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PubMed1996

The bryostatins inhibit growth of B16/F10 melanoma cells in vitro through a protein kinase C-independent mechanism: dissociation of activities using 26-epi-bryostatin 1.

Bryostatin 1 is a potential cancer chemotherapeutic agent in Phase II clinical trials, with positive responses observed for malignant melanoma, among other tumors. The bryostatins are known to be potent ligands for protein kinase C (PKC), functioning as partial antagonists. In the present study, we explore the mechanism by which the bryostatins inhibit growth to B16/F10 mouse melanoma cells in vitro. Three experimental approaches suggest that the growth inhibition is independent of PKC. First, we characterized in detail the translocation and down-regulation of the PKC isozymes alpha, delta, and epsilon in response to phorbol ester and bryostatin 1 in these cells. Although the dose-response curves obtained for the translocation-activation of PKC isozymes showed good correlation with the growth-enhancing activity of phorbol 12-myristate 13-acetate, for no PKC isozyme was there a good correlation with the growth-inhibitory activity of bryostatin 1. Second, inhibition PKC, inhibited the growth of the B16/F10 melanoma cell lines with potency similar to that of bryostatin 1. We confirmed here that 26-epi-bryostatin 1 showed 60-fold reduced affinity for PKC and 30-60-fold reduced potency to translocate and downregulate PKC isozymes compared with bryostatin 1. We presumed that the principal toxicity of bryostatin 1 reflects its interaction with PKC, and we would thus predict that epi-bryostatin 1 would be less toxic. Indeed, we found at least 10-fold reduced toxicity of 26-epi-bryostatin 1 in C57BL/6 mice compared with bryostatin 1. We conclude that the growth inhibition of the bryostatins, at least in this system, does not result from interaction with PKC. As exemplified by 26-epi-bryostatin 1, this insight permits the design of analogues with comparable growth inhibition to bryostatin 1 but with reduced toxicity.

Animals↗
PubMed1994

Immunomodulation in patients receiving intravenous Bryostatin 1 in a phase I clinical study: comparison with effects of Bryostatin 1 on lymphocyte function in vitro.

Bryostatin 1 is a protein kinase C activator that inhibits growth of tumour cells and activates lymphocytes in vitro, properties that have encouraged its use in phase 1 clinical studies as an anticancer agent. We investigated interleukin-2(IL-2)-induced proliferation and lymphokine-activated killer (LAK) cell activity in peripheral blood mononuclear cells (PBMC) from cancer patients receiving Bryostatin intravenously. After Bryostatin administration both LAK generation and proliferation were enhanced when patients' PBMC were stimulated with IL-2 in vitro. However, when normal donors' PBMC were cultured in vitro in the presence Bryostatin and IL-2, LAK induction was inhibited while IL-2-driven proliferation was increased. These effects were also seen following only 2 h exposure to Bryostatin and could be elicited by conditioned medium from Bryostatin-pretreated cells. Neither IL-4 nor interferon gamma was detected in the conditioned medium. Bryostatin in vitro was found to increase expression of IL-2 receptors on CD4+, CD8+ and CD56+ cells and augment the proportion of CD8+ cells in conjunction with IL-2. We conclude that Bryostatin in combination with IL-2 in vitro enhances proliferation and IL-2 receptor expression on lymphocytes, favouring CD8+ cells while suppressing the generation of LAK activity. Intravenous administration of Bryostatin increases the potential of IL-2 to induce proliferation and LAK activity in lymphocytes which, taken together with its putative direct antitumour effect, makes Bryostatin an interesting candidate for clinical trials in combination with IL-2.

Adjuvants, Immunologic↗
PubMed1991

Bryostatins selectively regulate protein kinase C-mediated effects on GH4 cell proliferation.

