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Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Morphological changes in the nucleus were observed, using a fluorescence microscope with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner, while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G
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Objective:To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. japonicus) on HT-29 cancer cells.Methods:The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Morphological changes in the nucleus were observed, using a fluorescence microscope with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting.Results:After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner, while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-GConclusions:Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from O. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.
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Objective To study the effect of quercetin on the apoptosis of human colon cancer HT-29 cells and observe the possible mechanism.Methods Human colon cancer HT-29 cells were cultured in vitro and divided into four groups, including the control group, the 16μg/ml quercetin group, the Traf6 inhibitor group and the 16μg/ml quercetin + inhibitor group. The cells in control group were cultured with complete medium and other groups were treated with quercetin or/and Traf6 inhibitor. Flow cytometry was used to observe the impact of quercetin on the apoptosis of HT-29 cells; Western Blot technology was used to detect the expression levels of Traf6, TAK1, p-TAK1, caspase-3, Bax and Bcl-2; RT-PCR was used to investigate the expression level of Traf6 mRNA after treating for 24 h.Results Compared with the 16μg/ml quercetin group, the expression levels of Traf6 (0.59 ± 0.03 vs. 0.96 ± 0.04), p-TAK1 (0.43 ± 0.02vs. 0.72 ± 0.04), caspase-3 (0.59 ± 0.03vs. 0.70 ± 0.04), and Bax (0.48 ± 0.03vs.0.67 ± 0.04) were significantly decreased in 16μg/ml quercetin + inhibitor group(P<0.05). while the expression levels of Bcl-2 (0.54 ± 0.03vs. 0.44 ± 0.02) was significantly increased (P<0.05).Conclusions Quercetin can induce the apoptosis of human colon cancer HT-29 cells and the effective mechanism may relate to the activation of Traf6/TAK1 signaling pathway.
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Objective To study the effect of quercetin on the apoptosis of human colon cancer HT-29 cells and observe the possible mechanism.Methods Human colon cancer HT-29 cells were cultured in vitro and divided into four groups, including the control group, the 16μg/ml quercetin group, the Traf6 inhibitor group and the 16μg/ml quercetin + inhibitor group. The cells in control group were cultured with complete medium and other groups were treated with quercetin or/and Traf6 inhibitor. Flow cytometry was used to observe the impact of quercetin on the apoptosis of HT-29 cells; Western Blot technology was used to detect the expression levels of Traf6, TAK1, p-TAK1, caspase-3, Bax and Bcl-2; RT-PCR was used to investigate the expression level of Traf6 mRNA after treating for 24 h.Results Compared with the 16μg/ml quercetin group, the expression levels of Traf6 (0.59 ± 0.03 vs. 0.96 ± 0.04), p-TAK1 (0.43 ± 0.02vs. 0.72 ± 0.04), caspase-3 (0.59 ± 0.03vs. 0.70 ± 0.04), and Bax (0.48 ± 0.03vs.0.67 ± 0.04) were significantly decreased in 16μg/ml quercetin + inhibitor group(P<0.05). while the expression levels of Bcl-2 (0.54 ± 0.03vs. 0.44 ± 0.02) was significantly increased (P<0.05).Conclusions Quercetin can induce the apoptosis of human colon cancer HT-29 cells and the effective mechanism may relate to the activation of Traf6/TAK1 signaling pathway.
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Objective To establish model of the chicken embryo transplantation of human colon cancer cells ,and investigate the effect of Solanine、VEGF antibody and Solanine combined with VEGF antibody on human colon cancer cells induce tumor angio‐genesis and tumor proliferation .Methods The model of the chicken embryo transplantation of human colon cancer HT‐29 cells were divided into three experimental group and control group .We added to the chick embryo chorioallantoic membrane with Sola‐nine、VEGF antibody and Solanine+ VEGF antibody mixture ,PBS was added to the control group .Then we analysed picture through the stereomicroscope and IPP 6 .0 image analysis software ,using immunohistochemistry envision method to detect of CD34 antigen and ki‐67 antigen ,and observing effect of Solanine group ,VEGF antibody group ,Solanine+ VEGF antibody group and the effect on the tumor angiogenesis and tumor proliferation .Results The tumor angiogenesis ,CD34 antigen and ki‐67 antigen of Sola‐nine+VEGF antibody group were significantly better than those of VEGF antibody group and Solanine group(P<0 .01);VEGF antibody group had statistical significant difference with Solanine group(P<0 .01);the effect of other three groups were better than that of the control group(P<0 .01) .Conclusion Solanine、VEGF antibody and Solanine combined with VEGF antibody could in‐hibit tumor angiogenesis and tumor proliferation of human colon cancer cell line HT‐29 to induce .It provides a new way for anti‐an‐giogenes .
