The DPA-derivative 11S, 17S-dihydroxy 7,9,13,15,19 (Z,E,Z,E,Z)-docosapentaenoic acid inhibits IL-6 production by inhibiting ROS production and ERK/NF-κB pathway in keratinocytes HaCaT stimulated with a fine dust PM10.

11 S, 17S-dihydroxy 7,9,13,15,19 (Z,E,Z,E,Z)-docosapentaenoic acid (DoPE) is a derivative of docosapentaenoic acid, a specialized pro-resolving mediator of inflammation such as lipoxins, resolvins, maresins, and protectins. PM10 is a fine dust particle that induces oxidative stress, DNA damage, inflammation, aging, and cancer. The anti-inflammatory mechanism of DoPE, however, has not yet been elucidated. In these studies, we investigated whether DoPE has anti-inflammatory effects in human keratinocyte HaCaT cells. We demonstrated that DoPE suppressed PM10-induced expressions of IL-6 mRNA and protein in human HaCaT keratinocytes. We also investigated the modulating effects of DoPE on reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK). ROS production, extracellular signal regulated kinase (ERK) phosphorylation, and translocation of nuclear factor-kappa B (NF-kB) p65 and NF-kB activity were suppressed by DoPE in PM10-stimulated HaCaT cells. Collectively, our results demonstrated that DoPE inhibited IL-6 expression by reducing ROS generation, suppressing ERK phosphorylation, and inhibiting translocation of NF-kB p65 and NF-kB activity in PM10-stimulated HaCaT cells, suggesting that DoPE can be useful for the resolution of the inflammation caused by IL-6.

IL-32γ suppressed atopic dermatitis through inhibition of miR-205 expression via inactivation of nuclear factor-kappa B.

BACKGROUND: IL-32 is a novel cytokine involved in many inflammatory diseases. However, the role of IL-32γ, an isotype of IL-32, in atopic dermatitis (AD) has not been reported. OBJECTIVE: We investigated the effects of IL-32γ on development of AD and its action mechanisms. METHODS: We used phthalic anhydride (PA) and an MC903-induced AD model using wild-type and IL-32γ transgenic mice. We conducted the therapy experiments by using recombinant IL-32γ protein in a reconstructed human skin model and PA-induced model. We conducted a receiver operating characteristic analysis of IL-32γ with new AD biomarkers, IL-31 and IL-33, in serum from patients with AD. RESULTS: Dermatitis severity and epidermal thickness were significantly reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. The concentration of AD-related cytokines was reduced in PA- and MC903-induced IL-32γ transgenic mice compared with in wild-type mice. Subsequent analysis showed that IL-32γ inhibits miR-205 expression in PA- and MC903-induced skin tissue samples and TNF-α/IFN-γ-treated HaCaT cells. IL-32γ reduced NF-κB activity in skin tissue samples from PA- and MC903-induced mice and TNF-α/IFN-γ-treated HaCaT cells. NF-κB inhibitor treatment with IL-32γ expression further suppressed expression of inflammatory mediators as well as miR-205 in TNF-α/IFN-γ-treated HaCaT cells. Furthermore, recombinant IL-32γ protein alleviated AD-like inflammation in in vivo and reconstructed human skin models. Spearman correlation analysis showed that serum levels of IL-32γ and miR-205 were significantly concordant in patients with AD. CONCLUSION: Our results indicate that IL-32γ reduces AD through the inhibition of miR-205 expression via inactivation of NF-κB.

Interleukin-32θ Triggers Cellular Senescence and Reduces Sensitivity to Doxorubicin-Mediated Cytotoxicity in MDA-MB-231 Cells.

