bryostatin-1 and Lung-Neoplasms

Bryostatin-1 is a macrocyclic lactone, which exhibits pleiotropic biological effects via protein kinase C and has shown preclinical synergy with paclitaxel for enhanced tumor cell apoptosis.. Patients had stage IIIB (pleural effusion)/IV non-small cell lung cancer, measurable disease, performance status 0-2 Eastern Cooperative Oncology Group, adequate organ function, and no prior chemotherapy. Patients received dexamethasone premedication followed by paclitaxel at a dose of 90 mg/m(2) on days 1, 8, and 15 along with bryostatin-1 50 microg/m(2) on days 2, 9, and 16 every 28 days until disease progression. Correlative assays measuring serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and T-lymphocyte numbers were performed based on a previous study showing cytokine induction in vivo by bryostatin-1. Fifteen patients were enrolled.. Thirty cycles of the bryostatin-1 and paclitaxel were delivered with a median of 2 per patient (range 1-4). Myalgia was the predominant non-hematologic toxicity encountered as 3 patients developed grade 4 and 1 patient developed grade 3 myalgia. Four patients were removed from the study during cycle 1 for rapid disease progression or myalgia. Eleven patients could be evaluated for response. Five patients had stable disease, two had a mixed response, and four had progressive disease. Ten patients received second-line chemotherapy after leaving the study. Median survival was 31 weeks (95% confidence interval: 5.4-49.3). Correlative data showed a trend towards decreased plasma IL-6 and TNF-alpha after each cycle of therapy presumably due to the dexamethasone premedication and/or paclitaxel.. This drug combination showed no significant clinical response and was associated with reproducible toxicity. The predominance of myalgia in the absence of elevated serum cytokines suggests a non-inflammatory etiology of this toxicity.

Topics: Adenocarcinoma; Aged; Antineoplastic Combined Chemotherapy Protocols; Bryostatins; Carcinoma, Non-Small-Cell Lung; Disease-Free Survival; Drug Administration Schedule; Female; Glucocorticoids; Humans; Interleukin-6; Lactones; Lung Neoplasms; Macrolides; Male; Middle Aged; Paclitaxel; Premedication; Survival Rate; T-Lymphocytes; Tumor Necrosis Factor-alpha

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apoptosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-gamma release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a "per-cell" basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Bryostatins; Cell Proliferation; Flow Cytometry; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-15; Interleukin-2; Interleukin-7; Interleukins; Ionomycin; Lung Neoplasms; Lymph Nodes; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tumor Cells, Cultured

Bryostatin 1 is currently in clinical trials as a cancer chemotherapeutic agent. Although bryostatin 1, like phorbol 12-myristate 13-acetate (PMA), is a potent activator of protein kinase C (PKC), it induces only a subset of those responses induced by PMA and antagonizes others. We report that, in the HOP-92 non-small cell lung cancer line, bryostatin 1 induced a biphasic proliferative response, with maximal proliferation at 1 to 10 nmol/L. This biphasic response mirrored a biphasic suppression of the level of PKCdelta protein, with maximal suppression likewise at 1 to 10 nmol/L bryostatin 1. The typical phorbol ester PMA, in contrast to bryostatin 1, had no effect on the level of PKCdelta and modest suppression of cell proliferation, particularly evident at later treatment times. Flow cytometric analysis revealed changes in the fraction of cells in the G0-G1 and S phases corresponding to the effects on proliferation. Cells overexpressing PKCdelta exhibited a lower rate of cell proliferation compared with control untreated cells and showed neither a proliferative response nor a loss of PKCdelta in response to bryostatin 1. Conversely, treatment with PKCdelta small interfering RNA significantly increased the cellular growth compared with controls. We conclude that the differential effect on cellular proliferation induced by bryostatin 1 compared with PMA reflects the differential suppression of PKCdelta.

