Antitumor effect of the selective hypoxia-inducible factor-1 inhibitors echinomycin and PX-478 on uterine fibroids.

OBJECTIVE: To investigate the antitumor effects of the selective hypoxia-inducible factor-1 (HIF-1) inhibitors echinomycin and PX-478 on uterine fibroids. DESIGN: Experimental study using in vitro primary culture systems and an in vivo mouse xenograft model. SETTING: Academic university center. PATIENT(S): Women with uterine fibroids who underwent hysterectomy or myomectomy. INTERVENTION(S): Administration of the selective HIF-1 inhibitors echinomycin and PX-478 to the media of the primary cultured uterine fibroid cells and to nonobese diabetic/severe combined immunodeficient mice bearing fibroid xenografts consisting of the primary cultured fibroid cells and type Ⅰ collagen gels beneath the kidney capsule. MAIN OUTCOME MEASURE(S): Cell proliferation was measured by Cell Counting Kit-8 assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by measuring caspase 3 and 7 activities. The xenografts were evaluated by gross appearance, surface area, and histology. The Ki-67 index was measured to evaluate proliferation of the xenografts. RESULT(S): Both echinomycin and PX-478 inhibited cell proliferation and induced apoptosis in fibroid cells cultured under hypoxia and normoxia. Enlargement of the fibroid xenografts was significantly attenuated. The Ki-67 index significantly decreased after the administration of the HIF-1 inhibitors in the xenograft model. Eight of 27 xenografts treated with the HIF-1 inhibitors contained calcification and hyalinizing components from 3 days after the grafting to 2 weeks, suggesting that the HIF-inhibitors induce degeneration of the fibroid xenografts. CONCLUSION(S): The selective HIF-1 inhibitors echinomycin and PX-478 show antitumor effects against uterine fibroids both in vitro and in vivo. These findings support the potential use of HIF-1 inhibitors for the treatment of uterine fibroids.

HIF-1α inhibitor PX-478 preserves pancreatic ß cell function in diabetes.

During progression of type 2 diabetes, pancreatic ß cells are subjected to sustained metabolic overload. We postulated that this state mediates a hypoxic phenotype driven by hypoxia-inducible factor-1α (HIF-1α) and that treatment with the HIF-1α inhibitor PX-478 would improve ß cell function. Our studies showed that the HIF-1α protein was present in pancreatic ß cells of diabetic mouse models. In mouse islets with high glucose metabolism, the emergence of intracellular Ca2+ oscillations at low glucose concentration and the abnormally high basal release of insulin were suppressed by treatment with the HIF-1α inhibitor PX-478, indicating improvement of ß cell function. Treatment of db/db mice with PX-478 prevented the rise of glycemia and diabetes progression by maintenance of elevated plasma insulin concentration. In streptozotocin-induced diabetic mice, PX-478 improved the recovery of glucose homeostasis. Islets isolated from these mice showed hallmarks of improved ß cell function including elevation of insulin content, increased expression of genes involved in ß cell function and maturity, inhibition of dedifferentiation markers, and formation of mature insulin granules. In response to PX-478 treatment, human islet organoids chronically exposed to high glucose presented improved stimulation index of glucose-induced insulin secretion. These results suggest that the HIF-1α inhibitor PX-478 has the potential to act as an antidiabetic therapeutic agent that preserves ß cell function under metabolic overload.

Hypoxia inducible factor 1α inhibitor PX-478 reduces atherosclerosis in mice.

