Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts (original) (raw)
Simvastatin affects Akt signaling, cell survival, and cholesterol levels in lipid rafts. In an initial series of experiments, we asked whether inhibition of endogenous cholesterol synthesis, rather than dispersion of cholesterol from rafts by an exogenous agent (23), alters Akt signaling and apoptosis rates in LNCaP cells. To address this question we used simvastatin, an FDA-approved cholesterol-lowering drug. Simvastatin, like other statins (34), is an inhibitor of the enzyme HMG-CoA reductase, which catalyzes the rate-limiting step (HMG-CoA to mevalonate) in cholesterol biosynthesis. Statin drug–induced apoptosis in PCa cells has been reported (35), but the mechanism of this effect is unknown. Treatment of LNCaP cells with simvastatin for 16 hours decreased phosphorylation of Akt in a dose-dependent manner (Figure 1A), with substantial inhibition evident at 20 μM simvastatin. Serum-free conditions were used because serum contains significant amounts of cholesterol. Simvastatin-induced inhibition of Akt phosphorylation was reversible by replenishment of cellular cholesterol levels; this is consistent with reformation of raft microdomains. Simvastatin also elicited dose-dependent effects on apoptosis in LNCaP cells (not shown). The 20-μM simvastatin dose was used in subsequent experiments.
Simvastatin treatment downregulates Akt phosphorylation and Akt kinase activity and induces apoptosis in LNCaP cells. (A) Cells were incubated with varying doses of simvastatin (sim) in the absence or presence of cholesterol complexes for 16 hours. Whole-cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies to total Akt or S473-phosphorylated Akt (S473-P). (B) LNCaP cells were incubated in the presence of 20 μM simvastatin or vehicle (control) in serum-free medium at 37°C for the indicated times, after which lysates were collected for Western blot and in vitro kinase assay. A GSK3 fusion protein was used as Akt substrate after immunoprecipitation with anti-Akt antibody. Kinase assay eluates were blotted with antibodies to total Akt, phospho-GSK3α/β (p-GSK3α/β), T308-P Akt, and S473-P Akt. (C) Cells in serum-free medium were treated with 20 μM simvastatin in the absence or presence of cholesterol (chol) complexes at 37°C for 12 hours, after which lysates were collected and kinase assay was performed as in B. (D) LNCaP cells were treated for varying times with 20 μM simvastatin. Apoptosis was determined by DNA fragmentation. The means ± SD of triplicate determinations are shown. (E) LNCaP cells were treated with 20 μM simvastatin with or without cholesterol complexes for 12 hours followed by DNA fragmentation analysis (*P < 0.05). (F) LNCaP cells were incubated with 20 μM simvastatin with or without cholesterol complexes for 12 hours, after which lysates were collected for immunoblot with the indicated antibodies. c-Caspase-7, cleaved caspase-7.
Consistent with published data that serum withdrawal stimulates Akt activity in LNCaP cells (36), we observed a time-dependent increase in Akt activity and phosphorylation at the 2 principal regulatory sites (T308 and S473) when cells were placed in serum-free conditions (Figure 1B). Simvastatin treatment under serum-free conditions resulted in a relative reduction in Akt kinase activity and a time-dependent decrease in phosphorylation at both regulatory sites (T308 and S473; Figure 1B). To test whether the effect of simvastatin on Akt activity was cholesterol-dependent, cholesterol complexes were added to cells simultaneously with simvastatin. Cholesterol repletion reversed the inhibition of Akt by simvastatin (Figure 1C), suggesting that this effect was mediated by a reduction in membrane cholesterol.
Consistent with the observation that constitutive PI3K/Akt pathway signaling is required for survival of LNCaP cells (37), simvastatin induced a time-dependent increase in apoptosis (Figure 1D). This effect was preceded by reductions in Akt activity as assessed in whole-cell lysates (Figure 1, A and B). To determine whether the apoptotic effect of simvastatin was cholesterol-dependent, cholesterol complexes were added to cells simultaneously with simvastatin, and apoptotic rates were measured. Following cholesterol repletion of cell membranes, apoptosis was inhibited (Figure 1E). Consistent with the simvastatin-induced inhibition of Akt phosphorylation and kinase activity (Figure 1, A–C), phosphorylation of the Akt effector mammalian target of rapamycin (mTOR) was also inhibited by simvastatin (Figure 1F). Phosphorylation of mTOR was restored to basal levels following cholesterol replacement (Figure 1F). We also determined the effect of simvastatin on caspase-7 and cyclin A. Levels of cleaved caspase-7 increased markedly in response to simvastatin treatment for 12 hours, but not following cholesterol repletion (Figure 1F). We also observed a modest decrease in cyclin A levels with simvastatin treatment; these returned to the basal level with re-addition of cholesterol (Figure 1F). Taken together, these data suggest that the apoptotic effect of simvastatin results from inhibition of Akt pathway signaling.
