance to 5-FU. HCT116/R, a HCT116 colon cancer cell subline carrying acquired resistance to 5-FU, showed increased expression and activation of HSP90's client proteins and transcriptional up-regulation of TYMS. Forced overexpression of HSP90 or constitutive active Src in HCT116 cells increased TYMS expression. Conversely, pharmacological blockade of HSP90 or Src in HCT116/R cells effectively suppressed the changes involved in 5-FU resistance in vitro and xenograft tumor growth, hematogenous spread, and metastatic tumor development in vivo. This study suggests a novel function of HSP90-Src pathway in regulation of TYMS expression and acquisition of 5-FU resistance. Thus, therapeutics targeting this pathway may be an effective clinical strategy to overcome 5-FU resistance in colon cancer. PMID:26416450

  17. Elisidepsin Interacts Directly with Glycosylceramides in the Plasma Membrane of Tumor Cells to Induce Necrotic Cell Death

    PubMed Central

    Molina-Guijarro, José Manuel; García, Carolina; Macías, Álvaro; García-Fernández, Luis Francisco; Moreno, Cristina; Reyes, Fernando; Martínez-Leal, Juan Fernando; Fernández, Rogelio; Martínez, Valentín; Valenzuela, Carmen; Lillo, M. Pilar; Galmarini, Carlos M.

    2015-01-01

    Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG gene in these deficient cells restored glycosylceramides synthesis, rendering them sensitive to elisidepsin, at a similar level than parental B16 cells. These results indicate that glycosylceramides act as membrane targets of elisidepsin, facilitating its insertion in the plasma membrane and the subsequent membrane permeabilization that leads to drug-induced cell death. They also indicate that cell membrane lipids are a plausible target for antineoplastic therapy. PMID:26474061

  18. De Novo Proteome Analysis of Genetically Modified Tumor Cells By a Metabolic Labeling/Azide-alkyne Cycloaddition Approach*

    PubMed Central

    Ballikaya, Seda; Lee, Jennifer; Warnken, Uwe; Schnölzer, Martina; Gebert, Johannes; Kopitz, Jürgen

    2014-01-01

    Activin receptor type II (ACVR2) is a member of the transforming growth factor type II receptor family and controls cell growth and differentiation, thereby acting as a tumor suppressor. ACVR2 inactivation is known to drive colorectal tumorigenesis. We used an ACVR2-deficient microsatellite unstable colon cancer cell line (HCT116) to set up a novel experimental design for comprehensive analysis of proteomic changes associated with such functional loss of a tumor suppressor. To this end we combined two existing technologies. First, the ACVR2 gene was reconstituted in an ACVR2-deficient colorectal cancer (CRC) cell line by means of recombinase-mediated cassette exchange, resulting in the generation of an inducible expression system that allowed the regulation of ACVR2 gene expression in a doxycycline-dependent manner. Functional expression in the induced cells was explicitly proven. Second, we used the methionine analog azidohomoalanine for metabolic labeling of newly synthesized proteins in our cell line model. Labeled proteins were tagged with biotin via a Click-iT chemistry approach enabling specific extraction of labeled proteins by streptavidin-coated beads. Tryptic on-bead digestion of captured proteins and subsequent ultra-high-performance LC coupled to LTQ Orbitrap XL mass spectrometry identified 513 proteins, with 25 of them differentially expressed between ACVR2-deficient and -proficient cells. Among these, several candidates that had already been linked to colorectal cancer or were known to play a key role in cell growth or apoptosis control were identified, proving the utility of the presented experimental approach. In principle, this strategy can be adapted to analyze any gene of interest and its effect on the cellular de novo proteome. PMID:25225355

  19. Down-regulation of malignant potential by alpha linolenic acid in human and mouse colon cancer cells.

    PubMed

    Chamberland, John P; Moon, Hyun-Seuk

    2015-03-01

    Omega-3 fatty acids (also called ω-3 fatty acis or n-3 fatty acid) are polyunsaturated fatty acids (PUFAs) with a double bond (C=C) at the third carbon atom from the end of the carbon chain. Numerous test tube and animal studies have shown that omega-3 fatty acids may prevent or inhibit the growth of cancers, suggesting that omega-3 fatty acids are important in cancer physiology. Alpha-linolenic acid (ALA) is one of an essential omega-3 fatty acid and organic compound found in seeds (chia and flaxseed), nuts (notably walnuts), and many common vegetable oils. ALA has also been shown to down-regulate cell proliferation of prostate, breast, and bladder cancer cells. However, direct evidence that ALA suppresses to the development of colon cancer has not been studied. Also, no previous studies have evaluated whether ALA may regulate malignant potential (adhesion, invasion and colony formation) in colon cancer cells. In order to address the questions above, we conducted in vitro studies and evaluated whether ALA may down-regulate malignant potential in human (HT29 and HCT116) and mouse (MCA38) colon cancer cell lines. We observed that treatment with 1-5 mM of ALA inhibits cell proliferation, adhesion and invasion in both human and mouse colon cancer cell lines. Interestingly, we observed that ALA did not decrease total colony numbers when compared to control. By contrast, we found that size of colony was significantly changed by ALA treatment when compared to control in all colon cancer cell lines. We suggest that our data enhance our current knowledge of ALA's mechanism and provide crucial information to further the development of new therapies for the management or chemoprevention of colon cancer. PMID:25336096

  20. Cell-host, LINE and environment

    PubMed Central

    Del Re, Brunella; Giorgi, Gianfranco

    2013-01-01

    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

  1. Identifying cancer origin using circulating tumor cells.

    PubMed

    Lu, Si-Hong; Tsai, Wen-Sy; Chang, Ying-Hsu; Chou, Teh-Ying; Pang, See-Tong; Lin, Po-Hung; Tsai, Chun-Ming; Chang, Ying-Chih

    2016-04-01

    Circulating tumor cells (CTCs) have become an established clinical evaluation biomarker. CTC count provides a good correlation with the prognosis of cancer patients, but has only been used with known cancer patients, and has been unable to predict the origin of the CTCs. This study demonstrates the analysis of CTCs for the identification of their primary cancer source. Twelve mL blood samples were equally dispensed on 6 CMx chips, microfluidic chips coated with an anti-EpCAM-conjugated supported lipid bilayer, for CTC capture and isolation. Captured CTCs were eluted to an immunofluorescence (IF) staining panel consisting of 6 groups of antibodies: anti-panCK, anti-CK18, anti-CK7, anti-TTF-1, anti-CK20/anti-CDX2, and anti-PSA/anti-PSMA. Cancer cell lines of lung (H1975), colorectal (DLD-1, HCT-116), and prostate (PC3, DU145, LNCaP) were selected to establish the sensitivity and specificity for distinguishing CTCs from lung, colorectal, and prostate cancer. Spiking experiments performed in 2mL of culture medium or whole blood proved the CMx platform can enumerate cancer cells of lung, colorectal, and prostate. The IF panel was tested on blood samples from lung cancer patients (n = 3), colorectal cancer patients (n = 5), prostate cancer patients (n = 5), and healthy individuals (n = 12). Peripheral blood samples found panCK(+) and CK18(+) CTCs in lung, colorectal, and prostate cancers. CTCs expressing CK7(+) or TTF-1(+), (CK20/ CDX2)(+), or (PSA/ PSMA)(+) corresponded to lung, colorectal, or prostate cancer, respectively. In conclusion, we have designed an immunofluorescence staining panel to identify CTCs in peripheral blood to correctly identify cancer cell origin. PMID:26828696

  2. Brain-derived neurotrophic factor regulates cell motility in human colon cancer.

    PubMed

    Huang, Ssu-Ming; Lin, Chingju; Lin, Hsiao-Yun; Chiu, Chien-Ming; Fang, Chia-Wei; Liao, Kuan-Fu; Chen, Dar-Ren; Yeh, Wei-Lan

