tretinoin and 8-chloro-cyclic-adenosine-monophosphate

Both cAMP and retinoids play a role in cell differentiation and the control of cell growth. A site-selective cAMP analog, 8-Cl-cAMP and retinoic acid synergistically inhibit growth and induce apoptosis in certain cancer cells. In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective even at doses that are toxic to the host. The objective of our present study was to examine the mechanism(s) of synergistic effects of retinoic acid (9-cis, 13-cis or all-trans RA) and 8-Cl-cAMP on apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. RA induced growth inhibition and apoptosis in OVCAR-3 and OVCAR-8 cells. 8-Cl-cAMP acted synergistically with RA in inducing and activating retinoic acid receptor beta (RARbeta) which correlates with growth inhibition and apoptosis in both cell types. In addition, induction of apoptosis by RA plus 8-Cl-cAMP requires caspase-3 activation followed by cleavage of anti-poly(ADP-ribose) polymerase. Furthermore, mutations in CRE-related motif within the RARbeta promoter resulted in loss of both transcriptional activation of RARbeta and synergy between RA and 8-Cl-cAMP. RARbeta expression appears to be associated with induction of apoptosis. Introduction of the RARbeta gene into OVCAR-3 cells resulted in gain of RA sensitivity. Loss of RARbeta expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. Thus, combined treatment with RA and 8-Cl-cAMP may provide an effective means for inducing RARbeta expression leading to apoptosis in ovarian cancer cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Division; Drug Synergism; Female; Humans; Ovarian Neoplasms; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

We investigated the intracellular mechanisms of retinoic acid (9-cis-RA, 13-cis-RA or all-trans-RA) and a cyclic AMP analog 8-Cl-cAMP on growth-inhibition and apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. The cyclic AMP analog, 8-Cl-cAMP, acted synergistically with RA in inducing and activating retinoic acid receptor beta (RARbeta) which correlated with the growth inhibition, cell cycle arrest, and apoptosis in both cell types. In addition, combined treatment of cells with RA plus 8-Cl-cAMP resulted in the release of cytochrome c, loss in mitochondrial membrane potential and activation of caspase-3 followed by cleavage of anti-poly(ADP-ribose)polymerase and DNA-dependent protein kinase (catalytic subunit). Interestingly, inhibition of caspase-3 activation blocked RA plus 8-Cl-cAMP induced apoptosis. Furthermore, mutations in a CRE-related motif within the RARbeta promoter resulted in loss of both transcriptional activation of RARbeta and synergy between RA and 8-Cl-cAMP. Thus, RARbeta can mediate RA and/or cyclic AMP action in ovarian cancer cells by promoting apoptosis. Loss of RARbeta expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion in inducing apoptosis via caspase-3 activation, and may have potential for combination biotherapy for the treatment of malignant disease such as ovarian cancer.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amino Acid Sequence; Apoptosis; Caspase 3; Caspases; Cell Cycle; Cell Survival; Cytochrome c Group; DNA-Activated Protein Kinase; DNA-Binding Proteins; Drug Synergism; Enzyme Activation; Female; Humans; Molecular Sequence Data; Nuclear Proteins; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Protein Serine-Threonine Kinases; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

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.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Survival; Cytochrome c Group; DNA-Activated Protein Kinase; DNA-Binding Proteins; Drug Synergism; Gene Expression Regulation, Neoplastic; Hormones; Humans; Mitochondria; Nuclear Proteins; Poly Adenosine Diphosphate Ribose; Protein Serine-Threonine Kinases; Receptors, Retinoic Acid; Tretinoin; Tumor Cells, Cultured

Vesicles, shed in the extracellular medium by several kinds of normal and tumoral cells, are known to play important roles in cell-cell and cell-matrix interactions and to participate in mechanisms by which tumoral cells acquire metastatic capability and evade immune surveillance. Regulation of the shedding phenomenon and molecular mechanisms involved in extracellular vesicle production are not known and are the subject of this investigation. Fetal calf serum stimulated shedding short after its addition and its stimulatory effect was dose dependent. This effect was reduced after gelatin-Sepharose adsorption indicating a possible involvement of gelatinases on its stimulatory effect. This conclusion was confirmed by the inhibitory effect of bathophenanthroline. Shedding of membrane vesicles decreased after treatment with all trans retinoic acid, a molecule known for its capability to induce cell differentiation. Brefeldin A, an inhibitor of intracellular vesicle movements, and methylamine, an inhibitor of exocytosis, did not abolish shedding. Quercetin, an inhibitor of phosphatidyl inositol 4 kinase and 1,4 phosphatidyl inositol 5 kinase, and 8-Cl-cAMP, a site selective cAMP analogous which induces growth inhibition and differentiation, significantly decreased the amount of shed vesicles.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Membrane; Extracellular Matrix; Female; Humans; Microscopy, Electron; Neoplasm Invasiveness; Phospholipases A; Quercetin; Second Messenger Systems; Signal Transduction; Tretinoin; Tumor Cells, Cultured

The enhanced expression of the regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase type I, RIalpha, has been correlated with cancer cell growth. Retinoic acid (RA) has been shown to play an important role in the regulation of proliferation and differentiation in neoplastic cells. In the present study, the effects of cAMP analogue 8-chlorocyclic-AMP (8-Cl-cAMP) and RA (both singly and combined) on growth inhibition and apoptosis in Ewing's sarcoma CHP-100 cells were evaluated. The inhibitory effects of 8-Cl-cAMP and RA (9-cis-RA, 13-cis-RA, and all-trans-RA) on cell viability were time and dose related. The degree of growth inhibition induced by 9-cis-RA was the greatest among all of the RA analogues (13-cis-RA and all-trans-RA) examined. The combined effects of 8-Cl-cAMP and RA on the induction of growth arrest at the G0-G1 stage of the cell cycle, apoptosis, down-regulation of RIalpha, and cleavage of poly(ADP-ribose) polymerase were synergistic. In conclusion, it is clear that RA and 8-Cl-cAMP act in a synergistic fashion and have potential for combination chemotherapy for the treatment of malignant disease.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Cycle; Cell Division; Cell Survival; Drug Synergism; Enzyme-Linked Immunosorbent Assay; Humans; Nucleosomes; Sarcoma, Ewing; Tretinoin; Tumor Cells, Cultured