RESUMO
Melanoma cells are resistant to transforming growth factor-ß (TGFß)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFß. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFß-mediated growth inhibition.
Assuntos
Melanoma/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos , Flavonoides/farmacologia , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/genética , MAP Quinase Quinase 2/metabolismo , Melanoma/genética , Melanoma/patologia , Mutação , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Pirimidinas/farmacologia , Proteína Smad2/genética , Proteína Smad3/genéticaRESUMO
The goal of this study was to examine the effects of metabotropic glutamate receptor-1 (GRM1) blockade on melanoma anchorage-independent growth and invasion. We performed colony and invasion assays using GRM1-expressing melanoma lines and the GRM1-negative UACC930 line. Using the glutamate-release inhibitor Riluzole or the non-competitive GRM1 antagonist BAY 36-7620 we were able to induce considerable inhibition of colony formation and invasion in GRM1-expressing melanoma lines. Neither pharmacological agent induced significant reduction in colony formation or invasion in the GRM1-negative melanoma line, UACC930. Additionally we assessed the efficacy of these inhibitors to inhibit the growth of fresh melanoma tumor samples cultured on a 74-mum nylon mesh. Both Riluzole and BAY 36-7620 significantly inhibited tumor cell growth into the interstitial spaces of the mesh. When repeated with normal mole samples both inhibitors were much less effective in preventing the outgrowth of cells. These experiments show that a specific antagonist of GRM1 (BAY 36-7620) or an inhibitor of glutamate release (Riluzole) can significantly suppress melanoma migration, invasion and colony formation as well as inhibit the proliferation of fresh melanoma cells. These findings, added to our previous work, strengthen the case that GRM1 is a valid therapeutic target in patients with melanoma.
