Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251)

Ayşe Mine Yılmaz; Burak Kızılca; Zafer Alparslan; Kadir Nokay; Şürkü Toprak Durkan; Ebrar Tutar; Betul Yılmaz

doi:10.12991/jrespharm.2031131

Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251)

Abstract

Glioblastoma multiforme (GBM) is an astrocytoma classified among the most aggressive brain tumors. Currently considered incurable, patients have a median survival of 15 months after diagnosis. Treatment typically involves tumor resection followed by radiotherapy and chemotherapy. Temozolomide is the most commonly used chemotherapeutic agent for GBM. Given the short median survival and the development of drug resistance to temozolomide, enhancing the efficacy of current treatments is of paramount importance. In this context, insulin is suggested as a supportive therapy to chemotherapy. It is hypothesized that insulin enhances the effectiveness of antimetabolite chemotherapies by prolonging the S phase. While insulin has been observed to extend the S phase in various cancers in the literature, its direct effect on the cell cycle in GBM has not been explored despite studies examining the impact of insulin on GBM by inhibiting insulin and IGF receptor gene function. In the MTT assay, cell survival decreased in groups treated with temozolomide (p<0.001), while no significant difference was found in the survival of groups containing insulin. In the cell cycle analysis, insulin in temozolomide-treated groups was observed to increase G0/G1 phase with increasing doses while shortening the S phase (p<0.008). The phenomenon of increasing the effectiveness of antimetabolite chemotherapies through S phase extension was not demonstrated in our data on the GBM U251 cell line; the S phase did not prolong. Considering temozolomide is an alkylating agent rather than an antimetabolite chemotherapy agent, the absence of this phenomenon should be expected. Therefore, insulin chemotherapy application is not expected to affect GBM based on the current hypothesis and was not observed in our study. If insulin enhances the efficacy of alkylating agents, it must act through a pathway other than the S phase, although no viability effect was observed in our study. In our study, the GBM model cell line U251 was cultured under appropriate conditions. In a total of 8 groups, insulin (at doses of 100 mU/L and 1 U/L) and temozolomide were administered according to literature-based protocols. MTT cell viability assay, cell cycle analysis via flow cytometry, and western blot experiments (assessing the expressions of p21, p27, p53, and beta-actin proteins) were conducted. Statistical analyses of the data were performed using GraphPad 9.0 software, with p<0.05 considered statistically significant.

Keywords

Supporting Institution

The experiments of the project were carried out at the Marmara University Faculty of Medicine, Department of Medical Biochemistry and the GEMHAM Research Center.

Project Number

TUBİTAK-2209A

Ethical Statement

The experiments performed in this project were obtained from cell culture studies; therefore, an ethics committee report is not necessary.

Thanks

This study has been supported by TÜBİTAK as project number 2209-A.

References

[1] Tamimi AF, Juweid M. Epidemiology and Outcome of Glioblastoma. In: De Vleeschouwer S, editor. Glioblastoma [Internet]. Brisbane (AU): Codon Publications. 2017;27.
[2] Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholtz-Sloan JS, Villano JL. Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Biomarkers Prev. 2014;23(10):1985-96. https://doi.org/10.1158/1055-9965.EPI-14-0275.
[3] Louis, DN, Perry A, Reifenberger G. et al. The 2016 World Health Organization Classification of Tumors of the Central Nervous System: a summary. Acta Neuropathol. 2016; 131: 803–820. https://doi.org/10.1007/s00401-016-1545-1.
[4] Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO; European Organisation for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005 ; 352(10):987-96. doi: 10.1056/NEJMoa043330.
[5] Darkes MJ, Plosker G L, Jarvis B. Temozolomide: a review of its use in the treatment of malignant gliomas, malignant melanoma and other advanced cancers. Am J Cancer. 2002; 1(1): 55-80. DOI:10.2165/00024669-200201010-00006.
[6] Villano JL, Seery TE, Bressler LR. Temozolomide in malignant gliomas: current use and future targets. Cancer Chemother Pharmacol. 2009; 64(4): 647–655. https://doi.org/10.1007/s00280-009-1050-5.
[7] Zhang AMY, Wellberg EA, Kopp JL, Johnson JD. Hyperinsulinemia in Obesity, Inflammation, and Cancer. Diabetes Metab J. 2021; 45(3): 285-311. doi: 10.4093/dmj.2020.0250.
[8] Rose DP, Vona-Davis L. The cellular and molecular mechanisms by which insulin influences breast cancer risk and progression. Endocr Relat Cancer. 2012. DOI: 10.1530/ERC-12-0203.

