Mechanism of thalidomide to enhance cytotoxicity of temozolomide in U251-MG glioma cells in vitro.

Background Glioma is the most common primary brain tumor with poor prognosis. Temozolomide has been used with thalidomide to treat gliomas. We investigated the synergistic mechanism of these two drugs in vitro. Methods Human malignant glioma cells U251-MG were cultured and assigned to four groups with different treatments for 3 days: temozolomide group (100 mu mol/L), thalidomide group (100 mu g/L), temozolomide (100 mu mol/L) plus thalidomide group (100 mu g/L) and control group. MTT assay was applied to evaluate the cell viability. Cell cycle was analyzed by flow cytometry. The ultra-structural features of autophagosomes were observed with electron microscope. Acridine orange and monodansylcadaverine were adopted to label autophagosomes and flow cytometry was applied for quantification of autophagosomes. The expression of autophagy-associated protein was detected by Western blotting. Results Proliferation of tumor cell was obviously suppressed by temozolomide with thalidomide treatment than by either drug used alone (P=0.000 for each day). The combination treatment induced cell cycle arrest at G0/G1 phase. Typical autophagic ultra-structural character was found after the combined treatment. Thalidomide promoted the autophagy induced by temozolomide. The autophagy-associated proteins - microtubule associated protein 1 light chain 3 (MAP1LC3) and Beclin1 were more significantly up-regulated by the combined treatment than temozolomide used alone (MAP1LC3, P=0.000; Beclin1, P=0.004). The expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN), which promoted autophagy by suppressing Pl3K/Akt/mTOR signaling pathway, was elevated by thalidomide (thalidomide group: P=0.000; combined group: P=0.002). Conclusions Thalidomide enhances the cytotoxicity of temozolomide by promoting the autophagy induced by temozolomide. Contributing to the up-regulation of PTEN by thalidomide, the expression of autophagy associated protein-MAP1LC3 and Beclin1 was enhanced, which leads to a reinforced autophagy in the combined treatment of temozolomide and thalidomide in vitro. Chin Med J 2009,122(11):1260-1266