Metastasis Of B16F10 Melanoma Cells Research Articles

Synthesis and pharmacological evaluation of the stereoisomers of 3-carba cyclic-phosphatidic acid.

Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a five-membered ring with the sn-3 phosphate. Here, we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA(5) GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA(5) compared to the R-stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.

1-Deoxynojirimycin Inhibits Metastasis of B16F10 Melanoma Cells by Attenuating the Activity and Expression of Matrix Metalloproteinases-2 and -9 and Altering Cell Surface Glycosylation

1-Deoxynojirimycin (1-DNJ), an iminosugar rich in mulberry, has been shown to possess antimetastatic potential. The antimetastatic mechanisms of 1-DNJ in melanoma B16F10 cells were studied, as were the antimetastatic activity (cell adhesion, migration, and invasion) of 1-DNJ, matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) mRNA, and flow cytometric analysis of cell surface in melanoma B16F10 cells. 1-DNJ significantly inhibited invasion, migration, and cell-matrix adhesion and markedly decreased MMP-2 and MMP-9 activity and mRNA expression. In contrast, 1-DNJ effectively enhanced the expression of TIMP-2 mRNA. In addition, 1-DNJ significantly decreased abnormal glycosylation and/or sialylation on B16F10 melanoma cell surface but increased the levels of α-mannose. Thus, the antimetastatic effects of 1-DNJ against B16F10 melanoma cells are likely associated with its attenuated activities and expression of MMP-2/9, enhancement of the TIMP-2 mRNA expression, and alterations of the cell surface-binding motif. These results suggest that 1-DNJ may be useful as an adjuvant of antimetastatic agents such as cisplatin.

Induction of tumor-specific acquired immunity against already established tumors by selective stimulation of innate DEC-205+ dendritic cells

Two major distinct subsets of dendritic cells (DCs) are arranged to regulate our immune responses in vivo; 33D1+ and DEC-205+ DCs. Using anti-33D1-specific monoclonal antibody, 33D1+ DCs were successfully depleted from C57BL/6 mice. When 33D1+ DC-depleted mice were stimulated with LPS, serum IL-12, but not IL-10 secretion that may be mediated by the remaining DEC-205+ DCs was markedly enhanced, which may induce Th1 dominancy upon TLR signaling. The 33D1+ DC-depleted mice, implanted with syngeneic Hepa1-6 hepatoma or B16-F10 melanoma cells into the dermis, showed apparent inhibition of already established tumor growth in vivo when they were subcutaneously (sc) injected once or twice with LPS after tumor implantation. Moreover, the development of lung metastasis of B16-F10 melanoma cells injected intravenously was also suppressed when 33D1+ DC-deleted mice were stimulated twice with LPS in a similar manner, in which the actual cell number of NK1.1+CD3− NK cells in lung tissues was markedly increased. Furthermore, intraperitoneal (ip) administration of a very small amount of melphalan (l-phenylalanine mustard; l-PAM) (0.25 mg/kg) in LPS-stimulated 33D1+ DC-deleted mice helped to induce H-2Kb-restricted epitope-specific CD8+ cytotoxic T lymphocytes (CTLs) among tumor-infiltrating lymphocytes against already established syngeneic E.G7-OVA lymphoma. These findings indicate the importance and effectiveness of selective targeting of a specific subset of DCs, such as DEC-205+ DCs alone or with a very small amount of anticancer drugs to activate both CD8+ CTLs and NK effectors without externally added tumor antigen stimulation in vivo and provide a new direction for tumor immunotherapy.

