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

Abstract

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-beta-cyclodextrin (MbetaCD) 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 M beta CD dramatically impaired their apoptotic resistance and capacity to generate tumors. Furthermore, weekly intraperitoneal injections of M beta CD 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.

Cholesterol-dependent induction of dendrite formation by ginsenoside Rh2 in cultured melanoma cells

“…MßCD depletes cholesterol from the cell membranes and has been used for disrupting lipid rafts in various cells (27,30), including B16 cells (37). Therefore, in order to examine whether Rh2 affects lipid rafts, we stained lipid-raft-associated molecules, such as gangliosides, caveolins, and flotillins, in cells and examined whether Rh2 showed any effect on the staining pattern of these molecules.…”

Ion Channels as Promising Therapeutic Targets for Melanoma

“…This link was supported by the correlation between of trans-mitochondrial Ca 2+ flux triggered by SOCs and SOCE itself and levels of AKT in malignant vs non-tumour melanoma cells (Feldman et al, 2010). Indeed, the high basal AKT activity in malignant melanoma cells is Ca 2+ dependant and can be reversed by decreasing cytosolic Ca 2+ concentration with Ca 2+ chelator (Fedida-Metula et al, 2008) giving a special importance of SOCE in the control of intracellular Ca 2+ concentration. Thus, down-regulation of the SOCs regulator Stim1 by shRNA leading to ~ ten-fold reduction of SOCE correlated to a significantly decreased of pAKT levels compared to control transfected cells (Feldman et al, 2010).…”

“…Elevated level of PKB activity is a common finding in lesions of advanced melanoma (Dai et al, 2005;Slipicevic et al, 2005) and inversely correlates with patient survival (Dai et al, 2005). One of the activation pathways of PKB involves the complex calcium (Ca 2+ )/calmodulin associated (Dong et al, 2007) with functional Store-Operated Channels (SOCs) (Fedida-Metula et al, 2008). In most non-excitable, cells depletion of endoplasmic reticulum (ER) stores elicits sustained Ca 2+ influx by SOCs in plasma membrane, defining the major Ca 2+ influx pathway (Putney, 1986).…”

RAGE and Its Ligands in Melanoma

“…All these proteins play important function in malignant melanoma [153][154][155] and increases in S100B levels could therefore favor increases in cell proliferation, migration and invasion through their modulation. S100B could also play a role in melanoma cell growth through the activation of the Nuclear Dbf2 related (ndr) kinase [156][157][158] and the interaction with the phosphoprotein AHNAK/desmoyokin [159][160][161].…”