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.
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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 modified eagle’s medium
- DMSO:
-
Dimethyl sulfoxide
- DNPH:
-
Dinitrophenyl hydrazine
- ETBR:
-
Ethidium bromide
- FBS:
-
Fetal bovine serum
- FITC:
-
Fluorescein isothiocyanate
- LPO:
-
Lipid peroxides
- MDA:
-
Malondialdehyde
- MMP:
-
Mitochondrial membrane depolarization
- MTT:
-
3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide
- NAC:
-
N-Acetyl cysteine
- NBT:
-
Nitro blue tetrazolium
- NCCS:
-
National Centre for Cell Science
- NSCLC:
-
Non-small cell lung cancer
- PBMC:
-
Peripheral Blood Mononuclear Cell
- PBS:
-
Phosphate-buffered saline
- PCC:
-
Protein carbonyl content
- PI:
-
Propidium Iodide
- pNA:
-
p-Nitroaniline
- PVDF:
-
Polyvinylidene fluoride
- ROS:
-
Reactive oxygen species
- SDS:
-
Sodium dodecyl sulphate
- SOD:
-
Superoxide dismutase
- TBA:
-
Thiobarbituric acid
- TBARS:
-
Thiobarbituric acid reactive substances
- TBST:
-
Tris-buffered saline with Tween 20
- TCA:
-
Trichloroacetic acid
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Acknowledgements
The authors wish to gratefully acknowledge the (i) DST-FIST (Grant No. SR/FST/LSI-639/2015(C)), (ii) UGC-SAP (Grant No. F.5-1/2018/DRS-II(SAP-II)), (iii) DST PURSE (Grant No. SR/PURSE Phase 2/38 (G)) (iv) University Science Instrumentation Centre (USIC), Alagappa University and (v) RUSA 2.0 [F. 24-51/2014-U, Policy (TN Multi-Gen), Dept of Edn, GoI]. The authors sincerely thank Dr. P. Kumar, Assistant Professor, Department of Animal Health and Management, Alagappa University for his help in taking the fluorescent images.
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Balan, D.J., Rajavel, T., Das, M. et al. Thymol induces mitochondrial pathway-mediated apoptosis via ROS generation, macromolecular damage and SOD diminution in A549 cells. Pharmacol. Rep 73, 240–254 (2021). https://doi.org/10.1007/s43440-020-00171-6
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DOI: https://doi.org/10.1007/s43440-020-00171-6