Sythesis of retinoic acid analogues & investigations into their ability to induce stem cell differentation

Abstract

Human embryogenesis and ensuing adult homeostasis are directed by the complex interplay of a wide range of exquisitely controlled signalling molecules and pathways. All-trans-retinoic acid (ATRA) is one such important hormone-like compound that regulates a wide range of processes. The endogenous effects of ATRA have the potential to be translated into treatment for numerous clinical indications; however administration at efficacious concentrations is associated with severe side effects. Consequently, a large group of synthetic molecules - known as retinoids - that are structurally and/or functionally analogous to ATRA have been prepared and tested in vitro in the search for a panacea and for use as pharmacological tools to elucidate the retinoid molecular pathway. A small library of stable synthetic retinoids was prepared and their biological activity investigated using TERA2.cl.SP12 human embryonal carcinoma (EC) stem cells. Three compounds, CEB16, CEB17 and CEB18 were found to inhibit cellular proliferation and induce neural and non-neural differentiation. These effects were thoroughly characterised and quantified by monitoring phenotypic changes and the expression of established antigenic markers. Compared to the ability of ATRA and its geometric isomers 9-cis-retinoic acid and 13-cis-retinoic acid, the order of efficacy of induction of neural differentiation was found to be: 13-cis-retinoic acid>9-cis-retinoic acid=ATRA>CEB 18>CEB 17>>CEB 16. The molecular mechanism of natural and synthetic retinoid action during TERA2.cl.SP12 differentiation was investigated by performing a detailed temporal analysis of the gene expression of eleven transcripts involved in retinoid transport, activation and metabolism. To date, limited data have been published on the effects of synthetic retinoids on the retinoid pathway during differentiation of human cells and no human studies have examined the activity of synthetic retinoids on the HOX genes. All of the retinoic acid inducible genes examined in this study were found to be modulated in TERA2.cl.SP12 cells in response to both ATRA and two isomeric synthetic retinoids, CEB16 and CEB17, albeit relatively slowly compared to other cell lines. These compounds are therefore believed to activate the same pathway as the natural metabolite in these cells and that this is responsible for at least some of the observed effects. Genes were regulated in a concentration and retinoid dependent manner and this modulation was often multi-phasic demonstrating the complex behaviour of the retinoid system. Interestingly, ATRA was not the most effective inductive agent in all gene analyses. For example 10 μM CEB17 induced up-regulation of RAR-ß more strongly than 10 μm ATRA and 10 μM CEB16 induced the greatest increase in RAR-y transcripts. Further experimentation is required to confirm an apparent relationship between the timing of addition of inductive agent and the expression pattern of several genes. This behaviour highlights the importance of retinoid degradation in culture, which is often overlooked. The three synthetic compounds described extensively herein should be more stable, and thus may be suitable and convenient alternatives for molecular biologists to use in place of ATRA.