h-89 and Prostatic-Neoplasms

Reference treatment of advanced prostate cancer (PCa) relies on pharmacological or surgical androgen deprivation therapy. However, it is only temporarily efficient as tumor cells inevitably adapt to the low testosterone environment and become hormone-refractory (HRPCa). We observed that androgen removal in HRPCa-derived LNCaP cells causes different alterations in their Ca(2+) homeostasis among which a reduction of ER Ca(2+) content. We show that the decrease in [Ca(2+)]ER is due to a modest overexpression of type 1 IP3R and a threefold increased phosphorylation of IP3R1 on Ser-1716, a protein kinase A (PKA) consensus site, both implicated in ER Ca(2+) leak. Accordingly, ER Ca(2+) content was restored by siRNA-mediated down-regulation of IP3R1 or by inhibition of its phosphorylation by competition with a permeant TAT-peptide containing the Ser-1716 consensus phosphorylation sequence or by treatment with the PKA inhibitor H89. Moreover, inhibition of the IP3R1 phosphorylation by both methods sensitized the LNCaP cells to androgen deprivation-induced apoptosis. In addition, SERCA2b overexpression precluded the effect of androgen deprivation on ER Ca(2+) store content and reduced resistance to androgen deprivation. Taken together, these results indicate that lowering the ER Ca(2+)-store content by increasing IP3R1 levels and IP3R1 phosphorylation by PKA is a protective mechanism by which HRPCa-derived cells escape cell death in the absence of androgenic stimulation.

Topics: Androgens; Apoptosis; Calcium; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Endoplasmic Reticulum; Gene Expression Regulation, Neoplastic; Humans; Inositol 1,4,5-Trisphosphate Receptors; Isoquinolines; Male; Peptide Fragments; Phosphorylation; Prostatic Neoplasms; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Testosterone; Transgenes; Tumor Escape

Prostate cancer is the most commonly diagnosed non-cutaneous cancer in American men. Unfortunately, few successful therapies for castration-resistant prostate cancer (CRPC) exist. The protein kinase A (PKA) pathway is a critical mediator of cellular proliferation and differentiation in various normal and cancerous cells. However, the PKA activity and the mechanism of regulation in CRPC remain unclear. Then, in this study, we intended to reveal the PKA activity and the mechanism of regulation in CRPC.. Western blotting, quantitative real-time polymerase chain reaction, cytotoxicity analysis, and cell proliferation assay were used to resolve the regulatory role of PKA in prostate cancer cell line, LNCaP and their derivatives.. cAMP-specific phosphodiesterase 4B (PDE4B) was downregulated and the PKA pathway was activated in castration-resistant LNCaP derivatives (CxR cells). Rolipram activated the PKA pathway via inhibition of PDE4B, resulting in AR transactivation while the PKA inhibitor, H89 reduced AR transactivation. In response to hydrogen peroxide and in hydrogen peroxide-resistant LNCaP derivatives (HPR50 cells) PDE4B was decreased and as a result PKA activity was increased. Moreover, PDE4B expression was reduced in advanced prostate cancer and PDE4B knockdown promoted castration-resistant growth of LNCaP cells.. Oxidative stress may suppress PDE4B expression and activate the PKA pathway. The PDE4B/PKA pathway contributed to progression of androgen-dependent prostate cancer to CRPC. This pathway may represent an attractive therapeutic molecular target.

Topics: Cell Line, Tumor; Cell Proliferation; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 4; Disease Progression; Down-Regulation; Humans; Hydrogen Peroxide; Isoquinolines; Male; Phosphodiesterase 4 Inhibitors; Prostatic Neoplasms; Protein Kinase Inhibitors; Rolipram; Sulfonamides

• To test whether β1-adrenoceptor activation leads to phosphorylation of the β2-adrenoceptor in human prostate tissue.. • Prostate tissue from patients undergoing radical prostatectomy was stimulated in vitro with the α1-adrenergic agonist phenylephrine (10 µM). • α2-adrenoceptor phosphorylation at serines 345/346 was studied using Western blot analysis with a phospho-specific antibody. • The role of second messenger kinases was assessed by studying the effects of the protein kinase C (PKC) inhibitor Ro 31-8425 and the protein kinase A (PKA) inhibitor H89 on phenylephrine-induced phosphorylation. • The expression of G protein-coupled receptor kinases (GRKs) 2/3 was analysed using quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR), Western blot analysis and immunohistochemistry.. • Stimulation of prostate tissue with phenylephrine resulted in phosphorylation of the β2-adrenoceptor (5, 10 and 20 min after stimulation). • This α1-adrenoceptor-induced phosphorylation of β2-adrenoceptors was resistant to inhibition of PKC and PKA. • Changes in phosphorylation levels were not attributable to changes in receptor levels, as these remained constant during stimulation. • RT-PCR and Western blot analysis showed expression of GRK2/3 in human prostate tissues. • Immunohistochemical staining showed that GRK2/3 expression in human prostate tissue is located to stromal and smooth muscle cells.. • Activation of α1-adrenoceptors causes phosphorylation of β2-adrenoceptors in the human prostate. This may enhance α1-adrenergic contraction and is possibly mediated by GRK2, which is expressed in prostate smooth muscle. • Mutual regulation between different adrenergic receptors might be involved in the therapeutic effects of α1-blockers in patients with benign prostate hyperplasia.

Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic beta-Agonists; G-Protein-Coupled Receptor Kinase 1; G-Protein-Coupled Receptor Kinase 2; GTP-Binding Proteins; Humans; Immunohistochemistry; Indoles; Isoquinolines; Male; Maleimides; Phenylephrine; Phosphorylation; Prostate; Prostatic Neoplasms; Protein Kinase Inhibitors; Receptors, Adrenergic, beta-1; Receptors, Adrenergic, beta-2; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides

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

The effect of vasoactive intestinal peptide (VIP) on intracellular Ca(2+) levels and its relationship with the expression of c-fos and vascular endothelial growth factor (VEGF) as well as with neuroendocrine (NE) differentiation were investigated in human prostate LNCaP cells. VIP induced the expression of c-fos mRNA as studied by reverse transcription polymerase chain reaction (RT-PCR). It was accompanied by VIP stimulation of c-fos protein synthesis, as measured by Western blot analysis. VIP enhanced intracellular Ca(2+) levels as evaluated using the calcium probe fura-2. VIP regulation of c-fos expression depended on [Ca(2+)](i) concentration since the intracellular calcium chelator BAPTA/AM decreased c-fos expression (both mRNA and protein) to basal levels. As shown by means of real-time RT-PCR, VIP stimulated VEGF mRNA expression: the effect was inhibited by 40% in the presence of curcumin (an inhibitor of AP-1 binding), and it was dependent on Ca(2+) since BAPTA/AM inhibited this VIP action by 43%. Similar observations were made on the effects of BAPTA/AM and curcumin on VIP stimulation of VEGF protein expression. Simultaneous treatment of cells with the protein kinase A inhibitor H89 and BAPTA/AM completely blocked this VIP effect, whereas each agent alone led only to a partial inhibition. In addition, the calcium chelator blocked by 37% the ability of VIP to induce NE cell differentiation as estimated by the observation of neurite development. These features support a VIP signalling pathway that could be mediated through both cAMP and [Ca(2+)](i) increase in prostate LNCaP cancer cells. Moreover, our data suggest the implication of c-Fos on the induction of the main angiogenic factor VEGF since the promoter region of the VEGF gene possesses AP-1 (i.e., c-Fos/c-Jun heterodimer) response elements. This feature represents a link between the nuclear oncogene c-fos, angiogenesis and NE differentiation by means of an initiating signal upon VIP receptors.

Topics: Calcium; Calcium Signaling; Cell Differentiation; Cell Line, Tumor; Chelating Agents; Cyclic AMP; Drug Interactions; Egtazic Acid; Gene Expression Regulation; Humans; Isoquinolines; Male; Neuropeptides; Prostatic Neoplasms; Proto-Oncogene Proteins c-fos; RNA, Messenger; Sulfonamides; Vascular Endothelial Growth Factor A; Vasoactive Intestinal Peptide

The nicotinamide adenine dinucleotide-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of 15 (S)-hydroxyl group of prostaglandins and lipoxins and participates along with cyclooxygenases and lipoxygenases in controlling the cellular levels of prostaglandins and lipoxins. 15-PGDH could be induced by IL-6 and forskolin in addition to androgens in a time- and dose-dependent manner but not by other cytokines and growth factors in LNCaP cells. Concurrent addition of IL-6 and forskolin showed additive effect in the induction of 15-PGDH activity. However, combined addition of dihydrotestosterone (DHT) and IL-6 or DHT plus forskolin exhibited synergistic induction of 15-PGDH activity. The increase in enzyme activity was correlated with the expression of the enzyme protein as shown by Western blot analysis. The induction by DHT or IL-6 or forskolin or their combinations was inhibited by antiandrogen, casodex, in a dose-dependent manner, indicating that a functional androgen receptor was required for the action of any of these three agents. The induction by forskolin plus DHT or by either agent or by IL-6 alone was greatly inhibited by H-89, indicating the involvement of protein kinase A in the actions of forskolin, DHT, and IL-6. The induction of 15-PGDH by IL-6 was also blocked by some other protein kinase inhibitors, indicating the participation of MAPK, MAPK/ERK kinase, and STAT3 in the signaling pathway of IL-6. These results indicate that the induction of 15-PGDH by DHT, IL-6, and forskolin in LNCaP cells may involve a functional androgen receptor and phosphorylation-dependent multiple signaling pathways.

