Zinc And P53 Induce Apoptosis In Prostate Cancer Cells Through Mitochondrial HK2

Backgroud:Metabolic reprogramming and apoptosis escape are two main characteristics of tumor cells.It has been found that some metabolic enzymes and apoptotic regulatory factors play an important role in regulating apoptosis through their structural or functional interactions.The interaction of tumor cell oncogenes,tumor suppressor genes,microelements in the microenvironment and other factors affect the expression or function of metabolic enzymes.This suggested that exploring the role of metabolic enzymes in the regulation of tumor cell apoptosis may be helpful to further elucidate the anti-tumor mechanism of tumor suppressor.In recent years,hexokinase 2（HK2）has attracted much attention due to its dual roles in metabolism and apoptosis regulation.HK2 expression was significantly increased in tumor cells.In tumor cells,HK2 binds to the voltage-dependent anion channel（VDAC）on the mitochondrial outer membrane by N-terminal binding domains.This suggests that HK2 is not only involved in the glycolysis of tumor cells,but also that mitochondrial localization of HK2 may be an intervention target for tumor therapy.Many factors play a central and pivotal role in regulation of the association of HK2 with VDAC.It was found that glycogen synthase kinase 3β（GSK3β）phosphorylates the specific HK2-docking site（Thr51）on VDAC,thereby disrupting the binding of HK2 to VDAC.Other studies have shown that protein kinase B（PKB/Akt）phosphorylates GSK3βon Ser9,resulting in inhibition of kinase activity.Therefore,we hypothesized that the Akt-GSK3β-VDAC pathway is involved in the regulation of HK2 mitochondrial localization.Prostate cancer（PCa）is the most common urological malignancy.Zinc concentration is high in healthy prostate,but significantly decreased in prostate cancer.Intracellular zinc levels were negatively correlated with prostate cancer progression.Many studies have shown that zinc can induce apoptosis in prostate cancer cells.It also has been found that zinc increased the sensitivity of lung cancer cells to apoptosis by down-regulating Akt,and zinc can also reverse the drug resistance of temozolomide in glioblastoma by inhibiting Akt.Therefore,we speculated that zinc-induced apoptosis might be mediated by Akt signaling pathway.Tumor suppressor p53 regulates both cell apoptosis and cell metabolism.Studies have found that HK2 promoter contains functionally active response elements for p53,so HK2 may be an important downstream molecule of p53 in regulating metabolism and apoptosis.Recent studies have found that p53 induces apoptosis by inhibiting Akt.In addition,it has been shown that zinc can not only increase p53-induced apoptosis,but also affect cell metabolism by changing the expression of metabolic enzymes,including HK2.This suggests that zinc and p53 may not only regulate the expression of HK2,but also induce apoptosis by affecting mitochondrial localization of HK2 via the Akt pathway.In this study,human prostate cancer cell lines and xenograft models of prostate cancer were selected as the research subjects.From the perspective of regulating cell apoptosis by metabolic enzymes in tumor cells,mitochondrial localization of HK2was taken as the entry point to explore the mechanism of tumor inhibition by zinc and p53,so as to provide new ideas and strategies for the treatment of prostate cancer.Method:（1）To observe the effects of zinc and p53 on HK2 mitochondrial localization and the role of Akt and GSK3βin this process.Prostate cancer cells were transfected with Pmp53 for 6h,and/or treated with 250μM ZnCl₂ for 24h.The mitochondrial localization of HK2 and its co-localization with VDAC1 were detected by immunofluorescence.The protein interaction between HK2 and VDAC1 was detected by immunoprecipitation.The phosphorylation level of VDAC1 was detected by immunoprecipitation and Western blotting.Phosphorylation levels of Akt and GSK3βwere detected by Western blotting.（2）To observe the effects of zinc and p53 on cell proliferation and apoptosis.Prostate cancer cells were transfected with Pmp53 for 6h,and/or treated with 250μM ZnCl₂ for 24h.The cell viability was detected by MTT assay.Western blotting was used to detect the expression levels of anti-apoptotic and pro-apoptotic proteins in bcl-2 family.The mitochondrial membrane potential was detected by flow cytometry with JC-1 staining.The protein expression level of cytochrome C in the cytoplasm was detected by Western blotting.Annexin V/PI staining flow cytometry was used to detect apoptosis rate.Protein expression levels of cleaved caspase-3 were detected by Western blotting.Nuclear morphological changes were detected by Hoechst33342staining.