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• W2019970740 abstract "Caveolin-1 (CAV1), a highly conserved membrane-associated protein, is a putative regulator of cellular transformation. CAV1 is localized in the plasmalemma, secretory vesicles, Golgi, mitochondria, and endoplasmic reticulum membrane and associates with the microtubule cytoskeleton. Taxanes such as paclitaxel (Taxol) are potent anti-tumor agents that repress the dynamic instability of microtubules and arrest cells in the G2/M phase. Src phosphorylation of Tyr-14 on CAV1 regulates its cellular localization and function. We report that phosphorylation of CAV1 on Tyr-14 regulates paclitaxel-mediated apoptosis in MCF-7 breast cancer cells. Befitting its role as a multitasking molecule, we show that CAV1 sensitizes cells to apoptosis by regulating cell cycle progression and activation of the apoptotic signaling molecules BCL2, p53, and p21. We demonstrate that phosphorylated CAV1 triggers apoptosis by inactivating BCL2 and increasing mitochondrial permeability more efficiently than non-phosphorylated CAV1. Furthermore, expression of p21, which correlates with taxane sensitivity, is regulated by CAV1 phosphorylation in a p53-dependent manner. Collectively, our findings underscore the importance of CAV1 phosphorylation in apoptosis and suggest that events that negate CAV1 tyrosine phosphorylation may contribute to anti-microtubule drug resistance. Caveolin-1 (CAV1), a highly conserved membrane-associated protein, is a putative regulator of cellular transformation. CAV1 is localized in the plasmalemma, secretory vesicles, Golgi, mitochondria, and endoplasmic reticulum membrane and associates with the microtubule cytoskeleton. Taxanes such as paclitaxel (Taxol) are potent anti-tumor agents that repress the dynamic instability of microtubules and arrest cells in the G2/M phase. Src phosphorylation of Tyr-14 on CAV1 regulates its cellular localization and function. We report that phosphorylation of CAV1 on Tyr-14 regulates paclitaxel-mediated apoptosis in MCF-7 breast cancer cells. Befitting its role as a multitasking molecule, we show that CAV1 sensitizes cells to apoptosis by regulating cell cycle progression and activation of the apoptotic signaling molecules BCL2, p53, and p21. We demonstrate that phosphorylated CAV1 triggers apoptosis by inactivating BCL2 and increasing mitochondrial permeability more efficiently than non-phosphorylated CAV1. Furthermore, expression of p21, which correlates with taxane sensitivity, is regulated by CAV1 phosphorylation in a p53-dependent manner. Collectively, our findings underscore the importance of CAV1 phosphorylation in apoptosis and suggest that events that negate CAV1 tyrosine phosphorylation may contribute to anti-microtubule drug resistance. Caveolin-1 (CAV1) 2The abbreviations used are: CAV1, caveolin-1; EV, empty vector; IMEM, improved minimal essential medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde phosphate dehydrogenase; wt, wild-type; ANOVA, analysis of variance; siRNA, small interfering RNA. is a 21-24-kDa protein and the prototype of a family of integral membrane proteins that associate with specific cholesterol- and sphingolipid-rich domains to form the structural foundation of membrane invaginations called caveolae. Caveolae act as sites of signal transduction in various cell types (1Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). CAV1 is thought to regulate the activity of proteins such as Src kinases, epidermal growth factor tyrosine kinase, Her2/neu (ErbB2) kinase, ERK (extracellular signal-regulated kinase), H-Ras, endothelial nitric-oxide synthase, and G proteins (1Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 2Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar) involved in survival pathways. In human breast tumors, CAV1 levels inversely correlate with tumor size (3Sagara Y. Mimori K. Yoshinaga K. Tanaka F. Nishida K. Ohno S. Inoue H. Mori M. Br. J. Cancer. 