[9]Vigneri R, Sciacca L, Vigneri P. Rethinking the Relationship between Insulin and Cancer. Trends Endocrinol Metab. 2020. https://doi.org/10.1016/j.tem.2020.05.004.
[10]Damyanov C, Gerasimova D, Maslev I, Gavrilov V. Low-dose chemotherapy with insulin (insulin potentiation therapy) in combination with hormone therapy for treatment of castration-resistant prostate cancer. ISRN Urol. 2012; 2012: 140182. https://doi.org/10.5402/2012/140182.
[11]Baserga R. The insulin receptor substrate-1: a biomarker for cancer? Exp Cell Res. 2009 https://doi.org/10.1016/j.yexcr.2008.09.017.
[12]Malaguarnera R, Belfiore A. The insulin receptor: a new target for cancer therapy. Front Endocrinol. 2011; 2: 93. https://doi.org/10.3389/fendo.2011.00093.
[13] Zou K, Ju JH, Xie H. Pretreatment with insulin enhances anticancer functions of 5-fluorouracil in human esophageal and colonic cancer cells. Acta Pharmacol Sinica. 2007;28(5):721–730. https://doi.org/10.1111/j.1745-7254.2007.00554.x.
[14] Iyikesici MS, Slocum AK, Slocum A, Berkarda F B, Kalamian M, Seyfried TN. Efficacy of Metabolically Supported Chemotherapy Combined with Ketogenic Diet, Hyperthermia, and Hyperbaric Oxygen Therapy for Stage IV Triple-Negative Breast Cancer. Cureus. 2017; 9(7): e1445. https://doi.org/10.7759/cureus.1445.
[15]Iyikesici MS. Survival outcomes of metabolically supported chemotherapy combined with ketogenic diet, hyperthermia, and hyperbaric oxygen therapy in advanced gastric cancer. Niger J Clin Pract. 2020; 23(5): 734–740. https://doi.org/10.4103/njcp.njcp_509_18.
[16] Iyikesici MS, Slocum AK, Winters N, Kalamian M, Seyfried TN. Metabolically Supported Chemotherapy for Managing End-Stage Breast Cancer: A Complete and Durable Response. Cureus. 2021;13(4):e14686. https://doi.org/10.7759/cureus.14686.
[17] Sha H, Li Y, Du X, Wang H. Insulin in endometrial carcinoma chemotherapy: a beneficial addition and not a problem. Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban. 2010; 30(5): 631–637. https://doi.org/10.1007/s11596-010-0555-9.
[18] Gong Y. Ma Y, Sinyuk M, Loganathan S, Thompson RC, Sarkaria JN, Chen W, Lathia J D, Mobley BC, Clark SW, Wang J. Insulin-mediated signaling promotes proliferation and survival of glioblastoma through Akt activation. Neuro-Oncol. 2016; 18(1): 48–57. https://doi.org/10.1093/neuonc/nov096.
[19] Wu D, Dong S, Zhou W. Insulin like growth factor in cancer: New perspectives (Review). Mol Med Rep. 2025; 32: 209. DOI: https://doi.org/10.3892/mmr.2025.13574.
[20] Wick W, Weller M, Weiler M, Batchelor T, Yung AW, Platten M. Pathway inhibition: emerging molecular targets for treating glioblastoma. Neuro-Oncol. 2011; 13(6): 566–579. https://doi.org/10.1093/neuonc/nor039.
[21] Skuli SJ, Alomari S, Gaitsch H, Bakayoko A, Skuli N, Tyler B. M. Metformin and Cancer, an Ambiguanidous Relationship. Pharmaceuticals (Basel). 2022; 15(5): 626. https://doi.org/10.3390/ph15050626.
[22] Ye L, Liu X, Jin K, Niu Y, Zuo Q, Song J, Han W, Chen G, Li B. Effects of Insulin on Proliferation, Apoptosis, and Ferroptosis in Primordial Germ Cells via PI3K-AKT-mTOR Signaling Pathway. Genes. 2023;14(10):1975. https://doi.org/10.3390/genes14101975.
[23] Singh N, Miner A, Hennis L, Mittal S. Mechanisms of temozolomide resistance in glioblastoma - a comprehensive review. Cancer Drug Resist. 2021; 4: 17-43. http://dx.doi.org/10.20517/cdr.2020.79.
[24] Akbarnejad Z, Eskandary H, Dini L, Vergallo C, Nematollahi-Mahani SN, Farsinejad A, Abadi M F S, Ahmadi M. Cytotoxicity of temozolomide on human glioblastoma cells is enhanced by the concomitant exposure to an extremely low-frequency electromagnetic field (100Hz, 100G). Biomed Pharmacother. 2017; 92:254–264. https://doi.org/10.1016/j.biopha.2017.05.050.
[25] Gallagher EJ, LeRoith D. The proliferating role of insulin and insulin-like growth factors in cancer. Trends Endocrinol Metab. 2010; 21(10): 610–618. https://doi.org/10.1016/j.tem.2010.06.007.
[26] Yung WK. Temozolomide in malignant gliomas. Semin Oncology. 2000;27 (3 Suppl 6): 27–34.
[27] D'Atri S, Tentori L, Lacal PM, Graziani G, Pagani E, Benincasa E, Zambruno G, Bonmassar E, Jiricny J. Involvement of the mismatch repair system in temozolomide-induced apoptosis. Mol Pharmacol. 1998; 54(2): 334–341. https://doi.org/10.1124/mol.54.2.334.
[28] Garcia JH, Jain S, Aghi MK. Metabolic Drivers of Invasion in Glioblastoma. Front. Cell Dev. Biol. 2021; 9:683276. doi: 10.3389/fcell.2021.683276.

