Reversion of the transformed phenotype of B16 mouse melanoma: involvement of an 83 kd cell surface glycoprotein in specific growth inhibition

Treating B16 mouse melanoma cells with monoclonal antibody NORM-2 reduces cell growth in tissue culture, agar, and syngeneic mice. We show that the NORM-2 antibody recognizes an integral 83 kd glycoprotein that is mobile in the plane of the plasma membrane of B16 melanoma cells. Expression of the glycoprotein is reduced under conditions that inhibit B16 growth, such as low serum, high cell density, and addition of transforming growth factor-beta. The glycoprotein reappears during S phase, when growth-arrested cells are restimulated. The NORM-2 antigen appears to be involved in growth regulation of B16 melanoma cells both in vitro and in vivo.     

T-cadherin suppresses the cell proliferation of mouse melanoma B16F10 and tumor angiogenesis in the model of the chorioallantoic membrane

The influence of T-cadherin on the pigmentation and proliferation of mouse melanoma B16F10 cells in vitro and on the growth and neovascularization of tumor cell masses formed by the B16F10 cells in a model of the chorioallantoic membrane of a chicken embryo is studied. It is found that the proliferative activity of the cells decreases in the cell culture of mouse melanoma upon the overexpression of T-cadherin in comparison with the cells in the control. It is shown in experiments in vitro that the B16F10 cells with the overexpression of T-cadherin are rarely settling are to the chorioallantoic membrane than the control cells. In addition, it is found that the control cells of mouse melanoma form tumors with area more 0.1 mm² more often than the cells with the overexpression of T-cadherin and the amount of the vessels growing to tumor cell masses formed by the cells with the overexpression of T-cadherin is significantly lower than the same index for the cells in the control. Thus, the overexpression of T-cadherin in the B16F10 cells suppresses the proliferation of these cells in vitro and the growth of the tumor masses formed by melanoma cells on the chorioallantoic membrane and their neovascularization in vivo.     

Skin tumor responsiveness to interleukin-2 treatment and CD8 Foxp3+ T cell expansion in an immunocompetent mouse model

Recombinant human interleukin-2 (rhIL-2) therapy is approved for treating patients with advanced melanoma yet significant responses are observed in only 10–15% of patients. Interleukin-2 induces Foxp3 expression in activated human CD8 T cells in vitro and expands circulating CD8 Foxp3+ T cells in melanoma patients. Employing IL-2 responsive (B16-F1, B16-BL6, JB/MS, MCA-205) and nonresponsive (JB/RH, B16-F10) subcutaneous tumor mouse models, we evaluated CD8 Foxp3+ T cell distribution and changes in response to rhIL-2 (50,000 U, i.p. or s.q., twice daily for 5 days). In tumor-free mice and subcutaneous tumor-bearing mouse models, CD8 Foxp3+ T cells were a rare but naturally occurring cell subset. Primarily located in skin-draining lymph nodes, CD8 Foxp3+ T cells expressed both activated T cell (CD28⁺, CD44⁺) and Treg (CTLA4⁺, PD1^lo/var, NKG2A^+/var) markers. Following treatment with rhIL-2, a dramatic increase in CD8 Foxp3+ T cell prevalence was observed in the circulation and tumor-draining lymph nodes (TD.LNs) of animals bearing IL-2 nonresponsive tumors, while no significant changes were observed in the circulation and TD.LNs of animals bearing IL-2 responsive tumors. These findings suggest expansion of CD8 Foxp3+ T cell population in response to rhIL-2 treatment may serve as an early marker for tumor responsiveness to immunotherapy in an immune competent model. Additionally, these data may provide insight to predict response in patients with melanoma undergoing rhIL-2 treatment.     

Human T cells targeted with anti-T3 cross-linked to antitumor antibody prevent tumor growth in nude mice

