Drug Res (Stuttg) 2022; 72(04): 220-225
DOI: 10.1055/a-1775-7908
Original Article

Thymoquinone Augments Methotrexate-Induced Apoptosis on Osteosarcoma Cells

Narjes Sanapour
1   Department of Clinical Biochemistry, Tabriz University of Medical Sciences, Tabriz, Iran
Faezeh Malakoti
1   Department of Clinical Biochemistry, Tabriz University of Medical Sciences, Tabriz, Iran
Darioush Shanebandi
2   Molecular Medicine Research Center, Tabriz University of Medical Sciences, Iran
Niloufar Targhazeh
2   Molecular Medicine Research Center, Tabriz University of Medical Sciences, Iran
Bahman Yousefi
2   Molecular Medicine Research Center, Tabriz University of Medical Sciences, Iran
Jafar Soleimanpour
2   Molecular Medicine Research Center, Tabriz University of Medical Sciences, Iran
3   Department of Orthopedics Surgery, Shohada Teaching Hospital, Tabriz University of Medical Sciences, Tabriz, Iran
Maryam Majidinia
4   Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran
› Author Affiliations


Background Osteosarcoma (OS) as the most frequent primary bone malignancy in children and adolescents has a short survival rate in advanced stages. Alternative herbal medicines with fewer side effects or the potency to protect common therapy’s side effects can be helpful in combinational therapies. Herein, we aim to explore the effects of Thymoquinone (TQ) combined with Methotrexate (MTX) on Saos-2 cells apoptosis.

Methods The effects of TQ and MTX alone or in combination on Saos-2 cell viability were measured by MTT assay. Real-time PCR was applied for the measurement of Bax, BCL-2, and caspase-9 mRNA expression. Apoptosis evaluation was conducted by flow cytometry.

Results TQ improves the cytotoxic effects of MTX on Saos-2 cells proliferation at lower doses. Indeed, the IC50 of MTX decreased from 26 μM to 15 μM when it combined with TQ. TQ and MTX can induce the expression level of pro-apoptotic factors, Bax and caspase-9 while inhibiting anti-apoptotic protein BCL-2. Moreover, the combination of TQ and MTX potentiates apoptosis to 73%, compared to either TQ (48%) or MTX (53%) treated cells.

Conclusion The co-treatment of TQ and MTX is associated with the up-regulation of apoptotic factors and down-regulation of anti-apoptotic factors, conducting apoptosis aggravation and OS cell death.

Publication History

Received: 05 December 2021

Accepted: 07 February 2022

Article published online:
06 April 2022

© 2022. Thieme. All rights reserved.