Topics: Bryostatins; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Macrolides; Protein Kinase C-delta; Tetradecanoylphorbol Acetate

Daphnane-type diterpene gnidimacrin (NSC 252940) shows significant antitumor activity against murine tumors and human tumor cell lines. This compound binds to and directly activates protein kinase C (PKC), arresting the cell cycle at the G(1) phase through inhibition of cdk2 activity in human K562 leukemia cells. In our study, we examined whether cellular PKC is involved in the antiproliferating effect of gnidimacrin. In a 24-hr exposure of K562 cells to high concentrations of bryostatin 1 (0.11-3.3 microM), both expression of PKC alpha and PKC betaII was downregulated, and thereafter these cells became resistant to gnidimacrin in response to the degree of PKC downregulation. In addition, PKC alpha and PKC betaII genes were transfected to gnidimacrin-resistant human hepatoma HLE cells that demonstrated positive expression of PKC alpha and negative expression of PKC betaII. PKC betaII gene-transfected cells became sensitive to gnidimacrin in relation to the degree of PKC betaII expression. The most sensitive clone to show 0.001 microg/mL (1.2 nM) as IC(50) in a continuous 4-day exposure was obtained. While PKC alpha gene-transfected cells exhibited an increase in PKC alpha expression and became sensitive to gnidimacrin, sensitivity was one-hundredth of that in PKC betaIotaIota gene-transfected cells. These results suggest that PKC, in particular PKC betaIotaIota, is necessary in the antitumor effect of gnidimacrin.

Topics: Antineoplastic Agents, Phytogenic; Bryostatins; Carcinoma, Small Cell; CDC2-CDC28 Kinases; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinases; Diterpenes; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Induction; G1 Phase; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Isoenzymes; K562 Cells; Lactones; Lung Neoplasms; Macrolides; Neoplasm Proteins; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured

Bryostatin 1 and phorbol esters reduce the intracellular melanin level in high metastatic overexpressing nPKCdelta BL6 (BL6T) cells, thereby inducing white experimental metastasis in syngeneic mice. We evaluate here the possible differences between white and black metastases induced by both treatments on the proliferative and metastatic potential as well as on the expression of some cytokines involved in the metastatic process such as TGFbeta, IL-10 and IFNgamma. The level of expression of gelatinase A is also considered. White and black metastases induced after the injection of bryostatin 1- or phorbol ester-treated cells into the tail vein of syngenic mice were isolated and analysed for the levels of LDH usually used as markers of cytotoxicity, for the levels of cytokines and gelatinase A or dissociated and cultured in vitro for a few passages. The cultured cells were analysed in vitro for the proliferative capacity and the melanin synthesis. The same cells were also re-injected into syngeneic mice and the number of experimental metastases were counted after 17 days or injected with matrigel in order to quantify the proliferative capacity in vivo. The results show only one significant difference between bryostatin I and phorbol ester, namely the cells obtained from white bryostatin 1-treated cells return to a black phenotype after a few passages in culture. This suggests that PKC mediates many of the biological effects of bryostatin 1 but that its effect is lost in vitro. On the other hand, white and black metastases (at least for metastases induced by BL6T cells treated with phorbol ester) do appear significantly different. In vivo white metastases show lower levels of LDH, lower levels of proliferative capacity into matrigel, higher levels of TGFbeta and IFNgamma and, when re-injected into syngeneic mice, give big black metastases. Therefore, in murine melanoma cells, the treatment with bryostatin I induces the appearance of a white population expressing different levels of TGFbeta, IFNgamma, IL-10 and gelatinase A. Such a white population is more difficult to diagnose and is capable of turning into a more aggressive phenotype under suitable environmental conditions.

Topics: Animals; Antineoplastic Agents; Bryostatins; Interferon-gamma; Interleukin-10; L-Lactate Dehydrogenase; Lactones; Lung Neoplasms; Macrolides; Matrix Metalloproteinase 2; Melanoma; Mice; Mice, Inbred C57BL; Protein Kinase C; Reverse Transcriptase Polymerase Chain Reaction; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

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.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Bryostatins; Cell Division; DNA, Recombinant; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Isoenzymes; Lactones; Lung Neoplasms; Macrolides; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mutation; Neoplasm Metastasis; Protein Kinase C; Protein Kinase C-alpha; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

