Metastatic tumor antigen 1 contributes to hepatocarcinogenesis posttranscriptionally through RNA-binding function.

BACKGROUND AND AIMS: Both nuclear and cytoplasmic overexpression of metastatic tumor antigen 1 (MTA1) contributes to tumorigenesis of HCC. Most studies have focused on nuclear MTA1 whose function is mainly a chromatin modifier regulating the expression of various cancer-promoting genes. By contrast, the molecular mechanisms of cytoplasmic MTA1 in carcinogenesis remain elusive. Here, we reveal a role of MTA1 in posttranscriptional gene regulation. APPROACH AND RESULTS: We conducted the in vitro and in vivo RNA-protein interaction assays indicating that MTA1 could bind directly to the 3'-untranslated region of MYC RNA. Mutation at the first glycine of the conserved GXXG loop within a K-homology II domain-like structure in MTA1 (G78D) resulted in the loss of RNA-binding activity. We used gain- and loss-of-function strategy showing that MTA1, but not the G78D mutant, extended the half-life of MYC and protected it from the lethal -7-mediated degradation. The G78D mutant exhibited lower activity in promoting tumorigenesis than wild-type in vitro and in vivo. Furthermore, RNA-immunoprecipitation sequencing analysis demonstrated that MTA1 binds various oncogenesis-related mRNAs besides MYC . The clinical relevance of cytoplasmic MTA1 and its interaction with MYC were investigated using HBV-HCC cohorts with or without early recurrence. The results showed that higher cytoplasmic MTA1 level and MTA1- MYC interaction were associated with early recurrence. CONCLUSIONS: MTA1 is a generic RNA-binding protein. Cytoplasmic MTA1 and its binding to MYC is associated with early recurrence in patients with HBV-HCC. This function enables it to regulate gene expression posttranscriptionally and contributes to hepatocarcinogenesis.

Expression of Metastatic Tumor Antigen 1 Splice Variant Correlates With Early Recurrence and Aggressive Features of Hepatitis B Virus-Associated Hepatocellular Carcinoma.

Overexpression of metastatic tumor antigen 1 (MTA1) was correlated with poor prognosis of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HBV-HCC). The aim of this study was to examine the clinical significance of the expression of MTA1 and its exon 4-excluded form (MTA1dE4), the most abundant spliced variant of MTA1, in patients receiving curative resection for HBV-HCC. We collected 102 patients with HBV-HCC and received curative resection retrospectively and examined the expressions level of total MTA1/MTA1dE4 in their paired nontumor and tumor liver tissues by using RT-qPCR. The association between MTA1/MTA1dE4 expression and various tumor features as well as tumor recurrence was analyzed. During the median follow-up period of 4 years, 25 patients (24.5%) showed early recurrence (within 12 months postresection) and 42 (54.5%) showed late recurrence. In Kaplan-Meier analysis, MTA1dE4 overexpression in tumor, but not MTA1, was associated with early recurrence (P = 0.0365), but not late recurrence. In multivariate analysis, only alpha-fetoprotein (AFP) ≥200 ng/mL (P = 0.006) and large tumor size (P = 0.027) were correlated with early recurrence. In the subgroup of patients with AFP <200 ng/mL, high MTA1dE4, but not total MTA1, expression could help predict early recurrence (P = 0.0195). In vitro, wound healing and invasion assays were performed in HCC cells, and MTA1dE4 was found to exhibit a higher ability in promoting migration and invasion of hepatoma cells than full-length MTA1. Conclusion: MTA1dE4 expression is correlated with more aggressive tumor characteristics and might serve as a more sensitive marker for early recurrence of HBV-HCC, especially for low-AFP patients.

Eicosapentaenoic acid reduces indoleamine 2,3-dioxygenase 1 expression in tumor cells.

