Synergistic Effects of Retinoic Acid and 8-Chloro-Adenosine 3*,5*- Cyclic Monophosphate on the Regulation of Retinoic Acid Receptor b and Apoptosis: Involvement of Mitochondria

In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective, even at doses that are toxic to the host. In late stages of breast cancer, patients do not respond to RA because the expression of RA receptor b (RARb) is lost. In the present study, the intracellular mechanism(s) of synergistic effects of RA and a site-selective cyclic AMP (cAMP) analogue, 8-chloro-adenosine 3*,5*-cyclic monophosphate (8-Cl-cAMP), on growth inhibition and apoptosis in breast cancer cells was examined. Our data demonstrated that hormone-dependent MCF-7 cells, but not hormoneindependent MDA-MB-231 cells, are sensitive to RA-induced growth inhibition and apoptosis. Introduction of the RARb gene into MDA-MB-231 cells resulted in a gain of RA sensitivity. 8-Cl-cAMP acted synergistically with alltrans-RA in inducing and activating RARb gene expression that correlates with the reduction in mitochondrial membrane potential, redistribution of cytochrome c, activation of caspases, cleavage of poly(ADP-ribose) polymerase and DNA-dependent protein kinase (catalytic subunit), and induction of apoptosis. Mutations in the cAMP response element-related motif within the RARb promoter resulted in loss of synergy in RARb transcription. In addition, inhibition of RARb expression by an antisense construct also blocked the antitumor effects of RA 1 8-Cl-cAMP. Thus, RARb can mediate RA and/or cAMP action in breast cancer cells by promoting apoptosis. Therefore, loss of RARb expression may contribute to the tumorigenicity of human mammary epithelial cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion and may have potential for combination biotherapy for the treatment of malignant diseases.