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Objective:To explore the regulatory effects of proliferation and apoptosis on THC-8307 by MMS2 siRNA and P53 siRNA.Methods:We experimentally suppressed the MMS2 and P53 expression in human colon cancer cells by the interference RNA technology ( RNAi) as monitored by Real-time qRT-PCR and Western blot.THC-8307 cells that express rate significantly reduced were collected as case group , while using untreated cells as the blank control group , and mock-treated cells as the negative control group.After separately interfering the target genes in each group ,test the relationship and expression level of the two genes.Utilizing flow cytometry techniques to test cells proliferation and apoptosis rate of each group.Results: Compared to the control group , colon cancer cells in which MMS2 and P53 were silenced displayed significant increase of P53,MMS2 mRNA and protein levels(P<0.05).MMS2-depleted cells displayed increase in apoptosis rates ,for both early and later stages ( P<0.05 ).Conclusion: MMS2 and P53 negatively regulate each other in colon cancer cells proliferation and apoptosis .
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[ ABSTRACT] AIM:To investigate the effects of sphingosine kinase l ( SphK1) and focal adhesion kinase ( FAK) on the epithelial-mesenchymal transition ( EMT) of human colon cancer HCT 116 cells.METHODS:Human colon cancer HCT116 cells were divided into 3 groups.N, N-dimethylsphingosine (DMS) was used to suppress the activity of SphK1. PF573228 was used to suppress the activation of FAK .The cells treated with equal volume of culture medium severed as control group.The cell viability was measured by MTT assay .The protein expression of SphK1, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and matrix metalloproteinase (MMP) 2 was analyzed by Western blot.The mR-NA expression of SphK1, sphingosine-1-phosphate (S1P), FAK, E-cadherin and vimentin was detected by real-time PCR. The ability of tumor cell migration was measured by wound-healing assay.RESULTS:The cell viability of HCT116 cells was suppressed by DMS and PF 573228 in dose and time dependent manners .DMS significantly suppressed the expression of SphK1, FAK, N-cadherin, vimentin and MMP2, meanwhile enhanced the expression of E-cadherin.PF573228 reduced the expression of FAK , SphK1, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin (P<0.01).In addition, the migration ability of HCT116 cells was significantly decreased by treating with DMS and PF573228 (P<0.01).Compared with control group , the mRNA expression of FAK, SphK1, S1P and vimentin was de-creased, while the expression of E-cadherin was increased significantly in PF573228 group and DMS group (P<0.05). CONCLUSION:SphK1 and FAK signaling pathways may play an important role in the occurrence of EMT in the colon cancer HCT116 cells.
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Objective:To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate (EtOAc) extract of Phellinus linteus grown on germinated brown rice (PB). Methods: EtOAc extract of PB was partitioned with n-hexane, EtOAc, and water-saturated n-butanol. Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR, respectively. Cytotoxicity against HT-29 cells was tested by SRB assay. Results: The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line. Atractylenolide I, a eudesmane-type sesquiterpene lactone, a major anticancer substance of PB, was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC. This structure was elucidated by one-and two-dimensional NMR spectroscopic data. Atractylenolide I has not been reported in mushrooms or rice as of yet. The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells. Conclusions:Atractylenolide I might contribute to the anticancer effect of PB.
