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Sample records for cell lines hct-116

  1. 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 - 50?g/mL, and then a plateau, whereas at 12.5 and 25 ?g/mL, there is a slight growth suppression in QBC-939 cells, but then a steady suppression from 50, 100 and 200?g/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

  2. Anti-cancer effects of 2-oxoquinoline derivatives on the HCT116 and LoVo human colon cancer cell lines.

    PubMed

    Fang, Feng-Qi; Guo, Hui-Shu; Zhang, Jie; Ban, Li-Ying; Liu, Ji-Wei; Yu, Pei-Yao

    2015-12-01

    The present study demonstrated the anti-tumor effects of the quinoline derivative [5-(3-chloro-oxo-4-phenyl-cyclobutyl)-quinoli-8-yl-oxy] acetic acid hydrazide (CQAH) against colorectal carcinoma. Substantial apoptotic effects of CQAH on HCT116 and LoVo human colon cancer cell lines were observed. Apoptosis was identified based on cell morphological characteristics, including cell shrinkage and chromatin condensation as well as Annexin V/propidium iodide double staining followed by flow cytometric analysis and detection of apoptosis-associated proteins by western blot analysis. CQAH induced caspase-3 and PARP cleavage, reduced the expression of the anti-apoptotic proteins myeloid cell leukemia-1 and B-cell lymphoma (Bcl) extra large protein and elevated the expression of the pro-apoptotic protein Bcl-2 homologous antagonist killer. In addition, pharmacological inhibition of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, significantly reduced CQAH-mediated cell death as well as cleavage of caspase-3 and PARP. Co-treatment of CQAH with the commercial chemotherapeutics 5-fluorouracil and camptothecin-11 significantly improved their efficacies. Comparison of the apoptotic effects of CQAH with those of two illustrated structure-activity associations for this compound type, indicating that substitution at position-4 of the azetidine phenyl ring is pivotal for inducing apoptosis. In conclusion, the results of the present study indicated CQAH and its analogues are potent candidate drugs for the treatment of colon carcinoma. PMID:26498992

  3. 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

  4. 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

  5. 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

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

    PubMed

    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

  7. 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-?B 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-?B 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

  8. JNK signaling pathway is involved in piperlongumine-mediated apoptosis in human colorectal cancer HCT116 cells

    PubMed Central

    LI, WEN; WEN, CHUANGYU; BAI, HAIYAN; WANG, XIAOYAN; ZHANG, XIAOLI; HUANG, LANLAN; YANG, XIANGLING; IWAMOTO, AIKICHI; LIU, HUANLIANG

    2015-01-01

    Piperlongumine (PPLGM), an alkaloid isolated from the long pepper (Piper longum L.), can selectively trigger cancer cell death in colorectal cancer cells. The present study investigated whether the c-Jun NH2-terminal kinase (JNK) signaling pathway is involved in PPLGM-induced apoptosis in the human colorectal cancer HCT116 cell line. The results demonstrated that PPLGM reduced the cell viability and induced cell apoptosis in a time- and concentration-dependent manner, without a significant effect on cell cycle distribution. Meanwhile, treatment with 10 µM PPLGM resulted in JNK activation within 1 h, and a marked and sustained increase in c-Jun phosphorylation in the HCT116 cells. In addition, SP600125, a general inhibitor of JNK, inhibited PPLGM-induced apoptosis in the HCT116 cells by inhibiting PPLGM-induced c-Jun phosphorylation. Altogether, it can be concluded that the JNK signaling pathway, at least in part, is involved in PPLGM-mediated apoptosis in HCT116 cells. PMID:26622558

  9. Cell specific apoptosis by RLX is mediated by NF?B 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 ?M and have further investigated the involvement of NF?B and caspase-3 in RLX action in CRC apoptosis. Following RLX and BEZ-235 treatment in HCT-116, we observed significant down-regulation of NF?B (1 to 0.1 fold) and up-regulation of caspase-3 (1 to 2 fold) protein expressions. Additionally, morphological studies revealed membrane blebbing, cell shrinkage, chromatin condensation and finally apoptosis in HCT-116 cells. Conclusions Overall, these findings indicate that RLX is a potent small molecule which triggers apoptosis, and promising potential candidate to be a chemotherapeutic agent. PMID:25303828

  10. Induction of apoptosis in HCT-116 colon cancer cells by polysaccharide of Larimichthys crocea swim bladder

    PubMed Central

    SUO, HUAYI; SONG, JIA-LE; ZHOU, YALIN; LIU, ZHENHU; YI, RUOKUN; ZHU, KAI; XIE, JIE; ZHAO, XIN

    2015-01-01

    Larimichthys crocea swim bladder is a traditional food and medicine widely used in China. The in vitro anticancer effects of polysaccharide of L. crocea swim bladder (PLCSB) in HCT-116 human colon cancer cells was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. At concentrations ranging between 0 and 800 ?g/ml PLCSB, cancer cell viability was decreased by PLCSB in a concentration-dependent manner. In particular, 400 ?g/ml PLCSB significantly (P<0.05) induced apoptosis, which was demonstrated by 4,6-diamidino-2-phenylindole staining and flow cytometry analysis. To elucidate the mechanisms underlying the anticancer effect of PLCSB in HCT-116 cancer cells, the expression of apoptosis and metastasis-associated genes was analyzed by reverse transcription-polymerase chain reaction and western blot analysis. A total of 400 ?g/ml PLCSB significantly induced apoptosis in HCT-116 cells (P<0.05) via the upregulation Bax, p53, p21, apoptotic protease activating factor 1, caspase-3, -8, and -9, as well as Fas and the downregulation of B-cell lymphoma 2 (Bcl-2), Bcl-extra large and Fas ligand (L). The results of this study demonstrated that PLCSB exhibits an anticancer effect on HCT-116 colon cancer cells, in vitro. PMID:25624917

  11. Mature microRNAs identified in highly purified nuclei from HCT116 colon cancer cells

    PubMed Central

    Park, Chang Won; Zeng, Yan; Zhang, Xiaoxiao; Subramanian, Subbaya

    2010-01-01

    MicroRNAs (miRNAs) have emerged as one of the major regulatory mechanisms of gene expression. A major function of miRNAs involves the post-transcriptional regulation of target mRNAs, which is reported to occur primarily in the cytoplasm. However, there is a significant amount of evidence demonstrating the existence of small non-coding RNAs, including small-interfering RNA (siRNA), miRNA and Piwi-interacting RNA (piRNA) in the nucleus. In order to elucidate the potential subcellular localizations and functions of miRNAs, we have identified numerous miRNAs that are present in isolated nuclei from human colon cancer HCT116 cells. MicroRNA profiles were compared between cytoplasmic and nuclear fractions of the HCT116 cell line on the basis of multiple microarray analyses. MicroRNA species showing significant existence in isolated and highly purified populations of nuclei were selected and further tested with RT-PCR. The nuclear localization of the mature form of miRNAs was verified again by control RT-PCR excluding the detection of premature forms of miRNA, such as pri-miRNA or pre-miRNA. The elevated levels of representative miRNAs identified in purified nuclei were confirmed by northern blot analysis, supporting the notion that significant numbers of mature miRNAs exist not only in the cytoplasm but also in the nucleus. These results will likely provide a basis for further studies concerning the intracellular trafficking and nuclear location of miRNAs. PMID:20864815

  12. Viability and oxidative response of human colorectal HCT-116 cancer cells treated with visfatin/eNampt in vitro.

    PubMed

    Buldak, R J; Gowarzewski, M; Buldak, L; Skonieczna, M; Kukla, M; Polaniak, R; Zwirska-Korczala, K

    2015-08-01

    Visfatin/eNampt is a novel adipokine, secreted by visceral and subcutaneous fat, which could be involved in the development of obesity-associated cancer. Only few studies revealed reactive oxygen species (ROS)-dependent action of visfatin in endothelial cells, myotubes and melanoma cells. The potential pro-apoptotic properties of visfatin/eNampt in human colorectal HCT-116 cells remain unknown. The aim of the study was to examine the effects of visfatin/eNampt on cell viability along with the determination of apoptosis/necrosis extent and ROS level in HCT-116 cells. Additionally antioxidant enzymes' activities (i.e catalase (CAT), gluthatione peroxidase (GSH-Px)), and lipid peroxidation intensity in HCT-116 cells line was evaluated. Viability of HCT-116 cells was decreased after visfatin/eNampt treatment for 24 hours. The number of apoptotic cells in tested cells treated with increasing visfatin/eNampt concentrations (10, 100, 250 ng/ml) was elevated compared to untreated cells (6.4%, 9.7%, 16% vs. 3.2%; respectively). After 24 hours in the visfatin/eNampt treated group (10 - 100 ng/ml) CAT and GSH-Px activities significantly increased and this observation was accompanied by the decrease of ROS level when compared to the control group. Interestingly ROS level (using DCF detection technique) and lipid peroxidation ratio were increased in cells stimulated by visfatin/eNampt in concentration of 250 ng/ml along with the decreased activity of selected antioxidant enzymes when compared to remaining study groups, including control. We concluded that visfatin/eNampt induces decrease of cell viability and apoptosis boost in human colorectal cancer HCT-116 cells line. Visfatin/eNampt affected the level of ROS as well as antioxidant capacity, however the association of ROS level and apoptosis rate was not linear. The role for visfatin/eNampt in cancer redox status in vitro may provide a greater insight into the association between fat derived visfatin/eNampt and its endocrine action in colorectal carcinoma cells. PMID:26348080

  13. 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.

  14. 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

  15. Benzylidenetetralones, cyclic chalcone analogues, induce cell cycle arrest and apoptosis in HCT116 colorectal cancer cells.

    PubMed

    Drutovic, David; Chripkova, Martina; Pilatova, Martina; Kruzliak, Peter; Perjesi, Pal; Sarissky, Marek; Lupi, Monica; Damia, Giovanna; Broggini, Massimo; Mojzis, Jan

    2014-10-01

    Colorectal cancer is the third most common cancer in the world, with 1.2 million new cancer cases annually. Chalcones are secondary metabolite precursors of flavonoids that exhibit diverse biological activities, including antioxidant and antitumor activities. The aim of this study was to investigate the antiproliferative effect of new synthetic chalcone derivatives on HCT116 cells. (E)-2-(2',4'-dimethoxybenzylidene)-1-tetralone (Q705) was found to be the most active (IC50?=?3.44?±?0.25 ?M). Based on these results, this compound was chosen for further analysis of its biochemical and molecular mechanisms. Our results showed that Q705 inhibited the growth and clonogenicity of HCT116 cells. The results of a flow cytometric analyses suggested that this compound caused a significant cell cycle arrest in G2/M phase and increased the proportion of cells in the subG0/G1 phase, marker of apoptosis. Q705-induced apoptosis was confirmed by TdT-mediated dUTP nick end labelling (TUNEL) assay. Treatment of HCT116 cells with this chalcone significantly increased the caspase-3,-7 activity and resulted in cleavage of poly-ADP-ribose polymerase (PARP). Changes in the nuclear morphology such as chromatin condensation were also observed. These effects were associated with a decreased expression of bcl-xL and increased overall ratio of bax/bcl-xL mRNA levels. Immunofluorescence and qRT-PCR analysis revealed that Q705 induced H2AX histone modifications characteristic of DNA damage, disruption of microtubule organization and downregulation of tubulins. In summary, these results suggest that the cyclic chalcone analogue Q705 has potential as a new compound for colorectal cancer therapy. PMID:25008568

  16. Lentivirus-mediated knockdown of eukaryotic translation initiation factor 3 subunit D inhibits proliferation of HCT116 colon cancer cells

    PubMed Central

    Yu, Xiaojun; Zheng, Bo’an; Chai, Rui

    2014-01-01

    Dysregulation of protein synthesis is emerging as a major contributory factor in cancer development. eIF3D (eukaryotic translation initiation factor 3 subunit D) is one member of the eIF3 (eukaryotic translation initiation factor 3) family, which is essential for initiation of protein synthesis in eukaryotic cells. Acquaintance with eIF3D is little since it has been identified as a dispensable subunit of eIF3 complex. Recently, eIF3D was found to embed somatic mutations in human colorectal cancers, indicating its importance for tumour progression. To further probe into its action in colon cancer, we utilized lentivirus-mediated RNA interference to knock down eIF3D expression in one colon cancer cell line HCT116. Knockdown of eIF3D in HCT116 cells significantly inhibited cell proliferation and colony formation in vitro. Flow cytometry analysis indicated that depletion of eIF3D led to cell-cycle arrest in the G2/M phase, and induced an excess accumulation of HCT116 cells in the sub-G1 phase representing apoptotic cells. Signalling pathways responsible for cell growth and apoptosis have also been found altered after eIF3D silencing, such as AMPK? (AMP-activated protein kinase alpha), Bad, PRAS40 [proline-rich Akt (PKB) substrate of 40 kDa], SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase), GSK3? and PARP [poly(ADP-ribose) polymerase]. Taken together, these findings suggest that eIF3D might play an important role in colon cancer progression. PMID:25370813

  17. 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

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

    PubMed Central

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

    2015-01-01

    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.

  19. Orexin A induces autophagy in HCT-116 human colon cancer cells through the ERK signaling pathway.

    PubMed

    Wen, Jing; Zhao, Yuyan; Guo, Lei

    2016-01-01

    Orexins are a class of peptides which have a potent influence on a broad variety of cancer cells. Autophagy is closely associated with tumors; however, its function is not yet completely understood. In this study, we aimed to determine whether orexin A induces autophagy in HCT?116 human colon cancer cells and to elucidate the molecular mechanisms involved. For this purpose, HCT?116 cells were treated with orexin A, and cell viability was then measured by MTT assay, and apoptosis was determined by flow cytometry. The expression levels of autophagy?related proteins were measured by western blot analysis. Quantitative analysis of autophagy following acridine orange (AO) staining was performed using fluorescence microscopy, and cellular morphology was observed under a transmission electron microscope. In addition, the HCT?116 cells were treated with the extracellular signal?regulated kinase (ERK) inhibitor, U0126, or the autophagy inhibitor, chloroquine, in combination with orexin A in order to examine the activation of ERK. We found that orexin A significantly inhibited the viability of the HCT?116 cells. Both autophagy and apoptosis were activated during the orexin A?induced death of HCT?116 cells. When the HCT?116 cells were treated with orexin A for 24 h, an accumulation of punctate microtubule-associated protein-1 light chain 3 (LC3) and an increase in LC3?? protein levels were also detected, indicating the activation of autophagy. Moreover, orexin A upregulated ERK phosphorylation; however, U0126 or chloroquine abrogated ERK phosphorylation and decreased autophagy, compared to treatment with orexin A alone. Therefore, our findings demonstratedm that orexin A induced autophagy through the ERK pathway in HCT?116 human colon cancer cells. The inhibition of autophagy may thus prove to be an effective strategy for enhancing the antitumor potential of orexin A as a treatment for colon cancer. PMID:26572581

  20. Balsalazide Potentiates Parthenolide-Mediated Inhibition of Nuclear Factor-?B 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-?B (NF-?B) activity. Parthenolide (PT) inhibits NF-?B and exerts promising anticancer effects by promoting apoptosis. The present investigated the antitumor effects of balsalazide, combined with PT, on NF-?B 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-?B p65 subunit and the phosphorylation of inhibitor of NF-?B. Moreover, PT and balsalazide dramatically enhanced NF-?B 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

  1. Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells.

    PubMed

    Hall, Emily H; Schoenbach, Karl H; Beebe, Stephen J

    2007-09-01

    Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53(+/+) and HCT116p53(-/-) colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53(+/+) and HCT116p53(-/-) cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax. PMID:17520193

  2. 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)

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

    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.

  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. 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

  5. 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.

  6. RasGAP-derived peptide GAP159 enhances cisplatin-induced cytotoxicity and apoptosis in HCT116 cells

    PubMed Central

    Zhang, Hao; Zhang, Shenghua; He, Hongwei; Zhang, Caixia; Chen, Yi; Yu, Dongke; Chen, Jianhua; Shao, Rongguang

    2014-01-01

    To increase the efficacy of currently used anti-cancer genotoxins, one of the current efforts is to find agents that can sensitize cancer cells to genotoxins so that the efficacious doses of genotoxins can be lowered to reduce deleterious side-effects. In this study, we reported that a synthetic RasGAP-derived peptide GAP159 could enhance the effect of chemotherapeutic agent cisplatin (CDDP) in human colon carcinoma HCT116 cells. Our results showed that GAP159 significantly increased the CDDP-induced cytotoxicity and apoptosis in HCT116 cells. This synergistic effect was associated with the inhibitions of phospho-AKT, phospho-ERK and NF-?B. In mouse colon tumor CT26 animal models, GAP159 combined with CDDP significantly suppressed CT26 tumor growth, and GAP159 alone showed slight inhibitory effect. Our data suggests that co-treatment of GAP159 and chemotherapeutics will become a potential therapeutic strategy for colon cancers.

  7. Synergistic Actions of Atorvastatin with ?-Tocotrienol and Celecoxib against Human Colon Cancer HT29 and HCT116 Cells

    PubMed Central

    Yang, Zhihong; Xiao, Hang; Jin, Huanyu; Koo, Phillip T.; Tsang, Dorothea J.; Yang, Chung S.

    2010-01-01

    The synergistic actions of atorvastatin (ATST) with ?-tocotrienol (?-TT) and celecoxib (CXIB) were studied in human colon cancer cell lines HT29 and HCT116. The synergistic inhibition of cell growth by ATST and ?-TT was demonstrated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and isobologram analysis. ?-TT exhibited a similar inhibitory action when combined with ATST. Mevalonate and geranylgeranyl pyrophosphate eliminated most of the growth inhibitory effect of ATST, but only marginally decreased that of ?-TT; whereas farnesyl pyrophosphate and squalene exhibited little effect on the inhibitory action of ATST and ?-TT, indicating protein geranylgeranylation, but not farnesylation are involved in the inhibition of colon cancer cell growth. Both mevalonate and squalene restored the cellular cholesterol level that was reduced by ATST treatment, but only mevalonate eliminated the cell growth inhibitory effect, suggesting that the cholesterol level in cells does not play an essential role in inhibiting cancer cell growth. Protein level of HMG-CoA reductase increased after ATST treatment, and the presence of ?-TT attenuated the elevated level of HMG-CoA reductase. ATST also decreased membrane-bound RhoA, possibly due to a reduced level of protein geranylgeranylation; addition of ?-TT enhanced this effect. The mediation of HMG-CoA reductase and RhoA provides a possible mechanism for the synergistic action of ATST and ?-TT. The triple combination of ATST, ?-TT, and CXIB showed a synergistic inhibition of cancer cell growth in MTT assays. The synergistic action of these three compounds was also illustrated by their induction of G0/G1 phase cell cycle arrest and apoptosis. PMID:19626588

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

    PubMed

    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 ?-catenin in nucleus and inhibits the binding of ?-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 ?-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/?-catenin inhibitor can be a putative agent for the treatment of colorectal cancers. PMID:25749342

  9. The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116.

    PubMed

    Dabkeviciene, Daiva; Jonusiene, Violeta; Zitkute, Vilmante; Zalyte, Egle; Grigaitis, Pranas; Kirveliene, Vida; Sasnauskiene, Ausra

    2015-12-01

    Colorectal cancer is one of the most common malignant diseases and is a leading cause of cancer mortality in the Western world. Primary or acquired resistance to chemotherapeutic drugs is a common phenomenon which causes a failure in cancer treatment. A diverse range of molecular mechanisms has been implicated in drug resistance: DNA damage repair, alterations in drug metabolism, mutation of drug targets, increased rates of drug efflux, and activation of survival signaling pathways. The aim of this study was to investigate the expression of CXCL8-CXCR1/2 pathway, its impact on cell proliferation and cytokine expression in human colorectal carcinoma HCT116 cells, and their chemotherapy-resistant subline. We found that IL-1 alpha stimulates the production of CXCL8 through IL-1 receptor signaling. Our data indicate that CXCL8 is upregulated in chemoresistant subline of colorectal cancer cells HCT116, and modulation of CXCR2 pathway can be a target for proliferation inhibition of chemoresistant colorectal cancer cells. PMID:26519257

  10. 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 (??m), 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

  11. 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 ...

  12. 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

  13. 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)-?(2)-olefin ?-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)-?(2)-olefin ?-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

  14. 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

  15. 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

  16. The effects of a novel aliphatic-chain hydroxamate derivative WMJ-S-001 in HCT116 colorectal cancer cell death

    PubMed Central

    Huang, Yu-Han; Huang, Shiu-Wen; Hsu, Ya-Fen; Ou, George; Huang, Wei-Jan; Hsu, Ming-Jen

    2015-01-01

    Hydroxamate derivatives have attracted considerable attention due to their broad pharmacological properties and have been extensively investigated. We recently demonstrated that WMJ-S-001, a novel aliphatic hydroxamate derivative, exhibits anti-inflammatory and anti-angiogenic activities. In this study, we explored the underlying mechanisms by which WMJ-S-001 induces HCT116 colorectal cancer cell death. WMJ-S-001 inhibited cell proliferation and induced cell apoptosis in HCT116 cells. These actions were associated with AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) activation, p53 phosphorylation and acetylation, as well as the modulation of p21cip/Waf1, cyclin D1, survivin and Bax. AMPK-p38MAPK signaling blockade reduced WMJ-S-001-induced p53 phosphorylation. Transfection with AMPK dominant negative mutant (DN) reduced WMJ-S-001’s effects on p53 and Sp1 binding to the survivn promoter region. Transfection with HDAC3-Flag or HDAC4-Flag also abrogated WMJ-S-001’s enhancing effect on p53 acetylation. WMJ-S-001’s actions on p21cip/Waf1, cyclin D1, survivin, Bax were reduced in p53-null HCT116 cells. Furthermore, WMJ-S-001 was shown to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. In summary, the death of HCT116 colorectal cancer cells exposed to WMJ-S-001 may involve AMPK-p38MAPK-p53-survivin cascade. These results support the role of WMJ-S-001 as a potential drug candidate and warrant the clinical development in the treatment of cancer. PMID:26510776

  17. 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

  18. Carnosine inhibits KRAS-mediated HCT116 proliferation by affecting ATP and ROS production.

    PubMed

    Iovine, Barbara; Iannella, Maria Luigia; Nocella, Francesca; Pricolo, Maria Rosaria; Bevilacqua, Maria Assunta

    2012-02-28

    Carnosine is a natural dipeptide that has generated particular interest for its antioxidant, anti-aging and especially for its antiproliferative properties. In this study, we demonstrate that carnosine inhibits the proliferation of human HCT116 colon cancer cells. In this cell line, the activating KRAS mutation induces mitochondrial ROS, the signaling molecules for cell proliferation. We observed that 50-100 mM carnosine decreases ATP and ROS concentration and induces cell cycle arrest in G1 phase. In HCT116 cells these effects are related to decreased ERK1/2 phosphorylation and increased p21waf1 protein. Our findings support the concept that carnosine could inhibit HCT116 cell growth via its antioxidant activity and its ability to affect glycolysis. PMID:22137144

  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. 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

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

    PubMed

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

    2014-04-22

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

  2. 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.

  3. Curcumin Induces Downregulation of E2F4 Expression and Apoptotic Cell Death in HCT116 Human Colon Cancer Cells; Involvement of Reactive Oxygen Species

    PubMed Central

    Kim, Kyung-Chan

    2010-01-01

    E2F transcription factors and their target genes have been known to play an important role in cell growth control. We found that curcumin, a polyphenolic phytochemical isolated from the plant Curcuma longa, markedly suppressed E2F4 expression in HCT116 colon cancer cells. Hydrogen peroxide was also found to decrease E2F4 protein level, indicating the involvement of reactive oxygen species (ROS) in curucmin-induced downregulation of E2F4 expression. Involvement of ROS in E2F4 downregulation in response to curcumin was confirmed by the result that pretreatment of cells with N-acetylcystein (NAC) before exposure of curcumin almost completely blocked the reduction of E2F4 expression at the protein as well as mRNA level. Anti-proliferative effect of curcumin was also suppressed by NAC which is consistent to previous reports showing curcumin-superoxide production and induction of poly (ADP-ribose) polymerase (PARP) cleavage as well as apoptosis. Expression of several genes, cyclin A, p21, and p27, which has been shown to be regulated in E2F4-dependent manner and involved in the cell cycle progression was also affected by curcumin. Moreover, decreased (cyclin A) and increased (p21 and p27) expression of these E2F4 downstream genes by curcumin was restored by pretreatment of cells with NAC and E2F4 overexpression which is induced by doxycycline. In addition, E2F4 overexpression was observed to partially ameliorate curcumin-induced growth inhibition by cell viability assay. Taken together, we found curcumin-induced ROS down-regulation of E2F4 expression and modulation of E2F4 target genes which finally lead to the apoptotic cell death in HCT116 colon cancer cells, suggesting that E2F4 appears to be a novel determinant of curcumin-induced cytotoxicity. PMID:21311680

  4. Raman micro-spectroscopic investigation of the interaction of cultured HCT116 colon cancer cells with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase

    NASA Astrophysics Data System (ADS)

    Akyuz, S.; Ozel, A. E.; Balci, K.; Akyuz, T.; Coker, A.; Arisan, E. D.; Palavan-Unsal, N.; Ozalpan, A.

    2011-05-01

    The interaction of cultured colon cancer cells with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been investigated, using Raman micro-spectroscopy, in order to investigate DFMO induced effects. Raman spectra of the cultured HCT116 colon cancer cells treated with DFMO at different concentrations (0, 1, 2.5, 5, and 7.5 mM) were recorded in the range 550-2300 cm -1. It has been shown that second derivative profile of the raw Raman spectrum of the colon cancer cells (i.e., the original experimental spectrum without any computational corrections) discriminates the weak but sharp bands from the strong, broad fluorescence background, and gives information about the position of the peaks and their band widths. The relative integrated intensities of the 781 cm -1 and 788 cm -1 DNA/RNA marker bands to that of 1451 cm -1 band are found to decrease by addition of DFMO. Up to 65% reduction in the magnitude of the 1003 cm -1 band, the characteristic phenylalanine ring breathing mode, in comparison to that of 1451 band, is observed. The results indicate DFMO induced apoptosis. On the other hand the intensity ratio of the tyrosine Fermi doubled around 830 cm -1 and 850 cm -1, which is a marker of hydrogen-bonding state of phenolic OH, is changed. The addition of DFMO may alter the tyrosine environment in cells, and parts of tyrosine residues are exposed. We also observed some modifications in amide I band, pointing out the alterations of the secondary structure of cell proteins by the presence of DFMO.

  5. Resveratrol induces AMPK-dependent MDR1 inhibition in colorectal cancer HCT116/L-OHP cells by preventing activation of NF-?B signaling and suppressing cAMP-responsive element transcriptional activity.

    PubMed

    Wang, Ziyuan; Zhang, Long; Ni, Zhenhua; Sun, Jian; Gao, Hong; Cheng, Zhuoan; Xu, Jianhua; Yin, Peihao

    2015-12-01

    Resveratrol, a natural polyphenolic compound found in foods and beverages, has attracted increasing attention in recent years because of its potent chemopreventive and anti-tumor effects. In this study, the effects of resveratrol on the expression of P-glycoprotein/multi-drug resistance protein 1 (P-gp/MDR1), and the underlying molecular mechanisms, were investigated in oxaliplatin (L-OHP)-resistant colorectal cancer cells (HCT116/L-OHP). Resveratrol downregulated MDR1 protein and mRNA expression levels and reduced MDR1 promoter activity. It also enhanced the intracellular accumulation of rhodamine 123, suggesting that resveratrol can reverse multi-drug resistance by downregulating MDR1 expression and reducing drug efflux. Resveratrol treatment also reduced nuclear factor-?B (NF-?B) activity, reduced phosphorylation levels of I?B?, and reduced nuclear translocation of the NF-?B subunit p65. Moreover, downregulation of MDR1 expression and promoter activity was mediated by resveratrol-induced AMP-activated protein kinase (AMPK) phosphorylation. The inhibitory effects of resveratrol on MDR1 expression and cAMP-responsive element-binding protein (CREB) phosphorylation were reversed by AMPK? siRNA transfection. We found that the transcriptional activity of cAMP-responsive element (CRE) was inhibited by resveratrol. These results demonstrated that the inhibitory effects of resveratrol on MDR1 expression in HCT116/L-OHP cells were closely associated with the inhibition of NF-?B signaling and CREB activation in an AMPK-dependent manner. PMID:26124005

  6. 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...

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

    PubMed

    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

  8. 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 ...

  9. 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...

  10. 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...

  11. Growth inhibition and antioxidative status induced by selenium-enriched broccoli extract and selenocompounds in DNA mismatch repair-deficient human colon cancer cells.

    PubMed

    Tsai, Cheng-Fang; Ou, Bor-Rung; Liang, Yu-Chuan; Yeh, Jan-Ying

    2013-08-15

    The effects of enzymatic-digested Se-enriched broccoli extracts (SeB) and selenocompounds on growth and antioxidative status in human colon cancer cells was investigated in this study. HCT116 and HCT116+Chr.3 cells were treated with selenocompounds (sodium selenite, sodium selenate, Se-Met, MeSeCys) or SeB [high-Se (H-SeB) or low-Se (L-SeB)]. The cytotoxicity induced by selenocompounds in HCT116 cells was not associated with cellular H2O2 level, while the differential cytotoxicity observed by sodium selenite between HCT116 and HCT116+Chr.3 cell lines was related to cellular H2O2 production with the change in antioxidative enzyme activity, and the restoration of chromosome 3. H-SeB was found to reduce the cellular H2O2 content in HCT116+Chr.3 cells. The results in this study indicate that regardless of Se content, the cytotoxicity in HCT116 cells of both SeB forms appeared to be H2O2-independent, whereas the cytotoxicity in HCT116+Chr.3 of either SeB form appeared to be H2O2-dependent with an increase in antioxidative ability for H-SeB. PMID:23561105

  12. The Dermal Layer of Sweet Sorghum (Sorghum bicolor) Stalk, a Byproduct of Biofuel Production and Source of Unique 3-Deoxyanthocyanidins, Has More Antiproliferative and Proapoptotic Activity than the Pith in p53 Variants of HCT116 and Colon Cancer Stem Cells.

    PubMed

    Massey, Aaron R; Reddivari, Lavanya; Vanamala, Jairam

    2014-03-31

    There is a growing interest in the utilization of sweet sorghum as a renewable resource for biofuels. During the biofuel production process, large quantities of biomass are generated, creating a rich source of bioactive compounds. However, knowledge of sweet sorghum stalk is lacking. We measured the phenolic content (Folin-Ciocalteu assay), antioxidant activity (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) assay), and phytochemical composition (LC-MS) in both the pith and dermal layer of the stalk. We further tested the antiproliferative (5-bromo-2'- deoxyuridine assay) and proapoptotic (terminal deoxynucleotidyl transferase dUTP nick end labeling assay) activities of these extracts using HCT116 cells and colon cancer stem cells (CCSCs) with and without the tumor suppressor gene p53. For the first time, we show that the dermal layer extract of sweet sorghum contains more of the 3-deoxyanthocyanidins apigeninidin and luteolinidin than the pith, and this is associated with more anticancer activity. Furthermore, luteolinidin suppressed CCSC proliferation more than apigeninidin. In addition to being renewable biofuel, sweet sorghum may also serve as a source of health-promoting compounds. PMID:24655033

  13. 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.

  14. 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

  15. Proteomic Analysis of Nuclei isolated from cancer cell lines treated with Indenoisoquinoline NSC 724998, a novel Topoisomerase I Inhibitor

    PubMed Central

    Han, Bingnan; Stockwin, Luke H.; Hancock, Chad; Yu, Sherry X.; Hollingshead, Melinda G.; Newton, Dianne L.

    2010-01-01

    The indenoisoquinoline NSC724998 is a novel topoisomerase I (Top1) inhibitor entering Phase I clinical trials at the National Cancer Institute, USA. In this study, 2-D PAGE analysis was performed on nuclear lysates prepared from HCT-116 and A375 cells treated with 1µM NSC724998 for 24 hrs and the differentially regulated spots identified by LC-MS/MS. 114 protein spot differentials were identified, 66 from A375 cells and 48 from HCT-116 cells. Proteins related to apoptosis changed specifically in A375 cells, whereas proteins involved in the ubiquitin-proteasome system were highly enriched in treated HCT-116 cells. Importantly, 12 differentially expressed proteins (ETFA, HCC1, HNRCL, KAP1, NPM, NUCL, PRDX1, PRP19, PSB6, RAE1L, RU2A and SFRS9) were common to both cell lines. Western blotting and immunocytochemistry confirmed significant nuclear upregulation of both the proteasome subunit PSB6 and the transcriptional repressor KAP1. Interestingly, increased KAP1 polypeptide was accompanied by enhanced phosphorylation at Ser824. Similar to ?H2AX, KAP1 phosphorylation was consistently enhanced in a panel of 12 cell lines and in A375 xenografts following NSC 724998 treatment. In summary, these data enhance our understanding of protein dynamics in the nucleus following DNA damage and provide an alternate marker (pKAP1) with potential for monitoring clinical responses to Top1 poisons. PMID:20515076

  16. [Corrigendum] Activation of platelet protease-activated receptor-1 induces epithelial-mesenchymal transition and chemotaxis of colon cancer cell line SW620.