    2015-06-01

    Brain-derived neurotrophic factor (BDNF) is a potent neurotrophic factor that has been shown to affect cancer cell metastasis and migration. In the present study, we investigated the mechanisms of BDNF-induced cell migration in colon cancer cells. The migratory activities of two colon cancer cell lines, HCT116 and SW480, were found to be increased in the presence of human BDNF. Heme oxygenase-1 (HO)-1 is known to be involved in the development and progression of tumors. However, the molecular mechanisms that underlie HO-1 in the regulation of colon cancer cell migration remain unclear. Expression of HO-1 protein and mRNA increased in response to BDNF stimulation. The BDNF-induced increase in cell migration was antagonized by a HO-1 inhibitor and HO-1 siRNA. Furthermore, the expression of vascular endothelial growth factor (VEGF) also increased in response to BDNF stimulation, as did VEGF mRNA expression and transcriptional activity. The increase in BDNF-induced cancer cell migration was antagonized by a VEGF-neutralizing antibody. Moreover, transfection with HO-1 siRNA effectively reduced the increased VEGF expression induced by BDNF. The BDNF-induced cell migration was regulated by the ERK, p38, and Akt signaling pathways. Furthermore, BDNF-increased HO-1 and VEGF promoter transcriptional activity were inhibited by ERK, p38, and AKT pharmacological inhibitors and dominant-negative mutants in colon cancer cells. These results indicate that BDNF increases the migration of colon cancer cells by regulating VEGF/HO-1 activation through the ERK, p38, and PI3K/Akt signaling pathways. The results of this study may provide a relevant contribution to our understanding of the molecular mechanisms by which BDNF promotes colon cancer cell motility. PMID:25876647

  3. Streptomyces malaysiense sp. nov.: A novel Malaysian mangrove soil actinobacterium with antioxidative activity and cytotoxic potential against human cancer cell lines

    PubMed Central

    Ser, Hooi-Leng; Palanisamy, Uma Devi; Yin, Wai-Fong; Chan, Kok-Gan; Goh, Bey-Hing; Lee, Learn-Han

    2016-01-01

    Actinobacteria from the unique intertidal ecosystem of the mangroves are known to produce novel, bioactive secondary metabolites. A novel strain known as MUSC 136T (=DSM 100712T = MCCC 1K01246T) which was isolated from Malaysian mangrove forest soil has proven to be no exception. Assessed by a polyphasic approach, its taxonomy showed a range of phylogenetic and chemotaxonomic properties consistent with the genus of Streptomyces. Phylogenetically, highest similarity was to Streptomyces misionensis NBRC 13063T (99.6%) along with two other strains (>98.9% sequence similarities). The DNA–DNA relatedness between MUSC 136T and these type strains ranged from 22.7 ± 0.5% to 46.5 ± 0.2%. Overall, polyphasic approach studies indicated this strain represents a novel species, for which the name Streptomyces malaysiense sp. nov. is proposed. The potential bioactivities of this strain were explored by means of antioxidant and cytotoxic assays. Intriguingly, MUSC 136T exhibited strong antioxidative activities as evaluated by a panel of antioxidant assays. It was also found to possess high cytotoxic effect against HCT-116 cells, which probably mediated through altering p53 protein and intracellular glutathione levels. Chemical analysis of the extract using GC-MS further affirms that the strain produces chemopreventive related metabolites. PMID:27072394

  4. Streptomyces malaysiense sp. nov.: A novel Malaysian mangrove soil actinobacterium with antioxidative activity and cytotoxic potential against human cancer cell lines.

    PubMed

    Ser, Hooi-Leng; Palanisamy, Uma Devi; Yin, Wai-Fong; Chan, Kok-Gan; Goh, Bey-Hing; Lee, Learn-Han

    2016-01-01

    Actinobacteria from the unique intertidal ecosystem of the mangroves are known to produce novel, bioactive secondary metabolites. A novel strain known as MUSC 136(T) (=DSM 100712(T) = MCCC 1K01246(T)) which was isolated from Malaysian mangrove forest soil has proven to be no exception. Assessed by a polyphasic approach, its taxonomy showed a range of phylogenetic and chemotaxonomic properties consistent with the genus of Streptomyces. Phylogenetically, highest similarity was to Streptomyces misionensis NBRC 13063(T) (99.6%) along with two other strains (>98.9% sequence similarities). The DNA-DNA relatedness between MUSC 136(T) and these type strains ranged from 22.7 ± 0.5% to 46.5 ± 0.2%. Overall, polyphasic approach studies indicated this strain represents a novel species, for which the name Streptomyces malaysiense sp. nov. is proposed. The potential bioactivities of this strain were explored by means of antioxidant and cytotoxic assays. Intriguingly, MUSC 136(T) exhibited strong antioxidative activities as evaluated by a panel of antioxidant assays. It was also found to possess high cytotoxic effect against HCT-116 cells, which probably mediated through altering p53 protein and intracellular glutathione levels. Chemical analysis of the extract using GC-MS further affirms that the strain produces chemopreventive related metabolites. PMID:27072394

  5. Carbon-Ion Beam Irradiation Kills X-Ray-Resistant p53-Null Cancer Cells by Inducing Mitotic Catastrophe

    PubMed Central

    Amornwichet, Napapat; Oike, Takahiro; Shibata, Atsushi; Ogiwara, Hideaki; Tsuchiya, Naoto; Yamauchi, Motohiro; Saitoh, Yuka; Sekine, Ryota; Isono, Mayu; Yoshida, Yukari; Ohno, Tatsuya; Kohno, Takashi; Nakano, Takashi

    2014-01-01

    Background and Purpose To understand the mechanisms involved in the strong killing effect of carbon-ion beam irradiation on cancer cells with TP53 tumor suppressor gene deficiencies. Materials and Methods DNA damage responses after carbon-ion beam or X-ray irradiation in isogenic HCT116 colorectal cancer cell lines with and without TP53 (p53+/+ and p53-/-, respectively) were analyzed as follows: cell survival by clonogenic assay, cell death modes by morphologic observation of DAPI-stained nuclei, DNA double-strand breaks (DSBs) by immunostaining of phosphorylated H2AX (γH2AX), and cell cycle by flow cytometry and immunostaining of Ser10-phosphorylated histone H3. Results The p53-/- cells were more resistant than the p53+/+ cells to X-ray irradiation, while the sensitivities of the p53+/+ and p53-/- cells to carbon-ion beam irradiation were comparable. X-ray and carbon-ion beam irradiations predominantly induced apoptosis of the p53+/+ cells but not the p53-/- cells. In the p53-/- cells, carbon-ion beam irradiation, but not X-ray irradiation, markedly induced mitotic catastrophe that was associated with premature mitotic entry with harboring long-retained DSBs at 24 h post-irradiation. Conclusions Efficient induction of mitotic catastrophe in apoptosis-resistant p53-deficient cells implies a strong cancer cell-killing effect of carbon-ion beam irradiation that is independent of the p53 status, suggesting its biological advantage over X-ray treatment. PMID:25531293

  6. HDAC4 Promotes Growth of Colon Cancer Cells via Repression of p21

    PubMed Central

    Wilson, Andrew J.; Byun, Do-Sun; Nasser, Shannon; Murray, Lucas B.; Ayyanar, Kanyalakshmi; Arango, Diego; Figueroa, Maria; Melnick, Ari; Kao, Gary D.; Augenlicht, Leonard H.

    2008-01-01

    The class II Histone deacetylase (HDAC), HDAC4, is expressed in a tissue-specific manner, and it represses differentiation of specific cell types. We demonstrate here that HDAC4 is expressed in the proliferative zone in small intestine and colon and that its expression is down-regulated during intestinal differentiation in vivo and in vitro. Subcellular localization studies demonstrated HDAC4 expression was predominantly nuclear in proliferating HCT116 cells and relocalized to the cytoplasm after cell cycle arrest. Down-regulating HDAC4 expression by small interfering RNA (siRNA) in HCT116 cells induced growth inhibition and apoptosis in vitro, reduced xenograft tumor growth, and increased p21 transcription. Conversely, overexpression of HDAC4 repressed p21 promoter activity. p21 was likely a direct target of HDAC4, because HDAC4 down-regulation increased p21 mRNA when protein synthesis was inhibited by cycloheximide. The importance of p21 repression in HDAC4-mediated growth promotion was demonstrated by the failure of HDAC4 down-regulation to induce growth arrest in HCT116 p21-null cells. HDAC4 down-regulation failed to induce p21 when Sp1 was functionally inhibited by mithramycin or siRNA-mediated down-regulation. HDAC4 expression overlapped with that of Sp1, and a physical interaction was demonstrated by coimmunoprecipitation. Chromatin immunoprecipitation (ChIP) and sequential ChIP analyses demonstrated Sp1-dependent binding of HDAC4 to the proximal p21 promoter, likely directed through the HDAC4–HDAC3–N-CoR/SMRT corepressor complex. Consistent with increased transcription, HDAC4 or SMRT down-regulation resulted in increased histone H3 acetylation at the proximal p21 promoter locus. These studies identify HDAC4 as a novel regulator of colon cell proliferation through repression of p21. PMID:18632985

  7. Binding of the sialic acid-binding lectin, Siglec-9, to the membrane mucin, MUC1, induces recruitment of β-catenin and subsequent cell growth.

