Curcumol Attenuates Endometriosis by Inhibiting the JAK2/STAT3 Signaling Pathway.

BACKGROUND Curcumol is a hydrogenated austenitic compound with hemiketal. In this study we evaluated the effects of curcumol on local inflammatory response, cell proliferation, and metastasis in endometriosis, and elucidated the underlying mechanisms. MATERIAL AND METHODS Ectopic endometrial stromal cells were treated with increasing doses of curcumol. The MTT assay was used to assess cell viability. FITC-labeled annexin-V/PI double-staining method and flow cytometry were used to determine cell apoptosis. Cell migration was evaluated using a wound healing assay. ELISA kits were used to detect the levels of TNF-alpha, IL-6, and IL-1ß. Western blot assay was used to examine the phosphorylation degree of JAK2 and STAT3 and the expression of Bax, Bcl2, and caspase-3 proteins. Autologous endometrial transplantation was used to establish a rat model to assess the anti-EMS effect of curcumol in vivo. RESULTS Curcumol can inhibit the proliferation of ectopic endometrial stromal cells, promote cell apoptosis, and weaken cell migration ability. Curcumol can reduce the expression of Bax and caspase-3 protein and increase the expression of Bcl2 protein. Curcumol also can inhibit the secretion of inflammatory cytokines, including tumor necrosis cytokines (TNF)-alpha, interleukin (IL)-6, and IL-1ß, by ectopic endometrial stromal cells. In addition, curcumol can also inhibit the phosphorylation of JAK2 and STAT3. In vivo experiments also proved that curcumol could inhibit the growth of ectopic lesions in EMS model rats. CONCLUSIONS Curcumol can inhibit the JAK2/STAT3 pathway, reduce the inflammatory cytokines secreted by ectopic endometrial stromal cells, inhibit cell proliferation and migration, and reduce the volume of ectopic lesions.

Hemistepsin a Induces Apoptosis of Hepatocellular Carcinoma Cells by Downregulating STAT3.

Hemistepta lyrata (Bunge) Bunge is a biennial medicinal plant possessing beneficial effects including anti-inflammation, and hemistepsin A (HsA) isolated from H. lyrata has been known as a hepatoprotective sesquiterpene lactone. In this report, we explored the cytotoxic effects of H. lyrata on hepatocellular carcinoma (HCC) cells and investigated the associated bioactive compounds and their relevant mechanisms. From the viability results of HCC cells treated with various H. lyrata extracts, HsA was identified as the major compound contributing to the H. lyrata-mediated cytotoxicity. HsA increased expression of cleaved PARP and cells with Sub-G1 phase, Annexin V binding, and TUNEL staining, which imply HsA induces apoptosis. In addition, HsA provoked oxidative stress by decreasing the reduced glutathione/oxidized glutathione ratio and accumulating reactive oxygen species and glutathione-protein adducts. Moreover, HsA inhibited the transactivation of signal transducer and activator of transcription 3 (STAT3) by its dephosphorylation at Y705 and glutathione conjugation. Stable expression of a constitutive active mutant of STAT3 prevented the reduction of cell viability by HsA. Finally, HsA enhanced the sensitivity of sorafenib-mediated cytotoxicity by exaggerating oxidative stress and Y705 dephosphorylation of STAT3. Therefore, HsA will be a promising candidate to induce apoptosis of HCC cells via downregulating STAT3 and sensitizing conventional chemotherapeutic agents.

310 nm UV-LEDs attenuate imiquimod-induced psoriasis-like skin lesions in C57BL/6 mice and inhibit IL-22-induced STAT3 expression in HaCaT cells.