For the relatively nonimmunogenic B16-F10 murine melanoma, it has been found that genetically engineered expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) but not interleukin (IL)-2, IL-4, or interferon-gamma (IFN-gamma) resulted in a vaccine that could induce resistance to rechallenge. Because T cells from lymph nodes draining the sites of some progressive tumors can mediate tumor regression after in vitro activation, it seemed possible that even apparently nonimmunogenic melanoma cells might induce similar preeffector cells in the vaccine-draining lymph nodes (DLNs).. C57BL/6 mice were vaccinated with B16-F10 cells that were either unmodified or genetically modified to produce IL-2, IL-4, GM-CSF, or IFN-gamma. DLNs were harvested 10 days after vaccination for adoptive immunotherapy (AIT). The DLN cells were activated with bryostatin 1 and ionomycin (B/I), expanded for 10 days in culture, and transferred to mice with 3-day pulmonary metastases. Pulmonary nodules were counted 14 days after AIT.. Adoptive transfer of expanded DLN lymphocytes sensitized by inoculation of WT B16-F10, or IL-4, GM-CSF, or IFN-gamma expressing cells significantly reduced pulmonary metastases. Despite the spontaneous regression of IL-2-transduced B16-F10 tumors, DLN from mice inoculated with IL-2 producing B16 cells had little or no antitumor activity.. B16-F10 vaccination strategies that apparently do not induce systemic immunity can effectively sensitize DLN preeffector cells.

Topics: Adjuvants, Immunologic; Animals; Bryostatins; Cancer Vaccines; Cytokines; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Immunotherapy, Adoptive; Lactones; Lung Neoplasms; Macrolides; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Regression, Spontaneous; T-Lymphocytes; Transduction, Genetic; Tumor Cells, Cultured

Bryostatin 1, a macrocyclic lactone, has undergone phase I trials as an anticancer agent. Because of the lipid solubility of this compound it must be delivered either in ethanol or in a PET formulation. During the trial, these vehicles caused a large number of treatment-related side effects. We have synthesized the triethanolamine salt of 26-succinylbryostatin 1 and find that this compound is approx. 100-fold more water soluble than bryostatin 1. Because of the potential for clinical use, we have evaluated the biologic activity of this compound. We find that in a concentration-dependent manner 26-succinylbryostatin 1 is capable of activating protein kinase C (PKC) in vitro and displacing [3H]PDBu from PKC. However, at all concentrations tested the activity was less than the parent compound bryostatin 1. Addition of bryostatin 1 but not 26-succinylbryostatin 1 to U937 leukemic cells in culture stimulated a drop in cytosolic PKC, secondary to translocation of PKC to the membrane. Although 26-succinylbryostatin 1 did not stimulate a drop in the cytosolic levels of PKC, addition to U937 cells activated transcription from an AP-1 enhancer construct and c-Jun protein phosphorylation in a similar fashion to bryostatin 1 and differentiation of U937 cells. Unlike bryostatin 1, 26-succinylbryostatin 1 was unable to cause aggregation of human platelets. Although injection of bryostatin-1 into mice carrying B16 melanoma inhibits tumor growth, there was no significant inhibition of melanoma growth when identical doses of 26-succinylbryostatin 1 were injected. Therefore, 26-succinylbryostatin 1 shares some but not all of the pharmacologic properities of bryostatin 1. This compound can activate protein phosphorylation without lowering cytosolic levels of PKC.

Topics: Animals; Antineoplastic Agents; Blood Platelets; Blotting, Western; Bryostatins; Cell Line; Cell Membrane; Chloramphenicol O-Acetyltransferase; Enzyme Activation; Glutathione Transferase; Humans; Kinetics; Lactones; Lung Neoplasms; Macrolides; Melanoma, Experimental; Mice; Mice, Inbred C3H; Phorbol 12,13-Dibutyrate; Platelet Aggregation; Protein Kinase C; Recombinant Fusion Proteins; Tumor Cells, Cultured

Protein kinase C (PKC) influences cellular sensitivity to cis-diamminedichloroplatinum(II) (cDDP). We have investigated whether the PKC signal transduction pathway is affected during the development of cellular resistance to cDDP. Activators of PKC, such as phorbol 12,13-dibutyrate (PDBu), enhanced the sensitivity of human small cell lung cancer H69 cells to cDDP by 2-fold but had no effect on the sensitivity of cDDP-resistant H69 cells (H69/CP) to cDDP. The maximum sensitization was achieved with 10 nM PDBu and blocked by down-regulation of PKC with higher concentrations of PDBu (1 microM) or bryostatin 1 (0.1 microM). PKC activity was decreased significantly in H69/CP cells compared to the drug-sensitive variant. A similar reduction in PKC activity was noted in ovarian carcinoma 2008 cells that were resistant to cDDP. A modest decrease in PKC activity was also observed in etoposide-resistant H69 (H69/VP-16) cells but not in Taxol-resistant H69 cells or bleomycin-resistant human head and neck carcinoma A-253 cells. H69 cells expressed conventional PKC alpha and-beta, novel PKC delta, atypical PKC zeta and-iota, and novel/atypical PKC mu. A decrease in cPKC alpha and-beta and an increase in nPKC delta were associated with the cDDP-resistant phenotype. The abundance of aPKC zeta or-iota was unaffected. H69/ VP-16 cells also displayed a reduction in cPKC beta and an increase in nPKC delta. Taxol-resistant H69 cells had no alteration in the expression of any of the PKC isozymes. Thus, a reduction in cPKCs and an increase in nPKC may be associated with cDDP resistance.