BACKGROUND AND AIMS: Hypoxia inducible factor 1α (HIF1α) plays a critical role in atherosclerosis as demonstrated in endothelial-targeted HIF1α -deficient mice. However, it has not been shown if specific pharmacological inhibitors of HIF1α can be used as potential drugs for atherosclerosis. PX-478 is a selective inhibitor of HIF1α, which was used to reduce cancer and obesity in animal models. Here, we tested whether PX-478 can be used to inhibit atherosclerosis. METHODS: We first tested PX-478 in human aortic endothelial cells (HAEC) and found that it significantly inhibited expression of HIF1α and its targets, including Collagen I. Next, two independent atherosclerosis models, C57BL/6 mice treated with AAV-PCSK9 and ApoE-/- mice, were used to test the efficacy of PX-478. Both mouse models were fed a Western diet for 3 months with bi-weekly treatment with PX-478 (40 mg/kg) or saline. RESULTS: PX-478 treatment reduced atherosclerotic plaque burden in the aortic trees in both mouse models, while plaque burden in the aortic sinus was reduced in the AAV-PCSK9 mouse model, but not in the ApoE-/- mice. Russell-Movat's Pentachrome and Picrosirius Red staining showed a significant reduction in extracellular matrix remodeling and collagen maturation, respectively, in the PX-478-treated mice. As expected, PX-478 treatment reduced diet-induced weight-gain and abdominal adipocyte hypertrophy. Interestingly, PX-478 reduced plasma LDL cholesterol by 69% and 30% in AAV-PCSK9 and ApoE-/- mice, respectively. To explore the cholesterol-lowering mechanisms, we carried out an RNA sequencing study using the liver tissues from the ApoE-/- mouse study. We found 450 genes upregulated and 381 genes downregulated by PX-478 treatment in the liver. Further, gene ontology analysis showed that PX-478 treatment upregulated fatty acid and lipid catabolic pathways, while downregulating lipid biosynthesis and plasma lipoprotein particle remodeling processes. Of interest, Cfd, Elovl3, and Insig2 were some of the most downregulated genes by PX-478, and have been implicated in fat storage, fatty acid elongation, and cholesterol metabolism. The downregulation of Cfd, Elovl3, and Insig2 was further validated by qPCR in the liver tissues of ApoE-/- mice treated with PX-478. CONCLUSIONS: These results suggest that PX-478 is a potential anti-atherogenic drug, which targets vascular endothelium and hepatic cholesterol pathways.

Dimeric G-Quadruplex: An Efficient Probe for Ultrasensitive Fluorescence Detection of Mustard Compounds.

Some mustard compounds (mustards) are highly toxic chemical warfare agents. Some are explored as new anticancer drugs. Therefore, the fast, selective, and sensitive detection of mustards is extremely important for public security and cancer therapy. Mustards mostly target the N7 position on the guanine bases of DNA. The guanine-rich G-quadruplex DNA (G4) has been widely studied in the sensing area, and it was found that dimeric G4 (D-G4) could dramatically light up the fluorescence intensity of thioflavin T (ThT). Based on this, we used for the first time the D-G4 DNA as a selective probe for ultrasensitive fluorescence detection of nitrogen mustard (NM). When NM occupies the N7 on guanine, it can block the formation of the D-G4 structure due to the steric hindrance, and hence, it inhibits the combination of D-G4 with ThT, leading to a sharp decrease of fluorescence intensity. The proposed reaction mechanism is proved using ultraviolet-visible (UV-Vis) spectra, circular dichroism (CD) spectra, and polyacrylamide gel electrophoresis. Herein, the concentration of D-G4/ThT used is as low as 50 nM due to its highly fluorescent performance, enabling both high sensitivity and low cost. NM can be detected with a wide linear range from 10 to 2000 nM. The detection limit of NM reaches a surprisingly low concentration of 6 nM, which is 2 or 3 orders of magnitude lower than that of previously developed fluorescence methods for mustards and simulants.

Toxic blister agents: Chemistry, mode of their action and effective treatment strategies.

