RESUMO
Retinoic acid (RA) induces differentiation of S91 melanoma cells through activation of RA receptor (RAR)gamma without affecting cell viability. The novel RARgamma-agonist CD437 (AHPN), however, also induces concomitant apoptosis through an unknown mechanism which was investigated here. By utilizing DNA microarray analysis, five apoptosis-associated, CD437-induced transcripts (CITs) were identified. Interestingly, all CITs are also regulated by p53 in a DNA damage response, and consistent with this interpretation, CD437 was found to cause DNA adduct-formation. However, p53 is not required for CD437-dependent regulation of CITs. Among this set of genes, induction of p21(WAF1/CIP1) is likely to be responsible for early S-phase growth-arrest of CD437-treated cells, whereas ei24 is a critical mediator of CD437-induced apoptosis in S91 cells. These data suggest an RAR-independent mechanism in which CD437 causes DNA adduct-formation, resulting in induction of a p53-independent DNA damage response, and subsequent growth-arrest and apoptosis. CD437-mediated DNA adduct-formation may also explain its apoptotic effects in other cell types.
Assuntos
Apoptose/efeitos dos fármacos , Melanoma Experimental/patologia , Receptores do Ácido Retinoico/metabolismo , Retinoides/farmacologia , Células 3T3 , Animais , Proteínas Reguladoras de Apoptose , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/metabolismo , Adutos de DNA/metabolismo , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma Experimental/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , Especificidade de Órgãos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/genética , Fase S/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Retinoic acid (RA) slows growth and induces differentiation of tumor cells through activation of RA receptors (RARs). However, melanoma cell lines display highly variable responsiveness to RA, which is a poorly understood phenomenon. By using Northern and Western blot analyses, we show that RA-resistant A375 and RA-responsive S91 melanoma cells express comparable levels of major components of RAR-signaling pathways. However, A375 cells have substantially higher intracellular reactive oxygen species (ROS) levels than S91 cells. Lowering ROS levels in A375 cells through hypoxic culture conditions restores RAR-dependent trans-activity, which could be further enhanced by addition of the antioxidant N-acetyl-cysteine. Hypoxia also enhances RAR activity in the moderately RA-responsive C32 cells, which have intermediate ROS levels. Conversely, increasing oxidative stress in highly RA-responsive S91 and B16 cells, which have low ROS levels, by treatment with H(2)O(2) impairs RAR activity. Consistent with these observations, RA more potently inhibited the proliferation of hypoxic A375 cells than that of normoxic cells. Oxidative states diminish, whereas reducing conditions enhance, DNA binding of retinoid X receptor/RAR heterodimers in vitro, providing a molecular basis for the observed inverse correlation between RAR activity and ROS levels. The redox state of melanoma cells provides a novel, epigenetic control mechanism of RAR activity and RA resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Melanoma/patologia , Receptores do Ácido Retinoico/fisiologia , Tretinoína/farmacologia , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Northern Blotting , Western Blotting , Hipóxia Celular , DNA/metabolismo , Dimerização , Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Luciferases/genética , Melanoma/tratamento farmacológico , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Proteínas Recombinantes de Fusão , Receptores X de Retinoides , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
Retinoic acid (RA) induces growth-arrest of many tumor cell lines but it is an ineffective therapeutic against melanoma. We investigated whether the histone deacetylase (HDAC)-inhibitor sodium butyrate (BUT) can restore or potentiate the RA-response of RA-resistant human A375, and RA-responsive S91 murine melanoma cells. BUT induced expression of RARbeta and p21(waf1/cip1) mRNA in A375 cells but in S91 cells only p21(waf1/cip1) was induced. RA and BUT synergistically activated transcription of an RA-dependent reporter gene in S91, but not A375 cells. BUT increased histone H4-acetylation in both cell types. RA potentiated BUT-mediated inhibition of S91 cell proliferation, whereas A375 cells remained largely resistant to both compounds. HDAC-inhibitors may enhance the activity of RA on RA-responsive melanoma cells.
