Wogonin ameliorates ER stress-associated inflammatory response, apoptotic death and renal fibrosis in a unilateral ureteral obstruction mouse model.

Wogonin, a vital bioactive compound extracted from the medicinal plant, Scutellaria baicalensis, has been wildly used for its potential in mitigating the progression of chronic diseases. Chronic kidney disease (CKD) represents a significant global health challenge due to its high prevalence, morbidity and mortality rates, and associated complications. This study aimed to assess the potential of wogonin in attenuating renal fibrosis and to elucidate the underlying molecular mechanisms using a unilateral ureteral obstruction (UUO) mouse model as a CKD mimic. Male mice, 8 weeks old, underwent orally administrated of either 50 mg/kg/day of wogonin or positive control of 5 mg/kg/day candesartan following UUO surgery. NRK52E cells were exposed to tumor growth factors-beta (TGF-ß) to evaluate the anti-fibrotic effects of wogonin. The results demonstrated that wogonin treatment effectively attenuated TGF-ß-induced fibrosis markers in NRK-52E cells. Additionally, administration of wogonin significantly improved histopathological alterations and downregulated the expression of pro-fibrotic factors (Fibronectin, α-smooth muscle actin, Collagen IV, E-cadherin, and TGF-ß), oxidative stress markers (Catalase, superoxide dismutase 2, NADPH oxidase 4, and thioredoxin reductase 1), inflammatory molecules (Cyclooxygenase-2 and TNF-α), and the infiltration of neutrophils and macrophages in UUO mice. Furthermore, wogonin treatment mitigated endoplasmic reticulum (ER) stress-associated molecular markers (GRP78, GRP94, ATF4, CHOP, and the caspase cascade) and suppressed apoptosis. The findings indicate that wogonin treatment ameliorates key fibrotic aspects of CKD by attenuating ER stress-related apoptosis, inflammation, and oxidative stress, suggesting its potential as a future therapeutic target.

Roles of oxidative stress/JNK/ERK signals in paraquat-triggered hepatic apoptosis.

Paraquat (PQ), a toxic and nonselective bipyridyl herbicide, is one of the most extensively used pesticides in agricultural countries. In addition to pneumotoxicity, the liver is an important target organ for PQ poisoning in humans. However, the mechanism of PQ in hepatotoxicity remains unclear. In this study, we found that exposure of rat hepatic H4IIE cells to PQ (0.1-2 mM) induced significant cytotoxicity and apoptosis, which was accompanied by mitochondria-dependent apoptotic signals, including loss of mitochondrial membrane potential (MMP), cytosolic cytochrome c release, and changes in the Bcl-2/Bax mRNA ratio. Moreover, PQ (0.5 mM) exposure markedly induced JNK and ERK1/2 activation, but not p38-MAPK. Blockade of JNK and ERK1/2 signaling by pretreatment with the specific pharmacological inhibitors SP600125 and PD98059, respectively, effectively prevented PQ-induced cytotoxicity, mitochondrial dysfunction, and apoptotic events. Additionally, PQ exposure stimulated significant oxidative stress-related signals, including reactive oxygen species (ROS) generation and intracellular glutathione (GSH) depletion, which could be reversed by the antioxidant N-Acetylcysteine (NAC). Buffering the oxidative stress response with NAC also effectively abrogated PQ-induced hepatotoxicity, MMP loss, apoptosis, and phosphorylation of JNK and ERK1/2 protein, however, the JNK or ERK inhibitors did not suppress ROS generation in PQ-treated cells. Collectively, these results demonstrate that PQ exposure induces hepatic cell toxicity and death via an oxidative stress-dependent JNK/ERK activation-mediated downstream mitochondria-regulated apoptotic pathway.

Chlorpyrifos induces neuronal cell death via both oxidative stress and Akt activation downstream-regulated CHOP-triggered apoptotic pathways.

