竹节香附素A通过抑制程序性细胞死亡配体1调节前列腺癌小鼠肿瘤免疫并发挥抗肿瘤作用

Raddeanin A regulates tumor immunity and exerts anti-tumor effects in prostate cancer mice by inhibiting programmed cell death ligand 1

目的:探讨竹节香附素A(RA)对前列腺癌移植瘤模型小鼠的治疗效果及潜在作用机制。方法:(1)采用Western blot实验检测不同浓度RA(0、0.5、1、2和4μmol/L)对前列腺癌细胞系PC-3、DU145和RM-1中程序性细胞死亡配体1(PD-L1)蛋白表达的影响。(2)将30只C57BL/6J小鼠随机分为空白对照组、低剂量RA组和高剂量RA组,每组10只。低、高剂量RA组小鼠分别腹腔注射2和4 mg/kg RA,连续给药24 d。记录小鼠体重,统计各组小鼠肿瘤体积和瘤重;采用免疫组化实验检测小鼠肿瘤组织中Ki67和PD-L1蛋白表达;通过流式细胞术检测肿瘤组织中CD8^(+)T细胞、CD4^(+)T细胞和调节性T细胞(Treg)比例,以及干扰素γ(IFN-γ)和颗粒酶B(GzmB)水平。结果:(1)RA处理显著降低PC-3、DU145和RM-1细胞中PD-L1表达水平(P<0.05或P<0.01)。(2)在体内实验中,RA处理组小鼠的肿瘤体积和重量显著减小,同时肿瘤组织中Ki67和PD-L1的表达水平显著降低(P<0.05或P<0.01)。此外,RA处理显著增加小鼠肿瘤内CD8^(+)T细胞和CD4^(+)T细胞的比例,提高IFN-γ和GzmB水平,同时减少活化的Treg数量(P<0.05或P<0.01)。结论:RA对前列腺癌小鼠肿瘤生长具有较好抑制作用,其机制可能与抑制PD-L1表达、增加肿瘤浸润T细胞及抑制Treg有关。

AIM:To investigate the therapeutic effect of raddeanin A(RA)on prostate cancer xenograft mouse model,and to explore its potential mechanisms.METHODS:(1)Western blot analysis was used to investigate the effects of different concentrations(0,0.5,1,2 and 4μmol/L)of RA on the expression of programmed cell death ligand 1(PD-L1)protein in prostate cancer cell lines PC-3,DU145 and RM-1.(2)Thirty C57BL/6J mice were randomly divided into blank group,low-dose RA group,and high-dose RA group,with 10 mice in each group.The mice in low-and high-dose RA groups were intraperitoneally injected with 2 and 4 mg/kg RA continuously for 24 d,respectively.Mouse body weight was recorded,and tumor volume and weight were measured.Immunohistochemistry experiments were conducted to detect the expression of Ki67 and PD-L1 proteins in mouse tumor tissues.Flow cytometry was used to determine the percentages of CD8^(+)T cells,CD4^(+)T cells and regulatory T cells(Treg),as well as the levels of interferon-γ(IFN-γ)and granzyme B(GzmB)in tumor tissues.RESULTS:Treatment with RA significantly reduced the expression of PD-L1 in PC-3,DU145 and RM-1 cells(P<0.05 or P<0.01).In vivo experiments showed that RA treatment led to significant decreases in tumor volume and weight(P<0.05 or P<0.01).Additionally,the expression levels of Ki67 and PD-L1 in tumor tissues were significantly reduced(P<0.05 or P<0.01).Furthermore,RA treatment significantly increased the percentages of CD8^(+)T cells and CD4^(+)T cells within mouse tumors,elevated the levels of IFN-γand GzmB,and reduced the number of activated Treg(P<0.05 or P<0.01).CONCLUSION:The RA exhibits potent inhibitory effects on tumor growth in a prostate cancer xenograft mouse model.Its mechanism may be associated with the inhibition of PD-L1 expression,increased infiltration of tumor-infiltrating T cells,and suppression of Treg.