Injection Of B16 Melanoma Cells Research Articles

Improved Anti-Tumour Adaptive Immunity Can Overcome the Melanoma Immunosuppressive Tumour Microenvironment.

Clinical benefits obtained from checkpoint blockade regimens demonstrate the importance of overcoming the immunosuppressive tumour microenvironment (TME) in cancer immunotherapy. Intravenous (i.v.) injection of B16 melanoma cells (H-2Kb) leads to lethal disseminated pulmonary metastasis in Balb/c recipients (H-2Kd). This lack of immune control is related to low major histocompatibility complex (MHC) expression on B16 cells which is associated with delayed and decreased anti-tumour adaptive immune responses (e.g., alloantibody formation) as: (i) other tumour types with normal H-2Kb expression are rejected with concomitant antibody production; (ii) preincubation of B16 with IFN-gamma to upregulate H-2Kb expression resulted in improved antibody production and anti-tumour activity. The delayed/decreased anti-tumour adaptive immune responses induced by B16 inoculation is not able to interrupt progression of primary metastases, while it is able to effectively eliminate secondary inoculated subcutaneously (s.c.) B16 cells from progression. This is due to the presence of an immunosuppressive TME within the primary metastases characterized by increased regulatory T cells (Tregs) and an increased T helper cells (Th) 2/1 profile. These tumour-induced immunosuppressive T cell populations are counteracted by improved adaptive immunity via active and passive immunization, resulting in effective elimination of the TME, destruction of the metastatic tumour and a reversal of Th2/1 profile in a time-sensitive manner. Thus, we here demonstrate that the TME is not irreversible and adaptive immunity is able to eradicate established solid tumour and its immunosuppressive TME. This study will help design treatments to overcome the immunosuppressive effect of the TME and improve efficacy of cancer immunotherapy.

Chronic Ly49H Receptor Engagement in vivo Decreases NK Cell Response to Stimulation Through ITAM-Dependent and Independent Pathways Both in vitro and in vivo.

Natural killer (NK) cells play an important role in the innate immune response. The summation of activation and inhibitory signals delivered through cell surface membrane receptors determines NK cell function. However, the continuous engagement of an activating receptor on NK cells appears to render the cells hyporesponsive to stimulation through other unrelated activating receptors. The mechanism by which this takes place remains unclear. Herein we demonstrate that continuous in vivo engagement of the Ly49H receptor with its ligand, m157, results in Ly49H+ NK cells that are hyporesponsive to further stimulation by other ITAM-dependent and independent receptors, while Ly49H− NK cells remain unaffected. The hyporesponsiveness of the NK cell correlates with the degree of Ly49H receptor downmodulation on its cell surface. We observe defects in calcium flux in the hyporesponsive NK cells following stimulation through the NK1.1 receptor. In addition, we observe differences in signaling molecules that play a role in calcium flux, including spleen tyrosine kinase (Syk) at baseline and phosphorylated phospholipase C gamma 2 (p-PLCγ2) at both baseline and following stimulation through NK1.1. We also demonstrate that various ITAM associated activation receptors, including Ly49H, remain associated with their respective adaptor molecules. With regard to in vivo NK cell function, we did not find differences in the formation of metastatic lung lesions following IV injection of B16 melanoma cells. However, we did observe defects in rejection of missing-self targets in vivo. The data suggest that continuous engagement of the Ly49H activating receptor on NK cells results in hyporesponsiveness of the NK cells to all of the ITAM-dependent and independent receptors we analyzed due to altered signaling pathways downstream of the receptor and adaptor molecule.

