Extract of Ganoderma sinensis spores induces cell cycle arrest of hepatoma cell via endoplasmic reticulum stress.

CONTEXT: Ganoderma sinensis Zhao, Xu et Zhang (Ganodermataceae) has been used for the prevention or treatment of a variety of diseases, including cancer. OBJECTIVE: We investigated the antitumor activity and mechanism of an extract from G. sinensis against hepatocellular carcinoma. MATERIALS AND METHODS: A G. sinensis extract (GSE) was obtained from sporoderm-broken G. sinensis spores by supercritical fluid carbon dioxide extraction. Hepatoma cells, HepG2 cells, were treated with emulsified sample of GSE at 12.5, 25, 50, 100 and 150 µg/mL for 24 h. The Alamar Blue assay was used to examine growth inhibitory effects. Changes in cell structure and morphology were assessed via transmission electron microscopy and confocal laser scanning microscope. Cell cycle distribution was analysed by flow cytometry. RESULTS: GSE suppressed the proliferation of HepG2 cells (IC50=70.14 µg/mL). Extensive cytoplasmic vacuolation originating from dilation of the endoplasmic reticulum (ER) was shown in GSE-treated HepG2 cells. GSE treatment also upregulated the expression of ER stress-related proteins in HepG2 cells. Cells tended to be arrested at the G2/M cell cycle stage after GSE treatment (30.8 ± 1.4% and 42.2 ± 2.6% at GSE with 50 µg/mL and 100 µg/mL vs. 21.03 ± 1.10%, control). Pre-treatment with salubrinal, an inhibitor of ER stress, effectively attenuated cell cycle arrest induced by GSE. DISCUSSION AND CONCLUSIONS: Our findings provide new evidence that GSE suppresses growth of cancer cells in vitro through activating the ER stress pathway. The GSE may be clinically applied in the prevention and/or treatment of cancer.

Glycolytic activation of peritumoral monocytes fosters immune privilege via the PFKFB3-PD-L1 axis in human hepatocellular carcinoma.

BACKGROUND & AIMS: Programmed cell death 1 ligand 1 (PD-L1) expression on antigen-presenting cells is essential for T cell impairment, and PD-L1-expressing macrophages may mechanistically shape and therapeutically predict the clinical efficacy of PD-L1 or programmed cell death 1 blockade. We aimed to elucidate the mechanisms underlying PD-L1 upregulation in human tumor microenvironments, which remain poorly understood despite the clinical success of immune checkpoint inhibitors. METHODS: Monocytes/macrophages were purified from peripheral blood, non-tumor, or paired tumor tissues of patients with hepatocellular carcinoma (HCC), and their possible glycolytic switch was evaluated. The underlying regulatory mechanisms and clinical significance of metabolic switching were studied with both ex vivo analyses and in vitro experiments. RESULTS: We found that monocytes significantly enhanced the levels of glycolysis at the peritumoral region of human HCC. The activation of glycolysis induced PD-L1 expression on these cells and subsequently attenuated cytotoxic T lymphocyte responses in tumor tissues. Mechanistically, tumor-derived soluble factors, including hyaluronan fragments, induced the upregulation of a key glycolytic enzyme, PFKFB3, in tumor-associated monocytes. This enzyme not only modulated the cellular metabolic switch but also mediated the increased expression of PD-L1 by activating the nuclear factor kappa B signaling pathway in these cells. Consistently, the levels of PFKFB3+CD68+ cell infiltration in peritumoral tissues were negatively correlated with overall survival and could serve as an independent prognostic factor for survival in patients with HCC. CONCLUSIONS: Our results reveal a mechanism by which the cellular metabolic switch regulates the pro-tumor functions of monocytes in a specific human tumor microenvironment. PFKFB3 in both cancer cells and tumor-associated monocytes is a potential therapeutic target in human HCC. LAY SUMMARY: Programmed cell death 1 ligand 1 (PD-L1) expressed on antigen-presenting cells, rather than tumor cells, has been reported to play an essential role in checkpoint blockade therapy. A fundamental understanding of mechanisms that regulate the expression of PD-L1 on tumor-infiltrating monocytes/macrophages will undoubtedly lead to the possibility of developing novel PD-L1 blockade strategies with high specificity and efficiency. The current study unveils a novel mechanism by which metabolic switching links immune activation responses to immune tolerance in the tumor milieu, identifying potential targets for future immune-based anti-cancer therapies.

