The immunomodulatory, antitumor and antimetastatic responses of melanoma-bearing normal and alcoholic mice to sunitinib and ALT-803: a combinatorial treatment approach.

ALT-803, a novel IL-15/IL-15 receptor alpha complex, and the tyrosine kinase inhibitor, sunitinib, were examined for their single and combined effects on the growth of subcutaneous B16BL6 melanoma and on lymph node and lung metastasis. The study was conducted in immunocompetent C57BL/6 mice drinking water (Water mice) and in mice that chronically consumed alcohol (Alcohol mice), which are deficient in CD8(+) T cells. Sunitinib inhibited melanoma growth and was more effective in Alcohol mice. ALT-803 did not alter tumor growth or survival in Water or Alcohol mice. Combined ALT-803 and sunitinib inhibited melanoma growth and increased survival, and these effects were greater than sunitinib alone in Water mice. ALT-803 and alcohol independently suppressed lymph node and lung metastasis, whereas sunitinib alone or in combination with ALT-803 increased lymph node and lung metastasis in Water and Alcohol mice. Initially, ALT-803 increased IFN-γ-producing CD8(+)CD44(hi) memory T cells and CD8(+)CD44(hi)CD62L(lo) effector memory T cells and sunitinib decreased immunosuppressive MDSC and T regulatory cells (Treg). However, the impact of these treatments diminished with time. Subcutaneous tumors from Water mice showed increased numbers of CD8(+) T cells, CD8(+)CD44(hi) T cells, NK cells, and MDSC cells and decreased Treg cells after ALT-803 treatment.

Amino acid-dependent NPRL2 interaction with Raptor determines mTOR Complex 1 activation.

We assign a new function to a tumor suppressor NPRL2 that activates the mTOR complex 1 (mTORC1) activity. The positive regulation of mTORC1 activity by NPRL2 is mediated through NPRL2 interaction with Raptor. While NPRL2 interacts with Rag GTPases, RagD in particular, to interfere with mTORC1 activity in amino acid scarcity, NPRL2 interacts with Raptor in amino acid sufficiency to activate mTORC1. A reciprocal relationship exists between NPRL2 binding to Rag GTPases and Raptor. NPRL2 majorly locates in the lysosomal membranes and has a higher binding affinity to the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) that inactivates mTORC1. However, the binding affinity of NPRL2 with Raptor is much less pronounced in cells expressing the dominant negative mutant heterodimer of RagA(GDP)/RagD(GTP) than in cells expressing the dominant positive mutant heterodimer, RagA(GTP)/RagD(GDP). The positive effect of NPRL2 on TORC1 pathway was also evidenced in Drosophila animal model. Here, we propose a 'seesaw' model in which the interactive behavior of NPRL2 with Raptor determines mTORC1 activation by amino acid signaling in animal cells.

Effects of Alcohol on Tumor Growth, Metastasis, Immune Response, and Host Survival.

Most research involving alcohol and cancer concerns the relationship between alcohol consumption and cancer risk and the mechanisms of carcinogenesis. This review relates the amount and duration of alcohol intake in humans and in animal models of cancer to tumor growth, angiogenesis, invasion, metastasis, immune response, and host survival in specific types and subtypes of cancer. Research on the influence of alcohol drinking on human cancer patients is limited. Although there is more information in animal models of cancer, many aspects still are ill defined. More research is needed to define the mechanisms that underlie the role of alcohol on cancer progression in both animals and humans. Activation of the immune system can play a positive role in keeping cancer under control, but this also can facilitate cancer progression. Additionally, a functional immune system is required for cancer patients to achieve an optimal response to conventional chemotherapy. Insight into the underlying mechanisms of these interactions could lead to effective immunotherapeutic approaches to treat alcoholics with cancer. Defining the epigenetic mechanisms that modulate cancer progression also has great potential for the development of new treatment options not only for treating alcoholics with cancer but also for treating other alcohol-induced diseases.

Chronic alcohol consumption inhibits melanoma growth but decreases the survival of mice immunized with tumor cell lysate and boosted with α-galactosylceramide.

