Synergistic effects of retinoic acid and 8-chloro-adenosine 3',5'-cyclic monophosphate on the regulation of retinoic acid receptor beta and apoptosis: involvement of mitochondria.

ABSTRACT

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 beta (RARbeta) 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 hormone-independent MDA-MB-231 cells, are sensitive to RA-induced growth inhibition and apoptosis. Introduction of the RARbeta gene into MDA-MB-231 cells resulted in a gain of RA sensitivity. 8-Cl-cAMP acted synergistically with all-trans-RA in inducing and activating RARbeta 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 RARbeta promoter resulted in loss of synergy in RARbeta transcription. In addition, inhibition of RARbeta expression by an antisense construct also blocked the antitumor effects of RA + 8-Cl-cAMP. Thus, RARbeta can mediate RA and/or cAMP action in breast cancer cells by promoting apoptosis. Therefore, loss of RARbeta 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.