The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate [TPA) or phorbol 12-myristate 13-acetate), directly activates the calcium- and phospholipid-dependent protein kinase C (protein kinase C), which, in turn, generates a number of cellular responses. The bryostatins, a family of macrocyclic lactones isolated from marine bryozoans, also bind to and active protein kinase C. However, they differ from TPA in the selectivity of their responses in that they behave either as agonists or antagonists of protein kinase C actions. We used several bryostatins and TPA to examine the role of protein kinase C in the regulation of GH4C1 rat pituitary tumor cell proliferation. TPA inhibited [3H]thymidine incorporation in GH4 cells in a stereoselective and concentration-dependent manner. Examination of cell cycle distribution by flow cytometry revealed that TPA decreased the percentage of cells in S-phase and proportionally increased the percentage of G1-phase cells. Bryostatin 1 alone did not affect cell proliferation, but prevented the TPA inhibition of cell proliferation. Bryostatin 1 treatment from 30 min to 6 h after TPA treatment also prevented the growth-inhibitory action of TPA, suggesting that prolonged stimulation of protein kinase C is necessary for growth inhibition. Both bryostatin 1 and TPA down-regulated protein kinase C, indicating that down regulation of the enzyme cannot account for the growth inhibitory action of TPA. Bryostatin 2, which differs from bryostatin 1 by a hydroxyl substitution for the acetyl group at the C-7 carbon of the macrocyclic lactone ring (R1), inhibited cell proliferation and did not reduce the growth-inhibitory action of TPA. Bryostatins 3 and 8 (each of which has an ester group in the R1 position, yet contains other structural modifications) are antagonists for TPA inhibition of GH4 cell proliferation like bryostatin 1. We next examined the effect of bryostatins 3 and 8 on cell-substratum adhesion, a cellular response observed after GH4 cells are treated with growth-inhibitory agents. Bryostatin 8 (like bryostatin 1) did not enhance cell-substratum adhesion and blocked the action of TPA. In contrast, bryostatin 3 enhanced cell-substratum adhesion. Because bryostatin 3 blocked TPA inhibition of cell proliferation, yet did not block TPA-enhanced cell-substratum adhesion, these responses are not interdependent. We next examined the effect of bryostatin on other growth-inhibitory agents for GH4 cells. Bryostatin 8 blocks the effect of TPA on [3H]thymidine incorporation and the entry of G1 cells into S-phase, but does not block the growth-inhibitory action of thyrotropin-releasing hormone or epidermal growth factor.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2000

The in vivo effect of bryostatin-1 on paclitaxel-induced tumor growth, mitotic entry, and blood flow.

Pretreatment of tumor cells with the protein kinase C (PKC) inhibitor bryostatin-1 enhances the cytotoxicity of most chemotherapeutic agents. However, in the case of paclitaxel, this effect has been shown in vitro to be best achieved when bryostatin-1 follows (rather than precedes) paclitaxel treatment. With combination trials of bryostatin-1 and paclitaxel planned for clinical trials and with only in vitro data available regarding drug sequence, we elected to undertake an in vivo study evaluating the effect of sequential bryostatin-1 and paclitaxel in a tumor-bearing mouse model and to correlate this effect to cell cycle events, tumor metabolism, and tumor blood flow. At the maximum tolerated i.p. dose, bryostatin-1 at 80 microg/kg resulted in a small but significant increase in tumor doubling time (4.2 +/- 0.3 days) compared with control tumors (3.0 +/- 0.3 days; P < 0.01). Mice treated with i.v. paclitaxel, administered at a dose of 12 mg/kg every 12 h for three doses, weekly for 3 weeks, had a tumor doubling time of 23.4 +/- 1.7 days. Mice pretreated with i.p. bryostatin-1 (80 microg/kg) followed 12 h later by i.v. paclitaxel (12 mg/kg every 12h for three doses) weekly for 3 weeks had a tumor doubling time of 9.7 +/- 1.1 days. This was significantly less (P < .001) than paclitaxel alone, which indicated an inhibitory effect by bryostatin-1 on paclitaxel therapy. In comparison, tumor-bearing mice that were treated with the same dose but with the sequence of paclitaxel followed by bryostatin-1 had a tumor doubling time of 29.6 +/- 0.6 days. This was significantly greater than the tumor doubling times for any condition tested (P < 0.01), demonstrating the sequence dependence of this combination. The efficacy of paclitaxel is dependent on mitotic entry, a step that requires activation of p34cdc2 kinase activity. Treatment with paclitaxel in vivo increased p34 cdc2 kinase activity in the mouse mammary tumors, whereas administration of bryostatin-1 before paclitaxel prevented the p34cdc2 kinase activation by paclitaxel. This was further evaluated in vitro by flow cytometry in MKN-74 human gastric cancer cells. As determined by MPM-2 labeling, which identifies cells in mitosis, pretreatment with bryostatin-1 prevented paclitaxel-treated cells from entering mitosis. Bryostatin-1 has been reported to induce changes in muscle metabolism and to decrease muscle blood flow. These events could impact on the interaction of bryostatin-1 with paclitaxel. Using proton-decoupled phosphorus nuclear magnetic resonance (31P-NMR) spectroscopy in vivo, bryostatin-1 at 80 micro1g/kg induced a decrease in both intratumoral pH and high-energy phosphates. In vivo perfusion studies, using dynamic enhanced NMR imaging with gadolinium diethylenetriamine pentaacetic acid, also demonstrated decreased tumor blood flow. These studies suggest that the inhibition of tumor response to paclitaxel by bryostatin-1 is multifactorial and includes such diverse factors as inhibition of cell entry into mitosis, a decrease in pH and energy metabolism, and a decrease in tumor blood flow. These results indicate that, as this combination enters Phase I clinical trials, the sequence of paclitaxel followed by bryostatin-1 will be critical in the clinical trial design.

