19th Annual Child Health Research Days

RBC Convention Center

Oct. 24 & 26, 2023


#42 Role of autophagy in temozolomide-induced apoptosis in Rhabdomyosarcoma cells.

Adel Rezaei Moghadam, University of Manitoba; Simone Cristina da Silva Rosa, University of Manitoba; Javad Alizadeh, University of Manitoba; Jared Field, University of Manitoba; Saeid Ghavami, University of Manitoba; Joseph W. Gordon, University of Manitoba


Rhabdomyosarcoma (RMS) is a muscle-derived tumor and is the most common pediatric soft tissue sarcoma representing 5% of all childhood cancers. 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 RMS cell lines.


MTT assay and Nicoletti analysis were used to measure cell death in SJCRH30 (RH30) human RMS cells and the mouse myoblast cell line C2C12, following TMZ treatment (100 µM). We monitored autophagy using electron microcopy and immunoblotting (LC3 lipidation, Atg15-12 conjugation, p62 degredation). We also confirmed TMZ induced autophagy flux induction in RH30 cells using bafilomycin A1 (100 nM, 3 hrs) in presence of TMZ treatment (100 μM). The effect of bafilomycin A1 (4,6 nM) on TMZ-induced cytotoxicity was also evaluated.


We showed that TMZ decreased the viability of RMS cells in a dose/time-dependent manner and induced accumulation of sub-G1 cell population, representing apoptotic cells. Interestingly, TMZ induced apoptosis by 16-fold in the RH30 cells, but only increased apoptosis by 63% in C2C12 cells. In RH30 cells, TMZ decreased the expression of antiapoptotic proteins Bcl-XL and Mcl-1, and increased the expression of the death gene Nix. Moreover, we showed that TMZ altered biochemical markers of autophagy, and induced morphological evidence of autophagosomes formation. Finally, treatment of RMS cells with Bafilomycin A1( 4 and 6 nM) significantly increased TMZ-induced cell death in RH30 cells.


In conclusion, our investigation showed that TMZ induced simultaneous autophagy and apoptosis in both RH30 and C2C12 cells and cell death induction by TMZ was dependent on Bafilomycin A1 inhibited processes, such as autophagosome-lysosome fusion or autolysosome acidification.