bryostatin-1 and Prostatic-Neoplasms

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Bryostatins; Cell Division; Humans; Lactones; Macrolides; Male; Neoplasm Proteins; Prostatic Neoplasms; Protein Kinase C; Protein Kinase C-alpha; RNA, Messenger

Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response.

Topics: Apoptosis; Bryostatins; Cell Division; Cell Line, Tumor; Down-Regulation; Humans; Male; Phosphorylation; Prostatic Neoplasms; Protein Kinase C; Subcellular Fractions; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; U937 Cells

Phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 are both potent protein kinase C (PKC) activators. In LNCaP human prostate cancer cells, PMA induces tumor necrosis factor alpha (TNFα) secretion and inhibits proliferation; bryostatin 1 does not, and indeed blocks the response to PMA. This difference has been attributed to bryostatin 1 not localizing PKCδ to the plasma membrane. Since phorbol ester lipophilicity influences PKCδ localization, we have examined in LNCaP cells a series of phorbol esters and related derivatives spanning some eight logs in lipophilicity (logP) to see if any behave like bryostatin 1. The compounds showed marked differences in their effects on proliferation and TNFα secretion. For example, maximal responses for TNFα secretion relative to PMA ranged from 97 % for octyl-indolactam V to 24 % for phorbol 12,13-dibenzoate. Dose-response curves ranged from monophasic for indolactam V to markedly biphasic for sapintoxin D. The divergent patterns of response, however, correlated neither to lipophilicity, to plasma membrane translocation of PKCδ, nor to the ability to interact with model membranes. In U937 human leukemia cells, a second system in which PMA and bryostatin 1 have divergent effects, viz. PMA but not bryostatin 1 inhibits proliferation and induces attachment, all the compounds acted like PMA for proliferation, but several induced a reduced level or a biphasic dose-response curve for attachment. We conclude that active phorbol esters are not all equivalent. Depending on the system, some might partially resemble bryostatin 1 in their behavior; this encourages the concept that bryostatin-like behavior may be obtained from other structural templates.

Topics: Antineoplastic Agents; Bryostatins; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Humans; Inhibitory Concentration 50; Leukemia; Male; Molecular Structure; Phorbol Esters; Prostatic Neoplasms

Bryostatin 1, a macrocyclic lactone that has been widely characterized as an ultrapotent protein kinase C (PKC) activator, displays marked pharmacological differences with the typical phorbol ester tumor promoters. Bryostatin 1 impairs phorbol 12-myristate 13-acetate (PMA)-induced tumor promotion in mice and is in clinical trials as an anticancer agent for a number of hematopoietic malignancies and solid tumors. In this study, we characterized the effect of bryostatin 1 on LNCaP prostate cancer cells, a cellular model in which PKC isozymes play important roles in the control of growth and survival. Although phorbol esters promote a strong apoptotic response in LNCaP cells via PKCdelta-mediated release of TNFalpha, bryostatin 1 failed to trigger a death effect even at high concentrations, and it prevented PMA-induced apoptosis in these cells. Mechanistic analysis revealed that bryostatin 1 is unable to induce TNFalpha release, and it impairs the secretion of this cytokine from LNCaP cells in response to PMA. Unlike PMA, bryostatin 1 failed to promote the translocation of PKCdelta to the plasma membrane. Moreover, bryostatin 1 prevented PMA-induced PKCdelta peripheral translocation. Studies using a membrane-targeted PKCdelta construct revealed that the peripheral localization of the kinase is a requisite for triggering apoptosis in LNCaP cells, arguing that mislocalization of PKCdelta may explain the actions of bryostatin 1. The identification of an antiapoptotic effect of bryostatin 1 may have significant relevance in the context of its therapeutic efficacy.

Topics: Antineoplastic Agents; Apoptosis; Bryostatins; Humans; Isoenzymes; Lactones; Male; Phorbol Esters; Prostatic Neoplasms; Protein Kinase C; Protein Kinase C-delta; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

We have previously shown that protein kinase D1 (PKD1), charter member of PKD protein family, is downregulated in advanced prostate cancer (PC) and influences androgen receptor (AR) function in PC cells. Other independent studies showed that serine 82 residue in heat shock protein 27 (Hsp27) undergoes substrate phosphorylation by PKD1 and is associated with nuclear transport of AR resulting in increased AR transcriptional activity. In this study, we show that PKD1 interacts and phosphorylates Hsp27 at Ser82 in PC cells, which is mediated by p38-dependent mitogen-activated protein kinase pathway and is necessary for PKD1 repression of AR transcriptional activity and androgen-dependent proliferation of PC cells. The study provides first in vivo evidence that Hsp27 is a mediator of repression of AR function by PKD1 in PC cells, thereby linking the data in the published literature.

