h-89 and Adenocarcinoma

The androgen-signaling pathway with the androgen receptor (AR) as its key molecule is widely understood to influence prostate tumor growth significantly even after androgen ablation. Under androgen-deprived conditions, the AR may be activated inappropriately through interaction with other molecules, including cyclic AMP-dependent protein kinase A (PKA). In a previous study, we have shown that knocking down the AR significantly inhibits prostate tumor growth. In this study, we show that combined inhibition of the AR and the regulatory subunit I alpha of PKA (RIalpha) with small interference RNAs significantly increased the growth-inhibitory and proapoptotic effects of AR knockdown. This treatment strategy was effective in androgen-sensitive and in androgen ablation-resistant prostate cancer cells. In addition, we report that downregulating PKA RIalpha was sufficient to inhibit PKA signaling and interestingly also impaired AR expression and activation. Vice versa, AR knockdown induced a decline in PKA RIalpha, associated with reduced PKA activity. This mutual influence on expression level was specific, because siRNAs against the AR did not affect expression of PKA RIalpha in AR negative DU-145 cells and a siRNA control did not affect protein expression. Another important finding of our study was that depletion of PKA RIalpha also potentiated the antiproliferative effect of the antiandrogen bicalutamide in androgen-sensitive LNCaP. We therefore concluded that combined inhibition of PKA RIalpha and AR may be a promising new therapeutic option for prostate cancer patients and might be superior to solely preventing AR expression.

Topics: Adenocarcinoma; Androgen Antagonists; Androgen Receptor Antagonists; Androgens; Anilides; Animals; Apoptosis; Bucladesine; Cell Division; Colforsin; Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; Enzyme Induction; Gene Knockdown Techniques; Humans; Isoquinolines; Male; Metribolone; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Nitriles; Prostatic Neoplasms; Receptors, Androgen; RNA, Small Interfering; Signal Transduction; Sulfonamides; Tosyl Compounds

We investigated the mechanism of ligand-independent activation of the estrogen receptor (ER) by 3,3'-diindolylmethane (DIM), a promising anticancer agent derived from vegetables of the Brassica genus, in Ishikawa and HEC-1B human endometrial cancer cells. DIM stimulated the activity of an ER-responsive reporter by over 40-fold, equivalent to the maximum induction produced by estradiol (E2), whereas cotreatment of cells with the ER antagonist, ICI-182,780 (ICI), abolished the stimulatory effect of DIM. DIM also induced the expressions of the endogenous genes, TGF-alpha, alkaline phosphatase, and progesterone receptor similar to levels induced by E2. Induction of gene expression by DIM was inhibited by the protein synthesis inhibitor, cycloheximide. In addition, cotreatment of cells with the protein kinase A (PKA) inhibitor, H89, or the MAPK inhibitor, PD98059, reduced DIM activation of the ER by 75% and 50%, respectively. Simultaneous treatment of cells with both inhibitors completely abolished the effect of DIM. DIM stimulated MAPK activity and induced phosphorylation of the endogenous PKA target, cAMP response element binding protein (CREB), in a PKA-dependent manner. Expression of MCREB, a nonphosphorylatable CREB mutant, partially abolished activation of the ER by DIM. These results demonstrate that DIM is a mechanistically novel activator of the ER that requires PKA-dependent phosphorylation of CREB.

Topics: Adenocarcinoma; Anticarcinogenic Agents; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Endometrial Neoplasms; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Female; Flavonoids; Fulvestrant; Humans; Indoles; Isoquinolines; Ligands; MAP Kinase Signaling System; Phosphoprotein Phosphatases; Phosphorylation; Point Mutation; Sulfonamides; Transcription Factors; Transcriptional Activation; Tumor Cells, Cultured

Estrogen acts to promote DNA synthesis in the MCF-7 human breast cancer cell line via its interaction with high levels of estrogen receptor. The primary mode of estrogen action has been considered to be through transcriptional activation of genes containing estrogen response elements, including the immediate early genes c-myc and fos. Recent reports have indicated that estrogen, acting through the estrogen receptor, is capable of inducing the mitogen-activated protein kinase (MAPK) cytoplasmic signaling cascade. In this study, specific small molecule inhibitors of MAPK and phosphatidylinositol 3-kinase activity were used to determine the influence of these cascades on estrogen-mediated mitogenesis. Phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, as well as inhibitors of MAPK kinase-1, PD098059 and U0126, decreased the fraction of cells entering DNA synthesis after treatment with 17beta-estradiol. These compounds did not inhibit expression of myc or fos. However, the drugs did prevent the accumulation of cyclin D1 and hyperphosphorylated retinoblastoma protein, indicating that the block occurred at, or prior to, this point in the cell cycle. Although these compounds were effective in preventing estrogen-mediated mitogenesis, the downstream kinases extracellular signal-regulated kinase 1, extracellular signal-regulated kinase 2, and protein kinase B were not activated over basal levels by estrogen treatment. These studies suggest that estrogen initiates mitogenesis by inducing the transcription of immediate early genes, but cytoplasmic signaling pathways play an important role in the control of subsequent events in the cell cycle.

