Inhibition of Balb/c 3T3 cell transformation by synthetic acyclic retinoid NIK-333; possible involvement of enhanced gap junctional intercellular communication

Background: In an attempt to develop effective and non-cytotoxic cancer chemopreventive derivatives of retinoids, a novel acyclic retinoid has previously been synthesized. This acyclic retinoid, NIK-333, had been reported to suppress recurrence of primary hepatocellular carcinomas. We explored the molecular mechanisms by which NIK-333 exerts anti-proliferative effects. Methods: We employed Balb/c 3T3 cells, since they can be used as a quantitative transformation assay and since we can study a possible involvement of gap-junctional intercellular communication (GJIC) in their growth control. We included all-trans-retinoic acid (ATRA) for comparison. Results: We first confirmed that these cells express the retinoid receptors, RARα, γ and RXRα, suggesting that they respond to NIK-333 and ATRA. When NIK-333 or ATRA was added to Balb/c 3T3 cells during the induction of cell transformation by a standard (3-methylcholanthrene (MCA) alone) or two-stage (low dose of MCA plus 12-O-tetradecanoylphorbol-13-acetate (TPA)) protocol, there was a significant decrease in the number of transformed foci. The extent of inhibition of transformation by NIK-333 was similar to that exerted by ATRA. Employing the microinjection dye-transfer assay, we found that both retinoids increase GJIC level when measured 24 h after treatment. The extent of GJIC increase by NIK-333 was similar to that of ATRA. These retinoids also increased the amount of connexin 43 (Cx43) on the plasma membrane as revealed by immunostaining. Conclusion: These data indicate that NIK-333 suppresses chemical carcinogenesis in vitro and support the hypothesis that enhancement of GJIC is involved in this process.