Alcohol consumption increases the incidence of multiple types of cancer. However, how chronic alcohol consumption affects tumor progression and host survival remains largely unexplored. Using a mouse B16BL6 melanoma model, we studied the effects of chronic alcohol consumption on s.c. tumor growth, iNKT cell antitumor immune response, and host survival. The results indicate that although chronic alcohol consumption inhibits melanoma growth, this does not translate into increased host survival. Immunizing mice with a melanoma cell lysate does not significantly increase the median survival of water-drinking, melanoma-bearing mice, but significantly increases the median survival of alcohol-consuming, melanoma-bearing mice. Even though survival is extended in the alcohol-consuming mice after immunization, the median survival is not different from the immunized mice in the water-drinking group. Immunization with tumor cell lysate combined with α-galatosylceramide activation of iNKT cells significantly increases host survival of both groups of melanoma-bearing mice compared to their respective non-immunized counterparts; however, the median survival of the alcohol-consuming group is significantly lower than that of the water-drinking group. Alcohol consumption increases NKT cells in the thymus and blood and skews NKT cell cytokine profile from Th1 dominant to Th2 dominant in the tumor-bearing mice. In summary, these results indicate that chronic alcohol consumption activates the immune system, which leads to the inhibition of s.c. melanoma growth and enhances the immune response to immunization with melanoma lysate. With tumor progression, alcohol consumption accelerates iNKT cell dysfunction and compromises antitumor immunity, which leads to decreased survival of melanoma-bearing mice.

Alcohol consumption and antitumor immunity: dynamic changes from activation to accelerated deterioration of the immune system.

The molecular mechanisms of how alcohol and its metabolites induce cancer have been studied extensively. However, the mechanisms whereby chronic alcohol consumption affects antitumor immunity and host survival have largely been unexplored. We studied the effects of chronic alcohol consumption on the immune system and antitumor immunity in mice inoculated with B16BL6 melanoma and found that alcohol consumption activates the immune system leading to an increase in the proportion of IFN-γ-producing NK, NKT, and T cells in mice not injected with tumors. One outcome associated with enhanced IFN-γ activation is inhibition of melanoma lung metastasis. However, the anti-metastatic effects do not translate into increased survival of mice bearing subcutaneous tumors. Continued growth of the subcutaneous tumors and alcohol consumption accelerates the deterioration of the immune system, which is reflected in the following: (1) inhibition in the expansion of memory CD8+ T cells, (2) accelerated decay of Th1 cytokine-producing cells, (3) increased myeloid-derived suppressor cells, (4) compromised circulation of B cells and T cells, and (5) increased NKT cells that exhibit an IL-4 dominant cytokine profile, which is inhibitory to antitumor immunity. Taken together, the dynamic effects of alcohol consumption on antitumor immunity are in two opposing phases: the first phase associated with immune stimulation is tumor inhibitory and the second phase resulting from the interaction between the effects of alcohol and the tumor leads to immune inhibition and resultant tumor progression.

Chronic alcohol consumption enhances iNKT cell maturation and activation.

Alcohol consumption exhibits diverse effects on different types of immune cells. NKT cells are a unique T cell population and play important immunoregulatory roles in different types of immune responses. The effects of chronic alcohol consumption on NKT cells remain to be elucidated. Using a mouse model of chronic alcohol consumption, we found that alcohol increases the percentage of NKT cells, especially iNKT cells in the thymus and liver, but not in the spleen or blood. Alcohol consumption decreases the percentage of NK1.1(-) iNKT cells in the total iNKT cell population in all of the tissues and organs examined. In the thymus, alcohol consumption increases the number of NK1.1(+)CD44(hi) mature iNKT cells but does not alter the number of NK1.1(-) immature iNKT cells. A BrdU incorporation assay shows that alcohol consumption increases the proliferation of thymic NK1.1(-) iNKT cells, especially the NK1.1(-)CD44(lo) Stage I iNKT cells. The percentage of NKG2A(+) iNKT cells increases in all of the tissues and organs examined; whereas CXCR3(+) iNKT cells only increases in the thymus of alcohol-consuming mice. Chronic alcohol consumption increases the percentage of IFN-γ-producing iNKT cells and increases the blood concentration of IFN-γ and IL-12 after in vivo α-galactosylceramide (αGalCer) stimulation. Consistent with the increased cytokine production, the in vivo activation of iNKT cells also enhances the activation of dendritic cells (DC) and NK, B, and T cells in the alcohol-consuming mice. Taken together the data indicate that chronic alcohol consumption enhances iNKT cell maturation and activation, which favors the Th1 immune response.

A novel approach to identify driver genes involved in androgen-independent prostate cancer.

