Assessment of effectiveness in stabilization/solidification of arsenic-contaminated soil: long-term leaching test and geophysical measurement.

This study focused on evaluating the effectiveness of stabilizer/binding agents in immobilizing arsenic (As) in contaminated soil using both geochemical and geophysical monitoring methods. The effluent from the stabilizer/binding agent's application and control columns was analyzed, and the status of the columns was monitored using electrical resistivity (ER) and induced polarization (IP) methods. As stabilizers/binder, acid mine drainage sludge (AMDS) and steel slag (SS) were used, which delayed As and Ca leaching time and significantly reduced As leaching amount. Determination coefficients for As and Fe leaching exhibited elevated values (control column, R2 = 0.955; AMDS column, R2 = 0.908; and SS column, R2 = 0.833). A discernible decline in the concentration of leached Fe was accompanied by a corresponding reduction in IP. The determination coefficients correlating IP and Fe leaching remained substantial (control column, R2 = 0.768; AMDS column, R2 = 0.807; and SS column, R2 = 0.818). Such IP measurements manifest as instrumental tools in monitoring and assessing the retention capacity of applied stabilizer/binding agents in As-affected soils, thereby furnishing crucial data for the enduring surveillance of stabilization/solidification locales. This research posits a swift and continuous monitoring method for solidification/stabilization locales in situ.

Integrative biohydrogen- and biomethane-producing bioprocesses for comprehensive production of biohythane.

The production of biohythane, a combination of energy-dense hydrogen and methane, from the anaerobic digestion of low-cost organic wastes has attracted attention as a potential candidate for the transition to a sustainable circular economy. Substantial research has been initiated to upscale the process engineering to establish a hythane-based economy by addressing major challenges associated with the process and product upgrading. This review provides an overview of the feasibility of biohythane production in various anaerobic digestion systems (single-stage, dual-stage) and possible technologies to upgrade biohythane to hydrogen-enriched renewable natural gas. The main goal of this review is to promote research in biohythane production technology by outlining critical needs, including meta-omics and metabolic engineering approaches for the advancements in biohythane production technology.

Inhibitory Effects of Porphyra tenera Extract on Oxidation and Inflammatory Responses.

Porphyra tenera (laver) has long been a popular and traditional seaweed food in Korea, Japan, and China. Historically, it was known as a marine medicinal herb to treat hemorrhoids and cholera morbus in Donguibogam. We investigated the effects of P. tenera extract (PTE) for its antioxidant and anti-inflammatory activities. These activities were measured using assays for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging and its superoxide dismutase- (SOD-) like activity, and through the inhibitory production of inflammatory mediators (prostaglandin E2 (PGE2), NO, tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6)) in lipopolysaccharide- (LPS-) stimulated Raw 264.7 cells. The antioxidant assay results showed that PTE displayed DPPH radical scavenging activity (46.44%), NO radical scavenging activity (67.14%), and SOD-like activity (80.29%) at a concentration of 5 mg/mL. In the anti-inflammatory assays, treatment with PTE (1 mg/mL) significantly inhibited expression levels of LPS-induced COX-2 and iNOS, as well as the production of PGE2, NO, TNF-α, and IL-6. These results show that PTE has antioxidant and anti-inflammatory properties and provide scientific evidence to explain the antioxidative and anti-inflammatory properties of PTE.

Astaxanthin Reduces Stemness Markers in BT20 and T47D Breast Cancer Stem Cells by Inhibiting Expression of Pontin and Mutant p53.

Astaxanthin (AST) is a product made from marine organisms that has been used as an anti-cancer supplement. It reduces pontin expression and induces apoptosis in SKBR3, a breast cancer cell line. Using Western blotting and qRT-PCR analyses, this study revealed that in the T47D and BT20 breast cancer cell lines, AST inhibits expression of pontin and mutp53, as well as the Oct4 and Nanog cancer stem cell (CSC) stemness genes. In addition, we explored the mechanism by which AST eradicates breast cancer cells using pontin siRNAs. Pontin knockdown by pontin siRNA reduced proliferation, Oct4 and Nanog expression, colony and spheroid formation, and migration and invasion abilities in breast cancer cells. In addition, reductions in Oct4, Nanog, and mutp53 expression following rottlerin treatment confirmed the role of pontin in these cells. Therefore, pontin may play a central role in the regulation of CSC properties and in cell proliferation following AST treatment. Taken together, these findings demonstrate that AST can repress CSC stemness genes in breast cancer cells, which implies that AST therapy could be used to improve the efficacy of other anti-cancer therapies against breast cancer cells.

