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Background:Ulcerative colitis-associated colorectal cancer(UcCRC)is the most serious complication of ulcerative colitis(UC). 5-Aminosalicylic acid(5-ASA)could reduce the risk of UC progressing to UcCRC. Aims:To investigate the effect of 5-ASA on cell proliferation,apoptosis and cell cycle in human colon cancer cell line HT-29 for verifying the inhibitory effect of 5-ASA on UcCRC. Methods:HT-29 cells were treated with different concentrations(0,10,20,30, 40 mmol/ L)of 5-ASA. Cell proliferation was measured by CCK-8 assay. Cell apoptosis was determined by TUNEL method. Cell cycle was analyzed by flow cytometry. Expressions of cell mitotic regulators AuroraB and BubR1 were determined by immunohistochemistry. Results:Proliferation inhibition rates and apoptosis rates of HT-29 cells in 5-ASA 10,20,30,40 mmol/ L groups were increased with increase in concentration of 5-ASA(P ﹤ 0. 05). Proportions of cells in phase G0 / G1 in 5-ASA 30,40 mmol/ L groups were significantly lower than those in 5-ASA 0,10,20 mmol/ L groups (P ﹤ 0. 05);proportions of cells in phase S in 5-ASA 0,10,20,30 mmol/ L groups were significantly lower than that in 5-ASA 40 mmol/ L group(P ﹤ 0. 05);while proportions of cells in phase G2 / M in 5-ASA 30,40 mmol/ L groups were significantly higher than those in 5-ASA 0,10,20 mmol/ L groups(P ﹤ 0. 05). Mean optical density(MOD)values of AuroraB and BubR1 were decreased with increase in concentration of 5-ASA(P ﹤ 0. 05). Concentration of 5-ASA was positively correlated with proliferation inhibition rate and apoptosis rate of HT-29 cells(P ﹤ 0. 05),but was negatively correlated with MOD values of AuroraB and BubR1( P ﹤ 0. 05). MOD values of AuroraB and BubR1 were negatively correlated with proliferation inhibition rate and apoptosis rate of HT-29 cells as well as proportion of cells in phase G2/ M (P ﹤0. 05),but were positively correlated with proportion of cells in phase G0/ G1(P ﹤0. 05). MOD value of AuroraB was positively correlated with MOD value of BubR1(P ﹤ 0. 05). Conclusions:5-ASA can inhibit proliferation and induce apoptosis in HT-29 cells,the mechanism may be related to inhibiting expressions of AuroraB and BubR1 and the resulted effect on cell cycle.
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Objective To explore the effects of silencing Livin gene by RNA interference mediated by lentiviral vector on colorectal cancer HT-29 cell xenograft growth and sensitivity to radiotherapy in nude mice.Methods BALB/c nude mice models were established by subcutaneously inoculating differently treated HT-29 cells into nude mice and the tumor growth situation of tumors was observed by measuring the volume of tumors and the weight of the nude mice at different time points after cell seeding.Livin expression was detected by RT-PCR and immunohistochemistry,respetively.Apoptosis rate was detected by TUNEL.Normal saline,lentivirus carring unrelated sequences,lentivirus caning Livin shRNA were injected intratumorally.All the nude mice were given 10 Gy of 6 MV X-ray irradiation.The changes of mice weight and the tumor volume were measured at different time points and the weight and tumor growth curves were drawn.Results The inhibition rate of tumor volume was(50.04 ± 0.07)％ and the tumor weight of the RNA interfering group was significantly less than that in experimental group compared to the blank and negative groups(F=4.85,P＜0.05),and the inhibition rate of tumor weight was(50.27 ±0.17)％.Relative Livin mRNA expression level in the RNA interfering experimental group was(17.75 ±0.08)％,and was significantly lower than that of the blank group(67.60 ± 0.05)％ and the negative group(68.54 ± 0.03)％(F=89.97,P＜0.01).Livin protein expression level in the RNA inferring group was also significantly lower[(36.00 ± 3.40)％ versus(85.00 ± 3.15)％,(80.33 ± 3.08)％,F=107.32,P＜0.01].The apoptosis rate in the RNA interfering experimental group was significantly higher than that in the blank and the negative groups[(23.67 ± 2.25)％ versus(5.00 ± 1.50)％,(8.33 ± 1.82)％,F=56.94,P＜0.01].Combined with radiotherapy,the tumor volume at different groups had significant difference(F=10.70,P＜0.01),and RNA interfering group was significantly less than negative group and blank group(F=7.01-9.32,P＜0.01).Conclusions Silencing of Livin gene expression by lentiviral vector-mediated RNA interference could inhibit the growth of colorectal HT-29 cell xenograft and increase the sensitivity of the transplanted tumors to radiotherapy.
