靶向USP2下调PD-L1表达的丹酚酸B抗肿瘤免疫作用研究

Salvianolic acid B exerts its anti-tumor immunity by targeting USP2 and reducing the PD-L1 level

以程序性死亡配体1/程序性死亡受体1(programmed cell death ligand 1/programmed cell death protein 1,PD-L1/PD-1)免疫检查点阻断治疗为代表的肿瘤免疫疗法在临床上取得了令人鼓舞的治疗效果。小分子肿瘤免疫治疗药物与免疫检查点抗体药物的联用,为肿瘤治疗提供了新策略。因此,研发能阻断PD-L1/PD-1免疫检查点相互作用的小分子药物是下一代肿瘤免疫疗法的新方向。本研究对丹酚酸B(salvianolic acid B,SAB)下调肿瘤细胞中PD-L1表达发挥抗肿瘤作用及机制进行了研究。利用Western blot、流式细胞术、PD-1/PD-L1相互作用分析SAB对结肠癌细胞RKO和前列腺癌细胞PC3细胞内和膜表面PD-L1蛋白表达水平的影响;荧光定量PCR检测SAB对PD-L1 mRNA的影响;细胞阻抗法和结晶紫法检测SAB对人类外周血单核细胞(peripheral blood mononuclear cell,PBMC)杀伤肿瘤细胞的效果;表面等离子共振技术分析SAB与泛素羧基末端水解酶2(ubiquitin carboxyl-terminal hydrolase 2,USP2)的直接相互作用;MC38荷瘤小鼠(所有动物实验均遵循中国医学科学院医药生物技术研究所伦理委员会的规定)检测SAB的体内抑瘤效果。结果表明,SAB能分别以浓度依赖性和时间依赖性方式下调RKO、PC3细胞中以及细胞膜表面PD-L1的水平,这与SAB抑制肿瘤细胞中去泛素化酶USP2的活性有关。机制研究发现,SAB可与USP2发生直接相互作用并抑制其去泛素化酶活性,从而促进PD-L1发生泛素-蛋白酶体途径降解。此外,SAB可促进共培养的PBMC对RKO细胞的杀伤作用。小鼠荷瘤实验证实,SAB可显著抑制C57BL/6小鼠中MC38移植瘤的生长。20 mg·kg^(-1) SAB处理荷瘤小鼠后,可使瘤体积减少63.2%。以上结果说明,SAB通过直接结合USP2并抑制其活性,促进PD-L1发生泛素-蛋白酶体途径降解,从而发挥抗肿瘤作用。本研究为将SAB研发成靶向USP2-PD-L1轴的小分子肿瘤免疫治疗药物奠定了基础。

With the development of small-molecule immunotherapy drugs,its combination with the programmed cell death ligand 1/programmed cell death protein 1(PD-L1/PD-1)antibodies would provide a new opportunity for cancer treatment.Therefore,targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity and considered as the next generation of tumor immunotherapy.In the present study,we investigated the anti-tumor role of salvianolic acid B(SAB)by regulating the PD-L1 level in tumors.Changes of total PD-L1 and membrane PD-L1 levels were determined by Western blot,flow cytometry and PD-1/PD-L1 interaction assays.The expression of mRNA level of PD-L1 was detected by real-time PCR.The cytotoxicity of activated peripheral blood mononuclear cell(PBMC)cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment.Surface plasma resonance technique was used to analyze the direct interaction between SAB and ubiquitin carboxyl-terminal hydrolase 2(USP2).The antitumor effect of SAB in vivo was examined by C57BL/6 mice bearing MC38 xenograft tumor(all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences).Western blot and flow cytometry assay showed that SAB can significantly downregulate the abundance of PD-L1 in RKO and PC3 cells in dose-and time-dependent manner.PD-1/PD-L1 binding assay revealed that SAB reduces the binding of tumor cells to recombinant PD-1 protein.Mechanism studies revealed that SAB can bind directly to USP2 protein and inhibit its activity,thus promote the ubiquitinproteasome pathway degradation of PD-L1 proteins.In addition,Cell impedance and crystal violet staining indicated that SAB enhances the killing activity of co-cultured PBMC cells toward tumor cells.MC38 tumor transplanted mouse experiments revealed that SAB treatment displayed significant suppression in the growth of MC38 tumor xenografts in C57BL/6 mice with an inhibition rate of 63.2%at 20 mg·kg^(-1).Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level.Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.