Hydroxysafflor yellow A induced ferroptosis of Osteosarcoma cancer cells by HIF-1α/HK2 and SLC7A11 pathway.

Osteosarcoma is a very serious primary bone cancer with a high death rate and a dismal prognosis. Since there is no permanent therapy for this condition, it is necessary to develop a cure. Therefore, this investigation was carried out to assess the impacts and biological functions of hydroxysafflor yellow A (HYSA) in osteosarcoma cell lines (MG63). In this investigational study, MG63 cells were utilized. Microarray experiments, quantitative polymerase chain reaction (qPCR), immunofluorescent staining, extracellular acidification rate (ECAR), oxygen consumption rate (OCR), glucose consumption, lactate production, and ATP levels, proliferation assay, 5-Ethynyl-2'-deoxyuridine (EDU) staining, and Western blot were performed. In MG63 cells, HYSA lowered cell proliferation and metastasis rates, suppressed EDU cell number, and enhanced caspase-3/9 activity levels. HYSA reduced the Warburg effect and induced ferroptosis (FPT) in MG63 cells. Inhibiting ferroptosis diminished HYSA's anti-cancer activities in MG63 cells. The stimulation of the HIF-1α/SLC7A11 pathway decreased HYSA's anti-cancer activities in MG63 cells. HIF-1α is one target spot for HYSA in a model of osteosarcoma cancer (OC). HYSA altered HIF-1α's thermophoretic activity; following binding with HYSA, HIF-1α's melting point increased from ~55°C to ~60°C. HYSA significantly enhanced the thermal stability of exogenous WT HIF-1α while not affecting Mut HIF-1α, suggesting that ARG-311, GLY-312, GLN-347, and GLN-387 may be involved in the interaction between HIF-1α and HYSA. Conclusively, our study revealed that HYSA induced FPT and reduced the Warburg effect of OC through mitochondrial damage by HIF-1α/HK2/SLC7A11 pathway. HYSA is a possible therapeutic option for OC or other cancers.

2-Deoxy-D-glucose ameliorates inflammation and fibrosis in a silicosis mouse model by inhibiting hypoxia-inducible factor-1α in alveolar macrophages.

Inhaling silica causes the occupational illness silicosis, which mostly results in the gradual fibrosis of lung tissue. Previous research has demonstrated that hypoxia-inducible factor-1α (HIF-1α) and glycolysis-related genes are up-regulated in silicosis. The role of 2-deoxy-D-glucose (2-DG) as an inhibitor of glycolysis in silicosis mouse models and its molecular mechanisms remain unclear. Therefore, we used 2-DG to observe its effect on pulmonary inflammation and fibrosis in a silicosis mouse model. Furthermore, in vitro cell experiments were conducted to explore the specific mechanisms of HIF-1α. Our study found that 2-DG down-regulated HIF-1α levels in alveolar macrophages induced by silica exposure and reduced the interleukin-1ß (IL-1ß) level in pulmonary inflammation. Additionally, 2-DG reduced silica-induced pulmonary fibrosis. From these findings, we hypothesize that 2-DG reduced glucose transporter 1 (GLUT1) expression by inhibiting glycolysis, which inhibits the expression of HIF-1α and ultimately reduces transcription of the inflammatory cytokine, IL-1ß, thus alleviating lung damage. Therefore, we elucidated the important regulatory role of HIF-1α in an experimental silicosis model and the potential defense mechanisms of 2-DG. These results provide a possible effective strategy for 2-DG in the treatment of silicosis.

Rosmarinic acid against cognitive impairment via RACK1/HIF-1α regulated microglial polarization in sepsis-surviving mice.

