The Role And Mechanism Of Gambogic Acid Against Hormone-resistant Prostate Cancer

Objectives:To investigate the anti-tumor efficacy of GA in metastatic castrate resistant prostate cancer(CRPC)and explore the underlying mechanism.Methods:Several cell lines from independent PbCre4;Ptenfl/flTp53fl/fl prostate adenocar-cinoma tumors,designated PCAP(Prostate Cancer Adenocarcinoma Pten/Tp53 null)and various LuCaPs from the genomically-representative tumors of mCRPC patient-derived xenografts(PDX)were used to investigate the anti-tumor effect of GA and analyze underly-ing mechanism.1.Growth inhibition of compounds derived from traditional Chinese medicine(TCM)towards prostate cancer cells.1.1 Drug screening of anti-tumor compounds selected based on moving stasis and detoxify toxin principle in the theory of TCM.PCAP-1 cells were treated with different concentrations of 11 compounds,including Cryptotanshinone(CT),Tanshinone IIA,Matrine,Oxymatrine,Curcumol,Curcumin,Berberine hydrochloride(BBH),Peimine,Peiminine;Quercetin and Gambogic acid for 48h.CTG assay was used to analyze the cell viability.1.2 Growth inhibition of GA in PCAPs bulk and stem/progenitor cells.PCAPs cells cultured in 2D and PCAP-1 in 3D were treated with different concentrations of GA for 24h and 48h as for 2D,7 days for 3D.CTG assay was used to determine the cell viability of 2D.SFA and CTG-3D were used to analyze the sphere forming rate and stem/progenitor cell viability,respectively.1.3 Growth inhibition of GA in human PrCa cell lines.LNCaP,PC-3 and DU145 cells were treated with different concentrations of GA for 24h and 48h.CTG assay was used to test the cell viability.1.4 Growth inhibition of GA in LuCaPs stem/progenitor cells.Genomically representa-tive LuCaPs from metastatic CRPC PDXs including LuCaP23.1,73,92,136,167 were cultured in 3D,after treating with different concentrations of GA for 2 weeks,SFA and CTG-3D were used to test the sphere forming rate and cell viability,respectively.2.Pro-apoptotic effect of GA in PCAP-1 cells.2.1 Cell cycle distribution analysis of GA treated PCAP-1 cells.Flow cytometer was used to analysis the cell cycle distribution of PCAP-1 cells treated with GA 500nM in different times with PI staining.2.2 Cleaved CASPs proteins levels in GA treated PCAP-1 cells.PCAP-1 cells were treated with GA 500nM in indicated times,and WB was performend to analyze the proteins levels of CASP-3,8,9 and corresponding cleaved CASPs comparing to B6WT cells.2.3 Synergistic effect of Z-VAD-FMK and GA.PCAP-1 cells were treated with GA in the presence of f Z-VAD-FMK or/and DFOM for 24h,CTG assay was used to test the cell viability of different groups to detect whether Z-VAD-FMK or/and DFOM can reverse the growth inhibition of GA or not.3.The role of ROS in GA's anti-proliferative and pro-apoptotic effect in PrCa cells.3.1 The effect of GA in ROS and MMP in PCAP-1 and LuCaPs cells.PCAP-1 and LuCaP92 organoids were treated with GA 500nM for indicated times.Flow cytometer analysis was used to determine the intracellular ROS level with CM-H2DCFDA probe.MMP were determined by confocal and flow cytometer analysis with JC-1 and TMRM probe,respectively.3.2 The reverse effect of NAC against GA,s growth inhibition towards PCAP-1 and LuCaPs.PCAP-1 and LuCaPs cells were treated with GA in the presence of NAC or not.Flow cytometer analysis was used to determine the ROS level in PCAP-1 cells.WB was used to analysis the protein level of CASP-3,8,9 and cleaved CASP-3,8,9.CTG and CTG-3D assay was used to test the cell viability of PCAP-1 and LuCaPs,respectively.4.The role of GA in the thioredoxin(Trx)antioxidant system.4.1 Exploring the possible mechanism of ROS accumulation of GA.Cellular ROS inducers based on diverse mechanisms were selected to be combined with GA to treat PCAP-1 cells and LuCaP136 organoids,cell viability was then determained with CTG and CTG-3D,respectively,to select the synergistic drug with GA.4.2 Effect of GA on thioredoxin