Molecular Mechanisms of Taxane Resistance

Abstract

Treatment with the taxanes (Paclitaxel or Docetaxel) is often the therapy of choice for women with breast cancer. In most cases, the taxanes can arrest cell proliferation at the G2/M phase of mitosis and cause cell death. However, some tumors develop resistance during the course of treatment, which is a major concern for patients, physicians and scientists. Several mechanisms have been proposed to explain how resistance to the taxanes occurs, but this phenomenon remains incompletely understood. We hypothesize that acquired taxane resistance in breast cancer cells may be associated with cellular transporters and when challenged with cytotoxic concentrations of taxane drugs may die through multiple cell death mechanisms.

Using the MDA-MB-231 breast cancer cell line, we developed cells that are resistant to either Docetaxel (DocR) or Paclitaxel (PacR). We then characterized the cell lines and found that DocR cells were cross resistant to Paclitaxel but PacR cells were only partially cross resistant to Docetaxel. It was confirmed by DNA fingerprinting that the genetic lineages of sensitive and resistant cell lines were similar. In addition, the resistant cell lines were found to proliferate more rapidly compared to sensitive cells, simultaneously having a greater number of cells in the S phase of the cell cycle.

While B-tubulin isotype expression changes and mutations have been implicated in conferring resistance to the taxanes, this was not the case in our cell lines. Of the transporters, Pgp, a member of the ABC superfamily of transporters was found to be upregulated in both resistant cell lines compared to sensitive cells, however siRNA experiments to inhibit expression of Pgp showed only a minor reduction of cell number in resistance cells, indicating the possibility that other ABC or SLC superfamily transporters may play a role in the resistance phenotypes.

Finally, levels of cell death and the response to IC50 concentrations of the taxane drugs were measured in sensitive and resistant cells. Clear differences in the use of alternative cell death pathways mechanisms emerged. Sensitive cells have higher levels of apoptosis compared to resistant cells determined by both SubG1 and Annexin V assays. There was no significant difference in caspase activation between sensitive and resistant cells by both western blots of protein expression and poly-caspase activation assays. Paclitaxel resistant cells had the highest levels of mitochondrial membrane permeability compared to CTL and DocR cells as was determined by JC-1 assays. Although both sensitive and resistant cells change their morphology consistent with what is described as induction of mitotic catastrophe at IC50 concentrations of either taxane drug, PacR cells followed by DocR were largest in cell size. Using propidium iodide dye, both resistant cell lines showed higher levels of necrosis compared to sensitive cells with the PacR cells having the highest levels. Finally, autophagy levels as determined by protein expression of cleaved LC3 and p62 were highest in PacR cells and when autophagy was inhibited with Beclin-1 siRNA, PacR cells showed a reduction in cell number in both Paclitaxel and Docetaxel compared to CTL and DocR cells.

In conclusion, it appears that several cell death pathways are used by sensitive and taxane resistant MDA-MB-231 breast cancer cells and that clinical use of inhibitors or inducers of different cell death pathways in conjunction with standard taxane chemotherapy may improve the success of taxane therapy in women with breast cancer in the clinic.