Topics: Androgen Antagonists; Anilides; Blotting, Western; Colforsin; Cyclic AMP-Dependent Protein Kinases; Dihydrotestosterone; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Induction; Enzyme Inhibitors; Humans; Hydroxyprostaglandin Dehydrogenases; Interleukin-6; Isoquinolines; Kinetics; Male; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; STAT3 Transcription Factor; Sulfonamides; Tosyl Compounds; Trans-Activators; Tumor Cells, Cultured

One of the causes of insensitivity to androgen ablation therapy in prostate cancer is thought to be attributable to elevated neuropeptides secreted by neuroendocrine cells in the tumor mass. Calcitonin (CT), one of these neuropeptides, is reported to be associated with the growth of prostate cancer. There is an increase in mitogen-activated protein (MAP) kinase activation as prostate cancer progresses to a more advanced and androgen-independent disease. We examined the effect of CT on signal transduction and the relation between CT and early-response genes in the human androgen-insensitive prostate cancer cell line, DU145. The basal phosphorylation level of extracellular signal-regulated kinase 1/2, which is a key kinase in the mediation of growth factor-induced mitogenesis in prostate cancer cells, was constitutively up-regulated. N-[2-(4-bromocinnamyl) aminoethyl]-5-isoquinoline-sulfonamide (H89), a specific inhibitor of protein kinase A, potentiated the effects of more increased phosphorylation of extracellular signal-regulated kinase 1/2. CT induced the inhibition of this MAP kinase phosphorylation, and this effect was completely abolished by pretreatment with H89. Our findings demonstrate that CT caused the inhibition of constitutive MAP kinase phosphorylation in a protein kinase A-dependent manner in DU145. The transient increase of c-fos expression was detected after CT treatment, whereas expression of c-jun RNA was down-regulated after CT treatment. These results suggest that CT may regulate early-response genes, c-fos and c-jun, via a MAP kinase cascade. In conclusion, these findings suggest that DU145 might be a useful model as a therapeutic approach of neuropeptides in androgen-independent prostatic carcinoma.

Topics: Calcitonin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Immediate-Early; Humans; Isoquinolines; Male; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptors, Calcitonin; RNA, Messenger; Sulfonamides; Tumor Cells, Cultured

Arachidonic acid (AA) is the precursor for prostaglandin E2 (PGE2) synthesis and increases growth of prostate cancer cells. To further elucidate the mechanisms involved in AA-induced prostate cell growth, induction of c-fos expression by AA was investigated in a human prostate cancer cell line, PC-3. c-fos mRNA was induced shortly after addition of AA, along with a remarkable increase in PGE2 production. c-fos expression and PGE2 production induced by AA was blocked by a cyclo-oxygenase inhibitor, flurbiprofen, suggesting that PGE2 mediated c-fos induction. Protein kinase A (PKA) inhibitor H-89 abolished induction of c-fos expression by AA, and partially inhibited PGE2 production. Protein kinase C (PKC) inhibitor GF109203X had no significant effect on c-fos expression or PGE2 production. Expression of prostaglandin (EP) receptors, which mediate signal transduction from PGE2 to the cells, was examined by reverse transcription polymerase chain reaction in several human prostate cell lines. EP4 and EP2, which are coupled to the PKA signalling pathway, were expressed in all cells tested. Expression of EP1, which activates the PKC pathway, was not detected. The current study showed that induction of the immediate early gene c-fos by AA is mediated by PGE2, which activates the PKA pathway via the EP2/4 receptor in the PC-3 cells.

Topics: Arachidonic Acid; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; Flurbiprofen; Gene Expression; Genes, fos; Humans; Isoquinolines; Male; Prostatic Neoplasms; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP4 Subtype; RNA, Messenger; Signal Transduction; Sulfonamides; Tumor Cells, Cultured

Melatonin, secreted nocturnally by the pineal gland, can bind to human benign prostate epithelial cells and attenuate their growth and viability. In the present study, melatonin binding and responses were explored in the human steroid-independent PC3 prostatic tumor cells. PC3 cells bound 125I-melatonin with low affinity (Kd ca. 0.9 nM) at high as well as low cell density. Melatonin enhanced cGMP and 3H-thymidine incorporation at low, but attenuated them at high cell density. In addition, melatonin inhibited cAMP at low, but augmented it at high cell density. These effects were associated with an increase in cell count at low- but not high-density cultures. Pertussis toxin treatment suppressed 125I-melatonin binding and ablated all the effects of melatonin on 3H-thymidine incorporation, cAMP, and cGMP at both cell densities. Cholera toxin treatment failed to block the effects of melatonin on 3H-thymidine incorporation, but prevented the modulation by melatonin of cAMP at low and cGMP at high cell density. The cGMP analog 8-Br-cGMP, inhibited melatonin's effects on 3H-thymidine incorporation at both cell densities. H89, a protein kinase A inhibitor, prevented melatonin's effects on 3H-thymidine incorporation at low but not high cell density. These results provide the first demonstration of direct interaction of melatonin with hormone-insensitive prostate tumor cells. The melatonin receptors in the PC3 cells are coupled to pertussis toxin-sensitive G proteins to induce cell density-dependent changes in cGMP, cAMP, and cell growth.

Topics: Binding Sites; Cell Count; Cell Survival; Cholera Toxin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; DNA; Humans; Isoquinolines; Male; Melatonin; Pertussis Toxin; Prostatic Neoplasms; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Melatonin; Serotonin Antagonists; Signal Transduction; Sulfonamides; Thymidine; Tumor Cells, Cultured; Virulence Factors, Bordetella