Bcl-2 Down-Regulation Is a Novel Mechanism of Paclitaxel Resistance

Abstract

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...

Erythropoietin treatment of human ovarian cancer cells results in enhanced signaling and a paclitaxel‐resistant phenotype

“…However, in this regard, Ferlini et al have reviewed the evidence for a potential dual role for Bcl-2 in paclitaxel-induced apoptosis and have provided experimental data supporting the concept that paclitaxel acts not only on microtubule turnover but also on the mitochondrion, and that Bcl-2 is its target. 57 A direct action of paclitaxel on isolated mitochondria had already been reported. 58 Ferlini et al showed that A2780 cells resistant to increasing concentrations of paclitaxel exhibited correspondingly reduced Bcl-2 expression, an effect that was reversible upon artificially increasing Bcl-2 expression.…”

Survivin expression in ovarian cancer and its correlation with clinico-pathological, surgical and apoptosis-related parameters

“…Sections were incubated with normal goat serum 20% for 30 min, then with polyclonal antibody ab496 (ABCAM Limited, Cambridge, UK) diluted according to the manufacturer's instructions, overnight. Bcl-2 and p53 were analysed using mouse monoclonal antibodies (clone 124 and DO-7, respectively, DAKO, Carpintera, CA, USA), according to the methods previously described (Ferrandina et al, 1999;Ferlini et al, 2003).…”

The establishment of two paclitaxel‐resistant prostate cancer cell lines and the mechanisms of paclitaxel resistance with two cell lines

“…Whereas bcl-2 expression was consistently down-regulated in T47-D breast cancer cells (12). In prostate cancer, although Bcl-2/Bcl-xL bispecific antisense oligonucleotide also enhanced paclitaxel chemosensitivity in PC-3 and LNCaP cells (13,14), involvement to paclitaxel-resistance of Bcl-2/Bcl-xL in prostate cancer is not clear.…”