Human peripheral blood T cells were tested for the ability to prevent tumor growth in nude mice when targeted with anti-T3 cross-linked to antitumor antibodies. LS174T human colon adenocarcinoma cells were mixed with human PBL coated either with anti-T3 (Fab) cross-linked to 315F6 (Fab) (an antitumor monoclonal antibody) or with no antibody, and were injected subcutaneously into nude mice. Tumor growth was totally inhibited at effector to target (E:T) ratios of 7.0:1 and 2.1:1, and was partially inhibited at 0.7:1 with antibody-coated PBL, but was not inhibited by uncoated PBL. T cell-mediated protection against tumor growth occurred when an antitumor was physically cross-linked to anti-T3. Neither a mixture of unlinked anti-T3 and antitumor antibodies nor anti-human MHC class I cross-linked to antitumor antibody prevented tumor growth. Whereas in vitro cytotoxicity was mediated exclusively by T8+ cells and was augmented by brief exposure of effector cells to IL 2, tumor neutralization in vivo was mediated by both T4+ and T8+ cells and was not significantly stimulated by prior exposure of the cells to IL 2. We conclude that human T cells, when targeted with appropriate antibody heteroaggregates, can specifically inhibit tumor growth at low E:T ratios, and that cells mediating tumor neutralization in vivo may differ from those mediating cytotoxicity in vitro.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. II. Novel immunotherapy of B16 melanomas by local and systemic adoptive transfer

Lyt-1+, L3T4a+ autoreactive cloned T cells, producing lymphotoxin (LT) and interferon-gamma (IFN-gamma) in response to self-class II major histocompatibility complex antigen in vitro were examined for their anti-tumor effect in vivo against B16 melanomas. Without the aid of exogenous interleukin 2, the autoreactive T cells, when injected immediately and at an equal cell number into the site of s.c. inoculated B16 melanoma cells inhibited tumor growth in sublethally irradiated and nonirradiated syngeneic mice. The autoreactive T cells also induced regression of tumors established 3 days earlier. Normal spleen cells or class II-restricted cloned T cells specific for chicken gamma-globulin (CGG) had no inhibitory effect on tumor growth. A single injection of autoreactive T cells delayed tumor growth and prolonged the survival of mice that had received a lethal dose of B16 melanoma cells. The autoreactive T cells caused extensive necrosis at the injection site. A treatment regime consisting of two successive injections of anti-I-Ab monoclonal antibody 3JP prevented the inhibition of tumor growth, supporting the hypothesis that the autoreactive T cells inhibited the growth of melanomas by releasing LT and IFN-gamma upon recognition of I-A antigen-bearing cells at the injection site. The CGG-specific control T cells did not cause necrosis and survived within the nests of uninhibited tumor cells. Autoreactive T cells administered i.v. immediately after i.v. injection of B16 melanoma cells markedly reduced pulmonary metastases, whereas CGG-specific T cells did not. These results indicate that autoreactive T cells can function in vivo as inhibitors of tumor growth.     

Inhibition of melanoma growth and metastasis by combination with (-)-epigallocatechin-3-gallate and dacarbazine in mice.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, was shown to have cancer chemopreventive activity. In this study, we examined the antimetastatic effects of EGCG or the combination of EGCG and dacarbazine on B16-F3m melanoma cells in vitro and in vivo. First, the antimetastatic potentials of five green tea catechins were examined by soft agar colony formation assay, and the results show that EGCG was more effective than the other catechins in inhibiting soft agar colony formation. Second, EGCG dose-dependently inhibited B16-F3m cell migration and invasion by in vitro Transwell assay. Third, EGCG significantly inhibited the spread of B16-F3m cells on fibronectin, laminin, collagen, and Matrigel in a dose-dependent manner. In addition, EGCG significantly inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activity of matrix metalloproteinase-9 (MMP-9). In animal experiments, EGCG alone reduced lung metastases in mice bearing B16-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice. These results demonstrate that combination treatment with EGCG and dacarbazine strongly inhibits melanoma growth and metastasis, and the action mechanisms of EGCG are associated with the inhibition of cell spreading, cell-extracellular matrix and cell-cell interactions, MMP-9 and FAK activities.     

Syngeneic monoclonal antimelanoma antibodies and their application for analysis of tumor antigens, gene cloning, and in vitro/in vivo diagnosis

