Advantages of the doxorubicin-resistant, acute myelogenous leukemia cell line, AML-2/DX100, for the screening of multidrug resistance protein inhibitors and pro-oxidants

The doxorubicin-resistant, acute myelogenous leukemia cell line, AML-2/DX100, characterized by the over-expression of multidrug resistance protein (MRP) and the down-regulation of catalase, has advantages for the screening of MRP inhibitors as well as for cytotoxic substances producing potential reactive oxygen species. The screening power of AML-2/DX100 cells for an MRP inhibitor, probenecid, was approximately 4-fold stronger than that of another resistant cell line, HL-60/Adr, over-expressing MRP. AML-2/DX100 was approximately 2- to 5-fold more sensitive to pro-oxidants such as Paraquat, H₂O₂ and t-butyl hydroperoxide, when compared with its parental cells.     

Trichostatin A,a histone deacetylase inhibitor suppresses NADPH Oxidase 4‐Derived Redox Signalling and Angiogenesis

Histone deacetylase (HDAC) inhibitors are known to suppress abnormal development of blood vessels. Angiogenic activity in endothelial cells depends upon NADPH oxidase 4 (Nox4)‐dependent redox signalling. We set out to study whether the HDAC inhibitor trichostatin A (TSA) affects Nox4 expression and angiogenesis. Nox4 expression was measured by real time PCR and Western blot analysis in endothelial cells. Hydrogen peroxide (H₂O₂) was measured by amplex^® red assay in endothelial cells. Nox4 was knocked down by Nox4 shRNA. In vitro angiogenic activities such migration and tubulogenesis were assessed using wound healing and Matrigel assays, respectively. In vivo angiogenic activity was assessed using subcutaneous sponge assay in C57Bl/6 and Nox4‐deficient mice. Trichostatin A reduced Nox4 expression in a time‐ and concentration‐dependent manner. Both TSA and Nox4 silencing decreased Nox4 protein and H₂O₂. Mechanistically, TSA reduced expression of Nox4 via ubiquitination of p300‐ histone acetyltransferase (p300‐HAT). Thus, blocking of the ubiquitination pathway using an inhibitor of ubiquitin‐activating enzyme E1 (PYR‐41) prevented TSA inhibition of Nox4 expression. Trichostatin A also reduced migration and tube formation, and these effects were not observed in Nox4‐deficient endothelial cells. Finally, transforming growth factor beta1 (TGFβ1) enhanced angiogenesis in sponge model in C57BL/6 mice. This response to TGFβ1 was substantially reduced in Nox4‐deficient mice. Similarly intraperitoneal infusion of TSA (1 mg/kg) also suppressed TGFβ1‐induced angiogenesis in C57BL/6 mice. Trichostatin A reduces Nox4 expression and angiogenesis via inhibition of the p300‐HAT‐dependent pathway. This mechanism might be exploited to prevent aberrant angiogenesis in diabetic retinopathy, complicated vascular tumours and malformations.     

Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H₂O₂) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H₂O₂-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.     

Anti-oxidant adaptation in the AML cells supersensitive to hydrogen peroxide

The purpose of this study was to investigate the adaptive mechanisms of hydrogen peroxide-supersensitive AML cells against the reactive oxygen species (ROS). Their scavenging capacity against ROS was determined using a fluorometric probe in the doxorubicin-resistant AML-2/DX100 cell characterized by the down-regulation of catalase. AML-2/DX100 cells had more scavenging capacity against endogenous pro-oxidants than did the parental cells AML-2/WT, suggesting that an anti-oxidant adaptation against ROS occurred. cDNA microarrays for 8000 human genes revealed that among 21 anti-oxidant genes, each four gene was up- and down-regulated more than 1.5-fold in AML-2/DX100 compared with AML-2/WT. The mRNA expression of glutathione S-transferase Pi, peroxiredoxin 2, thioredoxin 2, and glutaredoxin was elevated whereas that of peroxiredoxin 3, metallothionein-1F, superoxide dismutase 2, and thioredoxin reductase 1 was depressed. The result indicates that the down-regulation of certain anti-oxidant mechanisms can be compensated for by the up- and down-regulation of the other anti-oxidant mechanisms.     

