Human tumor bone metastasis model in athymic nude rats.
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Animals↗
Biomedical subjects
K Olden
Publications and source records attributed to K Olden.
Swainsonine modulation of protein kinase C activity in murine peritoneal macrophages.
The level of Ca2+, phospholipid-dependent, protein kinase C (PKC) activity in murine peritoneal macrophages treated with swainsonine, an indolizidine alkaloid, has been investigated. The present studies are based on our recent report that murine peritoneal macrophages are activated by swainsonine (Grzegorzewski, K.; Newton, S.A.; Akiyama, S.K.; Sharrow, S.; Olden, K.; White, S.L., Cancer Commun. 1:373-379, 1989). Presently, we have demonstrated that macrophages treated with swainsonine exhibited a substantial increase in PKC activity. The activity was enhanced as much as 4- to 5-fold over that obtained in untreated macrophages and was inhibited by H-7 (1-[5-isoquinoline sulphonyl]-2-methylpiperazine), D-sphingosine, or a monoclonal antibody specific for the active site of PKC. This represents the first report to demonstrate an effect of swainsonine on a second messenger system known to be involved in tumor promotion and macrophage activation. Elevation of PKC activity occurred much more slowly in swainsonine-treated cells than in cells treated with agents known to activate PKC directly, e.g., PMA (4-beta-phorbol-12-beta-myristate-13-gamma-acetate) or gamma-interferon (IFN-gamma). Furthermore the increase in PKC activity was inhibited by alpha-amanitine and cycloheximide, inhibitors of RNA and protein synthesis, respectively. These results suggest that swainsonine enhancement of PKC activity occurred by an indirect and possibly protein-synthesis-dependent mechanism. Whatever its precise mechanism of action, swainsonine provides a potentially important new probe to evaluate PKC mediated events. Selective enhancement of PKC activity may be important not only in elucidating the role of PKC in tumor promotion or macrophage activation but, also, in contributing to development of therapeutic regimens.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Swainsonine inhibition of spontaneous metastasis.
We have previously shown that swainsonine, administered systemically to C57BL/6 mice, inhibited the pulmonary metastasis of iv injected B16-F10 melanoma cells by a mechanism involving interleukin-2 production and augmentation of natural killer cell activity. From this finding, which uses an "experimental metastasis" model system, we considered: (a) whether swainsonine would be effective in the inhibition of authentic or spontaneous metastasis; (b) whether the drug would also inhibit metastasis formation in organs other than the lungs; and (c) whether the drug would block the metastasis of tumor cells of different histological type or origin. Our data indicated that swainsonine effectively inhibited the spontaneous metastasis of B16-BL6 melanoma (by 88%) and M5076 reticulum sarcoma (by 95%) murine tumor cells to the lung and liver, respectively. In both cases, the antimetastatic activity of the drug increased as a function of the concentration in drinking water up to 3 micrograms/mL. These findings indicate that the antimetastatic activity of swainsonine is not limited to artificial or experimentally induced metastasis nor to a single tumor type or specific organ. The inhibition of metastasis is likely due to a combination of events, which are currently under investigation.
Alkaloids↗
Bromoconduritol treatment delays intracellular transport of secretory glycoproteins in human hepatoma cell cultures.
Previous studies in our laboratory have shown that specific glycan structures are required for the normal secretion of some glycoproteins. Bromoconduritol is known to inhibit the removal of the innermost glucose moiety from the Glc3Man9(GlcNAc)2 precursor of N-linked glycoproteins. We have used this inhibitor to investigate the possible role of glycan structure in the intracellular transport of secretory glycoproteins of Hep G2 cultures. Cells were pretreated with 1mM bromoconduritol for 1h, pulsed with [35S]-methionine for 10min and chased for varying intervals. Specific glycoproteins and albumin were immunoprecipitated from the cell lysate and medium. We found that bromoconduritol-treatment inhibited the secretion of alpha 1-protease inhibitor, ceruloplasmin, alpha 2-macroglobulin, transferrin, and alpha-fetoprotein. Apparently, the glucosylated high-mannose intermediate is not secreted, since glycoproteins in the medium are of complex form. We conclude that the removal of the innermost glucose residue from secretory glycoprotein represents an important regulatory step in the intracellular transport pathway.
Carcinoma, Hepatocellular↗
Accumulation of unglycosylated liver secretory glycoproteins in the rough endoplasmic reticulum.
