Bcl-2 is an anti-apoptotic protein that plays a crucial role in the regulation of the mitochondrial apoptotic pathway. However, its exact role in tumor cell sensitivity to microtubule-targeted agents (MTAs) remains controversial. Bcl-2 overexpression is generally correlated to drug resistance, but its down-regulation has been shown to be involved in paclitaxel-resistance in human ovarian cancer cells (Ferlini, 2003). In this case, Bcl-2 may be a paclitaxel-binding protein, as suggested by phage-display experiments (Rodi, 1999), necessary for paclitaxel cytotoxicity. The aim of our study was to investigate the action of vinflunine, the newest Vinca alkaloid currently in phase III clinical trials, depending on Bcl-2 expression in human ovarian cancer cell lines. We showed that A2780 wild type (wt) cells, sensitive to paclitaxel, were also sensitive to vinflunine (IC50 = 25 nM at 72h-treatment). A2780 TC1 cell line, which was established to be resistant to paclitaxel without P-gp overexpression, did not express Bcl-2 protein. We determined that A2780 TC1 cells were 800-fold more resistant to vinflunine (IC50 = 20 μM) than A2780 wt cells. To confirm that Bcl-2 down-regulation was related to resistance, we tested vinflunine on A2780 TC1 cells stably transfected with Bcl-2 construct. We showed that sensitivity was highly increased in these cells (IC50 = 700 nM). Nevertheless, as sensitivity was not totally restored, we examinated the isotypic composition of tubulin and its post-translational modifications. Increased levels of acetylated α-tubulin, βII and βIII tubulin were observed in A2780 TC1 cells. We then investigated the microtubule network response to the drug. Tubulin paracrystals appeared in both sensitive and resistant cells for 20 μM of vinflunine. For a lower concentration (500 nM), we noticed a microtubule depolymerization in A2780 wt cells, but no structural change of the A2780 TC1 cells network. Moreover, while vinflunine treatment induced mitotic arrest and apoptosis in A2780 wt cells, it induced G2 block in A2780 TC1 cells that became multinucleated. Thus, cross-resistance to paclitaxel and vinflunine depends on Bcl-2 expression and tubulin/microtubule system modifications. Finally, as Bcl-2 and tubulin are both localized to mitochondria (Carré, 2002), we performed co-immunoprecipitation experiments on isolated mitochondria and demonstrated their association. Furthermore, by dot-blot experiments with purified tubulin and Bcl-2, we showed that the two proteins can directly interact. Altogether, our results strengthen the key role of Bcl-2 in MTA-resistance and clearly demonstrate a direct interaction between tubulin and Bcl-2. As we have previously shown that MTAs directly act on mitochondria to induce apoptosis (André, 2000), we are determining the involvement of both mitochondrial tubulin and Bcl-2 in the mechanism of action of these anticancer drugs.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]