| Introduction: Prostate cancer is the first high incidence and fatal tumor of urogenital system in China,among which castration-resistant prostate cancer(CRPC)advances rapidly,has poor prognosis and lacks effective treatment.At present,a single first-line endocrine therapy or immunotherapy can prolong the limited median survival time of CRPC patients,and the efficacy needs to be further improved.Studies have shown that androgen receptor(AR)mediated endocrine therapy resistance and decreased anti-tumor immune function mediated by suppression of immune signaling pathways such as c GAS-STING are key factors promoting the progression of CRPC.Therefore,to target and solve the key factors inducing the progression of CRPC is to solve the clinical treatment problems that need to be solved in CRPC research.Eliminating AR and completely blocking AR signaling pathway and activating c GAS-STING pathway are potential strategies for the efficient treatment of CRPC,which is of great significance for alleviating the treatment dilemma of CRPC.However,currently there are no drugs that can degrade AR and enhance anti-tumor immunity in the synergistic treatment of CRPC.In recent years,thanks to the rapid development of nanotechnology,nanocarriers increase the accumulation of drugs in tumors,reduce systemic toxicity,and thus improve anti-tumor effects due to their advantages in biological distribution,circulation and cell uptake.This study aims to solve the treatment problem of CRPC by designing and synthesizing arsenic nanomedicine with clinical conversion prospect.Methods: Arsenic nanosheets(ANs)were made from elemental arsenic using liquid stripping technology.Arsenic nanosheets(ANs)were synthesized PANs modified with polyethylene glycol(PEG).PANs were re-modified by prostate-specific membrane antigen(PSMA)targeting short peptide in PSMA-617 to obtain pegylated PSMA-targeted arsenic nanosheets(PMPANs)with better dispersion and tumor targeting ability,and the physicochemical characterization of the arsenic nanosheets were carried out by transmission electron microscopy,surface charge and infrared spectroscopy to verify their synthesis effect.Meanwhile,C4-2 cells(human prostate cancer cells,CRPC cell line)were incubated with PANs and PMPANs at the cellular level,and the targeting ability of PMPANs to C4-2 cells was tested by cell flow detection(FCM).The ability of PMPANs to kill and inhibit proliferation of C4-2 cells were detected by viability assay(MTT),plate cloning assay,etc.Secondly,the uptake and transport of PMPANs by C4-2 cells were observed by laser scanning confocal microscopy(LSCM),FCM,and immuno-fluorescence(IF),and the expression changes of AR key proteins and associated gene proteins were detected by Western blotting(WB),enzyme-linked immunosorbent assay(ELISA),and cell crawling tablet IF,and the potential mechanism of AR degradation was explored.Subsequently,the tumor bearing experiment of NCG mice xenografted with C4-2 cells was conducted,and the anti-tumor effect and biosafety of PMPANs at animal level were evaluated by blood biochemistry,hematoxylin-eosin staining(HE),immunohistochemistry(IHC)and IF.In addition,on the basis of PMPANs,combined with chloroquine(CQ)treatment,C4-2cells were observed to locate uptake and transport of PMPANs + CQ,and then WB was used to detect the changes of key proteins of c GAS-STING immune pathway.Besides,the changes of anti-tumor immune cells and immune factors were observed by FCM and IF,etc.,so as to evaluate the immune response after PMPANs + CQ,treatment and explore the potential anti-tumor immune mechanism.Finally,the tumor bearing model of normal immune mice with prostate cancer was established in C57BL/6 mice at animal level to verify the anti-tumor immune effect and biosafety of PMPANs + CQ.Results: ANs was successfully pegylated and targeted to produce PMPANs,and obtained good targeted anti-tumor ability,with higher tumor cell uptake rate,cell killing rate,and inhibition of cell proliferation.In the meanwhile,PMPANs effectively degraded AR through ubiquitin proteasome(UPS)pathway,blocked AR signaling pathway to the greatest extent,and showed significant anti-tumor effects(endocrine therapy)at the cellular and animal levels,improving the dilemma of non-response to common CRPC endocrine therapy.In addition,PMPANs can induce mitochondrial damage and promote the occurrence of mitochondrial autophagy mediated by PINK1/Parkin.After being combined with CQ,autophagy is blocked,and CRPC cells cannot clear damaged mitochondria through mitochondrial autophagy mediated by PINK1/Parkin,resulting in abnormal mitochondria that cannot be degraded by lysosomes and accumulate in the cell,thereby releasing a large amount of mitochondrial DNA,further activating the c GAS-STING signaling pathway,promoting the differentiation of spleen immune cells and recruit them to infiltrate tumor tissues(CD4+/CD8+ T cells,NK cells).The secretion of related anti-tumor immune cytokines in serum increased significantly,which together played a powerful antitumor effect.Through the comparison of the final results,it was found that PMPANs + CQ had better anti-tumor effect than PMPANs alone,and AR degradation-mediated endocrine therapy and c GAS-STING pathway activation modulated immunotherapy were combined to achieve the functional promotion effect of 1+1 > 2 and effectively inhibit the progression of prostate cancer.Finally,no obvious systemic toxicity was found in blood biochemistry and tissue safety evaluation.Conclusion: We have successfully designed and synthesized arsenic nanosheets PMPANs nanoparticle with targeted function,which can efficiently degrade AR to improve the endocrine therapy effect of CRPC.Through the combined application of CQ to inhibit autophagy and activate c GAS-STING pathway,a large number of anti-tumor immune cells are recruited from peripheral blood to continuously penetrate into tumor tissues and kill tumor cells,realizing the synergistic effect of endocrine therapy and immunotherapy in PCa,and providing new ideas and strategies for the clinical treatment of CRPC. |
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