NEK2 Promotes Hepatoma Metastasis and Serves as Biomarker for High Recurrence Risk after Hepatic Resection.

INTRODUCTION AND AIM: Developing reliable biomarkers for hepatocellular carcinoma (HCC) patients who are at a high risk of recurrence after curative hepatic resection is very important for determining subsequent therapeutic strategies. We investigated the role of the cell cycle factor NIMA-related kinase 2 (NEK2) in HCC progression in hepatoma cells and post-surgery patients. MATERIAL AND METHODS: The effects of NEK2 on proliferation, invasion and migration of hepatoma HuH7 and SK-Hep1 cells were evaluated. In a post-surgery HCC cohort (N = 97), the Nek2 induction levels in the tumors were examined with real-time RT-PCR analysis, and the results were analyzed for their correlations with recurrence. RESULTS: NEK2 promoted G1 to S phase cell cycle progression by causing increases in cyclin D1 and AKT phosphorylation and decreases in the cyclin-dependent kinase inhibitor p27, indicating that NEK2 plays an important role during interphase in addition to its previously identified role in M phase. NEK2 also enhanced the proliferation, migration and invasion of hepatoma cells and regulated the expression of E-cadherin and MMP9. The Nek2 mRNA levels in the tumors were highly correlated with recurrence rates in the post-surgery HCC patients. Combined evaluation of the tumor AJCC stage and the Nek2 level can serve as a reliable method for predicting the relative risk of HCC recurrence in these patients. CONCLUSIONS: NEK2 plays a significant role in cell cycle progression in the inter- and M-phases. NEK2 enhances HCC metastasis and is correlated with recurrence and thus can potentially serve a promising high-risk biomarker for HCC.

Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis.

Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGHs) harbouring the HBV pre-S2 mutant large surface protein (LHBS) have been recognized as a morphologically distinct hallmark of HCC in the advanced stages of chronic HBV infection. Considering its preneoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin α1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin α/ß-associated nuclear pore complex (NPC). By interacting with importin α1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transport of an essential DNA repair and recombination factor, Nijmegen breakage syndrome 1 (NBS1), upon DNA damage, thereby delaying the formation of nuclear foci at the sites of DNA double-strand breaks (DSBs). Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability, indicated by increased global gene copy number variations (CNVs), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA DSBs representing significant genomic instability. In conclusion, type II GGHs harbouring HBV pre-S2 mutant oncoprotein represent a high-risk marker for the loss of genome integrity in chronic HBV carriers and explain the complex chromosome changes in HCCs. Mouse array CGH raw data: GEO Accession No. GSE61378 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61378).

Zinc-dependent interaction between JAB1 and pre-S2 mutant large surface antigen of hepatitis B virus and its implications for viral hepatocarcinogenesis.

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The pre-S2 mutant large HBV surface protein (Δ2 LHBS), which contains an in-frame deletion of approximately 17 amino acids in LHBS, is highly associated with risks and prognoses of HBV-induced HCC. It was previously reported that Δ2 LHBS interacts with the Jun activation domain-binding protein 1 (JAB1), a zinc metalloprotease. This promotes the degradation of the cell cycle regulator p27(Kip1) and is believed to be the major mechanism for Δ2 LHBS-induced HCC. In this study, it was found that the interaction between JAB1 and Δ2 LHBS is facilitated by divalent metal Zn(2+) ions. The binding of JAB1 to Δ2 LHBS requires the JAB1/CSN5 MPN metalloenzyme (JAMM) motif and residue H138 that binds to Zn(2+) ions in JAB1. Isothermal titration calorimetry showed that Δ2 LHBS binds directly to Zn(2+) ions in a two-site binding mode. Residues H71 and H116 in Δ2 LHBS, which also contact Zn(2+) ions, are also indispensable for Δ2 LHBS-mediated p27(Kip1) degradation in human HuH7 cells. These results suggest that developing drugs that interrupt interactions between Δ2 LHBS and JAB1 can be used to mitigate Δ2 LHBS-associated risks for HCC.

Treatment of myopic choroidal neovascularization with posterior sub-Tenon's bevacizumab injection (Avastin ®).