    PubMed

    Jia, Yitao; Zhang, Suqiao; Miao, Lingling; Wang, Jingbao; Jin, Zujian; Gu, Bin; Duan, Zhihui; Zhao, Zhaolong; Ma, Shunmao; Zhang, Wenjin; Li, Zhongxin

    2016-02-01

    After the publication of the article, it has been brought to the authors' attention by an interested reader that we had made an error regarding the colon cancer cell line in the manuscript. The error relates to Materials and methods, as well as Results, the colon cancer cell line in the Transwell migration assay and Flow cytometric detection of CXCR4 expression is HCT-116 rather than SW620. Accordingly, the correct legends in Figs. 3 and 6 in the paper are HCT-116 cells. This error does not affect the overall conclusions reported in the present study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this error may have caused. [the original article was published in the Oncology Reports 33: 2681-2688, 2015; DOI: 10.3892/or.2015.3897]. PMID:26718652

  17. Mutations to Ku Reveal Differences in Human Somatic Cell Lines

    PubMed Central

    Fattah, Kazi R.; Ruis, Brian L.; Hendrickson, Eric A.

    2008-01-01

    NHEJ (non-homologous end joining) is the predominant mechanism for repairing DNA double-stranded breaks in human cells. One essential NHEJ factor is the Ku heterodimer, which is composed of Ku70 and Ku86. Here we have generated heterozygous loss-of-function mutations for each of these genes in two different human somatic cell lines, HCT116 and NALM-6 using gene targeting. Previous work had suggested that phenotypic differences might exist between the genes and/or between the cell lines. By providing a side-by-each comparison of the four cell lines, we demonstrate that there are indeed subtle differences between loss-of-function mutations for Ku70 versus Ku86, which is accentuated by whether the mutations were derived in the HCT116 or NALM-6 genetic background. Overall, however, the phenotypes of the four lines are quite similar and they provide a compelling argument for the hypothesis that Ku loss-of-function mutations in human somatic cells result in demonstrable haploinsufficiencies. Collectively, these studies demonstrate the importance of proper biallelic expression of these genes for NHEJ and telomere maintenance and they provide insights into why these genes are uniquely essential for primates. PMID:18387344

  18. 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 ?mol/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

  19. 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

  20. 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

  1. Transcriptomic profiling of radiation-resistant or -sensitive human colorectal cancer cells: acute effects of a single X-radiation dose.

    PubMed

    Ng, Cheng E; Liu, Qing Y; Qutob, Sami S; Scott, Bradley J; Tan, Wei L; Mesak, Felix M

    2007-06-01

    We previously isolated several clones that were closely-related genetically from a human colorectal tumor (HCT116) cell line. These clones displayed significantly different X-radiation response phenotypes. In this paper, we investigated how a single dose of X-radiation modulated the transcriptomic profiles of either the radiation-resistant (HCT116Clone2_XRR) or the radiation-sensitive (HCT116CloneK_XRS) clone when each was compared to a reference clone, HCT116Clone10_control. The latter represented a control clone that displayed a similar X-radiation response as the parental HCT116 cells. Pooled RNAs were obtained from HCT116Clone2_XRR, HCT116CloneK_XRS or HCT116Clone10_control cells either before or at 10 min, 6 or 24 h after treatment with 4-Gy X-radiation. Transcriptomic profiles were assessed by cDNA microarrays. At least three independent experiments were carried out for each time point and statistical analysis was performed by paired t-test (p<0.05). From 19,200 genes/ESTs examined, we identified only 120 genes/ESTs that were differentially expressed at any one of these four time points. Interestingly, different patterns of gene modulation were observed between the radiation-sensitive and radiation-resistant clones. However, the fold changes of gene modulation were generally small (2-3 fold). Surprisingly, only 12.7% of 79 genes involved in DNA damage sensor/repair and cell cycle and between 2.6 and 9.2% of 76 genes involved in apoptosis, were significantly modulated in these early time points following irradiation. By comparison, up to 10% of 40 known housekeeping genes were differentially expressed. Thus in our experimental model, we were able to detect the up-regulation or down-regulation of mostly novel genes and/or pathways in the acute period (up to 24 h) following a single dose of 4-Gy X-radiation. PMID:17487357

  2. MicroRNA-203 induces apoptosis by upregulating Puma expression in colon and lung cancer cells.

    PubMed

    Funamizu, Naotake; Lacy, Curtis R; Kamada, Minori; Yanaga, Katsuhiko; Manome, Yoshinobu

    2015-11-01

    The present study investigated the relationship between microRNA-203 (miR-203) and the p53 upregulated modulator of apoptosis (Puma) in colon (HCT116) and lung cancer (A549) cells. Colon and lung cancer cell lines were selected for this study since a relationship between p53/miR-203 and p53/Puma has been established in both cancers. In the present study, adriamycin and nutlin-3 were used to activate p53, which induced both miR-203 and Puma expression in HCT116 cells. In contrast, HCT 116 cells with downregulated p53 showed decreased miR-203 and Puma expression. Importantly, we found that overexpressed miR-203 in HCT116 cells resulted in significantly increased Puma expression (P<0.05). Based on these findings, we hypothesized that another limb of the p53/Puma axis depends on miR-203 expression. To further validate this relationship, we used lung cancer cells (A549) and found that activated p53 increased both miR-203 and Puma expression. In addition, we found that Puma expression remained elevated in cells with overexpressed miR-203 in the presence of p53 downregulation. Cumulatively, our data purport that p53 not only increased Puma expression directly, but that it may also do so through miR-203. Additionally, functional studies revealed that miR-203 overexpression induced apoptosis and inhibited cell invasiveness. PMID:26397233

  3. Sulforaphane inhibits hypoxia-induced HIF-1? 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-1? (HIF-1?) expression in HCT116 human colon cancer cells and AGS human gastric cancer cells. We found that hypoxia-induced HIF-1? protein expression in HCT116 and AGS cells, while treatment with sulforaphane markedly and concentration-dependently inhibited HIF-1? 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-1? stability. However, degradation of HIF-1? by sulforaphane was not mediated through the 26S proteasome pathway. We also found that the inhibition of HIF-1? 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-1? 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

  4. Cox-2 is needed but not sufficient for apoptosis induced by Cox-2 selective inhibitors in colon cancer cells.

    PubMed

    Agarwal, B; Swaroop, P; Protiva, P; Raj, S V; Shirin, H; Holt, P R

    2003-12-01

    The role of Cox-2 in NSAID-induced apoptosis is debated. We studied the role of Cox-2 inhibition in apoptosis induced by a selective Cox-2 inhibitor, SC236 (a structural analogue of celecoxib) in two colon cancer cell lines, HT29 (expressing Cox-2 protein) and HCT116 (not expressing Cox-2 protein). Apoptosis was quantified by flow cytometry. SC236 0-75 microM decreased cell numbers and induced apoptosis to identical levels in HT29 and HCT116 cells. However, SC236, concentrations >75 microM reduced Cox-2 protein expression in HT29 cells and induced greater levels of apoptosis in HT29 than in HCT116 cells. In contrast, sulindac sulfide (SSD) (which inhibits Cox-1 and Cox-2) 0-200 microM or sulindac sulfone (SSN) 0-500 microM (without significant activity against Cox-1 or Cox-2) caused identical decreases in cell number and increases in apoptosis in HT29 and HCT116 cells. Neither SSD nor SSN altered the expression of Cox-2 in HT29 cells. To determine that the higher levels of apoptosis in HT29 cells with SC236 >75 microM were related to decreased Cox-2 protein levels, we decreased Cox-2 protein expression in HT29 cells with curcumin (diferuloylmethane) and studied its effect on SC236-induced apoptosis. Curcumin augmented apoptosis induced by SC236 in HT29 cells but not in Cox-2 lacking HCT116 cells. In conclusion, selective Cox-2 inhibitors can induce apoptosis independent of Cox-2 expression. However they may selectively target cells that express Cox-2 by decreasing their Cox-2 protein expression. PMID:14739610

  5. Functional characterization of the nitrogen permease regulator-like-2 candidate tumor suppressor gene in colorectal cancer cell lines

    PubMed Central

    LIU, AI-YUN; LIU, MING-NA; PEI, FENG-HUA; CHEN, JING; WANG, XIN-HONG; LIU, DAN; DU, YA-JU; LIU, BING-RONG

    2015-01-01

    The nitrogen permease regulator-like-2 (NPRL2) gene is a candidate tumor suppressor gene, which has been identified in the 3p21.3 human chromosome region. Decreased expression levels of NPRL2 have been observed in colorectal cancer (CRC) tissues, however, the function of NPRL2 in CRC progression remains to be fully elucidated. The present study investigated the biological characteristics of the HCT116 and HT29 CRC cell lines overexpressing exogenous NPRL2. NPRL2 recombinant lentiviral vectors were also constructed and transfected in the present study. Cell growth was determined using a Cell Counting Kit-8 assay and a colony formation assay. The cell cycle and rate of apoptosis were assessed using flow cytometric analysis. Transwell assays were used to evaluate cell invasion. The protein expression of phosphorylated (p)-AKT and caspase 3, B-cell lymphoma 2 (Bcl2) and Bcl-2-associated X protein apoptosis-associated genes, were detected using western blotting. The results revealed that NPRL2 overexpression inhibited cell growth, induced cell cycle G1 phase arrest, promoted apoptosis and inhibited invasion in the two human CRC cell lines. Furthermore, the protein expression levels of p-AKT and Bcl2 were significantly reduced in the NPRL2-transfected HCT116 and HT29 cells, compared with the mock-transfected group and control group, while the protein expression of caspase-3 was increased. Therefore, NPRL2 acted as a functional tumor suppressor in the CRC cell lines. PMID:26044952

  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. 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 ?mol/L of omega-3 polyunsaturated fatty acids ?-linolenic acid (?LA) 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 ?-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 ?LA-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

  8. Oridonin triggers apoptosis in colorectal carcinoma cells and suppression of microRNA-32 expression augments oridonin-mediated apoptotic effects.

    PubMed

    Yang, Jie; Jiang, Hai; Wang, Chunyu; Yang, Bo; Zhao, Lijun; Hu, Dongling; Qiu, Guihua; Dong, Xiaolin; Xiao, Bin

    2015-05-01

    Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been found to exhibit various anti-tumor effects. In this work, to investigate its pharmacological effects on human colorectal carcinoma HCT-116 and LoVo cells, cell proliferation and apoptosis were respectively evaluated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC, and propidium iodide (PI) staining. Western blotting was used to detect the expression levels of Bim, Bax, Bcl-2, cytosolic cytochrome c, procaspase-9, cleaved caspase-9, procaspase-3, and caspase-3 proteins. Caspase-Glo-9 and Caspase-Glo-3 assays were applied to determine caspase-9 and caspase-3 activity. MicroRNA-32 (miR-32) expression level was detected by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The in vivo anti-tumor effects of oridonin were evaluated using cell lines HCT-116 and LoVo xenograft model. The results indicated that oridonin effectively inhibited cell proliferation and induced apoptosis in HCT-116 and LoVo cells in a concentration-dependent manner. Oridonin treatment upregulated the expression levels of Bim, Bax, cytosolic cytochrome c, cleaved caspase-9 and cleaved caspase-3 proteins, downregulated the expression levels of Bcl-2, procaspase-9 and procaspase-3 proteins, and meanwhile obviously activated caspase-9 and caspase-3 in a dose-dependent manner in HCT-116 and LoVo cells. The results of qRT-PCR demonstrated that oridonin treatment significantly decreased miR-32 expression, and furthermore, suppression of miR-32 expression by miR-32 inhibitors augmented oridonin-mediated inhibitory and apoptotic effects in HCT-116 and LoVo cells. In vivo results indicated that oridonin administration through intraperitoneal injection suppressed tumor growth in nude mice. Therefore, these findings suggest that oridonin maybe is a potential candidate for colorectal cancer treatment. PMID:26054686

  9. SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-CCL247 (HCT 116)

    Cancer.gov

    A. THAWING CELLS............................................................................................................................ 6 B. PROPAGATING CELLS .................................................................................................................... 7 C. SUBCULTURING CELLS................................................................................................................... 8 5. HARVESTING OF CELLS FOR CRYOPRESERVATION............................................................. 9 6. CRYOPRESERVATION OF CELLS ............................................................................................11 A. CRYOPRESERVATIONUSINGARATE-CONTROLLEDPROGRAMMABLEFREEZER ................................11 i. Using the Cryomed ...............................................................................................................11 B. CRYOPRESERVATION USING “MR. FROSTY” ..................................................................................12 7. STORAGE.

  10. 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.

  11. 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.

  12. 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.

  13. 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??g/mL), weak cytotoxicity against A549, Ca Ski, and HCT116 cells (with IC50 values of 46.3 ± 0.7, 32.5 ± 1.1, and 35.0 ± 0.3??g/mL, resp.), and no inhibitory effect against MCF7 cells. It exhibited mild cytotoxicity against a noncancerous human lung fibroblast cell line (MRC5), with an IC50 value of 25.2 ± 2.7??g/mL. This is the first report on the chemical composition of this rhizome's oil and its selective antiproliferative effect on HT29. The obtained data provided a basis for further investigation of the mode of cell death. PMID:25177723

  14. Zinc Finger Nuclease Mediated Knockout of ADP-Dependent Glucokinase in Cancer Cell Lines: Effects on Cell Survival and Mitochondrial Oxidative Metabolism

    PubMed Central

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

    2013-01-01

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

  15. 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

  16. 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

  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 (?H2AX) 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. Dihydroartemisinin is a Hypoxia-Active Anti-Cancer Drug in Colorectal Carcinoma Cells

    PubMed Central

    Ontikatze, Teona; Rudner, Justine; Handrick, René; Belka, Claus; Jendrossek, Verena

    2014-01-01

    Tumor hypoxia is one main biological factor that drives resistance to chemotherapy and radiotherapy. To develop a novel strategy for overcoming hypoxia-induced therapy resistance, we examined the anti-neoplastic activity of the reactive oxygen donor dihydroartemisinin (DHA) in human colon cancer cell lines in normoxia and severe hypoxia. In addition, we analyzed the involvement of the intrinsic apoptosis pathway for DHA-mediated cytotoxicity in HCT116 cells in short-term and long-term in vitro assays. When applied at lower concentrations (?25??M), DHA induced apoptosis in Colo205, HCT15, and HCT116 cells, whereas necrotic cell death was increased when cells were treated with higher DHA concentrations (50??M). However, no preference for DHA-induced apoptosis or necrosis could be detected between the treatment under normoxic or hypoxic conditions. Moreover, DHA potently reduced clonogenic survival of HCT116 cells in normoxia and hypoxia. Treatment of HCT116 cells with 25??M DHA resulted in activation of Bax under normoxic and hypoxic conditions. Interestingly, cytochrome c release from the mitochondria and caspase-activation were observed only under normoxic conditions, whereas, under hypoxic conditions DHA induced a caspase-independent apoptosis-like cell death. However, under both conditions, generation of reactive oxygen species was an important mediator of DHA-induced toxicity. Further molecular analysis suggests that DHA-mediated cell death involves different sets of pro-apoptotic Bcl-2 family members. The pronounced cytotoxic activity of DHA in severe hypoxia as well as normoxia offers new perspectives for targeting the hypoxic tumor cell fraction to improve treatment outcome for cancer patients. PMID:24904829

  19. Bone morphogenetic protein 2 inhibits the proliferation and growth of human colorectal cancer cells

    PubMed Central

    ZHANG, YUNYUAN; CHEN, XIAN; QIAO, MIN; ZHANG, BING-QIANG; WANG, NING; ZHANG, ZHONGLIN; LIAO, ZHAN; ZENG, LIYI; DENG, YOULIN; DENG, FANG; ZHANG, JUNHUI; YIN, LIANGJUN; LIU, WEI; ZHANG, QIAN; YAN, ZHENGJIAN; YE, JIXING; WANG, ZHONGLIANG; ZHOU, LAN; LUU, HUE H.; HAYDON, REX C.; HE, TONG-CHUAN; ZHANG, HONGYU

    2014-01-01

    Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/?-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells. PMID:24993644

  20. Multiple promoter elements govern expression of the human ornithine decarboxylase gene in colon carcinoma cells.

    PubMed Central

    Moshier, J A; Osborne, D L; Skunca, M; Dosescu, J; Gilbert, J D; Fitzgerald, M C; Polidori, G; Wagner, R L; Friezner Degen, S J; Luk, G D

    1992-01-01

    Overexpression of the ornithine decarboxylase (ODC) gene may be important to the development and maintenance of colonic neoplasms, as well as tumors in general. In this study, we examined the promoter elements governing constitutive expression of the human ODC gene in HCT 116 human colon carcinoma cells and, for comparison, K562 human erythro-leukemia cells. It was determined by functional analysis that the promoter elements responsible reside within the 378 bp immediately upstream from the transcription start site. Within this sequence, there are at least three regions that modulate the efficiency of the ODC promoter cooperatively. Both DNA bandshift and footprint assays demonstrated all three regions to be rich in sites that bind to nuclear proteins isolated from HCT 116 and K562 cells; the protein binding pattern of non-transformed, diploid fibroblasts was found to be much less complex. Several of the protein binding sequences have little or no homology to common regulatory elements. We suggest that the constitutive activity of the ODC gene in HCT 116 colon carcinoma cells, and perhaps transformed cells in general, involves a complex interaction of multiple regulatory sequences and their associated nuclear proteins. Finally, the saturation of the promoter in these transformed cell lines suggests that high levels of protein binding in the ODC promoter may contribute to elevated constitutive expression of this gene. Images PMID:1598217

  1. Magneto-controllable capture and release of cancer cells by using a micropillar device decorated with graphite oxide-coated magnetic nanoparticles.

    PubMed

    Yu, Xiaolei; He, Rongxiang; Li, Shasha; Cai, Bo; Zhao, Libo; Liao, Lei; Liu, Wei; Zeng, Qian; Wang, Hao; Guo, Shi-Shang; Zhao, Xing-Zhong

    2013-11-25

    Aiming to highly efficient capture and analysis of circulating tumor cells, a micropillar device decorated with graphite oxide-coated magnetic nanoparticles is developed for magneto-controllable capture and release of cancer cells. Graphite oxide-coated, Fe3 O4 magnetic nanoparticles (MNPs) are synthesized by solution mixing and functionalized with a specific antibody, following by the immobilization of such modified MNPs on our designed micropillar device. For the proof-of-concept study, a HCT116 colorectal cancer cell line is employed to exam the capture efficiency. Under magnetic field manipulation, the high density packing of antibody-modified MNPs on the micropillars increases the local concentration of antibody, as well as the topographic interactions between cancer cells and micropillar surfaces. The flow rate and the micropillar geometry are optimized by studying their effects on capture efficiency. Then, a different number of HCT116 cells spiked in two kinds of cell suspension are investigated, yielding capture efficiency >70% in culture medium and >40% in blood sample, respectively. Moreover, the captured HCT116 cells are able to be released from the micropillars with a saturated efficiency of 92.9% upon the removal of applied magnetic field and it is found that 78% of the released cancer cells are viable, making them suitable for subsequent biological analysis. PMID:23650272

  2. XIAP-targeting drugs re-sensitize PIK3CA-mutated colorectal cancer cells for death receptor-induced apoptosis

    PubMed Central

    Ehrenschwender, M; Bittner, S; Seibold, K; Wajant, H

    2014-01-01

    Mutations in the oncogenic PIK3CA gene are found in 10–20% of colorectal cancers (CRCs) and are associated with poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic TRAIL death receptor antibodies emerged as promising anti-neoplastic therapeutics, but to date failed to prove their capability in the clinical setting as especially primary tumors exhibit high rates of TRAIL resistance. In our study, we investigated the molecular mechanisms underlying TRAIL resistance in CRC cells with a mutant PIK3CA (PIK3CA-mut) gene. We show that inhibition of the constitutively active phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway only partially overcame TRAIL resistance in PIK3CA-mut-protected HCT116 cells, although synergistic effects of TRAIL plus PI3K, Akt or cyclin-dependent kinase (CDK) inhibitors could be noted. In sharp contrast, TRAIL triggered full-blown cell death induction in HCT116 PIK3CA-mut cells treated with proteasome inhibitors such as bortezomib and MG132. At the molecular level, resistance of HCT116 PIK3CA-mut cells against TRAIL was reflected by impaired caspase-3 activation and we provide evidence for a crucial involvement of the E3-ligase X-linked inhibitor of apoptosis protein (XIAP) therein. Drugs interfering with the activity and/or the expression of XIAP, such as the second mitochondria-derived activator of caspase mimetic BV6 and mithramycin-A, completely restored TRAIL sensitivity in PIK3CA-mut-protected HCT116 cells independent of a functional mitochondrial cell death pathway. Importantly, proteasome inhibitors and XIAP-targeting agents also sensitized other CRC cell lines with mutated PIK3CA for TRAIL-induced cell death. Together, our data suggest that proteasome- or XIAP-targeting drugs offer a novel therapeutic approach to overcome TRAIL resistance in PIK3CA-mutated CRC. PMID:25501831

  3. The farnesyl transferase inhibitor RPR-130401 does not alter radiation susceptibility in human tumor cells with a K-Ras mutation in spite of large changes in ploidy and lamin B distribution

    PubMed Central

    Mégnin-Chanet, Frédérique; Lavelle, François; Favaudon, Vincent

    2002-01-01

    Background Growth inhibition by RPR-130401, a non-peptidomimetic farnesyltransferase inhibitor, was investigated without or with combined exposure to ionizing radiation in three human tumor cell lines (HCT-116, MiAPaCa-2 and A-549) bearing a point mutation in the K-Ras gene. Results RPR-130401 inhibited cell growth with an IC50 of 50 nM (HCT-116), 120 nM (MiAPaCa-2) and 710 nM (A-549), with a poor incidence of apoptosis. The drug brought about G1 and S phase depletion together with arrest of cells in G2 phase and induced a significant accumulation of hyperploid cells showing active S phase DNA synthesis, with HCT-116 and A-549 cells being the most and least responsive, respectively. The drug also produced dramatic changes of the nuclear lamin B pattern, without lamin B cleavage and perturbation of the actin cytoskeleton. On the other hand, RPR-130401 elicited strictly additive interaction in combined treatment with ionizing radiation with regard to cell kill, altered cell cycle progression and induced hyperploidy. Conclusions The data suggest that disruption of orderly progression through mitosis and cytokinesis, is a major outcome of drug action and that this effect proceeds from inhibition of lamin B farnesylation. It is anticipated from the strict additivity of RPR-130401 and radiation that neither induced radiation resistance nor acute or late complications of radiotherapy, should occur in combined treatment with RPR-130401. PMID:11929613

  4. 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

  5. Activation of AKT signaling promotes epithelial-mesenchymal transition and tumor growth in colorectal cancer cells.

    PubMed

    Suman, Suman; Kurisetty, Vittal; Das, Trinath P; Vadodkar, Aditi; Ramos, Gabriel; Lakshmanaswamy, Rajkumar; Damodaran, Chendil

    2014-02-01

    Activation of the serine-threonine protein kinase AKT has emerged as a central feature of epithelial-mesenchymal transition (EMT), which is the initial step for metastasis in many cancer models, including colorectal cancer. The focus of our study was to dissect the role of AKT and its molecular regulation of EMT in colorectal cancer. HCT-116 colorectal cancer cells stably overexpressing AKT (AKT/HCT-116) showed significantly higher cell proliferation compared with vector-transfected cells (pCMV/HCT-116). Elevated expression of important EMT-related transcription factors and genes such as Snail, Slug, ?-catenin, vimentin, and MMP-9 correlated with increased migration and invasion by AKT/HCT-116 cells. Further, in vivo studies confirmed that AKT/HCT-116 xenografts were highly aggressive and angiogenic in nature compared with pCMV/HCT-116 xenografts. Molecular analysis of tumor samples revealed transcriptional regulation of Snail, Slug, ?-catenin, MMP-2, and MMP-9 in AKT/HCT-116 tumors. These results were supported by immunohistochemistry analysis. Low levels of E-cadherin expression with a concomitant increase in and nuclear localization of ?-catenin were evident in AKT/HCT-116 tumors compared with control tumors. Increased microvessel formation coincident with high expression of Factor VIII and increased numbers of reticulocytes confirmed the angiogenic property of AKT/HCT-116 tumors. Our results confirm the potential role of AKT signaling in regulating EMT and angiogenesis in colorectal cancer and suggest that inhibition of AKT can serve as an important therapeutic strategy in modulating EMT in colorectal cancer growth and metastasis. PMID:24000138

  6. 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 ?g/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 ?g/mL against the nine tested cancer cell lines. The lowest IC50 value of 8.40 ?g/mL was recorded with the extract of E. drupifera against MDA-MB231 breast cancer cell line. The IC50 values below 10 ?g/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

  7. Selective killing of G2 decatenation checkpoint defective colon cancer cells by catalytic topoisomerase II inhibitor.

    PubMed

    Jain, Chetan Kumar; Roychoudhury, Susanta; Majumder, Hemanta Kumar

    2015-05-01

    Cancer cells with defective DNA decatenation checkpoint can be selectively targeted by the catalytic inhibitors of DNA topoisomerase II? (topo II?) enzyme. Upon treatment with catalytic topo II? inhibitors, cells with defective decatenation checkpoint fail to arrest their cell cycle in G2 phase and enter into M phase with catenated and under-condensed chromosomes resulting into impaired mitosis and eventually cell death. In the present work we analyzed decatenation checkpoint in five different colon cancer cell lines (HCT116, HT-29, Caco2, COLO 205 and SW480) and in one non-cancerous cell line (HEK293T). Four out of the five colon cancer cell lines i.e. HCT116, HT-29, Caco2, and COLO 205 were found to be compromised for the decatenation checkpoint function at different extents, whereas SW480 and HEK293T cell lines were found to be proficient for the checkpoint function. Upon treatment with ICRF193, decatenation checkpoint defective cell lines failed to arrest the cell cycle in G2 phase and entered into M phase without proper chromosomal decatenation, resulting into the formation of tangled mass of catenated and under-condensed chromosomes. Such cells underwent mitotic catastrophe and rapid apoptosis like cell death and showed higher sensitivity for ICRF193. Our study suggests that catalytic inhibitors of topoisomerase II? are promising therapeutic agents for the treatment of colon cancers with defective DNA decatenation checkpoint. PMID:25746763

  8. Effect of new berberine derivatives on colon cancer cells.

    PubMed

    Guamán Ortiz, Luis Miguel; Croce, Anna Leta; Aredia, Francesca; Sapienza, Simone; Fiorillo, Gaetano; Syeda, Tanjia Monir; Buzzetti, Franco; Lombardi, Paolo; Scovassi, Anna Ivana

    2015-10-01

    The natural alkaloid berberine has been recently described as a promising anticancer drug. In order to improve its efficacy and bioavailability, several derivatives have been designed and synthesized and found to be even more potent than the lead compound. Among the series of berberine derivatives we have produced, five compounds were identified to be able to heavily affect the proliferation of human HCT116 and SW613-B3 colon carcinoma cell lines. Remarkably, these active compounds exhibit high fluorescence emission property and ability to induce autophagy. PMID:26341980

  9. Involvement of glutathione and glutathione metabolizing enzymes in human colorectal cancer cell lines and tissues.

    PubMed

    Kim, Areum Daseul; Zhang, Rui; Han, Xia; Kang, Kyoung Ah; Piao, Mei Jing; Maeng, Young Hee; Chang, Weon Young; Hyun, Jin Won

    2015-09-01

    Reduced glutathione (GSH) is an abundant tripeptide present in the majority of cell types. GSH is highly reactive and is often conjugated to other molecules, via its sulfhydryl moiety. GSH is synthesized from glutamic acid, cysteine, and glycine via two sequential ATP?consuming steps, which are catalyzed by glutamate cysteine ligase (GCL) and GSH synthetase (GSS). However, the role of GSH in cancer remains to be elucidated. The present study aimed to determine the levels of GSH and GSH synthetic enzymes in human colorectal cancer. The mRNA and protein expression levels of GSH, the catalytic subunit of GCL (GCLC) and GSS were significantly higher in the following five colon cancer cell lines: Caco?2, SNU?407, SNU?1033, HCT?116, and HT?29, as compared with the normal colon cell line, FHC. Similarly, in 9 out of 15 patients with colon cancer, GSH expression levels were higher in tumor tissue, as compared with adjacent normal tissue. In addition, the protein expression levels of GCLC and GSS were higher in the tumor tissue of 8 out of 15, and 10 out of 15 patients with colon cancer respectively, as compared with adjacent normal tissue. Immunohistochemical analyses confirmed that GCLC and GSS were expressed at higher levels in colon cancer tissue, as compared with normal mucosa. Since GSH and GSH metabolizing enzymes are present at elevated levels in colonic tumors, they may serve as clinically useful biomarkers of colon cancer, and/or targets for anti-colon cancer drugs. PMID:26059756

  10. Association of DNA methyltransferases expression with global and gene-specific DNA methylation in colorectal cancer cells.

    PubMed

    Sarabi, Mostafa Moradi; Naghibalhossaini, Fakhraddin

    2015-10-01

    There are conflicting reports regarding the association between DNA methyltransferases (DNMTs) expression and global or gene-specific DNA methylation in colorectal cancer (CRC) cells. To correlate DNMTs expression with DNA methylation, we quantified DNMT1, DNMT3A and DNMT3B mRNA levels in five CRC cell lines (HCT116, LS180, HT29/219, Caco2 and SW742) by real-time reverse-transcriptase polymerase chain reaction (PCR) assay. In addition, we examined the global 5-methyl cytosine levels and the methylation patterns of 12 CpG islands in these CRC cells by enzyme-linked immunosorbent assay and methylation-specific PCR methods, respectively. The average expression levels of three DNMTs in HCT116, Caco2, HT29/219 and SW742, relative to the expression level in LS180 (taken to be 1), were 90·1, 31·6, 2·66 and 1·86. Our data indicated that overall about 1·45%, 1·03%, 0·98%, 0·86% and 0·85% of the cytosines were methylated in the genome of HCT116, Caco2, HT29/219, SW742 and LS180 cells, respectively. The 5-mC percentages were positively correlated with the relative cellular DNMTs expression in five CRC cell lines as verified by Pearson correlation test. However, we found no positive correlation between mRNA expression of DNMTs and gene promoter hypermethylation in these cells. Our results suggest that cellular DNMT expression is positively correlated with global DNA methylation level but not with regional DNA hypermethylation at each locus. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26416384

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

    PubMed Central

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

    2014-01-01

    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 (50nM) 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

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

    PubMed

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

    2014-09-26

    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. PMID:25218158

  13. Curcumin inhibits the proteasome activity in human colon cancer cells in vitro and in vivo

    PubMed Central

    Milacic, Vesna; Banerjee, Sanjeev; Landis-Piwowar, Kristin R.; Sarkar, Fazlul H.; Majumdar, Adhip P.N.; Dou, Q. Ping

    2008-01-01

    Curcumin (diferuloylmethane) is the major active ingredient of turmeric (curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiological conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the N-terminal threonine of the proteasomal chymotrypsin-like subunit. Consistently, curcumin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50=1.85 µM) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor–bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression and apoptosis induction in tumor tissues. Our study demonstrates that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapaeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early stage and late stage/refractory colon cancer. PMID:18794115

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

    PubMed

    Nikoli?, Biljana; Vasilijevi?, Bojana; Miti?-?ulafi?, Dragana; Vukovi?-Ga?i?, Branka; Kneževi?-Vuk?evi?, 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 ?M in bacterial assay and 50 ?M 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

  15. 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 ?mol/L. Treatment of the cells with physalin B (2.5–10 ?mol/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

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

    PubMed

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

    2012-02-10

    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. PMID:22079639

  17. 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

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

    PubMed

    Morales, Fátima; Ramírez, Alberto; Conejo-García, Ana; Morata, Cynthia; Marchal, Juan A; Campos, Joaquín 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

  19. 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

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

    PubMed Central

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

    2014-01-01

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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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-1?, MIP-1?, MCP-1 and TNF-?) 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

  6. 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

  7. 3-Deoxy-3,4-dehydro analogs of XM462. Preparation and activity on sphingolipid metabolism and cell fate.

    PubMed

    Camacho, Luz; Simbari, Fabio; Garrido, Maria; Abad, José Luis; Casas, Josefina; Delgado, Antonio; Fabriàs, Gemma

    2012-05-15

    Three analogs of the dihydroceramide desaturase inhibitor XM462 are reported. The compounds inhibit both dihydroceramide desaturase and acid ceramidase, but with different potencies depending on the N-acyl moiety. Other enzymes of sphingolipid metabolism, such as neutral ceramidase, acid sphingomyelinase, acid glucosylceramide hydrolase, sphingomyelin synthase and glucosylceramide synthase, are not affected. The effect on the sphingolipidome of the two best inhibitors, namely (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)octanamide (RBM2-1B) and (R,E)-N-(1-hydroxy-4-(tridecylthio)but-3-en-2-yl)pivalamide (RBM2-1D), is in accordance with the results obtained in the enzyme assays. These two compounds reduce cell viability in A549 and HCT116 cell lines with similar potencies and both induced apoptotic cell death to similar levels than C8-Cer in HCT116 cells. The possible therapeutic implications of the activities of these compounds are discussed. PMID:22537678

  8. 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

  9. Antitumor Activity of Americanin A Isolated from the Seeds of Phytolacca americana by Regulating the ATM/ATR Signaling Pathway and the Skp2-p27 Axis in Human Colon Cancer Cells.

