Barun Poudel

Dioscin inhibits adipogenesis through the AMPK/MAPK pathway in 3T3-L1 cells and modulates fat accumulation in obese mice

Dioscin (DS) is a steroidal saponin present in a number of medicinal plants and has been shown to exert anticancer, antifungal and antiviral effects. The present study aimed to deternube the effects DS on the regulation of adipogenesis and to elucidate the underlying mechanisms. In vitro experiments were performed using differentiating 3T3-L1 cells treated with various concentrations (0-4 µM) of DS for 6 days. A cell viability assay was performed on differentiating cells following exposure to DS. Oil Red O staining and triglyceride content assay were performed to evaluate the lipid accumulation in the cells. We also carried out the following experiments: i) flow cytometry for cell cycle analysis, ii) quantitative reverse transcription polymerase chain reaction for measuring adipogenesis-related gene expression, and iii) western blot analysis to measure the expression of adipogenesis transcription factors and AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and mitogen-activated protein kinase (MAPK) phosphorylation. In vivo experiements were performed using mice with obesity induced by a high-fat diet (HFD) that were treated with or without DS for 7 weeks. DS suppressed lipid accumulation in the 3T3-L1 cells without affecting viability at a dose of up to 4 µM. It also delayed cell cycle progression 48 h after the initiation of adipogenesis. DS inhibited adipocyte differentiation by the downregulation of adipogenic transcription factors and attenuated the expression of adipogenesis-associated genes. In addition, it enhanced the phosphorylation of AMPK and its target molecule, ACC, during the differentiation of the cells. Moreover, the inhibition of adipogenesis by DS was mediated through the suppression of the phosphorylation of MAPKs, such as extracellular-regulated kinase 1/2 (ERK1/2) and p38, but not c-Jun-N-terminal kinase (JNK). DS significantly reduced weight gain in the mice with HFD-induced obesity; this was evident by the suppression of fat accumulation in the abdomen. the present study reveals an anti-adipogenic effect of DS in vitro and in vivo and highlights AMPK/MAPK signaling as targets for DS during adipogenesis.

Tools to Detect Influenza Virus

In 2009, pandemic influenza A (H1N1) virus (H1N1 09) started to spread quickly in many countries. It causes respiratory infection with signs and symptoms of common infectious agents. Thus, clinicians sometimes may miss the H1N1 patient. Clinical laboratory tests are important for the diagnosis of the H1N1 infection. There are several tests available, however, the rapid test and direct fluorescence antigen test are unable to rule out the influenza virus infection and viral culture test is time consuming. Therefore, nucleic acid amplification techniques based on reverse transcription polymerase chain reaction assays are regarded as a specific diagnosis to confirm the influenza virus infection. Although the nucleic acid-based techniques are highly sensitive and specific, the high mutation rate of the influenza RNA-dependent RNA polymerase could limit the utility of the techniques. In addition, their use depends on the availability, cost and throughput of the diagnostic techniques. To overcome these drawbacks, evaluation and development of the techniques should be continued. This review provides an overview of various techniques for specific diagnosis of influenza infection.

Collagen I enhances functional activities of human monocyte-derived dendritic cells via discoidin domain receptor 2.

We evaluated the involvement of collagen and their discoidin domain receptors (DDRs), DDR1 and DDR2, on the activation of human monocyte-derived dendritic cells (hDCs). DDR2 was markedly expressed on mature hDCs in comparison to immature ones. Collagen I enhanced the release of IL-12p40, TNF-α and IFN-γ by hDCs. Additionally, hDCs exhibited enhanced expression of costimulatory molecules, and potent functional activities which, in turn, has therapeutic value. Interestingly, DDR2 depletion showed decrease in capacity of hDCs to stimulate T cells proliferation, whereas DDR1 silencing had no significant affect. These data demonstrate that DDR2 enhances hDCs activation and contributes to their functional activities. In addition, application of collagen I treated dendritic cells (DCs) vaccine reduced tumor burden giving longer survival in melanoma mice. Our study suggests that collagen I may enhance functional activities of DCs in immune response.

