18β-甘草次酸诱导人乳腺癌细胞凋亡及其细胞内Ca^(2+)水平的变化

18β-glycyrrhetinic acid-induced apoptosis and changed intracellular Ca^(2+) concentration in human breast carcinoma cells

背景与目的:18-甘草次酸是甘草的重要成分,近年来的研究发现18-甘草次酸具有抑制人淋巴细胞性白血病、肝癌和肺癌的细胞增殖。本研究探讨18-甘草次酸对人乳腺癌MCF-7细胞诱导凋亡的作用。目前研究已证实细胞内游离Ca2+浓度([Ca2+]i)的动态变化在诱发细胞凋亡过程的多个环节中起重要作用,因此本研究也探讨由18-甘草次酸诱导的MCF-7细胞凋亡发生与[Ca2+]i变化的关系。方法:用50~250μmol/L浓度梯度的18β-甘草次酸处理MCF-7细胞24h,用MTT比色法测定细胞增殖能力。100μmol/L和150μmol/L 18β-甘草次酸处理细胞24 h,用末端脱氧核苷酸转移酶介导dUTP末端标记法、Annexin V流式细胞仪法和单细胞凝胶电泳法检测凋亡细胞。150μmol/L 18β-甘草次酸处理细胞24 h,用Fura-2荧光负载方法测定[Ca2+]i的变化。分别用100μmol/L BAPTA-AM和0.5 mmol/L EGTA与150μmol/L 18β-甘草次酸联合处理MCF-7细胞24 h,用单细胞凝胶电泳法检测凋亡细胞。结果:从100μmol/L 18β-甘草次酸浓度起对MCF-7细胞的增殖抑制率显著升高(P<0.01和P<0.05),呈剂量依赖性,半增殖抑制浓度(IC 50)为234.33μmol/L。100μmol/L和150μmol/L 18β-甘草次酸使细胞凋亡率显著升高(P<0.01和P<0.05);18β-甘草次酸处理组的[Ca2+]i也明显高于对照组(P<0.05)。18β-甘草次酸与BAPTA-AM联合处理组和18β-甘草次酸与EGTA联合处理组的凋亡率均明显低于单纯的150μmol/L18β-甘草次酸处理组(P<0.05和P<0.01)。结论:18-甘草次酸具有抑制MCF-7细胞增殖和诱导其凋亡的作用,而18-甘草次酸诱导MCF-7细胞凋亡的作用在抑制该细胞增殖方面起主要作用。18-甘草次酸诱导MCF-7细胞凋亡依赖于细胞内Ca2+水平上调,而细胞外Ca2+内流是导致细胞内Ca2+水平上调的原因之一。

Background and purpose： 18β-glyeyrrhetinic acid （GA） is one of the important components of glycyrrhiza. Recent years, studies showed that CA has the effect of proliferation inhibition in human acute lymphoblastic leukemia ceils, human liver carcinoma and lung cancer cells. We investigated the effects of GA on induction of apoptosis in human breast carcinoma （MCF-7） cells. The previous studies have demonstrated that the dynamic change of intracellular free Ca^2＋ concentration（ [ Ca^2＋ ] i） plays important roles in many links of apoptosis-induced process. Therefore we also researched the relationship between CA-induced apoptosis and [ Ca^2＋ ] i in MCF-7 cells. Methods： After MCF-7 cells were treated with 50- 250 p, mol/L CA for 24 h, cell viability for proliferation was assessed by MTT assay. MCF-7 cells treated with 100 μmol/L and 150 μmoL/L CA for 24 h, the apoptotic rates in MCF7 cells were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method, flow cytometry with Annexin V/ propidium iodide fluorescent stain and single cell gel eleetrophoresis assay （ SCGE）. For cells treated with 150 p, mol/L GA for 24 h, [ Ca2 ＋ ] i was measured by Fure-2 fluo- rescein load method. For cells treated with 150μmol/L GA combined with 100 μmol/L BAPTA-AM or 0.5 mmol/L EGTA for 24 h, cell apoptosis were examined by SCGE. Results： For cells treated with GA from 100 μmol/L to 250μmol/L, the rate of proliferative inhibition was increased significantly （P 〈 0.01 and P 〈 O. 05）. The degrees of inhibition were dose-dependent. IC50 of CA-inhibited proliferation was 234.33μmol/L. For cells treated with 100 μmol/L and 150 μmol/L GA, the rates of cell apoptosis rose remarkably （P 〈0. O1 and P 〈 0.05 ）. [Ca^2＋]i after treatment with 150μmol/L CA was much higher than that of control（ P 〈 0.05）. The rate of cell apoptosis after treatment with the combination of CA and BAP- TA-AM or EGTA was markedly lower than that treated with a single GA （ P 〈 0.05 and P 〈 0. 01 ）. Conclusions： GA has the effects of proliferation inhibition and apoptosis induction in MCF-7 cells. GA-inducted apoptosis plays main role in inhibiting MCF-7 cell proliferation. The apoptosis induced by GA depends on rise of [ Ca^2＋ ] i. It is the part cause of the rise of [ Ca^2＋ ] i that extracellular free Ca^2＋ flowed into MCF-7 cell.