Chlorpyrifos (CPF) is one of the most abundant and widely used organophosphate pesticides for agricultural, industrial, and household purposes in the world. Epidemiological studies have reported that CPF can induce neurotoxic impairments in mammalian, which is linked to an important risk factor for development of neurodegenerative diseases (NDs). However, limited information is available on CPF-induced neurotoxicity, with the underlying exact mechanism remains unclear. In this study, CPF exposure (10-400 µM) significantly reduced Neuro-2a cell viability and induced apoptotic events, including the increase in caspase-3 activity, apoptotic cell population, and cleavage of caspase-3/-7 and PARP. Exposure of Neuro-2a cells to CPF also triggered CHOP activation. Transfection with CHOP-specific siRNA markedly suppressed the expression of CHOP, and attenuated cytotoxicity and apoptotic events in CPF-exposed Neuro-2a cells. Furthermore, CPF exposure obviously evoked the phosphorylation of Akt as well as ROS generation in a time-dependent manner. Pretreatment with LY294002 (an Akt inhibitor) effectively attenuated the CPF-induced Akt phosphorylation, CHOP activation, and apoptotic events, but not that ROS production. Of note, buffering the ROS generation with antioxidant N-acetylcysteine effectively prevented the CPF-induced ROS generation, CHOP activation, and apoptotic events, but not that the Akt phosphorylation. Collectively, these findings indicate that CPF exposure exerts neuronal cytotoxicity via the independent pathways of ROS generation and Akt activation downstream-regulated CHOP-triggered apoptosis, ultimately leading to neuronal cell death.

Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca2+-Regulated ERK1/2-Activating Pathway.

Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01-0.5 mM) exhibited greater cytotoxicity than ketamine (0.1-3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca2+]i). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca2+ chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca2+]i in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca2+]i-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca2+]i/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis.

Methylmercury Induces Mitochondria- and Endoplasmic Reticulum Stress-Dependent Pancreatic ß-Cell Apoptosis via an Oxidative Stress-Mediated JNK Signaling Pathway.

Methylmercury (MeHg), a long-lasting organic pollutant, is known to induce cytotoxic effects in mammalian cells. Epidemiological studies have suggested that environmental exposure to MeHg is linked to the development of diabetes mellitus (DM). The exact molecular mechanism of MeHg-induced pancreatic ß-cell cytotoxicity is still unclear. Here, we found that MeHg (1-4 µM) significantly decreased insulin secretion and cell viability in pancreatic ß-cell-derived RIN-m5F cells. A concomitant elevation of mitochondrial-dependent apoptotic events was observed, including decreased mitochondrial membrane potential and increased proapoptotic (Bax, Bak, p53)/antiapoptotic (Bcl-2) mRNA ratio, cytochrome c release, annexin V-Cy3 binding, caspase-3 activity, and caspase-3/-7/-9 activation. Exposure of RIN-m5F cells to MeHg (2 µM) also induced protein expression of endoplasmic reticulum (ER) stress-related signaling molecules, including C/EBP homologous protein (CHOP), X-box binding protein (XBP-1), and caspase-12. Pretreatment with 4-phenylbutyric acid (4-PBA; an ER stress inhibitor) and specific siRNAs for CHOP and XBP-1 significantly inhibited their expression and caspase-3/-12 activation in MeHg-exposed RIN-mF cells. MeHg could also evoke c-Jun N-terminal kinase (JNK) activation and reactive oxygen species (ROS) generation. Antioxidant N-acetylcysteine (NAC; 1mM) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 100 µM) markedly prevented MeH-induced ROS generation and decreased cell viability in RIN-m5F cells. Furthermore, pretreatment of cells with SP600125 (JNK inhibitor; 10 µM) or NAC (1 mM) or transfection with JNK-specific siRNA obviously attenuated the MeHg-induced JNK phosphorylation, CHOP and XBP-1 protein expression, apoptotic events, and insulin secretion dysfunction. NAC significantly inhibited MeHg-activated JNK signaling, but SP600125 could not effectively reduce MeHg-induced ROS generation. Collectively, these findings demonstrate that the induction of ROS-activated JNK signaling is a crucial mechanism underlying MeHg-induced mitochondria- and ER stress-dependent apoptosis, ultimately leading to ß-cell death.