In this article, we summarized syngeneic monoclonal antimelanoma antibodies and their application for chemical characterization of mouse melanoma antigens, cloning of genomic DNA controlling antigen expression, and in vivo/in vitro tumor diagnosis. The melanoma antigen is composed of a protein complex in association with GM3(NeuAc)-like sugar moiety. The GM3 structure expresses the cross-species epitopes shared in various mammalian species, whereas the mouse specific melanoma epitope is present on protein molecules. By using the monoclonal antimelanoma reactive with GM3 epitope, we developed a very sensitive sandwich radioimmunoassay system detecting soluble melanoma antigens equivalent to 10(2)-10(3) cells/ml. The antibody was also useful in imaging tumor in vivo. These results indicate that the antibody with cross-species reactivity has a potential for tumor targeting. The monoclonal antibody M562 recognizing protein molecule with species specific epitope but not other antimelanoma antibodies, however, effectively inhibited experimental lung metastasis of melanoma cells, indicating that the M562 epitope seems to possess important biological functions. Recently, the genomic DNA controlling the antigen expression was successfully isolated by DNA transfection and expression technique with monoclonal anti-melanoma M562 and the fluorescence-activated cell sorter. We also found that genomic DNA possesses transformation-related activity in NIH3T3 cells.     

Cell binding and tumor inhibiting functions of a new antihuman melanoma murine monoclonal antibody.

Murine, antihuman melanoma cell monoclonal antibody (mAb) 16.C8 was generated by fusing the murine myeloma cell line P3X63/Ag8.653 with splenocytes from a nude mouse bearing a human melanoma xenograft, after reconstitution with splenocytes from syngeneic immunocompetent BALB/c mice. The antibody reacted strongly with fresh human melanoma cells and exhibited preferential reactivity with established human melanoma and neuroectodermal tumor cell lines. Electrophoresis and Western blotting experiments indicated that 16.C8 is directed against a sialoglycoprotein antigen with a molecular weight of 110-120 kDa. mAb 16.C8 mediated lysis of melanoma cells in vitro in antibody-dependent cellular cytotoxicity assays using human mononuclear effector cells isolated from normal volunteers or malignant melanoma patients. In addition, the administration of mAb 16.C8 to nude mice bearing established human melanoma lung and liver metastases resulted in significant inhibition of tumor growth as shown by gross and histologic examination. In contrast, animals treated with Hanks' balanced salt solution or nonspecific immunoglobulin exhibited a large tumor burden. These results suggest that mAb 16.C8 may be of value in treatment of metastatic melanoma in humans.     

Melanoma cell expression of CD200 inhibits tumor formation and lung metastasis via inhibition of myeloid cell functions

CD200 is a cell surface glycoprotein that functions through engaging CD200 receptor on cells of the myeloid lineage and inhibits their functions. Expression of CD200 has been implicated in a variety of human cancer cells including melanoma cells and has been thought to play a protumor role. To investigate the role of cancer cell expression of CD200 in tumor formation and metastasis, we generated CD200-positive and CD200-negative B16 melanoma cells. Subcutaneous injection of CD200-positive B16 melanoma cells inhibited tumor formation and growth in C57BL/6 mice but not in Rag1^−/−C57BL/6 mice. However, i.v. injection of CD200-positive B16 melanoma cells dramatically inhibited tumor foci formation in the lungs of both C57BL/6 and Rag1^−/−C57BL6 mice. Flow cytometry analysis revealed higher expression of CD200R in Gr1⁺ myeloid cells in the lung than in peripheral myeloid cells. Depletion of Gr1⁺ cells or stimulation of CD200R with an agonistic antibody in vivo dramatically inhibited tumor foci formation in the lungs. In addition, treatment with tumor antigen specific CD4 or CD8 T cells or their combination yielded a survival advantage for CD200 positive tumor bearing mice over mice bearing CD200-negative tumors. Taken together, we have revealed a novel role for CD200-CD200R interaction in inhibiting tumor formation and metastasis. Targeting CD200R may represent a novel approach for cancer immunotherapy.     

Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture

The effects of vitamin K on the morphology and the growth of mouse neuroblastoma (P₂), mouse melanoma (B-16) and rat glioma (C-6) cells in culture were studied. Vitamin K₃ inhibited the growth (due to cell death and partial or complete inhibition of cell division) of all three cell types without causing any morphological differentiation. Vitamin K₃ was more effective than vitamin K₁. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma or glioma cells. Glioma cells did not grow in hormone-supplemented serum-free medium; however, both neuroblastoma and melanoma cells grew to a level 70–80% of that found in serum-supplemented medium. Neuroblastoma cells and melanoma cells cultured in serum-free medium exhibited a 2–3 fold higher sensitivity to vitamin K₃ than those cultured in serum-supplemented medium. This suggests that serum factors attenuate the growth inhibitory effect of vitamin K₃ on tumor cells in culture, probably by reducing the availability of this vitamin to the cells. Neuroblastoma cells were more sensitive to vitamin K₃ than were melanoma cells even when they were treated in serum-free medium. The fact that micromolar concentrations of vitamin K₃ inhibit the growth of tumor cells in culture suggests that this vitamin may be a potentially useful anticancer agent.     