Topics: Antineoplastic Agents; Bleomycin; Bryostatins; Carcinoma; Carcinoma, Small Cell; Cisplatin; Drug Resistance, Neoplasm; Enzyme Activation; Etoposide; Female; Head and Neck Neoplasms; Herpes Simplex Virus Protein Vmw65; Humans; Lactones; Lung Neoplasms; Macrolides; Ovarian Neoplasms; Paclitaxel; Phorbol 12,13-Dibutyrate; Protein Kinase C; Signal Transduction; Tumor Cells, Cultured

Lymphocytes obtained from tumor-draining lymph nodes (DLN) can have potent in vivo antitumor activity after in vitro activation with bryostatin 1 and ionomycin. However, the presence of visceral metastases in the donor can inhibit the effectiveness of such lymphocytes. In the present study, we tested the ability of low-dose cyclophosphamide to overcome metastasis-induced immunosuppression in a murine model.. Mice were injected with MCA-105 sarcoma cells in the footpad alone or in the footpad and the tail vein to establish lung metastases. Cyclophosphamide was given i.p. 1 day before harvesting the draining popliteal lymph nodes. For all donor groups, DLN cells were activated with 5 nM bryostatin 1 and 1 microM ionomycin and cultured for 7 days in 20 U/ml IL-2. Activated DLN cells were then adoptively transferred to syngeneic mice with 3-day lung metastases.. The adoptive transfer of DLN cells from mice with footpad tumors only significantly reduced the number of lung metastases compared to untreated mice. However, activated DLN cells obtained from mice with both footpad and lung tumors were significantly less effective. Treatment of similar donor mice with 10 mg/kg cyclophosphamide significantly improved the anti-tumor activity of adoptively transferred cells. This dose of cyclophosphamide did not reduce the number of cells obtained from each lymph node or the expansion of cell numbers in vitro.. These results suggest that the administration of low-dose cyclophosphamide prior to harvesting DLN cells may improve the success of adoptive immunotherapy in cancer patients.

Topics: Animals; Bryostatins; Cyclophosphamide; Female; Immune Tolerance; Immunotherapy, Adoptive; Ionomycin; Lactones; Lung Neoplasms; Lymphocyte Activation; Lymphocytes, Tumor-Infiltrating; Macrolides; Mice; Mice, Inbred C57BL; Mitogens; Neoplasm Metastasis; Neoplasm Transplantation; Sarcoma, Experimental