Since their use during the First World War, Blister agents have posed a major threat to the individuals and have caused around two million casualties. Major incidents occurred not only due to their use as chemical warfare agents but also because of occupational hazards. Therefore, a clear understanding of these agents and their mode of action is essential to develop effective decontamination and therapeutic strategies. The blister agents have been categorised on the basis of their chemistry and the biological interactions that entail post contamination. These compounds have been known to majorly cause blisters/bullae along with alkylation of the contaminated DNA. However, due to the high toxicity and restricted use, very little research has been conducted and a lot remains to be clearly understood about these compounds. Various decontamination solutions and detection technologies have been developed, which have proven to be effective for their timely mitigation. But a major hurdle seems to be the lack of proper understanding of the toxicological mechanism of action of these compounds. Current review is about the detailed and updated information on physical, chemical and biological aspects of various blister agents. It also illustrates the mechanism of their action, toxicological effects, detection technologies and possible decontamination strategies.

Inhibition of hypoxia-inducible factor-1α alleviates acinar cell necrosis in a mouse model of acute pancreatitis.

Hypoxia-inducible factor-1α (Hif1α) is activated in hypoxia and is closely related to oxidative stress, immunity and cell metabolism. Recently, it is reported that Hif1α is involved in atherosclerosis, ischemia-reperfusion (I/R) injury, alcoholic liver disease and pancreatic tumors. In this study, we found that Hif1 signal pathway is significantly changed in pancreas of acute pancreatitis (AP) mice. Meanwhile, we verified that the high expression of Hif1α injured pancreatic tissues of cerulean-induced AP mice, which prompting that Hif1α participated in the progress of histopathology on AP. We applied a Hif1α inhibitor PX478 and observed that it could alleviate histological injury of pancreas as well as the levels of serum amylase, lipase and proinflammatory cytokine in the murine model of AP induced by caerulein. In addition, PX478 could reduce the formation of necrosome (RIP3 and p-MLKL) and the generation of reactive oxygen species (ROS) in AP mice. Correspondingly, we further confirmed the effectiveness of PX478 in vitro and found that inhibiting Hif1α could mitigated the necrosis of pancreatic acinar cells via reducing the RIP3 and p-MLKL expression and the ROS production. In conclusion, inhibiting Hif1α could protect against acinar cells necrosis in AP, which may provide a new target for the prevention and treatment of AP clinically.

PTEN protects kidney against acute kidney injury by alleviating apoptosis and promoting autophagy via regulating HIF1-α and mTOR through PI3K/Akt pathway.

Phosphatase and tensin homolog (PTEN) deleted on human chromosome 10 is a tumor suppressor with bispecific phosphatase activity, which is often involved in the study of energy metabolism and tumorigenesis. PTEN is recently reported to participate in the process of acute injury. However, the mechanism of PTEN in Ischemia-Reperfusion Injury (IRI) has not yet been clearly elucidated. In this study, mice with bilateral renal artery ischemia-reperfusion and HK-2 cells with hypoxia/reoxygenation (H/R) were used as acute kidney injury models. We demonstrated that PTEN was downregulated in IRI-induced kidney as well as in H/R-induced HK-2 cells. By silencing and overexpressing PTEN with si-PTEN RNA and PHBLV-CMV-PTEN-flag lentivirus before H/R, we found that PTEN protected HK-2 cells against H/R-induced injury reflected by the change in cell activity and the release of LDH. Furthermore, we inhibited HIF1-α with PX-478 and inactivated mTOR with Rapamycin before the silence of PTEN in H/R model. Our data indicated that the renoprotective effect of PTEN worked via PI3K/Akt/mTOR pathway and PI3K/Akt/HIF1-α pathway, hence alleviating apoptosis and improving autophagy respectively. Our findings provide valuable insights into the molecular mechanism underlying renoprotection of PTEN on autophagy and apoptosis induced by renal IRI, which offers a novel therapeutic target for the treatment of AKI.

Dichloroacetate and PX-478 exhibit strong synergistic effects in a various number of cancer cell lines.

