大黄素-8-O-β-D-葡萄糖苷联合去甲斑蝥素体内外抗肿瘤作用及其机制

Anti-tumor effect and mechanism of emodin-8-O-β-D-glucopyrano⁃side combined with norcantharidin in vitro and in vivo

目的探究大黄素-8-O-β-D-葡萄糖苷(EG)与去甲斑蝥素(NCTD)联用的抗肿瘤作用及机制。方法①EG和NCTD与人肝癌HepG2细胞和人肺癌A549细胞作用48 h。给药方案:EG和NCTD分别单用(终浓度均为60~960μmol·L-1)、同时联用(EG+NCTD,1∶1同时给药,终浓度分别为30~480μmol·L-1)、序贯联用方案Ⅰ(EG→NCTD,EG作用24 h后除去EG,加入NCTD继续作用24 h,终浓度均为60~960μmol·L-1)和序贯联用方案Ⅱ(NCTD→EG,NCTD作用24 h后除去NCTD,加入EG继续作用24 h,终浓度均为60~960μmol·L-1)。MTT法检测细胞存活率,并计算相应的联合指数(CI)值,判断药物联合作用效应。随后利用反向找靶技术并构建蛋白-蛋白互作(PPI)网络筛选两药联用潜在的关键靶点。流式细胞术分析EG和NCTD单用及NCTD→EG序贯联用对HepG2和A549细胞凋亡的影响,Western印迹法检测胱天蛋白酶3(CASP3)和活化CASP3蛋白表达。②建立肝癌H22移植瘤小鼠模型,设模型对照(给予PBS)、5-氟脲嘧啶(5-Fu)阳性对照(30 mg·kg^(-1))、EG单用(4 mg·kg^(-1))、NCTD单用(4 mg·kg^(-1))、两药同时联用(EG+NCTD,各2 mg·kg^(-1))及序贯联用(NCTD→EG,NCTD 4 mg·kg^(-1)6 d,EG 4 mg·kg^(-1)6 d)组,均ip给药,每天1次,共12 d。末次给药后24 h小鼠称重后处死,剥离移植瘤组织称重,计算肿瘤抑制率。结果①EG和NCTD单用、同时联用及2种方案序贯联用均呈浓度依赖性抑制HepG2和A549细胞存活(P<0.01),在一定浓度范围内具有协同作用。EG和NCTD单用及同时联用三者比较,NCTD单用IC50最低,同时联用未表现出更好的作用。与两者单用比较,NCTD→EG序贯联用IC50最低。基于反向找靶及PPI分析筛选出关键靶点CASP3,表明两药联用可能涉及细胞凋亡通路。流式细胞术和Western印迹实验结果表明,与EG和NCTD单用组相比,NCTD→EG序贯联用明显增加HepG2和A549细胞凋亡率(P<0.01),且活化CASP3蛋白表达增加(P<0.01),而CASP3蛋白表达无明显变化。②肝癌H22移植瘤模型小鼠体内实验结果表明,与模型对照组相比,NCTD单用、同时联用、NCTD→EG序贯联用和5-Fu阳性对照组瘤重均显著降低(P<0.05),抑瘤率分别为42.4%,47.8%,50.4%和69.0%;EG单用抑瘤作用不明显。同时联用组小鼠体重较模型对照组升高(P<0.05),5-Fu组显著降低(P<0.01)。结论EG与NCTD联用具有协同抗肿瘤作用,其机制与诱导肿瘤细胞凋亡有关。

OBJECTIVE To investigate the anti-tumor effects and mechanism of emodin-8-O-β-Dglucoside(EG)combined with norcantharidin(NCTD).METHODS①EG and NCTD were adminis⁃tered with human liver cancer HepG2 cells and human lung cancer A549 cells for 48 h.Administration schedule:EG and NCTD were used alone(the final concentration was 60-960μmol·L-1),EG and NCTD were used in combination(EG+NCTD,1∶1 ratio concomitant administration,the final concentra⁃tion was 30-480μmol·L-1),the sequential schemeⅠ(EG→NCTD,EG was removed after 24 h,NCTD was added for 24 h,the final concentration was 60-960μmol·L-1)and the sequential schemeⅡ(NCTD→EG,NCTD was removed after 24 h,EG was added for 24 h,the final concentration was 60-960μmol·L-1).MTT assay was used to detect the cell viability and the combination index(CI)value was converted to evaluate the interactions between the two drugs.Potential key targets for the combination of the two drugs were screened using reverse targeted fishing technology and protein-protein interaction(PPI)network.Flow cytometry was used to analyze the effects of EG,NCTD and NCTD→EG administration on tumor cell apoptosis.The expressions of caspase 3(CASP3)and cleaved CASP3 were detected by Western blotting.②The H22 liver tumor-bearing model mice were divided into the model control(PBS),5-fluoro⁃uracil(5-Fu)positive control(30 mg·kg^(-1)),EG alone(4 mg·kg^(-1)),NCTD alone(4 mg·kg^(-1)),concomitant administration of EG and NCTD(EG+NCTD,each 2 mg·kg^(-1)),and sequential administration(NCTD→EG,NCTD 4 mg·kg^(-1)6 d,EG 4 mg·kg^(-1)6 d)groups,all ip administered,once a day,for 12 d.24 h after the last administration,the mice were weighed and sacrificed.The transplanted tumor tissues were stripped and the tumor inhibition rate was calculated.RESULTS①EG,NCTD,concomitant and sequen⁃tial administration decreased the viability of HepG2 and A549 cells in a concentration-dependent manner(P<0.01),with synergistic effects at some concentrations.Compared with EG,NCTD alone and EG+NCTD,NCTD alone had the lowest IC50,while EG+NCTD showed no better effect.Compared with the two drugs alone,NCTD→EG sequential administration had the lowest IC50.The key target CASP3 was screened out based on reverse target fishing and PPI analysis of EG and NCTD.Flow cytometry and Western blotting showed that NCTD→EG sequential administration significantly increased the apoptosis rates of HepG2 and A549 cells(P<0.01),and that cleaved CASP3 expressions were increased(P<0.01),but CASP3 expression was not significantly changed.②The liver cancer H22 tumor mouse model showed that compared with the model control group,the tumor mass of NCTD,NCTD+EG,NCTD→EG and 5-Fu positive control groups were significantly decreased(P<0.05),and the tumor inhibitory rates were 42.4%,47.8%,50.4%and 69.0%,respectively.EG alone had no obvious antitumor effect.Compared with model control group,the body mass of mice in NCTD+EG group was increased(P<0.05),but was significantly decreased in the 5-Fu group(P<0.01).CONCLUSION Combination of EG and NCTD shows anti-tumor activity synergistically via induction of tumor cell apoptosis.