Bryostatin I is a natural product currently under clinical evaluation as an antitumor agent. Like the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) it activates protein kinase C (PKC). Bryostatin I inhibits the growth of the human-derived A549 lung and MCF-7 adenocarcinoma cell lines, but much more weakly than TPA. The hypotheses were tested that differences between cell lines in their response to bryostatin I are related to cellular PKC isotype content, and that differences between TPA and bryostatin I in their effects on cell growth are associated with differential abilities to modulate specific PKC isoenzymes. PKC isozyme profiles were studied by Western-blot analysis in the cytosol, particulate and nuclear fractions of A549 and MCF-7 cells. PKCs-alpha, -epsilon and -zeta were detected in both cell types with predominant location in the cytosol. Separation of cytosolic PKC isoenzymes in A549 cells by hydroxylapatite column chromatography and determination of PKC activity in fractions yielded a major peak which contained PKC-alpha. Exposure of cells to bryostatin I or TPA for 30 min caused the redistribution of PKCs-alpha and -epsilon from the cytosol to the particulate and nuclear fractions in a concentration-dependent fashion. PKC-epsilon was completely down-regulated by exposure to 10 nM bryostatin I for 18 hr or to TPA for 24 hr. Down-regulation of PKC-alpha was partial at 10 nM and complete at 1 microM of either agent. Bryostatin I inhibited incorporation of [3H]-labelled thymidine into cells only transiently, whereas TPA arrested growth for several days in A549 cells and irreversibly in MCF-7 cells. A549 cells, in which PKC was depleted by exposure to phorbol ester for 9 weeks, were resistant towards bryostatin-induced inhibition of DNA synthesis. The results suggest that the susceptibility of adenocarcinoma cells towards bryostatin-induced growth delay are determined by cellular levels of PKCs-alpha and/or -epsilon. However, differences between bryostatin I and TPA in their abilities to inhibit cell growth do not seem to be intrinsically related to differences in redistribution or down-regulation of specific PKC isoenzymes.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Bryostatins; Cell Division; Chromatography; Durapatite; Enzyme Activation; Humans; Isoenzymes; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Subcellular Fractions; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Recent studies have demonstrated that noncytolytic T-cells can mediate regression of murine tumors. In this report, we demonstrate that MCA-105 tumor-draining lymph node cells (DLN) activated with the protein kinase C activator, bryostatin 1, plus a calcium ionophore are capable of inducing specific tumor regression in vivo when adoptively transferred to mice with established metastases. However, these activated DLN cells lack in vitro cytotoxicity against autologous tumor. Antibody against gamma-interferon (IFN-gamma) markedly inhibited the therapeutic efficacy of these activated DLN cells. Anti-tumor necrosis factor produced a statistically significant but weaker inhibition of tumor regression. IFN-gamma, but not tumor necrosis factor alpha, could be shown to be secreted by activated DLN cells in vitro in response to specific tumor. Secretion of IFN-gamma was primarily a function of CD8+ T-cells. IFN-gamma was not directly cytotoxic to sarcoma cells in vitro. Moreover, tumor cells incubated with IFN-gamma were not more susceptible to lysis by activated DLN cells. However, recombinant murine IFN-gamma had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [3H]thymidine uptake assay. Similarly, supernatants obtained from DLN/autologous tumor cocultures markedly inhibited MCA-105 proliferation; this antiproliferative effect was abrogated by the addition of anti-IFN-gamma antibody to the cultures. These results suggest that secretion of IFN-gamma by adoptively transferred DLN cells plays an essential role in tumor rejection. The dominant effect of IFN-gamma may be its demonstrated antiproliferative activity.

Topics: Adjuvants, Immunologic; Animals; Antibodies; Bryostatins; Female; Immunoglobulin G; Immunotherapy, Adoptive; Interferon-gamma; Ionomycin; Lactones; Lung Neoplasms; Lymph Nodes; Lymphocyte Activation; Macrolides; Methylcholanthrene; Mice; Mice, Inbred C57BL; Sarcoma, Experimental; T-Lymphocytes; Tumor Necrosis Factor-alpha

Adoptive immunotherapy in humans may be limited by the lack of autologous tumor cells to activate and expand tumor-specific T cells. Pharmacologic manipulation of protein kinase C (PKC) and intracellular calcium may substitute for tumor antigen and stimulate T cells for adoptive immunotherapy. In the present study, we evaluated the ability of the PKC activator Bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to activate lymphocytes obtained from popliteal lymph nodes (DLN) draining an MCA-105 footpad tumor. The adoptive transfer of B/I-stimulated DLN cells eradicated MCA-105 pulmonary metastases. These lymphocytes do not require concomitant IL-2 administration to mediate regression of lung metastases. Three days after intrasplenic injection of tumor cells and splenectomy, mice were given iv injections of B/I-stimulated DLN cells. Adoptive immunotherapy with these cells induced regression of established liver metastases. In an intradermal tumor model, the adoptive transfer of B/I-stimulated MCA-105 DLN cells cured mice of MCA-105 intradermal (id) tumors, but did not induce regression of MCA-206 tumors. Mice cured of MCA-105 id tumors were protected against MCA-105, but not MCA-203, tumor challenge in the footpad 7 weeks after adoptive immunotherapy.