2004; 91: 959-965Crossref PubMed Scopus (84) Google Scholar), and CAV1 expression reduces the growth of mouse mammary tumors and their spontaneous metastasis to lung and bone (4Sloan E.K. Stanley K.L. Anderson R.L. Oncogene. 2004; 23: 7893-7897Crossref PubMed Scopus (139) Google Scholar). However, in breast cancer cell culture models, CAV1 is down-regulated in non-invasive human breast cancer cells but upregulated in cells with an invasive phenotype (5Lee S.W. Reimer C.L. Oh P. Campbell D.B. Schnitzer J.E. Oncogene. 1998; 16: 1391-1397Crossref PubMed Scopus (399) Google Scholar, 6Zajchowski D.A. Bartholdi M.F. Gong Y. Webster L. Liu H.L. Munishkin A. Beauheim C. Harvey S. Ethier S.P. Johnson P.H. Cancer Res. 2001; 61: 5168-5178PubMed Google Scholar, 7Xie Z. Zeng X. Waldman T. Glazer R.I. Cancer Res. 2003; 63: 5370-5375PubMed Google Scholar). Taxanes are potent anti-tumor agents that function by binding to the β subunits of tubulin and repressing the dynamic instability of spindles (8Manfredi J.J. Horwitz S.B. Pharmacol. Ther. 1984; 25: 83-125Crossref PubMed Scopus (475) Google Scholar, 9Yvon A.M. Wadsworth P. Jordan M.A. Mol. Biol. Cell. 1999; 10: 947-959Crossref PubMed Scopus (459) Google Scholar), activities that lead to cell cycle arrest in the G2/M phase (10Dumontet C. Sikic B. J. Clin. Oncol. 1999; 17: 1061-1070Crossref PubMed Google Scholar). Taxanes such as paclitaxel (Taxol) or docetaxel (Taxotere) are routinely used in the first-line treatment of metastatic breast, lung, ovarian, and digestive cancers (11Gligorov J. Lotz J.P. Oncologist. 2004; 9: 3-8Crossref PubMed Scopus (200) Google Scholar). In primary breast cancer, inclusion of taxane in adjuvant chemotherapy reduces the relative risk of recurrence and improves overall survival (12Clavarezza M. Del Mastro L. Venturini M. Ann Oncol. 2006; 17 (Suppl. 7): vii22-vii26Abstract Full Text PDF PubMed Scopus (9) Google Scholar). Acquired resistance through cellular adaptations or mutations in neoplastic cells remains a major problem in chemotherapy. Although taxanes are substrates for ABC transporters, other resistance mechanisms are clearly important (13Trock B.J. Leonessa F. Clarke R. J. Natl. Cancer Inst. 1997; 89: 917-931Crossref PubMed Scopus (401) Google Scholar). Therefore, it is important to improve our understanding of the mechanisms of drug responsiveness and to identify better predictors of drug efficacy. CAV1 is essential for the formation and movement of caveolae through the cytoplasm along microtubule tracks, and it is localized in the microtubule-organizing center or peri-centrosomal region in Chinese hamster ovary cells (14Mundy D.I. Machleidt T. Ying Y.S. Anderson R.G. Bloom G.S. J. Cell Sci. 2002; 115: 4327-4339Crossref PubMed Scopus (260) Google Scholar). These findings suggest an essential relationship between CAV1 and microtubules or microtubule-associated proteins and their function. Treatment with a cytostatic dose of paclitaxel blocks lung cancer cells in G2/M and causes an up-regulation of CAV1, implicating CAV1 in taxane-mediated cell death and perhaps drug resistance (15Roussel E. Belanger M.M. Couet J. Anticancer Drugs. 2004; 15: 961-967Crossref PubMed Scopus (17) Google Scholar, 16Yang C.P. Galbiati F. Volonte D. Horwitz S.B. Lisanti M.P. FEBS Lett. 1998; 439: 368-372Crossref PubMed Scopus (143) Google Scholar, 17Couet J. Belanger M.M. Roussel E. Drolet M.C. Adv. Drug Deliv. Rev. 2001; 49: 223-235Crossref PubMed Scopus (107) Google Scholar, 18Belanger M.M. Roussel E. Couet J. Anticancer Drugs. 