Tumor endothelial marker 8 enhances tumor immunity in conjunction with immnization against differentiation Ag

BACKGROUND: We have previously shown that xenogenic DNA vaccines encoding rat neu and melanosomal differentiation Ag induce tumor immunity. Others have developed vaccines targeting tumor neovasculature. Tumor endothelial marker 8 (TEM8) is expressed in the neovasculature of human tumors, and in the mouse melanoma B16, but its expression is limited in normal adult tissues. We describe a DNA vaccine combining xenogeneic tumor Ag and TEM8. METHODS: In-situ hybridization was used to detect TEM8 RNA in mouse tumors. Mice transgenic for the rat neu proto-oncogene were immunized with DNA vaccines encoding TEM8 and the extracellular domain of rat neu and challenged with the 233-VSGA1 breast cancer cell line. In parallel experiments, C57BL/6 mice were immunized with TEM8 and human tyrosinase-related protein 1 (hTYRP1/hgp75) and challenged with B16F10 melanoma. RESULTS: TEM8 was expressed in the stroma of transplantable mouse breast and melanoma tumors. In both model systems, TEM8 DNA had no activity as a single agent but significantly enhanced the anit-tumor immunity of neu and hTYRP1/hgp75 DNA vaccines when given in concert. The observed synergy was dependent upon CD8+ T cells, as depletion of this cell population just prior to tumor challenge obviated the effect of the TEM8 vaccine in both tumor models. DISCUSSION: A local immune responses to TEM8 may increase inflammation or tumor necrosis within the tumor, resulting in improved Ag presentation of HER2/neu and hTYRP1/hgp75. Alternatively, TEM8 expression in host APC may act more as an adjuvant than an immunologic target.     

Antitumor activity of IFN-lambda in murine tumor models

IFN-lambda 1, -lambda 2 and -lambda 3 have been discovered as the latest members of the class II cytokine family and shown to possess antiviral activity. Murine B16 melanoma and Colon26 cancer cells were transduced with mouse IFN-lambda to determine whether IFN-lambda possesses antitumor activity. Overexpression of IFN-lambda induced cell surface MHC class I expression and Fas/CD95 Ag, induced significant caspase-3/7 activity, and increased p21(Waf1/Cip1) and dephosphorylated Rb (Ser(780)) in B16 cells in vitro. IFN-lambda expression in tumor cell lines markedly inhibited s.c. and metastatic tumor formation in vivo compared with mock transfections (p < 0.05). Moreover, IFN-lambda expression induced lymphocytic infiltrates, and an Ab-mediated immune cell depletion assay showed that NK cells were critical to IFN-lambda-mediated tumor growth inhibition. Hydrodynamic injection of IFN-lambda cDNA successfully targeted liver metastatic foci of Colon26 cells, and moderately decreased the mortality of mice with tumors. IFN-lambda overexpression in the liver increased NK/NKT cells and enhanced their tumor-killing activity, and suggested the activation of innate immune responses. Thus, IFN-lambda induced both tumor apoptosis and NK cell-mediated immunological tumor destruction through innate immune responses. These findings suggested that local delivery of IFN-lambda might prove a useful adjunctive strategy in the clinical treatment of human malignancies.     

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls.?These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.     

