Co-Inoculation with Arbuscular Mycorrhizal Fungi and Dark Septate Endophytes under Drought Stress: Synergistic or Competitive Effects on Maize Growth, Photosynthesis, Root Hydraulic Properties and Aquaporins?

Arbuscular mycorrhizal fungi (AMF) and dark septate fungi (DSE) were simultaneously colonized in the root cells of maize. Single AMF and DSE symbiosis have been proven to improve the drought tolerance of maize. However, the effects of both fungi coexisting in maize roots under drought stress are not yet known. In this study, pot experiments of maize seedlings were conducted through four inoculation treatments (single AMF inoculation of Rhizophagus irregularis, single DSE inoculation of Exophiala pisciphila, co-inoculation of AMF + DSE and non-mycorrhizal inoculation) under well-watered (WW) and drought-stressed (DS) conditions. AMF and DSE colonization status, maize physiology and aquaporin gene expression in maize roots were investigated. The objective of this paper was to evaluate whether AMF and DSE had competitive, independent or synergistic effects on regulating the drought tolerance of maize. When maize seedlings of three inoculation treatments were subjected to drought stress, single AMF inoculation had the highest shoot and root dry weight, plant height, root length, osmotic root hydraulic conductivity and hydrostatic root hydraulic conductivity in maize seedlings. However, co-inoculation of AMF + DSE induced the highest stomatal conductance in maize leaves and the lowest H2O2 and O2•- concentration, membrane electrolyte leakage, intercellular CO2 concentration and gene expression level of ZmPIP1;1, ZmPIP1;2, ZmPIP2;1, ZmPIP2;5 and ZmPIP2;6. In addition, co-inoculation of AMF + DSE also obviously down-regulated the GintAQPF1 and GintAQPF2 expression in R. irregularis compared with single AMF inoculation treatment. Under DS stress, there were competitive relationships between AMF and DSE with regard to regulating mycorrhizal colonization, maize growth, root hydraulic conductivity and the gene expression of aquaporins in R. irregularis, but there were synergistic relationships with regard to regulating membrane electrolyte leakage, oxidative damage, photosynthesis and the aquaporin gene expression of maize seedlings. The obtained results improve our knowledge about how the mechanisms of AMF and DSE coexist, promoting the drought tolerance of host plants.

Exogenous Melatonin Enhances the Yield and Secondary Metabolite Contents of Prunella vulgaris by Modulating Antioxidant System, Root Architecture and Photosynthetic Capacity.

Melatonin (MT) plays a number of key roles in regulating plant growth and secondary metabolite accumulation. Prunella vulgaris is an important traditional Chinese herbal medicinal plant which is used for the treatment of lymph, goiter, and mastitis. However, the effect of MT on the yield and medicinal component content of P. vulgaris remains still unclear. In this research, we have examined the influence of different concentrations of MT (0, 50, 100, 200, 400 µM) on the physiological characteristics, secondary metabolite contents, and yield of P. vulgaris biomass. The results showed that 50-200 µM MT treatment had a positive effect on P. vulgaris. MT treatment at 100 µM greatly increased the activities of superoxide dismutase and peroxidase, the contents of soluble sugar and proline, and obviously decreased the relative electrical conductivity, the contents of malondialdehyde and hydrogen peroxide of leaves. Furthermore, it markedly promoted the growth and development of the root system, increased the content of photosynthetic pigments, improved the performance of photosystems I and II and the coordination of both photosystems, and enhanced the photosynthetic capacity of P. vulgaris. In addition, it significantly increased the dry mass of whole plant and spica and promoted the accumulation of total flavonoids, total phenolics, caffeic acid, ferulic acid, rosmarinic acid, and hyperoside in the spica of P. vulgaris. These findings demonstrated that the application of MT could effectively activate the antioxidant defense system of P. vulgaris, protect the photosynthetic apparatus from photooxidation damage, and improve the photosynthetic capacity and the root absorption capacity, thereby promoting the yield and accumulation of secondary metabolites in P. vulgaris.

Rhizoglomus intraradices Improves Plant Growth, Root Morphology and Phytohormone Balance of Robinia pseudoacacia in Arsenic-Contaminated Soils.