2003; 14: 281-287Crossref PubMed Scopus (41) Google Scholar). However, the role of CAV1 function in cell death remains unclear. In macrophages, induction of apoptosis by different apoptotic agents such as simvastatin and camptothecin leads to a large increase in CAV1 expression. As an early event, this increase in CAV1 is independent of caspase activation or DNA fragmentation but is associated with the plasma membrane translocation of phosphatidylserine (19Gargalovic P. Dory L. J. Lipid Res. 2003; 44: 1622-1632Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Originally identified as a substrate for v-Src (20Glenney Jr., J.R. Zokas L. J. Cell Biol. 1989; 108: 2401-2408Crossref PubMed Scopus (360) Google Scholar), CAV1 is phosphorylated on Tyr-14 by c-Src (21Lee H. Volonte D. Galbiati F. Iyengar P. Lublin D.M. Bregman D.B. Wilson M.T. Campos-Gonzalez R. Bouzahzah B. Pestell R.G. Scherer P.E. Lisanti M.P. Mol. Endocrinol. 2000; 14: 1750-1775Crossref PubMed Google Scholar). Mounting evidence suggests that phosphorylated CAV1 regulates caveolae formation and function (21Lee H. Volonte D. Galbiati F. Iyengar P. Lublin D.M. Bregman D.B. Wilson M.T. Campos-Gonzalez R. Bouzahzah B. Pestell R.G. Scherer P.E. Lisanti M.P. Mol. Endocrinol. 2000; 14: 1750-1775Crossref PubMed Google Scholar, 22Ko Y.G. Liu P. Pathak R.K. Craig L.C. Anderson R.G. J. Cell. Biochem. 1998; 71: 524-535Crossref PubMed Scopus (36) Google Scholar, 23Shajahan A.N. Tiruppathi C. Smrcka A.V. Malik A.B. Minshall R.D. J. Biol. Chem. 2004; 279: 48055-48062Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Beliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 25Kiss A.L. Turi A. Mullner N. Kovacs E. Botos E. Greger A. Mol. Cell. Endocrinol. 2005; 245: 128-137Crossref PubMed Scopus (28) Google Scholar). The precise role of Src kinase in taxane-mediated cytotoxicity is unclear. Src can increase Taxotere sensitivity by mediating downstream apoptotic events through BCL2 phosphorylation in v-Src-transformed human gall bladder epithelial cells (26Boudny V. Nakano S. Br. J. Cancer. 2002; 86: 463-469Crossref PubMed Scopus (27) Google Scholar). However, in human ovarian cancer cells, an inhibition of Src activity increases paclitaxel-induced cytotoxicity (27Chen T. Pengetnze Y. Taylor C.C. Mol. Cancer Ther. 2005; 4: 217-224Crossref PubMed Scopus (80) Google Scholar). Thus, the precise role of CAV1, and particularly that of phospho-CAV1(Y14), in affecting breast cancer cell responsiveness to taxanes is unknown. The current study was undertaken to determine whether CAV1 is involved in the cytotoxic and proapoptotic actions of paclitaxel in MCF-7 human breast adenocarcinoma cells. We overexpressed wild type (wt), a phosphorylation-defective CAV1 mutant (Y14F), or empty vector (EV) in MCF-7 cells that normally express low levels of endogenous CAV1. The effects of wtCAV1, Y14F, or EV expression on cell growth, apoptosis, and p53/p21 transcription in response to low dose (10 nm) paclitaxel were analyzed. This study provides novel insights into the function of CAV1 in paclitaxel sensitivity and the role of phosphorylation on Tyr-14 in CAV1-mediated effects on mitochondrial permeability and apoptosis. Cell Culture and Reagents—MCF-7 cells (obtained from the LCCC Tissue Culture Shared Resource) were cultured in improved minimal essential medium (IMEM; Biofluids, Rockville, MD) supplemented with 5% fetal bovine serum (FBS). Cells were maintained in a humidified atmosphere at 37 °C and 95% air/5% CO2. Paclitaxel was obtained from Sigma and was dissolved in ethanol (which was used as the vehicle control). PP2 was purchased from Calbiochem. All other reagents were obtained from Sigma unless otherwise indicated. Generation of Stable Cell Lines—MCF-7 cells were grown to 50-60% confluence and transfected with either human wt CAV1 or Tyr-14 → Phe-14 phosphorylation-deficient mutant (Y14F) in pcDNA6 or EV using the FuGENE 6 transfection reagent (Roche Applied Sciences). Medium was replaced 24 h later with complete growth medium, and the cells were allowed to grow for 5 days. Complete growth medium containing blasticidin S HCl (Invitrogen) (10 μg/ml) was used for stable selection. For all experiments, pooled populations of stable cell lines were used. Western Blot