Anti-Apoptotic Effects of Docosahexaenoic Acid in IL-1ß-Induced Human Chondrosarcoma Cell Death through Involvement of the MAPK Signaling Pathway.

Osteoarthritis (OA) is a degenerative disease characterized by progressive articular cartilage destruction and joint marginal osteophyte formation with different degrees of synovitis. Docosahexaenoic acid (DHA) is an unsaturated fatty acid with anti-inflammatory, antioxidant, and antiapoptotic functions. In this study, the human chondrosarcoma cell line SW1353 was cultured in vitro, and an OA cell model was constructed with inflammatory factor IL-1ß stimulation. After cells were treated with DHA, cell apoptosis was measured. Western blot assay was used to detect protein expression of apoptosis-related factors (Bax, Bcl-2, and cleaved caspase-3) and mitogen-activated protein kinase (MAPK) signaling pathway family members, including extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), and p38 MAPK. Our results show that IL-1ß promotes the apoptosis of SW1353 cells, increases the expression of Bax and cleaved caspase-3, and activates the MAPK signaling pathway. In contrast, DHA inhibits the expression of IL-1ß, inhibits IL-1ß-induced cell apoptosis, and has a certain inhibitory effect on the activation of the MAPK signaling pathway. When the MAPK signaling pathway is inhibited by its inhibitors, the effects of DHA on SW1353 cells are weakened. Thus, DHA enhances the apoptosis of SW1353 cells through the MAPK signaling pathway.

Vitamins D3 and K2 may partially counterbalance the detrimental effects of pentosidine in ex vivo human osteoblasts.

Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D3, are prerequisites for optimal osteoblasts functioning. Vitamin K2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D3 and K2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D3 and vitamin K2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might interfere with the maturation of osteoblasts, leading to morphological modifications, cellular malfunctioning, and inhibition of the calcification process. However, these processes may be all partially counterbalanced by vitamins D3 and K2. Therefore, detrimental AGE accumulation in bone might be attenuated and/or reversed by the presence or supplementation of vitamins D3 and K2.

Lupalbigenin from Derris scandens Sensitizes Detachment-induced Cell Death in Human Lung Cancer Cells.

BACKGROUND/AIM: The ability of cancer cells to resist to anoikis has been shown to augment cancer cell metastasis in many cancers. In search for potential substances for anti-metastatic approaches, this study aimed to investigate anoikis-sensitizing activity of lupalbigenin, extracted from Derris scandens. MATERIALS AND METHODS: Human lung cancer cells were treated with non-cytotoxic concentrations of lupalbigenin in a detachment condition. Anoikis was evaluated at various time points using MTT viability assays. The effect of lupalbigenin on anchorage-independent growth was performed by soft-agar assay. The survival signaling proteins, as well as regulatory proteins of apoptosis and metastasis, were examined by western blot analysis. RESULTS: Lupalbigenin treatment significantly down-regulated survival proteins, including protein kinase B (pAKT/AKT) and extracellular signal-regulated kinase (pERK/ERK), as well as anti-apoptotic protein B-cell lymphoma 2 (BCL-2), resulting in the enhancement of the cellular response to anoikis and the decrease of growth and survival in an anchorage-independent condition. CONCLUSION: Lupalbigenin sensitizes detachment-induced cell death in human lung cancer cell through down-regulation of pro-survival proteins.

Arabinosylated lipoarabinomannan (Ara-LAM) mediated intracellular mechanisms against tuberculosis infection: involvement of protein kinase C (PKC) mediated signaling.

Tuberculosis causes severe immunosuppression thereby ensuring the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates TLR-2 receptor down-stream signaling, indicating the possible involvement of TLR-2 in the regulation of the host immune response. Moreover, different PKC isoforms are also involved in the course of infection. Arabinosylated lipoarabinomannan (Ara-LAM) possesses immuno-modulatory properties which induce the pro-inflammatory responses via induction of TLR-2-mediated signaling. Here, we found that pretreatment of M. tuberculosis-infected macrophages with Ara-LAM caused a significant increase in the conventional PKC expression along with their active association with TLR-2. This association activated the TLR-2 -mediated downstream signaling, facilitating the activation of MAP kinase P38. All these events culminated in the up-regulation of proinflammatory response, which was abrogated by treatment with PKC-α and P38 inhibitors. Moreover, pretreatment of macrophages with Ara-LAM abrogated the IL-10 production while restored MHC-II expression in the infected macrophages. This study demonstrates that Ara-LAM confers protection against tuberculosis via TLR-2/PKC signaling crosstalk which is responsible for the induction of host protective immune response against tuberculosis.

