Up-regulation of Caveolae and Caveolar Constituents in Multidrug-resistant Cancer Cells

Abstract

Cancer chemotherapy often results in the development of multidrug resistance (MDR), which is commonly associated with overexpression of P-glycoprotein (P-gp), a plasma membrane drug efflux ATPase. It was shown recently that glycosphingolipids are elevated in MDR cells. Sphingolipids are major constituents of caveolae and of detergent-insoluble, glycosphingolipid-rich membrane domains. Here we report that multidrug-resistant HT-29 human colon adenocarcinoma cells exhibit a 12-fold overexpression of caveolin-1, a 21-kDa coat/adaptor protein of caveolae. Similar observations were made in adriamycin-resistant MCF-7 human breast adenocarcinoma cells. Caveolin-2 expression is also up-regulated in MCF-7-AdrR cells, but neither caveolin-1 nor caveolin-2 were detected in MCF-7 cells stably transfected with P-gp. The up-regulation of caveolins is associated with a 5-fold increase in the number of caveolae-like structures observed in plasma membrane profiles of HT-29-MDR cells and with the appearance of a comparable number of caveolae in MCF-7-AdrR cells. A significant fraction (∼40%) of cellular P-gp is localized in low density detergent-insoluble membrane fractions derived from either HT-29-MDR or MCF-7-AdrR cells. The distribution of recombinant P-gp in stably transfected MCF-7 cells was similar, even though these cells do not express caveolins and are devoid of caveolae. The possibility that caveolae contribute to the multidrug resistance and thus are co-selected with P-gp during the acquisition of this phenotype is discussed. Cancer chemotherapy often results in the development of multidrug resistance (MDR), which is commonly associated with overexpression of P-glycoprotein (P-gp), a plasma membrane drug efflux ATPase. It was shown recently that glycosphingolipids are elevated in MDR cells. Sphingolipids are major constituents of caveolae and of detergent-insoluble, glycosphingolipid-rich membrane domains. Here we report that multidrug-resistant HT-29 human colon adenocarcinoma cells exhibit a 12-fold overexpression of caveolin-1, a 21-kDa coat/adaptor protein of caveolae. Similar observations were made in adriamycin-resistant MCF-7 human breast adenocarcinoma cells. Caveolin-2 expression is also up-regulated in MCF-7-AdrR cells, but neither caveolin-1 nor caveolin-2 were detected in MCF-7 cells stably transfected with P-gp. The up-regulation of caveolins is associated with a 5-fold increase in the number of caveolae-like structures observed in plasma membrane profiles of HT-29-MDR cells and with the appearance of a comparable number of caveolae in MCF-7-AdrR cells. A significant fraction (∼40%) of cellular P-gp is localized in low density detergent-insoluble membrane fractions derived from either HT-29-MDR or MCF-7-AdrR cells. The distribution of recombinant P-gp in stably transfected MCF-7 cells was similar, even though these cells do not express caveolins and are devoid of caveolae. The possibility that caveolae contribute to the multidrug resistance and thus are co-selected with P-gp during the acquisition of this phenotype is discussed. multidrug resistance detergent-insoluble, glycosphingolipid-rich membrane domains P-glycoprotein sterol regulatory element-binding protein 4-morpholineethanesulfonic acid. Although chemotherapy improves long term survival in cancer patients, the treatment often results in the development of tumors that are resistant to most cytotoxic drugs commonly used in chemotherapy, leading to an untreatable and incurable disease (1Gottesman M.M. Cancer Res. 1993; 53: 747-754PubMed Google Scholar). Known as multidrug resistance (MDR),1 this phenomenon may be defined as the ability of cancer cells exposed to a given drug to resist the cytotoxic actions of a broad range of structurally and functionally unrelated drugs. MDR is often caused by overexpression of a plasma membrane ATPase called