[Progress of regulation of leukemia stem cells of chronic myeloid leukemia by autophagy].

Leukemia stem cells (LSC) that were found in chronic myeloid leukemia (CML) responsible for the abnormal proliferation with the potential of self-renewal and multi-directional differentiation are involved in the pathophysiological process for drug resistance and relapse of CML. Autophagy, a conservative lysosomal degradation process that mediates cell degradation and recycling process, plays crucial roles in maintaining the homeostasis and function of intracellular environment. Recent studies suggested that autophagy is involved in the regulation of LSC differentiation and also closely related to the chemo-sensitivity of CML. In this review, we focused on the role of autophagy on chemotherapy sensitivity of CML as well as the leukemia stem cell function for the development of new anti-leukemia drugs.

A combined experimental and computational investigation on the unusual molecular mechanism of the Lossen rearrangement reaction activated by carcinogenic halogenated quinones.

The classic Lossen rearrangement is a well-known reaction describing the transformation of an O-activated hydroxamic acid into the corresponding isocyanate. In this study, we found that chlorinated benzoquinones (CnBQ) serve as a new class of agents for the activation of benzohydroxamic acid (BHA), leading to Lossen rearrangement. Compared to the classic one, this new kind of CnBQ-activated Lossen rearrangement has the following unique characteristics: (1) The stability of CnBQ-activated BHA intermediates was found to depend not only on the degree but also on the position of Cl-substitution on CnBQs, which can be divided into two subgroups. (2) It is the relative energy of the anionic CnBQ-BHA intermediates that determine the rate of this CnBQ-activated rearrangement, which is the rate-limiting step, and the Cl or H ortho to the reaction site at CnBQ is crucial for the stability of the anionic intermediates. (3) A pKa-activation energy correlation was observed, which can explain why the correlation exists between the rate of the rearrangement and the acidity of the conjugate acid of the anionic leaving group, the hydroxlated quinones. These findings may have broad implications for future research on halogenated quinoid carcinogens and hydroxamate biomedical agents.

Serum advanced glycation end products are associated with insulin resistance in male nondiabetic patients with obstructive sleep apnea.

PURPOSE: Advanced glycation end products (AGEs) play an important role in the pathogenesis of diabetic vascular complications. Recently, growing evidence has shown that AGEs could be involved in the pathogenesis of insulin resistance. It has also been suggested that circulating AGE are associated with insulin resistance in nondiabetic patients. This study investigated whether serum AGEs levels are associated with insulin resistance in nondiabetic patients with obstructive sleep apnea (OSA). METHODS: A total of 139 male nondiabetic patients with OSA were recruited for participation in the study. Serum AGE levels were examined using an enzyme-linked immunosorbent assay. Insulin resistance was determined using the homeostasis model assessment index (HOMA-IR). RESULTS: There was a significant correlation between serum AGEs and the apnea-hypopnea index (AHI) (r = 0.281, p = 0.014), duration of SaO2 < 90% (r = 0.267, p = 0.018), minimum SaO2 (r = -0.188, p = 0.046), high-sensitivity C-reactive protein (hsCRP) (r = 0.274, p = 0.012), and HOMA-IR (r = 0.303, p < 0.001). Multiple regression analysis showed that serum AGEs (p = 0.011), AHI (p = 0.024), waist circumference (p = 0.040), and hsCRP (p = 0.046) were independently associated with HOMA-IR (R(2) = 0.392). In addition, the strength of the correlation between serum AGEs and HOMA-IR was related to the severity of OSA. CONCLUSIONS: The present study indicated that serum AGE levels were associated with insulin resistance in male nondiabetic patients with OSA. These findings suggest that AGEs may play a role in insulin resistance in OSA and may also be a biomarker for patients with OSA with high risk of developing type 2 diabetes.