Microglial polarization modulation has been considered the potential therapeutic strategy for relieving cognitive impairment in sepsis survivors. Rosmarinic acid (RA), a water-soluble polyphenolic natural compound, processes a strong protective effect on various types of neurological disorders including Parkinson's disease, depression, and anxiety. However, its role and potential molecular mechanisms in sepsis-associated cognitive impairment remain unclear. To investigate the preventive and therapeutic effect of RA on sepsis-associated cognitive impairment and elucidate the potential mechanism of RA on regulating microglial polarization, we established a CLP-induced cognitive impairment model in mice and a lipopolysaccharide-induced microglia polarization cell model in BV-2. RACK1 siRNA was designed to identify the potential molecular mechanism of RACK1 on microglial polarization. The preventive and therapeutic effect of RA on cognitive impairment followed by PET-CT and behavioral tests including open-field test and tail suspension test. RACK1/HIF-1α pathway and microglial morphology in the hippocampus or BV-2 cells were measured. The results showed that RA significantly ameliorated the CLP-induced depressive and anxiety-like behaviors and promoted whole-brain glucose uptake in mice. Moreover, RA markedly improved CLP-induced hippocampal neuron loss and microglial activation by inhibiting microglial M1 polarization. Furthermore, experiments showed RACK1 was involved in the regulation of LPS-induced microglial M1 polarization via HIF-1α, and RA suppressed lipopolysaccharide or sepsis-associated microglial M1 polarization via RACK1/HIF-1α pathway (rescued the decrease of RACK1 and increase of HIF-1α). Taken together, RA could be a potential preventive and therapeutic medication in improving cognitive impairment through RACK1/HIF-1α pathway-regulated microglial polarization.

Metformin and thymoquinone co-treatment enhance 5-fluorouracil cytotoxicity by suppressing the PI3K/mTOR/HIF1α pathway and increasing oxidative stress in colon cancer cells.

This study investigated the chemotherapeutic effects of 5-fluorouracil (5-FU), metformin (Met), and/or thymoquinone (TQ) single/dual/triple therapies in the HT29, SW480 and SW620 colon cancer (CRC) cell lines. Cell cycle/apoptosis were measured by flow cytometry. The gene and protein expression of apoptosis (PCNA/survivin/BAX/Cytochrome-C/Caspase-3) and cell cycle (CCND1/CCND3/p21/p27) molecules, the PI3K/mTOR/HIF1α oncogenic pathway, and glycolysis regulatory enzymes were measured by quantitative-PCR and Western blot. Markers of oxidative stress were also measured by colorimetric assays. Although all treatments induced anti-cancer effects related to cell cycle arrest and apoptosis, the triple therapy showed the highest pro-apoptotic actions that coincided with the lowest expression of CCND1/CCND3/PCNA/survivin and the maximal increases in p21/p27/BAX/Cytochrome-C/Caspase-3 in all cell lines. The triple therapy also revealed the best suppression of the PI3K/mTOR/HIF1α pathway by increasing its endogenous inhibitors (PTEN/AMPKα) in all cell lines. Moreover, the lowest expression of lactate dehydrogenase and pyruvate dehydrogenase kinase-1 with the highest expression of pyruvate dehydrogenase were seen with the triple therapy, which also showed the highest increases in oxidative stress markers (ROS/RNS/MDA/protein carbonyl groups) alongside the lowest antioxidant levels (GSH/CAT) in all cell lines. In conclusion, this is the first study to reveal enhanced anti-cancer effects for metformin/thymoquinone in CRC that were superior to all monotherapies and the other dual therapies. However, the triple therapy approach showed the best tumoricidal actions related to cell cycle arrest and apoptosis in all cell lines, possibly by enhancing oxidative glycolysis and augmenting oxidative stress through stronger modulation of the PI3K/mTOR/HIF1α oncogenic network.

GDF11 promotes wound healing in diabetic mice via stimulating HIF-1ɑ-VEGF/SDF-1ɑ-mediated endothelial progenitor cell mobilization and neovascularization.