Topics: Animals; Antineoplastic Agents; Bryostatins; Female; Immunotherapy, Adoptive; Lactones; Liver Neoplasms, Experimental; Lung Neoplasms; Lymphocyte Activation; Macrolides; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; T-Lymphocytes

12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin 1 are activators of protein kinase C (PKC). TPA is a potent inhibitor of the growth of A549 cells, while bryostatin 1 exerts a weak antiproliferative effect upon this cell line. We tested the hypothesis that the PKC inhibitor staurosporine (STAU) can interfere with the effects of TPA or bryostatin 1 on A549 cells. STAU alone arrested A549 cell growth effectively with an IC50 of 0.65 nM as determined by cell counting after incubation for 96 hr. It also caused the release of lactate dehydrogenase from cells with an IC50 of 18.4 nM. On incubation with cells for up to 8 hr, STAU (100 nM) alone did not reduce thymidine incorporation into cells. However, it partially abrogated the inhibition of DNA synthesis caused by TPA or bryostatin 1 (10 nM). The IC50 for inhibition by STAU of the activity of PKC purified from A549 cells was 6.1 nM. Localization and levels of PKC were studied by Western blot and phorbol ester receptor binding analyses. STAU (100 nM) did not prevent the TPA-induced rapid redistribution of PKC to the cell membrane, but instead increased it by 25%. The PKC downregulation caused by TPA was not reduced in the presence of STAU. The results suggest that (i) PKC activation is involved in growth inhibition caused by TPA or bryostatin 1 in A549 cells, and (ii) subcellular localization or levels of PKC can be pharmacologically manipulated even under conditions of inhibited kinase function.

Topics: Alkaloids; Bryostatins; Cell Division; DNA; Down-Regulation; Enzyme Activation; Humans; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate

We examined the ability of bryostatin 1 (Bryo), a novel protein kinase C activator, plus ionomycin (Io), a calcium ionophore, to activate T-cells with specific antitumor activity. Lymphocytes from the draining lymph nodes (DLN) of MCA-105 tumor-bearing host mice were stimulated with Bryo/Io, either fresh or after in vitro stimulation with autologous tumor, and then were incubated in interleukin-2 at 20 units/ml. Lymphocytes sensitized with tumor cells in vitro and then stimulated with Bryo/Io exhibited significant expansion (12-fold) after a total of 3 weeks in culture and moderate cytolytic activity (40% at an effector:tumor cell ratio of (80:1) and were exclusively CD8+ T-cells. DLN cells activated immediately with Bryo/Io, without tumor antigen sensitization in vitro, displayed marked growth (130-fold expansion) over 3 weeks in culture, had weak cytolytic activity (8% at an effector:tumor ratio of 80:1), and were a mixed population of CD8+ and CD4+ cells. Despite the differences in phenotypes and in cytotoxicity, both groups of DLN cells were highly effective in vivo against MCA-105 pulmonary metastases. Bryo/Io-activated DLN cells from MCA-105 tumor-bearing hosts had no therapeutic efficacy against B16 melanoma or MCA-203 sarcoma metastases. Lymph node cells from normal mice and non-draining lymph node cells from tumor-bearing hosts could be expanded with Bryo/Io to a degree similar to that of DLN cells but had no antitumor activity. Phenotypic analyses and in vitro and in vivo depletion studies demonstrate that CD8+ cells mediated tumor regression.

Topics: Animals; Antibodies, Monoclonal; Antigens, Surface; Antineoplastic Agents; Bryostatins; Cytotoxicity, Immunologic; Female; Immunotherapy, Adoptive; Lactones; Lung Neoplasms; Lymphocyte Activation; Lymphocyte Depletion; Macrolides; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasms, Experimental; Phenotype; T-Lymphocyte Subsets; T-Lymphocytes

The growth of human-derived A549 lung carcinoma cells is inhibited by activators of protein kinase C (PKC) such as 12-O-tetradecanoylphorbol- 13-acetate (TPA). In this study, the effect of serum deprivation on TPA-induced growth retardation has been investigated. Cells cultured with 10% FCS and TPA (10(-8) M) stopped growing for 6 days, whereas inhibition of DNA synthesis caused by TPA in cells which were grown in medium containing the serum substitute ultraser lasted for less than 48 hr. The ability of cells to respond to the growth-inhibitory potential of TPA decreased with decreasing amounts of FCS in the cellular medium. Addition of fetuin or epidermal growth factor (EGF) to incubates with serum-deprived cells increased the ability of TPA to affect growth, but addition of platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) or retinoic acid (RA) was without effect. Growth arrest caused by bryostatin I, another PKC activator, was equally transitory in serum-supplemented and serum-deprived cells. Cytosol of serum-deprived cells contained only 32% of specific phorbol ester binding sites compared to cells grown with FCS; PKC enzyme activity and immunodectable protein were similarly reduced in cells grown without FCS. There was no difference in rate of TPA-induced down-regulation of PKC activity and cytosolic phorbol ester receptor sites between cells grown with or without serum.

