Manon Carré

We have previously reported that anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. In this study, we show that tubulin is present in mitochondria isolated from different human cancerous and non-cancerous cell lines. The absence of polymerized microtubules and cytosolic proteins was checked to ensure that this tubulin is an inherent component of the mitochondria. In addition, a salt wash did not release the tubulin from the mitochondria. By using electron microscopy, we then showed that tubulin is localized in the mitochondrial membranes. As compared with cellular tubulin, mitochondrial tubulin is enriched in acetylated and tyrosinated α-tubulin and is also enriched in the class III β-tubulin isotype but contains very little of the class IV β-tubulin isotype. The mitochondrial tubulin is likely to be organized in α/β dimers and represents 2.2 ± 0.5% of total cellular tubulin. Lastly, we showed by immunoprecipitation experiments that the mitochondrial tubulin is specifically associated with the voltage-dependent anion channel, the main component of the permeability transition pore. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with the permeability transition pore. We have previously reported that anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. In this study, we show that tubulin is present in mitochondria isolated from different human cancerous and non-cancerous cell lines. The absence of polymerized microtubules and cytosolic proteins was checked to ensure that this tubulin is an inherent component of the mitochondria. In addition, a salt wash did not release the tubulin from the mitochondria. By using electron microscopy, we then showed that tubulin is localized in the mitochondrial membranes. As compared with cellular tubulin, mitochondrial tubulin is enriched in acetylated and tyrosinated α-tubulin and is also enriched in the class III β-tubulin isotype but contains very little of the class IV β-tubulin isotype. The mitochondrial tubulin is likely to be organized in α/β dimers and represents 2.2 ± 0.5% of total cellular tubulin. Lastly, we showed by immunoprecipitation experiments that the mitochondrial tubulin is specifically associated with the voltage-dependent anion channel, the main component of the permeability transition pore. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with the permeability transition pore. permeability transition pore adenine nucleotide translocator voltage-dependent anion channel neuron specific enolase 1,4-piperazinediethanesulfonic acid Mitochondria are key players in cell death. Proteins present in the mitochondrial intermembrane space have been shown to induce apoptosis (1Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1302) Google Scholar). They are released into the cytosol in response to a variety of apoptotic stimuli (2Zamzani N. Kroemer G. Nat. Rev. Mol. Cell Biol. 2001; 2: 67-71Crossref PubMed Scopus (896) Google Scholar). Cytochrome c initiates caspase activation when released from mitochondria during apoptosis. Cytosolic cytochrome c binds to apaf-1, ATP, and multiple procaspase-9 molecules to induce formation of the apoptosome. The caspase-9, in turn, activates downstream executioner caspases such as caspase-3, leading to characteristic morphological changes in apoptosis. Other mitochondrial proteins are released from mitochondria such as the apoptosis-inducing factor, which triggers caspase-independent cell death, or Smac/Diablo, which promotes apoptosis by inactivation of the inhibitors of apoptotic proteins (1Ferri K.F. Kroemer G. Nat. Cell Biol. 2001; 3: E255-E263Crossref PubMed Scopus (1302) Google Scholar). Mitochondrial membrane permeabilization is critical for the release of proapoptotic factors from mitochondria (2Zamzani N. Kroemer G. Nat. Rev. Mol. Cell Biol. 2001; 2: 67-71Crossref PubMed Scopus (896) Google Scholar). The exact mode of permeabilization of mitochondria has not been elucidated, and several hypotheses have been proposed. According to the main hypothesis, the permeability transition pore (PTP),1 a multiprotein complex that is formed at the contact sites between the inner and outer membrane, is responsible for mitochondrial permeabilization. The PTP contains transmembranous proteins such as the adenine