藤梨根提取物通过Akt-mTOR介导的自噬信号通路抑制乳腺癌细胞增殖、迁移、侵袭和糖酵解

Actinidia Chinensis Root Extract Inhibits the Proliferation, Migration, Invasion and Glycolysis of Breast Cancer Cells Through the Akt-mTOR Mediated Autophagy Signaling Pathway

关键词:  藤梨根提取物  乳腺癌  增殖  迁移  侵袭  糖酵解  Akt-mTOR

Key words:  Actinidia chinensis root extract  breast cancer  proliferation  migration  invasion  glycolysis  Akt-mTOR

摘要:

目的 探究藤梨根提取物对乳腺癌细胞MDA-MB-231增殖、迁移、侵袭及糖酵解的影响。方法 MDA-MB-231细胞经藤梨根提取物处理后，MTT试验与克隆形成试验检测细胞增殖能力；划痕试验和Transwell试验检测细胞迁移和侵袭，Western blotting检测基质金属蛋白酶2（MMP-2）和基质金属蛋白酶9（MMP-9）蛋白表达情况，采用葡萄糖摄取试剂盒和乳酸检测试剂盒检测细胞葡萄糖摄取量和乳酸生成量，采用Western blotting检测细胞内M2型丙酮酸激酶（PKM2），葡萄糖转运体1（GLUT1）和乳酸脱氢酶A （LDHA）以及Akt-mTOR介导的自噬信号通路相关蛋白表达情况。结果 与对照组比较，10 mg·mL^-1藤梨根提取物能显著抑制MDA-MB-231细胞的细胞活性（P<0.05），而浓度为0.1和1 mg·mL^-1的藤梨根提取物则对细胞活性无明显影响。藤梨根提取物能够剂量依赖性地抑制MDA-MB-231细胞的克隆形成率、迁移率、侵袭细胞数量、葡萄糖摄取量和乳酸生成量（P均<0.05）。藤梨根提取物（1 μg·mL^-1和10 μg·mL^-1）能够减少细胞迁移标记蛋白（MMP-2和MMP-9）的表达，抑制糖酵解相关蛋白PKM2，GLUT1和LDHA的表达（P均<0.05）。此外，藤梨根提取物抑制了Akt/mTOR信号通路中关键蛋白的活性，减少了p-Akt和p-mTOR表达，促进LC3Ⅰ转化为LC3Ⅱ，上调自噬相关蛋白Beclin1，下调自噬标记蛋白p62，增强MDA-MB-231细胞自噬活性（P均<0.05）。结论 藤梨根提取物抑制细胞增殖、迁移、侵袭和糖酵解，其作用可能与失活Akt-mTOR途径诱导的自噬有关。

Abstract:

OBJECTIVE To investigate the effects of the Actinidia chinensis root extract(acRoots) on the proliferation, migration, invasion and glycolysis of breast cancer cells MDA-MB-231. METHODS MTT assay and clony formation assay were used to detect the proliferation ability of MDA-MB-231 cells treated with acRoots extract. The wound healing and Transwell asssays were performed to detect cell migration and invasion. Western blotting was used to detect the protein levels of matrix metalloproteinase 2(MMP-2) and matrix metalloproteinase 9(MMP-9). Glucose uptake kit and lactate colorimetry kit were used to detect glucose intake and lactate production in cells. Western blotting was used to detect the expression of the level of glycolysis-related proteins pyruvate kinase M2(PKM2), glucose transporter1(GLUT1), lactate dehydrogenase A(LDHA) and Akt-mTOR mediated autophagy signaling pathway. RESULTS Compared with the control group, the acRoots extract (10 mg·mL^-1) could significantly inhibit the cell viability of MDA-MB-231 cells, while the cell activity of MDA-MB-231 cells was not affected by 0.1 and 1 mg·mL^-1 acRoots extract. The acRoots extract could significantly reduce the colony formation, migration rate, number of invasion cells, glucose uptake and lactic acid production of MDA-MB-231 cells in a dose-dependent manner(all P<0.05). The extract of acRoots(1 μg·mL^-1 and 10 μg·mL^-1) could reduce the expression of cell migration marker proteins(MMP-2 and MMP-9), and inhibit the expression of glycolysis-related proteins PKM2, GLUT1 and LDHA(all P<0.05). In addition, the extract of acRoots inhibited the activity of key proteins in the Akt/mTOR signaling pathway, decreased the expression of p-Akt and p-mTOR, promoted the conversion of LC3Ⅰ to LC3Ⅱ, up-regulated the autophagy-related protein Beclin1, down-regulated the autophagic marker protein p62, enhanced MDA-MB-231 cell autophagy activity(all P<0.05). CONCLUSION The acRoots extract inhibit cell proliferation, migration, invasion and glycolysis, and its effect may be related to autophagy induced by inactivating Akt-mTOR pathway.