摘要
研发能阻断免疫检查点程序性死亡分子1/程序性死亡配体1(PD-1/PD-L1)相互作用的小分子药物是下一代肿瘤免疫疗法的新方向。本研究对金丝桃苷下调非小细胞肺癌(NSCLC)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相互作用分析检测金丝桃苷对NSCLC细胞内和细胞膜表面PD-L1蛋白表达水平的影响;荧光定量PCR和Western blot检测金丝桃苷对PD-L1 mRNA和c-Myc蛋白的影响;细胞阻抗法和结晶紫法检测金丝桃苷对共培养T细胞杀伤肿瘤细胞的效果;Lewis荷瘤小鼠检测金丝桃苷的体内抑瘤效果。结果表明,金丝桃苷能分别以浓度依赖性和时间依赖性方式下调H1975、HCC827细胞中以及细胞膜表面PD-L1的水平,这与其抑制NSCLC细胞中PD-L1 mRNA的表达水平有关。机制研究发现,金丝桃苷可降低H1975、HCC827细胞中转录因子c-Myc的蛋白表达量,从而在转录水平下调PD-L1的表达。此外,金丝桃苷可促进共培养的T细胞对H1975细胞的杀伤作用。小鼠荷瘤实验证实(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),金丝桃苷可显著抑制C57BL/6小鼠中Lewis移植瘤的生长。25 mg·kg^(-1)金丝桃苷处理荷瘤小鼠后,可使瘤体积减小48.3%。以上结果说明,金丝桃苷通过降低NSCLC细胞中c-Myc的蛋白表达量,在转录水平抑制PD-L1的表达,从而发挥抗肿瘤作用。本研究为金丝桃苷在肿瘤免疫治疗中的潜在应用奠定了基础。
Blocking the binding of programmed death 1(PD-1)on the T cells and programmed death ligand 1(PD-L1)on the tumor cells has become a hotspot in the field of tumor immunotherapy.Small-molecule checkpoint inhibitor targeting PD-1/PD-L1 axis is the new direction of tumor immunotherapy.In the present study,we investigated the anti-tumor role of hyperoside by regulating the PD-L1 level in non-small cell lung cancer(NSCLC).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 human T cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment.The antitumor effect of hyperoside in vivo was examined by C57BL/6 mice bearing Lewis xenograft tumor.Western blot and flow cytometry assay showed that hyperoside significantly downregulated the abundance of PD-L1 in H1975 and HCC827 cells in dose-and time-dependent manner.PD-1/PD-L1 binding assay revealed that hyperoside reduced the binding of tumor cells to recombinant PD-1 protein.In addition,hyperoside decreased the abundance of c-Myc,a key transcriptional regulator of PD-L1,in H1975 and HCC827 cells.Cell impedance and crystal violet staining indicated that hyperoside enhanced the killing activity of co-cultured T cells toward tumor cells.Animal experiments(all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences)revealed that hyperoside treatment displayed significant suppression in the growth of Lewis tumor xenografts in C57BL/6 mice with an inhibition rate of 48.3%at 25 mg·kg^(-1).Our results demonstrate that hyperoside exerts its anti-NSCLC activity by reducing the PD-L1 level.Our study provides an important material basis and scientific basis for developing hyperoside into a new small molecule drug for tumor immunotherapy.
作者
董靖雯
况泽安
殷明晓
刘晓嘉
刘阳
邓洪斌
DONG Jing-wen;KUANG Ze-an;YIN Ming-xiao;LIU Xiao-jia;LIU Yang;DENG Hong-bin(Institute of Medicinal Biotechnology,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China)
出处
《药学学报》
CAS
CSCD
北大核心
2021年第10期2817-2824,共8页
Acta Pharmaceutica Sinica
基金
国家自然科学基金资助项目(81973366,81773782)。