Animals↗
PubMed1991

Successful treatment of murine melanoma with bryostatin 1.

Bryostatins are a novel class of protein kinase C activators which were isolated from the marine bryozoan Bugula neritina and found to possess both antineoplastic and immunoenhancing properties. In this report, we examined the relationship between the in vivo and in vitro antineoplastic effects of bryostatin 1. The in vivo antitumor activity of bryostatin 1 was demonstrated in a B16 melanoma pulmonary metastases model, in which treatment of tumor-bearing C57BL/6 mice with 5 days of bryostatin 1 resulted in a significant reduction in of the number of lung nodules (control, 87; bryostatin, 7). There was a clear dose-response effect, with the optimal antimelanoma dose being 100 micrograms/kg/day, but even low doses of bryostatin 1 of 1 micrograms/kg/day resulted in a 53% reduction in the number of metastases. Although bryostatin 1 shares many biological properties with the phorbol esters, parallel treatment with 12-O-tetradecanoyl 13-phorbol acetate was ineffective against B16 melanoma in vivo. Using a clonogenic assay, bryostatin 1 was found to have a direct antiproliferative effect against B16 melanoma. This inhibition occurred at relatively high bryostatin 1 concentrations (10(-6) M), in comparison with a sensitive cell line REH (10(-10) M). Treatment of mice with bryostatin 1 or preincubation of normal spleen cells with bryostatin 1 failed to enhance nonspecific cell-mediated cytotoxicity against B16 melanoma in vitro. Moreover, bryostatin 1 was found to inhibit both natural killer cell activity and interleukin 2 generation of lymphokine-activated killer cells. Thus, a role for an in vivo immune enhancement mechanism as the basis for the antimelanoma activity observed with bryostatin 1 cannot be invoked from these experiments. These findings indicate that bryostatin 1 may act directly on the B16 melanoma pulmonary metastases. The precise mechanism whereby bryostatin exerts its antimelanoma effects remains unclear.

Animals↗
PubMed1992

In vivo administration of bryostatin 1, a protein kinase C activator, decreases murine resistance to Salmonella typhimurium.

Bryostatin 1, a potent activator of protein kinase C, has antitumor activity against murine lymphoma, leukemia, and melanoma. In vitro, this compound stimulates the release of gamma-interferon, interleukins, and hematopoietic growth factors from accessory cells and activates both T- and B-cells. Bryostatin 1 is also able to stimulate neutrophils to undergo oxidative burst and degranulation. Because of the ability of this compound to stimulate the immune system, cause release of immune mediators, and activate neutrophils, we have examined its effect on bacterial infection by using the gram-negative bacterium Salmonella typhimurium in mice. We find that animals given injections i.v. of S. typhimurium have a shortened life span if they are also given injections i.p. of nonlethal doses of bryostatin 1. There is a dose-response relationship with 100 micrograms/kg bryostatin 1 having a greater effect on survival than 40 micrograms/kg. Below 40 micrograms/kg there are no effects on survival. Analysis of the first 4 h of Salmonella infection demonstrates that bryostatin 1 does not affect the blood clearance of the bacterium. However, by day 2 of infection greater numbers of bacteria are found in the livers and spleens of mice given injections of bryostatin 1. By day 5, 10-fold more S. typhimurium bacteria are found in the livers and spleens of mice receiving 40 micrograms/kg of bryostatin 1. To determine whether bryostatin 1 was affecting growth or causing the death of bacteria, we used a Salmonella carrying a plasmid which has a temperature-sensitive origin of replication and is unable to replicate when the bacteria are in mice. This experiment demonstrates that bryostatin 1 represses bacterial killing but does not affect bacterial growth. Bryostatin 1 given i.p. stimulates a transient syndrome of weight loss and diarrhea from which the mice recover and regain weight, suggesting that bryostatin 1 may release a number of important humoral mediators in vivo. The weight loss is exacerbated by Salmonella infection with mice receiving bryostatin 1 and S. typhimurium, in that they lose approximately 33% of body weight prior to death. Thus, at doses used to treat murine tumors, bryostatin 1 treatment does not affect the clearance of S. typhimurium from the blood but does decrease the killing of bacteria in the liver and spleen, leading to early animal death. Such potential effects of bryostatin 1 on the outcome of bacterial infections should be evaluated in ongoing human trials of this agent.

