| ObjectiveProstate cancer(PCa)is one of the most common malignant tumors of the male reproductive system.Currently,the commonly used PCa-treatment drugs are androgen receptor antagonists such as bicalutamide and enzalutamide.Inevitably,the patient develops drug resistance for long-term administration,and the increasing-dose enlarge the side effects of drugs.Therefore,we should keep on finding high-efficiency and low-toxicity prostate cancer treatment drugs.The cellular physiology of endoplasmic reticulum(ER)is mainly the synthesis,process and transportation of protein and lipid.When misfolded or unfolded proteins accumulate in ER organelle,the cell activates the endoplasmic reticulum stress(ER stress),following triggers unfolded protein response(UPR)pathway.UPR contains three pathways: IRElα,ATF6 and PERK pathway to remove the accumulation of these "waste proteins".The ER stress response is an important physiological process of balancing cell microenviroment homeostasis.Short-term and low-level ER stress is benefit to cell survival,but long-term high-intensity ER insult can deteriorate the process of cleaning proteins which results in cell death.Our previous research found that the selective PERK/eIF2α pathway activator—CCT020312 could inhibit the development of breast cancer.As we know,prostate cancer and breast cancer have a high degree of biological similarity.Therefore,this research intends to explore whether CCT020312 inhibit PCa,and could CCT020312 be used in the treatment of PCa?Methods1.The pharmacodynamic effect of selective PERK/eIF2α pathway activator—CCT020312 on PCa cells in vitro and in vivo(1)CCK-8 assay was used to detect the effect of CCT020312 on cell viabitlity in C4-2 and LNCaP cells;(2)The x CELLigence real-time cell analysis system and cell colony formation assay were performed to observe the effect of CCT020312 on cell proliferation in C4-2 and LNCaP cells;(3)Subcutaneous xenograft model of C4-2 cell was established to observe the anti-tumor and toxicity effects of CCTT020312(40mg/kg,ip)on tumor size and mice body weight.2.The mechanism of selective PERK/eIF2α pathway activator—CCT020312 surpressing PCa cells(1)Flow cytometry was used to detect cell cycle in PCa cells;(2)Flow cytometry and Western blotting were used to detect the proteins of apoptosis related signalling molecules such as Bcl-2,Bax,Cleaved-PARP,PARP,Cleaved-caspase3,Caspase3 to explore the effect of CCT020312 on apoptosis in C4-2 and LNCaP cells;(3)RT-qPCR was used to examine genes of BIP m RNA,ATF4 m RNA,CHOP m RNA;Western blotting was used to detect the proteins of PERK pathway related proteins like BIP,PERK,p-PERK,eIF2α,p-eIF2α,ATF4,CHOP;then C4-2 and LNCaP cells were transfected with CHOP RNAi,CCK-8 assay and flow cytometry were used to detect the effect of CCT020312 on cell viability,apoptosis.These experiments were performed to explore the effect of CCT020312 on PERK pathway in C4-2 and LNCaP cells;(4)RT-qPCR was used to examine genes of LC3Ⅱ/Ⅰ m RNA,Atg5 m RNA and Atg8 m RNA;Western blotting was used to detect the proteins of LC3Ⅱ/Ⅰ,Atg12-Atg5,Beclin1 and p62;Laser confocal scanning were used to investigate autophagy in stable GFP-LC3 B transfected C4-2 cell and LNCaP cell lines after CCT020312 treatment;Transmission electron microscopy were applied to observe the existence of autophagesomes and autolysosomes.These experiments were taken to explore the effect of CCT020312 on autophagy pathway in C4-2 and LNCaP cells;(5)PCa cells were treated with autophagy inhibitors Baf-A1 to detect the effect of CCT020312 on cell viability,exploring the role of CCT020312-induced autophagy in C4-2 and LNCaP cells;(6)After PCa cells were transfected with CHOP RNAi,Western blotting was performed to detect the the effect of CCT020312 on proteins of CHOP,LC3Ⅱ/Ⅰ,Beclin1;and to detect the effect of autophagy inhibitor Baf-A1 on CCT020312-upregulated CHOP,LC3Ⅱ/Ⅰ proteins,exploring the relationship between PERK pathway and CCT020312-induced autophagy.(7)Western blotting was used to detect the proteins of BIP,PERK,p-PERK,eIF2α,p-eIF2α,ATF4,CHOP,LC3Ⅱ/Ⅰ,Atg12-Atg5,Beclin1 in subcutenous xenograft tumors;Immunohistochemistry was taken to examine the expression of Ki67,LC3Ⅱ/Ⅰ and Beclin1;TUNEL assay was obtained to detect the apoptosis in subcutaneous xenograft.Results1.The pharmacodynamic effect of selective PERK/eIF2α pathway activator—CCT020312 on PCa cells in vitro and in vivo(1)The results of CCK-8 showed that CCT020312 obviously inhibited cell viability of C4-2 and LNCaP cells in 24 h and 48 h,which was dose-dependent and time-dependent.(2)The results of RTCA-S16 and colon assay indicated that CCT020312 could retarded cell proliferation of C4-2 and LNCaP cells.(3)The results of subcutaneous xenograft tumor model exhibited that CCT020312 delayed tumor growth and has little effect on body weight.2.The mechanism of selective PERK/eIF2α pathway activator—CCT020312 in PCa cells(1)The results of flow cytometer showed that CCT020312 induced PCa cells G1 phase arrest.(2)The results of flow cytometer and western blotting indicated that CCT020312 downregulated Bcl-2 protein and upregulated proteins of Bax,Cleaved-PARP,Cleaved-caspase3,significantly increased cell apoptosis in C4-2 and LNCaP cells.(3)The results of RT-qPCR showed that CCT020312 elevated the m RNA expression of BIP,ATF4,CHOP;also,CCT020312 significantly promoted molecules expression of BIP,p-PERK,p-eIF2α,ATF4,CHOP;besides,CHOP RNAi could reverse the effect of CCT020312 on cell viability inhibition and induced-apoptosis in C4-2 and LNCaP cells.(4)The results of RT-qPCR indicated that CCT020312 activated the m RNA expression of LC3,Atg5 and Atg8;and Western blotting showed that CCT020312 upregulated LC3Ⅱ/Ⅰ,Atg12-Atg5,Beclin1 and p62 proteins;Laser confocal scanning demonstrated that green fluorescence accumulated and the spots were greater in cells treated with CCT020312 than in untreated C4-2 cells and LNCaP cells with stable GFP-LC3 B transfection;autolysosomes were observed in CCT020312-treated cells using scanning electron microscope.(5)The results of CCK8 exhibited that autophagy inhibitor Baf-A1 could reverse the effect of CCT020312 on cell viability inhibition.(6)The results of Western blotting indicated that CHOP RNAi could reverse the effect of CCT020312 on the upregulation of CHOP,LC3Ⅱ/Ⅰ,Beclin1 proteins,but Autophagy inhibitor Baf-A1 could not reverse the effect of CCT020312 on the upregulation of CHOP,LC3Ⅱ/Ⅰ.(7)The results of Western blotting indicated that CCT020312 downregulated p-m TOR and upregulated BIP,p-PERK,p-eIF2α,ATF4,CHOP,LC3Ⅱ/Ⅰ,Atg12-Atg5,Beclin1 proteins in vivo.Immunohistochemistry showed the the expression of Ki67 was lower and LC3Ⅱ/Ⅰ,Beclin1 was higher in CCT020312 group than in control group.TUNEL assay results demonstrated the TUNEL positive red apoptosis-related fluorescence in CCT020312 group was lighter than control group.ConclusionCCT020312 exerts an anti-tumor effect on PCa cells characterized by viability and proliferation hindrance.The mechanism is involved in CCT020312 inducing cell cycle G1 arrest,promoting apoptosis and autophagy mediated cell death. |
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