The Effects And Mechanisms Of Proscillaridin A On Prostate Cancer Cells

Prostate cancer is one of the most commonly diagnosed malignancies in male.The incidence of prostate cancer in China has been increasing year by year,and it is usually found in the middle and late stages because of the inconspicuousearly symptoms.At present,the treatment of early localized prostate cancer is surgical treatment and local radiotherapy;the treatment of advanced prostate cancer is castration therapy,including surgical castration and drug castration,which has a significant effect in the early stage of treatment.However,most patients developed to highly aggressive castration-resistant prostate cancer few months after castration therapy.At this time,castration therapy has no effect and the only treatment that can be chosen is chemotherapy.Although chemotherapy drugs such as paclitaxel increase the survival rate of patients with metastatic androgen-independent prostate cancer,the prognosis of this disease is still poor.Therefore,there is an urgent need to explore new therapeutic methods or drugs/bioactive molecules to improving the outcomes of prostate cancer treatment and improve the prognosis of prostate cancer patients.Cancer chemoprevention and treatment by natural compounds has gained increasing attention because it is considered as safe,cost effective and alternative form of modern healthcare system.Natural products have significantly contributed in discovery of anticancer drugs.At present,more than 60% FDA approved anticancer drugs were derived from natural products including medicinal plants.Plants' and animals' secondary metabolites such as alkaloids and terpenes,and they have been extensively studied for their potential use in the treatment of cancer.Cardiacglycosides are plants' and animals' secondary metabolites which have long been used to treat cardiac congestions and cardiac arrhythmias and now have been reported to exhibit anticancer activity at non-toxic concentration in various in vitro and in vivo cancer models through multiple mechanisms including inhibition of proliferation,induction of apoptosis and augmentation of chemotherapy drugs.Signal transducer and activator of transcription3(STAT3)has been shown activated in many tumors including prostate cancer and associated with tumor progression and drug resistance,which is a predictor of poor prognosis.Doxorubicin(DOX)is a clinically routine chemotherapeutic drug and is widely used to treat a variety of malignancies including prostate cancer.DOX exerts anti-tumor effects by inducing apoptosis of tumor cells mainly by inducing DNA damage and interfering with DNA metabolism.However DOX has a strong side effect on the heart,and is usually used in combination with other types of chemotherapeutic drugs to reduce its toxic and side effects,and at the same time enhancing the anti-tumor effect.DOX induces STAT3 activation may be one of the main reasons that cause resistance in anti-tumor therapy.Therefore inhibition of STAT3 activation can enhance the anti-tumor effect.At the same time,there are reports in the literature that natural compounds with inhibitory effects on STAT3 can enhance the anti-cancer activity of DOX in A549 lung adenocarcinoma cells and MDA-MB-231 breast cancer cells as well as in gastric cancer SGC-7901 and HGC-27 cells.Proscillaridin A(PSN-A),a cardiac glycoside constituent of urginea maritima,exerts a cardiotonic effect by inhibiting Na+-K+-ATPase and increasing intracellular Na+and Ca2+andhas been shown to exhibit anticancer activity.PSN-A can effectively inhibit proliferation and induce apoptosis in lung cancer cells and breast cancer cells,and enhance anti-tumor activity by inhibiting the JAK/STAT3 pathway in combination with other anti-tumor drugs.However,the cellular targets and anticancer mechanism of PSN-A in various cancers including prostate cancer remain largely unexplored.The aim of this study is to investigate the effect of PSN-A on human prostatic carcinoma androgen-dependent LNCa P cells and androgen-independent DU145 cells in vitro and the possible mechanisms,we will also explore the combition effect and mechanism of PSN-A with DOX on prostate cancer cells.Providing theoretical support to develop PSN-A into a potential compound for the treatment of prostate cancer.In this study,the morphology change of LNCa P and DU-145 cells will be observed by inverted phase contrast microscope.Effect of PSN-A on cell viability was measured by MTT assay and colony formation assay.Apoptosis,reactive oxygen species(ROS)production and mitochondrial membrane potential(MMP)changes were detect by flow cytometry.The anti-apoptosis proteins and JAK/STAT3 pathway proteins were detected by western blot to further analyze the anti-tumor mechanism of PSN-A.The use of a multi-functional microplate reader to detect the change of intracellular free