Downregulation of ERK signaling impairs U2OS osteosarcoma cell migration in collagen matrix by suppressing MMP9 production

The present study investigated the role of extracellular signal-regulated kinase (ERK) activation in the migratory phenotype of human U2OS osteosarcoma (OS) cells in a collagen matrix. The activation of ERK was inhibited by PD98059, a specific inhibitor of ERK kinase. Additionally, no significant differences were observed in the adhesion and proliferation of the cells with or without PD98059 treatment in collagen-coated dishes. The migratory capacity of the U2OS cells was then examined in non-coated and collagen-coated dishes, and the results depicted that collagen I enhanced the migration of the U2OS cells, the effect of which was significantly blocked by the treatment of the cells with PD98059. Furthermore, enhanced gene and protein expression of matrix metalloproteinase 9 (MMP9), but not MMP2, was observed to be involved in the enhanced migratory phenotype of the U20S cells in the collagen-coated plates. This effect was partially abolished by the treatment of the cells in the collagen-coated dishes with ERK inhibitor. Collectively, the data demonstrate that ERK signaling is important for the migration of U2OS cells through the extracellular matrix (ECM), which is comprised mostly of collagen, by enhancing MMP9 production. These results may contribute to the regulation of MMP9 production in metastatic OS.

DDR2 inhibition reduces migration and invasion of murine metastatic melanoma cells by suppressing MMP2/9 expression through ERK/NF-κB pathway

Metastatic melanoma is one of the most deadly and evasive cancers. Collagen I in the extracellular matrix promotes the migration and invasion of tumor cells through the production of matrix metalloproteinase (MMP) 2 and 9. Discoidin domain receptor (DDR) 2 is a collagen receptor that is implicated in several cancer types including breast and prostate cancers. However, the role of DDR2 in the migration and invasion of murine melanoma cells is less studied. In the present study, we investigated the effects and underlying mechanisms of DDR2 in migration and invasion of B16BL6 melanoma cells in response to collagen I. Results demonstrated that DDR2 is expressed and is phosphorylated by collagen I in the cells. Upon down-regulation of DDR2 using small-interfering RNA (siRNA) approach, both of the cell migratory and invasive phenotypes were significantly attenuated when compared with the control cells. This effect was mediated via suppression of MMP2/9 upon DDR2 inhibition. Furthermore, inhibition of DDR2 by specific siRNA markedly reduced the activation of extracellular regulated kinase (ERK) 1 and 2 and nuclear factor of kappa B (NF-κB) in the cells when compared with the control cells. Overall, these data demonstrated that DDR2 siRNA-mediated suppression of ERK1/2 and NF-κB could down-regulate the expressions of MMP2/9 in response to collagen I to reduce the migratory and invasive phenotypes of the cells.

Luteolin is a bioflavonoid that attenuates adipocyte-derived inflammatory responses via suppression of nuclear factor-κB/mitogen-activated protein kinases pathway

Background: Inflammation of adipocytes has been a therapeutic target for treatment of obesity and metabolic disorders which cause insulin resistance and hence lead to type II diabetes. Luteolin is a bioflavonoid with many beneficial properties such as antioxidant, antiproliferative, and anti-cancer. Objectives: To elucidate the potential anti-inflammatory response and the underlying mechanism of luteolin in 3T3-L1 adipocytes. Materials and Methods: We stimulated 3T3-L1 adipocytes with the mixture of tumor necrosis factor-α, lipopolysaccharide, and interferon-γ (TLI) in the presence or absence of luteolin. We performed Griess’ method for nitric oxide (NO) production and measure mRNA and protein expressions by real-time polymerase chain reaction and western blotting, respectively. Results: Luteolin opposed the stimulation of inducible nitric oxide synthase and NO production by simultaneous treatment of adipocytes with TLI. Furthermore, it reduced the pro-inflammatory genes such as cyclooxygenase-2, interleukin-6, resistin, and monocyte chemoattractant protein-1. Furthermore, luteolin improved the insulin sensitivity by enhancing the expression of insulin receptor substrates (IRS1/2) and glucose transporter-4 via phosphatidylinositol-3K signaling pathway. This inhibition was associated with suppression of Iκ-B-α degradation and subsequent inhibition of nuclear factor-κB (NF-κB) p65 translocation to the nucleus. In addition, luteolin blocked the phosphorylation of ERK1/2, c-Jun N-terminal Kinases and also p38 mitogen-activated protein kinases (MAPKs). Conclusions: These results illustrate that luteolin attenuates inflammatory responses in the adipocytes through suppression of NF-κB and MAPKs activation, and also improves insulin sensitivity in 3T3-L1 cells, suggesting that luteolin may represent a therapeutic agent to prevent obesity-associated inflammation and insulin resistance.