    PubMed

    Jung, Cholomi; Hong, Ji-Young; Bae, Song Yi; Kang, Sam Sik; Park, Hyen Joo; Lee, Sang Kook

    2015-12-24

    The antiproliferative and antitumor activities of americanin A (1), a neolignan isolated from the seeds of Phytolacca americana, were investigated in human colon cancer cells. Compound 1 inhibited the proliferation of HCT116 human colon cancer cells both in vitro and in vivo. The induction of G2/M cell-cycle arrest by 1 was concomitant with regulation of the ataxia telangiectasia-mutated/ATM and Rad3-related (ATM/ATR) signaling pathway. Treatment with 1 activated ATM and ATR, initiating the subsequent signal transduction cascades that include checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), and tumor suppressor p53. Another line of evidence underlined the significance of 1 in regulation of the S phase kinase-associated protein 2 (Skp2)-p27 axis. Compound 1 targeted selectively Skp2 for degradation and thereby stabilized p27. Therefore, compound 1 suppressed the activity of cyclin B1 and its partner cell division cycle 2 (cdc2) to prevent entry into mitosis. Furthermore, prolonged treatment with 1 induced apoptosis by producing excessive reactive oxygen species. The intraperitoneal administration of 1 inhibited the growth of HCT116 tumor xenografts in nude mice without any overt toxicity. Modulation of the ATM/ATR signaling pathway and the Skp2-p27 axis might be plausible mechanisms of action for the antiproliferative and antitumor activities of 1 in human colon cancer cells. PMID:26595875

  10. 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

  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. Multiple Effects of Berberine Derivatives on Colon Cancer Cells

    PubMed Central

    Guamán Ortiz, Luis Miguel; Dutto, Ilaria; Arcamone, Andrea G.; Buzzetti, Franco

    2014-01-01

    The pharmacological use of the plant alkaloid berberine is based on its antibacterial and anti-inflammatory properties; recently, anticancer activity has been attributed to this compound. To exploit this interesting feature, we synthesized three berberine derivatives, namely, NAX012, NAX014, and NAX018, and we tested their effects on two human colon carcinoma cell lines, that is, HCT116 and SW613-B3, which are characterized by wt and mutated p53, respectively. We observed that cell proliferation is more affected by cell treatment with the derivatives than with the lead compound; moreover, the derivatives proved to induce cell cycle arrest and cell death through apoptosis, thus suggesting that they could be promising anticancer drugs. Finally, we detected typical signs of autophagy in cells treated with berberine derivatives. PMID:25045712

  13. Lipoic acid induces p53-independent cell death in colorectal cancer cells and potentiates the cytotoxicity of 5-fluorouracil.

    PubMed

    Dörsam, Bastian; Göder, Anja; Seiwert, Nina; Kaina, Bernd; Fahrer, Jörg

    2015-10-01

    Alpha-lipoic acid (LA), which plays a pivotal role in mitochondrial energy metabolism, is an endogenous dithiol compound with an array of antioxidative functions. It has been shown that LA triggers cell death in tumor cell lines, whereas non-transformed cells are hardly affected. In the present study, we analyzed the cytotoxicity of LA on colorectal cancer (CRC) cells differing in their p53 status and investigated a putative synergistic effect with the anticancer drug 5-fluorouracil (5-FU). We show that LA induces a dose-dependent decrease in cell viability, which was independent of the p53 status as attested in isogenic p53-proficient and p53-deficient cell lines. This effect was largely attributable to cell death induction as revealed by Annexin-V/PI staining. LA-treated HCT116 cells underwent caspase-dependent and caspase-independent cell death, which was blocked by the pan-caspase inhibitor zVAD and the RIP-kinase inhibitor Necrostatin-1, respectively. In CaCO-2 and HT29 cells, LA induced caspase-dependent cell demise via activation of caspase-9, caspase-3 and caspase-7 with subsequent PARP-1 cleavage as demonstrated by immunoblot analysis, activity assays and pan-caspase inhibition. Interestingly, LA treatment did neither activate p53 nor induced genotoxic effects as shown by lack of DNA strand breaks and phosphorylation of histone 2AX. Finally, we provide evidence that LA increases the cytotoxic effect induced by the anticancer drug 5-FU as revealed by significantly enhanced cell death rates in HCT116 and CaCO-2 cells. Collectively, these findings demonstrate that LA induces CRC cell death independent of their p53 status and potentiates the cytotoxicity of 5-FU without causing DNA damage on its own, which makes it a candidate for tumor therapy. PMID:25526924

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

    PubMed

    Ragusa, Marco; Statello, Luisa; Maugeri, Marco; Barbagallo, Cristina; Passanisi, Roberta; Alhamdani, Mohamed S; Li Destri, Giovanni; Cappellani, Alessandro; Barbagallo, Davide; Scalia, Marina; Valadi, Hadi; Hoheisel, Jörg D; Di Pietro, Cinzia; Purrello, Michele

    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

  15. 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

  16. 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.

  17. 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

  18. 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 ?M 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 ?-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

  19. 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 ?M 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 ?-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

  20. 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.

  1. Homeostatic Maintenance of Allele-Specific p16 Methylation in Cancer Cells Accompanied by Dynamic Focal Methylation and Hydroxymethylation

    PubMed Central

    Qin, Sisi; Li, Qiang; Zhou, Jing; Liu, Zhao-jun; Su, Na; Wilson, James; Lu, Zhe-ming; Deng, Dajun

    2014-01-01

    Aim p16 Methylation frequently occurs in carcinogenesis. While it has been hypothesized that the p16 methylation states are dynamically maintained in cancer cells, direct evidence supporting this hypothesis has not been available until now. Methods A fusion cell model was established which reprogrammed the native DNA methylation pattern of the cells. The methylation status of the p16 alleles was then repeatedly quantitatively analyzed in the fusion monoclonal, parental cancer cell lines (p16-completely methylated-AGS and unmethylated-MGC803), and HCT116 non-fusion cell using DHPLC and bisulfite sequencing. Histone methylation was analyzed using chromatin immuno-precipitation (ChIP)-PCR. P16 expression status was determined using immuno-staining and RT-PCR. Results The methylation status for the majority of the p16 alleles was stably maintained in the fusion monoclonal cells after up to 60 passages. Most importantly, focal de novo methylation, demethylation, and hydroxymethylation were consistently observed within about 27% of the p16 alleles in the fusion monoclones, but not the homozygously methylated or unmethylated parental cells. Furthermore, subclones of the monoclones consistently maintained the same p16 methylation pattern. A similar phenomenon was also observed using the p16 hemi-methylated HCT116 non-fusion cancer cell line. Interestingly, transcription was not observed in p16 alleles that were hydroxymethylated with an antisense-strand-specific pattern. Also, the levels of H3K9 and H3K4 trimethylation in the fusion cells were found to be slightly lower than the parental AGS and MGC803 cells, respectively. Conclusion The present study provides the first direct evidence confirming that the methylation states of p16 CpG islands is not only homeostatically maintained, but also accompanied by a dynamic process of transient focal methylation, demethylation, and hydroxymethylation in cancer cells. PMID:24828678

  2. 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 ?g/ml, respectively. Analysis of the cytotoxic activity showed that wild celery oil and isofuranodiene are able to induce apoptosis in colon cancer cells in a time and concentration-dependent manner suggesting a potential role as models for the development of chemopreventive agents. PMID:24924290

  3. Differential Regulation of Specific Sphingolipids in Colon Cancer Cells during Staurosporine-Induced Apoptosis.

    PubMed

    Del Solar, Virginia; Lizardo, Darleny Y; Li, Nasi; Hurst, Jerod J; Brais, Christopher J; Atilla-Gokcumen, G Ekin

    2015-12-17

    Apoptosis is accompanied by distinct morphological changes at the plasma and organelle membrane level. Involvement of certain lipids in apoptosis has been established; however, we have limited understanding of the specific lipid structures that participate in this process. We used untargeted comparative lipidomics to study the changes in lipid composition during staurosporine-induced apoptosis in HCT-116. Our results revealed that ceramides, dihydroceramides, and sphingomyelins, with defined acyl chains, constitute the majority of changes in the lipidome. Expression levels and activities of enzymes responsible for the biosynthesis of lipids that change suggest that de novo synthesis causes these specific changes. Further analysis of the lipidome during apoptosis in other cancer and non-cancer cell lines suggested that accumulation of ceramides and dihydroceramides is specific to cancer cells. Taken together, our data propose that these molecules are regulated at the lipid-specific level during apoptosis and that this regulation differs between cancer and non-cancer cells. PMID:26687483

  4. 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

  5. Anticancer effects of fucoxanthin and fucoxanthinol on colorectal cancer cell lines and colorectal cancer tissues

    PubMed Central

    TAKAHASHI, KAZUTO; HOSOKAWA, MASASHI; KASAJIMA, HIROYUKI; HATANAKA, KAZUTERU; KUDO, KAZUHIRO; SHIMOYAMA, NORIHIKO; MIYASHITA, KAZUO

    2015-01-01

    Colorectal cancer is one of the most malignant neoplasms worldwide. Fucoxanthin is a carotenoid present in the chloroplasts of brown seaweeds. In the present study, the anticancer effects of fucoxanthin and its metabolite, fucoxanthinol, on 6 colorectal cancer cell lines and 20 tissue samples from surgically resected clinical colorectal cancer specimens were examined using a collagen-gel droplet embedded culture drug sensitivity test (CD-DST). The in vitro sensitivity to fucoxanthin, fucoxanthinol and the anticancer drugs is expressed as T/C (%), where T is the absorbance of cells which stained by neutral red treated with carotenoids and C is the absorbance of non-staining cells. Fucoxanthin and fucoxanthinol decreased the T/C (%) of Caco-2, WiDr, HCT116, and DLD-1 cell lines at doses of 20 µM. Fucoxanthinol also decreased the T/C (%) of SW620 cells, while the T/C (%) of Colo205 cells was not reduced by treatment with either carotenoid. Specifically, the T/C (%) of Caco-2 and WiDr cells, which were incubated in carotenoid-free medium for 6 days following treatment with 20 µM fucoxanthinol for 24 h, was markedly decreased to 1.4±0.2 and 12.0±0.3%, respectively. Furthermore, fucoxanthin and fucoxanthinol decreased the T/C (%) in colorectal cancer tissue samples. Notably, 20 µM fucoxanthinol treatment resulted in a higher proportion of colorectal cancer samples with a T/C (%) of <50% (13/20, 65%) compared with samples treated with 20 µM fucoxanthin (2/20, 10%). The median T/C (%) value of 35.1% for the 20 cancers specimens treated with 20 µM fucoxanthinol was lower than the median T/C (%) values of 86.3% and 75.8% for those treated with fluorouracil and paclitaxel, respectively. These results suggested that fucoxanthin and fucoxanthinol may be of use as chemotherapeutic agents in colorectal cancer. PMID:26622691

  6. 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 (?-H2AX) were higher in PES1-silenced cells than in control cells. Besides, etoposide-induced ?-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.

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

    PubMed

    Ivanovska, Jelena; Zlobec, Inti; Forster, Stefan; 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

    2015-11-01

    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

  8. OSU-CG5, a novel energy restriction mimetic agent, targets human colorectal cancer cells in vitro

    PubMed Central

    Arafa, El-shaimaa A; Abdelazeem, Ahmed H; Arab, Hany H; Omar, Hany A

    2014-01-01

    Aim: Energy-restriction mimetic agents (ERMAs) are small-molecule agents that target various aspects of energy metabolism, which has emerged as a promising approach in cancer therapy. In the current study, we tested the ability of OSU-CG5, a novel ERMA, to target human colorectal cancer (CRC) in vitro. Methods: Two human CRC cell lines (HCT-116 and Caco-2) were tested. Cell viability was assessed using MTT assay. Caspase-3/7 activities were measured using Caspase-Glo 3/7 assay kit. Western blot analysis was used to measure the expression of relevant proteins in the cells. Glucose consumption of the cells was detected using glucose uptake cell-based assay kit. Results: OSU-CG5 dose-dependently inhibited HCT-116 and Caco-2 cell proliferation with the IC50 values of 3.9 and 4.6 ?mol/L, respectively, which were 20–25-fold lower than those of resveratrol, a reference ERMA. Both OSU-CG5 (5, 10, and 20 ?mol/L) and resveratrol (50, 100, and 200 ?mol/L) dose-dependently increased caspase-3/7 activity and PARP level in the cells. Furthermore, both OSU-CG5 and resveratrol induced dose-dependent energy restriction in the cells: they suppressed glucose uptake and Akt phosphorylation, decreased the levels of p-mTOR and p-p70S6K, increased the levels of ER stress response proteins GRP78 and GADD153, and increased the level of ?-TrCP, which led to the downregulation of cyclin D1 and Sp1. Conclusion: OSU-CG5 exhibits promising anti-cancer activity against human CRC cells in vitro, which was, at least in part, due to energy restriction and the consequent induction of ER stress and apoptosis. PMID:24464048

  9. HuR Regulates Alternative Splicing of the TRA2 Gene in Human Colon Cancer Cells under Oxidative Stress

    E-print Network

    Dever, Jennifer A.

    HuR Regulates Alternative Splicing of the TRA2 Gene in Human Colon Cancer Cells under Oxidative the translation of target mRNAs. The human TRA2 gene encodes splicing factor transformer 2 (Tra2 ) and generates 5 mRNA isoforms (TRA2 1 to -5) through alternative splicing. Exposure of HCT116 colon cancer cells

  10. 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...

  11. 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 ?-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 ?-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

  12. TMPRSS4 correlates with colorectal cancer pathological stage and regulates cell proliferation and self-renewal ability

    PubMed Central

    Huang, Ao; Zhou, Houmin; Zhao, Hongchao; Quan, Yingjun; Feng, Bo; Zheng, Minhua

    2014-01-01

    Transmembrane protease/serine 4 (TMPRSS4) is a member of the type II transmembrane serine protease (TTSP) family and it was found highly expressed in several cancers. This study aims to evaluate the expression of TMPRSS4 in colorectal cancer (CRC) and investigate its role in proliferation and self-renewal of colon cancer cells. qRT-PCR and immunohistochemistry were used to detect the mRNA and protein expression level of TMRPSS4 in CRC samples respectively. Loss of function assay was conducted with RNAi technique. Cell proliferation was done with WST-8 assay; cell apoptosis and cell cycle analysis were performed with flow cytometry; invasion and migration were done with transwell assay. Plate and soft agarose clonogenic assays were used to detect clone-formation ability. CD44 and CD133 expressions were analyzed by flow cytometry and western blot. We found that TMPRSS4 was highly expressed in CRC tissues both at mRNA and protein level and correlated with pathological stage. Knockdown of TMPRSS4 in highly expressed colon cancer cell line HCT116 resulted in inhibition of cell proliferation, induction of cell apoptosis and suppression of invasion and migration; moreover, knockdown of TMPRSS4 suppressed the in vitro clone-formation ability of HCT116 and reduced the expressions of CD44 and CD133. The findings in this research showed that TMPRSS4 was associated with CRC stage and regulated the proliferation and self-renewal ability of colon cancer cells; TMRPSS4 was involved in the development and progression of CRC. PMID:24335200

  13. 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.

  14. Defective autophagosome formation in p53-null colorectal cancer reinforces crocin-induced apoptosis.

    PubMed

    Amin, Amr; Bajbouj, Khuloud; Koch, Adrian; Gandesiri, Muktheshwar; Schneider-Stock, Regine

    2015-01-01

    Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53(-/-) cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G1 (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53(-/-) after 24 h. Crocin induced inefficient autophagy in HCT116 p53(-/-) cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53(-/-) after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53(-/-) cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, ?H2AX indicating that crocin induced an autophagy-independent classical programmed cell death. PMID:25584615

  15. 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

  16. Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

    PubMed Central

    Le François, B G; Maroun, J A; Birnboim, H C

    2007-01-01

    Thymidylate synthase (TS) is the enzyme that catalyses the last step in de novo thymidylate synthesis. It is of interest clinically because it is an effective target for drugs such as 5-fluorouracil, often used in combination therapy. Despite a number of earlier reports indicating that TS is a cell cycle-dependent enzyme, this remains equivocal. Here, we show that in HCT116 cells synchronised by serum starvation, there is a clear dissociation between the expression of cyclin E (a well-characterised cell-cycle protein) and TS. Although both cyclin E and TS mRNA and protein increased during G1, TS upregulation was delayed. Moreover, TS levels did not decrease following S-phase completion while cyclin E decreased sharply. Similarly, clear differences were seen between cyclin E and TS as asynchronously growing HCT116 cells were growth-inhibited by low-serum treatment. In contrast to previous reports using rodent cells, adenovirus-mediated over-expression of E2F1 and cyclin E in three human cell lines had no effect on TS. Cell-cycle progression was blocked by treatment of cells with pharmacological inhibitors of CDK2 and CDK4 and by ectopic expression of p16INK4A. Whereas CDK2 inhibition had no effect on TS levels, inhibition of CDK4 was associated with decreased TS protein levels. These results provide the first evidence that drugs targeting CDK4 may be useful with anti-TS drugs as combination therapy for cancer. PMID:17923872

  17. 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.

  18. Involvement of autophagy inhibition in Brucea javanica oil emulsion-induced colon cancer cell death

    PubMed Central

    YAN, ZHENG; ZHANG, BEI; HUANG, YUANYUAN; QIU, HUIJUAN; CHEN, PING; GUO, GUI-FANG

    2015-01-01

    Brucea javanica oil emulsion (BJOE), the petroleum ether extract of B. javanica emulsified by phospholipid, is widely used in China as an anticancer agent. The extracts from B. javanica induce cancer cell death by various mechanisms; however, it is not known whether these mechanisms involve autophagy, which is an important process in cancer development and treatment. Thus, the current study aimed to investigate whether BJOE modulates autophagy in HCT116 human colon cancer cells and whether modulation of autophagy is an anticancer mechanism of BJOE. Immunoblotting was employed to analyze the protein expression levels of microtubule-associated protein light-chain 3 (LC3), a specific protein marker of autophagy, in HCT116 cancer cells following exposure to BJOE. The apoptosis rate of the HCT116 cancer cells was detected by performing an Annexin V-fluorescein isothiocyanate/propidium iodide assay. According to the effect of BJOE administration on autophagy in the HCT116 cancer cells (induction or suppression), a functionally opposite agent (autophagy suppressor or inducer) was applied to counteract this effect, and the apoptosis rate of the cancer cells was detected again. The role of autophagy (pro-survival or pro-death) was demonstrated by comparing the rates of apoptotic cancer cells prior to and following the counteraction. The results revealed that BJOE suppressed the protein expression levels of LC3, including the LC3-I and LC3-II forms, and induced apoptosis in the HCT116 cancer cells with a high level of basal LC3. The apoptosis-inducing activity of BJOE was significantly attenuated when autophagy was induced by the administration of trehalose, an autophagy inducer. The data indicates that autophagy inhibition is involved in BJOE-induced cancer cell death, and that this inhibition may be a potential anticancer mechanism of BJOE. PMID:25663926

  19. 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

  20. 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

    Zhao, Quan; Huo, Xue-Chen; Sun, Fu-Dong; Dong, Rui-Qian

    2015-10-01

    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 (??m) were evaluated in MiapaCa?2 human PC cells. The effects of the extract on cell cycle phase distribution and ??m 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 ??m of the PC cells. PMID:26165362

  1. 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 (??m) were evaluated in MiapaCa-2 human PC cells. The effects of the extract on cell cycle phase distribution and ??m 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 ??m of the PC cells. PMID:26165362

  2. 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 ...

  3. 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.

  4. 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

  5. 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.

  6. 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. ?H2AX and neutral comet assays were performed to assess the effect of therapy on DSB formation/repair. In vivo assessments were made by use of HCT116 cells in a xenograft mouse model. Tumor growth delay was measured at a volume of 500 mm{sup 3}. Results: Both lines were radiosensitized by ABT alone, and ABT further increased chemotherapy dose-modifying factors to the 1.6 to 1.8 range. All combinations were synergistic (combination indices <0.9). ABT treatment significantly increased DSB after RT (?H2AX, 69% vs 43%; P=.017) and delayed repair. We found tumor growth delays of 7.22 days for RT; 11.90 days for RT and ABT; 13.5 days for oxaliplatin, RT, and ABT; 14.17 days for 5-fluorouracil, RT, and ABT; and 23.81 days for irinotecan, RT, and ABT. Conclusion: ABT-888 radiosensitizes at similar or higher levels compared with classic chemotherapies and acts synergistically with these chemotherapies to enhance RT effects. In vivo confirmation of these results indicates a potential role for combining its use with existing chemoradiation regimens.

  7. 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.

  8. 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.

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

    PubMed

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

    2013-10-01

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

  11. 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

  12. 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

  13. 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.

  14. The CDX1–microRNA-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 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

  15. 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-1? or TNF-? this effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1?. 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

  16. The impact of ATRA on shaping human myeloid cell responses to epithelial cell-derived stimuli and on T-lymphocyte polarization.

    PubMed

    Chatterjee, Arunima; 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-1? or TNF-? this effect was inhibited by ATRA but had no impact on CXCL1, CXCL8, and CCL20 secretion in response to IL-1?. 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

  17. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro.

    PubMed

    Seeram, Navindra P; Adams, Lynn S; Zhang, Yanjun; Lee, Rupo; Sand, Daniel; Scheuller, Henry S; Heber, David

    2006-12-13

    Berry fruits are widely consumed in our diet and have attracted much attention due to their potential human health benefits. Berries contain a diverse range of phytochemicals with biological properties such as antioxidant, anticancer, anti-neurodegerative, and anti-inflammatory activities. In the current study, extracts of six popularly consumed berries--blackberry, black raspberry, blueberry, cranberry, red raspberry and strawberry--were evaluated for their phenolic constituents using high performance liquid chromatography with ultraviolet (HPLC-UV) and electrospray ionization mass spectrometry (LC-ESI-MS) detection. The major classes of berry phenolics were anthocyanins, flavonols, flavanols, ellagitannins, gallotannins, proanthocyanidins, and phenolic acids. The berry extracts were evaluated for their ability to inhibit the growth of human oral (KB, CAL-27), breast (MCF-7), colon (HT-29, HCT116), and prostate (LNCaP) tumor cell lines at concentrations ranging from 25 to 200 micro g/mL. With increasing concentration of berry extract, increasing inhibition of cell proliferation in all of the cell lines were observed, with different degrees of potency between cell lines. The berry extracts were also evaluated for their ability to stimulate apoptosis of the COX-2 expressing colon cancer cell line, HT-29. Black raspberry and strawberry extracts showed the most significant pro-apoptotic effects against this cell line. The data provided by the current study and from other laboratories warrants further investigation into the chemopreventive and chemotherapeutic effects of berries using in vivo models. PMID:17147415

  18. 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

  19. 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

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

    PubMed

    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

  1. 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

  2. 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 ?-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

  3. A fluorescent probe for specific detection of cysteine in the lipid dense region of cells.

    PubMed

    Ali, Firoj; H A, Anila; Taye, Nandaraj; Gonnade, Rajesh G; Chattopadhyay, Samit; Das, Amitava

    2015-12-11

    A new cysteine (Cys) specific chemodosimetric reagent () is used in imaging of endogenous Cys localized in the lipid dense region of the live Hct116 cells and the release of Cys within HepG2 cells from a drug following a biochemical transformation. A silica surface, modified with , could be used for quantitative estimation of Cys present in aqueous solution (pH 7.2) and in a human blood plasma (HBP). PMID:26442642

  4. Pediatric brain tumor cell lines.

    PubMed

    Xu, Jingying; Margol, Ashley; Asgharzadeh, Shahab; Erdreich-Epstein, Anat

    2015-02-01

    Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research. PMID:25211508

  5. Synergistic apoptosis-inducing effect of aspirin and isosorbide mononitrate on human colon cancer cells.

    PubMed

    Wang, Xiaodong; Diao, Yuwen; Liu, Yu; Gao, Ningning; Gao, Dong; Wan, Yanyan; Zhong, Jingjing; Jin, Guangyi

    2015-09-01

    Aspirin and isosorbide mononitrate (ISMN) are two commonly used drugs, which are clinically applied for the treatment of inflammatory and cardiovascular diseases, respectively. Recently, aspirin has attracted interest due to its potential application for the treatment of cancer, particularly colon cancer. NO-aspirin, an aspirin derivative containing a covalently bound NO-donating moiety, has been proven to be an effective anti?tumor agent with apoptosis-inducing ability. In the present study, ISMN was used as an NO donor and its synergic effect with aspirin was assessed in human colon cancer cells. In vitro, an MTT assay demonstrated that ISMN had a synergistic effect on the growth inhibitory effects of aspirin on HCT116 and SW620 colon cancer cells, while the growth of EA.hy926 normal endothelial cells was unaffected. This synergistic anti?tumor effect was further validated in vivo using nude mouse HCT116 cell xenograft model. Observation of nuclear morphology, Annexin V-fluorescein isothiocyanate/propidium iodide double staining and a caspase-3 activity assay suggested that the combination of the two drugs induced apoptosis in HCT116 cells. Furthermore, the molecular mechanisms of the apoptotic effect of the drugs was assessed using an NO release assay, reverse transcription quantitative polymerase chain reaction analysis, western blot analysis and a luciferase reporter assay. It was certified that the increase in the amount of NO release, the decrease in the luciferase promoter activity and the expression of cyclin D1 and c-myc in HCT116 cells were affected by aspirin and ISMN in a synergistic manner. In conclusion, the present study was the first, to the best of our knowledge, to report on the synergistic apoptosis-inducing effects of aspirin and ISMN in human colon cancer cells, which were mediated via Wnt and NO signaling pathways. The results of the present study will facilitate the development of future therapeutic strategies. PMID:26094902

  6. hcrcn81 promotes cell proliferation through Wnt signaling pathway in colorectal cancer.

    PubMed

    Chen, Yao; Jiang, Tingting; Shi, Lihong; He, Kunyan

    2016-01-01

    The objective of the study was to investigate the role of hcrcn81 gene in Wnt/?-catenin signaling pathway related to human colorectal cancer. A total of 30 pairs of human colorectal cancer tissues with control normal tissues were analyzed by qRT-PCR. The proliferation, apoptosis, cell cycle, cell colony and metastasis of LS174T(-hcrcn81), HCT116(-hcrcn81), LoVo(+hcrcn81) and SMMC-7721(+hcrcn81) cells were tested, of which hcrcn81 was knockdown in LS174T, HCT116 cells and hcrcn81 was overexpressed in LoVo, SMMC-7721 cells. Besides, the mRNA and protein levels of hcrcn81, ?-catenin, c-Myc, cyclinD1, GSK-3? and survivin in colon cancer cell lines were evaluated by qRT-PCR and western blot. The mRNA levels of ?-catenin and Survivin were up-regulated in 76.7 % (23/30) and 63.3 % (19/30) of the tumor samples, respectively. hcrcn81 and GSK-3? mRNA expression levels were down-regulated in 20/30 (66.7 %) and 21/30 (70.0 %) of the tumor samples as compared to the adjacent normal tissues, respectively. Furthermore, in LoVo(+hcrcn81) and SMMC-7721(+hcrcn81) cells, the mRNA and protein levels of ?-catenin, c-Myc, cyclinD1 and Survivin were up-regulated, whereas those of GSK-3 were down-regulated. In LS174T(-hcrcn81) and HCT116(-hcrcn81) cells, the mRNA levels of ?-catenin, c-Myc, cyclinD1 and Survivin were down-regulated, whereas GSK-3?mRNA was up-regulated. Cell proliferation in LoVo(+hcrcn81) and SMMC-7721(+hcrcn81) groups was significantly enhanced (P < 0.05). Proliferation index in both LoVo(+hcrcn81) and SMMC-7721(+hcrcn81) groups was significantly higher than that in the control groups (P < 0.05). The number of colony in LoVo(+hcrcn81) and SMMC-7721(+hcrcn81) cells were significantly higher than that in the control groups (P < 0.05). In addition, the percentage of apoptotic cells in LoVo(+hcrcn81) and SMMC-7721(+hcrcn81) groups were significantly lower than that in the control groups (P < 0.01, P < 0.01). Finally, the number of migrating cells was significantly higher in LoVo(+hcrcn81) and SMMC-7721(+hcrcn81) groups than that in the control group (P < 0.05). hcrcn81 might promote carcinogenesis and progression through regulation of the Wnt/?-catenin signaling pathway and plays an important role in the carcinogenesis of colorectal cancer. PMID:26607374

  7. Differential proteomics identifies PDIA3 as a novel chemoprevention target in human colon cancer cells.

    PubMed

    Ménoret, Antoine; Drew, David A; Miyamoto, Shingo; Nakanishi, Masako; Vella, Anthony T; Rosenberg, Daniel W

    2014-02-01

    Chemoprevention offers a promising strategy to prevent or delay the development of various cancers. Critical to this approach is the identification of molecular targets that may track with chemopreventive efficacy. To address this issue, we screened a panel of chemoprevention agents, including resveratrol, epigallocatechin-3-gallate, ursodeoxycholic acid, and sulindac sulfide for their effects on human colon cancer cell viability. Resveratrol elicited the most potent effect in HCT116 cells and was selected for further study. Proteomic PF 2D maps were generated from HCT116 cells treated with resveratrol versus vehicle alone. Analysis of proteomic maps using tandem mass spectrometry (MS) identified a panel of differentially modified proteins. Two proteins, actin and Hsp60, were previously shown in other cell culture systems to be affected by resveratrol, validating our approach. PDIA3, RPL19, histone H2B and TCP1? were uniquely identified by our proteomic discovery platform. PDIA3 was of particular interest given its potential role in regulating chemosensitivity of cancer cells. Total levels of PDIA3 in HCT116 cells were unchanged following 24 h of resveratrol treatment, confirmed by Western blot analysis. Immunoprecipitation of PDIA3 revealed a new set of client proteins following resveratrol treatment, including ?, ?, and ?-catenins, and cellular fractionation identified decreased nuclear localization of ?-catenin by resveratrol. These data establish differential proteomic mapping as a powerful tool for identifying novel molecular targets of chemopreventive agents. PMID:23255428

  8. Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation.

    PubMed

    Kim, Wun-Jae; Lee, Se-Jung; Choi, Young Deuk; Moon, Sung-Kwon

    2010-04-01

    Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer. PMID:20198313

  9. The silence of p66(Shc) in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway.

    PubMed

    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 p66(Shc) protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66(Shc) in the progress of colon cancer still unknown. In this study, we found that p66(Shc) highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO2 cells. The silence of p66(Shc) 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 p66(Shc) siRNA. Furthermore, after HCT8 cells treated with p66(Shc) 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 p66(Shc) 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

  10. 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

  11. 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

  12. 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-?B)-p65. An increase in double strand breaks as determined by gamma-H2A histone family member X (?-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.

  13. 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.

  14. p53 interferes with microtubule-stabilizing agent-induced apoptosis in prostate and colorectal cancer cells.

    PubMed

    Kim, Ji Young; Chung, Jin-Yong; Lee, Seung Gee; Kim, Yoon-Jae; Park, Ji-Eun; Yun, Jeanho; Park, Young Chul; Kim, Byeong Gee; Yoo, Young Hyun; Kim, Jong-Min

    2013-06-01

    Taxanes are microtubule-stabilizing agents that have anticancer activity against several types of human solid tumors. Although the primary mechanism of action of these drugs is well understood, the signaling pathways that confer resistance to these agents in certain types of cancer remain poorly understood. In particular, the association of p53 with the mechanism(s) of taxane-mediated cell death is still controversial. In this study, we showed that p53 has a profound inhibitory effect on docetaxel (Doc)-induced apoptosis in prostate and colorectal cancer cells and that caspases play a critical role in this process. Doc induced prostate cancer cell apoptosis at high levels in p53-null PC3 cells, at intermediate levels in p53-mutant DU145 cells and at low levels in p53 wild-type LNCaP cells. While transient overexpression of p53 in PC3 cells suppressed Doc-induced apoptosis, knockdown of p53 in LNCaP cells increased apoptosis. This finding was further confirmed using an isogenic pair of colorectal cancer cell lines, HCT-116 p53-/- and p53+/+, indicating that p53 inhibits induction of apoptosis by Doc. To our knowledge, this is the first report describing that chemical or genetic knockout of p53 enhances the susceptibility of both prostate and colorectal cancer cells to Doc-induced apoptosis. These results may suggest an approach to stratify patients for regimens involving Doc. PMID:23563592

  15. 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

  16. 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.