BACKGROUND: Insertional mutagenesis screens have been used with great success to identify oncogenes and tumor suppressor genes. Typically, these screens use gammaretroviruses (γRV) or transposons as insertional mutagens. However, insertional mutations from replication-competent γRVs or transposons that occur later during oncogenesis can produce passenger mutations that do not drive cancer progression. Here, we utilized a replication-incompetent lentiviral vector (LV) to perform an insertional mutagenesis screen to identify genes in the progression to androgen-independent prostate cancer (AIPC). METHODS: Prostate cancer cells were mutagenized with a LV to enrich for clones with a selective advantage in an androgen-deficient environment provided by a dysregulated gene(s) near the vector integration site. We performed our screen using an in vitro AIPC model and also an in vivo xenotransplant model for AIPC. Our approach identified proviral integration sites utilizing a shuttle vector that allows for rapid rescue of plasmids in E. coli that contain LV long terminal repeat (LTR)-chromosome junctions. This shuttle vector approach does not require PCR amplification and has several advantages over PCR-based techniques. RESULTS: Proviral integrations were enriched near prostate cancer susceptibility loci in cells grown in androgen-deficient medium (p < 0.001), and five candidate genes that influence AIPC were identified; ATPAF1, GCOM1, MEX3D, PTRF, and TRPM4. Additionally, we showed that RNAi knockdown of ATPAF1 significantly reduces growth (p < 0.05) in androgen-deficient conditions. CONCLUSIONS: Our approach has proven effective for use in PCa, identifying a known prostate cancer gene, PTRF, and also several genes not previously associated with prostate cancer. The replication-incompetent shuttle vector approach has broad potential applications for cancer gene discovery, and for interrogating diverse biological and disease processes.

Synthesis and anticancer activity of new flavonoid analogs and inconsistencies in assays related to proliferation and viability measurements.

Flavonoids have been studied intensely for their ability to act as anti-carcinogenic, anti-inflammatory, anti-viral and anti-aging agents and are often marketed as supplements related to their anti-inflammatory activity. Previous studies have primarily focused on the effects of polar natural flavonoids. We examined the activity of novel hydrophobic and lipophilic flavonols against human DU-145 and PC-3 prostate cancer cell lines. All flavonol analogs were more active than the naturally occurring flavonols quercetin, kaempferol, kaempferide and galangin. The most potent analogs were 6.5-fold more active against DU-145 and PC-3 cells than quercetin and fell within the biologically relevant concentration range (low micromolar). We also evaluated the potential toxic effects of flavonol analogs on normal cells, an assessment that has frequently been ignored when studying the anticancer effects of flavonoids. During these analyses, we discovered that various metabolic and DNA staining assays were unreliable methods for assessing cell viability of flavonoids. Flavonoids reduce colorimetric dyes such as MTT and Alamar Blue in the absence of cells. We showed that flavonol-treated prostate cancer cells were stained less intensely with crystal violet than untreated cells at non-toxic concentrations. The trypan blue exclusion assay was selected as a reliable alternative for measuring cell viability.

Amelioration of behavioral abnormalities in BH(4)-deficient mice by dietary supplementation of tyrosine.

This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4)-deficient Spr (-/-) mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr (-/-) mice. We found that Spr (-/-) mice display variable 'open-field' behaviors, impaired motor functions on the 'rotating rod', and dystonic 'hind-limb clasping'. In this study, we report that these aberrant motor deficits displayed by Spr (-/-) mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr (-/-) mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA) and its metabolites in Spr (-/-) mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr (-/-) mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency.

Alcohol consumption suppresses mammary tumor metastasis in a syngeneic tumor transplantation model.

Epidemiological studies indicate a positive correlation between alcohol consumption and the risk of developing breast cancer. However, little is known about whether alcohol consumption affects breast cancer metastasis. Considering that the primary cause of death in breast cancer patients is due to metastasis, further insight into whether alcohol consumption influences disease progression and survival is needed. We tested the effect of alcohol consumption on breast cancer metastasis using the 4T1.2 syngeneic mammary tumor model in Balb/c mice. The treatment groups included a High-consuming group (18 % w/v alcohol in drinking water), a Moderate-consuming group (5 % w/v), a Low-consuming group (1 % w/v), and a Water-drinking control group. 4T1.2 mammary tumor cells were injected orthotopically into the mammary fat pad. Metastases were enumerated in lungs and in distant mammary glands 4 weeks after injection. Consumption of High alcohol protected against metastasis, as High-consuming mice typically had 65-75 % fewer metastases compared to Water-drinking controls. A suggestive reduction in tumor spread was observed in the Moderate-drinking group, although the effects did not reach statistical significance. Consumption of the Low alcohol dose did not affect metastasis. CXCR4 expression in the primary tumors was significantly reduced by High alcohol consumption; however, expression of this chemokine receptor in the primary tumor did not correlate with metastatic potential. Additional studies were conducted to test for possible direct effects of 0.3 % w/v ethanol on tumor cell proliferation, migration, invasion, and colony formation of 4T1.2 cells in vitro. Our results indicate that, for this murine model, alcohol consumption does not exacerbate tumor metastasis, and that High alcohol consumption reduces tumor spread.

