Gemcitabine, PI3kinase-Akt Pathway Inhibition and Radiation in Human Glioma Cell Lines

ABSTRACT Introduction Glioblastoma multiforme (GBM) is the most common primary brain tumor. Standard treatment is surgery and Radiotherapy (RT) with temozolomide. The median survival is ranging from 12-14 months. However, patients with an unmethylated MGMT gene promoter do not show a survival advantage for temozolomide, and might be eligible for alternative treatment. Gemcitabine is an excellent radiosensitizer and is investigated as an alternative for temozolomide in GBM patients. Activation of the PI3Kinase-Akt pathway may preclude effective RT; therefore we investigated the radiosensitizing effect of gemcitabine in combination with the Akt inhibitor MK-2206. Materials and methods Three malignant glioma cell lines (U87, U251, and D384) were tested on cell proliferation (SRB assay) cell survival (clonogenic assay) and spheroid growth following treatment by the allosteric Akt-inhibitor MK-2206 (1 uM, 10 uM), gemcitabine and γ-irradiation (4 Gy single dose or 5 fractions of 2 Gy). Expression of Akt and pAkt was assessed by Western blot. Results The U87 cell line was most sensitive and D384 was the most resistant one. MK-2206 decreased the IC50 of gemcitabine in all cell lines from 86, 237, 903 nM for gemcitabine alone to 57, 93, 860 nM, respectively. MK-2206 down regulated pAkt expression in a concentration and time dependent manner; at 0.1 µM pAkt was partly inhibited at 30 min and completely at 1, 2, 4 and 24 h, but started to recover at 48 h. At 1 µM pAkt inhibition was immediate and lasted longer. Since 25 nM gemcitabine and 4 GY RT alone stimulated pAKT expression, cells were preincubated with MK-2206 (1 µM for 1h). MK-2206 was additive to Gemcitabine and RT in reducing clonogenic cell survival, but MK-2206 was synergistic with gemcitabine alone and with RT in inhibition of spheroid growth The effect was more pronounced with RT.Conclusion: Addition of MK-2206 to gemcitabine significantly enhanced inhibition of cell proliferation and spheroid growth and inhibited the gemcitabine and RT mediated pAkt stimulation. The use of gemcitabine in combination with PI3kinase-Akt pathway inhibitors is a promising tool in GBM therapy and might be an alternative for patients resistant to temozolomide. Disclosure All authors have declared no conflicts of interest.