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Exploring the Apoptotic and Cytotoxic Effects of Thymol-Induced Apoptosis in C6 Glioma

Articles in Press

Document Type : Original Article(s)

Authors

1 Student Research Committee, North Khorasan University of Medical Sciences, Bojnurd, Iran

2 Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran

3 Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences, Bojnurd, Iran

4 Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

5 Department of Ophthalmology, Khatam-Ol-Anbia Hospital, Mashhad University of Medical Sciences, Mashhad, Iran

6 Department of Physiology and Pharmacology, Faculty of Medicine, North Khorasan University of Medical ‎Sciences, Bojnurd, Iran

10.30476/mejc.2026.106361.2261

Abstract

Background: Glioblastoma (GBM) is the most common and aggressive brain tumor associated with a poor prognosis, highlighting the need for new therapeutic strategies. The present study aimed to evaluate the anticancer properties of thymol in rat GBM C6 malignant cells.
Method: This in-vitro study, conducted to evaluate the effects of thymol on cell viability and apoptosis using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and Annexin V-FITC/PI staining kit, respectively. Moreover, reactive oxygen species (ROS) generation was evaluated using the Dichlorodihydrofluorescein diacetate assay, and the mRNA levels of Bax, Bcl2, and p53 were assessed using real-time. Data from three independent experiments were analyzed using GraphPad Prism 8.2.1 and compared for statistically significant differences (P < 0.05) using a one-way ANOVA with a Tukey post-hoc test.
Results: The half-maximal inhibitory concentration (IC50) of thymol for C6 cells was 350 μM at 24 h and 260 μM at 48 h post-treatment. At the same time, lower cytotoxic effects were observed on normal human foreskin fibroblast cells (IC50 = 675 μM). 350 Μm of thymol also caused a significant G2/M cell cycle arrest and early and late apoptosis compared with the non-treated control group. Thymol also caused a significant increase in Bax and p53 expression, as well as ROS generation, when cells were treated with thymol compared with the non-treated control group.
Conclusion: The results indicate the anticancer properties of thymol including cytotoxic, and pro-oxidant effects with selectivity towards cancer cells. These preclinical findings provide a strong rationale for further exploration of thymol as a potential phytochemical agent in combating GBM.

Highlights

Amir R. Afshari (google scholar)

Reza Salarinia (google scholar)

Keywords

Main Subjects

Please cite this article as: Ghazvini N, Hosseinzadeh P, Afshari S, Bibak B, Jalali M, Biglari GH, et al. Exploring the Apoptotic and Cytotoxic Effects of Thymol-Induced Apoptosis in C6 Glioma. Middle East J Cancer. 2026: in press. doi: 10.30476/mejc.2026.106361.2261.