Ultraviolet light-emitting diodes (UV-LEDs) are a novel light source for phototherapy. This study aimed to evaluate the therapeutic effects of UV-LEDs on psoriasis. Importantly, 310 nm UV-LEDs have not been studied in psoriasis in vitro and in vivo. Effects due to 310 nm UV-LED and 311 nm narrowband ultraviolet B (NBUVB) irradiation were compared for suppressing IL-22-induced activation of STAT3 expression using cell viability assay, western blotting, and immunocytochemistry. C57BL/6 mice were topically treated with imiquimod (IMQ) for 6 consecutive days and degenerative changes were observed. Test groups were irradiated with a 310 nm UV-LED and 311 nm NBUVB. Phenotypic observations, histopathological examinations, and ELISA were conducted with skin and blood samples. STAT3-dependent IL-22 signalling and effects in keratinocytes are negatively regulated by the 310 nm UV-LED, which significantly ameliorated IMQ-induced psoriasis-like dermatitis development and reduced Th17 cytokine levels (IL-17A, IL-22) in serum and dorsal skin. Histopathological findings showed decreases in epidermal thickness and inflammatory T-cell infiltration in the UV-LED-irradiated groups. Quantitative PCR confirmed a UV radiation energy-dependent decrease in IL-17A and IL-22 mRNA levels. The results demonstrated that UV-LEDs had anti-inflammatory and immunoregulatory effects. So, UV-LED phototherapy inhibits psoriasis development by suppressing STAT3 protein and inflammatory cytokines and could be useful in treating psoriasis.

Persistent Leptin Signaling in the Arcuate Nucleus Impairs Hypothalamic Insulin Signaling and Glucose Homeostasis in Obese Mice.

BACKGROUND: Obesity is associated with reduced physiological responses to leptin and insulin, leading to the concept of obesity-associated hormonal resistance. OBJECTIVES: Here, we demonstrate that contrary to expectations, leptin signaling not only remains functional but also is constantly activated in the arcuate nucleus of the hypothalamus (ARH) neurons of obese mice. This state of persistent response to endogenous leptin underpins the lack of response to exogenous leptin. METHODS AND RESULTS: The study of combined leptin and insulin signaling demonstrates that there is a common pool of ARH neurons responding to both hormones. More importantly, we show that the constant activation of leptin receptor neurons in the ARH prevents insulin signaling in these neurons, leading to impaired glucose tolerance. Accordingly, antagonising leptin signaling in diet-induced obese (DIO) mice restores insulin signaling in the ARH and improves glucose homeostasis. Direct inhibition of PTP1B in the CNS restores arcuate insulin signaling similarly to leptin inhibition; this effect is likely to be mediated by AgRP neurons since PTP1B deletion specifically in AgRP neurons restores glucose and insulin tolerance in DIO mice. CONCLUSIONS: Finally, our results suggest that the constant activation of arcuate leptin signaling in DIO mice increases PTP1B expression, which exerts an inhibitory effect on insulin signaling leading to impaired glucose homeostasis.

Evaluation of the renoprotective effect of nano turmeric against toxic dose of copper sulfate: Role of vascular cell adhesion molecule-1, kidney injury molecule-1, and signal transducer and activator of transcription 3 protein expressions.

The aim of this study was to compare the potential renoprotective effects of turmeric (TM) and nano turmeric (NTM) with those of desferrioxamine (DSM) against copper sulfate (CS)-induced toxicity. Rats were administered a toxic dose of CS with TM, NTM, and DSM for 1 week. Next, serum-urea creatinine, uric acid, interleukin (IL)-10, c-reactive protein (CRP), and caspase-3 levels; renal nitric oxide (NO), glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), vascular cell adhesion molecule-1 (VCAM-1), kidney injury molecule (KIM)-1, signal transducer and activator of transcription 3 (STAT-3) protein expression; and nuclear factor (NF)-κB and B-cell lymphoma -2 (Bcl-2) messenger RNA expression levels were estimated. Administration of the investigated antioxidants downregulated the marked increase in urea, creatinine, uric acid, CRP, caspase-3, NO, MDA, VCAM-1, kidney injury molecule (KIM-1), STAT-3, NF-κB, and DNA fragmentation, and increased Bcl-2, IL-10, GSH, and SOD levels induced by CS. The histopathological examination confirmed the effects of the antioxidants on the investigated biochemical parameters. Interestingly, NTM exhibited a superior renoprotective effect, which was comparable with that of DSM. In conclusion, NTM was shown to be a promising candidate against CS-induced toxicity, and several molecular mechanisms were implicated in the CS-induced renotoxicity as well as the treatment effects of NTM.

S1PR1 predicts patient survival and promotes chemotherapy drug resistance in gastric cancer cells through STAT3 constitutive activation.