Resveratrol improves diabetes-induced cognitive dysfunction in part through the miR-146a-5p/TXNIP axis.

Resveratrol (RSV) has been shown to have a neuroprotective effect in various central nervous system disorders, although the role of RSV in diabetes-induced cognitive dysfunction is still not fully elucidated. Here, we investigated whether RSV improved diabetes-related cognitive dysfunction in vivo and in vitro. We induced a rat diabetic model with a high-fat and high-sucrose diet followed by intraperitoneal injection of streptozotocin and a diabetic neuron cell model by stimulation with high levels of glucose. We observed that RSV improved impairment in spatial learning and memory in the Morris water maze test (MWM) and novel object recognition test (ORT) in diabetic rats. RSV reversed the reduced miR-146a-5p and upregulated thioredoxin-interacting protein (TXNIP) and inhibited the diabetes-induced increase in interleukin (IL)-1ß and tumor necrosis factor (TNF)-α levels in vivo and in vitro. RSV also inhibited diabetes-induced endoplasmic reticulum stress (ESR) by reducing ESR-related protein expression in vivo and in vitro. Moreover, inhibition of miR-146a-5p partially abolished the protective effects of RSV in HG-treated primary neurons. Additionally, we used starBase to predict that miR-146a-5p interacts with TXNIP, which we then verified using a luciferase reporter gene assay. We further observed that miR-146a-5p regulates the mRNA and protein expression of TXNIP in vitro, indicating that the miR-146a-5p/TXNIP axis is involved in the regulation of cognitive dysfunction in a rat diabetic model. Collectively, these results demonstrate that RSV plays a neuroprotective role in diabetes-associated cognitive dysfunction at least in part through regulation of the miR-146a-5p/TXNIP axis.

Interleukin-17-educated monocytes suppress cytotoxic T-cell function through B7-H1 in hepatocellular carcinoma patients.

Substantial evidence indicates that inflammation is a critical component of tumor progression. The proinflammatory IL-17-producing cells have recently been detected in tumors, but the effect of IL-17 on antigen-presenting cells in tumors is presently unknown. We recently found that B7-H1(+) macrophages (Mφs) were enriched predominantly in the peritumoral stroma of hepatocellular carcinomas (HCCs). Here, we found a positive correlation between IL-17-producing cells and B7-H1-expressing Mφs in the same area. The B7-H1(+) monocytes/Mφs from HCC tissues expressed significantly more HLA-DR, CD80, and CD86 than B7-H1(-) cells. Accordingly, IL-17 could activate monocytes to express B7-H1 in a dose-dependent manner. Although culture supernatants derived from hepatoma cells also induced B7-H1 expression on monocytes, IL-17 additionally increased hepatoma-mediated B7-H1 expression. Autocrine inflammatory cytokines released from IL-17-activated monocytes stimulated B7-H1 expression. Moreover, these IL-17-exposed monocytes effectively suppressed cytotoxic T-cell immunity in vitro; the effect could be reversed by blocking B7-H1 on those monocytes. Consistent with this, cytotoxic T cells from HCC tissues expressed significant B7-H1 receptor programmed death 1 (PD-1) and exhibited an exhausted phenotype. These data reveal a fine-tuned collaborative action between different stromal cells to counteract T-cell responses in tumors. Such IL-17-mediated immune tolerance should be considered for the rational design of effective immune-based anti-cancer therapies.

Tumor-activated monocytes promote expansion of IL-17-producing CD8+ T cells in hepatocellular carcinoma patients.