Georg Thieme Verlag
Rüdigerstraße 14, 70469 Stuttgart, Germany

  • References

  • 1 Eaton BR, Schwarz R, Vatner R. et al. Osteosarcoma. Pediatr. Blood Cancer 2021; 68: e28352
  • 2 Sadykova LR, Ntekim AI, Muyangwa-Semenova M. et al. Epidemiology and Risk Factors of Osteosarcoma. Cancer Investig 2020; 38: 259-269
  • 3 Jafari F, Javdansirat S, Sanaie S. et al. Osteosarcoma: A comprehensive review of management and treatment strategies. Ann Diagn Pathol 2020; 49: 151654
  • 4 Lee DH, Thoennissen NH, Goff C. et al. Synergistic effect of low-dose cucurbitacin B and low-dose methotrexate for treatment of human osteosarcoma. Cancer Lett 2011; 306: 161-170
  • 5 van Dalen EC, van As JW, de Camargo B. Methotrexate for high-grade osteosarcoma in children and young adults. Cochrane Database Syst Rev 2011; 2011: Cd006325
  • 6 Yang XN, Zeng JC, Song YC. et al. Targeted antiosteosarcoma methotrexate-bisphosphonate conjugate induces apoptosis of osteosarcoma cells in vitro. Eur Rev Med Pharmacol Sci 2014; 18: 2116-2123
  • 7 Negrão MV, da Silva Rocha LS, da Motta Girardi D. et al. Perioperative chemotherapy with and without high-dose methotrexate in adult osteosarcoma. Anticancer Drugs 2017; 28: 915-921
  • 8 Fox E, Busch C, DeBernardo A. et al. A pharmacologically-based approach to high dose methotrexate administration to investigate nephrotoxicity and acute kidney injury biomarkers in children and adolescents with newly diagnosed osteosarcoma. Cancer Chemother Pharmacol 2021; 87: 807-815
  • 9 Valer JB, Curra M, Gabriel AF. et al. Oral mucositis in childhood cancer patients receiving high-dose methotrexate: Prevalence, relationship with other toxicities and methotrexate elimination. Int J Paediatr Dent 2021; 31: 238-246
  • 10 Dos Santos Cavalcanti A, Meohas W, Ribeiro GO. et al. Patient-derived osteosarcoma cells are resistant to methotrexate. PloS One 2017; 12: e0184891
  • 11 Yan JP, Xiang RM. Effect assessment of methotrexate in combination with other chemotherapeutic agents for osteosarcoma in children: A protocol for systematic review and meta-analysis. Medicine 2021; 100: e25534
  • 12 Zhang B, Zhang Y, Li R. et al. The efficacy and safety comparison of first-line chemotherapeutic agents (high-dose methotrexate, doxorubicin, cisplatin, and ifosfamide) for osteosarcoma: a network meta-analysis. J Orthop Surg Res 2020; 15: 51
  • 13 Yu D, Zhang S, Feng A. et al. Methotrexate, doxorubicin, and cisplatinum regimen is still the preferred option for osteosarcoma chemotherapy: A meta-analysis and clinical observation. Medicine 2019; 98: e15582
  • 14 Imran M, Rauf A, Khan IA. et al. Thymoquinone: A novel strategy to combat cancer: A review. Biomed Pharmacother 2018; 106: 390-402
  • 15 El-Far AH, Tantawy MA, Al Jaouni SK. et al. Thymoquinone-chemotherapeutic combinations: new regimen to combat cancer and cancer stem cells. Naunyn Schmiedebergs Arch. Pharmacol 2020; 393: 1581-1598
  • 16 Junaid M, Akter Y, Afrose SS. et al. Biological Role of AKT and Regulation of AKT Signaling Pathway by Thymoquinone: Perspectives in Cancer Therapeutics. Mini Rev Med Chem 2021; 21: 288-301
  • 17 Bhattacharjee M, Upadhyay P, Sarker S. et al. Combinatorial therapy of Thymoquinone and Emodin synergistically enhances apoptosis, attenuates cell migration and reduces stemness efficiently in breast cancer. Biochim Biophys Acta Gen Subj 2020; 1864: 129695
  • 18 El-Far AH, Godugu K, Noreldin AE. et al. Thymoquinone and Costunolide Induce Apoptosis of Both Proliferative and Doxorubicin-Induced-Senescent Colon and Breast Cancer Cells. Integr Cancer Ther. 2021 20. 15347354211035450
  • 19 Guler EM, Sisman BH, Kocyigit A. et al. Investigation of cellular effects of thymoquinone on glioma cell. Toxicol Rep 2021; 8: 162-170
  • 20 Peng L, Liu A, Shen Y. et al. Antitumor and anti-angiogenesis effects of thymoquinone on osteosarcoma through the NF-κB pathway. Oncol Rep 2013; 29: 571-578
  • 21 Roepke M, Diestel A, Bajbouj K. et al. Lack of p53 augments thymoquinone-induced apoptosis and caspase activation in human osteosarcoma cells. Cancer Biol Ther 2007; 6: 160-169
  • 22 Samarghandian S, Azimi-Nezhad M, Farkhondeh T. Thymoquinone-induced antitumor and apoptosis in human lung adenocarcinoma cells. J Cell Physiol 2019; 234: 10421-10431
  • 23 Malakoti F, Alemi F, Karimzadeh H. et al. Long noncoding RNA-miRNA-mRNA axes multiple roles in Osteosarcoma. Gene Rep 2021; 101090
  • 24 Smeland S, Bielack SS, Whelan J. et al. Survival and prognosis with osteosarcoma: outcomes in more than 2000 patients in the EURAMOS-1 (European and American Osteosarcoma Study) cohort. Eur J Cancer 2019; 109: 36-50
  • 25 Duchman KR, Gao Y, Miller BJ. Prognostic factors for survival in patients with high-grade osteosarcoma using the Surveillance, Epidemiology, and End Results (SEER) Program database. Cancer Epidemiol 2015; 39: 593-599
  • 26 Lilienthal I, Herold N. Targeting Molecular Mechanisms Underlying Treatment Efficacy and Resistance in Osteosarcoma: A Review of Current and Future Strategies. Int J Mol Sci 2020; 21: 6885
  • 27 Rajeswari B, Guruprasad CS, Nair M. et al. High dose methotrexate containing regimen in pediatric non-metastatic extremity osteosarcoma patients: experience from a tertiary cancer center in India. Pediatr Hematol Oncol. 2021: 1-8
  • 28 Hurkmans EGE, Klumpers MJ, Vermeulen SH. et al. Analysis of Drug Metabolizing Gene Panel in Osteosarcoma Patients Identifies Association Between Variants in SULT1E1, CYP2B6 and CYP4F8 and Methotrexate Levels and Toxicities. Front Pharmacol 2020; 11: 1241
  • 29 Yousefi B, Azimi A, Majidinia M. et al. Balaglitazone reverses P-glycoprotein-mediated multidrug resistance via upregulation of PTEN in a PPARγ-dependent manner in leukemia cells. Tumor Biol. 2017 39. 1010428317716501
  • 30 Mihanfar A, Aghazadeh Attari J, Mohebbi I. et al. Ovarian cancer stem cell: A potential therapeutic target for overcoming multidrug resistance. J Cell Physiol 2019; 234: 3238-3253
  • 31 Yousefi B, Samadi N, Baradaran B. et al. Differential effects of peroxisome proliferator-activated receptor agonists on doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. Cell Mol Biol 2015; 61: 118-122
  • 32 Niu G, Yousefi B, Qujeq D. et al. Melatonin and doxorubicin co-delivered via a functionalized graphene-dendrimeric system enhances apoptosis of osteosarcoma cells. Mater Sci Eng 2021; 119: 111554
  • 33 Sarman H, Bayram R, Benek SB. Anticancer drugs with chemotherapeutic interactions with thymoquinone in osteosarcoma cells. Eur Rev Med Pharmacol Sci 2016; 20: 1263-1270