Elevated levels of cholesterol-rich lipid rafts in cancer cells are correlated with apoptosis sensitivity induced by cholesterol-depleting agents

Abstract

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-beta 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.

Figure 1

The effects of MBCD on cell viability. A: Serum-starved A431 cells were treated with 0 to 10 mmol/L MBCD for 24 hours, and cells were subjected to MTS assay as described in Materials and Methods. B: Serum-starved A431 cells were treated with 5 mmol/L MBCD for 0 to 8 hours, and cells were subjected MTS assay. Error bars represent the means ± SD of three independent experiments. Similar results were observed in two independent experiments.

Figure 2

Apoptotic features of MBCD-induced cell death. A: A431 cells were treated with 5 mmol/L MBCD, stained with annexin V-FITC and PI, and subjected to flow cytometry analysis. Fluorescence dot blots of annexin V-positive (horizontal axis) and PI-positive (vertical axis) cells are shown. Cells that were positively stained by annexin V-FITC only (early apoptosis) and positive for both annexin V-FITC and PI (late apoptosis) were quantitated, and both subpopulations were considered as overall apoptotic cells. B: A431 cells were treated with 2, 5, and 10 mmol/L MBCD for 4 hours, and DNA was isolated and analyzed by 2% agarose gel electrophoresis. C: Cells were treated with 5 mmol/L MBCD for 4 hours, stained with DAPI, and analyzed by fluorescence microscopy. D: A431 cells were treated without or with 5 mmol/L MBCD for 4 and 6 hours and then incubated with DiOC₆ to monitor ΔΨ_m by FACS. The loss of mitochondrial membrane potential was equated with decreased fluorescence and shift to the left. These experiments were repeated three separate times with comparable results.

Figure 3

Effect of MBCD on basal and EGF-induced Akt activation. A: Serum-starved A431 cells were treated with 1, 5, or 10 mmol/L MBCD for 1 or 2 hours or with 30 nmol/L EGF for 5 minutes, followed by cell lysis with 2× sample buffer. Aliquots (30 μg of protein) from each treatment were subjected to immunoblotting analysis using anti-phospho-EGF receptor antibodies that specifically recognize tyrosine-phosphorylated EGF receptors at 845 or 1068 residues. B: Serum-starved A431 cells were treated with 5 mmol/L MBCD for 0 to 6 hours or with 30 nmol/L EGF for 5 minutes, and cells were lysed with 2× sample buffer. Thirty micrograms of protein from each treatment was subjected to immunoblotting analysis using antibodies specific for phospho-Akt, Bcl-xL, caspase-3, PARP, active-ERK1/2, and ERK1/2 (loading control). C: Serum-starved A431 cells were pretreated with 5 mmol/L MBCD and then washed with medium, followed by 30 nmol/L EGF treatment for 10 minutes. Thirty micrograms of protein from each treatment was subjected to immunoblotting analysis using anti-phospho Akt and anti-total Akt antibodies. D: Serum-starved A431 cells were pretreated with 5 mmol/L MBCD for 1 or 2 hours and then washed with medium, followed by 30 nmol/L EGF treatment for 2 hours. Cell viability was measured by MTS assay. Values represent the means ± SD of three independent experiments. These experiments were performed two separate times with comparable results.

Figure 4

Effect of cholesterol repletion on cell morphology, Akt activation, cell viability, and raft/caveolae distribution. A: Serum-starved A431 cells were treated either without or with 5 mmol/L MBCD or 1 mmol/L cholesterol for 1 hour. Some MBCD-treated cells were washed with medium, and then cells were incubated without or with 1 mmol/L cholesterol for 1 hour. Cells were then stained with Texas Red-labeled phalloidin to visualize actin. B: Serum-starved A431 cells were treated as described in A, and then cell lysates were subjected to immunoblotting analysis using anti-phospho-Akt antibodies. C: Serum-starved A431 cells were treated as described in A, and cell viability was measured by MTS assay. Values represent the means ± SD of three independent experiments. D: Serum-starved A431 cells were treated as described in B, and cells were stained with filipin and Alexa Fluo568-CTXB and then subjected to confocal microscopy analysis. Similar results were obtained in three different experiments.

Figure 5

Levels of rafts/caveolae in prostate cancer and breast cancer cell lines and their normal cell lines and their sensitivity to MBCD-induced apoptosis. A and B: Cells were cultured on aseptic coverslip overnight, and cells were stained with CTXB-Alexa 568 and filipin to detect plasma membrane cholesterol and lipid rafts, followed by confocal microscopy analysis. C and D: Serum-starved cells were treated with various concentrations of MBCD (0, 1, 3, 5, and 10 mmol/L) for 24 hours, and cells were subjected to MTS assay as described in Material and Methods. Values represent the means ± SD of three independent experiments. Similar observations were made in three different experiments.

Figure 6

Simvastatin-induced apoptosis in A431 cells. A: Serum-starved A431 cells were treated with 0 to 60 μmol/L simvastatin for 24 hours, and cells were subjected to MTS assay as described in Materials and Methods. B: Serum-starved A431 cells were treated with 30 μmol/L simvastatin for 0 to 16 hours, and cells were subjected to MTS assay. Values represent the means ± SD of three independent experiments. C: Serum-starved A431 cells were treated either without or with 10 and 30 μmol/L simvastatin for 24 hours, and then cells were fixed and stained with propidium iodide, followed by sub-G1 analysis (values indicate the percentage of cells with sub-G1 DNA content). Values represent the means ± SD of three independent experiments. Similar results were obtained in three different experiments.

Figure 7

The effect of simvastatin on raft/caveolae levels and Akt activity in A431 cells. A: A431 cells were cultured on aseptic coverslip overnight. Serum-starved A431 cells were treated without or with 1 mmol/L cholesterol for 2 hours, 30 μmol/L simvastatin for 4 hours, or 1 mmol/L cholesterol for 2 hours after pretreatment with 30 μmol/L simvastatin for 4 hours. Cells were then stained with CTXB-Alexa 568 and filipin to detect plasma membrane cholesterol and lipid rafts, followed by confocal microscopy analysis. B: Serum-starved A431 cells were treated without or with 30 μmol/L simvastatin for indicated times, or 1 mmol/L cholesterol for 2 hours after pretreatment with 30 μmol/L simvastatin for 4 hours or with 30 nmol/L EGF for 5 minutes. Cells were lysed with 2× sample buffer, and 30 μg of protein from each treatment was subjected to immunoblotting analysis using anti-phospho-Akt and anti-ERK1/2 antibodies (loading control). C: Serum-starved A431 cells were treated without or with 30 μmol/L simvastatin for indicated times, 1 mmol/L cholesterol for 2 hours, or 1 mmol/L cholesterol for 2 hours after pretreatment with 30 μmol/L simvastatin for 4 hours. MTS assay was performed to assess cell viability. Values represent the means ± SD of three independent experiments. Similar results were obtained in three different experiments.