Marine plants and animals have omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA is required for biological processes, but humans are unable to synthesize them and must be obtained from dietary sources. EPA has been used as an antitumor agent but the molecular mechanisms for the regulation of tumor microenvironment immunity by EPA are still unknown. The indoleamine 2,3-dioxygenase 1 (IDO) catalyzes conversion of tryptophan to kynurenine to induce immune evasion in tumor microenvironment. In this study, EPA inhibited the expression of IDO via downregulation of protein kinase B (Akt)/mammalian targets of rapamycin (mTOR) signaling pathway in tumor cells. Meanwhile, a significant decrease in kynurenine levels and increase in T cell survival were observed after tumor cells treated with EPA. The results demonstrated that EPA can activate host antitumor immunity by inhibiting tumor IDO expression. Therefore, our finding suggests that EPA can be enormous potential for cancer immunotherapy.

Protective effect of N-acetylcysteine on progression to end-stage renal disease: Necessity for prospective clinical trial.

BACKGROUND: We aimed to evaluate the potential benefits of N-acetylcysteine (NAC) on the risk of chronic kidney disease (CKD) progression to dialysis-requiring end-stage renal disease (ESRDd). METHODS: In a population-based cohort study of 145,062 individuals, 123,608 CKD patients who were followed up for 10years were included, and CKD patients treated with NAC (ICD-9-CM) were compared with those who were not treated. Using propensity score matching, we analyzed the predictors of CKD progression to ESRDd by Cox proportional hazards regression with adjustments for sex, age, and comorbidities, and evaluated the effect of NAC using cumulative defined daily dose (cDDD). RESULTS: NAC use was associated with a reduced risk for progression to ESRDd [hazard ratio (HR), 0.819; 95% confidence interval (CI), 0.781-0.965; P=0.017]. Risk reduction was proportional to cDDD in NAC users compared with that in NAC non users (HR, 0.835, 0.811, and 0.799 for cDDD 91-180, 181-360, and >360, respectively; P for trend=0.018). Risk reduction was apparent in women (P=0.001) and in younger-aged patients of 18-29years (P=0.021) and 30-39years (P=0.033), in the presence of hypertension (P=0.003), and in the absence of diabetes mellitus (P=0.042) and congestive heart failure (P=0.036). CONCLUSION: NAC use was associated with a reduced risk for progression to ESRDd. These results, obtained from retrospective data, indicate that a prospective study is warranted.

Pegylated IFN-α suppresses hepatitis C virus by promoting the DAPK-mTOR pathway.

Death-associated protein kinase (DAPK) has been found to be induced by IFN, but its antiviral activity remains elusive. Therefore, we investigated whether DAPK plays a role in the pegylated IFN-α (peg-IFN-α)-induced antiviral activity against hepatitis C virus (HCV) replication. Primary human hepatocytes, Huh-7, and infectious HCV cell culture were used to study the relationship between peg-IFN-α and the DAPK-mammalian target of rapamycin (mTOR) pathways. The activation of DAPK and signaling pathways were determined using immunoblotting. By silencing DAPK and mTOR, we further assessed the role of DAPK and mTOR in the peg-IFN-α-induced suppression of HCV replication. Peg-IFN-α up-regulated the expression of DAPK and mTOR, which was associated with the suppression of HCV replication. Overexpression of DAPK enhanced mTOR expression and then inhibited HCV replication. In addition, knockdown of DAPK reduced the expression of mTOR in peg-IFN-α-treated cells, whereas silencing of mTOR had no effect on DAPK expression, suggesting mTOR may be a downstream effector of DAPK. More importantly, knockdown of DAPK or mTOR significantly mitigated the inhibitory effects of peg-IFN-α on HCV replication. In conclusion, our data suggest that the DAPK-mTOR pathway is critical for anti-HCV effects of peg-IFN-α.

Invasive liver abscess syndrome predisposed by Klebsiella pneumoniae related prostate abscess in a nondiabetic patient: a case report.

