Loss of metastatic responsiveness to cell shape modulation in a newly characterized B16 melanoma adhesive cell variant

Repeated selection of an adherent subpopulation from B16-F1 melanoma cells growing in suspension culture on poly(hydroxyethylmethacrylate) [poly(HEMA)] coated plates resulted in the isolation of an adherent variant designated B16-A10. B16-A10 cells are more adherent to poly(hydroxyethylmethacrylate) coated plates than are B16-F1 cells and express an organized actin structure characteristic of highly adherent low metastatic cells as opposed to the poor cytoskeletal organization of B16-F1 cells. Upon growth in suspension, B16-A10 cells do not acquire the enhanced metastatic capability characteristic of B16-F1 cells and they express similar lung colonizing ability irrespective of the culture conditions. The increased metastatic ability of B16-F1 cells in suspension culture has previously been associated with the decreased accessibility of surface proteins to lactoperoxidase catalyzed iodination and with the increased expression of sialylated peanut agglutinin-binding oligosaccharides on these proteins. B16-A10 cells which show no cell shape induced increase in metastatic ability do not undergo alteration in either of these two properties in suspension culture. The absence of these two phenomena on B16-A10 cells grown in suspension indicates that they are interrelated and involved in the increased metastatic ability of B16-F1 cells grown in suspension.     

Inhibition of spontaneous and experimental metastasis by a new derivative of camptothecin,CPT-11, in mice

Summary The antimetastatic effect of a new water-soluble derivative of camptothecin, 7-ethyl-10-(4-(1-piperidino)-1-piperidino) carbonyloxy-camptothecin (CPT-11), were examined in several metastatic murine tumor systems. Intravenous (i.v.) injection of CPT-11 into BALB/c mice inhibited lung metastasis by i.v. inoculated, metastatic colonic adenocarcinoma 26 (C26) cells, C26NL-17, in BALB/c mice. This treatment was also effective in C57BL/6 mice against lung metastasis by i.v. inoculated B16-F10 and B16-BL6 cells, highly metastatic variants of the B16 melanoma. Furthermore, intraperitoneal (i.p.) injection of CPT-11 significantly inhibited the growth of C26NL-22 cells, a highly metastatic variant of C26, inoculated subcutaneously (s.c.) into the left front footpads of BALB/c mice. Also, i.p. or i.v. injection of CPT-11 effectively inhibited the growth of 3LL tumors inoculated s.c. into the hind footpads of C57BL/6 mice. Moreover, following s.c. inoculation of either C26NL-22 or 3LL cells, combined surgical excision of the primary tumor and either i.p. or i. v. CPT-11 injections given before or after surgery markedly inhibited the formation of pulmonary metastases. These results show that a new derivative of camptothecin, CPT-11, has a potent inhibitory effect against both spontaneous and experimental lung metastasis.     

Inhibition of macrophage Ia antigen expression by shed plasma membrane vesicles from metastatic murine melanoma lines

Shed plasma membrane-derived vesicles from metastatic variants of the murine B16 melanoma were examined for their ability to inhibit the induction of murine immune region-associated (Ia) antigen expression on macrophages, the initial step in the formation of an immune response. Membrane material that appears as a greater than 50 million-dalton fraction on column chromatography is found only in conditioned media from tumor cells and not in culture media from normal cells, such as murine 3T3 cells. Membrane vesicles from both metastatic variants B16-F1 (low lung colonizing) and B16-F10 (high lung colonizing) were taken up by macrophages; however, only membrane vesicles isolated from the B16-F10 cultures exhibited significant inhibitory activity for Ia induction. This inhibition appears to result from enhanced prostaglandin synthesis, since treatment with aspirin can reverse the membrane vesicle-induced inhibition. The inhibitory component(s) released into the media was demonstrated to be predominantly associated with membrane vesicles; however, the component(s) retained its activity after Triton X-100 treatment, indicating that the intact membrane vesicle was not necessary for the action of the inhibitory material. Treatments with heat (65 degrees C) and proteases (papain) indicated that the inhibitory component(s) is a heat-labile protein.     