To determine whether simvastatin treatment alters the cholesterol composition of lipid raft microdomains, LNCaP cells were treated with simvastatin for 16 hours, with and without cholesterol repletion, and lipid raft fractions were isolated by sucrose gradient ultracentrifugation. Raft fractions were identified by immunoblot of gradient fractions with antibodies against the raft markers G protein α inhibitory subunit (Giα2) and flotillin-2 (Figure 2A). In the example shown, flotillin-2 was present in fractions 5–8, while Giα2 was largely restricted to fraction 6; this indicates that lipid raft components were located within fractions 5–8 (primarily fraction 6 in the figure). Consistent with this interpretation, the EGFR was found predominantly outside the raft compartment, a result previously reported for LNCaP cells (23). Using this methodology, we isolated verified raft fractions from cells treated with vehicle, simvastatin, and simvastatin plus cholesterol. Cholesterol content was subsequently measured after extraction of lipids with chloroform and methanol. In comparison with control cells, cholesterol content (normalized to protein) in rafts was significantly decreased by simvastatin treatment, while repletion of membrane cholesterol restored raft cholesterol to normal levels (Figure 2B). This result indicates that cholesterol content of membrane rafts is significantly reduced by pharmacologic inhibition of endogenous cholesterol synthesis.
Simvastatin treatment reduces the cholesterol content of lipid rafts of LNCaP cells and inhibits Akt phosphorylation in rafts. (A) Immunoblot results obtained following fractionation of Triton X-100–insoluble material by sucrose gradient ultracentrifugation. This panel demonstrates how lipid raft fractions used for the cholesterol determinations shown in B were obtained. Flotation fractions demonstrating enrichment in the raft markers Giα2 and flotillin-2 (i.e., fraction 6 in this example) were designated as raft fractions. (B) Cells were incubated in serum-free medium in the absence (control) or presence of 10 μM simvastatin overnight at 37°C. After the drug treatment, 1 group was incubated with cholesterol complexes (sim + chol) at 37°C for 1 hour. The cholesterol/protein ratio was determined in lipid raft fractions prepared as shown in A and under the conditions described in the text. Values shown are means ± SD of triplicate determinations (*P < 0.01). (C) Cells were incubated in the presence of 20 μM simvastatin or vehicle in serum-free medium at 37°C for the indicated times. C+M and raft fractions were isolated by successive detergent extraction, resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. (D) Cells were treated with 20 μM simvastatin with or without cholesterol complexes for 4 hours, followed by raft extraction and analysis as in C.
In view of the marked effect of simvastatin treatment on Akt phosphorylation and activity in whole-cell lysates (Figure 1) as well as on raft composition (Figure 2B), we determined the effect of simvastatin on raft-localized Akt. Raft and cytosolic plus nonraft membrane (C+M) fractions were isolated from LNCaP cells treated with simvastatin for different times. In these preparations, the raft fraction consists of detergent-resistant membranes that are insoluble in cold Triton X-100, from which cytoskeletal and nuclear structures are removed by pelleting of the insoluble material. Raft material is solubilized in buffer containing octylglucoside (OCG) (38). These preparations are essentially identical to raft preparations obtained by flotation of Triton X-100–insoluble material in sucrose gradients (39).
Akt was present in both C+M and raft fractions of control and simvastatin-treated cells but was more abundant in the C+M fraction (Figure 2C). Akt localization was not detectably altered with simvastatin treatment, nor was Akt phosphorylation altered in the C+M fraction (Figure 2C). In contrast, Akt phosphorylation was decreased in rafts of simvastatin-treated cells by 4 hours and remained suppressed for the duration of the experiment. No such decrease was evident in cells under control conditions (Figure 2C). Replacement of membrane cholesterol partially restored Akt phosphorylation in rafts (Figure 2D). We conclude from these experiments that the effects of simvastatin are substantially more evident on raft-resident Akt than on Akt in the nonraft membrane and that this suppressive effect can be reversed with cholesterol repletion.