Non-healing diabetic wounds (DW) are a serious clinical problem that remained poorly understood. We recently found that topical application of growth differentiation factor 11 (GDF11) accelerated skin wound healing in both Type 1 DM (T1DM) and genetically engineered Type 2 diabetic db/db (T2DM) mice. In the present study, we elucidated the cellular and molecular mechanisms underlying the action of GDF11 on healing of small skin wound. Single round-shape full-thickness wound of 5-mm diameter with muscle and bone exposed was made on mouse dorsum using a sterile punch biopsy 7 days following the onset of DM. Recombinant human GDF11 (rGDF11, 50 ng/mL, 10 µL) was topically applied onto the wound area twice a day until epidermal closure (maximum 14 days). Digital images of wound were obtained once a day from D0 to D14 post-wounding. We showed that topical application of GDF11 accelerated the healing of full-thickness skin wounds in both type 1 and type 2 diabetic mice, even after GDF8 (a muscle growth factor) had been silenced. At the cellular level, GDF11 significantly facilitated neovascularization to enhance regeneration of skin tissues by stimulating mobilization, migration and homing of endothelial progenitor cells (EPCs) to the wounded area. At the molecular level, GDF11 greatly increased HIF-1ɑ expression to enhance the activities of VEGF and SDF-1ɑ, thereby neovascularization. We found that endogenous GDF11 level was robustly decreased in skin tissue of diabetic wounds. The specific antibody against GDF11 or silence of GDF11 by siRNA in healthy mice mimicked the non-healing property of diabetic wound. Thus, we demonstrate that GDF11 promotes diabetic wound healing via stimulating endothelial progenitor cells mobilization and neovascularization mediated by HIF-1ɑ-VEGF/SDF-1ɑ pathway. Our results support the potential of GDF11 as a therapeutic agent for non-healing DW.

HIF-1α promotes paraquat induced acute lung injury and implicates a role NF-κB and Rac2 activity.

Paraquat (PQ) is a bipyridine herbicide and oral exposure is the main way of PQ exposure with a very high mortality. At present, it is believed that large number of oxygen free radicals are generated and cause lipid peroxidation of tissue and organ cell membranes after PQ is absorbed. PQ exposure could cause multiple organ dysfunction, among which acute lung injury is the most common and most serious. However, its specific mechanism is still unclear. In this study, the C57BL/6J mouse (alveolar epithelial cell-specific knockout HIF-1α) model of acute lung injury (40 mg/kg PQ) at several time pointes and a model of acute type II alveolar epithelial cell (A549, 800 µM PQ) injury constructed. The oxidative stress (ROS, MDA) and inflammatory response (IL-1ß, IL-6, TNF-α) were significantly inhibited in the alveolar epithelial cell-specific knockout of HIF-1α mice and siRNA technology to inhibit HIF-1α in alveolar epithelial cells. Further proteomic analysis showed that the expression of Rac2 protein, which is closely related to oxidative stress, was significantly increased after PQ exposure. And the inhibition of Rac2 expression in vitro significantly alleviated PQ-induced oxidative stress and inflammatory response. The expression of Rac2 protein was regulated by HIF-1α. The above suggests that HIF-1α may promote oxidative stress and inflammatory response in alveolar epithelial cells by regulating the expression of Rac2, and then participate in the promotion of PQ exposure-induced acute lung injury.

Dihydromyricetin improves LPS-induced sickness and depressive-like behaviors in mice by inhibiting the TLR4/Akt/HIF1a/NLRP3 pathway.