BACKGROUND: S1PR1-STAT3 inter-regulatory loop was initially suggested to be oncogenic in several cancer cells. However, the clinical relevance of this mechanism in tumor progression, disease prognosis and drug response was not established. METHODS: The correlations between S1PR1 transcription, overall survival and chemotherapy response of GC patients were tested using a large clinical database. The relevance of S1PR1 expression and STAT3 activation in both tumor tissues and cancer cell lines was also tested. The effect of S1PR1 high expression achieved by persistent STAT3 activation on tumor cell drug resistance was investigated in vitro and in vivo. FINDINGS: An enhanced S1PR1 expression was highly related with a reduced overall survival time and a worse response to chemotherapy drug and closer correlation to STAT3 in gastric cancer patients. The issue chip analysis showed that the expressions of S1PR1 and STAT3 activation were increased in higher graded gastric cancer (GC) tissues. Cellular studies supported the notion that the high S1PR1 expression was responsible for drug resistance in GC cells through a molecular pattern derived by constitutive activation of STAT3. The disruption of S1PR1-STAT3 signaling significantly re-sensitized drug resistance in GC cells in vitro and in vivo. INTERPRETATION: S1PR1-STAT3 signaling may participate drug resistance in GC, thus could serve as a drug target to increase the efficacy of GC treatment. FUND: This work was supported by the National Natural Science Foundation of China (No. 81570775, 81471095), the grant from the research projects in traditional Chinese medicine industry of China (No. 201507004-2).

Early life stress experience may blunt hypothalamic leptin signalling.

The aim of this study was to investigate whether neonatal maternal separation (MS) - chronic stress experience in early life - affects the anorectic efficacy of leptin in the offspring at adolescence. Sprague-Dawley pups were separated from the dam daily for 3 h during postnatal day 1-14 or left undisturbed as non-handled controls (NH). NH and MS male pups received an intraperitoneal leptin (100 µg/kg) or saline on postnatal day (PND) 28, and then food intake and body weight gain were recorded. The hypothalamic levels of leptin-signalling-related genes, phosphorylated signal transducer and activator of transcription-3 (pSTAT3) and protein-tyrosine phosphatase 1B (PTP1B) were examined at 40 min after a single injection of leptin on PND 39 by immunohistochemistry and Western blot analysis. Leptin-induced suppressions in food intake and weight gain was observed in NH pups, but not in MS. Leptin increased pSTAT3 in the hypothalamic arcuate nucleus of NH pups, but not of MS. Interestingly, basal levels of the hypothalamic PTP1B and pSTAT3 were increased in MS pups compared with NH controls. The results suggest that neonatal MS experience may blunt the anorectic efficacy of leptin later in life, possibly in relation with increased expressions of PTP1B and/or pSTAT3 in the hypothalamus.

Antitumor activity of Annona squamosa seed oil.

CONTEXT: Custard apple (Annona squamosa Linn.) is an edible tropical fruit, and its seeds have been used to treat "malignant sore" (cancer) and other usage as insecticide. A comparison of extraction processes, chemical composition analysis and antitumor activity of A. squamosa seed oil (ASO) were investigated. MATERIALS AND METHODS: The optimal extraction parameters of ASO were established by comparing percolation, soxhlet, ultrasonic and SFE-CO2 extraction methods. The chemical composition of fatty acid and content of total annonaceous acetogenins (ACGs) of ASO was investigated by GC-MS and colorimetric assay, and anti-tumor activity of ASO was tested using H22 xenografts bearing mice. RESULTS: The optimal extraction parameters of ASO were obtained as follows: using soxhlet extraction method with extraction solvent of petroleum ether, temperature of 80°C, and extraction time of 90min. Under these conditions, the yield of ASO was 22.65%. GC-MS analysis results showed that the main chemical compositions of fatty acid of ASO were palmitic acid (9.92%), linoleic acid (20.49%), oleic acid (56.50%) and stearic acid (9.14%). The total ACGs content in ASO was 41.00mg/g. ASO inhibited the growth of H22 tumor cells in mice with a maximum inhibitory rate of 53.54% by oral administration. Furthermore, it was found that ASO exerted an antitumor effect via decreasing interleukin-6 (IL-6), janus kinase (Jak) and phosphorylated signal transducers and activators of transcription (p-Stat3) expression. DISCUSSION AND CONCLUSION: The results demonstrated that ASO suppressed the H22 solid tumor development may due to its main chemical constituents unsaturated fatty acid and ACGs via IL-6/Jak/Stat3 pathway. ASO may be a potential candidate for the treatment of cancer.

