奥拉帕利联合IL-1β抑制剂对HRD阴性卵巢上皮性癌细胞生长的抑制作用  

IL-1βinhibitor sensitizes to olaparib in homologous recombination deficiency proficient ovarian cancer cells

作　　者：许君芬[1] 岑以璇 汤桑桑 任妍 吕卫国[1] Junfen Xu;Yixuan Cen;Sangsang Tang;Yan Ren;Weiguo Lyu(Department of Gynecologic Oncology,Women′s Hospital,Zhejiang University School of Medicine,Women′s Reproductive Health Laboratory of Zhejiang Province,Hangzhou 310006,China)

机构地区：[1]浙江大学医学院附属妇产科医院妇瘤科浙江省女性生殖健康研究重点实验室,杭州310006

出　　处：《中华妇产科杂志》2022年第7期519-529,共11页Chinese Journal of Obstetrics and Gynecology

基　　金：北京康华中西医发展基金会妇科肿瘤专项研究基金(KH-2021-LLZX-016)。

摘　　要：目的:探讨聚二磷酸腺苷核糖聚合酶(PARP)抑制剂联合白细胞介素1β(IL-1β)抑制剂对同源重组修复缺陷(HRD)阴性卵巢上皮性癌(卵巢癌)细胞的抑制作用。方法:(1)使用HRD阴性的卵巢癌细胞系OVCAR3、CAOV3细胞,先分别给予PARP抑制剂奥拉帕利(122 μmol/L)和二甲基亚砜(作为对照)处理OVCAR3细胞,RNA测序及筛选差异表达基因;随后采用酶联免疫吸附试验(ELISA)和蛋白印迹(western blot)法验证奥拉帕利处理后OVCAR3、CAOV3细胞中IL-1β蛋白的表达。(2)根据不同浓度(0、1.25、2.5、5、10、20、40、80、160、320 μmol/L)IL-1β抑制剂diacerein(为蒽醌类化合物)在OVCAR3和CAOV3细胞中的药物剂量反应曲线,确定两种细胞IL-1β抑制剂的50%抑制浓度(IC 50)。细胞实验分为4组,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组,活细胞计数(CCK-8)法检测4组OVCAR3、CAOV3细胞的存活率。(3)将稳定表达荧光素酶基因luciferase的OVCAR3细胞(OVCAR3-Luc细胞)按照1×10 7个/只接种于裸鼠腹腔中,建立裸鼠腹腔移植瘤模型。动物实验将16只成瘤裸鼠采用随机表法随机分为4组,对照组、奥拉帕利组、IL-1β抑制剂组、奥拉帕利+IL-1β抑制剂组,每组4只。采用荧光素酶小动物活体成像测定4组裸鼠移植瘤的荧光信号强度,免疫组化法检测移植瘤组织中增殖相关核抗原(Ki-67)蛋白的表达。(4)药物毒副反应:腹腔注射后第29天,裸鼠称重后取其外周血进行血常规和肝肾功能检测;随后处死裸鼠,取裸鼠重要器官进行HE染色评估药物对器官的损害情况。 结果:(1)RNA测序及差异表达基因筛选,共获得25个显著差异表达基因,尤以IL-1β mRNA的表达差异最显著。ELISA法检测显示,奥拉帕利处理后OVCAR3、CAOV3细胞分泌的IL-1β蛋白含量分别为(36.2±3.5)和(49.5±3.5)pg/ml,均显著高于对照OVCAR3、CAOV3细胞[分别为(5.3±0.7)和(14.7±0.7)pg/ml;P均<0.001];western blot检测显示,�Objective To investigate the inhibitory effect of combined strategy of poly adenosine diphosphate ribose polymerase(PARP)inhibitor and interleukin-1β(IL-1β)inhibitor on homologous recombination deficiency(HRD)-proficient ovarian cancer cells.Methods(1)HRD-proficient ovarian cancer cell lines OVCAR3 and CAOV3 were treated with PARP inhibitor olaparib.Screening by RNA sequencing analysis,the expression level of IL-1βwas validated by enzyme-linked immunosorbent assay(ELISA)and western blot.(2)The dose-response curves of IL-1βinhibitor diacerein were evaluated by cell counting kit-8(CCK-8)assays in OVCAR3 and CAOV3 cells.CCK-8 assays were further applied to determine the viabilities of OVCAR3 and CAOV3 cells.(3)To evaluate the synergistic effects of olaparib and IL-1βinhibitor in vivo,the transplanted ovarian cancer model was constructed.BALB/c-nude mice(n=16)were injected intraperitoneally with 1×107 OVACR3 cells labelled with luciferase(OVCAR3-Luc).Immunohistochemistry(IHC)assay was performed to determine nuclear antigen associated with cell proliferation(Ki-67)expression.(4)Blood routine tests,kidney and liver function tests were performed to analyze the toxic reaction of different drug treatments.The potential drug-induced injuries of vital organs including heart,liver,spleen,lungs and kidneys of nude mice were determined by hematoxylin-eosin(HE)staining.Results(1)The RNA sequencing results showed that the mRNA level of IL-1βwas the most significantly increased among the 25 differentially expressed genes in OVCAR3 cells treated with olaparib,compared to the negative control group.Olaparib treatment significantly promoted the secretion and expression of IL-1βprotein in both OVACR3 and CAOV3 cells by ELISA[(36.2±3.5)and(49.5±3.5)pg/ml,respectively;all P<0.001]and western bolt(2.87±0.37 and 2.05±0.08,respectively;all P<0.01).(2)The half maximal inhibitory concentration(IC50)value of IL-1βinhibitor was determined as follows:75μmol/L for OVACR3 cells and 100μmol/L for CAOV3 cells.The treatments were div

关 键 词：卵巢肿瘤 酞嗪类 哌嗪类 白细胞介素1Β 细胞系 肿瘤 

分 类 号：R737.31[医药卫生—肿瘤]