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

    PubMed Central

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

    2014-01-01

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

  18. Enhancement of CD3AK cell proliferation and killing ability by ?-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 ?-Thujone to human immune cells is still unknown. Our study focuses on investigating the effects and mechanism of ?-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 ?-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-? and FasL detected with ELISA assay were not significantly changed. Mechanically, the study shows that ?-Thujone could enhance the expression of p-ERK1/2 and p-Akt. In addition, ?-Thujone has no cytotoxicity to HCT116 and SW620 cells proliferation. In a word, ?-Thujone enhances CD3AK cell proliferation and cytotoxicity via the improvement of expression of CD107a, p-Akt and p-ERK1/2. PMID:26655741

  19. Anti-Colon Cancer Effects of 6-Shogaol Through G2/M Cell Cycle Arrest by p53/p21-cdc2/cdc25A Crosstalk.

    PubMed

    Qi, Lian-Wen; Zhang, Zhiyu; Zhang, Chun-Feng; Anderson, Samantha; Liu, Qun; Yuan, Chun-Su; Wang, Chong-Zhi

    2015-01-01

    Chemopreventive agents can be identified from botanicals. Recently, there has been strong support for the potential of 6-shogaol, a natural compound from dietary ginger (Zingiber officinale), in cancer chemoprevention. However, whether 6-shogaol inhibits the growth of colorectal tumors in vivo remains unknown, and the underlying anticancer mechanisms have not been well characterized. In this work, we observed that 6-shogaol (15 mg/kg) significantly inhibited colorectal tumor growth in a xenograft mouse model. We show that 6-shogaol inhibited HCT-116 and SW-480 cell proliferation with IC50 of 7.5 and 10 ?M, respectively. Growth of HCT-116 cells was arrested at the G2/M phase of the cell cycle, primarily mediated by the up-regulation of p53, the CDK inhibitor p21(waf1/cip1) and GADD45?, and by the down-regulation of cdc2 and cdc25A. Using p53(-/-) and p53(+/+) HCT-116 cells, we confirmed that p53/p21 was the main pathway that contributed to the G2/M cell cycle arrest by 6-shogaol. 6-Shogaol induced apoptosis, mainly through the mitochondrial pathway, and the bcl-2 family might act as a key regulator. Our results demonstrated that 6-shogaol induces cancer cell death by inducing G2/M cell cycle arrest and apoptosis. 6-Shogaol could be an active natural product in colon cancer chemoprevention. PMID:26119958

  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-? 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-?. 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-? 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-? protein. PMID:26472352

  1. 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-? 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-?. 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-? 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-? protein. PMID:26472352

  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. Black Raspberry-Derived Anthocyanins Demethylate Tumor Suppressor Genes Through the Inhibition of DNMT1 and DNMT3B in Colon Cancer Cells

    PubMed Central

    Wang, Li-Shu; Kuo, Chieh-Ti; Cho, Seung-Ju; Seguin, Claire; Siddiqui, Jibran; Stoner, Kristen; Weng, Yu-I; Huang, Tim H.-M.; Tichelaar, Jay; Yearsley, Martha; Stoner, Gary D.; Huang, Yi-Wen

    2013-01-01

    We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 ?g/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of ?-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers. PMID:23368921

  4. Resveratrol induces DNA damage in colon cancer cells by poisoning topoisomerase II and activates the ATM kinase to trigger p53-dependent apoptosis.

    PubMed

    Demoulin, Benjamin; Hermant, Maryse; Castrogiovanni, Cédric; Staudt, Catherine; Dumont, Patrick

    2015-08-01

    Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural polyphenol synthesized by various plants such as grape vine. Resveratrol (RSV) is a widely studied molecule, largely for its chemopreventive effect in different mouse cancer models. We propose a mechanism underlying the cytotoxic activity of RSV on colon cancer cells. Our data show that resveratrol induces apoptosis, as observed by the cleavage of PARP-1 and chromatin condensation. We show that the tumor suppressor p53 is activated in response to RSV and participates to the apoptotic process. Additionally, we show that HCT-116 p53 wt colon carcinoma cells are significantly more sensitive than HCT-116 p53-/- cells to RSV. RSV induces DNA damage including double strand breaks, as evidenced by the presence of multiple ?-H2AX foci in 50% of cells after a 24 h treatment with 25 ?M RSV. The formation of DNA damage does not appear to rely on a pro-oxidant effect of the molecule, inhibition of topoisomerase I, or DNA intercalation. Rather, we show that DNA damage is the consequence of type II topoisomerase poisoning. Exposure of HCT-116 cells to RSV leads to activation of the Ataxia Telangiectasia Mutated (ATM) kinase, and ATM is required to activate p53. PMID:25952326

  5. Derricin and Derricidin Inhibit Wnt/?-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/?-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/?-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/?-catenin pathway. Both chalcones are able to affect the cell distribution of ?-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/?-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/?-catenin pathway and colon cancer cell growth in vitro. PMID:25775405

  6. Tussilagone suppresses colon cancer cell proliferation by promoting the degradation of ?-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/?-catenin pathway. •TSL suppressed the ?-catenin/T-cell factor transcriptional activity. •The proteasomal degradation of ?-catenin was induced by TSL. •TSL suppressed the Wnt/?-catenin target genes, cyclin D1 and c-myc. •TSL inhibit the proliferation of colon cancer cells. -- Abstract: Abnormal activation of the Wnt/?-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 ?-catenin dependent Wnt pathway. TSL suppressed ?-catenin/T-cell factor transcriptional activity and down-regulated ?-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-3?. Since the mRNA level was not changed by TSL, proteasomal degradation might be responsible for the decreased level of ?-catenin. In SW480 and HCT116 colon cancer cell lines, TSL suppressed the ?-catenin activity and also decreased the expression of cyclin D1 and c-myc, representative target genes of the Wnt/?-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. Acetyl-keto-?-boswellic acid inhibits cellular proliferation through a p21-dependent pathway in colon cancer cells

    PubMed Central

    Liu, Jian-Jun; Huang, Baohua; Hooi, Shing Chuan

    2006-01-01

    Although there is increasing evidence showing that boswellic acid might be a potential anticancer agent, the mechanisms involved in its action are unclear. In the present study, we showed that acetyl-keto-?-boswellic acid (AKBA) inhibited cellular growth in several colon cancer cell lines. Cell cycle analysis by flow cytometry showed that cells were arrested at the G1 phase after AKBA treatment. Further analysis showed that cyclin D1 and E, CDK 2 and 4 and phosphorylated Rb were decreased in AKBA-treated cells while p21 expression was increased. The growth inhibitory effect of AKBA was dependent on p21 but not p53. HCT-116 p53?/? cells were sensitized to the apoptotic effect of AKBA, suggesting that p21 may have protected cells against apoptosis by inducing a G1 arrest. In conclusion, we have demonstrated that AKBA inhibited cellular growth in colon cancer cells. These findings may have implications to the use of boswellic acids as potential anticancer agents in colon cancer. PMID:16783403

  8. Acetyl-keto-beta-boswellic acid inhibits cellular proliferation through a p21-dependent pathway in colon cancer cells.

    PubMed

    Liu, Jian-Jun; Huang, Baohua; Hooi, Shing Chuan

    2006-08-01

    1. Although there is increasing evidence showing that boswellic acid might be a potential anticancer agent, the mechanisms involved in its action are unclear. 2. In the present study, we showed that acetyl-keto-beta-boswellic acid (AKBA) inhibited cellular growth in several colon cancer cell lines. Cell cycle analysis by flow cytometry showed that cells were arrested at the G1 phase after AKBA treatment. 3. Further analysis showed that cyclin D1 and E, CDK 2 and 4 and phosphorylated Rb were decreased in AKBA-treated cells while p21 expression was increased. 4. The growth inhibitory effect of AKBA was dependent on p21 but not p53. HCT-116 p53(-/-) cells were sensitized to the apoptotic effect of AKBA, suggesting that p21 may have protected cells against apoptosis by inducing a G1 arrest.5. In conclusion, we have demonstrated that AKBA inhibited cellular growth in colon cancer cells. These findings may have implications to the use of boswellic acids as potential anticancer agents in colon cancer. PMID:16783403

  9. 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

  10. 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

  11. 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 4?-[(4"-benzamido)-amino]-4'-O-demethyl-epipodophyllotoxin derivative. Previous reports have shown that NPRL-Z-1 possesses anticancer activity. Here NPRL-Z-1 displayed cytotoxic effects against four human cancer cell lines (HCT 116, A549, ACHN, and A498) and exhibited potent activity in A498 human renal carcinoma cells, with an IC50 value of 2.38 µM via the MTT assay. We also found that NPRL-Z-1 induced cell cycle arrest in G1-phase and detected DNA double-strand breaks in A498 cells. NPRL-Z-1 induced ataxia telangiectasia-mutated (ATM) protein kinase phosphorylation at serine 1981, leading to the activation of DNA damage signaling pathways, including Chk2, histone H2AX, and p53/p21. By ICE assay, the data suggested that NPRL-Z-1 acted on and stabilized the topoisomerase II (TOP2)-DNA complex, leading to TOP2cc formation. NPRL-Z-1-induced DNA damage signaling and apoptotic death was also reversed by TOP2? or TOP2? knockdown. In addition, NPRL-Z-1 inhibited the Akt signaling pathway and induced reactive oxygen species (ROS) generation. These results demonstrated that NPRL-Z-1 appeared to be a novel TOP2 poison and ROS generator. Thus, NPRL-Z-1 may present a significant potential anticancer candidate against renal carcinoma. PMID:25372714

  12. New Arylthioindoles and Related Bioisosteres at the Sulfur Bridging Group. 4. Synthesis, Tubulin Polymerization, Cell Growth Inhibition, and Molecular Modeling Studies

    PubMed Central

    La Regina, Giuseppe; Sarkar, Taradas; Bai, Ruoli; Edler, Michael C.; Saletti, Roberto; Coluccia, Antonio; Piscitelli, Francesco; Minelli, Lara; Gatti, Valerio; Mazzoccoli, Carmela; Palermo, Vanessa; Mazzoni, Cristina; Falcone, Claudio; Scovassi, Anna Ivana; Giansanti, Vincenzo; Campiglia, Pietro; Porta, Amalia; Maresca, Bruno; Hamel, Ernest; Brancale, Andrea; Novellino, Ettore; Silvestri, Romano

    2009-01-01

    New arylthioindoles along with the corresponding ketone and methylene compounds were potent tubulin assembly inhibitors. As growth inhibitors of MCF-7 cells, sulfur derivatives were superior or sometimes equivalent to the ketones, while methylene derivatives were substantially less effective. Esters 24, 27–29, 36, 39,and 41 showed ~50% of inhibition on human HeLa and HCT116/chr3 cells at 0.5 ?M, and these compounds inhibited the growth of HEK, M14, and U937 cells with IC50's in the 78–220 nM range. While murine macrophage J744.1 cell growth was significantly less affected (20% at higher concentrations), four other nontransformed cell lines remained sensitive to these esters. The effect of drug treatment on cell morphology was examined by time-lapse microscopy. In a protocol set up to evaluate toxicity on the Saccharomyces cerevisiae BY4741 wild type strain, compounds 24 and 54 strongly reduced cell growth, and 29, 36, and 39 also showed significant inhibition. PMID:19601594

  13. 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 NF?BIA 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 NF?B pathway after irradiation but another mechanism of activation must operate in bystander cells. PMID:26099456

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

    PubMed Central

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

    2014-01-01

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

  15. 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

  16. Induction of mitosis delay, apoptosis and aneuploidy in human cells by phenyl hydroquinone, an Ames test-negative carcinogen.

    PubMed

    Imai, Masaru; Matsuno, Ryo; Komura, Jun-ichiro; Ono, Tetsuya; Yamamoto, Kazuo

    2009-02-01

    Ortho-phenyl phenol and its hepatic derivative, phenyl hydroquinone, do not generate base-substitution-type mutations, but cause bladder cancer in rats and mice. The mechanism of their carcinogenic effect is unknown. We have previously shown that o-phenyl phenol and phenyl hydroquinone induce mitotic arrest and aneuploidy in Saccharomyces cerevisiae. To further delineate the mechanism of action of phenyl hydroquinone, we examined its effect on human cells. Treatment of the colon cancer cell line HCT116 with 0 to 150 microM phenyl hydroquinone caused a concentration-dependent inhibition of growth, accumulation of cells having G2/M DNA content, and an increase in the mitotic index. Moreover, a dose-dependent increase in apoptotic cells was observed. Finally, a high frequency of aneuploid cells was found. On the other hand, no increase in gamma-H2AX foci was observed. The results show that phenyl hydroquinone does induce mitotic arrest, apoptosis and aneuploidy in the absence of DNA damage. Our results may be useful to understand the mechanisms of action of chemical substances that are Ames test-negative carcinogens. PMID:19420803

  17. RASSF10 suppresses colorectal cancer growth by activating P53 signaling and sensitizes colorectal cancer cell to docetaxel.

    PubMed

    Guo, Jing; Yang, Yage; Yang, Yunsheng; Linghu, Enqiang; Zhan, Qimin; Brock, Malcolm V; Herman, James G; Zhang, Bingyong; Guo, Mingzhou

    2015-02-28

    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

  18. 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 ?EGFR 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

  19. Platinum(II/IV) complexes containing ethylenediamine-N,N'-di-2/3-propionate ester ligands induced caspase-dependent apoptosis in cisplatin-resistant colon cancer cells.

    PubMed

    Kalu?erovi?, Goran N; Mijatovi?, Sanja A; Zmejkovski, Bojana B; Bulatovi?, Mirna Z; Gómez-Ruiz, Santiago; Moji?, Marija K; Steinborn, Dirk; Miljkovi?, Djordje M; Schmidt, Harry; Stoši?-Gruji?i?, Stanislava D; Sabo, Tibor J; Maksimovi?-Ivani?, Danijela D

    2012-08-01

    Several new R(2)eddp (R = i-Pr, i-Bu; eddp = ethylenediamine-N,N'-di-3-propionate) esters and corresponding platinum(ii) and platinum(iv) complexes of the general formula [PtCl(n)(R(2)edda-type)] (n = 2, 4) were synthesized and characterized by spectroscopic methods (IR, (1)H and (13)C NMR) and elemental analysis. The crystal structure of platinum(iv) complex [PtCl(4){(c-Pe)(2)eddip}] (3a) was resolved and is given herein. Ligand precursors, platinum(ii), and platinum(iv) complexes were tested against eight tumor cell lines (CT26CL25, HTC116, SW620, PC3, LNCaP, U251, A375, and B16). Selectivity in the action of those compounds between tumor and two normal primary cells (fibroblasts and keratinocytes) are discussed. A structure-activity relationship of these compounds is discussed. Furthermore, cell cycle distribution, induction of necrosis, apoptosis, autophagy, anoikis, caspase activation, ROS, and RNS are presented on the cisplatin-resistant colon carcinoma HCT116 cell line. PMID:22820831

  20. Half-sandwich ruthenium(II) biotin conjugates as biological vectors to cancer cells.

    PubMed

    Babak, Maria V; Pla?uk, Damian; Meier, Samuel M; Arabshahi, Homayon John; Reynisson, Jóhannes; Rychlik, B?a?ej; B?au?, Andrzej; Szulc, Katarzyna; Hanif, Muhammad; Strobl, Sebastian; Roller, Alexander; Keppler, Bernhard K; Hartinger, Christian G

    2015-03-23

    Ruthenium(II)-arene complexes with biotin-containing ligands were prepared so that a novel drug delivery system based on tumor-specific vitamin-receptor mediated endocytosis could be developed. The complexes were characterized by spectroscopic methods and their in vitro anticancer activity in cancer cell lines with various levels of major biotin receptor (COLO205, HCT116 and SW620 cells) was tested in comparison with the ligands. In all cases, coordination of ruthenium resulted in significantly enhanced cytotoxicity. The affinity of Ru(II) -biotin complexes to avidin was investigated and was lower than that of unmodified biotin. Hill coefficients in the range 2.012-2.851 suggest strong positive cooperation between the complexes and avidin. To estimate the likelihood of binding to the biotin receptor/transporter, docking studies with avidin and streptavidin were conducted. These explain, to some extent, the in vitro anticancer activity results and support the conclusion that these novel half-sandwich ruthenium(II)-biotin conjugates may act as biological vectors to cancer cells, although no clear relationship between the cellular Ru content, the cytotoxicity, and the presence of the biotin moiety was observed. PMID:25676245

  1. 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 ?-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.

  2. 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

  3. ATAD2 Overexpression Identifies Colorectal Cancer Patients with Poor Prognosis and Drives Proliferation of Cancer Cells.

    PubMed

    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

  4. 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

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

    PubMed

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

    2011-12-01

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

  6. BI-69A11 enhances susceptibility of colon cancer cells to mda-7/IL-24-induced growth inhibition by targeting Akt

    PubMed Central

    Pal, I; Sarkar, S; Rajput, S; Dey, K K; Chakraborty, S; Dash, R; Das, S K; Sarkar, D; Barile, E; De, S K; Pellecchia, M; Fisher, P B; Mandal, M

    2014-01-01

    Background: Akt and its downstream signalling pathways contribute to the aetiology and progression of colorectal carcinoma (CRC). Targeting the Akt pathway is an attractive strategy but few chemotherapeutic drugs have been used to treat CRC with only limited success. BI-69A11, a small molecule inhibitor of Akt, efficiently inhibits growth in melanoma cells. Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 promotes cancer-selective apoptosis when delivered by a tropism-modified replication incompetent adenovirus (Ad.5/3-mda-7). However, Ad.5/3-mda-7 displays diminished antitumour efficacy in several CRC cell lines, which correlates with the expression of K-RAS. Methods: The individual and combinatorial effect of BI-69A11 and Ad.5/3-mda-7 in vitro was studied by cell viability, cell cycle, apoptosis and invasion assays in HT29 and HCT116 cells containing wild type or mutant K-ras, respectively. In vivo HT29 tumour xenografts were used to test the efficacy of the combination treatment. Results: BI-69A11 inhibited growth and induced apoptosis in CRC. However, combinatorial treatment was more effective compared with single treatment. This combination showed profound antitumour and anti angiogenic effects in vitro and in vivo by downregulating Akt activity. Conclusions: BI-69A11 enhances the antitumour efficacy of Ad.5/3-mda-7 on CRC overexpressing K-RAS by inducing apoptosis and regulating Akt activity thereby warranting further evaluation in treating CRC. PMID:24892445

  7. INCREASED LEVELS OF SUPEROXIDE AND HYDROGEN PEROXIDE MEDIATE THE DIFFERENTIAL SUSCEPTIBILITY OF CANCER CELLS VS. NORMAL CELLS TO GLUCOSE DEPRIVATION

    PubMed Central

    Aykin-Burns, Nùkhet; Ahmad, Iman M.; Zhu, Yueming; Oberley, Larry W.; Spitz, Douglas R.

    2009-01-01

    Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose deprivation-induced cytotoxicity. To determine if oxidative stress mediated by O2•? and hydroperoxides contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, oxidation of dihydroethidine (DHE; for O2•?) and 5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (CDCFH2; for hydroperoxides) were measured in human colon and breast cancer cells (HT29, HCT116, SW480, MB231) and compared to normal human cells (FHC, 33Co, HMEC). Cancer cells showed significant increases in DHE (2–20 fold) and CDCFH2 (1.8–10 fold) oxidation, relative to normal cells that were more pronounced in the presence of the mitochondrial electron transport chain blocker, antimycin A. Furthermore, HCT116 and MB231 cells were more susceptible to glucose deprivation-induced cytotoxicity and oxidative stress, relative to 33Co and HMEC. HT-29 cells were also more susceptible to 2-deoxyglucose-(2DG)-induced cytotoxicity, relative to FHC. Over expression of manganese superoxide dismutase and mitochondrially targeted catalase significantly protected HCT116 and MB231 cells from glucose deprivation-induced cytotoxicity and oxidative stress, as well as protecting HT-29 cells from 2DG-induced cytotoxicity. These results show cancer cells (relative to normal cells) demonstrate increased steady-state levels of reactive oxygen species (ROS, i.e. O2•? and H2O2) that contribute to differential susceptibility to glucose deprivation-induced cytotoxicity and oxidative stress. These studies support the hypotheses that cancer cells increase glucose metabolism to compensate for excess metabolic production of ROS as well as that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells. PMID:18937644

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

    PubMed

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

    2013-01-01

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

  9. Confocal spectral imaging by microspectrofluorometry using two-photon excitation: application to the study of anticancer drugs within single living cancer cells

    NASA Astrophysics Data System (ADS)

    Chourpa, Igor; Pereira, Manuela; Millot, Jean-Marc; Morjani, Hamid; Manfait, Michel

    1999-06-01

    The use of the two-photon excitation (TPE) is believed to be prominent for fluorometric studies with cells. We evaluated the advantages and limitations of the two-photon technique compared to the single photon one when it used to detect potent anticancer drugs, camptothecins (CPTs), within single living cancer cells. The technique we used was confocal microspectrofluorometry amplified with possibility of the spectral imaging analysis. We have previously reported the use of the florescence emission of CPTs to study them qualitatively and quantitatively, namely, to follow the status of their hydrolyzable lactone moiety. However, the intracellular investigation of CPTs using microspectrofluorometry with single photon UV excitation (SPE) is hindered by significant interference of their fluorescence emission with cellular autofluorescence. We attempted to overcome these problems using the two-photon excitation. The intracellular single-photon- and two-photon-excited emission spectra from treated and control cells (HCT-116 line) were recorded using a spectral imaging approach. The obtained data demonstrate that, apart from intrinsically increased three- dimensional resolution, the two-photon approach was advantageous over the single-photon method with respect to selective fluorometric detection of intracellular CPTs. Nevertheless, much attention should be paid to avoid any excessive irradiation of the cells with UV and even NIR light.

  10. 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

  11. Synthesis of a DNA-targeting nickel (II) complex with testosterone thiosemicarbazone which exhibits selective cytotoxicity towards human prostate cancer cells (LNCaP).

    PubMed

    Heng, Mok Piew; Sinniah, Saravana Kumar; Teoh, Wuen Yew; Sim, Kae Shin; Ng, Seik Weng; Cheah, Yoke Kqueen; Tan, Kong Wai

    2015-11-01

    Testosterone thiosemicarbazone, L and its nickel (II) complex 1 were synthesized and characterized by using FTIR, CHN, (1)H NMR, and X-ray crystallography. X-ray diffraction study confirmed the formation of L from condensation of testosterone and thiosemicarbazide. Mononuclear complex 1 is coordinated to two Schiff base ligands via two imine nitrogens and two tautomeric thiol sulfurs. The cytotoxicity of both compounds was investigated via MTT assay with cisplatin as positive reference standard. L is more potent towards androgen-dependent LNCaP (prostate) and HCT 116 (colon). On the other hand, complex 1, which is in a distorted square planar environment with L acting as a bidentate NS-donor ligand, is capable of inhibiting the growth of all the cancer cell lines tested, including PC-3 (prostate). It is noteworthy that both compounds are less toxic towards human colon cell CCD-18Co. The intrinsic DNA binding constant (Kb) of both compounds were evaluated via UV-Vis spectrophotometry. Both compounds showed Kb values which are comparable to the reported Kb value of typical classical intercalator such as ethidium bromide. The binding constant of the complex is almost double compared with ligand L. Both compounds were unable to inhibit the action topoisomerase I, which is the common target in cancer treatment (especially colon cancer). This suggest a topoisomerase I independent-cell death mechanism. PMID:26057090

  12. 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

  13. LYTAK1, a novel TAK1 inhibitor, suppresses KRAS mutant colorectal cancer cell growth in vitro and in vivo.

    PubMed

    Zhou, Jundong; Zheng, Bing; Ji, Jiansong; Shen, Fei; Min, Han; Liu, Biao; Wu, Jinchang; Zhang, Shuyu

    2015-05-01

    KRAS mutation in colorectal cancer (CRC) activates transforming growth factor-? (TGF-?)-activated kinase 1 (TAK1) to promote tumor progression. In the current study, we explored the potential effect of LYTAK1, a novel TAK1 inhibitor, against KRAS mutant CRC cells in vitro and in vivo. We found that LYTAK1 dose-dependently inhibited KRAS mutant CRC cell (HT-29 and SW-620 lines) growth, and induced cell cycle G1-S arrest. Further, LYTAK1 activated apoptosis in HT-29 cells and SW-620 cells, and apoptosis inhibitors almost reversed LYTAK1-mediated growth inhibition. While in KRAS wild-type (WT) CRC cell lines (DLD-1 and HCT-116), LYTAK1 had almost no effect on cell growth, cell cycle progression, or cell apoptosis. In KRAS mutant HT-29 cells and SW-260 cells, LYTAK1 blocked TAK1 activation or phosphorylation at Thr-184/187. Activation of nuclear factor ?B (NF-?B) in these cells, detected by phosphorylations of p65 and I?B kinase ? (IKK?) as well as expression of NF-?B-regulated gene cyclin D1, was significantly inhibited by LYTAK1. Further, LYTAK1 treatment resulted in downregulation of ?-catenin and Wnt response gene Axin 2, indicating Wnt inactivation. In vivo, oral LYTAK1 significantly inhibited HT-29 xenograft growth in nude mice. Together, these results show that LYTAK1 inhibits KRAS mutant CRC cell growth both in vitro and in vivo. LYTAK1 might be investigated as a novel agent against CRC with KRAS mutation. PMID:25524577

  14. 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.

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

    PubMed

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

    2014-11-01

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

  16. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    Hahm, Eun-Ryeong; Singh, Shivendra V

    2012-06-01

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

  19. Immortalization of Mouse Germ Line Stem Cells

    PubMed Central

    Hofmann, Marie-Claude; Braydich-Stolle, Laura; Dettin, Luis; Johnson, Eric; Dym, Martin

    2011-01-01

    In the mammalian testis, the germ line stem cells are a small subpopulation of type A spermatogonia that proliferate and ultimately differentiate into sperm under the control of both endocrine and paracrine factors. To study the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby germ line stem cells can be either cultured for a prolonged period of time or expanded as cell lines. In the study reported here, we chose to immortalize type A spermatogonia using the Simian virus large T-antigen gene (LTAg) under the control of an ecdysone-inducible promoter. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow and the cells exhibited morphological features typical of spermatogonia at the light microscopic level. Moreover, the cells expressed detectable levels of protein markers specific for germ cells such as Dazl, and specific for germ line stem cells such as Oct-4, a transcription factor, and GFR?-1, the receptor for glial cell line–derived neurotrophic factor (GDNF). Further analysis confirmed the spermatogonial phenotype and also revealed the expression of markers expressed in stem cells such as Piwi12 and Prame11. Since the cells respond to GDNF by a marked increase in their rate of proliferation, this cell line represents a good in vitro model for studying aspects of mouse germ line stem cell biology. PMID:15671143

  20. Negative regulation of chemokine receptor CXCR4 by tumor suppressor p53 in breast cancer cells: implications of p53 mutation or isoform expression on breast cancer cell invasion.

    PubMed

    Mehta, S A; Christopherson, K W; Bhat-Nakshatri, P; Goulet, R J; Broxmeyer, H E; Kopelovich, L; Nakshatri, H

    2007-05-17

    Chemokine receptor CXCR4 and its ligand CXCL12 are suggested to be involved in migration, invasion and metastasis of breast cancer cells. Mutation of the tumor suppressor gene p53 in breast cancer is associated with metastasis and aggressive clinical phenotype. In this report, we demonstrate that wild type but not the dominant-negative mutant (V143A) or cancer-specific mutants (R175H or R280K) of p53 repress CXCR4 expression. Recently described cancer-specific p53 isoform, Delta133p53, also failed to repress CXCR4 promoter activity. Short-interfering RNA-mediated depletion of p53 increased endogenous CXCR4 expression in MCF-7 breast cancer cells that contain wild-type p53. Basal CXCR4 promoter activity in HCT116 colon carcinoma cells deleted of p53 [HCT116(p53KO)] was 10-fold higher compared to that in parental HCT116 cells with functional wild-type p53. Deletion analysis of CXCR4 promoter identified a seven-base pair p53-repressor element homologous to cyclic AMP/AP-1 response (CRE/AP-1) element. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed binding of ATF-1 and cJun to the CRE/AP-1 element. The p53 rescue drug PRIMA-1 reduced CXCR4 mRNA and cell surface expression in MDA-MB-231 cells, which express R280K mutant p53. CP-31398, another p53 rescue drug, similarly reduced cell surface levels of CXCR4. PRIMA-1-mediated decrease in CXCR4 expression correlated with reduced invasion of MDA-MB-231 cells through matrigel. These results suggest a mechanism for elevated CXCR4 expression and metastasis of breast cancers with p53 mutations or isoform expression. We propose that p53 rescue drugs either alone or in combination with chemotherapeutic drugs may be effective in reducing CXCR4-mediated metastasis. PMID:17130833

  1. 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.

  2. Krüppel-like factor 4 prevents centrosome amplification following gamma-irradiation-induced DNA damage.

    PubMed

    Yoon, Hong S; Ghaleb, Amr M; Nandan, Mandayam O; Hisamuddin, Irfan M; Dalton, William Brian; Yang, Vincent W

    2005-06-01

    Centrosome duplication is a carefully controlled process in the cell cycle. Previous studies indicate that the tumor suppressor, p53, regulates centrosome duplication. Here, we present evidence for the involvement of the mammalian Krüppel-like transcription factor, KLF4, in preventing centrosome amplification following DNA damage caused by gamma-irradiation. The colon cancer cell line HCT116, which contains wild-type p53 alleles (HCT116 p53+/+), displayed stable centrosome numbers following gamma-irradiation. In contrast, HCT116 cells null for the p53 alleles (HCT116 p53-/-) exhibited centrosome amplification after irradiation. In the latter cell line, KLF4 was not activated following gamma-irradiation due to the absence of p53. However, centrosome amplification could be suppressed in irradiated HCT116 p53-/- cells by conditional induction of exogenous KLF4. Conversely, in a HCT116 p53+/+ cell line stably transfected with small hairpin RNA (shRNA) designed to specifically inhibit KLF4, gamma-irradiation induced centrosome amplification. In these cells, the inability of KLF4 to become activated in response to DNA damage was directly associated with an increase in cyclin E level and Cdk2 activity, both essential for regulating centrosome duplication. Cotransfection experiments showed that KLF4 overexpression suppressed the promoter activity of the cyclin E gene. The results of this study demonstrated that KLF4 is both necessary and sufficient in preventing centrosome amplification following gamma-radiation-induced DNA damage and does so by transcriptionally suppressing cyclin E expression. PMID:15806166

  3. Biophysical Profiling of Tumor Cell Lines

    PubMed Central

    Coffman, Frederick; Hamid, Rachid; Cohen, Marion C.; Garippa, Ralph; Cohen, Stanley

    2011-01-01

    Despite significant differences in genetic profiles, cancer cells share common phenotypic properties, including membrane-associated changes that facilitate invasion and metastasis. The Corning Epic® optical biosensor was used to monitor dynamic mass rearrangements within and proximal to the cell membrane in tumor cell lines derived from cancers of the colon, bone, cervix, lung and breast. Data was collected in real time and required no exogenously added signaling moiety (signal-free technology). Cell lines displayed unique profiles over the time-courses: the time-courses all displayed initial signal increases to maximal values, but the rate of increase to those maxima and the value of those maxima were distinct for each cell line. The rate of decline following the maxima also differed among cell lines. There were correlations between the signal maxima and the observed metastatic behavior of the cells in xenograft experiments; for most cell types the cells that were more highly metastatic in mice had lower time-course maxima values, however the reverse was seen in breast cancer cells. The unique profiles of these cell lines and the correlation of at least one profile characteristic with metastatic behavior demonstrate the potential utility of biophysical tumor cell profiling in the study of cancer biology. PMID:21988886

  4. p14(ARF) Prevents Proliferation of Aneuploid Cells by Inducing p53-Dependent Apoptosis.

    PubMed

    Veneziano, Lorena; Barra, Viviana; Lentini, Laura; Spatafora, Sergio; Di Leonardo, Aldo

    2016-02-01

    Weakening the Spindle Assembly Checkpoint by reduced expression of its components induces chromosome instability and aneuploidy that are hallmarks of cancer cells. The tumor suppressor p14(ARF) is overexpressed in response to oncogenic stimuli to stabilize p53 halting cell progression. Previously, we found that lack or reduced expression of p14(ARF) is involved in the maintenance of aneuploid cells in primary human cells, suggesting that it could be part of a pathway controlling their proliferation. To investigate this aspect further, p14(ARF) was ectopically expressed in HCT116 cells after depletion of the Spindle Assembly Checkpoint MAD2 protein that was used as a trigger for aneuploidy. p14(ARF) Re-expression reduced the number of aneuploid cells in MAD2 post-transcriptionally silenced cells. Also aberrant mitoses, frequently displayed in MAD2-depleted cells, were decreased when p14(ARF) was expressed at the same time. In addition, p14(ARF) ectopic expression in MAD2-depleted cells induced apoptosis associated with increased p53 protein levels. Conversely, p14(ARF) ectopic expression did not induce apoptosis in HCT116 p53KO cells. Collectively, our results suggest that the tumor suppressor p14(ARF) may have an important role in counteracting proliferation of aneuploid cells by activating p53-dependent apoptosis. J. Cell. Physiol. 231: 336-344, 2016. © 2015 Wiley Periodicals, Inc. PMID:25752701

  5. Mitochondrial p53 phosphorylation induces Bak-mediated and caspase-independent cell death

    PubMed Central

    Luo, Na; Nie, Chunlai; Zhao, Xinyu; Yuan, Zhu; Liu, Xinyu; Wei, Yuquan

    2015-01-01

    Chemoresistance in cancer has previously been attributed to gene mutations or deficiency. Caspase mutations or Bax deficiency can lead to resistance to cancer drugs. We recently demonstrated that Bak initiates a caspase/Bax-independent cell death pathway. We show that Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone that is known to have anti-tumor activity in a variety of models, induces caspase-independent cell death in HCT116 Bax knockout (KO) or MCF-7 Bax knockdown (KD) cells that express wild-type (WT) Bak. The re-expression of Bax in HCT116 Bax KO cells fails to enhance the PL-induced cell death. Additionally, Bak knockdown by shRNA efficiently attenuates PL-induced cell death. These results suggest that PL-induced cell death depends primarily on Bak, not Bax, in these cells. Further experimentation demonstrated that p53 Ser15 phosphorylation and mitochondrial translocation mediated Bak activation and subsequent cell death. Knockdown of p53 or a p53 Ser15 mutant significantly inhibited p53 mitochondrial translocation and cell death. Furthermore, we found that Akt mediated p53 phosphorylation and the subsequent mitochondrial accumulation. Taken together, our data elaborate the role of Bak in caspase/Bax-independent cell death and suggest that PL may be an effective agent for overcoming chemoresistance in cancer cells with dysfunctional caspases. PMID:25980443

  6. 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

  7. MSH3 Mismatch Repair Protein Regulates Sensitivity to Cytotoxic Drugs and a Histone Deacetylase Inhibitor in Human Colon Carcinoma Cells

    PubMed Central

    Park, Jae Myung; Huang, Shengbing; Tougeron, David; Sinicrope, Frank A.