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The aim of the study was to investigate the inhibitory effects of calcium against intestinal cancer in vitro and in vivo. We first investigated the effects of calcium treatment in HCT116 and HT29 human colon cancer cells. At the concentration range of 0.8-2.4 mM, calcium significantly inhibited cell growth (by 9-29%), attachment (by 12-26%), invasion (by 15-31%), and migration (by 19-61%). An immunofluorescence microscope analysis showed that the treatment with calcium (1.6 mM) for 24 h increased plasma membrane beta-catenin but decreased nuclear beta-catenin levels in HT29 cells. We then investigated the effect of dietary calcium on intestinal tumorigenesis in ApcMin/+ mice. Mice received dietary treatment starting at 6 weeks of age for the consecutive 8 weeks. The basal control diet contained high-fat (20% mixed lipids by weight) and low-calcium (1.4 mg/g diet) to mimic the average Western diet, while the treatment diet contained an enriched level of calcium (5.2 mg calcium/g diet). The dietary calcium treatment decreased the total number of small intestinal tumors (by 31.4%; P or = 2 mm in diameter, showing a 75.6% inhibition in the small intestinal tumor multiplicity (P < 0.001). Immunohistochemical analysis showed significantly reduced nuclear staining of beta-catenin (expressed as nuclear positivity), but increased plasma membrane staining of beta-catenin, in the adenomas from the calcium-treated groups in comparison to those from the control group (P < 0.001). These results demonstrate intestinal cancer inhibitory effects of calcium both in human colon cancer cells and Apc Min/+ mice. The decreased beta-catenin nuclear localization caused by the calcium treatment may contribute to the inhibitory action.
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Animals , Humans , Mice , Adenoma , beta Catenin , Calcium , Calcium, Dietary , Cell Membrane , Cell Transformation, Neoplastic , Colon , Colonic Neoplasms , Diet , Fluorescent Antibody Technique , HT29 Cells , Hydrazines , Intestinal NeoplasmsABSTRACT
BACKGROUND AND AIMS: Interferon-α (IFN-α) treatment is associated with up-regulation of epidermal growth factor receptor (EGFR) expression and marked growth inhibition of colon cancer cell lines. We aimed to determine the effect of combining IFN-α and gefitinib in the growth of human colon cancer cell lines. METHODS: Two human colon cancer cell lines SW480 and LOVO were treated with IFN-α alone or gefitinib alone or IFN-α plus gefitinib. Proliferation of colon cancer cells was measured by methyl thiazolyl tetrazolium (MTT) assay; the apoptosis rate was analysed by flow cytometry (FCM). The expression of XIAP, XAF1 mRNA was detected by RT-PCR and the expression of XIAP, XAF1 protein was detected by western blotting. RESULTS: Methyl thiazolyl tetrazolium showed that IFN-α, gefitinib and IFN-α plus gefitinib significantly inhibited SW480 and LOVO cells in a dose-dependent manner (p < 0.05). The FCM revealed that IFN α, gefitinib and IFN-α plus gefitinib could markedly upgrade the apoptosis rate (p < 0.05). The expression of XIAP mRNA down-regulated markedly (p < 0.05) while the expression of XAF1 mRNA up-regulated significantly (p < 0.05). The expression of XIAP protein was down-regulated markedly (p < 0.05) while the expression of XAF1 protein was up-regulated significantly (p < 0.05). CONCLUSION: IFN-α promotes the antiproliferaative effect of gefitinib on human colon cancer cell lines and the mechanism may be related to up-regulation expression of EGFR by IFN-α.
