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Carcinogenesis Advance Access originally published online on June 25, 2008
Carcinogenesis 2008 29(8):1546-1554; doi:10.1093/carcin/bgn146
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Targeting lipid rafts inhibits protein kinase B by disrupting calcium homeostasis and attenuates malignant properties of melanoma cells

Shlomit Fedida-Metula1, Shira Elhyany1, Sylvia Tsory1, Shraga Segal1, Michal Hershfinkel2, Israel Sekler3 and Daniel Fishman2,*

1 Department of Microbiology and Immunology
2 Department of Morphology
3 Department of Physiology, Faculty of Health Sciences, Ben-Gurion University Cancer Research Center, Ben-Gurion University of the Negev, PO Box 653, Beer-Sheva 84105, Israel

* To whom correspondence should be addressed. Tel: +972 8 6477250; Fax: +972 8 6477626; Email: dmitrif{at}bgu.ac.il


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Failure of current therapeutic modalities to treat melanoma remains a challenge for clinical and experimental oncology. The aggressive growth and apoptotic resistance of this tumor are mediated, in part, by aberrantly activated protein kinase B/Akt (PKB). In many cells, PKB signaling depends on integrity of cholesterol-enriched membrane microdomains (rafts). However, it is still unclear if rafts support deregulated PKB activity in melanoma. In this study, ablation of rafts in murine (B16BL6-8, JB/RH1) and human (GA) melanoma lines by cholesterol-chelating methyl-β-cyclodextrin (MβCD) reduced levels of constitutively active PKB in a dose- and time-dependent manner, while reconstitution of microdomains restored PKB activity. PKB was sensitive to the membrane-permeable Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra (acetocymethyl) ester and to the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) implying the contribution of Ca2+ signaling to PKB deregulation. Indeed, malignant and apoptosis-resistant clone of B16BL6 melanoma (B16BL6-8) displayed significantly higher [Ca2+]i and store-operated Ca2+ influx (SOC) relative to non-malignant apoptosis-sensitive B16BL6 clone (Kb30) expressing barely detectable basal levels of active PKB. Raft ablation in B16BL6-8 cells robustly inhibited SOC and decreased [Ca2+]i to levels comparable with those detected in Kb30 cells. Treating cells by PKB-inhibiting doses of MβCD dramatically impaired their apoptotic resistance and capacity to generate tumors. Furthermore, weekly intraperitoneal injections of MβCD to mice grafted with melanoma cells at doses of 300 and 800 mg/kg significantly attenuated tumor development. Our data implicate membrane rafts in enhancing the resistance of melanoma to apoptosis and indicate that targeting raft microdomains is a potentially effective strategy to cure this frequently fatal form of cancer.

Abbreviations: BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra (acetocymethyl) ester; MβCD, methyl-β-cyclodextrin; PBS, phosphate-buffered saline; PKB, protein kinase B; SOC, store-operated Ca2+ influx; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfoneyl)-2H-tetrasolium-5-carboxanilide


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Malignant melanoma is a ubiquitous type of malignancy characterized by high mortality rates due to its aggressive growth and extreme resistance to cell death-inducing factors (1,2). The molecular derangements underlying the relatively low susceptibility of melanoma cells to apoptosis ultimately include an aberrant activation of protein kinase B/Akt (PKB) (1–10). Elevated levels of constitutively phosphorylated (active) PKB is a common finding in lesions of advanced melanoma (7,8) and inversely correlate with patient survival (7), making this enzyme an attractive target for therapeutic intervention. It is generally accepted that deregulated PKB activity in melanoma is associated with the overexpression of its particular isoforms (4–6), loss of a negative PKB regulator phosphatase and tensin homolog (3), mutations affecting Ras oncogenes (9) and other abnormalities in the phosphatidylinositol-3,4,5-phosphate/PKB pathway. In addition to this pathway, stimulation of many tyrosine kinase- and G-coupled receptors is accompanied by a massive release of calcium (Ca2+) from intracellular stores followed by store-operated Ca2+ influx (SOC) (11). The resulting increase of Ca2+ concentration in the cytosol ([Ca2+]i) may also activate PKB along with phosphatidylinositol-3,4,5-phosphate pathway (12–16), though possible contribution of Ca2+ signaling to PKB deregulation in melanoma cells has not been explored previously.