Membrane lipids and enzymes of cultured high- and low-metastatic B16 melanoma variants

B16 melanoma cell variants were used to determine if the metastatic properties of these cells could be correlated to distinct plasma membrane, microsome, and mitochondrial membrane lipid compositions and membrane-bound enzyme activities in high- and low-metastatic cell variants, respectively. The high-metastatic B16-F10 melanoma cell membranes had lower cholesterol/phospholipid ratios, lower arachidonic acid content, lower polyunsaturated fatty acid content, higher phosphatidylcholine/phosphatidylethanolamine ratios, and higher succinate cytochrome c reductase activity than those of B16-F1 melanoma cell membranes. No differences in cholesterol/phospholipid ratio were noted in the mitochondria. Na+-K+-adenosinetriphosphatase activity and solubility of 5'-nucleotidase activity were also similar. The data indicate that the membrane lipid composition of B16-F10 melanoma cells is distinct from that of B16-F1 melanoma cells and may help to elucidate the molecular basis for the different metastatic properties of these cell lines in vivo.     

Inhibitory effect of Cordyceps sinensis on experimental hepatic metastasis of melanoma by suppressing tumor cell invasion

We investigated the anti-metastatic activity of a water extract of Cordyceps sinensis (WECS) using a model of mice injected with B16-F0 mouse melanoma cells into the spleen. WECS administered intraperitoneally reduced the number of metastatic surface nodules of B16-F0 cells in the liver of C57BL/6Cr mice in a dose-dependent manner, and significantly prolonged their survival. To identify the mechanism of the anti-metastatic effect of WECS, we examined its effects on hepatocyte growth factor (HGF)-accelerated invasion of B16-F0 cells using a chemo-invasion assay in vitro. As a result, WECS reduced HGF-accelerated B16-F0 cell invasion in a concentration-dependent manner. These findings suggest that WECS exerts an anti-metastatic action, in part by inhibiting the HGF-accelerated tumor invasiveness of mouse melanoma cells.     

Effects of tertiary amine local anesthetics on the blood-borne implantation and cell surface properties of metastatic mouse melanoma cells

Tertiary amine local anesthetics, such as dibucaine and tetracaine, have been found to reversibly modify the blood-borne implantation and experimental metastatic properties of B16 melanoma cells in syngeneic C57BL/6 mice. Local anesthetic treatment in vitro of low (B16-F1) and high (B16-F10) lung-colonizing melanoma variants reduced significantly their abilities to form lung tumor colonies, but the cells recovered their original in vivo colonizing properties within several hours after drug removal. Low drug concentrations (0.5 mM tetracaine) that altered the metastatic properties of these cells did not modify cell plating efficiencies or tumor growth rates at subcutaneous sites. With the use of [125]-5-iodo-2'-deoxyuridine-labeled B16 cells, the kinetic distributions of viable tumor cells in mice were also shown to be altered. Fewer cells were initially localized and retained in the lungs, and more cells reached extrapulmonary sites. Local anesthetics modified B16 cell morphology, inducing cell rounding and loss of microvilli. These effects occurred concomitant with alterations in microfilament organization. Anesthetic-treated cells also exhibited decreased cell-adhesion characteristics in both homotypic and heterotypic (endothelial and subendothelial matrix) cell-adhesion assays. Despite these changes, cell surface lactoperoxidase-catalyzed iodination of external proteins and labeling of cellular glycoproteins on polyacrylamide gels with 125I-labeled lectins did not reveal differences in the displays or quantities of cell surface glycoproteins after drug treatment. The data are consistent with the idea that reversible disruptions in transmembrane cytoskeletal control induced by local anesthetics result in alterations in cell shape, loss of stable adhesive interactions, and a decrease in blood-borne arrest and metastatic properties.     

Relationship between secreted urokinase plasminogen activator activity and metastatic potential in murine B16 cells transfected with human urokinase sense and antisense genes.

