All-trans-retinoic acid (ATRA), a potent differentiating agent and critical for development, is effective in cancer chemoprevention and treatment. In addition, ATRA and its analogues have been investigated in several systems as potential agents of chemoprevention and treatment of number of cancers including breast cancer. As a differentiating agent, ATRA redirects cells towards their normal phenotype ameliorating tumor proliferation and invasion. ATRA also exerts its effect through the induction of apoptosis. In spite of these benefits, its clinical use has been hindered, due to the emergence of resistance and side effects. ATRA is responsible for the self-induction of cytochrome P450 dependent enzymes (ATRA 4-hydroxylase) present in different tissues as well as in cancer cells, which rapidly metabolize ATRA resulting in resistance. High therapeutic doses of ATRA result in side effects and are not recommended. We have designed and synthesized retinoic acid metabolism blocking agents (RAMBAs) (VN/14-1, VN/50-1, VN/66-1 and VN/69-1) with the hypothesis that RAMBAs act by increasing the level of endogenous ATRA, thereby avoiding the toxicities of exogenously administered high dose of ATRA. We have shown previously that these RAMBAs are exceptionally potent inhibitors of ATRA metabolism in vitro in estrogen receptor positive (ER+) breast cancer cells and they (VN/14-1 and VN/50-1) also enhance the anti-proliferative action of ATRA in MCF-7 and T47D cells and in addition possess multiple biological activities. Here, we show that VN/14-1 (5 and 10 μM, six-day treatment) is able to increase the expression of cytokeratin 18 - a well known differentiation marker, in MCF-7 and T47D cells as determined by Western immunoblotting and this effect was comparable to ATRA. In T47D cells, VN/14-1 (1, 5 and 10 μM, six-day treatment) induces the active form of caspase-9, which suggests that apoptosis in T47D cells implicates the caspase-9 pathway. ATRA mediates its action through the retinoic acid receptors, which can heterodimerize with other steroid receptors such as the estrogen receptor. VN/14-1, which also has retinoidal activity binds to retinoic acid receptor (RAR α) and was shown to increase the expression of estrogen receptor protein when MCF-7 cells were treated with 1, 5 and 10 μM of VN/14-1 for 6 days. When female ovariectomized nude mice bearing MCF-7 tumor xenografts (n=5) were treated once daily (s.c. inj.), with 0.033 μmol/kg (equivalent to 12.2 mg/kg) VN/14-1 for six weeks, VN/14-1 caused a significant reduction (85.2%) in the mean final tumor volume compared with the vehicle-treated control animals (P = 0.022). There was no apparent toxicity at this dose. VN/14-1 has promising in vitro and in vivo effects against hormone dependent (ER+) breast cancer models and is a strong candidate for the development of therapeutics for breast cancer.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]