CSE1L/CAS, the cellular apoptosis susceptibility protein, enhances invasion and metastasis but not proliferation of cancer cells

BackgroundThe cellular apoptosis susceptibility (CAS) protein is regarded as a proliferation-associated protein that associates with tumour proliferation as it associates with microtubule and functions in the mitotic spindle checkpoint. However, there is no any actual experimental study showing CAS (or CSE1 and CSE1L) can increase the proliferation of cancer cells. Previous pathological study has reported that CAS was strongly positive stained in all of the metastasis melanoma that be examined. Thus, CAS may regulate the invasion and metastasis of cancers. CAS is highly expressed in cancers; if CAS is associated with cancer proliferation, then increased CAS expression should be able to increase the proliferation of cancer cells. We studied whether increased CAS expression can increase cancer cell proliferation and whether CAS regulates the invasion of cancer cells.MethodsWe enhanced or reduced CAS expression by transfecting CAS or anti-CAS expression vectors into human MCF-7 breast cancer cells. The proliferations of cells were determined by trypan blue exclusion assay and flow cytometry analysis. Invasion of cancer cells were determined by matrigel-based invasion assay.ResultsOur studies showed that increased CAS expression was unable to enhance cancer cell proliferation. Immunofluorescence showed CAS was distributed in cytoplasm areas near cell membrane and cell protrusions. CAS was localized in cytoplasmic vesicle and immunogold electronmicroscopy showed CAS was located in vesicle membrane. CAS overexpression enhanced matrix metalloproteinase-2 (MMP-2) secretion and cancer cell invasion. Animal experiments showed CAS reduction inhibited the metastasis of B16-F10 melanoma cells by 56% in C57BL/6 mice.ConclusionOur results indicate that CAS increases the invasion but not the proliferation of cancer cells. Thus, CAS plus ECM-degradation proteinases may be used as the markers for predicting the advance of tumour metastasis.

RNA interference of metastasis‐associated gene 1 inhibits metastasis of B16F10 melanoma cells in a C57BL/6 mouse model

MTA1 (metastasis-associated gene 1) has been reported to be overexpressed in cancers with high potential to metastasize. Studies of the molecular mechanisms revealed that MTA1 plays an important role in the process of metastasis of many types of cancer. However, the role of MTA1 in melanoma development is unclear. We have investigated the therapeutic value of MTA1 in the B16F10 melanoma cell line with the C57BL/6 mouse model. Studies in vitro showed that MTA1 promoted the metastatic ability of B16F10 cancer cells. MTA1 down-regulation by RNA interference greatly reversed the malignant phenotypes of cancer cells. Immunohistochemical staining of MTA1 in human melanoma samples confirmed the up-regulation of MTA1 in the process of carcinogenesis. Studies in vivo confirmed down-regulation of MTA1 suppressed the growth and experimental metastasis of B16F10 melanoma cells. MTA1 plays an important role in melanoma development and metastasis. It has a promising potential as a target for in cancer gene therapy or chemotherapy.

Inhibition of tumor invasion and metastasis by aqueous extract of the radix of Platycodon grandiflorum

Platycodon grandiflorum is a traditional oriental herbal medicine that is known for its immunostimulatory and anti-tumor effects. This study examined the anti-metastatic activities of an aqueous extract from the root of P. grandiflorum (Changkil: CK) using in vitro and in vivo metastasis assays. CK inhibited the invasion of B16-F10 melanoma cells through a reconstituted basement membrane (Matrigel)-coated filter, and strongly inhibited the adhesion of B16-F10 melanoma cell to extracellular matrices such as Matrigel, fibronectin and laminin substrates. CK also inhibited an experimentally induced lung cancer and prolonged the survival time in vivo. In addition, CK augmented NK cell activity. These results show that CK can reduce the extent of a lung metastasis of B16-F10 melanoma cells by inhibiting the adhesion of tumor cells to the basement membrane possibly and activating NK cells.