Honokiol protects against renal ischemia/reperfusion injury via the suppression of oxidative stress, iNOS, inflammation and STAT3 in rats.

Honokiol is the predominant active ingredient in the commonly used traditional Chinese medicine, Magnolia, which has been confirmed in previous studies to exhibit anti-oxidation, antimicrobial, antitumor and other pharmacological effects. However, its effects on renal ischemia/reperfusion injury (IRI) remain to be elucidated. The present study aimed to examine the effects of honokiol on renal IRI, and to investigate its potential protective mechanisms in the heart. Male adult Wistar albino rats were induced into a renal IRI model. Subsequently, the levels of serum creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), and the levels of serum nitrite and the kidney nitrite were examined in the IRI group. The levels of oxidative stress, inducible nitric oxide synthase (iNOS), inflammatory factors and caspase-3 were evaluated using a series of commercially available kits. The levels of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the protein expression levels of STAT3 were determined using western blotting. Pretreatment with honokiol significantly reduced the levels of serum creatinine, BUN, ALT, AST and ALP, and the level of nitrite in the kidney of the IRI group, compared with the control group. The levels of malondialdehyde, the activity of myeloperoxidase, and the gene expression and activity of iNOS were reduced in the IRI rats, compared with the sham-operated rats, whereas the levels of superoxide dismutase and catalase were increased following treatment with honokiol in the IRI rats. In addition, the expression levels of tumor necrosis factor-α and interleukin-6 in the IRI rats were increased by honokiol. Treatment with honokiol suppressed the protein expression levels of p-STAT3 and caspase-3 in the IRI rats. These findings indicated that honokiol protects against renal IRI via the suppression of oxidative stress, iNOS, inflammation and STAT3 in the rat.

Isolinderalactone enhances the inhibition of SOCS3 on STAT3 activity by decreasing miR-30c in breast cancer.

Development of an efficient treatment for triple-negative breast cancer is an urgent issues. Compounds from plant extracts are a potential source of novel cancer treatment. Isolinderalactone, a kind of sesquiterpenoids compound, was purified from the root of Lindera strychnifolia and Neolitsea daibuensis and shows anti-inflammatory and anticancer capacity. In the present study, isolinderalactone induced apoptosis in MDA-MB-231 cells which is a kind of triple-negative breast cancer cell line through induction of an intrinsic mitochondria-mediated and caspase-independent cell death. Treatment of isolinderalactone increased the protein level of the suppressor of cytokine signaling 3 (SCOS3), decreased phosphorylation of the signal transducer and activator of transcription 3 (STAT3), and suppressed expression of the down-stream genes of the X-linked inhibitor of apoptosis protein in MDA-MB-231 cells. Our results further showed that the level of SOCS3 expression was induced by isolinderalactone due to inhibiting the microRNA hsa-miR-30c-5p (miR-30c) expression. In addition, intraperitoneal injection of isolinderalactone induced apoptosis in a xenograft breast tumor while it did not significantly affect the histology of liver, kidney and lung of the treated mice. In conclusion, isolinderalactone induces apoptosis in MDA-MB­231 cells and suppresses STAT3 signaling pathway through regulation of SOCS3 and miR-30c. It may become a novel treatment for triple-negative breast cancer in the future.

Research on the bioactivity of isoquercetin extracted from marestail on bladder cancer EJ cell and the mechanism of its occurrence.

Research studies in recent years have found that isoquercetin has an inhibiting effect on multiple carcinogens, but research studies filed on isoquercetin in bladder cancer are quite few. This paper observed the influence of isoquercetin on biological activity of the EJ cell of bladder cancer through HC dyeing and trypan blue counting, studied the EJ cell cycle by flow cytometry (FCM), and then analyzed the influence of isoquercetin and its effect on the protein expression of STAT3 and STAT3-inhibiting factors (PIAS3) in EJ cells. Research has shown that isoquercetin has an inhibitory effect on the EJ cells of bladder cancer, but it is not obvious.

Effect of Rhizoma paridis total saponins on apoptosis of colorectal cancer cells and imbalance of the JAK/STAT3 molecular pathway induced by IL-6 suppression.