Influence of Alloying Element Mg on Na and Sr Modifying Al-7Si Hypoeutectic Alloy.

The influence of alloying element Mg on Na and Sr modifying Al-7Si hypoeutectic alloys was investigated. The residual content of Na and the morphology of modified eutectic silicon were characterized. It was found that the alloying element Mg had an enhanced effect on the uptake of sodium in the Al-7Si hypoeutectic alloy modified by the Na-contained modifier. Moreover, the morphology of eutectic silicon of the modified Al-7Si alloys was significantly different from that of Al-7Si-0.4Mg alloys in the present research. When the addition of the modifier is enough, both modifiers could entirely modify the eutectic silicon phase of Al-7Si alloys, while incompletely modified eutectic silicon was observed in both Na-modified and Sr-modified Al-7Si-0.4Mg alloy. It was observed that there was an adhering relationship between the partially modified eutectic silicon with Mg-rich phases. According to the results, it can be proposed that the addition of Mg will affect the solidification behavior of alloys, thereby, leading to the incomplete modification of eutectic silicon phases.

Quercetin induces tongue squamous cell carcinoma cell apoptosis via the JNK activation-regulated ERK/GSK-3α/ß-mediated mitochondria-dependent apoptotic signaling pathway.

Tongue squamous cell carcinoma (SCC) is a most common type of oral cancer. Due to its highly invasive nature and poor survival rate, the development of effective pharmacological therapeutic agents is urgently required. Quercetin (3,3',4',5,7-pentahydroxyflavone) is a polyphenolic flavonoid found in plants and is an active component of Chinese herbal medicine. The present study investigated the pharmacological effects and possible mechanisms of quercetin on apoptosis of the tongue SCC-derived SAS cell line. Following treatment with quercetin, cell viability was assessed via the MTT assay. Apoptotic and necrotic cells, mitochondrial transmembrane potential and caspase-3/7 activity were analyzed via flow cytometric analyses. A caspase-3 activity assay kit was used to detect the expression of caspase-3 activity. Western blot analysis was performed to examine the expression levels of proteins associated with the MAPKs, AMPKα, GSK3-α/ß and caspase-related signaling pathways. The results revealed that quercetin induced morphological alterations and decreased the viability of SAS cells. Quercetin also increased apoptosis-related Annexin V-FITC fluorescence and caspase-3 activity, and induced mitochondria-dependent apoptotic signals, including a decrease in mitochondrial transmembrane potential and Bcl-2 protein expression, and an increase in cytosolic cytochrome c, Bax, Bak, cleaved caspase-3, cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase protein expression. Furthermore, quercetin significantly increased the protein expression levels of phosphorylated (p)-ERK, p-JNK1/2 and p-GSK3-α/ß, but not p-p38 or p-AMPKα in SAS cells. Pretreatment with the pharmacological JNK inhibitor SP600125 effectively reduced the quercetin-induced apoptosis-related signals, as well as p-ERK1/2 and p-GSK3-α/ß protein expression. Both ERK1/2 and GSK3-α/ß inhibitors, PD98059 and LiCl, respectively, could significantly prevent the quercetin-induced phosphorylation of ERK1/2 and GSK3-α/ß, but not JNK activation. Taken together, these results suggested that quercetin may induce tongue SCC cell apoptosis via the JNK-activation-regulated ERK1/2 and GSK3-α/ß-mediated mitochondria-dependent apoptotic signaling pathway.

Integration of traditional Chinese medicine and nibble debridement and dressing method reduces thrombosis and inflammatory response in the treatment of thromboangiitis obliterans.