Suppressive effects of young radish cultivated with sulfur on growth and metastasis of B16-F10 melanoma cells

The oral administration of extracts of young radishes cultivated with sulfur after intravenous tumor cell injection achieved a marked reduction of pulmonary colonization in mice. Treatment of the mice with extracts of young radish cultivated with sulfur did not show any increase in the number of CD8+ or NK T cells in the spleen, indicating no influence on host immunity. Sulforaphane, which could be a candidate for an active compound from young radishes cultivated with sulfur, inhibited cell growth of B16-F10 melanoma cells. In addition, extracts of the young radish cultivated with sulfur-fed group showed enhanced quinine reductase (QR) activities in the liver and lung and a slight increase of glutathione S-transferase (GST) activity in the liver. These results suggested that the administration of extracts of young radishes cultivated with sulfur suppressed pulmonary tumorigenesis, possibly due to increased activity of detoxification enzymes in the liver and lung, and partly due to cell cytotoxicity.

Interaction between integrin α 5 and fibronectin is required for metastasis of B16F10 melanoma cells

In this study, we report the role of integrin α 5 in promoting melanoma metastasis. The α 5 expression was remarkably elevated in highly metastatic B16F10 melanoma cells compared to lowly metastatic B16F1 cells, whereas no significant changes were detected in those of integrin α 4, α v, and β 1 subunits. Neutralization of α 5 with anti-α 5 antibody significantly suppressed the potential of B16F10 cells for pulmonary metastasis in mice and inhibited cell adhesion or spreading to fibronectin in vitro. Furthermore, loss of the interaction between α 5 and fibronectin diminished cell survival and induced apoptosis in B16F10 cells. Above results provide clear evidence that integrin α 5 is positively correlated with melanoma metastasis and might be an anti-melanoma target.

Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice.

We examined the antitumour effect of a combination of betulinic acid (BA) and vincristine (VCR) on murine melanoma B16F10 cells in vitro and in vivo. Betulinic acid, a pentacyclic triterpene, showed a synergistic cytotoxic effect on melanoma cells by combinational use of VCR. Betulinic acid and VCR induced cell cycle arrest at different points (BA at G1 phase and VCR at G2/M phase) and caused apoptosis in B16F10 melanoma cells. In the in vivo study, VCR inhibited metastasis of tumour cells to the lung. The addition of BA to VCR augmented suppression of the experimental lung metastasis of melanoma cells in C57BL/6 mice. The number of lung nodules of more than 1 mm in diameter in mice treated with BA and VCR was less than that in mice treated with VCR alone. These results suggest that BA is an effective supplement for enhancing the chemotherapeutic effect on malignant melanoma.

Effect of Withania somnifera on B16F‐10 melanoma induced metastasis in mice

Withania somnifera, a plant with known immunopotentiating activity and its bioactive fraction-Withanolide D were studied for their anti-metastatic activity using B16F-10 melanoma cells in C57BL/6 mice. Simultaneous administration of Withania extract (122 +/- 10 tumour nodules) and Withanolide (126 +/- 9 lung tumour nodules) could significantly (p < 0.001) inhibit the metastatic colony formation of the melanoma in lungs. 72.58% by extract and 69.84% by Withanolide treated, as compared to the untreated control animals also increased the survival days. Lung collagen hydroxyproline content was highly elevated in the control animals (23.5 +/- 0.9 micro g/mg protein), which was reduced by the simultaneous administration of both the extract (16.3 +/- 2.0 micro g/mg protein) and Withanolide (15.3 +/- 1.8 micro g/mg protein). The level of lung hexosamines (4.85 +/- 0.20 mg/100 mg tissue) and uronic acids (330.1 +/- 23.7 micro g/100 mg tissue) content was also elevated in the control animals. The elevated level of hexosamine was significantly reduced by the treatment with extract (1.92 +/- 0.05) and Withanolide (1.85 +/- 0.05). Similarly, the uronic acid content was also been reduced by the simultaneous administration of both Withania extract (194.2 +/- 17.4) and Withanolide (183.2 +/- 8.8). The control animals had 35.3 +/- 3.8 U/L gamma-glutamyl transpeptidase (gamma-GT), which was reduced by 50% by the treatment of extract and Withanolide to 17.5 +/- 4.0 U/L and 16.3 +/- 4.4 U/L respectively. There was a significant reduction in the levels of sialic acid in the serum of Withania extract (60.7 +/- 7.7) and Withanolide (67.16 +/- 5.8) treated animals compared to the higher level (102.2 +/- 8.7) in the control animals. Histopathological analysis of the lung tissues also correlated with these findings. Prophylactic administrations of both extract as well as Withanolide were ineffective in inhibiting the metastasis of B16F-10 melanoma cells.

