P53-PGC-1? Mediated Mitochondrial Dysfunction Promotes Apoptosis Of PC3 Prostate Cancer Cells

Prostate cancer seriously threatens the health of the majority of men.At present,castration resistance of prostate cancer has become a major problem in clinical treatment.Therefore,finding new anti-cancer targets based on the biological characteristics of prostate cancer is of great significance for the treatment of prostate cancer.Previous studies showed that normal prostate epithelial cells are primarily provide ATP by glycolysis,whereas prostate cancer cells are mainly provide ATP by oxidative phosphorylation.Mitochondria are the main site of oxidative phosphorylation,which is abundant in prostate cancer cells.This study aims to identify therapeutic targets with mitochondria as the core and provide an important theoretical basis for the development of new anticancer drugs.Maintenance of mitochondrial function is dependent on intracellular mitochondrial quality control,which includes: mitochondrial biogenesis,mitochondrial fission/fusion,and mitochondrial autophagy.Studies have shown that peroxisome proliferator-activated receptor gamma coactivator-1alpha(PGC-1alpha)is a major regulator of mitochondrial function.It can bind to a variety of transcription factors with histone acetyltransferase activity in the nucleus,thereby regulating cellular processes such as mitochondrial biogenesis and mitochondrial fission/fusion.Nuclear Respiratory Factor(NRF1)is the main transcription factor downstream of PGC-1alpha,which promotes mitochondrial gene copy by activating mitochondrial transcription factor(TFAM).The PGC-1?/NRF1/TFAM signaling pathway is a classical pathway for mitochondrial biogenesis and a major source of intracellular mitochondria.In addition,studies have reported that PGC1-? in human muscle can transcriptionally promote the expression of mitochondrial fusion protein Mfn2 after exercise;PGC1-? can also transcriptionally promote the expression of mitochondrial fusion protein Mfn1 during cardiac development in mice.Furthermore,PGC1-alpha may also be involved in the regulation of dynamin related protein 1(DRP1).Taken together,PGC-1? is involved in the regulation of mitochondrial biogenesis and fission/fusion to maintain normal mitochondrial function.It has been reported that PGC-1? is highly expressed in prostate cancer cells compared with normal prostate epithelial cells,and promotes the growth of prostate cancer cells by activating androgen receptor(AR).However,whether the regulation of mitochondrial function by PGC-1? plays a role in promoting cancer in prostate cancer cells remains to be explored.The tumor suppressor p53 is a classical tumor suppressor,and mutations or deletions of p53 are present in more than 50% of tumor cells including prostate cancer cells(e g,PC3 and DU145).In recent years,the regulation of p53 on tumor cell metabolism has attracted the attention of scientists.It has been found that p53 inhibits the aerobic glycolysis(Warburg effect)and promotes oxidative phosphorylation in most tumor cells to exert a tumor suppressor effect.However,in the prostate cancer cells with oxidative phosphorylation as the main energy source,the mechanism of p53 interfering with energy metabolism and affecting tumor cell survival needs to be further studied.In summary,the three biological characteristics of prostate cancer cells include:(1)abundant mitochondria and supply energy by oxidative phosphorylation;(2)high expression of PGC-1alpha;(3)p53 mutation or deletion exists in most cancer cells.PGC-1alpha is an important safeguard for maintaining mitochondrial function.Is there any correlation between p53 and PGC-1alpha or mitochondrial function? A number of studies have demonstrated that PGC-1? is expressed at high levels in p53-mutant or p53-deleted tumor cells and plays a role in cancer progression.For example,Ogasawara et al.found that PGC-1?,which is highly expressed in p53-deficient chronic lymphocytic leukemia cells,maintains normal mitochondrial function.In addition,the R72 variant of mutant p53 promotes tumor metabolism and metastasis by enhancing the function of PGC-1?.These findings suggest that the deletion or mutation of p53 may contribute to the enhancement of PGC-1? expression and function.It has been reported that p53 can damage the mitochondrial function of Hep G2 cells,but the detailed mechanism is not elucidated.Given the above background,the p53-PGC-1? pathway may be involved in the regulation of mitochondrial function.In order to confirm that p53 deletion is the main cause of high expression of PGC-1alpha and enhancement of mitochondrial function,we used transient transfection technology to transfer exogenous p53 into p53-deleted prostate cancer PC3 cells.Cell experiments were used to observe the effect of p53 on mitochondrial function of prostate cancer PC3 cells,and to detect the expression of PGC-1alpha and its downstream mitochondrial biogenesis and mitochondrial fission/fusion-related proteins.Finally,the effect of p53/PGC-1alpha pathway on apoptosis was detected.Experimental method:(1)Western blot and indirect immunofluorescence techniques were used to detect the transfection efficiency of p53 in prostate cancer PC3 cells.(2)The effect of p53 on the oxygen consumption rate of prostate cancer PC3 cells was detected by OCR;Mitotracker-RED staining was used to detect the effect of p53 on mitochondrial mass in prostate cancer PC3 cells.(3)Western blot was used to detect the effect of p53 on the expression of PGC-1 alpha in PC3 cells of prostate cancer;indirect immunofluorescence was used to detect the effect of p53 on the nuclear localization of PGC-1 alpha in PC3 cells of prostate cancer;MTT was used to detect the effect of ZLN005,an activator of PGC-1 alpha,on the survival rate of PC3 cells of prostate cancer;Western blot was used to detect the effect of ZLN005 on the expression of PGC-1 alpha in PC3 cells of prostate cancer;The effect of ZLN005 on the oxygen consumption rate of each group was firstly detected by OCR.Mitotracker-RED staining was used to detect the effect of ZLN005 on mitochondrial mass of each group.(4)q-PCR was used to detect the expression of genes downstream of PGC-1a involved in the regulation of mitochondrial biogenesis and mitochondrial fission/fusion.Western blot was used to detect the expression of proteins downstream of PGC-1a involved in mitochondrial biogenesis and mitochondrial fission/fusion.(5)Flow cytometry was used to detect apoptosis and Western blot was used to detect the expression of apoptosis-related proteins.Results:(1)Compared with negative control(nc),p53 group successfully overexpressed p53,and its transfection efficiency reached 70%.(2)Compared with negative control(nc),the oxygen consumption rate and mitochondrial mass in p53 group decreased.(3)Compared with negative control(nc),p53 induced a decrease of PGC-1? protein expression and nuclear localization;the optimal dose of ZLN005 was 15 u M;Compared with p53 group,the oxygen consumption rate and the mitochondrial mass in ZLN005+p53 group increased.(4)Compared with p53 group,the expression of mitochondrial biogenesis and mitochondrial fission/fusion-related genes was increased in ZLN005+p53 group,and the corresponding protein of each gene was highly expressed.(5)Compared with p53 group,the apoptotic rate and the expression of apoptotic related proteins in ZLN005+p53 group decreased.Conclusion: p53 reduces the expression of mitochondrial biogenesis and fission/fusionrelated genes by inhibiting PGC-1?,resulting in mitochondrial dysfunction and apoptosis of PC3 cells.