Arbuscular mycorrhizal fungi (AMF) are known to improve the resistance of host plants against various heavy metal stresses. However, the arsenic (As) resistance mechanism of AMF-inoculated woody legumes remains unclear. In this study, black locust (Robinia pseudoacacia L.) seedlings were cultivated in potted soils inoculated with or without AMF Rhizoglomus intraradices under three different levels of As stress (0, 100, and 200 mg As kg-1 soil) over 4 months. The objective of this paper was to investigate the effects of AMF on plant growth, root morphology, and the content and ratio of endogenous phytohormones and soil glomalin under As stress condition. As stress toxicity suppressed the AM spore germination and colonization, plant growth, and the content of soil glomalin and changed the morphological characteristics of the roots and the balance of endogenous hormone levels in plants. However, R. intraradices inoculation improved the shoot and root dry weights, total root length, root surface area, root volume, and the number of root forks and tips across all As treatments. R. intraradices inoculation obviously decreased the percentage of root length in the 0- to 0.2-mm diameter class and increased those in the 0.5- to 1.0-mm and >1.0-mm diameter classes; the percentages in the 0.2- to 0.5-mm diameter class were less affected by R. intraradices inoculation. The concentrations of the easily extractable glomalin-related (EE-GRSP) and total glomalin-related soil protein (T-GRSP) were higher in the of R. intraradices-inoculated seedlings than those in the non-inoculated seedlings. Furthermore, R. intraradices inoculation increased the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA), but decreased the concentrations of gibberellic acid (GA) and zeatin riboside (ZR). The phytohormone ratios of IAA/ABA, GA/ABA, ZR/ABA, and (IAA + GA + ZR)/IAA in the R. intraradices-inoculated seedlings were lower than those in the non-inoculated seedlings. These results indicated that R. intraradices alleviated As toxicity in R. pseudoacacia seedlings by improving their plant growth, altering root morphology, regulating the concentrations and ratios of phytohormones, and increasing the concentration of soil glomalin. The results suggested that AMF-inoculated R. pseudoacacia seedlings would be a critical factor in successful vegetation restoration and soil development in As-contaminated soils.

Characterization of Arsenic-Induced Cancer Stem-Like Cells.

Arsenic is a well-known human carcinogen. However, the mechanisms underlying arsenic-induced carcinogenesis remain elusive. Here we show that chronic and low level of arsenic stress induces transformation of the human bronchial epithelial cells, BEAS-2B, and that some of the transformed cells show characteristics of cancer stem-like cells (CSCs). Meanwhile, we demonstrate that arsenic stress dedifferentiates CD61+ BEAS-2B cells into CSC-like CD61- cells featured with noncanonical epithelial-mesenchymal transition (EMT), enhanced chemoresistance, and metastasis. Finally, we show that oncogene c-Myc expression is associated with arsenic-induced tumor initiation and progression. Altogether, our findings highlight a unique mechanism of arsenic-induced transformation of human bronchial epithelial cells and provide a novel therapeutic target for arsenic-initiated lung cancer.

Taurocholic acid is an active promoting factor, not just a biomarker of progression of liver cirrhosis: evidence from a human metabolomic study and in vitro experiments.

