Abstract:
Peloruside A (PLA) and laulimalide (LAU) are potent microtubule-stabilizing natural products that are effective at nanomolar concentrations in cultured cancer cells. The drugs have a mechanism of action similar to that of paclitaxel (PTX), a clinically useful anticancer drug, but have a number of advantages that make them unique to PTX. They also bind to a distinct site on tubulin that differs from the classical taxoid site.
The development of chemoresistance in cancer cells is a major problem to the successful treatment of cancer in the clinic. The role of β-tubulin alterations in the resistance to microtubule-stabilizing agents that bind to the taxoid site has been reported previously. In an attempt to understand the mechanisms of resistance to PLA and LAU, we used two drug-resistant sublines (R1 and L4) of the 1A9 human ovarian carcinoma cell line. L4 cells were 20-fold resistant to PLA and 30-fold resistant to LAU; whereas, R1 cells were 7-fold resistant to PLA and 2-fold resistant to LAU. Neither cell line showed resistance to PTX or other drugs that bind to the taxoid site on β-tubulin, nor was there resistance to microtubule-destabilizing drugs. The cells exhibit a βI-tubulin mutation at amino acid positions 296 (in R1) and 306 (in L4).
Analysis of PLA- and LAU-induced effects on the R1 and L4 resistant cells showed that there was an impaired ability of PLA and LAU to bind and stabilize microtubules. Analysis of β-tubulin isotype expression showed that there was increased mRNA and protein abundance of βII- and βIII-tubulin isotypes in L4 cells but not in R1 cells. Evidence of posttranslational modification of βII- and βIII-tubulin isotypes was also identified in L4 cells by 2D-electrophoresis. Interestingly, siRNA-mediated knock-down of both βII- and βIII-tubulin partially sensitized L4 cells to both PLA and LAU. Proteomic analysis of the parental and the resistant cells also showed downregulation of intermediate filament protein, vimentin, in the L4 cells. In addition to the studies with PLA and LAU, the microtubule-stabilizing activity of a novel marine natural product, zampanolide, and its ability to overcome P-gp-mediated multidrug resistance, was also investigated.
To conclude, these findings revealed an important role for βI-tubulin mutations, βII- and βIII-tubulin isotypes, and vimentin in PLA- and LAU-resistance and may have implications for the improved targeting of anticancer drugs whose mechanisms of action involve targeting of microtubules.