BACKGROUND: One key approach for anticancer therapy is drug combination. Drug combinations can help reduce doses and thereby decrease side effects. Furthermore, the likelihood of drug resistance is reduced. Distinct alterations in tumor metabolism have been described in past decades, but metabolism has yet to be targeted in clinical cancer therapy. Recently, we found evidence for synergism between dichloroacetate (DCA), a pyruvate dehydrogenase kinase inhibitor, and the HIF-1α inhibitor PX-478. In this study, we aimed to analyse this synergism in cell lines of different cancer types and to identify the underlying biochemical mechanisms. METHODS: The dose-dependent antiproliferative effects of the single drugs and their combination were assessed using SRB assays. FACS, Western blot and HPLC analyses were performed to investigate changes in reactive oxygen species levels, apoptosis and the cell cycle. Additionally, real-time metabolic analyses (Seahorse) were performed with DCA-treated MCF-7 cells. RESULTS: The combination of DCA and PX-478 produced synergistic effects in all eight cancer cell lines tested, including colorectal, lung, breast, cervical, liver and brain cancer. Reactive oxygen species generation and apoptosis played important roles in this synergism. Furthermore, cell proliferation was inhibited by the combination treatment. CONCLUSIONS: Here, we found that these tumor metabolism-targeting compounds exhibited a potent synergism across all tested cancer cell lines. Thus, we highly recommend the combination of these two compounds for progression to in vivo translational and clinical trials.

FIH-1-modulated HIF-1α C-TAD promotes acute kidney injury to chronic kidney disease progression via regulating KLF5 signaling.

Incomplete recovery from episodes of acute kidney injury (AKI) can predispose patients to develop chronic kidney disease (CKD). Although hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the response to hypoxia/ischemia, the role of HIF-1α in CKD progression following incomplete recovery from AKI is poorly understood. Here, we investigated this issue using moderate and severe ischemia/reperfusion injury (I/RI) mouse models. We found that the outcomes of AKI were highly associated with the time course of tubular HIF-1α expression. Sustained activation of HIF-1α, accompanied by the development of renal fibrotic lesions, was found in kidneys with severe AKI. The AKI to CKD progression was markedly ameliorated when PX-478 (a specific HIF-1α inhibitor, 5 mg· kg-1·d-1, i.p.) was administered starting on day 5 after severe I/RI for 10 consecutive days. Furthermore, we demonstrated that HIF-1α C-terminal transcriptional activation domain (C-TAD) transcriptionally stimulated KLF5, which promoted progression of CKD following severe AKI. The effect of HIF-1α C-TAD activation on promoting AKI to CKD progression was also confirmed in in vivo and in vitro studies. Moreover, we revealed that activation of HIF-1α C-TAD resulted in the loss of FIH-1, which was the key factor governing HIF-1α-driven AKI to CKD progression. Overexpression of FIH-1 inhibited HIF-1α C-TAD and prevented AKI to CKD progression. Thus, FIH-1-modulated HIF-1α C-TAD activation was the key mechanism of AKI to CKD progression by transcriptionally regulating KLF5 pathway. Our results provide new insights into the role of HIF-1α in AKI to CKD progression and also the potential therapeutic strategy for the prevention of renal diseases progression.

Mustards-Derived Terpyridine-Platinum Complexes as Anticancer Agents: DNA Alkylation vs Coordination.

The development of bifunctional platinum complexes with the ability to interact with DNA via different binding modes is of interest in anticancer metallodrug research. Therefore, we report platinum(II) terpyridine complexes to target DNA by coordination and/or through a tethered alkylating moiety. The platinum complexes were evaluated for their in vitro antiproliferative properties against the human cancer cell lines HCT116 (colorectal), SW480 (colon), NCI-H460 (non-small cell lung), and SiHa (cervix) and generally exhibited potent antiproliferative activity although lower than their respective terpyridine ligands. 1H NMR spectroscopy and/or ESI-MS studies on the aqueous stability and reactivity with various small biomolecules, acting as protein and DNA model compounds, were used to establish potential modes of action for these complexes. These investigations indicated rapid binding of complex PtL3 to the biomolecules through coordination to the Pt center, while PtL4 in addition alkylated 9-ethylguanine. PtL3 was investigated for its reactivity to the model protein hen egg white lysozyme (HEWL) by protein crystallography which allowed identification of the Nδ1 atom of His15 as the binding site.