Animals↗
PubMed1992

Sensitization of human cervical carcinoma cells to cis-diamminedichloroplatinum(II) by bryostatin 1.

Bryostatins are an important class of protein kinase C (PKC) activators. We have investigated the effect of bryostatin 1 on the antiproliferative activity of cis-diamminedichloroplatinum(II) (CP). A 24-h pretreatment of HeLa cells with 1 nM bryostatin 1 increased cellular sensitivity to CP by 4-fold. The effect of bryostatin 1 on the IC50 of CP (concentration of drug required to inhibit cell proliferation by 50%) was concentration-dependent and biphasic; the maximum effect of bryostatin 1 was seen with 1 nM, but higher concentrations of bryostatin 1 (greater than or equal to 10 nM) produced less CP sensitization. Although bryostatin 1 and phorbol esters caused an equivalent stimulation of HeLa cell PKC in cell-free systems, bryostatin 1 was less effective than phorbol esters in sensitizing cells to CP. Additionally, higher concentrations of bryostatin 1 (greater than or equal to 10 nM) antagonized CP sensitization by phorbol esters. Bryostatin 1 was even more potent than 12-O-tetradecanoylphorbol-13-acetate in inducing PKC down-regulation, and the maximum down-regulation was achieved with 10 nM bryostatin 1. Bryostatin 1 also increased cellular sensitivity to a CP analogue, cis-dichloro(ethylenediamine)platinum(II). A 24-h pretreatment with 1 nM bryostatin 1 increased cellular cis-[3H]DEP by 60%. The concentration- and time-dependent enhancement in CP sensitivity by bryostatin 1 was related to the increase in cis-[3H]DEP level. Thus, cellular accumulation of CP may be regulated by a PKC-dependent phosphorylation event.

Antineoplastic Agents↗
PubMed2000

The transient increase of tight junction permeability induced by bryostatin 1 correlates with rapid downregulation of protein kinase C-alpha.

The role of PKC-alpha in altered epithelial barrier permeability following the activation of PKC by TPA (12-O-tetradecanoyl phorbol 13-acetate) and bryostatin 1 in LLC-PK1 cells was investigated in this study. Like TPA, bryostatin 1 binds to and activates PKC but unlike TPA, it is not a tumor promoter. TPA at 10(-7) M induced a sustained 95% decrease in transepithelial electrical resistance (R(t)) across LLC-PK1 epithelial cell sheets, while 10(-7) M bryostatin 1 caused only a 30% decrease in R(t), which spontaneously reversed after 5 h. Simultaneous exposure of cell sheets to 10(-7) M TPA and 10(-7) M bryostatin 1 blunted the increase in epithelial permeability observed with 10(-7) M TPA alone. Co-incubation of cell sheets with bryostatin 1 and MG-132, a proteasomal inhibitor, caused a further decrease in R(t) at the 6-h time point and inhibited the recovery in R(t) seen with bryostatin 1 alone at this time point. TPA caused a rapid translocation of PKC-alpha from the cytosol to the membrane of the cell where it remained elevated. Bryostatin 1 treatment resulted in a slower translocation of PKC-alpha from the cytosol to the membrane and a much more rapid downregulation of PKC-alpha, with disappearance from this compartment after only 6 h. The classical PKC inhibitor Go6976 prevented the decrease in R(t) seen with TPA. Treatment of cells with TPA and bryostatin 1 resulted in a PKC-alpha translocation and downregulation profile which more closely resembled that seen with bryostatin 1 alone. Co-incubation of cells with MG-132 and bryostatin 1 caused a slower downregulation of PKC-alpha from the membrane fraction. Bryostatin 1 treatment of cells expressing a dominant/negative form of PKC-alpha resulted in a slower and less extensive decrease in R(t) compared to the corresponding control cells. For both TPA and bryostatin 1, the level of PKC-alpha in the membrane-associated fraction of the treated cells correlated closely with increased transepithelial permeability. Due to its transient effect on tight junction permeability, bryostatin 1 offers a novel pharmacological tool to investigate junctional physiology.

