[Inhibitory effect of artesunate on bone destruction in rheumatoid arthritis: an exploration based on AhR/ARNT/NQO1 signaling pathway].

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 µmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 µmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.

Antiproliferative effect of 2-Hydroxy-6-tridecylbenzoic acid from ginkgo biloba sarcotestas through the aryl hydrocarbon receptor pathway in triple-negative breast cancer cells.

This study aims to isolate the potential antiproliferative and cytotoxic compounds from ginkgo biloba sarcotestas (GBS) and investigates the underlying mechanism in human MDA-MB-231 and mouse 4T-1 triple-negative breast cancer cells. Our results showed that 2-Hydroxy-6-tridecylbenzoic acid was isolated by cytotoxicity-guided fractionation where different fractions were assessed using MTT assay against MDA-MB-231 and 4T-1 cells. Colony formation assay showed that 2-Hydroxy-6-tridecylbenzoic acid significantly inhibited cell proliferation. The inhibition was associated with the enhancement of cytochrome P450 (CYP) 1B1 expression in a dose- and time-dependent manner and no significant change of CYP1A1 expression by qPCR and Western blot assays in MDA-MB-231 and 4T-1 cells. The mechanism was further demonstrated by the activation of aryl hydrocarbon receptor (AhR) pathway with the upregulation of AhR, AhR nuclear translocator (ARNT) and AhR-dependent xenobiotic response elements (XRE) activity. These findings may have implications for development of anticancer agents containing 2-Hydroxy-6-tridecylbenzoic acid as functional additives.

Effects of sequential exposure to water accommodated fraction of crude oil and chlorpyrifos on molecular and biochemical biomarkers in rainbow trout.

Fish can be simultaneously or sequentially exposed to various kinds of pollutants, resulting in combined effects. Polycyclic aromatic hydrocarbons induce cytochrome P450 monooxygenase 1A (CYP1A) expression, which catalyzes the conversion of the organophosphorus insecticide chlorpyrifos (CPF) into its most active derivative, CPF-oxon. CPF-oxon inhibits CYP1A and other enzymes, including carboxylesterases (CEs) and acetylcholinesterase (AChE). We studied the effects of an in vivo exposure to crude oil water accommodated fraction (WAF) followed by an ex vivo exposure of liver tissue to CPF on the expression of Cyp1a, AhR and ARNT mRNA, CYP1A protein and on the activity of biomarker enzymes in the rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were exposed to WAF (62 µg L-1 TPH) for 48 h. Then, liver was dissected out, sliced and exposed to 20 µg L-1 CPF ex vivo for 1 h. Liver tissue was analyzed for mRNA and protein expression and for CEs, AChE, glutathione S-transferase (GST) and CYP1A (EROD) activity. WAF induced Cyp1a mRNA and CYP1A protein expression by 10-fold and 2.5-8.3-fold, respectively, with no effect of CPF. WAF induced AhR expression significantly (4-fold) in control but not in CPF treated liver tissue. ARNT mRNA expression was significantly lowered (5-fold) by WAF. CPF significantly reduced liver EROD activity, independently of WAF pre-treatment. CEs activity was significantly inhibited in an additive manner following in vivo exposure to WAF (42%) and ex vivo exposure to CPF (19%). CPF exposure inhibited AChE activity (37%) and increased GST activity (42%).

Downregulation of HIF complex in the hypothalamus exacerbates diet-induced obesity.

Hypothalamic hypoxia-inducible factor-1 (HIF-1) can regulate whole-body energy homeostasis in response to changes in blood glucose, suggesting that it acts as a sensor for systemic energy stores. Here, we hypothesized that hypothalamic HIF-1 could be affected by diet-induced obesity (DIO). We used eight-week old, male C57Bl6 mice, fed normal chow diet or with high fat diet for 1, 3, 7, 14 and 28 days. The expression of HIF-1alpha and HIF-1beta was measured by PCR and western blotting and its hypothalamic distribution was evaluated by fluorescence microscopy. Inhibition of HIF-1beta in arcuate nucleus of hypothalamus was performed using stereotaxic injection of shRNA lentiviral particles and animals were grouped under normal chow diet or high fat diet for 14 days. Using bioinformatics, we show that in humans, the levels of HIF-1 transcripts are directly correlated with those of hypothalamic transcripts for proteins involved in inflammation, regulation of apoptosis, autophagy, and the ubiquitin/proteasome system; furthermore, in rodents, hypothalamic HIF-1 expression is directly correlated with the phenotype of increased energy expenditure. In mice, DIO was accompanied by increased HIF-1 expression. The inhibition of hypothalamic HIF-1 by injection of an shRNA resulted in a further increase in body mass, a decreased basal metabolic rate, increased hypothalamic inflammation, and glucose intolerance. Thus, hypothalamic HIF-1 is increased during DIO, and its inhibition worsens the obesity-associated metabolic phenotype. Thus, hypothalamic HIF-1 emerges as a target for therapeutic intervention against obesity.