Murine melanoma B16-F1 cells of low metastatic potential were transfected with the human gene for the prepro form of urokinase in an SV40 expression vector (plasmid pSV2-uPA), and cells expressing high amounts of the human urokinase gene product were selected for by an enzyme-linked immunosorbent assay specific for human high molecular weight urokinase. Southern analysis showed one of the cell lines (clone 7) had incorporated 150 copies of the pSV2-uPA plasmid into its genomic DNA. The human urokinase synthesized by the pSV2-uPA-transfected murine B16 cells was found to be glycosylated and did not bind to the murine cell surface urokinase receptor sites. In an in vivo assay that measures metastasis from a primary tumor (spontaneous metastatic assay), clone 7 cells showed an increased ability to metastasize (12 of 12 mice showed metastatic tumors), while control cells showed a lower ability to metastasize (only 2 of 11 mice showed metastatic tumors). In a second in vivo assay, which measures only the steps of the metastatic migration process during which tumor cells extravasate from the blood and then grow into pulmonary tumors (lung colonization assay), a significant multifold increase in the ability to form lung tumors was shown by the high human urokinase-secreting B16-F1 cells. In B16-F10 cells incorporating an antisense sequence to preprourokinase (plasmid pSV1-ASuPA-265) and secreting significantly decreased amounts of murine urokinase, a corresponding significant decrease in lung colonization was observed. These results provide direct experimental support for a role of secreted (non-surface-bound) urokinase in the colonization steps of the metastatic process. Furthermore, the data indicate that the higher lung colonization ability of the B16-F10 line than of the B16-F1 line is primarily based on the quantitative differences in their abilities to produce urokinase.     

Forskolin: a potential antimetastatic agent

Forskolin, a diterpene from the roots of an Indian plant, Coleus forskohlii, is a potent platelet aggregation inhibitor and has been examined for its effects on (a) tumor-induced human platelet aggregation and (b) pulmonary tumor colonization in mice. These studies employed a subline of B16 murine melanoma, B16-F10 (highly metastatic to lungs). Forskolin (2 microM) strongly inhibits the melanoma cell-induced human platelet aggregation. A single dose of forskolin (82 micrograms/mouse) administered intraperitoneally 30 or 60 min prior to tail vein injection of cultured B16-F10 cells (2 or 3 X 10(5) cells/mouse) reduced tumor colonization in the lungs by more than 70%. Similar results were obtained in three separate experiments. These findings raise the possibility that forskolin could prove of value in the clinic for the prevention of cancer metastasis.     

NK and CD8+ T cell-mediated eradication of poorly immunogenic B16-F10 melanoma by the combined action of IL-12 gene therapy and 4-1BB costimulation

In previous reports, systemic administration of a stimulatory monoclonal antibody directed against the 4-1BB receptor had no effect on survival or tumor burden in mice inoculated with the poorly immunogenic B16-F10 melanoma. We combined IL-12 gene transfer with 4-1BB costimulation to explore a previously noted cooperative anti-tumor effect against this model tumor. We hypothesize that the innate immune response mediated by IL-12-activated natural killer (NK) cells initiates the activation of the immune system, leading to the priming of T cells, whereas 4-1BB costimulation enhances the function of primed tumor-specific T cells. The effect of the combination therapy on the growth of subcutaneous (s.c.) tumors and pulmonary metastasis was examined. The combination therapy significantly retarded the growth of subcutaneously-inoculated tumors, and 50% of tumor-bearing mice survived with complete tumor regression. In contrast, neither IL-12 gene transfer nor anti-4-1BB antibody administration alone was as effective. Enhanced CTL activity against both B16-F10 tumor cells and TRP-2-pulsed EL4 syngeneic tumor cells was observed in tumor-bearing animals treated with the combination therapy 2 weeks after treatment and, in long-term survivors from this combination therapy, at >120 days. In a pulmonary metastatic model, only the combination therapy generated significant protection against metastasis. In vivo depletion of NK or CD8(+) but not CD4(+) subsets eliminated the protective immunity. Furthermore, NK cell depletion significantly reduced both tumor-specific CTL activity and the number of tumor-specific IFN-gamma-producing cells, suggesting that this synergistic effect requires the participation of both NK and CD8(+) T cells.     

