Studies Of The Anticancer Effect Of Picropodophyllin And Its Influence On IGF-I System To Human Colorectal Cancer HCT-15 Cells

Colorectal cancer is one of the most common malignant tumors of digestive system with high mortality in the world,which has been a serious threat to human health.According to current studies,it is agreed that the occurrence and development of tumor is not a single molecular event,but a pathological process in which multiple factors interact,and the process is extremely complex and changeable.Aging of the population,genetic factors,increased risk factors?such as obesity,smoking,drinking?and adverse eating habits are closely related to the occurrence and development of colorectal cancer.In recent years,with the rapid development of economy and the improvement of living standards,compared with the dietary structure decades ago,compared with the diet structure decades ago,there are many changes now.The incidence and mortality of cancer are also increasing year by year,and the population with cancer tends to be younger.Therefore,improving survival is of great significance for the delay of colorectal cancer patients in the effective antitumor therapy.However,due to the severe side effects and poor tolerance of traditional chemotherapeutic drugs,the expected effect could not be achieved.Picropodophyllin is a natural active substance isolated and purified from the Chinese medicinal plant Podophyllum emodi Wall.Previous studies have shown that picropodophyllin can significantly inhibit the proliferation of ovarian cancer,breast cancer,glioblastoma and uveal melanoma.However,the role of picropodophyllin in the treatment of colorectal cancer has not been reported.In this study,we use human colorectal cancer HCT-15 cells as the research object,to explore the effect of PPP on the proliferation and cell cycle of colorectal cancer HCT-15 cells.Further analysis,whether PPP plays an antitumor role is related to the regulation of IGF-I signaling pathway.In order to provide evidence for better treatment of colorectal cancer.Part ? Effect of picropodophyllin on proliferation and cell cycle of human colorectal cancer HCT-15 cellsObjective:In this study,we use human colorectal cancer HCT-15 cells as the research object,to explore the effect of PPP on the proliferation and cell cycle of colorectal cancer HCT-15 and its possible mechanism.Methods:1.Colorectal cancer HCT-15 cells were treated with different concentrations of PPP for 24h?48h and 72h,and the effect of PPP on the proliferation of HCT-15 cells was detected by CCK-8 assay.2.Colorectal cancer HCT-15 cells were treated with different concentrations of PPP for 48h,morphological changes and the cell number of HCT-15 cells were observed under inverted microscope.3.Colorectal cancer HCT-15 cells were treated with different concentrations of PPP for 48h,and the changes of HCT-15 cell cycle were detected by flow cytometry.4.Colorectal cancer HCT-15 cells were treated with different concentrations of PPP for 48h,and the expression levels of PCNA and CyclinD1 protein was detected by Western Blot.5.Experimental data were statistically analyzed by SPSS 19.0 software.Results:1.The results of CCK-8 assay showed that picropodophyllin could significantly inhibit the proliferation of HCT-15 cells with dose-time dependent.2.Under inverted microscope,it was found that the cells lost their original shape and retracted into a circle,the refractive index decreased,and the number of adherent growth cells decreased significantly.3.Flow cytometry showed that the proportion of G₂/M phase cells was significantly increased.The proportion of G₀/G₁ and S phase cells was significantly decreased.4.Western blot results showed that the expression of PCNA,CyclinD1protein was significantly decreased.Conclusion:PPP can significantly inhibit the proliferation of human colorectal cancer HCT-15 cells.It is speculated that the mechanism of the inhibition may be through down-regulating the expression of PCNA,CyclinD1 protein,and then blocking the cells in G₂/M phase to play an anti-tumor effect.Part ? The effect of picropodophyllin on the growth of human colorectal cancer HCT-15 cells and its relationship with IGF-I signaling pathwayObjective:According to the results of the first part,we further analyzed whether the inhibitory effect of PPP on human colorectal cancer HCT-15 cells was related to the regulation of IGF-I signaling pathway.Methods:1.The proliferation activity of HCT-15 cells was detected with control,IGF-I,PPP,?IGF-I+PPP?treatment for 48h by CCK-8 assay.2.The morphological changes of HCT-15 cells was observed with control,IGF-I,PPP,?IGF-I+PPP?treatment for 48h by inverted microscope.3.The expression levels of IGF-IR and pIGF-IR protein was detected with control,IGF-I,PPP,?IGF-I+PPP?treatment for 48h by Western Blot.4.Experimental data were statistically analyzed by SPSS 19.0 software.Results:1.The results of CCK-8 showed that compared with control group,the cell survival rate in IGF-I group was significantly increase,while that in PPP group and?IGF-I+PPP?group was significantly decrease.There was no difference in cell viability between PPP group and?IGF-I+PPP?group.2.Under the inverted microscope,it was found that the cells in the group control were well attached,the cells in the IGF-I group were dense adherent growth and the number of cells was the highest.The cells in the PPP group and?IGF-I+PPP?groups were severely restricted in growth,showing that some of the cells retracted or even ruptured in abnormal cell morphology.3.The results of Western Blot showed that compared with the control group IGF-I group could significantly up-regulate the expression of pIGF-IR protein,and both PPP group and?IGF-I+PPP?group could turn down the expression of pIGF-IR protein,and there was no difference between PPP group and?IGF-I+PPP?group.There was no difference in the expression of IGF-IR protein among the four groups.Conclusion:PPP can antagonize the effect of IGF-I on cell proliferation.At the protein molecular level,it can also decrease the high expression of pIGF-IR induced by IGF-I which indicates that PPP can regulate the signal pathway of IGF-I.