Recent studies demonstrated that elevated levels of aberrantly activated PKB in prostate (17,18), epidermoid (17) and breast (17) carcinomas correlate with increased amounts of cholesterol-enriched microdomains (lipid rafts) in the membranes of cells derived from these tumors, compared with their respective non-neoplastic tissues (17). Lipid rafts are small dynamic aggregates of membrane molecules stabilized by selective accumulation of cholesterol and glycosphingolipids (19,20). These structures specifically accommodate cohorts of membrane receptors and non-receptor molecules, stabilize the formation of functional complexes and promote signal transduction (20). Stimulation of PKB by insulin (21), epidermal (22) and platelet-derived (23) growth factors requires intact rafts, as the receptors for these growth factors and/or associated post-receptor signaling elements reside in the rafts (21–23). However, the manner in which cholesterol-enriched microdomains support deregulated PKB activity in aforementioned types of cancer cells (17) has not been defined and requires further investigation. In malignant melanoma, lipid rafts are involved in regulation of cell adhesion and motility (24,25), but their role in PKB-mediated survival has not been established. Interestingly, the inhibitors of cholesterol biosynthesis (statins), which destabilize raft microdomains when applied at high doses, were reported to exert in vitro cytotoxic and cytostatic effects on melanoma cells (26). However, these effects may not necessarily be mediated by elimination of rafts, since statins also inhibit prenylation of membrane GTPases and other potential upstream regulators of PKB at concentrations significantly lower than those required to disrupt microdomains (27). Statins may also alter signal transduction elements by binding inter-cellular adhesion molecule-1 and other cytoadhesion molecules expressed at the plasma membrane (28). Therefore, to assess the involvement of lipid rafts in controlling melanoma cell survival and tumorigenicity, we utilized membrane-impermeable cyclic oligosaccharide methyl-β-cyclodextrin (MβCD), which specifically extracts cholesterol from plasma membrane and alters raft integrity (29). Our data demonstrate that targeting lipid rafts in melanoma cells by this compound abolishes constitutively active PKB by altering cellular Ca2+ homeostasis, as manifested by the robust inhibition of SOC and decreased [Ca2+]i, renders these cells susceptible to apoptosis and attenuates their tumorigenicity.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell lines, cell culture reagents and laboratory animals
B16BL6 melanoma clones (B16BL6-8 and Kb30), JB/RH1 and GA cells described previously in (30–32) were kindly provided by Prof. E.Gorelik (Department of Pathology, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA) and Prof. J.Gopas (Department of Immunology, Ben-Gurion University, Beer-Sheva, Israel) and grown in RPMI medium supplemented by 10% bovine serum and antibiotics (Biological Industries, Beth Haemek, Israel). MβCD and water-soluble cholesterol were purchased from Sigma (Rehovot, Israel), 1,2-bis(o-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid tetra (acetocymethyl) ester (BAPTA-AM) and N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) from Calbiochem Merck Biosciences (Schwalbach, Germany). To generate experimental tumors, B16BL6-8 cells (2.5 x 105 cells/30 µl of sterile saline) were engrafted into the footpad of 8-week-old C57BL/J mice (Harlan, Jerusalem, Israel). About 300 or 800 mg/kg of MβCD dissolved in 0.3 ml of sterile saline was administered to mice by weekly intraperitoneal injection. The first dose was applied 8 days after inoculation of tumor cells. Serum aspartate transaminase (AST), alanine transaminase (ALT), creatinine and urea were tested using commercial reagents and AU 2700 Olympus analyzer (Olympus Life and Material Science Europe, GmbH, Hamburg, Germany). All in vivo studies were approved by Institutional Committee for Ethical Care of Laboratory Animals (Ben-Gurion University of the Negev).

Immunoblotting and immunoprecipiation
Protein extracts for immunoblotting were prepared using a buffer (10 mM Tris–HCl, pH 8.0, 1% vol/wt sodium dodecyl sulfate) preheated to 100°C and cells homogenized by repeated passage through a syringe equipped with 27 gauge. To prepare protein extracts from tumors, melanin-producing lesions were excised from euthanized mice, minced in ice-cold 0.1 M phosphate-buffered saline (PBS) containing protease inhibitors cocktail (Roche Diagnostics, Mannheim, Germany), suspended in a preheated lysis buffer and homogenized by repeated passage through syringes equipped with 19 and 21 gauge needles. Debris was removed by centrifugation at 12 000g for 5 min at room temperature. Samples were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to immunoblotting with anti-Akt1 (Santa Cruz Biotechnology, Santa Cruz, CA, diluted 1:200), anti-Akt2 (Cell Signaling, Beverly, MA, diluted 1:300), anti-Akt3 (Santa Cruz Biotechnology, diluted 1:200) and p-Akt (Ser 473) (Cell Signaling, diluted 1:800) antibodies. For immunoprecipitation, cells were lysed using a buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate and protease inhibitors, incubated for 20 min at 4°C, homogenized by passing several times through a syringe equipped with 27 gauge needle and clarified by centrifugation (12 000g, 15 min, 4°C). Samples (500 µg of protein in 0.2 ml of lysis buffer/each) were incubated with anti-Akt1 (diluted 1:33), anti-Akt2 (diluted 1:40) and anti-Akt3 antibodies (diluted 1:30) and protein A-sepharose (Amersham Biosciences, Uppsala, Sweden) on rocking platform (12 h, 4°C). Immune complexes were resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted to anti-Akt1, anti-Akt2, anti-Akt3 and anti-p-Akt antibodies.

Isolation of lipid rafts
Assay was performed as described in ref. (33). Briefly, 0.3 ml of cell lysate prepared using a buffer (25 mM 4-(2-hydroxyethyl)-1-piperasineethanesulfonic acid, pH 6.9, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 1% Brij 58, 100 mM NaCl, 2 mM ethylenediaminetetraacetic acid, 10 mg/ml aprotinin, 100 mg/ml leupeptin, 10 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate and protease inhibitors) was gently mixed with 0.3 ml of 85% sucrose (wt/vol in lysis buffer), overlaid with 1 ml of 35% sucrose and 0.3 ml of 5% sucrose. Following centrifugation (200 000g, 16 h, 4°C), nine fractions (0.2 ml) were collected from the top of the gradient and subjected to the dot blot analysis with horseradish peroxidase-conjugated cholera toxin B subunit (Molecular Probes, Eugene, OR).

