β-amyrin modulates P38 MAPK and Jnk pathway to inhibit cell proliferation and induce ROS-mediated apoptosis in HeLa cells

Abstract

It is aimed to investigate the effect of β-amyrin on p38 mitogen-activated protein kinase and Jun N-terminal kinase pathways and apoptosis in HeLa cells. HeLa treated cells were divided into 6 groups, group I-HeLa untreated cells as control, group II- dimethyl sulfoxide serve as vehicle control, group III- cisplatin as standard drug, group IV- β-amyrin-treated HeLa cells, group V- cells were pretreated with 100 μm N-acetyl-L-cystein for 1 hour and then treated with cisplatin and group VI- cells were pretreated with 100 μm N-acetyl-L-cystein for 1 hour and then treated with β-amyrin. The antiproliferative effect was measured using the MTT assay. Genotoxic effects were studied using micronucleus assay. Total reactive oxygen species, nitric oxide and caspase 3 level were determined on a spectrofluorimeter and colorimeter. Protein expression was analyzed by immunoblotting. β-Amyrin (10-200 μm) and cisplatin (0.01-100 μm) had an inhibitory effect on the proliferation of cancer cells in a dose-dependent manner, with the IC50 values at 100 μm and 10 μm for β-amyrin and cisplatin, respectively. Western blot analysis revealed expressions of apoptotic pathway related proteins, Bcl-2, caspase-3, caspase-9, phospho-p38 mitogen-activated protein kinase and phospho-Jun N-terminal kinase, growth arrest and deoxyribonucleic acid-damage-inducible, beta in all groups. Genotoxic effects were observed after treatment with β-amyrin as well as with cisplatin. It was observed that HeLa cells showed significant elevation of total reactive oxygen species after β-amyrin treatment. Protein expression analysis showed that the β-amyrin upregulated phospho-p38 mitogen-activated protein kinase, phospho-Jun N-terminal kinase and growth arrest and deoxyribonucleic acid-damage-inducible, beta on HeLa cells. Increased phospho-Jun N-terminal kinase directly activated caspases and decreased Bcl-2 in HeLa cells. These results indicated that β-amyrin induced the apoptosis through reactive oxygen species-mediated mechanism by activating p38 mitogen-activated protein kinase and Jun N-terminal kinase through transcriptional factor, GADD45β. In turn, activated Jun N-terminal kinase directly activated caspase-9 and caspase-3 and destined the HeLa cells to apoptosis.