Cholesterol regulates Akt signaling, but not apoptosis, in normal prostate epithelial cells. To determine the effect of lipid raft disruption on Akt signaling in a relevant normal cell type, we evaluated Akt phosphorylation in early-passage normal prostate epithelial cells (PrECs) in response to the cholesterol-binding agents filipin and 2-hydroxypropyl-β-cyclodextrin, as well as simvastatin. As anticipated, EGF stimulated Akt phosphorylation in PrECs (Figure 3A); however, this effect was potently amplified by increasing membrane cholesterol levels (Figure 3A, lanes 1, 5, and 6). Filipin, cyclodextrin, and simvastatin inhibited EGF-induced Akt phosphorylation (Figure 3A, lanes 2–5). Filipin and cyclodextrin both inhibited the ability of cholesterol to potentiate EGF-dependent Akt phosphorylation (Figure 3A, lanes 7 and 8). Cholesterol repletion following filipin and cyclodextrin treatment restored Akt phosphorylation to levels that were comparable to those seen with EGF alone (Figure 3A, lanes 9 and 10). In the absence of EGF, cholesterol, used singly, modestly activated Akt (Figure 3, lane 11). These data indicate that plasma membrane cholesterol is a component of Akt pathway signaling in PrECs as it is in LNCaP cells.
Cholesterol depletion inhibits Akt phosphorylation but does not induce apoptosis in PrECs. (A) Immunodetection of Akt in cell lysates following treatments. PrECs were treated with 20 ng/ml EGF, in the presence or absence of cholesterol complexes, under the following conditions: 1 hour of pretreatment with 2 μg/ml filipin (lane 2), 1 hour of pretreatment with 20 mM cyclodextrin (lane 3), 16 hours of pretreatment with 20 μM simvastatin (lane 4), 1 hour of pretreatment with vehicle (lane 5). After 1 hour of cholesterol pretreatment (lane 6), some groups were incubated with 2 μg/ml filipin (lane 7) or 20 mM cyclodextrin (lane 8). Other groups treated identically to conditions in lanes 7 and 8 were repleted (asterisk) with cholesterol for 1 hour (lanes 9 and 10). (B) PrECs were treated with varying concentrations of simvastatin or vehicle for 24 hours, and the extent of apoptosis was determined by DNA fragmentation. Values shown are means ± SD of triplicate determinations.
To determine whether PrECs were susceptible to HMG-CoA reductase inhibitor–induced apoptosis, PrECs were treated for 24 hours with up to 100 μM simvastatin. Apoptosis rates were not demonstrably affected by these treatments (Figure 3B). These observations are consistent with published findings describing the triggering of apoptosis in LNCaP cells with PI3K inhibitors under conditions where PrECs were unaffected (37).
Elevated circulating cholesterol alters tumor lipid rafts in vivo. An implication of the finding that the Akt survival function is regulated by cholesterol-rich microdomains is that membrane cholesterol is a mediator of tumor cell survival in vivo. To test this hypothesis, we carried out experiments in which tumor xenografts were created in SCID mice under normal conditions and under conditions where serum cholesterol was chronically elevated. Circulating cholesterol was raised by feeding with a hypercholesterolemic diet, an established method of raising serum cholesterol in rodents (40). We also attempted to lower cholesterol in vivo with simvastatin. However, treatment of SCID mice over a 4-week period with daily i.p. injections of simvastatin (20 mg/kg) did not lower cholesterol levels (not shown); this is consistent with previous reports that statin drugs at physiologically relevant doses do not lower serum lipids in mice, even under hyperlipidemic conditions (41–44).
After 4 weeks on the high-cholesterol regimen, total serum cholesterol concentrations were significantly elevated (Figure 4A). Tumor xenografts were created using LNCaP/sHB cells (45), an LNCaP subline engineered to constitutively secrete the processed form of the EGFR/ErbB1 activator heparin-binding EGF-like growth factor (HB-EGF). LNCaP/sHB cells efficiently form tumors in SCID mice (45). Tumor incidence 6 weeks after cell injection, and tumor volumes measured at 5 and 6 weeks after injection, were both significantly increased in the high-cholesterol group in comparison with the normal group (Figure 4, B–D).
High levels of serum cholesterol increase tumor aggressiveness. (A) Serum cholesterol levels in venous blood after stable elevation using dietary modification for 4 weeks. Values are means ± SD of determinations from 5 animals (P < 0.001). (B) Subcutaneous xenograft tumors were created by subcutaneous injection of LNCaP/sHB cells after stable cholesterol elevation was demonstrated. The tumor take was significantly different between normal and high-cholesterol groups (P < 0.0001). (C) Mice were sacrificed 6 weeks after tumor cell injection. Four representative xenograft tumors from each group are shown. (D) Volume measurements were made at 5 weeks and 6 weeks after tumor cell injection. Median tumor volumes (horizontal lines) for the normal group were 0.077 cm3 (5 weeks) and 0.099 cm3 (6 weeks); median tumor volumes for the hypercholesteremic group were 0.135 cm3 (5 weeks) and 0.141 cm3 (6 weeks) (*P < 0.01; **P < 0.005).