The NLRP3 inflammasome activation and neuroinflammation play a crucial role in nerve damage, which can lead to sickness and depressive-like behavior. Dihydromyricetin (DMY) is an important flavanone extracted from Ampelopsis grossedentata. It has been shown to have a significant anti-inflammatory effect in multiple disease models. However, its protective effects on sickness and depressive-like behavior caused by neuroinflammation and its underlying mechanism are still unclear. In this study, we investigated the effects and mechanism of DMY on lipopolysaccharide (LPS)-treated mice with sickness behavior and BV2 cells in Vitro. The effects of LPS treatment and DMY administration on behavioral changes were determined by using behavioral tests including an open field test, tail suspension test and a sucrose preference test. The anti-inflammatory effects of DMY in conditions of neuroinflammatory injury in Vitro and in Vivo were analyzed by using real-time PCR analysis and western blot. The results indicated that DMY improved sickness and depressive-like behaviors in mice induced by LPS. DMY suppressed the expression of microglia markers CD11b, accompanied by reduced expression of pro-inflammatory cytokines, such as TNFα, IL-6, IL-1ß, COX-2, and iNOS in a dose-dependent manner. Interestingly, DMY dramatically inhibited the expression of TLR4/Akt/HIF1a/NLRP3 signaling pathway-related proteins both in Vitro and in Vivo, including TLR4, CD14, PDPk1, p-Akt, p-NF-κB p65, p-GSK-3ß, HIF1a, NLRP3, ASC, and caspase-1. The above results suggested that DMY suppressed the activation of the TLR4/Akt/HIF1a/NLRP3 pathway, which may contribute to its anti-depressive effects.

A tiered approach to investigate the inhalation toxicity of cobalt substances. Tier 2a: Grouping cobalt compounds based on their capacity to stabilize HIF-1α in human alveolar epithelial cells in vitro.

Excessive inhalation of cobalt (Co) dust can have harmful effects on the respiratory tract, yet all cobalt substances do not have the same potential for inducing toxicity. The prevalent hypothesis is that the potential of Co substances to release Co2+ ions in the organism and in cells drives their toxicity profile. Here, we explored the possibility of grouping Co substances for predicting inhalation toxicity based on in vitro data using the stabilization of hypoxia-inducible factor (HIF)-1α as a read out for intracellular Co ion content. We evaluated the potential of 11 inorganic Co compounds and two Co metal powder samples to stabilize intracellular HIF-1α in alveolar epithelial cells (A549) after 24 h exposure to 250-1000 µM Co equivalents. Cytotoxic activity of the substances was assessed in parallel after 72 h at the same doses. Two groups were identified: (1) substances with high intracellular bioavailability (n=9), causing cytotoxicity and stabilizing HIF-1α and (2) substances with low intracellular bioavailability (n = 4), and not inducing these effects. This study provides a link between screening-level data (solubility in artificial lung fluids, Tier 1) and hypothesized biological key events.

Celastrol Protects against Cerebral Ischemia/Reperfusion Injury in Mice by Inhibiting Glycolysis through Targeting HIF-1α/PDK1 Axis.

Cerebral ischemia/reperfusion (I/R) injury is closely related to dysfunctional glucose metabolism. Celastrol is a bioactive compound that has been found to exhibit neuroprotective effects in cerebral ischemia, while whether it can protect against cerebral I/R injury by regulating glycolysis remains unclear. The goal of this study is to investigate the role of celastrol on cerebral I/R injury and its underlying mechanisms in transient middle cerebral artery occlusion (tMCAO) mice. Methods. To observe the protective effect of celastrol and select its optimal dosage for further study, neurological score, TTC staining, and HE staining were used to evaluate neurological function, cerebral infarct volume, and cortical cell damage, respectively. QRT-PCR and Western blot were used to detect the mRNA and protein expression of hypoxia inducible factor-1α (HIF-1α), pyruvate dehydrogenasekinase1 (PDK1), lactate dehydrogenase A (LDHA), glucose transporter1 (GLUT1), and hexokinase2 (HK2), respectively. The lactate production, ATP level, and glucose content were assessed by assay kits. Results. Our results indicated that celastrol dose-dependently improved neurological function and reduced cerebral infarct volume and cortical cell death of tMCAO mice, and its optimal dosage was 4.5 mg/kg. In addition, celastrol significantly blocked I/R-induced increase of LDHA, GLUT1, HK2, and lactate production as well as decrease of ATP level and glucose content. Moreover, celastrol inhibited the I/R-induced upregulation of HIF-1α and PDK1. Overexpression of HIF-1α by DMOG reversed the protective effect of celastrol on cerebral I/R injury and blocked celastrol-induced suppression of glycolysis. Conclusions. Taken together, these results suggested that celastrol protected against cerebral I/R injury through inhibiting glycolysis via the HIF-1α/PDK1 axis.

