Interestingly, the invasion capacity of metformin treated TMZ-resistant cells was even lower than the two parental lines, indicating that metformin might exert direct inhibition for cell invasion in addition to working via the pathways that are activated by TMZ. == Metformin down-regulates SOX2 expression and reduces the formation of neurospheres in TMZ-resistant glioblastoma cells == SOX2 has been reported to play a pivotal role in developing drug resistance during glioblastoma treatment [24, 25]. which will in turn yield potentially translational value for clinical applications. Keywords: glioblastoma, metformin, temozolomide resistance, SOX2, global gene expression == INTRO == Glioblastoma multiforme (GBM) is the most prevalent and deadly primary malignant brain tumor, with a median overall survival of 14. 6 months [1]. Current treatments of GBM include maximum safe craniotomy, radiotherapy, and chemotherapy. Temozolomide (TMZ) is the main drug in chemotherapy regimens intended for newly diagnosed GBM. TMZ induces double-stranded DNA breaks in tumor cells and acts via interference with the DNA mismatch repair (MMR) pathway [2, 3]. Despite intense therapies, GBM recurrence is inevitable in most GBM patients [4]. This is partly due to the development of TMZ resistance, which is mediated by multiple possible mechanisms, including increased activity of DNA repair enzyme, O6-methylguanine DNA methyltransferase (MGMT) [5, 6], amplification or mutations of the epidermal growth element receptor (EGFR) gene [7], mutations of tumor suppressor genes TP53 and PTEN [8, 9], and inepte miRNA expression [10]. Although PSMA617 TFA different therapeutic molecules have been developed to target these pathways and have demonstrated promising results from bothin vitroandin vivoexperimental models, these strategies often showed limited efficacy and/or intolerable side effects when applied to GBM patients. Recent work has shown that metformin, an FDA approved anti-diabetic medication , could have anti-cancer effects in patients with a variety of cancer types including breast cancer, pancreatic cancer, colon cancer, and ovarian cancer [1114]. In addition , metformin might have synergistic effects with TMZ treatment and enhance chemotherapy efficacy in GBM [1517], which opens a new avenue to conquer TMZ resistance in glioma treatment. As an inexpensive, well-tolerated, first-line anti-diabetic oral drug, metformin continues to be reported to significantly reduce gluconeogenesis in the liver and increase insulin receptor sensitivity and glucose uptake in peripheral tissues. In addition , metformin also functions along the fatty acid metabolism pathway by de-repressing fatty acid oxidation. Several potential mechanisms have been investigated attempting to explain the anti-cancer effects of metformin. Previous reports have identified metformin playing a role in activating AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling pathway, which is important in regulating cancer cell survival, proliferation and apoptosis, as well as the process of epithelial-to-mesenchymal cells transition (EMT) phenotype [1214]. As AKT phosphorylation is implicated in TMZ drug resistance [18, 19], it is possible that metformin might take action via inhibition of AKT phosphorylation in cancer cells, thus inhibiting cancer proliferation, metastasis, PSMA617 TFA and drug resistance [16, 20]. Metformin has also been found to reverse or reduce drug resistance through inhibition of insulin-like growth factor-1-receptor (IGF1R) [21, 22]. To investigate the potential mechanisms of how metformin functions with TMZ and identify molecular changes in gene expression regulatory networks in GBM, we developed two TMZ-resistant glioblastoma cell lines, and compared proliferation, neurosphere formation, and invasion capacity of metformin treated, TMZ-resistant cells with their corresponding parental cells. Our results demonstrate that metformin might function through multiple pathways in partial restoration of TMZ sensitivity in glioblastoma cells, which subsequently enhances chemotherapy effects of TMZ. == RESULTS == == Generation of TMZ-resistant glioblastoma HBEGF cell lines == Glioblastoma cell lines U87 and U251 (named as U87P and U251P intended for parental cell lines. Nomenclature of all cell lines is listed inSupplementary Table S1) were treated with TMZ with gradually increasing doses, starting from 50 M to 600 M, over a period of 810 months. IC50(half or 50% minimal inhibitory concentration) was used to monitor the change of their resistance properties. Before TMZ treatment induction, IC50of U87P was 325 M. At the end of the treatment, IC50has increased by 2 . 6 folds and reached 1, 165 M. Similarly, IC50for U251P was 722 M, while the cells obtained after TMZ induction showed an IC50of 1, 994 M, a PSMA617 TFA nearly 2-fold increase compared to the parental line. It is worth noting that once established, both TMZ-resistant cell lines maintained strong resistance to further TMZ treatment. These.