Analyses—To determine the effects of paclitaxel on CAV1 protein expression, cells were treated with vehicle or 10 nm paclitaxel in FBS-IMEM for 24 h. Controls were treated with vehicle alone (0.02% v/v ethanol). For Western blot analysis, cells were lysed for 30 min at 4 °C in lysis buffer (50 mm Tris-HCl, pH 7.5, containing 150 mm NaCl, 1 mm EDTA, 0.5% sodium deoxycholate, 1% Igepal CA-630, 0.1% SDS, 1 mm Na3VO4, 44 μg/ml phenylmethylsulfonyl fluoride) supplemented with Complete Mini protease inhibitor mixture tablets and 1 mm sodium orthovanadate phosphatase inhibitor (Roche Applied Science). Total protein was quantified using the bicinchoninic acid assay (Pierce). Whole cell lysate (20-50 μg) was resolved by SDS-PAGE. The following primary antibodies were used for immunoblotting: monoclonal antibody against phospho-CAV1(Y14) and polyclonal antibody against CAV1 (BD Biosciences); monoclonal cleaved poly(ADP-ribose) polymerase, polyclonal phospho-BCL2(S70), polyclonal p53, polyclonal Src, and phospho-Src(pY418) antibodies (Cell Signaling, Danvers, MA); and monoclonal BCL2 (Stressgen Corp., Ann Arbor, MA); monoclonal β-tubulin (Sigma); monoclonal p21 (Calbiochem). Equal protein loading of gels was confirmed by immunostaining with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunostaining and Confocal Microscopy—Cells grown on coverslips were washed with phosphate-buffered saline and incubated at least 3 h in serum-free and phenol red-free medium. Cells were then fixed, permeabilized, and incubated with primary antibody. Fluorophore conjugates and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Molecular Probes, Inc. (Eugene, OR). Where appropriate, DAPI was added to visualize the nucleus, and non-confocal DAPI images were acquired using Hg lamp excitation and a UV filter set. Confocal microscopy was performed using an Olympus IX-70 confocal microscope with 405-, 488-, and 543-nm excitation lasers. Fluorescence emission was separately detected for each fluorophore in optical sections <1 μm in thickness (pinhole set to achieve 1 Airy unit). Transcriptional Reporter Assays—Cells were transfected with 0.4 μg of p53 (Panomics, Fremont, CA) and p21 luciferase reporter plasmid (a gift from Dr. Jane Trepel, National Institutes of Health) and 0.1 μg of pCMV-Renilla (Promega, Madison, WI) per well using the FuGENE 6 transfection reagent. The next day, cells were treated with 10 nm paclitaxel for 24 h. Activation of the luciferase constructs was measured using the Dual Luciferase assay kit (Promega). Luciferase values were normalized to Renilla luminescence. Three independent experiments were performed in quadruplicate. Data are presented as the mean ± S.E. for all experiments. Cell Proliferation Assays—Cells were seeded at a density of 1-2 × 104 cells/well in 24-well plates. For Src inactivation, cells were incubated with 10 μm PP2 for 2-3 h before adding paclitaxel. For small interfering RNA (siRNA)-mediated knock down of CAV1, cells were transiently transfected with CAV1 siRNA for 48 h before adding paclitaxel. To assess paclitaxel-induced growth inhibition, cells were treated with 10 nm paclitaxel (in FBS-IMEM) for 24 h. Cells were then trypsinized, resuspended in phosphate-buffered saline, and counted using a Z1 Single Coulter Counter (Beckman Coulter, Miami, FL). At least three independent experiments were done in sextuplicate. Data were normalized to vehicle-treated cells and are presented as the mean ± S.E. from a representative experiment. Cell Cycle and Apoptosis Assays—Following treatment of cells with 10 nm paclitaxel in FBS-IMEM for 24 h, cells were fixed in 70% ethanol for 20 min at 4 °C. Cell cycle distribution was measured by fluorescence-activated cell sorting in the Lombardi Comprehensive Cancer Center Flow Cytometry Shared Resource facility. Annexin V and propidium iodide staining was done using an Annexin V-fluorescein isothiocyanate kit (Trevigen, Gaithersburg, MD). Mitochondrial permeability was detected using the ApoAlert mitochondrial membrane sensor kit (Clontech, Mountain View, CA). Quantitative Real-time Polymerase Chain Reaction—Primer for CAV1 (Hs00971716_m1) and the housekeeping gene ribosomal protein, large, P0 (RPLPO) (Hs99999902_m1) was purchased from Applied Biosystems (Foster City, CA). MCF-7 cells were allowed to grow to 60-70% confluence in 75-cm2 flasks and were then treated with either different concentrations of paclitaxel (0, 10, 100 nm) for 24 h or 10 nm paclitaxel for 0, 24, and 48 h. RNA was extracted using the TRIzol reagent (Invitrogen), cleaned using the RNeasy kit (Qiagen, Valencia, CA), and analyzed by the Agilent Bioanalyzer 2100 (Santa Clara, CA). About 1 μg of DNase I (Invitrogen)-treated RNA was reverse transcribed with SuperScript II reverse transcriptase (Invitrogen) using Oligo(dT)16 (Applied Biosystems). Real-time absolute quantitative PCR for each cDNA sample and a standard curve were established using TaqMan PCR mastermix in the presence of CAV1 primer or the internal control RPLP0 primer. Reactions (10 μl) were run in triplicate in 384-well plates on an ABI Prism 7900 HT sequence detection system using the protocol suggested by the manufacturer. The ratio of CAV1 induction was estimated in comparison with RPLPO expression; data presented are the mean ± S.E. Transfection of siRNA—Cells were plated in 12- or 24-well plates in complete medium and allowed to grow to 50% confluence. Approximately, 100 nm p21 siRNA, p53 siRNA (Cell Signaling), CAV1 siRNA (Dharmacon, Lafayette, CO), or their respective control siRNA were transfected using the TransIT-siQUEST (Mirus, Madison, WI) transfection reagent according to the manufacturer’s protocol. At 24 h, 10 nm paclitaxel or vehicle was added to the siRNA-transfected cells. Cells were lysed at 48 h post-transfection and subjected to Western blot analysis or cell proliferation assay as described above. Statistical Analyses—Statistical analyses were performed using the Sigmastat software package (Jandel Scientific, SPSS, Chicago, IL). Where appropriate, protein expression, cell growth, and apoptosis were compared using Student’s t test or ANOVA with a post hoc t test for multiple comparisons. Where several groups were compared with the same control, we used Dunnett’s test. Differences were considered significant at p ≤ 0.05; all tests were two-tailed. Exposure to Taxane Increased CAV1 Expression in MCF-7 Cells—CAV1 expression is very low but remains detectable in MCF-7 human breast cancer cells. To evaluate the effect of paclitaxel on CAV1 expression, MCF-7 cells were grown to 50-70% confluence and treated with 10 nm paclitaxel for 0, 24, or 48 h or with vehicle alone, 10 nm, or 100 nm paclitaxel for 24 h. Real-time PCR showed a significant increase in CAV1 expression in MCF-7 cells within 24 or 48 h following paclitaxel treatment compared with control (untreated) (Fig. 1A, p ≤ 0.004, one-way ANOVA). Western blot analysis showed a corresponding increase in CAV1 protein within 24 or 48 h (Fig. 1B). Treatment of MCF-7 cells with 10 and 100 nm for 24 h showed a significant increase in both CAV1 transcription (Fig. 1C, p ≤ 0.001, one-way ANOVA) and protein expression (Fig. 1D) compared with controls (vehicle). Thus, in MCF-7 cells, a significant induction of CAV1 occurs within 24 h following treatment with 10 nm paclitaxel. The induction of CAV1 following paclitaxel treatment suggests a role for CAV1 in drug responsiveness. Moreover, attenuating levels of endogenous CAV1 in MCF-7 cells with siRNA correlates with reduction in paclitaxel-induced inhibition of cell growth (Fig. 2, A and B).FIGURE 2CAV1 is required for paclitaxel-induced cell growth inhibition. A, paclitaxel-induced up-regulation of CAV1 expression was inhibited with siRNA in MCF-7 cells. MCF-7 cells were transfected with either control or CAV1 siRNA for 48 h. Next, paclitaxel