The proinflammatory IL-17-producing CD8(+) T cells (Tc17 cells) have recently been detected in tumors, but the nature and regulation of these cells in human tumors are presently unknown. We have recently found that IL-17(+) cells are accumulated in human hepatocellular carcinomas (HCC), where they promote disease progression by fostering angiogenesis. In this study, we showed that Tc17 cells constitute a remarkable portion of IL-17-producing cells in human HCC. Although most circulating Tc17 cells were negative for IFN-gamma, >80% of Tc17 cells in HCC tissues were positive for IFN-gamma, and they were enriched predominantly in invading tumor edge. Most CD68(+) cells located in invading tumor edge exhibited an activated phenotype and, accordingly, the activated monocytes isolated from HCC tissues were significantly superior to those isolated from nontumor tissues in inducing expansion of Tc17 cells in vitro with phenotypic features similar to those isolated from tumors. Compared with IL-17(-)IFN-gamma(+)CD8(+) cells, these IFN-gamma(+)Tc17 cells have significantly higher expression of proinflammatory cytokines (IL-2, IL-22, and TNF-alpha), but reduced expression of granzyme B and perforin. Moreover, we found that tumor-activated monocytes secreted a set of key cytokines (IL-1beta, IL-6, and IL-23) to trigger the proliferation of Tc17 cells. These data reveal an intriguing mechanism in which human Tc17 cells are generated by a fine-tuned collaborative action between different types of immune cells in distinct tumor microenvironments.

Safety and Efficacy of Placenta-Derived Mesenchymal Stem Cell Treatment for Diabetic Patients with Critical Limb Ischemia: A Pilot Study.

AIM: Diabetic foot has become the main cause of non-traumatic amputation. Stem cell therapy, especially mesenchymal stem cells (MSCs), holds a great promise as a therapy for diabetic foot with ischemia limb arterial disease. The aim of this pilot study is to evaluate the safety and efficacy of placenta-derived MSCs (P-MSCs) treatment for diabetic patients with critical limb ischemia (CLI). METHODS: Four eligible diabetic patients with CLI were consecutively enrolled in this pilot study. On the base of the standard-of-care treatment, these patients accepted P-MSCs treatment by intramuscular injection for successive 3 times at an interval of 4 weeks, and the safety and efficacy of placenta-derived MSCs (P-MSCs) treatment were evaluated. RESULTS: There were no serious adverse events during the period of P-MSCs injection and the 24-weeks follow-up period. The clinical ischemic features of patients were improved 24 weeks after P-MSCs treatment. The scores of resting pain and limb coldness significantly decreased, and pain-free walking distance significantly increased from baseline to 24 weeks after P-MSCs therapy. The resting ankle brachial index increased, but no statistically significant difference was found. The findings of magnetic resonance angiography showed the increase of collateral vessel formation in one patient, but there were no significant changes observed in the other patients. CONCLUSIONS: The data in this pilot study indicated that multiple intramuscular P-MSCs injections may be a safe and effective alternative therapy for diabetic patients with CLI, and larger, placebo-controlled, perspective studies are needed to prove these results.

Fast analysis of polybrominated diphenyl ethers in eggs using selective pressurized liquid extraction coupled with online GPC-GC/MS.

This article reports the applicability of online gel permeation chromatography (GPC)-GC/MS for the determination of seven predominant polybrominated diphenyl ethers (PBDEs) in eggs to effectively eliminate matrix interference. Selective pressurized liquid extraction using acidic alumina as a fat retainer was used for cleanup of the PBDEs in eggs. It was selected because of its advantages: simpler operation, minimum time spent on sample handing to get fat-free extracts, and low volume of solvent consumption. After concentration, the extract was directly injected for online GC/MS operated in the negative ion chemical ionization mode with a 15 m capillary column. Recoveries of spiked samples were between 75.1 and 102.0%, with RSDs (n=3) ranging from 3.69-11.47% when spiked at levels of 2 and 20 ng/g, dry mass. The LOD varied from 0.25-34 ng/g, dry mass. The proposed method was proven to be rapid, efficient, and reliable for the trace determination of PBDEs in eggs.

Targeting adenosinergic pathway enhances the anti-tumor efficacy of sorafenib in hepatocellular carcinoma.

