Antitumor Activity And Mechanism Of Action Of Herbal Extract Raddeanin A And Novel Imipridone ONC213 In Prostate Cancer

Androgen and androgen receptor?AR?play an important role in the progression of prostate cancer.Therefor,androgen receptor is a major therapeutic target for the treatment of prostate cancer.To date,Androgen deprivation therapy?ADT?is the main treatment for advanced and metastatic prostate cancer.ADT refers to the use of surgical castration to reduce androgen levels in patients,or to block AR activity by interrupting the binding of androgen to AR via AR antagonists.This treatment is effective at the initial stage.However,most patients will relapse within 2-3 years and progress to castration resistant prostate cancer?CRPC?,which is in curable.Studies have shown that CRPC also relies on the AR signaling pathway for survival and progression.The US FDA has approved several AR targeting new drugs,including abiraterone?inhibitor of the key enzyme CYP17A1?and enzalutamide?novel antagonists of AR?,for the treatment of CRPC.These treatments are effective initially,however the tumors eventually acquire resistance to these drugs,resulting in disease progression and unsatifying clinical outcome.It has been reported that a variety of mechanisms can promote the emergence of resistance to AR-targeting agents,such as the synthesis of intratumoral androgen,abnormal expression of AR co-regulators,mutation and amplification of AR gene,and the generation of AR splice variants?AR-Vs?.AR-Vs are considered the main cause of CRPC.Compared with full-length AR?AR-FL?,most AR-Vs contain N-terminal domain and DNA-binding domain but lack a ligand binding domain,indicating that AR-Vs have constitutive transcriptional activity and are not regulated by androgens.AR-V7 and AR^v567es are the widely studied splice variants that can reduce the median survival of patients and lead to poor prognosis.Therefore,understanding the mechanism of action of AR-Vs and developing drugs that can target both AR-FL and AR-Vs may represent a better therapeutic approach to improve the treatment of CRPC.In this study,we investigated the antitumor activities and mechanism of action of two drugs in prostate cancer,Raddeanin A?RA?and the novel imipridone derivative,ONC213.Raddeanin A is the triterpenoid saponin extracted from the roots of Anemone raddeana Regel,a traditional Chinese medicinal herb.It was mainly used to treat rheumatism in the ancient times.In our study,RA was found to inhibit the growth of AR-expressing prostate cancer cell lines within the plasma concentration range,while its antitumor activity against AR-null cells was minimal,suggesting that its antitumor activity may be related to AR in prostate cancer.Further studies revealed that RA reduced the expression of AR-FL and AR-Vs proteins,decreased the activity of the AR signaling pathway and the transcription of genes downstream of AR-FL and AR-Vs.RA also reduced transcription of the AR gene,shortened the half-life of AR protein,and accelerated the degradation of AR protein through the proteasome pathway.This was accompanied by enhancement of the therapeutic effect of first-line drug docetaxel in prostate cancer.We also studied the antitumor activity and the underlying molecular mechanism of the new imipridone analog ONC213.The imipridone ONC201 have inhibitory effects on various cancers and are undergoing clinical trials for various solid tumors.ONC213 is a structural analog of ONC201.We first tested the antitumor activity of ONC213 in vitro and in vivo.Our results showed that ONC213 significantly decreased the number of viable prostate cancer cells in vitro and the tumor volume of a 22Rv1-dervied xenograft mouse model.Further studies demonstated that ONC213inactivated AKT/mTOR and ERK signaling pathways reflected by downregulation of p-AKT,the AKT downstream p-S6,and p-ERK.In addition,ONC213 down-regulated Mcl-1 protein,and the Mcl-1 inhibitor significantly enhanced ONC213-induced apoptosis.Based on the reported negative feedback regulation between PI3K and AR signaling pathways,we investigated the effect of ONC213 on AR.We found that ONC213 decreased AR-FL and AR-V protein levels.This was accompanied by decreased AR-driven luciferase reporter gene activities and mRNA levels of AR-FL or AR-Vs target genes.ONC213 treatment also caused downregulation of the transcripts of AR-FL and AR-Vs.These results suggest that ONC213 exerts its antitumor activity against prostate cancer cells through downregulation of AR.However,overexpression of AR-FL in LNCaP cells could only partially rescue the cells,suggesting that there must exist other molecular mechanisms responsible for the antitumor activity of ONC213 against prostate cancer.Interestingly,we found that ONC213down-regulated c-Myc.When combined with the c-Myc inhibitor 10058-F4,apoptosis induced by ONC213 was significantly enhanced,suggesting that c-Myc also plays an important role in the antitumor activity of ONC213 in prostate cancer.Furthermore,ONC213 treatment also induced the protein levels of ATF4,a biomarker of mitochondrial stress.Switching protstate cancer cells from glucose media to galactose media significantly enhanced apoptosis induced by ONC213.These results suggest that ONC213 may target oxidative phosphorylation to kill prostate cancer cells,however the precise molecular mechanism needs to be established.In summary,the AR signaling pathway remains crucial for CRPC,and AR-Vs cause resistance to current AR-targeting therapies.The results in this study demonstrate that both Raddeanin A and ONC213 inactivate the AR signaling pathway and inhibit prostate cancer growth.Our results of our study form a solid foundation for the clinical development of Raddeanin A and ONC213 for treating CRPC.