Thymol induces mitochondrial pathway-mediated apoptosis via ROS generation, macromolecular damage and SOD diminution in
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ARTICLE
Thymol induces mitochondrial pathway‑mediated apoptosis via ROS generation, macromolecular damage and SOD diminution in A549 cells Devasahayam Jaya Balan1 · Tamilselvam Rajavel1,2 · Mamali Das1 · Sethuraman Sathya1 · Mahalingam Jeyakumar1 · Kasi Pandima Devi1 Received: 10 June 2020 / Revised: 1 October 2020 / Accepted: 7 October 2020 © Maj Institute of Pharmacology Polish Academy of Sciences 2020
Abstract Background Thymol is a monoterpene phenol found in thyme species plants. The present study was carried out to investigate the effect of thymol and its molecular mechanism on non-small lung cancer (A549) cells. Methods The cytotoxic effect of thymol on A549 cells was assessed via MTT assay. ROS production, macromolecular damage, apoptosis were determined using DCF-DA, PI, AO/EtBr stains, respectively. ROS-dependent effect of thymol was confirmed using NAC. The expression of caspase-9, Bcl-2, Bax and cell cycle profile was analyzed via western blot and FACS, respectively. Results The antiproliferative effect of thymol on A549 cells was found to be both dose and time dependent with IC50 values of 112 μg/ml (745 μM) at 24 h. Thymol treatment favored apoptotic cell death and caused G0/G1 cell cycle arrest. It mediated cellular and nuclear morphological changes, phosphatidylserine translocation, and mitochondrial membrane depolarization. Additionally, upregulation of Bax, downregulation of Bcl-2, and apoptotic fragmented DNA were also observed. Thymol induced ROS by reducing the SOD level which was confirmed via in vitro and in silico analysis. Furthermore, the levels of lipid peroxides and protein carbonyl content were elevated in thymol-treated groups. Notably, N-acetyl cysteine pretreatment reversed the efficacy of thymol on A549 cells. Moreover, thymol-treated human PBMC cells did not show any significant cytotoxicity. Conclusion Overall, our results confirmed that thymol can act as a safe and potent therapeutic agent to treat NSCLC.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s43440-020-00171-6) contains supplementary material, which is available to authorized users. * Kasi Pandima Devi [email protected]; [email protected] Devasahayam Jaya Balan [email protected]
Sethuraman Sathya [email protected] Mahalingam Jeyakumar [email protected]
Tamilselvam Rajavel [email protected]
1
Department of Biotechnology, Alagappa University [Science Campus], Karaikudi, Tamil Nadu 630 003, India
Mamali Das [email protected]
2
Present Address: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA
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Graphic abstract
Keywords Thymol · Monoterpene phenol · NSCLC · ROS · NAC · Apoptosis · SOD · A549
Abbreviations AO Acridine orange BCIP 5-Bromo-4-chloro-3-indolyl phosphate CLSM Confocal laser scanning microscopy DAPI 4′,6-Diamidino-2-phenylindole DCF-DA 2′,7′-Dichlorodihydrofluorescein diacetate DMEM Dulbecco’s
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