| Breast cancer is a common malignant tumor in women,accounting for a quarter of all malignant tumors in the body,and the incidence is increasing year by year.According to biological behavior,molecular and clinicopathological characteristics,breast cancer can be divided into many subtypes.Triple negative breast cancer(TNBC)refers to the estrogen receptor(ER),erogesterone receptor(PR)and human epidermal growth factor receptor 2(HER-2)all express negative subtypes of breast cancer.TNBC is characterized by high tumor heterogeneity,strong invasiveness,high recurrence rate,rapid distant metastasis and poor prognosis.Due to the negative expressions of ER and PR and the lack of HER-2 over expression,TNBC has not responded effectively to trastuzumab and other relevant targeted therapies.Currently,chemotherapy is the main treatment for TNBC,but it cannot resist drug resistance of TNBC.Studies have demonstrated that insulin-like growth factor 1 receptor(IGF-1R)can play a role in mitogenicity when activated,which has a profound impact on cell atypia and tumor differentiation.Therefore,interference with IGF-1R mediated signaling pathways may be a new option for treatment of TNBC.Picropodophyllin(PPP)is a kind of IGF-1R tyrosine kinase inhibitor.Related studies have shown that PPP significantly inhibits the proliferation of colon cancer,ovarian cancer,acute lymphoblastic leukemia and other tumor cells.However,since there is no relevant report on the effect of PPP on TNBC cells,this study explored the correlation between the IGF-1R inhibitor PPP and the proliferation of MDA-MB-231 cells,we hope that we could provide the experimental evidence for the treatment of malignant breast cancer with PPP.Part I Effect of picropodophyllin on proliferation and cell cycle of MDA-MB-231 cells in triple negative breast cancerObjective:In this study,the human TNBC cancer MDA-MB-231 cell line was taken as the experimental object to investigate the effect of PPP on the proliferation and cell cycle of TNBC MDA-MB-231 cells,providing experimental basis for the development of PPP as a drug for the treatment of TNBC.Methods:1.MTT was used to detect the effect of PPP on the proliferation ability of MDA-MB-231 cells with different concentrations(0.2?mol/L,0.4?mol/L,0.8?mol/L,1.6?mol/L,3.2?mol/L,6.4?mol/L)and different time(24h,48 h,72h).PPP solutions with concentrations of 0.4?mol/L,0.8?mol/L,1.6?mol/L were selected for subsequent experiments.2.Inverted microscope was used to observed the changes in cell morphology,structure and number of MDA-MB-231 cells under different concentration PPP for 48 h.3.Flow cytometry was used to detect the effects of PPP on the cell cycle of MDA-MB-231 cells after 48 h.4.Western blot was used to detect the expression of Cyclin D1 and PCNA in MDA-MB-231 cells treated with PPP at different concentrations for 48 h.5.SPSS 19.0 was used for statistical analysis of experimental data.Results:1.MTT showed that PPP significantly inhibited the proliferation activity of MDA-MB-231 cells,and showed a time-dose dependence.2.Observation under an inverted microscope showed that the cellswas treated with PPP,compared with the control group,the cells lost their original epithelial-like morphology and shrank back to a circular state with decreased refractive index,and the number of cells decreased significantly.3.Flow cytometry showed that MDA-MB-231 cells treated with PPP after 48 h,the proportion of cells in S phase increased significantly,G0/G1 phase decreased,and G2/M phase did not change significantly.4.Western Blot showed that the protein expression levels of PCNA and Cyclin D1 could be significantly decreased after PPP treatment for48 h in MDA-MB-231 cells.Conclusion:PPP can effectively inhibit the proliferation of human TNBC MDA-MB-231 cell line and induce tumor cells to block in S phase,which may be related to the down-regulation of PCNA and Cyclin D1 protein expression levels.Part II The effect of picropodophyllin on mda-mb-231 cells and its relationship with IGF-I signaling pathwayObjective:Based on the results of the first part,we investigated the correlation between inhibition of picropodophyllin on the proliferation of mda-mb-231 cells and IGF-IR regulated cell signaling pathways.Methods:1.According to the relevant results of the first part,the action time was selected as 48 h,and the concentration of PPP was 0.8?mol/L,the concentration of IGF-1 was 100ng/ml.The control group,IGF-1 group,PPP group and(IGF-1+PPP)group were treated for 48 h,and cell proliferation activity was detected by MTT.2.After 48 h,the IGF-1 group,PPP group and(IGF-1+PPP)group,protein expression levels of IGF-1R and p IGF-1R were detected byWestern Blot.3.SPSS 19.0 statistical software was used to analyze the experimental data.Results:1.MTT results showed that the cell proliferation rate of IGF-1 group increased significantly compared with the control group.The proliferation rate of in PPP group and IGF-1+PPP group was significantly decreased.There was no significant difference in cell survival rate between the PPP group and the IGF-1+PPP group.2.Western Blot results showed that the protein expression level of p IGF-1R in IGF-1 group was significantly up-regulated compared with the control group.The protein expression of p IGF-1R in PPP group and IGF-1+PPP group was significantly down-regulated,and there was no significant difference between PPP group and IGF-1+PPP group.There was no significant difference in IGF-1R protein expression in the control group,IGF-1 group,PPP group and(IGF-1+PPP)group.Conclusion:PPP can antagonize the effect of IGF-1 on promoting cell proliferation,and can affect cell proliferation by lowering the protein expression of p IGF-1R,suggesting that PPP can block IGF-1 cell signaling pathways by inhibiting the high expression of p IGF-1R. |
PDF Full Text Request