Tumor Metastasis in VWF Deficient Mice: The Contributions of PGE2 and VEGF

Dysregulation of the hemostatic system in malignancy leads to a hypercoagulable state and thrombotic complications. In addition to thrombosis, growing evidence from both human and animal studies, suggests that constituents of the hemostatic system also play an important role in tumor progression and metastasis. Platelets contribute to metastasis by protecting tumor cells from immune detection and inducing epithelial-mesenchymal transition, thereby facilitating the extravasation of circulating tumor cells out of the vasculature. Moreover, platelet-tumor cell interactions have been shown to promote prostaglandin E2 (PGE2) synthesis, an important modulator of angiogenesis and proliferation. The role of von willebrand factor (VWF) in malignancy has been of particular interest as it serves as an adhesive link between platelets and endothelium, and could possibly facilitate platelet interactions with tumor cells. Yet, the absence of VWF is associated with increased metastatic potential in a B16 melanoma mouse model, suggesting that the primary contribution of VWF to the metastatic model may not be platelet mediated. We employed a combined approach using cell lines and mouse models to explore the interaction of platelets and VWF in metastasis.In vitro studies using lung and melanoma cells lines show that the interaction of resting and activated platelets with tumor cells leads to upregulation of COX-2 expression and PGE2 biosynthesis but does stimulate VEGF-A production. In an experimental metastasis model of C57BL/6 wild type and VWF null mice, the injection of B16 melanoma cells by tail vein yielded a significant increase in the number of pulmonary metastatic foci. PGE2 measurements from mouse serum showed reduced levels of prostaglandin in VWF deficient mice injected with tumor, suggesting that the absence of VWF precludes an important tumor cell-platelet interaction that facilities PGE2 production. Moreover, preliminary results show increased VEGF-A in VWF deficient mice, suggesting that increased metastasis in this model may be the result of dysregulated angiogenesis.In conclusion, platelet-tumor cell interaction yield a pro-metastatic effect, in part mediated by PGE2 production. Despite its unique role in platelet adhesion and thrombus formation, VWF does not facilitate the interaction of platelets and tumor cells to produce PGE2. Its protective effect may lie in its ability to modulate angiogenesis via VEGF-specific pathways. DisclosuresNo relevant conflicts of interest to declare.

Biomarkers for Detection and Monitoring of B16 Melanoma in Mouse Urine and Feces.

Melanoma is the most malignant type of skin cancer. Early detection of melanoma is thus critical for patient prognosis and survival. At present, examination by a skilled dermatologist followed by biopsy of suspicious lesions is the diagnostic gold standard. The aim of the present study was to examine an alternative and noninvasive method for the diagnosis of melanoma at an early stage. We identified and compared the volatile organic compounds (VOCs) in mouse urine and feces, before and after a subcutaneous injection of B16 melanoma cells. We identified a total of 16 VOCs in urine and 13 VOCs in feces that could serve as potential biomarkers. Statistical analysis significantly discriminated between the cancer and control groups. These results should be validated in a larger-scale animal study, after which a study could be designed in patients to develop a melanoma biomarker.

Tissue transglutaminase activity protects from cutaneous melanoma metastatic dissemination: an in vivo study

The role of tissue transglutaminase (TG-2, TGase-2) in cancer development is still a fascinating field of research. The available reports do not elucidate fully its mechanism of action, due to the limitations of in vitro approaches. Therefore, to understand TG-2 role in cancer, we carried out an in vivo study with a more direct approach. TG-2 was in vivo overexpressed in a murine model of melanoma (intravenous injection of B16 melanoma cells in C57BL/6N mice) by means of a plasmid carrying the TG-2 cDNA. The evaluation of the frequency and size of the metastases indicated that the number of melanoma lung foci was more markedly reduced by TG-2 overexpression than the metastatic size. Then, TG-2 overexpressing mice showed a prolonged survival with respect to control mice. Further analyses were carried by means of proteomic analysis of melanoma cell lysates and meta-analysis of published transcriptomic datasets. Proteomic analysis of cell lysates from a human melanoma cell line compared to human keratinocytes showed significant differences in the expression of TG-2 substrates known to be involved in proliferation/differentiation and cancer progression. Taken together, these findings indicate a protective role of TG-2 enzymatic activity in melanoma progression in vivo.