Melatonin Exerts Anticancer Effects in Human Tongue Squamous Cell Carcinoma Cells by Promoting Autophagy.

BACKGROUND/AIM: The global prevalence of head and neck squamous cell carcinoma (HNSCC) remains high, and its prognosis poor. We investigated the anticancer effects of melatonin in human tongue squamous cell carcinoma cells (SCC-25) and its mechanisms of action. MATERIALS AND METHODS: MTT assay was used to determine cell viability. To assess the effects of melatonin on SCC-25 cell metastasis, we conducted cell formation, wound healing, transwell migration and invasion assay. Western blot analysis was performed to measure the levels of autophage marker proteins. RESULTS: We found that melatonin treatment significantly reduced the viability and colony formation ability of SCC-25 cells, impairing cell migration and invasion. Western blotting assay revealed that melatonin increased the levels of autophagy markers, such as LC-3B and Beclin-1. Consequently, melatonin induces autophage in SCC-25 cells. CONCLUSION: Melatonin may be a promising anticancer agent for the treatment of human tongue squamous cell carcinoma.

Astaxanthin Modulates Apoptotic Molecules to Induce Death of SKBR3 Breast Cancer Cells.

Astaxanthin (AST) is related to apoptosis but the details of the mechanism of how AST makes apoptosis is not clear. The present study investigated apoptotic effects of AST to SKBR3, a breast cancer cell line in detail. Cell viability assay showed cellular proliferation and morphological changes of the cells were observed under AST treatment. FACS analysis indicated that AST blocked cell cycle progression at G0/G1, suppressed proliferation dose-dependently, and induced apoptosis of the cells. The apoptosis of the cells by AST was further demonstrated through the decreased expression level of mutp53 and cleaved a PARP-1 fragment, respectively. In addition, AST induced the intrinsic apoptosis of the cells by activation of Bax/Bcl2, cleaved caspase-3, and cleaved caspase-9 as well as the phosphorylation of ERK1/2, JNK, and p38. Furthermore, AST decreased production of intracellular reactive oxygen species as well as modulated expressions of superoxide dismutases and Pontin, an anti-apoptotic factor. Co-immunoprecipitation assay revealed AST reduced interaction between Pontin and mutant p53. Taken together, these studies proved that AST regulates the expression of apoptotic molecules to induce intrinsic apoptosis of the cells, suggesting AST therapy might provide an alternative for improving the efficacies of other anti-cancer therapies for breast cancer.

Systemic Pharmacological Approach to Identification and Experimental Verification of the Effect of Anisi Stellati Fructus Extract on Chronic Myeloid Leukemia Cells.

Anisi stellati fructus (ASF) is the dried fruit of the Illicium verum Hook.f. tree. The aim of this research was to evaluate the antileukemic effect of ASF on chronic myeloid leukemia (CML) cells, which was hypothesized from the systemic pharmacological analysis of ASF, focusing on the combined effect of ASF extract (ASFE) and imatinib (IM). The compounds of ASF were identified using the Traditional Chinese Medicine Systems Pharmacology database and analysis platform. The target gene information was acquired from the UniProt database. The compound and target interaction network was generated from Cytoscape 3.7.1. Using this analysis, 10 compounds effective against CML cells were obtained. ASFE was prepared and analyzed by high-pressure liquid chromatography to provide experimental proof for the relationship between ASF and CML. The anti-p210Bcr-Abl effects of ASFE and ASFE + IM combination were evaluated by western blotting. Either ASFE alone or in combined treatment with IM on K-562 CML cells resulted in a significant reduction of the Bcr-Abl levels. As expected from the systemic analysis results, ASF had antileukemic activity, showing that it is a potential therapy for CML.