Confocal microscopy
Cells were grown on glass coverslips, washed by 0.1 M PBS and reacted with 1 µg/ml of Alexa 647-cholera toxin B subunit (Molecular Probes) in PBS containing 1% of bovine serum albumin for 1 h at room temperature and analyzed by confocal microscopy (LSM 510, Carl Zeiss, Jena, Germany).

Calcium measurements by flow cytometry
Assays were performed as described in refs (14,34) with modifications. For evaluation of [Ca2+]i, cells were counted, suspended in Ringer’s solution (6 x 105 cell/ml) containing 2.5 µM of Fluo-4 (Molecular Probes) and 0.1% bovine serum albumin, incubated in the dark for 30 min at room temperature, washed by centrifugation, resuspended in Ringer’s solution and analyzed by flow cytometry using FACSCallibur instrument (BD Bioscience, San Jose, CA). For evaluation of store-operated Ca2+ influx, cells were treated, as mentioned previously, except that they were finally suspended in calcium-free Ringer’s solution and fluorescent changes were monitored as a function of time. Intracellular Ca2+ stores were depleted by addition of thapsigargin to a final concentration of 100 nM. At the end of thapsigargin-induced calcium peak, store-operated Ca2+ channels were stimulated by addition of CaCl2 to a final concentration of 2 mM. Data were analyzed by FlowJo software and calcium concentrations were calculated using the equation [Ca2+i] = Fluo4 kd (Ca2+) x (F – Fmin)/(Fmax – F) (35), where Fluo4 kd (Ca2+) is 345 nM, F is instantaneous fluorescence intensity, Fmax is maximal fluorescence intensity determined after addition of CaCl2 and ionomycin to final concentrations of 2 mM and of 3 µg/ml, respectively, whereas Fmin is a minimal fluorescence intensity measured after addition of ethylene glycole tetraacetate to a final concentration of 4 mM.

Evaluation of cell viability (XTT assay, measurements of cellular DNA content and inner mitochondrial membrane potential)
For 2,3-bis(2-methoxy-4-nitro-5-sulfoneyl)-2H-tetrasolium-5-carboxanilide (XTT) assay, cells were plated onto 96-well tissue culture plate (2.5–5 x 103 cells per well), subjected to various treatments and viability was determined using a commercial kit (Biological Industries) according to the protocol supplied by the manufacturer. To measure cellular DNA content, cells were fixed in 70% ethanol, washed by PBS, resuspended in PBS containing 0.1% Triton X-100 and 30 µg/ml of DNAse-free RNAse A (Sigma) and incubated for 12 h at 4°C. Propidium iodide (Sigma) was added to a final concentration of 10 µg/ml and cells analyzed by flow cytometry. To evaluate inner mitochondrial membrane potential ({Delta}{psi}m), 5 x 105 cells were incubated with 500 nM of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzmidazolylcarbocyanine iodide (JC-1) (Molecular Probes) in serum-free RPMI medium for 10 min at 37°C in the dark and immediately analyzed by flow cytometry. Data were processed with FlowJo software.

Statistical analysis
Statistical significance was examined by Student’s t-test and SPSS software.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Elimination of lipid rafts abolished deregulated PKB activity in melanoma cells
To assess the consequence of raft disintegration on PKB activity and expression of its specific isoforms in melanoma, cells of independently isolated murine (B16BL6-8 and JB/RH1) and human (GA) melanoma lines were exposed to increasing doses of MβCD for different time intervals and cellular levels of active PKB (p-Akt), Akt1, Akt2 and Akt3 PKB isoforms were examined by immunoblotting. All of the cell lines displayed high basal levels of p-Akt, whereas the addition of MβCD to the culture medium at concentrations of 7 and 14 mmol for 1 and 2 h significantly reduced p-Akt in a dose- and time-dependent manner (Figure 1A). Lower concentration of MβCD and shorter incubation times did not markedly alter p-Akt levels, whereas no further inhibition was achieved by higher doses or longer exposure times (data not shown). Of three PKB isoforms detected in B16BL6-8, JB/RH1 and GA cells, protein levels of Akt1 were highest and predominated over Akt2 and Akt3. MβCD did not significantly change the expression of Akt1 and Akt3, whereas Akt2 levels in B16BL6-8 and GA cells were decreased in the course of treatment (Figure 1A). To evaluate phosphorylation status of each particular isoform, we precipitated Akt1, Akt2 and Akt3 by the appropriate antibodies and probed the resulting immune complexes with anti-p-Akt antibodies. High levels of constitutively phosphorylated Akt1 and Akt3 were detected, whereas Akt2-containing immune complexes did not display anti-p-Akt immune reactivity. Phosphorylation of both Akt1 and Akt3 was inhibited by MβCD (Figure 1B). To confirm that the inhibitory effect of MβCD on PKB was specifically mediated by ablation of cholesterol-enriched rafts, we assessed p-Akt in MβCD-treated melanoma cells after replenishment of membrane cholesterol achieved by incubating the cells in culture medium supplemented with water-soluble cholesterol. The replenishment procedure restored PKB phosphorylation to levels detected in control samples, while no significant change in expression of major Akt1 isoform was detected (Figure 1C). The analysis of raft integrity in this experiment was performed using cholera toxin B subunit, which specifically binds to the raft-residing ganglioside GM1. In MβCD-treated cells, GM1 was redistributed from the low-density cellular fractions obtained by ultracentrifugation in a gradient of sucrose (Figure 1D, fractions 1–3) to more dense fractions (fractions 3–6) and underexpressed at the plasma membrane (Figure 1E), thereby confirming elimination of rafts by MβCD. Cholesterol replenishment reconstituted raft structures manifested by reappearance of GM1 in the buoyant fractions (Figure 1D) and its restored membrane expression (Figure 1E). Taken together, data indicate that the constitutive activity of PKB in melanoma cells depends on the integrity of cholesterol-enriched lipid rafts and can be efficiently abolished by altering raft microdomains with a cholesterol-chelating agent such as MβCD.