Effect of Diallyl Sulphide, Diallyl Disulphide, and Allyl Methyl Sulphide on the Inhibition of Lung Metastasis of B16F-10 Melanoma Cells in Mice.

Effect of naturally occurring sulphur-containing compounds such as diallyl sulphide (DAS), diallyl disulphide (DADS), and allyl methyl sulphide (AMS) on the inhibition of lung metastasis of B16F-10 melanoma cells was studied in C57BL/6 mice. Simultaneous administration of the compounds after tumor induction produced a significant reduction in tumor nodule formation (DAS 55%, DADS 90%, and AMS 54%). The effect was lower when the compounds were administered after tumor development, whereas prophylactic administration of the compounds did not have any effect on lung tumor nodule formation. Increased lung collagen hydroxyproline, serum sialic acid, and gamma glutamyl transpeptidase activity in the metastasized lungs of control animals was significantly reduced in the animals treated with the sulphur-containing compounds. Pathology of the lung tissue also correlated with the above findings. Our results are indicative of the antimetastatic activity of these sulphur-containing compounds.

The field bean protease inhibitor has the potential to suppress B16F10 melanoma cell lung metastasis in mice

Metastasis is a characteristic and fatal feature of human malignancies. Its regulation is therefore of the utmost significance to clinicians. The present study was undertaken to determine whether a legume-derived protease inhibitor (PI) of trypsin/chymotrypsin, the field bean PI (FBPI), also has plasmin inhibitory activity and can inhibit pulmonary metastasis of B16F10 melanoma cells systemically injected into BDF 1 mice. Two approaches to the problem were made. In the first, the melanoma cells were exposed to two different concentrations of the FBPI prior to their inoculation into animals. In the second, the mice were treated intraperitoneally with FBPI at a dose of 100 mg/kg body weight once daily for 10 days, the treatment being started soon after the systemic injection of the tumour cells. The study revealed that both modes of FBPI treatment could effectively block lung cell metastasis by the melanoma cells and that FBPI has plasmin blocking activity. Since urokinase type plasminogen activator and plasmin are known to play significant roles in tumour cell metastasis, the dose-dependent inhibitory effect of FBPI with antiplasmin activity on tumour cell metastasis suggests that its antimetastatogenic action is probably mediated through its plasmin inhibitory action.

Acidic polysaccharide from Panax ginseng, ginsan, induces Th1 cell and macrophage cytokines and generates LAK cells in synergy with rIL-2.

We previously reported that an acidic polysaccharide from Panax ginseng named ginsan inhibits the incidence of benzo[a]pyrene-induced autochthonous lung tumors in mice. To elucidate the mechanism of antineoplastic activity, ginsan was tested for its ability to generate LAK cells and to produce cytokines. Spleen cells became cytotoxic to a wide range of tumor cells after 5 days of culture with ginsan in a non-major histocompatibility restricted manner and the activity of ginsan was 12 times higher than that of lentinan. The generation of killer cells by rIL-2 was neutralized only in the presence of anti-IL-2, whereas by ginsan it was neutralized in the presence of anti-IL-2 as well as anti-IFN gamma, or anti-IL-1 alpha. It was confirmed that ginsan induces the expression of mRNA for IL-2, IFN gamma, IL-1 alpha, and GM-CSF. Depletion of AsGM1+ cells from spleen cells reduced the generation of LAK by rIL-2. In contrast, depletion of AsGM1+ as well as Thy1+ cells, CD4+ cells, or DC8+ cells reduced the generation of LAK cells by ginsan. The serologic phenotype of rIL-2 induced LAK cells was CD8- cells, whereas the ginsan induced LAK cells, were CD8+ cells. Ginsan synergized with rIL-2 to generate LAK cells (2.0-15 fold) and the most dramatic synergy was seen at rIL-2 concentrations below 3 U/ml. Ginsan alone inhibited pulmonary metastasis of B16-F10 melanoma cells and enhanced the inhibition of lung colonies by rIL-2. These findings demonstrate that ginsan generates LAK cells from both NK and T cells through endogeneously produced multiple cytokines. This property may contribute to its effectiveness in the immunoprevention and immunotherapy of cancer.