过表达核糖核酸酶抑制因子通过调节ILK/PI3K/AKT信号途径抑制小鼠黑色素瘤B16-F10细胞体内外生长

核糖核酸酶抑制因子（ribonuclease inhibitor,RI）是胞浆内的一种酸性蛋白质.已有研究证明,RI与核糖核酸酶A（RNaseA）和血管生成素（angiogenin,ANG）结合可抑制其活性.本室前期实验证实,RI可有效抑制某些肿瘤的生长和转移. 然而,RI抑制肿瘤的分子机制尚不清楚. 本研究探讨RI对小鼠黑色素瘤B16-F10细胞生长和凋亡的影响及其机制. MTT法结合流式细胞术分析结果证明,RI基因稳定转染导致B16+F10黑色素瘤细胞S期阻滞,抑制B16-F10黑色素瘤细胞增殖. Annexin V/PI结合流式细胞术结果显示,RI过表达引起细胞凋亡.与此相一致,蛋白质印迹分析显示,过表达RI引起抗凋亡分子Bcl-2表达下调,而Bax上调,同时伴有Pro-casepase 3激活. C57BL/ 6小鼠移植成瘤实验显示,与对照相比,转染RI的B16-F10细胞形成的肿瘤重量显著减少,同时伴有肿瘤组织微血管密度降低.提示RI过表达能抑制微血管生成. 此外,体内外组织/细胞免疫化学和蛋白质印迹结果揭示,过表达RI可显著抑制整合素连接激酶(integrin-linked kinase,ILK)下游靶分子Akt和GSK-3β的磷酸化,并降低β-联蛋白的表达.研究结果证明,过表达RI可通过抑制ILK/ PI3K/AKT信号通路,促进细胞凋亡,引起S期阻滞,并抑制血管生成,从而显著抑制小鼠黑色素瘤B16-F10细胞在体内、外的生长.上述结果提示,RI可能是治疗黑色素瘤的有效分子靶点.     

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine

We have shown previously that loxoribine exhibits adjuvant activity for B cells, activates natural killer (NK) cells, and enhances the activation of lymphokine-activated killer cells by interleukin-2 (IL-2). In this study, we examined loxoribine for protective effects in a B16 melanoma lung tumor metastasis model. Significant inhibition of B16 metastasis was seen in mice given a single injection of 2 mg loxoribine as late as day 3 of tumor growth but the greatest inhibition (96%) was seen in mice given four injections of loxoribine on alternate days starting the day before tumor injection. In experiments in which both IL-2 and loxoribine were administered, both agents were active when tested alone, but the combination of IL-2 and loxoribine gave significantly greater inhibition of metastasis. Loxoribine partially inhibited the development of tumors in mice that had been depleted of NK cells by the administration of anti-asialo-GM1 or anti-NK1. 1 antibodies and in NK-deficient beige mice. In all cases, protection was seen only when smaller tumor inocula were injected. Taken together, these data suggest that both NK and non-NK cell populations or effector mechanisms with antitumor activity were activated by loxoribine. Since substituted guanosine analogs have been shown to have adjuvant activity in B cell systems, we evaluated whether loxoribine was active as an adjuvant in a tumor protection model. Mice immunized with both irradiated tumor cells and loxoribine developed a significantly lower number of lung tumors when challenged by live B16 tumor cells, whereas mice injected with either vaccine or loxoribine alone were not protected. There was a clear dose response seen with both loxoribine and the vaccine preparations. These data suggest that loxoribine may be useful in tumor therapy as an immunomodulator or as an adjuvant for use with tumor vaccines.     

Elevation of intracellular cyclic AMP inhibits NF-κB-mediated thymosin β4 expression in melanoma cells

Thymosin β4 (Tβ4) is a major actin-sequestering protein that has been implicated in the growth, survival, motility, and metastasis of certain tumors and is considered an indicator for malignant progression. Therefore, identifying compounds that can downregulate Tβ4 expression is very important for the development of anti-cancer chemotherapies. In this study, we investigated the effects of elevated cAMP on Tβ4 expression and the metastatic potential of murine B16 melanoma cells. In addition, we also dissected the mechanism underlying cAMP-mediated Tβ4 suppression. We found that treatment with the cAMP-inducing compounds α-MSH (α-melanocyte stimulating hormone) and IBMX (3-isobutyl-1-methylxanthine) significantly suppressed Tβ4 expression and regulated EMT-associated genes through the suppression of NF-κB activation in B16F10 cells. Along with decreased Tβ4 expression, the in vitro invasiveness and anchorage-independent growth in a semi-solid agar of these cells were also inhibited. In animal experiments, the metastatic potential of the α-MSH- or IBMX-treated B16F10 melanoma cells was decreased compared to untreated control cells. Collectively, our data demonstrate that elevated intracellular cAMP significantly suppresses Tβ4 expression and reduces MMP-9 activity, which leads to decreased metastatic potential. Moreover, suppression of NF-κB activation by α-MSH or IBMX is critical for inhibiting Tβ4 expression.     