The recently discovered interleukin (IL)- 32 isoform IL-32θ exerts anti-metastatic effects in the breast tumor microenvironment. However, the involvement of IL-32θ in breast cancer cell proliferation is not yet fully understood; therefore, the current study aimed to determine how IL-32θ affects cancer cell growth and evaluated the responses of IL-32θ-expressing cells to other cancer therapy. We compared the functions of IL-32θ in triple-negative breast cancer MDA-MB-231 cells that stably express IL-32θ, with MDA-MB-231 cells transfected with a mock vector. Slower growth was observed in cells expressing IL-32θ than in control cells, and changes were noted in nuclear morphology, mitotic division, and nucleolar size between the two groups of cells. Interleukin-32θ significantly reduced the colony-forming ability of MDA-MB-231 cells and induced permanent cell cycle arrest at the G1 phase. Long-term IL-32θ accumulation triggered permanent senescence and chromosomal instability in MDA-MB-231 cells. Genotoxic drug doxorubicin (DR) reduced the viability of MDA-MB-231 cells not expressing IL-32θ more than in cells expressing IL-32θ. Overall, these findings suggest that IL-32θ exerts antiproliferative effects in breast cancer cells and initiates senescence, which may cause DR resistance. Therefore, targeting IL-32θ in combination with DR treatment may not be suitable for treating metastatic breast cancer.

Peroxiredoxin I deficiency increases keratinocyte apoptosis in a skin tumor model via the ROS-p38 MAPK pathway.

Keratinocyte hyperproliferation is an essential link in skin cancer pathogenesis. Peroxiredoxin I (Prx I) is known to regulate cancer cell proliferation, differentiation, and apoptosis, but its role in skin cancer remains unclear. This study aimed to elucidate the role and mechanism of Prx I in skin cancer pathogenesis. Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were used to create a skin tumor model of the initiation/promotion stage of cancer. The role of Prx I in H2O2-induced keratinocyte apoptosis was also investigated. After DMBA/TPA treatment, Prx I deficiency was significantly associated with less skin tumors, lower Bcl-2 expression, and higher p-p38 and cleaved caspase-3 expressions in Prx I knockout tumors than in wild-type controls. H2O2 stimulation caused more cellular apoptosis in Prx I knockdown HaCaT cells than in normal HaCaT cells. The signaling study revealed that Bcl-2, p-p38, and cleaved caspase-3 expressions were consistent with the results in the tumors. In conclusion, the deletion of Prx I triggered the DMBA/TPA-induced skin tumor formation in vivo and in vitro by regulating the reactive oxygen species (ROS)-p38 mitogen-activated protein kinase (MAPK) pathway. These findings provide a theoretical basis for treating skin cancer.

Methyl lucidone induces apoptosis and G2/M phase arrest via the PI3K/Akt/NF-κB pathway in ovarian cancer cells.

Context: Methyl lucidone (ML) from the dried fruit of Lindera erythrocarpa Makino (Lauraceae) exhibits cytotoxic effects in various cancer cell lines. However, its effects on ovarian cancer cells remain unknown.Objective: This study evaluates the mechanism of ML-induced apoptosis, cell cycle distribution in ovarian cells.Materials and methods: The cytotoxic effect of ML (2.5-80 µM) on OVCAR-8 and SKOV-3 cells was evaluated by MTS assay for 24 and 48 h. Apoptosis and cell cycle arrest were analysed by flow cytometry. PCR, western blot analyses were performed to examine the related signalling pathways.Results: ML induced significant cellular morphological changes and apoptosis in ovarian cancer cells, leading to an antiproliferative effect (IC50 = 33.3-54.7 µM for OVCAR-8 and 48.8-60.7 µM for SKOV-3 cells). Treatment with ML induced cleavage of caspase-3/9 and PARP and release of cytochrome c from the mitochondria. Moreover, ML downregulated the expression of Bcl-2 and Bcl-xL and induced cell cycle arrest in the G2/M phase. Additionally, ML suppressed the expression of cyclin-A/B and promoted that of the cyclin-dependent kinase inhibitors p21 and p27. The expression of death receptors was not altered. Interestingly, ML also inhibited the activity of PI3K/Akt and NF-κB.Discussion and conclusions: ML caused G2/M phase arrest and apoptosis in ovarian cancer cells by activating intrinsic apoptotic pathways and suppressing the PI3K/Akt survival pathway. ML may be a potential anticancer agent to suppress ovarian cancer proliferation; thus, to improve the survival rate of cancer patients.

