Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts
Liyan Zhuang, Jayoung Kim, Rosalyn M. Adam, Keith R. Solomon, Michael R. Freeman
Liyan Zhuang, Jayoung Kim, Rosalyn M. Adam, Keith R. Solomon, Michael R. Freeman
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Article Oncology

Cholesterol targeting alters lipid raft composition and cell survival in prostate cancer cells and xenografts

Abstract

Lipid rafts are cholesterol- and sphingolipid-enriched microdomains in cell membranes that regulate phosphorylation cascades originating from membrane-bound proteins. In this study, we tested whether alteration of the cholesterol content of lipid rafts in prostate cancer (PCa) cell membranes affects cell survival mechanisms in vitro and in vivo. Simvastatin, a cholesterol synthesis inhibitor, lowered raft cholesterol content, inhibited Akt1 serine-threonine kinase (protein kinase Bα)/protein kinase B (Akt/PKB) pathway signaling, and induced apoptosis in caveolin- and PTEN-negative LNCaP PCa cells. Replenishing cell membranes with cholesterol reversed these inhibitory and apoptotic effects. Cholesterol also potentiated Akt activation in normal prostate epithelial cells, which were resistant to the apoptotic effects of simvastatin. Elevation of circulating cholesterol in SCID mice increased the cholesterol content and the extent of protein tyrosine phosphorylation in lipid rafts isolated from LNCaP/sHB xenograft tumors. Cholesterol elevation also promoted tumor growth, increased phosphorylation of Akt, and reduced apoptosis in the xenografts. Our results implicate membrane cholesterol in Akt signaling in both normal and malignant cells and provide evidence that PCa cells can become dependent on a cholesterol-regulated Akt pathway for cell survival.

Authors

Liyan Zhuang, Jayoung Kim, Rosalyn M. Adam, Keith R. Solomon, Michael R. Freeman

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Simvastatin treatment reduces the cholesterol content of lipid rafts of ...
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.