Targeting rhabdomyosarcoma with temozolomide: how autophagy regulates TMZ-induced apoptosis in rhabdomyosarcoma cells

Thumbnail Image
Rezaei Moghadam, Adel
Journal Title
Journal ISSN
Volume Title
Rhabdomyosarcoma (RMS) is a muscle-derived tumor and is the most common pediatric soft tissue sarcoma representing 5% of all childhood cancers. Statistically, RMS is a major clinical problem in pediatric oncology. Treatment of RMS with the oral alkylating agent temozolomide (TMZ), alone or in combination with other drugs, has recently received considerable interest. However, the mechanism of action of TMZ remains unclear. The aim of this investigation was to determine if autophagy modulates TMZ-induced cell death in an RMS cell line (RH30 cells), and determine if the cellular response to TMZ is different in RMS cells compared to non-transformed mouse myoblast cell line (C2C12 cells). We show that TMZ decreased the viability of RMS cells in a dose- and time-dependent manner and induced accumulation of sub-G1 cell population, representing apoptotic cells. Interestingly, TMZ induced apoptosis by 17-fold in the RH30 cells, (2.11% vs 36.94%; p<0.05), but only by 3-fold in C2C12 cells (10.95% vs 29.64%; not significant). In RH30 cells, TMZ decreased the expression of antiapoptotic proteins BCL-XL and MCL-1. Moreover, we show that TMZ induced biochemical markers of autophagy, such as LC3 lipidation and P62 degradation (Immunoblotting), and induced morphological evidence autophagy, including accumulation of autophagosomes and autophagolysosome in both cell lines, determined by transmission electron microscopy. Treatment of RMS cells with the autophagy inhibitor Bafilomycin A1 significantly increased TMZ-induced cell death in RMZ cell line. We have demonstrated that autophagy inhibition increased TMZ induced apoptosis. Our investigation showed that TMZ induced simultaneous autophagy and apoptosis in both RH30 and C2C12s; however, the regulation of apoptotic cell death induction by TMZ appears to be dependent on autophagy processes.
Temozolomide, Rhabdomyosarcoma, Cell Death, Apoptosis, Targeting, Autophagy