The association of N-palmitoylethanolamine with the FAAH inhibitor URB597 impairs melanoma growth through a supra-additive action

BackgroundThe incidence of melanoma is considerably increasing worldwide. Frequent failing of classical treatments led to development of novel therapeutic strategies aiming at managing advanced forms of this skin cancer. Additionally, the implication of the endocannabinoid system in malignancy is actively investigated.MethodsWe investigated the cytotoxicity of endocannabinoids and their hydrolysis inhibitors on the murine B16 melanoma cell line using a MTT test. Enzyme and receptor expression was measured by RT-PCR and enzymatic degradation of endocannabinoids using radiolabeled substrates. Cell death was assessed by Annexin-V/Propidium iodine staining. Tumors were induced in C57BL/6 mice by s.c. flank injection of B16 melanoma cells. Mice were injected i.p. for six days with vehicle or treatment, and tumor size was measured each day and weighted at the end of the treatment. Haematoxylin-Eosin staining and TUNEL assay were performed to quantify necrosis and apoptosis in the tumor and endocannabinoid levels were quantified by HPLC-MS. Tube formation assay and CD31 immunostaining were used to evaluate the antiangiogenic effects of the treatments.ResultsThe N-arachidonoylethanolamine (anandamide, AEA), 2-arachidonoylglycerol and N- palmitoylethanolamine (PEA) reduced viability of B16 cells. The association of PEA with the fatty acid amide hydrolase (FAAH) inhibitor URB597 considerably reduced cell viability consequently to an inhibition of PEA hydrolysis and an increase of PEA levels. The increase of cell death observed with this combination of molecules was confirmed in vivo where only co-treatment with both PEA and URB597 led to decreased melanoma progression. The antiproliferative action of the treatment was associated with an elevation of PEA levels and larger necrotic regions in the tumor.ConclusionsThis study suggests the interest of targeting the endocannabinoid system in the management of skin cancer and underlines the advantage of associating endocannabinoids with enzymatic hydrolysis inhibitors. This may contribute to the improvement of long-term palliation or cure of melanoma.

Endogenous μ-opioid peptides modulate immune response towards malignant melanoma

Opioids exert major effects not only in the central nervous system but also in immune responses. We investigated the effects of μ-opioid peptides, secreted by tumor cells, on anti-tumor immune responses. For this purpose, tumor growth was studied in wild-type and μ-opioid receptor-deficient (MOR-/-) mice injected with B16 melanoma cells. The ability of these cells to produce opioids was studied by Western blots in vitro. Finally, biopsy material from human melanomas was investigated by immunohistochemistry for ß endorphin expression. Injection of B16 melanoma cells, producing endogenous ß endorphin, in the flank of MOR-/- mice revealed a profound reduction in tumor growth, paralleled by a significantly higher infiltration of immune cells into the tumors, when compared to tumor growth after injection of B16 melanoma cells into wild-type mice. Opioids present in B16 cell supernatant significantly reduced the proliferation of normal but not MOR-/- leucocytes. Immunohistochemical analyses of biopsies from human melanoma tissues showed a positive correlation between expression of ß endorphin and tumor progression. Our data provide evidence that μ-opioid peptides may play a major role in cancer progression by modulating immune response. This finding may have implications for the future optimization of immunointerventions for cancer.

The Foxc2 transcription factor regulates tumor angiogenesis

The Forkhead/Fox transcription factor Foxc2 is a critical regulator of vascular development. However, the role of Foxc2 in pathological angiogenesis in cancer remains unknown. Here we show that FoxC2 is highly expressed in human breast and colonic tumors and in the tumor endothelium in human and mouse melanomas. Using the B16 melanoma tumor model, we investigated the function of Foxc2 in tumor angiogenesis. After subcutaneous injection of B16 melanoma cells, primary tumor growth as well as neovascularization was markedly reduced in mice lacking one copy of the Foxc2 gene ( Foxc2+/−). Consistently, expression levels of several angiogenic factors, including vascular endothelial growth factor ( Vegf), matrix metallopeptidase 2 ( Mmp2), and platelet-derived growth factor-B ( Pdgfb), were significantly decreased in B16 tumors grown in Foxc2+/− mice, and tumor blood vessels formed in Foxc2+/− mice showed reduced coverage of mural cells and endothelial cell apoptosis. In addition, the tumor tissue in Foxc2+/− mice had an accumulation of necrotic cells. Taken together, these findings demonstrate that haplodeficiency of Foxc2 results in impaired formation of tumor blood vessels as well as reduced tumor growth and thereby provide evidence that Foxc2 is critical for tumor development and angiogenesis.