BACKGROUND: Previous studies have indicated that bile acid is associated with progression of liver cirrhosis. However, the particular role of specific bile acid in the development of liver cirrhosis is not definite. The present study aims to identify the specific bile acid and explore its possible mechanisms in promoting liver cirrhosis. METHODS: Thirty two cirrhotic patients and 27 healthy volunteers were enrolled. Age, gender, Child-Pugh classification and serum of patients and volunteers were collected. Liquid chromatography tandem mass spectrometry (LC-MS) was utilized to determine concentrations of 12 bile acids in serum. Principal component analysis, fold change analysis and heatmap analysis were used to identify the most changed bile acid. And pathway analysis was used to identify the most affected pathway in bile acid metabolism. Spearman rank correlation analysis was employed to assess correlation between concentrations of bile acids and Child-Pugh classification. Hepatic stellate cells (LX-2) were cultured in DMEM. LX-2 cells were also co-cultured with HepG2 cells in the transwell chambers. LX-2 cells were treated with Na+/taurocholate in different concentrations. Western blot was used to evaluate the expression of alpha smooth muscle actin (α-SMA), type I collagen, and Toll-like receptor 4 (TLR4) in LX-2 cells. RESULTS: Concentrations of 12 bile acids in serum of patients and healthy volunteers were determined with LC-MS successively. Principal component analysis, fold change analysis and heatmap analysis identified taurocholic acid (TCA) to be the most changed bile acid. Pathway analysis showed that TCA biosynthesis increased significantly. Spearman rank correlation analysis showed that concentration of TCA in serum of cirrhotic patients was positively associated with Child-Pugh classification. TCA increased the expression of α-SMA, type I collagen, and TLR4 in LX-2 cells. Moreover, the above effect was strengthened when LX-2 cells were co-cultured with HepG2 cells. CONCLUSIONS: Increased TCA concentration in serum of liver cirrhotic patients is mainly due to increased bile acid biosynthesis. TCA is an active promoter of the progression of liver cirrhosis. TCA promoting liver cirrhosis is likely through activating hepatic stellate cells via upregulating TLR4 expression. TCA is a potential therapeutic target for the prevention and treatment of liver cirrhosis.

Arsenic-induced sub-lethal stress reprograms human bronchial epithelial cells to CD61¯ cancer stem cells.

In the present report, we demonstrate that sub-lethal stress induced by consecutive exposure to 0.25 µM arsenic (As3+) for six months can trigger reprogramming of the human bronchial epithelial cell (BEAS-2B) to form cancer stem cells (CSCs) without forced introduction of the stemness transcription factors. These CSCs formed from As3+-induced sub-lethal stress featured with an increased expression of the endogenous stemness genes, including Oct4, Sox2, Klf4, Myc, and others that are associated with the pluripotency and self-renewal of the CSCs. Flow cytometry analysis indicated that 90% of the CSC cells are CD61¯, whereas 100% of the parental cells are CD61+. These CD61¯ CSCs are highly tumorigenic and metastatic to the lung in xenotransplantation tests in NOD/SCID Il2rγ-/- mice. Additional tests also revealed that the CD61¯ CSCs showed a significant decrease in the expression of the genes important for DNA repair and oxidative phosphorylation. To determine the clinical relevance of the above findings, we stratified human lung cancers based on the level of CD61 protein and found that CD61low cancer correlates with poorer survival of the patients. Such a correlation was also observed in human breast cancer and ovarian cancer. Taken together, our findings suggest that in addition to the traditional approaches of enforced introduction of the exogenous stemness circuit transcription factors, sub-lethal stress induced by consecutive low dose As3+ is also able to convert non-stem cells to the CSCs.

Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells.

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-l-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H2O2, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H2O2. Furthermore, both arsenic and H2O2 were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation.

Cold acclimation induces freezing tolerance via antioxidative enzymes, proline metabolism and gene expression changes in two chrysanthemum species.

Cold acclimation is necessary for chrysanthemum to achieve its genetically determined maximum freezing tolerance, but the underlying physiological and molecular mechanisms are unclear. The aim of this study was to discover whether changes in antioxidative enzymes, proline metabolism and frost-related gene expression induced by cold acclimation are related to freezing tolerance. Our results showed that the semi-lethal temperature (LT50) decreased from -7.3 to -23.5 °C in Chrysanthemum dichrum and -2.1 to -7.1 °C in Chrysanthemum makinoi, respectively, after cold acclimation for 21 days. The activities of SOD, CAT and APX showed a rapid and transient increase in the two chrysanthemum species after 1 day of cold acclimation, followed by a gradual increase during the subsequent days and then stabilization. qRT-PCR analysis showed that the expression levels of some isozyme genes (Mn SOD, CAT and APX) were upregulated, which was consistent with the SOD, CAT and APX activities, while others remained relatively constant (Fe SOD and Cu/Zn SOD). P5CS and PDH expression were increased under cold acclimation and the level of P5CS presented similar trends as proline content, indicating proline accumulation was via P5CS and PDH cooperation. Cold acclimation also promoted DREB, COR413 and CSD gene expression. The activities of three enzymes and gene expression were higher in C. dichrum than in C. makinoi after cold acclimation. Our data suggested that cold-inducible freezing-tolerance could be attributed to higher activity of antioxidant enzymes, and increased proline content and frost-related gene expression during different periods.