activity.PCAP-lcells and LuCaP136 organoids was incubated with indicated concentrations of GA in indicated times,then the activity of Trx,thiorexocin reductase and the ratio of NADPH/NADP+ were determined by Trx assay kit,TrxR assay kit and NADPH/NAPH+ Glo assay kit,respectively.5.Synergy of GA and Enzalutamide(En)or Docetaxel(Doc)towards CRPC.LuCaP73?LuCaP92?LuCaP 136 cells cultured in 3D were incubated with GA,En and Doc alone or in combination with each other for 2 weeks,cell viability was measured with CTG-3D.Results:Growth inhibition of GA and undelying mechanism against metastatic CRPC was as following:1.GA is with potent activity towards PrCa bulk and Stem/progenitor cells.1.1 Preliminary screening of the 11 compounds on PCAP-1 cells selected GA as the most potency anti-PC compound which was 50 times more potent than the next two most active compounds,curcumin and CT.1.2 GA inhibited cell proliferation in a concentration-and time-dependent manner in PCAP-1 and three additional PCAP lines,demonstrating a generalizable vulnerability,while nontumorigenic luminal epithelial cells(B6WT)were relatively resistant to GA.SFA and CTG-3D assay of GA-treated towards PCAP-1 organoids showed that GA concentration-dependently inhibited the stem/progenitor cell growth,and GA gained complete inhibition with a concentration of 200nM.1.3 GA demonstrated a concentration-and time-dependent growth inhibition towards various human prostate cancer cell lines,which showed slight more resistant comparing to PCAP-1.1.4 LuCaPs organoid growth were generally sensitive to sub-micromolar range of GA with different sensitivity,while 500nM GA showed a complete organoids inhibition towards tested LuCaPs.2.GA induced apoptotic and ferroptotic cell death.2.1 GA 500nM 4h induced a significant increased subGl population in PCAP-1 cells rapidly,but not in B6WT cells.2.2 Cleaved CASPs were observed rapidly(6h)following GA 500nM treatment towards PCAP-1 cells but not in B6WT cells.2.3 GA-initiated inhibition was partially reversible with either Z-VAD-FMK and DFOM alone or together,Z-VAD-FMK showed more potency relative to DFOM.3.ROS accumulation is causal for anti-proliferative and pro-apoptotic effects of GA in PC cells.3.1 GA induced a time and concentration-dependent rapid accumulation of ROS with decreased MMP in PCAP-1 but not B6WT cells.In addition,LuCaP92 and LuCaP136 cells demonstrated concentration-dependent MMP decrease following GA treatment.3.2 The presence of NAC with GA neutralized intracelluar ROS accumulation,complet-ely alleviated growth inhibition,even following 1?M GA treatment and inhibited GA-induced cleavde CASPs in PCAP-1 cells.In addition,NAC partially protected LuCaPs organoids from GA-mediated growth inhibition in LuCaPs with a more pronounced effect in LuCaP92.4.GA inhibited thioredoxin activity.4.1 Auranofin was the one compound that demonstrated additive growth inhibition together with GA but not BSO in PCAP-1 cells and LuCaP136 organoids.4.2 Trx activity was consistently decreased over time in both PCAP-1 and LuCaP136 cells and their extracts following GA treatment with slightly increased NADPH/NADH ratio and TrxR activity.5.Submicromolar concentrations of GA enhanced the cell killing observed for either Doc or En in LuCaP73?92?136.Conclusions:Based on different CRPC models,we all determined that GA inhibited thioredoxin,a necessary component of cellular antioxidative to disrupt the intra-cellular ROS balance and induced ROS accumulation,consequently leading to apoptotic and ferroptotic cell death.Besides,GA displayed synergistic inhibition with Docetaxel and Enzalutamide in LuCaPs.NAC partially reversed growth inhibition in CRPC organoids,indicated that GA may have additional mechanisms of action in heterogeneous models.In summary,all the data suggest that GA may be useful,especially in combination therapies,for treating the inherent heterogeneity and plasticity that contributes to the therapeutic resistance of CRPC.