Interleukin-32θ inhibits tumor-promoting effects of macrophage-secreted CCL18 in breast cancer.

BACKGROUND: Tumor-associated macrophages can promote breast cancer metastasis by secreting cytokines and growth factors. Interleukin (IL)-32θ, a newly identified IL-32 isoform, was previously shown to down-regulate various proinflammatory factors of macrophages. Here, we report the presence of IL-32θ in breast cancer tissues and evaluate its effects on macrophage-regulated breast cancer metastasis. METHODS: RT-qPCR was used to analyze the mRNA expression of IL-32θ, Chemokine (C-C motif) ligand 18 (CCL18) in breast cancer tissues. In vitro cell-based experiments using IL-32θ-expressing MDA-MB-231 cells were conducted to examine the effects of IL-32θ on metastasis and its molecular signaling. In vivo xenograft, immunohistochemistry, and optical imaging models were generated to support in vitro and clinical findings. RESULTS: The clinical data displayed opposite expression patterns of CCL18 and IL-32θ mRNA in macrophage-infiltrated breast tumor tissues compared with those in the other tissues tested. In MDA-MB-231 cells, IL-32θ overexpression attenuated migration, invasion, tumor-promoting factors, and increased epithelial markers levels upon treatment with conditioned media from THP-1-derived macrophages. Additionally, IL-32θ expression in a xenograft model led to a remarkable decrease in tumor size and macrophage-stimulated tumor promotion. This inhibition was mediated through a direct interaction with protein kinase C-δ (PKCδ), subsequently eliminating the downstream factors STAT3 and NF-κB. Blocking CCL18 during co-culture of macrophages and breast cancer cells reduced the levels of breast cancer progression-related factors and PKCδ downstream signaling suggesting CCL18 as the main macrophage-secreted factors triggering the signaling pathway inhibited by IL-32θ. CONCLUSIONS: Our findings demonstrate a novel role of IL-32θ as an intracellular modulator to suppress macrophage-promoted breast cancer progression by targeting CCL18-dependent signaling.

Development of a new 15-valent pneumococcal conjugate vaccine (PCV15) and evaluation of its immunogenicity.

A new 15-valent pneumococcal conjugate vaccine (PCV15) against serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 11A, 14, 18C, 19A, 19F, 22F, and 23F has been developed using aluminum phosphate as an adjuvant. Using the rabbit model, immunogenicity of each serotype was evaluated by measuring antigen specific antibodies and functional antibody titers and comparing them to a control vaccine, Prevnar13®. Among the shared serotypes in both PCV15 and Prevnar13®, Type 3 and 23F in PCV15 exhibited a lower opsonic index than Prevnar13®. Conversely, the other types showed greater or nearly the same immunogenic effects. Type 11A and 22F are two additional serotypes included in PCV15, and only 22F showed a reasonable opsonic index compared with other types. Type 11A exhibited a basal level fold-increase in OPA; thus, we further optimized 11A as well as 3 and 23F by controlling the polysaccharide-to-protein conjugation ratio as a variable. Antibody levels and functional antibody activities were evaluated by ELISA and OPA, and improved levels of immunogenic activities were observed for all three serotypes. In this study, we propose a new PCV15 candidate, in which the common 13 serotypes and a licensed control vaccine have equivalent efficacy while two additional serotypes showed adequate immunogenicity in the rabbit model.

Calotropis gigantea extract induces apoptosis through extrinsic/intrinsic pathways and reactive oxygen species generation in A549 and NCI-H1299 non-small cell lung cancer cells.

