Characterization of novel retinoic acid metabolism blocking agents (RAMBAs) in breast cancer model systems

Abstract

All-trans-retinoic acid (ATRA) and other retinoids, potent modulators of a variety of important cellular functions are effective in prevention and therapy of many cancers including breast cancer. Increased metabolism of ATRA by ATRA inducible cytochrome P450 enzymes present in different cells including cancer cells is one of the reasons for the emergence of ATRA resistance and hampering its clinical use. Additionally, high therapeutic doses of ATRA have adverse effects and are not recommended.Retinoic acid metabolism blocking agents (RAMBAs) prevent rapid metabolism of ATRA. Use of RAMBAs is an emerging approach to increase the endogenous levels and thus potentiate the effect of ATRA without administering high exogenous doses. The objective of this project was to investigate the in vitro and in vivo role of novel C-4 azolyl RAMBAs rationally developed in our laboratory, against breast cancer; determine how they would potentiate the effectiveness of ATRA as well as study the molecular mechanisms involved. Novel RAMBAs (VN/14-1, VN/50-1, VN/66-1 and VN/69-1), structural analogs of ATRA/13-cis-retinoic acid, inhibited ATRA metabolism in hormone dependent MCF-7 cells and T47D cells; and microsomes with IC50 values in nanomolar range as determined by HPLC analysis. RAMBAs (0.0001--10 muM) had dose dependent antiproliferative action, suggesting that they have retinoidal activity as well. VN/50-1 and VN/14-1 enhanced ATRA's antiproliferative action in MCF-7 cells. RAMBAs induced differentiation in these cell lines as determined by analysis of differentiation markers cytokeratin 8/18 and E-cadherin. Induction of apoptosis by RAMBAs was determined by TUNEL assay and Western blotting of apoptosis related proteins. Cell cycle analysis indicated that RAMBAs arrest cells in the G1 phase and reduce cells in the S phase. RAMBAs reduced the expression of Cyclin D1 in these cell lines. VN/14-1 like ATRA increased the expression of estrogen receptor-alpha in MCF-7 cells. VN/14-1 besides being a potent RAMBA, has pleiotropic role, is well tolerated and significantly suppressed MCF-7 tumor growth (∼90.35% vs. control, p < 0.0005; at 66.0 mumole/kg/day dose) in female ovariectomized nude mice. Promising RAMBAs endowed with multiple biological activities should be suitable for further clinical investigations for chemoprevention and therapy of breast cancer.