Monitoring the gene expression profiles of doxorubicin-resistant acute myelocytic leukemia cells by DNA microarray analysis

Acquired drug-resistance phenotype is a key factor in the relapse of patients suffering hematological malignancies. In order to investigate the genes involved in drug resistance, a human leukemia cell line that is resistant to doxorubicin, an anthracycline anticancer agent (AML-2/DX100), was selected and its gene expression profile was analyzed using a cDNA microarray. A number of genes were differentially expressed in the AML-2/DX100 cells, compared with the wild type (AML-2/WT). Pro-apoptotic genes such as TNFSF7 and p21 (Cip1/Waf1) were significantly down-regulated, whereas the IKBKB, PCNA, stathmin 1, MCM5, MMP-2 and MRP1 genes, which are involved in anti-apoptotic or cell cycle progression, were over-expressed. The AML-2/DX100 cells were also resistant to other anticancer drugs, including daunorubicin and camptothecin, and the expression levels of the differentially regulated genes such as STMN1, MMP-2 and CTSG, were constantly maintained. This suggests that the deregulated genes obtained from the DNA microarray analysis in a cell line model of drug resistance might contribute to the acquired drug resistance after chronic exposure.     

Resveratrol-mediated reversal of doxorubicin resistance in acute myeloid leukemia cells via downregulation of MRP1 expression

Chemo-resistance to anti-cancer drugs is a major obstacle in efforts to develop a successful treatment of acute myeloid leukemia (AML). In this study, we investigate whether resveratrol, a common ingredient in a broad variety of fruits and vegetables, can reverse drug resistance in AML cells. Three doxorubicin-resistant AML cell lines (AML-2/DX30, AML-2/DX100, AML-2/DX300) were prepared via long-term exposure to doxorubicin for more than 3 months. DNA microarray analysis demonstrated that many genes were differentially expressed in the resistant cells, as compared with the wild type AML-2/WT cells. In particular, the expression level of the MRP1 gene was significantly increased in the AML-2/DX300 cells, as compared to that detected in AML-2 cells. Importantly, the resveratrol was shown not only to induce cell growth arrest and apoptotic death in doxorubicin-resistant AML cells, but was also shown to downregulate the expression of an MRP1 gene. Furthermore, resveratrol treatment induced a significant increase in the uptake of 5(6)-carboxyfluorescein diacetate, a MRP1 substrate, into the doxorubicin-resistant AML-2/DX300 cells. The results of this study show that resveratrol may facilitate the cellular uptake of doxorubicin via an induced downregulation of MRP1 expression, and also suggest that it may prove useful in overcoming doxorubicin resistance, or in sensitizing doxorubicin-resistant AML cells to anti-leukemic agents.     

Double-edged sword of chemosensitizer: increase of multidrug resistance protein (MRP) in leukemic cells by an MRP inhibitor probenecid

The multidrug resistance protein (MRP) is a drug efflux membrane pump conferring multidrug resistance to tumor cells. Clinical trials have been undertaken to improve the effectiveness of chemotherapy by adding an MRP inhibitor to the treatment regimen. This study attempted not only to determine novel resistance mechanisms in MRP-overexpressing AML cells (AML-2/DX100) by chronic exposure to doxorubicin in the presence of an MRP inhibitor probenecid but also to find out whether probenecid could increase MRP levels. AML-2/DXPBA cultured in the presence of probenecid (600 microM) and doxorubicin (100 ng/ml) showed a higher level of the multidrug resistance (MDR) phenotype when compared to AML-2/DX100. AML-2/DXPBA showed increased levels of MRP compared to those of AML-2/DX100. Probenecid increased the MRP levels without an increase in MRP mRNA in AML-2/WT in both a time- and dose-dependent manner. Of the MRP inhibitors including probenecid, ofloxacin, erythromycin, and rifampicin used in this study, only probenecid showed a marked chemosensitizing effect in AML-2/DX100 but not in HL-60/Adr, suggesting that the chemosensitizing effects of the MRP inhibitors vary according to the type of resistant cells. The maximum noncytotoxic concentrations of these MRP inhibitors increased the MRP levels to various degrees in both AML-2/WT and HL-60/WT. However, the chemosensitizing effects of the MRP inhibitors were not correlated with their MRP-increasing effects. Altogether, MRP inhibitors such as probenecid have been shown to function as a double-edged sword, indicating that they are not only an effective chemosensitizer of MRP-associated MDR tumor cells but also an MRP activator. Therefore caution should be taken whenever using MRP inhibitors to reverse MRP-mediated multidrug resistance in clinical cancer chemotherapy as well as when used to inhibit MRP expression in vitro.     

