Study on mechanism of thalidomide combined with temozolomide to suppress proliferation of U251 glioma cell in vitro

Objective To provide a more reasonable regimen of temozolomide and thalidomide, and study the mechanism of these 2 drugs in inhibiting the proliferation and growth of U251 glioma cell in vitro. Methods Human glioma cell line U251 was cultured in vitro and divided into different treatment groups for 3 d: temozolomide group (100 μmol/L), thalidomide group (100 μg/L), temozolomide (100 μmol/L) with thalidomide group (100 μg/L) and vehicle control group. After different treatment for 3 d, 3⁃(4, 5⁃ dimethylthiazol⁃2⁃yl)⁃2, 5⁃diphenyltetrazolium bromide (MTT) was adopted for the determination of cell viability, and cell cycle was analysed by flow cytometry. After labeled with acridine orange (AO), autophagy vesicles were quantitatively detected by flow cytometry. TdT⁃ mediated dUTP⁃biotin nick end labeling (TUNEL) was employed in observing and detecting the apoptosis of treated cells. Western blotting was used in examining the autophagy and apoptosis⁃related proteins. Results Compared with the 2 drugs used alone, temozolomide with thalidomide imposed more obvious inhibition on tumor cell growth (P = 0.000, for all). Combination of 2 drugs induced tumor cell cycle arrest in G0-G1. Both of autophagic and apoptotic cell death could be induced by temozolomide with thalidomide in U251. In two⁃drug treatment group, the expression of autophagy⁃related microtubule⁃associated protein 1 light chain 3 (MAP1LC3) and apoptosis⁃related Caspase⁃3 was significantly higher in transcriptional level in comparing with single drug treatment group (P = 0.000, for all). Conclusion Temozolomide combined with thalidomide may induce 2 types of programmed cell death—apoptotic and autophagic cell death by up ⁃regulating the expression of related genes in U251 glioma cell, thereby, the combination of these 2 drugs may suppress the proliferation and growth of U251 glioma cell in vitro. DOI：10.3969/j.issn.1672-6731.2010.05.007