Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of ⌬ m after stimulation of the respiratory state. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A (A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.Taxanes are natural products derived from trees of the genus Taxoidaceae. The first taxane introduced in cancer therapy was paclitaxel, firstly isolated from Taxus brevifolia (Schiff et al., 1979). The clinical success of taxanes is dependent on the excellent response rate in second-line treatment of relapsing/resistant cancers and on the efficacy of taxanes in the multichemotherapeutic approach of ovarian and breast cancer (Verweij et al., 1994).In earlier studies, the microtubule network appeared as the main target of paclitaxel (Schiff et al., 1979;Manfredi et al., 1982). In fact, taxanes bind to ␤-tubulin subunits, thereby disrupting normal turnover of the microtubules. The final consequence is the arrest of the cell cycle in M phase with formation of aberrant mitosis and the activation of cell death pathways (Jordan et al., 1993). Along with arrest in M phase of the cell cycle, taxanes have also been reported to induce post-translational serine phosphorylation of the Bcl-2 protein (Haldar et al., 1995). The BCL2 gene is the homologous of the nematode CED-9 gene product (Hengartner and Horvitz, 1994) and is capable of prolonging cell survival by inhibiting apoptotic cell death. Overexpression of Bcl-2 has been observed in follicular lymphoma, where this protein is deregulated by chromosomal translocation, and in a large number of human tumors, including breast, lung, and prostate cancer.Disagreement exists on the levels of Bcl-2 and resistance to taxanes. A strong suggestion for a direct role of Bcl-2 in mediating paclitax...

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Class III B-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TUBB3. Following trypsin digestion, these proteins were characterized by mass spectrometry analysis. Functional analysis revealed that these proteins are involved in adaptation to oxidative stress and glucose deprivation, thereby suggesting that TUBB3 is a survival factor able to directly contribute to drug resistance. Moreover, glycosylation of TUBB3 could represent an attractive pathway whose inhibition could hamper cytoskeletal compartmentalization and TUBB3 function.

The supply of oxygen and nutrients to solid tumors is inefficient because cancer tissues have an inadequate number of microvessels, thus inducing the selective growth of the most aggressive cancer cells. This explains why many of the factors underlying a poor prognosis are induced in hypoxic/hypoglycemic conditions. Among these factors, a prominent role in several solid tumors is played by the class III β-tubulin gene (TUBB3). The study described here reveals that glucose deprivation enhances TUBB3 expression at both the gene and protein levels in A2780 ovarian cancer cells. In silico analysis of TUBB3 mRNA sequence predicted a putative binding site for the RNA-binding protein Hu antigen (HuR) in the 3′ flanking untranslated region. A hypoglycemic-dependent engagement of this site was shown using RNA pull-down and ribonucleoimmunoprecipitation techniques. Thereafter, HuR gene silencing revealed that TUBB3 translation is HuR dependent in hypoglycemia because HuR silencing inhibited the entry of TUBB3 mRNA into cytoskeletal and free polysomes. Finally, the clinical value of this finding was assessed in a clinical cohort of 46 ovarian cancer patients in whom it was found that HuR cytoplasmic staining was associated with high levels of TUBB3 and poor survival. CancerRes; 70(14); 5891-900. ©2010 AACR.