Figure 1
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  		Fig. 1. MβCD-mediated ablation of rafts reduces levels of constitutively active PKB in melanoma cells. (A) Left panel: cells were treated by indicated doses of MβCD in serum-free RPMI for depicted time intervals. Aliquots of protein lysates (50 µg per lane) were subjected to immunoblotting with anti-p-Akt, -Akt1, -Akt2, -Akt3 and -actin antibodies. Right panel: intensities of p-Akt bands determined by densitometry were normalized to those of actin (mean ± SD of three independent experiments). (B) Protein extracts prepared from MβCD-treated (14 mM, 2 h) B16BL6-8 cells were subjected to immunoprecipitation (IP) with anti-Akt1, -Akt2 and -Akt3 antibodies; immune complexes were analyzed by immunoblotting (IB) with anti-p-Akt, -Akt1, -Akt2 and -Akt3 antibodies. Extracts incubated with protein A-sepharose beads without antibodies were used as a negative control for IP. (C) Left panel: MβCD-treated B16BL6-8 cells (14 mM, 2 h) were maintained for additional 1 h in serum-free medium containing indicated concentrations of water-soluble cholesterol (Ch). p-Akt, Akt1 and actin expressed were tested by immunoblotting. Right panel: intensities of p-Akt bands normalized to those of actin (mean ± SD of three independent experiments). (D) MβCD-treated B16BL6-8 cells (14 mM, 2 h) were replenished with Ch (50 µg/ml, 1 h) and cell lysates fractionated using centrifugation in a sucrose gradient. GM1 expression in these fractions was tested using horseradish peroxidase-conjugated cholera toxin B subunit and dot blot. (E) B16BL6-8 cells were treated, as described in (D) and membrane expression of GM1 was analyzed using Alexa 647-conjugated cholera toxin B subunit and confocal microscopy. Scale bar corresponds to 20 µm. (*P < 0.05, **P < 0.01, Student's t-test).

 
Lipid rafts contribute to PKB deregulation by maintaining Ca2+homeostasis
Ca2+ is an essential second messenger modulating PKB activity in many types of cells (12–16). Since the integrity of lipid rafts has been implicated previously in the regulation of Ca2+ homeostasis (36–39), we hypothesized that ablation of microdomains may alter availability of intracellular Ca2+ required to maintain PKB in an active state. Membrane-permeable chelator of intracellular Ca2+ BAPTA-AM significantly reduced p-Akt levels in a dose- and time-dependent manner without markedly altering expression of the major PKB isoform Akt1 (Figure 2A), indicating the contribution of Ca2+ signaling to PKB deregulation. Similarly, the exposure of cells to the potent antagonist of Ca2+/calmodulin W7 also led to PKB hypophosphorylation (Figure 2B). To further address the role of Ca2+, we compared steady state [Ca2+]i and extent of SOC in malignant and apoptosis-resistant B16BL6-8 cells to those detected in a non-malignant apoptosis-sensitive clone of B16BL6 melanoma (Kb30) expressing low basal p-Akt (Figure 2C, insert). Since Kb30 cells are loosely adherent, we utilized flow cytometry and fluorescent indicator Fluo4-AM for Ca2+ measurements (8,32). Both [Ca2+]i (Figure 2C) and SOC function expressed, as a slope of the curve fragment corresponding to the peak detected after addition of Ca2+, were significantly higher in B16BL6-8 cells (Figure 2D), implying that the augmented Ca2+ influx is associated with the increased malignant potential of melanoma cells. Treating B16BL6-8 cells with MβCD produced >10-fold inhibition of SOC (Figure 2E) and decreased [Ca2+]i to levels comparable with those in Kb30 cells and BAPTA-AM-treated B16BL6-8 cells (Figure 2C), whereas the cholesterol replenishment restored both [Ca2+]i (Figure 2C) and SOC function (Figure 2E). Given the critical role of store-operated pathway in determining [Ca2+]i, the results of these experiments suggest that aberrant PKB activity is supported by SOC-mediated Ca2+ permeation in a pathway that involves Ca2+/calmodulin, whereas inhibition of SOC specifically triggered by disruption of cholesterol-enriched rafts abolished this deregulation by decreasing [Ca2+]i.


