冬凌草甲素逆转BEL-7402/5-FU化疗耐药作用的体内研究

Oridonin A reverses the chemoresistance of BEL-7402/5-FU in vivo

目的探讨冬凌草甲素逆转人肝癌耐药细胞株BEL-7402/5-FU裸鼠移植瘤耐药效应及机制。方法取4~6周龄BALB/c雌性裸小鼠40只,皮下接种BEL-7402/5-FU细胞,建立BEL-7402/5-FU裸鼠移植瘤耐药模型,按照随机数字表法分成八组,即模型组,冬凌草甲素高、中、低剂量组,5-FU组和5-FU+冬凌草甲素高、中、低剂量组,每组5只。分组当天开始灌胃给药,模型组给予生理盐水灌胃,冬凌草甲素高、中、低剂量组给予75、50、25 mg/(kg·d)冬凌草甲素溶液灌胃。5-FU组给予20 mg/(kg·d)5-FU腹腔注射。5-FU+冬凌草甲素高、中、低剂量组给予20 mg/(kg·d)5-FU腹腔注射,联合75、50、25 mg/(kg·d)冬凌草甲素溶液灌胃,每日1次,连续5 d。观察各组瘤重及抑瘤率,实时荧光定量PCR法检测各组肿瘤组织中Yes相关蛋白(YAP)、具有PDZ结合域的转录共刺激因子(TAZ)的mRNA表达水平,Western blot法检测各组肿瘤组织中细胞周期蛋白D1(CyclinDl)、结缔组织生长因子(CTGF)、生存素(Survivin)、B淋巴细胞瘤-2(Bcl-2)蛋白表达水平。结果40只裸鼠均成瘤,成瘤率100%。和模型组比较,各组均有明显的抑瘤率[(1.079±0.307)g、(1.240±0.250)g、(1.386±0.329)g、(0.783±0.194)g、(0.382±0.214)g、(0.603±0.204)g、(0.897±0.317)g比(1.852±0.584)g,P<0.05或P<0.01],其中5-FU+冬凌草甲素高剂量组抑瘤率最为明显(P<0.01)。与模型组比较,5-FU组、5-FU+冬凌草甲素高、中、低剂量组的YAP和TAZ mRNA相对表达量均显著降低[YAP:(0.319±0.142)、(0.242±0.120)、(0.231±0.114)、(0.267±0.053)比(1.003±0.093);TAZ:(0.542±0.222)、(0.225±0.114)、(0.416±0.185)、(0.446±0.147)比(1.003±0.096),P<0.05或P<0.01],而5-FU+冬凌草甲素高剂量组的TAZ mRNA相对表达量降低最为明显(P<0.01)。与模型组相比,冬凌草甲素中剂量组、5-FU+冬凌草甲素高、中剂量组的CyclinDl、CTGF、Survivin、Bcl-2蛋白表达量不同程度降低[CyclinDl:(0.333±0.050)、(0.284±0.033)、(0.336±0.053)比(0.445±0.087);CTGF:(0.089±0.007)、(0.041±0.013)、(0.049±0.007)比(0.171±0.025);Survivin:(0.293±0.062)、(0.185±0.056)、(0.289±0.026)比(0.386±0.060);Bcl-2:(0.356±0.053)、(0.175±0.034)、(0.374±0.057)比(0.472±0.049),P<0.05或P<0.01],而5-FU+冬凌草甲素高剂量组的蛋白相对表达量显著降低(P<0.01)。结论冬凌草甲素可抑制BEL-7402/5-FU裸鼠移植瘤生长,其逆转耐药机制可能与降低YAP、TAZ的mRNA表达,降低CyclinDl、CTGF、Survivin、Bcl-2蛋白表达有关。

Objective To assess effects and explore the underlying molecular events of oridonin reversal of drug-resistant liver cancer cells(BEL-7402/5-FU)in nude mouse xenografts.Methods Forty female BALB/c mice of 4 to 6 weeks of age were subcutaneously inoculated with BEL-7402/5-FU cells to establish tumor cell xenografts and after that,these mice were randomly divided into eight groups(n=5 per group),i.e.,the model,oridonin at high,medium,and low doses,5-FU,and 5-FU+oridonin at a high,medium,or low dose.The model group of mice was given normal saline,while treatment groups of mice received oridonin at 75,50,or 25 mg/kg·d,5-FU at 20 mg/kg·d,or 5-FU at 20 mg/kg·d plus oridonin at 75,50,or 25 mg/kg·d once a week for 5 weeks.Mice were monitored daily and tumor xenografts were measured every three days.At the end of experiments,mice were killed using CO2 and the cervical translocation.Tumor xenografts were resected for the real-time fluorescent quantitative PCR analysis of YAP and TAZ mRNA levels or Western blot analysis of CyclinDl,CTGF,Survivin,and Bcl-2 proteins.Results The model showed 100%tumorigenesis rate(40/40 mice).The treatments suppressed tumor xenograft growth compared to the model group of mice(1.079±0.307,1.240±0.250,1.386±0.329,0.783±0.194,0.382±0.214,0.603±0.204,and 0.897±0.317 vs.1.852±0.584 g,respectively;P<0.05 or P<0.01),while the mice treated with 5-FU+high dose of oridonin had the most evident inhibitory rate(0.382±0.214 vs.1.852±0.584 g;P<0.01).Moreover,compared with those of the model mice,expression of YAP(0.319±0.142,0.242±0.120,0.231±0.114,and 0.267±0.053 vs.1.003±0.093)and TAZ(0.542±0.222,0.225±0.114,0.416±0.185,and 0.446±0.147 vs.1.003±0.096)mRNA was significantly reduced in mice received 5-FU and 5-FU+oridonin at a high,medium,or low dose(P<0.05 or P<0.01).Similarly,oridonin at a medium dose or 5-FU plus oridonin at a high or medium dose also significantly decreased expression of Cyclin Dl(0.333±0.050,0.284±0.033,and 0.336±0.053 vs.0.445±0.087),CTGF(0.089±0.007,0.041±0.013,and 0.049±0.007 vs.0.171±0.025),Survivin(0.293±0.062,0.185±0.056,and 0.289±0.026 vs.0.386±0.060),and Bcl-2(0.356±0.053,0.175±0.034,and 0.374±0.057 vs.0.472±0.049)proteins(P<0.05 or P<0.01).Conclusion Oridonin treatment can suppress growth of drug-resistant BEL-7402/5-FU cell xenografts in nude mice and oridonin reversal of liver cancer cell drug resistance may be attributed to the role of oridonin in the downregulated expression of YAP and TAZ mRNA and Cyclin Dl,CTGF,Survivin,and Bcl-2 proteins.