PI3K/Akt signalling is required for the attachment and spreading, and growth in vivo of metastatic scirrhous gastric carcinoma.

BACKGROUND: PI3K/Akt (PKB) pathway has been shown in several cell types to be activated by ligands to cell surface integrins, leading to the metastasis of tumour cells. The signalling pathways involved in the metastatic spread of human scirrhous gastric carcinoma cells have not been defined. METHODS: The role of the PI3K/Akt pathway in an extensive peritoneal-seeding cell line, OCUM-2MD3 and a parental cell line, OCUM-2M, was investigated by assessing in vitro adhesion and spreading assay, and in vivo peritoneal metastatic model. We also examined the correlation of PI3K/Akt pathway with integrin signals by immunoprecipitations, using cells by transfection with mutant p85 (Δp85). RESULTS: Adhesiveness and spreading of OCUM-2MD3 cells on collagen type IV was significantly decreased by PI3K inhibitors and expression of mutant p85, but not by inhibitors of protein kinase C (PKC) or extracellular signal-regulated kinase (ERK). Immunoprecipitation studies indicated that the PI3K/Akt pathway was associated with integrin signalling through Src and vinculin. In an in vivo experimental metastasis model, p85 inhibition reduced peritoneal metastasis of OCUM-2MD3 cells. CONCLUSION: PI3K/Akt signalling may be required for integrin-dependent attachment and spreading of scirrhous gastric carcinoma cells, and would be translated into generating better strategies to optimise their use in cancer clinical trials.

Inhibition of transforming growth factor-ß-activated kinase-1 blocks cancer cell adhesion, invasion, and metastasis.

BACKGROUND: Tumour cell metastasis involves cell adhesion and invasion, processes that depend on signal transduction, which can be influenced by the tumour microenvironment. N-6 polyunsaturated fatty acids, found both in the diet and in response to inflammatory responses, are important components of this microenvironment. METHODS: We used short hairpin RNA (shRNA) knockdown of TGF-ß-activated kinase-1 (TAK1) in human tumour cells to examine its involvement in fatty acid-stimulated cell adhesion and invasion in vitro. An in vivo model of metastasis was developed in which cells, stably expressing firefly luciferase and either a control shRNA or a TAK1-specific shRNA, were injected into the mammary fat pads of mice fed diets, rich in n-6 polyunsaturated fatty acids. Tumour growth and spontaneous metastasis were monitored with in vivo and in situ imaging of bioluminescence. RESULTS: Arachidonic acid activated TAK1 and downstream kinases in MDA-MB-435 breast cancer cells and led to increased adhesion and invasion. Knockdown of TAK1 blocked this activation and inhibited both cell adhesion and invasion in vitro. Tumour growth at the site of injection was not affected by TAK1 knockdown, but both the incidence and extent of metastasis to the lung were significantly reduced in mice injected with TAK1 knockdown cells compared with mice carrying control tumour cells. CONCLUSION: These data demonstrate the importance of TAK1 signalling in tumour metastasis in vivo and suggest an opportunity for antimetastatic therapies.

Linoleic acid enhances angiogenesis through suppression of angiostatin induced by plasminogen activator inhibitor 1.

BACKGROUND: The intake of dietary fatty acids is highly correlated with the risk of various cancers. Linoleic acid (LA) is the most abundant polyunsaturated fat in the western diet, but the mechanism(s) by fatty acids such as LA modulate cancer cells is unclear. In this study, we examined the role of LA in various steps in gastric cancer progression. METHODS: The difference in gene expression between LA-treated and untreated OCUM-2MD3 gastric carcinoma cells was examined by mRNA differential display. The involvement of candidate genes was examined by oligo- and plasmid-mediated RNA interference. Biological functions of several of these genes were examined using in vitro assays for invasion, angiogenesis, apoptosis, cell viability, and matrix digestion. Angiogenesis in vivo was measured by CD-31 immunohistochemistry and microvessel density scoring. RESULTS: LA enhanced the plasminogen activator inhibitor 1 (PAI-1) mRNA and protein expression, which are controlled by PAI-1 mRNA-binding protein. LA-stimulated invasion depended on PAI-1. LA also enhanced angiogenesis by suppression of angiostatin, also through PAI-1. LA did not alter cell growth in culture, but increased dietary LA-enhanced tumour growth in an animal model. CONCLUSION: Our findings suggest that dietary LA impacts multiple steps in cancer invasion and angiogenesis, and that reducing LA in the diet may help slow cancer progression.

Elevated dietary linoleic acid increases gastric carcinoma cell invasion and metastasis in mice.