Efficient removal of arsenic by strategically designed and layer-by-layer assembled PS@+rGO@GO@Fe3O4 composites.

The PS@+rGO@GO@Fe3O4 (PG-Fe3O4) hybrid composites for Arsenic removal were successfully fabricated and well dispersed using layer-by-layer assembly and a hydrothermal method. The PG-Fe3O4 hybrid composites were composed of uniformly coated Fe3O4 nanoparticles on graphene oxide layers with water flow space between 3D structures providing many contact area and adsorption sites for Arsenic adsorption. The PG-Fe3O4 hybrid composite has large surface adsorption sites and exhibits high adsorption capacities of 104 mg/g for As (III) and 68 mg/g for As (V) at 25 °C and pH 7 comparison with pure Fe3O4 and P-Fe3O4 samples.

Effects of astaxanthin on dinitrofluorobenzene-induced contact dermatitis in mice.

Astaxanthin (AST) is known to exhibit antioxidative and antitumor properties, therefore, the present study investigated its other potential medical applications. AST was observed to exhibit anti­allergic and anti­inflammatory effects in a dinitrofluorobenzene (DNFB)­induced contact dermatitis (CD) mouse model and RBL­2H3 cell lines. The topical application of AST effectively inhibited the enlargement of ear thickness and increase in weight, which occurred following repeated application of DNFB. Furthermore, topical application of different concentrations of AST inhibited inflammatory hyperplasia, edema, spongiosis, and the infiltration of mononuclear cells and mast cells in the ear tissue. In addition, the levels of TNF­α and IFN­Î³ produced were decreased by application of AST in vivo, and treatment of RBL­2H3 cells with AST inhibited the release of histamine and ß­hexosaminidase in vitro. Taken together, these data suggested that AST may be used to treat patients with allergic skin diseases through a mechanism, which may be associated with that involved in anti­inflammatory or anti-allergic activities.

7-Ketocholesterol induces the reduction of KCNMB1 in atherosclerotic blood vessels.

Hypertension is a high-risk symptom in atherosclerotic patients, and vascular rigidity is one of the main factors leading to hypertension. ß1-Subunit of BKCa channel (KCNMB1; MaxiKß1) has been reported as a modulator of vascular flexibility. To determine the relationship between atherosclerosis and KCNMB1, we studied some atherogenic factors affecting vascular tone. Blood of atherosclerotic patients shows increased concentration of 7-ketocholesterol (7K), which has been studied as a harmful lipid to blood vessels. Our data showed that KCNMB1 was significantly down-regulated in the presence of 7K, in a dose-/time-dependent manner in vascular smooth muscle cells (VSMCs). And, the reduction of KCNMB1 was confirmed in cell images of 7K-stimulated VSMCs and in vessel tissue images of ApoE knock-out mice. To determine whether aryl hydrocarbon receptor (AhR) was involved in the reduction of KCNMB1 by 7K-stimulation, protein level of AhR was analyzed by Western blot. Our data showed that the reduction of KCNMB1 was modulated through the AhR pathway. In conclusion, results of our study suggest that 7K induces the reduction of KCNMB1 through the AhR pathway.