Trichostatin a modulates intracellular reactive oxygen species through SOD2 and FOXO1 in human bone marrow‐mesenchymal stem cells

Engraft cells are often exposed to oxidative stress and inflammation; therefore, any factor that can provide the stem cells resistance to these stresses may yield better efficacy in stem cell therapy. Studies indicate that histone deacetylase (HDACs) inhibitors alleviate damage induced by oxidative stress. In this study, we investigated whether regulation of reactive oxygen species (ROS) occurs through the HDAC inhibitor trichostatin A (TSA) in human bone marrow‐mesenchymal stem cells (hBM‐MSCs). Intracellular ROS levels increased following exposure to hydrogen peroxide (H₂O₂), and were suppressed by TSA treatment. Levels of the antioxidant enzyme superoxide dismutase 2 (SOD2) increased following treatment with 200 nM TSA and to a lesser level at 1–5 μM TSA. Cell protective effects against oxidative stress were significantly increased in TSA‐MSCs after treatment with low doses of TSA (50–500 nM) and decreased with high doses of TSA (5–10 μM). Consistent results were obtained with immunoblot analysis for caspase3. Investigation of Forkhead box O1 (FOXO1), superoxide dismutase 2 (SOD2), and p53 levels to determine intracellular signaling by TSA in oxidative stress‐induced MSCs demonstrated that expression of phosphorylated‐FOXO1 and phosphorylated‐SOD2 decreased in H₂O₂‐treated MSCs while levels of p53 increased. These effects were reversed by the treatment of 200 nM TSA. These results suggest that the main function of ROS modulation by TSA is activated through SOD2 and FOXO1. Thus, optimal treatment with TSA may protect hBM‐MSCs against oxidative stress. Copyright © 2014 John Wiley & Sons, Ltd.     

Hydrogen peroxide permeability of plasma membrane aquaporins of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Although aquaporins have been known to transport hydrogen peroxide (H₂O₂) across cell membranes, the H₂O₂-regulated expression patterns and the permeability of every family member of the plasma membrane intrinsic protein (PIP) toward H₂O₂ have not been determined. This study investigates the H₂O₂-regulated expression levels of all plasma membrane aquaporins of Arabidopsis thaliana (AtPIPs), and determines the permeability of every AtPIP for H₂O₂ in yeast. Hydrogen peroxide treatment of Arabidopsis down-regulated the expression of AtPIP2 subfamily in roots but not in leaves, whereas the expression of AtPIP1 subfamily was not affected by H₂O₂ treatment. The growth and survival of yeast cells that expressed AtPIP2;2, AtPIP2;4, AtPIP2;5, or AtPIP2;7 was reduced in the presence of H₂O₂, while the growth of yeast cells expressing any other AtPIP family member was not affected by H₂O₂. These results show that only certain isoforms of AtPIPs whose expression is regulated by H₂O₂ treatment are permeable for H₂O₂ in yeast cells, and suggest that the integrated regulation of aquaporin expression by H₂O₂ and the capacity of individual aquaporin to transport H₂O₂ are important for plant response to H₂O₂.     

