Differential regulation of proapoptotic bcl-2 family genes in human glioblastoma cell line in response to cisplatin treatment
Mahdieh sadat Taghavi
,1,* Reza mahdian
,2 Azim akbarzadeh
1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2. Biotechnology Research Center, Molecular Medicine Department, Pasteur Institute of Iran, Tehran, Iran
3. Pilot Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran
Malignant gliomas are the most common primary malignancies in the brain. in an adult population, this type of tumor accounts for about 1% of all cancers, with more than 2% of deaths being attributed to malignant gliomas. cis-diamminedichloroplatinum ii (cisplatin) is one of the most effective chemotherapeutic agents used against different human cancers including malignant gliomas genes. cisplatin affects many dna-dependent cellular functions leading to dna damage and apoptosis-mediated cell death. however, its mechanism of apoptosis induction is not fully understood and may involve regulation of expression of multiple genes. bak, bax and bad are proapoptotic members of the bcl2 family that play key role in the regulation of apoptosis. in the present study, we investigated the expression of bak, bax and bad genes in u87mg cells treated with cisplatin.
U87mg was treated with various concentrations of cisplatin (3.125, 6.25, 12.5, 25, 50, and 100 μm/ml) for 48 hours. control experiments were carried out using the complete growth culture medium. cell viability was assessed using mtt assay and ic50 was determined. the u87mg glioma cell line was treated with ic50 dose of cisplatin for 48 hours. rna was extracted and cdna was synthesized. gene expression study was performed on bak, bax and bad as targets and tbp as internal control gene using very sensitive quantitative real-time pcr. the gene expression ratios was calculated using the formula 2-ΔΔct.
Different concentrations of cisplatin at 48 hours had a cytotoxic effect on glioblastoma cell lines ic50 of cisplatin after 48 hours was 19.66 μg/ml for u87mg. melting curve analysis, and gel electrophoresis confirmed the specific amplification of fragments of interest. the relative gene expressions between two samples (treated and untreated) were calculated as 1.02, 1.09 and 0.71 for bak, bax and bad.
Gene expression studies can determine expression profiling for bcl2 family genes and their effects on apoptosis pathways in different cell lines after cisplatin treatment. our results showed that cisplatin treatment induced different regulation of bak, bax and bad genes, in u87mg cell line.
Bcl2 family, cisplatin, , glioma, real-time pcr