nucleotide translocator (ANT), the voltage-dependent anion channel (VDAC), and the peripheral benzodiazepine receptor as well as the mitochondrial membrane-associated proteins (hexokinase, creatine kinase and cyclophilin D). ANT and VDAC are the most abundant PTP components of the inner and outer mitochondrial membranes, respectively. An alternative hypothesis for mitochondrial permeabilization involves proapoptotic members of the Bcl-2 family, which can form pores in the outer membrane by oligomerization (2Zamzani N. Kroemer G. Nat. Rev. Mol. Cell Biol. 2001; 2: 67-71Crossref PubMed Scopus (896) Google Scholar). However, the involvement of the Bcl-2 family members in the early events of the release of cytochromec (3Scorrano L. Ashiya M. Buttle K. Weiler S. Oakes S.A. Mannella C.A. Korsmeyer S.J. Dev. Cell. 2002; 2: 55-67Abstract Full Text Full Text PDF PubMed Scopus (884) Google Scholar) and their interaction with PTP or isolated PTP components such as VDAC (4Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1919) Google Scholar) remain controversial. Microtubules are dynamic components of the cytoskeleton. They are critical for a wide variety of functions in eukaryotic cells. They are involved in the maintenance of cell shape, cell signaling, mitosis by the mediation of chromosome migration, mRNA localization, and vesicle and organelle trafficking mechanisms (5Dutcher S.K. Curr. Opin. Cell Biol. 2001; 13: 49-54Crossref PubMed Scopus (173) Google Scholar). Microtubules are the principal target in cells for a family of anticancer drugs, the so-called anti-tubulin agents. These agents strongly suppress microtubule dynamics that are essential for progression throughout mitosis (6Gonçalves A. Braguer D. Kamath K. Martello L. Briand C. Horwitz S. Wilson L. Jordan M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11737-11742Crossref PubMed Scopus (229) Google Scholar), leading to sustained mitotic arrest and apoptosis (7Gonçalves A. Braguer D. Carles G. Andreá N. Prevot C. Briand C. Biochem. Pharmacol. 2000; 60: 1579-1584Crossref PubMed Scopus (87) Google Scholar). However, the molecular pathways involved in the apoptotic process induced by these agents are still not elucidated. We have recently shown (8Andreá N. Braguer D. Brasseur G. Gonçalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar) that paclitaxel acts directly on mitochondria isolated from human cancer cells to release cytochrome c. The release of cytochrome c is prevented by cyclosporin A, which prevents PTP opening by binding to cyclophilin D (9Crompton M. Ellinger H. Costi A. Biochem. J. 1988; 255: 357-360PubMed Google Scholar). This demonstrates the involvement of this pore. The release was also observed with other anti-tubulin agents, either microtubule-stabilizing agents like docetaxel or microtubule-depolymerizing agents like nocodazole, CI 980, and vinorelbine (10Braguer D. Andreá N. Carreá M. Carles G. Goncalves A. Briand C. 92nd Annual Meeting of the American Association for Cancer Research, New Orleans, LA, March 24–28, 2001. 42. American Association for Cancer Research, Philadelphia, PA2001: 369Google Scholar). Anti-mitochondrial agents such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and arsenic trioxide induce the release of cytochrome c from isolated mitochondria (11Lim M.L. Minamikawa T. Nagley P. FEBS Lett. 2001; 503: 69-74Crossref PubMed Scopus (97) Google Scholar, 12Carreá M. Carles G. Andreá N. Douillard S. Ciccolini J. Briand C. Braguer D. Biochem. Pharmacol. 2002; 63: 1831-1842Crossref PubMed Scopus (61) Google Scholar) and, interestingly, also act like anti-tubulin agents by depolymerizing microtubules in intact cells and in vitro experiments (12–14.). In contrast, doxorubicin, an anticancer drug that acts on DNA independently of microtubules, does not induce the release of cytochrome c (8Andreá N. Braguer D. Brasseur G. Gonçalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar). Consequently, we hypothesized that tubulin could be involved in the mechanism of the release of cytochrome c and should be present in mitochondria. More than twenty years ago the existence of membrane-associated tubulin (15Bhattacharyya B. Wolff J. Nature. 