Figure 2
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  		Fig. 2. Raft microdomains support Ca2±-dependent deregulation of PKB. (A and B) Left panels: melanoma cells were treated by indicated concentrations of BAPTA-AM (A) and W7 (B) for depicted time intervals. The expression of p-Akt, Akt1 and actin was examined by immunoblotting. Right panels: intensities of p-Akt bands normalized to those of actin (mean ± SD of three independent experiments). (C) Steady state [Ca2+]i was determined in control, MβCD-treated (14 mM, 2 h), cholesterol-replenished (Ch, 50 µg/ml, 1 h) and BAPTA-AM-treated (50 µM, 3 h) B16BL6-8 cells and Kb30 cells using Fluo4-AM and flow cytometry. Mean ± SD of three independent experiments. Insert: levels of p-Akt and Akt1 in control and insulin-treated (10–6 M, 15 min) B16BL6-8 and Kb30 cells. (D) SOC function in B16BL6-8 and Kb30 cells. Left panel: each curve shows changes of Fluo4 mean fluorescence intensity (mean ± SD of triplicates in one representative experiment of three performed) as a function of time (left panel). Right panel: the rates of SOC expressed as a slope of the curve fragment corresponding to the peak detected after addition of Ca2+ (mean ± SD of three independent experiments). (E) SOC function B16BL6-8 cells subjected to treatments described in (C). (*P < 0.05, **P < 0.01, Student's t-test).

 
Altering raft integrity renders melanoma cells susceptible to apoptosis
We have reported previously that deregulated PKB activity promotes serum-independent growth of highly malignant melanoma cells and protects them from apoptosis induced by starvation for serum-derived growth factors (40). To examine whether the deactivation of PKB achieved by raft ablation impairs the resistance of cells to apoptosis, cultures of B16BL6-8, JB/RH1 and GA cells pretreated with 7 and 14 mM MβCD for different time intervals were deprived of serum for 24, 48 or 72 h and subjected to XTT assay. Control samples displayed a steady increase of XTT values corresponding to numbers of metabolically active cells even in the absence of serum, whereas MβCD pretreatment markedly decreased XTT values in a dose- and time-dependent manner compared with the controls (Figure 3). The reduced XTT values were accompanied by increased percentages of cells with sub-G1 DNA content (apoptotic cells) detected in MβCD-pretreated and serum-starved cultures of B16BL6-8, JB/RH1 and GA cells, as was determined by propidium iodide and flow cytometry. In contrast, no increased cell death was observed in serum-starved control samples (Figure 4A–C). The extent of apoptosis was also evaluated by assessing inner mitochondrial membrane potential ({Delta}{psi}m) using the fluorescent indicator JC-1. As depicted in Figure 4D and E, percentages of cells with collapsed {Delta}{psi}m (apoptotic cells) in MβCD-pretreated and serum-starved cultures were similar to percentages of cells with sub-G1 DNA content detected in these cultures. In the course of this experiment, we also monitored raft integrity, p-Akt levels and SOC function. In MβCD-treated cells, GM1 reappeared in buoyant fractions only after 48 h of serum starvation (Figure 5A) coinciding with p-Akt levels (Figure 5B) and SOC activity (Figure 5C), which were partially recovered at this time point. In contrast, serum-starved controls did not display any change of GM1 distribution in the sucrose gradient (Figure 5A). However, the activities of p-Akt (Figure 5B) and SOC (Figure 5C) gradually declined in control cells, presumably, due to the lack of growth factors, though both parameters did not reach values detected in cells after 2 h of MβCD treatment. These data indicate that raft microdomains are not restored immediately after removal of cholesterol-depleting agent and PKB remains inhibited for prolonged time interval sensitizing cells to apoptosis. However, if MβCD-pretreated cells were cultured in serum-supplemented medium, no increase in apoptosis relative to control cultures was observed (data not shown) implying that raft ablation by itself is not cytotoxic but, rather, impairs melanoma cell survival.


Figure 3
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  		Fig. 3. Progressive decrease of XTT values corresponding to amounts of metabolically active cells in raft-ablated melanoma cultures deprived from serum. Cells were treated by indicated concentrations of MβCD for depicted time intervals and then cultivated in MβCD-free medium deprived of serum for 24, 48 and 72 h. XTT assay was performed as described in Materials and Methods. Mean ± SD of octaplicates obtained in one representative experiment of three performed are shown. (*P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test).

 


Figure 4
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  		Fig. 4. Altering raft integrity renders melanoma cells susceptible to apoptosis. MβCD-treated (14 mM, 2 h) B16BL6-8 (A and D), JB/RH1 (B and E) and GA (C and F) cells were maintained in serum-free medium for time intervals. Proportions of cells with sub-G1 DNA content (A–C) and collapsed {Delta}{psi}m (D–F) were determined using propidium iodide and JC-1, respectively, with the analysis by flow cytometry. Upper panels: typical histograms (A–C) and dot plots (D–E). In each histogram, x-axis shows fluorescence intensity corresponding to cellular DNA content and y-axis shows cell numbers. In each dot plot, X and Y axes show intensities of green and red JC-1-emmitted signals, respectively; gates were created to discriminate viable cells emitting higher red and lower green fluorescence from dead cells with collapsed {Delta}{psi}m emitting higher green and lower red fluorescence. Lower panels: bars represent mean ± SD of triplicate obtained in one experiment of three performed are shown. (*P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test).