Activated T cell exosomes promote tumor invasion via Fas signaling pathway

Activated T cells release bioactive Fas ligand (FasL) in exosomes, which subsequently induce self-apoptosis of T cells. However, their potential effects on cell apoptosis in tumors are still unknown. In this study, we purified exosomes expressing FasL from activated CD8(+) T cell from OT-I mice and found that activated T cell exosomes had little effect on apoptosis and proliferation of tumor cells but promoted the invasion of B16 and 3LL cancer cells in vitro via the Fas/FasL pathway. Activated T cell exosomes increased the amount of cellular FLICE inhibitory proteins and subsequently activated the ERK and NF-κB pathways, which subsequently increased MMP9 expression in the B16 murine melanoma cells. In a tumor-invasive model in vivo, we observed that the activated T cell exosomes promoted the migration of B16 tumor cells to lung. Interestingly, pretreatment with FasL mAb significantly reduced the migration of B16 tumor cells to lung. Furthermore, CD8 and FasL double-positive exosomes from tumor mice, but not normal mice, also increased the expression of MMP9 and promoted the invasive ability of B16 murine melanoma and 3LL lung cancer cells. In conclusion, our results indicate that activated T cell exosomes promote melanoma and lung cancer cell metastasis by increasing the expression of MMP9 via Fas signaling, revealing a new mechanism of tumor immune escape.     

Biochemical analysis of metastasis-related Ax actin in B16 mouse melanoma cells after chemical reversional modulation and of tumor progression-related A' actin in the ontogeny of human malignant melanoma

To examine the correlation between tumor metastasis and Ax actin in mouse melanoma and between tumor progression and A'.actin in human melanoma and further to investigate whether or not it is a generally existing principle, we studied the effects of reversion agents, which distinctly decrease metastatic ability of melanoma cells, on the appearance of Ax actin. Will an induced decrease in metastasis of established highly metastatic B16-F10 mouse melanoma cells cause the appearance of Ax actin? We also examined the appearance of A' actin in eight human benign pigment cell tumors and nine human malignant melanoma tissues or cells in relation to tumor progression. In vitro treatment of B16-F10 cells with each of these agents suppressed metastatic ability of the cells injected intravenously into syngenic mice; however, none of the treated cells represented Ax actin in vitro. These results suggest that the appearance of Ax actin may be a result of long-term tumor cell progression leading to changes in gene level, but because the treatments with these agents were only carried out over a short period, they could not effect changes in gene level; thus, Ax actin appearance remained unchanged. Appearance of A' actin was detected only in human benign pigment cell tumors such as nevus cell nevi, but not in malignant melanomas, which were also formed in a long period of tumor progression in vivo. These results suggest that A' actin is a clinically useful marker to determine the prognosis and level of tumor progression of human pigment cell tumors.     

Antiproliferative activity of IL-27 on melanoma.

IL-27 is a member of the IL-6/IL-12 family and activates both STAT1 and STAT3 through its receptor, which consists of WSX-1 and gp130. We previously demonstrated that IL-27 has potent antitumor activities, which are mediated through CD8(+) T cells, NK cells, or its own antiangiogenic activity. In this study, we demonstrate that IL-27 also possesses a direct antiproliferative activity on melanoma. Although WSX-1 expression was hardly detected in parental mouse melanoma B16F10 cells, IL-27 activated STAT1 and STAT3 and up-regulated MHC class I in B16F10 transfectants expressing wild-type WSX-1. In contrast, IL-27 failed to activate STAT1 and up-regulate MHC class I in those expressing mutant WSX-1, in which the putative STAT1-binding Tyr-609 of the cytoplasmic region was replaced by Phe. IL-27 inhibited the tumor growth of transfectants expressing wild-type WSX-1 in a dose-dependent manner. IL-27 augmented the expression of IFN regulatory factor (IRF)-1 and IRF-8, which possess tumor suppressor activities, in B16F10 transfectants expressing wild-type WSX-1. Down-regulation of IRF-1 but not IRF-8 with small interfering RNA partially blocked the IL-27-induced growth inhibition. A small, but significant, direct antiproliferative effect of IL-27 was also observed in vivo. Moreover, several human melanoma cells were revealed to express both IL-27 receptor subunits, and activation of STAT1 and STAT3 and growth inhibition by IL-27 were detected. These results suggest that IL-27 has an antiproliferative activity on melanomas through WSX-1/STAT1 signaling. Thus, IL-27 may be an attractive candidate as an antitumor agent applicable to cancer immunotherapy.