1976; 264: 576-577Crossref PubMed Scopus (72) Google Scholar, 16Stephens R.E. Biol. Cell. 1986; 57: 95-110Crossref PubMed Scopus (57) Google Scholar), including that of mitochondria (17Bernier-Valentin F. Rousset B. J. Biol. Chem. 1982; 257: 7092-7099Abstract Full Text PDF PubMed Google Scholar), was described but has remained controversial. The current study presents evidence that tubulin is an inherent component of mitochondrial membranes specifically interacting with the VDAC. The human neuroblastoma SK-N-SH and IMR-32 cell lines, the human pulmonary cancer cell line A549, and the human breast cancer cell line MCF-7 were routinely cultured as described previously (8Andreá N. Braguer D. Brasseur G. Gonçalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar, 18Chadebech P. Brichese L. Baldin V. Vidal S. Valette A. Exp. Cell Res. 2000; 254: 241-248Crossref PubMed Scopus (18) Google Scholar). The human breast non-tumor cell line HBL-100 was cultured as described previously (19Bourgarel-Rey V. Vallee S. Rimet O. Champion S. Braguer D. Desobry A. Briand C. Barra Y. Mol. Pharmacol. 2001; 59: 1165-1170Crossref PubMed Scopus (50) Google Scholar). The human cervical cancer HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 2 mml-glutamine. The nasal septum carcinoma RPMI 2650 cells were cultured in Eagle’s minimum essential medium with Earle’s buffer supplemented with 10% fetal calf serum, 2 mm glutamine, and 1 mm pyruvate. The antibodies used were VDAC (anti-porin, 31 HL France Biochem, Meudon, France), α-tubulin (clone DM1A Sigma), β-tubulin (clone TU-06, Euromedex, Souffelweyersheim, France), tyrosinated α-tubulin (clone TUB-1A2, Sigma), acetylated α-tubulin (clone 6-11B-1, Sigma), class I β-tubulin isotype (gift from Charles Dumontet), class II β-tubulin isotype (N178101B, BioGenex, San Ramon, CA), class III β-tubulin isotype (gift from Anthony Frankfurter), class IV β-tubulin isotype (N176101B, BioGenex), neuron-specific enolase (NSE) (Zymed Laboratories Inc.), dynein (clone 70.1, Sigma), kinesin (clone IBII, Sigma), and ANT (Q18, Santa Cruz Biotechnology). Mitochondria were isolated from SK-N-SH, IMR-32, and A549 cells as described previously (8Andreá N. Braguer D. Brasseur G. Gonçalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar) with slight modifications. Briefly, 9 × 107 cells (36 ± 4 mg of protein) were suspended in a sucrose buffer (250 mmsucrose, 1 mm dithiothreitol, 10 mm KCl, 1 mm EDTA, 1 mm EGTA, 1.5 mmMgCl2, phenylmethylsulfonyl fluoride, protease inhibitors, 20 mm Hepes, pH 7.4) at 4 °C, homogenized with 50–100 strokes (depending on the cell line) in a glass homogenizer (Kimble Kontes, Vineland, NJ), and centrifuged twice at 800 ×g for 10 min. The supernatants were then centrifuged at 15,000 × g for 10 min at 4 °C. The mitochondrial pellets were immediately washed three times in the sucrose buffer. The protein content of the isolated mitochondria obtained by this procedure was determined according to the Bradford method (0.71 ± 0.05 mg). Mitochondria from HeLa, MCF-7, and RPMI 2650 cells were isolated using a similar extraction procedure except for the composition of the isolation buffer (210 mm mannitol, 70 mm sucrose, 1 mm EDTA, protease inhibitors, 10 mm Hepes, pH 7.4). To calculate the yield of mitochondrial isolation under our experimental conditions, we determined the percentage of mitochondria that were not isolated and pelleted after the first centrifugation at 800 × g. The quantity of mitochondria was estimated by quantification of the VDAC by Western blotting. The pellet obtained after the 800 × gcentrifugation was lysed and loaded on a gel together with the lysate of mitochondria isolated during the same experiment. The gel was processed for immunoblotting with VDAC, and the yield of the mitochondria isolation procedure was defined as the ratio of the quantity of VDAC in the isolated mitochondria preparation to the total quantity of VDAC in both the cells and the isolated mitochondria. Nuclei were isolated from SK-N-SH as described (20Fulda S. Jeremias I. Steiner H.H. Pietsch T. Debatin K.M. J. 1999; PubMed Scopus Google Scholar). Briefly, × cells were washed with in buffer mm KCl, 2 mm 1 mm phenylmethylsulfonyl fluoride, mm Hepes, pH for 10 and centrifuged for 1 min at the pellet was in a buffer mm KCl, 10 mm sucrose, mm