BACKGROUND: Thromboangiitis obliterans (TAO), also known as Buerger's disease, is an occlusive arterial disease; however, the pathogenesis of TAO is still unclear. Research has shown that traditional Chinese medicine (TCM) has significant advantages in the treatment of TAO. Our purpose was to explore the underlying roles of TCM in combination with nibble debridement and dressing method (NDDM) in a TAO rat model. METHODS: We administered rats with 10 mg/mL sodium laurate to establish a TAO model, and then the TAO model rats were treated with notoginseng powder (NP), maifusheng (MFS), or the combination of NP or MFS and NDDM. Gangrene classification and blood rheology were evaluated; the pathological characteristics of rat limbs were examined by hematoxylin and eosin (H&E) staining and Masson staining; and cluster of differentiation 3+ (CD3+) and cluster of differentiation 20+ (CD20+) levels were measured by immunohistochemistry (IHC) and flow cytometry. In addition, inflammation-associated cytokines were analyzed by quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Integration of NP or MFS and NDDM dramatically reduced the gangrene classification and affected blood rheology parameters of TAO model rats compared with NP and MFS alone. Meanwhile, NP or MFS in combination with NDDM decreased CD3+CD20+ T cells, reduced thrombosis and inflammatory cell infiltration, and dramatically decreased the levels of inflammation-associated cytokines. CONCLUSIONS: Our results suggested that integration of NP or MFS and NDDM could relieve the symptoms of TAO model rats induced by sodium laurate, which might provide a new management strategy for TAO.

Arsenic induces autophagy-dependent apoptosis via Akt inactivation and AMPK activation signaling pathways leading to neuronal cell death.

Inorganic arsenic (As3+), a well-known worldwide industrial and environmental pollutant, has been linked to neurodegenerative disorders (NDs). Autophagy plays an important role in controlling neuronal cell survival/death. However, limited information is available regarding the toxicological mechanism at the interplay between autophagy and As3+-induced neurotoxicity. The present study found that As3+ exposure induced a concomitant activation of apoptosis and autophagy in Neuro-2a cells, which was accompanied with the increase of phosphatidylserine exposure on outer membrane leaflets and apoptotic cell population, and the activation of caspase-3, -7, and PARP as well as the elevation of protein expressions of LC3-II, Atg-5, and Beclin-1, and the accumulation of autophagosome. Pretreatment of cells with autophagy inhibitor 3-MA, but not that of Z-VAD-FMK (a pan-caspase inhibitor), effectively prevented the As3+-induced autophagic and apoptotic responses, indicating that As3+-triggered autophagy was contributing to neuronal cell apoptosis. Furthermore, As3+ exposure evoked the dephosphorylation of Akt. Pretreatment with SC79, an Akt activator, could significantly attenuated As3+-induced Akt inactivation as well as autophagic and apoptotic events. Expectedly, inhibition of Akt signaling with LY294002 obviously enhanced As3+-triggered autophagy and apoptosis. Exposure to As3+ also dramatically increased the phosphorylation level of AMPKα. Pretreatment of AMPK inhibitor (Compound C) could markedly abrogate the As3+-induced phosphorylated AMPKα expression, and autophagy and apoptosis activation. Taken together, these results indicated that As3+ exerted its cytotoxicity in neuronal cells via the Akt inactivation/AMPK activation downstream-regulated autophagy-dependent apoptosis pathways, which ultimately lead to cell death. Our findings suggest that the regulation of Akt/AMPK signals may be a promising intervention to against As3+-induced neurotoxicity and NDs.

4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, a Major Active Metabolite of Bisphenol A, Triggers Pancreatic ß-Cell Death via a JNK/AMPKα Activation-Regulated Endoplasmic Reticulum Stress-Mediated Apoptotic Pathway.