BACKGROUND: Dietary (n-6)-polyunsaturated fatty acids influence cancer development, but the mechanisms have not been well characterised in gastric carcinoma. METHODS: We used two in vivo models to investigate the effects of these common dietary components on tumour metastasis. In a model of experimental metastasis, immunocompromised mice were fed diets containing linoleic acid (LA) at 2% (LLA), 8% (HLA) or 12% (VHLA) by weight and inoculated intraperitoneally (i.p.) with human gastric carcinoma cells (OCUM-2MD3). To model spontaneous metastasis, OCUM-2MD3 tumours were grafted onto the stomach walls of mice fed with the different diets. In in vitro assays, we investigated invasion and ERK phosphorylation of OCUM-2MD3 cells in the presence or absence of LA. Finally, we tested whether a cyclooxygenase (COX) inhibitor, indomethacin, could block peritoneal metastasis in vivo. RESULTS: Both the HLA and VHLA groups showed increased incidence of tumour nodules (LA: 53%; HLA: 89%; VHLA: 100%; P<0.03); the VHLA group also displayed increased numbers of tumour nodules and higher total volume relative to LLA group in experimental metastasis model. Both liver invasion (78%) and metastasis to the peritoneal cavity (67%) were more frequent in VHLA group compared with the LLA group (22% and 11%, respectively; P<0.03) in spontaneous metastasis model. We also found that the invasive ability of these cells is greatly enhanced when exposed to LA in vitro. Linoleic acid also increased invasion of other scirrhous gastric carcinoma cells, OCUM-12, NUGC3 and MKN-45. Linoleic acid effect on OCUM-2MD3 cells seems to be dependent on phosphorylation of ERK. The data suggest that invasion and phosphorylation of ERK were dependent on COX. Indomethacin decreased the number of tumours and total tumour volume in both LLA and VHLA groups. Finally, COX-1, which is known to be an important enzyme in the generation of bioactive metabolites from dietary fatty acids, appears to be responsible for the increased metastatic behaviour of OCUM-2MD3 cells in the mouse model. CONCLUSION: Dietary LA stimulates invasion and peritoneal metastasis of gastric carcinoma cells through COX-catalysed metabolism and activation of ERK, steps that compose pathway potentially amenable to therapeutic intervention.

Collagen can modulate cell interactions with fibronectin.

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.

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.

Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites.

Virus-transformed fibroblasts show an increased production of proteases as well as loss of extracellular adhesive proteins. To determine whether these transformation-associated events are related, we investigated the capacity of Rous sarcoma virus-transformed cells (embryonic chick fibroblasts and mouse BALB/c 3T3) to degrade fibronectin by using a novel cross-linked protein substratum: fluorescence-labeled or radiolabeled fibronectin covalently linked to the surface of a fixed gelatin film. In serum-containing medium, the coupled fibronectin was not released when incubated without cells, and only a small amount was released when incubated with nontransformed cells. However, when transformed cells were seeded on the radiolabeled fibronectin-coupled substratum, there was a threefold increase in the time-dependent release of radioactivity into the medium. The released material was characterized as peptides with molecular sizes of less than 30,000 daltons. Correspondingly, growth of transformed cells on the rhodamine-fibronectin substratum resulted in the appearance of discrete negative fluorescent spots beneath the cells and along their migratory paths, whereas a uniform fluorescent carpet was detected with nontransformed cells. The release of radioactivity was partially inhibited by protease inhibitors, including alpha 2-macroglobulin, leupeptin, and benzamidine, but the negative fluorescent spots appeared unaffected by any of these inhibitors. However, both the release of radiolabeled peptides and the appearance of fluorescence-negative spots were inhibited by 1,10-phenanthroline at concentrations that did not affect cellular attachment and protein synthesis, thus supporting a role for proteases in localized degradation of fibronectin substratum. These fluorescence-negative spots coincided with sites of fibronectin disappearance as judged by indirect labeling with antibodies to cellular fibronectin. In addition, immunofluorescent analyses showed a correlation between vinculin localization and the negative fibronectin spots found under transformed cells, indicating that degradation occurs at cell substratum contact sites. These results can be correlated with other transformation-associated phenotypic changes, and are discussed in terms of the invasion of tumor cells into the extracellular matrix.

Inhibition of fusion of embryonic muscle cells in culture by tunicamycin is prevented by leupeptin.

The carbohydrate requirement for alignment and fusion of embryonic quail muscle cells has been examined in tissue culture by use of tunicamycin (TM). The mononucleated, spindle-shaped proliferating myoblasts were treated with TM at various times before fusion and differentiation into multinucleated muscle fibers capable of spontaneous contraction. Tm blocked protein glycosylation and expression of glycoproteins on the cell surface, and strongly inhibited fusion when added to cultures of differentiating muscle cells before the fusion "burst," but had no apparent effect on cell alignment. The inhibition of fusion was partially prevented when TM was administered in the presence of protease inhibitors such as leupeptin and pepstatin, but the inhibition of glycosylation was not prevented. Both glycosylation and fusion were completely restored to normal by the removal of the antibiotic from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by glycoproteins with asparagine-linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation. Our findings do not rule out the possibility that oligosaccharide units of surface glycolipids have some role in myoblast fusion.