EGTA, mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, mm pH 7.4) and centrifuged for 2 min at × g. The pelleted were immediately in and for min at for electron mitochondria were first in 2 or in the for 2 at (20Fulda S. Jeremias I. Steiner H.H. Pietsch T. Debatin K.M. J. 1999; PubMed Scopus Google Scholar). Mitochondria and the were then by centrifugation at 4 15,000 and Western was To microtubules were present on isolated from isolated mitochondria or polymerized tubulin were with and loaded on Microtubules and mitochondria were by using electron by electron microscopy, cells on glass isolated and isolated were using the were and according to or were with or β-tubulin in serum for 1 at °C. with and were according to the 2 or mitochondria were in in in and into electron mitochondria and cells were in buffer mm EDTA, 1 mm and mm pH was and was using an quantification was then using a as described previously G. Braguer D. C. V. Gonçalves A. M. Briand C. J. 1999; PubMed Scopus Google Scholar). three experiments were was using are as ± The of tubulin quantity in isolated mitochondria and cells was using a of tubulin previously from as described P. C. F. M. V. 2001; PubMed Scopus Google Scholar). tubulin is of α/β the quantity of the and represents of the loaded quantity of tubulin. The of mitochondrial tubulin by with cellular tubulin was by on of isolated mitochondria and cell of tubulin. α-tubulin and β-tubulin were also mitochondria were in buffer mm pH 1 mm EDTA, mm 10 mm 10 mm and protease and for min at × g at 4 °C. The supernatants were by with of protein for 1 with and were by centrifugation for 2 min at × g. The supernatants were with 4 of antibodies at 4 were by with protein for 2 and centrifugation for 2 min at × g. The pellets were washed twice with buffer mm and twice in the same buffer mm to and binding of proteins to The protein with were then suspended in and by Western as described We have shown previously that α-tubulin is present in isolated mitochondria from human neuroblastoma SK-N-SH cells (8Andreá N. Braguer D. Brasseur G. Gonçalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar, 12Carreá M. Carles G. Andreá N. Douillard S. Ciccolini J. Briand C. Braguer D. Biochem. Pharmacol. 2002; 63: 1831-1842Crossref PubMed Scopus (61) Google Scholar, B. M. J. 1982; Scopus Google Scholar, PubMed Scopus (97) Google Scholar). In to the of tubulin in SK-N-SH we also tubulin in the mitochondria isolated from human neuroblastoma cell line and from carcinoma breast nasal septum carcinoma and cancerous breast cells Thus, tubulin is present in mitochondria from a wide of cancerous and non-cancerous cells. The of mitochondrial tubulin to according to the cell from which mitochondria were We then our study on the SK-N-SH cell line to tubulin is an component of the mitochondria. we checked that the tubulin by Western was not present in the form of microtubules associated with the mitochondria. electron microscopy, we at the same of isolated mitochondria and in microtubules microtubules were the isolated mitochondria we showed by Western that proteins that are associated with microtubules such as the of the proteins dynein and kinesin were also from the isolated mitochondria These that the mitochondrial tubulin by Western does not from microtubules pelleted during the mitochondrial isolation procedure or to the mitochondria and that mitochondrial tubulin is not to mitochondria binding with the cytosolic a of the enolase P. O. PubMed Scopus Google Scholar) that is present throughout the but not in mitochondria S.A. P. J. PubMed Scopus Google Scholar), was not in our mitochondrial preparation This that cytosolic protein was present in the isolated mitochondria. To the of tubulin with we a salt wash of the isolated mitochondria K. J. F. C. A. G. C. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar) with 2 for 2 at °C. was in the of the mitochondria. The quantity of both tubulin and VDAC in the from mitochondria is from mitochondria. is that the of tubulin in the mitochondria is not to that the tubulin is an inherent protein component of the mitochondria. To the of the mitochondrial tubulin, we used with and antibodies and electron We and β-tubulin in the membranes of isolated mitochondria but not in their We then the of tubulin in mitochondrial membranes in intact which the that the of tubulin with the mitochondria is not to the extraction the membranes were not in intact we then isolated and the same as for isolated mitochondria. was not in membranes the of the tubulin on mitochondrial membranes. these show that is specifically associated with mitochondrial membranes. The quantity of tubulin in cells and in mitochondria was determined by using a with tubulin Mitochondrial represents ± of total mitochondrial and cellular represents ± of cell Thus, is a of tubulin in the mitochondria. Mitochondrial protein represents ± of the total cellular protein as determined by cellular and mitochondrial protein into the yield of the mitochondrial isolation procedure ± these to that mitochondrial tubulin represents of the total of cellular tubulin in SK-N-SH cells. and β-tubulin were present at in mitochondria ± and ± of the mitochondrial strongly that mitochondrial tubulin is present as α/β To the of of α-tubulin and β-tubulin between mitochondrial and cellular tubulin, we immunoblotting using of isolated mitochondria and cell of tubulin. were between cellular and mitochondrial Mitochondrial α-tubulin was tyrosinated and acetylated than cellular α-tubulin ± and a ± in the of β-tubulin were also The main were a ± in the class III and a ± in the class IV in mitochondrial tubulin compared with cell tubulin these that mitochondrial tubulin is a different of cellular tubulin and that it could have a specific several anti-tubulin agents, target is tubulin, cytochrome c release from mitochondria the opening of the PTP (8Andreá N. Braguer D. Brasseur G. Gonçalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar), we mitochondrial tubulin could be associated with the proteins of this we for an interaction between mitochondrial tubulin and both VDAC and the proteins of the of isolated mitochondria from SK-N-SH cells were with an to and the were for the of VDAC or ANT by mitochondrial were with an to VDAC, and the were for the of tubulin. These experiments that the to α-tubulin tubulin and VDAC and that the immunoprecipitation of VDAC tubulin Thus, mitochondrial tubulin with the PTP binding to the outer membrane protein VDAC. were under the same mm on mitochondria isolated from A549 cells not the interaction between tubulin and VDAC in cancer cell experiments with mitochondrial tubulin and ANT did not an interaction between these proteins the of between VDAC and mitochondrial tubulin. In this study, we showed that tubulin is an inherent component of mitochondrial membranes in several human cancer cell as well as in a non-cancerous cell in the of of α-tubulin and β-tubulin between the mitochondrial and cellular were We also that mitochondrial tubulin is specifically associated with VDAC, the main PTP component in the mitochondrial outer The of tubulin in mitochondria was tubulin was to be a cytosolic protein involved in microtubule formation by an between and several reported the of tubulin in organelle or membranes 20 years ago (15Bhattacharyya B. Wolff J. Nature. 1976; 264: 576-577Crossref PubMed Scopus (72) Google Scholar, F. Rousset B. J. Biol. Chem. 1982; 257: 7092-7099Abstract Full Text PDF PubMed Google Scholar). C. Cell 1999; PubMed Scopus Google Scholar) the existence of dimers in the of and K. J. F. C. A. G. C. Mol. Biol. Cell. 2001; PubMed Scopus Google Scholar) showed that is associated with the that the could play the role of a Thus, the are a protein (5Dutcher S.K. Curr. Opin. Cell Biol. 2001; 13: 49-54Crossref PubMed Scopus (173) Google Scholar) with members in cells is the first of mitochondrial tubulin has been R.E. Biol. Cell. 1986; 57: 95-110Crossref PubMed Scopus (57) Google Scholar). Mitochondrial tubulin was as a of the cell To such a in this study, we determined that our mitochondrial preparation did not microtubules, or a cytosolic By using electron microscopy, we then showed that tubulin is an inherent component of mitochondrial membranes. Mitochondrial tubulin represents 0.5% of mitochondrial which is with previously (17Bernier-Valentin F. Rousset B. J. Biol. Chem. 1982; 257: 7092-7099Abstract Full Text PDF PubMed Google Scholar, M. Biochem. J. PubMed Scopus Google Scholar). and β-tubulin is present in in strongly that mitochondrial