    2013-01-01

    Background MSH3 is a DNA mismatch repair (MMR) gene that undergoes frequent somatic mutation in colorectal cancers (CRCs) with MMR deficiency. MSH3, together with MSH2, forms the MutS? heteroduplex that interacts with interstrand cross-links induced by drugs such as cisplatin. To date, the impact of MSH3 on chemosensitivity is unknown. Methods We utilized isogenic HCT116 (MLH1?/MSH3?) cells where MLH1 is restored by transfer of chromosome 3 (HCT116+ch3) and also MSH3 by chromosome 5 (HCT116+3+5). We generated HCT116+3+5, SW480 (MLH1+/MSH3+) and SW48 (MLH1?/MSH3+) cells with shRNA knockdown of MSH3. Cells were treated with 5-fluorouracil (5-FU), SN-38, oxaliplatin, or the histone deacetylase (HDAC) inhibitor PCI-24781 and cell viability, clonogenic survival, DNA damage and apoptosis were analyzed. Results MSH3-deficient vs proficient CRC cells showed increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, but not 5-FU, as shown in assays for apoptosis and clonogenic survival. In contrast, suppression of MLH1 attenuated the cytotoxic effect of 5-FU, but did not alter sensitivity to SN-38 or oxaliplatin. The impact of MSH3 knockdown on chemosensitivity to SN-38 and oxaliplatin was maintained independent of MLH1 status. In MSH3-deficient vs proficient cells, SN-38 and oxaliplatin induced higher levels of phosphorylated histone H2AX and Chk2, and similar results were found in MLH1-proficient SW480 cells. MSH3-deficient vs proficient cells showed increased 53BP1 nuclear foci after irradiation, suggesting that MSH3 can regulate DNA double strand break (DSB) repair. We then utilized PCI-24781 that interferes with homologous recombination (HR) indicated by a reduction in Rad51 expression. The addition of PCI-24781 to oxaliplatin enhanced cytotoxicity to a greater extent compared to either drug alone. Conclusion MSH3 status can regulate the DNA damage response and extent of apoptosis induced by chemotherapy. The ability of MSH3 to regulate chemosensitivity was independent of MLH1 status. PCI-24781-mediated impairment of HR enhanced oxaliplatin sensitivity, suggesting that reduced DSB repair capacity may be contributory. PMID:23724141

  8. ApoG2 inhibits the antiapoptotic protein, Mcl?1, and induces mitochondria?dependent apoptosis in human colorectal cancer cells.

    PubMed

    Li, Tianxiao; Yuan, Gang; Zhang, Lin; Ye, Lijun; Li, Shuxia; Fan, Yuhua; Sun, Jian

    2015-11-01

    Colorectal cancer (CRC) is a worldwide malignancy of high incidence and mortality. At present, there is a lack of effective drugs against CRC. The B?cell leukemia/lymphoma 2 (Bcl?2) protein family members are considered to be closely associated with tumorigenesis and the chemoresistance of CRC. As a novel gossypol derivative targeting antiapoptotic proteins of the Bcl?2 family, apogossypolone (ApoG2) exhibits antitumor properties in various cancer types, although its effects against CRC remain to be fully elucidated. In the present study, the cytotoxicity of ApoG2 in vitro on CRC cells was investigated, with the aim of elucidating the underlying mechanism. Using an MTT assay, ApoG2 was revealed to inhibit the growth of the HT29, SW480 and HCT116 CRC cell lines in a dose? and a time?dependent manner. Hoechst staining revealed that ApoG2 induced CRC cell apoptosis, marked by morphological changes, including cell shrinkage and nuclear fragmentation. Flow cytometric analysis also detected a higher apoptotic ratio following treatment with ApoG2. The ratio was dependent upon the concentration of ApoG2, which the cells were exposed to, and the duration of the exposure. Western blot analysis and immunoprecipitation experiments revealed that ApoG2 treatment led to the downregulation of the protein expression of Mcl?1, and the interruption of the binding of Mcl?1 to the protein Bax. Furthermore, treatment with ApoG2 led to the release of cytochrome c into the cytoplasm and the activation of caspases 3 and 7. The present study revealed that ApoG2 inhibited the proliferation of the CRC cell lines through mitochondrial signaling pathway?dependent apoptosis, which may be associated with the disruption of the function of the Mcl?1 protein by ApoG2. PMID:26352605

  9. 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.; O’Leary, 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 5 µM 5Z-7-oxozeaenol, cell viability, growth, and clonogenic survival were significantly decreased. Consistent with TAK1 inhibition being causally related to thiol-mediated oxidative stress, 10 mM 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

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

    PubMed Central

    2014-01-01

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

  11. 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 transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes.

  12. Interaction of the Clostridium difficile Binary Toxin CDT and Its Host Cell Receptor, Lipolysis-stimulated Lipoprotein Receptor (LSR).

    PubMed

    Hemmasi, Sarah; Czulkies, Bernd A; Schorch, Björn; Veit, Antonia; Aktories, Klaus; Papatheodorou, Panagiotis

    2015-05-29

    CDT (Clostridium difficile transferase) is a binary, actin ADP-ribosylating toxin frequently associated with hypervirulent strains of the human enteric pathogen C. difficile, the most serious cause of antibiotic-associated diarrhea and pseudomembranous colitis. CDT leads to the collapse of the actin cytoskeleton and, eventually, to cell death. Low doses of CDT result in the formation of microtubule-based protrusions on the cell surface that increase the adherence and colonization of C. difficile. The lipolysis-stimulated lipoprotein receptor (LSR) is the host cell receptor for CDT, and our aim was to gain a deeper insight into the interplay between both proteins. We show that CDT interacts with the extracellular, Ig-like domain of LSR with an affinity in the nanomolar range. We identified LSR splice variants in the colon carcinoma cell line HCT116 and disrupted the LSR gene in these cells by applying the CRISPR-Cas9 technology. LSR truncations ectopically expressed in LSR knock-out cells indicated that intracellular parts of LSR are not essential for plasma membrane targeting of the receptor and cellular uptake of CDT. By generating a series of N- and C-terminal truncations of the binding component of CDT (CDTb), we found that amino acids 757-866 of CDTb are sufficient for binding to LSR. With a transposon-based, random mutagenesis approach, we identified potential LSR-interacting epitopes in CDTb. This study increases our understanding about the interaction between CDT and its receptor LSR, which is key to the development of anti-toxin strategies for preventing cell entry of the toxin. PMID:25882847

  13. 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

  14. Mechanism of Alternariol monomethyl ether-induced mitochondrial apoptosis in human colon carcinoma cells.

    PubMed

    Bensassi, Fatma; Gallerne, Cindy; el Dein, Ossama Sharaf; Hajlaoui, Mohamed Rabeh; Bacha, Hassen; Lemaire, Christophe

    2011-12-18

    Alternariol monomethyl ether (AME) is a major mycotoxin produced by fungi of the genus Alternaria and a common contaminant of food products such as fruits and cereals worldwide. AME can cause serious health problems for animals as well as for humans. In this study, human colon carcinoma cells (HCT116) were used to explore the mechanisms of cell death induced by AME. Exposure of HCT116 cells to AME resulted in significant cytotoxicity manifested by a loss in cell viability mainly mediated by activation of apoptotic process. AME activated the mitochondrial apoptotic pathway evidenced by the opening of the mitochondrial permeability transition pore (PTP), loss of the mitochondrial transmembrane potential (??m) downstream generation of O(2)(-), cytochrome c release and caspase 9 and 3 activation. Experiments conducted on isolated organelles indicated that AME does not directly target mitochondria to induce PTP-dependent permeabilization of mitochondrial membranes. Moreover, no difference was observed in Bax-KO cells in comparison to parental cells, suggesting that the pro-apoptotic protein Bax is not involved in AME-induced mitochondrial apoptosis. Our findings demonstrate for the first time that AME induces cell death in human colon carcinoma cells by activating the mitochondrial pathway of apoptosis. PMID:22001388

  15. MicroRNA-148b suppresses cell growth by targeting cholecystokinin-2 receptor in colorectal cancer.

    PubMed

    Song, Yongxi; Xu, Yingying; Wang, Zhenning; Chen, Yue; Yue, Zhenyu; Gao, Peng; Xing, Chengzhong; Xu, Huimian

    2012-09-01

    MicroRNAs (miRNAs) play an important role in the regulation of a variety of cellular processes, including cell growth, differentiation, apoptosis and carcinogenesis. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in colorectal cancer. The expression of miR-148b was significantly downregulated in 96 pairs of human colorectal cancer tissues (p<0.0001) and three cell lines (p<0.01) compared with non-tumor adjacent tissues by quantitative real-time PCR. The results of in situ hybridization highlighted that miR-148b was important in the cancer transformation process. Using statistical analysis, we found that the expression level of miR-148b was associated with tumor size (p=0.033) in colorectal cancer patients. Moreover, overexpression of miR-148b in HCT-116 and HT-29 cells could inhibit cell proliferation in vitro and suppress tumorigenicity in vivo. Importantly, the result of luciferase activity assay and western blot showed that the cholecystokinin-2 receptor gene (CCK2R) was a target of miR-148b and was downregulated by miR-148b at the translational level. Then, we used siRNA, radioimmunoassay and ELISA to demonstrate that miR-148b might have an effect on cell proliferation by regulating the expression of CCK2R which functioned depending on the gastrin in colorectal cancer. Taken together, our data provides the first evidences that miR-148b acts as a tumor suppressor in colorectal cancer and should be further evaluated as a biomarker and therapeutic tool against colorectal cancer. PMID:22020560

  16. SHORT REPORT Open Access Establishment of three cell lines from

    E-print Network

    Gray, Matthew

    , thymus cell line (GSTC), spleen cell line (GSSC) and kidney cell line (GSKC) were initially established, killed and wiped with 75 v/v ethanol, and the thymus, spleen and kidney tissues were removed for primary

  17. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ...part of the Identification of Human Cell Lines Project. All data and corresponding...database. The use of misidentified cell lines in cancer and other biomedical research continues...literature describing studies employing cell lines may be misleading or...

  18. Non-thermal Plasma Causes p53-Dependent Apoptosis in Human Colon Carcinoma Cells.

    PubMed

    Tuhvatulin, A I; Sysolyatina, E V; Scheblyakov, D V; Logunov, D Yu; Vasiliev, M M; Yurova, M A; Danilova, M A; Petrov, O F; Naroditsky, B S; Morfill, G E; Grigoriev, A I; Fortov, V E; Gintsburg, A L; Ermolaeva, S A

    2012-07-01

    Non-thermal plasma (NTP) consists of a huge amount of biologically active particles, whereas its temperature is close to ambient. This combination allows one to use NTP as a perspective tool for solving different biomedical tasks, including antitumor therapy. The treatment of tumor cells with NTP caused dose-dependent effects, such as growth arrest and apoptosis. However, while the outcome of NTP treatment has been established, the molecular mechanisms of the interaction between NTP and eukaryotic cells have not been thoroughly studied thus far. In this work, the mechanisms and the type of death of human colon carcinoma HCT 116 cells upon application of non-thermal argon plasma were studied. The effect of NTP on the major stress-activated protein p53 was investigated. The results demonstrate that the viability of HCT116 cells upon plasma treatment is dependent on the functional p53 protein. NTP treatment caused an increase in the intracellular concentration of p53 and the induction of the p53-controlled regulon. The p53-dependent accumulation of active proapoptotic caspase-3 was shown in NTP-treated cells. The study was the first to demonstrate that treatment of human colon carcinoma cells with NTP results in p53-dependent apoptosis. The results obtained contribute to our understanding of the applicability of NTP in antitumor therapy. PMID:23150806

  19. 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.

  20. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    PubMed Central

    Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

    1988-01-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:3414781

  1. Cytotoxicity of the bisphenolic honokiol from Magnolia officinalis against multiple drug-resistant tumor cells as determined by pharmacogenomics and molecular docking.

    PubMed

    Saeed, Mohamed; Kuete, Victor; Kadioglu, Onat; Börtzler, Jonas; Khalid, Hassan; Greten, Henry Johannes; Efferth, Thomas

    2014-10-15

    A main problem in oncology is the development of drug-resistance. Some plant-derived lignans are established in cancer therapy, e.g. the semisynthetic epipodophyllotoxins etoposide and teniposide. Their activity is, unfortunately, hampered by the ATP-binding cassette (ABC) efflux transporter, P-glycoprotein. Here, we investigated the bisphenolic honokiol derived from Magnolia officinalis. P-glycoprotein-overexpressing CEM/ADR5000 cells were not cross-resistant to honokiol, but MDA-MB-231 BRCP cells transfected with another ABC-transporter, BCRP, revealed 3-fold resistance. Further drug resistance mechanisms analyzed study was the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). HCT116 p53(-/-) did not reveal resistance to honokiol, and EGFR-transfected U87.MG EGFR cells were collateral sensitive compared to wild-type cells (degree of resistance: 0.34). To gain insight into possible modes of collateral sensitivity, we performed in silico molecular docking studies of honokiol to EGFR and EGFR-related downstream signal proteins. Honokiol bound with comparable binding energies to EGFR (-7.30 ± 0.01 kcal/mol) as the control drugs erlotinib (-7.50 ± 0.30 kcal/mol) and gefitinib (-8.30 ± 0.10 kcal/mol). Similar binding affinities of AKT, MEK1, MEK2, STAT3 and mTOR were calculated for honokiol (range from -9.0 ± 0.01 to 7.40 ± 0.01 kcal/mol) compared to corresponding control inhibitor compounds for these signal transducers. This indicates that collateral sensitivity of EGFR-transfectant cells towards honokiol may be due to binding to EGFR and downstream signal transducers. COMPARE and hierarchical cluster analyses of microarray-based transcriptomic mRNA expression data of 59 tumor cell lines revealed a specific gene expression profile predicting sensitivity or resistance towards honokiol. PMID:25442261

  2. Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models

    PubMed Central

    Bhave, Madhura Satish; Hassanbhai, Ammar Mansoor; Anand, Padmaja; Luo, Kathy Qian; Teoh, Swee Hin

    2015-01-01

    Traditional cancer treatments, such as chemotherapy and radiation therapy continue to have limited efficacy due to tumor hypoxia. While bacterial cancer therapy has the potential to overcome this problem, it comes with the risk of toxicity and infection. To circumvent these issues, this paper investigates the anti-tumor effects of non-viable bacterial derivatives of Clostridium sporogenes. These non-viable derivatives are heat-inactivated C. sporogenes bacteria (IB) and the secreted bacterial proteins in culture media, known as conditioned media (CM). In this project, the effects of IB and CM on CT26 and HCT116 colorectal cancer cells were examined on a 2-Dimensional (2D) and 3-Dimensional (3D) platform. IB significantly inhibited cell proliferation of CT26 to 6.3% of the control in 72?hours for the 2D monolayer culture. In the 3D spheroid culture, cell proliferation of HCT116 spheroids notably dropped to 26.2%. Similarly the CM also remarkably reduced the cell-proliferation of the CT26 cells to 2.4% and 20% in the 2D and 3D models, respectively. Interestingly the effect of boiled conditioned media (BCM) on the cells in the 3D model was less inhibitory than that of CM. Thus, the inhibitive effect of inactivated C. sporogenes and its conditioned media on colorectal cancer cells is established. PMID:26507312

  3. Covalent coupling of gum arabic onto superparamagnetic iron oxide nanoparticles for MRI cell labeling: physicochemical and in vitro characterization.

    PubMed

    Palma, Susana I C J; Carvalho, Alexandra; Silva, Joana; Martins, Pedro; Marciello, Marzia; Fernandes, Alexandra R; del Puerto Morales, Maria; Roque, Ana C A

    2015-01-01

    Gum arabic (GA) is a hydrophilic composite polysaccharide derived from exudates of Acacia senegal and Acacia seyal trees. It is biocompatible, possesses emulsifying and stabilizing properties and has been explored as coating agent of nanomaterials for biomedical applications, namely magnetic nanoparticles (MNPs). Previous studies focused on the adsorption of GA onto MNPs produced by co-precipitation methods. In this work, MNPs produced by a thermal decomposition method, known to produce uniform particles with better crystalline properties, were used for the covalent coupling of GA through its free amine groups, which increases the stability of the coating layer. The MNPs were produced by thermal decomposition of Fe(acac)3 in organic solvent and, after ligand-exchange with meso-2,3-dimercaptosuccinic acid (DMSA), GA coating was achieved by the establishment of a covalent bond between DMSA and GA moieties. Clusters of several magnetic cores entrapped in a shell of GA were obtained, with good colloidal stability and promising magnetic relaxation properties (r2 /r1 ratio of 350). HCT116 colorectal carcinoma cell line was used for in vitro cytotoxicity evaluation and cell-labeling efficiency studies. We show that, upon administration at the respective IC50 , GA coating enhances MNP cellular uptake by 19 times compared to particles bearing only DMSA moieties. Accordingly, in vitro MR images of cells incubated with increasing concentrations of GA-coated MNP present dose-dependent contrast enhancement. The obtained results suggest that the GA magnetic nanosystem could be used as a MRI contrast agent for cell-labeling applications. PMID:25766788

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

    PubMed

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

    2014-02-01

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

  6. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton (Palos Park, IL); Cook, Glenn M. (Naperville, IL)

    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.

  7. 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 ?g/ml and that IC50 value on Hep 2 cell line was 112 ?g whereas 94 ?g 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

  8. Antiproliferative activity of New Zealand propolis and phenolic compounds vs human colorectal adenocarcinoma cells.

    PubMed

    Catchpole, Owen; Mitchell, Kevin; Bloor, Stephen; Davis, Paul; Suddes, Amanda

    2015-10-01

    New Zealand propolis is a "European" type propolis obtained by honey bees mainly from exudates of poplar. European type propolis is known to have anti-inflammatory and anti-cancer properties and this activity has been attributed to some of the main constituents such as chrysin and CAPE (caffeic acid phenethyl ester). As part of our studies on how New Zealand propolis might benefit gastro-intestinal health, we carried out in vitro bioactivity-guided fractionation of "Bio30™" propolis using both anti-inflammatory (TNF-?, COX-1, COX-2) and anti-colon cancer (DLD-1 colon cancer cell viability) assays; and determined the phenolic compounds responsible for the activity. The New Zealand wax-free Bio30™ propolis tincture solids had very high levels of the dihydroflavonoids pinocembrin and pinobanksin-3-O-acetate, and high levels of the dimethylallyl, benzyl and 3-methyl-3-butenyl caffeates relative to CAPE. The DLD-1 assays identified strong anti-proliferative activity associated with these components as well as chrysin, galangin and CAPE and a number of lesser known or lower concentration compounds including benzyl ferulate, benzyl isoferulate, pinostrobin, 5-phenylpenta-2,4-dienoic acid and tectochrysin. The phenolic compounds pinocembrin, pinobanksin-3-O-acetate, tectochrysin, dimethylallyl caffeate, 3-methyl-3-butenyl caffeate, benzyl ferulate and benzyl isoferulate also showed good broad spectrum activity in anti-proliferative assays against three other gastro-intestinal cancer cell lines; HCT-116 colon carcinoma, KYSE-30 oesophageal squamous cancer, and NCI-N87 gastric carcinoma. Activity is also observed in anti-inflammatory assays although it appears to be limited to one of the first cytokines in the inflammatory cascade, TNF-?. PMID:26347954

  9. The Clinical Relevance of Cancer Cell Lines

    PubMed Central

    2013-01-01

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

  10. 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....

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

    PubMed

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

    2014-01-01

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

  12. LZ-207, a Newly Synthesized Flavonoid, Induces Apoptosis and Suppresses Inflammation-Related Colon Cancer by Inhibiting the NF-?B Signaling Pathway

    PubMed Central

    Sun, Jie; Li, Fanni; Zhao, Yue; Zhao, Li; Qiao, Chen; Li, Zhiyu; Guo, Qinglong; Lu, Na

    2015-01-01

    Flavonoids and flavonoid derivatives, which have significant biological and pharmacological activities, including antitumor and anti-inflammatory activities, have been widely used in human healthcare. To design a more effective flavonoid antitumor agent, we altered the flavonoid backbone with substitutions of piperazine and methoxy groups to synthesize a novel flavonoid derivative, LZ-207. The anticancer effect of LZ-207 against HCT116 colon cancer cells and the underlying mechanism of this effect were explored in this study. Specifically, LZ-207 exhibited inhibitory effects on growth and viability in several human colon cancer cell lines and induced apoptosis in HCT116 cells both in vitro and in vivo. LZ-207 treatment also suppressed the nuclear translocation of NF-?B and the phosphorylation of I?B and IKK?/? in a dose-dependent manner in both HCT116 cells and human acute monocytic leukemia THP-1 cells. Moreover, LZ-207 also reduced the secretion of the pro-inflammatory cytokine interleukin-6 (IL-6) in LPS-induced THP-1 cells, and this effect was confirmed at the transcriptional level. Furthermore, LZ-207 significantly inhibited HCT116 cell proliferation that was elicited by LPS-induced THP-1 cells in a co-culture system. These findings elucidated some potential molecular mechanisms for preventing inflammation-driven colon cancer using the newly synthesized flavonoid LZ-207 and suggested the possibility of further developing novel therapeutic agents derived from flavonoids. PMID:26023926

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

    PubMed

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

    2014-01-01

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

  14. Effects of Oplopanax horridus on Human Colorectal Cancer Cells

    PubMed Central

    LI, XIAO-LI; SUN, SHI; WANG, CHONG-ZHI; WILLIAMS, STAINLEY; YUAN, CHUN-SU

    2011-01-01

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

  15. Antioxidant activity and cytotoxicity on tumour cells of the essential oil from Cedronella canariensis var. canariensis (L.) Webb & Berthel. (Lamiaceae).

    PubMed

    Zorzetto, Christian; Sánchez-Mateo, Candelaria C; Rabanal, Rosa M; Lupidi, Giulio; Bramucci, Massimo; Quassinti, Luana; Iannarelli, Romilde; Papa, Fabrizio; Maggi, Filippo

    2015-01-01

    Cedronella canariensis is a lemon-scented species of the family Lamiaceae endemic to the Canary Islands where it is used in the traditional medicine to prepare infusions or inhalations for anti-catarrhal, tonic, diuretic, hypoglycaemiant, hypotensive, anti-inflammatory and decongestant of the respiratory tract. In this work we investigated for the first time the antioxidant activity of the essential oil and its inhibitory effects on tumour cells (A375, MDA-MB-231, HCT 116) proliferation by DPPH, ABTS, FRAP and MTT assays, respectively. The oil, analysed by GC-ionisation flame detector and GC-MS, was characterised by pinocarvone (58.0%) and ?-pinene (10.8%) as the major constituents, being typical of the chemotype 'canariensis'. Noteworthy was the cytotoxic activity of the oil against the tumour cells examined, with IC50 values of 4.3, 7.3 and 11.4 ?g/mL on A375, MDA-MB-231 and HCT 116 tumour cells, respectively, as well as the scavenging activity against the ABTS radical (IC50 of 10.5 ?g/mL). PMID:25560780

  16. Upregulation of long non-coding RNA PRNCR1 in colorectal cancer promotes cell proliferation and cell cycle progression.

    PubMed

    Yang, Liu; Qiu, Mantang; Xu, Youtao; Wang, Jie; Zheng, Yanyan; Li, Ming; Xu, Lin; Yin, Rong

    2016-01-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide. Long non-coding RNAs (lncRNAs) have been confirmed to play a critical regulatory role in various biological processes including carcinogenesis, which indicates that lncRNAs are valuable biomarkers and therapeutic targets. The novel lncRNA prostate cancer non?coding RNA 1 (PRNCR1) is located in the susceptible genomic area of CRC, however the functional role of PRNCR1 remains unknown. Thus, we aimed to investigate the clinical significance and biological function of PRNCR1 in CRC. Quantitative real?time polymerase chain reaction (qRT?PCR) was used to assess the expression profile of PRNCR1 in CRC tissues and cell lines. An antisense oligonucleotide (ASO) was designed to knock down PRNCR1. In a cohort of 63 patients, PRNCR1 was significantly overexpressed in CRC tissues compared with the expression in adjacent tissues, with an average fold increase of 10.55 (P=0.006). Additionally, a high level of PRNCR1 was associated with large tumor volume (P<0.05). Based on receiver operating characteristic curve (ROC), we found that the area under the curve (AUC) of PRNCR1 was 0.799 while the AUC of conventional biomarker CEA?CA199 was 0.651, indicating that PRNCR1 could be a sensitive diagnostic biomarker of CRC. Compared with the normal human colorectal epithelial cell line (FHC), PRNCR1 was upregulated in most CRC cell lines (HCT116, SW480, LoVo and HT?29). After knockdown of PRNCR1 by ASO, CRC cell proliferation ability was significantly inhibited. We further found that PRNCR1 knockdown induced cell cycle arrest in the G0/G1 phase and a significant decrease in the proportion of cells in the S phases. In contrast, PRNCR1 knockdown did not affect cell apoptosis or invasive ability. Hence, these data indicate that PRNCR1 promotes the proliferation of CRC cells and is a potential oncogene of CRC. PMID:26530130

  17. MYC-repressed long noncoding RNAs antagonize MYC-induced cell proliferation and cell cycle progression

    PubMed Central

    Jeon, Young-Jun; Fadda, Paolo; Alder, Hansjuerg; Croce, Carlo M.

    2015-01-01

    The transcription factor MYC is a proto-oncogene regulating cell proliferation, cell cycle, apoptosis and metabolism. The recent identification of MYC-regulated long noncoding RNAs (lncRNAs) expands our knowledge of the role of lncRNAs in MYC functions. Here, we identify MYC-repressed lncRNAs named MYCLo-4, -5 and -6 by comparing 3 categories of lncRNAs (downregulated in highly MYC-expressing colorectal cancer, up-regulated by MYC knockdown in HCT116, upregulated by MYC knockdown in RKO). The MYC-repressed MYCLos are implicated in MYC-modulated cell proliferation through cell cycle regulation. By screening cell cycle-related genes regulated by MYC and the MYC-repressed MYCLos, we identified the MYC-repressed gene GADD45A as a target gene of the MYC-repressed MYCLos such as MYCLo-4 and MYCLo-6. PMID:26003165

  18. Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

    PubMed Central

    Link, Alexander; Balaguer, Francesc; Shen, Yan; Lozano, Juan Jose; Leung, Hon-Chiu E.; Boland, C. Richard; Goel, Ajay

    2013-01-01

    Aim Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells. Materials and Methods To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2?-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR. Results As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean ?-values of 0.12), however, non-significant changes in mean ?-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines. Conclusions Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical. PMID:23460897

  19. Regulatory networks define phenotypic classes of human stem cell lines

    E-print Network

    Shamir, Ron

    LETTERS Regulatory networks define phenotypic classes of human stem cell lines Franz-Josef Mu¨ller1 called stem cells, even though they range from pluripotent cells--typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differenti- ation--to adult stem cell lines, which can

  20. Optimum 3D Matrix Stiffness for Maintenance of Cancer Stem Cells Is Dependent on Tissue Origin of Cancer Cells

    PubMed Central

    Jabbari, Esmaiel; Sarvestani, Samaneh K.; Daneshian, Leily; Moeinzadeh, Seyedsina

    2015-01-01

    Introduction The growth and expression of cancer stem cells (CSCs) depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells’ tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA) hydrogel without the interference of other factors in the microenvironment. Methods Human MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x106 cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers. Results The optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 ?m. Conclusion The marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells’ tissue origin. PMID:26168187

  1. On the ontology based representation of cell lines.

    PubMed

    Ganzinger, Matthias; He, Shan; Breuhahn, Kai; Knaup, Petra

    2012-01-01

    Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT) integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed. PMID:23144907

  2. A resource for cell line authentication, annotation and quality control.

    PubMed

    Yu, Mamie; Selvaraj, Suresh K; Liang-Chu, May M Y; Aghajani, Sahar; Busse, Matthew; Yuan, Jean; Lee, Genee; Peale, Franklin; Klijn, Christiaan; Bourgon, Richard; Kaminker, Joshua S; Neve, Richard M

    2015-04-16

    Cell line misidentification, contamination and poor annotation affect scientific reproducibility. Here we outline simple measures to detect or avoid cross-contamination, present a framework for cell line annotation linked to short tandem repeat and single nucleotide polymorphism profiles, and provide a catalogue of synonymous cell lines. This resource will enable our community to eradicate the use of misidentified lines and generate credible cell-based data. PMID:25877200

  3. Triphala Extract Suppresses Proliferation and Induces Apoptosis in Human Colon Cancer Stem Cells via Suppressing c-Myc/Cyclin D1 and Elevation of Bax/Bcl-2 Ratio

    PubMed Central

    Vadde, Ramakrishna; Radhakrishnan, Sridhar; Reddivari, Lavanya; Vanamala, Jairam K. P.

    2015-01-01

    Colon cancer is the second leading cause of cancer related deaths in the USA. Cancer stem cells (CSCs) have the ability to drive continued expansion of the population of malignant cells. Therefore, strategies that target CSCs could be effective against colon cancer and in reducing the risk of relapse and metastasis. In this study, we evaluated the antiproliferative and proapoptotic effects of triphala, a widely used formulation in Indian traditional medicine, on HCT116 colon cancer cells and human colon cancer stem cells (HCCSCs). The total phenolic content, antioxidant activity, and phytochemical composition (LC-MS-MS) of methanol extract of triphala (MET) were also measured. We observed that MET contains a variety of phenolics including naringin, quercetin, homoorientin, and isorhamnetin. MET suppressed proliferation independent of p53 status in HCT116 and in HCCSCs. MET also induced p53-independent apoptosis in HCCSCs as indicated by elevated levels of cleaved PARP. Western blotting data suggested that MET suppressed protein levels of c-Myc and cyclin D1, key proteins involved in proliferation, and induced apoptosis through elevation of Bax/Bcl-2 ratio. Furthermore, MET inhibited HCCSCs colony formation, a measure of CSCs self-renewal ability. Anticancer effects of triphala observed in our study warrant future studies to determine its efficacy in vivo. PMID:26167492

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

    PubMed

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

    2012-10-01

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

  5. Involvement of ROS in Curcumin-induced Autophagic Cell Death

    PubMed Central

    Lee, Youn Ju; Kim, Nam-Yi; Suh, Young-Ah

    2011-01-01

    Many anticancer agents as well as ionizing radiation have been shown to induce autophagy which is originally described as a protein recycling process and recently reported to play a crucial role in various disorders. In HCT116 human colon cancer cells, we found that curcumin, a polyphenolic phytochemical extracted from the plant Curcuma longa, markedly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II and degradation of sequestome-1 (SQSTM1) which is a marker of autophagosome degradation. Moreover, we found that curcumin caused GFP-LC3 formation puncta, a marker of autophagosome, and decrease of GFP-LC3 and SQSTM1 protein level in GFP-LC3 expressing HCT116 cells. It was further confirmed that treatment of cells with hydrogen peroxide induced increase of LC3 conversion and decrease of GFP-LC3 and SQSTM1 levels, but these changes by curcumin were almost completely blocked in the presence of antioxidant, N-acetylcystein (NAC), indicating that curcumin leads to reactive oxygen species (ROS) production, which results in autophagosome development and autolysosomal degradation. In parallel with NAC, SQSTM1 degradation was also diminished by bafilomycin A, a potent inhibitor of autophagosome-lysosome fusion, and cell viability assay was further confirmed that cucurmin-induced cell death was partially blocked by bafilomycin A as well as NAC. We also observed that NAC abolished curcumin-induced activation of extracelluar signal-regulated kinases (ERK) 1/2 and p38 mitogen-activated protein kinases (MAPK), but not Jun N-terminal kinase (JNK). However, the activation of ERK1/2 and p38 MAPK seemed to have no effect on the curcumin-induced autophagy, since both the conversion of LC3 protein and SQSTM1 degradation by curcumin was not changed in the presence of NAC. Taken together, our data suggest that curcumin induced ROS production, which resulted in autophagic activation and concomitant cell death in HCT116 human colon cancer cell. However, ROS-dependent activation of ERK1/2 and p38 MAPK, but not JNK, might not be involved in the curcumin-induced autophagy. PMID:21461234

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

    PubMed Central

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

    2014-01-01

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

  7. Many are called MDS cell lines: one is chosen.

    PubMed

    Drexler, Hans G; Dirks, Willy G; Macleod, Roderick A F

    2009-08-01

    Myelodysplastic syndromes (MDS) comprise a heterogenous group of clonal disorders of hematopoietic progenitors, showing genetic instability and in many cases progression to acute myeloid leukemia (AML). When MDS progress towards AML (AML/MDS), additional genetic lesions cause a block in differentiation and an accumulation of blast cells. Hence, both pathophysiologically and clinically the MDS and AML/MDS phases are distinguishable. Leukemia cell lines are key resources for modelling hematological malignancies. Characterization of these cell lines has provided a rich vein of insights into the mechanisms underlying malignant transformation. Some 31 cell lines have been described in the literature purportedly established from patients with MDS. However, a significant minority of these has proved false after DNA profiling which revealed their cross-contamination with older established leukemia cell lines. Most remaining ("authentic") MDS cell lines were established during the leukemic phase of the disease progression rather than during the MDS phase. Based on these data we have assigned the 31 candidate MDS cell lines to one of the three categories: (1) false (cross-contaminated) cell lines and non-malignant cell lines; (2) malignant cell lines established in the AML/MDS leukemic phase; and (3) apparently legitimate MDS cell lines established during the MDS phase. While MDS and AML/MDS cell lines both provide singular resources for modelling pathology, mining oncogenically modified macromolecules, and testing druggability, we contend these groups should be considered separately. PMID:19344951

  8. Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses

    PubMed Central

    Dobrzy?ski, Maciej; Nguyen, Lan K.; Birtwistle, Marc R.; von Kriegsheim, Alexander; Blanco Fernández, Alfonso; Cheong, Alex; Kolch, Walter; Kholodenko, Boris N.