P-glycoprotein (P-gp) (2Bosch I. Croop J. Biochim. Biophys. Acta. 1996; 1288: F37-F54PubMed Google Scholar). P-gp acts as an energy-dependent drug efflux pump, increasing outward transport of active drugs and thereby decreasing their intracellular concentration and reducing their cytotoxic efficacy. However, additional mechanisms that contribute to MDR have been described (see Ref. 3Kaye S.B. Am. J. Med. 1995; 99: 40S-44SAbstract Full Text PDF PubMed Scopus (8) Google Scholar for review). Recent studies have indicated that glucosylceramide accumulates to a major extent in various types of MDR cells (4Lavie Y. Cao H.-T. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Glucosylceramide and other glycosphingolipids are important constituents of detergent-insoluble membrane domains termed DIGs (5Parton R.G. Simons K. Science. 1995; 269: 1398-1399Crossref PubMed Scopus (295) Google Scholar) that are enriched also in sphingomyelin and cholesterol (6Harder T. Simons K. Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (716) Google Scholar). DIGs are related in their lipid composition and their insolubility in cold non-ionic detergents to nonclathrin-coated, plasma membrane vesicular invaginations termed caveolae (reviewed in Ref. 7Parton R.G. Curr. Op. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (495) Google Scholar). Caveolin-1, a 21-kDa integral membrane protein, is a major caveolar coat protein (8Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G. Cell. 1992; 68: 673-682Abstract Full Text PDF PubMed Scopus (1863) Google Scholar) that has the ability to engage in complex interactions with other caveolin molecules, as well as other proteins (9Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1343) Google Scholar). Heterologous expression of caveolin-1 induces the appearance of caveolae-like vesicles in cells that normally lack caveolae, e.g. lymphocytes, Sf9 insect cells, and transformed fibroblasts (10Fra A.M. Williamson E. Simons K. Parton R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8655-8659Crossref PubMed Scopus (525) Google Scholar, 11Li S. Song K.S. Koh S.S. Kikuchi A. Lisanti M.P. J. Biol. Chem. 1996; 271: 28647-28654Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 12Engelman J.A. Wykoff C.C. Yasuhara S. Song K.S. Okamoto T. Lisanti M.P. J. Biol. Chem. 1997; 272: 16374-16381Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Caveolae have been implicated in a number of plasma membrane transport processes such as endocytosis (13Tran D. Carpentier J.L. Sawano F. Gorden P. Orci L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7957-7961Crossref PubMed Scopus (198) Google Scholar), transcytosis (14Schnitzer J.E. Oh P. Pinney E. Allard J. J. Cell Biol. 1994; 127: 1217-1232Crossref PubMed Scopus (770) Google Scholar), and cholesterol efflux (15Fielding C.J. Fielding P.E. J. Lipid Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar). In addition, caveolae are enriched in signaling molecules, and certain receptor, transducer, and effector proteins are recruited onto caveolae upon cell surface receptor activation (9Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1343) Google Scholar, 16Lisanti M.P. Tang Z. Scherer P.E. Kubler E. Koleske A.J. Sargiacomo M. Mol. Membr. Biol. 1995; 12: 121-124Crossref PubMed Scopus (131) Google Scholar). The accumulation of glucosylceramide in MDR cells (4Lavie Y. Cao H.-T. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) prompted us to examine the possible role of caveolae in multidrug resistance. Human colon adenocarcinoma HT-29-wt and HT-29-MDR cells (17Breuer W. Slotki I.N. Ausiello D.A. Cabantchik I.Z. Am. J. Physiol. 1993; 265: C1711-C1715Crossref PubMed Google Scholar) were kindly provided by Prof. I. Cabantchik (Hebrew University, Jerusalem). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum. HT-29-MDR cells were maintained in the same medium supplemented with colchicine (300 ng/ml). HT-29-MDR cells were plated in drug-free medium prior to experiments. MCF-7 human breast adenocarcinoma cells, adriamycin-resistant MCF-7 cells (MCF-AdrR), and MCF-7 cells stably transfected with P-gp (BC-19) were kindly provided by Dr. Merrill E. Goldsmith (National Cancer Institute, Bethesda, MD). The MCF-7 and derived cell lines were grown according to published procedures (18Fairchild C.R. Ivy S.P. Kao-Shan C.S. Whang-Peng J. Rosen N. Israel M.A. Melera P.W. Cowan K.H. Goldsmith M.E. Cancer Res. 1987; 47: 5141-5148PubMed Google Scholar,19Fairchild C.R. Moscow J.A. O'Brien E.E. Cowan K.H. Mol. Pharmacol. 1990; 37: 801-809PubMed Google Scholar). Caveolin-rich membrane domains were purified from cultured cells as a low density, Triton-insoluble complex, essentially as described (20Lisanti M.P. Tang Z. Scherer P.E. Sargiacomo M. Methods Enzymol. 1995; 250: 655-668Crossref PubMed Scopus (117) Google Scholar). The protein content of each fraction was determined according to the modified Lowry procedure (21Markwell M.A. Haas S.M. Bieber L. Tolbert L.L. Anal. Biochem. 1978; 87: 206-211Crossref PubMed Scopus (5307) Google Scholar). Aliquots taken from each of the sucrose density gradient fractions were separated by electrophoresis on 7.5% or 15% SDS-polyacrylamide gels (for resolution of P-gp and caveolin-1, respectively). Proteins were transferred to nitrocellulose membranes and blocked by incubation for 1 h with 5% skim milk (w/v) in phosphate-buffered saline containing 0.1% Triton X-100. Immunoblot analysis was carried out with monoclonal antibodies to caveolin-1 (clone 2297; Transduction Laboratories), caveolin-2 (clone 65; Transduction Laboratories), or anti-P-gp (C219; Signet), all utilized in a dilution of 1:1000 in the blocking buffer. The blots were then washed extensively and reacted with horseradish peroxidase-linked goat anti-mouse IgG. Bands were visualized by enhanced chemiluminescence using a commercially available kit (Amersham Pharmacia Biotech). Quantitation of the caveolin and P-gp was carried out by quantitative densitometry using an image densitometer (PDI Inc.), and the data were processed using Quantity One image analysis software. [3H]Serine labeling of cell lipids and analysis of glycosphingolipids by thin-layer chromatography were performed essentially as described before (4Lavie Y. Cao H.-T. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Confluent monolayer cell cultures were fixed in glutaraldehyde, post-fixed in OsO4, stained with uranylacetate and lead citrate, dehydrated in increasing concentrations of ethanol, and embedded in Epon. Sections were examined utilizing a Philips 410 transmission electron microscope, and cell profiles were routinely photographed at a magnification of 24,000×. Caveolae-like structures were identified by their characteristic round shape, size (50–100 nm), and location at or near the plasma membrane. The number of caveolae/10 μm of plasma membrane was determined by counting morphologically identifiable caveolae in individual cell surface membrane profiles of each cell line (n = 20). The specific sphingolipid- and cholesterol-enriched composition of caveolae facilitates their purification on sucrose density gradients together with other detergent-insoluble, low density membrane domains (20Lisanti M.P. Tang Z. Scherer P.E. Sargiacomo M. Methods Enzymol. 1995; 250: 655-668Crossref PubMed Scopus (117) Google Scholar, 22Brown D. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2604) Google Scholar). Triton X-100-based lysates were prepared from HT-29 human adenocarcinoma cells and fractionated in a discontinuous sucrose density gradient, and the fractions were analyzed for protein concentration and caveolin-1 immunoreactivity. The distribution of protein along the gradient was characteristically skewed, with most protein concentrated at high density sucrose fractions 8–12 (Fig. 1 A). There was no apparent difference between parental HT-29-wt and multidrug-resistant HT-29-MDR cells in either the level or distribution of protein. Caveolin-1 was concentrated in low density fractions 4 and 5 (Fig. 1, B and C). The level of caveolin-1 in HT-29-MDR cells was greatly increased (12-fold) as compared with the parental cell line (Fig. 1,B and C). An even more dramatic up-regulation of caveolin-1 immunoreactivity was observed in multidrug-resistant MCF-7 breast adenocarcinoma cells. In the parental, drug-sensitive cells caveolin-1 expression was undetectable (Fig. 2 A, top panel); in contrast, there was a massive caveolin-1-immunoreactive 21-kDa band in the adriamycin-resistant MCF-7-AdrR cells (Fig. 2 A, middle panel). To test the possibility that the up-regulation of caveolin-1 is a consequence of overexpression of P-gp in the MDR cells, caveolin-1 levels were examined also in MCF-7 cells stably transfected with P-gp (BC-19 cell line). These cells express P-gp levels that are comparable with those found in MCF-7-AdrR (Ref.19Fairchild C.R. Moscow J.A. O'Brien E.E. Cowan K.H. Mol. Pharmacol. 1990; 37: 801-809PubMed Google Scholar; cf. Fig. 4). As shown in Fig. 2 A(bottom panel), caveolin-1 was undetectable in these cells. These results indicate that the up-regulation of caveolin-1 in MDR cancer cells might be a general phenomenon in MDR cancer cells and that the higher level of caveolin-1 is not a secondary cellular response to the overexpression of P-gp.Figure 2Up-regulation of caveolin-1 and caveolin-2 in multidrug-resistant MCF-7 human breast adenocarcinoma cells.Parental MCF-7 cells, multidrug-resistant MCF-7-AdrR cells and BC-19 cells (MCF-7 cells stably transfected with P-gp) were lysed and fractionated by flotation in a discontinuous sucrose density gradient. The fractions were analyzed for caveolin-1 (A) and caveolin-2 (B) immunoreactivity. The locations of the caveolin-1 and caveolin-2 immunoreactive bands (arrowheads) and of the 21-kDa molecular mass marker are indicated. Lane C denotes the position of a caveolin-1 or -2 standard (top and bottom panels) or an aliquot of rat adipocyte membranes (middle panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Localization of P-glycoprotein in low density, Triton-insoluble membrane domains. A, Triton X-100 lysates prepared from HT-29-MDR cells were fractionated by flotation in a discontinuous sucrose density gradient, and 50-μl aliquots were separated by SDS-PAGE and immunoblotted with an antiserum directed to P-glycoprotein as described under “Experimental Procedures” (inset). The locations of the P-gp-immunoreactive band and of molecular mass markers are indicated. Data obtained from three independent experiments similar to the one shown were quantitated by densitometry, and the relative distribution of P-gp in the fractions is presented as the percentage of the total (mean ± S.D., n = 3). Western blots of P-gp in gradient fractions were prepared from MCF-7-AdrR (B) and BC-19 cells (C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Caveolin-2 is a homolog of caveolin-1 (23Scherer P.E. Okamoto T. Chun M. Nishimoto I. Lodish H.F. Lisanti M.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 131-135Crossref PubMed Scopus (491) Google Scholar). The two proteins are known to be co-expressed in most cell types (24Scherer P.E. Lewis R.Y. Volonte D. Engelman J.A. Galbiati F. Couet J. Kohtz D.S. van Donselaar E. Peters P. Lisanti M.P. J. Biol. Chem. 1997; 272: 29337-29346Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) and are thought to form hetero-oligomers in basolaterally destined caveolae (25Scheiffele P. Verkade P. Fra A.M. Virta H. Simons K. Ikonen E. J. Cell Biol. 1998; 140: 795-806Crossref PubMed Scopus (263) Google Scholar). It was therefore of interest to determine whether the up-regulation of caveolin-1 is accompanied by overexpression of Fig. 2 B that this is the caveolin-2 is undetectable in parental MCF-7 (Fig. 2 top panel), there is a band of caveolin-2 immunoreactive protein in MCF-7-AdrR cells (Fig. 2 middle panel). In contrast, the expression of caveolin-2 in BC-19 cells was low to be detected (Fig. 2 bottom panel). results have indicated that of caveolae, the is elevated in various MDR cell lines (4Lavie Y. Cao H.