BACKGROUND: Prostate abscess is usually a complication of acute urinary tract infection. Invasive liver abscess syndrome is characterized with Klebsiella pneumoniae related multiple organ metastasis. Concomitant pyogenic liver abscess and prostate abscess have rarely been reported. Recurrent episode of liver abscess is even rarer. CASE PRESENTATION: We report a 71-year-old male with acute bacterial prostate abscess and urinary tract infection caused by K. pneumoniae associated with multiple liver abscess, psoas muscle abscess and osteomyelitis. Blood culture and urine culture yielded K. pneumoniae, which confirmed the diagnosis of invasive liver abscess syndrome caused by K. pneumoniae. The patient was successfully treated with empirical antibiotics for 6 weeks. CONCLUSIONS: This case emphasizes the importance of timely and accurate diagnosis followed by appropriate treatment in disseminated K. pneumoniae infection to prevent significant morbidity and mortality.

Characterization of metastatic tumor antigen 1 and its interaction with hepatitis B virus X protein in NF-κB signaling and tumor progression in a woodchuck hepatocellular carcinoma model.

The metastatic tumor antigen 1 (MTA1) protein is associated with tumor invasiveness and poor prognosis in patients with hepatocellular carcinoma (HCC), particularly in those with hepatitis B virus (HBV)-related HCC. Chronically woodchuck hepatitis virus (WHV)-infected woodchuck is an ideal animal model for studying the pathogenesis of HBV-associated liver diseases, including HCC. To investigate the roles of MTA1 in HBV-associated hepatocarcinogenesis in the woodchuck model, we cloned the woodchuck MTA1 (wk-MTA1) complementary (c)DNA and characterized its molecular functions. The sequence and organization of the wk-MTA1 protein were highly conserved among different species. Similar to its expression in human HCC, wk-MTA1 was upregulated in woodchuck HCC, as determined at RNA and protein levels. Furthermore, an MTA1-spliced variant, wk-MTA1dE4, was overexpressed in woodchuck HCC, and it was attributed to approximately 50% of the total transcripts. The percentage of wk-MTA1dE4-overexpressed woodchuck HCCs was higher than that of the total wk-MTA1-overexpressed HCCs (77.8% vs 61.1%) and wk-MTA1dE4 may represent a more sensitive marker than the total wk-MTA1 in woodchuck HCC. We overexpressed or knocked down wk-MTA1 in a woodchuck HCC cell line and demonstrated that wk-MTA1 could interact with the WHV X protein (WHx) and play indispensable roles in WHx-mediated NF-κB activation and tumor cell migration- and invasion-promoting activities. In conclusion, our results support the hypothesis that woodchuck HCC recapitulates HBV-associated HCC with respect to the molecular characteristics of MTA1 and provides new clues for conducting mechanistic studies of MTA1 in HBV-associated hepatocarcinogenesis, including the possible clinical significance of wk-MTA1dE4.

Emphysematous cholecystitis presenting as gas-forming liver abscess and pneumoperitoneum in a dialysis patient: a case report and review of the literature.

BACKGROUND: Emphysematous cholecystitis is a rare variant of acute cholecystitis with a high mortality rate. The combination of emphysematous cholecystitis, liver abscess and pneumoperitoneum are even rarer. Herein we present a case of emphysematous cholecystitis in a senile diabetic lady who had worsening hemodynamics while undergoing hemodialysis. CASE PRESENTATION: A 64-year-old woman with history of type 2 diabetes mellitus and end stage renal disease with regular hemodialysis presented to the emergency department with a 1-day history of sudden onset of lassitude and hypotension during hemodialysis. The result of a computed tomography (CT)-scan revealed air encircling the gallbladder, liver parenchymal and minimal pneumoperitoneal and liver abscess with no cholelithiasis. The patient had received empirical antibiotics with piperacillin-tazobactam 2.25 g intravenous route every 6 h for 14 days and cholecystectomy with surgical debridement and lead an uneventful postoperative hospital course. Escherichia coli was demonstrated as well as blood culture and peritoneal fluid culture. CONCLUSION: In a senile diabetic and dialysis patient, we should take emphysematous cholecystitis into consideration once vague abdominal pain occurrs. Empirical antibiotic therapy and adequate surgical intervention should take place as soon as possible.