Details

Primary Language

English

Subjects

Basic Pharmacology, Medical Pharmacology

Journal Section

Research Article

Authors

Ayşe Mine Yılmaz ^*
0000-0003-0802-523X
Türkiye

Burak Kızılca
0009-0006-9929-8514
Türkiye

Zafer Alparslan
0009-0004-1037-6594
Türkiye

Kadir Nokay
0009-0006-2600-6605
Türkiye

Şürkü Toprak Durkan
0009-0007-5264-4253
Türkiye

Ebrar Tutar
0000-0003-2273-7487
Türkiye

Betul Yılmaz
0000-0003-1762-0284
Türkiye

Publication Date

October 6, 2026

Submission Date

September 3, 2026

Acceptance Date

September 21, 2026

Published in Issue

Year 2026 Volume: 30 Number: 5

DOI

https://doi.org/10.12991/jrespharm.2031131

IZ

https://izlik.org/JA63TE96NF

Cite

RIS / Bibtex

APA

Yılmaz, A. M., Kızılca, B., Alparslan, Z., Nokay, K., Durkan, Ş. T., Tutar, E., & Yılmaz, B. (2026). Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251). Journal of Research in Pharmacy, 30(5), 1281-1291. https://doi.org/10.12991/jrespharm.2031131

AMA

1.Yılmaz AM, Kızılca B, Alparslan Z, et al. Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251). J. Res. Pharm. 2026;30(5):1281-1291. doi:10.12991/jrespharm.2031131

Chicago

Yılmaz, Ayşe Mine, Burak Kızılca, Zafer Alparslan, et al. 2026. “Insulin and Temozolomide Effects in a Glioblastoma Multiforme Cell Model (U-251)”. Journal of Research in Pharmacy 30 (5): 1281-91. https://doi.org/10.12991/jrespharm.2031131.

EndNote

Yılmaz AM, Kızılca B, Alparslan Z, Nokay K, Durkan ŞT, Tutar E, Yılmaz B (October 1, 2026) Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251). Journal of Research in Pharmacy 30 5 1281–1291.

IEEE

[1]A. M. Yılmaz et al., “Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251)”, J. Res. Pharm., vol. 30, no. 5, pp. 1281–1291, Oct. 2026, doi: 10.12991/jrespharm.2031131.

ISNAD

Yılmaz, Ayşe Mine - Kızılca, Burak - Alparslan, Zafer - Nokay, Kadir - Durkan, Şürkü Toprak - Tutar, Ebrar - Yılmaz, Betul. “Insulin and Temozolomide Effects in a Glioblastoma Multiforme Cell Model (U-251)”. Journal of Research in Pharmacy 30/5 (October 1, 2026): 1281-1291. https://doi.org/10.12991/jrespharm.2031131.

JAMA

1.Yılmaz AM, Kızılca B, Alparslan Z, Nokay K, Durkan ŞT, Tutar E, Yılmaz B. Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251). J. Res. Pharm. 2026;30:1281–1291.

MLA

Yılmaz, Ayşe Mine, et al. “Insulin and Temozolomide Effects in a Glioblastoma Multiforme Cell Model (U-251)”. Journal of Research in Pharmacy, vol. 30, no. 5, Oct. 2026, pp. 1281-9, doi:10.12991/jrespharm.2031131.

Vancouver

1.Ayşe Mine Yılmaz, Burak Kızılca, Zafer Alparslan, Kadir Nokay, Şürkü Toprak Durkan, Ebrar Tutar, Betul Yılmaz. Insulin and Temozolomide effects in a glioblastoma multiforme cell model (U-251). J. Res. Pharm. 2026 Oct. 1;30(5):1281-9. doi:10.12991/jrespharm.2031131