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Objective To study the effect of a selective COX-2 inhibitor Celecoxib on cell proliferation and apoptosis of human coloretal cancer cell line HT-29 to seek an effective and safe drug for colon cancer chemoprevention. Methods Using MTT assay, flow cytometry(FCM), acridine orange and ethidium bromide staining, the effect of celecoxib on the proliferation and apoptosis of HT-29 cells were investigated. Results The growth of HT-29 cells was inhibited by celecoxib in a dose- and time- dependent manners. FCM analysis showed that the treated HT-29 cells had typical Sub-G 1 peak, the apoptotic rate of which was (7 31?2 37)%～(48 3?2 86)%. The cell ratio of G 0/G 1 phase increased, whereas the cell ratio of S and G 2/M phases decreased after treatment, which was in a dose-dependent manner as well. The treated HT-29 cells exhibited some morphological features of apoptosis, including cell shrinkage, nuclear condensation, DNA fragmentation, and the formation of apoptosis bodies, the apoptotic index of which was in a dose- and time- dependent manners. Conclusions Celecoxib inhibited proliferation and induced apoptosis of human colorectal cancer cell line HT-29, which may be related to blocking the cell cycle progress of HT-29 cells.
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Objective To investigate the effects and mechanisms of inositol hexaphosphate on diffe-rentiation of HT-29 human colon carcinoma cell line. Method Cells were exposed to various concentrations (control,1.8 mmol/L,3.3 mmol/L) of IP6 for different time (1d,3d,5d). Its effects on the cells were evaluated by detecting follow indices:(1) Observing ultrastructure of HT-29 cells by transmission electron microscopy. (2) Detecting alkaline phosphate (ALP) activity to evaluate cell differentiation. (3) Expression of c-Myc mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR). (4) Immunocytochemical stain was used to detect expression of Rb protein. Results (1) After having been treated at 3.3mmol/L concentration,the cell structure changed,and appeared tendency of differentiation,such as karyopyknosis,abundant cellular organs and microvillus increase. (2) The activity of alkaline phosphate increased along with the concentration and time. (3) RT-PCR showed that different concentrations of IP6 decreased the expression of c-Myc mRNA. (4) The immunocytochemical stain showed that different concentrations of IP6 increased expression of Rb protein. Conclusion IP6 can induce differentiation of HT-29 cell by augmenting expression of Rb,decreasing expression of c-Myc and blocking cell cycle.
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Objective: To investigate the effects and mechanisms of inositol hexaphosphate(IP6)on cell cycle of HT-29 human colon carcinoma cell line. Method: HT-29 cells were exposed to various concentrations of IP6 for certain time. The effect of IP6 on cells proliferation was evaluated by MTT assay. Flow cytometric analysis was performed for cell cycle progression. Immunocytochemical stain was used to detect the expression of p53 and p21 protein. Results: A significant dose-dependent as well as time-dependent growth inhibition was observed in IP6-treated HT-29 cells, and associated with an increase in G1 arrest; Different concentrations of IP6 decreased the abnormal expressions of p53 gene and strongly increased the expression of p21. Conclusions: IP6 had an inhibitory effect on proliferation of HT-29 cells .and its mechanisms might be related to many factors, such as inhibiting the abnormal expression of p53 gene, inducing the expression of cell cycle inhibitor p21 which block the cell cycle.