 


Figure 5
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  		Fig. 5. Lipid rafts, SOC and p-Akt are not recovered immediately after removal of cholesterol-depleting agent. (A) MβCD-treated B16BL6-8 cells (14 mM, 2 h) were maintained in serum-free conditions for depicted time intervals and cell lysates fractionated by centrifugation in a sucrose gradient. The expression of GM1 in cell fractions was tested using horseradish peroxidase-conjugated cholera toxin B subunit and dot blot. (B) Left panel: B16BL6-8 cells treated as described in (A) were examined for p-Akt, Akt1 and actin expression by immunoblotting (upper panel). Right panel: intensities of p-Akt bands normalized to those of actin (mean ± SD of two independent experiments). (C) SOC function assessed in B16BL6-8 subjected to treatments described in (A). Upper panel: curves represent changes of Fluo4 mean fluorescence intensity (mean ± SD of triplicates in one representative experiment) as a function of time. Right panel: the rates of SOC expressed (mean ± SD of two independent experiments). (*P < 0.05, **P < 0.001, Student's t-test).

 
Raft-ablating compound MβCD inhibits melanoma tumor growth
Deregulated PKB activity and associated resistance of melanoma cells to apoptosis are critical for tumorigenicity (5,10,40,41). Therefore, having observed that ablation of rafts impaired melanoma cell survival, we examined whether MβCD will affect their tumorigenicity. For that purpose, B16BL6-8 cells, which rapidly generate characteristic melanin-producing tumors in syngeneic C57BL/J mice (30), were treated by either 7 or 14 mM MβCD for 2 h and then grafted into the footpad of mice. None of the depicted doses was cytotoxic and the cells were viable before their inoculation into the animals, as was assessed microscopically or by performing XTT assay (data not shown). In the control group, lesions grew progressively and started to expand into the thigh by day 28, when the experiment was terminated due to the large size of tumors (Figure 6A). Tumors developed in mice inoculated with cells pretreated with 7 mM of MβCD were significantly smaller compared with the control group [0.29 ± 0.02 cm (mean ± SD) versus 0.42 ± 0.04 cm, P < 0.05, 0.34 ± 0.01 versus 0.51 ± 0.01, P < 0.05 and 0.36 ± 0.02 versus 0.65 ± 0.06, P < 0.001, for days 20, 24 and 28, respectively]. Pretreatment of cells with 14 mM of MβCD completely arrested their in vivo growth (0.27 ± 0.03 versus 0.42 ± 0.04 cm, P < 0.05, 0.29 ± 0.02 versus 0.51 ± 0.01, P < 0.01 and 0.28 ± 0.01 versus. 0.65 ± 0.06, P < 0. 01, for days 20, 24 and 28, respectively) (Figure 6A), indicating that intact rafts are crucial for the in vivo growth of melanoma cells. To evaluate the possible therapeutic potential of raft-ablating cyclodextrin derivative in melanoma treatment, mice were inoculated with B16BL6-8 cells and then treated by weekly intraperitoneal injection of MβCD administered at doses of 300 and 800 mg/kg. The duration of this experiment was extended to 36 days in order to clearly distinguish the tumor-inhibitory effect of MβCD. As shown in Figure 6B, large tumors expanding into the thigh were detected in control animals by day 36. In contrast, treatment with 300 mg/kg of MβCD abolished invasiveness of lesions into the thigh and tumors assessed on days 28, 32 and 36 were significantly smaller than in the control group (0.43 ± 0.3 versus 0.62 ± 0.05 cm, P < 0.05, 0.62 ± 0.06 versus 1.1 ± 0.15, P < 0.05, 0.6 ± 0.07 versus 1.35 ± 0.19, P < 0.001, for days 28, 32 and 36, respectively). Treatment with 800 mg/kg of MβCD further inhibited tumor growth (0.3 ± 0.013 versus 0.42 ± 0.07, P < 0.05, 0.32 ± 0.03 versus 0.62 ± 0.05 cm, P < 0.001, 0.36 ± 0.03 versus 1.1 ± 0.15, P < 0.001, 0.37 ± .02 versus 1.35 ± 0.19, P < 0.001, for days 24, 28, 32 and 36). A significant difference in tumor size was also observed between mice treated with 800 and 300 mg/kg of MβCD (P < 0.001, P < 0.01 and P < 0.01 for days 28, 32 and 36, respectively) (Figure 6B). It must be noted, that although the higher dose of MβCD (800 mg/kg) was somewhat more efficient in restraining tumor growth, it exerted a marked toxicity in the animals (60% of mice died in the course of the experiment). The lower dose of MβCD (300 mg/kg) was well tolerated with no mortality and weight loss observed. However, in these animals there was a moderate elevation of ALT and AST sera activities relative to controls (102.33 ± 22.3 versus 47.66 + 17.01 IU/l, P < 0.05 and 223.33 ± 58.07 versus 94 ± 21.65 IU/l, P < 0.05, respectively), though isolated livers were of normal appearance with no histological changes observed (data not shown), implying a mild hepatotoxicity. No nephrotoxicity was recorded, since serum creatinine and urea levels in treated mice were comparable with those in control animals (56.66 ± 17.95 versus 53 ± 11.19 mg/dl and 0.14 ± 0.06 versus 0.19 ± 0.03 mg/dl, respectively). To assess the effect of tolerated dose of MβCD on PKB status in tumors, we excised tumors from representative treated and control animals on day 28 of the experiment and assessed p-Akt levels by immunoblotting. As shown in Figure 6B (insert), p-Akt levels in tumors obtained from MβCD-treated mice were significantly lower relative to the control group. Thus, the inhibitory effect mediated by the tolerated dose of MβCD supports our hypothesis that altering the integrity of membrane rafts in melanoma can be an efficient strategy to inhibit melanoma growth.