The aim of this study was to report the successful treatment of choroidal neovascularization (CNV) in pathologic myopia (PM) with a posterior sub-Tenon bevacizumab (PSTB; Avastin(®)) injection. The study was a prospective case series including nine eyes of eight patients with PM and CNV. All nine eyes were injected with PSTB (12.5 mg/0.5 ml). Treatment effectiveness was evaluated with optical coherence tomography (OCT). If intraretinal edema or subretinal fluid were detected, injections were repeated after 2 weeks. The main outcome measures were logMAR best-corrected visual acuity (BCVA) and central foveal thickness. The mean follow-up time was 77.56 weeks. BCVA improved by a mean of -0.38 logMAR (>3 lines). The average reduction in absolute central foveal thickness was 25.67 µm. OCT revealed marked CNV volume reduction and fluid-free status in seven eyes. The fluid-free status remained for ≥ 1 year in these eyes. Fluorescein angiography revealed CNV resolution in three eyes. Corneal stromal penetration of subconjunctival bevacizumab has been demonstrated in animal studies. PSTB may be an equally effective, yet less invasive alternative for the treatment of myopic CNV.

A novel natural Nrf2 activator with PPARγ-agonist (monascin) attenuates the toxicity of methylglyoxal and hyperglycemia.

Methylglyoxal (MG) is a toxic-glucose metabolite and a major precursor of advanced glycation endproducts (AGEs). MG has been reported to result in inflammation by activating receptor for AGEs (RAGE). We recently found that Monascus-fermented metabolite monascin acts as a novel natural peroxisome proliferator-activated receptor-γ (PPARγ) agonist that improves insulin sensitivity. We investigated the metabolic, biochemical, and molecular abnormalities characteristic of type 2 diabetes in MG-treated Wistar rats treated with oral administration of monascin or rosiglitazone. Monascin (a novel PPARγ agonist) activated nuclear factor-erythroid 2-related factor 2 (Nrf2) and down-regulated hyperinsulinmia in oral glucose tolerance test (OGTT). Monascin was able to elevate glyoxalase-1 expression via activation of hepatic Nrf2, hence, resulting in MG metabolism to d-lactic acid and protected from AGEs production in MG-treated rats. Rosiglitazone did not activate Nrf2 nor glyoxalase expression to lower serum and hepatic AGEs levels. Monascin acts as a novel natural Nrf2 activator with PPARγ-agonist activity were confirmed by Nrf2 and PPARγ reporter assays in Hep G2 cells. These findings suggest that monascin acts as an anti-diabetic and anti-oxidative stress agent to a greater degree than rosiglitazone and thus may have therapeutic potential for the prevention of diabetes.

Characterization of the role of protein-cysteine residues in the binding with sodium arsenite.

To better characterize the interaction of protein-cysteines with sodium arsenite, arsenic-binding proteins were identified from the arsenic-resistant Chinese hamster ovary cell line SA7 using a p-aminophenylarsine oxide (PAO)-agarose matrix in combination with proteomic techniques. Twenty of the isolated arsenic-binding proteins were further peptide-mapped by MALDI-Q-TOF-MS. The binding capacity of PAO-agarose-retained proteins was then verified by re-applying Escherichia coli overexpressed recombinant proteins with various numbers of cysteine residues onto the PAO-agarose matrix. The results showed that recombinant heat shock protein 27 (HSP27, with one cysteine residue), reticulocalbin-3 (RCN3, with no cysteine residue), galectin-1 (GAL1, with six cysteine residues), but not peroxiredoxin 6 (Prdx6, with one cysteine residue but not retained by the PAO-agarose matrix), were bound to the PAO-agarose matrix. The six free cysteine residues in GAL1 were individually or double-mutated to alanine by means of site-directed mutagenesis and subjected to CD and ICP-MS analysis. The binding capacity of GAL1 for sodium arsenite was significantly attenuated in C16A, C88A and all double mutant clones. Taken together, our current data suggest that the cysteine residues in GAL1 may play a critical role in the binding of arsenic, but that in the case of RCN3 and Prdx6, this interaction may be mediated by other factors.

Antigenic Cross-Reactivity Between SARS-CoV-2 S1-RBD and Its Receptor ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein-protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2.

Extraction of magnetic circular dichroism effects from blended mixture of magnetic linear dichroism signals in the cobalt/Scotch tape system.

