Bcl-2 down-regulation is a novel mechanism of paclitaxel resistance.
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 deltapsim after stimulation of the respiratory state. To evaluate the binding of [14C]paclitaxel to isolated mitochondria, mitochondrial proteins were precipitated yielding 18.6 +/- 2.1 cpm/microg of protein. After stimulation of the respiratory state, binding of [14C]paclitaxel increased up to 163.2 +/- 46.7 cpm/microg of protein. 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.
Ferlini
Cristiano
C
Department of Obstetrics and Gynaecology, Università Cattolica Sacro Cuore, L.go A. Gemelli 8, 00168, Rome, Italy. cferlini@rm.unicatt.it
Raspaglio
Giuseppina
G
Mozzetti
Simona
S
Distefano
Mariagrazia
M
Filippetti
Flavia
F
Martinelli
Enrica
E
Ferrandina
Gabriella
G
Gallo
Daniela
D
Ranelletti
Franco Oreste
FO
Scambia
Giovanni
G
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D015153:Blotting, Western; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; D019253:Proto-Oncogene Proteins c-bcl-2; D017239:Paclitaxel