Animals↗
PubMed1990

Bryostatin 1, a unique biologic response modifier: anti-leukemic activity in vitro.

Bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, has demonstrated both antineoplastic activity against the murine P388 leukemia line in vivo and stimulatory activity against mouse and human hematopoietic progenitors. We studied the effects of bryostatin 1 on the growth of human leukemias in vitro. Bryostatin 1 inhibited 1 to 4 logs of clonogenic leukemia cell growth from three of four leukemia cell lines. Bryostatin 1 also inhibited, by at least 1 log, the proliferation of clonogenic acute nonlymphocytic leukemia (ANLL) cells from 10 to 12 patients with newly diagnosed or relapsed ANLL. Maximal inhibition of leukemic growth occurred at 10(-9) to 10(-7) mol/L bryostatin 1. Interestingly, bryostatin 1 also inhibited the growth of hematopoietic progenitors from eight patients with myelodysplastic syndromes (MDS). Leukemia cells exposed to bryostatin 1 for up to 96 hours and then washed, demonstrated no substantial inhibition of clonogenic growth, indicating that the anti-leukemic effect of bryostatin 1 is cytostatic. The phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) produced more potent inhibition of clonogenic leukemia growth, and this inhibition was blocked by bryostatin 1. Thus, the anti-leukemic activity of bryostatin 1 may be mediated through activation of protein kinase C. Bryostatin 1 inhibits clonogenic leukemia cells at concentrations that stimulate normal hematopoietic progenitors. The differential effects of bryostatin 1 on normal and abnormal hematopoiesis suggest that bryostatin 1 may have value in the treatment of leukemias and MDS.

Antineoplastic Agents↗
PubMed1990

Effects of bryostatins 1 and 2 on morphological and functional differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate to mature ganglion cells when treated with the phorbol ester tetradecanoylphorbol acetate (TPA). Bryostatins are a new class of protein kinase C activators that are structurally unrelated to phorbol esters. This paper describes the effects of bryostatins 1 and 2 on morphological and functional differentiation of SH-SY5Y cells. Both bryostatins induced a rapid translocation of protein kinase C from the cytosol to the membrane fraction. Within 24 h, the bryostatins had caused a nearly complete down-regulation of the enzyme. Bryostatin 1 competed for [3H]phorbol-12,13-dibutyrate binding in intact cells with potency equal to that of TPA, in contrast to bryostatin 2, which exhibited a Ki value 1 order of magnitude higher than those of the two other agents. Bryostatins induced morphological changes similar to those induced by TPA. These changes were, however, only transient, occurring during the first 6 h of incubation in the presence of these compounds. By 72 h, the cells had acquired a morphology typical of untreated cells and, although a wide range of bryostatin concentrations were used, morphological changes characteristic of differentiated SH-SY5Y cells were not detected at 72 h. Bryostatin 1 at 5 nM and bryostatin 2 at 100 nM inhibited DNA synthesis, as measured by incorporation of [3H]thymidine by SH-SY5Y cells, although to a significantly lesser degree than TPA. In spite of the fact that bryostatins failed to induce morphological differentiation in SH-SY5Y cells, these compounds down-regulated c-myc mRNA expression. Bryostatins were significantly weaker in stimulating noradrenaline synthesis, compared with TPA, and high concentrations of these agents blocked the effect of the phorbol ester when they were included together with TPA. When SH-SY5Y cells were incubated in the presence of high concentrations of bryostatins, a decreased sensitivity of cells to muscarinic agonist-induced increases in cytosolic free Ca2+ was observed. The results suggest that down-regulation of protein kinase C activity and c-myc mRNA expression do not necessarily correlate with the morphological differentiation of SH-SY5Y cells.

Antineoplastic Agents↗
PubMed1995

The antineoplastic bryostatins affect human basophils and mast cells differently.