Circular dichroism (CD) signals revealed in some materials may arise from different origins during measurements. Magnetic field dependent CD (MCD) emanating from the spin-polarized band provides direct insight into the spin-spin interband transitions in magnetic materials. On the contrary, natural CD effects which are artefactual signals resulting from the linear polarization (LP) components during the polarization modulation with a photo-elastic modulator in anisotropic polymer systems were usually observed. There is no simple method to reliably distinguish MCD effect due to spin polarized band structures from natural CD effect, which limits our understanding of the magnetic material/polymer hybrid structures. This paper aims to introduce a general strategy of averaging out the magnetic linear dichroism (MLD) contributions due to the anisotropic structure and disentangling MCD signal(s) from natural MCD signal(s). We demonstrate the effectiveness of separating MCD from natural MCD using rotational MCD measurement and presented the results of a sample with Co thin film on polymer Scotch tape (unplasticized polyvinyl chloride) glued on a quartz substrate. We demonstrate that the proposed method can be used as an effective tool in disentangling MCD and natural MCD effects, and it opens prospects to study the magnetic material /polymer hybrid systems.

[Expression and significance of mucosal beta-defensin-2, TNFalpha and IL-1beta in ulcerative colitis].

OBJECTIVE: To investigate the expression and significance of human beta-defensin-2 (HBD2), TNFalpha and IL-1beta in ulcerative colitis (UC). METHODS: Thirty-five patients with active UC diagnosed by the department of gastroenterology in West China Hospital were included in this study. Ulcerative colitis disease activity index (UCAI) was assessed and the pathological grades of UC were classified. Immunohistochemistry assay and real-time quantitative PCR were used for the expression of HBD2, TNFalpha, IL-1beta in colonic mucosa of UC. RESULTS: Among the 35 patients with UC, 10 cases were mild, 13 moderate and 12 severe. Of the 35 cases, there were 11 with grade I, 13 grade II and 11 grade III lesion according to Truelove criteria. The score of UCAI had positive correlation with pathological grading (r = 0.890, P < 0.01). The expressions of HBD2, TNFalpha, IL-1beta in colonic mucosa of UC with immunohistochemistry and real-time quantitative PCR were significantly higher than those in healthy control (P < 0.05); the expressions increased gradually with the severity of pathological grade and there was a higher expression of them in inflamed area than in non-inflamed (P < 0.05). A good positive correlation was also found between HBD2 and other inflammatory cytokines. CONCLUSIONS: It is shown that there is a higher expression of HBD2 in colonic mucosa as compared with healthy control, a higher expression of it in inflamed area than in non-inflamed area and a positive correlation of expression between HBD2 and pro-inflammatory cytokines such as TNFalpha and IL-1beta, implying that HBD2 and pro-inflammatory cytokines are interdependent and interactive playing an important role in magnifying and aggravating inflammatory injury in UC.

Interface-induced spin Hall magnetoresistance enhancement in Pt-based tri-layer structure.

In this study, we integrated bilayer structure of covered Pt on nickel zinc ferrite (NZFO) and CoFe/Pt/NZFO tri-layer structure by pulsed laser deposition system for a spin Hall magnetoresistance (SMR) study. In the bilayer structure, the angular-dependent magnetoresistance (MR) results indicate that Pt/NZFO has a well-defined SMR behavior. Moreover, the spin Hall angle and the spin diffusion length, which were 0.0648 and 1.31 nm, respectively, can be fitted by changing the Pt thickness in the longitudinal SMR function. Particularly, the MR ratio of the bilayer structure (Pt/NZFO) has the highest changing ratio (about 0.135%), compared to the prototype structure Pt/Y3Fe5O12 (YIG) because the NZFO has higher magnetization. Meanwhile, the tri-layer samples (CoFe/Pt/NZFO) indicate that the MR behavior is related with CoFe thickness as revealed in angular-dependent MR measurement. Additionally, comparison between the tri-layer structure with Pt/NZFO and CoFe/Pt bilayer systems suggests that the SMR ratio can be enhanced by more than 70%, indicating that additional spin current should be injected into Pt layer.

Suppression of HSP27 increases the anti­tumor effects of quercetin in human leukemia U937 cells.

