PI3K/AKT/mTOR pathway inhibition and miR-210-mediated suppression of Atg7 promote autophagy in TOPC-induced apoptosis of H1975 cells

doi:10.5246/jcps.2023.11.071

Abstract

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:

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.

Figures/Tables 8

Table 1. The sequences of qRT-PCR primers.

References 44

[1]	Hua, M.; Ji, X.W.; Li, R.T.; Ji, S.M.; Li, J.; Yao, Q.Y.; Wang, L.J.; Hao, F.R.; Lu, W.; Zhou, T.Y. Pharmacokinetic-pharmacodynamic modeling of the anticancer effect of TM208 on non-small cell lung cancer xenograft. J. Chin. Pharm. Sci. 2016, 25, 502–511.
[2]	Wang, Z.; Wu, X.E.; Liang, Y.N.; Wang, L.; Song, Z.X.; Liu, J.L.; Tang, Z.S. Cordycepin induces apoptosis and inhibits proliferation of human lung cancer cell line H1975 via inhibiting the phosphorylation of EGFR. Molecules. 2016, 21, 1267.
[3]	Huang, Z.F.; Su, W.H.; Lu, T.; Wang, Y.Y.; Dong, Y.T.; Qin, Y.; Liu, D.H.; Sun, L.L.; Jiao, W.J. First-line immune-checkpoint inhibitors in non-small cell lung cancer: current landscape and future progress. Front. Pharmacol. 2020, 11, 578091.
[4]	Han, Y.H.; Mun, J.G.; Jeon, H.D.; Kee, J.Y.; Hong, S.H. Betulin inhibits lung metastasis by inducing cell cycle arrest, autophagy, and apoptosis of metastatic colorectal cancer cells. Nutrients. 2019, 12, 66.
[5]	Boulares, A.H.; Yakovlev, A.G.; Ivanova, V.; Stoica, B.A.; Wang, G.P.; Iyer, S.; Smulson, M. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. J. Biol. Chem. 1999, 274, 22932–22940.
[6]	Kim, W.K.; Pyee, Y.; Chung, H.J.; Park, H.J.; Hong, J.Y.; Son, K.H.; Lee, S.K. Antitumor activity of spicatoside A by modulation of autophagy and apoptosis in human colorectal cancer cells. J. Nat. Prod. 2016, 79, 1097–1104.
[7]	Maiuri, M.C.; Zalckvar, E.; Kimchi, A.; Kroemer, G. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 2007, 8, 741–752.
[8]	Wang, S.H.; Xu, X.L.; Hu, Y.L.; Lei, T.; Liu, T.X. Sotetsuflavone induces autophagy in non-small cell lung cancer through blocking PI3K/akt/mTOR signaling pathway in vivo and in vitro. Front. Pharmacol. 2019, 10, 1460.
[9]	Liu, M.W.; Su, M.X.; Tang, D.Y.; Hao, L.; Xun, X.H.; Huang, Y.Q. Ligustrazin increases lung cell autophagy and ameliorates paraquat-induced pulmonary fibrosis by inhibiting PI3K/Akt/mTOR and hedgehog signalling via increasing miR-193a expression. BMC Pulm. Med. 2019, 19, 35.
[10]	Zhang, H.Y.; Liang, H.X.; Wu, S.H.; Zhang, Y.Y.; Yu, Z.J. MicroRNA-638 induces apoptosis and autophagy in human liver cancer cells by targeting enhancer of zeste homolog 2 (EZH2). Environ. Toxicol. Pharmacol. 2021, 82, 103559.
[11]	Wang, N.; Feng, T.; Liu, X.; Liu, Q. Curcumin inhibits migration and invasion of non-small cell lung cancer cells through up-regulation of miR-206 and suppression of PI3K/AKT/mTOR signaling pathway. Acta Pharmaceutica. 2020, 70, 399–409.
[12]	Li, H.; Huang, D.L.; Hang, S.Y. RETRACTED: Salidroside inhibits the growth, migration and invasion of Wilms’ tumor cells through down-regulation of miR-891b. Life Sci. 2019, 222, 60–68.