Incubation of antigen-sensitized T lymphocytes activated with bryostatin 1 + ionomycin in IL-7 + IL-15 increases yield of cells capable of inducing regression of melanoma metastases compared to culture in IL-2

Regression of established tumors can be induced by adoptive immunotherapy (AIT) with tumor draining lymph node (DLN) lymphocytes activated with bryostatin and ionomycin (B/I). We hypothesized that B/I-activated T cells cultured in IL-7 + IL-15 might proliferate and survive in culture better than cells cultured in IL-2, and that these cells would have equal or greater anti-tumor activity in vivo. Tumor antigen-sensitized DLN lymphocytes from either wild-type or T cell receptor transgenic mice were harvested, activated with B/I, and expanded in culture with either IL-2, IL-7 + IL-15 or a regimen of alternating cytokines. Cell yields, proliferation, apoptosis, phenotypes, and in vitro responses to tumor antigen were compared for cells grown in different cytokines. These T cells were also tested for anti-tumor activity against melanoma lung metastases established by prior i.v. injection of B16 melanoma cells. IL-7 + IL-15 or alternating cytokines resulted in much faster and prolonged proliferation and much less apoptosis of B/I-activated T cells than culturing the same cells in IL-2. This resulted in approximately tenfold greater yields of viable cells. Culture in IL-7 + IL-15 yielded higher proportions of CD8+ T cells and a higher proportion of cells with a central memory phenotype. Despite this, T cells grown in IL-7 + IL-15 had higher IFN-gamma release responses to tumor antigen than cells grown in IL-2. Adoptive transfer of B/I-activated T cells grown in IL-7 + IL-15 or the alternating regimen had equal or greater efficacy on a "per-cell" basis against melanoma metastases. Activation of tumor antigen-sensitized T cells with B/I and culture in IL-7 + IL-15 is a promising modification of standard regimens for production of T cells for use in adoptive immunotherapy of cancer.

STAT1 Ser727 – key regulator for NK cell-mediated cytotoxicity and tumor surveillance

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling cascade plays an important role in hematopoiesis. A constitutive activation of this pathway is found in a broad variety of diverse human and murine leukemias and lymphomas. Whereas STAT3 and STAT5 accelerate and initiate tumor formation, STAT1 is generally considered a tumor suppressor. As shown by Kovacic et al. [1], STAT1 accelerates leukemia formation by up-regulation of MHC class I on leukemic cells allowing them to escape natural killer (NK) cell-mediated lysis. In the present study we investigate the role of the STAT1 serine727 phosphorylation site for leukemia initiation and progression. After retroviral leukemia induction STAT1S727A knock-in mice show enhanced disease latency compared to wild type (WT) mice. Upon injection of B16 melanoma cells, numbers of lung metastases were increased in STAT1-/- recipients, but significantly reduced in STAT1S727A mice when compared to WT mice. This indicates that the effect of STAT1S727A may not be cell-intrinsic but rather results from altered interactions with the host immune system. Clearance of leukemic and B16 cells is mainly mediated by NK cells. Therefore, differences in NK cells might explain our in vivo data. As determined in a [3H]thymidine incorporation assay, STAT1S727A NK cells showed enhanced proliferation rates compared to WT cells. Moreover, in a classical [51Cr]-release assay STAT1S727A NK cells were able to lyse leukemic cells more efficiently than WT NK cells. NK cells can be divided into different functional subsets according to cell surface expression of DX5, Mac-1 and CD27. Ex vivo fluorescence-activated cell sorting (FACS)-based analysis revealed a distinct expression pattern in STAT1S727A NK cells (Mac-1+, DX5+, CD27+), that is characteristic for a pre-activated NK cell phenotype [2] and is found in STAT1S727A mice in spite of the absence of any activating stimulus. These experiments indicate that phosphorylation of STAT1 serine727 in NK cells is relevant for activation, cytolytic function and as a consequence for their ability to eradicate tumor cells in vivo.