Quercetin, a natural flavonoid, inhibits the growth of leukemia cells and induces apoptosis. Heat shock protein 27 (HSP27) has been reported to promote the development of leukemia by protecting tumor cells from apoptosis through various mechanisms. The present study investigated the effects of small hairpin (sh)RNA-mediated HSP27 knockdown on the anti­cancer effects of quercetin in U937 human leukemia cells. Cells were transfected with recombinant lentiviral vector pCMV­G­NR­U6­shHSP27 (shHSP27), which expressed shRNA specifically targeting the HSP27 gene, alone or in combination with quercetin. The results showed that shHSP27 and quercetin synergistically inhibited U937 cell proliferation and induced apoptosis by decreasing the Bcl2-to-Bax ratio. Furthermore, this combined treatment significantly suppressed the infiltration of tumor cells and the expression of angiogenesis­associated proteins HIF1α and VEGF. Compared with shHSP27 or quercetin alone, shHSP27 plus quercetin markedly decreased the protein expression of cyclinD1 and thus blocked the cell cycle at G1 phase. The Notch/AKT/mTOR signaling pathway is important in tumor aggressiveness; quercetin plus shHSP27 significantly decreased Notch 1 expression and the phosphorylation levels of the downstream signaling proteins AKT and mTOR. The inhibitory effects of quercetin plus shHSP27 on this pathway may thus have been responsible for the cell cycle arrest, inhibition of proliferations and infiltration as well as enhancement of apoptosis. Therefore, these findings collectively suggested that suppression of HSP27 expression amplified the anti­cancer effects of quercetin in U937 human leukemia cells, and that quercetin in combination with shHSP27 represents a promising therapeutic strategy for human leukemia.

Monascus secondary metabolites monascin and ankaflavin inhibit activation of RBL-2H3 cells.

Monascus-fermented products have been used as dietary food and traditional medicine due to their beneficial effects on circulation and digestive systems in Asia for thousands of years. Besides, monascin and ankaflavin, secondary metabolites from Monascus-fermented products, have proven anti-inflammatory and immunomodulatory effects. In previous research, monascin and ankaflavin ameliorated ovalbumin-induced airway allergic reaction often used as a type I allergy asthma model. Additionally, mast cells play critical roles in type I allergy. Therefore, RBL-2H3 cells were used as the mast cell model to determine whether the improving effects on asthma of monascin and ankaflavin came from influencing mast cells. PMA and ionomycin are common activators of mast cells because they stimulate the main signaling molecules during mast cell activation. Forty micromolar monascin and ankaflavin inhibited PMA/ionomycin-induced mast cell degranulation and TNF-α secretion through suppressing the phosphorylation of PKC and MAPK family ERK, JNK, and p38. Consequently, monascin and ankaflavin affected the activation of mast cells and may have the potential to improve type I allergy.

Increased Standardised Incidence Ratio of Malignant Pleural Mesothelioma in Taiwanese Asbestos Workers: A 29-Year Retrospective Cohort Study.

OBJECTIVE: This paper aimed to determine the standardised incidence ratio (SIR) of malignant pleural mesothelioma (MPM) in workers exposed to asbestos in Taiwan. METHODS: All workers employed in asbestos-related factories and registered by the Bureau of Labour Insurance between 1 March, 1950, and 31 December, 1989, were included in the study and were followed from 1 January, 1980, through 31 December, 2009. Incident cases of all cancers, including MPM (ICD-9 code: 163), were obtained from the Taiwan Cancer Registry. SIRs were calculated based on comparison with the incidence rate of the general population of Taiwan and adjusted for age, calendar period, sex, and duration of employment. RESULTS: The highest SIR of MPM was found for male workers first employed before 1979, with a time since first employment more than 30 years (SIR 4.52, 95% CI: 2.25-8.09). After consideration of duration of employment, the SIR for male MPM was 5.78 (95% CI: 1.19-16.89) for the workers employed for more than 20 years in asbestos-related factories. CONCLUSIONS: This study corroborates the association between occupational asbestos exposure and MPM. The highest risk of MPM was found among male asbestos workers employed before 1979 and working for more than 20 years in asbestos-related factories.

The effect of galectin 1 on 3T3 cell proliferation on chitosan membranes.

Galectin-1 (GAL1), a beta-galactoside-binding protein, functions in cell adhesion, development, and growth regulation. A number of studies suggest that GAL1 play an important role in enhancing cell adhesion to extracellular matrix and inducing cell proliferation. Chitosan is a derivative of chitin extracted from lobsters, crabs and shrimps' exoskeletons. In clinical medicine, chitosan membrane had been used as a semi-permeable biological dressing. Although chitosan membranes show no cytotoxicity, some cell types (e.g. 3T3 cells) fail to attach and proliferate on their surface. In these studies, we show that over-expression of GAL1 does not enhance 3T3 cell proliferation on chitosan membranes. However, coating the chitosan membrane with recombinant GAL1 proteins significantly expedites 3T3 cells proliferation. The enhanced cell growth was inhibited by thiodigalactoside (TDG, a potent inhibitor of beta-galactoside binding) and GAL1 monoclonal antibodies, suggesting GAL1's specific effect on the proliferation of 3T3 cells upon chitosan membranes. Moreover, immunoblotting detected a markedly suppressed tyrosine phosphorylation in several proteins on 3T3 cell growths upon GAL1-coated chitosan membrane. Pretreating the cells with sodium fluoride (NaF, a phosphatase inhibitor) inhibits the attachment and proliferation of 3T3 cells. These findings support a proposed role for altered levels of protein phosphorylation in GAL1-mediated cell attachment and proliferation on chitosan membranes.