Biomolecular evidence of anti-inflammatory effects by Clematis mandshurica Ruprecht root extract in rodent cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Clematis mandshurica Ruprecht root is widely used in Asia as an analgesic and anti-inflammatory agent. This research investigated the anti-inflammatory effects of Clematis mandshurica Ruprecht root extract (CRE) using RAW 264.7 macrophage cells and carrageenan- (CA-) induced rat paw edema. MATERIALS AND METHODS: Production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6, nitric oxide (NO) and prostaglandin E2 (PGE2) in the culture supernatant, mRNA expression of TNF-α, IL-1ß, IL-6, iNOS and COX-2, protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in the extract were assayed. In addition, the effect of CRE on acute inflammation in vivo was observed using CA-induced rat hind paw edema assay. The changes on the histopathology and histomorphometry of hind paw skins-dorsum and ventrum pedis were observed using CA-treated rats. RESULTS: Treatment with CRE (0.25, 0.5, and 1 mg/mL) resulted in inhibited levels of protein expression of lipopolysaccharide- (LPS-) induced iNOS, COX-2, NF-κB, and MAPKs (ERK, JNK, and p38) as well as production of TNF-α, IL-1ß, IL-6, NO, and PGE2 induced by LPS. Consistent with these results, CRE reduced the LPS-induced expressions of these cytokines, iNOS and COX-2 at the mRNA levels in a dose-dependent manner. In particular, results of the CA-induced rat hind paw edema assay showed an anti-edema effect of CRE. In addition, treatment with CRE resulted in dose-dependent inhibition of CA-induced increases of skin thickness, mast cell degranulation, and infiltrated inflammatory, TNF-α, IL-1ß, iNOS, and COX-2-positive cells in both dorsum and ventrum pedis skin, respectively. CONCLUSIONS: These results demonstrate that CRE exhibits anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the pathways of NF-κB and MAPKs in LPS-induced macrophage cells. In addition, results of the CA-induced rat hind paw edema assay show an anti-edema effect of CRE. Our findings also support the traditional use of CRE in the inflammatory symptoms of rheumatic arthritis and acute icteric hepatitis. Thus, CRE may have therapeutic potential for a variety of inflammation-mediated diseases and may be developed into potent anti-inflammatory drugs.

Antiasthmatic effects of schizandrae fructus extract in mice with asthma.

BACKGROUND: Schizandrae fructus (SF), the fruit of Schisandra chinensis, has been used for the treatment of cough, wheezing, dry mouth, hepatitis, cardiovascular disease, and as a tonic and astringent in China, Japan, and Korea. OBJECTIVE: Investigation of the antiasthmatic effects of SF. MATERIALS AND METHODS: We investigated the effects of SF on airway hyperresponsiveness (AHR) to methacholine, production levels of antigen-specific antibodies, and histopathological changes in the lung tissue in a mouse model (Balb/c) of asthma induced by repeated intranasal instillation of an antigen. RESULTS: SF lowered AHR to methacholine (P < 0.05), antigen-specific immunoglobulin E (IgE) level (P < 0.01), and immune cell infiltration in mice with asthma. Prednisolone (PD) effectively decreased AHR (P < 0.01), total antibody (P < 0.01) and IgE (P < 0.01) levels, and immune cell infiltration. SF and PD did not affect the levels of antigen-specific IgG1 and IgG2a antibodies. CONCLUSION: Our data suggest that SF has possible application as an antiasthmatic drug. We also suggest that SF could be used as a complementary or alternative medicine to glucocorticoids.

Counteracting the activation of pAkt by inhibition of MEK/Erk inhibition reduces actin disruption-mediated apoptosis in PTEN-null PC3M prostate cancer cell lines.

The actin cytoskeleton is important in the maintenance of cellular homeostasis and in signal transduction pathways leading to cell growth and apoptotic cell death in eukaryotic cells. Disruption of actin dynamics is associated with morphological changes in cancer cells. Deletion of phosphatase and tensin homolog (PTEN), a tumor suppressor gene involved in the regulation of the cell cycle and apoptosis, leads to cytoskeleton disruption and double-strand breaks (DSBs). To study the mechanism(s) of actin disruption-mediated apoptosis and its potential application for anticancer therapy, PTEN-null PC3M prostate cancer cells were treated with latrunculin B (LB). LB induced destabilization of the actin microfilament and apoptosis in a dose-dependent manner, as demonstrated by morphological changes and nuclear condensation in the PC3M cells. In addition, it resulted in an increase in the levels of γH2AX recruitment, implicating the induction of DNA damage, including DSBs. Induction of Bax, with little effect on Bcl-2 expression, indicated that actin disruption causes apoptosis through activation of Bax signaling in PC3M cells. Treatment with U20126, a mitogen-activated protein kinase kinase (MEK) inhibitor, resulted in attenuated induction of DSBs and apoptosis through activation of protein kinase B (Akt), suggesting that LB-mediated actin dysfunction induces DSBs via the MEK/extracellular signal-regulated kinase (Erk) pathway in cells. Therefore, counteracting activation of phosphorylated Akt stemming from the inhibition of MEK/Erk resulted in attenuation of actin disruption-induced apoptotic events in the PC3M cells. The results of this study provide information not only for use in delineation of the molecular association between actin disruption and tumorigenesis, but also for the development of a strategy for actin-based anticancer chemotherapy against highly metastatic prostate cancer.