Novel Investigations of Flavonoids as Chemopreventive Agents for Hepatocellular Carcinoma.

We would like to highlight the application of natural products to hepatocellular carcinoma (HCC). We will focus on the natural products known as flavonoids, which target this disease at different stages of hepatocarcinogenesis. In spite of the use of chemotherapy and radiotherapy in treating HCC, patients with HCC still face poor prognosis because of the nature of multidrug resistance and toxicity derived from chemotherapy and radiotherapy. Flavonoids can be found in many vegetables, fruits, and herbal medicines that exert their different anticancer effects via different intracellular signaling pathways and serve as antioxidants. In this review, we will discuss seven common flavonoids that exert different biological effects against HCC via different pathways.

Ribavirin enhances the action of interferon-α against hepatitis C virus by promoting the p53 activity through the ERK1/2 pathway.

BACKGROUND/AIMS: Ribavirin significantly enhances the antiviral response of interferon-α (IFN-α) against Hepatitis C virus (HCV), but the underlying mechanisms remain poorly understood. Recently, p53 has been identified as an important factor involving the suppression of HCV replication in hepatocytes. We, therefore, decided to investigate whether and how ribavirin inhibits the replication of HCV by promoting the activity of p53. METHODS: HepG2 and HCV replicons (JFH1/HepG2) were utilized to study the relationship between ribavirin and p53. The effect of ribavirin on cell cycles was analyzed by flow cytometry. The activation of p53 and the signaling pathways were determined using immunoblotting. By knocking down ERK1/ERK2 and p53 utilizing RNA interference strategy, we further assessed the role of ERK1/2 and p53 in the suppression of HCV replication by ribavirin in a HCV replicon system. RESULTS: Using HepG2 and HCV replicons, we demonstrated that ribavirin caused the cell cycle arrest at G1 phase and stabilized and activated p53, which was associated with the antiviral activity of ribavirin. Compared to either ribavirin or IFN-α alone, ribavirin plus IFN-α resulted in greater p53 activation and HCV suppression. We further identified ERK1/2 that linked ribavirin signals to p53 activation. More importantly, knockdown of ERK1/2 and p53 partially mitigated the inhibitory effects of ribavirin on the HCV replication, indicating that ERK1/2-p53 pathway was involved in the anti-HCV effects of ribavirin. CONCLUSION: Ribavirin stimulates ERK1/2 and subsequently promotes p53 activity which at least partly contributes to the enhanced antiviral response of IFN-α plus ribavirin against HCV.

Hypokalemic paralysis as primary presentation of Fanconi syndrome associated with Sjögren syndrome.

Hypokalemic paralysis is a rare presentation of Fanconi syndrome (FS) caused by Sjögren Syndrome (SS). We describe a 39-year-old man who manifested flaccid paralysis of 4 limbs. Laboratory investigations showed profound hypokalemia (1.6 mmol/L) with renal K wasting, hyperchloremic metabolic acidosis with positive urine anion gap, hypophosphatemia with hyperphosphaturia, hypouricemia with hyperuricosuria, normoglycemic glycosuria, and abnormal serum creatinine concentration 2.2 mg/dL. A thorough survey for the cause of FS revealed that he had xerophthalmia and xerostomia accompanied by high anti-Ro antibody, positive Schirmer test, and delayed saliva excretion on sialoscintigraphy, confirming the diagnosis of SS. Potassium citrate, active vitamin D3, and high phosphate diet for his FS coupled with mycophenolate mofetil for SS resolved clinical symptoms and ameliorated renal function. Early recognition of HP due to the underlying SS-related FS with prompt therapy not only could terminate potentially life-threatening hypokalemia, but also improve renal outcome.

Hypercalcemic crisis successfully treated with prompt calcium-free hemodialysis.