Fermentation improves anti-inflammatory effect of sipjeondaebotang on LPS-stimulated RAW 264.7 cells.

Sipjeondaebotang (SJ) has been used as a traditional drug in east-Asian countries. In this study, to provide insight into the biological effects of SJ and SJ fermented by Lactobacillus, we investigated their effects on lipopolysaccharide (LPS)-mediated inflammation in macrophages. The investigation was focused on whether SJ and fermented SJ could inhibit the production of pro-inflammatory mediators such as prostaglandin (PG) E(2) and nitric oxide (NO) as well as the expressions of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in LPS-stimulated RAW 264.7 cells. We found that SJ modestly inhibited LPS-induced PGE(2), NO and TNF-α production as well as the expressions of COX-2 and iNOS. Interestingly, fermentation significantly increased its inhibitory effect on the expression of all pro-inflammatory mediators. Furthermore, fermented SJ exhibited increased inhibition of p38 MAPK and c-Jun NH(2)-terminal kinase (JNK) MAPK phosphorylation as well as NF-κB p65 translocation by reduced IκBα degradation compared with either untreated controls or unfermented SJ. High performance liquid chromatography (HPLC) analysis showed fermentation by Lactobacillus increases liquiritigenin and cinnamyl alcohol contained in SJ, which are known for their anti-inflammatory activities. Finally, SJ fermented by Lactobacillus exerted potent anti-inflammatory activity by inhibiting MAPK and NF-κB signaling in RAW 264.7 cells.

Induction of MAP kinase homologues during growth and morphogenetic development of Karnal bunt (Tilletia indica) under the influence of host factor(s) from wheat spikes.

Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) in response to certain environmental conditions, play important role in mating type switching (Fus3) and pathogenicity (Pmk1) in many fungi. In order to determine the roles of such regulatory genes in Tilletia indica, the causal pathogen of Karnal bunt (KB) of wheat, semi-quantitative and quantitative RT-PCR was carried out to isolate and determine the expression of MAP kinase homologues during fungal growth and development under in vitro culture. Maximum expression of TiFus3 and TiPmk1 genes were observed at 14th and 21st days of culture and decreased thereafter. To investigate whether the fungus alters the expression levels of same kinases upon interaction with plants, cultures were treated with 1% of host factors (extracted from S-2 stage of wheat spikes). Such treatment induced the expression of MAPks in time dependent manner compared to the absence of host factors. These results suggest that host factor(s) provide certain signal(s) which activate TiFus3 and TiPmk1 during morphogenetic development of T. indica. The results also provides a clue about the role of host factors in enhancing the disease potential due to induction of MAP kinases involved in fungal development and pathogenecity.

Dichloromethane fraction of Laminaria japonica ethanolic extract inhibits lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 cells via NF-κB pathway.

Strong anti-inflammatory activity has been found in Laminaria japonica dichloromethane fraction (LDF); however, the molecular mechanisms underlying its anti-inflammatory activity are not reported. Our results indicated that LDF inhibited LPS-induced nitric oxide and prostaglandin E(2) production in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW 264.7 cells. Also, levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1ß and IL-6 were remarkably reduced by LDF in LPS-treated RAW 264.7 cells. LDF greatly inhibited promoter activity of nuclear factor-κB (NF-κB) and translocation of NF-κB subunits by prevention of the degradation of inhibitor κB-α in LPS-treated RAW 264.7 cells (p < 0.05). Moreover, LDF inhibited activation of mitogen-activated protein kinases and AKT in LPS-treated RAW 264.7 cells. These results indicate that the LDF downregulates iNOS and COX-2 expressions through the suppression of NF-κB pathway associated with inhibition of multiple signaling proteins.

Inhibitory effects of the transgenic Panax ginsengs on phorbol ester plus A23187-induced IL-6 production and cyclooxygenase-2 via suppression of NF-κB and MAPKs in HMC-1.

