Study On Activities Of Viaminate And Its Two Metabolites M₁, M₂ In Vitro

ObjectiveTo study anti-proliferative activities of viaminate(VA) and its two metabolites M₁, M₂ by using All-trans retinoic acid(RA) as a control, to compare the transcriptional activities of these four compounds on their receptors, therefore to investigate the effect modes of the similarities and differences of these substances for exploring the underlying mechanism of viaminate and developing new drugs.MethodsHeLa cells were cultured in vitro. The anti-proliferative effects of the four compounds at various concentrations on HeLa cells were measured by XTT assays. Transcriptional activities of the four compounds on their receptors were determined in HeLa cells that were transiently transfected withβRARE-tk-CAT for RAR (α,β, orγ) and with TREpal-tk-CAT for RXRαby liposome-based method. CAT ELISA was employed to evaluate the amount of CAT protein induced in response to 24hr exposure to 1μM of the four substances.Results1. Anti-proliferative activities of the four compounds: HeLa cells were resistant to up to 50μM RA or VA, whereas the growth of cells were significantly inhibited (P＜0.01) than that in controls at 100μM treatment. At this concentration, the inhibition ratio of RA and VA was about 37%, 83%, respectively. The 500μM treatment resulted in cell death. The cells were resistant to up to 10μM M₁ or M₂, whereas the growth of cells were significantly inhibited (P＜0.05, P＜0.01 respectively) than that in controls at 50μM treatment. At this concentration, the inhibition ratio of M₁ and M₂ was about 29%, 36%, respectively. The 100μM treatment resulted in cell death.2. Transcriptional activities of the four compounds: The relative CAT amount in HeLa cells that were transfected by RARα, RARβand RARγ, plasmids followed by RA treatment was 117.7 times(P＜0.01), 53.8 times(P＜0.01) and 44.3 times(P＜0.01) greater than that of control, respectively, and that followed by VA treatment was 2.1 times(P＜0.05), 2.1 times and 1.4 times(P＜0.05) greater than that of control, respectively, while that were transfected by RXRαplasmid followed by RA or VA treatment was not greater than that of control. The relative CAT amount in transfected HeLa cells treated with M₁ or M₂ was not greater than that of control.ConclusionThe rank of anti-proliferative activities of these four compounds is as follows: M₂＞M₁＞VA＞RA. The result indicates that the anti-proliferative activity of VA is not only accomplished by the drag itself, but also accomplished by its two effective metabolites M₁ and M₂.At the level of receptor, VA has limited activation to RARs as compared with RA, while M₁ and M₂ have no obvious activation to RARs and RXRα. This may indicate that the anti-proliferative activities of M₁ and M₂ are accomplished through a RAR-independent pathway, implying VA has a multi-pathway effect m vivo. It also has guiding significance for developing new generation retinoids.