We sought to determine whether lipid raft membranes isolated from LNCaP/sHB xenograft tumors exhibited alterations attributable to the high-cholesterol regimen. Cholesterol content of lipid rafts isolated from tumor xenografts by sucrose gradient ultracentrifugation (as shown in Figure 2) was significantly elevated in the high-cholesterol group in comparison with the normal group (Figure 5A). This demonstrates that elevated circulating cholesterol increases the cholesterol content of lipid raft membranes in tumors. Lipid raft protein fractions were also isolated from xenografts by differential solubilization in Triton X-100 and OCG (38) in order to evaluate levels of protein phosphorylation on tyrosine residues. Tyrosine phosphorylation of lipid raft proteins was significantly increased in the high-cholesterol group (Figure 5B).
Elevated cholesterol and protein tyrosine phosphorylation in lipid rafts isolated from xenograft tumors exposed to high circulating cholesterol. (A) Cholesterol level in lipid rafts isolated from LNCaP/sHB xenograft tumors by sucrose density gradient ultracentrifugation (n = 4 tumors per condition) (P < 0.01). (B) Evidence for increased tyrosine phosphorylation of lipid raft proteins isolated from LNCaP/sHB xenograft tumors by differential solubilization in Triton X-100 and OCG (38). OCG-soluble proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and blotted with anti-phosphotyrosine antibody (right panel). Top panel: Quantitative evaluation by scanning densitometry of immunoblot shown on the bottom (P < 0.0005). MW, molecular weight.
Increased Akt activation and decreased apoptosis in xenograft tumors exposed to high circulating cholesterol. To determine whether high circulating cholesterol alters cell survival within tumors, we analyzed LNCaP/sHB tumors harvested from animals in the high-cholesterol and normal groups for Akt activation status and apoptotic frequency. In order to perform this analysis we first confirmed that our anti–phospho-Akt (S473-P) antibody was suitable to identify activated Akt by immunofluorescence microscopy. To test this, serum-starved LNCaP cells were treated with pervanadate, one of the most potent known Akt activators (46, 47), and Akt and phospho-Akt were localized by immunofluorescence (Figure 6A). As shown in the figure, staining with the anti–phospho-Akt antibody increased dramatically after pervanadate treatment. In addition, Akt was localized predominantly at the cell membrane, consistent with the literature on the effects on Akt activation of pervanadate and other agents (46–49) (Figure 6, A–C). Tissue sections from LNCaP/sHB xenograft tumors taken from mice in normal and high-cholesterol cohorts were subsequently analyzed. Using the same staining protocol used for the cultured cells, we observed that Akt activation was significantly increased in tumors harvested from the high-cholesterol group (Figure 6D). Consistent with this result, the percentage of apoptotic cells in tumors obtained from the high-cholesterol group was significantly reduced in comparison with that in tumors obtained from the normal group (Figure 7), strongly suggesting a suppressive effect of elevated cholesterol on apoptotic signaling within the tumors.
Increased Akt activation in xenograft tumors from mice with high circulating cholesterol. (A–C) Tests of S473-P antibody specificity for the experiment shown in D. LNCaP cells were starved in serum-free medium at 37°C for 16 hours and then treated with 0.5 mM pervanadate for 15 minutes at 37°C. Original magnification: A, ×200; B and C, ×600. (D) Anti–phospho-Akt1 antibody was used to detect the status of Akt activation in LNCaP/sHB xenograft tumors from normal and high-cholesterol animals with the immunofluorescence procedures used for A–C. Two representative images are shown (original magnification, ×400). Optical intensities of the images were determined automatically with computer-controlled software (n = 4 in each group). The values shown are mean signal intensity (INT) per square millimeter ± SD versus group (P < 0.001).
Reduced apoptosis in xenograft tumors from mice with high circulating cholesterol. Apoptosis rates in LNCaP/sHB xenograft tumors from normal (n = 3) and high-cholesterol (n = 4) groups as evaluated by TUNEL (original magnification, ×200). Fluorescence originates from condensed nuclei in apoptotic cells. The graph is presented as percent apoptotic cells (apoptotic cells/total cells) ± SD versus group (P < 0.001).