BACKGROUND: Sorafenib is the most widely used first-line treatment for patients with advanced hepatocellular carcinoma (HCC), but such treatment provides only limited survival benefits that might be related to the immune status of distinct tumor microenvironments. A fundamental understanding of the distribution and phenotypes of T lymphocytes in tumors will undoubtedly lead to the development of novel immunotherapeutic strategies that could possibly enhance the efficacy of sorafenib treatments. METHODS: Flow cytometry, immunohistochemistry and immunofluorescence analyses were performed to detect the infiltration and distribution of various leukocyte populations, and the expression of different immune checkpoint molecules in fresh HCC tumor tissues. Correlations among indicating genes were calculated in 365 patients with HCC from The Cancer Genome Atlas (TCGA) data set, and the cumulative overall survival time was calculated using the Kaplan-Meier method. Moreover, role of adenosinergic pathway on sorafenib anti-tumor efficacy was investigated using both subcutaneous and orthotopic transplantation tumor model in immune competent C57BL/6 mice. RESULTS: We revealed that levels of CD3+ and CD8+ T cells were significantly downregulated in HCC tumor tissue, so were the infiltration of CD169+ cells (a Mφ subpopulation with T cell activation capacities) and their contact with CD8+ cells in tumor milieus. Moreover, levels of PD-1 and CD39 expression were significantly upregulated in human HCC-infiltrating CD4+ and CD8+ T cells, and CD39+CD8+ T cells exhibited a CD69+PD-1+perforinlowIFNγlow "exhausted" phenotype. Levels of both CD39+ T cells infiltration and adenosine receptor ADORA2B expression in tumor tissues were negatively correlated with overall survival of patients with HCC. Accordingly, mice treated with sorafenib in combination with adenosine A2B receptor blockage reagents exhibited significantly reduced tumor progression compared with control groups. CONCLUSIONS: These results suggest that adenosinergic pathway might represent an applicable target for sorafenib-combined-therapies in human HCC.

Increased intratumoral regulatory T cells are related to intratumoral macrophages and poor prognosis in hepatocellular carcinoma patients.

Immunosuppression mediated by regulatory T cells (Tregs) is a key facilitator of tumor immune evasion, but the source of these Tregs and their contribution to human cancer progression remains unclear. This study investigated the properties of FoxP3(+) Tregs, their prognostic value in patients with hepatocellular carcinoma (HCC) and the underlying mechanisms of FoxP3(+) Treg intratumoral accumulation. In addition to an increased number of circulating FoxP3(+) Tregs, the results also showed that FoxP3(+) Tregs gathered in the tumor site, where they suppressed tissue-derived CD4(+)CD25(-) T-cell activation (p < 0.001), promoting disease progression and poor prognosis in HCC patients (< 0.01). The intratumoral prevalence of FoxP3(+) Tregs was associated with a high density of macrophages (Mvarphi) (p < 0.001). Depletion of tissue Mvarphi thus attenuated the increase of liver FoxP3(+) Treg frequency attributed to in vivo inoculation with hepatoma (p = 0.01), whereas Mvarphi exposed to tumor culture supernatants from hepatoma-derived cell lines increased FoxP3(+) Treg frequency in vitro (p < 0.001). This increase was partially blocked by antiinterleukin-10 antibody (p < 0.01). In conclusion, tumor-associated Mvarphi may trigger a rise of the intratumoral FoxP3(+) Treg population, which in turn may promote HCC progression.

MicroRNA-155 inhibition attenuates endoplasmic reticulum stress-induced cardiomyocyte apoptosis following myocardial infarction via reducing macrophage inflammation.