Topics: Bombesin; Bryostatins; Down-Regulation; Enzyme Activation; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; Male; Molecular Chaperones; Multiprotein Complexes; Neurotensin; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostatic Neoplasms; Protein Binding; Protein Kinase C; Receptors, Androgen; Signal Transduction; Transcriptional Activation; Tumor Cells, Cultured

Phorbol 12-myristate 13-acetate (PMA)-induced apoptosis of androgen sensitive LNCaP human prostate cancer cells is a well known phenomenon that involves prolonged translocation of multiple protein kinase C (PKC) isozymes to nonnuclear membranes. We have shown recently that PMA-induced death of C4-2 cells, androgen hypersensitive derivatives of LNCaP cells, requires both PKCdelta and a redundant pathway that includes PKCs alpha and epsilon. In contrast, it has been reported that overexpression of murine PKCepsilon in LNCaP cells renders those cells resistant to PMA-induced death, as well as androgen insensitive. Here we report that inducible or constitutive overexpression of human PKCepsilon does not alter the sensitivity of LNCaP cells to either PMA or androgen, nor does it alter expression of caveolin-1 or phosphorylated Rb, reported effects of overexpression of murine PKCepsilon. Moreover, overexpression of very high amounts of PKCepsilon sensitized LNCaP cells to induction of apoptosis by bryostatin 1, a non tumor-promoting activator and down-regulator of PKC isozymes that blocks PMA-induced apoptosis of parental LNCaP cells, mimicked our previous results with overexpression of PKCalpha in LNCaP cells. Given reports that overexpression of PKCepsilon is frequent in human prostate tumors, our results may have important implications for a potential prostate cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Bryostatins; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dihydrotestosterone; Dose-Response Relationship, Drug; Humans; Immunoblotting; Macrolides; Male; Phosphorylation; Prostatic Neoplasms; Protein Kinase C-epsilon; Retinoblastoma Protein; Tetradecanoylphorbol Acetate; Transfection

Previously, we reported that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced apoptosis of LNCaP human prostate cancer cells was accompanied by prolonged translocation of protein kinase C (PKC)alpha to non-nuclear membranes and that TPA-resistant LNCaP cells had down-regulated PKCalpha. Here we show that 10 nM bryostatin 1 induced transient membrane translocation and down-regulation of PKCalpha, prolonged translocation of PKCdelta and epsilon to non-nuclear membranes, and did not induce cell death but blocked TPA-induced apoptosis. To test the hypothesis that inhibition of TPA-induced apoptosis by bryostatin 1 was due to down-regulation of PKCalpha, we inducibly overexpressed PKCalpha in LNCaP cells. Overexpression of PKCalpha alone did not induce apoptosis, even in clones that contained much more membrane-bound, active PKCalpha than was observed in TPA-treated untransfected LNCaP cells. However, the addition of 10 nM bryostatin 1 to PKCalpha-overexpressing LNCaP cells did not yield down-regulation of PKCalpha and induced extensive apoptosis. Immunoblot analysis revealed that TPA induced prolonged hyperphosphorylation of Raf-1 and activation of extracellular-regulated/mitogen-activated protein kinases 1 and 2 in untransfected LNCaP cells, as did bryostatin 1 in PKCalpha-overexpressing cells. On the other hand, bryostatin 1 induced only transient hyperphosphorylation of Raf-1 and activation of extracellular-regulated/mitogen-activated protein kinases 1 and 2 in untransfected LNCaP cells. These results confirm a role of prolonged membrane-associated PKCalpha in PKC activator-mediated LNCaP apoptosis and suggest involvement of the mitogen-activated protein kinase pathway.

Topics: Antineoplastic Agents; Apoptosis; Biological Transport; Bryostatins; Carcinogens; Cell Division; Down-Regulation; Enzyme Activation; Humans; Isoenzymes; Lactones; Macrolides; Male; Mitogen-Activated Protein Kinases; Phosphorylation; Prostatic Neoplasms; Protein Kinase C; Protein Kinase C-alpha; Proto-Oncogene Proteins c-raf; Subcellular Fractions; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured