Mechanism Of Neriifolin Contributes To Inhibition Of DNA Damage Repair Through Endoplasmic Reticulum Stress In Prostate Cancer Cells

IntroductionProstate cancer is one of the most common malignancies in male genitourinary system.Usually,prostate cancer cells acquire the ability to survive in low androgen conditions gradually after endocrine therapies,eventually transforming into castration-resistant prostate cancer(CRPC).Conventional chemotherapy drugs to treat CRPC have low response rate,large side effects,and a certain risk of drug resistance.Cardiac glycosides are clinically used in the treatment of heart failure.More and more studies have found the inhibitory effect of cardiac glycosides on a variety of tumors in recent years.Moreover,tumor cells are more sensitive than normal somatic cells,which points out a promising direction for the study of tumor therapy.However,although cardiac glycosides have shown definite anticancer activity,the mechanism of their anticancer effect is still not clear,which limites their further development in tumor therapy.This study aimed to explore the effects of neriifolin,a cardiac glycoside compound,on the growth of prostate cancer cells and its molecular mechanism through transcriptome sequencing analysis and functional studies in vitro and in vivo,so as to provide some experimental basis for the application of cardiac glycosides in the treatment of CRPC.Methods and ResultsCCK8 cell proliferation toxicity assay,flow cytometry and Western-blotting were used to find that neriifolin could effectively inhibit the growth and induce apoptosis of prostate cancer cells.Then RNA of the drug-treated cells was extracted for transcriptome sequencing analysis.The target genes were screened from the sequencing results and verified at RNA and protein levels.A large amount of DNA damage and down-regulation of BRCA1 and BRCA2 were found in the cells.Based on the principle of drug and intermolecular interaction,we demonstrated that neriifolin can increase Ca2+ level and endoplasmic reticulum stress,increase the expression of CHOP and decrease the C/EBP-α in prostate cancer cells by flow cytometry,Western-blotting,immunofluorescence,chromatin immunoprecipitation and other methods.Sequence alignment and chromatin immunoprecipitation confirmed the regulation of C/EBP-α on BRCA1 and BRCA2.Rescue experiments were designed for CHOP and C/EBP-α.Then in vivo experiments were performed on mice inoculated subcutaneously with tumors.The experimental results above showed the mechanism of neriifolin inducing apoptosis by CHOP and C/EBP-α.ConclusionThis study demonstrated that the cardiac glycoside compounds neriifolin increases CHOP level through endoplasmic reticulum stress in prostate cancer cells,thereby inhibiting the expression of C/EBP-α which involved in the transcription of BRCA1/2.So BRCA1 and BRCA2,the DNA damage repair protein,were reduced,eventually lead to the accumulation of DNA double strand breaks,inhibition of proliferation and increase of apoptosis in prostate cancer cells.These findings suggest that the novel role of neriifolin,a cardiac glycoside compound,in regulating DNA damage repair in prostate cancer cells may provide an optimized strategy for the treatment of CRPC.