Previous studies in this laboratory (1) have shown that tunicamycin-treatment inhibits the secretion of three secretory glycoproteins--alpha 2-macroglobulin, ceruloplasmin, and alpha 1-protease inhibitor in human hepatoma (Hep G2) cell cultures. In the present study, we have investigated (i) their site of accumulation within the endoplasmic reticulum/Golgi pathway, and (ii) the solubility characteristics of these unglycosylated proteins. Using percoll density gradient centrifugation, we found that tunicamycin-treatment markedly inhibited the transport of alpha 2-macroglobulin, ceruloplasmin and alpha 1-protease inhibitor from the rough endoplasmic reticulum. However, there was no detectable changes in their solubility properties as both the glycosylated and unglycosylated species were associated with the 100,000 xg supernatant fraction following disruption of the microsomal fraction (i) with 0.2% Triton X-100 and (ii) by repeated freeze-thaw cycles. Also no evidence of protein aggregation was detected by liquid chromatography of the unglycosylated proteins on Bio-Gel A-1.5 column.
Biological Transport↗
Asparagine-linked oligosaccharides and tumor metastasis.
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Animals↗
Role of fibronectin in adhesion, migration, and metastasis.
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Amino Acid Sequence↗
Induction of macrophage tumoricidal activity, major histocompatibility complex class II antigen (Iak) expression, and interleukin-1 production by swainsonine.
Previous studies in our laboratory have shown that the reported antitumor activity of systemically administered swainsonine, an indolizidine alkaloid, is due at least in part to immune modulation involving effector cells (Humphries, M.J.; Matsumoto, K; White, S.L.; Olden, K. Cancer Res. 48:1410-1415; 1988 and White, S. L.; Schweitzer, K.; Humphries, M.J.; Olden, K. Biochem. Biophys. Res. Commun. 150:615-625; 1988). In this report, studies are presented to show that swainsonine was effective in activating peritoneal macrophages to cytotoxicity against tumor cells. Stimulation of tumoricidal activity of macrophages was associated with increased secretion of interleukin-1 (IL-1) and expression of the Iak major histocompatibility complex (MHC) antigen on the cell surface. The 3-fold stimulation of cytotoxicity observed in these in vivo studies was comparable to that obtained with Corynebacterium parvum, a commonly used in vivo activating agent. The in vitro incubation of thioglycollate-elicited peritoneal macrophages with swainsonine consistently resulted in levels of activation (6- to 8-fold) comparable to that obtained by treatment with known in vitro macrophage activating agents such as lipopolysaccharide (LPS) or recombinant gamma-interferon (rIFN-gamma). The stimulation observed by using swainsonine in combination with LPS was additive, suggesting different mechanisms of action. These studies have important implications not only for treatment of cancer, infectious diseases, and immune suppressive disorders, but also for elucidation of the mechanism of macrophage activation.
Alkaloids↗
Swainsonine: a new antineoplastic immunomodulator.
Swainsonine, an indolizidine alkaloid with immunomodulatory activity, has been found to be effective in inhibiting metastatic dissemination and growth of primary tumors of both murine and human origins. The unique ability of swainsonine to exhibit antimetastatic, anti-proliferative, and immunomodulatory activity imparts this drug a promising future in cancer therapy.
Adjuvants, Immunologic↗
Experimental approaches for the prevention of hematogenous metastasis.
With improvements in surgical procedures and cure rates, the probable course of cancer for the majority of patients is now largely determined by metastasis rather than growth of the primary tumor itself. Thus, metastasis has received increasing attention over the past decade. These studies have led to the identification of several of the molecular events crucial for metastatic dissemination, information which is now being used to design therapeutic strategies to inhibit metastasis formation. Even though the molecular events involved in the dissemination of malignant disease are only partially known, several promising agents are now being tested for their capacity to limit the spread of cancer. A few clinical trials have shown benefit in prolonging survival and disease-free state, particularly when such therapy is employed on an adjuvant basis.
Antineoplastic Agents↗
Augmentation of murine natural killer cell activity by swainsonine, a new antimetastatic immunomodulator.
Swainsonine, an indolizidine alkaloid, has been found to inhibit the experimental metastasis of B16-F10 melanoma cells when administered systemically to syngeneic C57BL/6 mice. The inhibition was both potent and dose dependent with greater than or equal to 80% reduction in pulmonary colonization being observed after only 24-h exposure to 3 micrograms/ml of swainsonine in drinking water. In contrast, the inhibitory activity of swainsonine was completely abrogated when assays were performed in mice depleted of their natural killer (NK) cell activity either experimentally (anti-asialo-GM1 antibody- or cyclophosphamide-treated C57BL/6 mice) or as a result of genetic mutation (homozygous C57BL/6bg/bg beige mice). Swainsonine elicited a 32.0% increase in spleen cell number 2 days after administration and induced a concomitant 2- to 3-fold increase in splenic NK cell activity. These results indicate (a) an absolute requirement for a functional NK cell population in order for swainsonine to exert its inhibitory effects on experimental metastasis, and (b) that the antimetastatic activity of swainsonine is mediated primarily through the ability of the drug to augment NK cell reactivity. On the basis of these findings, swainsonine can be classified as a new immunomodulator that has the ability, at least in a prophylactic setting, to block tumor metastasis.
