Paclitaxel-resistant MCF-7 cells show a caspase-less phenotype but retain sensitivity to many anticancer agents

2657 Anticancer drug resistance occurs as a result of altered response to cytotoxic insult, particularly via inhibition or inactivation of apoptosis (programmed cell death type I, PCDI), and plays a major role in tumour development and progression. An alternative form of cell death - non-apoptotic or autophagic cell death (PCDII) has recently emerged as a factor contributing to the cytotoxic response of cancer cells. We have studied cell death in a drug resistant model MCF-7 human breast cancer cells with acquired resistance (c. 20-fold) to paclitaxel termed MCF-7TaxR. This novel cell line was generated by us in-house. It has been reported that the absence of caspase-3 in parent MCF-7 cells (due to chromosome deletion) may explain why this cell line recruits both apoptotic and autophagic cell death mechanisms following cytotoxic insult. Considering the mitochondrial pathway of apoptosis we detected caspase-9 protein in MCF-7 cells but a total absence in MCF-7TaxR (western blotting), confirmed by PCR analysis for mRNA levels. Interestingly, MCF-7TaxR was shown to over-express the MDR transporter P-glycoprotein (western blot). However, MTT cell viability assay indicated that MCF-7TaxR cells showed a marked lack of cross-resistance to a range of agents such as VP-16, doxorubicin and vinblastine, contrary to the MDR phenotype. In addition, MCF-7TaxR displayed collateral sensitivity to carboplatin and cisplatin relative to the parental MCF-7 cells i.e. IC50values for carboplatin: MCF-7TaxR 26.0 µM; MCF-7 84.0 µM; for cisplatin: MCF-7TaxR 7.5 µM; MCF-7 17.0 µM. Apoptotic response was examined by flow cytometry (Annexin-V with propidium iodide) by treating MCF-7, MCF-7TaxR and MB-231 (caspase-proficient control) breast cancer cells with 0.5 µM staurosporine together with the pan-caspase inhibitor Z-VAD (100 µM ). Results showed the ability of staurosporine to induce caspase-mediated apoptosis in breast cancer cells was of the order: MB-231>>MCF-7>> MCF-7TaxR. Further western blot analysis revealed an absence of caspase-7 in MCF-7TaxR, with DNA microarray analysis and q-PCR confirming the absence of caspase mRNA. Given the constitutive absence of caspase-3 in MCF-7 and the variant MCF-7TaxR cells, we would describe the MCF-7TaxR cells as “caspase-less”. Hence, we have tried to look for the presence of autophagic cell death in our MCF-7TaxR line. Flow cytometry with acridine orange to detect the presence of autophagic cell death (preincubation with 0.5µM staurosporine and 200-500nM bafilomycin as autophagy inhibitor) showed evidence of autophagy in MCF-7 cells, with a greater response in MCF-7TaxR cells. Collectively, these finding indicate for the first time the lack of involvement of caspase-mediated cell death in a taxane-resistant cancer cell line, with otherwise retained drug sensitivity, due to alternative cell death mechanisms.