The Synergistic Effect Of VD₃ Combined With Metformin Against Human Prostate Cancer Cells DU145 And Its Molecular Mechanism

Objective:The human prostate cancer is one of the most common clinical tumors,which is seriously harmful for people's health.When it progresses to androgen-independent prostate cancer,the treatment is more difficult.Therefore,it is necessary to find a promising clinical therapeutic strategy.Based on the cells are in different cycle phases in a cell line,the action mechanism of anticancer drugs and the cell proliferation kinetics principle,the doctors usually design combination drug scheme to improve the effect of treatment,to delay the drug resistance and to reduce drug toxicity.On the basis of the successful experiences of clinical chemical drug combination treatment,our experiments were to explore the effects of metformin hydrochloride,Vitamin D₃?VD₃?and the combination of two drugs on the growth of human prostate cancer DU 145 cells in vitro and the possible action mechanism of the synergistic effect.Methods:1.MTT method was used to detect the effects of single medicine and the combination of the two drugs on the growth of human prostate cancer DU 145 cells in logarithmic phase in vitro.Firstly we assessed the concentration range of metformin hydrochloride and VD₃,and then the drugs were diluted proportionally within the scope to add in the cells alone or in combination,the inhibition rates were determined at 24 h,48 h and 72 h.So the concentration dependence and time dependence of the two medicines' inhibitory effect alone on cell growth were assessed.The IRs of different concentrations of metformin hydrochloride,VD₃ and the combination groups were calculated respectively.At the same time we calculated coefficient of drug interaction to judge whether the two drugs had synergy effect.The concentration that was the combination of the two drugs with strongest synergy effect was chosen to explore the possible mechanism of synergy in the follow-up studies.Then the experiments were divided into four groups,which were the negative control group,metformin hydrochloride group,VD₃ group and the combination group.2.Hoechst 33342 staining was used to detect cell apoptosis.The cell morphology of the four groups was respectively observed under inverted fluorescence microscope,and then we calculated the apoptosis rates of different groups.3.Flow cytometry was adopted to detect cell cycle phase distribution of the four groups.4.We detected the expression of p-AMPK,p-mTOR,p-S6P,c-Myc and p-Bcl-2 protein by Western Blotting.Results:Both metformin hydrochloride and VD₃ caused a significant concentration-dependent and time-dependent increase in the IR on human prostate cancer DU 145 cells,respectively in 1000-10000 ?g/ml and 400-800 ?g/ml.The combination groups had stronger inhibitory effects.Except for the VD₃ 400 ?g/ml group for 24 h,all the other groups resulted in significant difference compared with the negative control group?P<0.05?.The concentration of metformin hydrochloride 1585 ?g/ml combinined with VD₃400 ?g/ml had the strongest synergy,which was used in subsequent experiments.There was obvious reduction in the total number of cells as well as morphologic changes was concurrent.The apoptosis rate of the conbination treatment groups resulted in significant difference compared with the negative control group?P<0.01?.The combination played a significant role in G1/S cell cycle arrest of the cells.We found there exsited activation of phospho-AMPK with subsequent inhibition of downstream p-mTOR and p-S6P.The changes of the above proteins showed that the direct antiproliferative effects of these treatments were mediated via AMPK/mTOR signaling pathway.The phosphorylation B'cl-2 protein of the combination group decreased significantly,which protects cells from apoptosis,compared with the negative control group,indicating that suppression of p-Bcl-2 expression was related to the increase cellar apoptosis in combination treatment group.At the same time we detected the decreased c-Myc protein,which was also a possible molecular mechanism of synergistic inhibition proliferation effect.Conclusion:These results suggest metformin and VD₃ can synergismly inhibit proliferation,cause cell cycle G1/S arrest and induce apotosis in DU 145 due to increasing p-AMPK and reducing p-mTOR,p-S6P protein by AMPK/mTOR signaling pathway.This can also reduce expression of oncogenic protein c-Myc.Combine treatment of the two drugs has synergistic inhibition on DU145,which supplies a valued choice for AIPC.