4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), a major active metabolite of bisphenol A (BPA), is generated in the mammalian liver. Some studies have suggested that MBP exerts greater toxicity than BPA. However, the mechanism underlying MBP-induced pancreatic ß-cell cytotoxicity remains largely unclear. This study demonstrated the cytotoxicity of MBP in pancreatic ß-cells and elucidated the cellular mechanism involved in MBP-induced ß-cell death. Our results showed that MBP exposure significantly reduced cell viability, caused insulin secretion dysfunction, and induced apoptotic events including increased caspase-3 activity and the expression of active forms of caspase-3/-7/-9 and PARP protein. In addition, MBP triggered endoplasmic reticulum (ER) stress, as indicated by the upregulation of GRP 78, CHOP, and cleaved caspase-12 proteins. Pretreatment with 4-phenylbutyric acid (4-PBA; a pharmacological inhibitor of ER stress) markedly reversed MBP-induced ER stress and apoptosis-related signals. Furthermore, exposure to MBP significantly induced the protein phosphorylation of JNK and AMP-activated protein kinase (AMPK)α. Pretreatment of ß-cells with pharmacological inhibitors for JNK (SP600125) and AMPK (compound C), respectively, effectively abrogated the MBP-induced apoptosis-related signals. Both JNK and AMPK inhibitors also suppressed the MBP-induced activation of JNK and AMPKα and of each other. In conclusion, these findings suggest that MBP exposure exerts cytotoxicity on ß-cells via the interdependent activation of JNK and AMPKα, which regulates the downstream apoptotic signaling pathway.

Roles of ERK/Akt signals in mitochondria-dependent and endoplasmic reticulum stress-triggered neuronal cell apoptosis induced by 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene, a major active metabolite of bisphenol A.

Bisphenol A (BPA) is recognized as a harmful pollutant in the worldwide. Growing studies have reported that BPA can cause adverse effects and diseases in human, and link to a potential risk factor for development of neurodegenerative diseases (NDs). 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which generated in the mammalian liver after BPA exposure, is a major active metabolite of BPA. MBP has been suggested to exert greater toxicity than BPA. However, the molecular mechanism of MBP on the neuronal cytotoxicity remains unclear. In this study, MBP exposure significantly reduced Neuro-2a cell viability and induced apoptotic events that MBP (5-15 µM) exhibited greater neuronal cytotoxicity than BPA (50-100 µM). The mitochondria-dependent apoptotic signals including the decrease in mitochondrial membrane potential (MMP) and the increase in cytosolic apoptosis-induced factor (AIF), cytochrome c release, and Bax protein expression were involved in MBP (10 µM)-induced Neuro-2a cell death. Exposure of Neuro-2a cells to MBP (10 µM) also triggered endoplasmic reticulum (ER) stress through the induction of several key molecules including glucose-regulated protein (GRP)78, C/EBP homologous protein (CHOP), X-box binding protein (XBP)-1, protein kinase R-like ER kinase (PERK), eukaryotic initiation factor 2α (eIF2α), inositol-requiring enzyme(IRE)-1, activation transcription factor(AFT)4 and ATF6, and caspase-12. Pretreatment with 4-PBA (an ER stress inhibitor) and specific siRNAs for GRP78, CHOP, and XBP-1 significantly suppressed the expression of these ER stress-related proteins and the activation of caspase-12/-3/-7 in MBP-exposed Neuro-2a cells. Furthermore, MBP (10 µM) exposure dramatically increased the activation of extracellular regulated protein (ERK)1/2 and decreased Akt phosphorylation. Pretreatment with PD98059 (an ERK1/2 inhibitor) and transfection with the overexpression of activation of Akt1 (myr-Akt1) effectively suppressed MBP-induced apoptotic and ER stress-related signals. Collectively, these results demonstrate that MBP exposure exerts neuronal cytotoxicity via the interplay of ERK activation and Akt inactivation-regulated mitochondria-dependent and ER stress-triggered apoptotic pathway, which ultimately leads to neuronal cell death.

Oxidative storm in a patient with acute rotenone-containing plant poisoning.