Expression of collagenase IV (basement membrane collagenase) activity in murine tumor cell hybrids that differ in metastatic potential

Expression of a basement membrane collagen-degrading metalloprotease activity (collagenase IV) was studied in a series of murine cell hybrids derived from fusions between highly metastatic cells (B16-F10RR) or moderately metastatic cells (UV-2237RR) and tumorigenic cells (K-1735 clone 16) or normal cells [peritoneal macrophages (PEC) or C3H mouse embryo fibroblasts (C3H-F)]. The collagenase IV activity of the parent cells and the hybrids was assayed in vitro and compared to the metastatic propensity of the same cells evaluated in both syngeneic (C57BL/6 X C3H/HeN)F1 mice and BALB/c nude mice. The level of collagenase IV activity secreted by the parent lines correlated with their metastatic capacity. The highly metastatic B16-F10RR line secreted the highest enzyme activity, whereas the tumorigenic but nonmetastatic K-1735 clone 16 and the normal parents PEC and C3H-F secreted the lowest enzyme activity. The enzyme activity was completely inhibited with EDTA. The hybrid derived from fusion of cells from two metastatic cell lines as well as hybrids derived from a metastatic and a nonmetastatic tumor cell line expressed higher levels of collagenase IV activity than either parent, and this expression was associated with a high ability to produce metastases in both nude and syngeneic mice. Fusion of metastatic cells with normal cells produced hybrid cells that exhibited suppression of both collagenase IV activity and metastatic capacity. Collagenase IV activity and metastatic propensity can, therefore, be altered by somatic cell hybridization; in the series of hybrids examined in these experiments the expression of type IV collagen-degrading metalloprotease activity and the metastatic ability were closely correlated, which suggests that collagenase IV activity and other properties required for metastasis are genetically linked.     

Control of melanogenesis in mouse melanoma cells of varying metastatic potential.

The control of melanin production, tyrosinase activity, and cell replication by melanocyte-stimulating hormone (MSH) and cyclic AMP (cAMP) was examined in differentially metastasizing B16 mouse melanoma variants. In B16-F1 cells (low metastatic potential), MSH or cAMP greatly elevated tyrosinase activity and melanin content while inhibiting cell replication. The same parameters in B16-F5 cells (intermediate metastatic potential) were altered to a much lesser degree, whereas B16-F10 cells (high metastatic potential) were not significantly affected by MSH or cAMP. Therefore, a correlation exists between loss of hormonal regulation and increased metastatic potential.     

Effect of retinoic acid on tumor-mediated immunologic alterations in mice bearing a variant of the B16 melanoma

This investigation examined the effect of retinoic acid on tumor progression and immunological status of mice bearing the B16-F10 melanoma (previously selected for high lung-colonizing capacity). Tumor cells were implanted s.c. in syngeneic C57BL/6 mice, half of which were treated with beta-all trans retinoic acid (RA). Although RA failed to exhibit direct toxicity on this variant at the concentration used, the immunologic aberrations induced by the tumors were diminished by i.p. RA administration (at 45 micrograms twice/week for 3 weeks). In mice bearing B16-F10 tumors, tumor burdens were decreased from 2.9% of body weight to 1.6%. The mitogenic responses of splenic lymphocytes to concanavalin A (ConA) were increased in tumor-bearing mice following this RA treatment. The presence of these tumor cells decreased the absolute number of CD4- and CD8-positive splenic lymphocytes. Following RA treatment, the CD8-positive population was increased in tumor-bearing mice, while the CD4+ population was not significantly altered. Since previous studies indicated that plasma membrane fragments (or vesicles) could alter lymphocyte distributions and proliferative capacities, the in vitro shedding of membrane fragments from B16-F10 tumor cells was assayed and observed to be decreased after continuous treatment of cultures with 10(-6) M RA for 21 days. Membrane shedding from B16-F10 cells was inhibited by 48.5% following RA treatment. Based on these in vivo and in vitro results, we suggest that RA treatment may diminish tumor growth by decreasing tumor-induced immunosuppressive events.     

Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.     