Gefitinib resistance resulted from STAT3-mediated Akt activation in lung cancer cells.

Hyperactivation of Epidermal Growth Factor Receptor (EGFR) tyrosine kinase is prevalent in human lung cancer and its inhibition by the tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib, initially controls tumor growth. However, most patients ultimately relapse due to the development of drug resistance. In this study, we have discovered a STAT3-dependent Akt activation that impairs the efficacy of gefitinib. Mechanistically, gefitinib increased association of EGFR with STAT3, which de-repressed STAT3 from SOCS3, an upstream suppressor of STAT3. Such a de-repression of STAT3 in turn fostered Akt activation. Genetic or pharmacological inhibition of STAT3 abrogated Akt activation and combined gefitinib with STAT3 inhibition synergistically reduced the growth of the tumor cells. Taken together, this study suggests that activation of STAT3 is an intrinsic mechanism of drug resistance in response to EGFR TKIs. Combinational targeting on both EGFR and STAT3 may enhance the efficacy of gefitinib or other EGFR TKIs in lung cancer.

Mdig de-represses H19 large intergenic non-coding RNA (lincRNA) by down-regulating H3K9me3 and heterochromatin.

Mineral dust-induced gene (mdig) had been linked to the development of human lung cancers associated with environmental exposure to mineral dust, tobacco smoke or other carcinogens. In the present studies, we demonstrated that the overexpression of mdig in A549 adenocarcinomic human alveolar type II epithelial cells decreases the heterochromatin conformation of the cells and de-represses the transcription of genes in the tandemly repeated DNA regions. Although mdig can only cause a marginal decrease of the total histone H3 lysine 9 trimethylation (H3K9me3), a significant reduction of H3K9me3 in the promoter region of H19, the paternally imprinted but maternally expressed gene transcribing a large intergenic non-coding RNA (lincRNA), was observed in the cells with mdig overexpression. Silencing mdig by either shRNA or siRNA not only increased the level of H3K9me3 in the promoter region of H19 but also attenuated the transcription of H19 long non-coding RNA. Demethylation assays using immunoprecipitated mdig and histone H3 peptide substrate suggested that mdig is able to remove the methyl groups from H3K9me3. Clinically, we found that higher levels of mdig and H19 expression correlate with poorer survival of the lung cancer patients. Taken together, our results imply that mdig is involved in the regulation of H3K9me3 to influence the heterochromatin structure of the genome and the expression of genes important for cell growth or transformation.

Anatomical and physiological differences and differentially expressed genes between the green and yellow leaf tissue in a variegated chrysanthemum variety.

The leaves of the chrysanthemum variety 'NAU04-1-31-1' are variegated with distinct green and yellow sectors. The chlorophyll content of the yellow leaf tissue is less than that in the green one. The chloroplasts in the yellow leaf tissue were vacuolated, lacked thylakoid membrane structure and contained clusters of plastoglobuli with few or no starch grains. The yellow leaf tissue was more sensitive to photo-inhibition than the green leaf tissue. Suppression subtractive hybridization (SSHs) libraries were constructed to identify genes which were differentially transcribed in the two tissue types. The sequencing of 339 SSH clones identified 150 unigenes (93 singletons and 57 contigs), of which 85 were differentially transcribed in the green leaf tissue and 65 in the yellow leaf tissue. Unigenes associated with photosynthesis were particularly frequent, and many of these genes were up-regulated in the yellow leaf tissue. Both CmChlH which encodes the large subunit of Mg-chelatase and CmFtsH (ATP-dependent metalloprotease) were up-regulated in the yellow leaf tissue, and their transcription was regulated by light.

JNK and STAT3 signaling pathways converge on Akt-mediated phosphorylation of EZH2 in bronchial epithelial cells induced by arsenic.