References
  1. Afshari AR, Sanati M, Ahmadi SS, Kesharwani P, Sahebkar A. Harnessing the capacity of phytochemicals to enhance immune checkpoint inhibitor therapy of cancers: A focus on brain malignancies. Cancer Lett. 2024;593:216955. doi: 10.1016/j.canlet.2024.216955. PMID: 38750720.
  2. Hanif F, Muzaffar K, Perveen K, Malhi SM, Simjee Sh U. Glioblastoma multiforme: A review of its epidemiology and pathogenesis through clinical presentation and treatment. Asian Pac J Cancer Prev. 2017;18:3-9. doi: 10.22034/APJCP.2017.18.1.3. PMID: 28239999; PMCID: PMC5563115.
  3. Sanati M, Binabaj MM, Ahmadi SS, Aminyavari S, Javid H, Mollazadeh H, et al. Recent advances in glioblastoma multiforme therapy: A focus on autophagy regulation. Biomed Pharmacother. 2022;155:113740. doi: 10.1016/j.biopha.2022.113740. PMID: 36166963.
  4. Mohtashami E, Shafaei-Bajestani N, Mollazadeh H, Mousavi SH, Jalili-Nik M, Sahebkar A, et al. The current state of potential therapeutic modalities for glioblastoma multiforme: A clinical review. Curr Drug Metab. 2020;21:564-78. doi: 10.2174/1389200221666200714101038. PMID: 32664839.
  5. Ostrom QT, Price M, Neff C, Cioffi G, Waite KA, Kruchko C, et al. CBTRUS Statistical Report: Primary brain and other central nervous system tumors diagnosed in the United States in 2015-2019. Neuro Oncol. 2022;24:v1-v95. doi: 10.1093/neuonc/noac202. PMID: 36196752; PMCID: PMC9533228.
  6. Ostrom QT, Gittleman H, Farah P, Ondracek A, Chen Y, Wolinsky Y, et al. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2006-2010. Neuro Oncol. 2013;15 Suppl 2:ii1-56. doi: 10.1093/neuonc/not151. PMID: 24137015; PMCID: PMCPMC3798196.
  7. Sanati M, Afshari AR, Ahmadi SS, Jamialahmadi T, Sahebkar A. Application of RNA-based therapeutics in glioma: A review. Prog Mol Biol Transl Sci. 2024;204:133-61. doi: 10.1016/bs.pmbts.2023.12.001. PMID: 38458736.
  8. Sanati M, Afshari AR, Ahmadi SS, Moallem SA, Sahebkar A. Modulation of the ubiquitin-proteasome system by phytochemicals: Therapeutic implications in malignancies with an emphasis on brain tumors. Biofactors. 2023;49:782-819. doi: 10.1002/biof.1958. PMID: 37162294.
  9. Hosseini A, Alavi MS, Shahraki AR, Rajabian A, Afshari AR, Kesharwani P, et al. Psidium guajava induces cytotoxicity in human malignant glioblastoma cell line: Role of reactive oxygen species. Toxicol In Vitro. 2023;89:105567. doi: 10.1016/j.tiv.2023.105567. PMID: 36758825.
  10. Bagherniya M, Mahdavi A, Abbasi E, Iranshahy M, Sathyapalan T, Sahebkar A. The effects of phytochemicals and herbal bio-active compounds on tumour necrosis factor-α in overweight and obese individuals: a clinical review. Inflammopharmacology. 2022. doi: 10.1007/s10787-021-00902-y.
  11. Luthra R, Roy A. Role of Medicinal Plants against Neurodegenerative Diseases. Curr Pharm Biotechnol. 2022;23:123-39. doi: 10.2174/1389201022666210211123539.
  12. Jalili-Nik M, Soltani A, Moussavi S, Ghayour-Mobarhan M, Ferns GA, Hassanian SM, et al. Current status and future prospective of Curcumin as a potential therapeutic agent in the treatment of colorectal cancer. J Cell Physiol. 2018;233:6337-45. doi: 10.1002/jcp.26368. PMID: 29219177.
  13. Soltani A, Salmaninejad A, Jalili-Nik M, Soleimani A, Javid H, Hashemy SI, Sahebkar A. 5'-Adenosine monophosphate-activated protein kinase: A potential target for disease prevention by curcumin. J Cell Physiol. 2019;234(3):2241-51. doi: 10.1002/jcp.27192. PMID: 30146757.
  14. Qoorchi Moheb Seraj F, Heravi-Faz N, Soltani A, Ahmadi SS, Shahbeiki F, Talebpour A, et al. Thymol has anticancer effects in U-87 human malignant glioblastoma cells. Mol Biol Rep. 2022;49:9623-32. doi: 10.1007/s11033-022-07867-3. PMID: 35997850.
  15. Islam MT, Khalipha ABR, Bagchi R, Mondal M, Smrity SZ, Uddin SJ, et al. Anticancer activity of thymol: A literature-based review and docking study with Emphasis on its anticancer mechanisms. IUBMB Life. 2019;71:9-19. doi: 10.1002/iub.1935. PMID: 30308112.