    PubMed

    Tanida, Shuhei; Akita, Kaoru; Ishida, Akiko; Mori, Yugo; Toda, Munetoyo; Inoue, Mizue; Ohta, Mariko; Yashiro, Masakazu; Sawada, Tetsuji; Hirakawa, Kosei; Nakada, Hiroshi

    2013-11-01

    Because MUC1 carries a variety of sialoglycans that are possibly recognized by the siglec family, we examined MUC1-binding siglecs and found that Siglec-9 prominently bound to MUC1. An immunochemical study showed that Siglec-9-positive immune cells were associated with MUC1-positive cells in human colon, pancreas, and breast tumor tissues. We investigated whether or not this interaction has any functional implications for MUC1-expressing cells. When mouse 3T3 fibroblast cells and a human colon cancer cell line, HCT116, stably transfected with MUC1cDNA were ligated with recombinant soluble Siglec-9, β-catenin was recruited to the MUC1 C-terminal domain, which was enhanced on stimulation with soluble Siglec-9 in dose- and time-dependent manners. A co-culture model of MUC1-expressing cells and Siglec-9-expressing cells mimicking the interaction between MUC1-expressing malignant cells, and Siglec-9-expressing immune cells in a tumor microenvironment was designed. Brief co-incubation of Siglec-9-expressing HEK293 cells, but not mock HEK293 cells, with MUC1-expressing cells similarly enhanced the recruitment of β-catenin to the MUC1 C-terminal domain. In addition, treatment of MUC1-expressing cells with neuraminidase almost completely abolished the effect of Siglec-9 on MUC1-mediated signaling. The recruited β-catenin was thereafter transported to the nucleus, leading to cell growth. These findings suggest that Siglec-9 expressed on immune cells may play a role as a potential counterreceptor for MUC1 and that this signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent pathway. PMID:24045940

  8. Innate Response to Human Cancer Cells with or without IL-2 Receptor Common γ-Chain Function in NOD Background Mice Lacking Adaptive Immunity.

    PubMed

    Nishime, Chiyoko; Kawai, Kenji; Yamamoto, Takehiro; Katano, Ikumi; Monnai, Makoto; Goda, Nobuhito; Mizushima, Tomoko; Suemizu, Hiroshi; Nakamura, Masato; Murata, Mitsuru; Suematsu, Makoto; Wakui, Masatoshi

    2015-08-15

    Immunodeficient hosts exhibit high acceptance of xenogeneic or neoplastic cells mainly due to lack of adaptive immunity, although it still remains to be elucidated how innate response affects the engraftment. IL-2R common γ-chain (IL-2Rγc) signaling is required for development of NK cells and a subset of dendritic cells producing IFN-γ. To better understand innate response in the absence of adaptive immunity, we examined amounts of metastatic foci in the livers after intrasplenic transfer of human colon cancer HCT116 cells into NOD/SCID versus NOD/SCID/IL-2Rγc (null) (NOG) hosts. The intravital microscopic imaging of livers in the hosts depleted of NK cells and/or macrophages revealed that IL-2Rγc function critically contributes to elimination of cancer cells without the need for NK cells and macrophages. In the absence of IL-2Rγc, macrophages play a role in the defense against tumors despite the NOD Sirpa allele, which allows human CD47 to bind to the encoded signal regulatory protein α to inhibit macrophage phagocytosis of human cells. Analogous experiments using human pancreas cancer MIA PaCa-2 cells provided findings roughly similar to those from the experiments using HCT116 cells except for lack of suppression of metastases by macrophages in NOG hosts. Administration of mouse IFN-γ to NOG hosts appeared to partially compensate lack of IL-2Rγc-dependent elimination of transferred HCT116 cells. These results provide insights into the nature of innate response in the absence of adaptive immunity, aiding in developing tumor xenograft models in experimental oncology. PMID:26170385

  9. Potential anticancer agents. I. Synthesis of isoxazole moiety containing quinazoline derivatives and preliminarily in vitro anticancer activity.

    PubMed

    Yong, Jian-Ping; Lu, Can-Zhong; Wu, Xiaoyuan

    2015-01-01

    14 new structures of isoxazole-moiety-containing quinazoline derivatives(3a~3n) were synthesized for the first time and characterized by IR, (1)H NMR, (13)C NMR, ESI-MS. Subsequently, their in vitro anticancer activity against A549, HCT116 and MCF-7 cell lines was preliminarily evaluated using the MTT method. Among them, most compounds showed good to excellent anticancer activity, especially 3d, 3i, 3k and 3m exhibited the more potent anticancer activity against A549, HCT116 and MCF-7 cell lines, which can be regarded as the promising drug candidates for development of anticancer drugs. PMID:25142319

  10. Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by inducing G1 arrest and apoptosis

    PubMed Central

    Jada, S R; Matthews, C; Saad, M S; Hamzah, A S; Lajis, N H; Stevens, M F G; Stanslas, J

    2008-01-01

    Background and purpose: Andrographolide, the major phytoconstituent of Andrographis paniculata, was previously shown by us to have activity against breast cancer. This led to synthesis of new andrographolide analogues to find compounds with better activity than the parent compound. Selected benzylidene derivatives were investigated for their mechanisms of action by studying their effects on the cell cycle progression and cell death. Experimental approach: Microculture tetrazolium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulphorhodamine B (SRB) assays were utilized in assessing the in vitro growth inhibition and cytotoxicity of compounds. Flow cytometry was used to analyse the cell cycle distribution of control and treated cells. CDK1 and CDK4 levels were determined by western blotting. Apoptotic cell death was assessed by fluorescence microscopy and flow cytometry. Key results: Compounds, in nanomolar to micromolar concentrations, exhibited growth inhibition and cytotoxicity in MCF-7 (breast) and HCT-116 (colon) cancer cells. In the NCI screen, 3,19-(2-bromobenzylidene) andrographolide (SRJ09) and 3,19-(3-chloro-4-fluorobenzylidene) andrographolide (SRJ23) showed greater cytotoxic potency and selectivity than andrographolide. SRJ09 and SRJ23 induced G1 arrest and apoptosis in MCF-7 and HCT-116 cells, respectively. SRJ09 downregulated CDK4 but not CDK1 level in MCF-7 cells. Apoptosis induced by SRJ09 and SRJ23 in HCT-116 cells was confirmed by annexin V-FITC/PI flow cytometry analysis. Conclusion and implications: The new benzylidene derivatives of andrographolide are potential anticancer agents. SRJ09 emerged as the lead compound in this study, exhibiting anticancer activity by downregulating CDK4 to promote a G1 phase cell cycle arrest, coupled with induction of apoptosis. PMID:18806812

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

    SciTech Connect

    Simoes, Maria L.; Hockley, Sarah L.; Schwerdtle, Tanja; Schmeiser, Heinz H.; Phillips, David H.; Arlt, Volker M.

    2008-10-01

    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.

  12. Triptolide abrogates growth of colon cancer and induces cell cycle arrest by inhibiting transcriptional activation of E2F.