A 64-year-old woman presented with coma, seizure, and lactic acidosis after ingesting 80 yam bean seeds. This rotenone-containing seeds cause cellular asphyxia via blockage of the mitochondrial electron transport. Subsequent oxidative stress results in the formation of lipid peroxidation (LPO). Rotenone analysis via liquid chromatography mass spectrometry revealed the following: 31,590 ng/mL in cooked yam bean seed and 100 ng/mL in the blood. We attempted to use N-acetylcysteine to alleviate oxidative stress and documented the continuous decline in the plasma concentration of LPO.

Cadmium exposure induces pancreatic ß-cell death via a Ca2+-triggered JNK/CHOP-related apoptotic signaling pathway.

Cadmium (Cd) is known to be ranked the 7th hazardous substance in the Substance Priority List by Agency for Toxic Substances and Disease Registry. The experimental and epidemiological data have suggested that Cd is linked to the development of diabetes mellitus (DM). The molecular mechanism of Cd on the pancreatic ß-cell cytotoxicity still remains unclear. Evidence has pointed toward that Ca2+ is an important regulator of toxic insult-induced ß-cell cytotoxicity. The role of Ca2+ in the Cd-induced ß-cell cytotoxicity is still unknown. In this study, we found that Cd exposure significantly inhibited insulin secretion and cell viability in the pancreatic ß-cell-derived RIN-m5F cells. Cd exposure induced apoptotic events, including the increased populations of apoptotic cells and sub-G1 hypodiploid cells, and caspase-3/-7/-9 and poly (ADP-ribose) polymerase (PARP) activation, which largely depended on the activation of c-Jun N-terminal kinase (JNK) and C/EBP homologous protein (CHOP). Transfection with siRNAs for JNK and CHOP or pretreatment with specific pharmacological inhibitor of JNK (SP600125) in ß-cells effectively prevented the Cd-induced insulin secretion dysfunction and apoptosis. JNK-specific siRNA dramatically suppressed Cd-induced JNK phosphorylation and CHOP protein expression, but JNK phosphorylation could not be inhibited by CHOP-specific siRNA. Furthermore, Cd exposure significantly increased the intracellular calcium ([Ca2+]i) levels. Buffering the Ca2+ response with BAPTA/AM effectively abrogated the Cd-induced [Ca2+]i elevation, insulin secretion dysfunction, apoptosis, and protein expression of JNK phosphorylation and CHOP activation. Taken together, these findings demonstrated that Cd exposure exerts ß-cell death via a [Ca2+]i-dependent JNK activation-activated downstream CHOP-related apoptotic signaling pathway.

Myeloid translocation gene CBFA2T3 directs a relapse gene program and determines patient-specific outcomes in AML.

CBFA2T3 is a master transcriptional coregulator in hematopoiesis. In this study, we report novel functions of CBFA2T3 in acute myeloid leukemia (AML) relapse. CBFA2T3 regulates cell-fate genes to establish gene expression signatures associated with leukemia stem cell (LSC) transformation and relapse. Gene set enrichment analysis showed that CBFA2T3 expression marks LSC signatures in primary AML samples. Analysis of paired primary and relapsed samples showed that acquisition of LSC gene signatures involves cell type-specific activation of CBFA2T3 transcription via the NM_005187 promoter by GCN5. Short hairpin RNA-mediated downregulation of CBFA2T3 arrests G1/S cell cycle progression, diminishes LSC gene signatures, and attenuates in vitro and in vivo proliferation of AML cells. We also found that the RUNX1-RUNX1T1 fusion protein transcriptionally represses NM_005187 to confer t(8;21) AML patients a natural resistance to relapse, whereas lacking a similar repression mechanism renders non-core-binding factor AML patients highly susceptible to relapse. These studies show that 2 related primary AML-associated factors, the expression level of CBFA2T3 and the ability of leukemia cells to repress cell type-specific CBFA2T3 gene transcription, play important roles in patient prognosis, providing a paradigm that differential abilities to repress hematopoietic coregulator gene transcription are correlated with patient-specific outcomes in AML.

