Effects Of Temozolomide In Combination With Metformin On Properties Of Glioma Cells And Glioma Stem Cells And Mechanism

Background: Temozolomide(TMZ) is the major chemotherapeutic drug used clinically in the treatment of glioblastoma(GBM), the prognosis of patients with GBM has remained dismal. The fast recurrence and multi-drug resistance are some of the key challenges in combating brain tumors. Glioma stem cells(GSCs) which are considered the source of relapse and chemoresistance, the need for more effective therapeutic options is overwhelming. Recent studies suggested that one of the cytotoxic mechanisms of TMZ on GBM goes through an AMP-activated protein kinase(AMPK) activation step. Accumulating evidence shows that GBM frequently displays hyperactivation of the Akt pathway and endogenous Akt kinase activity can be activated in response to clinically relevant concentrations of TMZ. Epidemiologic data have revealed that Metformin(MET), a first-line treatment for type-2 diabetes, can selectively kill cancer stem cells with minor adverse effects. Its mechanism of antiproliferative mainly by activating AMPK and inhibiting the activity of Akt. But whether MET can potentiate the cytotoxicity of TMZ for GSCs is still unknown.Objective: The purpose of the present study was to explore the effects of temozolomide in combination with metformin on properties of glioma cells and glioma stem cells in vitro, and to clarify the mechanism.Methods: The human glioma U87 and U251 cells were cultured in the neural stem cell medium, and the GSCs were identified by Immunofluorescence methods and flow cytometry; the GSCs were elected and were treated with(control group) different concentrations of TMZ(TMZ group) and MET(MET group) or TMZ plus MET(TMZ+MET group); the number of secondary neurosphere formation were countedunder microscope, proliferation inhibition of GSCs was assessed by CCK-8 assay and the combined effect of two drugs was analyzed with Chou-Talalay combination index(CI); flow cytometry, Annexin V and PI staining were used to detect the percentage of cell apoptotic rate of GSCs, invasion rate was evaluated in transwell culture system; p-AMPK/AMPK, p-ACC/ACC, p-Akt/Akt, p-m TOR/m TOR, p-4EBP1/4EBP1, p-S6K/S6 K and β-actin were detected by Western blotting.Results: 1. The cells within the sphere were positive for neural stem cell markers CD133 and nestin, and lack of immunoreactivity for markers of differentiated neural cell types such as GFAP for astrocytes and β-tubulin III for neurons. 2. TMZ plus MET acts synergistically in inhibiting glioma cells and GSCs proliferation(P<0.05, compared with single agent; CI<1). TMZ plus MET contribute more effectively to inhibit GSCs self-renewal and inhibit gliosphere formation and expansion(P<0.05, compared with single agent). The combinatorial treatment significantly inducing GSCs apoptosis compared with single drug(P< 0.05), and the expression of Bcl-2 was significantly decreased, meanwhile the activities of Bax and cleaved caspase-3 were markedly elevated, compared with the TMZ or MET groups. The combination of TMZ and MET inhibits invasion of GSCs in vitro. 3. TMZ induced Akt phosphorylation in a time-dependent manner and MET decreased phosphorylation of Akt in a dose- and time-dependent manner. MET effectively reversed the Akt activation induced by TMZ after 48 hours treatment. We although found that single-agent treatment with TMZ or MET showed obvious inhibition of phosphorylated m TOR, 4EBP1, and S6 K, under combination of the two drugs, the activated forms of these proteins were significantly suppressed under their combination. Compared with single drug, treatment with dual PI3K/m TOR inhibitor NVP-BEZ235 significantly increases cell apoptosis in combination with TMZ or MET under standard growth conditions after 48 hours(P < 0.05). In addition, we found that the combination of NVP-BEZ235 with both TMZ and MET effectively promoted extensive cell apoptosis within 48 hours in U87 GSCs and U251 GSCs(P <0.05). These results show that TMZ combined with MET synergize to inhibit GSCs proliferation through downregulation of Akt/m TOR signaling pathway, since addition of Akt-m TOR inhibitor such as NVP-BEZ235 promotes massive and rapid cell death. 4. Both TMZ and MET clearly induced AMPK phosphorylation in a dose-and time-dependent manner. When AMPK activity was inhibited with its inhibitor compound C, TMZ-induced cell apoptosis and death were largely repressed in AMPK inhibiting cells(P < 0.05, TMZ vs TMZ+compound C), but there was only a slight effect on MET(P > 0.05, MET vs MET+compound C) and the MET alone still induced a significant level of cell apoptosis and death. Although AMPK activation inhibited by compound C, the combination treatment of TMZ and MET still produced synergistic cell apoptosis compared with any single drug. Moreover, compound C mildly inhibited TMZ- and MET-induced GSCs apoptosis(P>0.05, TMZ+MET+compound C vs TMZ+MET). These findings along with the data from compound C suggest that alteration of AMPK pathway may be one of the mechanisms for the synergism between TMZ and MET combination, but AMPK activation is not solely or definitively responsible for the synergistic effect for the combination of TMZ and MET.Conclusion: 1. Serum free culture method can be used to develop GSCs which have the ability of self-renewal and multi-direction differentiation. 2. The combination of TMZ and MET acted synergistically to inhibit the serial self-renewal capability of GSCs as well as elimination GSCs. 3. TMZ in combination with MET synergistically inhibits the GSCs proliferation through downregulation of Akt-m TOR signaling pathway. 4. The combination of TMZ and MET was accompanied with a powerful AMPK activation, while this pathway is not determinant.