Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: Evidence for cytotoxic mechanisms different from inhibition of protein synthesis

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase, Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-VaI-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis, Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis, However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria, In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bar from the cytosol onto mitochondria in response to onconase, This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated tells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent. [References: 68]

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