Endoplasmic reticulum stress (ERS)-induced cardiomyocyte apoptosis plays an important role in the pathological process following myocardial infarction (MI). Macrophages that express microRNA-155 (miR-155) mediate cardiac inflammation, fibrosis, and hypertrophy. Therefore, we investigated if miR-155 regulates ERS-induced cardiomyocyte apoptosis after MI using a mouse model, lipopolysaccharide (LPS)-induced rat bone marrow derived macrophages (BMDMs)and hypoxia-induced neonatal rat cardiomyocytes (NRCMs). In vivo, miR-155 levelswere significantly higher in the MI group compared to the sham group. MI increasedmacrophage infiltration, nuclear factor-κB (NF-κB) activation, ERS induced-apoptosis, and SOCS1 expression, all of which were attenuated by the miR-155 antagomir, with the exception of SOCS1 expression. Additionally, post-MI cardiac dysfunction was significantly improved by miR-155 inhibition. In vitro, LPS upregulated miR-155 expression in BMDMs, and the miR-155 antagomir decreased LPS-induced macrophage inflammation and NF-κB pathway activation, but increased expression of SOCS1. Hypoxia increased NF-κB pathway activation, ERS marker expression, and apoptosis in NRCMs. Interestingly, conditioned medium from LPS-induced macrophages in combination with the miR-155 antagomir decreased, while the miR-155 agomir increased, the hypoxia-induced effects in NRCM's. The miR-155 agomir effects were reversed by inhibiting the NF-κB pathway in cardiomyocytes. Moreover, SOCS1 knockdown in LPS-induced macrophages promoted NF-κB pathway activation and ERS-induced cardiomyocyte apoptosis in the hypoxia-induced NRCMs, but the SOCS1-siRNA-induced effects were markedly decreased by miR-155 antagomir treatment. These data suggest that miR-155 inhibition attenuates ERS-induced cardiomyocyte apoptosis after MI via reducing macrophage inflammation through the SOCS1/NF-κB pathway.

Involvement of histone hypoacetylation in INH-induced rat liver injury.

This study explores the mechanism of histone acetylation under the effect of oxidative stress in rat liver injury induced by isoniazid (INH). Fifty-six adult SD rats were selected and divided randomly into INH groups (48) and control (8). Rats in INH groups were intragastrically injected with 55 mg kg-1 day-1 for 3, 7, 10, 14, 21, and 28 days, and control rats were given an equal volume of distilled water. Pathological changes in liver tissues were observed by HE staining. Western blot analysis was conducted to measure the expression levels of H3k14ac and H4k8ac. The activities of HAT, HDAC and IL-1ß, and TNF-α were detected by ELISA in liver tissues. Real-time RT-PCR analysis was performed to determine the protein expression levels of HAT, HDAC, and IL-1ß and the mRNA expression of TNF-α. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were assayed by biochemical methods in liver tissues. At different time points, the SOD activity decreased, whereas the MDA content significantly increased after 14 days (FSOD = 11.15, FMDA = 7.42, P < 0.01). During this period, the expression of histone acetylated H3K14 and H4K8 acetylation decreased compared with the control group (FH3K14 = 4.18, FH4K8 = 3.87, P < 0.05); by contrast, HDAC1 and HDAC2 showed a high expression level compared with those in the control group (FHDAC1 = 29.13, FHDAC2 = 58.34, P < 0.01). Moreover, the expression of CBP/P300 was lower than that in the control group (FCBP/P300 = 12.18, P = 0.001), and the protein contents of IL-1ß and TNF-α in rat liver tissues were up-regulated (FIL-1ß = 44.88, FTNF-α = 41.56, P < 0.01). These results suggest that histone acetylation is involved in INH-induced rat liver injury. Furthermore, the hypoacetylation of histones H3K14 and H4K8 is negatively correlated with oxidative stress-mediated rat liver injury.

[Plasma mannan?binding lectin and MBL?associated serine protease 2 in patients with hepatocellular carcinoma].