We observed the influence of different concentrations of Rhizoma paridis total saponins (RPTS) on the apoptosis of colorectal cancer cells and explored the internal mechanism involved. We determined whether RPTS influences the interleukin-6 (IL-6)/Janus kinase (JAK)-signal transducer and activator of transcription-3 (STAT3) apoptosis molecular pathway and looked for colon cancer-related signal transduction pathways or targets inducing apoptosis. We also cultured SW480 colorectal cancer cells using different concentrations of RPTS (10, 20, 40, and 80 µg/ mL), and observed the effect of RPTS on SW480 cell morphology under a fluorescence inverted microscope. We detected serum IL-6 using the polymerase chain reaction and the expression of JAK-STAT3 protein by western blot. After treating SW480 with RPTS and Hoechst 33258 dyeing, we found that the typical apoptosis morphology had changed. Secretion of IL-6 in the serum decreased significantly (P < 0.05), and STAT3 levels were reduced. RPTS can significantly promote apoptosis in SW480 colorectal cancer cells. The mechanism may be that it suppresses the secretion of IL-6 and inhibits the IL-6/JAK-STAT3 protein signaling pathway.

Inhibition of the JAK2/STAT3 signaling pathway exerts a therapeutic effect on osteosarcoma.

Osteosarcoma (OS) is the most common type of malignant bone tumor. Despite aggressive multimodal treatments, including surgical resection, chemotherapy and adjunctive immunotherapies, patients with OS with high-grade malignancy have a poor five-year survival rate that has remained unchanged over the past two decades, highlighting the urgent requirement for novel therapeutic approaches. Signal transducers and activators of transcription 3 (STAT3) has been implicated as an oncogene and therapeutic target in a variety of neoplastic diseases. The aim of the present study was to determine whether inhibition of the janus kinase 2 (JAK2)/STAT3 pathway by FLLL32, a specific JAK2/STAT3 inhibitor, is able to provide a potential therapy for OS. FLLL32 inhibited OS cell growth in vitro and delayed OS growth in an OS xenograft nude mouse model. STAT3 knockdown by short hairpin RNA delayed OS formation in vivo. Thus, the JAK2/STAT3 pathway is important in OS formation. Efficacy of the FLLL32 pharmacological inhibitor in delaying OS growth suggests that targeting JAK2/STAT3 may be a potential therapeutic strategy for patients with OS.

Molecular markers to assess short-term disease local recurrence in nasopharyngeal carcinoma.

An important challenge in nasopharyngeal carcinoma (NPC) research is to develop effective predictors of tumor recurrence following treatment to determine whether immediate adjuvant therapy is necessary. We retrospectively analyzed archived specimens collected from 45 patients with paired samples of primary NPC (pNPC) and recurrent NPC (rNPC). Clinical samples were collected from the Cancer Center Databases of the First People's Hospital of Foshan and Shantou Central Hospital (affiliates of Sun Yat-Sen University) between 2001 and 2012. Expression levels of phosphor-Stat3 (p-Stat3), signalosome complex subunit 5 (Jab1/Csn5), Akt1, C/EBP homologous protein (CHOP), Ki-67, and apoptosis were determined by immunohistochemistry in pNPC and rNPC samples from the same patients. Differences in these markers between the short-term interval to recurrence (ITR) group (ITR <18 months) and long-term ITR group (ITR ≥18 months) were further analyzed. In Cox's regression analysis, the ITR was significantly associated as an independent­negative prognostic factor for overall survival (hazard ratio, 0.211; 95% confidence interval, 0.053-0.841; P=0.027). p-Stat3 was increased in the short-term ITR group (ITR <18 months) and tended to be lower in the long-term ITR group (ITR ≥18 months). In the short-term ITR group, nuclear Akt expression was significantly increased in paired rNPC (P=0.028). In the long-term ITR group, the expression of nuclear Jab1/Csn5 (P=0.047) and assessment of apoptosis measured with TdT-mediated dUTP nick end­labeling (TUNEL) (P=0.003) was significantly increased in paired rNPC. The results suggest that differences between short- and long-term ITR may predict outcome in rNPC. Furthermore, the overexpression of Jab1/Csn5 and Akt may contribute to the carcinogenesis of rNPC, and Akt seems to promote the progression of short-term ITR. Intra-individual changes of Jab1/Csn5, Akt, and TUNEL may help to identify short-term ITR.

Aberrantly activated pSTAT3-Ser727 in human endometrial cancer is suppressed by HO-3867, a novel STAT3 inhibitor.