Enhanced suppression of pulmonary metastasis of malignant melanoma cells by combined administration of α‐galactosylceramide and interleukin‐18

alpha-Galactosylceramide (alpha-GalCer) shows antitumor effects by activating natural killer (NK) cells indirectly through stimulation of the secretion of cytokines by NKT cells, whereas interleukin (IL)-18 shows antitumor effects by activating NK cells directly. In the present study, we examined the antitumor effect of the combined administration of alpha-GalCer and IL-18. An injection of NK cell-sensitive mouse B16 melanoma cells into a mouse tail vein produced pulmonary metastasis. The daily administration of alpha-GalCer or IL-18 alone for 4 days starting 1 day after the injection of B16 melanoma cells markedly suppressed the number of pulmonary metastatic foci, and their combined administration enhanced the antitumor effect compared with single administration. The antitumor effect of their combined administration was completely abolished by treatment of mice with anti-asialo GM1 serum, which depletes NK cells but not NKT cells. Combined administration of alpha-GalCer and IL-18 enhanced the cytotoxicity of NK cells and increased the number of NK cells in the lung. Analysis of NKT cell-dependent and NK cell-independent secretion of cytokines, to which NK cells can respond, showed that the administration of alpha-GalCer increased the secretion of IL-2, IL-4, interferon-gamma, IL-12, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-alpha, and IL-10, and the combined administration of alpha-GalCer and IL-18 enhanced the secretion of IL-2, IL-4, interferon-gamma, and granulocyte-macrophage colony-stimulating factor further but only slightly. These results show that IL-18 in combination with alpha-GalCer exerts an antitumor effect on NK cell-sensitive tumors primarily by the direct stimulation of NK cells by IL-18 and the indirect stimulation of NK cells by alpha-GalCer through its activation of NKT cells.

Osteopontin in Melanocytic Lesions—A First Step Towards Invasion?

Determination of the earliest molecular changes that occur during the process of cellular transformation is of intense interest to the field of tumor biology but represents a considerable technical challenge in that it is not normally amenable to pathological examination. As the transition from melanocyte hyperplasia to primary cutaneous melanoma often occurs within dysplastic nevi at the dermal–epidermal junction on sun exposed sites of the skin, it presents a unique opportunity to examine these initial stages of tumor progression. Aberrant changes associated with this transition can be identified via gene expression profiling using high-density DNA microarrays probed with samples prepared from melanocytic lesions or cell lines (Carr et al, 2003) or conversely using immunohistochemical staining to determine expression in tissue section microarrays from defined stages (Pacifico et al, 2004). Genes identified as being differentially expressed in this way provide prognostic markers as well as insight into the process of cellular transformation. The combination of both of these techniques were used in the report ofZhou et al (2005), in order to discover genes highly upregulated in melanocytic lesions. Comparison of metastatic tumor nodules with benign nevi has found over 190 genes potentially implicated in this process of early stage transformation, with the osteopontin gene already recognized for its involvement in transformation of a number of diverse tumor types (Wai and Kuo, 2004) showing the highest abundance of differential expression.

Anti-tumoral effect of native and immobilized bovine serum amine oxidase in a mouse melanoma model