Effect of treatment with baicalein on the intracerebral tumor growth and survival of orthotopic glioma models.

Baicalein, a widely used Chinese herbal medicine, has been proved as a promising chemopreventive compound for many cancers. The aim of this work was to assess the anti-tumor effect of baicalein in the orthotopic glioma models. It was found that treatment of mice with U87 gliomas with baicalein (20 and 40 mg/kg/day, i.p.) significantly inhibited the intracerebral tumor growth and prolonged the survival. Furthermore, treatment with baicalein suppressed cell proliferation, promoted apoptosis, and arrested cell cycle in U87 gliomas. In addition, treatment with baicalein reduced tumor permeability, attenuated edema of tumors and brains, and improved tight junctions in gliomas. Finally, treatment with baicalein reduced the expression of HIF-1α, VEGF, and VEGFR2 in U87 gliomas. In addition, treatment with baicalein also markedly suppressed tumor growth and prolonged the survival of rats with 9L gliomas. In conclusion, baicalein has an obvious anti-tumor activity in the orthotopic glioma models. Our results suggested that treatment with baicalein might be an effective therapy for recurrent malignant brain cancers through suppressing tumor growth and alleviating edema.

O-glycosylation regulates polarized secretion by modulating Tango1 stability.

Polarized secretion is crucial in many tissues. The conserved protein modification, O-glycosylation, plays a role in regulating secretion. However, the mechanisms by which this occurs are unknown. Here, we demonstrate that an O-glycosyltransferase functions as a novel regulator of secretion and secretory vesicle formation in vivo by glycosylating the essential Golgi/endoplasmic reticulum protein, Tango1 (Transport and Golgi organization 1), and conferring protection from furin-mediated proteolysis. Loss of the O-glycosyltransferase PGANT4 resulted in Tango1 cleavage, loss of secretory granules, and disrupted apical secretion. The secretory defects seen upon loss of pgant4 could be rescued either by overexpression of Tango1 or by knockdown of a specific furin (Dfur2) in vivo. Our studies elucidate a novel regulatory mechanism whereby secretion is influenced by the yin/yang of O-glycosylation and proteolytic cleavage. Moreover, our data have broader implications for the potential treatment of diseases resulting from the loss of O-glycosylation by modulating the activity of specific proteases.

Increased metastatic potential of residual carcinoma after transarterial embolization in rat with McA-RH7777 hepatoma.

Transarterial chemoembolization represents a first-line non-curative therapy for hepatocellular carcinoma (HCC), although the biological changes in the remaining cancer after embolization are not completely understood. In the present study, we examined whether transarterial embolization (TAE) enhances the metastatic potential of residual HCC and investigated the mechanisms underlying embolization. The hepatoma cell line McA-RH7777, which is marked by green fluorescent protein (GFP), was used in the study. The invasion of cells cultured under hypoxia and normoxia was observed using the Transwell assay. Twenty male buffalo rats were implanted with GFP transfected McA-RH7777 tumors in the left lateral lobe of the liver. After laparotomy and retrograde placement of a catheter into the gastroduodenal artery (on the 14th day after implantation), TAE using lipiodol (0.2 ml/kg) was performed. Tumor volumes were measured before and after treatment using magnetic resonance imaging (MRI). Lung metastases were observed using fluorescence imaging, and the molecular changes of residual tumor cells were evaluated by western blotting or immunohistochemistry. The invasion assays indicated that the number of invading hypoxic cells was significantly higher than that of normoxic cells (30.2 ± 2.46 vs. 20.4 ± 1.89, P=0.013). Accompanying an increase in hypoxia-inducible factor-1α (HIF-1α) expression, the metastatic potential of tumor cells following hypoxia or TAE was enhanced. This enhanced metastatic potential was indicated by a significant reduction in the expression of E-cadherin and an upregulation of N-cadherin and vimentin expression. The number of lung metastases in the TAE group was 19.20 ± 1.76, whereas this number was 11.30 ± 1.54 in the control group, which represented a statistically significant difference (P=0.003). In conclusion, hypoxia in the residual tumor after TAE can increase the invasiveness and metastatic potential of HCC and may be responsible for the failure of TAE.

Low-level laser therapy induces the expressions of BMP-2, osteocalcin, and TGF-ß1 in hypoxic-cultured human osteoblasts.

The aim of this study was to examine the effect of low-level laser therapy (LLLT) on the cell viability and the expression of hypoxia-inducible factor-1s (HIF-1s), bone morphogenic protein-2 (BMP-2), osteocalcin, type I collagen, transforming growth factor-ß1 (TGF-ß1), and Akt in hypoxic-cultured human osteoblasts. Human fetal osteoblast cells (cell line 1.19) were cultured under 1 % oxygen tension for 72 h. Cell cultures were divided into two groups. At the experimental side, low-level laser (808 nm, GaAlAs diode) was applied at 0, 24, and 48 h. After irradiation, each cell culture was incubated 24 h more under hypoxia. Total energy was 1.2, 2.4, and 3.6 J/cm(2), respectively. Non-irradiated cultures served as controls. Comparisons between the two groups were analyzed by t test; a p value <0.05 was considered statistically significant. Hypoxia resulted in a decrease in the expression of type I collagen, osteocalcin, and TGF-ß1 (p < 0.001, p < 0.001, and p < 0.01, respectively). Cell viability and BMP-2 expression were not decreased by hypoxic condition. On the other hand, LLLT on hypoxic-cultured osteoblast promoted the expression of BMP-2, osteocalcin, and TGF-ß1 (p < 0.05, p < 0.01, and p < 0.001, respectively). Cell proliferation was also increased time-dependently. However, hypoxia decreased in type I collagen expression (p < 0.001), and LLLT did not affect type I collagen expression in hypoxic-cultured osteoblasts. Furthermore, LLLT inhibited HIF-1 and Akt expression in hypoxic conditioned osteoblasts. We concluded that LLLT induces the expression of BMP-2, osteocalcin, and TGF- ß1 in 1 % hypoxic-cultured human osteoblasts.

Cloning and characterization of hypoxia-inducible factor-1 subunits from Ascaris suum - a parasitic nematode highly adapted to changes of oxygen conditions during its life cycle.

The parasitic nematode Ascaris suum successfully adapts to a significant decrease in oxygen availability during its life cycle by altering its metabolic system dramatically. However, little is known about the regulatory mechanisms of adaptation to hypoxic environments in A. suum. In multicellular organisms, hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1ß subunits, is a master regulator of genes involved in adaptation to hypoxia. In the present study, cDNAs encoding HIF-1α and HIF-1ß were cloned from A. suum and characterized. The full-length A. suum hif-1α and hif-1ß cDNAs contain open reading frames encoding proteins with 832 and 436 amino acids, respectively. In the deduced amino acid sequences of A. suum HIF-1α and HIF-1ß, functional domains essential for DNA-binding, dimerization, and oxygen-dependent prolyl hydroxylation were conserved. The interaction between A. suum HIF-1α and HIF-1ß was confirmed by the yeast two-hybrid assay. Both A. suum hif-1α and hif-1ß mRNAs were expressed at all stages examined (fertilized eggs, third-stage larvae, lung-stage larvae, young adult worms, and adult muscle tissue), and most abundantly in the aerobic free-living third-stage larvae, followed by a gradual decrease after infection of the host. hif-1 mRNA transcription was not sensitive to the oxygen environment in either third-stage larvae or adult worms (muscle tissue), and was regulated in a stage-specific manner. High expression of hif-1 mRNAs in third-stage larvae suggests its contribution to pre-adaptation to a hypoxic environment after infection of their host. Sequence analysis of 5'-upstream regions of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) genes, which show stage-specific expression and play an important role in oxygen adaptation during the life cycle, revealed that all subunits except for the adult-type flavoprotein subunit (Fp) possess putative hypoxia-responsive elements (HREs), suggesting that they are hif-1 target genes.

Reduced cholesterol and triglycerides in mice with a mutation in Mia2, a liver protein that localizes to ER exit sites.

Through forward genetic screening in the mouse, a recessive mutation (couch potato, cpto) has been discovered that dramatically reduces plasma cholesterol levels across all lipoprotein classes. The cpto mutation altered a highly conserved residue in the Src homology domain 3 (SH3) domain of the Mia2 protein. Full-length hepatic Mia2 structurally and functionally resembled the related Mia3 protein. Mia2 localized to endoplasmic reticulum (ER) exit sites, suggesting a role in guiding proteins from the ER to the Golgi. Similarly to the Mia3 protein, Mia2's cytosolic C terminus interacted directly with COPII proteins Sec23 and Sec24, whereas its lumenal SH3 domain may facilitate interactions with secretory cargo. Fractionation of plasma revealed that Mia2(cpto/cpto) mice had lower circulating VLDL, LDL, HDL, and triglycerides. Mia2 is thus a novel, hepatic, ER-to-Golgi trafficking protein that regulates cholesterol metabolism.