Our previous studies have that demonstrated the overexpression of the squalene synthase gene enhances the biosynthesis of triterpene and phytosterol in Panax ginseng. The total ginsenoside contents in adventitious roots of transgenic P. ginseng were about 1.6-3-fold higher than those in the wild-type. In the present work, we have evaluated the anti-inflammatory effects of two types of transgenic P. ginseng (BS and SS) and the wild-type P. ginseng (GS) in a stimulated human mast cell line 1 (HMC-1). GS, BS, and SS inhibited not only the production of interleukin 6 (IL-6), but also the expression of cyclooxygenase-2 in phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187 (PMACI)-stimulated HMC-1. Additionally, GS, BS, and SS suppressed the expression of the nuclear transcription factor κB and mitogen-activated protein kinases induced by PMACI. The anti-inflammatory effects of BS and SS were higher than that of GS. These results provide new insights into the pharmacological actions of transgenic P. ginseng as a potential molecule for use in therapy in mast cell-mediated inflammatory diseases.

Regulation of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 cells by 2''-hydroxy-3''-en-anhydroicaritin involves down-regulation of NF-kappaB and MAPK expression.

2''-hydroxy-3''-en-anhydroicaritin, a flavone, was isolated from the Chinese medicinal herb Epimedium brevicornum for the first time. In our previous study, we have carried out a screening program to identify the anti-inflammatory potentials of 2''-hydroxy-3''-en-anhydroicaritin. In the present study, we further found that this compound regulated lipopolysaccharide (LPS)-induced levels of nitric oxide (NO), and prostaglandin E(2) (PGE(2)) (**p<0.01 or *p<0.05), and reduced levels of iNOS and COX-2 in RAW 264.7 macrophages in a concentration-dependent manner. We further investigated signal transduction mechanisms to determine how 2''-hydroxy-3''-en-anhydroicaritin affects RAW264.7 macrophages pretreated with 0.5, 2.5, or 12.5mg/L of 2''-hydroxy-3''-en-anhydroicaritin 1h prior to treatment with 1mg/L of LPS. Thirty minutes later, cells were harvested and mitogen-activated protein kinases (MAPK) activation and I kappaB alpha were measured by western blotting. Alternatively, the macrophages were fixed and nuclear factor-kappaB (NF-kappaB) activation was measured by immunocytochemical analysis. Signal transduction studies showed that the flavone significantly inhibited extracellular signal-regulated kinase (ERK), p38, and c-jun NH2-terminal kinase (JNK) phosphorylation protein expression. The flavone also inhibited p65-NF-kappaB translocation into the nucleus by I kappaB alpha degradation. Therefore, 2''-hydroxy-3''-en-anhydroicaritin may inhibit LPS-induced production of inflammatory cytokines by blocking NF-kappaB and MAPK signaling in RAW264.7 cells.

Skin tumor promotion by argemone oil/alkaloid in mice: evidence for enhanced cell proliferation, ornithine decarboxylase, cyclooxygenase-2 and activation of MAPK/NF-kappaB pathway.

Consumption of argemone oil (AO) contaminated edible oil causes "Epidemic Dropsy". Previously, we have shown that AO and isolated sanguinarine possess genotoxicity and skin tumor initiating activity. Here, we evaluate tumor-promoting potential of AO/sanguinarine alkaloid and investigate the molecular mechanisms involved therein. Single topical application of AO (50-400 microl/mouse) or sanguinarine alkaloid (1.5-12.0 micromol/mouse) afforded significant increase in (i) ornithine decarboxylase (ODC) activity, (ii) uptake of [(3)H]-thymidine in DNA, (iii) cyclooxygenase-2 (COX-2), proliferating cell nuclear antigen (PCNA) and ODC protein expressions, (iv) phosphorylation of extracellular signal-regulated kinase (ERK)1/2, c-jun-N-terminal kinase (JNK)1/2 and p38 mitogen-activated protein (MAP) kinases, (v) increased NF-kappaB activation and (vi) no significant increase in dark basal keratinocytes. Subsequently, when AO and sanguinarine alkaloid was tested either as complete or stage I or stage II tumor promoter in 7, 12-dimethyl benz(a)anthracene (DMBA)-initiated mice, there was enhanced tumor incidence, tumor body burden and higher % of mice with tumors, when AO (0.1 ml) or isolated sanguinarine (1.5 micromol) was tested as stage II tumor promoter. However, no tumors were found when AO or sanguinarine alkaloid was tested either as complete or stage I tumor promoter. These results indicate that AO/ sanguinarine alkaloid possesses tumor-promoting potential at stage II level involving MAPK/NF-kappaB pathway.

