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【摘要】
Lipid rafts/caveolae are membrane platforms for signaling molecules that regulate various cellular functions, including cell survival. To better understand the role of rafts in tumor progression and therapeutics, we investigated the effect of raft disruption on cell viability and compared raft levels in human cancer cell lines versus their normal counterparts. Here, we report that cholesterol depletion using methyl-ß cyclodextrin caused anoikis-like apoptosis, which in A431 cells involved decreased raft levels, Bcl-xL down-regulation, caspase-3 activation, and Akt inactivation regardless of epidermal growth factor receptor activation. Cholesterol repletion replenished rafts on the cell surface and restored Akt activation and cell viability. Moreover, the breast cancer and the prostate cancer cell lines contained more lipid rafts and were more sensitive to cholesterol depletion-induced cell death than their normal counterparts. These results indicate that cancer cells contain increased levels of rafts and suggest a potential use of raft-modulating agents as anti-cancer drugs.
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Lipid rafts are low-density, detergent-resistant microdomains of plasma membrane that are enriched in cholesterol and glycosphingolipids. Caveolae, a subclass of rafts, are characterized by flask-like invaginations of the plasma membrane that are distinguished from bulk lipid rafts by the presence of caveolin-1. Rafts/caveolae are known to be abundant in various signaling molecules, such as cell surface receptors and intracellular signaling molecules, and thus, these microdomains have been involved in many cellular functions, including the regulation of apoptosis and cell proliferation.1 Growth factor receptors, T-cell receptors, and the tumor necrosis factor receptor superfamily have been shown to interact with rafts/caveolae, and some intracellular signaling molecules are redistributed to rafts/caveolae after the activation of those receptors.2-6 Moreover, this redistribution plays an important role in the regulation of receptor-mediated cellular function, and accordingly, the disruption of rafts/caveolae results in the impairment of signaling events and receptor function. Therefore, it has been proposed that rafts/caveolae serve as molecular platforms that spatially organize appropriate molecules for specific signaling pathways.7
Cholesterol is an abundant component of the plasma membranes of eukaryotic cells and plays an essential role in maintaining membrane integrity and fluidity.8 It is also critical for liquid-ordered raft/caveolae formation by serving as a spacer between the hydrocarbon chains of sphingolipids.9,10 Therefore, it has been speculated that alterations in the cholesterol contents of cells should modify the properties of these domains. In fact, several lines of study have demonstrated that the depletion of cholesterol from the plasma membrane causes disruption of rafts/caveolae and release of raft/caveolae constituents into a non-raft/caveola membrane, which renders them nonfunctional.9,11 These studies indicate that cholesterol is crucial for maintaining intact raft/caveola structure and function.
The cholesterol contents of cell membranes are tightly regulated, and this process involves the uptake of cholesterol-rich low-density lipoprotein both from plasma and from synthetic pathways. Interestingly, cholesterol accumulation has been reported in various solid tumors, including prostate cancer and oral cancer.12,13 In addition, cholesterol metabolism is dysregulated in many malignancies, including myeloid leukemia, lung, and breast cancers.14-17 For example, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in cholesterol biosynthesis that catalyzes mevalonate formation, and HMG-CoA reductase activity is up-regulated in certain tumors. Moreover, malignant cells have been reported to have elevated levels of mevalonate, a cholesterol precursor, and mevalonate treatment was found to promote tumor growth in vivo and to stimulate the proliferation of breast cancer cells.15
Akt/protein kinase B (PKB) is a serine/threonine kinase that is a critical regulator for cell survival and proliferation, especially in human malignant cancer. Activated Akt phosphorylates pro-apoptotic proteins, thereby inactivating their activities. Akt activation also up-regulates anti-apoptotic genes such as Bcl-xL and FLICE-inhibitory protein (FLIP).18-25 Akt activation involves phosphorylation of Ser473 and Thr308 by phosphoinositide-dependent kinases and integrin-linked kinase. Recent studies have suggested that rafts are implicated in Akt activation.26-28
Given that cholesterol is an essential lipid component of rafts/caveolae implicated in Akt activation and that cholesterol is accumulated in several tumors, the present study investigated whether changes in cholesterol level alter raft/caveola levels, which are critical for Akt activation and tumor cell survival. We found that cholesterol depletion results in apoptosis with Akt inactivation and that this occurs in parallel with reduced raft/caveola formation. The present study shows that cancer cell types with higher membrane cholesterol levels have more rafts/caveolae and are more sensitive to the apoptosis induced by cholesterol-depleting agents. We also discuss the implication of cholesterol accumulation in tumors on raft/caveola formation and provide a biological basis for the potential therapeutic applications of cholesterol regulation in cancer therapy.
【关键词】 elevated cholesterol-rich correlated apoptosis sensitivity cholesterol-depleting
Materials and Methods
Cell Culture
The human epidermoid carcinoma cell line A431, human normal prostate and breast epithelial cell lines PZ-HPV7 and MCF-10A, human breast cancer cell lines MCF-7 and MDA-MB-231, and human prostate cancer cell lines PC-3 and LNCaP were obtained from the American Type Culture Collection (Rockville, MD). Dulbecco??s modified Eagles medium (DMEM) and RPMI 1640 with L-glutamine were purchased from JEIL Biotech Services, Inc. (Daegu, Korea); fetal bovine serum (FBS) and antibiotic-antimycotic (100x) were obtained from GIBCO Laboratories (Grand Island, NY). Human A431 cells were grown at 37??C in DMEM supplemented with 10% FBS and antibiotic-antimycotic (1x). PC-3, LNCaP, MCF-7, and MDA-MB-231 were grown in RPMI 1640 containing 10% FBS and antibiotic-antimycotic (1x). Human normal prostate epithelial cell line PZ-HPV7 was grown in keratinocyte serum-free medium with 5 ng/ml human recombinant epidermal growth factor (EGF) and 50 µg/ml bovine pituitary extract. MCF-10A was maintained in DMEM with 5% horse serum, 10 µg/ml insulin, 10 ng/ml EGF, 0.5 ng/ml hydrocortisone, and 100 ng/ml cholera toxin. The cells were grown to approximately 70% confluence and were then serum-starved for 4 hours using 0.1% bovine serum albumin (BSA) in medium before treatment.
Antibodies and Reagents
Alexa Fluor 568-conjugated cholera toxin subunit B and Texas Red-labeled goat anti-rabbit IgG were from Molecular Probes (Eugene, OR). Anti-caspase-3, anti-Bcl-xL, horseradish peroxidase-conjugated goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-phospho-Akt (Ser473) antibody was from Cell Signaling Technology (Beverly, MA); and anti-Erk1/2, anti-active Erk1/2, and anti-poly (ADP-ribose) polymerase were from Upstate (Lake Placid, NY). Annexin V-fluorescein isothiocyanate (FITC) was purchased from BD Pharmingen (San Diego, CA). 3,3'-Diethyloxacabocyanine iodide (DiOC6) and 4'6-diamidino-2-phenylindole?
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作者单位：From the Division of Specific Organs Cancer,* Pediatric Oncology Division, National Cancer Center, Ilsan-gu, Goyang-si, Gyeonggi-do; and the Department of Pathology, Tumor Immunity Medical Research Center and Cancer Research Institute, and the Department of Pharmacology, Seoul National University Co