All-trans retinoic acid-and N-(4-hydroxyphenil)-retinamide-induced growth arrest and apoptosis in orbital fibroblasts in Graves' disease

In this study, we evaluated by reverse transcription-polymerase chain reaction (RT-PCR) the expression pattern of retinoic acid receptors (RAR) alpha, beta, and gamma and cellular retinoic acid binding protein-I (CRBP-I) genes in 12 primary cultures of fibroblasts (F) from orbital tissue of Graves' ophthalmopathy (GO) patients. We also studied the in vitro effects of all-trans retinoic acid (RA) and N-(4-hydroxyphenil)-retinamide (4HPR), a less toxic and better tolerated synthetic derivative of RA, on cell morphology, growth, apoptosis, and cyclic adenosine monophosphate (cAMP) accumulation. All primary cultures expressed RAR alpha, beta, gamma, and CRBP-I. FGO treated with RA and 4HPR (10(-7) mol/L) presented morphologic changes and significantly inhibited cell growth after 72 hours. At 96 hours of drug exposure, apoptosis was detected in 15% and 50% of RA- and 4HPR (10(-7) mol/L)-treated cells, and p53 protein increased in cell lysates. 4HPR induced a 70% decrease of Bcl-2 protein. After 30 minutes of RA and 4HPR (10(-7) mol/L) exposure, a 20% decrease of basal cAMP accumulation was seen, and forskolin cAMP-induced increase was abolished. The expression of RAR alpha, beta, gamma, and CRBP-I in primary cultures of FGO indicates that they are targets for retinoids. Moreover, we show that RA and 4HPR are able to induce morphologic changes, inhibition of cell growth, and apoptosis in FGO exerting their effects through RAR-modulated pathways. The rapid inhibition of cAMP accumulation indicates that a novel nonclassic retinoid pathway may also be involved. Finally, the potent in vitro effects of 4HPR, a retinoid derivative with fewer adverse reactions in vivo, could justify further investigations on a clinical application of retinoids in GO.