Topics: Adenocarcinoma; Androstadienes; Breast Neoplasms; Butadienes; Chromones; Culture Media, Serum-Free; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Depression, Chemical; DNA Replication; Enzyme Activation; Enzyme Inhibitors; Estradiol; Estrogen Receptor Modulators; Estrogens; Female; Flavonoids; Fulvestrant; Humans; Isoquinolines; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Mitosis; Morpholines; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Retinoblastoma Protein; Sulfonamides; Wortmannin

The regulation of expression of cyclooxygenase 2 (COX-2) was investigated by treatment with PGE(2) in human endometrial adenocarcinoma cell line HEC-1B. One microM PGE(2) could stimulate the expression of COX-2 approximately twofold in this cell line. The same concentration of PGE(2) also stimulated activation of mitogen-activated protein kinase (MAP kinase) and protein kinase B (PKB). PGE(2)-induced MAP kinase activation was sensitive to a MAP kinase kinase (MEK) inhibitor, PD098059, and a protein kinase A inhibitor, H-89. PD098059 and H-89 also partially inhibited the expression of COX-2 stimulated by PGE(2). PGE(2) could stimulate the activation of PKB, which was sensitive to phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. Whereas wortmannin alone partially inhibited the expression of COX-2, a combination of wortmannin and PD098059 totally inhibited PGE(2)-mediated COX-2 expression. These results suggest that MAP kinase and PI3K pathways are stimulated with PGE(2), and that both of these pathways are involved in the expression of COX-2. In addition, they also suggest that protein kinase A remains upstream of PGE(2)-induced activation of MAP kinase in HEC-1B cells.

Topics: Adenocarcinoma; Cyclooxygenase 2; Dinoprostone; Endometrial Neoplasms; Enzyme Activation; Enzyme Inhibitors; Female; Flavonoids; Gene Expression; Humans; Immunohistochemistry; Isoenzymes; Isoquinolines; Kinetics; Membrane Proteins; Mitogen-Activated Protein Kinases; Prostaglandin-Endoperoxide Synthases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sulfonamides; Tumor Cells, Cultured

The activation of mitogen-activated protein kinase (MAP kinase) and the regulation of cyclooxygenase 2 (COX-2) were investigated in the human endometrial adenocarcinoma cell line HEC-1B by treatment with platelet-activating factor (PAF) and hCG. Pre-treatment of the cells with both oestradiol and medroxyprogesterone acetate was required for MAP kinase activation and COX-2 expression to respond to PAF and hCG. PAF-induced MAP kinase activation was sensitive to MAP kinase kinase (MEK) inhibitor, PD098059, and phosphatidylinositol-3-OH kinase (PI3K) inhibitor, wortmannin. In contrast, hCG-induced MAP kinase activation was sensitive to PD098059 and protein kinase A inhibitor, H-89, but not to wortmannin. PAF-induced COX-2 expression was insensitive to PD098059 but sensitive to wortmannin, whereas hCG-induced COX-2 expression was sensitive to PD098059 and H-89 but insensitive to wortmannin. 8-(4-chlorophenylthio)-cAMP, a potent cAMP analogue, induced activation of MAP kinase and expression of COX-2. These results indicate that MAP kinase is activated with PAF and hCG in HEC-1B cells. In addition, COX-2 expression is stimulated through the MAP kinase activation pathway with hCG and the wortmannin sensitive pathway with PAF in HEC-1B cells. These results also imply that protein kinase A remains upstream of hCG-induced activation of MAP kinase in HEC-1B cells.

Topics: Adenocarcinoma; Androstadienes; Antineoplastic Agents, Hormonal; Chorionic Gonadotropin; Cyclic AMP; Cyclooxygenase 2; Endometrial Neoplasms; Enzyme Activation; Enzyme Inhibitors; Estradiol; Female; Flavonoids; Humans; Isoenzymes; Isoquinolines; Medroxyprogesterone Acetate; Membrane Proteins; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Mitogens; Platelet Activating Factor; Prostaglandin-Endoperoxide Synthases; Protein Kinase Inhibitors; Signal Transduction; Sulfonamides; Thionucleotides; Tumor Cells, Cultured; Wortmannin