Induction of IL-12 production by the activation of discoidin domain receptor 2 via NF-κB and JNK pathway

We investigated the mechanism involving discoidin domain receptor 2 (DDR2) mediated production of interleukin 12 (IL-12). When compared to control, collagen I upregulated the IL-12 luciferase activity on DDR2 expressing cells. Collagen I induced the phosphorylation of DDR2 and enhanced the phosphorylation of mitogen activated protein kinase (MAPK) kinases. In addition, NF-κB binding activity was enhanced when the cells expressing NF-κB reporter were exposed to collagen I. Moreover, when IL-12 reporter transfected cells were treated with biochemical inhibitors of c-Jun N-terminal kinase (JNK) and NF-κB, collagen-induced IL-12 promoter activity was significantly downregulated in comparison to non-treated cells. Similarly, confirmatory experiments on murine dendritic cells revealed that IL-12 promoter activity is dose dependently downregulated upon NF-κB and JNK inhibitor treatment on collagen I stimulation. In summary, DDR2 is involved in the collagen I-induced IL-12 production via NF-κB and JNK pathway.

Triticumoside induces apoptosis via caspase-dependent mitochondrial pathway and inhibits migration through downregulation of MMP2/9 in human lung cancer cells

Non-small cell lung cancer (NSCLC) is the major cancer-related death worldwide with only 14% five-year survival rate. Triticumoside, a phenolic compound present in Triticum aestivum sprout extract, has been recognized to have antiobesity and anti-inflammatory effects. However, the effect of triticumoside on cancer cell proliferation and migration has not been studied. In order to elucidate whether triticumoside exhibits an anticancer effect, cells were incubated with different doses of triticumoside, and apoptosis was assessed by observing cell viability, cellular morphological changes, and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Cell cycle analysis, western blotting, wound healing assay, and quantitative-polymerase chain reaction were also performed. Triticumoside exhibited marked cytotoxicity in the cells in dose- and time-dependent manner. Triticumoside caused morphological changes, including cellular rounding, nuclear condensation, and shrinkage. Likewise, triticumoside enhanced the sub-G1 proportion of cells. Additionally, triticumoside regulated expression of apoptosis-associated proteins, such as B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X, and procaspase-3/9. Triticumoside also inhibited migration of the cells through downregulation of matrix metalloproteinase-2/9 (MMP2/9). Collectively, these results suggest that triticumoside induces apoptosis through caspase-dependent mitochondrial pathway and suppresses migration via inhibition of MMP2/9 in NSCLC A549 cells.

Collagen I-induced dendritic cells activation is regulated by TNF-α production through down-regulation of IRF4

Previously we have shown that collagen I enhances the maturation and function of dendritic cells (DCs). Inflammatory mediators such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β and lipopolysaccharide (LPS) are also known to activate DCs. Here we investigated the involvement of TNF-α on the collagen I-induced DCs activation. TNF-a neutralization inhibited collagen I-induced IL-12 secretions by DCs. Additionally, we observed suppression of collagen I-induced costimulatory molecules expression along with down-regulation of genes involved in DCs activation pathway. Furthermore, TNF-α inhibition upon collagen Istimulation up-regulated the expression of interferon regulatory transcription factor IRF4, when compared to collagen I only treated cells. Collectively, our data demonstrate that collagen I induce TNF-α production, which is crucial for the activation and function of DCs, through down-regulation of IRF4, and implicates the importance in development of anti- TNF-α therapeutics for several inflammatory diseases.

In vitro and in vivo anti-cancer activity of dichloromethane fraction of Triticum aestivum sprouts

Triticum aestivum sprouts (TA) contain significant amounts of chlorophyll, minerals, enzymes, and other functional entities. Furthermore, TA extracts have been shown to possess anti-obesity, anti-diabetic and hepatoprotective effects and are believed to help blood flow, digestion, and general detoxification of the body. In this study, the mechanism underlying the anti-cancer effects of a dichloromethane fraction of TA (TDF) was investigated in vitro and in vivo. In vitro study was done by examining cancer cells growth, morphological changes, cell cycles, expressions of death receptors and apoptosis-linked proteins in wide range of human cancer cell lines. To investigate the effect of TDF in vivo, C57BL/6 mice were injected with B16 melanoma cells and orally administered TDF. TDF markedly inhibited cancer cell growth and induced cellular morphological alterations, cell cycle arrest and apoptosis, and enhanced the expressions of death receptors (DR)-4, 5, and 6 in cell lines. In addition, TDF regulated the expressions mitochondrial apoptosis-linked proteins and induced caspase-dependent cell death. It also significantly enhanced phosphorylation of ERK1/2 and JNK, but not p38, whereas inhibited the activation of NF-κB in cancer cells. In our mouse model, TDF significantly suppressed B16 melanoma growth, to an extent similar to cisplatin (reference control) and augmented immunomodulatory cytokines. In brief, this study presents the mechanism responsible for the anti-cancer effects of TDF in vitro and in vivo.