BACKGROUND: Calotropis gigantea (CG) is a tall and waxy flower that is used as a traditional remedy for fever, indigestion, rheumatism, leprosy, and leukoderma. However, the precise mechanisms of its anticancer effects have not yet been examined in human non-small cell lung cancer (NSCLC) cells. In this study, we investigated whether CG extract exerted an apoptotic effect in A549 and NCI-H1299 NSCLC cells. METHODS: The ethanol extract of CG was prepared, and its apoptotic effects on A549 and NCI-H1299 NSCLC cells were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining, cell cycle analysis, real-time polymerase chain reaction (RT-PCR), western blotting, JC-1 staining, and ROS detection assay. RESULTS: The CG extract induced apoptosis through the stimulation of intrinsic and extrinsic signaling pathways in A549 and NCI-H1299 lung cancer cells. Cell cycle arrest was induced by the CG extract in both cell lines. Reactive oxygen species (ROS), which can induce cell death, were also generated in the CG-treated A549 and NCI-H1299 cells. CONCLUSIONS: These data confirmed that CG caused apoptosis through the activation of extrinsic and intrinsic pathways, cell cycle arrest, and ROS generation in A549 and NCI-H1299 lung cancer cells. Thus, CG can be suggested as a potential agent for lung cancer therapy.

Epimagnolin A inhibits IL-6 production by inhibiting p38/NF-κB and AP-1 signaling pathways in PMA-stimulated THP-1 cells.

Epimagnolin A is a lignan obtained from the flower buds of Magnolia fargesii, which is traditionally used in Asian medicine for treating headache and nasal congestion. A herbal compound fargesin obtained from M. fargesii, has exerted anti-inflammatory effects in human monocytic THP-1 cells in the previous study. The anti-inflammatory effects of epimagnolin A, however, have been not elucidated yet. In this study, it was demonstrated that epimagnolin A reduced phorbol-12-myristate-13-acetate (PMA)-induced IL-6 promoter activity and IL-6 production in human monocytic THP-1 cells. Furthermore, it was investigated the modulating effects of epimagnolin A on mitogen-activated protein kinase, nuclear factor-kappa B (NF-κB), and activator protein 1 (AP-1) activities. Phosphorylation of p38 and nuclear translocation of p50 and c-Jun were down-regulated by epimagnolin A in the PMA-stimulated THP-1 cell. The results revealed that epimagnolin A attenuated the binding affinity of NF-κB and AP-1 transcription factors to IL-6 promoter and IL-6 production through p38/NF-kB and AP-1 signaling pathways in the PMA-stimulated THP-1 cells. These results suggest that epimagnolin A can be a useful drug for the treatment of inflammatory diseases.

Inhibition of IL-13 and IL-13Rα2 Expression by IL-32θ in Human Monocytic Cells Requires PKCδ and STAT3 Association.

Interleukin (IL)-32θ, a newly identified IL-32 isoform, has been reported to exert pro-inflammatory effects through the association with protein kinase C delta (PKCδ). In this study, we further examined the effects of IL-32θ on IL-13 and IL-13Rα2 expression and the related mechanism in THP-1 cells. Upon stimulating IL-32θ-expressing and non-expressing cells with phorbol 12-myristate 13-acetate (PMA), the previous microarray analysis showed that IL-13Rα2 and IL-13 mRNA expression were significantly decreased by IL-32θ. The protein expression of these factors was also confirmed to be down-regulated. The nuclear translocation of transcription factors STAT3 and STAT6, which are necessary for IL-13Rα2 and IL-13 promoter activities, was suppressed by IL-32θ. Additionally, a direct association was found between IL-32θ, PKCδ, and signal transducer and activator of transcription 3 (STAT3), but not STAT6, revealing that IL-32θ might act mainly through STAT3 and indirectly affect STAT6. Moreover, the interaction of IL-32θ with STAT3 requires PKCδ, since blocking PKCδ activity eliminated the interaction and consequently limited the inhibitory effect of IL-32θ on STAT3 activity. Interfering with STAT3 or STAT6 binding by decoy oligodeoxynucleotides (ODNs) identified that IL-32θ had additive effects with the STAT3 decoy ODN to suppress IL-13 and IL-13Rα2 mRNA expression. Taken together, our data demonstrate the intracellular interaction of IL-32θ, PKCδ, and STAT3 to regulate IL-13 and IL-13Rα2 synthesis, supporting the role of IL-32θ as an inflammatory modulator.