Bryostatins, macrocyclic lactones from the marine bryozoan Bugula neritina, are potent antineoplastic agents and multi-potential stimulators of immune cells. We have examined the effects of bryostatins on mediator release from human basophilic leukocytes and human tissue mast cells. Bryostatins 1, 2, and 5 (10 to 3,000 nmol/L) induced histamine secretion from purified and unpurified peripheral blood basophils, whereas they caused no release of peptide-leukotriene C4 from these cells. The rate of histamine release caused by bryostatin 1 was slower than that caused by anti-IgE (t1/2 +/- SEM = 38.2 +/- 4.7 minutes v 8.9 +/- 0.2 minutes; P < .01), whereas the temperature dependence was similar (optimum release at 37 degrees C, approximately 30% less at 30 degrees C, and no release at 22 degrees C or 4 degrees C). The addition of increasing concentrations of extracellular Ca2+ to the medium caused histamine release in the presence of bryostatins. Subeffective concentrations of bryostatins and anti-IgE produced a synergistic effect on histamine release from basophils. Staurosporine, chelerythrine, and calphostin C (0.1 to 10 nmol/L), which are protein kinase C inhibitors, inhibited the histamine secretion activated by bryostatin 1 and tetradecanoylphorbol-acetate (TPA). Preincubation with granulocyte-monocyte colony-stimulating factor (GM-CSF; 1 and 5 nmol/L) and interleukin-3 (IL-3; 10 ng/mL) potentiated the activation of human basophils induced by bryostatin 1. Neither bryostatin 1 nor bryostatin 2 induced the release of histamine from mast cells isolated from human lung or skin tissues. However, brief (10 minutes) preincubation with bryostatin 1 (3 to 300 nmol/L) potently inhibited the histamine secretion induced by anti-IgE from skin or lung mast cells. Bryostatin 1 was a more potent (by approximately 30 times) inhibitor of IgE-mediated histamine release than was TPA. The heterogeneous effects exerted by bryostatins on human basophils and mast cells can be of interest for those designing therapeutic trials using these agents.

Antibodies, Anti-Idiotypic↗
PubMed1996

Comparison of the antitumor activity of bryostatins 1, 5, and 8.

Bryostatin 1, a macrocyclic natural lactone isolated from a marine Bryozoan, has undergone phase I testing in humans. Side effects of treatment have included muscle pain and joint aches, a transient decrease in platelets, and the release of tumor necrosis factor alpha (TNF alpha) and IL-6 into the blood stream. In animals, anticancer activity has been demonstrated against murine leukemias, lymphomas, melanomas, and sarcomas. The mechanism of action of this compound depends in part on its ability to activate protein kinase C. To determine the biologic activity and toxicity of other members of the family of bryostatin compounds, we studied the ability of bryostatins 5 and 8 to inhibit the growth of murine melanoma K1735-M2. Bryostatins 1, 5, and 8 induced equivalent inhibition of melanoma growth, but bryostatins 5 and 8 induced less weight loss than bryostatin 1 (P < 0.001). Neither the injection of an antimurine TNF alpha antibody nor an adenovirus, which produces a mutated TNF receptor inhibiting TNF alpha activity, into mice had any effect on either bryostatin-induced weight loss or melanoma tumor growth inhibition. Using a novel competition assay, the levels of bryostatin in the plasma were measured. The approximate half-life (t1/2) of bryostatin was 8.62 min, the clearance (Cl) 3.53 ml/min and the AUC 322.20 nmol/l min. A similar result was obtained with each bryostatin analog. These results suggest that human testing of additional bryostatin analogs may yield compounds with similar antitumor activity but decreased side effects. A novel assay to measure the level of all bryostatins in the plasma of patients undergoing treatment is described.

Animals↗
PubMed1992

Binding of [3H]bryostatin 4 to protein kinase C.

The bryostatins represent a unique class of activators of protein kinase C (PKC) which induce only a subset of the responses typical of the phorbol esters and block those responses to the phorbol esters which they themselves do not induce. To better understand the interaction of the bryostatins with PKC, we have synthesized [26-3H]bryostatin 4 and characterized its binding to PKC. [3H]Bryostatin 4 and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) differed markedly in their binding to PKC reconstituted with phosphatidylserine (PS). The binding affinity of [3H]bryostatin 4 under these conditions was too high to measure and the rate of release of bound bryostatin was much slower than that of the phorbol esters, with a half-time of several hours. These properties caused bryostatin 1 to appear to inhibit [3H]PDBu binding under these conditions in a non-competitive fashion. Both the high potency and the slow rate of release of the bryostatins may contribute to their unique pattern of biological activity. By reconstituting PKC in a mixture of 1.5% Triton X-100:0.3% PS, we were able to establish reversible conditions for [3H]bryostatin 4 binding. Under these latter conditions, binding of [3H]bryostatin 4 was competitively inhibited by PDBu, consistent with both the bryostatin and phorbol esters binding to PKC in a qualitatively similar fashion. Binding affinities to PKC isozymes alpha, beta, and gamma were compared and little difference was found, suggesting that differential recognition by these isozymes does not account for the unique biological activity of the bryostatins.