    PubMed

    Oliveira, Amanda R; Beyer, Georg; Chugh, Rohit; Skube, Steven J; Majumder, Kaustav; Banerjee, Sulagna; Sangwan, Veena; Li, Lihua; Dawra, Rajinder K; Subramanian, Subbaya; Saluja, Ashok K; Dudeja, Vikas

    2015-06-01

    Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1-S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models. PMID:25893635

  13. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

    Apparatus for processing a metallic fluid containing iron oxide, container for a molten metal including an electrically conductive refractory disposed for contact with the molten metal which contains iron oxide, an electrolyte in the form of a basic slag on top of the molten metal, an electrode in the container in contcat with the slag electrically separated from the refractory, and means for establishing a voltage across the refractory and the electrode to reduce iron oxide to iron at the surface of the refractory in contact with the iron oxide containing fluid. A process is disclosed for refining an iron product containing not more than about 10% by weight oxygen and not more than about 10% by weight sulfur, comprising providing an electrolyte of a slag containing one or more of calcium oxide, magnesium oxide, silica or alumina, providing a cathode of the iron product in contact with the electrolyte, providing an anode in contact with the electrolyte electrically separated from the cathode, and operating an electrochemical cell formed by the anode, the cathode and the electrolyte to separate oxygen or sulfur present in the iron product therefrom.

  14. Differential SELEX in Human Glioma Cell Lines

    PubMed Central

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

    2009-01-01

    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

  15. Synergistic inhibition of colon cancer cell growth with nanoemulsion-loaded paclitaxel and PI3K/mTOR dual inhibitor BEZ235 through apoptosis

    PubMed Central

    Zou, Hong; Li, Li; Garcia Carcedo, Ines; Xu, Zhi Ping; Monteiro, Michael; Gu, Wenyi

    2016-01-01

    Colon cancer is the third most common cancer in the world, with drug resistance and metastasis being the major challenges to effective treatments. To overcome this, combination therapy with different chemotherapeutics is a common practice. In this study, we demonstrated that paclitaxel (PTX) together with BEZ235 exhibited a synergetic inhibition effect on colon cancer cell growth. Furthermore, nanoemulsion (NE)-loaded PTX and BEZ235 were more effective than the free drug, and a combination treatment of both NE drugs increased the efficiency of the treatments. BEZ235 pretreatment before adding PTX sensitized the cancer cells further, suggesting a synergistic inhibition effect through the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin pathway. The 50% inhibitory concentrations for BEZ235 were 127.1 nM and 145.0 nM and for PTX 9.7 nM and 9.5 nM for HCT-116 and HT-29 cells, respectively. When loaded with NE the 50% inhibitory concentrations for BEZ235 decreased to 52.6 nM and 55.6 nM and for PTX to 1.9 nM and 2.3 nM for HCT-116 and HT-29 cells, respectively. Combination treatment with 10 nM NE-BEZ235 and 0.6 nM and 1.78 nM NE-PTX could kill 50% of HCT-116 and HT-29, respectively. The cell death caused by the treatment was through apoptotic cell death, which coincided with decreased expression of anti-apoptotic protein B-cell lymphoma 2. Our data indicate that the combination therapy of PTX with the phosphatidylinositol-3-kinases/protein kinase B/mammalian target of rapamycin dual inhibitor BEZ235 using NE delivery may hold promise for a more effective approach for colon cancer treatment. PMID:27226714

  16. The natural product peiminine represses colorectal carcinoma tumor growth by inducing autophagic cell death

    SciTech Connect

    Lyu, Qing; Tou, Fangfang; Su, Hong; Wu, Xiaoyong; Chen, Xinyi; Zheng, Zhi

    2015-06-19

    Autophagy is evolutionarily conservative in eukaryotic cells that engulf cellular long-lived proteins and organelles, and it degrades the contents through fusion with lysosomes, via which the cell acquires recycled building blocks for the synthesis of new molecules. In this study, we revealed that peiminine induces cell death and enhances autophagic flux in colorectal carcinoma HCT-116 cells. We determined that peiminine enhances the autophagic flux by repressing the phosphorylation of mTOR through inhibiting upstream signals. Knocking down ATG5 greatly reduced the peiminine-induced cell death in wild-type HCT-116 cells, while treating Bax/Bak-deficient cells with peiminine resulted in significant cell death. In summary, our discoveries demonstrated that peiminine represses colorectal carcinoma cell proliferation and cell growth by inducing autophagic cell death. - Highlights: • Peiminine induces autophagy and upregulates autophagic flux. • Peiminine represses colorectal carcinoma tumor growth. • Peiminine induces autophagic cell death. • Peiminine represses mTOR phosphorylation by influencing PI3K/Akt and AMPK pathway.

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

    PubMed Central

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

    2016-01-01

    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 [1]-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 either senescence or 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-α 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-β or IFN-γ, suggesting that IFNs may have potential as therapeutic agents in cancer cells with BRCA2 mutations. PMID:25043256

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

    PubMed

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

    2014-11-01

    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 IFNα-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 IFNβ or IFNγ, suggesting that IFNs may have potential as therapeutic agents in cancer cells with BRCA2 mutations. The GEO Accession No. for microarray analysis is GSE54830. PMID:25043256

  19. CCL21 Facilitates Chemoresistance and Cancer Stem Cell-Like Properties of Colorectal Cancer Cells through AKT/GSK-3β/Snail Signals

    PubMed Central

    Lu, Lin-Lin; Chen, Xiao-Hui; Zhang, Ge; Liu, Zong-Cai; Wu, Nong; Wang, Hao; Qi, Yi-Fei; Wang, Hong-Sheng; Cai, Shao Hui; Du, Jun

    2016-01-01

    Some evidence indicated that chemoresistance associates with the acquisition of cancer stem-like properties. Recent studies suggested that chemokines can promote the chemoresistance and stem cell properties in various cancer cells, while the underling mechanism is still not completely illustrated. In our study, we found that CCL21 can upregulate the expression of P-glycoprotein (P-gp) and stem cell property markers such as Bmi-1, Nanog, and OCT-4 in colorectal cancer (CRC) HCT116 cells and then improve the cell survival rate and mammosphere formation. Our results suggested that Snail was crucial for CCL21-mediated chemoresistance and cancer stem cell property in CRC cells. Further, we observed that CCL21 treatment increased the protein but not mRNA levels of Snail, which suggested that CCL21 upregulates Snail via posttranscriptional ways. The downstream signals AKT/GSK-3β mediated CCL21 induced the upregulation of Snail due to the fact that CCL21 treatment can obviously phosphorylate both AKT and GSK-3β. The inhibitor of PI3K/Akt, LY294002 significantly abolished CCL21 induced chemoresistance and mammosphere formation of HCT116 cells. Collectively, our results in the present study revealed that CCL21 can facilitate chemoresistance and stem cell property of CRC cells via the upregulation of P-gp, Bmi-1, Nanog, and OCT-4 through AKT/GSK-3β/Snail signals, which suggested a potential therapeutic approach to CRC patients. PMID:27057280

  20. The pivotal role of intracellular calcium in oxaliplatin-induced inhibition of neurite outgrowth but not cell death in differentiated PC12 cells.

    PubMed

    Takeshita, Miki; Banno, Yoshiko; Nakamura, Mitsuhiro; Otsuka, Mayuko; Teramachi, Hitomi; Tsuchiya, Teruo; Itoh, Yoshinori

    2011-11-21

    The antineoplastic efficacy of oxaliplatin, a widely used anticancer drug, is restricted by its adverse effects such as peripheral neuropathy. Infusing a combination of calcium gluconate and magnesium sulfate (Ca/Mg) suppresses the acute neurotoxic side effects of oxaliplatin, although the mechanism is unclear. To elucidate the molecular mechanisms of oxaliplatin-induced neurotoxicity and the effects of Ca/Mg against this toxicity, we examined the effect of Ca/Mg on oxaliplatin-induced inhibition of neurite outgrowth in PC12 cells, a commonly used neuronal cell model. Oxaliplatin and oxalate suppressed nerve growth factor (NGF)-induced neurite outgrowth and reduced the NGF-mediated increase in the intracellular calcium concentration [Ca(2+)](i). A calcium-chelating agent, BAPTA/AM, also exhibited similar inhibitory effects on neurite outgrowth and [Ca(2+)](i). The addition of Ca/Mg attenuated these inhibitions induced by oxaliplatin and oxalate. The NGF-induced upregulation of growth-associated protein-43 (GAP-43) was suppressed by oxaliplatin and oxalate. Oxaliplatin, but not oxalate, suppressed NGF-stimulated extracellular signal-regulated kinase activation, and this inhibition was not affected by Ca/Mg. Ca/Mg did not modify the oxaliplatin-induced loss of cell viability or apoptosis in PC12 or HCT-116 cells, a human colorectal cancer cell line. These results suggest that the inhibition of neurite outgrowth but not tumor cell death induced by oxaliplatin is partly associated with reductions in [Ca(2+)](i) and GAP-43 expression, and this inhibition was suppressed by the addition of Ca/Mg. Therefore, it may be assumed that Ca/Mg is useful for protecting against oxaliplatin-induced neurotoxicity without reducing the antitumor activity of oxaliplatin. PMID:21981408