Bovine serum amine oxidase (BSAO) oxidatively deaminates polyamines containing primary amine groups, spermidine and spermine, to form the cytotoxic products hydrogen peroxide and aldehyde(s). Polyamines are present at elevated levels in many tumor tissues. The aims of the study were to evaluate the anti-tumoral activities of native and immobilized BSAO in mouse melanoma and also to determine the mechanism of tumor cell death. C57BL mice received a subcutaneous injection of B16 melanoma cells to induce formation of tumors, prior to antitumor treatments with native and immobilized BSAO. The enzyme was immobilized in a poly(ethylene glycol) (PEG) biocompatible matrix. Antitumor treatments consisted of a single injection of enzyme into the tumor. When immobilized BSAO (2.5 mU) was injected into the tumor, there was a marked decrease of 70% of the tumor growth. This was compared with a decrease of only 32% of tumor size when the same amount of native BSAO was administered. The type of cell death was analysed in tumors that were treated with native or immobilized BSAO. When tumors were treated with immobilized BSAO, there was induction of a high level of apoptosis (around 70%), compared to less than 10% with the native enzyme. Apoptotic cell death was assessed by nuclear chromatin condensation using Hoechst staining and labelling of externalized phosphatidylserine using Annexin V. However, native BSAO, probably due to a burst of cytotoxic products, induced a high level of necrosis of about 40%, compared to less than 10% with immobilized BSAO. In conclusion, the advantage is that immobilized BSAO can act by allowing the slow release of cytotoxic products, which induces tumor cell death by apoptosis rather than necrosis.

ANTI-TUMOR EFFECT OF INTRAVENOUS TNFα GENE DELIVERY NAKED PLASMID DNA USING A HYDRODYNAMICS-BASED PROCEDURE

High levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of naked plasmid (pDNA) into animals via the tail vein, the so-called hydrodynamics-based procedure. In this study, we evaluated the efficacy of hydrodynamics-based tumor necrosis factor alpha (TNFα) transfer for tumor treatment, in which the naked pDNA encoding TNFα was administered into the tail vein following an intravenous injection of B16 melanoma cells. The mice treated with TNFα-expressing pDNA displayed a profound reduction in lung metastasis. These results suggest that the hydrodynamics-based transfer of naked pDNA is a convenient and efficient method of TNFα gene therapy against metastatic tumors.

Anti-tumor effects of naked plasmid DNA using a hydrodynamics-based procedure

High levels of foreign gene expression in mouse hepatocytes can be achieved by the rapid injection of a large volume of naked plasmid (pDNA) into animals via the tail vein, the so-called hydrodynamics-based procedure. In this study, we evaluated the efficacy of hydrodynamics-based tumor necrosis factor alpha (TNFalpha) transfer for tumor treatment, in which the naked pDNA encoding TNFalpha was administered into the tail vein following a intravenous injection of B16 melanoma cells. The mice treated with TNFalpha-expressing pDNA displayed a profound reduction in lung metastasis.

Potent anti‐Metastatic Activity of Dimeric Sesquiterpene Thioalkaloids from the Rhizome of Nuphar pumilum.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals

AbstractMesenchymal stem cells (MSCs) are largely studied for their potential clinical use. Recently, they have gained further interest after demonstration of an immunosuppressive role. In this study, we investigated whether in vivo injection of MSCs could display side effects related to systemic immunosuppression favoring tumor growth. We first showed in vitro that the murine C3H10T1/2 (C3) MSC line and primary MSCs exhibit immunosuppressive properties in mixed lymphocyte reaction. We demonstrated that this effect is mediated by soluble factors, secreted only on “activation” of MSCs in the presence of splenocytes. Moreover, the immunosuppression is mediated by CD8+ regulatory cells responsible for the inhibition of allogeneic lymphocyte proliferation. We then demonstrated that the C3 MSCs expressing the human bone morphogenetic protein 2 (hBMP-2) differentiation factor were not rejected when implanted in various allogeneic immunocompetent mice and were still able to differentiate into bone. Importantly, using a murine melanoma tumor model, we showed that the subcutaneous injection of B16 melanoma cells led to tumor growth in allogeneic recipients only when MSCs were coinjected. Although the potential side effects of immunosuppression induced by MSCs have to be considered in further clinical studies, the usefulness of MSCs for various therapeutic applications still remains of great interest. (Blood. 2003;102:3837-3844)

Platelet and osteoclast beta3 integrins are critical for bone metastasis.