Matrix metalloproteinase-1 inhibitory activity of Kaempferia pandurata Roxb.

Matrix metalloproteinase (MMP)-1 is a superfamily of zinc-dependent endopeptidases that are capable of degrading all components of the extracellular matrix. Kaempferia pandurata extract (0.01-0.5 microg/mL) significantly reduced the expression of MMP-1 and induced the expression of type 1 procollagen at the protein and mRNA levels in a dose-dependent manner. Ultraviolet (UV)-induced MMP-1 initiates cleavage of fibrillar collagen. Once cleaved by MMP-1, collagen can be further degraded by elevated levels of MMP-3 and MMP-9. It was found that increased MMP-1 expression due to UV irradiation was mediated by activation of mitogen-activated protein kinases such as extracellular-regulated kinase (ERK), Jun N-terminal kinase (JNK), and p38 kinase. Treatment of K. pandurata extract in the range of 0.01-0.5 microg/mL inhibited the UV-induced phosphorylations of ERK, JNK, and p38, respectively. Moreover, inhibition of phosphorylated ERK, JNK, and p38 by K. pandurata extract resulted in decreased c-Fos expression and c-Jun phosphorylation induced by UV light. The results strongly suggest that K. pandurata is potentially useful for the prevention and treatment of skin aging.

Mitogen-activated protein kinases mediate upregulation of hypothalamic angiotensin II type 1 receptors in heart failure rats.

In heart failure (HF), angiotensin II type 1 receptor (AT(1)-R) expression is upregulated in brain regions regulating sympathetic drive, blood pressure, and body fluid homeostasis. However, the mechanism by which brain AT(1)-R are upregulated in HF remains unknown. The present study examined the hypothesis that the angiotensin II (Ang II)-triggered mitogen-activated protein kinases (MAPKs) p44/42, p38, and c-Jun N-terminal kinase contribute to upregulation of the AT(1)-R in the hypothalamus of rats with HF. AT(1)-R protein, AT(1)-R mRNA, and AT(1)-R immunoreactivity increased in the paraventricular nucleus of hypothalamus and the subfornical organ of rats with ischemia-induced HF compared with sham-operated controls. Phosphorylated p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK also increased in paraventricular nucleus and subfornical organ. A 4-week ICV infusion of the AT(1)-R antagonist losartan decreased AT(1)-R protein and phosphorylation of p44/42 MAPK, c-Jun N-terminal kinase, and p38 MAPK in the HF rats. A 4-week ICV infusion of the p44/42 MAPK inhibitor PD98059 or the c-Jun N-terminal kinase inhibitor SP600125 significantly decreased AT(1)-R protein and AT(1)-R immunoreactivity in the paraventricular nucleus and subfornical organ, but the p38 MAPK inhibitor SB203580 did not. Treatment with ICV losartan, PD98059, and SP600125 had no effect on AT(1)-R expression by Western blot in sham-operated rats. In untreated HF rats 4 weeks after coronary ligation, a 3-hour ICV infusion of PD98059, SP600125, or losartan reduced AT(1)-R mRNA in paraventricular nucleus and subfornical organ. These data indicate that MAPK plays an important role in the upregulation of AT(1)-R in the rat forebrain in HF and suggest that Ang II upregulates its own receptor by this mechanism.

Hibiscus sabdariffa L. water extract inhibits the adipocyte differentiation through the PI3-K and MAPK pathway.

Hibiscus sabdariffa L., a tropical beverage material and medical herb, is used commonly as in folk medicines against hypertension, pyrexia, inflammation, liver disorders, and obesity. This report was designed to investigate the inhibitory mechanisms of hibiscus extract on adipocyte differentiation in 3T3-L1 preadipocytes. The possible inhibitory pathways that regulate the adipocyte differentiation contain the adipogenic transcription factors, C/EBPalpha and PPARgamma, PI3-kinase, and MAPK pathway. In this study, we examined whether hibiscus extract affected the adipogenesis via these three pathways. To differentiate preadipocyte in adipocyte, confluent 3T3-L1 preadipocytes were treated with the hormone mixture including isobutylmethylxanthine, dexamethasone, and insulin (MDI). Hibiscus extract inhibited significantly the lipid droplet accumulation by MDI in a dose-dependent manner and attenuated dramatically the protein and mRNA expressions of adipogenic transcriptional factors, C/EBPalpha and PPARgamma, during adipogenesis. The increase of phosphorylation and expression of PI3-K/Akt during adipocytic differentiation was markedly inhibited by treatment with hibiscus extract or PI3-K inhibitors. Furthermore, the phosphorylation and expression of MEK-1/ERK known to regulate the early phase of adipogenesis were clearly decreased with the addition of hibiscus extract. Taken together, this report suggests that hibiscus extract inhibits the adipocyte differentiation through the modulation of PI3-K/Akt and ERK pathway that play pivotal roles during adipogenesis.