  1. IDO1 Metabolites Activate β-catenin Signaling to Promote Cancer Cell Proliferation and Colon Tumorigenesis in Mice

    PubMed Central

    Thaker, Ameet I.; Rao, M Suprada; Bishnupuri, Kumar S.; Kerr, Thomas A; Foster, Lynne; Marinshaw, Jeffrey M.; Newberry, Rodney D.; Stenson, William F.; Ciorba, Matthew A

    2013-01-01

    BACKGROUND & AIMS Indoleamine 2,3 dioxygenase-1 (IDO1) catabolizes tryptophan along the kynurenine pathway. Though IDO1 is expressed in inflamed and neoplastic epithelial cells of the colon, its role in colon tumorigenesis is not well understood. We used genetic and pharmacologic approaches to manipulate IDO1 activity in mice with colitis-associated cancer and human colon cancer cell lines. METHODS C57Bl6 wild type (control), IDO1−/−, Rag1−/−, Rag1/IDO1 double knockout mice were exposed to azoxymethane and dextran sodium sulfate (DSS) to induce colitis and tumorigenesis. Colitis severity was assessed by measurements of disease activity, cytokine levels and histologic analysis. In vitro experiments were conducted using HCT116 and HT29 human colon cancer cells. 1-methyl tryptophan and small interfering RNA were used to inhibit IDO1. Kynurenine pathway metabolites were used to simulate IDO1 activity. RESULTS C57Bl6 mice given pharmacologic inhibitors of IDO1 and IDO1−/− mice had lower tumor burdens and reduced proliferation in the neoplastic epithelium following administration of DSS and azoxymethane than control mice. These reductions were also observed in Rag1/IDO1 double knockout mice compared to Rag1−/− mice (which lack mature adaptive immunity). In human colon cancer cells, blockade of IDO1 activity reduced nuclear and activated β-catenin, transcription of its target genes (cyclin D1 and Axin2), and ultimately proliferation. Exogenous administration of IDO1 pathway metabolites kynurenine and quinolinic acid led to activation of β-catenin and proliferation of human colon cancer cells, and increased tumor growth in mice. CONCLUSIONS IDO1, which catabolizes tryptophan, promotes colitis-associated tumorigenesis in mice, independent of its ability to limit T-cell mediated immune surveillance. The epithelial cell-autonomous survival advantage provided by IDO1 to colon epithelial cells indicate its potential as a therapeutic target. PMID:23669411

  2. Disruption of thioredoxin metabolism enhances the toxicity of transforming growth factor β-activated kinase 1 (TAK1) inhibition in KRAS-mutated colon cancer cells.

    PubMed

    Hrabe, Jennifer E; O'Leary, Brianne R; Fath, Melissa A; Rodman, Samuel N; Button, Anna M; Domann, Frederick E; Spitz, Douglas R; Mezhir, James J

    2015-08-01

    Transforming growth factor β-activated kinase 1 (TAK1) is critical for survival of many KRAS mutated colorectal cancer cells, and TAK1 inhibition with 5Z-7-oxozeaenol has been associated with oxidative stress leading to tumor cell killing. When SW 620 and HCT 116 human colon cancer cells were treated with 5µM 5Z-7-oxozeaenol, cell viability, growth, and clonogenic survival were significantly decreased. Consistent with TAK1 inhibition being causally related to thiol-mediated oxidative stress, 10mM N-acetylcysteine (NAC) partially reversed the growth inhibitory effects of 5Z-7-oxozeaenol. In addition, 5Z-7-oxozeaenol also increased steady-state levels of H2DCFDA oxidation as well as increased levels of total glutathione (GSH) and glutathione disulfide (GSSG). Interestingly, depletion of GSH using buthionine sulfoximine did not significantly potentiate 5Z-7-oxozeaenol toxicity in either cell line. In contrast, pre-treatment of cells with auranofin (Au) to inhibit thioredoxin reductase activity significantly increased levels of oxidized thioredoxin as well as sensitized cells to 5Z-7-oxozeaenol-induced growth inhibition and clonogenic cell killing. These results were confirmed in SW 620 murine xenografts, where treatment with 5Z-7-oxozeaenol or with Au plus 5Z-7-oxozeaenol significantly inhibited growth, with Au plus 5Z-7-oxozeaenol trending toward greater growth inhibition compared to 5Z-7-oxozeaenol alone. These results support the hypothesis that thiol-mediated oxidative stress is causally related to TAK1-induced colon cancer cell killing. In addition, these results support the hypothesis that thioredoxin metabolism is a critical target for enhancing colon cancer cell killing via TAK1 inhibition and could represent an effective therapeutic strategy in patients with these highly resistant tumors. PMID:26114584

  3. Disruption of thioredoxin metabolism enhances the toxicity of transforming growth factor ?-activated kinase 1 (TAK1) inhibition in KRAS-mutated colon cancer cells

    PubMed Central

    Hrabe, Jennifer E.; OLeary, Brianne R.; Fath, Melissa A.; Rodman, Samuel N.; Button, Anna M.; Domann, Frederick E.; Spitz, Douglas R.; Mezhir, James J.

    2015-01-01

    Transforming growth factor ?-activated kinase 1 (TAK1) is critical for survival of many KRAS mutated colorectal cancer cells, and TAK1 inhibition with 5Z-7-oxozeaenol has been associated with oxidative stress leading to tumor cell killing. When SW 620 and HCT 116 human colon cancer cells were treated with 5M 5Z-7-oxozeaenol, cell viability, growth, and clonogenic survival were significantly decreased. Consistent with TAK1 inhibition being causally related to thiol-mediated oxidative stress, 10mM N-acetylcysteine (NAC) partially reversed the growth inhibitory effects of 5Z-7-oxozeaenol. In addition, 5Z-7-oxozeaenol also increased steady-state levels of H2DCFDA oxidation as well as increased levels of total glutathione (GSH) and glutathione disulfide (GSSG). Interestingly, depletion of GSH using buthionine sulfoximine did not significantly potentiate 5Z-7-oxozeaenol toxicity in either cell line. In contrast, pre-treatment of cells with auranofin (Au) to inhibit thioredoxin reductase activity significantly increased levels of oxidized thioredoxin as well as sensitized cells to 5Z-7-oxozeaenol-induced growth inhibition and clonogenic cell killing. These results were confirmed in SW 620 murine xenografts, where treatment with 5Z-7-oxozeaenol or with Au plus 5Z-7-oxozeaenol significantly inhibited growth, with Au plus 5Z-7-oxozeaenol trending toward greater growth inhibition compared to 5Z-7-oxozeaenol alone. These results support the hypothesis that thiol-mediated oxidative stress is causally related to TAK1-induced colon cancer cell killing. In addition, these results support the hypothesis that thioredoxin metabolism is a critical target for enhancing colon cancer cell killing via TAK1 inhibition and could represent an effective therapeutic strategy in patients with these highly resistant tumors. PMID:26114584

  4. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

    1995-07-30

    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.