Topics: alpha-Fetoproteins; Animals; Antineoplastic Agents; Blood; Bryostatins; Cattle; Cell Division; Culture Media; DNA Replication; Enzyme Activation; Epidermal Growth Factor; Humans; Kinetics; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin

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.

Topics: Animals; Antineoplastic Agents; Bryostatins; Cytotoxicity, Immunologic; Female; Killer Cells, Lymphokine-Activated; Lactones; Lung Neoplasms; Macrolides; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Spleen; Tetradecanoylphorbol Acetate

Activators of protein kinase C (PKC), such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatins 1 and 2, inhibit the growth of A549 cells. At high concentrations the bryostatins do not affect cell growth. Here the hypothesis has been tested that modulation of A549 cell growth is the consequence of agent-induced changes in location or extent of cellular PKC activity. PKC activity was measured after semi-purification with nondenaturing polyacrylamide gel electrophoresis in the cytosol and the particulate fraction of A549 cells. When cells were exposed to TPA or mezerein, PKC activity underwent rapid and concentration-dependent translocation from the cytosol to the membrane. TPA at 0.1 microM or mezerein at 1 microM caused almost complete translocation within 30 min. Incubation with bryostatins 1 or 2 also led to enzyme translocation, which was, however, much weaker than that observed with the tumor promoters. Neither 4 alpha-phorboldidecanoate nor the synthetic diacylglycerols 1,2-sn-dioctanoylglycerol or 1-oleoyl-2-acetyl-sn-glycerol mimicked TPA in this way. Exposure of cells to TPA or the bryostatins for longer than 30 min caused the gradual disappearance of total cellular PKC activity. PKC downregulation was concentration dependent and complete after 24 h. A549 cells which had acquired temporary resistance toward the growth-arresting potential of TPA were completely devoid of any measurable PKC activity. The bryostatins were potent inhibitors of the binding of [3H]phorbol-12,13-dibutyrate to its receptors in intact cells, and the inhibition was dependent on bryostatin concentration. The results support the contention that PKC is involved in the mediation of growth inhibition caused by TPA or the bryostatins. However, the relationship between growth arrest and PKC translocation or downregulation seems to be a complex one.

Topics: Antineoplastic Agents; Bryostatins; Cell Line; Cytosol; Humans; Kinetics; Lactones; Lung Neoplasms; Macrolides; Membranes; Protein Kinase C; Tetradecanoylphorbol Acetate

Phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit the growth of A549 human lung carcinoma cells at non-toxic concentrations, whereas 1-oleoyl-2-acetylglycerol and 1,2-dioctanoylglycerol, synthetic analogues of the physiological ligands of protein kinase C (PKC), do not. Experiments were conducted to test the hypothesis that other activators of PKC are capable of interfering with A549 cell growth. The non-phorboid tumour promotor mezerein mimicked the growth-inhibitory effect of TPA in that it arrested growth for 5 days, after which cells proliferated again in the continued presence of the agent. TPA was 20 times more potent as a growth inhibitor than was mezerein. Bryostatin 1 at 10 nM and bryostatin 2 at 100 nM also arrested A549 cell growth and inhibited DNA replication as measured by incorporation of [methyl-3H]-thymidine into cells. Inhibition of DNA synthesis to between 90 and 75% of control values developed during the first hour of incubation of the cells with TPA, mezerein or the bryostatins. The extent of inhibition changed little during the subsequent 5 hr of incubation, after which it increased further to reach maximal values within 12 hr. At concentrations above those which caused maximal growth inhibition, the bryostatins abolished both their own inhibition of DNA synthesis and the anti-replicative effect of TPA and mezerein. The results show that activators of PKC other than phorbol esters are capable of inhibiting the growth of A549 cells. The bryostatins not only interfere with A549 cell growth but can also counter the growth-inhibitory effect of PKC activators, presumably via interaction with a target separate from the phorbol ester receptor site.

Topics: Animals; Bryostatins; Cell Division; Diterpenes; DNA Replication; Enzyme Activation; Lactones; Lung Neoplasms; Macrolides; Protein Kinase C; Terpenes; Tetradecanoylphorbol Acetate; Time Factors