Dl-3-N-Butylphthalide Attenuates Hypoxic Injury of Neural Stem Cells by Increasing Hypoxia-Inducible Factor-1alpha.

OBJECTIVE: To assess the potential effect of dl-3-N-butylphthalide (dl-NBP) for the proliferation and differentiation of neural stem cells (NSCs) against hypoxia and the underlying mechanism. MATERIALS AND METHODS: Hippocampal NSCs were obtained from fetal rats. NSCs combined with dl-NBP and single NSCs were cultured. The impact of siRNA-mediated hypoxia-inducible factor-1alpha (HIF-1α) knockdown on NSCs was detected with western blotting (WB) and quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR). Cell-counting kit-8 assay was used for evaluating the viability of NSCs. Levels of HIF-1α protein were measured using WB, and vascular endothelial growth factor (VEGF) expression was quantified using RT-qPCR and enzyme-linked immunosorbent assay. RESULTS: Compared with 7 different concentrations of dl-NBP, 0.25 g/L was determined as the optimal concentration to significantly increase the viability of NSCs (p < 0.001). Dl-NBP can significantly increase the viability of hypoxic NSCs (p < 0.001) and improve the differentiation of hypoxic NSCs into astrocytes (p = 0.001) and oligodendrocytes (p < 0.001). Meanwhile, Dl-NBP can significantly elevate levels of HIF-1α protein (p < 0.001) and VEGF mRNA (p = 0.001) / protein (p < 0.001) in NSCs in the hypoxic environment. However, after transfection with HIF-1α siRNA in NSCs, the viability and differentiation of NSCs was not recovered using dl-NBP under the hypoxic condition, as well as levels of HIF-1α and VEGF. CONCLUSION: Dl-NBP can reverse the weaker proliferation and differentiation power of NSCs in the hypoxic environment. The HIF-1α - VEGF pathway may be implicated in this protective effect of dl-NBP.

Suppression of collagen IV alpha-2 subunit by prolyl hydroxylase domain inhibition via hypoxia-inducible factor-1 in chronic kidney disease.

Elevation of hypoxia-inducible factor 1 protein has been shown to be protective in acute kidney injury and HIF1α enhancing drug therapies are currently in clinical trials for the treatment of anemia of chronic kidney disease. Despite its benefits, long-term HIF1 elevation seems to be associated with additional effects in the kidneys such as tubulointerstitial fibrosis. To better understand the effects of prolonged HIF1 exposure, assessment of baseline and post-therapy levels of HIF1α and other related biomarkers is essential. In this study, we assessed the effect of HIF1α enhancement using prolyl hydroxylase inhibitor (PHD-I) DMOG, on a key profibrotic marker of kidney disease. In specific, we examined the change in expression of Collagen 4 subunit A2 in cultured urinary cells of CKD patients pre and post 24-hour exposure to 1mM DMOG. Our results show that besides HIF1α enhancement, COL4A2 protein is suppressed in presence of DMOG. To determine if this effect is mediated by HIF1, we used HIF1α gene silencing in HEK293 cells and examined the effect of DMOG on protein and gene expression of COL4A2 post 24-hour exposure. We showed that silencing HIF1α reverses and amplifies the expression of COL4A2 in HEK293 cells. Our data suggest that HIF1 directly regulates the expression of COL4A2 in kidney cells and that HIF1α enhancing therapy has suppressive effects on COL4A2 that may be clinically relevant and must be considered in determining the safety and efficacy of these drugs in the treatment of anemia.

Hypoxia and Hypoxia-Inducible Factor Signaling in Muscular Dystrophies: Cause and Consequences.