Ca2+in the fluorescent probe Fluo-3 AM was used to analyze whether the intracellular Ca2+change was related to the apoptotic effects of PSN-A.The results are as follows:1.The results of MTT and colony formation assay showed that PSN-A inhibited the proliferation of LNCa P cells and DU-145 cells in a dose-dependent manner.And the inhibition effect of PSN-A on DU145 cells was found to be relatively lower than LNCa P cells.2.Flow cytometry showed that PSN-A induced apoptosis of LNCa P cells and DU145 cells in a dose-dependent manner while DU145 cells were found to be less insensitive to PSN-A treatment than LNCa P cells.The apoptosis rate of LNCa P cells treated with the same concentration of PSN-A was significantly higher than DU145 cells.3.Flow cytometry showed that PSN-A induces ROS generation and decreased MMP in prostate cancer cells.PSN-A treatment increased the level of ROS generation in both cell lines in a dose-dependent manner,however,higher level of ROS was observed in LNCa P cells compared to DU145 cells.PSN-A treatment dissipated MMP significantly in LNCa P cells in a dose-dependent manner.AlthoughPSN-A decreased MMP in DU145 cells,however,dose-dependent effect was not significant.4.Western blot results showed that PSN-A inhibited the expression of anti-apoptotic Bcl-2 protein in LNCa P cells in a dose-dependent manner,and increased the expression of pro-apoptotic Bax protein,and decreased the Bcl-2/Bax ratio.PSN-A had no obvious effect on the regulation of Bax protein expression in DU145 cells,but inhibited the expression of Bcl-2 and decreased the Bcl-2/Bax ratio.Both prostate cancer cells induced caspase-3 activation and PARP-1 cleavage in a dose-dependent manner.5.Western blot results showed that PSN-A inhibited STAT3 phosphorylation in both cell lines in a dose-dependent manner.By detecting tyrosine kinase(positive regulator)and protein tyrosine phosphatase(negative regulator),the upstream of STAT3,PSN-A inhibited the phosphorylation of the positive regulator JAK2 in a dose-dependent manner.PSN-A slightly increased the expression of SHP-1 but no significant difference,while the expression of SHP-2 and PTEN was unaffected.It was shown that PSN-A inhibited the phosphorylation of STAT3 by inhibiting the phosphorylation of the upstream tyrosine kinase JAK2.6.PSN-A augmented DOX toxicity in prostate cancer cells.PSN-A remarkably enhanced the apoptotic effect of DOX in LNCa P cells compared to DU145 cells as evident from flow cytometry analysis of apoptosis and Western blot detection of PARP-1 cleavage.DOX increased the phosphorylation of STAT3 while PSN-A effectively suppressed DOX-induced STAT3 phosphorylation in prostate cancer cells.7.PSN-A increased intracellular Ca2+in both LNCa P and DU145 cells in a dose-dependent manner.We measured the effect of PSN-Aon cell viability in the presence and absence of BAPTA-AM(10?M),and found that BAPTA-AM failed to protect the cells from toxic effect of PSN-A in prostate cancer cells indicating that increase in intracellular Ca2+is not associated with anticancer activity of PSN-Ain our study model.Based on the above findings,our present study achieved the following conclusions:1.We demonstrated for the first time that PSN-A inhibited growth,induced apoptosis of prostate cancer LNCa P and DU145 cellsin a dose-dependent manner.2.PSN-A increased ROS generation,decreased the level of MMP and Bcl-2/Bax ratio,increased expressions of cleaved caspase-3 and PARP-1cleavge in LNCa P and DU145 cells,and validating the involvement of mitochondrial apoptosis,indicating that cardiac glycosides could effectively initiate mitochondrial apoptosis by modulating Bcl-2 family proteins and capase-3 protein in prostate cancer cells3.We demonstrated for the first time that PSN-A inhibited STAT3 activation by inhibiting JAK2 phosphorylation in prostate cancer LNCa P cells and DU145 cells.4.PSN-A enhanced the cytotoxicity of DOX in prostate cancer cells by enhancing the pro-apoptotic effects as well as inhibiting DOX-induced STAT3 phosphorylation in LNCa P cells and DU145 cells.5.PSN-A increased intracellular free Ca2+in prostate cancer LNCa P cells and DU145 cells,and the increased Ca2+was found to be not associated with anticancer activity of PSN-A.6.LNCa P cells were found to be more sensitive to PSN-A treatment while DU145 cells exhibited less sensitive to PSN-A treatment.This differential effect of PSN-A might be associated with the androgen sensitivity of the two cells.In summary,our results confirmed the anticancer activity and the possible underlying molecular mechanisms of PSN-A on human prostate cancer LNCa P cells and DU145 cells for the first time,providing a theoretical basis for the development of PSN-A as an effective therapeutic agent for prostate cancer.Further studies are needed to develop PSN-A as a potential compound for the treatment of prostate cancer.