Molecular characterization of a potato MAP kinase transcriptionally regulated by multiple environmental stresses.

The MAPK cascade is an evolutionary conserved signaling pathway that links external stimuli with cellular responses. Using polymerase chain reaction (PCR), a DNA fragment corresponding to a Solanum tuberosum MAPK, StMPK1, was isolated. StMPK1 amino acid sequence displayed over 90% identity with tomato MPK1 (LeMPK1) and tobacco SIPK. Southern blot analysis indicated that the gene encoding StMPK1 is present in a single copy in the potato genome. StMPK1 mRNA levels differentially accumulated in potato tuber in response to wounding and to wounding plus Fusarium solani f. sp. eumartii. Transcript accumulation after infection was transient and started earlier than what was observed in wounded tubers. StMPK1 mRNA levels also increased in potato tuber after 24 h of treatment with jasmonic acid (JA) and abscicic acid (ABA), but not in response to ethylene or salicylic acid. In addition, StMPK1 transcript levels increased after a heat-shock treatment at 42 degrees C. The results suggest that StMPK1 may participate in the cellular responses against multiple environmental stimuli in potato tubers.

Effects of bee venom on the pro-inflammatory responses in RAW264.7 macrophage cell line.

The purpose of this study is to elucidate the molecular mechanism of anti-inflammatory effect of bee venom (BV), which has been used for the treatment of various inflammatory diseases in oriental medicine. With this aim, we examined the effects of BV on the nitric oxide (NO) production by lipopolysaccharide (LPS) or sodium nitroprusside in RAW264.7 macrophages. We further investigated the effects of BV on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) with RT-PCR in LPS-stimulated RAW264.7 cells. BV suppressed the NO production and decreased the levels of iNOS, COX-2, NF-kappaB and MAPK mRNA in a dose-dependent manner. These results suggest that BV has an anti-inflammatory effect by inhibiting iNOS and COX-2 expression, possibly through suppression of NF-kappaB and MAPK expression.

[Effects of Yiqi Huayu Recipe on gene expression pattern of normal and apoptotic chondrocytes in cervical intervertebral disc in rats].

OBJECTIVE: To study the effects of Yiqi Huayu Recipe on the gene expression pattern in normal and apoptotic chondrocytes in the cervical intervertebral disc of rats. METHODS: The intervertebral disc endplates of rats were digested enzymatically. The apoptosis of the chondrocytes was induced by anti-Fas antibody. BiostarR-40s microarray chips were used to investigate the gene expression pattern in chondrocytes in the normal control, apoptosis and Yiqi Huayu Recipe groups. The results were scanned with Scan Array 4000 and analyzed with GenePix Pro 3.0, and then subjected to standardization and ratio analysis. RESULTS: In the apoptosis group, 30 kinds of genes expressed in cervical intervertebral disc chondrocytes were screened out, in which 10 were up-regulated (ratio>2) and 20 down-regulated (ratio<0.5) as compared with the normal control group. On the other hand, in the Yiqi Huayu Recipe group, 97 kinds of genes expressed in cervical intervertebral disc chondrocytes were screened out, in which 44 were up-regulated (ratio>2) and 53 down-regulated (ratio<0.5) as compared with the apoptosis group. CONCLUSION: There are some signal transduction pathways that control the apoptosis of the intervertebral disc chondrocytes. Yiqi Huayu Recipe regulates the gene express of the apoptotic chondrocytes in intervertebral disc. The study gives a further understanding to the mechanism of the cervical spondylosis and enriches the theories of qi and blood of traditional Chinese medicine.

Pentameric procyanidins isolated from Theobroma cacao seeds selectively downregulate ErbB2 in human aortic endothelial cells.