OBJECTIVE: To detect the plasma levels of mannan?binding lectin (MBL) and MBL?associated serine protease?2 (MASP-2) in patients with hepatocellular carcinoma (HCC) and explore their role in the tumorigenesis and progression of HCC. METHODS: The plasma levels of MBL and MASP?2 were detected by enzyme?linked immunosorbent assay in 64 HCC patients and 30 healthy control subjects. The correlation of MBL and MASP?2 with the clinical parameters of HCC patients were analyzed. RESULTS: The plasma levels of MBL (P=0.014) and MASP?2 (P=0.002) were significantly higher in HCC patients than in the healthy controls, but the MBL?to?MASP?2 ratio did not differ significantly between the two groups. In HCC patients, plasma MBL level was positively correlated with vascular invasion (r=0.253, P=0.047) and total bilirubin level (r=0.283, P=0.024). The plasma level of MASP?2 was positively correlated with TNM stage (r=0.276, P=0.027) and negatively correlated with plasma albumin level (r=0.?0.317, P=0.015). ROC curve analysis revealed an area under curve of 0.665 for MBL (P=0.010) and 0.694 for MASP?2 (P=0.003). The sensitivities of MBL and MASP?2 were 50% and 89.1% in the diagnosis of HCC, respectively. CONCLUSION: MBL and MASP?2 are associated with the inflammatory state and disease progression in patients with HCC.

Metformin Use Is Associated with Reduced Incidence and Improved Survival of Endometrial Cancer: A Meta-Analysis.

Studies have suggested that metformin can potentially decrease the incidence of cancer and improve survival outcomes. However, the association between metformin use and the incidence and survival of endometrial cancer (EC) remains controversial. So, a meta-analysis was performed. An electronic search was conducted using PubMed, EMBASE, and Web of Science. The outcome measures were relative risks (RRs) or hazard ratios (HRs) with 95% confidence intervals (CIs) comparing the EC incidence and survival in patients treated with and without metformin. Eleven studies involving 766,926 participants were included in this study. In the pooled analysis of five studies which evaluated the association of metformin use with the incidence of EC, we found that metformin use was associated with a 13% reduction in EC risk among patients with diabetes (RR = 0.87, 95% CI: 0.80-0.95; p = 0.006). In the pooled analysis of six retrospective cohort studies evaluating the effect of metformin on the survival of EC patients, we found that, relative to nonuse, metformin use significantly improved the survival of EC patients (HR = 0.63, 95% CI: 0.45-0.87; p = 0.006). This study showed that metformin use was significantly associated with a decreased incidence of EC in diabetes and a favorable survival outcome of EC patients.

Insulin autoimmune syndrome in a pregnant female: A rare case report.

RATIONALE: Insulin autoimmune syndrome (IAS) is an uncommon disorder characterized by hyperinsulinemic hypoglycemia related to insulin-binding autoantibodies. To the best of our knowledge, we report the first case of a pregnant female with IAS. PATIENT CONCERNS: The 26-year-old patient with Graves disease and 10 weeks pregnant developed IAS after approximately 6 months treatment with methimazole. The patient exhibited recurrent spontaneous hypoglycemia. DIAGNOSES: On evaluation, laboratory findings detected both high fasting insulin (>1000 mIU/L) and insulin autoantibodies. An oral glucose tolerance test showed elevated insulin concentrations with disproportionately elevated C-peptide levels. The imaging study showed nomasslesionsinthepancreas,and the patient was clinically diagnosed with IAS. INTERVENTIONS: The patient had an abortion, discontinued methimazole and switched to oral prednisone (30 mg once daily) and propylth- iouracil (100 mg 3 times daily) for 3 months. OUTCOMES: At the 3-month follow-up visit, hypoglycemic episodes had disappeared and insulin antibody levels were no longer detectable. LESSONS: We have described this case and reviewed the relevant literature concerning diagnosis and treatment of IAS. Importantly, this case indicates that clinicians should view pregnancy as another factor of hypoglycemia in IAS.

A supercritical-CO2 extract of Ganoderma lucidum spores inhibits cholangiocarcinoma cell migration by reversing the epithelial-mesenchymal transition.