7-Methoxy-luteolin-8-C-ß-6-deoxy-xylo-pyranos-3-uloside exactly (mLU8C-PU) isolated from Arthraxon hispidus inhibits migratory and invasive responses mediated via downregulation of MMP-9 and IL-8 expression in MCF-7 breast cancer cells.

7-Methoxy-luteolin-8-C-ß-6-deoxy-xylo-pyranos-3-uloside (mLU8C-PU) is a glycosylflavone of luteolin isolated from Arthraxon hispidus (Thunb.). Luteolin is known to exert anti-migratory and anti-invasive effects on tumor cells. However, there are no reports on the effects of mLU8C-PU on tumor invasiveness and associated signaling pathways. In this study, we demonstrated the anti-migratory and anti-invasive effects of mLU8C-PU in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 breast cancer cells. We also investigated the effect of mLU8C-PU on invasion- related signal transducers, including protein kinase Cα (PKCα), c-Jun N terminal kinase (JNK), activator protein-1 (AP-1), and nuclear factor-kappa B (NF-ĸB). TPA-induced membrane translocation of PKCα, phosphorylation of JNK, and the nuclear translocations of AP-1 and NF-κB were downregulated by mLU8C-PU in MCF-7 cells. In addition, mLU8C-PU also inhibited matrix metalloproteinase-9 (MMP-9) and interleukin-8 (IL-8) expression. These results indicate that mLU8C-PU inhibits migratory and invasive responses in MCF-7 breast cancer cells by suppressing MMP-9 and IL-8 expression through mitigating TPA-induced PKCα, JNK activation, and the nuclear translocation of AP-1 and NF-κB. These results suggest that mLU8C-PU may be used as an anti-metastatic agent.

Peroxiredoxin II Is Essential for Maintaining Stemness by Redox Regulation in Liver Cancer Cells.

Redox regulation in cancer stem cells (CSCs) is viewed as a good target for cancer therapy because redox status plays an important role in cancer stem-cell maintenance. Here, we investigated the role of Peroxiredoxin II (Prx II), an antioxidant enzyme, in association with maintenance of liver CSCs. Our study demonstrates that Prx II overexpressed in liver cancer cells has high potential for self-renewal activity. Prx II expression significantly corelated with expression of epithelial-cell adhesion molecules (EpCAM) and cytokerain 19 in liver cancer tissues of hepatocellular carcinoma (HCC) patients. Downregulation of Prx II in Huh7 cells with treatment of siRNA reduced expression of EpCAM and CD133 as well as Sox2 in accordance with increased ROS and apoptosis, which were reversed in Huh7-hPrx II cells. Huh7-hPrx II cells exhibited strong sphere-formation activity compared with mock cells. Vascular endothelial growth factor (VEGF) exposure enhanced sphere formation, cell-surface expression of EpCAM and CD133, and pSTAT3 along with activation of VEGF receptor 2 in Huh7-hPrx II cells. The result also emerged in Huh7-H-ras(G12V) and SK-HEP-1-H-ras(G12V) cells with high-level expression of Prx II. Prx II was involved in regulation of VEGF driving cancer stem cells through VEGFR-2/STAT3 signaling to upregulate Bmi1 and Sox2. In addition, knockdown of Prx II in Huh7-H-ras(G12V) cells showed significant reduction in cell migration in vitro and in tumorigenic potential in vivo. Taken together, all the results demonstrated that Prx II plays a key role in the CSC self-renewal of HCC cells through redox regulation. Stem Cells 2016;34:1188-1197.

