Abstract
Objective: This is an in vitro experimental study designed to analyze the role of alternative splicing of mRNA in the apoptotic process of the cancer cells. Here we induced apoptosis in the glioblastoma multiforme (GBM) T-98G cell line to obtain a better understanding in the regulation of mRNA expression of the soluble Tumor Necrosis factor-related Apoptosis-Inducing Ligand (sTRAIL) gene.
 Methods: Cells were induced to undergo apoptosis by treatment with rotenone at 10, 20 and 40 µM for 6 h. Dimethylsulphoxide (DMSO) was used to dissolve rotenone and as a negative control. The morphology of the GBM-T98G cells was viewed with an inverted microscope. DNA, RNA and protein extractions were performed to analyse apoptotic DNA fragmentation by a DNA laddering assay, a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for TRAIL mRNA expression and ELISA for caspase-9 protein expression. Electrophoresis was also performed on TRAIL complementary DNA (cDNA) produced from TRAIL qRT-PCR mRNA.
 Results: Nucleosomal DNA degradation was confirmed by DNA laddering, whereas the TRAIL melting curve and the cDNA electrophoresis showed a shift in the balance of the TRAIL mRNA isoform to the pro-apoptotic mRNA isoform, in conjunction with a significant increase in expression of TRAIL mRNA and caspase-9 protein.
 Conclusion: These findings indicate the regulation of apoptotic events at the level of TRAIL mRNA expression, as indicated by the shift in the balance of mRNA expression of the TRAIL isoform towards the pro-apoptotic isoform.
Highlights
Glioblastoma multiforme (GBM) is the most aggressive and invasive malignant subset of brain tumours and is associated with dismal prognosis [1]
Observation of the cell morphology showed membrane budding changes in glioblastoma T-98G cells treated with 10 μM rotenone
Cells treated with 40 μM rotenone were swollen, indicating that the cell membranes were no longer intact; these hydropic degeneration picture leads to the process of necrotic degradation
Summary
Glioblastoma multiforme (GBM) is the most aggressive and invasive malignant subset of brain tumours and is associated with dismal prognosis [1]. The standard first-line treatment for glioblastoma includes surgery, followed by focal fractionated radiotherapy with concomitant and adjuvant administration of the alkylating chemotherapy temozolomide. The addition of temozolomide significantly improves the median 2-and 5 y survival when compared to radiotherapy alone in patients with newly diagnosed glioblastoma. Regardless of treatment, glioblastoma patients have a poor prognosis, with a median survival of 14.6 mo [1, 2]. Malignant gliomas show resistance to apoptosis—a characteristic that underlies both tumourigenesis and the inherent resistance of cancer cells to radiotherapy and chemotherapy. A full understanding of the cell death mechanism and the genetic regulation behind it is obviously of great medical interest
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