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Thymol induces mitochondrial pathway-mediated apoptosis via ROS generation, macromolecular damage and SOD diminution in A549 cells

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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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Correspondence to Kasi Pandima Devi.

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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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