tubulin is organized in α/β we not these tubulin dimers in mitochondrial membranes are or in study of α-tubulin and β-tubulin that mitochondrial tubulin has a specific of that is different from that of total cellular tubulin. Mitochondrial tubulin contains very little of the class IV β-tubulin isotype but is in the class III β-tubulin isotype. the class III β-tubulin the it is to and it is not that mitochondria are enriched for this isotype mitochondria are a of Pharmacol. PubMed Scopus Google Scholar). Thus, mitochondrial tubulin is in the a to the inherent of tubulin and the mitochondrial to be leading to a of tubulin These that mitochondrial tubulin have specific on mitochondrial tubulin were first mitochondria were as cell the functions of mitochondrial tubulin were to of mitochondria with microtubules in cells been described S.J. Proc. Natl. Acad. Sci. U. S. A. 1982; PubMed Scopus Google Scholar), and mitochondrial tubulin was as an interaction for the of mitochondria microtubules via proteins R.E. Biol. Cell. 1986; 57: 95-110Crossref PubMed Scopus (57) Google Scholar). K. Wilson L. Jordan M.A. Mol. Pharmacol. 2001; 60: PubMed Scopus Google Scholar) hypothesized be the cell to the between the of and the observed on the microtubule in the cells. mitochondrial tubulin represents of the total cellular tubulin, mitochondria at in in drug binding of the anti-tubulin agents to mitochondrial tubulin. However, it that the main functions of mitochondrial tubulin be to the involvement of mitochondria in apoptosis the PTP In the present study, we that mitochondrial tubulin is specifically associated with VDAC, the main outer membrane PTP an between PTP and mitochondrial tubulin anti-tubulin such as vinorelbine and paclitaxel directly induce the PTP opening and the release of cytochrome c from isolated mitochondria (8Andreá N. Braguer D. Brasseur G. Gonçalves A. Lemesle-Meunier D. Guise S. Jordan M.A. Briand C. Cancer Res. 2000; 60: 5349-5353PubMed Google Scholar). could to mitochondrial tubulin and the of the tubulin that the of leading to As mitochondria are as for agents 2001; 2: Scopus Google Scholar) and as Pharmacol. Sci. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Kroemer G. 2000; PubMed Scopus Google Scholar), the existence of mitochondrial tubulin and with PTP could the of stimuli that can be by mitochondria. the role of mitochondrial tubulin should not be to during apoptosis induced by Mitochondrial tubulin in a for from the to the mitochondria via the VDAC Y. S. 2002; PubMed Scopus Google Scholar) or could be involved on in the apoptotic the of mitochondrial tubulin with VDAC is or is not a interaction to be elucidated. M. Biochem. J. PubMed Scopus Google Scholar) previously showed that the immunoprecipitation of VDAC protein 2 VDAC, tubulin, and could form a complex in which or tubulin be an we have shown that mitochondrial tubulin is not associated with we an interaction with component of the PTP such as which can vitro G. J. P. F. J. FEBS Lett. 2001; PubMed Scopus Google Scholar), or with mitochondrial In in the present we the of tubulin in mitochondrial membranes and with the main PTP outer membrane VDAC. very likely that mitochondrial tubulin could act as a of the PTP However, the of mitochondrial tubulin have to be of mitochondrial tubulin is in and could to the of specific as for We Wilson and Jordan for on the and from the of

Eddy Pasquier 1,5 , Joseph Ciccolini 2 , Manon Carre 3 , Sarah Giacometti 2 , Raphaelle Fanciullino 2 , Charlotte Pouchy 1,‡ , Marie-Pierre Montero 3 , Cindy Serdjebi 2 , Maria Kavallaris 1 and Nicolas André 3,4,5 1 Children’s Cancer Institute Australia, Lowy Cancer Research Centre, UNSW, Randwick, NSW, Australia 2 Pharmacokinetics Unit, UMR-MD3, Aix-Marseille Univ, Marseille, France 3 INSERM UMR 911, Centre de Recherche en Oncologie biologique et en Oncopharmacologie, Aix-Marseille Univ, Marseille, France 4 Hematology & Pediatric Oncology Department, La Timone University Hospital of Marseille, France 5 Metronomics Global Health Initiative, Marseille, France ‡ Current Address: Université Pierre et Marie Curie - Univ Paris 6, CNRS UMR 7211, INSERM U959, Paris, France Received: October 13, 2011; Accepted: October 14, 2011; Published: October 17, 2011; Keywords: breast