BACKGROUND: Ganoderma lucidum (G. lucidum) is an oriental medical mushroom that has been widely used in Asian countries for centuries to prevent and treat different diseases, including cancer. HYPOTHESIS/PURPOSE: The objective of this study was to investigate the effect of A supercritical-CO2 extract of G. lucidum spores on the transforming growth factor beta 1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) of cholangiocarcinoma cells. STUDY DESIGN: This was an in vitro study with human cholangiocarcinoma TFK-1 cells treated with varying concentrations of G. lucidum. METHODS: A supercritical-CO2 extract of G. lucidum spores (GLE) was obtained from completely sporoderm-broken germinating G. lucidum spores by supercritical fluid carbon dioxide (SCF-CO2) extraction. GLE pre-incubated with human cholangiocarcinoma TFK-1 cells prior to TGF-ß1 treatment (2ng/ml) for 48h. Changes in EMT markers were analyzed by western blotting and immunofluorescence. The formation of F-actin stress fibers was assessed via immunostaining with phalloidin and examined using confocal microscopy. Additionally, the effect of the GLE on TGF-ß1-induced migration was investigated by a Boyden chamber assay. RESULTS: TGF-ß1-induced reduction in E-cadherin expression was associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin and Fibronectin were evident in predominantly elongated fibroblast-like cells. The GLE suppressed the TGF-ß1-induced morphological changes and the changes in cadherin expression, and also inhibited the formation of F-actin stress fibers, which are a hallmark of EMT. The GLE also inhibited TGF-ß1-induced migration of TFK-1 cells. CONCLUSION: Our findings provide new evidence that GLE suppress cholangiocarcinoma migration in vitro through inhibition of TGF-ß1-induced EMT. The GLE may be clinically applied in the prevention and/or treatment of cancer metastasis.

Polymorphism 2184A/G in the AGER gene is not associated with diabetic retinopathy in Han Chinese patients with type 2 diabetes.

OBJECTIVE: To assess the association between the 2184A/G polymorphism in the advanced glycosylation end product-specific receptor (AGER) gene and the susceptibility to diabetic retinopathy (DR) in Han Chinese patients with type 2 diabetes mellitus (T2DM). METHODS: This cross-sectional genotyping study included patients with T2DM with and without DR. Genotype and allele frequencies of the 2184A/G polymorphism were detected using polymerase chain reaction-restriction fragment-length polymorphism analysis. RESULTS: This study included 943 patients with T2DM (285 with DR [DR group] and 658 without DR [NDR group]). There were no significant differences in age, sex, body mass index, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, diastolic blood pressure, glycosylated haemoglobin, fasting blood glucose, postprandial 2-hour blood glucose, and triglycerides between the two groups. The duration of T2DM and systolic blood pressure were significantly increased in the DR group compared with the NDR group. No significant differences were found in allele (A and G) and genotype (AA, AG and GG) frequencies of the 2184A/G polymorphism between the two groups. CONCLUSION: The 2184A/G polymorphism in the AGER gene is not associated with DR in Han Chinese patients with T2DM.

Effects of 90-Day Feeding of Transgenic Maize BT799 on the Reproductive System in Male Wistar Rats.

BT799 is a genetically modified (GM) maize plant that expresses the Cry1Ac gene from Bacillus thuringiensis (Bt). The Cry1Ac gene was introduced into maize line Zhen58 to encode the Bt crystal protein and thus produce insect-resistant maize BT799. Expression of Bt protein in planta confers resistance to Lepidopteran pests and corn rootworms. The present study was designed to investigate any potential effects of BT799 on the reproductive system of male rats and evaluate the nutritional value of diets containing BT799 maize grain in a 90-day subchronic rodent feeding study. Male Wistar rats were fed with diets containing BT799 maize flours or made from its near isogenic control (Zhen58) at a concentration of 84.7%, nutritionally equal to the standard AIN-93G diet. Another blank control group of male rats were treated with commercial AIN-93G diet. No significant differences in body weight, hematology and serum chemistry results were observed between rats fed with the diets containing transgenic BT799, Zhen58 and the control in this 13-week feeding study. Results of serum hormone levels, sperm parameters and relative organ/body weights indicated no treatment-related side effects on the reproductive system of male rats. In addition, no diet-related changes were found in necropsy and histopathology examinations. Based on results of the current study, we did not find any differences in the parameters tested in our study of the reproductive system of male rats between BT799 and Zhen58 or the control.