Effects of monascin on anti-inflammation mediated by Nrf2 activation in advanced glycation end product-treated THP-1 monocytes and methylglyoxal-treated wistar rats.

Hyperglycemia is associated with advanced glycation end products (AGEs). This study was designed to evaluate the inhibitory effects of monascin on receptor for advanced glycation end product (RAGE) signal and THP-1 monocyte inflammation after treatment with S100b, a specific ligand of RAGE. Monascin inhibited cytokine production by S100b-treated THP-1 monocytes via up-regulation of nuclear factor-erythroid 2-related factor-2 (Nrf2) and alleviated p47phox translocation to the membrane. Methylglyoxal (MG, 600 mg/kg bw) was used to induce diabetes in Wistar rats. Inhibitions of RAGE and p47phox by monascin were confirmed by peripheral blood mononuclear cells (PBMCs) of MG-induced rats. Silymarin (SM) was used as a positive control group. It was found that monascin promoted heme oxygenase-1 (HO-1) expression mediated by Nrf2. Suppressions of AGEs, tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-ß) in serum of MG-induced rats were attenuated in the monascin administration group treated with retinoic acid (RA). RA treatment resulted in Nrf2 inactivation by increasing RA receptor-α (RARα) activity, suggesting that RA acts as an inhibitor of Nrf2. The results showed that monascin exerted anti-inflammatory and antioxidative effects mediated by Nrf2 to prevent the development of diseases such as type 2 diabetes caused by inflammation.

Inhibition of heat shock protein (Hsp) 27 potentiates the suppressive effect of Hsp90 inhibitors in targeting breast cancer stem-like cells.

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44 + cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. In the present study, we discovered that the expression of Hsp90α was increased in ALDH + human breast cancer cells. Geldanamycin (GA), a Hsp90 inhibitor, could suppress ALDH + breast cancer cells in a dose dependent manner. We are interesting in the insufficiently inhibitory effect of low dose GA treatment. It was correlated with the upregulation of Hsp27 and Hsp70. By co-treatment with HSP inhibitors, quercetin or KNK437 potentiated BCSCs, which determined with ALDH+ population or mammosphere cells, toward GA inhibition, as well as anti-proliferation and anti-migration effects of GA. With siRNA mediated gene silencing, we found that knockdown of Hsp27 could mimic the effect of HSP inhibitors to potentiate the BCSC targeting effect of GA. In conclusion, combination of HSP inhibitors with Hsp90 inhibitors could serve as a potential solution to prevent the drug resistance and avoid the toxicity of high dose of Hsp90 inhibitors in clinical application. Furthermore, Hsp27 may play a role in chemoresistant character of BCSCs.

Ankaflavin: a natural novel PPARγ agonist upregulates Nrf2 to attenuate methylglyoxal-induced diabetes in vivo.