ANTECEDENTES Y OBJETIVOS: El tratamiento con interferón α (IFN-α) se halla asociado con la regulación por incremento de la expresión del receptor del factor de crecimiento epidérmico y la acentuada inhibición del crecimiento de las líneas celulares del cáncer colorrectal. El presente trabajo tuvo por objetivo determinar el efecto que se produce al combinar el IFN-α y el gefitinib en el crecimiento de las líneas celulares del cáncer de colon. MÉTODOS: Dos líneas celulares de cáncer del colon en humanos - SW480 y LOVO - fueron tratadas con IFN-α solamente, gefitinib solamente, o IFN-α más gefitinib. La proliferación de las células cancerosas del colon se midió mediante ensayo de metil tiazolil tetrazolio (MTT); la tasa de apoptosis se analizó mediante citometría de flujo (CMF); la expresión de XIAP/XAF1 mRNA fue detectada mediante RT-PCR y la expresión de la proteína XIAP/XAF1 fue detectada mediante inmunoblot (western blot). RESULTADOS: El MTT mostró que el IFN-α, el gefitinib, y el IFN-α más gefitinib inhibían de forma significativa las células SW480 y LOVO en dependencia de la dosis (p < 0.05). La CMF reveló que el IFN-α, el gefitinib, y el IFN-α más gefitinib podían aumentar notablemente la tasa de apoptosis (p < 0.05). La expresión de XIAP mRNA tuvo una marcada regulación por decremento (p < 0.05) mientras que la expresión de XAF1 mRNA tuvo una significativa regulación por incremento (p < 0.05); la expresión de la proteína XIAP fue notablemente regulada por decremento (p < 0.05) mientras que la expresión de la proteína XAF1 fue regulada por incremento de manera significativa (p < 0.05). CONCLUSIÓN: El IFN-α promueve el efecto antiproliferativo del gefitinib sobre las líneas celulares del cáncer colorrectal, y el mecanismo puede hallarse relacionado con la expresión de la regulación por incremento del EGFR mediante el IFN-α.
Subject(s)
Humans , Antineoplastic Agents/pharmacology , Colonic Neoplasms/pathology , Interferon-alpha/pharmacology , Quinazolines/pharmacology , ErbB Receptors/antagonists & inhibitors , Apoptosis/drug effects , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Colonic Neoplasms/metabolism , Dose-Response Relationship, Drug , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasm Proteins/metabolism , ErbB Receptors/metabolismABSTRACT
Objective To construct an siRNA expression plasmid against tumor susceptibility gene 101(tsg101) and investigate its effect of RNA interference on oxaliplatin(L-OHP)-resistant human colon cancer cell line HT29/L-OHP.Methods The targeting fragments specifically against tsg101 were designed according to the principle of small interfering RNA designation using an online software.The sequences were cloned into siRNA expression plasmids,and the plasmids were transfected into HT29/L-OHP cells.The expression of tsg101 mRNA after the transfection of TSG101-siRNA plasmid was detected by RT-PCR.The expression of TSG101 after TSG101-siRNA transfection was detected by Western blotting.MTT test was applied to measure the inhibition combined with L-OHP on the proliferation of HT29/L-OHP cells.Distribution of cell cycle was analyzed using flow cytometry after RNA interference to HT29/L-OHP cells.Results The expected fragments were designed,and the TSG101-siRNA plasmid was confirmed by DNA sequencing.The TSG101-siRNA plasmid inhibited the expression of tsg101 mRNA,and the expression of TSG101 protein,and suppressed the proliferation of HT29/L-OHP cells obviously when combined with oxaliplatin.The analysis of cell cycle indicated that the TSG101-siRNA plasmid reduced the cells in G2/M phases and increased the cells in G0/G1 and S phases.Conclusion The expression vector of TSG101-siRNA combined with oxaliplatin inhibits the proliferation of HT29/L-OHP cells and increases the sensitivity to chemotherapy.
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Objective To establish a drug-resistant human colon cancer cell line to oxaliplatin(L-OHP)based on cell line HT29.Methods A resistant cell line-HT29/L-OHP was established by gradually increasing the dose of L-OHP and intermittent administration.The growth curves,multidrug resistance and resistance index of HT29/L-OHP cell line to anticancer agents were detected by MTT assay.The changes of its biological characteristics were determined by light microscopy,electron microscopy,and flow cytometry.Results HT29/L-OHP cell line was established after 3 months,which had stable resistance to L-OHP and a resistance index of 10.64.HT29/L-OHP cells exhibited cross resistance to many other chemotherapeutic agents.As compared with parental cells,the morphological and chromatosome number of HT29/L-OHP were changed;its doubling time was prolonged;and the number of cells in S phase and G0/G1 phase were decreased while in G2/M phase increased.Conclusion HT29/L-OHP cell line shows the typical multidrug-resistant phenotype.