（3）To investigate the effects of zinc and p53 in prostate cancer cells xenograft models.A nude mouse xenograft model of prostate cancer cell DU145 was established.The tumor-targeted attenuated Salmonella（Ty21a）carrying the co-expression plasmid Pmp53 was injected into the tail vein,and zinc was treated with gavage for 1 month.The tumor growth curve was recorded.After sacrifice,tumor quality was recorded.Western blotting was used to detect the protein expression levels of cleaved-caspase3 and Bcl-2 family proteins.（4）To observe the effect of zinc and p53 on HK2 expression.Prostate cancer cells were transfected with Pmp53 for 6h,and/or treated with 250μM ZnCl₂ for 24h.The expression level of HK2 mRNA was detected by qPCR.Western blotting was used to detect the protein expression level of HK2.The protein expression of HK2and p53 in 85 prostate cancer patients was observed by immunohistochemical staining,and the correlation between them was analyzed by Spearman rank correlation analysis.Western blotting was used to detect the protein expression levels of p53 and HK2 in prostate cancer cell xenograft tissues.（5）To observe the effects of zinc and p53 on glycolysis and oxidative phosphorylation.Prostate cancer cells were transfected with Pmp53 for 6h,and/or treated with 250μM ZnCl₂ for 24h.Glucose consumption,lactate secretion,cell oxygenation rate（OCR）,extracellular acidification rate（ECAR）and ATP levels were detected.Result:（1）In prostate cancer cells DU145 and PC3,zinc and p53 reduced HK2 binding to VDAC1.The phosphorylation level of VDAC1 increased,while the phosphorylation level of Akt and GSK3βdecreased.Zinc and p53 have a synergistic effect.（2）In prostate cancer cells DU145 and PC3,Zinc and p53 inhibited cell proliferation.After zinc and Pmp53 treatment,the anti-apoptotic proteins bcl-2 and Mcl-1 in mitochondria were decreased,while the pro-apoptotic proteins Bax and Bak were increased.Mitochondrial membrane potential decreased and cytochrome C release increased.The cells apoptosis rate increased.Cleaved caspase-3 expression was increased.Hoechst staining increased and nuclear fragmentation occurred.Zinc and p53 have a synergistic effect.（3）In the xenograft model of prostate cancer,compared with the control group,the proliferation of the tumor in the zinc-treated group and Pmp53-treated group were inhibited,and the inhibition effect was more obvious in the combination treatment group.After the mice were sacrificed,the tumors were weighed.The quality of the tumors in the zinc-treated group and Pmp53-treated group were decreased.And the decrease in the combination group was the more obvious.In tumor tissues,the protein expression levels of Bax,Bak and cleaved-caspase3 were increased in the zinc-and Pmp53-treated groups compared with the control group,and the combination treatment group was further increased.However,the protein expression levels of Bcl-2 and Mcl-1 decreased,and the combination treatment group decreased further.Zinc and p53 have a synergistic effect.（4）In prostate cancer cells DU145 and PC3,zinc and p53 increased HK2expression at mRNA and protein levels.In prostate cancer patients,the expression of p53 protein in cell nucleus is significantly correlated with the expression of HK2protein.In the xenograft model of prostate cancer,HK2 protein expression was increased after treatment with Pmp53 and zinc.Zinc and p53 have a synergistic effect.（5）In prostate cancer cells DU145 and PC3,zinc and p53 reduced glucose consumption,lactate secretion and ATP levels,and inhibited glycolysis and oxidative phosphorylation.Conclusion:1.In vitro and in vivo experiments found that zinc combined with p53 induced mitochondrial apoptosis.Zinc and p53 have synergistic inhibitory effect on prostate cancer.2.In prostate cancer cells,zinc and p53 inhibit phosphorylation of AKT,resulting in a decrease in phosphorylation of GSK3β,but its activity is enhanced.Increased activity of GSK3βincreases phosphorylation of VDAC1,resulting in dissociation of HK2 from VDAC1.After dissociation with HK2,VDAC interacts with Bcl-2 family proteins,leading to changes in mitochondrial membrane permeability,increased release of cytochrome C,and finally induced apoptosis.3.Zinc and p53 increased HK2 mRNA and protein expression,but inhibited glycolysis and oxidative phosphorylation in prostate cancer cells.These results suggest that increased HK2 expression may not be sufficient to reverse inhibited glucose metabolism in prostate cancer cells after zinc and p53 treatment.In conclusion,zinc and p53 have synergistic tumor inhibition effects in vivo and in vitro.Zinc and p53 reduce HK2 mitochondrial localization through the Akt-GSK3β-VDAC pathway,leading to changes in mitochondrial membrane permeability and ultimately inducing mitochondrial apoptosis.HK2 mitochondrial localization may be more critical in determining cell fate than its protein expression.Therefore,zinc and p53 interfering with the interaction between VDAC and HK2 to reduce the mitochondrial localization of HK2 may provide a new strategy for the treatment of prostate cancer.