Hypercalcemic crisis, a life-threatening emergency, is defined as decompensated hypercalcemia presented with characteristic symptoms such as oliguria, cardiac arrhythmia, or coma. We report the case of a 63-year-old man diagnosed with mucosa-associated lymphoid tissue lymphoma and multiple bony metastases, who presented to the emergency department (ED) with coma and severe hypercalcemia (4.15 mmol/L). Prompt hydration with normal saline and intravenous pamidronate failed to correct his hypercalcemic coma. Calcium-free hemodialysis rapidly decreased the level of serum total calcium to 2.15 mmol/L after a 2-hour session, and the patient dramatically regained consciousness shortly after hemodialysis. Calcium- free hemodialysis has proved favorable for rapidly correcting hypercalcemia in the presence of severe hypercalcemic symptoms, congestive heart failure, renal failure, or other conditions that contraindicate adequate hydration. This case highlights the fact that for all patients with comas of questionable cause in the ED, hypercalcemia- induced coma must be considered, especially in patients with malignancies. Early diagnosis and prompt treatment with calcium-free hemodialysis not only rapidly improve patient consciousness but also prevent the fatal complication of hypercalcemia.

Regulatory T cells negatively regulate neovasculature of airway remodeling via DLL4-Notch signaling.

Regulatory T cells (Treg) have been shown to prevent the development of allergic asthma; however, the role of Treg in asthma with established airway remodeling is unknown. To address this, we exploited an OVA-induced chronic asthma mouse model wherein Treg were adoptively transferred to the mice at chronic stage of the model. We found that among the structural alterations of airway remodeling, Treg selectively reduced the vessel numbers in both peritracheal and peribronchial regions and the lung parenchyma. Extracellular matrix deposition, mucus metaplasia, muscular hyperplasia, and vasodilation, as were also induced by chronic allergen challenge, were not affected by Treg. TUNEL staining of the lung sections revealed an increased endothelial cell (EC) apoptosis in mice receiving Treg transfers compared with their asthmatic counterparts. By using Matrigel angiogenesis assays, we showed that Treg inhibited EC angiogenesis both in vitro and in vivo. Treg preferentially expressed Notch ligand DLL4, and an anti-DLL4 blocking Ab abrogated the inhibitory effect of Treg on EC tube formation. In vivo, decreased airway and lung vessel numbers as well as ameliorated airway hyperresponsiveness after Treg transfers were reverted when Treg-derived DLL4 signal was blocked by the anti-DLL4 Ab. Our findings demonstrate a novel function of Treg whereby Treg down-regulate remodeling angiogenesis via proapoptotic DLL4-Notch signaling, and suggest a therapeutic potential of Treg in alleviating airway hyperresponsiveness of chronic asthma.

Prompt plasmapheresis successfully rescue pulmonary-renal syndrome caused by ANCA-negative microscopic polyangiitis.

Pulmonary-renal syndrome (PRS) associated with antineutrophil cytoplasmic antibodies (ANCA)-negative microscopic polyangiitis (MPA) is relatively rare, and the effects of plasmapheresis on these patients remain unclear. Here, we report the case of a 66-year-old man who presented with fever, acute renal failure, thrombocytopenia, and sudden onset of diffuse pulmonary hemorrhage. Prompt plasmapheresis and concurrent pulse therapy with methylprednisolone effectively rescued his pulmonary-renal syndrome. The patient was then diagnosed with MPA on the basis of typical histological findings and the absence of surrogate markers of Wegener's granulomatosis and Churg-Strauss syndrome. This case demonstrates the therapeutic effects of plasmapheresis on ANCA-negative MPA and highlights the necessity of prompt plasmapheresis for not only resolving pulmonary hemorrhage but also increasing the likelihood of renal function restoration in patients with PRS.

Oncostatin M-induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: an implication for the pathogenesis of arthritis.