cancer, angiogenesis, propranolol, beta-adrenergic receptor antagonist, chemotherapy, combination therapy Correspondence: Eddy Pasquier, PhD, email: // Nicolas André, MD, PhD, email: // // Abstract Recent clinical evidence revealed that the use of beta-blockers such as propranolol, prior to diagnosis or concurrently with chemotherapy, could increase relapse-free and overall survival in breast cancer patients. We therefore hypothesized that propranolol may be able to increase the efficacy of chemotherapy either through direct effects on cancer cells or via anti-angiogenic mechanisms. In vitro proliferation assay showed that propranolol (from 50-100 μM) induces dose-dependent anti-proliferative effects in a panel of 9 human cancer and “normal” cell lines. Matrigel assays revealed that propranolol displays potent anti-angiogenic properties at non-toxic concentrations (<50 μM) but exert no vascular-disrupting activity. Combining chemotherapeutic drugs, such as 5-fluorouracil (5-FU) or paclitaxel, with propranolol at the lowest effective concentration resulted in synergistic, additive or antagonistic effects on cell proliferation in vitro depending on the cell type and the dose of chemotherapy used. Interestingly, breast cancer and vascular endothelial cells were among the most responsive to these combinations. Furthermore, Matrigel assays indicated that low concentrations of propranolol (10 – 50 μM) potentiated the anti-angiogenic effects of 5-FU and paclitaxel. Using an orthotopic xenograft model of triple-negative breast cancer, based on s.c injection of luciferase-expressing MDA-MB-231 cells in the mammary fat pad of nude mice, we showed that propranolol, when used alone, induced only transient anti-tumor effects, if at all, and did not increase median survival. However, the combination of propranolol with chemotherapy resulted in more profound and sustained anti-tumor effects and significantly increased the survival benefits induced by chemotherapy alone (+19% and +79% in median survival for the combination as compared with 5-FU alone and paclitaxel alone, respectively; p<0.05). Collectively our results show that propranolol can potentiate the anti-angiogenic effects and anti-tumor efficacy of chemotherapy. The current study, together with retrospective clinical data, strongly suggests that the use of propranolol concurrently with chemotherapy may improve the outcome of breast cancer patients, thus providing a strong rationale for the evaluation of this drug combination in prospective clinical studies.

Angiogenesis is a critical event in tumor growth and metastasis, which can be inhibited by conventional anticancer drugs such as the microtubule-damaging agent paclitaxel (Taxol). In this study, we investigate the mechanism of action of paclitaxel on human endothelial cells. We characterize two distinct effects of paclitaxel on human umbilical vein endothelial cell and human microvascular endothelial cell-1 proliferation according to drug concentration: a cytostatic effect at low concentrations and a cytotoxic effect at concentrations > or =10 nmol/L. The cytotoxic effect involves signaling pathways similar to those described in tumor cells (i.e., microtubule network disturbance, G(2)-M arrest, increase in Bax/Bcl-2 ratio, and mitochondria permeabilization) that result in apoptosis. In sharp contrast, the cytostatic effect involves an inhibition of endothelial cell proliferation without apoptosis induction and without any structural modification of the microtubule network. This cytostatic effect is due to a slowing of the cell cycle rather than to an arrest in a specific phase of the cell cycle. In addition, paclitaxel, at cytostatic concentrations, early initiates an apoptotic signaling pathway associated with increases in the mitochondrial reducing potential, mitochondrial membrane potential, p53 expression, and Bax/Bcl-2 ratio. However, this apoptotic pathway is stopped upstream of mitochondria permeabilization and it does not lead to endothelial cell death. Finally, we found that paclitaxel inhibits endothelial cell morphogenesis on Matrigel at all tested concentrations. In conclusion, we describe the mechanism of action of low concentrations of paclitaxel related to the antiangiogenic properties of this drug.