Adjuvants, Immunologic↗
Stimulation of DNA synthesis in murine lymphocytes by the drug swainsonine: immunomodulatory properties.
Swainsonine, an inhibitor of Golgi alpha-mannosidase II, has recently been shown to have potent antimetastatic activity in experimental metastasis assays. In the case of systemic administration, the possible mechanism of action is unknown; the results reported here indicate that it can be explained at least in part by swainsonine stimulation of lymphocyte proliferation. In the present experiments, the standard in vitro mitogenic stimulation assay was used to test the effect of swainsonine on spleen cells. Treatment of spleen cell cultures with the optimum concentrations of the drug enhanced proliferation by 80-146%, as measured by (3H) thymidine incorporation, relative to untreated cultures. Similarly, when spleen cell cultures were prepared from mice maintained on swainsonine-supplemented drinking water, proliferation was stimulated at least 3-fold relative to cultures derived from animals maintained on regular water. The enhanced mitogenesis is apparently not directly related to increased expression of Concanavalin A (Con A) binding sites, since swainsonine induced mitogenesis is not inhibited by alpha-methyl-mannoside in contrast to Con A induced mitogenesis which is completely inhibited. These results suggest that the antimetastatic effect of systemically administered swainsonine is at least in part related to its ability to enhance proliferation of those specific cell populations involved in immune surveillance. This represents the first demonstration of a mechanism of action of swainsonine on the immune system.
Adjuvants, Immunologic↗
The cell interaction sites of fibronectin in tumour metastasis.
Adhesion to specific extracellular matrix molecules appears to be an important prerequisite for successful target organ colonization by metastasizing tumour cells. Interference in the adhesive function of malignant cells with antiadhesive agents is therefore one potential approach for preventing metastasis. Recently, synthetic peptides taken from the cell interaction sites of fibronectin have been characterized as inhibitors of cellular adhesion in vitro. Using these antiadhesive probes we have examined the role of cell adhesion to fibronectin in tumour metastasis using the B16-F10 murine melanoma model system. Two sequences from the IIICS cell-binding domain, the 25-mer CS1 peptide and the tetrapeptide Arg-Glu-Asp-Val (REDV), had no detectable activity, but the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS), an active sequence from the central cell-binding domain, exhibited potent, dose-dependent inhibition, indicating a role for this cell recognition determinant in tumour metastasis. Under appropriate conditions GRGDS treatment afforded remarkable protection to the host; mice injected with melanoma cells and peptide were still alive 15 months after injection whereas mice injected with melanoma cells alone died within six weeks. Kinetic analyses of the retention of tumour cells in the lungs and of the vascular clearance rate of labelled GRGDS predict an early time frame of activity for the peptide. From the results of a variety of in vitro invasion and migration assays it appears that GRGDS may interfere with multiple, fibronectin-mediated adhesive and migratory events at different points of the metastatic cascade. In preliminary studies designed to optimize the therapeutic usefulness of GRGDS-like agents, peptide conjugates have been found to possess enhanced antiadhesive activity as well as an extended vascular clearance rate. In the future, therefore, these or related peptide derivatives may be potentially useful agents for the prevention of tumour metastasis.
Animals↗
Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment.
Fibronectin contains at least two domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types, including fibroblasts. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in fibronectin-mediated neurite extension of neurons from chick embryo dorsal root and sympathetic ganglia. In studies using explanted ganglia, both fl3, a 75-kD tryptic fragment of human plasma fibronectin containing the central cell-binding domain, and CS1-IgG, a synthetic peptide-IgG conjugate containing the principal cell adhesion site from the IIICS, supported neurite outgrowth after adsorption onto the substrate. The maximal activities of fl3 and CSl-IgG were 45-55% and 25-30% that of intact fibronectin, respectively. Co-coating of the substrate with f13 and CS1-IgG produced an additive stimulation of neurite outgrowth, the extent of which approached that obtained with fibronectin. Similar results were obtained with purified neuronal cell preparations isolated by tryptic dissociation of dorsal root ganglia. In complementary studies, blockage of the adhesive function of either the central cell-binding domain (with mAb 333, an antiadhesive monoclonal antibody) or the IIICS (with CS1 peptide), resulted in approximately 60 or 30% reduction in fibronectin-mediated neurite outgrowth, respectively. When tested in combination, the inhibitory activities of mAb 333 and CSl were additive. From these results, we conclude that neurons from the peripheral nervous system can extend neurites on both the central cell-binding domain and the IIICS region of fibronectin, and that these cells are therefore the first normal, embryonic cell type shown to adhere to the IIICS. These results suggest that spatiotemporal fluctuations in the alternative mRNA splicing of the IIICS region of fibronectin may be important in regulation of cell adhesive events during development of the peripheral nervous system.
