A Novel Deubiquitinase Inhibitor B-AP15 Triggers Apoptosis In Both Androgen Receptor-dependent And-independent Prostate Cancers

Background Since a subset of patients progressing to lethal and incurable metastatic castrate-resistant prostate cancer(C RPC),prostate cancer(PCa)remains a leading cause of cancer-related death in men.The ubiquitin-proteasome system(UPS),a highly specific and selective route for cellular protein degradation,controls the fate of most proteins by striking a dynamic balance between ubiquitination and deubiquitination of substrates.Deubiquitinating enzymes(DUBs)which have recently emerged as novel targets of anti-cancer therapy,regulate multiple cellular processes,including cell cycle control,DNA damage response and repair,apoptosis,chromatin modification,and various signal transduction pathways.With the use of FDA-approved drug bortizomib/Velcade as a 20 S proteasome inhibitor in the clinical treatment of multiple myeloma,the proteasome inhibition has proven to be a promising strategy for cancer therapy.Intriguingly,androgen receptor(AR)plays a critical role in the growth and progression of human prostate cancer.AR is aligand-dependent transcriptionfactor,belonging to the nuclear receptor superfamily.Generally,prostate cancer occurs with AR gene amplification and mutation which in reverse dominates the survival,proliferation and growth of prostate cancer.In our previous study,we have identified ubiquitin-specific protease 14(USP14)as a novel regulator of AR,inhibiting the degradation of AR via deubiquitinating thisoncoprotein in the androgen-responsive prostate cancer cells,and androgen-independent show insusceptibility.Recently,a novel small molecule b-AP15 is identified as the inhibitor of the USP14/UCHL5(DUBs)of the 19 S proteasome,resulting in cell growth inhibition a nd apoptosis in several human cancer cell lines.Our objectives and significance is to find out the evaluation of AR and anti-tumor effects on both AR-dependent and-independent prostate cancers under the inhibition of USP14 and UCHL5.Here,we provide evidence that treatment with b-AP15 induces AR surpression.Furtherly,it inhibits the growth of PCa cells and enhances apoptosis of both androgen receptor-dependent and-independent prostate cancer cells in cultures and xenograft models,associated with induction of UPS inhibition,caspase activation,ER stress,generation of reactive oxygen species(ROS).Methods 1.Cell viability assay: MTS assay and CCK-8 assay were performed.2.Colony formation assay: The colonies> 60 μm were counted under a light microscope.3.Cell death assay: Cells with Annexin V-fluoroisothio-cyanate(FITC)/PI double staining determined by flow cytometry and inverted fluorescence microscope.4.Si RNA transfection: Two si RNAs against human USP14 and UCHL5 were used to transfect LNCa P cells.Cells were collected for Western blot assay.5.Protein assay was performed by Western blot and co-IP analysis.6.Mitochondrial membrane integrity measurement: The mitochondrial membrane potential of b-AP15 and untreated cells was assayed by using Rhodamine-123 staining.7.Measurement of ROS generation: Cancer cells with 10 μM of DCFH-DA staining were analyzed through flow cytometer.8.Quantitative real-time polymerase chain reaction(q RT-PCR): According to the manufacturer’s instructions of the Ta Ka Ra kit,a melting curve analysis was performed to demonstrate the PCR product specificity after PC R.Every samplewas analyzed in triplicate.The relative expression level of a target gene was presented as the sample versus the control.9.Establishment of PC-3 nude mouse xenograft model: Approximately 1×106 of PC-3 cells were inoculated subcutaneously in the left armpit of each mouse.72 h after cell inoculation,mice were randomly divided into 2 groups and treated with either vehicle(DMSO,cremophor,and 0.85% Na Cl at 1:3:6 ratio,v:v:v)and b-AP15(5 mg/kg/2d)for totally 14 days.10.Tissue protein expression was detected by Immunohistochemical staining.Results 1.b-AP15 treatment suppressed cell proliferation and colony