Inhibitory effects of traditional herbal formula pyungwi-san on inflammatory response in vitro and in vivo.

Pyungwi-san (PWS) is a traditional basic herbal formula. We investigated the effects of PWS on induction of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines (interleukin-6 (IL-6) and tumor necrosis factor- α (TNF- α )) and nuclear factor-kappa B (NF- κ B) as well as mitogen-activated protein kinases (MAPKs) in lipopolysaccharide-(LPS-) induced Raw 264.7 cells and on paw edema in rats. Treatment with PWS (0.5, 0.75, and 1 mg/mL) resulted in inhibited levels of expression of LPS-induced COX-2, iNOS, NF- κ B, and MAPKs as well as production of prostaglandin E2 (PGE2), nitric oxide (NO), IL-6, and TNF- α induced by LPS. Our results demonstrate that PWS possesses anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the signaling pathways of NF- κ B and MAPKs in LPS-induced macrophage cells. More importantly, results of the carrageenan-(CA-) induced paw edema demonstrate an anti-edema effect of PWS. In addition, it is considered that PWS also inhibits the acute edematous inflammations through suppression of mast cell degranulations and inflammatory mediators, including COX-2, iNOS and TNF- α . Thus, our findings may provide scientific evidence to explain the anti-inflammatory properties of PWS in vitro and in vivo.

Anti-inflammatory effects of bangpungtongsung-san, a traditional herbal prescription.

Bangpungtongsung-san (BPTS), a traditional oriental herbal prescription, is widely used for expelling wind, draining heat, and providing general improvement to the immune system. In this study, we investigated the effects of BPTS on induction of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), proinflammatory cytokines, nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinases (MAPKs) in lipopolysaccharide- (LPS- ) stimulated Raw 264.7 cells, and on paw edema in rats. At concentrations of 0.5, 0.75, and 1 mg/mL, treatment with BPTS inhibited levels of expression of LPS-induced NF-κB and MAPKs (ERK, JNK, and p38) as well as production of proinflammatory mediators, such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) by LPS. These results suggest that BPTS may exert anti-inflammatory effects via reduction of proinflammatory mediators, including NO, PGE(2), TNF-α, and IL-6 through suppression of the signaling pathways of NF-κB and MAPKs in LPS-induced macrophages. In addition, using the carrageenan-induced paw edema assay, an antiedema effect of BPTS was observed in rats. These findings may provide scientific evidence validating the use of BPTS in treatment of patients with heat syndrome in Korean oriental medicine.

Hypoxia-inducible factor-1α regulates KCNMB1 expression in human pulmonary artery smooth muscle cells.

Previously, we observed that hypoxia increases the expression of the ß1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BK(Ca)). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-α (HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1α transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the ß1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.

Actin disruption agents induce phosphorylation of histone H2AX in human breast adenocarcinoma MCF-7 cells.

Modified actin dynamics are a unique feature of transformed cancer cells and thereby promising targets for cancer chemotherapy. While latrunculin B (LB) and pectenotoxin-2 (PTX-2), both derived from natural sources, inhibit actin polymerization, jasplakinolide (JSP) prevents actin depolymerization. The purpose of this study was to examine the detailed molecular action of actin disruption inducing apoptosis via double strand breaks (DSBs). Actin disruption induced phosphorylation of H2AX, a well known DSB marker leading to G2 arrest and consequently resulted in apoptosis on MCF-7 cancer cells. Cells impaired by actin disruption activated Erk (extracellular signal-related kinase) and p53 protein was involved in DNA damage responses, but did not change the levels of p21Cip1/WAF1 protein in MCF-7 cells. To overcome the DSBs by actin disruption, MCF-7 cells set the repair system through the homologous recombination (HR) pathway. These results indicate that actin is involved in the signaling inducing DSBs and HR repair as well as G2 cell cycle arrest in human cancer. Therefore, the results suggest that actin disruption might be a potential candidate for developing anti-cancer therapies in human breast cancer.