Protective effects of PX478 on gut barrier in a mouse model of ethanol and burn injury.

Ethanol remains a confounder in postburn pathology, which is associated with an impaired intestinal barrier. Previously, we demonstrated that ethanol and burn injury reduce intestinal oxygen delivery (hypoxia) and alters microRNA (miR) expression in small intestinal epithelial cells. Hypoxia has been shown to influence expression of miRs and miR biogenesis components. Therefore, we examined whether hypoxia influences expression of miR biogenesis components (drosha, dicer, and argonaute-2 [ago-2]) and miRs (-7a and -150) and whether these changes impacted other parameters following ethanol and burn injury. Mice were gavaged with ethanol (∼2.9 g/kg) 4 h before receiving a ∼12.5% total body surface full thickness burn. Mice were resuscitated at the time of injury with normal saline with or without 5 mg/kg PX-478, a hypoxia-inducible factor-1α inhibitor. One day following injury mice were euthanized, and the expression of miRs and their biogenesis components as well as bacterial growth, tight junction proteins, intestinal transit, and permeability were assessed. Ethanol combined with burn injury significantly reduced expression of drosha, ago-2, miRs (-7a and -150), occludin, zonula occludens-1, claudin-4, zonula occludens-1, mucins-2 and -4, and intestinal transit compared to shams. Furthermore, there was an increase in intestinal permeability, total bacteria, and Enterobacteriaceae populations following the combined injury compared to shams. PX-478 treatment improved expression of drosha, ago-2, miRs (-7a and -150), occludin, claudin-4, zonula occludens-1, and mucin-2. PX-478 treatment also improved intestinal transit and reduced dysbiosis and permeability. These data suggest that PX-478 improves miR biogenesis and miR expression, and restores barrier integrity while reducing bacterial dysbiosis following ethanol and burn injury.

Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro†.

Various metabolic and hormonal factors expressed in cumulus cells are positively correlated with the in vitro maturation (IVM) of oocytes. However, the role of hypoxia sensing both during maturation of cumulus-oocyte complexes (COCs) as well as during the resumption of meiosis remains uncertain. HIF1alpha plays major roles in cellular responses to hypoxia, and here we investigated its role during bovine COC maturation by assessing the expression of related genes in cumulus cells. COCs were divided into the following groups: immature (control), in vitro matured (IVM/control), or matured in the presence of a blocker of HIF1alpha activity (echinomycin, IVM/E). We found an inhibition of cumulus cell expansion in IVM/E, compared with the IVM/control. Transcript levels of several factors (n = 13) were assessed in cumulus cells. Decreased expression of HAS2, TNFAIP6, TMSB4, TMSB10, GATM, GLUT1, CX43, COX2, PTGES, and STAR was found in IVM/E (P < 0.05). Additionally, decreased protein levels were detected for STAR, HAS2, and PCNA (P < 0.05), while activated-Caspase 3 remained unaffected in IVM/E. Progesterone output decreased in IVM/E. The application of PX-478, another blocker of HIF1alpha expression, yielded identical results. Negative effects of HIF1alpha suppression were further observed in the significantly decreased oocyte maturation and blastocyst rates from COCs matured with echinomycin (P < 0.05) or PX-478 (P < 0.05). These results support the importance of HIF1alpha for COC maturation and subsequent embryo development. HIF1alpha is a multidirectional factor controlling intercellular communication within COCs, steroidogenic activity, and oocyte development rates, and exerting effects on blastocyst rates.

Adduct of the blistering warfare agent sesquimustard with human serum albumin and its mass spectrometric identification for biomedical verification of exposure.