Animals↗
PubMed2005

Enhancement of cisplatin sensitivity of cisplatin-resistant human cervical carcinoma cells by bryostatin 1.

PURPOSE: Bryostatin 1, a unique protein kinase C (PKC) activator, is already in the clinical trials. An understanding of complex regulation of PKC by bryostatin 1 is essential for effective use of bryostatin 1 in the clinic. We have previously shown that the ability of bryostatin 1 to enhance cisplatin sensitivity correlated with its ability to down-regulate PKCdelta in HeLa cells. We have investigated how bryostatin 1 influences PKCdelta regulation in cisplatin-resistant HeLa (HeLa/CP) cells, and if bryostatin 1 could be used to reverse cisplatin resistance. EXPERIMENTAL DESIGN: Phorbol 12,13-dibutyrate (PDBu), bryostatin 1, and small interfering RNA were used to manipulate PKC level/activation status. Cell death was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Annexin V dye-binding assay, and analysis of hypodiploid peak in a flow cytometer. RESULTS: Bryostatin 1 elicited a biphasic concentration response on PKCdelta down-regulation and cisplatin-induced cell death in HeLa/CP cells; the maximum effect was achieved with 1 nmol/L bryostatin 1. Down-regulation of PKCalpha increased with increasing concentrations of bryostatin 1. PDBu induced down-regulation of PKCalpha in HeLa and HeLa/CP cells but it had little effect on PKCdelta down-regulation in HeLa/CP cells. However, both PDBu and bryostatin 1 enhanced the sensitivity of HeLa/CP cells to cisplatin. Knockdown of PKCdelta by small interfering RNA inhibited cisplatin-induced apoptosis but knockdown of PKCalpha enhanced cisplatin-induced cell death. CONCLUSIONS: These results suggest that although PKCdelta acts as a proapoptotic protein, full-length PKCdelta may inhibit cisplatin-induced cell death. Thus, persistent activation/down-regulation of PKCdelta by bryostatin 1 was associated with cisplatin sensitization. Furthermore, PKCalpha acts as an antiapoptotic protein and down-regulation of PKCalpha by PDBu was associated with cellular sensitization to cisplatin.

Antineoplastic Agents↗
PubMed1998

Potentiation of 2-chlorodeoxyadenosine activity by bryostatin 1 in the resistant chronic lymphocytic leukemia cell line (WSU-CLL): association with increased ratios of dCK/5'-NT and Bax/Bcl-2.

The activities of 2-chlorodeoxyadenosine (2-CdA) metabolizing enzymes, deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase (5'-NT) were measured in control and bryostatin 1 treated CLL cells using an EBV-negative WSU-CLL cell line. This cell line was established from a patient with CLL resistant to fludarabine. The results revealed a significant increase in dCK activity in bryostatin 1 treated cells at 48 and 72 h compared with the control. 5'-NT activity decreased significantly at 48 h. The ratio of dCK to 5'-NT activity was significantly increased in bryostatin 1 treated WSU-CLL cells after 48 h. WSU-CLL cells treated with bryostatin 1 exhibited an increase in the percentage of apoptotic and dead cells from control levels of 16% to 40%. This percentage was further increased to 67% following the addition of 11.2 microM 2-CdA to WSU-CLL cells pretreated with bryostatin 1. Results from Western blot analysis indicate that WSU-CLL cells express high levels of Bcl-2, Bcl-xL and c-myc, and a low level of Bax. p53 in untreated WSU-CLL cells is undetectable. WSU-CLL cells treated with bryostatin 1 showed a significant increase in the ratio of Bax to Bcl-2. To demonstrate that the bryostatin 1 mediated enhancement of 2-CdA efficacy was not restricted to in vitro cell culture, we have studied the tumor growth delay of WSU-CLL xenografts treated with placebo, bryostatin 1, 2-CdA, and bryostatin 1 followed by 2-CdA. SCID mice given bryostatin 1 at 75 microg x kg(-1) x d(-1) for 5 days followed by 30 mg x kg(-1) x d(-1) 2-CdA for 5 days in two cycles, had significantly improved tumor growth delay (P = 0.05). We conclude that bryostatin 1 is not only capable of inducing apoptosis by itself, but also sensitizes de novo resistant WSU-CLL cells to the chemo-therapeutic effects of 2-CdA. The bryostatin 1-induced increased ratio of dCK/5'-NT activity and an increased ratio of Bax/Bcl-2 are at least two mechanisms through which this natural compound is able to potentiate the anti-tumor activity of 2-CdA in otherwise resistant CLL cells.