Modified expression of aryl hydrocarbon receptor-related genes by deoxymiroestrol, a phytoestrogen, in mouse hepatocytes in primary culture.

ETHNOPHARMACOLOGICAL RELEVANCE: Deoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation. MATERIALS AND METHODS: The expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation. RESULTS: The mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of ß-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, ß-naphthoflavone (ßNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by ßNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2. CONCLUSION: The findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.

Red ginseng deregulates hypoxia-induced genes by dissociating the HIF-1 dimer.

Water extract of Korean red ginseng (KRGW) contains numerous bioactive ginsenosides and is very popular as a multi-purpose medicine for health improvement. KRGW has been in the limelight because of its clinical benefit in cancer control. A growing body of evidence suggests that hypoxia-inducible factor-1 (HIF-1) plays critical roles in tumor promotion under hypoxia and that it is a compelling target for cancer therapy. In this paper we investigated the effect of KRGW on HIF-1-mediated adaptation to hypoxia. In both Hep3B cancer and HEK293 immortalized normal cell lines, KRGW attenuated the expression of hypoxia-induced genes without apparent cytotoxicity. Mechanistically, KRGW did not affect the synthesis, degradation, and translocation of HIF-1 in hypoxia. Interestingly, KRGW was found to repress the transcriptional activity of HIF-1 by interfering with the dimerization between HIF-1α and aryl hydrocarbon receptor nuclear translocator. To identify the HIF-inhibiting ingredient(s), we examined the effects of major ginsenosides on HIF-1 activity, but all ginsenosides tested failed to inactivate HIF-1. Based on these results, we propose that HIF-1 inhibition underlies the anticancer effect of ginseng. It is also proposed that KRGW could be an anticancer drug targeting hypoxic tumors.

Curcumin inhibits hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear translocator: a mechanism of tumor growth inhibition.

Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT), plays a key role in cell survival and angiogenesis in hypoxic tumors, and many efforts have been made to develop anticancer agents that target HIF-1alpha. However, although ARNT is also required for HIF-1 activity, ARNT has been disregarded as a therapeutic target. Curcumin is a commonly used spice and coloring agent with a variety of beneficial biological effects, which include tumor inhibition. In the present study, we tested the possibility that curcumin inhibits tumor growth by targeting HIF-1. The effects of curcumin on HIF-1 activity and expression were examined in cancer cell lines and in xenografted tumors. We found that curcumin inhibits HIF-1 activity and that this in turn down-regulates genes targeted by HIF-1. Moreover, of the two HIF-1 subunits, only ARNT was found to be destabilized by curcumin in several cancer cell types, and furthermore, ARNT expression rescued HIF-1 repression by curcumin. We also found that curcumin stimulated the proteasomal degradation of ARNT via oxidation and ubiquitination processes. In mice bearing Hep3B hepatoma, curcumin retarded tumor growth and suppressed ARNT, erythropoietin, and vascular endothelial growth factor in tumors. These results suggest that the anticancer activity of curcumin is attributable to HIF-1 inactivation by ARNT degradation.

Ontogenetic development, sexual differentiation, and effects of Aroclor 1254 exposure on expression of the arylhydrocarbon receptor and of the arylhydrocarbon receptor nuclear translocator in the rat hypothalamus.

Interaction of polychlorinated biphenyls (PCBs) with the aryl hydrocarbon receptor (AhR)/nuclear translocator (ARNT) system might interfere with the mechanisms controlling the sexual differentiation of the developing hypothalamus. The aim of this study was to evaluate the presence of AhR/ARNT in brain cells and the developmental profile of their expression in the hypothalamus of male and female rats during the perinatal period. Brain accumulation of the main PCB congeners after prenatal exposure to Aroclor 1254 and its influence on hypothalamic expression of AhR/ARNT was also assessed. The results show that: (a) AhR and ARNT are expressed both in neurons and in glia; (b) AhR expression progressively increases in the developing hypothalamus particularly in males, while ARNT is relatively constant in both sexes; (c) the prenatal administration of Aroclor to dams produces a differential accumulation of PCBs, depending on the chlorine atom number, and stimulates AhR expression only in the male hypothalamus. In conclusion, the developing male hypothalamus might be more sensitive to disrupting potential of PCBs.