OBJECTIVE: Constitutive activation of STAT3 is a hallmark of various human cancers, however an increased pSTAT3 expression in high grade human endometrial cancer has not been reported. In the present study, we examine the expression of STAT family of proteins in endometrial cancer cell lines and the efficacy of HO-3867, a novel STAT3 inhibitor designed in our lab. METHODS: Expression of STAT family proteins was evaluated via Western blot. The cell viability, post-treatment with HO-3867, was assessed using MTT, cell-cycle profile and Annexin assay. In vivo efficacy of HO-3867 was evaluated using xenograft mice. RESULTS: Expression of activated STATs was inconsistent among the cell lines and 18 human endometrial cancer specimens tested. While pSTAT3 Tyr705 was not expressed in any of the cell lines, pSTAT3 Ser727 was highly expressed in endometrial cancer cell lines and tumor specimens. HO-3867 decreased the expression of pSTAT3 Ser727 while total STAT3 remained constant; cell viability decreased by 50-80% and induced G2/M arrest in 55% of Ishikawa cells at the G2/M cell cycle checkpoint. There was an increase in p53, a decrease in Bcl2 and Bcl-xL, and cleavage of caspase-3, caspase-7 and PARP. HO-3867 mediated a dosage-dependent inhibition of the growth of xenografted endometrial tumors. CONCLUSIONS: HO-3867 treatment decreases the high levels of pSTAT3 Ser727 in endometrial cancer cells by inducing cell cycle arrest and apoptosis. This suggests a specific role of serine-phosphorylated STAT3, independent of tyrosine phosphorylation in the oncogenesis of endometrial cancer. HO-3867 could potentially serve as an adjunctive targeted therapy.

PTPRT regulates high-fat diet-induced obesity and insulin resistance.

Obesity is a risk factor for many human diseases. However, the underlying molecular causes of obesity are not well understood. Here, we report that protein tyrosine phosphatase receptor T (PTPRT) knockout mice are resistant to high-fat diet-induced obesity. Those mice avoid many deleterious side effects of high-fat diet-induced obesity, displaying improved peripheral insulin sensitivity, lower blood glucose and insulin levels. Compared to wild type littermates, PTPRT knockout mice show reduced food intake. Consistently, STAT3 phosphorylation is up-regulated in the hypothalamus of PTPRT knockout mice. These studies implicate PTPRT-modulated STAT3 signaling in the regulation of high-fat diet-induced obesity.

Coinjection of CCK and leptin reduces food intake via increased CART/TRH and reduced AMPK phosphorylation in the hypothalamus.

CCK and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important for elucidating the mechanisms by which energy balance is maintained. We found here that coadministration of subthreshold CCK and leptin, which individually have no effect on feeding, dramatically reduced food intake in rats. Phosphorylation of AMP-activated protein kinase (AMPK) in the hypothalamus significantly decreased after coinjection of CCK and leptin. In addition, coadministration of these hormones significantly increased mRNA levels of anorectic cocaine- and amphetamine-regulated transcript (CART) and thyrotropin-releasing hormone (TRH) in the hypothalamus. The interactive effect of CCK and leptin on food intake was abolished by intracerebroventricular preadministration of the AMPK activator AICAR or anti-CART/anti-TRH antibodies. These findings indicate that coinjection of CCK and leptin reduces food intake via reduced AMPK phosphorylation and increased CART/TRH in the hypothalamus. Furthermore, by using midbrain-transected rats, we investigated the role of the neural pathway from the hindbrain to the hypothalamus in the interaction of CCK and leptin to reduce food intake. Food intake reduction induced by coinjection of CCK and leptin was blocked in midbrain-transected rats. Therefore, the neural pathway from hindbrain to hypothalamus plays an important role in transmitting the anorectic signals provided by coinjection of CCK and leptin. Our findings give further insight into the mechanisms of feeding and energy balance.

Glucosamine attenuates cigarette smoke-induced lung inflammation by inhibiting ROS-sensitive inflammatory signaling.