  16. Elbe H, Yigitturk G, Cavusoglu T, Uyanikgil Y, Ozturk F. Apoptotic effects of thymol, a novel monoterpene phenol, on different types of cancer. Bratisl Lek Listy. 2020;121:122-8. doi: 10.4149/BLL_2020_016. PMID: 32115964.
  17. Herrera-Bravo J, Belén LH, Reyes ME, Silva V, Fuentealba S, Paz C, et al. Thymol as adjuvant in oncology: molecular mechanisms, therapeutic potentials, and prospects for integration in cancer management. Naunyn Schmiedebergs Arch Pharmacol. 2024;397:8259-84. doi: 10.1007/s00210-024-03196-3. PMID: 38847831.
  18. Taibi M, Elbouzidi A, Haddou M, Baraich A, Ou-Yahia D, Bellaouchi R, et al. Evaluation of the interaction between carvacrol and thymol, major compounds of ptychotis verticillata essential oil: Antioxidant, anti-inflammatory and anticancer activities against breast cancer lines. Life. 2024;14:1037. PMID: doi:10.3390/life14081037.
  19. Satooka H, Kubo I. Effects of thymol on B16-F10 melanoma cells. J Agric Food Chem. 2012;60:2746-52. doi: 10.1021/jf204525b. PMID: 22352891.
  20. Anvarbatcha R, Kunnathodi F, Islam M. Induction of G0/G1 phase cell cycle arrest and apoptosis by thymol through ROS generation and caspase-9/-3 activation in breast and colorectal cancer cell lines. J Cancer Res Ther. 2023;19:1915-24. doi: 10.4103/jcrt.jcrt_308_22. PMID: 38376297
  21. Pouyamanesh G, Ameli N, Metanat Y, Khorrami A, Abbasinezhad-Moud F, Qoorchi Moheb Seraj F, et al. Thymol enhances 5-fluorouracil cytotoxicity by reducing migration and increasing apoptosis and cell cycle arrest in esophageal cancer cells: An in-vitro study. Indian J Clin Biochem. 2025;40(3):422-33. doi: 10.1007/s12291-024-01219-7. PMID: 40625606; PMCID: PMC12229301.
  22. Gunes-Bayir A, Kocyigit A, Guler EM, Kiziltan HS. Effects of thymol, a natural phenolic compound, on human gastric adenocarcinoma cells in vitro. Altern Ther Health Med. 2019;25:12-21. PMID: 29477139.
  23. Li Y, Wen JM, Du CJ, Hu SM, Chen JX, Zhang SG, et al. Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis. Biochem Biophys Res Commun. 2017;491(2):530-6. doi: 10.1016/j.bbrc.2017.04.009. PMID: 28389245.
  24. Anvarbatcha R, Kunnathodi F, Islam M. Induction of G0/G1 phase cell cycle arrest and apoptosis by thymol through ROS generation and caspase-9/-3 activation in breast and colorectal cancer cell lines. J Cancer Res Ther. 2023;19. doi: 10.4103/jcrt.jcrt_308_22. PMID: 38376297
  25. Mollazadeh H, Afshari AR, Hosseinzadeh H. Review on the potential therapeutic roles of Nigella sativa in the treatment of patients with cancer: Involvement of apoptosis: - Black cumin and cancer. J Pharmacopuncture. 2017;20:158-72. doi: 10.3831/KPI.2017.20.019. PMID: 30087792; PMCID: PMCPMC5633668.
  26. Seresht HR, Albadry BJ, Al-mosawi AKM, Gholami O, Cheshomi H. The cytotoxic effects of thymol as the major component of Trachyspermum ammi on breast cancer (MCF-7) cells. Pharm Chem J. 2019;53:101-7. doi: 10.1007/s11094-019-01961-w.
  27. Liu Q, Cao Y, Zhou P, Gui S, Wu X, Xia Y, et al. Panduratin A inhibits cell proliferation by inducing G0/G1 phase cell cycle arrest and induces apoptosis in breast cancer cells. Biomol Ther (Seoul). 2018;26:328-34. doi: 10.4062/biomolther.2017.042. PMID: 29301388; PMCID: PMCPMC5933901.
  28. Saleh AM, Al-Qudah MA, Nasr A, Rizvi SA, Borai A, Daghistani M. Comprehensive analysis of the chemical composition and in vitro cytotoxic mechanisms of Pallines Spinosa flower and leaf essential oils against breast cancer cells. Cell Physiol Biochem. 2017;42:2043-65. doi: 10.1159/000479900. PMID: 28803233.
  29. Simon HU, Haj-Yehia A, Levi-Schaffer F. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis. 2000;5:415-8. doi: 10.1023/A:1009616228304.
  30. Jalili-Nik M, Sadeghi MM, Mohtashami E, Mollazadeh H, Afshari AR, Sahebkar A. Zerumbone promotes cytotoxicity in human malignant glioblastoma cells through reactive oxygen species (ROS) generation. Oxid Med Cell Longev. 2020;2020:3237983. doi: 10.1155/2020/3237983. PMID: 32454937. PMCID: PMC7225859