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion.

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH-terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.

A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells.

Adhesive interactions between cells and the extracellular matrix occur at several stages of metastasis. Such interactions might be inhibited by synthetic peptide probes derived from the cell-binding regions of matrix molecules. Gly-Arg-Gly-Asp-Ser (GRGDS) is a pentapeptide sequence that appears to be critical for cell interaction with fibronectin. Coinjection of GRGDS with B16-F10 murine melanoma cells dramatically inhibited the formation of lung colonies in C57BL/6 mice. Two closely related control peptides, in which specific amino acids within the GRGDS sequence were transposed or substituted, displayed little or no activity. Inhibition by GRGDS was dose-dependent, noncytotoxic, and did not result from an impairment of cellular tumorigenicity. GRGDS may function by inhibiting tumor cell retention in the lung since radiolabeled B16-F10 tumor cells injected with the peptide were lost at a substantially greater rate than control cells.

Assessment of isocenter alignment during CT colonography: Implications for clinical practice.

INTRODUCTION: Optimization of image quality and patient radiation dose is achieved in part by positioning the patient at the isocenter of the CT gantry. The aim of this study was to establish whether there was increased isocenter misalignment (IM) in CT colonography (CTC) scans by comparing patient position during the prone part of a CTC to patient position during renal stone protocol CT (CT-KUB) and patient position during the supine part of a CTC to patient position during abdominopelvic CT (CT-AP). METHODS: Two hundred and twenty two consecutive outpatient adult CTC studies performed between January and December 2016 were retrospectively analyzed. Automated dose-tracking software was used to quantify IM in the x and y planes. Renal stone CT-KUB (n = 100) and standard CT-AP (n = 100) were used as comparison studies. RESULTS: IM during CTC was significantly greater in the y-axis compared with the x-axis for both prone (p = 0.002) and supine (p < 0.001) scanning. IM was significantly greater during prone CTC compared with CT-KUB (p = 0.008) and during supine CTC compared with CT-AP (p = 0.0001). IM was shown to be slightly greater in studies performed by more experienced radiographers (p = 0.04). IM was not associated with patient age, gender or size (p > 0.05 for all). CONCLUSION: Isocenter misalignment is greater during CT colonography compared with CT-KUB or CT-AP. Strategies for improving patient positioning could include radiographer education and automated patient centering solutions.

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.

Treatment of gastroparesis: a multidisciplinary clinical review.

This clinical review on the treatment of patients with gastroparesis is a consensus document developed by the American Motility Society Task Force on Gastroparesis. It is a multidisciplinary effort with input from gastroenterologists and other specialists who are involved in the care of patients with gastroparesis. To provide practical guidelines for treatment, this document covers results of published research studies in the literature and areas developed by consensus agreement where clinical research trials remain lacking in the field of gastroparesis.

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.

Protective effects of swainsonine on murine survival and bone marrow proliferation during cytotoxic chemotherapy.

We have investigated the ability of swainsonine, an indolizidine alkaloid with pleiotropic in vivo effects, to confer protection against the cytotoxic effects of both cell cycle-specific and cell cycle-nonspecific cytotoxic anticancer agents. The intraperitoneal administration of swainsonine decreased the lethality of methotrexate (MTX), fluorouracil (5-FU), cyclophosphamide (CPM), and doxorubicin (DOX) in non-tumor-bearing C57BL/6 mice. The increased survival rate was found to correlate with stimulation of bone marrow cell proliferation, as measured by increases in 1) bone marrow cellularity, 2) in vivo and in vitro colony-forming activity, and 3) engraftment efficiency. These responses were critically dependent on the dose, sequence, and timing of swainsonine administration. If these results are confirmed in humans, swainsonine may offer promise in future intensive chemotherapy programs, allowing increased dosage and/or frequency of administration of cytotoxic agents without increasing toxic effects in bone marrow.

Inhibition of experimental metastasis by castanospermine in mice: blockage of two distinct stages of tumor colonization by oligosaccharide processing inhibitors.