Hydrogen peroxide production is an early event during bicarbonate induced stomatal closure in abaxial epidermis of <Emphasis Type="Italic">Arabidopsis</Emphasis>

The presence of 2 mM bicarbonate in the incubation medium induced stomatal closure in abaxial epidermis of Arabidopsis. Exposure to 2 mM bicarbonate elevated the levels of H₂O₂ in guard cells within 5 min, as indicated by the fluorescent probe, dichlorofluorescein diacetate (H₂DCF-DA). Bicarbonate-induced stomatal closure as well as H₂O₂ production were restricted by exogenous catalase or diphenylene iodonium (DPI, an inhibitor of NAD(P)H oxidase). The reduced sensitivity of stomata to bicarbonate and H₂O₂ production in homozygous atrbohD/F double mutant of Arabidopsis confirmed that NADP(H) oxidase is involved during bicarbonate induced ROS production in guard cells. The production of H₂O₂ was quicker and greater with ABA than that with bicarbonate. Such pattern of H₂O₂ production may be one of the reasons for ABA being more effective than bicarbonate, in promoting stomatal closure. Our results demonstrate that H₂O₂ is an essential secondary messenger during bicarbonate induced stomatal closure in Arabidopsis.     

Isolation, Purification, and Characterization of Catalase from the Methylotrophic Yeast Pichia pastoris

Catalase (CAT_pp) with molecular weight 223 kD was isolated from the methylotrophic yeast Pichia pastoris and purified 90-fold by ion-exchange chromatography and gel filtration. Quantitative parameters of absorption and CD spectra of CAT_pp solutions and of its membrane-concentrated form (CAT_pp-conc) were studied. Rates of H₂O₂ decomposition and kinetic characteristics K _m and k _cat of CAT_pp and CAT_pp-conc were determined in 10 mM phosphate buffer (pH 7.4) at 30°C, as well as the effective constant k _in of the enzyme inactivation rate during the catalysis and the constant k ₂ of the interaction rate of the Complex I catalases with H₂O₂. Thermal inactivation of CAT_pp in solutions at 45°C was characterized by the effective rate constant k _in ^*, and the low-frequency (27 kHz) ultrasonic inactivation of CAT_pp at 20°C was characterized by the firstorder rate constant k _in (US). All spectral and kinetic characteristics of CAT_pp and CAT_pp-conc were compared with the corresponding values for catalase from bovine liver (CAT) and for catalase from the methylotrophic yeast Candida boidinii (CAT_cb). All three catalases were rather similar in their spectral properties but strongly varied in their kinetic parameters, and their comparison suggests that CAT_pp should be the best enzyme in its overall properties as it displayed the maximal efficiency in terms of k _cat/K _m, thermal stability comparable with the thermal stability of CAT in terms of k _in ^*, the minimal k _in, and high stability in the ultrasonic cavitation field at the US power of 60 W/cm².     

Effects of light-emitting diodes on thermally-induced oxidative stress in the bay scallop Argopecten irradians

ABSTRACT

Water temperature is an important stressor that affects the physiological and biochemical responses of scallops. In this study, we investigated the effect of different light-emitting diodes (LEDs; red, green and blue) on oxidative stress in Argopecten irradians. PCR revealed MnSOD mRNA expression in the digestive diverticula, gill, adductor muscle and eye. CAT and HSP70 mRNA were expressed in the digestive diverticula, gill and adductor muscle. Additionally, we measured the changes in the expression of HSP70, MnSOD and CAT as well as H₂O₂ levels during thermal/laboratory stress. In the digestive diverticula, gill and adductor muscle, the mRNA expressions and activities and H₂O₂ levels significantly increased in response to thermal changes. The gene expressions and activities and H₂O₂ levels were significantly lower in scallops that received green LED light than in those that received no mitigating treatment. A comet assay revealed that thermal change groups had increased rates of nuclear DNA damage; however, treatment with green LED reduced the frequency of damage. The results indicated that low or high water temperature conditions induced oxidative stress in A. irradians but that green LED significantly reduced this stress.     

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H₂O₂) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H₂O₂ treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H₂O₂ was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H₂O₂ induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.