formation in prostate cancer cel s Increasing concentrations of b-AP15(0–5μM)treatment for 24 h,48 h and 72 h inhibited the proliferation of LNCa P,22Rv1,PC-3,DU145 and WPMY-1 cells which indicating no obvious selectivity on both cancer cells and normal cell.Additionally,the dose-dependent suppression of long-term colony formation in LNCa P and PC-3 exposed to b-AP15 for 7 days was observed.2.b-AP15 induces cell cycle arrest in vitro Cells were exposed to various concentrations of b-AP15(0,0.5,1,2 μM)and subsequently subject to cell cycle analysis;and we found t hat increasing dosage of b-AP15 dramatically induced G0/G1 cell cycle arrest in both cell lines at 24 h.As western blot analyses,we found that b-AP15 significantly decreased the expression of cyclin D1,CDK6,CDK4,CDK2 and phosphorylation/inactivation of Rb—marker proteins from G1 to S phase,and up-regulated the expression of p27,which is a known tumour suppressor via inhibiting the cyclin-CDK function.3.Induction of caspase-dependent apoptosis by b-AP15 It was found that the Annexin-V/PI-positive population increased significantly and the morphological changes of apoptosis appeared in b-AP15 treated cells.Our data show that b-AP15 elicited significant cleavage of PARP,a hall mark of apoptosis,and activation of caspase-3,-8,-9 in LNCa P and PC-3 cells,as detectedwith western blot analyses.4.Induction of apoptosis by b-AP15 is associated with mitochondrial dysfunction In addition to triggering apoptotic signals,b-AP15 treatment caused mitochondria to undergo loss of mitochondrial membrane potential,as detected by rhodamine-123 staining and flow cytometry.b-AP15 triggered a remarkable decline in the expression of anti-apoptotic protein(Bcl-2)in both dose-and time-response experiments of the two cell lines,while the pro-apoptotic proteins(Bim,Bax,Noxa)were increased significantly.Moreover,when LNCa P and PC-3 cells were exposed to escalating doses of b-AP15 for 48 h,the pro-apoptotic factors(cytochrome C)and apoptosis-inducing factor(AIF)levels in the cytosol were significantly increased as revealed by western blotting following with the extraction of cytosolic and mitochondrial fractions.5.N-acetyl-cysteine(NAC)reversed b-AP15-induced ROS generation and apoptosis 6.b-AP15 significantly inhibited the proteasome function and the expression of androgen receptor(AR)in LNCa P and PC-3 cells with endoplasmic reticulum stress(ER stress)generation as well Low concentration of b-AP15 triggers accumulation of ubiquitinated proteins(Ub-prs)which means the apoptosis induced by b-AP15 occurs after the proteasome inhibition.Further,b-AP15 results in generation of ER stress and suppression of androgen receptor(AR).These results of co-IP suggest that b-AP15 destabilizes AR proteins via suppressing the deubiquitination of AR but not on transcription level.After the transfection of UCHL5 on LNCa P cells with si RNA,no obvious changes on AR expression suggesting that USP14 is critical for the stabilization of AR.7.Anti-cancer activity of b-AP15 in vivo Compared with the control group,we found that the tumor size and tumor weight of the group treated with b-AP15 were strikingly reduced.Importantly,there was no significant difference in body weight of the nude mice between the two groups.Additionally,we found that the levels of proteasome substrates,includingtotal or K48-linked ubiquitin proteins and p27,were markedly increased in the b-AP15-treated tumor tissues.Moreover,the immunohistochemistry also revealed that cleaved caspase-3 was also increased in b-AP15 treated tumor cells.As the detection of CK,ALT and creatine on nude mouse xenograft models displayed,there were no significant dysfunctional difference of heart/cardiac tissues,liver and kidney compared with the control group which means the toxic impact by b-AP15 can be effectively controlled.Conclution We have demonstrated the antitumor activity of b-AP15 in both androgen receptor-dependent and-independent prostate cancer cells,which identifies a new promising therapeutic strategy for prostate cancer.