  5. Circulating and tumor-infiltrating mucosal associated invariant T (MAIT) cells in colorectal cancer patients

    PubMed Central

    Ling, Limian; Lin, Yuyang; Zheng, Wenwen; Hong, Sen; Tang, Xiuqi; Zhao, Pingwei; Li, Ming; Ni, Jingsong; Li, Chenguang; Wang, Lei; Jiang, Yanfang

    2016-01-01

    Mucosal associated invariant T (MAIT) cells are important for immune defense against infectious pathogens and regulate the pathogenesis of various inflammatory diseases. However, their roles in the development of colorectal cancer (CRC) are still unclear. This study examined the phenotype, distribution, clinical relevance and potential function of MAIT cells in CRC patients. We found that the percentages of circulating memory CD8+ MAIT cells were significantly reduced while tumor infiltrating MAIT cells were increased, especially in patients with advanced CRC. The serum CEA levels were positively correlated with the percentages of tumor infiltrating MAIT cells in CRC patients, but negatively correlated with the percentages of circulating MAIT in advanced CRC patients. Activated circulating MAIT cells from CRC patients produced lower IFN-γ, but higher IL-17. Furthermore, higher levels of Vα7.2-Jα33, IFN-γ and IL-17A were expressed in the CRC tissues. Co-culture of activated MAIT cells with HCT116 cells enhanced IL-17 expression and induced HCT116 cell cycle arrest at G2/M phase in a contact- and dose-dependent manner, which was abrogated by treatment with anti-MR1. Therefore, MAIT cells preferably infiltrate into the solid tumor in CRC patients and may participate in the immune surveillance of CRC. PMID:26837580

  6. Circulating and tumor-infiltrating mucosal associated invariant T (MAIT) cells in colorectal cancer patients.

    PubMed

    Ling, Limian; Lin, Yuyang; Zheng, Wenwen; Hong, Sen; Tang, Xiuqi; Zhao, Pingwei; Li, Ming; Ni, Jingsong; Li, Chenguang; Wang, Lei; Jiang, Yanfang

    2016-01-01

    Mucosal associated invariant T (MAIT) cells are important for immune defense against infectious pathogens and regulate the pathogenesis of various inflammatory diseases. However, their roles in the development of colorectal cancer (CRC) are still unclear. This study examined the phenotype, distribution, clinical relevance and potential function of MAIT cells in CRC patients. We found that the percentages of circulating memory CD8(+) MAIT cells were significantly reduced while tumor infiltrating MAIT cells were increased, especially in patients with advanced CRC. The serum CEA levels were positively correlated with the percentages of tumor infiltrating MAIT cells in CRC patients, but negatively correlated with the percentages of circulating MAIT in advanced CRC patients. Activated circulating MAIT cells from CRC patients produced lower IFN-γ, but higher IL-17. Furthermore, higher levels of Vα7.2-Jα33, IFN-γ and IL-17A were expressed in the CRC tissues. Co-culture of activated MAIT cells with HCT116 cells enhanced IL-17 expression and induced HCT116 cell cycle arrest at G2/M phase in a contact- and dose-dependent manner, which was abrogated by treatment with anti-MR1. Therefore, MAIT cells preferably infiltrate into the solid tumor in CRC patients and may participate in the immune surveillance of CRC. PMID:26837580

  7. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been widely done using labor intensive and cytotoxic liposome-based transfection reagents....

  8. Comprehensive analysis for histone acetylation of human colon cancer cells treated with a novel HDAC inhibitor.

    PubMed

    Zhao, Yunlong; Fang, Xiuli; Wang, Ye; Zhang, Junmei; Jiang, Sheng; Liu, Zhe; Ma, Zhenyi; Xu, Liyan; Li, Enmin; Zhang, Kai

    2014-01-01

    Extensive evidence suggests that dysregulation of histone lysine acetylation is intimately linked with the development of cancer in epigenetic level. Histone acetylation on lysine is regulated mainly by the "pencil"--Histone acetyltransferases (HATs) and the "eraser"--Histone deacetylases HDACs. Dramatic elevation of global histone deacetylation is considered as a biomarker for cancer. Therefore, current antitumor drug design often targets HDACs, inhibiting overexpressed HDAC in tumor cells with natural or synthesized small molecules like largazole. Recently, a novel largazole derivative (largazole-7) was designed and prepared by replacement of Val 1 with tyrosine, and this modification increases selectivity toward human cancer cells over normal cells more than 100-fold. However, it is unclear about the dynamic level of histone acetylation under the treatment of this drug. It is also unclear whether the other modifications are also affected by largazole-7 treatment. Therefore, a global mapping of modifications on the histone proteins of cancer cell line treated by this drug may be of great benefit to elucidating its molecular mechanisms and exploring its potent as an antitumor drug. To realize the goal, we combined stable isotope labeling by amino acids in cell culture (SILAC) and high resolution MS for comprehensive identification and quantitative analysis of histone lysine acetylation and other modifications of Human Colon Cancer Cells (HCT-116) with and without treatment of largazole-7. In this analysis, we identified 68 histone PTMs in 38 sites on core histones, including lysine acetylation, methylation and butyrylation, a novel lysine modification. Further quantitative analysis not only discovered the global increased acetylated lysines, but also observed the changes of abundance of lysine methylation and butyrylation under stimulation of the drug. To our knowledge, it is the first report that regulation of largazole-7 against lysine butyrylation. Our study expands the catalog of histone marks in cancer, and provides an approach for understanding the known and new epigenetic marks under treatment of drugs. PMID:23888955

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

    SciTech Connect

    Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg ; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

    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.

  10. Antiproliferative efficacy of Tabernaemontana divaricata against HEP2 cell line and Vero cell line

    PubMed Central

    Kumar, Arvind; Selvakumar, S.

    2015-01-01

    Background: Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2). Materials and Method: The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability. Results: The extract on Hep 2 cell line up to 7.8 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Conclusion: Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line). PMID:26109773

  11. Characterization of swine testicular cell line as immature porcine Sertoli cell line.

    PubMed

    Ma, Changping; Song, Huibin; Guan, Kaifeng; Zhou, Jiawei; Xia, Xuanyan; Li, Fenge

    2016-04-01

    Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions. PMID:26744029

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

    PubMed Central

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

    2014-01-01

    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 (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, 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-κB, MMP-13), TGF-β3, 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-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis. PMID:25238234

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

    SciTech Connect

    Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra; De Ridder, Mark

    2013-03-01

    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 transcriptionpolymerase 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 oxideproducing 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.

  14. Cannabinoid receptor-independent cytotoxic effects of cannabinoids in human colorectal carcinoma cells: synergism with 5-fluorouracil.

    PubMed

    Gustafsson, Sofia B; Lindgren, Theres; Jonsson, Maria; Jacobsson, Stig O P

    2009-03-01

    Cannabinoids (CBs) have been found to exert antiproliferative effects upon a variety of cancer cells, including colorectal carcinoma cells. However, little is known about the signalling mechanisms behind the antitumoural effect in these cells, whether the effects are shared by endogenous lipids related to endocannabinoids, or whether such effects are synergistic with treatment paradigms currently used in the clinic. The aim of this preclinical study was to investigate the effect of synthetic and endogenous CBs and their related fatty acids on the viability of human colorectal carcinoma Caco-2 cells, and to determine whether CB effects are synergistic with those seen with the pyrimidine antagonist 5-fluorouracil (5-FU). The synthetic CB HU 210, the endogenous CB anandamide, the endogenous structural analogue of anandamide, N-arachidonoyl glycine (NAGly), as well as the related polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid showed antiproliferative and cytotoxic effects in the Caco-2 cells, as measured by using [(3)H]-thymidine incorporation assay, the CyQUANT proliferation assay and calcein-AM fluorescence. HU 210 was the most potent compound examined, followed by anandamide, whereas NAGly showed equal potency and efficacy as the polyunsaturated fatty acids. Furthermore, HU 210 and 5-FU produced synergistic effects in the Caco-2 cells, but not in the human colorectal carcinoma cell lines HCT116 or HT29. The compounds examined produced cytotoxic, rather than antiproliferative effects, by a mechanism not involving CB receptors, since the CB receptor antagonists AM251 and AM630 did not attenuate the effects, nor did pertussis toxin. However, alpha-tocopherol and the nitric oxide synthase inhibitor L-NAME attenuated the CB toxicity, suggesting involvement of oxidative stress. It is concluded that the CB system may provide new targets for the development of drugs to treat colorectal cancer. PMID:18629502