The extent of maturation of the oligosaccharide subunits of tumor cell glycoproteins appears to correlate with malignant potential, suggesting that modification of oligosaccharide structures may alter metastatic capacity. Castanospermine, a recently discovered inhibitor of glucosidase I, was tested for its effect on experimental metastasis of B16-F10 murine melanoma cells and was compared to treatment with swainsonine and tunicamycin. All three drugs block different steps in the pathway of glycoprotein processing yet each was a potent inhibitor of pulmonary colonization after i.v. injection of treated cells into C57BL/6 mice (greater than or equal to 80% inhibition). This result indicates a generality of inhibition of experimental metastasis by blockage of protein glycosylation or oligosaccharide processing and strongly implicates carbohydrate residues in at least one critical step of the metastatic cascade. Cytotoxic side effects could not account for the inhibitory activity. In order to identify a possible mechanism of inhibition of colonization, the adhesive behavior and pulmonary retention properties of B16-F10 cells treated with the above inhibitors were examined. Tunicamycin-treated B16-F10 cells exhibited poor adhesion to substrate-adsorbed fibronectin and laminin, whereas both castanospermine- and swainsonine-treated cells possessed near normal adhesive capacity; furthermore, the initial rate of loss of tunicamycin-treated cells from the lungs of mice was substantially greater than either control, castanospermine- or swainsonine-treated cells. These data suggest that these processing inhibitors can block experimental metastasis by at least two different mechanisms. The antimetastatic effect of tunicamycin may be related to interference in tumor cell-extracellular matrix interactions, whereas treatment with castanospermine or swainsonine appears to block at a stage distal to initial tumor cell arrest.

Regulation of the adhesion of a human breast carcinoma cell line to type IV collagen and vitronectin: roles for lipoxygenase and protein kinase C.

We have investigated the regulation of adhesion of metastatic human breast carcinoma cells to various protein substrates in the presence or absence of the protein kinase C (PKC) activator, 12-tetradecanoyl phorbol 13-acetate (TPA) or calcium ionophore A23187 (A23187). Both TPA and A23187 dramatically enhanced MDA-MB-435 cell adhesion to type IV collagen (collagen IV), vitronectin, and, to some extent, fibronectin and laminin. Adhesion to BSA and polylysine were not affected. TPA and A23187 induced substantial dose-dependent effects that were apparent after 30- and 60-min incubations, respectively, whereas a phorbol ester, which does not activate PKC, had no effect. A23187, but not TPA, induced a release of arachidonic acid (AA) from MDA-MB-435 cells. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, prevented A23187 and exogenous AA, but not TPA, from stimulating cell adhesion to collagen IV. In contrast, the increase in adhesion to vitronectin induced by A23187 and AA was, at best, only partially inhibited by nordihydroguaiaretic acid treatment. Calphostin C, a PKC inhibitor, blocked the stimulation of adhesion by A23187, exogenous AA, and TPA to both collagen IV and vitronectin. Together, these results suggest that calcium mobilization activates the release of AA and its metabolism through a lipoxygenase pathway leading to a rapid increase of MDA-MB-435 cell adhesion to collagen IV, whereas other mechanisms regulate adhesion to vitronectin. Finally, PKC activation, occurring downstream from calcium mobilization or the AA effects, is a key event involved in the regulation of adhesion to both proteins.

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.

Cis-polyunsaturated fatty acids stimulate beta1 integrin-mediated adhesion of human breast carcinoma cells to type IV collagen by activating protein kinases C-epsilon and -mu.

We have investigated the effects of various fatty acids (FAs) on integrin-mediated MDA-MB-435 breast carcinoma cell adhesion to type IV collagen (collagen IV) in vitro. Arachidonic acid (AA) and linoleic acid both induced a dose-dependent increase in cell adhesion to collagen IV with no significant increase in nonspecific adhesion to polylysine and BSA. Oleic acid (a monounsaturated FA), AA methyl ester, and linoelaidic acid (a trans-isomer of linoleic acid) failed to stimulate adhesion to collagen IV, suggesting that these effects required cis-polyunsaturation and a free carboxylic moiety and that they were not due to membrane perturbations. Calphostin C, a protein kinase C (PKC) inhibitor, blocked cis-polyunsaturated FA (cis-PUFA)-induced cell adhesion in a dose-dependent manner, suggesting a role for a calcium-dependent PKC in this signal transduction pathway. Immunoblotting revealed that cis-PUFAs induced the translocation of PKCepsilon and PKCmu, two of the novel PKC isozymes, from the cytosol to the membrane. In contrast, a conventional PKC isozyme, PKCalpha, as well as the atypical isozymes, PKCzeta and PKCiota, did not translocate after cis-PUFA treatment. Function-blocking antibodies specific for alpha1, alpha2, and beta1, integrin subunits inhibited cell adhesion to collagen IV, whereas antibodies to alpha3 and alpha5 did not. No increase in the expression of these integrins on the cell surface was detected after the incubation of cells with cis-PUFAs, suggesting that there is an increase in the activity, but not in the amount, of these beta1, integrins. Altogether, these data suggest that cis-PUFAs enhance human breast cancer cell adhesion to collagen IV by selectively activating specific PKC isozymes, which leads to the activation of beta1 integrins.