The Effects Of Simvastatin Combined With Paclitaxel On Hippo Signaling Pathway In Breast Cancer Stem Cells

Background: breast cancer is one of the major threats to the health of women in the world,and it is a major cause of the high mortality of breast cancer,because of its low recurrence and poor chemotherapy effect.Breast cancer stem cells(BCSCs)are considered to be the root cause of recurrence,because they can resist the effects of radiotherapy and chemotherapy.There are the characteristics of self-renewal,multilineage differentiation,high tumorigenicity and drug resistance to radiotherapy and chemotherapy of breast cancer stem cells.Breast cancer stem cells become a hot research topic in recent years.Breast cancer stem cells are potential therapeutic targets.Hippo signaling pathway was found to inhibit proliferation and promote apoptosis.However,much evidences suggests that the Hippo signaling pathway can also r egulate stem cell and progenitor cell self-renewal and amplification.Hippo signali ng pathway plays a role in the occurrence and progression.For exmple breast ca ncer,Liver cancer,non small cell lung cancer,Endometrial carcinoma.The Hippo signaling pathway is activated by a kinase cascade,which ultimately acts on its core transcription factor TAZ/YAP,which is blocked by the phosphorylation of TAZ/YAP and 14-3-3 proteinin the cytoplasm by ubiquitin dependent proteasome degradation.No-phosphorylated TAZ/YAP is transferred to the nucleus,and interact with TEAD1-4 or other transcription factors Smad and sp73,in order to induced the expression of CTGF,AX1,BMP4 gene,of the In breast cancer,Hippo signal ing pathway,which based on the core factors,TAZ/YAP was the most obvious.It is more clear that reast cancer stem cells into the ball,self-renewal,differentiati on.Simvastatin is an inhibitor of HMG-Co A.In recent years,it has been shown that simvastatin can inhibit cancer as an anticancer drug.Studies have shown tha t simvastatin can inhibit the translocation of Hippo-TAZ from the cytoplasm to t he nucleus,and thus inhibit the biological role of TAZ in the manipulation of d ownstream transcription factors in the nucleus.Taxol is a new type of anti micro tubule drugs by promoting tubulin polymerization and inhibitiing microtubule dep olymerization and maintaining stability,inhibiting of cell mitosis.As the first-line drug to chemotherapy breast cancer and ovarian cancer,in recent years,the drug efficacy and reduced resistance phenomenon are increased simvastatin can increa se the synergy of paclitaxel on tumor the effects or not have not been reported.In this study,we investigated the effects of simvastatin and paclitaxel on invasion and metastasis of breast cancer cell line MDA-MB-231,EMT and their effects on breast cancer stem cells.Methods: the experiment was divided into four groups: blank treatment group,simvastatin treatment group,paclitaxel treatment group,simvastatin + paclitaxel treatment group.(1): CCK8 detection of tumor inhibition rate of each treatment group after trypsin digestion of MDA-MB-231 cells by 5×103 / 24 h after inoculated,change the culture medium with different concentration gradient(gradient),drug culture 48 h,CCK8 light,light at 37C°2H incubation with eliasa measured OD values Gradpad is used to calculate the drug IC50.Adding two kinds of drugs and IC50 when measured separately before the same concentration gradient(two drugs proportion 1:1)after 48 h,value OD,(2)the change of migration ability of MDA-MB-231 cells was detected by scratch test:MDA-MB-231 cells were cultured in serum-free 48 h in each treatment group,and were photographed at 0h and 48 h respectively.The changes were detected by Transwell MDA-MB-231 apoptosis invasion and metastasis in each treatment group: cells were cultured after 24 h sampling and counting chamber film camera,cell number.Each treatment group by gelatin zymography detection in MDA-MB-231 cells of MMP-2expression.(3)Western blot technique was used to detect the changes of protein levels in MDA-MB-231 cells.Detection indicators include: Hippo signaling critical factor-TAZ,stem cell markers-OCT4,ALDH1.Matrix metalloproteinase-2-MMP-2,Vimentin,E-cadherin,apoptosis related protein Bax,P53,Caspsase3,Bcl-2.The expression of TAZ,OCT4,E-cadherin and Vimentin protein in the cells were also observed by immunocytochemistry.(4)CD44+/CD24-staining was used to detect the proportion of stem cells in each treatment group.(5)stem cells into the ball to test the composition of the ball ability and quantity of each group treatment.(6)after each group of cells treated with 48 h,the same number of cells were cultured in each hole,and after two weeks,the crystal violet was observed.Results:(1)The experimental results showed that simvastatin combined with paclitaxel could inhibit the migration distance,invasion and metastasis.Western blot and gelatinzymography expression and detection of MMP-2 results showed that: compared with the blank control group and single dosing group,simvastatin combined with paclitaxel group MMP-2 expression significantly decreased.(2)The detection of EMT markers were found: compared with the blank control group and single dosing group,simvastatin combined with paclitaxel group increased E-cadherin protein,reduce the protein content of Vimentin in cells.(3)marker detection results of apoptosis related signal pathway showed that: compared with the blank control group and single dosing group,simvastatin combined with paclitaxel group,P53,Bcl-2,Caspsase3 expression decreased,Bax expression was significantly increased,colony formation test also indicate: compared with the blank control group and single dosing group,simvastatin combined with paclitaxel group,significantly inhibited cell proliferation.(4)Western blot and immunofluorescence technique.The results showed that: compared with the blank control group and single dosing group,simvastatin combined with paclitaxel group,the expression of TAZ,OCT4 and ALDH1 protein decreased significantly,(5)Flow cytometry CD44+/CD24-staining was used to detect the percentage of stem cells and stem cells into the ball experiment results show that: the blank control group compared with the single drug group,simvastatin combined with paclitaxel group more obvious inhibition of breast cancer stem cells and its proportion of the ball.Conclusion: simvastatin combined with paclitaxel can inhibit the metastasis and invasion of MDA-MB-231,promote apoptosis and inhibit proliferation.The effect of simvastatin combined with paclitaxel on the proliferation and self-renewal of breast cancer stem cells through down-regulation of Hippo-TAZ and its downstream target Oct4 may provide new ideas for clinical chemotherapy.