茜草对人神经胶质瘤U87细胞的生长及丙酮酸代谢的影响

Effects of Radix Rubiae on Cell Growth and Pyruvate Metabolism of Human Glioblastoma U87 Cell Line

目的考察茜草对人神经胶质瘤U87细胞生长的影响,并从抗肿瘤作用分子机制及丙酮酸代谢角度探讨作用机理。方法体外培养U87细胞,以MTT法考察茜草不同提取物对U87细胞的生长抑制作用;人全基因组表达谱芯片初步筛选茜草醇提物抑制U87细胞的可能机制;逆转录PCR验证部分差异表达基因;碘化丙啶(PI)单染法检测细胞周期分布;分光光度法检测细胞内H_2O_2含量、丙酮酸含量、乳酸含量、Caspase-3活力、丙酮酸激酶(PK)活力、乳酸脱氢酶(LDH)活力、苹果酸脱氢酶(MDH)活力、琥珀酸脱氢酶(SDH)活力;Western Blot检测超氧化物歧化酶1(SOD1)、过氧化氢酶(Catalase)、p-Histone H2A.X(Ser139)、p-乳腺癌易感蛋白1(BRCA1)(Ser1524)、人mutS同源蛋白6(MSH6)、增殖细胞核抗原(PCNA)、单羧酸转运蛋白1(MCT1)的蛋白表达。结果以不同剂量的茜草醇提物、水提物(终浓度分别为200,100,50,25,12.5 mg·L^(-1))作用于U87细胞24,48,72 h后,U87细胞生长受到明显抑制,抑制作用具有剂量依赖和时间依赖性,醇提物优于水提物。茜草醇提物50 mg·L^(-1)作用于U87 24 h后,表达谱芯片结果揭示其作用机制与细胞周期、凋亡、氧化-还原平衡、DNA损伤及修复、单羧酸转运等有关。茜草醇提物100,50 mg·L^(-1)处理U87 24 h进行后续实验,逆转录PCR验证MSH6、Catalase、BAX、硫氧还蛋白相互作用蛋白(TXNIP)、PCNA mRNA表达与芯片结果一致,而DNA复制解旋酶/核酸酶2(DNA2)mRNA表达仅100 mg·L^(-1)组与芯片结果相符。茜草醇提物可使U87细胞阻滞在S期、提高Caspase-3活力;还增加了细胞内H_2O_2含量,而Catalase和SOD1蛋白表达减少;细胞内DNA双链断裂的标志p-Histone H2A.X(Ser139)蛋白表达增加,DNA修复相关的p-BRCA1(Ser1524)、MSH6、PCNA蛋白表达减少。茜草醇提物处理后,U87细胞内丙酮酸含量减少、乳酸含量增加;PK、LDH、MDH、SDH活力增加、MCT1蛋白表达减少。结论茜草能有效抑制人神经胶质瘤U87细胞在体外的生长活力,作用呈现一定剂量依赖和时间依赖,且醇提优于水提。其作用机制涉及多方面,与细胞周期阻滞、Caspase-3活化、影响氧化-还原平衡、DNA损伤及修复、细胞内丙酮酸代谢有关。茜草是一味具有开发潜力的抗肿瘤中药。

Objective To investigate the inhibitory effects of Radix Rubiae on the growth of human glioblastoma U87 cell line and to explore the underlying mechanism from the perspectives of conventional experimental anticancer mechanism and pyruvate metabolism. Methods U87 cells cultured in vitro were used for the study. The effective ingredients were extracted from Radix Rubiae with water or ethanol. MTT assay was applied to determine the growth inhibition. To explore the preliminary mechanism, the whole human genome oligo microarray was used. Several differentially expressed genes found by microarray were further verified by reverse transcription PCR（RT- PCR）. Cell cycle distribution was analyzed by propidium iodide（PI）staining and flow cytometry. Spectrophotometry was performed to measure the intracellular content of H2O2,pyruvate and lactate as well as the activities of Caspase- 3, pyruvate kinase（PK）, lactate dehydrogenase（LDH）, malate dehydrogenase（MDH） and succinate dehydrogenase（SDH）.Western blot was employed to detect the protein expression level of superoxide dismutase 1（SOD1）, Catalase,p- Histone H2 A.X（Ser139）, p- breast cancer type 1 susceptibility protein（BRCA1）（Ser1524）, mut S homolog 6（MSH6）,proliferating cell nuclear antigen（PCNA）,and monocarboxylate transporter 1（MCT1）. Results After treating U87 cells with different final concentrations（200, 100, 50, 25, 12.5 mg·L^-1）of the water extract and ethanol extract of Radix Rubiae for 24, 48 and 72 h, the cell viability was significantly reduced in a time- and dose- dependent manner. And the ethanol extract had stronger inhibitory effect than water extract. After the cells were treated with ethanol extract for 24 h in final concentration of 50 mg·L^-1,the results of microarray assay showed that the mechanism of growth inhibition was related to cell cycle distribution,apoptosis,redox balance,DNA damage,DNA damage repair and monocarboxylic acid transport,etc. In the following experiments,the cells were treated with ethanol extract respectively in final concentration of 100 and 50 mg·L^（-1） for 24 h. The regulation of m RNA expression of MSH6,Catalase,BCL2- associated X protein（BAX）,thioredoxin interacting protein（TXNIP） and PCNA showed by RT- PCR was consistent with the microarray data. However,the change of DNA replication helicase/ nuclease 2（DNA2） mRNA expression only in cells treated with 100 mg·L^（-1） of ethanol extract was similar to the microarray results. The ethanol extract of Radix Rubiae could induce S phase arrest and up- regulation of Caspase- 3 activity,up- regulate intracellular H2O2 content and down- regulate protein expression of Catalase and SOD1 in U87 cells. The increased p- Histone H2 A.X（Ser139） and reduced p- BRCA1（Ser1524）,MSH6 and PCNA protein level demonstrated that treatment of Radix Rubiae induced DNA double- strand breaks and attenuated DNA damage repair. Moreover,the intracellular content of pyruvate was decreased while that of lactate was increased, the intracellular PK, LDH, MDH, SDH activity was up- regulated and MCT1 protein expression was down- regulated. Conclusion These data demonstrated that Radix Rubiae significantly inhibited the growth of human glioblastoma U87 cell line in vitro in a time- and dose- dependent manner. Extracting with ethanol was recommended since it had stronger inhibitory effect. The underlying mechanism was implicated in diverse aspects including cell cycle arrest, Caspase- 3 activation, impact on redox balance, DNA damage and repair,and cellular pyruvate metabolism. The present study revealed a promising value of Radix Rubiae as an anticancer herb in treating human glioblastoma.