Promotion of adipogenesis by 15-(S)-hydroxyeicosatetraenoic acid.

Excess adipogenesis is a characteristic of obesity, which is associated with serious health problem, including type 2 diabetes. Here, to better understand the mechanisms for the development of adipocytes, we investigated the regulatory role of 15-(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) in adipogenesis by treating 3T3-L1 murine preadipocytes and human bone marrow mesenchymal stem cells (hBMSCs) with 15(S)-HETE. In the 3T3-L1 study, 15(S)-HETE stimulated lipid accumulation and enhanced expression of adipogenic markers such as peroxisome proliferator-activated receptor gamma (PPARγ), yet reduced the activity of factor negatively controlling adipogenesis, such as the 5'-AMP-activated protein kinase. In the early stage of adipogenesis, 15(S)-HETE increased activation of protein kinase B and expression of CCAAT/enhancer-binding protein (C/EBP)ß and C/EBPδ. Finally, 15(S)-HETE promoted adipogenesis in hBMSCs, and resulted in increased lipid accumulation and expression of adipogenic markers. In conclusion, 15(S)-HETE functions as a natural PPARγ agonist and enhances adipogenesis. Our findings may provide the basis for the development of novel therapeutic measures to treat obesity.

CCR5 deficiency increased susceptibility to lipopolysaccharide-induced acute renal injury.

C-C chemokine receptor 5 (CCR5) regulates leukocyte chemotaxis and activation, and its deficiency exacerbates development of nephritis. Therefore, we investigated the role of CCR5 during lipopolysaccharide (LPS)-induced acute kidney injury. CCR5-deficient (CCR5-/-) and wild-type (CCR5+/+) mice, both aged about 10 months, had acute renal injury induced by intraperitoneal injection of LPS (10 mg/kg). Compared with CCR5+/+ mice, CCR5-/- mice showed increased mortality and renal injury, including elevated creatinine and blood urea nitrogen levels, following LPS challenge. Compared to CCR5+/+ mice, CCR5-/- mice also exhibited greater increases in the serum concentrations of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1ß following LPS challenge. Furthermore, infiltration of macrophages and neutrophils, expression of intracellular adhesion molecule (ICAM)-1, and the number of apoptotic cells were more greatly increased by LPS treatment in CCR5-/- mice than in CCR5+/+ mice. The concentrations of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1ß were also significantly increased in the kidney of CCR5-/- mice after LPS challenge. Moreover, primary kidney cells from CCR5-/- mice showed greater increases in TNF-α production and p38 MAP kinase activation following treatment with LPS compared with that observed in the cells from CCR5+/+ mice. LPS-induced TNF-α production and apoptosis in the primary kidney cells from CCR5-/- mice were inhibited by treatment with p38 MAP kinase inhibitor. These results suggest that CCR5 deficiency increased the production of TNF-α following LPS treatment through increased activation of the p38 pathway in the kidney, resulting in renal apoptosis and leukocyte infiltration and led to exacerbation of LPS-induced acute kidney injury.

A1E reduces stemness and self-renewal in HPV 16-positive cervical cancer stem cells.