Establishment of an immunodeficient alcohol mouse model to study the effects of alcohol on human cells in vivo.

OBJECTIVE: The effects of alcohol (ethanol) are well documented and contribute to significant health problems and financial burden on the health care system. Several mouse models have been described that facilitate studies of the effects of alcohol on the mouse immune system. Our goal was to establish a chronic alcohol mouse model using the immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse. This severely immunodeficient model has previously been shown to allow efficient engraftment of human hematopoietic repopulating cells and cancer cells, thereby facilitating diverse studies on human hematopoiesis, immune cell function, and oncogenesis in vivo. METHOD: NSG mice were provided ethanol in their drinking water as the only available fluid, starting at 5% weight/volume (w/v) and subsequently were increased to 10%, 15%, and 20% w/v. Mice were then maintained at 20% w/v, a level that models chronic alcohol use in humans. Alcohol consumption and weight were monitored. RESULTS: NSG mice readily consumed alcohol throughout the study and showed no adverse effects. No significant difference between group mean weights was identified the day before increasing the ethanol dose or at the end of 5 weeks at 20% w/v (p > .28). While the mice were maintained at 20% w/v ethanol, the mean daily ethanol intake was 27.2 g/kg of body weight, 32% of caloric intake. CONCLUSIONS: Here we have established a chronic alcohol mouse model using the powerful immunodeficient NSG mouse. This model should allow for novel studies on the effects of alcohol on engrafted human cells, including studies on the effects of alcohol on hematopoiesis, immunity, and cancer.

Chronic alcohol consumption impairs distribution and compromises circulation of B cells in B16BL6 melanoma-bearing mice.

Accumulating research indicates that B cells are involved in anti-tumor immunity. Chronic alcohol consumption is associated with decreased survival of cancer patients. The effect of alcohol consumption on B cells in tumor-bearing hosts is unknown. Results in melanoma-bearing mice showed that chronic alcohol consumption did not alter the percentage and number of B cells in bone marrow, spleen, and lymph nodes but dramatically decreased B cells in the peripheral blood. Alcohol consumption did not alter the development of B cells in the bone marrow and did not affect follicular B cells in the spleen; however, it increased T1 B cells and decreased marginal zone B cells in the spleen. Alcohol consumption also decreased mature B cells in the blood. It did not alter the chemotactic capacity of plasma to facilitate migration of splenocytes or the chemotactic response of splenocytes to CXCL13 and CCL21. However, the response of splenocytes to sphingosine-1-phosphate was impaired in alcohol-consuming, melanoma-bearing mice. The expression of sphingosine-1-phosphate receptor-1 (S1PR1) and sphingosine-1-phosphate lyase-1 (SPL1) in splenocytes was downregulated. Taken together, these results indicate that chronic alcohol consumption decreases peripheral blood B cells by compromising B cell egress from the spleen. The downregulation of S1PR1 and SPL1 expression in alcohol-consuming, melanoma-bearing mice could be associated with compromised egress of B cells from the spleen.

Diet, nutrients, phytochemicals, and cancer metastasis suppressor genes.

The major factor in the morbidity and mortality of cancer patients is metastasis. There exists a relative lack of specific therapeutic approaches to control metastasis, and this is a fruitful area for investigation. A healthy diet and lifestyle not only can inhibit tumorigenesis but also can have a major impact on cancer progression and survival. Many chemicals found in edible plants are known to inhibit metastatic progression of cancer. While the mechanisms underlying antimetastatic activity of some phytochemicals are being delineated, the impact of diet, dietary components, and various phytochemicals on metastasis suppressor genes is underexplored. Epigenetic regulation of metastasis suppressor genes promises to be a potentially important mechanism by which dietary components can impact cancer metastasis since many dietary constituents are known to modulate gene expression. The review addresses this area of research as well as the current state of knowledge regarding the impact of diet, dietary components, and phytochemicals on metastasis suppressor genes.