Ankaflavin (AK) is an active compound having anti-inflammatory, anti-cancer, antiatherosclerotic, and hypolipidemic effects. We have previously reported that AK acts as an antioxidant and antidiabetic drug; however, the mechanism by which AK prevents diabetes remains unknown. Hyperglycemia is associated with protein glycation, which produces advanced glycation end-products (AGEs). Methylglyoxal (MG)-a metabolite of carbohydrates-is believed to cause insulin resistance by inducing inflammation and pancreas damage. In this work, diabetes was induced in Wistar rats (4 weeks of age) by treating them with MG (600 mg/kg bw) for 4 weeks. We observed that AK (10mg/kg bw) exerted peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity, thereby enhancing insulin sensitivity (as indicated by hepatic GLUT2 translocation, PTP1B suppression, and glucose uptake) by downregulating blood glucose and upregulating pancreatic and duodenal homeobox-1 and Maf-A expression and increasing insulin production in MG-induced rats. However, these effects were abolished by the administration of GW9662 (PPARγ antagonist), but the expression of hepatic heme oxygenase-1 (HO-1) and glutamate-cysteine ligase (GCL) was not suppressed in MG-induced rats. Therefore, the nuclear factor erythroid-related factor-2 (Nrf2) activation was investigated. AK did not affect hepatic Nrf2 mRNA or protein expression but significantly increased Nrf2 phosphorylation (serine 40), which was accompanied by increased transcriptional activation of hepatic HO-1 and GCL. These data indicated that AK protected rats from oxidative stress resulting from MG-induced insulin resistance. In contrast, these effects were not detected when the rats were treated with the antidiabetic drug rosiglitazone (10mg/kg bw). Moreover, we found that AK did not inhibit the generation of AGEs in vitro; however, the glutathione (GSH) levels in liver and pancreas of MG-induced rats were elevated in rats administered AK. Therefore, we believe that GSH may lower the MG level, which attenuates the formation of AGEs in the serum, kidney, liver, and pancreas of MG-induced rats. We also found that AK treatment reduced the production of inflammatory factors, such as tumor necrosis factor-α and interleukin-1ß. Taken together, the results of our mechanistic study of MG-induced rats suggest that the protective effects of AK against diabetes are mediated by the upregulation of the signaling pathway of Nrf2, which enhances antioxidant activity and serves as a PPARγ agonist to enhance insulin sensitivity.

The down-regulation of galectin-1 expression is a specific biomarker of arsenic toxicity.

Galectin-1 (GAL1) is known as a ß-galactoside-binding protein that also can bind with arsenic to regulate cell functions. Using RNA interference technique, we investigated the possible mechanism involved in GAL1 modulation of arsenite-inhibited cell survival in 3T3 fibroblast and KB oral cancer cells. GAL1 gene knockdown significantly attenuated sodium arsenite (NaAsO(2)) and arsenic trioxide (As(2)O(3)) inhibition of cell survival. However, GAL1 gene knockdown did not alter the inhibition of cell survival by antimony chloride, cadmium chloride or nickel sulfate. These results suggested the GAL1 selectively affects particular types of heavy metal elements. Flow cytometric analysis indicated GAL1 gene knockdown also suppressed As(III)-stimulated levels of sub-G1 and G2/M growth arrest in both cells. Moreover, atomic absorption spectrophotometric results showed that GAL1 gene knockdown reduced the total arsenic accumulation of both cells after the NaAsO(2) and As(2)O(3) treatment. These results suggested that GAL1 gene knockdown mediates the apoptotic effects of arsenic in 3T3 and KB cells via regulation of the cellular arsenic levels. We propose that down-regulation of GAL1 expression may be a useful and specific biomarker in assessing the toxicity of arsenic exposure.

Characterization of the interaction of galectin-1 with sodium arsenite.

We previously showed that galectin-1 (GAL1) is an arsenic-binding protein. In the current study, we further characterize the interaction of GAL1 with sodium arsenite (As(III)). The GALl-As(III) complex was prepared from the cell extracts of GAL1-transfected Escherichia coli (E. coli) that were pretreated with As(III). The results of the circular dichroism (CD) spectrum of GAL1-As(III) exhibited a negative signal at around 205-210 nm, whereas that of GAL1 showed a negative signal at around 215-220 nm. This shift in the CD spectrum is indicative of a substantial change in the secondary structure arising from the binding of As(III) to the GAL1 protein. The UV absorptive spectrum of the GAL1-As(III) complex was significantly lower than that of GAL1 itself. A mobility shift binding assay showed that the GAL1-As(III) complex migrated closer than GAL1 toward the anode. Capillary electrophoretic analysis also showed that As(III) binding decreased the mobility of GAL1. These results further confirmed the structural change of the GAL1 complex with As(III). Furthermore, isothermal titration microcalometric studies showed that As(III) titration into the GAL1 protein solution was an endothermic process with absorption enthalpy (DeltaH(abs)) around 8-10 kJ/mol As(III). The affinity constant (K(d)) of As(III) toward GAL1 was around 8.239 +/- 2.627 microM as estimated by tryptophan (Trp) fluorescence quenching. However, the binding of As(III) did not significantly affect the biological activity of GAL1, since the GAL1-As(III) complex only partially lost its lectin activity. In addition, we show that GAL1-transfected KB cells accumulated more arsenic than did the parental cells. Taken together, these results suggest that GAL1 might serve as a target protein of As(III) in vivo, and the binding of GAL1 with As(III) could interfere with the excretion of As(III).