  15. Effect of dapagliflozin on colon cancer cell [Rapid Communication].

    PubMed

    Saito, Tsugumichi; Okada, Shuichi; Yamada, Eijiro; Shimoda, Yoko; Osaki, Aya; Tagaya, Yuko; Shibusawa, Ryo; Okada, Junichi; Yamada, Masanobu

    2015-12-27

    Dapagliflozin is a SGLT2 (Sodium/Glucose cotransporter 2) inhibitor that reduces circulating glucose levels in type 2 diabetic patients by blocking the SGLT2-dependent reabsorption of glucose in the kidney. Dapagliflozin is metabolized by UGT1A9 (UDP Glucuronosyltransferase 1 family, Polypeptidase A9), suppressing its SGLT2 inhibitor activity. However little information is available on whether dapagliflozin acts in the absence of dapagliflozin metabolism. Treatment with 0.5?M dapagliflozin significantly reduced the number of HCT116 cells, which express SGLT2 but not UGT1A9. This was independent of SGLT2 inhibition, as the SGLT2 inhibitor phlorizin had no effect. Dapagliflozin also enhanced Erk phosphorylation but without changing levels of uncleaved and cleaved PPAR and uncleaved caspase-3, suggesting that the cause of the decrease in HCT116 cell number was apoptosis independent cell death. Taken together, these data indicate a new potential role for dapagliflozin as an anticancer reagent in tumor cell populations that do not express UGT1A9. PMID:26522271

  16. Epithelial-Mesenchymal Transition Associates with Maintenance of Stemness in Spheroid-Derived Stem-Like Colon Cancer Cells

    PubMed Central

    Fang, Jia-Feng; Zhang, Shi; Zhang, Fu-Cheng; Zhang, Hai-Bo; Lan, Tian-Yun; Lu, Hui-Qiong; Wei, Hong-Bo

    2013-01-01

    Despite earlier studies demonstrating characteristics of colon cancer stem cells (CCSCs) and the role of epithelial-mesenchymal transition (EMT) in tumor development, it remains controversial as to the relationship between CCSCs and EMT. In this study, in order to present an insight into this relationship in colon cancer, we developed HCT116 and HT29 sphere models, which are known to be the cells enriching cancer stem cells. Compared to their parental counterparts, spheroid cells displayed lower homotypic/heterotypic adhesion but higher in vitro migratory/invasive capacity, as well as higher tumorigenic and metastatic potential in vivo. The spheroid cells also demonstrated down-regulated E-cadherin and up-regulated α-SMA and Vimentin expression, which is the typical phenotype of EMT. In order to explore whether this phenomenon is associated to activation of Wnt/β-catenin pathway, we detected several key signaling molecules. Compared with their parental cells, HCT116 and HT29 spheroid cells demonstrated down-regulated expression of GSK3β, but up-regulated expression of Slug and Snail. And also, the up-regulation of nucleus β-catenin in spheroid cells indicated that the free β-catenin transferred from cytoplasm to cell nucleus. Our findings indicate that spheroid cells have the characteristics of colon cancer stem cells, and EMT may account for their stemness and malignancy. And persistent activation of Wnt/β-catenin pathway may play an important role in the EMT of CCSCs. PMID:24039918

  17. Stem cell factor suppresses apoptosis in neuroblastoma cell lines.

    PubMed

    Timeus, F; Crescenzio, N; Valle, P; Pistamiglio, P; Piglione, M; Garelli, E; Ricotti, E; Rocchi, P; Strippoli, P; Cordero di Montezemolo, L; Madon, E; Ramenghi, U; Basso, G

    1997-11-01

    Stem cell factor (SCF) is a glycoprotein growth factor produced by marrow stromal cells that acts after binding to its specific surface receptor, which is the protein encoded by the protooncogene c-kit. SCF synergizes with specific lineage factors in promoting the proliferation of primitive hematopoietic progenitors, and has been administered to expand the pool of these progenitors in cancer patients treated with high-dose chemotherapy. SCF and its c-kit receptor are expressed by some tumor cells, including myeloid leukemia, breast carcinoma, small cell lung carcinoma, melanoma, gynecological tumors, and testicular germ cell tumors. Previous studies of SCF in neuroblastoma have produced conflicting conclusions. To explore the role of SCF in neuroblastoma, we studied five neuroblastoma lines (IMR-5, SK-N-SH, SK-N-BE, AF8, and SJ-N-KP) and the neuroepithelioma line CHP-100. All lines expressed mRNA for c-kit and c-kit protein at low intensity as measured by flow cytometry, and secreted SCF in medium culture as shown by ELISA. Exogenous SCF did not modify 3H thymidine uptake in the neuroblastoma and neuroepithelioma cell lines. After 6 days' culture in the presence of anti-c-kit, the number of viable neuroblastoma cells was significantly lower than the control, and terminal deoxynucleotidyl transferase assay showed a substantial increase of apoptotic cells: The percentage of positive cells was 1-3% in the control lines, whereas in the presence of anti c-kit it varied from 29% of SK-N-BE to 92% of CHP-100. After 9 days' culture in the presence of anti-c-kit, no viable cells were detectable. These data indicate that SCF is produced by some neuroblastoma cell lines via an autocrine loop to protect them from apoptosis. PMID:9357969

  18. Spontaneous Cell Competition in Immortalized Mammalian Cell Lines

    PubMed Central

    Penzo-Méndez, Alfredo I.; Chen, Yi-Ju; Li, Jinyang; Witze, Eric S.; Stanger, Ben Z.

    2015-01-01

    Cell competition is a form of cell-cell interaction by which cells compare relative levels of fitness, resulting in the active elimination of less-fit cells, “losers,” by more-fit cells, “winners.” Here, we show that in three routinely-used mammalian cell lines – U2OS, 3T3, and MDCK cells – sub-clones arise stochastically that exhibit context-dependent competitive behavior. Specifically, cell death is elicited when winner and loser sub-clones are cultured together but not alone. Cell competition and elimination in these cell lines is caspase-dependent and requires cell-cell contact but does not require de novo RNA synthesis. Moreover, we show that the phenomenon involves differences in cellular metabolism. Hence, our study demonstrates that cell competition is a common feature of immortalized mammalian cells in vitro and implicates cellular metabolism as a mechanism by which cells sense relative levels of “fitness.” PMID:26200654

  19. Functional features of cancer stem cells in melanoma cell lines

    PubMed Central

    2013-01-01

    Background Recent evidence suggests a subset of cells within a tumor with "stem-like" characteristics. These cells are able to transplant tumors in immunodeficient hosts. Distinct from non-malignant stem cells, cancer stem cells (CSC) show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumor cells, and resistance to chemotherapy or radiation. They are often characterized by elevated expression of stem cell surface markers, in particular CD133, and sets of differentially expressed stem cell-associated genes. CSC are usually rare in clinical specimens and hardly amenable to functional studies and gene expression profiling. In this study, a panel of heterogenous melanoma cell lines was screened for typical CSC features. Methods Nine heterogeneous metastatic melanoma cell lines including D10 and WM115 were studied. Cell lines were phenotyped using flow cytometry and clonogenic assays were performed by limiting dilution analysis on magnetically sorted cells. Spheroidal growth was investigated in pretreated flasks. Gene expression profiles were assessed by using real-time rt-PCR and DNA microarrays. Magnetically sorted tumor cells were subcutaneously injected into the flanks of immunodeficient mice. Comparative immunohistochemistry was performed on xenografts and primary human melanoma sections. Results D10 cells expressed CD133 with a significantly higher clonogenic capacity as compared to CD133- cells. Na8, D10, and HBL cells formed spheroids on poly-HEMA-coated flasks. D10, Me39, RE, and WM115 cells expressed at least 2 of the 3 regulatory core transcription factors SOX2, NANOG, and OCT4 involved in the maintenance of stemness in mesenchymal stem cells. Gene expression profiling on CD133+ and CD133- D10 cells revealed 68 up- and 47 downregulated genes (+/-1.3 fold). Two genes, MGP and PROM1 (CD133), were outstandingly upregulated. CD133+ D10 cells formed tumors in NSG mice contrary to CD133- cells and CD133 expression was detected in xenografts and primary human melanoma sections using immunohistochemistry. Conclusions Established melanoma cell lines exhibit, to variable extents, the typical features of CSCs. The tumorigenic cell line D10, expressing CD133 and growing in spheroids and might qualify as a potential model of melanoma CSCs. PMID:23915418