OBJECTIVE: To examine the roles of STATs 1 and 3 in CCL2 production in human osteoblastic cells and their influences on arthritis development. METHODS: The expression of CCL2 in primary human osteoblasts and U2OS human osteoblastic cells was examined by Northern blotting and enzyme-linked immunosorbent assay. The roles of STAT-1/3 and c-Fos were assessed using short hairpin RNAs (shRNA) to silence their functions. Serine phosphorylation of STATs was assessed by Western blotting. Promoter activities of c-Fos and CCL2 were assessed by chloramphenicol acetyltransferase and luciferase assays, respectively. Interactions of STAT-1, STAT-3, and c-Fos with DNA were evaluated by electrophoretic mobility shift assay (EMSA) and immunoprecipitation. The effect of the JAK inhibitor AG-490 on collagen-induced arthritis (CIA) in rats was examined using immunohistochemistry. RESULTS: Oncostatin M (OSM) stimulated CCL2 expression in primary human osteoblasts and U2OS cells. In U2OS cells, STAT-1 and STAT-3 were involved in OSM-stimulated CCL2 expression, and both the phosphatidylinositol 3-kinase/Akt and MEK/ERK pathways were implicated in the activation of these STATs. STAT-1 and STAT-3 modulated the expression of c-Fos and directly transactivated the CCL2 promoter. Moreover, EMSA showed formation of a DNA-protein complex containing STAT-1, STAT-3, and interestingly, c-Fos. Immunoprecipitation confirmed the binding between c-Fos and STAT-1/3. Reporter assay revealed synergistic attenuation of CCL2 promoter activity by shRNA targeting of STAT-1, STAT-3, and c-Fos. AG-490 suppressed OSM-stimulated activation of STAT-1/3 and synthesis of CCL2 in vitro and diminished the severity of CIA and the number of CCL2-synthesizing osteoblasts in vivo. CONCLUSION: These findings show that multiple levels of STAT-1/3 signaling modulate OSM-stimulated CCL2 expression in human osteoblastic cells. Clinically, this pathway may be related to the pathogenesis of arthritis.

Simvastatin as a novel strategy to alleviate periapical lesions.

Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used cholesterol-lowering agents that also possess anti-inflammatory activities. Cysteine-rich 61 (Cyr61) and CCL2 are potential osteolytic mediators in inflammatory bone diseases. The study assessed the effect of simvastatin on tumor necrosis factor alpha (TNF- alpha)-induced synthesis of Cyr61 and CCL2 in MG-63 human osteoblastic cells. The therapeutic effect of simvastatin on rat apical periodontitis was also examined. The synthesis of Cyr61 in MG-63 was assessed by Western analysis. Expression of CCL2 was examined by an enzyme-linked immunosorbent assay. The effect of simvastatin on induced rat periapical lesion was examined radiographically and immunohistochemically. Western blot showed that TNF-alpha stimulated Cyr61 synthesis in MG-63, whereas simvastatin attenuated this effect in a dose-dependent manner. Simvastatin also reduced the levels of TNF-alpha-induced CCL2, and exogenous Cyr61 restored the inhibitory effects. Radiography and histopathology revealed that the administration of simvastatin markedly diminished the severity of induced rat periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and CD-68-positive macrophages were also decreased. Simvastatin suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.

An extract of green tea, epigallocatechin-3-gallate, reduces periapical lesions by inhibiting cysteine-rich 61 expression in osteoblasts.

Recent investigations indicate that epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has anti-inflammatory properties. This study assessed the effect of EGCG on oncostatin M (OSM)-induced synthesis of cysteine-rich 61 (Cyr61), a potential osteolytic mediator, in MG-63 human osteoblastic cells. The therapeutic effect of EGCG in apical periodontitis in rats was also examined. Western blot analysis showed that OSM stimulated Cyr61 synthesis in MG-63 in a time-dependent manner, whereas EGCG readily attenuated this effect. On the other hand, Cyr61 treatment of MG-63 cells induced the release of CCL2, a chemokine responsible for macrophage chemotaxis. In a rat model of induced apical periodontitis, radiography and histopathology revealed that administration of EGCG markedly diminished the severity of periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and infiltrating macrophages were also decreased. Thus, EGCG suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.

Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

OBJECTIVE: To assess the effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)-induced CCL2 synthesis and the associated signaling pathways in human osteoblastic cells. The therapeutic effect of EGCG on collagen-induced arthritis (CIA) in rats was also studied. METHODS: CCL2 and c-Fos messenger RNA expression was analyzed by Northern blotting. The modulating effects of EGCG on the activation of Raf-1, Akt, and phosphatidylinositol 3-kinase (PI 3-kinase) were examined by coimmunoprecipitation, Western blotting, and PI 3-kinase activity assay. Interactions between c-Fos and CCL2 promoter were evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay. The effect of EGCG on CIA in rats was examined clinically and immunohistochemically. RESULTS: EGCG inhibited OSM-stimulated CCL2 expression in primary human osteoblasts and MG-63 cells. In MG-63 cells, EGCG alleviated the OSM-induced phosphorylation of Raf-1 at Ser338 but restored the dephosphorylation of Raf-1 at Ser259. EGCG increased the activity of PI 3-kinase, the level of phosphorylated Akt (Ser473), and binding between Raf-1 and active Akt. EMSA and ChIP assay revealed that EGCG attenuated activator protein 1 (AP-1)-CCL2 promoter interaction, possibly by reducing c-Fos synthesis. Codistribution of CD68+ macrophages and CCL2+ osteoblasts in osteolytic areas was obvious in the CIA model. Administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the amount of CCL2-synthesizing osteoblasts. CONCLUSION: By stimulating PI 3-kinase activity, EGCG promoted Akt/Raf-1 crosstalk, resulting in decreased AP-1 binding to CCL2 promoter, and finally reduced CCL2 production in osteoblasts. EGCG alleviated the severity of CIA, probably by suppressing CCL2 synthesis in osteoblasts to diminish macrophage infiltration. Our data support the therapeutic potential of EGCG on arthritis.

Hypoxia-stimulated vascular endothelial growth factor production in human nasal polyp fibroblasts: effect of epigallocatechin-3-gallate on hypoxia-inducible factor-1 alpha synthesis.

OBJECTIVE: To verify the inhibitory effects of epigallocatechin-3-gallate (EGCG) on the synthesis of hypoxia-induced vascular endothelial growth factor (VEGF) in nasal polyp fibroblasts (NPFs). DESIGN: Eight primary cultures of NPFs were established from nasal polyps. Effects of EGCG on the production of hypoxia-inducible factor (HIF)-1 alpha (the most potent VEGF stimulant) and VEGF by NPFs under hypoxic conditions were measured by Western blot analysis. Immunohistochemical staining was used to examine the in vivo expressions of HIF-1 alpha and VEGF in 20 sections of nasal polyps. RESULTS: Western blot analysis showed that cobalt chloride induced HIF-1 alpha and VEGF synthesis in NPFs in a time-dependent manner, reaching a plateau at 4 and 8 hours, respectively, following treatment. Epigallocatechin-3-gallate attenuated the level of HIF-1 alpha induced by cobalt chloride and also reduced cobalt chloride-stimulated VEGF production by suppressing HIF-1 alpha synthesis. Furthermore, oligomycin (a specific HIF-1 alpha inhibitor) combined with EGCG resulted in a more profound inhibition of VEGF synthesis compared with oligomycin or EGCG treatment alone. Nevertheless, the synergistic effect seemed smaller than the sum of their individual actions. Immunohistochemical analysis revealed the presence of HIF-1 alpha and VEGF in NPFs and mononuclear round cells. Intimate alignment of VEGF-positive fibroblasts and proliferating small capillaries was frequently found. CONCLUSIONS: Nasal polyp fibroblasts contribute to the pathogenesis of nasal polyps by producing VEGF to promote angiogenesis under hypoxic conditions. Epigallocatechin-3-gallate substantially diminishes HIF-1 alpha and VEGF synthesis in NPFs.