The molecular mechanisms by which arsenic (As ( 3+) ) causes human cancers remain to be fully elucidated. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb-repressive complexes 2 (PRC2) that promotes trimethylation of lysine 27 of histone H3, leading to altered expression of tumor suppressors or oncogenes. In the present study, we determined the effect of As ( 3+) on EZH2 phosphorylation and the signaling pathways important for As ( 3+) -induced EZH2 phosphorylation in human bronchial epithelial cell line BEAS-2B. The involvement of kinases in As ( 3+) -induced EZH2 phosphorylation was validated by siRNA-based gene silencing. The data showed that As ( 3+) can induce phosphorylation of EZH2 at serine 21 in human bronchial epithelial cells and that the phosphorylation of EZH2 requires an As ( 3+) -activated signaling cascade from JNK and STAT3 to Akt. Transfection of the cells with siRNA specific for JNK1 revealed that JNK silencing reduced serine727 phosphorylation of STAT3, Akt activation and EZH2 phosphorylation, suggesting that JNK is the upstream kinase involved in As ( 3+) -induced EZH2 phosphorylation. Because As ( 3+) is capable of inducing miRNA-21 (miR-21), a STAT3-regulated miRNA that represses protein translation of PTEN or Spry2, we also tested the role of STAT3 and miR-21 in As ( 3+) -induced EZH2 phosphorylation. Ectopic overexpression of miR-21 promoted Akt activation and phosphorylation of EZH2, whereas inhibiting miR-21 by transfecting the cells with anti-miR-21 inhibited Akt activation and EZH2 phosphorylation. Taken together, these results demonstrate a contribution of the JNK, STAT3 and Akt signaling axis to As ( 3+) -induced EZH2 phosphorylation. Importantly, these findings may reveal new molecular mechanisms underlying As ( 3+) -induced carcinogenesis.

Detection of carboxyhemoglobin in patients with hepatic encephalopathy due to hepatitis B virus-related cirrhosis.

BACKGROUND: The heme oxygenase/carbon monoxide (HO/CO) system plays an important role in the development of hepatic fibrosis. The level of the HO/CO can be directly obtained by determining the carboxyhemoglobin (COHb) level. The aims of this study were to reveal the significance of COHb in patients with hepatitis B virus-related cirrhosis (HBC) complicated by hepatic encephalopathy (HE), and to further investigate the influence of the HO/CO pathway on the end-stage cirrhosis, hoping to find a reliable indicator to evaluate the course of HBC. METHODS: According to the diagnostic criteria, 63 HBC inpatients with HE were enrolled in group H. Patients regaining awareness with current therapies were categorized into group P-H. Comparisons were made with a control group (group N) consisting of 20 health volunteers. The levels of COHb, partial pressure of oxygen (PaO2) and oxygen saturation (SaO2) were determined by arterial blood gas analysis method. The incidences of hepatorenal syndrome (HRS), upper gastrointestinal bleeding, esophagogastric varices and spontaneous bacterial peritonitis (SBP) in group H were recorded. COHb levels in different groups were compared, and the correlations of COHb levels with HE grades (I, II, III, and IV), PaO2, SaO2 and hypoxemia were analyzed. RESULTS: The COHb level in group P-H ((1.672 ± 0.761)%) was significantly higher than that in group N ((0.983 ± 0.231)%) (P < 0.01), and the level in group H ((2.102 ± 1.021)%) was significantly higher than groups P-H and N (P < 0.01). A positive correlation was observed between the COHb concentration and the grade of HE (r(s) = 0.357, P = 0.004). There were no significant differences of COHb levels between HE patients with and without complications such as esophagogastric varices ((2.302 ± 1.072)% vs. (1.802 ± 1.041)%, P > 0.05) or the occurrence of SBP ((2.960 ± 0.561)% vs. (2.030 ± 1.021)%, P > 0.05). Compared with HE patients with HRS, the level of COHb was significantly higher in HE patients without HRS ((2.502 ± 1.073)% vs. (1.981 ± 1.020)%, P = 0.029). The COHb level had a negative correlation with PaO2 (r = -0.335, P = 0.007) while no statistically significant relationship was found with SaO2 (r = -0.071, P > 0.05). However, when the above two parameters met the diagnostic criteria of hypoxemia, the COHb concentration increased ((2.621 ± 0.880)% vs. (1.910 ± 0.931)%, P = 0.011). CONCLUSIONS: COHb is a potential candidate to estimate the severity and therapeutic effect of HE. The levels of COHb may be tissue-specific in cirrhotic patients with different complications.

