| Background:The incidence and mortality rate of prostate cancer is increasing every year.Androgen Deprivation Therapy(ADT)is one of the common treatments for prostate cancer.Although ADT is effective,the disease always deteriorates after a short period of control and evolves into Castration-Resistant Prostate Cancer(CRPC).The exact mechanism of progression is still unclear.It has been shown that the adrenal androgen,Dehydroepiandrosterone Sulfate(DHEAS),is transported into CRPC by Organic Anion Transporting Polypeptides 2B1(OATP2B1)after ADT treatment.DHEAS reacts to produce a potent androgen,dihydrotestosterone,which activates the androgen receptor(AR)to induce prostate-specific antigen secretion and promote proliferation,resulting in resistance.Many herbal active components such as18β-Glycyrrhizic acid(18β-GA)are known to significantly inhibit OATP2B1-mediated transport.Enzalutamide(ENZ)is an AR antagonist that blocks the binding of androgens to AR.However,a significant proportion of patients eventually develop drug resistance during ENZ treatment.Therefore,an in-depth study of the active component of Chinese medicine,18β-GA,blocking OATP2B1 uptake of DHEAS as a pathway to reduce androgen production and synergizing with androgen receptor inhibitor ENZ to delay tumor progression will provide new approaches and ideas for the prevention and treatment of CRPC.Objective:This study aime to investigate the mechanism of 18β-GA in reducing intracellular androgen production by inhibiting the uptake of DHEAS by OATP2B1 and thus synergizing with androgen receptor antagonist ENZ against castration-resistant prostate cancer,and to explore new methods and strategies for the prevention and treatment of CRPC.Methods:1.RT-q PCR and Western blotting were used to study the expression of the cell transporter OATP2B1 under androgen deprivation culture conditions,and cytotoxicity and clone formation assays were performed to investigate the effect of DHEAS on cell proliferation in prostate cancer LNCa P and 22Rv1 cells.2.Triple quadrupole tandem mass spectrometry(LC-MS/MS)method was used to investigate the concentration of DHEAS in HEK293 cells.3.LC-MS/MS was used to investigate the effect of 18β-GA on OATP2B1 mediated transport inhibition of DHEAS uptake and its inhibitory mode in OATP2B1-HEK293 cell model.4.LC-MS/MS was used to investigate the inhibitory effect of 18β-GA on OATP2B1-mediated DHEAS uptake under androgen-deprived culture conditions.Western blotting,cytotoxicity assay,clone formation assay,Transwell and scratch assay,and TUNEL assay were used to verify that 18β-GA combined with androgen receptor antagonist ENZ synergistically against the biological activity of tumor cells in prostate cancer 22RV1 cells.5.The nude mouse xenograft model under castration conditions was used to investigate the effect of 18β-GA on DHEAS uptake by tumor cells in vivo was assessed by biochemical indexes,hematoxylin eosin staining,and immunohistochemistry revealing 18β-GA is based on the interaction mechanism of OATP2B1 mediating DHEAS uptake inhibition with ENZ to synergize with castration resistant prostate cancer.Results:1.Under androgen deprivation culture conditions,the m RNA levels of OATP2B1 were upregulated by 458.74 % and 108.48 % in LNCap and 22RV1 cells,respectively,and the protein levels were upregulated by 89.51 % and 68.89 %,respectively.DHEAS promoted cell proliferation and increased cell viability in the LNCap and 22RV1 cell groups compared to the normal group by 21.78 % and20.79 %,respectively.2.The LC-MS/MS quantitative method for the determination of DHEAS concentration established in this experiment was sensitive,stable and reliable,and its specificity,linear range,extraction recovery,matrix effect,intra-batch inter-batch precision and accuracy were all in accordance with the corresponding acceptance criteria.3.In OATP2B1-HEK293 cells,Cyclosporin A(a classical inhibitor of OATP2B1)significantly inhibited the uptake transport of DHEAS,further demonstrating that DHEAS is a substrate of OATP2B1 with uptake kinetic parameters Km(μM)of 10.393±2.33 and Vmax(pmol/mg protein/min)of24.25±2.03.18β-GA significantly inhibited the uptake of DHEAS by OATP2B1,and the IC50 value was 3.51±1.19 μM.The addition of 18β-GA(100 μM)to serial concentrations of DHEAS revealed a significant decrease in the kinetic parameter Vmax of DHEAS uptake(from 44.82±1.28 to 34.87±0.74 pmol/mg protein /min),but Km values were almost unchanged,suggesting that 18β-GA may inhibit DHEAS uptake by OATP2B1 through a non-competitive inhibition mode.4.Under androgen deprivation culture conditions,DHEAS(10 μM)promotes the proliferation of prostate cancer cells 22RV1,and 18β-GA(40 μM)counteracts the proliferative effect of DHEAS-induced cells.18β-GA in combination with ENZ synergistically counteracts castration-resistant prostate cancer,showing some synergistic counteracting effects in proliferation,migration,and apoptosis.5.In xenograft nude mice,intraperitoneal injection of DHEAS under castration conditions promoted tumor growth,and its promoting effect was counteracted by18β-GA on top of DHEAS administration;the combination of 18β-GA and ENZ significantly inhibited tumor growth,suppressed tumor cell proliferation and promoted tumor cell apoptosis.Conclusion:1.18β-GA is an inhibitor of OATP2B1 which significantly inhibits OATP2B1-mediated uptake transport of DHEAS,and an OATP2B1-mediated interaction may occur in castration-resistant prostate cancer.2.18β-GA inhibits the uptake of DHEAS transport via OATP2B1,which in turn reduces androgen production.18β-GA in combination with the androgen receptor antagonist ENZ combats castration resistant prostate cancer progression by reducing ligand production and inhibiting androgen receptor. |
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