Erastin Downregulates Androgen Receptor And Its Splice Variants In Prostate Cancer

Prostate cancer(PCa)accounts for 13.5% of male tumors,which is only lower than that in lung cancer,and is the second-leading cause of cancer-related mortality in men.Although varieties of treatments could slow the progression of cancer,most of the patients become non-responsive to these treatments and inevitably progress to castration-resistant prostate cancer(CRPC).Androgen Receptor(AR)and its' splice variants(AR-Vs)play a critical role in the development and progression of PCa.Several drugs targeting androgen receptor reactivation can prolong the overall survival of patients with castration-resistant prostate cancer for about 4 months.However,even with these new drugs,the time to disease progression and overall survival still remain relatively short because of AR and AR-Vs.Erastin,as an inducer of ferroptosis,has been manifested to inhibit many kinds of cancer cells effectively and the mechanisms are not the same.However there are few studies about Erastin on prostate cancer and it is unclear whether Erastin affects the AR activation during inducing ferroptosis.In order to verify the inhibitory effect of Erastin on CRPC cells,both 22Rv1 and LNCa P95 cell lines were chosen as objects in this research.Firstly the SRB assay was used to test the survival rate of 22Rv1 and LNCa P95 cells after different doses of Erastin treatment in 24,48 and 72 hours.Flow cytometry was used to examine the effect of Erastin on CRPC cell death.The results showed that Erastin could inhibit these two CRPC cells survival rate in a time-and dose-dependent manner.Then we chose 10?M and 20?M Erastin for the next experiments.Flow cytometry showed that the effect of Erastin could increase the proportion of cells in sub-G1 phase and cause cell death.In order to verify that the effect caused by Erastin is ferroptosis,we firstly treated with erastin with or without a ferroptosis inhibitors(e.g.,ferrostatin-1 and liproxstatin-1),the apoptosis inhibitor(e.g.,ZVAD–FMK),necroptosis inhibitor(e.g.,necrosulfonamide)and autophagy inhibitor(e.g.,chloroquine).The results showed that in both cell lines,the apoptosis inhibitor,necroptosis inhibitor and autophagy inhibitor had no effect on erastin induced cell death.Then Western blot was used to detect the expression of ferroptosis marker protein GPX4,the results manifested that Erastin down-regulated the protein level of GPX4 in both cell lines.Given that ferroptosis is characterized by lipid peroxidation,we next investigated the level of GSH,ROS and MDA(an end product of lipid peroxidation).The levels of GSH were decreased,and the levels of both ROS and MDA increased in both cells.These results indicated that ferroptosis mainly occurred in the two types of cells after adding Erastin.Then to investigate the effect of Erastin on AR activity,Western blot assay was used to detect AR-FL and AR-Vs protein in CRPC cells and reporter gene assay was used to assess the AR promoter,AR-FL and AR-Vs activity.The q RT-PCR assay was used to examine AR-FL,AR-Vs m RNA and their downstream genes.The results show that the expression of AR-FL and AR-Vs were significantly down-regulated and AR promoter activity was inhibited.In addition,the levels of AR-FL and AR-Vs and their target genes m RNA were down-regulated after be treated with Erastin.These results showed that erastin could downregulate AR-FL and AR-Vs expression and transactivation.In addition,up-regulation tne expression of AR-FL and AR-Vs could reverse growth inhibition induced by Erastin in 22Rv1 cells.It was suggested that the inhibitory effect of Erastin on CRPC cells attributed to inhibiting the AR signaling pathway.In vivo experiments,the effects of Erastin on tumor size and body weight in tumorbearing mice were recorded.The serum PSA levels were measured by ELISA.The effects of Erastin on heart,liver,spleen and kidney in mice were examined by HE staining.It was found that Erastin could delay the growth rate of transplanted tumors,down-regulate the serum PSA levels and the levels of AR-FL and AR-Vs both in protein and m RNA compared with the control group.Finally,we measured the growth of 22Rv1 and LNCa P95 cells in the combination of Erastin and docetaxel,and calculated the combined index values.The results showed that combination therapy significantly inhibited the growth of cancer cells compared with monotherapy.In summary,we have demonstrated that erastin can significantly down-regulate the expression and activities of AR-FL and AR-Vs in prostate cancer in vitro and in vivo,and Erastin could enhance the growth inhibitory efficacy of docetaxel in CRPC cells.These results indicate that Erastin combined with docetaxel may be a promising therapeutic strategy for the treatment of human prostate cancer in the future.