Abstract
Background: We previously reported that Epac, a guanine nucleotide exchange factor that is directly activated by cAMP, increases melanoma cell migration. Although intracellular Ca2+ elevation is known to enhance cancer cell migration, the role of Epac in Ca2+ regulation in melanoma cells remains unknown. We thus examined whether Epac increases intracellular Ca2+ to enhance melanoma cell migration. Methods: SK-Mel-2, a human melanoma cell line, was used in this study. To examine the effect of Epac on Ca2+ regulation, 8-pMeOPT-2'-O-Me-cAMP (8-pMeOPT), an Epac specific cAMP agonist, was used. Intracellular Ca2+ measurements were performed by using Fura-2AM fluorescent dye and the monochromator-equipped imaging system. Migration assays were performed by using the 24-well Boyden chambers. Results: We first examined Ca2+ involvement in Epac-induced migration, and found that Ca2+ depletion, by EGTA to chelate extracellular and BAPTA-AM to chelate intracellular Ca2+, significantly decreased Epac-induced migration (adenoviral Epac1 overexpression (OE) 211±30 % of control; Epac1 OE+EGTA+BAPTA-AM 126±16 % of control, p<0.01 vs Epac1 OE, n=4), implying the role of Ca2+ in Epac-induced migration. In support of this result, we found that 8-pMeOPT elevated cytosolic Ca2+ levels in melanoma cells. We next explored the mechanism by which Epac elevates cytosolic Ca2+ level. When inositol triphosphate (IP3) receptors, which release Ca2+ from the endoplasmic reticulum, were inhibited by xestopongin C or 2-Aminoethoxydiphenyl borate (2-APB), 8-pMeOPT failed to induce cytosolic Ca2+ elevation. In contrast, L-type Ca2+ channel blockers, such as verapamil or nifedipine, did not inhibit Epac-mediated Ca2+ elevation. These data suggested that Epac-mediated cytosolic Ca2+ elevation is IP3 receptor-dependant. In addition, when melanoma cells were incubated with xestopongin C, Epac-induced migration was inhibited (xestopongin C 155±11 % of control, p<0.01 vs Epac1 OE; 2-APB 161±16% of control, p<0.01 vs Epac1 OE, n=4), suggesting that Epac increases melanoma cell migration via the IP3 receptor-Ca2+ signal. We next examined whether phospholipase C (PLC), which is activated by Epac to generate IP3, is involved in Epac-mediated cytosolic Ca2+ elevation. U73122, a PLC inhibitor, negated Epac-induced Ca2+ elevation, and migration (Epac1 OE+U73122 (10\#956;M) 161±7 % of control, p<0.01, n=4), indicating that Epac increases cytosolic Ca2+ via the PLC-IP3-IP3 receptor pathway to enhace migration. Conclusion: Epac increases melanoma cell migration via Ca2+ release from the endoplasmic reticulum. Since the role of the intracellular Ca2+ signaling in melanoma metastasis has not been well examined, the present study may provide significant insights to understand the molecular mechanism of melanoma migration and thus metastasis, and help to find a new therapeutic strategy for treating human melanoma metastasis.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4950.
Footnotes
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO
- American Association for Cancer Research