Effect of 3-methyladenine on cisplatin induced apoptosis in human glioma U251 cells

Abstract

Objective 3-methyl adenine (3-methyladenine, 3-MA) was used to test the effect on cisplatin induced U251 cell apoptosis. Methods MTT assay was used to test the effect of 3-MA on cisplatin cytotoxicity in U251 cells. Hoechst33342 staining was used to test the effect of 3-MA on cisplatin induced apoptosis. Western bolt was used to detect the expression of ER stress associated proteins, ubiquitinated protein, protein disulfide isomerase (PDI), glucose-regulated protein78 (Grp78), C/EBP homologous protein (CHOP), and activation of caspase-4 and caspase-3. The expression of microtubule-associated protein 1 light chain 3 (LC3) and PDI were observed by immunofluorescence staining and confocal microscopy. Results MTT assay showed that 3-MA could enhance cisplatin induced growth inhibition rate in human glioma U251 cells (P=0.00324 cisplatin troup vs. control group; P=0.02249 3-MA+ cisplatin group vs. cisplatin group). Hoechst33342 staining showed that 3-MA could increase cisplatin induced apoptotic effect in U251 cells. Western blot showed that 3-MA increases the expressions of ubiquitinated proteins, ERS marker proteins PDI, Grp78(P=0.00294, 0.0012, 0.00179 cisplatin group vs. control group; P=0.01896, 0.01515, 0.00734 3-MA+ cisplatin group vs cisplatin group), CHOP, and active Caspase-4 and active Caspase-3 (P=0.00081, 0.00017, 0.00018 cisplatin group vs. control group; P=0.00671, 0.00934, 0.01124 3-MA+ cisplatin group vs. cisplatin group). 3-MA could inhibit the expression of LC3II induced by cisplatin in U251 cell (P=0.00447 cisplatin group vs. control group; P=0.01588 3-MA+ cisplatin group vs. cisplatin group). Indirect immunofluorence staining showed that 3-MA inhibited the aggregation of LC3 and increased the expression of PDI. Conclusion 3-MA combined with cisplatin enhances cisplatin induced apoptosis by increasing ER stress. Key words: Cisplatin; Autophagy; ER stress; Apoptosis; Glioma