Prenatal alcohol exposure alters the course and severity of adjuvant-induced arthritis in female rats.

Prenatal alcohol exposure (PAE) has adverse effects on the development of numerous physiological systems, including the hypothalamic-pituitary-adrenal (HPA) axis and the immune system. HPA hyper-responsiveness and impairments in immune competence have been demonstrated. The present study investigated immune function in PAE females utilizing an adjuvant-induced arthritis (AA) model, widely used as a model of human rheumatoid arthritis. Given the effects of PAE on HPA and immune function, and the known interaction between HPA and immune systems in arthritis, we hypothesized that PAE females would have heightened autoimmune responses, resulting in increased severity of arthritis, compared to controls, and that altered HPA activity might play a role in the immune system changes observed. The data demonstrate, for the first time, an adverse effect of PAE on the course and severity of AA in adulthood, indicating an important long-term alteration in functional immune status. Although overall, across prenatal treatments, adjuvant-injected animals gained less weight, and exhibited decreased thymus and increased adrenal weights, and increased basal levels of corticosterone and adrenocorticotropin, PAE females had a more prolonged course of disease and greater severity of inflammation compared to controls. In addition, PAE females exhibited blunted lymphocyte proliferative responses to concanavalin A and a greater increase in basal ACTH levels compared to controls during the induction phase, before any clinical signs of disease were apparent. These data suggest that prenatal alcohol exposure has both direct and indirect effects on inflammatory processes, altering both immune and HPA function, and likely, the normal interactions between these systems.

Activation of the aryl hydrocarbon receptor by TCDD inhibits mammary tumor metastasis in a syngeneic mouse model of breast cancer.

Treatment with aryl hydrocarbon receptor (AhR) agonists can slow or reverse the growth of primary mammary tumors in rodents, which has fostered interest in developing selective AhR modulators for treatment of breast cancer. However, the major goal of breast cancer therapy is to inhibit metastasis, the primary cause of mortality in women with this disease. Studies conducted using breast cancer cell lines have demonstrated that AhR agonists suppress proliferation, invasiveness, and colony formation in vitro; however, further exploration using in vivo models of metastasis is warranted. To test the effect of AhR activation on metastasis, 4T1.2 mammary tumor cells were injected into the mammary gland fat pad of syngeneic Balb/c mice treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Primary tumor growth was monitored for 4 weeks, at which time metastasis was determined. TCDD treatment suppressed metastasis by approximately 50%, as measured both in the lung and in mammary glands at sites distant from the primary tumor. Primary tumor growth was not suppressed by TCDD exposure nor was proliferation of 4T1.2 cells affected by TCDD treatment in vitro. Taken together, these results suggest that the protective effect of AhR activation was selective for the metastatic process and not simply the result of a direct decrease in tumor cell proliferation or survival at the primary site. These observations in immunologically intact animals warrant further investigation into the mechanism of the protective effects of AhR activation and support the promise for use of AhR modulators to treat breast cancer.

Autophagy induction by tetrahydrobiopterin deficiency.

Tetrahydrobiopterin (BH4) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH4 deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH4-deficient Spr(-/-) mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH4 synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr(-/-) mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr(-/-) mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr(-/-) mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pah(enu2) mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH4 deficiency.

Differential effects of specific amino acid restriction on glucose metabolism, reduction/oxidation status and mitochondrial damage in DU145 and PC3 prostate cancer cells.

Selective amino acid restriction targets mitochondria to induce apoptosis of DU145 and PC3 prostate cancer cells. Biochemical assays and flow cytometry were uitilized to analyze the glucose consumption, lactate production, pyruvate dehydrogenase (PDH), nicotinamide adenine dinucleotide (NAD)/NADH and nicotinamide adenine dinucleotide phosphate (NADP)/NADPH ratios, mitochondrial glutathione peroxidase (GPx), manganese superoxide dismutase (SOD), glutathione, reactive oxygen species (ROS) and DNA damage in DU145 and PC prostate cancer cells cultured under various amino acid deprived conditions. Restriction of tyrosine and phenylalanine (Tyr/Phe), glutamine (Gln) or methionine (Met) differentially modulated glucose metabolism and PDH and antioxidant enzyme activity in the mitochondria of the two prostate cancer cell lines. In DU145 cells, Gln and Met restriction increased glucose consumption and decreased lactate production, but Tyr/Phe restriction did not. The examined restrictions increased mitochondrial PDH activity and accumulation of ROS. Gln and Met restriction increased GPx activity. Tyr/Phe and Met restriction increased SOD during the first 2 days of the restriction, and the activity returned to the basal level on day 4. All amino acid restrictions decreased reduced glutathione (GSH) and induced mitochondrial DNA damage. In PC3 cells, all amino acid restrictions reduced glucose consumption and lactate production. Gln restriction increased ROS and elevated GPx activity. Tyr/Phe restriction increased SOD activity. The amino acid restriction decreased GSH, but did not cause mitochondrial DNA damage. Specific amino acid dependency differentially regulates glucose metabolism, oxidation-reduction reactions of mitochondria and mitochondrial damage in DU145 and PC3 prostate cancer cell lines.