Muscular dystrophies (MDs) are a group of inherited degenerative muscle disorders characterized by a progressive skeletal muscle wasting. Respiratory impairments and subsequent hypoxemia are encountered in a significant subgroup of patients in almost all MD forms. In response to hypoxic stress, compensatory mechanisms are activated especially through Hypoxia-Inducible Factor 1 α (HIF-1α). In healthy muscle, hypoxia and HIF-1α activation are known to affect oxidative stress balance and metabolism. Recent evidence has also highlighted HIF-1α as a regulator of myogenesis and satellite cell function. However, the impact of HIF-1α pathway modifications in MDs remains to be investigated. Multifactorial pathological mechanisms could lead to HIF-1α activation in patient skeletal muscles. In addition to the genetic defect per se, respiratory failure or blood vessel alterations could modify hypoxia response pathways. Here, we will discuss the current knowledge about the hypoxia response pathway alterations in MDs and address whether such changes could influence MD pathophysiology.

Pregabalin Protects Brain Tissue from Subarachnoid Hemorrhage by Enhancing HIF-1α/eNOS Signaling and VEGF Production.

OBJECTIVE: We investigated the effects of different doses of pregabalin on the pathophysiologic changes in early brain injury after subarachnoid hemorrhage (SAH) in rats. METHODS: Thirty-eight Wistar albino rats were divided into 4 groups: control (n = 8), SAH (n = 10), SAH plus 30 mg/kg/day of pregabalin (n = 10), and SAH plus 60 mg/kg/day of pregabalin (n = 10). SAH was induced with 0.3 mL of autologous blood injected to the cisterna magna of rats. Pregabalin was administered intraperitoneally. Oxidative stress markers, mRNA expression of endothelial nitric oxide synthase, hypoxia-inducible factor-1α, and vascular endothelial growth factor, and histopathological changes were evaluated. RESULTS: Pregabalin increased mRNA expression of endothelial nitric oxide synthase, hypoxia-inducible factor-1α, and vascular endothelial growth factor in a dose-dependent manner. Significant improvement in the histopathological parameters was observed at 60 mg/kg, including a decrease in diffuse hemorrhagic areas, edema and apoptotic bodies in the associated cortical area, evident vacuolization in the hippocampal area, and apoptotic bodies. However, these improvements were not observed with the lower dose (30 mg/kg). In contrast, the antioxidant effect was greater with 30 mg/kg of pregabalin than with 60 mg/kg. CONCLUSIONS: Although the antioxidant effect was significant with the lower dose of pregabalin, the anti-inflammatory effects via vasodilatation were more marked with the higher dose. Significant improvements in the histopathological changes were observed with the higher dose of pregabalin. The dose-dependent effects of pregabalin on SAH should be evaluated in animal studies as a function of time and in the acute and chronic phases.

Long noncoding RNA ZFPM2-AS1 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by regulating the miR-576-3p/HIF-1α axis.

Long noncoding RNA (LncRNA) zinc finger protein multitype 2 antisense RNA 1 (ZFPM2-AS1) is highly expressed in a variety of tumors and is involved in promoting the malignant biological behaviors of cancer cells. However, the mechanism of ZFPM2-AS1 in the progression of hepatocellular carcinoma (HCC) remains to be explored. The ZFPM2-AS1 expression in HCC was measured by quantitative real-time PCR (qRT-PCR); cell counting kit-8, 5-bromo-2'-deoxyuridine (BrdU), and transwell assays were used to confirm the biological functions of ZFPM2-AS1 in regulating the malignant biological behaviors of HCC cells; the luciferase reporter gene assay was employed to detect whether ZFPM2-AS1 could bind to microRNA (miR)-576-3p; the regulatory relationship between ZFPM2-AS1 and miR-576-3p was probed by qRT-PCR; the effects of ZFPM2-AS1 and miR-576-3p on the expression of hypoxia-inducible factor 1α (HIF-1α) were detected by qRT-PCR and Western blot. The expression of ZFPM2-AS1 in HCC tissues, compared with that in normal liver tissues, was significantly upregulated. Knockdown of ZFPM2-AS1 markedly inhibited HCC cell proliferation, migration, and invasion while the overexpression of ZFPM2-AS1 worked oppositely. miR-576-3p could reverse the effects of ZFPM2-AS1 on the biological behaviors of HCC cells. Besides, ZFPM2-AS1 could bind to miR-576-3p and positively regulate the expression of HIF-1α, a target gene of miR-576-3p, by adsorbing miR-576-3p. ZFPM2-AS1 is abnormally highly expressed in HCC and facilitates proliferation, migration, and invasion of HCC cells by adsorbing miR-576-3p and upregulating HIF-1α expression.