Functional characterization of a Chrysanthemum dichrum stress-related promoter.

A 1,474-bp stress-inducible CdDREBa promoter was identified from Chrysanthemum dichrum, revealing several candidate stress-related cis-acting elements (MYC-box, MYB site, GT-1, and W-box) within it. In Arabidopsis leaf tissues transformed with a CdDREBa promoter-ß-glucuronidase (GUS) gene fusion, serially 5'-deleted CdDREBa promoters were differentially activated by cold and salinity. Histochemical and quantitative assays of GUS expression allowed us to localize a critical part of the promoter located between upstream 430 and 351 nt. This 80-bp fragment enhanced GUS expression under salinity stress when fused to -90/+8 CaMV 35S minimal promoter. Further promoter internal-deletion assays indicated that a low temperature-responsive element was located between positions -430 and -390, and a salinity inducible one between -385 and -351. Our results showed that there was a novel stress-related critical region except for the known cis-acting element (MYC-box, GT-1) in CdDREBa promoter.

Activation of Akt/GSK3ß and Akt/Bcl-2 signaling pathways in nickel-transformed BEAS-2B cells.

The Akt signaling pathway has been implicated in a wide range of cellular functions involving cell survival and proliferation, angiogenesis, metabolism and cell migration. Accumulating evidence suggests that Akt perturbations play an important role in human malignancy. Here, we investigated Akt perturbation in nickel-transformed cells. Chronic treatment of human bronchial epithelial BEAS-2B cells with low doses of nickel chloride resulted in cell transformation demonstrated by anchorage-independent (AI) growth, increased cell growth and alterations of cell growth pattern. Western blot assays show that phosphorylation of Akt at Ser473, but not that of p38, JNK and ERK, was increased in nickel-transformed cells compared with controls. Inhibition of Akt or PI3K by pharmacological or biochemical interference suppressed nickel AI growth and cell growth of transformed cells. Activation of Akt led to inhibition of GSK3ß by phosphorylation at Ser9 in nickel-transformed cells. In addition, two major anti-apoptotic proteins of the Bcl family, Bcl-2 and Bcl-XL, were increased in nickel-transformed cells. By employing the small interfering RNA technique (siRNA), our results showed that siRNA Akt attenuated the expression of Bcl-2 and Bcl-XL in nickel-transformed cells, indicating that induction of Bcl-2 and Bcl-XL was likely mediated through Akt. ROS generation was decreased in nickel-transformed cells compared with controls. Moreover, down-regulation of retinoblastoma protein (Rb) was observed in nickel-transformed cells. Taken together, these findings demonstrate that activation of Akt, followed by GSK3ß inhibition and Bcl-2, Bcl-XL up-regulation and decrease of ROS generation, along with a synergistic effect of Rb down-regulation may cause apoptosis resistance, contributing to the overall mechanism of nickel carcinogenesis.

NADPH oxidase activation is required in reactive oxygen species generation and cell transformation induced by hexavalent chromium.

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Although overproduction of reactive oxygen species (ROS) has been suggested to play a major role in its carcinogenicity, the mechanisms of Cr(VI)-induced ROS production remain unclear. In this study, we investigated the role of NADPH oxidase (NOX), one of the major sources of cellular ROS, in Cr(VI)-induced oxidative stress and carcinogenesis. We found that short-term exposure to Cr(VI) (2µM) resulted in a rapid increase in ROS generation in Beas-2B cells, and concomitantly increased NOX activity and expression of NOX members (NOX1-3 and NOX5) and subunits (p22(phox), p47(phox), p40(phox), and p67(phox)). Cr(VI) also induced phosphorylation of p47(phox) and membrane translocation of p47(phox) and p67(phox), further confirming NOX activation. Knockdown of p47(phox) with a short hairpin RNA attenuated the ROS production induced by Cr(VI). Chronic exposure (up to 3 months) to low doses of Cr(VI) (0.125, 0.25, and 0.5µM) also promoted ROS generation and the expression of NOX subunits, such as p47(phox) and p67(phox), but inhibited the expression of main antioxidant enzymes, such as superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Chronic Cr(VI) exposure resulted in transformation of Beas-2B cells, increasing cell proliferation, anchorage independent growth in soft agar, and forming aggressive tumors in nude mice. Stable knockdown of p47(phox) or overexpression of SOD1, SOD2, or catalase (CAT) eliminated Cr(VI)-induced malignant transformation. Our results suggest that NOX plays an important role in Cr(VI)-induced ROS generation and carcinogenesis.