Apart from the well-known sulfur mustard (SM), additional sulfur-containing blistering chemical warfare agents exist. Sesquimustard (Q) is one of them and five times more blistering than SM. It is a common impurity in mustard mixtures and regularly found in old munitions but can also be used in pure form. Compared to the extensive literature on SM, very little experimental data is available on Q and no protein biomarkers of exposure have been reported. We herein report for the first time the adduct of Q with the nucleophilic Cys34 residue of human serum albumin (HSA) formed in vitro and introduce two novel bioanalytical procedures for detection. After proteolysis of this HSA adduct catalyzed either by pronase or by proteinase K, two biomarkers were identified by high-resolution tandem mass spectrometry (MS/HR MS), namely a dipeptide and a tripeptide, both alkylated at their Cys residue, which we refer to as HETETE-CP and HETETE-CPF. HETETE represents the Q-derived thio-alkyl moiety bearing a terminal hydroxyl group: "hydroxyethylthioethylthioethyl." Targeting both peptide markers from plasma, a micro liquid chromatography-electrospray ionization tandem mass spectrometry method working in the selected reaction monitoring mode (µLC-ESI MS/MS SRM) was developed and validated as well suited for the verification of exposure to Q. Fulfilling the quality criteria defined by the Organisation for the Prohibition of Chemical Weapons, the novel methods enable the detection of exposure to Q alone or in mixtures with SM. We further report on the relative reactivity of Q compared to SM. Based on experiments making use of partially deuterated Q as the alkylating agent, we rule out a major role for six-membered ring sulfonium ions as relevant reactive species in the alkylation of Cys34. Furthermore, the results of molecular dynamics simulations are indicative that the protein environment around Cys34 allows adduct formation with elongated but not bulky molecules such as Q, and identify important hydrogen bonding interactions and hydrophobic contacts. Graphical abstract.

Synergisms of genome and metabolism stabilizing antitumor therapy (GMSAT) in human breast and colon cancer cell lines: a novel approach to screen for synergism.

BACKGROUND: Despite an improvement of prognosis in breast and colon cancer, the outcome of the metastatic disease is still severe. Microevolution of cancer cells often leads to drug resistance and tumor-recurrence. To target the driving forces of the tumor microevolution, we focused on synergistic drug combinations of selected compounds. The aim is to prevent the tumor from evolving in order to stabilize disease remission. To identify synergisms in a high number of compounds, we propose here a three-step concept that is cost efficient, independent of high-throughput machines and reliable in its predictions. METHODS: We created dose response curves using MTT- and SRB-assays with 14 different compounds in MCF-7, HT-29 and MDA-MB-231 cells. In order to efficiently screen for synergies, we developed a screening tool in which 14 drugs were combined (91 combinations) in MCF-7 and HT-29 using EC25 or less. The most promising combinations were verified by the method of Chou and Talalay. RESULTS: All 14 compounds exhibit antitumor effects on each of the three cell lines. The screening tool resulted in 19 potential synergisms detected in HT-29 (20.9%) and 27 in MCF-7 (29.7%). Seven of the top combinations were further verified over the whole dose response curve, and for five combinations a significant synergy could be confirmed. The combination Nutlin-3 (inhibition of MDM2) and PX-478 (inhibition of HIF-1α) could be confirmed for all three cell lines. The same accounts for the combination of Dichloroacetate (PDH activation) and NHI-2 (LDH-A inhibition). Our screening method proved to be an efficient tool that is reliable in its projections. CONCLUSIONS: The presented three-step concept proved to be cost- and time-efficient with respect to the resulting data. The newly found combinations show promising results in MCF-7, HT-29 and MDA-MB231 cancer cells.

Effects and Mechanism of Action of PX-478 in Oxygen-Induced Retinopathy in Mice.

