| 童焰,肖沁,赖智清,刘朝霞.双氢青蒿素经AMPK/mTOR通路逆转卵巢癌细胞A2780/DDP顺铂耐药的机制研究[J].井冈山大学自然版,2025,(1):51-57 |
| 双氢青蒿素经AMPK/mTOR通路逆转卵巢癌细胞A2780/DDP顺铂耐药的机制研究 |
| REVERSAL EFFECT AND ITS MECHANISM OF DIHYDROARTEMISININ ON CISPLATIN RESISTANCE IN OVARIAN CANCER CELL LINES A2780/DDP BY AMPK/MTOR PATHWAY |
| 投稿时间：2024-10-12 修订日期：2024-12-16 |
| DOI：10.3969/j.issn.1674-8085.2025.01.008 |
| 中文关键词: 双氢青蒿素 卵巢癌 顺铂耐药 AMPK/mTOR信号通路 |
| 英文关键词: DHA ovarian cancer cisplatin resistance AMPK/mTOR signaling pathway |
| 基金项目:国家自然科学基金项目(82260298)；江西省中医药管理局科技计划项目(2021A228） |
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| 中文摘要: |
| 为了探讨双氢青蒿素（dihydroartemisinin，DHA）通过AMPK/mTOR通路逆转卵巢癌细胞A2780/DDP顺铂耐药的分子机制。通过体外培养人卵巢癌A2780和顺铂耐药A2780/DDP细胞，给予不同浓度的DHA（5、10、20、40、80 μmol/L），培养48 h后，采用CCK-8法计算不同浓度DHA对A2780和A2780/DDP细胞增殖的抑制作用，选取最适的DHA浓度进行后续实验；选取单药DDP组（0.625、1.25、2.5、5、10 mg/L），最适DHA浓度与顺铂各浓度联用，计算顺铂对A2780和A2780/DDP细胞的IC50，并计算DHA对A2780和A2780/DDP细胞的耐药逆转倍数；DAPI法检测DHA对A2780/DDP细胞凋亡的影响；转染AMPKα siRNA后，Western blot法检测DHA对A2780/DDP细胞AMPKα、mTOR、p-mTOR、HIF-1α、P-gp蛋白的影响。实验结果显示，随DHA浓度升高，A2780/DDP细胞的生长受到明显抑制，且具有浓度依赖性，10 μmol/L是DHA最佳逆转耐药浓度；DDP+DHA组中顺铂对A2780/DDP的IC50值为4.95 mg/L，单用DDP组的IC50值为20.33 mg/L，DHA的逆转耐药倍数为4.11；DDP+DHA组呈现出凋亡形态，体积缩小，出现核固缩或碎裂，呈亮蓝色，且细胞凋亡率明显增加；敲低AMPK基因的表达后，p-mTOR、HIF-1α、P-gp表达明显增加，A2780/DDP细胞的生长速度明显增加。DHA可能通过调控AMPK/mTOR信号通路，下调HIF-1α、P-gp的表达，抑制A2780/DDP细胞的增殖并促进其凋亡，逆转耐药的发生。 |
| 英文摘要: |
| In order to explore the molecular mechanism of dihydroartemisinin (DHA) reversing the cisplatin resistance of ovarian cancer cells A2780/DDP through AMPK/mTOR pathway. Human ovarian cancer A2780 and cisplatin-resistant A2780/DDP cells were cultured with DHA under different concentrations (5, 10, 20, 40, 80 μmol/L) in vitro. The CCK-8 method was used to calculate the inhibitory effect of DHA on the proliferation of A2780 and A2780/DDP cells after 48 hours, and the most appropriate DHA concentration was selected for the subsequent experiments. There were single DDP groups (0.625, 1.25, 2.5, 5 and 10 mg/L) and the groups of DHA combined with cisplatin of each concentration. The IC50 of cisplatin and the resistance reversal fold of DHA on A2780 and A2780/DDP cells were calculated. DAPI assay was used to detect the effect of DHA on apoptosis of A2780/DDP. Effect of DHA on AMPKα, mTOR, p-mTOR, HIF-1α, and P-gp in A2780/DDP cells were detected by Western blot after treated with AMPKα siRNA and the agonist of AMPK. The results showed that the growth of A2780/DDP cells was significantly inhibited with increasing DHA concentration in a concentration-dependent manner. And the optimal concentration of DHA to reverse drug resistance was 10 μmol/L. The IC50 of cisplatin against A2780/DDP in the DDP+DHA group was 4.95 mg/L and 20.33 mg/L in the DDP group. And the resistance reversal fold of DHA was 4.11. The DDP+DHA group showed apoptotic morphology with reduced size, nuclear consolidation or fragmentation, bright blue color and significantly increased apoptosis rate. After knocking down the expression of AMPK gene, the expression of mTOR phosphorylation, HIF-1α and P-gp was increased, and the growth rate of A2780/DDP cells was significantly increased. Meanwhile, after using mTOR, HIF-1α and P-gp agonists respectively, the expression of their downstream HIF-1α and P-gp increased significantly, and the growth rate of A2780/DDP cells increased significantly compared with the DHA group. DHA may inhibit the proliferation and promote the apoptosis of A2780/DDP cells by regulating AMPK/mTOR signaling pathway and down-regulating the expression of HIF-1α and P-gp to reverse the drug resistance. |
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