Evodiamine inhibits vasculogenic mimicry in HCT116 cells by suppressing hypoxia-inducible factor 1-alpha-mediated angiogenesis.

Evodiamine (Evo), a quinazoline alkaloid and one of the most typical polycyclic heterocycles, is mainly isolated from Evodia rugulosa. Vasculogenic mimicry (VM) is a newly identified way of angiogenesis during tumor neovascularization, which is prevalent in a variety of highly invasive tumors. The purpose of this study was to investigate the effect and mechanism of Evo on VM in human colorectal cancer (CRC) cells. The number of VM structures was calculated by the three-dimensional culture of human CRC cells. Wound-healing was used to detect the migration of HCT116 cells. Gene expression was detected by reverse transcription-quantitative PCR assay. CD31/PAS staining was used to identify VM. Western blotting and immunofluorescence were used to detect protein levels. The results showed that Evo inhibited the migration of HCT116 cells, as well as the formation of VM. Furthermore, Evo reduced the expression of hypoxia-inducible factor 1-alpha (HIF-1α), VE-cadherin, VEGF, MMP2, and MMP9. In a model of subcutaneous xenotransplantation, Evo also inhibited tumor growth and VM formation. Our study demonstrates that Evo could inhibit VM in CRC cells HCT116 and reduce the expression of HIF-1α, VE-cadherin, VEGF, MMP2, and MMP9.

Discovery of a Napabucasin PROTAC as an Effective Degrader of the E3 Ligase ZFP91.

Napabucasin, undergoing multiple clinical trials, was reported to inhibit the signal transducer and transcription factor 3 (STAT3). To better elucidate its mechanism of action, we designed a napabucasin-based proteolysis targeting chimera (PROTAC), XD2-149 that resulted in inhibition of STAT3 signaling in pancreatic cancer cell lines without inducing proteasome-dependent degradation of STAT3. Proteomics analysis of XD2-149 revealed the downregulation of the E3 ubiquitin-protein ligase ZFP91. XD2-149 degrades ZFP91 with DC50 values in the nanomolar range. The cytotoxicity of XD2-149 was significantly, but not fully, reduced with ZFP91 knockdown providing evidence for its multi-targeted mechanism of action. The NQO1 inhibitor, dicoumarol, rescued the cytotoxicity of XD2-149 but not ZFP91 degradation, suggesting that the NQO1-induced cell death is independent of ZFP91. ZFP91 plays a role in tumorigenesis and is involved in multiple oncogenic pathways including NF-κB and HIF-1α.

Resveratrol alleviates bleomycin-induced pulmonary fibrosis via suppressing HIF-1α and NF-κB expression.

The absence of a gold standard for treating pulmonary fibrosis makes its management challenging. We established a rat model to study the effect of resveratrol (Res) on bleomycin-induced pulmonary fibrosis. Rats were randomly divided into control, model, low-Res, middle-Res, high-Res, and dexamethasone groups and treated with various concentrations of these drugs. Rats showed typical features of pulmonary fibrosis; i.e., alveolitis, fibrous hyperplasia, and fibrosis on days 7, 14, and 28, respectively. Expression of HIF-1α and NF-κB was higher in the middle-Res, high-Res, and dexamethasone groups than in the control group, but was less than that in the model and low Res groups. We conclude that different levels of HIF-1α and NF-κB expression at different stages of pulmonary fibrosis in rats is positively correlated with the disease severity. Furthermore, resveratrol alleviates bleomycin-induced pulmonary fibrosis by suppressing HIF-1α and NF-κB expression, indicating its potential as a promising therapeutic drug candidate.