Removal of nitrate and ammonium ions from livestock wastewater by hybrid systems composed of zero-valent iron and adsorbents.

The feasibility of hybrid systems for simultaneous removal of nitrate (NO3-) and ammonium ions (NH4+) from livestock wastewater was examined in batch experiments. As a part of efforts to remove nitrate and ammonium simultaneously, Fe0 and adsorbents including coconut-based granular activated carbon (GAC), sepiolite and filtralite were used. Various parameters such as adsorbent dosages and temperature were studied. Removal of NO3- increased with increase in temperature. Maximum NO3- removal (85.3%) was observed for the Fe0-filtralite hybrid system at 45 degrees C for a 24 h reaction time. Increase in GAC and sepiolite dosages had significant (P < 0.01) effect on the NH4+ removal efficiency, which was primarily due to the net negative surface charge of the adsorbents. The efficiency of hybrid systems for the removal of NO3- was in the order of filtralite > sepiolite > GAC, and the order of the removal of NH4+ was GAC > sepiolite > filtralite. The results of the present study suggest that the use of hybrid systems could be a promising innovative technology for achieving simultaneous removal of NO3- and NH4 from livestock wastewater.

Rodent-specific hypoxia response elements enhance PAI-1 expression through HIF-1 or HIF-2 in mouse hepatoma cells.

Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of numerous pathophysiological processes such as inflammation, thrombosis, angiogenesis and tumor metastasis. Its expression is induced by hypoxia at the transcriptional level, via the hypoxia inducible factor-1 (HIF-1) or -2 (HIF-2). In this study, we elucidated the mechanism of transcriptional regulation of mouse PAI-1 gene by hypoxia in mouse hepatoma cells. We searched for hypoxia response elements (HREs) of murine PAI-1 promoter using several molecular biological assays. DNAse I hypersensitivity assay first suggested that PAI-1 gene expression is up-regulated by protein-DNA interactions at the -3.6- and -3-kb upstream regions of the PAI-1 gene transcription start site. An approximately 6.4-kb region of DNA containing the 5'-flanking promoter region of the PAI-1 gene was isolated, mapped, and cloned into reporter gene assay vectors and sequenced. Luciferase reporter gene assay subsequently identified two functional HREs, located around -3.6 kb of the 5'-flanking promoter region of PAI-1 gene that were responsible for the enhancement of luciferase reporter gene activity. Mutation of the HREs in this fragment abolished luciferase reporter gene activity. Finally, in vitro and in vivo protein-DNA interaction assays confirmed binding of the two HREs to HIF-1 or HIF-2 protein. Our results show that two HREs located around -3.6 kb of the 5'-flanking promoter region of the mouse PAI-1 gene function as hypoxia enhancers, which, alongside other regulatory regions, control PAI-1 gene transcription by HIF-1 or HIF-2 under hypoxic environments in mouse hepatoma cells.

Actin disruption inhibits hypoxia inducible factor-1α expression via inactivity of Mdm2-mediated p70S6K.

The intracellular actin cytoskeleton is a central player in tumor cell migration and adhesion, and interacts with the extracellular matrix during the progression to metastasis. Although recent reports on motility events have revealed that the destabilization of actin affects cancer progression and hypoxia inducible factor-1α (HIF-1α) activity, little is known about the responsive activity of HIF-1α following actin disruption. Here, we demonstrate that the inhibition of actin polymerization or depolymerization attenuates HIF-1α expression independently of proteasomal degradation. The disruption of actin dynamics inactivates HIF-1α translational expression through p70S6K translational signaling; this is independent of p53 activation, suggesting that actin dysfunction-mediated HIF-1α destabilization may lead to the development of novel anticancer chemotherapeutic targets.