Inhibition of caspase-9 activity and Apaf-1 expression in cisplatin-resistant head and neck squamous cell carcinoma cells.

We have previously reported that cisplatin induces caspase-9 (Casp9) activation in head and neck squamous cell carcinoma cells in vitro (HNSCCs). Our purpose here was to examine whether HNSCCs selected for resistance to cisplatin fail to exhibit Casp9 activation in response to cisplatin. The cisplatin-resistant HNSCCs (HSC-2CR) were selected from cisplatin-sensitive HNSCCs (HSC-2) for growth in the presence of cisplatin. Following cisplatin treatment, protelyzed Casp9 subunits were detected in HSC-2, but not detected in HSC-2CR. Using a direct enzymatic assay measuring cleavage of the synthetic peptide substrate (LEHD-AFC), Casp9 activity in cisplatin-treated HSC-2CR was less than that in cisplatin-treated HSC-2. Apoptotic protease-activating factor 1 (Apaf-1) has been shown to participate as an adaptor molecule in Casp9 activation. In the presence of cytochrome c (Cyt c) released from mitochondria, Apaf-1 binds to Casp9 and causes its activation. HSC-2 expressed 2-fold higher levels of Apaf-1 compared with HSC-2CR. On the other hand, following cisplatin treatment, the same degree of increase in cytoplasmic Cyt c was detected in both HSC-2 and HSC-2CR. These results suggest that in a certain type of HNSCCs, the inhibition of Casp9 activity and Apaf-1 expression may represent a mechanism of acquired cisplatin resistance.