Cigarette smoking causes persistent lung inflammation that is mainly regulated by redox-sensitive pathways. We have reported that cigarette smoke (CS) activates a NADPH oxidase-dependent reactive oxygen species (ROS)-sensitive AMP-activated protein kinase (AMPK) signaling pathway leading to induction of lung inflammation. Glucosamine, a dietary supplement used to treat osteoarthritis, has antioxidant and anti-inflammatory properties. However, whether glucosamine has similar beneficial effects against CS-induced lung inflammation remains unclear. Using a murine model we show that chronic CS exposure for 4 weeks increased lung levels of 4-hydroxynonenal (an oxidative stress biomarker), phospho-AMPK, and macrophage inflammatory protein 2 and induced lung inflammation; all of these CS-induced events were suppressed by chronic treatment with glucosamine. Using human bronchial epithelial cells, we demonstrate that cigarette smoke extract (CSE) sequentially activated NADPH oxidase; increased intracellular levels of ROS; activated AMPK, mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription proteins 3 (STAT3); and induced interleukin-8 (IL-8). Additionally, using a ROS scavenger, a siRNA that targets AMPK, and various pharmacological inhibitors, we identified the signaling cascade that leads to induction of IL-8 by CSE. All these CSE-induced events were inhibited by glucosamine pretreatment. Our findings suggest a novel role for glucosamine in alleviating the oxidative stress and lung inflammation induced by chronic CS exposure in vivo and in suppressing the CSE-induced IL-8 in vitro by inhibiting both the ROS-sensitive NADPH oxidase/AMPK/MAPK signaling pathway and the downstream transcriptional factors NF-κB and STAT3.

Hypoxia-reoxygenation affects whole-genome expression in the newborn eye.

PURPOSE: Resuscitation of newborns is one of the most frequent procedures in neonatal medicine. The use of supplementary oxygen during resuscitation of the asphyxiated newborn has been shown to be detrimental to vulnerable tissues. We wanted to assess transcriptional changes in ocular tissue after the acute use of oxygen in the delivery room in a hypoxia-reoxygenation model of the newborn mouse. METHODS: C57BL/6 mice (n = 57), postnatal day 7, were randomized to receive either 120 minutes of hypoxia, at 8% O2, followed by 30 minutes of reoxygenation with 21, 40, 60, or 100% O2 or to normoxia followed by 30 minutes of 21% or 100% O2. Whole ocular homogenates were analyzed by Affymetrix 750k expression array, and RT-PCR was performed for validation. Bayesian analysis of variance for microarray data (BAMarray) was used to identify single significant genes, and Gene Set Enrichment Analysis (GSEA) was applied to reveal significant pathway systems. RESULTS: In total, ∼ 92% of the gene expression changes were altered in response to reoxygenation with 60% or 100% O2 compared to expression at the lower percentages of 21% and 40%. After 100% O2 treatment, genes involved in inflammation (Ccl12), angiogenesis (Igfr1, Stat3), and metabolism (Hk2) were upregulated. Pathway analyses after hypoxia-reoxygenation revealed significant alterations of six pathways which included apoptosis, TGF-beta signaling, oxidative phosphorylation, voltage-gated calcium channel complex, mitochondrion, and regulation of RAS protein signal transduction. CONCLUSIONS: Hypoxia-reoxygenation can induce immediate transcriptional responses in ocular tissue involving inflammation, angiogenesis, energy failure, and Ras signaling.

2-Methoxystypandrone inhibits signal transducer and activator of transcription 3 and nuclear factor-κB signaling by inhibiting Janus kinase 2 and IκB kinase.

Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) or the nuclear factor-κB (NF-κB) pathway occurs frequently in cancer cells and contributes to oncogenesis. The activation of Janus kinase 2 (JAK2) and IκB kinase (IKK) are key events in STAT3 and NF-κB signaling, respectively. We have identified 2-methoxystypandrone (2-MS) from a traditional Chinese medicinal herb Polygonum cuspidatum as a novel dual inhibitor of JAK2 and IKK. 2-MS inhibits both interleukin-6-induced and constitutively-activated STAT3, as well as tumor necrosis factor-α-induced NF-κB activation. 2-MS specifically inhibits JAK and IKKß kinase activities but has little effect on activities of other kinases tested. The inhibitory effects of 2-MS on STAT3 and NF-κB signaling can be eliminated by DTT or glutathione and can last for 4 h after a pulse treatment. Furthermore, 2-MS inhibits growth and induces death of tumor cells, particularly those with constitutively-activated STAT3 or NF-κB signaling. We propose that the natural compound 2-MS, as a potent dual inhibitor of STAT3 and NF-κB pathways, is a promising anticancer drug candidate.