[13]	Chen, Y.; Fu, L.L.; Wen, X.; Liu, B.; Huang, J.; Wang, J.H.; Wei, Y.Q. Oncogenic and tumor suppressive roles of microRNAs in apoptosis and autophagy. Apoptosis. 2014, 19, 1177–1189.
[14]	Chan, Y.C.; Banerjee, J.; Choi, S.Y.; Sen, C.K. miR-210: the master hypoxamir. Microcirculation. 2012, 19, 215–223.
[15]	Wang, C.; Zhang, Z.Z.; Yang, W.; Ouyang, Z.H.; Xue, J.B.; Li, X.L.; Zhang, J.; Chen, W.K.; Yan, Y.G.; Wang, W.J. miR-210 facilitates ECM degradation by suppressing autophagy via silencing of ATG7 in human degenerated NP cells. Biomed. Pharmacother. 2017, 93, 470–479.
[16]	Matulja, D.; Wittine, K.; Malatesti, N.; Laclef, S.; Turks, M.; Markovic, M.K.; Ambrožić, G.; Marković, D. Marine natural products with high anticancer activities. Curr. Med. Chem. 2020, 27, 1243–1307.
[17]	Rodrigues, T.; Reker, D.; Schneider, P.; Schneider, G. Counting on natural products for drug design. Nat. Chem. 2016, 8, 531–541.
[18]	Huang, D.F.; Yang, Y.F.; Ai, L.Q.; Lu, Y.; Wu, H.Z. Studies on the chemical constituents of Coleus forskohlii transplanted in Tongcheng and their antitumor activity. J. Chin. Med. Mater. 2011. 34, 375–378.
[19]	Arsakhant, P.; Sirion, U.; Chairoungdua, A.; Suksen, K.; Piyachaturawat, P.; Suksamrarn, A.; Saeeng, R. Design and synthesis of C-12 dithiocarbamate andrographolide analogues as an anticancer agent. Bioorg. Med. Chem. Lett. 2020, 30, 127263.
[20]	Ouyang, J.; Sun, F.J.; Feng, W.; Xie, Y.H.; Ren, L.J.; Chen, Y.C. Antimicrobial activity of galangin and its effects on murein hydrolases of vancomycin-intermediate staphylococcus aureus (VISA) strain Mu50. Chemotherapy. 2018, 63, 20–28.
[21]	Yu, J.Y.; Li, X.Q.; Wei, M.X. Synthesis and biological activities of artemisinin-piperazine-dithiocarbamate derivatives. Eur. J. Med. Chem. 2019, 169, 21–28.
[22]	Xiang, Y.H.; Li, H.P.; Wang, J.; Peng, X.Z.; Hu, C.L.; Luo, L.C. Design, synthesis, and anticancer activities of 8, 9-substituted Luotonin A analogs as novel topoisomerase I inhibitors. Med. Chem. Res. 2021, 30, 1512–1522.
[23]	Friedlaender, A.; Addeo, A.; Russo, A.; Gregorc, V.; Cortinovis, D.; Rolfo, C. Targeted therapies in early stage NSCLC: hype or hope? Int. J. Mol. Sci. 2020, 21, 6329.
[24]	Liu, F.R.; Gao, S.; Yang, Y.X.; Zhao, X.D.; Fan, Y.M.; Ma, W.X.; Yang, D.R.; Yang, A.M.; Yu, Y. Antitumor activity of curcumin by modulation of apoptosis and autophagy in human lung cancer A549 cells through inhibiting PI3K/Akt/mTOR pathway. Oncol. Rep. 2018, 39, 1523–1531.
[25]	Wang, C.; Jiang, L.P.; Wang, S.Q.; Shi, H.G.; Wang, J.W.; Wang, R.; Li, Y.M.; Dou, Y.H.; Liu, Y.; Hou, G.Q.; Ke, Y.; Liu, H.M. The antitumor activity of the novel compound jesridonin on human esophageal carcinoma cells. PLoS One. 2015, 10, e0130284.
[26]	Wang, H.L.; Ge, W.; Jiang, W.; Li, D.; Ju, X.L. SRPK1‑siRNA suppresses K562 cell growth and induces apoptosis via the PARP‑caspase3 pathway. Mol. Med. Rep. 2018, 17, 2070–2076.
[27]	Zhou, L.; Wang, S.S.; Cao, L.N.; Ren, X.M.; Li, Y.H.; Shao, J.H.; Xu, L.C. Lead acetate induces apoptosis in Leydig cells by activating PPARγ/caspase-3/PARP pathway. Int. J. Environ. Health Res. 2021, 31, 34–44.
