TOPC通过抑制PI3K/AKT/mTOR通路, 抑制miR-210, Atg7诱导H1975细胞凋亡

doi:10.5246/jcps.2023.11.071

摘要/Abstract

摘要：

TOPC [2-(2,5,5,8a-四甲基-3,4,4a,5,6,7,8,8a-八氢萘-1-基)乙基哌嗪-1-碳二硫酸酯]是我们团队合成的一种结肠酸-二硫氨基甲酸酯衍生物。该化合物对人非小细胞肺癌(NSCLC)细胞系A549和H1975的体外增殖抑制作用优于结肠酸。本研究的主要目的是探讨TOPC对H1975细胞可能的分子机制。MTT法检测细胞增殖, Hoechst 33342染色和Western blotting方法检测TOPC诱导的细胞凋亡, Western blotting检测自噬蛋白表达及相关PI3K/AKT/mTOR信号通路, qRT-PCR检测miR-210 mimic、inhibitor、inhibitor NC的转染率以及miR-210和Atg7的表达。结果表明, TOPC显著抑制A549和H1975细胞增殖。Hoechst 33342染色和Western blotting分析显示TOPC诱导H1975细胞凋亡。Western blotting检测显示, TOPC诱导H1975细胞发生自噬, 同时自噬相关蛋白Beclin-1、Atg5、Atg7、Atg12和LC3-II表达上调。此外, qRT-PCR结果显示TOPC显著下调了H1975细胞中miR-210的表达。进一步研究表明, 抑制PI3K/AKT/mTOR信号通路和抑制miR-210功能在Atg7介导的topc诱导的自噬中起作用。结果表明TOPC能显著抑制A549和H1975细胞的生长，是一种很有前途的非小细胞肺癌治疗药物, 值得进一步开发。

关键词: TOPC, 细胞凋亡, 自噬, PI3K/AKT/mTOR, miR-210

Abstract:

TOPC (2-(2,5,5,8a-tetramethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalen-1-yl) ethyl piperazine-1-carbodithioate) is a coleolic acid-dithiocarbamate derivative synthesized by our team. Notably, this compound exhibits superior inhibitory effects on the proliferation of human non-small-cell lung cancer (NSCLC) cell lines A549 and H1975 compared to coleolic acid. The primary objective of this study was to investigate the potential molecular mechanisms of TOPC against H1975 cells. Cell proliferation was assessed using the MTT assay, while apoptosis induced by TOPC was evaluated using Hoechst 33342 staining and Western blotting analysis. The expressions of autophagy proteins and the associated PI3K/AKT/mTOR signaling pathway were determined through Western blotting analysis. The transfection efficiency of the miR-210 mimic, inhibitor, and inhibitor NC, as well as the expressions of miR-210 and Atg7, were assessed using qRT-PCR. TOPC demonstrated significant inhibition of A549 and H1975 cell proliferation. Hoechst 33342 staining and Western blotting analysis revealed that TOPC induced apoptosis in H1975 cells. Moreover, TOPC induced autophagy in H1975 cells, as evidenced by increased expressions of autophagy-related proteins, such as Beclin-1, Atg5, Atg7, Atg12, and LC3-II. Additionally, qRT-PCR demonstrated that TOPC significantly downregulated the expression of miR-210 in H1975 cells. Further investigation suggested that TOPC-induced autophagy was mediated by inhibiting the PI3K/AKT/mTOR signaling pathway and suppressing the function of miR-210 on Atg7. The findings clearly demonstrated that TOPC substantially suppressed the growth of A549 and H1975 cells, making it a promising therapeutic agent for NSCLC. Its potential merits further development.

Key words: TOPC, Apoptosis, Autophagy, PI3K/AKT/mTOR, miR-210

Supporting:

郑吴殷晓, 李海平, 罗来春, 胡春玲, 尤朋涛. TOPC通过抑制PI3K/AKT/mTOR通路, 抑制miR-210, Atg7诱导H1975细胞凋亡[J]. 中国药学(英文版), 2023, 32(11): 881-892.

Wuyinxiao Zheng, Haiping Li, Laichun Luo, Chunling Hu, Pengtao You. PI3K/AKT/mTOR pathway inhibition and miR-210-mediated suppression of Atg7 promote autophagy in TOPC-induced apoptosis of H1975 cells[J]. Journal of Chinese Pharmaceutical Sciences, 2023, 32(11): 881-892.

图/表 8

Figure 1. Structures of coleolic acid, DTC, and TOPC.

Table 1. The sequences of qRT-PCR primers.

Figure 2. Effects of coleolic acid and TOPC on the viability of A549 and H1975 cells (A, B). A549 cells treated with different concentrations of coleolic acid or TOPC for 48 h vs. the control (C, D). H1975 cells treated with different concentrations of coleolic acid or TOPC for 48 h vs. the control. Data are represented as mean ± SD, in three separate experiments performed in triplicate. **P < 0.01 vs. the control group.

Figure 3. TOPC induces apoptosis of H1975 cells in the presence of 1, 2, and 4 μg/mL TOPC for 48 h. (A) Fluorescence images (200× magnification) of Hoechst 33342-stained cells. (B, C) The expression levels of cleaved-Caspase 3 and cleaved-Caspase PARP were detected using Western blotting analysis. Data are represented as mean ± SD, in three separate experiments performed in triplicate. *P < 0.05 and **P < 0.01 vs. the control group.

Figure 4. Effects of TOPC on autophagy and autophagy-related PI3K/AKT/mTOR proteins in H1975 cells (A, B). The expressions of autophagy-related proteins were detected using Western blotting analysis (C, D). The expressions of autophagy-related PI3K/AKT/mTOR signaling pathway proteins were detected using Western blotting analysis. Data are represented as mean ± SD, in three separate experiments performed in triplicate. *P < 0.05 and **P < 0.01 vs. the control group.

Figure 5. Autophagy inhibition promotes TOPC-induced apoptosis in H1975 cells. (A, B) The expression levels of LC3-II and cleaved-Caspase 3 were detected using Western blotting analysis. Data are represented as mean ± SD, in three separate experiments performed in triplicate. *P < 0.05 and **P < 0.01 vs. the control group. #P < 0.05 and ##P < 0.01 vs. the TOPC group.

Figure 6. TOPC represses miR-210 expression in H1975 cells. After treatment with different concentrations of TOPC for 48 h, miR-210 expression was analyzed by qRT-PCR in H1975 cells. Data are represented as mean ± SD, in three separate experiments performed in triplicate. **P < 0.01 vs. control group.

Figure 7. TOPC promotes autophagy by inhibiting miR-210 function on Atg7. Transfected or non-transfected H1975 cells were incubated with or without 2 μg/mL TOPC for 48 h. (A) qRT-PCR determined the relative miR-210 expression in H1975 cells after transfection. (B) qRT-PCR determined the relative miR-210 expression in H1975 cells after the corresponding transfection or administration. (C, D) qRT-PCR and Western blotting methods determined the relative expression of Atg7 in H1975 cells after the corresponding transfection or administration. Data are represented as mean ± SD, in three separate experiments performed in triplicate. *P < 0.05 and **P < 0.01 vs. control group. #P < 0.05 and ##P < 0.01 vs. TOPC group.

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