    2014-01-01

    We show theoretically and experimentally a mechanism behind the emergence of wide or bimodal protein distributions in biochemical networks with nonlinear input–output characteristics (the dose–response curve) and variability in protein abundance. Large cell-to-cell variation in the nonlinear dose–response characteristics can be beneficial to facilitate two distinct groups of response levels as opposed to a graded response. Under the circumstances that we quantify mathematically, the two distinct responses can coexist within a cellular population, leading to the emergence of a bimodal protein distribution. Using flow cytometry, we demonstrate the appearance of wide distributions in the hypoxia-inducible factor-mediated response network in HCT116 cells. With help of our theoretical framework, we perform a novel calculation of the magnitude of cell-to-cell heterogeneity in the dose–response obtained experimentally. PMID:24966234

  9. Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses.

    PubMed

    Dobrzy?ski, Maciej; Nguyen, Lan K; Birtwistle, Marc R; von Kriegsheim, Alexander; Blanco Fernández, Alfonso; Cheong, Alex; Kolch, Walter; Kholodenko, Boris N

    2014-09-01

    We show theoretically and experimentally a mechanism behind the emergence of wide or bimodal protein distributions in biochemical networks with nonlinear input-output characteristics (the dose-response curve) and variability in protein abundance. Large cell-to-cell variation in the nonlinear dose-response characteristics can be beneficial to facilitate two distinct groups of response levels as opposed to a graded response. Under the circumstances that we quantify mathematically, the two distinct responses can coexist within a cellular population, leading to the emergence of a bimodal protein distribution. Using flow cytometry, we demonstrate the appearance of wide distributions in the hypoxia-inducible factor-mediated response network in HCT116 cells. With help of our theoretical framework, we perform a novel calculation of the magnitude of cell-to-cell heterogeneity in the dose-response obtained experimentally. PMID:24966234

  10. Personalized chemotherapy profiling using cancer cell lines from selectable mice

    PubMed Central

    Kamiyama, Hirohiko; Rauenzahn, Sherri; Shim, Joong Sup; Karikari, Collins A.; Feldmann, Georg; Hua, Li; Kamiyama, Mihoko; Schuler, F. William; Lin, Ming-Tseh; Beaty, Robert M.; Karanam, Balasubramanyam; Liang, Hong; Mullendore, Michael E.; Mo, Guanglan; Hidalgo, Manuel; Jaffee, Elizabeth; Hruban, Ralph H.; Jinnah, H. A.; Roden, Richard B. S.; Jimeno, Antonio; Liu, Jun O.; Maitra, Anirban; Eshleman, James R.

    2013-01-01

    Purpose High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult, because contaminating stromal cells overgrow the malignant cells. Experimental Design We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified seventy-seven FDA approved drugs with activity, including two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. PMID:23340293

  11. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  12. Stable mammalian producer cell lines for structural biology.

    PubMed

    Büssow, Konrad

    2015-06-01

    The mammalian cell lines HEK293 and CHO have become important expression hosts in structural biology. Generating stable mammalian cell lines remains essential for studying the function and structure of recombinant proteins, despite the emergence of highly efficient transient transfection protocols. Production with stable cell lines can be scaled up easily and high volumetric product yield can be achieved. Protein structure reports of the past two years that used stable cell lines were surveyed for this review. Well-established techniques and novel approaches for generating stable cell lines and stable cell pools are presented, including cell sorting, site-specific recombination, transposons, the Lentivirus system and phage integrases. Host cell line optimization by endoglycosidase overexpression and sequence-specific genome engineering is highlighted. PMID:25804355

  13. Reporter cell lines for skin sensitization testing.

    PubMed

    Natsch, Andreas; Emter, Roger

    2015-10-01

    Skin sensitization has been described as an adverse outcome pathway (AOP), comprising a number of molecular events leading to the final adverse effect. In a new paradigm of toxicology, attempts are made to collect information using single mechanistic tests addressing different targets along such an AOP and to then integrate this information to arrive at a final toxicological prediction. This proposal is strongly influenced by the availability of methods for high-throughput screening of cellular events. Reporter cell lines are a particularly useful tool in such screening paradigms, as they can deliver highly reproducible and easily measureable results, and they can be designed to quantify induction or suppression at the transcription level of very specific molecular targets within cells. The first cell-based assay for skin sensitization, which has recently received ECVAM and OECD endorsement, is the reporter cell assay KeratinoSens™, reflecting activation of the Nrf2 pathway, and other assays measuring the Nrf2 pathway are under development or validation. An alternative approach (THP-G8) was recently developed based on activation of the Interleukin-8 gene. Here, we review these assays, their role in the AOP, their mechanistic interrelationships, their use for hazard and risk assessment, and their application in integrated testing strategies. At the same time, this study reviews (1) other cellular markers for sensitizers, and the potential to develop new reporter gene assays providing additional, non-redundant information, and (2) it presents approaches and new experimental data on attempts to further improve the predictivity of the existing assay. PMID:26194644

  14. Mechanism of Action of Two Flavone Isomers Targeting Cancer Cells with Varying Cell Differentiation Status

    PubMed Central

    Parsons, Laura B.; Miller, Gerald E.; Whitted, Crystal; Lynch, Kayla E.; Ramsauer, Robert E.; Patel, Jasmine U.; Wyatt, Jarrett E.; Street, Doris S.; Adams, Carolyn B.; McPherson, Brian; Tsui, Hei Man; Evans, Julie A.; Livesay, Christopher; Torrenegra, Ruben D.; Palau, Victoria E.

    2015-01-01

    Apoptosis can be triggered in two different ways, through the intrinsic or the extrinsic pathway. The intrinsic pathway is mediated by the mitochondria via the release of cytochrome C while the extrinsic pathway is prompted by death receptor signals and bypasses the mitochondria. These two pathways are closely related to cell proliferation and survival signaling cascades, which thereby constitute possible targets for cancer therapy. In previous studies we introduced two plant derived isomeric flavonoids, flavone A and flavone B which induce apoptosis in highly tumorigenic cancer cells of the breast, colon, pancreas, and the prostate. Flavone A displayed potent cytotoxic activity against more differentiated carcinomas of the colon (CaCo-2) and the pancreas (Panc28), whereas flavone B cytotoxic action is observed on poorly differentiated carcinomas of the colon (HCT 116) and pancreas (MIA PaCa). Apoptosis is induced by flavone A in better differentiated colon cancer CaCo-2 and pancreatic cancer Panc 28 cells via the intrinsic pathway by the inhibition of the activated forms of extracellular signal-regulated kinase (ERK) and pS6, and subsequent loss of phosphorylation of Bcl-2 associated death promoter (BAD) protein, while apoptosis is triggered by flavone B in poorly differentiated colon cancer HCT 116 and MIA PaCa pancreatic cancer cells through the extrinsic pathway with the concomitant upregulation of the phosphorylated forms of ERK and c-JUN at serine 73. These changes in protein levels ultimately lead to activation of apoptosis, without the involvement of AKT. PMID:26606169

  15. The pursuit of ES cell lines of domesticated ungulates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated state, have the ability to self-renew, and exhibit pluripotency, i.e., they can give rise to most if not all somatic cell types and to the germ cells, egg and sperm. These characteristics make ES cell lines...

  16. Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells

    SciTech Connect

    Chiu, Shu-Jun; Institute of Radiation Sciences, Tzu Chi Technology College, Hualien, Taiwan ; Hsaio, Ching-Hui; Tseng, Ho-Hsing; Su, Yu-Han; Shih, Wen-Ling; Lee, Jeng-Woei; Chuah, Jennifer Qiu-Yu

    2010-04-09

    Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors {gamma} used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G{sub 2}/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.

  17. RPS24 knockdown inhibits colorectal cancer cell migration and proliferation in vitro.

    PubMed

    Wang, Yue; Sui, Jinke; Li, Xu; Cao, Fuao; He, Jian; Yang, Bo; Zhu, Xiaoming; Sun, Yongsheng; Pu, Y D

    2015-10-25

    Besides new proteins synthesis, ribosomal protein has a role in extra-ribosomal functions, which are related to many diseases, such as Diamond-Blackfan anemia, hypoplasia, and cell apoptosis. However, the importance of RPS24 in human colon cancer is largely unknown. In this study, RPS24 gene expression was significantly inhibited in human colon cancer HCT116 and HT-29 cells using a lentivirus shRNA approach. Knockdown of RPS24 expression significantly inhibited cell proliferation, colony formation, cell migration and arrested cell in S phase. The results demonstrated for the first time that RPS24 gene had a critical role in human colon cancer. Therefore, our findings indicated that RPS24 gene may be a promising biomarker for therapy in human colon cancer and may have a potential application in the diagnosis or treatment of human colon cancer. PMID:26149657

  18. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R. (Oakland, CA)

    1989-01-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

  19. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

  20. PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

    PubMed

    Bertonha, Fernanda Bernardi; Barros Filho, Mateus de Camargo; Kuasne, Hellen; Dos Reis, Patricia Pintor; da Costa Prando, Erika; Muñoz, Juan José Augusto Moyano; Roffé, Martín; Hajj, Glaucia Noeli Maroso; Kowalski, Luiz Paulo; Rainho, Claudia Aparecida; Rogatto, Silvia Regina

    2015-02-01

    A significant association between DNA losses on 22q13.31 and head and neck squamous cell carcinomas (HNSCC) was previously reported by our group. Our data indicated that PHF21B gene, mapped on 22q13.31 and encoding a protein with function of chromatin-mediated transcriptional regulation, might be a putative tumor suppressor gene. To test this hypothesis, gene copy number was assessed in 75 HNSCC and 49 matched peripheral blood samples. PHF21B losses were detected in 43 tumors and were significantly associated with patients with familial history of cancer (P < 0.0001); i.e., 36/43 cases showed a positive family history of cancer and 22/36 had first-degree relatives with cancer (P = 0.049). In attempt to investigate other mechanisms for PHF21B loss of function, DNA sequencing was performed and no mutations were detected. We next evaluated the gene expression levels after inhibition of DNA methylation in nine HNSCC and breast carcinoma cell lines. Additionally, PHF21B expression levels were evaluated in colon cancer HCT116 cells as well as in its counterpart DKO (double knockout of DNMT1 and DNMT3B). The higher expression levels of PHF21B gene detected in DKO cells were inversely correlated with the DNA methylation. Further, DNA methylation in the specific promoter-associated CpG Island was investigated. Interestingly, gene hypermethylation was detected in 13/37 tumors: 5/13 HNSCC cases had family history of cancer in first-degree relatives and 8/13 showed both, DNA methylation and PHF21B losses in the tumor sample. One patient had PHF21B loss in the peripheral blood cells and PHF21B methylation in the tumor sample. Additionally, overexpression of PHF21B in cell lines drastically reduces clonogenic and migratory abilities. These data suggest that PHF21B is a novel tumor suppressor gene that can be inactivated by genetic and epigenetic mechanisms in the human cancer. PMID:25454821

  1. GREG cells, a dysferlin-deficient myogenic mouse cell line

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; Morree, Antoine de; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

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

  2. GREG cells, a dysferlin-deficient myogenic mouse cell line.

    PubMed

    Humphrey, Glen W; Mekhedov, Elena; Blank, Paul S; de Morree, Antoine; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency. PMID:22020321

  3. GREG cells, a dysferlin-deficient myogenic mouse cell line

    PubMed Central

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; de Morree, Antoine; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-01

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency. PMID:22020321

  4. Analysis of gene amplification in human tumor cell lines

    SciTech Connect

    Fukumoto, M.; Shevrin, D.H.; Roninson, I.B.

    1988-09-01

    Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.

  5. The transcriptional diversity of 25 Drosophila cell lines

    SciTech Connect

    Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu; Yang, Li; Zou, Yi; Eads, Brian D.; Carlson, Joseph W.; Landolin, Jane M.; Kapranov, Philipp; Dumais, Jacqueline; Samsonova, Anastasia; Choi, Jeong-Hyeon; Roberts, Johnny; Davis, Carrie A.; Tang, Haixu; van Baren, Marijke J.; Ghosh, Srinka; Dobin, Alexander; Bell, Kim; Lin, Wei; Langton, Laura; Duff, Michael O.; Tenney, Aaron E.; Zaleski, Chris; Brent, Michael R.; Hoskins, Roger A.; Kaufman, Thomas C.; Andrews, Justen; Graveley, Brenton R.; Perrimon, Norbert; Celniker, Susan E.; Gingeras, Thomas R.; Cherbas, Peter

    2010-11-15

    Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal discderived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. Wereport the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what those patterns reveal about the origins of the lines and the stability of spatial expression patterns. We also offer an initial analysis of previously unannotated transcripts in the cell lines.

  6. PI Control of Gene Expression in Tumorous Cell Lines 

    E-print Network

    Mendonca, Rouella J.

    2010-01-16

    in different genes in the Human Embryonic Kidney and Human Colon Adenocarcinoma Grade II cell lines. The difference in the gene expressions of the two cell lines motivates the problem in this thesis. The thesis provided intervention methods to make the colon...

  7. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... Cell Lines Project. All data and corresponding information will be posted in a publically held database... will be posted in a publically held database. The use of misidentified cell lines in cancer and other... suitable for use in a standard reference database. STR analysis involves simultaneous amplification...

  8. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines

    PubMed Central

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

    2014-01-01

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

  9. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma

    Cancer.gov

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

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

    PubMed Central

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

    2014-01-01

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

  11. Rhamnogalacturonan I containing homogalacturonan inhibits colon cancer cell proliferation by decreasing ICAM1 expression.

    PubMed

    Maxwell, Ellen G; Colquhoun, Ian J; Chau, Hoa K; Hotchkiss, Arland T; Waldron, Keith W; Morris, Victor J; Belshaw, Nigel J

    2015-11-01

    Pectin modified with pH, heat or enzymes, has previously been shown to exhibit anti-cancer activity. However, the structural requirements for modified pectin bioactivity have rarely been addressed. In this study several pectin extracts representing different structural components of pectin were assessed for effects against colon cancer cells. Rhamnogalacturonan I (RGI) extracts reduced proliferation of DLD1 and HCT116 colon cancer cells in a dose- and time-dependent manner. RGI reduced ICAM1 gene expression and siRNA-mediated knockdown of ICAM1 expression decreased cell proliferation providing a potential novel mechanism for the anti-cancer activity of pectin. Structural analysis of bioactive and non-bioactive RGIs suggested that a homogalacturonan component is maybe essential for the anti-proliferative activity, furthering the understanding of the structural requirements for pectin bioactivity. PMID:26256381

  12. Development of a conditionally immortalized human pancreatic ? cell line

    PubMed Central

    Scharfmann, Raphaël; Pechberty, Severine; Hazhouz, Yasmine; von Bülow, Manon; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Guez, Fanny; Rachdi, Latif; Lohmann, Matthias; Czernichow, Paul; Ravassard, Philippe

    2014-01-01

    Diabetic patients exhibit a reduction in ? cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human ? cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human ? cell line (EndoC-?H1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-?H1 cells display many functional properties of adult ? cells, including expression of ? cell markers and insulin secretion following glucose stimulation; however, unlike primary ? cells, EndoC-?H1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human ? cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-?H2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of ? cell–specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-?H2 cells are highly representative of human ? cells and should be a valuable tool for further analysis of human ? cells. PMID:24667639

  13. Histone Deacetylase Inhibitors Activate Tristetraprolin Expression through Induction of Early Growth Response Protein 1 (EGR1) in Colorectal Cancer Cells

    PubMed Central

    Sobolewski, Cyril; Sanduja, Sandhya; Blanco, Fernando F.; Hu, Liangyan; Dixon, Dan A.

    2015-01-01

    The RNA-binding protein tristetraprolin (TTP) promotes rapid decay of mRNAs bearing 3' UTR AU-rich elements (ARE). In many cancer types, loss of TTP expression is observed allowing for stabilization of ARE-mRNAs and their pathologic overexpression. Here we demonstrate that histone deacetylase (HDAC) inhibitors (Trichostatin A, SAHA and sodium butyrate) promote TTP expression in colorectal cancer cells (HCA-7, HCT-116, Moser and SW480 cells) and cervix carcinoma cells (HeLa). We found that HDAC inhibitors-induced TTP expression, promote the decay of COX-2 mRNA, and inhibit cancer cell proliferation. HDAC inhibitors were found to promote TTP transcription through activation of the transcription factor Early Growth Response protein 1 (EGR1). Altogether, our findings indicate that loss of TTP in tumors occurs through silencing of EGR1 and suggests a therapeutic approach to rescue TTP expression in colorectal cancer. PMID:26343742

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

    SciTech Connect

    Bae, Soo Kyung; Gwak, Jungsug; Song, Im-Sook; Park, Hyung-Soon; Oh, Sangtaek

    2011-05-27

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

  15. Hepatitis C virus infection of neuroepithelioma cell lines

    PubMed Central

    Fletcher, Nicola F; Yang, Jian Ping; Farquhar, Michelle J; Hu, Ke; Davis, Christopher; He, Qiuchen; Dowd, Kimberly; Ray, Stuart C; Krieger, Sophie E; Neyts, Johan; Baumert, Thomas F; Balfe, Peter; McKeating, Jane A; Wong-Staal, Flossie

    2012-01-01

    Background & Aims Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication. Methods We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism. Results Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas non-permissive neural cell lines lacked CLDN1 and in some cases SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1 and HCV E2. Furthermore, anti-CD81, interferon and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells. Conclusions Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at comparable levels to that of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo. PMID:20538002

  16. Antineoplastic activity of rinvanil and phenylacetylrinvanil in leukaemia cell lines.

    PubMed

    Luviano, Axel; Aguiñiga-Sánchez, Itzen; Demare, Patricia; Tiburcio, Reynaldo; Ledesma-Martínez, Edgar; Santiago-Osorio, Edelmiro; Regla, Ignacio

    2014-05-01

    In the search for novel chemotherapeutic agents for cancer treatment, capsaicin has been shown to inhibit proliferation and induce apoptosis in various types of cancer cell line, including leukaemia cell lines. The capsaicin analogues, rinvanil and phenylacetylrinvanil (PhAR), share a binding affinity for vanilloid receptors and may have biological activities similar to capsaicin; however, their anticancer potential has not yet been reported. This study analyses the antineoplastic activities of rinvanil and PhAR in leukaemia versus normal cells. P388, J774 and WEHI-3 leukaemia cell lines, as well as mouse bone marrow mononuclear cells, were cultured with varying concentrations of rinvanil and PhAR. Following this, proliferation and apoptosis were determined by the sulforhodamine B (SRB) assay and DNA ladder. Cultured leukaemia cell lines and mouse bone marrow mononuclear cells demonstrated a dose-dependent inhibition of proliferation, while non-diseased cells were less sensitive to the cytotoxic effect of capsaicin, rinvanil and PhAR. Rinvanil and PhAR also induced apoptosis in leukaemia cell lines but not in bone marrow. Given the lower IC50 values for apoptosis induction in leukaemia cells compared with that of normal cells, PhAR is a promising selective anticancer agent. PMID:24765194

  17. A methylation profile of in vitro immortalized human cell lines.

    PubMed

    Liu, Limin; Zhang, Jingmei; Bates, Steven; Li, Jian-Jian; Peehl, Dana M; Rhim, Johng S; Pfeifer, Gerd P

    2005-01-01

    Normal human diploid cells have a limited life span and undergo replicative senescence after various limited population doublings. Cells must pass the senescence barrier to become immortal. The exact mechanisms of immortalization are not clear, although inactivation of the RB pathway, and/or the p53 pathway and activation of telomerase has been shown to be necessary for immortalization of certain cell types with DNA viruses or hTERT. Methylation-associated inactivation of tumor suppressor genes plays an important role in tumor progression. To test if gene-specific methylation contributes to the immortalized and transformed phenotype, we analyzed the methylation status of 17 genes in normal cells immortalized with SV40, hTERT, Ad5, Ad12-SV40 or HPV-18. Some of these immortalized lines were progressively transformed and tumorigenic in nude mice. We observed gene-specific methylation in the in vitro immortalized and transformed cells. SV40 and HPV18 immortalization resulted in different methylation spectra. In SV40- and h-TERT-immortalized prostate epithelial cells, the most frequently methylated gene was RASSF1A, while in HPV18-immortalized cell lines, the RAR-beta2 gene was universally methylated. Immortalization with SV40 resulted in methylation of a greater number of genes than immortalization with HPV. Furthermore, in SV40-immortalized cell lines, methylation affected different genes in fibroblasts compared with epithelial cells, suggesting that different mechanisms may be used by SV40 to immortalize cell lines of different origins. In HPV18-immortalized and subsequently transformed cell lines, the most commonly methylated genes were hormone responsive genes, such as AR, ER-beta and RAR-beta2. In general, more genes were methylated in neoplastically-transformed cell lines than in only immortalized cell lines, indicating that accumulation of epigenetic abnormalities may contribute to oncogenesis. PMID:15586250

  18. Thermoradiotherapy in human head and neck squamous cell carcinoma cell lines.

    PubMed

    Asaumi, Jun-Ichi; Higuchi, Yuzuru; Murakami, Jun; Kuroda, Masahiro; Shibuya, Koichi; Konouchi, Hironobu; Hisatomi, Miki; Matsuzaki, Hidenobu; Shigehara, Hiroshi; Kawasaki, Shoji; Kishi, Kanji; Hiraki, Yoshio

    2002-09-01

    Thermoradiosensitivity of 8 cell lines of head and neck squamous cell carcinoma (HO-1-u-1, HSC2, HSC3, HSC4, SAS, KB, Hep2, and Ca9-22) was investigated. The differences of radiosensitivity between the cell line with the highest radiosensitivity and the cell line with the lowest radiosensitivity were 1.7-, 7.7-, and 41-fold at 2, 6 and 8 Gy, respectively. The differences between the cell line with the highest thermosensitivity and the cell line with the lowest thermosensitivity were 2.4-, 6.2- and 34.4-fold at 43 degrees C for 40, 60 and 100 min, and 2.6-, 4.9- and 127-fold at 44 degrees C for 20, 30 and 50 min, respectively. These findings indicated that there were large differences in both radiosensitivity and thermosensitivity among the 8 cell lines. There was a negative relationship between radiosensitivity and thermosensitivity (43 degrees C: r=-0.600, 44 degrees C: r=-0.848) in 7 of 8 cell lines, the exception being the HSC4 cell line, which was resistant to both therapies. Four of the 8 cell lines at 43 degrees C and 5 at 44 degrees C in the radiotherapy combined with thermotherapy showed actual survival rates smaller than the theoretical survival rates. Thus, thermoradiotherapy was deemed effective in the head and neck carcinoma cell lines, although 1 of 8 cell lines was resistant to both radiotherapy and thermotherapy. PMID:12165802

  19. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions

    NASA Astrophysics Data System (ADS)

    Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

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

    PubMed Central

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

    2014-01-01

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

  1. CHARACTERIZATION OF A SPONTANEOUSLY TRANSFORMED CHICKEN MONONUCLEAR CELL LINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 C or 41 C...

  2. Evaluation of Antioxidative and Cytotoxic Activities of Streptomyces pluripotens MUSC 137 Isolated from Mangrove Soil in Malaysia

    PubMed Central

    Ser, Hooi-Leng; Ab Mutalib, Nurul-Syakima; Yin, Wai-Fong; Chan, Kok-Gan; Goh, Bey-Hing; Lee, Learn-Han

    2015-01-01

    Streptomyces pluripotens MUSC 137 was isolated from mangrove soil obtained from Tanjung Lumpur, Pahang, Malaysia. We investigated the phylogenetic, genomic, biochemical, and phenotypic characteristics of this strain. Uniquely adapted microorganisms from mangrove habitats have previously yielded compounds of biopharmaceutical interest. In order to examine the bioactivities possessed by the strain, fermentation extract was prepared through solvent extraction method prior to bioactivities screenings. Antioxidant activity was examined via DPPH assay while the cytotoxic effect was assessed by means of examining the activity of the extract against selected human cancer cell lines, namely colon cancer cells (HCT-116, Caco-2, SW480, and HT-29), breast cancer cell (MCF-7), lung cancer cell (A549), prostate cancer cell (DU145), and cervical cancer cell (Ca Ski). The results revealed MUSC 137 possesses significant antioxidant activity and demonstrates cytotoxic effect against several cancer cell lines tested. The results indicated MCF-7 cells were most susceptible to the extract with the lowest IC50 (61.33 ± 17.10 ?g/mL), followed by HCT-116 and A549. Additionally, selective index (SI) showed that MUSC 137 extract was less toxic against normal cell lines when compared to MCF-7 and HCT-116 cells. The extract was further subjected to chemical analysis using GC–MS and revealed the presence of deferoxamine and pyrrolizidines related compounds which may account for the antioxidant and cytotoxic properties observed.

  3. A Stable Cranial Neural Crest Cell Line from Mouse

    PubMed Central

    Ishii, Mamoru; Arias, Athena C.; Liu, Liqiong; Chen, Yi-Bu; Bronner, Marianne E.

    2012-01-01

    Cranial neural crest cells give rise to ectomesenchymal derivatives such as cranial bones, cartilage, smooth muscle, dentin, as well as melanocytes, corneal endothelial cells, and neurons and glial cells of the peripheral nervous system. Previous studies have suggested that although multipotent stem-like cells may exist during the course of cranial neural crest development, they are transient, undergoing lineage restriction early in embryonic development. We have developed culture conditions that allow cranial neural crest cells to be grown as multipotent stem-like cells. With these methods, we obtained 2 independent cell lines, O9-1 and i10-1, which were derived from mass cultures of Wnt1-Cre; R26R-GFP-expressing cells. These cell lines can be propagated and passaged indefinitely, and can differentiate into osteoblasts, chondrocytes, smooth muscle cells, and glial cells. Whole-genome expression profiling of O9-1 cells revealed that this line stably expresses stem cell markers (CD44, Sca-1, and Bmi1) and neural crest markers (AP-2?, Twist1, Sox9, Myc, Ets1, Dlx1, Dlx2, Crabp1, Epha2, and Itgb1). O9-1 cells are capable of contributing to cranial mesenchymal (osteoblast and smooth muscle) neural crest fates when injected into E13.5 mouse cranial tissue explants and chicken embryos. These results suggest that O9-1 cells represent multipotent mesenchymal cranial neural crest cells. The O9-1 cell line should serve as a useful tool for investigating the molecular properties of differentiating cranial neural crest cells. PMID:22889333

  4. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

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

  5. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    SciTech Connect

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  6. Mitochondria-acting hexokinase II peptides carried by short-length carbon nanotubes with increased cellular uptake, endosomal evasion, and enhanced bioactivity against cancer cells

    NASA Astrophysics Data System (ADS)

    Yoong, Sia Lee; Lau, Wei Liang; Liu, Ang Yu; Prendergast, D'arcy; Ho, Han Kiat; Yu, Victor Chun Kong; Lee, Chengkuo; Ang, Wee Han; Pastorin, Giorgia

    2015-08-01

    Type II hexokinase (HKII) has emerged as a viable therapeutic target due to its involvement in metabolic reprogramming and also apoptosis prevention. The peptide derived from the fifteen amino acid sequence in the HKII N-terminal region [HKII(pep)] can compete with endogenous proteins for binding on mitochondria and trigger apoptosis. However, this peptide is not cell-permeable. In this study, multi-walled carbon nanotubes (MWCNTs) were used to effectively deliver HKII(pep) across cellular barriers without compromising their bioactivity. The peptide was conjugated on either oxidized MWCNTs or 2,2'-(ethylenedioxy)bis(ethylamine)-functionalized MWCNTs, yielding MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep), respectively. Both conjugates were shown to be internalized by breast cancer MCF-7 cells using confocal microscopy. Moreover, these nanoconjugates seemed to have escaped from endosomes and be in the vicinity of mitochondria. The WST-1 cytotoxicity assay conducted on MCF-7 and colon carcinoma HCT116 cells revealed that MWCNT-peptide conjugates were significantly more effective in curbing cancer cell growth compared to a commercially available cell permeable HKII fusion peptide. In addition, both nanoconjugates displayed an enhanced ability in eliciting apoptosis and depleting the ATP level in HCT116 cells compared to the mere HKII peptide. Importantly, hexokinase II release from mitochondria was demonstrated in MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep) treated cells, highlighting that the structure and bioactivity of HKII(pep) were not compromised after covalent conjugation to MWCNTs.Type II hexokinase (HKII) has emerged as a viable therapeutic target due to its involvement in metabolic reprogramming and also apoptosis prevention. The peptide derived from the fifteen amino acid sequence in the HKII N-terminal region [HKII(pep)] can compete with endogenous proteins for binding on mitochondria and trigger apoptosis. However, this peptide is not cell-permeable. In this study, multi-walled carbon nanotubes (MWCNTs) were used to effectively deliver HKII(pep) across cellular barriers without compromising their bioactivity. The peptide was conjugated on either oxidized MWCNTs or 2,2'-(ethylenedioxy)bis(ethylamine)-functionalized MWCNTs, yielding MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep), respectively. Both conjugates were shown to be internalized by breast cancer MCF-7 cells using confocal microscopy. Moreover, these nanoconjugates seemed to have escaped from endosomes and be in the vicinity of mitochondria. The WST-1 cytotoxicity assay conducted on MCF-7 and colon carcinoma HCT116 cells revealed that MWCNT-peptide conjugates were significantly more effective in curbing cancer cell growth compared to a commercially available cell permeable HKII fusion peptide. In addition, both nanoconjugates displayed an enhanced ability in eliciting apoptosis and depleting the ATP level in HCT116 cells compared to the mere HKII peptide. Importantly, hexokinase II release from mitochondria was demonstrated in MWCNT-HKII(pep) and MWCNT-TEG-HKII(pep) treated cells, highlighting that the structure and bioactivity of HKII(pep) were not compromised after covalent conjugation to MWCNTs. Electronic supplementary information (ESI) available: Additional TEM images, UV-Vis scanning characterisation, WST-1 assay results, and immunoblotting of HKII in the total cell lysate. See DOI: 10.1039/c5nr00980d

  7. Design, Synthesis and In Vitro Activity of Anticancer Styrylquinolines. The p53 Independent Mechanism of Action

    PubMed Central

    Mrozek-Wilczkiewicz, Anna; Rams-Baron, Marzena; Kryštof, Vladimír; Musiol, Robert

    2015-01-01

    A group of styrylquinolines were synthesized and tested for their anti-proliferative activity. Anti-proliferative activity was evaluated against the human colon carcinoma cell lines that had a normal expression of the p53 protein (HCT116 p53+/+) and mutants with a disabled TP53 gene (HCT116 p53-/-) and against the GM 07492 normal human fibroblast cell line. A SAR study revealed the importance of Cl and OH as substituents in the styryl moiety. Several of the compounds that were tested were found to have a marked anti-proliferative activity that was similar to or better than doxorubicin and were more active against the p53 null than the wild type cells. The cellular localization tests and caspase activity assays suggest a mechanism of action through the mitochondrial pathway of apoptosis in a p53-independent manner. The activity of the styrylquinoline compounds may be associated with their DNA intercalating ability. PMID:26599982

  8. Human Cell Line for Studies on Signaling and Endocrine Cancer

    Cancer.gov

    The first known immortalized cell line with a naturally-occurring inactivating mutation in PRKAR1A, the regulatory subunit type 1A (R1alpha) of protein kinase A (PKA), which is associated with tumor formation.