Flavonoids isolated from cocoa have biological activities relevant to oxidant defenses, vascular health, tumor suppression, and immune function. The intake of certain dietary flavonoids, along with other dietary substances such as tocopherols, ascorbate, and carotenoids, is epidemiologically associated with a reduced risk of cardiovascular disease. Flavonoids have also been shown to modulate tumor pathology in vitro and in animal models. We took advantage of the conserved sequences found in tyrosine kinases to study the influence of cocoa fractions and controls on gene expression. We report that the pentameric procyanidin (molecular weight of 1442 daltons) fraction isolated from cocoa was a potent inhibitor of tyrosine kinase ErbB2 expression, a receptor important in angiogenesis regulation. Consistent with this primary observation, the cocoa flavonoid fraction also suppressed human aortic endothelial cell (HAEC) growth and decreased expression of two tyrosine kinases responsive to ErbB2 modulation, namely VEGFR-2/KDR and MapK 11/p38beta2. These inhibitory effects were observed when HAECs were treated with the flavonol fraction (molecular weight 280 daltons) isolated from cocoa, which comprise the structural subunits from which the procyanidin flavonoid subclass is biosynthetically constructed. Down-regulation of ErbB2 and inhibition of HAEC growth by cocoa procyanidins may have several downstream implications, including reduced vascular endothelial growth factor (VEGF) activity and angiogenic activity associated with tumor pathology. These results suggest specific dietary flavonoids are capable of selectively inhibiting ErbB2 and therefore may offer important insight into the design of therapeutic agents that target tumors overexpressing ErbB2.

Inhibition of RANTES expression by indirubin in influenza virus-infected human bronchial epithelial cells.

The human bronchial epithelial cells are the primary sites of influenza virus infection. In this study, the effect of indirubin on the expression of the chemokine regulated on activation, normal T cell expressed and secreted (RANTES) by the influenza virus-infected H292 human epithelial cell line was examined. The expression of RANTES mRNA was analyzed using reverse transcription polymerase chain reaction and the concentration of RANTES production was determined by the enzyme-linked immunosorbent assay. At the non-cytotoxic concentrations, indirubin was found to reduce both the expression and production of RANTES in influenza A/NWS/33-infected H292 cells. Inhibition was also observed in influenza virus B/Lee-infected cells. Significant reduction of the expression of IL-8 was not observed after the infection. Indirubin-3'-oxime, a recently developed derivative with kinase inhibitory activity, also mediates a potent inhibitory effect on the expression of RANTES. The influenza virus infection-induced phosphorylation of the nuclear transcription NF-kB regulatory molecule IkBalpha and the p38 MAP kinase were also found to be inhibited by indirubin-3'-oxime. This finding suggests that indirubin is one of the components in the Chinese medicinal herbs Isatis indigotica and Strobilanthes cusia with immunomodulatory activity on the expression of RANTES.

Molecular and functional resistance to insulin in hypothalamus of rats exposed to cold.

Insulin and leptin act in the hypothalamus, providing robust anorexigenic signals. The exposure of homeothermic animals to a cold environment leads to increased feeding, accompanied by sustained low levels of insulin and leptin. In the present study, the initial and intermediate steps of the insulin-signaling cascade were evaluated in the hypothalamus of cold-exposed Wistar rats. By immunohistochemistry, most insulin receptor (IR) and insulin receptor substrate-2 (IRS-2) immunoreactivity localized to the arcuate nucleus. Basal levels of tyrosine phosphorylation of IR and IRS-2 were increased in cold-exposed rats compared with rats maintained at room temperature. However, after an acute, peripheral infusion of exogenous insulin, significantly lower increases of IR and IRS-2 tyrosine phosphorylation were detected in the hypothalamus of cold-exposed rats. Insulin-induced association of p85/phosphatidylinositol 3-kinase with IRS-2, Ser473 phosphorylation of Akt, and tyrosine phosphorylation of ERK was significantly reduced in the hypothalamus of cold-exposed rats. To test the hypothesis of functional impairment of insulin signaling in the hypothalamus, intracerebroventricularly cannulated rats were acutely treated with insulin, and food ingestion was measured over a period of 12 h. Cold-exposed animals presented a significantly lower insulin-induced reduction in food consumption compared with animals maintained at room temperature. Hence, the present studies reveal that animals exposed to cold are resistant, both at the molecular and the functional level, to the actions of insulin in the hypothalamus.