Effects of temozolomide on anti-apoptosis of glioma stem cells, multiple drug-resistant genes and cell cycle

Objective To analyze the effects of temozolomide (TMZ) on glioma U251 cells and their stem cell livin, MRP gene expression and cell cycle. Methods After U251 cells and their stem cells being isolated and cultured, the different concentrations of TMZ (0, 25, 50, 100, 200, and 400 μmol/L) were used to intervene for 72 h. CCK-8 was used to detect the change trend of cell proliferation. Flow cytometry (FCM) was used to detect the changes of cell cycle. RT-PCR technique was used to detect the expression levels of livin, MRP1, and MRP3 mRNA. Results Our study successfully isolated stem cells U251from U251 cells. Compared with a non-intervention group, after 25 μmol/L TMZ intervention of U251 cells and their stem cells, the proliferation rate were 0.952±0.011 (t=-8.219, P=0.001) and 0.937±0.029 (t=-3.822, P=0.019) respectively, and after 100 μmol/L TMZ intervention of U251 cells and their stem cells, the proliferation rate were 0.750±0.018 and 0.887±0.039 (t=5.480, P=0.005) respectively. Under the normal circumstances, the expression level of livin gene in U251 cells and their stem cells were 0.411±0.025×10-3 and 0.571±0.040×10-3 (t=-3.348, P=0.004) respectively; the expression level of MRP1 gene were 0.295±0.018×10-3 and 0.481±0.034×10-3 (t=-8.439, P=0.001) respectively; the expression level of MRP3 gene were 1.128±0.117×10-3 and 0.963±0.059 ×10-3 (t=2.176, P=0.095) respectively. After 200 μmol/L TMZ intervention of U251 cells and their stem cells, the expression levels of livin mRNA were 0.020±0.002×10-3 (t=27.123, P=0) and 0.066±0.007×10-3 (t=21.345, P=0) respectively. After the intervention of 200 μmol/L, the expression levels of MRP1 mRNA were 0.556±0.041×10-3 (t=-10.214, P=0.001) and 0.609±0.044×10-3 (t=-3.98, P=0.016); after the intervention of 200 μmol/L, the expression levels of MRP3 mRNA were 1.397±0.192×10-3 (t=-2.073, P=0.107) and 1.496±0.302×10-3 (t=-2.993, P=0.040); after the intervention of 100 μmol/L, the percentage of U251 cells in the cell cycle G2/M phase were 11.353±0.516 and 39.1±1.2 (t=-37.1, P=0), and the percentage of U251 stem cells in S phase were 21.1±1.0 and 30.2±1.7 (t=-8.07, P=0.001). Conclusions TMZ can effectively decrease the livin gene expression in U251 and its stem cells, and arrest cell division cycle, decrease cell proliferation rate, and promote cell apoptosis. MRP1 and MRP3 may be respectively one of the main reasons of primary glioma and recurrent glioma resistance. Key words: Glioma; Neoplastic stem cells; Neuronal apoptosis-inhibitory protein; Multidrug resistance-associated proteins; Cell cycle; Temozolomide