-T. Bursten S.L. Giuliano A.E. Cabot M.C. J. Biol. Chem. 1996; 271: 19530-19536Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). of glucosylceramide levels in HT-29 and HT-29-MDR cells that in those colon cancer cells, glucosylceramide is elevated ± ± of total lipid in HT-29 and HT-29-MDR cells, respectively). glucosylceramide levels found in the HT-29-MDR cells were to an of glucosylceramide which caused a in levels in the MDR cells, that the is active in these cells. The that three specific constituents of caveolae, caveolin-1, and are up-regulated in MDR cells, the possibility that acquisition of the MDR phenotype in cancer cells is associated with an increase in the number of caveolae. of profiles from HT-29-MDR cells the of vesicular in that are to the plasma membrane and are morphologically from caveolae (Fig. A, and In contrast, profiles of HT-29-wt cells caveolae-like structures (Fig. analysis of profiles that the HT-29-MDR cells 5-fold more caveolae-like structures as compared with the parental cells ± ± caveolae/10 respectively). Similar results were obtained in MCF-7 and MCF-7-AdrR cells. the parental MCF-7 cells no caveolae, the MCF-7-AdrR cells ± caveolae/10 with the data presented the up-regulation of caveolin-1, caveolin-2 and these results indicate that MDR cells have more caveolae and that these structures may a role in multidrug resistance. As in most other cell lines that MDR (1Gottesman M.M. Cancer Res. 1993; 53: 747-754PubMed Google Scholar, Am. J. Med. 1995; 99: Full Text PDF PubMed Scopus Google Scholar), the multidrug-resistant phenotype of HT-29-MDR cells is caused by overexpression of the MDR drug efflux ATPase the of the human Immunoblot analysis of fractions derived from HT-29-MDR cells with antibodies to P-gp that of cellular P-gp is associated with the low density fractions that caveolae (Fig. 4 A). The level of P-gp in HT-29-wt cells was of total cellular P-gp was localized in low density fractions of MCF-7-AdrR cells (Fig. 4 These data that a significant fraction of cellular P-gp is localized in detergent-insoluble low density membrane domains. To whether the fraction of P-gp in such domains is localized in caveolae or in the related membrane domains termed we examined the distribution of P-gp in BC-19 cells. In these cells, which do not express levels of caveolin-1 and caveolin-2 Fig. the distribution of P-gp is similar to that of P-gp found in MCF-7-AdrR cells, with a significant of P-gp localized in low density detergent-insoluble membranes (Fig. 4 C). that the of P-gp to these membrane domains is not on the of caveolin or caveolae. It may therefore be that cellular P-gp is in at two membrane One is the other one has the of DIGs and caveolae, and low The expression of caveolin is not a for P-gp to the An that is the relative of those two P-gp to drug efflux and drug resistance in the MDR cells. The results the and important possibility that caveolae a role in multidrug resistance of cancer cells. The up-regulation of caveolar protein and and lipid with the of caveolae-like all indicate that MDR cancer cells more caveolae. might be the role of caveolae in the to caveolae, their role in cholesterol efflux might be most in the of Fielding P.E. 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In with these we have found that the of HT-29-MDR cells (mean of is that of their parental drug-sensitive cells (mean of one consequence of the up-regulation of caveolins in MDR cells is a of which may MDR cells with the of cytotoxic drugs and thus contribute to their under of the high caveolin expression levels in MDR cells are but additional to be and for and Dr. for and for of this are to Prof. I. Cabantchik for the HT-29 cells and to Dr. Merrill E. Goldsmith for the cells.