  31. Sanati M, Afshari AR, Kesharwani P, Sukhorukov VN, Sahebkar A. Recent trends in the application of nanoparticles in cancer therapy: The involvement of oxidative stress. J Control Release. 2022;348:287-304. doi: 10.1016/j.jconrel.2022.05.035. PMID: 35644289.
  32. Glorieux C, Liu S, Trachootham D, Huang P. Targeting ROS in cancer: rationale and strategies. Nat Rev Drug Discov. 2024;23:583-606. doi: 10.1038/s41573-024-00979-4. PMID: 38982305.
  33. Balan DJ, Rajavel T, Das M, Sathya S, Jeyakumar M, Devi KP. Thymol induces mitochondrial pathway-mediated apoptosis via ROS generation, macromolecular damage and SOD diminution in A549 cells. Pharmacol Rep. 2021;73:240-54. doi: 10.1007/s43440-020-00171-6. PMID: 33095436.
  34. Altintas F, Tunc-Ata M, Secme M, Kucukatay V. The anticancer effects of thymol on HepG2 cell line. Med Oncol. 2023;40:260. doi: 10.1007/s12032-023-02134-2. PMID: 37542527.
  35. Günes-Bayir A, Kocyigit A, Güler EM, Kiziltan HS. Effects of thymol, a natural phenolic compound, on human gastric adenocarcinoma cells in vitro. Altern Ther Health Med. 2019;25:12-21. PMID: 29477139.
  36. Elbe H, Yigitturk G, Cavusoglu T, Baygar T, Ozgul Onal M, Ozturk F. Comparison of ultrastructural changes and the anticarcinogenic effects of thymol and carvacrol on ovarian cancer cells: which is more effective? Ultrastruct Pathol. 2020;44:193-202. doi: 10.1080/01913123.2020.1740366. PMID: 32183603
  37. Tekbas A, Huebner J, Settmacher U, Dahmen U. Plants and surgery: The protective effects of thymoquinone on hepatic injury-a systematic review of in vivo studies. Int J Mol Sci. 2018;19:1085. doi: 10.3390/ijms19041085. PMID: 29621129; PMCID: PMCPMC5979411.
  38. Wu H, Jiang K, Yin N, Ma X, Zhao G, Qiu C, et al. Thymol mitigates lipopolysaccharide-induced endometritis by regulating the TLR4- and ROS-mediated NF-kappaB signaling pathways. Oncotarget. 2017;8:20042-55. doi: 10.18632/oncotarget.15373. PMID: 28223539; PMCID: PMCPMC5386742.
  39. Afshari AR, Karimi Roshan M, Soukhtanloo M, Ghorbani A, Rahmani F, Jalili-Nik M, et al. Cytotoxic effects of auraptene against a human malignant glioblastoma cell line. Avicenna J Phytomed. 2019;9:334-46. PMID: 31309072; PMCID: PMCPMC6612248.
  40. Li Y, Wen JM, Du CJ, Hu SM, Chen JX, Zhang SG, et al. Thymol inhibits bladder cancer cell proliferation via inducing cell cycle arrest and apoptosis. Biochem Biophys Res Commun. 2017;491:530-6. doi: 10.1016/j.bbrc.2017.04.009. PMID: 28389245.
  41. Zeng Q, Che Y, Zhang Y, Chen M, Guo Q, Zhang W. Thymol isolated from thymus vulgaris L. inhibits colorectal cancer cell growth and metastasis by suppressing the Wnt/beta-catenin pathway. Drug Des Devel Ther. 2020;14:2535-47. doi: 10.2147/DDDT.S254218. PMID: 32669835; PMCID: PMCPMC7335897.
  42. Kang SH, Kim YS, Kim EK, Hwang JW, Jeong JH, Dong X, et al. Anticancer effect of thymol on AGS human gastric carcinoma cells. J Microbiol Biotechnol. 2016;26:28-37. doi: 10.4014/jmb.1506.06073. PMID: 26437948.
  43. Pouyamanesh G, Ameli N, Metanat Y, Khorrami A, Abbasinezhad-Moud F, Qoorchi Moheb Seraj F, et al. Thymol enhances 5-fluorouracil cytotoxicity by reducing migration and increasing apoptosis and cell cycle arrest in esophageal cancer cells: An in-vitro study. Indian J Clin Biochem. 2025;40:422-33. doi: 10.1007/s12291-024-01219-7. PMID: 40625606; PMCID: PMCPMC12229301.
  44. Nagoor Meeran MF, Javed H, Al Taee H, Azimullah S, Ojha SK. Pharmacological properties and molecular mechanisms of thymol: Prospects for its therapeutic potential and pharmaceutical development. Front Pharmacol. 2017;8:380. doi: 10.3389/fphar.2017.00380. PMID: 28694777; PMCID: PMCPMC5483461.
  45. dos Santos JG, Biavatti M, Mansani GC, Cruz LS, Kanunfre CC. Effect of thymol vapor phase on lung cancer cells exposed to radiation Efeito da fase vapor do timol em células de câncer de pulmão expostas à radiação. Braz J Dev. 2021;7:65323-34. doi: 10.34117/bjdv7n7-009.
Middle East Journal of Cancer

Articles in Press, Accepted Manuscript
Available Online from 01 October 2026
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