Oolong Tea Extract Induces DNA Damage and Cleavage and Inhibits Breast Cancer Cell Growth and Tumorigenesis.

BACKGROUND/AIM: Breast cancer is the most prevalent and devastating malignant disease among women worldwide. Green tea has been extensively studied for its anti-cancer effects, however, existing literature on the correlation of other types of tea with breast cancer is very limited. MATERIALS AND METHODS: We used six different breast cancer cell lines (ER+, PR+ or HER2+ and triple-negative), treated under different concentrations of green, oolong, black and dark tea extracts, and determined their biological effects. RESULTS: We determined cell viability, observed the changes of cell morphology, measured DNA damage and cleavage, and analyzed the effect on soft agar colony formation and growth. CONCLUSION: Oolong tea, same as green tea, can induce DNA damage and cleavage, play an inhibitory role in breast cancer cell growth, proliferation and tumorigenesis, and was a great potential as a chemo-preventive agent against breast cancer.

Phosphorylated OmpR Is Required for Type 3 Fimbriae Expression in Klebsiella pneumoniae Under Hypertonic Conditions.

OmpR/EnvZ is a two-component system that senses osmotic signals and controls downstream gene expression in many species of Enterobacteriaceae. However, the role of OmpR/EnvZ in Klebsiella pneumoniae remains unknown. In this study, we found that production of MrkA, the major subunit of type 3 fimbriae, was decreased under hypertonic conditions. A deletion mutant of ompR and a site-directed mutant with a single amino acid substitution of aspartate 55 to alanine (D55A), which mimics the unphosphorylated form of OmpR, markedly reduced MrkA production under hypertonic conditions. These results indicate that K. pneumoniae type 3 fimbriae expression is activated by the phosphorylated form of OmpR (OmpR∼P). Although no typical OmpR∼P binding site was found in the P mrkA sequence, mrkA mRNA levels and P mrkA activity were decreased in the ΔompR and ompR D55A strains compared with the wild type (WT) strain, indicating that OmpR∼P mediates type 3 fimbriae expression at the transcriptional level. Previous reports have demonstrated that a cyclic-di-GMP (c-di-GMP) related gene cluster, mrkHIJ, regulates the expression of type 3 fimbriae. We found that both the ompR and ompR D55A mutants exhibited decreased mrkHIJ mRNA levels, intracellular c-di-GMP concentration, and bacterial biofilm amount, but increased total intracellular phosphodiesterase activity in response to hypertonic conditions. These results indicate that OmpR∼P regulates type 3 fimbriae expression to influence K. pneumoniae biofilm formation via MrkHIJ and modulation of intracellular c-di-GMP levels. Taken together, we herein provide evidence that OmpR∼P acts as a critical factor in the regulation of the c-di-GMP signaling pathway, type 3 fimbriae expression, and biofilm amount in K. pneumoniae in response to osmotic stresses.

Fludarabine-resistance associates with ceramide metabolism and leukemia stem cell development in chronic lymphocytic leukemia.