IMPORTANCE: Retinopathy of prematurity (ROP) is an important risk factor for blindness in children due to neovascularization (NV). Hypoxia stimulates the formation of NV, as retinal hypoxia affects the stability and function of hypoxia-inducible factor (HIF) transcription factors. The purpose of this study is to study the mechanism of ROP and provide theoretical basis for clinical treatment of ROP. OBJECTIVE: In the present study, we used a mouse model of oxygen-induced retinopathy (OIR) to demonstrate the effects of the HIF-1α inhibitor PX-478 on OIR, and to determine its mechanism of action, to provide a theoretical basis for the clinical treatment of ROP. MATERIALS AND METHODS: The OIR mouse model was induced by exposing neonatal mouse pups and their mothers to 75 ± 5% oxygen from postnatal day 7 (P7) to P12, before being returned to room air from P12 to P17. Flat mount analyses were performed at P12 and P17. Hif1a, Hif2a, Hif3a, and Vegfa mRNA were detected by reverse transcription-polymerase chain reaction in OIR mice at P12 and P17. Hif1a and Vegfa mRNA were detected in OIR mice at P12 and P17 treatment with PX-478. Western blot analyses were used to assess the levels of HIF-1α, VEGF-A, and EPO before and after treatment with PX-478 at P12 and P17. RESULTS: Hif1a mRNA was increased in OIR mice at P12 and P17, while Vegfa mRNA was increased at P12 and P17. HIF-1α, VEGF-A, and EPO protein levels were increased in OIR mice at P12 and P17, as compared to control mice at the same age (all p < 0.05). Inhibition of HIF-1α by injection of PX-478 in OIR mice (P9-P16) caused a decrease in the retinal avascular area at P12 and P17 (both p < 0.05), NV areas at P17 (p < 0.05), Vegfa mRNA decreased at P12 and P17, as compared to control mice (p < 0.05), and VEGF-A and EPO protein levels at P12 and P17, as compared to control mice. Our study found that there were PX-478 both retina and vitreous body of OIR. Inhibition of HIF-1α by injection of PX-478 in OIR mice caused a decrease in the retinal avascular area at P12 and P17, NV areas decreased at P17, VEGF-A and EPO protein levels at P12 and P17. Endothelial cell migration assays and cell tube formation indication PX-478 attenuate cell migration and significantly weakened the cell cavity formation under the condition of hypoxia. CONCLUSION: HIF-1α plays a main role in OIR and can be considered a therapeutic target in OIR by suppressing downstream angiogenic factors, PX-478 decreasing the retinal avascular area and NV.

Long-term Respiratory Effects of Mustard Vesicants.

Sulfur mustard and related vesicants are cytotoxic alkylating agents that cause severe damage to the respiratory tract. Injury is progressive leading, over time, to asthma, bronchitis, bronchiectasis, airway stenosis, and pulmonary fibrosis. As there are no specific therapeutics available for victims of mustard gas poisoning, current clinical treatments mostly provide only symptomatic relief. In this article, the long-term effects of mustards on the respiratory tract are described in humans and experimental animal models in an effort to define cellular and molecular mechanisms contributing to lung injury and disease pathogenesis. A better understanding of mechanisms underlying pulmonary toxicity induced by mustards may help in identifying potential targets for the development of effective clinical therapeutics aimed at mitigating their adverse effects.

Influence of miR-199a on rats with non-small cell lung cancer via regulating the HIF-1α/VEGF signaling pathway.

OBJECTIVE: Micro-ribonucleic acids (miRNAs) are involved in the occurrence of various cancers, and the hypoxia-inducible factor 1-α (HIF-1α) is the main regulator of cell proliferation induced by hypoxia. The relationships of miR-199a and HIF-1α expressions with non-small cell lung cancer (NSCLC) remain unclear, so they were explored in this work. MATERIALS AND METHODS: On the basis of establishing the rat model of NSCLC, the messenger RNA (mRNA) expressions of miR-199a, HIF-1α and the vascular endothelial growth factor (VEGF) were analyzed in NSCLC rats, and the correlations of miR-199a with the mRNAs of HIF-1α and VEGF and cancer staging were investigated. To further study the role of miR-199a in NSCLC cell proliferation via the HIF-1α/VEGF signaling pathway, NSCLC cells were treated with the signaling pathway inhibitor and transfected with miR-199a mimics, respectively. Also,  the roles of the inhibitor PX-478 and miR-199a mimics in the expressions of miR-199a, HIF-1α, and VEGF proteins, as well as their influences on cell proliferation ability were detected. RESULTS: In NSCLC rats, the expression of miR-199a was substantially decreased (p<0.01), but the expressions of HIF-1α and VEGF were notably raised (p<0.01). MiR-199a was negatively correlated with the expression of VEGF. As cancer developed, the expression of miR-199a was gradually lowered, but the expressions of HIF-1α and VEGF were gradually increased. Both HIF-1α/VEGF signaling pathway inhibitor PX-478 and miR-199a mimics significantly reduced the expressions of HIF-1α and VEGF proteins (p<0.01) and suppressed the cell proliferation activity. CONCLUSIONS: MiR-199a prevents the proliferation of NSCLC cells via the targeted down-regulation of the HIF-1α/VEGF signaling pathway.