was added to the transfection medium to attain a final concentration of 10 nm; cells were incubated with paclitaxel for 24 h before Western blot analysis or cell proliferation assay. B, attenuation of CAV1 expression in MCF-7 cells correlated with reduction in paclitaxel-induced inhibition of cell growth. *, p < 0.05 versus control siRNA with vehicle alone. C, incubation of MCF-7 cells with 10 μm PP2 inhibited Src activation as detected by Western blot analysis of phosphor-Src(Y418). Although the level of CAV1 protein expression increased following treatment of MCF-7 cells with 10 nm paclitaxel, phospho-CAV1(Y14) was undetectable using our Western blot analysis protocol (for up to 50 μg of protein loading/gel). Incubation of MCF-7 cells with 10 μm PP2 in addition to 10 nm paclitaxel significantly reduced cell growth inhibition effect of paclitaxel compared with paclitaxel alone (panel D, p ≤ 0.05). *, p < 0.05 versus MCF-7 with paclitaxel alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Phosphorylation of CAV1 on Tyr-14 by Src is known to regulate CAV1 functions (21Lee H. Volonte D. Galbiati F. Iyengar P. Lublin D.M. Bregman D.B. Wilson M.T. Campos-Gonzalez R. Bouzahzah B. Pestell R.G. Scherer P.E. Lisanti M.P. Mol. Endocrinol. 2000; 14: 1750-1775Crossref PubMed Google Scholar, 22Ko Y.G. Liu P. Pathak R.K. Craig L.C. Anderson R.G. J. Cell. Biochem. 1998; 71: 524-535Crossref PubMed Scopus (36) Google Scholar, 23Shajahan A.N. Tiruppathi C. Smrcka A.V. Malik A.B. Minshall R.D. J. Biol. Chem. 2004; 279: 48055-48062Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 24Labrecque L. Nyalendo C. Langlois S. Durocher Y. Roghi C. Murphy G. Gingras D. Beliveau R. J. Biol. Chem. 2004; 279: 52132-52140Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 25Kiss A.L. Turi A. Mullner N. Kovacs E. Botos E. Greger A. Mol. Cell. Endocrinol. 2005; 245: 128-137Crossref PubMed Scopus (28) Google Scholar). To show whether Src inactivation reduces paclitaxel-induced inhibition of cell growth, we used PP2 to inhibit Src activation as detected by phosphorylation on Tyr-418 by Western blot analysis (Fig. 2C). Whereas CAV1 protein expression increases following treatment of MCF-7 cells with 10 nm paclitaxel, phospho-CAV1 (p-CAV1) is undetectable using our Western blot analysis protocol (for up to 50 μg of protein loading/gel). However, incubation of MCF-7 cells with 10 μm PP2 in addition to 10 nm paclitaxel significantly reduces the cell growth inhibition effect of paclitaxel compared with paclitaxel alone (Fig. 2D, p ≤ 0.05). wtCAV1 Expression Enhanced Paclitaxel-induced Growth Inhibition and Cell Cycle Arrest at G2/M—To establish the functional relevance of CAV1 in paclitaxel sensitivity, we generated MCF-7 cell lines that stably express either the full-length wild-type CAV1 (MCF-7/wtCAV1), Tyr-14 → Phe phosphorylation-deficient (MCF-7/Y14F) cells, or empty vector (MCF-7/EV) (Fig. 3A). Expression levels of CAV1 in MCF-7/wtCAV1 and MCF-7/Y14F were measured by Western blot analysis using specific antibodies for CAV1 or p-CAV1. CAV1α contains residues 1-178, whereas CAV1β contains residues 32-178. Because Tyr-14 is the principal substrate for Src kinase, only CAV1α undergoes tyrosine phosphorylation (28Li S. Seitz R. Lisanti M.P. J. Biol. Chem. 1996; 271: 3863-3868Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). CAV1 is thought to interact with Src and inhibit its activation (2Li S. Couet J. Lisanti M.P. J. Biol. Chem. 1996; 271: 29182-29190Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). Although not fully inactive, Src kinase activity is decreased in untreated MCF-7/wtCAV1 cells compared with MCF-7/EV and MCF-7/Y14F cells (Fig. 3A). Expression of CAV1 in MCF-7 cells can reduce the rate of proliferation (29Fiucci G. Ravid D. Reich R. Liscovitch M. Oncogene. 