Dimethyloxalylglycine (DMOG) and the Caspase Inhibitor "Ac-LETD-CHO" Protect Neuronal ND7/23 Cells of Gluocotoxicity.

It well known that long-lasting hyperglycaemia disrupts neuronal function and leads to neuropathy and other neurodegenerative diseases. The α-ketoglutarate analogue (DMOG) and the caspase-inhibitor "Ac-LETD-CHO are potential neuroprotective molecules. Whether their protections may also extend glucotoxicity-induced neuropathy is not known. Herein, we evaluated the possible cell-protective effects of DMOG and Ac-LETD-CHO against hyperglycaemia-induced reactive oxygen species and apoptosis in ND7/23 neuronal cells. The impact of glucotoxicity on the expression of HIF-1α and a panel of micro-RNAs of significance in hyperglycaemia and apoptosis was also investigated.ND7/23 cells cultured under hyperglycaemic conditions showed decreased cell viability and elevated levels of ROS production in a dose- and time-dependent manner. However, presence DMOG (500 µM) and/or Ac-LETD-CHO (50 µM) counteracted this effect and increase cell viability concomitant with reduction in ROS production, DNA damage and apoptosis. AcLETD-CHO suppressed hyperglycaemia-induced caspase 3 activation in ND7/23 cells. Both DMOG and Ac-LETD-CHO increased HIF-1α expression paralleled with the suppression of miR-126-5p, miR-128-3p and miR-181 expression and upregulation of miR-26b, 106a-5p, 106b-5p, 135a-5p, 135b-5p, 138-5p, 199a-5p, 200a-3p and 200c-3p expression.We demonstrate a mechanistic link for the DMOG and Ac-LETD-CHO protection against hyperglycaemia-induced neuronal dysfunction, DNA damage and apoptosis and thereby propose that pharmacological agents mimicking these effects may represent a promising novel therapy for the hyperglycaemia-induced neuropathy.

MGMT-inhibitor in combination with TGF-ßRI inhibitor or CDK 4/6 inhibitor increases temozolomide sensitivity in temozolomide-resistant glioblastoma cells.

BACKGROUND: Glioblastoma (GB) remains an incurable and deadly brain malignancy that often proves resistant to upfront treatment with temozolomide. Nevertheless, temozolomide remains the most commonly prescribed FDA-approved chemotherapy for GB. The DNA repair protein methylguanine-DNA methyl transferase (MGMT) confers resistance to temozolomide. Unsurprisingly temozolomide-resistant tumors tend to possess elevated MGMT protein levels or lack inhibitory MGMT promotor methylation. In this study, cultured human temozolomide resistance GB (43RG) cells were introduced to the MGMT inhibitor O6-benzylguanine combined with temozolomide and either LY2835219 (CDK 4/6 inhibitor) or LY2157299 (TGF-ßRI inhibitor) seeking to overcome GB treatment resistance. METHODS: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot, cell viability, and cell cycle progression. RESULTS: Our in vitro study demonstrated that sequential treatment of O6-Benzylguanine with either LY2385219 or LY2157299-enhanced temozolomide enhanced sensitivity in MGMT+ 43RG cells. Importantly, normal human neurons and astrocytes remained impervious to the drug therapies under these conditions. Furthermore, LY2835219 has additional anti-proliferative effects on cell cycling, including induction of an RB-associated G (1) arrest via suppression of cyclin D-CDK4/6-Rb pathway. LY2157299 enhances anti-tumor effect by disrupting TGF-ß-dependent HIF-1α signaling and by activating both Smad and PI3K-AKT pathways towards transcription of S/G2 checkpoints. CONCLUSION: This study establishes the groundwork for the development of a combinatorial pharmacologic approach by using either LY2385219 or LY2157299 inhibitor plus O6-Benzylguanine to augment temozolomide response in temozolomide-resistant GB cells.