IFN-γ is essential for the inhibition of B16BL6 melanoma lung metastasis in chronic alcohol drinking mice.

We previously found that chronic alcohol consumption (20% w/v in drinking water) that models the level consumed by human alcoholics, when administered to female C57BL/6 mice inhibits B16BL6 melanoma metastasis to the lung; however, the mechanism is not known. Chronic alcohol consumption increases IFN-γ producing NK, NKT, CD4(+), and CD8(+) T cells. To examine the impact of IFN-γ on metastasis, we inoculated B16BL6 melanoma cells i.v. into control and chronic alcohol drinking IFN-γ knockout (KO) mice. Knockout of the ifn-γ gene abrogated the anti-metastatic effects associated with alcohol consumption. We examined metastasis in common gamma-chain (γC) KO mice, which are deficient in NK, NKT and CD8(+) T cells, and in Vα14Jα281(-/-) KO mice, which are deficient in invariant NKT (iNKT) cells, in order to assess the importance of specific IFN-γ producing cell types to this effect. We found that the antimetastatic effect of alcohol was still present in γC KO mice and also in γC KO mice depleted of Gr-1(+) cells. Knockout of iNKT cells reduced the degree but not the antimetastatic effect associated with alcohol. These results indicate that the antimetastatic effect induced by chronic alcohol consumption is IFN-γ dependent and that multiple IFN-γ producing cell types contribute to this effect.

Chronic alcohol consumption decreases the percentage and number of NK cells in the peripheral lymph nodes and exacerbates B16BL6 melanoma metastasis into the draining lymph nodes.

NK cells in the lymph nodes play important roles in inhibiting tumor metastasis into draining lymph nodes. Previously, we reported that chronic alcohol consumption interferes with NK cell trafficking from the bone marrow to the spleen. Herein, we found that alcohol consumption decreases the numbers of NK cells in lymph nodes. Adoptive transfer experiments indicated that continued exposure of donor splenocytes to alcohol inhibits NK but not T cell trafficking to lymph nodes. Alcohol did not negatively affect CCR7(+) and CXCR3(+) NK cells, but decreased the percentage and number of CD62L(+) NK cells in the spleen, which are an important source of NK cell trafficking into the lymph nodes. These data suggest that modulation of the microenvironment associated with alcohol consumption impairs the trafficking of NK cells to lymph nodes. The decreased number of NK cells in the lymph nodes was associated with increased melanoma metastasis into the draining lymph nodes.

Laboratory models available to study alcohol-induced organ damage and immune variations: choosing the appropriate model.

The morbidity and mortality resulting from alcohol-related diseases globally impose a substantive cost to society. To minimize the financial burden on society and improve the quality of life for individuals suffering from the ill effects of alcohol abuse, substantial research in the alcohol field is focused on understanding the mechanisms by which alcohol-related diseases develop and progress. Since ethical concerns and inherent difficulties limit the amount of alcohol abuse research that can be performed in humans, most studies are performed in laboratory animals. This article summarizes the various laboratory models of alcohol abuse that are currently available and are used to study the mechanisms by which alcohol abuse induces organ damage and immune defects. The strengths and weaknesses of each of the models are discussed. Integrated into the review are the presentations that were made in the symposium "Methods of Ethanol Application in Alcohol Model-How Long is Long Enough" at the joint 2008 Research Society on Alcoholism (RSA) and International Society for Biomedical Research on Alcoholism (ISBRA) meeting, Washington, DC, emphasizing the importance not only of selecting the most appropriate laboratory alcohol model to address the specific goals of a project but also of ensuring that the findings can be extrapolated to alcohol-induced diseases in humans.