Department of Oncology and Hematology, University Hospital of Berlin, Berlin, Germany.
Anticancer Res. 2012 May;32(5):1627-37.
Metformin appears to interfere directly with cell proliferation and apoptosis in cancer cells in a non-insulin-mediated manner. One of the key mechanisms of metformin's action is the activation of adenosine monophosphate activated protein kinase (AMPK). AMPK is linked with the phosphatidylinositol 3-kinase (PI3K)/ phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) pathway and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) cascades--all known for being frequently dysregulated in breast cancer. Therefore, simultaneously targeting AMPK through metformin and the PI3K/AKT/mTOR pathway by an mTOR inhibitor could become a therapeutic approach. The aim of this study was to evaluate the anticancer effect of metformin alone and in combination with chemotherapeutic drugs and the mTOR inhibitor RAD001.
The proliferation of breast cancer cells was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; and the cell apoptosis with enzyme-linked immunosorbent assay (ELISA). Gene expression at the protein level was determined by western blot.
We tested metformin alone and in combination with RAD001 and/or chemotherapeutic agents (carboplatin, paclitaxel and doxorubicin, respectively) on several human breast cancer cell lines with respect to cell proliferation, apoptosis and autophagy. Metformin alone inhibited cell proliferation and induced apoptosis in different breast cancer cell lines (ERα-positive, HER2-positive, and triple-negative). The cytotoxic effect of metformin was more remarkable in triple-negative breast cancer cell lines than in other cell lines. The cell apoptosis induced by metformin is, at least partly, caspase-dependent and apoptosis inducing factor (AIF)-dependent. Interestingly, we demonstrated that metformin induced cell autophagy. Inhibiting autophagy with chloroquine, enhanced the treatment efficacy of metformin, indicating that autophagy induced by metformin may protect breast cancer cells from apoptosis. We further demonstrated that co-administration of metformin with chemotherapeutic agents and RAD001 intensified the inhibition of cell proliferation. The analysis of cell cycle-regulating proteins cyclin D, cyclin E and p27 by western blot indicated that the synergistic inhibition of G1 phase of the cell cycle by the combination treatment of metformin, chemotherapeutic drugs and/or RAD001 contributed to the synergistic inhibition of cell proliferation.
Our investigation provides a rationale for the clinical application of metformin within treatment regimens for breast cancer.
二甲双胍似乎通过非胰岛素介导的方式直接干扰癌细胞的增殖和凋亡。二甲双胍作用的一个关键机制是激活腺苷单磷酸激活蛋白激酶（AMPK）。AMPK 与磷酸肌醇 3-激酶（PI3K）/磷酸酶和张力蛋白同源物（PTEN）/蛋白激酶 B（AKT）途径和丝裂原激活蛋白激酶（MAPK）/细胞外信号调节激酶（ERK）级联反应相关，所有这些途径在乳腺癌中经常失调。因此，通过二甲双胍同时靶向 AMPK 以及通过 mTOR 抑制剂靶向 PI3K/AKT/mTOR 途径可能成为一种治疗方法。本研究旨在评估二甲双胍单独使用以及与化疗药物和 mTOR 抑制剂 RAD001 联合使用的抗癌作用。
使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定法测量乳腺癌细胞的增殖；通过酶联免疫吸附测定（ELISA）测量细胞凋亡。通过 Western blot 确定蛋白质水平的基因表达。
我们在几种人乳腺癌细胞系中单独测试了二甲双胍以及与 RAD001 和/或化疗药物（分别为卡铂、紫杉醇和多柔比星）联合使用，以评估细胞增殖、凋亡和自噬。二甲双胍单独使用可抑制不同乳腺癌细胞系（ERα 阳性、HER2 阳性和三阴性）的细胞增殖并诱导细胞凋亡。二甲双胍对三阴性乳腺癌细胞系的细胞毒性作用比其他细胞系更明显。二甲双胍诱导的细胞凋亡至少部分依赖于半胱天冬酶和凋亡诱导因子（AIF）。有趣的是，我们证明二甲双胍诱导细胞自噬。用氯喹抑制自噬增强了二甲双胍的治疗效果，表明二甲双胍诱导的自噬可能使乳腺癌细胞免于凋亡。我们进一步证明，二甲双胍与化疗药物和 RAD001 联合使用可增强对细胞增殖的抑制作用。通过 Western blot 分析细胞周期调节蛋白周期蛋白 D、周期蛋白 E 和 p27 的表达，表明二甲双胍、化疗药物和/或 RAD001 的联合治疗协同抑制细胞周期 G1 期，从而协同抑制细胞增殖。
我们的研究为临床应用二甲双胍治疗乳腺癌提供了依据。