BACKGROUND: Cervical cancer is the second most common cancer in females. Recent reports have revealed the critical role of cervical cancer stem cells (CSCs) in tumorigenicity and metastasis. Previously we demonstrated that A1E exerts an anti-proliferative action, which inhibits the growth of cervical cancer cells. METHODS: A1E is composed of 11 oriental medicinal herbs. Cervical cancer cell culture, wund healing and invasion assay, flow cytometry, sheroid formation assay, and wstern blot assays were performed in HPV 16-positive SiHa cell and HPV 16-negative C33A cells. RESULTS: A1E targets the E6 and E7 oncogenes; thus, A1E significantly inhibited proliferation of human papilloma virus (HPV) 16-positive SiHa cells, it did not inhibit the proliferation of HPV-negative C33A cells. Accordingly, we investigated whether A1E can regulate epithelial-to-mesenchymal transition (EMT), CSC self-renewal, and stemness-related gene expression in cervical cancer cells. Down rgulation of cell migration, cell invasion, and EMT was observed in A1E-treated SiHa cells. Specifically, A1E-treated SiHa cells showed significant decreases in OCT-3/4 and Sox2 expression levels and in sphere formation. Moreover, CSCs makers ALDH+ and ALDH, CD133 double positive cell were significantly decreased in A1E-treated SiHa cells. However, A1E treatment did not down regulate ALDH+ expression and the number of ALDH/CD133 double positive cells in C33A cells. CONCLUSIONS: Taken together, A1E can inhibit CSCs and reduce the expression of stemness markers. Treating CSCs with A1E may be a potential therapy for cervical cancer.

Hepatitis B virus X promotes hepatocellular carcinoma development via nuclear protein 1 pathway.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and chronic hepatitis B virus (HBV) infection is a major risk factor for HCC. Hepatitis B virus X (HBx) protein relates to trigger oncogenesis. HBx has oncogenic properties with a hyperproliferative response to HCC. Nuclear protein 1 (NUPR1) is a stress-response protein, frequently upregulated in several cancers. Recent data revealed that NUPR1 is involved in tumor progression, but its function in HCC is not revealed yet. Here we report HBx can induce NUPR1 in patients, mice, and HCC cell lines. In an HBx transgenic mouse model, we found that HBx overexpression upregulates NUPR1 expression consistently with tumor progression. Further, in cultured HBV positive cells, HBx knockdown induces downregulation of NUPR1. Smad4 is a representative transcription factor, regulated by HBx, and we showed that HBx upregulates NUPR1 by Smad4 dependent way. We found that NUPR1 can inhibit cell death and induce vasculogenic mimicry in HCC cell lines. Moreover, NUPR1 silencing in HepG2-HBx showed reduced cell motility. These results suggest that HBx can modulate NUPR1 expression through the Smad4 pathway and NUPR1 has a role in hepatocellular carcinoma progression.

Interleukin-32γ attenuates ethanol-induced liver injury by the inhibition of cytochrome P450 2E1 expression and inflammatory responses.

Alcohol abuse and alcoholism lead to alcoholic liver disease (ALD), which is a major type of chronic liver disease worldwide. Interleukin-32 (IL-32) is a novel cytokine involved in inflammation and cancer development. However, the role of IL-32 in chronic liver disease has not been reported. In the present paper, we tested the effect of IL-32γ on ethanol-induced liver injury in IL-32γ-overexpressing transgenic mice (IL-32γ mice) after chronic ethanol feeding. Male C57BL/6 and IL-32γ mice (10-12 weeks old) were fed on a Lieber-DeCarli diet containing 6.6% ethanol for 6 weeks. IL-32γ-transfected HepG2 and Huh7 cells, as well as primary hepatocytes from IL-32γ mice, were treated with or without ethanol. The hepatic steatosis and damage induced by ethanol administration were attenuated in IL-32γ mice. Ethanol-induced cytochrome P450 2E1 expression and hydrogen peroxide levels were decreased in the livers of IL-32γ mice, primary hepatocytes from IL-32γ mice and IL-32γ-overexpressing human hepatic cells. The ethanol-induced expression levels of cyclo-oxygenase-2 (COX-2) and IL-6 were reduced in the livers of IL-32γ mice. Because nuclear transcription factor κB (NF-κB) is a key redox transcription factor of inflammatory responses, we examined NF-κB activity. Ethanol-induced NF-κB activities were significantly lower in the livers of IL-32γ mice than in wild-type (WT) mice. Furthermore, reduced infiltration of natural killer cells, cytotoxic T-cells and macrophages in the liver after ethanol administration was observed in IL-32γ mice. These data suggest that IL-32γ prevents ethanol-induced hepatic injury via the inhibition of oxidative damage and inflammatory responses.