Antigenic differences among B16 melanoma variants selected for their differing abilities to metastasize: a possible mechanism for effective adjuvant immunotherapy

Most C57BL/6J mice will develop pulmonary metastases four to six weeks after excision of B16 melanoma isografts and begin to die within 40 days. When applied in an adjuvant setting after isograft excision, specific B16 immune RNA (I-RNA) therapy prevents pulmonary metastases and prolongs survival in 50% of treated animals. Since increased in vitro cell-mediated cytotoxicity (CMC) against B16 targets has been demonstrated in animals whose survival is improved by B16 I-RNA therapy, we have proposed that the treatment functioned, at least in part, by specifically altering host CMC in vivo. In this study, C57BL/6J splenocytes were specifically sensitized in vitro by B16 I-RNA exposure and examined for cytotoxic effect on variants of B16 melanoma selected for their differing metastatic potential in vivo. F10 tumor targets (explanted from a B16 melanoma vairant producing frequent pulmonary metastases in vivo) were consistently more sensitive to specific in vitro cytotoxic effect than F1 target cells (from a B16 melanoma variant with low metastatic potential in vivo). Lung metastases (F1 mets) were explanted after intravenous (IV) injection of F1 and used as target cells in the in vitro CMC assays. F1 mets demonstrated greater cytotoxic effect than F1 targets after exposure to B16 I-RNA-treated syngeneic splenocytes. C57BL/6J mice were sacrificed at weekly intervals following adjuvant in vivo B16 I-RNA therapy (after B16 isograft excision), and their splenocytes were shown to be consistently more cytotoxic in vitro to B16 and F10 than to F1 target cells. Splenocytes harvested from mice treated with I-RNA specific to antigenically distinct Lewis lung carcinoma (3LL) after 3LL isograft excision had no cytotoxic effect on any of the B16 variants. When nonsensitized C57BL/6J splenocytes were examined for cytotoxic effect in natural killer (NK) assays or in NK inhibition assays, differences in cytotoxic sensitivity of B16, F10, F1, and F1 mets could not be demonstrated. We conclude, therefore, that specifically sensitized effector cells could distinguish antigenic differences among B16 variants selected for differing in vivo metastatic potiential. These differences were tumor specific and could be demonstrated by cytotoxic splenocytes after either in vivo B16 I-RNA treatment or in vitro B16 I-RNA exposure. The relationship of these antigenic differences to in vivo metastatic potential and to the effectiveness of adjuvant B16 I-RNA therapy is discussed.     

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.     

Comparative studies on the quantitative analysis of experimental metastatic capacity

The purpose of these studies was to establish a procedure for determining the relative experimental metastatic potential of unrelated murine tumors. We used three tumors (the B16-F10 melanoma, which is syngeneic to the C57BL/6N mouse, and the K-1735 melanoma and the UV-2237 fibrosarcoma, which are syngeneic to the C3H/HeN mouse). Various numbers of tumor cells were injected into normal or immunosuppressed syngeneic recipients and into 3-week-old BALB/c nude mice. At appropriate intervals, the recipient mice were killed, and the metastatic burden was determined. The number of experimental metastases was not linearly correlated with cell input. Thus, simply comparing the incidence of metastasis resulting from the injection of one predetermined dose of tumor cells did not allow for determination of their relative metastatic capacities. More reproducible and meaningful results were obtained by introducing increasing numbers of viable tumor cells admixed with a constant number of nontumorigenic (X-irradiated) tumor cells serving as carrier. The incidence of metastasis by few or many injected cells is influenced by host factors such as immune status, and therefore determinations of the true metastatic nature of any given tumor necessitate the choice of an appropriate recipient.     

Comparison of the in vitro and in vivo effects of retinoids either alone or in combination with cisplatin and 5-fluorouracil on tumor development and metastasis of melanoma