  9. Embryonic germ cell lines and their derivation from mouse primordial germ cells.

    PubMed

    Labosky, P A; Barlow, D P; Hogan, B L

    1994-01-01

    When primordial germ cells of the mouse are cultured on feeder layers with the addition of the polypeptide signalling molecules leukaemia inhibitory factor, Steel factor and basic fibroblast growth factor they give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells (EG cells) can be induced to differentiate extensively in culture and also form teratocarcinomas when injected into nude mice. Additionally, they contribute to chimeras when injected into host blastocysts. We have derived multiple EG cell lines from 8.5 days post coitum (dpc) embryos of C57BL/6 inbred mice. Four independent EG cell lines with normal male karyotypes have formed chimeras (up to 70% coat colour chimerism) when injected into BALB/c host blastocysts. Chimeric mice from all four cell lines are fertile, but only those from one line have transmitted coat colour markers through the germline. Studies have also been carried out to determine whether gonadal primordial germ cells can give rise to pluripotent EG cells. Germ cells from gonads of 15.5 dpc C57BL/6 embryos and newborn mice failed to produce EG cell lines. EG cell lines capable of forming teratocarcinomas and coat colour chimeras have been established from primordial germ cells of 12.5 dpc genital ridges. We are currently testing the genomic imprinting status of the insulin-like growth factor type 2 receptor gene (Igf2r) in our different EG cell lines. PMID:7835148

  10. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    PubMed

    Sadowska, Anna; Kr?towski, Rafa?; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50??g/mL after 24 hours; 0.1, 10, 50, and 250??g/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test. PMID:23777253

  11. Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

    PubMed Central

    Sadowska, Anna; Kr?towski, Rafa?; Szynaka, Beata; Cechowska-Pasko, Marzanna

    2013-01-01

    Abstract The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50??g/mL after 24 hours; 0.1, 10, 50, and 250??g/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test. PMID:23777253

  12. Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

    SciTech Connect

    Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A. )

    1991-01-01

    1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.

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

    Cancer.gov

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

  14. Pharmacogenomic agreement between two cancer cell line data sets.

    PubMed

    2015-12-01

    Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics resources: The Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer databases. PMID:26570998

  15. Inducible human immunodeficiency virus type 1 packaging cell lines.

    PubMed Central

    Yu, H; Rabson, A B; Kaul, M; Ron, Y; Dougherty, J P

    1996-01-01

    Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. PMID:8676479

  16. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

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

  17. Two small cell lung cancer cell lines established from rigid bronchoscope biopsies.

    PubMed

    Postmus, P E; de Ley, L; van der Veen, A Y; Mesander, G; Buys, C H; Elema, J D

    1988-04-01

    Two new, good growing cell lines (GLC-8, GLC-11) have been established from biopsies of small cell lung cancer (SCLC). Tumor biopsies were procured by rigid bronchoscopy from tumor recurrences at the site of the primary lesions. Both tumors were clinically resistant to chemotherapy. Cytogenetic analysis revealed deletions in the short arm of chromosome 3. GLC-8 shows amplification of N-myc. Both cell lines show SCLC differentiations; neurosecretory granules were present and the SCLC related hormones dopa-decarboxylase and creatine kinase were elevated. Both cell lines behave as so-called 'classic' SCLC cell lines. PMID:2838297

  18. Curcumin-induced mitotic arrest is characterized by spindle abnormalities, defects in chromosomal congression and DNA damage.

    PubMed

    Blakemore, Louise M; Boes, Christoph; Cordell, Rebecca; Manson, Margaret M

    2013-02-01

    The chemopreventive agent curcumin has anti-proliferative effects in many tumour types, but characterization of cell cycle arrest, particularly with physiologically relevant concentrations, is still incomplete. Following oral ingestion, the highest concentrations of curcumin are achievable in the gut. Although it has been established that curcumin induces arrest at the G(2)/M stage of the cell cycle in colorectal cancer lines, it is not clear whether arrest occurs at the G(2)/M transition or in mitosis. To elucidate the precise stage of arrest, we performed a direct comparison of the levels of curcumin-induced G(2)/M boundary and mitotic arrest in eight colorectal cancer lines (Caco-2, DLD-1, HCA-7, HCT116p53+/+, HCT116p53(-)/(-), HCT116p21(-)/(-), HT-29 and SW480). Flow cytometry confirmed that these lines underwent G(2)/M arrest following treatment for 12h with clinically relevant concentrations of curcumin (5-10 ?M). In all eight lines, the majority of this arrest occurred at the G(2)/M transition, with a proportion of cells arresting in mitosis. Examination of the mitotic index using fluorescence microscopy showed that the HCT116 and Caco-2 lines exhibited the highest levels of curcumin-induced mitotic arrest. Image analysis revealed impaired mitotic progression in all lines, exemplified by mitotic spindle abnormalities and defects in chromosomal congression. Pre-treatment with inhibitors of the DNA damage signalling pathway abrogated curcumin-induced mitotic arrest, but had little effect at the G(2)/M boundary. Moreover, pH2A.X staining seen in mitotic, but not interphase, cells suggests that this aberrant mitosis results in DNA damage. PMID:23125222

  19. Far-red-absorbing cationic phthalocyanine photosensitizers: synthesis and evaluation of the photodynamic anticancer activity and the mode of cell death induction.

    PubMed

    Machacek, Miloslav; Cidlina, Antonin; Novakova, Veronika; Svec, Jan; Rudolf, Emil; Miletin, Miroslav; Ku?era, Radim; Simunek, Tomas; Zimcik, Petr

    2015-02-26

    Novel zinc, magnesium, and metal-free octasubstituted phthalocyanine photosensitizers bearing [(triethylammonio)ethyl]sulfanyl substituents in the peripheral or nonperipheral positions were synthesized and investigated for their photophysical properties (?? value up to 0.91, ?max up to 750 nm) and photodynamic anticancer activity. The photodynamic treatment of 3T3, HeLa, SK-MEL-28, and HCT 116 cancer cells revealed that the magnesium complexes were not active (IC50 > 100 ?M), whereas the IC50 values of the zinc complexes typically reached values in the submicromolar range with low toxicity in the dark (TC50 ? 1500 ?M). The subcellular changes upon photodynamic treatment of the HeLa cells indicated that the studied photosensitizers induced damage primarily to the lysosomes, which was followed by a relocalization and damage to other organelles. The time-lapse morphological changes along with the flow cytometry and caspase activity measurements indicated a predominant involvement of necrosis-like cell death. PMID:25599409

  20. Exometabolom analysis of breast cancer cell lines: Metabolic signature

    PubMed Central

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-01-01

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach. PMID:26293811

  1. Critical role of aquaporin-3 in epidermal growth factor-induced migration of colorectal carcinoma cells and its clinical significance.

    PubMed

    Li, Ang; Lu, Dehong; Zhang, Yupeng; Li, Jia; Fang, Yu; Li, Fei; Sun, Jiabang

    2013-02-01

    Aquaporins (AQPs) are a family of small, integral membrane proteins that have been shown to play an important role in tumor development and metastasis. Several studies have demonstrated that expression of AQP3 contributes to the enhanced migration of epithelial cells and is related to differentiation, metastasis and vascular invasion in lung and gastric cancer. Therefore, we investigated whether AQP3 could enhance human colorectal carcinoma cell migration and we examined the role of AQP3 in the prognosis of colorectal carcinoma. Our results showed that human epidermal growth factor (hEGF) increased the expression of AQP3 and, subsequently, the migration ability of human colorectal carcinoma cells HCT116 in a dose- and time-dependent manner. The enhanced migration ability of HCT116 cells was blocked by the AQP3 inhibitor, CuSO(4). Overexpression of AQP3 induced by hEGF was inhibited by a PI3K/AKT inhibitor, LY294002, but the ERK inhibitor U0126 had a minor effect on the hEGF-induced AQP3 upregulation. Immunohistochemical staining of the cancer tissues and corresponding normal tissues showed that AQP3 expression in cancer tissue was higher compared to that in normal tissue. The expression intensity of AQP3 was associated with the differentiation, lymph node and distant metastasis of colorectal carcinoma patients. Our results suggest that AQP3 overexpression could facilitate colorectal carcinoma cell migration and AQP3 may be considered a potential indicator and therapeutic target for colon tumor metastasis and prognosis. PMID:23165320

  2. BHD Tumor Cell Line and UOK257-2 wild type FLCN-restored Renal Cell Line

    Cancer.gov

    Center for Cancer Research, Urologic Oncology Branch is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize kidney cancer tumor cell lines.

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

    SciTech Connect

    Cha, Seho; Lim, Chunghun; Lee, Jae Young; Song, Yoon-Jae; Park, Junsoo; Choe, Joonho; Seo, Taegun

    2010-04-16

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

  4. Induction of apoptosis by ubenimex (Bestatin) in human non-small-cell lung cancer cell lines.

    PubMed

    Ezawa, K; Minato, K; Dobashi, K

    1996-01-01

    We studied the direct anti-tumor effects of ubenimex on five human lung cancer cell lines; ABC-1, RERF-LC-OK, RERF-LC-MS (adenocarcinoma) and SQ-5, EBC-1 (squamous cell carcinoma). Ubenimex dose-dependently inhibited the growth of these cancer cell lines except RERF-LC-MS. The results indicated that lung squamous cell carcinoma cell lines were more sensitive to ubenimex than lung adenocarcinoma cell lines. Coincidentally, histological observation by Hematoxylin eosine (HE) staining revealed that ubenimex induced nuclear condensation and apoptic body in the cancer cell lines. Immunohistochemical study showed ubenimex-treated cells expressed LeY antigen which is a useful phenotypic marker predictive of apoptosis. The induction of DNA fragmentation was also observed in the ubenimex treated cancer cell lines by ELISA. We conclude that ubenimex exhibits its direct anti-tumor effect against non-small-cell lung cancer cell lines, more effectively against squamous carcinoma cell lines, through the induction of apoptosis. PMID:8952869

  5. DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

    PubMed Central

    Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

    2009-01-01

    Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M?1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

  6. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    EPA Science Inventory

    We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

  7. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    SciTech Connect

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

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

    PubMed Central

    2010-01-01

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

  9. Guidelines for the use of cell lines in biomedical research

    PubMed Central

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-01-01

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

  10. Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

    PubMed Central

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

    2009-01-01

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

  11. Skin Biopsy and Patient-Specific Stem Cell Lines.

    PubMed

    Li, Yao; Nguyen, Huy V; Tsang, Stephen H

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  12. Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

    1989-06-01

    The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

  13. Comparative antibiotic eradication of mycoplasma infections from continuous cell lines.

    PubMed

    Uphoff, Cord C; Drexler, Hans G

    2002-02-01

    Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method. PMID:11929000

  14. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

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

  15. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  16. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

  17. Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines

    PubMed Central

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Chen, Wei-Ching; Fang, Jung-Hua; Weng, Tzu-Yang; Chen, Yi-Ling

    2015-01-01

    Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines. PMID:25928182

  18. Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines.

    PubMed

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Chen, Wei-Ching; Fang, Jung-Hua; Weng, Tzu-Yang; Chen, Yi-Ling

    2015-01-01

    Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines. PMID:25928182

  19. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

    Vergara, D; Simeone, P; De Matteis, S; Carloni, S; Lanuti, P; Marchisio, M; Miscia, S; Rizzello, A; Napolitano, R; Agostinelli, C; Maffia, M

    2015-12-15

    Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies. PMID:26588820

  20. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (inventor); Marley, Garry M. (inventor)

    1991-01-01

    Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

  1. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

    PubMed Central

    Cherbas, Lucy; Hackney, Jennifer; Gong, Lei; Salzer, Claire; Mauser, Eric; Zhang, Dayu; Cherbas, Peter

    2015-01-01

    We describe an adaptation of ?C31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. PMID:26450921

  2. Increased Growth Inhibitory Effects on Human Cancer Cells and Anti-Inflammatory Potency of Shogaols from Zingiber officinale Relative to Gingerols

    PubMed Central

    Sang, Shengmin; Hong, Jungil; Wu, Hou; Liu, Jing; Yang, Chung S.; Pan, Min-Hsiung; Badmaev, Vadimir; Ho, Chi-Tang

    2009-01-01

    Ginger, the rhizome of the plant Zingiber officinale, has received extensive attention due to its antioxidant, anti-inflammatory, and anti-tumor activities. Most researchers have considered gingerols as the active principles and have paid little attention to shogaols, the dehydration products of corresponding gingerols during storage or thermal processing. In this study, we have purified and identified eight major components including three major gingerols and corresponding shogaols from ginger extract and compared their anti-carcinogenic and anti-inflammatory activities. Our results showed that shogaols ([6]-, [8]-, and [10]-) had much stronger growth inhibitory effects than gingerols ([6]-, [8]-, and [10]-) on H-1299 human lung cancer cells and HCT-116 human colon cancer cells, especially when comparing [6]-shogaol with [6]-gingerol (IC50: ~8 µM vs. ~150 µM). In addition, we found that [6]-shogaol had much stronger inhibitory effects on arachidonic acid release and nitric oxide (NO) synthesis than [6]-gingerol. PMID:19877681

  3. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

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

    2012-05-01

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

  4. Ginger compound [6]-shogaol and its cysteine-conjugated metabolite (M2) activate Nrf2 in colon epithelial cells in vitro and in vivo.

    PubMed

    Chen, Huadong; Fu, Junsheng; Chen, Hao; Hu, Yuhui; Soroka, Dominique N; Prigge, Justin R; Schmidt, Edward E; Yan, Feng; Major, Michael B; Chen, Xiaoxin; Sang, Shengmin

    2014-09-15

    In this study, we identified Nrf2 as a molecular target of [6]-shogaol (6S), a bioactive compound isolated from ginger, in colon epithelial cells in vitro and in vivo. Following 6S treatment of HCT-116 cells, the intracellular GSH/GSSG ratio was initially diminished but was then elevated above the basal level. Intracellular reactive oxygen species (ROS) correlated inversely with the GSH/GSSG ratio. Further analysis using gene microarray showed that 6S upregulated the expression of Nrf2 target genes (AKR1B10, FTL, GGTLA4, and HMOX1) in HCT-116 cells. Western blotting confirmed upregulation, phosphorylation, and nuclear translocation of Nrf2 protein followed by Keap1 decrease and upregulation of Nrf2 target genes (AKR1B10, FTL, GGTLA4, HMOX1, and MT1) and glutathione synthesis genes (GCLC and GCLM). Pretreatment of cells with a specific inhibitor of p38 (SB202190), PI3K (LY294002), or MEK1 (PD098059) attenuated these effects of 6S. Using ultra-high-performance liquid chromatography-tandem mass spectrometry, we found that 6S modified multiple cysteine residues of Keap1 protein. In vivo 6S treatment induced Nrf2 nuclear translocation and significantly upregulated the expression of MT1, HMOX1, and GCLC in the colon of wild-type mice but not Nrf2(-/-) mice. Similar to 6S, a cysteine-conjugated metabolite of 6S (M2), which was previously found to be a carrier of 6S in vitro and in vivo, also activated Nrf2. Our data demonstrated that 6S and its cysteine-conjugated metabolite M2 activate Nrf2 in colon epithelial cells in vitro and in vivo through Keap1-dependent and -independent mechanisms. PMID:25148906

  5. Genomic alterations in oral squamous cell carcinoma cell lines detected by two-dimensional gel analysis.

    PubMed

    Yamamoto, K; Konishi, N; Inui, T; Kitahori, Y; Hiasa, Y; Kirita, T; Sugimura, M

    1998-11-01

    To initially analyze the genomic abnormalities in human oral squamous cell carcinoma, DNA extracted from each of four oral carcinoma cell lines (Ca9-22, HO-1-u-1, HSC-2, KB) was examined using restriction landmark genomic scanning (RLGS), a method especially conducive to detection of amplifications and rearrangements of genomic DNA. Isolated cell line and normal oral epithial DNAs were sequentially cleaved with specific restriction enzymes, radiolabelled and separated in two-dimensional gel electrophoreses. Thirteen distinct fragments were commonly amplified in the oral cancer cell lines, six of which were evident in all samples. These results suggest genetic alterations characteristic of oral squamous cell carcinogenesis. PMID:9930363

  6. Engineering retina from human retinal progenitors (cell lines).

    PubMed

    Dutt, Kamla; Cao, Yang

    2009-06-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell-cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr(2)e(3), expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription-polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell line retains its capacity to differentiate into multiple cell types holds great promise for the use of tissue-specific adult stem cells for therapy. PMID:19113950

  7. Validating classical line profile analyses using microbeam diffraction from individual dislocation cell walls and cell interiors

    SciTech Connect

    Levine, Lyle E.; Geantil, P.; Larson, Ben C; Tischler, Jonathan Zachary; Kassner, Michael E.; Liu, Wenjun

    2012-01-01

    Dislocation structures in deformed metals produce broad asymmetric diffraction line profiles. During analysis, these profiles are generally separated into two nearly symmetric subprofiles corresponding to diffraction by dislocation cell walls and cell interiors. These subprofiles are then interpreted using complex models of dislocation-based line broadening. Until now, it has not been possible to test the many assumptions that are made in such an analysis. Here, depth-resolved microbeam diffraction was used to measure diffraction line profiles from numerous individual dislocation cell walls and cell interiors in a heavily deformed Cu single crystal. Summing these profiles directly constructed the cell-interior and cell-wall subprofiles that have been approximated in the line profile analysis literature for the past 30 years. Direct comparison between the reconstructed subprofiles and the macroscopic asymmetric line profile from the same sample allows the first direct tests of many of the assumptions that have been used for interpreting these X-ray measurements.

  8. Comparative proteome analysis across non-small cell lung cancer cell lines.

    PubMed

    Grundner-Culemann, Kathrin; Dybowski, J Nikolaj; Klammer, Martin; Tebbe, Andreas; Schaab, Christoph; Daub, Henrik

    2016-01-01

    Non-small cell lung cancer (NSCLC) cell lines are widely used model systems to study molecular aspects of lung cancer. Comparative and in-depth proteome expression data across many NSCLC cell lines has not been generated yet, but would be of utility for the investigation of candidate targets and markers in oncogenesis. We employed a SILAC reference approach to perform replicate proteome quantifications across 23 distinct NSCLC cell lines. On average, close to 4000 distinct proteins were identified and quantified per cell line. These included many known targets and diagnostic markers, indicating that our proteome expression data represents a useful resource for NSCLC pre-clinical research. To assess proteome diversity within the NSCLC cell line panel, we performed hierarchical clustering and principal component analysis of proteome expression data. Our results indicate that general proteome diversity among NSCLC cell lines supersedes potential effects common to K-Ras or epidermal growth factor receptor (EGFR) oncoprotein expression. However, we observed partial segregation of EGFR or KRAS mutant cell lines for certain principal components, which reflected biological differences according to gene ontology enrichment analyses. Moreover, statistical analysis revealed several proteins that were significantly overexpressed in KRAS or EGFR mutant cell lines. PMID:26361996

  9. Phase transitions in tumor growth: II prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Llanos-Pérez, J. A.; Betancourt-Mar, A.; De Miguel, M. P.; Izquierdo-Kulich, E.; Royuela-García, M.; Tejera, E.; Nieto-Villar, J. M.

    2015-05-01

    We propose a mechanism for prostate cancer cell lines growth, LNCaP and PC3 based on a Gompertz dynamics. This growth exhibits a multifractal behavior and a "second order" phase transition. Finally, it was found that the cellular line PC3 exhibits a higher value of entropy production rate compared to LNCaP, which is indicative of the robustness of PC3, over to LNCaP and may be a quantitative index of metastatic potential tumors.

  10. A new anthraquinone from seed of Cassia obtusifolia.

    PubMed

    Shi, Bao-Jun; Zhang, Wei-Dong; Jiang, Hong-Fang; Zhu, Yu-Yao; Chen, Lei; Zha, Xiao-Ming; Lu, Yuan-Yuan; Zhang, Wei-Ming

    2016-01-01

    Seeds of Cassia obtusifolia L. are known as homology of medicine and food material, which is a commonly consumed beverage in China. One new compound, 8-hydroxy-1,7-dimethoxy-3-methylanthracene-9,10-dione-2-O-?-d-glucoside (1), together with 11 known compounds, including seven anthraquinones (2-8), was isolated from the seeds. The 2D NMR data of compound 2 are reported for the first time. The structures of the compounds were established on the basis of 1D and 2D NMR, IR and HR-ESI-MS spectra. The cytotoxic activities of all the compounds against five cell lines (LO2, HCT-116, A549, HepG2 and SGC7901) were evaluated by using CCK8 methods. Compounds 1, 3 and 7 show moderate cytotoxicity towards HCT-116 cells compared with oxaliplatin. PMID:25894611

  11. New cytotoxic compounds from flowers of Lawsonia inermis L.

    PubMed

    Li, Qian; Gao, Wenqin; Cao, Jiaqing; Bi, Xiuli; Chen, Gang; Zhang, Xiaoshu; Xia, Xichun; Zhao, Yuqing

    2014-04-01

    Three new compounds, a bicoumarin A (1), a biflavonoid A (2), and a biquinone A (3), as well as 12 other known compounds, were isolated from the flower of Lawsonia inermis L. The structures were elucidated by spectral analysis and new compounds 2 and 3 then were further confirmed by ECD calculations and single-crystal X-ray diffraction crystallography respectively. The cytotoxicity of the compounds against four cancer cell lines, including MCF-7, Hela, HCT-116, and HT-29 were evaluated using MTT assay. The IC50 values of compounds 3 and 5 against MCF-7, Hela, HCT-116, and HT-29 were 2.24, 1.42, 24.29, and 7.02 ?M and 6.1, 2.44, 5.58, and 10.21 ?M respectively. The two compounds exhibited stronger inhibitory activities than the positive control 5-fluorouracil (IC50=7.34, 11.50, 36.17, 18.83 ?M) against the four tested cell lines. These results demonstrated that compounds from the flowers of L. inermis L. showed cytotoxic activity on MCF-7, Hela, HCT-116, and HT-29 cell lines. PMID:24565962

  12. Triazole linked mono carbonyl curcumin-isatin bifunctional hybrids as novel anti tubulin agents: Design, synthesis, biological evaluation and molecular modeling studies.

    PubMed

    Sharma, Sahil; Gupta, Manish K; Saxena, Ajit K; Bedi, Preet Mohinder S

    2015-11-15

    Keeping in view the limitations associated with currently available anticancer drugs, molecular hybrids of mono carbonyl curcumin and isatin tethered by triazole ring have been synthesized and evaluated for in vitro cytotoxicity against THP-1, COLO-205, HCT-116, A549, HeLa, CAKI-I, PC-3, MiaPaca-2 human cancer cell lines. The results revealed that the compounds SA-1 to SA-9, SB-2, SB-3, SB-4, SB-7 and SC-2 showed a good range of IC50 values against THP-1, COLO-205, HCT-116 and PC-3 cell lines, while the other four cell lines among these were found to be almost resistant. Structure activity relationship revealed that the nature of Ring X and substitution at position R influences the activity. Methoxy substituted phenyl ring as Ring X and H as R were found to be the ideal structural features. The most potent compounds (SA-2, SA-3, SA-4, SA-7) were further tested for tubulin inhibition. Compound SA-2 was found to significantly inhibit the tubulin polymerization (IC50=1.2?M against HCT-116). Compound SA-2, moreover, lead to the disruption of microtubules as confirmed by immunofluorescence technique. The significant cytotoxicity and tubulin inhibition by SA-2 was streamlined by molecular modeling studies where it was docked at the curcumin binding site of tubulin. PMID:26515041

  13. The antiproliferative effect of coumarins on several cancer cell lines.

    PubMed

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y

    2001-01-01

    Twenty-one coumarins were examined for their antiproliferative activity towards several cancer cell lines, namely lung carcinoma (A549), melanin pigment producing mouse melanoma (B16 melanoma 4A5), human T-cell leukemia (CCRF-HSB-2), and human gastric cancer, lymph node metastasized (TGBC11TKB). The structure-activity relationship established from the results revealed that the 6,7-dihydroxy moiety had an important role for their antiproliferative activity. Analysis of cell cycle distribution indicated that esculetin-treated cells accumulated in the G1 (at 400 microM) or in S phase (at 100 microM). PMID:11396185

  14. Fluorescence Assay 2. http://www.tgrbio.com/cancer-cell-lines-primary-cell-

    E-print Network

    Collins, Gary S.

    Fluorescence Assay References 1. 2. http://www.tgrbio.com/cancer-cell-lines-primary-cell- cultures/cell-models-hek-293-cells.html Conclusion A Novel Fluorescence Assay for the Determination of Ligand Binding Constant-therapies in cancer patients. This makes the study of both agonist and antagonist ligands important as the knowledge

  15. Osmotic stress affects functional properties of human melanoma cell lines

    E-print Network

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  16. Mouse DRG Cell Line with Properties of Nociceptors

    PubMed Central

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C.; Grundy, David; Nassar, Mohammed A.

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons. PMID:26053673

  17. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  18. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    SciTech Connect

    Heaton, D.; Mustafi, R.; Schwartz, J.L. |

    1992-06-01

    The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

  19. Differential expression of Ia antigens by rheumatoid synovial lining cells.

    PubMed Central

    Burmester, G R; Jahn, B; Rohwer, P; Zacher, J; Winchester, R J; Kalden, J R

    1987-01-01

    The differential expression of Ia antigens was studied in freshly isolated rheumatoid nonlymphoid synovial lining cells (SLC) and rheumatoid synovial fibroblast cell lines cultured in the presence of Interferon-gamma, using a large panel of anti-Ia reagents with monomorphic or polymorphic specificities. All the HLA-DR or -DQ specificities detectable on the corresponding peripheral blood B cells were also expressed in freshly isolated SLC. However, in all instances, the number of DR-positive SLC exceeded the percentage of cells expressing DQ antigens. In addition, the epitope expression of Ia antigens varied within the DR or DQ populations of Ia molecules as revealed by polymorphic reagents. Double-label experiments or using the ingestion of Latex particles as a marker demonstrated that the synovial macrophages (type I SLC) primarily bear the DR+DQ+ phenotype, while there is an additional population of nonphagocytic SLC (previously termed type II SLC) that has a DR+ and monocyte marker negative phenotype but did not have detectable levels of DQ antigens as analyzed by both fluorescence microscopy and cell sorter analysis. This latter population frequently had a morphology showing dendritic processes and rapidly lost the expression of Ia antigens upon culture. Cells with a similar, primarily DR+ phenotype were readily obtained in synovial fibroblast cultures after treatment with Interferon-gamma. These data suggest that there are two populations of Ia+ synovial lining cells: the synovial macrophages (type I cells) with the DR+DQ+ phenotype, and cells probably related to fibroblasts with a DR+ phenotype without detectable DQ antigens (type II cells). The fact that the latter phenotype could be induced by Interferon-gamma treatment of cultured synovial fibroblasts suggests that this mediator may have a similar role in vivo in the activation of certain synovial cell populations. Images PMID:2442194

  20. Resveratrol induces chemosensitization to 5-fluorouracil through up-regulation of intercellular junctions, Epithelial-to-mesenchymal transition and apoptosis in colorectal cancer.

    PubMed

    Buhrmann, Constanze; Shayan, Parviz; Kraehe, Patricia; Popper, Bastian; Goel, Ajay; Shakibaei, Mehdi

    2015-11-01

    5-Fluorouracil (5-FU), a common chemotherapeutic agent used for the treatment of colorectal cancer (CRC), by itself has inadequate response rates; highlighting the need for novel and improved treatment regimens for these patients. Resveratrol, a naturally-occurring polyphenol, has been linked with chemosensitizing potential and anticancer properties; however, the underlying mechanisms for these effects remain poorly understood. The effect of resveratrol in parental CRC cell lines (HCT116, SW480) and their corresponding isogenic 5-FU-chemoresistant derived clones (HCT116R, SW480R) was examined by MTT assays, intercellular junction formation and apoptosis by electron- and immunoelectron microscopy, nuclear factor-kappaB (NF-?B) and NF-?B regulated gene products by western blot analysis in a 3D-alginate microenvironment. Resveratrol blocked the proliferation of all four CRC cell lines and synergized the invasion inhibitory effects of 5-FU. Interestingly, resveratrol induced a transition from 5-FU-induced formation of microvilli to a planar cell surface, which was concomitant with up-regulation of desmosomes, gap- and tight junctions (claudin-2) and adhesion molecules (E-cadherin) expression in HCT116 and HCT116R cells. Further, resveratrol significantly attenuated drug resistance through inhibition of epithelial-mesenchymal transition (EMT) factors (decreased vimentin and slug, increased E-cadherin) and down-regulation of NF-?B activation and its translocation to the nucleus and abolished NF-?B-regulated gene end-products (MMP-9, caspase-3). Moreover, this suppression was mediated through inhibition of I?B? kinase and I?B? phosphorylation and degradation. Our results demonstrate that resveratrol can potentiate the anti-tumor effects of 5-FU on CRC cells by chemosensitizing them, inhibiting an EMT phenotype via up-regulation of intercellular junctions and by down-regulation of NF-?B pathway. PMID:26310874

  1. Propagation of Bombyx mori Nucleopolyhedrovirus in nonpermissive insect cell lines.

    PubMed

    Woo, Soo-Dong; Roh, Jong Yul; Choi, Jae Young; Jin, Byung Rae

    2007-04-01

    This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via beta-galactosidase expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs. PMID:17483798

  2. [Establishment and biological characterization of human medulloblastoma cell lines].

    PubMed

    Yamada, M; Shimizu, K; Tamura, K; Okamoto, Y; Matsui, Y; Moriuchi, S; Park, K; Mabuchi, E; Yamamoto, K; Hayakawa, T

    1989-07-01

    Two cell lines of human medulloblastoma (ONS-76 and ONS-81) were established, and their biological characteristics were investigated. The cell line, ONS-76, was established from a tumor specimens obtained from a large cerebellar tumor of a 2-year-old girl. The pathological diagnosis was a typical medulloblastoma. The other cell line, ONS-81, was derived from a metastatic tumor in right frontal lobe of a 9-year-old girl. The tumor specimens were minced into fragments approximately 1 mm in diameter and cultured in plastic culture flasks in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50% patients serum. The cells growing as a monolayer were subcultured in RPMI 1640 supplemented with 10% FCS and initially with L-glutamine, sodium pyruvate, and nonessential amino acid. Microscopically, both cultured cells exhibited various morphological appearances, and this morphological heterogeneity seemed to be specific for medulloblastoma cells. The in vitro population doubling time of ONS-76 and ONS-81 were 18.6 and 19.2 hr, respectively. The ONS-76 and ONS-81 cells formed subcutaneous tumors in nude mice as serial transplantable xenograft, and these tumors had a microscopic appearance similar to that of the original medulloblastoma. Ultrastructurally++, the cultured cells showed primitive, undifferentiated appearance, and no neuronal or glial structures were not seen. Immunohistochemical studies showed that both cells expressed neuron-specific enolase (NSE) and neurofilament protein (NFP 200 K, 145 K), but glial fibrillary acidic protein (GFAP) and S-100 protein were not detected. The NFP immunoreactivities of both cultured cells were demonstrated as abnormal perinuclear deposits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2818910

  3. Induction of cytotoxicity of Pelagia noctiluca venom causes reactive oxygen species generation, lipid peroxydation induction and DNA damage in human colon cancer cells

    PubMed Central

    2011-01-01

    Background The long-lasting and abundant blooming of Pelagia noctiluca in Tunisian coastal waters compromises both touristic and fishing activities and causes substantial economic losses. Determining their molecular mode of action is, important in order to limit or prevent the subsequent damages. Thus, the aim of the present study was to investigate the propensity of Pelagia noctiluca venom to cause oxidative damage in HCT 116 cells and its associated genotoxic effects. Results Our results indicated an overproduction of ROS, an induction of catalase activity and an increase of MDA generation. We looked for DNA fragmentation by means of the comet assay. Results indicated that venom of Pelagia noctiluca induced DNA fragmentation. SDS-PAGE analysis of Pelagia noctiluca venom revealed at least 15 protein bands of molecular weights ranging from 4 to 120 kDa. Conclusion Oxidative damage may be an initiating event and contributes, in part, to the mechanism of toxicity of Pelagia noctiluca venom. PMID:22151830

  4. Identification of lymphoid cell lines bearing receptors for somatostatin.

    PubMed Central

    Nakamura, H; Koike, T; Hiruma, K; Sato, T; Tomioka, H; Yoshida, S

    1987-01-01

    The MT-2, derived from an adult T-cell leukaemia (ATL) cell, the Molt-4F, a human T-cell line, and the Isk, an EB virus-transformed B-cell line, were found to have high-affinity receptors for somatostatin, a cyclic tetradecapeptide that inhibits the release of substances such as growth hormone, TSH, glucagon, insulin, secretin, gastrin and cholecystokinin. The quantity of radioactivity bound varied linearly with the number of cells, and was displaced by non-radioactive somatostatin in a concentration-dependent manner. Specific binding of 125I-somatostatin was time- and temperature-dependent and at 22 degrees reached equilibrium within 120 min. Scatchard analysis demonstrated one class of specific-binding sites on MT-2 cells, Isk cells and Molt-4F cells that had respective densities and dissociation constants of 109 pM and 0.64 nM, 102 pM and 1.1 nM, and 5.8 pM and 0.22 nM. PMID:2892785

  5. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  6. USING NEUROBLASTOMA CELL LINES TO EXAMINE ORGANOPHOSPHATE NEUROTOXICITY

    EPA Science Inventory

    The need to deploy IN VITRO models to test neurotoxic scribes the use of by industry and government regulatory agencies. his research describes the neuroblastoma cell lines to address the relationship between esterase inhibition and neurotoxic outcome following exposure to organo...