Hypoxia inhibits TNF-α-induced TSLP expression in keratinocytes.

The expression of thymic stromal lymphopoietin (TSLP), a cytokine which greatly contributes to the induction of type I allergy, is upregulated in chronic inflammation such as atopic dermatitis and psoriasis. As hypoxia in the epidermis is important for maintaining skin homeostasis, we examined the regulation of TSLP expression by hypoxic conditions in normal skin epithelial tissues. TNF-α-induced expression of TSLP in human keratinocyte HaCaT and in mouse keratinocyte PAM212 cell lines were inhibited under hypoxic condition (1% O2), although the mRNA expressions of TNF-α, IL-6, IL-8, MCP-1, and VEGF-A were not inhibited. Hypoxia-mimicking conditions, which include NiCl2, CoCl2, and DMOG, an inhibitor of 2-oxoglutarate-dependent enzymes, also selectively inhibited TNF-α-induced TSLP expression. These results suggested that inactivation of prolyl hydroxylase by hypoxia and hypoxia-mimicking conditions is involved in the repression of TNF-α-induced TSLP expression. Interestingly, the inhibition of TSLP production by hypoxic treatment was significantly reversed by treatment with the HIF-2α antagonist but not with the HIF-1α inhibitor. DMOG-induced inhibition of TSLP promoter activity was dependent on the -71 to +185 bp promoter region, suggesting that the binding of HIF-2 to hypoxia response element (HRE) in this region repressed the TSLP expression. These results indicated that hypoxia and hypoxia-mimicking conditions inhibited TSLP expression via HIF-2 and HRE-dependent mechanisms. Therefore, PHD and HIF-2α could be a new strategy for treatment of atopic dermatitis and psoriasis.

A quantitative method to detect human exposure to sulfur and nitrogen mustards via protein adducts.

Sulfur and nitrogen mustards are internationally banned vesicants listed as Schedule 1 chemical agents in the Chemical Weapons Convention. These compounds are highly reactive electrophiles that form stable adducts to a variety of available amino acid residues on proteins upon exposure. We present a quantitative exposure assay that simultaneously measures agent specific protein adducts to cysteine for sulfur mustard (HD) and three nitrogen mustards (HN1, HN2, and HN3). Proteinase K was added to a serum or plasma sample to digest protein adducts and form the target analyte, the blister agent bound to the tripeptide cysteine-proline-phenylalanine (CPF). The mustard adducted-tripeptide was purified by solid phase extraction and analyzed using isotope dilution LC-MS/MS. Product ion structures were identified using high-resolution product ion scan data for HD-CPF, HN1-CPF, HN2-CPF, and HN3-CPF. Thorough matrix comparison, analyte recovery, ruggedness, and stability studies were incorporated during method validation to produce a robust method. The method demonstrated long term-stability, precision (RSD < 15%), and intra- and inter-day accuracies > 85% across the reportable range of 3.00-200 ng/mL for each analyte. Compared to previously published assays, this method quantitates both sulfur and nitrogen mustard exposure biomarkers, requires only 10 µL of sample volume, and can use either a liquid sample or dried sample spot.