Purpose  Retinoids have previously been reported to inhibit proliferation of melanoma cell lines in vitro. However, the relative antimetastatic efficacy of various retinoids on melanoma in vivo is unknown. Therefore, we investigated the effects of different retinoids on the invasion and metastasis of murine melanoma B16-F10 cells in vitro and in vivo. Based on the findings, the antitumor effects of a selected retinoid either alone or in combination with cisplatin were also investigated in a preclinical mouse melanoma model. Methods  Cell proliferation and invasion analyses of murine melanoma B16-F10 cells were assessed in the presence of different retinoids, either alone or in combination with cisplatin (CDDP) or 5-fluorouracil (5-FU). Experimental lung metastasis assay was performed in this study to investigate the antimetastatic efficacy of retinoids. Additionally, a mouse melanoma model was used to assess the antitumor efficacy of a selected retinoid in combination with cisplatin. Results  Retinoids showed significant antiproliferation and anti-invasion effects on murine melanoma B16-F10 cells. Pretreatment with retinoids increased the sensitivity to CDDP but not to 5-FU in in-vitro. Moreover, the number of metastatic colonies formed in the lungs of mice injected intravenously with B16-F10 cells was significantly reduced by injecting the respective retinoid once a day for 10 days. Treatment with a combination of cisplatin and 13-cis-retinoic acid resulted in a significant reduction in primary tumor size and the number of lung metastatic nodules in melanoma-bearing mice. Conclusion  These results suggest that retinoids not only exhibit antimetastatic effect, but also enhance the antitumor activity of cisplatin in vivo.     

Triflavin, an Arg-Gly-Asp-containing antiplatelet peptide inhibits cell-substratum adhesion and melanoma cell-induced lung colonization.

Triflavin, an Arg-Gly-Asp (RGD) containing peptide purified from Trimeresurus flavoviridis snake venom, inhibits human platelet aggregation by blocking fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex. In this study, we show that triflavin (1-30 micrograms/mouse) inhibits B16-F10 melanoma cell-induced lung colonization in C57BL/6 mice in a dose-dependent manner. In vitro, triflavin dose-dependently inhibits adhesion of B16-F10 melanoma cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, vitronectin, and collagen type I). Triflavin is approximately 600-800 times more potent than GRGDS at inhibiting cell adhesion. In addition, triflavin dose-dependently inhibits B16-F10 cell-induced platelet aggregation. These results imply that the inhibitory effect of triflavin on the adhesion of tumor cells to ECMs (e.g., fibronectin, vitronectin and collagen type I) and/or tumor cell-induced platelet aggregation may be partially responsible for its antimetastatic activity in C57BL/6 mice.     

Reduced tumorigenicity of B16-F10 mouse melanoma cells transfected with mycobacterial antigen 85A

Immunotherapy based on the administration of the mycobacterium bacillus Calmette-Guerin has been successfully used in the treatment of in situ transitional cell bladder cancer, and may be applicable to the treatment of cutaneous malignant melanoma. Antigen 85A (Ag85A) and heat shock protein 65 kDa (hsp65) are major secreted proteins of Mycobacterium species and potent stimulators of cell-mediated immunity. This study evaluated the ability of Ag85A and hsp65 gene transfection to limit tumor growth by B16-F10 mouse melanoma cells. Immunoblotting confirmed protein expression and secretion by B16-F10 cells that were transiently transfected with plasmid DNA containing the Ag85A or hsp65 gene. Groups of syngeneic C57BL/6 mice were injected subcutaneously with 1x10(5) untransfected B16-F10 cells or B16-F10 cells transiently transfected with either empty vector or vector containing the Ag85A or hsp65 gene. Ag85A-expressing B16-F10 cells exhibited a dramatic 76% reduction (p<0.05, Mann-Whitney U test) in tumor weight in comparison to empty vector controls at 14 days post-inoculation. In contrast, hsp65-transfected B16-F10 cells did not show any change in tumorigenicity. Decreased tumorigenicity by Ag85A-transfected B16-F10 cells was not due to a reduced ability of Ag85A-transfected B16-F10 cells to proliferate since both mock- and Ag85A-transfected B16-F10 cells showed increased in vitro proliferation in comparison to untransfected cells. Hematoxylin and eosin staining revealed that Ag85A-transfected B16-F10 tumors contained an inflammatory leukocyte infiltrate that was not present in hsp65-transfected tumors. Reduced tumor progression by Ag85A-transfected B16-F10 melanoma cells suggests that immunotherapy based on the transient induction of Ag85A expression may be an effective approach for the treatment of cutaneous malignant melanoma.