Adiponectin deficiency suppresses lymphoma growth in mice by modulating NK cells, CD8 T cells, and myeloid-derived suppressor cells.

Previously, we found that adiponectin (APN) suppresses IL-2-induced NK cell activation by downregulating the expression of the IFN-γ-inducible TNF-related apoptosis-inducing ligand and Fas ligand. Although the antitumor function of APN has been reported in several types of solid tumors, with few controversial results, no lymphoma studies have been conducted. In this study, we assessed the role of APN in immune cell function, including NK cells, CTLs, and myeloid-derived suppressor cells, in EL4 and B16F10 tumor-bearing APN knockout (KO) mice. We observed attenuated EL4 growth in the APNKO mice. Increased numbers of splenic NK cells and splenic CTLs were identified under naive conditions and EL4-challenged conditions, respectively. In APNKO mice, splenic NK cells showed enhanced cytotoxicity with and without IL-2 stimulation. Additionally, there were decreased levels of myeloid-derived suppressor cell accumulation in the EL4-bearing APNKO mice. Enforced MHC class I expression on B16F10 cells led to attenuated growth of these tumors in APNKO mice. Thus, our results suggest that EL4 regression in APNKO mice is not only due to an enhanced antitumor immune response but also to a high level of MHC class I expression.

Interleukin (IL)-32ß-mediated CCAAT/enhancer-binding protein α (C/EBPα) phosphorylation by protein kinase Cδ (PKCδ) abrogates the inhibitory effect of C/EBPα on IL-10 production.

We previously reported that IL-32ß promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32ß abrogated the inhibitory effect of CCAAT/enhancer-binding protein α (C/EBPα) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBPα Ser-21 was inhibited by a PKCδ-specific inhibitor, rottlerin, or IL-32ß knockdown by siRNA and that IL-32ß shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32ß suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32ß may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32ß interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32ß suppressed the inhibitory effect of C/EBPα on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBPα serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBPα S21G on IL-10 promoter activity was not modulated by IL-32ß. Taken together, our results show that IL-32ß-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.

IL-32γ enhances TNF-α-induced cell death in colon cancer.

Interleukin (IL)-32 is a recently discovered cytokine that appears to play an important role in human colon cancer growth. We investigated that IL-32γ in combination with TNF-α remarkably inhibited cell growth of human colon cancer cells (HCT116 and SW620) and tumor growth in xenograft-bearing nude mice. The transient enforced overexpression of IL-32γ potentiated the inhibitory effect of TNF-α on DNA synthesis, cell number and protein content, and enhanced apoptosis in colon cancer cells. We also found that knockdown of IL-32γ by siRNA showed the abolishment of cell growth inhibitory effect of TNF-α. The IL-32γ-overexpressing colon cancer cells further increased TNF-α-mediated expression of p38 MAPK as well as that of Bax, cleaved caspase-3 and -9, but decreased that of antiapoptotic proteins such as Bcl-2, cellular inhibitor of apoptosis protein (IAP) and X chromosome IAP. In xenograft model, the lipopolysaccharide (LPS)-injected (1.25 mg/kg) mice inoculated with IL-32γ-transfected HCT116 colon cancer cells were more decrease tumor volume and weight than inoculated with vector. Tumor tissues isolated from LPS-injected mice inoculated with IL-32γ-overexpressing colon cancer cells potentiated the expression levels of pro-apoptotic proteins such as cleaved caspase-3, 9 and Bax, but decreased that of Bcl-2. Furthermore, the mice increased IL-10 production, but decreased IL-6 levels in serum. In conclusion, our results suggest that IL-32γ may potentiate TNF-α-induced cell growth inhibition through activation of p38 MAPK pathways.