Long exposure of non-cytotoxic concentrations of methylselenol suppresses the invasive potential of B16F10 melanoma

To assess the inhibitory effects of methylselenol on the invasion of murine B16F10 melanoma cells, we carried out in vivo and in vitro experiments using Se-methylselenocysteine (Se-MSC) and selenomethionine (SeMet), respectively. In an animal experiment, the supplementation of drinking water with Se-MSC (4 ppm Se) led to a significant increase in Se levels in the lung, liver and serum in mice. Mice given a mash diet or water supplemented with Se-MSC (2, 4 and 6 ppm Se in the mash diet, and 2 and 4 ppm Se in the drinking water) displayed an almost completely diminished pulmonary metastasis of B16F10 melanoma cells and an enhanced survival, compared to the control mice which were given a basal diet. Treatment with non-cytotoxic concentrations of SeMet (2.5, 5 and 10 microM plus 0.02 U/ml METase, methioninase) induced a substantial decrease in the expression of integrin alphavbeta3, the FN receptor and adhesion ability to vitronectin (VN) and fibronectin (FN) in B16F10 melanoma cells. Moreover, these compounds suppressed gelatinase activity, invasive ability and wound migration in the culture system. SeMet-METase prevented the conversion of pro-MMP-9 to its active form and decreased pro-MMP-2 activities in a zymogram. The pre-treatment of B16F10 melanoma cells with SeMet-METase led to a decrease in pulmonary metastasis and extended survival in mice injected with tumor cells. Collectively, our results indicate that integrin expression is crucial in promoting the metastatic phenotype in murine B16F10 melanoma cells by supporting specific adhesive and invasive properties, suggesting that Se-MSC effectively reduces the metastasis of B16F10 melanoma cells as a nutritional adjuvant. Methylselenol may also contribute to the suppression of integrin expression.

Role of asparagine-linked carbohydrates in pulmonary metastasis of B16-F10 murine melanoma cells: implication through glycosylation inhibition by nojirimycin.

N-linked oligosaccharide moieties of cell surface glycoconjugates may be involved in the metastatic potential of tumour cells. Many studies have examined the anti-metastatic properties of inhibitors of carbohydrate synthesis or processing in vitro. However, there has so far been little evidence of such inhibitory factors on metastasis in vivo because of the general high toxicity of the inhibitors. In this study, we found nojirimycin (NM) to be a substantially non-toxic carbohydrate synthesis inhibitor that inhibited the experimental metastasis of B16-F10 melanoma cells not only in vitro but also in vivo. When NM was administered intraperitoneally to syngeneic C57BL/6 mice, the inhibition was dose-dependent, with 40-75% suppression of pulmonary colonization observed after 5 days exposure to NM (at 0.4 or 4 mg/day). An in vitro lectin sensitivity assay showed that NM-treated B16-F10 cells were less susceptible to the lectin concanavalin-A, suggesting alteration or decrease of high mannose-type carbohydrate moieties on the cell surface. Further in vitro analysis of adhesiveness between B16-F10 cells and endothelial cells demonstrated that NM treatment causes reduced binding of B16-F10 cells to endothelial cells. We have also studied the effect of NM on natural killer (NK) cells in mice. NM treatment elicited no substantial increase in the cytotoxic activity of splenic NK cells against YAC-1 target cells. These results indicate that NM acts on the process of adhesion and that specific structures of the cell surface carbohydrate moieties may be involved in the colonization phase of metastasis in vivo.