Figure 6
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  		Fig. 6. MβCD markedly diminishes tumorigenicity of highly malignant melanoma cells. (A) B16BL6-8 cells treated by indicated doses of MβCD (2 h) or untreated controls were grafted to C57BL6/J mice (n = 5, each group). Foot diameter was measured and values of mean ± SD deviation of three independent experiments are presented (# significant difference, refer to the text). (B) Indicated doses of MβCD were administered to C57BL/J mice engrafted with B16BL6-8 cells by weekly intraperitoneal injection. First dose was applied 8 days after inoculation of tumor cells. Foot diameter was measured and values of mean ± SD deviation of three independent experiments are presented. (# significant difference, refer to the text). Insert: levels of p-Akt, Akt1 and actin in tumors excised at day 28 from three representative mice of the control and MβCD-treated group (300 mg/kg/weekly) were assessed by immunoblotting. Intensities of p-Akt bands normalized to actin (mean ± SD of triplicate, *P < 0.05, Student's t-test).

 

   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
There is compelling evidence that aberrantly activated PKB is responsible for the extreme resistance of melanoma to apoptosis-inducing stimuli and augments its aggressive growth. We have previously raised the possibility to inactivate PKB in malignant clones of B16BL6 melanoma using cholesterol chelator MβCD (42). However, the effects of this agent on integrity of lipid rafts, the reproducibility of MβCD-mediated PKB inactivation in melanoma lines unrelated to B16BL6 and, most important, the consequences of raft disruption on melanoma cell survival and tumorigenicity have not been considered. These unresolved issues are addressed in our present study demonstrating that altering rafts is an effective approach to abolish PKB deregulation in melanoma cells and to attenuate their malignant properties. Indeed, elimination of rafts by MβCD significantly reduced levels of constitutively active PKB, whereas reconstitution of microdomains by replenishing membrane cholesterol restored PKB activity to its basal levels (Figure 1). The inhibitory effect of raft ablation on PKB activity was mediated by abnormalities in cellular Ca2+ homeostasis. Using membrane-permeable Ca2+ chelator BAPTA-AM, we demonstrated that the basal PKB activation depends on [Ca2+]i (Figure 2). MβCD significantly decreased [Ca2+]i and this decrease was associated with the attenuation of SOC (Figure 2), which is a major source of extracellular Ca2+ in non-excitable cells (11). Both SOC activity and [Ca2+]i were restored by cholesterol replenishment (Figure 2), indicating that disintegration of lipid rafts specifically inhibits SOC function and inactivates PKB by decreasing [Ca2+]i. The importance of Ca2+ signaling for maintaining PKB deregulation were further supported by the detection of less intense SOC activity and lower [Ca2+]i in non-malignant and apoptosis-sensitive Kb30 clone of B16BL6 melanoma. These cells exhibited barely detectable basal PKB activity, compared with the malignant and cell death-resistant B16BL6-8 clone, which displayed augmented SOC function, higher [Ca2+]i and constitutively active PKB (Figure 2). Although the particular signaling network linking [Ca2+]i to PKB in melanoma cells was not fully elucidated, our results imply the involvement of Ca2+/calmodulin in this process, since both BAPTA-AM and the antagonist of calmodulin W7 abrogated PKB activity (Figure 2). The proposed mechanism is consistent with previous studies demonstrating that Ca2+/calmodulin stimulates PKB due to its ability to associate with this enzyme (12) and its upstream regulatory molecules (13,16,43). Previous studies implicated lipid rafts in the regulation of SOC activity in endothelial cells (36), leukocytes (15,37) and platelets (38). Cells derived from certain solid tumors tend to accumulate cholesterol and, therefore, express higher amounts of rafts compared with their respective non-neoplastic counterparts (17). However, the dependence of SOC on raft integrity in cancer cells is still poorly understood, though the relevance of store-operated pathway for tumor cell growth and survival has been reported in glioblastoma (44), hepatoma (45), leukemia (46) and colon carcinoma (47). Our data, therefore, provide for the first time evidence that intact rafts are required for SOC function in melanoma and demonstrate that SOC supports deregulated PKB activity by maintaining [Ca2+]i. At present, we do not know the exact mode in which lipid rafts regulate SOC activity in highly malignant melanoma cells. Rafts may provide a ‘platform’ for proper assembly of functional channels composed of TRPC1 and other proteins (15,36–39,48). However, it is also possible that microdomains act upstream of SOC by regulating activities of raft-associated tyrosine kinase- and G-coupled membrane receptors (11,21–23). Notably, the aggressive growth of melanoma is contributed by raft-residing Hedgehog receptor (49,50) and Wnt5a receptor (51,52) that elicit their signals by increasing [Ca2+]i and activating PKC. It would be, therefore, of the particular interest to determine if Ca2+ influx triggered by these receptors is mediated by the raft-dependent SOC activity demonstrated in our study. Recent reports have shown that [Ca2+]i rise stimulates the motility of melanoma cells in a PKC-dependent manner (52) and protects them from apoptosis (53). Therefore, the disruption of rafts attenuating Ca2+ flux and abolishing deregulated PKB activity appears to be an attractive strategy that may impair apoptotic resistance of melanoma and to restrain its aggressive growth.

