Synergistic effects of retinoic acid and 8-chloro-adenosine 3 ',5 '-cyclicmonophosphate on the regulation of retinoic acid receptor beta and apoptosis: Involvement of mitochondria

In advanced or recurrent malignant diseases, retinoic acid (RA) is not effe ctive, even at doses that are toxic to the host, In late stages of breast c ancer, patients do not respond to RA because the expression of RA receptor beta (RAR beta) is lost. In the present study, the intracellular mechanism( s) of synergistic effects of RA and a site-selective cyclic AMP (cAMP) anal ogue, 8-chloro-adenosine 3',5'-cyclic monophosphate (8-Cl-cAMP), on growth inhibition and apoptosis in breast cancer cells was examined, Our data demo nstrated that hormone-dependent MCF-7 cells, but not hormone-independent MD A-MB-231 cells, are sensitive to RA-induced growth inhibition and apoptosis . Introduction of the RAR beta gene into MDA-MB-231 cells resulted in a gai n of RA sensitivity. 8-Cl-cAMP acted synergistically with all-trans-RA in i nducing and activating RAR beta gene expression that correlates with the re duction 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, M utations in the cAMP response element-related motif within the RAR beta pro moter resulted in loss of synergy in RAR beta transcription. In addition, i nhibition of RAR beta expression by an antisense construct also blocked the antitumor effects of RA + 8-Cl-cAMP, Thus, RAR beta can mediate RA and/or cAMP action in breast cancer cells by promoting apoptosis, Therefore, loss of RAR beta expression may contribute to the tumorigenicity of human mammar y epithelial cells, These findings suggest that RA and 8-Cl-cAMP act in a s ynergistic fashion and may have potential for combination biotherapy for th e treatment of malignant diseases.