2002; 21: 2365-2375Crossref PubMed Scopus (250) Google Scholar). Comparison of growth curves in basal medium shows a significant decrease in the rate of cell proliferation for MCF-7/wtCAV1 cells (p ≤ 0.05) at day 4 through day 6 compared with that for MCF-7/EV cells. In contrast, the difference in rates of proliferation between MCF-7/EV and MCF-7/Y14F cells is not significant (Fig. 3B). Cells were treated with paclitaxel to determine whether CAV1 tyrosine phosphorylation affects growth inhibition by the drug. MCF-7 cells expressing wtCAV1 are more sensitive than cells expressing either EV or Y14F (Fig. 3C). These data suggest that CAV1 phosphorylation on Tyr-14 plays a key role in paclitaxel-induced growth inhibition. Furthermore, the effects of CAV1 on paclitaxel sensitivity are not simply a consequence of changes in the rate of proliferation; cells that have a higher rate of proliferation are generally more sensitive to cell cycle-specific cytotoxic drugs like taxanes (30Matsuoka H. Furusawa M. Tomoda H. Seo Y. Anticancer Res. 1994; 14: 163-167PubMed Google Scholar). Additionally, we did not see any difference in the partitioning of caveolar and intercellular compartments following subcellular fractionation (data not shown) between wtCAV1 or the Y14F mutant-expressing cell under control conditions or following paclitaxel treatment for 24 h. Thus, in terms of paclitaxel sensitivity, caveolar localization is unlikely to account for any differences between MCF-7 cells expressing wtCAV1 and Y14F. Taxanes stabilize microtubules and block sensitive cells in the G2/M cell cycle phase (31Schiff P.B. Horwitz S.B. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1561-1565Crossref PubMed Scopus (1758) Google Scholar). To determine whether CAV1 expression alters G2/M cell cycle arrest following treatment with paclitaxel, MCF7-EV, MCF7-wtCAV1, or MCF-7/Y14F cells were treated with either vehicle alone or 10 nm paclitaxel for 24 h prior to fluorescence-activated cell sorter analysis of cell cycle distribution (Fig. 4A). MCF-7/Y14F cells exhibited a significant increase (p ≤ 0.05) in the proportion of cells in the S-phase relative to MCF-7/EV. Following treatment with 10 nm paclitaxel, the percentage of cells arrested in the G2/M phase was significantly higher in MCF7-wtCAV1 compared with MCF-7/EV (p ≤ 0.05). In MCF-7/Y14F cells, the percentage of cells in the G2/M phase was comparable with that in MCF-7/EV cells (Fig. 4B). Thus, the taxane-induced decrease in cell proliferation in MCF7-wtCAV1 cells is likely to be a consequence of increased cell cycle arrest in G2/M phase. To assess cellular morphology in vehicle (control) or drug-treated cells, MCF-7/EV, MCF-7/wtCAV1, and MCF-7/Y14F cells were treated with either vehicle or 10 nm paclitaxel for 24 h. Cells were fixed and permeabilized and co-stained for both β-tubulin and CAV1. In all three cell lines, vehicle-treated cells displayed an organized tubulin network that excludes the nucleus and extends throughout the cytoplasm (Fig. 4C). After 24 h of paclitaxel treatment, microtubule disruption was more distinct in MCF-7 cells expressing wtCAV1 compared with MCF-7 cells expressing EV or Y14F, as evident from clusters of microtubules or asters in the respective cells (Fig. 4D). Increased Apoptosis in MCF-7/wtCAV1-expressing Cells in Response to Paclitaxel—Paclitaxel can induce apoptosis in some breast epithelial cells (32Yeung T.K. Germond C. Chen X. Wang Z. Biochem. Biophys. Res. Commu. 