[28]	Chen, L.A.; Xiong, Y.Q.; Xu, J.; Wang, J.P.; Meng, Z.L.; Hong, Y.Q. Juglanin inhibits lung cancer by regulation of apoptosis, ROS and autophagy induction. Oncotarget. 2017, 8, 93878–93898.
[29]	Shi, L.; Tu, Y.J.; Xia, Y.; Ye, S.Q.; Ma, C.Z.; Liu, Y.W.; You, P.T. TEEG induced A549 cell autophagy by regulating the PI3K/AKT/mTOR signaling pathway. Anal. Cell Pathol. 2019, 2019, 1–6.
[30]	Nakatogawa, H. Two ubiquitin-like conjugation systems that mediate membrane formation during autophagy. Essays Biochem. 2013, 55, 39–50.
[31]	Clague, M.J.; Urbé, S. Ubiquitin: same molecule, different degradation pathways. Cell. 2010, 143, 682–685.
[32]	Parzych, K.R.; Klionsky, D.J. An overview of autophagy: morphology, mechanism, and regulation. Antioxid. Redox Signal. 2014, 20, 460–473.
[33]	Klionsky, D.J. The molecular machinery of autophagy: unanswered questions. J. Cell Sci. 2005, 118, 7–18.
[34]	Shen, F.G.; Ge, C.P.; Yuan, P. Aloe-emodin induces autophagy and apoptotic cell death in non-small cell lung cancer cells via Akt/mTOR and MAPK signaling. Eur. J. Pharmacol. 2020, 886, 173550.
[35]	Fu, L.J.; Wu, W.; Sun, X.J.; Zhang, P. Glucocorticoids enhanced osteoclast autophagy through the PI3K/akt/mTOR signaling pathway. Calcif. Tissue Int. 2020, 107, 60–71.
[36]	Yang, J.L.; Pi, C.C.; Wang, G.H. Inhibition of PI3K/Akt/mTOR pathway by apigenin induces apoptosis and autophagy in hepatocellular carcinoma cells. Biomed. Pharmacother. 2018, 103, 699–707.
[37]	Sundarraj, K.; Raghunath, A.; Panneerselvam, L.; Perumal, E. Fisetin inhibits autophagy in HepG2 cells via PI3K/akt/mTOR and AMPK pathway. Nutr. Cancer. 2021, 73, 2502–2514.
[38]	Petiot, A.; Ogier-Denis, E.; Blommaart, E.F.C.; Meijer, A.J.; Codogno, P. Distinct classes of phosphatidylinositol 3’-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J. Biol. Chem. 2000, 275, 992–998.
[39]	Wu, Y.T.; Tan, H.L.; Shui, G.H.; Bauvy, C.; Huang, Q.; Wenk, M.R.; Ong, C.N.; Codogno, P.; Shen, H.M. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J. Biol. Chem. 2010, 285, 10850–10861.
[40]	Sun, W.; Zu, Y.K.; Fu, X.N.; Deng, Y. Knockdown of lncRNA-XIST enhances the chemosensitivity of NSCLC cells via suppression of autophagy. Oncol. Rep. 2017, 38, 3347–3354.
[41]	Tian, J.; An, X.J.; Niu, L. Role of microRNAs in cardiac development and disease. Exp. Ther. Med. 2017, 13, 3–8.
[42]	Wang, D.F.; Gu, J.; Wang, T.; Ding, Z.J. OncomiRDB: a database for the experimentally verified oncogenic and tumor-suppressive microRNAs. Bioinformatics. 2014, 30, 2237–2238.
[43]	Ju, S.; Liang, Z.P.; Li, C.; Ding, C.; Xu, C.; Song, X.Y.; Zhao, J. The effect and mechanism of miR-210 in down-regulating the autophagy of lung cancer cells. Pathol. Res. Pract. 2019, 215, 453–458.
[44]	Li, G.; Wang, B.; Ding, X.C.; Zhang, X.H.; Tang, J.; Lin, H.Q. Plasma extracellular vesicle delivery of miR-210-3p by targeting ATG7 to promote sepsis-induced acute lung injury by regulating autophagy and activating inflammation. Exp. Mol. Med. 2021, 53, 1180–1191.