  20. Tumor Cells Switch to Mitochondrial Oxidative Phosphorylation under Radiation via mTOR-Mediated Hexokinase II Inhibition - A Warburg-Reversing Effect

    PubMed Central

    Lu, Chung-Ling; Qin, Lili; Liu, Hsin-Chen; Candas, Demet; Fan, Ming; Li, Jian Jian

    2015-01-01

    A unique feature of cancer cells is to convert glucose into lactate to produce cellular energy, even under the presence of oxygen. Called aerobic glycolysis [The Warburg Effect] it has been extensively studied and the concept of aerobic glycolysis in tumor cells is generally accepted. However, it is not clear if aerobic glycolysis in tumor cells is fixed, or can be reversed, especially under therapeutic stress conditions. Here, we report that mTOR, a critical regulator in cell proliferation, can be relocated to mitochondria, and as a result, enhances oxidative phosphorylation and reduces glycolysis. Three tumor cell lines (breast cancer MCF-7, colon cancer HCT116 and glioblastoma U87) showed a quick relocation of mTOR to mitochondria after irradiation with a single dose 5 Gy, which was companied with decreased lactate production, increased mitochondrial ATP generation and oxygen consumption. Inhibition of mTOR by rapamycin blocked radiation-induced mTOR mitochondrial relocation and the shift of glycolysis to mitochondrial respiration, and reduced the clonogenic survival. In irradiated cells, mTOR formed a complex with Hexokinase II [HK II], a key mitochondrial protein in regulation of glycolysis, causing reduced HK II enzymatic activity. These results support a novel mechanism by which tumor cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics from predominantly aerobic glycolysis to mitochondrial oxidative phosphorylation. Such a “waking-up” pathway for mitochondrial bioenergetics demonstrates a flexible feature in the energy metabolism of cancer cells, and may be required for additional cellular energy consumption for damage repair and survival. Thus, the reversible cellular energy metabolisms should be considered in blocking tumor metabolism and may be targeted to sensitize them in anti-cancer therapy. PMID:25807077

  1. Tumor cells switch to mitochondrial oxidative phosphorylation under radiation via mTOR-mediated hexokinase II inhibition--a Warburg-reversing effect.

    PubMed

    Lu, Chung-Ling; Qin, Lili; Liu, Hsin-Chen; Candas, Demet; Fan, Ming; Li, Jian Jian

    2015-01-01

    A unique feature of cancer cells is to convert glucose into lactate to produce cellular energy, even under the presence of oxygen. Called aerobic glycolysis [The Warburg Effect] it has been extensively studied and the concept of aerobic glycolysis in tumor cells is generally accepted. However, it is not clear if aerobic glycolysis in tumor cells is fixed, or can be reversed, especially under therapeutic stress conditions. Here, we report that mTOR, a critical regulator in cell proliferation, can be relocated to mitochondria, and as a result, enhances oxidative phosphorylation and reduces glycolysis. Three tumor cell lines (breast cancer MCF-7, colon cancer HCT116 and glioblastoma U87) showed a quick relocation of mTOR to mitochondria after irradiation with a single dose 5 Gy, which was companied with decreased lactate production, increased mitochondrial ATP generation and oxygen consumption. Inhibition of mTOR by rapamycin blocked radiation-induced mTOR mitochondrial relocation and the shift of glycolysis to mitochondrial respiration, and reduced the clonogenic survival. In irradiated cells, mTOR formed a complex with Hexokinase II [HK II], a key mitochondrial protein in regulation of glycolysis, causing reduced HK II enzymatic activity. These results support a novel mechanism by which tumor cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics from predominantly aerobic glycolysis to mitochondrial oxidative phosphorylation. Such a "waking-up" pathway for mitochondrial bioenergetics demonstrates a flexible feature in the energy metabolism of cancer cells, and may be required for additional cellular energy consumption for damage repair and survival. Thus, the reversible cellular energy metabolisms should be considered in blocking tumor metabolism and may be targeted to sensitize them in anti-cancer therapy. PMID:25807077

  2. Overexpression of KiSS-1 reduces colorectal cancer cell invasion by downregulating MMP-9 via blocking PI3K/Akt/NF-κB signal pathway.

    PubMed

    Chen, Shaoqin; Chen, Wei; Zhang, Xiang; Lin, Suyong; Chen, Zhihua

    2016-04-01

    Metastasis of colorectal cancer (CRC) depends critically on MMP-9. KiSS-1 is a human malignant melanoma metastasis-suppressor gene. Thus, the interaction between MMP-9 and KiSS-1 has drawn considerable attention in recent years. In the present study, it was hypothesized that KiSS-1 gene could repress the metastatic potential of colorectal cancer cells by inhibiting the expression of MMP-9. Stable transfection of KiSS-1 specific siRNA and KiSS-1 expression vector in human CRC cell line HCT-116 was achieved by lentivirus infection. Moreover, the cell proliferation, invasiveness, and apoptosis were evaluated by CCK-8 method, transwell experiment, and fluorescence activated cell sorter, respectively. We also investigated the expression of MMP-9, PI3K, Akt, pAKt, and NF-кB subunit p65 using western blotting. KiSS-1 overexpression significantly decreased the cell proliferation and invasiveness of HCT-119 cells, while apoptosis was enhanced. The result of western blotting showed that synthesis of MMP-9, PI3K, p65, and phosphorylation of Akt were significantly blocked by overexpression of KiSS-1. Concatenated treatment of KiSS-1 overexpression vector with PI3K and Akt agonists attenuated the effect of KiSS-1 on the biological activity of CRC cells and also released the expression of MMP-9, PI3K, p65, and phosphorylation of Akt from the influence of overexpression of KiSS-1. Overexpression of KiSS-1 suppressed the invasiveness of CRC cells, and the gene exerted its function by reducing the expression of MMP-9 via blocking of tge PI3K/Akt/NF-κB pathway. PMID:26847533

  3. Profilin potentiates chemotherapeutic agents mediated cell death via suppression of NF-κB and upregulation of p53.

    PubMed

    Zaidi, Adeel H; Raviprakash, Nune; Mokhamatam, Raveendra B; Gupta, Pankaj; Manna, Sunil K

    2016-04-01

    The molecular mechanism by which Profilin acts as a tumor suppressor is still unclear. Several chemotherapeutic agents, used till date either have unfavorable side effects or acquired resistance in tumor cells. Our findings show that Profilin enhances cell death mediated by several chemotherapeutic-agents. The activation of NF-κB and its dependent genes, mediated by paclitaxel and vinblastine, was completely inhibited in Profilin overexpressing cells. This inhibition was due to the Profilin mediated attenuation of IκBα degradation, thereby preventing p65 nuclear translocation and low NF-κB DNA binding activity.Moreover, Profilin increases level of p53 in the presence of known inducers, such as doxorubicin, vinblastine, and benzofuran. This increased p53 level leads to enhanced cell death as indicated by activation of caspases 3, 8, 9, which results in cleavage of PARP.Furthermore, knocking down of p53 in Profilin overexpressing cells leads to decreased cell death. Ectopic expression of Profilin in HCT116 p53 knock out cells showed lesser cell death as compared to the HCT116 p53 wild type cells. For the first time, we provide evidences, which suggest that Profilin synergizes with chemotherapeutic drugs to induce tumor cell death by regulating NF-κB and p53. Thus, modulation of Profilin may be a useful strategy for effective combination therapy. PMID:26842845

  4. Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

    PubMed Central

    Sundaramoorthy, Pasupathi; Sim, Jae Jun; Jang, Yeong-Su; Mishra, Siddhartha Kumar; Jeong, Keun-Yeong; Mander, Poonam; Chul, Oh Byung; Shim, Won-Sik; Oh, Seung Hyun; Nam, Ky-Youb; Kim, Hwan Mook

    2015-01-01

    Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calp