1999; 263: 398-404Crossref PubMed Scopus (132) Google Scholar). To determine whether CAV1 expression affects apoptosis, we treated MCF-7/EV, MCF-7/wtCAV1, and MCF-7/Y14F cells with 10 nm paclitaxel for 24 h. Apoptosis was detected by flow cytometry after staining for fluorescein isothiocyanate-conjugated Annexin V and for propidium iodide. At 10 nm paclitaxel for 24 h, only 1% of the MCF-7/EV or MCF-7/Y14F cells underwent apoptosis compared with 3% in MCF-7/wtCAV1 cells (Fig. 5A, p ≤ 0.05). Additionally, cells were analyzed for apoptosis by Western blot analysis of anti-poly(ADP-ribose) polymerase cleavage to a 85-89-kDa fragment (Fig. 5B). Paclitaxel-induced poly(ADP-ribose) polymerase cleavage appeared in MCF-7/wtCAV1 within 24 h following drug treatment and was more pronounced at 48 h relative to MCF-7/EV or MCF-7/Y14F cells. Although the poly(ADP-ribose) polymerase antibody (according to the manufacturer) should detect only a single band for cleaved poly(ADP-ribose) polymerase, we detected a nonspecific higher molecular band at ∼100-110 kDa that did not accurately reflect levels of full-length poly(ADP-ribose) polymerase in our experiments. These findings suggest that tyrosine phosphorylation of CAV1 accelerates apoptosis in response to paclitaxel treatment and/or could be essential to signaling pathway(s) that are required for the induction of apoptosis. Microtubule disruption following treatment with taxanes increases phosphorylation of BCL2(S70) in the G2/M phase of the cell cycle, abrogating the normal anti-apoptotic function of BCL2 and initiating an apoptotic program in cycling cancer cells (33Haldar S. Basu A. Croce C.M. Cancer Res. 1997; 57: 229-233PubMed Google Scholar). To determine whether activation of BCL2 phosphorylation correlates with the degree of apoptosis, Western blot analyses were done with whole cell lysate cells following treatment with vehicle or 10 nm paclitaxel for 24 or 48 h. At 24 h, we detected a significant increase in the level of BCL2(S70) phosphorylation in MCF-7/wtCAV1 cells in comparison with MCF-7/EV cells (p ≤ 0.05). In cells expressing Y14FCAV1, the level of BCL2(S70) phosphorylation remains comparable with cells expressing EV (Fig. 6A). Furthermore, measurement of mitochondrial permeability, which is tightly associated with the release" @default.
• W2019970740 created "2016-06-24" @default.
• W2019970740 creator A5001200228 @default.
• W2019970740 creator A5023487576 @default.
• W2019970740 creator A5027858713 @default.
• W2019970740 creator A5029233987 @default.
• W2019970740 creator A5067194798 @default.
• W2019970740 creator A5070975916 @default.
• W2019970740 date "2007-02-01" @default.
• W2019970740 modified "2023-09-28" @default.
• W2019970740 title "Caveolin-1 Tyrosine Phosphorylation Enhances Paclitaxel-mediated Cytotoxicity" @default.
• W2019970740 cites W1557888154 @default.
• W2019970740 cites W1972361063 @default.
• W2019970740 cites W1976507689 @default.
• W2019970740 cites W1977936920 @default.
• W2019970740 cites W1986303065 @default.
• W2019970740 cites W1989261260 @default.
• W2019970740 cites W1990839004 @default.
• W2019970740 cites W1997726565 @default.
• W2019970740 cites W2005623678 @default.
• W2019970740 cites W2023284036 @default.
• W2019970740 cites W2027740621 @default.
• W2019970740 cites W2029967111 @default.
• W2019970740 cites W2033799417 @default.
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• W2019970740 cites W2069348059 @default.
• W2019970740 cites W2074406830 @default.
• W2019970740 cites W2081406767 @default.
• W2019970740 cites W2084325612 @default.
• W2019970740 cites W2092437783 @default.
• W2019970740 cites W2095480781 @default.
• W2019970740 cites W2100412031 @default.
• W2019970740 cites W2103741737 @default.
• W2019970740 cites W2125575916 @default.
• W2019970740 cites W2127127403 @default.
• W2019970740 cites W2131193695 @default.
• W2019970740 cites W2133408654 @default.
• W2019970740 cites W2145881880 @default.
• W2019970740 cites W2158118486 @default.
• W2019970740 cites W2160499160 @default.
• W2019970740 cites W2160516150 @default.
• W2019970740 cites W2162643935 @default.
• W2019970740 cites W2162960688 @default.
• W2019970740 cites W2169384475 @default.
• W2019970740 cites W2170402088 @default.
• W2019970740 cites W2172064884 @default.
• W2019970740 cites W2320141410 @default.
• W2019970740 cites W2326953798 @default.
• W2019970740 cites W4300345082 @default.
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