5'-Nucleotidase↗
PubMed2006

Bryostatin enhancement of memory in Hermissenda.

Bryostatin, a potent agonist of protein kinase C (PKC), when administered to Hermissenda was found to affect acquisition of an associative learning paradigm. Low bryostatin concentrations (0.1 to 0.5 ng/ml) enhanced memory acquisition, while concentrations higher than 1.0 ng/ml down-regulated the pathway and no recall of the associative training was exhibited. The extent of enhancement depended upon the conditioning regime used and the memory stage normally fostered by that regime. The effects of two training events (TEs) with paired conditioned and unconditioned stimuli, which standardly evoked only short-term memory (STM) lasting 7 min, were--when bryostatin was added concurrently--enhanced to a long-term memory (LTM) that lasted about 20 h. The effects of both 4- and 6-paired TEs (which by themselves did not generate LTM), were also enhanced by bryostatin to induce a consolidated memory (CM) that lasted at least 5 days. The standard positive 9-TE regime typically produced a CM lasting at least 6 days. Low concentrations of bryostatin (<0.5 ng/ml) elicited no demonstrable enhancement of CM from 9-TEs. However, animals exposed to bryostatin concentrations higher than 1.0 ng/ml exhibited no behavioral learning. Sharp-electrode intracellular recordings of type-B photoreceptors in the eyes from animals conditioned in vivo with bryostatin revealed changes in input resistance and an enhanced long-lasting depolarization (LLD) in response to light. Likewise, quantitative immunocytochemical measurements using an antibody specific for the PKC-activated Ca2+/GTP-binding protein calexcitin showed enhanced antibody labeling with bryostatin. Animals exposed to the PKC inhibitor bisindolylmaleimide-XI (Ro-32-0432) administered by immersion prior to 9-TE conditioning showed no training-induced changes with or without bryostatin exposure. However, if animals received bryostatin before Ro-32, the enhanced acquisition and demonstrated recall still occurred. Therefore, pathways responsible for the enhancement effects induced by bryostatin were putatively mediated by PKC. Overall, the data indicated that PKC activation occurred and calexcitin levels were raised during the acquisition phases of associative conditioning and memory initiation, and subsequently returned to baseline levels within 24 and 48 h, respectively. Therefore, the protracted recall measured by the testing regime used was probably due to bryostatin-induced changes during the acquisition and facilitated storage of memory, and not necessarily to enhanced recall of the stored memory when tested many days after training.

Animals↗
PubMed1998

p53 and protein kinase C independent induction of growth arrest and apoptosis by bryostatin 1 in a highly metastatic mammary epithelial cell line: In vitro versus in vivo activity.

We have evaluated the effects of bryostatin 1 on growth of a highly malignant p53-null mouse mammary tumor line, 4T1, and the mechanism by which bryostatin 1 inhibits in vitro growth and in vivo development of tumor and metastases from the orthotopic site. Bryostatin 1 at 20-400 nM concentrations inhibits growth of 4T1 cells by approximately 60% in two-day cultures. Inhibition of growth is associated with an increase in the number of cells undergoing apoptosis with concomitant elevation in the steady state levels of bax protein and drop in bcl-2 levels. The cytotoxic effect of bryostatin 1 on 4T1 cells occurs independently of p53, since there was no evidence of p53-mediated transcriptional activity in 4T1 cells following treatment with bryostatin 1.4T1 cells respond in vivo to bryostatin 1 therapy (75 microg/kg body weight). Intraperitoneal administration of bryostatin 1 inhibits both primary and secondary tumor growth by approximately 50%. However, although bryostatin 1 has a remarkable capacity to slow tumor growth and progression, it is unable to completely eradicate tumor growth and progression due to in vivo development of tumor resistance to bryostatin 1. Levels and cellular distribution of PKCalpha and delta do not correlate with the growth inhibitory effects of bryostatin 1 on 4T1 cells; however, reduction in cytosolic PKCalpha and delta without associated increase in membrane compartment appear to correlate with bryostatin-resistance. Our results suggest that the therapeutic effects of bryostatin 1 in our system do not involve alterations in levels and distribution of PKC but rather a direct upregulation of bax/ bcl-2 ratios that is independent of p53.

Animals↗