Nickel-induced down-regulation of ΔNp63 and its role in the proliferation of keratinocytes.

Epidemiological, animal, and cell studies have demonstrated that nickel compounds are human carcinogens. The mechanisms of their carcinogenic actions remain to be investigated. p63, a close homologue of the p53 tumor suppressor protein, has been linked to cell fate determination and/or maintenance of self-renewing populations in several epithelial tissues, including skin, mammary gland, and prostate. ΔNp63, a dominant negative isoform of p63, is amplified in a variety of epithelial tumors including squamous cell carcinomas and carcinomas of the prostate and mammary glands. The present study shows that nickel suppressed ΔNp63 expression in a short-time treatment (up to 48 h). Nickel treatment caused activation of NF-κB. Blockage of NF-κB partially reversed nickel-induced ΔNp63 suppression. Nickel decreased interferon regulatory factor (IRF) 3 and IRF7, IKKε, and Sp100. Over-expression of IRF3 increased ΔNp63 expression suppressed by nickel. Nickel was able to activate p21, and its activation was offset by the over-expression of ΔNp63. In turn, elevated p63 expression counteracted the ability of nickel to restrict cell growth. The present study demonstrated that nickel decreased interferon regulatory proteins IRF3 and IRF7, and activated NF-κB, resulting in ΔNp63 suppression and then p21 up-regulation. ΔNp63 plays an important role in nickel-induced cell proliferation.

Reduced reactive oxygen species-generating capacity contributes to the enhanced cell growth of arsenic-transformed epithelial cells.

Reactive oxygen species (ROS) have been implicated in the activation of protein kinases, DNA damage responses, and cell apoptosis. The details of how ROS regulate these intracellular biochemical and genetic processes remain to be fully understood. By establishing transformed bronchial epithelial cells through chronic low-dose arsenic treatment, we showed that the capacity of ROS generation induced by arsenic is substantially reduced in the transformed cells relative to the nontransformed cells. Such a reduction in ROS generation endows cells with premalignant features, including rapid growth, resistance to arsenic toxicity, and increased colony formation of the transformed cells. To validate these observations, the capability of ROS generation was restored in the transformed cells by treatment with inhibitors or siRNAs to silence the function of superoxide dismutase (SOD) or catalase and cell growth was determined following these treatments. Enhancement in ROS generation suppressed cell growth and colony formation of the transformed cells significantly. Despite the fact that the transformed cells showed a decreased expression of NF-kappaB signaling proteins IKKbeta and IKKgamma, the proteolytic processing p105 and p100 and NF-kappaB DNA binding activity were elevated in these cells. Increasing ROS generation by silencing SOD and catalase reduced the DNA binding activity of NF-kappaB in the transformed cells. Taken together, the transformed cells induced by arsenic exhibited a decrease in ROS generation, which is responsible for the enhanced cell growth and colony formation of the transformed cells, most likely through a sustained alternative activation of the NF-kappaB transcription factor.

Arsenic inhibits neurite outgrowth by inhibiting the LKB1-AMPK signaling pathway.

BACKGROUND: Arsenic (As) is an environmental pollutant that induces numerous pathological effects, including neurodevelopmental disorders. OBJECTIVES AND METHODS: We evaluated the role of the LKB1-AMPK pathway in As-induced developmental neurotoxicity using Neuro-2a (N2a) neuroblastoma cells as a model of developing neurons. RESULTS: The addition of low concentrations of As (

Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells.

Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.