{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:47:58Z","timestamp":1787028478700,"version":"build-2736575974"},"reference-count":61,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2022,7,25]],"date-time":"2022-07-25T00:00:00Z","timestamp":1658707200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Tri-Service General Hospital","award":["TSGH-E-111223"],"award-info":[{"award-number":["TSGH-E-111223"]}]},{"name":"Tri-Service General Hospital","award":["VTA111-T-2-1"],"award-info":[{"award-number":["VTA111-T-2-1"]}]},{"name":"the Taipei, Taichung, Kaohsiung Veterans General Hospital, Tri-Service General Hospital, Academia Sinica Joint Research Program","award":["TSGH-E-111223"],"award-info":[{"award-number":["TSGH-E-111223"]}]},{"name":"the Taipei, Taichung, Kaohsiung Veterans General Hospital, Tri-Service General Hospital, Academia Sinica Joint Research Program","award":["VTA111-T-2-1"],"award-info":[{"award-number":["VTA111-T-2-1"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Glioblastoma is the most frequent and lethal primary central nervous system tumor in adults, accounting for around 15% of intracranial neoplasms and 40\u201350% of all primary malignant brain tumors, with an annual incidence of 3\u20136 cases per 100,000 population. Despite maximum treatment, patients only have a median survival time of 15 months. Metformin is a biguanide drug utilized as the first-line medication in treating type 2 diabetes. Recently, researchers have noticed that metformin can contribute to antineoplastic activity. The objective of this study is to investigate the mechanism of metformin as a potential adjuvant treatment drug in glioblastoma. Glioblastoma cell lines U87MG, LNZ308, and LN229 were treated with metformin, and several cellular functions and metabolic states were evaluated. First, the proliferation capability was investigated using the MTS assay and BrdU assay, while cell apoptosis was evaluated using the annexin V assay. Next, a wound-healing assay and mesenchymal biomarkers (N-cadherin, vimentin, and Twist) were used to detect the cell migration ability and epithelial\u2013mesenchymal transition (EMT) status of tumor cells. Gene set enrichment analysis (GSEA) was applied to the transcriptome of the metformin-treated glioblastoma cell line. Then, DCFH-DA and MitoSOX Red dyes were used to quantify reactive oxygen species (ROS) in the cytosol and mitochondria. JC-1 dye and Western blotting analysis were used to evaluate mitochondrial membrane potential and biogenesis. In addition, the combinatory effect of temozolomide (TMZ) with metformin treatment was assessed by combination index analysis. Metformin could decrease cell viability, proliferation, and migration, increase cell apoptosis, and disrupt EMT in all three glioblastoma cell lines. The GSEA study highlighted increased ROS and hypoxia in the metformin-treated glioblastoma cells. Metformin increased ROS production, impaired mitochondrial membrane potential, and reduced mitochondrial biogenesis. The combined treatment of metformin and TMZ had U87 as synergistic, LNZ308 as antagonistic, and LN229 as additive. Metformin alone or combined with TMZ could suppress mitochondrial transcription factor A, Twist, and O6-methylguanine-DNA methyltransferase (MGMT) proteins in TMZ-resistant LN229 cells. In conclusion, our study showed that metformin decreased metabolic activity, proliferation, migration, mitochondrial biogenesis, and mitochondrial membrane potential and increased apoptosis and ROS in some glioblastoma cells. The sensitivity of the TMZ-resistant glioblastoma cell line to metformin might be mediated via the suppression of mitochondrial biogenesis, EMT, and MGMT expression. Our work provides new insights into the choice of adjuvant agents in TMZ-resistant GBM therapy.<\/jats:p>","DOI":"10.3390\/ijms23158171","type":"journal-article","created":{"date-parts":[[2022,7,25]],"date-time":"2022-07-25T21:20:59Z","timestamp":1658784059000},"page":"8171","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":33,"title":["Exploring the Mechanism of Adjuvant Treatment of Glioblastoma Using Temozolomide and Metformin"],"prefix":"10.3390","volume":"23","author":[{"given":"Shao-Wei","family":"Feng","sequence":"first","affiliation":[{"name":"Department of Neurologic Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pei-Chi","family":"Chang","sequence":"additional","affiliation":[{"name":"Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan"},{"name":"Department of Biochemistry, National Defense Medical Center, Taipei 114, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hsuan-Yu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Radiology, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7868-3161","authenticated-orcid":false,"given":"Dueng-Yuan","family":"Hueng","sequence":"additional","affiliation":[{"name":"Department of Neurologic Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan"},{"name":"Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9601-8149","authenticated-orcid":false,"given":"Yao-Feng","family":"Li","sequence":"additional","affiliation":[{"name":"Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan"},{"name":"Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9305-921X","authenticated-orcid":false,"given":"Shih-Ming","family":"Huang","sequence":"additional","affiliation":[{"name":"Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan"},{"name":"Department of Biochemistry, National Defense Medical Center, Taipei 114, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1093\/neuonc\/noab106","article-title":"The 2021 WHO Classification of Tumors of the Central Nervous System: A summary","volume":"23","author":"Louis","year":"2021","journal-title":"Neuro Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"803","DOI":"10.1007\/s00401-016-1545-1","article-title":"The 2016 World Health Organization Classification of Tumors of the Central Nervous System: A summary","volume":"131","author":"Louis","year":"2016","journal-title":"Acta Neuropathol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1807","DOI":"10.1126\/science.1164382","article-title":"An integrated genomic analysis of human glioblastoma multiforme","volume":"321","author":"Parsons","year":"2008","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1056\/NEJMoa0808710","article-title":"IDH1 and IDH2 mutations in gliomas","volume":"360","author":"Yan","year":"2009","journal-title":"N. 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