Animals↗
Use of antiadhesive peptide and swainsonine to inhibit metastasis.
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Alkaloids↗
Investigation of the biological effects of anti-cell adhesive synthetic peptides that inhibit experimental metastasis of B16-F10 murine melanoma cells.
The experimental metastasis of B16-F10 murine melanoma cells is blocked by the anti-cell adhesive pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) derived from the central cell-binding domain of fibronectin. In this report, we show that peptide treatment substantially extends the survival time for mice injected intravenously with B16-F10 cells (8/8 vs. 0/8 mice alive at 150 d), thereby demonstrating the potential efficacy of GRGDS treatment in protection against metastatic colonization. We have also examined the specificity of GRGDS activity by testing a series of related homologues for their effects on experimental metastasis. The overall profile of the relative inhibitory activities of these peptides closely matched their previously established capacity to disrupt adhesion in vitro. Lung retention studies with radiolabeled B16-F10 cells revealed an accelerated rate of cell loss from the lung 0-6 h after coinjection with the active peptide GRGDS. This early effect of GRGDS was consistent with its short circulatory half-life, which was found to be 8 min. Taken together, these results suggest that peptide-mediated inhibition of pulmonary colonization is due to interference with B16-F10 cell adhesion to structures in the target organ. Possible peptide interference in tumor cell-blood cell interactions was examined in order to assess (a) possible biological side-effects of peptide treatment and (b) whether such interactions might be an alternative mechanism for GRGDS-mediated inhibition of pulmonary colonization. GRGDS was found to retain full inhibitory activity when coinjected with B16-F10 cells into mice in which platelet function was impaired by acetylsalicylic acid treatment or into thrombocytopenic mice treated with antiplatelet serum (76-93% inhibition of colony formation). These data suggest that platelet involvement in the effects of the peptide is minimal. Similarly, GRGDS was also found to be a potent inhibitor of experimental metastasis in natural killer (NK) cell-deficient beige mice (86% inhibition), thereby discounting the possibility that GRGDS artifactually enhanced NK cell activity. We conclude as a result of these studies that cell-binding fibronectin peptides are specific inhibitors of experimental metastasis that prolong survival, that they appear to function by blocking the adhesion of B16-F10 cells to structures in the target organ, and that they do not appear to act through side effects on certain metastasis-related blood cell functions. In the future, derivatives of fibronectin peptides may be potentially useful prophylactic agents for interfering with the process of metastasis.
Animals↗
Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion.
The principal region of the human plasma fibronectin molecule mediating the adhesion of melanoma cells appears to be the alternatively spliced type III connecting segment (IIICS (Humphries, M. J., Akiyama, S. K., Komoriya, A., Olden, K., and Yamada, K. M. (1986a) J. Cell Biol., in press]. A series of overlapping synthetic peptides spanning the entire IIICS (CS peptides) were examined for their effects on B16-F10 melanoma cell adhesion to the parent fibronectin molecule. Two nonadjacent CS peptides, designated CS1 and CS5, were inhibitory. In contrast, neither inhibited fibronectin-mediated spreading of fibroblastic baby hamster kidney cells. When N-terminal cysteine derivatives of the CS peptides were conjugated to IgG by covalent cross-linking with N-succinimidyl-3(2-pyridyldithio)propionate, both the CS1 and CS5 conjugates promoted B16-F10 melanoma cell spreading. All conjugates were inactive for spreading of baby hamster kidney cells, confirming the cell type specificity of the IIICS adhesion site. Determination of the amounts of CS peptide required to support melanoma cell adhesion revealed that the activity of CS1 was only 2.4-fold lower than that of the intact fibronectin molecule. CS5 was approximately 320-fold less active than fibronectin, suggesting that the CS1 region may be the major site of interaction with the melanoma cell surface. The adhesion-promoting activities of CS1-IgG and CS5-IgG were additive as were the inhibitory activities of the free peptides for B16-F10 cell spreading on fibronectin. These findings suggest that both regions of the IIICS can function separately or together in mediating the interaction of melanoma cells with fibronectin. Since CS1 and CS5 are each found in separate alternatively spliced regions of the IIICS, it is conceivable that the adhesion-promoting activity of fibronectin for different cell types may be under complex regulation.
Amino Acid Sequence↗
Investigation of the antimetastatic effects of agents that inhibit cell adhesion or protein glycosylation.
In this overview the authors describe their recent attempts to specifically interfere with the metastatic spread of B16-F10 melanoma cells. Using the experimental metastasis model system, inhibitory effects of (1) coinjection of cells with synthetic peptides derived from the glycoprotein fibronectin, which possess the ability to disrupt cell adhesion, and (2) treatment of cells with inhibitors of protein glycosylation and oligosaccharide processing have been examined.
Animals↗