Fludarabine (flu) -containing regimens such as flu, cyclophosphamide and rituximab have been established as one of the standard first line therapy in medically-fit chronic lymphocytic leukemia (CLL) patients. Therefore, flu-refractory (primary flu-insensitivity or flu-caused relapse) remains a major problem causing treatment failure for CLL patients. We isolated the peripheral blood mononuclear cells (PBMCs) from CLL patients and treated with flu to find flu-refractory cases, and established flu-resistant clonal cells to study molecular mechanism of flu-resistance. By comparing parental MEC-2 cells, a human CLL cell line, we found that flu-resistant clonal cells were significantly increased lethal dose 50 of flu concentration, and up-regulated expression of P-glycoprotein, a drug-resistant marker, glucosylceramide synthase (GCS), an enzyme that can convert ceramide to glucosylceramide, and CD34, a leukemia stem cell marker. Overexpression of GCS leads to promptly elimination of cellular ceramide levels and accumulation of glucosylceramide, which reduces apoptosis and promotes survival and proliferation of flu-resistant clonal cells. Furthermore, we demonstrated that the accumulation of glucosylceramide can be blocked by PDMP to restore flu-sensitivity in flu-resistant clonal cells. We also found that elevating glucosylceramide levels in flu-resistant clonal cells was associated with up-regulation of GCS and CD34 expression. Importantly, overexpression of GCS or CD34 was also determined in flu-refractory PBMCs. Our results show that flu-resistance is associated with the alteration of ceramide metabolism and the development of leukemia stem cell-like cells. The flu-resistance can be reversed by GCS inhibition as a novel strategy for overcoming drug resistance.

Disrupting cholesterol esterification by bitter melon suppresses triple-negative breast cancer cell growth.

Triple negative breast cancer (TNBC) is aggressive with a worse prognosis. We have recently shown that bitter melon extract (BME) treatment was more effective in inhibition of TNBC tumor growth in mouse models as compared to ER positive breast tumor growth. Aberrant dysregulation of lipid metabolism is associated with breast cancer progression, however, anti-cancer mechanism of BME linking lipid metabolism in breast cancer growth remains unexplored. Here, we observed that accumulation of esterified cholesterol was reduced in BME treated TNBC cell lines as compared to control cells. We next evaluated expression levels of acyl-CoA: cholesterol acyltransferase 1 (ACAT-1) in TNBC cells treated with BME. Our results demonstrated that BME treatment inhibited ACAT-1 expression in TNBC cells. Subsequently, we found that sterol regulatory element-binding proteins-1 and -2, and FASN was significantly reduced in BME treated TNBC cell lines. Low-density lipoprotein receptor was also downregulated in BME treated TNBC cells as compared to control cells. We further demonstrated that BME feeding reduced tumor growth in TNBC mammospheres implanted into NSG mice, and inhibits ACAT-1 expression. To our knowledge, this is the first report demonstrating BME suppresses TNBC cell growth through ACAT-1 inhibition, and have potential for additional therapeutic regimen against human breast cancer.

Low-dose tributyltin exposure induces an oxidative stress-triggered JNK-related pancreatic ß-cell apoptosis and a reversible hypoinsulinemic hyperglycemia in mice.

Tributyltin (TBT), an endocrine disrupting chemical, can be found in food (particular in fish and seafood) and drinking water by contamination. Here, we elucidated the effects and possible mechanisms of low-dose TBT on the growth and function of pancreatic ß-cells and glucose metabolism in mice. Submicromolar-concentration of TBT significantly induced ß-cell cytotoxicity and apoptosis, which were accompanied by poly (ADP-ribose) polymerase cleavage and mitogen-activated protein kinases-JNK and ERK1/2 phosphorylation. TBT could also suppress the glucose-stimulated insulin secretion in ß-cells and isolated mouse islets. TBT increased reactive oxygen species production. TBT-induced ß-cell cytotoxicity and apoptosis were significantly prevented by antioxidant N-acetylcysteine (NAC) and JNK inhibitor SP600125, but not ERK1/2 inhibitor PD98059 and p38 inhibitor SB203580. Both NAC and SP600125 inhibited JNK phosphorylation and reduced cell viability in TBT-treated ß-cells. Four-week exposure of TBT (0.25 mg/kg) to mice revealed the decreased plasma insulin, increased blood glucose and plasma malondialdehyde, suppressed islet insulin secretion, and increased islet caspase-3 activity, which could be reversed by NAC treatment. After removing the TBT exposure for 2 weeks, the TBT-induced glucose metabolism alteration was significantly reversed. These results suggest that low-dose TBT can induce ß-cell apoptosis and interfere with glucose homeostasis via an oxidative stress-related pathway.