Mice with a targeted deletion of beta3 integrin were used to examine the process by which tumor cells metastasize and destroy bone. Injection of B16 melanoma cells into the left cardiac ventricle resulted in osteolytic bone metastasis in 74% of beta3+/+ mice by 14 days. In contrast, only 4% of beta3-/- mice developed bone lesions. Direct intratibial inoculation of tumor resulted in marrow replacement by tumor in beta3-/- mice, but no associated trabecular bone resorption as seen inbeta3+/+ mice. Bone marrow transplantation studies showed that susceptibility to bone metastasis was conferred by a bone marrow-derived cell. To dissect the roles of osteoclast and platelet beta3 integrins in this model of bone metastasis, osteoclast-defective src-/- mice were used. Src-null mice were protected from tumor-associated bone destruction but were not protected from tumor cell metastasis to bone. In contrast, a highly specific platelet aggregation inhibitor of activated alphaIIbbeta3 prevented B16 metastases. These data demonstrate a critical role for platelet alphaIIbbeta3 in tumor entry into bone and suggest a mechanism by which antiplatelet therapy may be beneficial in preventing the metastasis of solid tumors.

Inhibition of indoleamine 2,3-dioxygenase suppresses NK cell activity and accelerates tumor growth.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, is induced under various pathological conditions, including viral and bacterial infection, allograft rejection, cerebral ischemia, and tumor growth. We have previously reported that the expression of IDO mRNA was increased in some clinical cases of hepatocellular carcinoma in which the recurrence-free survival rate in these IDO-positive patients was significantly higher than that in patients without IDO mRNA induction in tumors. Additionally, IDO expressed in tumors was localized not to the tumor cells but instead to tumor-infiltrating cells by immunohistochemistry. In this study, in order to elucidate the mechanisms underlying anti-tumor effect of IDO, we investigated whether IDO inhibitor (1-methyl-dl-tryptophan, 1MT) affects the growth of subcutaneous B16 tumors in mice. Subsequently, the activity of natural killer (NK) cells was investigated under the conditions of inhibited IDO activity in vivo and in vitro. IDO mRNA expression of B16 cells, B16 subcutaneous tumor, sprenocytes of mice, and human NK cells were studied by reverse transcription-polymerase chain reaction. B16 subcutaneous tumor growth with or without IDO inhibition was observed and cytotoxic activity of NK cells were investigated under the conditions of inhibited IDO activity in vivo and in vitro. IDO mRNA was expressed in B16 subcutaneous tumor, splenocytes of tumor bearing mice, co-cultured splenocytes with B16, and human NK cells. On day 14, after injection of B16 melanoma cells, the sizes of tumors in IDO-inhibited mice were significantly larger than those in control mice. The cytotoxic activity of mice NK cells was reduced by IDO inhibition in vivo. In vitro inhibition of IDO, NK activity was reduced in dose-dependent manner of 1MT. In conclusion, these results indicated that IDO plays an important role in anti-tumor immunity by regulating cytotoxic activity of NK cells.

Potent anti-metastatic activity of dimeric sesquiterpene thioalkaloids from the rhizome of Nuphar pumilum

The methanolic extract and its alkaloid fraction from the rhizomes of Nuphar pumilum inhibited invasion of B16 melanoma cells across collagen-coated filters in vitro. Dimeric sesquiterpene thioalkaloids with the 6-hydroxyl group, 6-hydroxythiobinupharidine, 6,6′-dihydroxythiobinupharidine, and 6-hydroxythionuphlutine B, showed potent activity with IC50 values of 0.029, 0.087, and 0.36 μM, respectively, but dimeric sesquiterpene thioalkaloids lacking the 6-hydroxyl group (thiobinupharidine, neothiobinupharidine, syn-thiobinupharidine sulfoxide, thionuphultine B β-sulfoxide, and neothiobinupharidine β-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, deoxynupharidine, 7-epideoxynupharidine, and nupharolutine) showed weak activity. The alkaloid fraction (20 mg/kg/d, po) and the principal dimeric sesquiterpene thioalkaloid 6-hydroxythiobinupharidine (5 mg/kg/d, po) significantly inhibited lung tumor formation by more than 90% 10 days after injection of B16 melanoma cells in mice.