In our study, feasibility of this strategy was examined by experiments addressing in vitro and in vivo effects of MβCD on melanoma cell survival and growth. Disintegration of rafts, which by itself did not exert cytotoxicity, rendered cells susceptible to apoptosis (Figures 3 and 4), while PKB activity remained compromised for prolonged time interval after MβCD treatment coinciding with slow recovery of rafts and SOC function (Figure 5). Our previous study demonstrated that the tumorigenicity of melanoma cells is closely associated with the constitutive overactivation of PKB and the ability of cells to resist apoptosis (40). Therefore, having observed that inactivation of PKB by raft disruption impaired cell survival, we tested whether this procedure would also affect tumorigenicity. Highly malignant B16BL6 cells were unable to generate tumors when pretreated by MβCD before their inoculation into the mice (Figure 6A), implying that lipid rafts are crucial for both melanoma cell survival and in vivo growth. The major role of raft microdomains in augmenting malignant potential of malignant cells has been addressed previously using a prostate cancer model (18). In this study, elevating levels of plasma cholesterol in mice engrafted with prostate carcinoma cells increased the amounts of rafts in cancer cells, augmented PKB activity, reduced apoptosis and promoted xenograft growth. In our present work employing yet another model, i.e. malignant melanoma, we further demonstrate that tumor development is significantly attenuated by administration of raft-ablating agent to mice. The delayed growth of melanoma lesions, their attenuated spread to the thigh and the diminished levels of activated PKB in the tumor mass detected in mice treated by repeated i.p injections of MβCD (Figure 6B) clearly indicate the efficacy of raft-ablating approach against melanoma. It would be, therefore, interesting to evaluate if targeting rafts will also attenuate the development of prostate cancer and other types of malignancies displaying PKB abnormalities. A major concern connected to the potential clinical application of raft-ablating chemicals is that these agents may also non-selectively alter microdomains and interfere with function in cells of vital organs like heart, liver, kidney, pancreas, etc. Completely opposite to this notion, certain β-cyclodextrin derivatives are widely utilized as carriers for water-insoluble drugs for parenteral use (29), implying that low doses of these compounds do not ultimately exert marked systemic toxicity. However, such doses may still suppress tumor growth, given the critical role of lipid rafts in supporting deregulated PKB activity (17,18). To the best of our knowledge, only one report has previously addressed the potential application of β-cyclodextrins as anticancer agents (53). In this study, weekly injections of 300 and 800 mg/kg MβCD to nude mice inhibited growth of human breast and ovarian carcinoma xenografts without producing marked systemic toxicity (54). In our hands, however, only 300 mg/kg was well tolerated by the mice though this dose produced a mild hepatotoxicity. The reason for this discrepancy is not clear and, probably, it may be related to distinct sensitivities of different animal strains to MβCD or to lot-to-lot variations of this agent. Nevertheless, the efficacy of the tolerated dose in inhibiting in vivo growth and invasion of melanoma (Figure 6B) provides a strong support for our hypothesis that altering lipid rafts represents a potentially safe and effective strategy to treat this malignant disease. A broad list of available β-cyclodextrin derivatives can now be screened for compounds less toxic than MβCD and their efficacy against melanoma cells when applied alone or in combination with chemo- and radiotherapeutic modalities evaluated. It must be noted, as well, that distinct types of rafts have been identified that differ in their biochemical composition, compartmentalization and function (19,20). It is, therefore, possible that only a specific subset of microdomains supports SOC and PKB deregulation. Identification and characterization of these microdomains and development of drugs that selectively target raft components specifically associated with SOC and PKB deregulation appear critical at this stage, and increasingly, an achievable aim in melanoma research.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Israel Science Foundation (425/03); Israeli Ministry of Science and Technology/Deutsches krebsforschungszentrum (Ca-112) to D.F. and S.S.


   Acknowledgments
 
We thank Prof. E.Gorelik (Department of Pathology, University of Pittsburgh, Pittsburgh, PA) and Prof. J.Gopas (Department of Microbiology and Immunology, Ben-Gurion University of the Negev, Beer-Sheva, Israel) for providing us with melanoma lines and Prof. W.F.Silverman (Department of Morphology, Ben-Gurion University of the Negev, Beer-Sheva, Israel) for critical reading of this manuscript. We mourn the loss of our esteemed colleague and mentor Prof. Shraga Segal, who passed away during performance of this study; our manuscript is dedicated to him in loving memory.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received December 9, 2007; revised May 19, 2008; accepted June 13, 2008.


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