  7. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  8. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... identification as part of this project will undergo STR profiling, a DNA profiling method that examines/screens for STRs (DNA elements 2-6 bps long repeated in tandem) in the human chromosomes, that has been shown... are expected between cell line DNA samples originating from unrelated individuals. Each unique...

  9. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  10. Choosing the right chondrocyte cell line: Focus on nitric oxide.

    PubMed

    Santoro, Anna; Conde, Javier; Scotece, Morena; Abella, Vanessa; López, Verónica; Pino, Jesús; Gómez, Rodolfo; Gómez-Reino, Juan Jesús; Gualillo, Oreste

    2015-12-01

    Nitric oxide (NO) has been considered a catabolic factor that contributes to OA pathology by inducing chondrocytes apoptosis, matrix metalloproteinases synthesis, and pro-inflammatory cytokines expression. Thus, the research on NO regulation in chondrocytes represents a relevant field which needs to be explored in depth. However, to date, only the murine ATDC-5 cell line and primary chondrocytes are well-established cells to study NO production in cartilage tissues. The goal of this study is to determine whether two commonly used human chondrocytic cell lines: SW-1353 and T/C-28a2 cell lines are good models to examine lipopolysaccharide and/or pro-inflammatory cytokine-driven NO release and iNOS expression. To this aim, we carefully examined NO production and iNOS protein expression in human T/C-28a2 and SW-1353 chondrocytes stimulated with LPS and interleukin (IL)-1 alone or in combination. We also use ATDC-5 cells as a positive control for NO production. NO accumulation has been determined by colorimetric Griess reaction, whereas NOS type II expression was determined by Western Blot analysis. Our results clearly demonstrated that neither human T/C-28a2 nor SW-1353 chondrocytes showed a detectable increase in NO production or iNOS expression after bacterial endotoxin or cytokines challenge with IL-1. Our study demonstrated that T/C-28a2 and SW-1353 human cell lines are not suitable for studying NO release and iNOS expression confirming that ATDC5 and human primary cultured chondrocytes are the best in vitro cell system to study the actions derived from this mediator. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1784-1788, 2015. PMID:26016689

  11. The Sponge-Derived Fijianolide Polyketide Class: Further Evaluation of Their Structural and Cytotoxicity Properties

    PubMed Central

    Johnson, Tyler A.; Tenney, Karen; Cichewicz, Robert H.; Morinaka, Brandon I.; White, Kimberly N.; Amagata, Taro; Subramanian, Balanehru; Media, Joseph; Mooberry, Susan L.; Valeriote, Frederick A.; Crews, Phillip

    2009-01-01

    The sponge derived polyketide macrolides fijianolides A (1) and B (2) (a.k.a. isolaulimalide and laulimalide) have taxol-like microtubule-stabilizing activity and the latter exhibits potent cytotoxicity. Insight on the biogeographical and phenotypic variations of Cacospongia mycofijiensis is presented that will enable future study of the biosynthetic pathway that produces the fijianolides. In addition to fijianolides A and B, six new fijianolides, D–I (7–12), were isolated, each with modifications to the C-20 side chain of the macrolide ring. Compounds 7–12 exhibited a range of in vitro activities against HCT-116 and MDA-MB-435 cell lines. Fijianolides 8 and 10 were shown to disrupt interphase and mitotic division but were less potent than 2. An in vivo evaluation of 2 using tumor-bearing SCID mice demonstrated significant inhibition of growth in HCT-116 tumors over 28 days. PMID:17622130

  12. Human small cell lung cancer cell lines expressing the proopiomelanocortin gene have aberrant glucocorticoid receptor function.

    PubMed Central

    Ray, D W; Littlewood, A C; Clark, A J; Davis, J R; White, A

    1994-01-01

    Some human small cell lung carcinomas (SCLC) secrete proopiomelanocortin (POMC) derived peptides, but in contrast to the pituitary, glucocorticoids fail to inhibit this hormone production. We have previously described an in vitro model using human SCLC cell lines that express POMC and are resistant to glucocorticoids. We have now identified the glucocorticoid receptor (GR) in the SCLC cell line COR L24 using a whole cell ligand binding assay (Kd = 5.7 nM; Bmax = 11 fmol/million cells), while another cell line, DMS 79, lacked significant glucocorticoid binding. To analyze GR function both positive (GMCO) and negative (TRE)3-tkCAT), glucocorticoid-regulated reporter gene constructs were transfected into COR L24 cells. In the SCLC cell line, neither hydrocortisone nor dexamethasone (500-2,000 nM) significantly induced chloramphenicol acetyltransferase expression from GMCO; in addition, they did not suppress chloramphenicol acetyltransferase expression from (TRE)3-tkCAT. Similar results were obtained with two other POMC-expressing SCLC cell lines. Expression of wild type GR in COR L24 cells restored glucocorticoid signaling, with marked induction of GMCO reporter gene expression by dexamethasone (9,100 +/- 910%; n = 3), and an estimated EC50 of 10 nM. This failure of the GR explains the resistance of the POMC gene to glucocorticoid inhibition and may have implications for cell growth in SCLC. Images PMID:8163665

  13. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    PubMed

    Kniss, Douglas A; Summerfield, Taryn L

    2014-02-11

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. PMID:24520087

  14. Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells.

    PubMed

    Lawandi, Janet; Tao, Chang; Ren, Binhai; Williams, Paul; Ling, Dora; Swan, M Anne; Nassif, Najah T; Torpy, Fraser R; O'Brien, Bronwyn A; Simpson, Ann M

    2015-01-01

    As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of ?-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-?, IL-1?, and IFN-?) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known ?-cell cytotoxins was associated with the expression of NF-?B and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial ?-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D. PMID:26029722

  15. Generation and characterization of a mouse lymphatic endothelial cell line.

    PubMed

    Sironi, Marina; Conti, Annarita; Bernasconi, Sergio; Fra, Anna M; Pasqualini, Fabio; Nebuloni, Manuela; Lauri, Eleonora; De Bortoli, Maida; Mantovani, Alberto; Dejana, Elisabetta; Vecchi, Annunciata

    2006-07-01

    Lymphatic vessels, by channeling fluid and leukocytes from the periphery into lymph nodes, play a central role in the development of the immune response. Despite their importance in homeostasis and disease, the difficulties in enriching and culturing lymphatic endothelial cells limit studies of their biology. Here, we report the isolation, stabilization, and characterization of a mouse lymphatic endothelial cell line (MELC) and the generated clones thereof. Cells were isolated from benign lymphangiomas induced by intraperitoneal injections of incomplete Freund's adjuvant. The MELC line expressed molecules typical of lymphatic endothelium, including VEGFR3/Flt-4, podoplanin, Prox-1, and D6, but not LYVE-1. It also expressed CD34, ICAM-1, VCAM, and JAM-A, but not CD31, VE-cadherin, E-selectin, or CX3CL1/fractalkine (both TNFalpha-induced), at variance with vascular endothelial cells tested in parallel. The inflammatory cytokines TNFalpha and IL-4 regulated production of selected adhesion molecules (VCAM), cytokines (IL-6), and chemokines (CCL2/JE). Whole genome transcriptional profiling identified a set of 150 known genes differentially expressed in MELC versus vascular endothelial cells. Thus, the MELC line may represent an invaluable source of lymphatic endothelium. PMID:16534603

  16. Alkylphosphocholines and curcumin induce programmed cell death in cutaneous T-cell lymphoma cell lines.

    PubMed

    Yosifov, Deyan Y; Kaloyanov, Kaloyan A; Guenova, Margarita L; Prisadashka, Kamelia; Balabanova, Maria B; Berger, Martin R; Konstantinov, Spiro M

    2014-01-01

    While most patients with early-stage cutaneous T-cell lymphomas (CTCL) have a very good prognosis, the survival of patients with extensive tumour stage and visceral involvement remains extremely poor and necessitates the development of more effective treatment modalities. In this study, we evaluated the in vitro effects of two alkylphosphocholines (APCs, miltefosine and erufosine) and the polyphenolic compound curcumin on 5 human CTCL cell lines (Hut-78, HH, MJ, My-La CD4+ and My-La CD8+). All tested drugs showed considerable cytotoxic activity, as determined by the MTT dye reduction assay. The IC50 values of both APCs ranged from the low micromolar level (Hut-78 cells) to 60-80?M (HH cells). The IC50 values of curcumin ranged from 12 to 24?M. All tested drugs induced apoptosis, as ascertained by morphological changes, DNA fragmentation and activation of caspase cascades. Miltefosine and erufosine induced dephosphorylation of Akt in My-La CD8+ cells and phosphorylation of JNK in Hut-78 and My-La CD8+ cells. APCs increased the level of the autophagic marker LC3B in Hut-78 and MJ cells. Results from co-treatment with autophagy modulators suggested that the cytotoxicity of APCs in CTCL cells is mediated, at least in part, by induction of autophagy. PMID:24225136

  17. Establishment of lal-/- Myeloid Lineage Cell Line That Resembles Myeloid-Derived Suppressive Cells

    PubMed Central

    Ding, Xinchun; Wu, Lingyan; Yan, Cong; Du, Hong

    2015-01-01

    Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions. PMID:25807535

  18. Targeted genetic modification of cell lines for recombinant protein production

    PubMed Central

    Piskareva, Olga; Muniyappa, Mohan

    2007-01-01

    Considerable increases in productivity have been achieved in biopharmaceutical production processes over the last two decades. Much of this has been a result of improvements in media formulation and process development. Though advances have been made in cell line development, there remains considerable opportunity for improvement in this area. The wealth of transcriptional and proteomic data being generated currently hold the promise of specific molecular interventions to improve the performance of production cell lines in the bioreactor. Achieving this—particularly for multi-gene modification—will require specific, targeted and controlled genetic manipulation of these cells. This review considers some of the current and potential future techniques that might be employed to realise this goal. PMID:19003191

  19. Plasmids and packaging cell lines for use in phage display

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  20. Patient sues UCLA over patent on cell line.

    PubMed

    Culliton, B J

    1984-09-28

    A patient's lawsuit against the University of California is raising questions about an individual's rights of ownership in relation to body tissues that have been turned over for biomedical research but are subsequently used commercially. In 1976, John Moore had his spleen removed at the University of California, Los Angeles, in connection with his leukemia treatment. The university and researchers David Golde and Shirley Quan recently received a patent on the biologically interesting Mo cell line, which was derived from Moore's spleen cells. Moore's suit claims that the university misappropriated his tissues and that the researchers failed to obtain a valid informed consent because they did not formally tell him about the potential commercial applications of the cell line. PMID:6474185

  1. A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

    PubMed

    Auffray, I; Dubart, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-05-01

    In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or results from synergistic interactions on the MS-5 cell surface between extracellular matrix proteins and cytokines will require further investigation. PMID:7513651

  2. Immunoglobulin G Locus Events in Soft Tissue Sarcoma Cell Lines

    PubMed Central

    Chen, Zhengshan; Li, Jing; Xiao, Yanna; Zhang, Junjun; Zhao, Yingying; Liu, Yuxuan; Ma, Changchun; Qiu, Yamei; Luo, Jin; Huang, Guowei; Korteweg, Christine; Gu, Jiang

    2011-01-01

    Recently immunoglobulins (Igs) have been found to be expressed by cells other than B lymphocytes, including various human carcinoma cells. Sarcomas are derived from mesenchyme, and the knowledge about the occurrence of Ig production in sarcoma cells is very limited. Here we investigated the phenomenon of immunoglobulin G (IgG) expression and its molecular basis in 3 sarcoma cell lines. The mRNA transcripts of IgG heavy chain and kappa light chain were detected by RT-PCR. In addition, the expression of IgG proteins was confirmed by Western blot and immunofluorescence. Immuno-electron microscopy localized IgG to the cell membrane and rough endoplasmic reticulum. The essential enzymes required for gene rearrangement and class switch recombination, and IgG germ-line transcripts were also identified in these sarcoma cells. Chromatin immunoprecipitation results demonstrated histone H3 acetylation of both the recombination activating gene and Ig heavy chain regulatory elements. Collectively, these results confirmed IgG expression in sarcoma cells, the mechanism of which is very similar to that regulating IgG expression in B lymphocytes. PMID:21731691

  3. Transgenic cell lines for detection of animal viruses.

    PubMed Central

    Olivo, P D

    1996-01-01

    Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses. PMID:8809463

  4. Ecdysone and The Cell Cycle: Investigations in a Mosquito Cell Line

    PubMed Central

    Fallon, Ann M.; Gerenday, Anna

    2010-01-01

    Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen. PMID:20303973

  5. Establishment of an epithelioid malignant schwannoma cell line (YST-1).

    PubMed

    Nagashima, Y; Ohaki, Y; Tanaka, Y; Sumino, K; Funabiki, T; Okuyama, T; Watanabe, S; Umeda, M; Misugi, K

    1990-01-01

    A novel cell line, YST-1, was established from an epithelioid malignant schwannoma (EMS) that occurred in the upper arm of an 8-year-old girl. YST-1 cells were polygonal and stellate in shape, contained abundant free ribosomes, mitochondria, lysosomes and rough-surfaced endoplasmic reticulum, and grew stably with a population doubling time of 40 h. Immunohistochemically, vimentin, S100 protein and S100 protein beta subunit were positive in the cytoplasm. The xeno-transplanted tumor in nude mice was composed of cells with an epithelioid arrangement similar to the original tumor. The borders of the tumor cells were connected intimately without desmosomal junctions, and there were abundant organelles in the cytoplasm. YST-1 cells were considered to be of value for studying the nature and histogenesis of EMS. PMID:1980563

  6. Heat induces gene amplification in cancer cells

    SciTech Connect

    Yan, Bin; Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 ; Ouyang, Ruoyun; Huang, Chenghui; Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 ; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and telomere functions are denatured. To our knowledge, this is the first study to provide direct evidence of hyperthermia induced gene amplification.

  7. Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

    PubMed

    Ricordy, R; Gensabella, G; Cacci, E; Augusti-Tocco, G

    2002-05-01

    Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression. PMID:11971996

  8. Bioenergetic Analysis of Ovarian Cancer Cell Lines: Profiling of Histological Subtypes and Identification of a Mitochondria-Defective Cell Line

    PubMed Central

    Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L. P. Madhubhani P.; Uusitalo, Larissa M.; Hempel, Nadine

    2014-01-01

    Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1? in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs. PMID:24858344

  9. STAT1 signaling is associated with acquired crossresistance to doxorubicin and radiation in myeloma cell lines

    E-print Network

    the 8226/Dox40 cell line to its parental line was performed to identify the underlying molecular mechanisms correlates positively with doxorubicin resistance in a human tumor cell line panel.8 STAT1 signaling has also mechanism that contributes to acquired crossresistance to both drugs and radiation in myeloma cell lines

  10. Proteomic Analysis of HepaRG Cells: A Novel Cell Line That Supports Hepatitis B Virus Infection

    E-print Network

    Proteomic Analysis of HepaRG Cells: A Novel Cell Line That Supports Hepatitis B Virus Infection, the only cell line that is susceptible to hepatitis B virus (HBV) infection and supports a complete virus cellular response to HBV infection. Keywords: Hepatitis B virus · HepaRG cell line · Two dimensional

  11. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  12. Serial analysis of gene expression in a microglial cell line.

    PubMed

    Inoue, H; Sawada, M; Ryo, A; Tanahashi, H; Wakatsuki, T; Hada, A; Kondoh, N; Nakagaki, K; Takahashi, K; Suzumura, A; Yamamoto, M; Tabira, T

    1999-12-01

    We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte-activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN-7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695-696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT-PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology. PMID:10559785

  13. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts.

    PubMed

    Mai, Qingyun; Yu, Yang; Li, Tao; Wang, Liu; Chen, Mei-jue; Huang, Shu-zhen; Zhou, Canquan; Zhou, Qi

    2007-12-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting. PMID:18071366

  14. Tumorigenic Potential of Mononucleated Small Cells of Hodgkin Lymphoma Cell Lines

    PubMed Central

    Ikeda, Jun-ichiro; Mamat, Suhana; Tian, Tian; Wang, Yi; Rahadiani, Nur; Aozasa, Katsuyuki; Morii, Eiichi

    2010-01-01

    Tumor cells with tumorigenic potential are limited to a small cell population known as cancer stem cells (CSCs). CSCs yield both CSCs and non-CSCs, whereas non-CSCs do not yield CSCs. CSCs have not been identified in any malignant lymphomas. Hodgkin lymphoma (HL) is a mostly B-cell neoplasm that can be diagnosed by the presence of multinucleated (Reed-Sternberg; RS) cells admixed with Hodgkin cells with distinct nucleoli and various inflammatory cells. Here, the tumorigenic potential of cells with a single nucleus (S) and cells with multiple nuclei (M), which may be equivalent to Hodgkin and RS cells, respectively, was examined in HL cell lines L1236 and L428. Cultures of single S cells yielded both S and M cells, whereas M cell cultures yielded only M cells. When either cultured in methylcellulose or inoculated into NOD/SCID mice, the colony number and tumor size were both larger in S than in M cells. Concentrations of intracellular reactive oxygen species (ROS) were at low levels in a portion of S cells that abundantly expressed FoxO3a, a transcription factor that regulates ROS-degrading enzymes. In clinical samples of HL, FoxO3a was expressed in mononuclear Hodgkin cells but not in multinucleated RS cells. These findings suggest that smaller cells or Hodgkin cells that show low-ROS concentrations and high FoxO3a expression levels might be candidates for HL CSCs. PMID:20952592

  15. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  16. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    PubMed Central

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-01-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer. PMID:24728108

  17. Biotechnology and the chicken B cell line DT40.

    PubMed

    Bachl, J; Caldwell, R B; Buerstedde, J-M

    2007-01-01

    Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information (random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest within living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection. PMID:17675859

  18. Hepatitis C virus infection of cholangiocarcinoma cell lines

    PubMed Central

    Fletcher, Nicola F.; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K.; van IJzendoorn, Sven C. D.; Baumert, Thomas F.; Balfe, Peter; Afford, Simon

    2015-01-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo. PMID:25701818

  19. Pseudoislet of hybrid cellular spheroids from commercial cell lines.

    PubMed

    Jo, Y H; Nam, B M; Kim, B Y; Nemeno, J G; Lee, S; Yeo, J E; Yang, W; Park, S H; Kim, Y S; Lee, J I

    2013-10-01

    Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 ?m. Their morphology was similar to naïve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy. PMID:24157046

  20. HIV-1 latency in actively dividing human T cell lines

    PubMed Central

    Jeeninga, Rienk E; Westerhout, Ellen M; van Gerven, Marja L; Berkhout, Ben

    2008-01-01

    Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNF? treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus. PMID:18439275

  1. New Model for Gastroenteropancreatic Large-Cell Neuroendocrine Carcinoma: Establishment of Two Clinically Relevant Cell Lines

    PubMed Central

    Krieg, Andreas; Mersch, Sabrina; Boeck, Inga; Dizdar, Levent; Weihe, Eberhard; Hilal, Zena; Krausch, Markus; Möhlendick, Birte; Topp, Stefan A.; Piekorz, Roland P.; Huckenbeck, Wolfgang; Stoecklein, Nikolas H.; Anlauf, Martin; Knoefel, Wolfram T.

    2014-01-01

    Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup. PMID:24551139

  2. New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

    PubMed

    Krieg, Andreas; Mersch, Sabrina; Boeck, Inga; Dizdar, Levent; Weihe, Eberhard; Hilal, Zena; Krausch, Markus; Möhlendick, Birte; Topp, Stefan A; Piekorz, Roland P; Huckenbeck, Wolfgang; Stoecklein, Nikolas H; Anlauf, Martin; Knoefel, Wolfram T

    2014-01-01

    Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup. PMID:24551139

  3. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  4. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    SciTech Connect

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young; Hwang, Meeyul; Kim, Ji-Hyun; Han, Bok-Ghee; Jeon, Jae-Pil

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  5. A process-line for large area organic solar cells

    NASA Astrophysics Data System (ADS)

    Alstrup, Jan; Krebs, Frederik C.; Kjær, Torben; Biancardo, Matteo; Spanggaard, Holger

    2005-10-01

    In this paper we would like to address the key role of fabrication in the performance and lifetime of organic photovoltaics. The realization of a complete process line for the construction of large area organic photovoltaics (250 x 400 mm) is described. Among many of the factors that influence organic solar cell lifetime, oxygen and water exposure is the most important. Multiple processes have to be performed under controlled atmosphere and a glove box (or glove boxes), which involves more volume than commercially available glove boxes, needs to house different instruments. The processes housed in the glove boxes were spin coating, evaporation, lamination/sealing and testing, under an inert atmosphere. The main strategy employed multiply connected glove boxes with one load lock. The first glove box was used for spin coating and lamination/sealing, the second will house a screen printer and the third one accommodate an evaporator completely build in house. The evaporator has 2 thermal evaporation sources and 2 e-beams with 4 and 1 crucibles. The process line should allow the entire device realization from substrate coating, to electrode evaporation including the sealing process avoiding air and water exposure. Organic solar cells from small test cells on ITO glass to big modules (250 x 400 mm) of 91 connected cells on ITO PET substrates were fabricated and characterized.

  6. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; Juntendo University School of Medicine, Tokyo ; Kishimoto, Koshi N.; Okuno, Hiroshi; Sano, Hirotaka; Kaneko, Kazuo; Itoi, Eiji

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  7. SilenciX cell lines for valuable insights into cellular SilenciX cell lines offer a fast and economical approach for performing studies of biological

    E-print Network

    Cai, Long

    that will create cell lines with any silenced gene of a researcher's interest. The cell lines may be HeLa cells in HeLa cells for proteins involved in DNA damage and repair path- ways1. Here we illustrate | Immunocytochemical staining of DNA-PKcs HeLa SilenciX cells 163 days after transfection. (a­d) Control HeLa Silenci

  8. Selected human T cell lines respond to thymopoietin with intracellular cyclic GMP elevations.

    PubMed

    Baker, B; Viamontes, G; Audhya, T; Goldstein, G

    1988-01-01

    Ten established human cell lines were tested for their responsiveness to thymopoietin by measuring their intracellular cyclic nucleotide levels. Three T cell lines (CCRF-CEM, MOLT-4 and CCRF-HSB-2) responded to thymopoietin with elevations of intracellular cGMP but not cAMP; seven other human cell lines did not respond to thymopoietin (three T cell lines, three B cell lines and one erythropoietic stem cell line). Interestingly, only one cell line (MOLT-4) was also responsive to the closely related polypeptide splenin, and this reactivity was restricted to human and not bovine splenin. The detection of human cell lines with distinctive patterns of response to immunoregulatory peptides should provide support for understanding the immunopharmacological mechanisms by which these molecules act. PMID:2849599

  9. Establishment of an ASPL-TFE3 renal cell carcinoma cell line (S-TFE).

    PubMed

    Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tsukamoto, Taiji

    2013-06-01

    Xp11 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. Various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome X cause the tumor. We established an Xp11 translocation renal cell carcinoma cell line from a renal tumor in a 18-y-old Japanese female and named it "S-TFE." The cell line and its xenograft demonstrated definite gene fusion including TFE3. They showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis and ASPL-TFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. S-TFE will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma. PMID:23760492

  10. Cytotoxic effects of mistletoe (Viscum album L.) in head and neck squamous cell carcinoma cell lines.

    PubMed

    Klingbeil, Ma Fátima G; Xavier, Flávia C A; Sardinha, Luiz R; Severino, Patricia; Mathor, Monica B; Rodrigues, Rodrigo V; Pinto, Décio S

    2013-11-01

    Head and neck squamous cell carcinoma is a complex disease with several etiologic factors and different molecular changes that may trigger certain events; it is also globally one of the most common malignancies in this topography. Extracts from Viscum album L. (VA) (mistletoe) have been used as adjuvant therapies with promising results in several types of cancer, mainly in European countries. In vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells, activating the apoptotic cascade or leading cells to necrosis. This study aimed to verify the effects of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in squamous cell carcinoma of the tongue cell lines SCC9 and SCC25, not previously studied. A concentration of 0.3 mg/ml (IC50) of the drugs induced apoptosis, affecting gene expression and protein levels of AKT, PTEN and CYCLIN D1. It was concluded that VA extracts have a cytotoxic effect on SCC9 and SCC25 cell lines, but while SCC9 cell line was more resistant to the action of the drugs, Iscador Qu Spezial and Iscador M have higher cytotoxic potential in both cell lines compared to Iscador P. PMID:24026291

  11. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    SciTech Connect

    Qin, J.-Z.; Xin, H.; Nickoloff, B.J.

    2010-05-28

    Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

  12. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye. PMID:25905107

  13. Complementation analysis of the murine scid cell line

    SciTech Connect

    Zdzienicka, M.Z. |; Priestly, A.; Jeggo, P.A.

    1995-09-01

    It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene. 53 refs., 4 figs., 1 tab.

  14. Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line

    SciTech Connect

    Kirchheimer, J.C.; Wojta, J.; Christ, G.; Binder, B.R. )

    1989-07-01

    Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-Pa in its active form and expresses high-affinity u-PA receptors. Approximately 70% of the u-Pa receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal antiu-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of ({sup 3}H)thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.

  15. Discriminating Normal and Cancerous Thyroid Cell Lines using Implicit Context Representation Cartesian Genetic Programming

    E-print Network

    Fernandez, Thomas

    Discriminating Normal and Cancerous Thyroid Cell Lines using Implicit Context Representation a method for discrimi- nating between thyroid cell lines. Five commercial thyroid cell lines were obtained common thyroid malignancy, followed by follicular carcinoma. Both of these cancers have a high chance

  16. Request for Human Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research

    E-print Network

    Bandettini, Peter A.

    Request for Human Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research NIH Form for use in NIH funded research in accord with the NIH Guidelines for Human Stem Cell Research (74 FR 32170 in NIH funded research, the stem cell lines, and provider information if the lines are available

  17. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  18. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  19. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  20. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  1. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  2. LETTER doi:10.1038/nature11003 The Cancer Cell Line Encyclopedia enables predictive

    E-print Network

    Kaski, Samuel

    LETTER doi:10.1038/nature11003 The Cancer Cell Line Encyclopedia enables predictive modelling and pharmacological annotation is available1 . Here we describe the Cancer Cell Line Encyclopedia (CCLE cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479of

  3. CHALLENGES AND PROSPECTS FOR THE ESTABLISHMENT OF EMBRYONIC STEM CELL LINES OF DOMESTICATED UNGULATES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The establishment of embryonic stem (ES) cell lines of domesticated ungulates, e.g., the pig, sheep, goat, cow or horse, is of interest for similar reasons to those of mouse and primate ES cell lines. Several applied research initiatives await the establishment of ungulates ES cell lines. These inc...

  4. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines.

    PubMed

    Aschacher, T; Wolf, B; Enzmann, F; Kienzl, P; Messner, B; Sampl, S; Svoboda, M; Mechtcheriakova, D; Holzmann, K; Bergmann, M

    2016-01-01

    A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer. PMID:25798839

  5. The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

    PubMed Central

    Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

    2013-01-01

    Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

  6. Susceptibility of human bone marrow cells and hematopoietic cell lines to coxsackievirus B3 infection.

    PubMed Central

    Vuorinen, T; Vainionpää, R; Vanharanta, R; Hyypiä, T

    1996-01-01

    Viremia is commonly observed in association with enterovirus infections, and during this phase viruses can be transmitted to secondary target organs in the body. It is not known, however, whether blood cells play a role in the pathogenesis of enterovirus infection supporting virus replication. Our earlier work (T. Vuorinen, R. Vainionpää, H. Kettinen, and T. Hyypiä, Blood 84:823-829, 1994) demonstrated that coxsackievirus B3 is able to replicate in representatives of B- and T-cell lines but not in a monocytic cell line or peripheral blood mononuclear cells, indicating that virus replication may depend on the differentiation and maturation stages of the cells. Therefore, we have broaden our studies and analyzed the susceptibility of granulocyte-macrophage CFU and hematopoietic cell lines with various differentiation and maturation stages to coxsackievirus B3 infection. Virus replication was detected in B- and T-cell lines with no direct correlation to the maturation stage. Granulocyte-macrophage CFU were also able to support virus multiplication. PMID:8971035

  7. Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736.

    PubMed

    Restelli, Valentina; Chilà, Rosaria; Lupi, Monica; Rinaldi, Andrea; Kwee, Ivo; Bertoni, Francesco; Damia, Giovanna; Carrassa, Laura

    2015-11-10

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL. PMID:26439697

  8. Establishment and characterization of triple drug resistant head and neck squamous cell carcinoma cell lines.

    PubMed

    Govindan, Sindhu Valiyaveedan; Kulsum, Safeena; Pandian, Ramanan Somasundara; Das, Debashish; Seshadri, Mukund; Hicks, Wesley; Kuriakose, Moni Abraham; Suresh, Amritha

    2015-08-01

    Resistance to chemotherapy leading to poor outcome and survival remains a challenge for developing strategies for therapeutic interventions in all types of cancer, including head and neck cancer. In vitro chemoresistant cell line models are an indispensable resource towards delineating the mechanisms involved in drug resistance/response and for the development of novel drugs. Current treatment for head and neck cancer includes chemotherapy with cisplatin, docetaxel and 5-fluorouracil (5-FU) and the response rates to these drugs in patients is 60-80%. The present study aimed to generate head and neck cancer triple drug-resistant cell lines in an effort towards elucidating the mechanisms underlying chemoresistance and providing a resourceful tool for drug design. Using two head and neck squamous cell carcinoma cell lines, Hep-2 (larynx) and CAL-27 (oral cavity), the present study sequentially exposed these cells to increasing concentrations of the combination of docetaxel, cisplatin and 5-FU (TPF) to generate triple drug-resistant cells, termed Hep-2 TPF resistant (TPFR) and CAL-27 TPFR. The effect of the drug treatments on the cell viability, apoptosis, cell cycle and the expression of genes associated with multidrug resistance were analyzed in the parental cells and drug-resistant counterparts. PMID:25962396

  9. PACAP protects against TNF?-induced cell death in olfactory epithelium and olfactory placodal cell lines

    PubMed Central

    Kanekar, Shami; Gandham, Mahendra; Lucero, Mary T

    2010-01-01

    In mouse olfactory epithelium (OE), pituitary adenylate cyclase activating peptide (PACAP) protects against axotomy-induced apoptosis. We used mouse OE to determine whether PACAP protects neurons during exposure to the inflammatory cytokine TNF?. Live slices of neonatal mouse OE were treated with 40 ng/ml TNF? ± 40 nM PACAP for 6 hours and dying cells were live-labeled with 0.5% propidium iodide. TNF? significantly increased the percentage of dying cells while co-incubation with PACAP prevented cell death. PACAP also prevented TNF?-mediated cell death in the olfactory placodal (OP) cell lines, OP6 and OP27. Although OP cell lines express all three PACAP receptors (PAC1, VPAC1,VPAC2), PACAP’s protection of these cells from TNF? was mimicked by the specific PAC1 receptor agonist maxadilan and abolished by the PAC1 antagonist PACAP6–38. Treatment of OP cell lines with blockers or activators of the PLC and AC/MAPKK pathways revealed that PACAP-mediated protection from TNF? involved both pathways. PACAP may therefore function through PAC1 receptors to protect neurons from cell death during inflammatory cytokine release in vivo as would occur upon viral infection or allergic rhinitis-associated injury. PMID:20654718

  10. Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines.

    PubMed

    Racusen, L C; Monteil, C; Sgrignoli, A; Lucskay, M; Marouillat, S; Rhim, J G; Morin, J P

    1997-03-01

    Few model systems exist for the study of injury to human renal proximal tubule epithelium. Optimized differentiated human renal epithelial cell lines with extended in vitro growth potential would provide an alternative model system to primary culture or other available non-human mammalian kidney cell lines. For this purpose, human renal tubule epithelial cells were isolated from normal kidney cortex and exposed in culture to a hybrid immortalizing virus, adenovirus 12-SV40. Cell lines were developed by limiting dilution, and three selected cell lines were screened for growth pattern, production of immortalizing virus, tumorigenicity, and ploidy. Cell lines were also monitored for response to inducer agents and matrix factors and were screened for expression of biochemical properties and differentiation markers of renal epithelium. All three are nonproducers of the immortalizing virus and are nontumorigenic. They grow in monolayer, have intermediate growth kinetics, and express markers of renal proximal tubular epithelium by immunohistochemistry. They also express biochemical properties comparable to other widely used proximal tubular cell lines including LLC-RK1, OK, and HK-2 and comparable to human tubular cells in stable culture. Growth medium containing low levels of fetal calf serum, or epidermal growth factor combined with parathyroid hormone, produced optimal growth characteristics, brush border enzyme expression, biochemical properties, and glucose transport in a selected cell line. The addition of dimethyl sulfoxide allows maintenance in morphologically intact monolayers for prolonged periods. These cell lines should be useful model systems for the study of human renal proximal tubular injury or disease. PMID:9042817

  11. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    PubMed

    Mehta, Rajvi H

    2014-11-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' e