Natural Product-Based Glycolysis Inhibitors as a Therapeutic Strategy for Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. Targeted therapy against the epidermal growth factor receptor (EGFR) is a promising treatment approach for NSCLC. However, resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a major challenge in its clinical management. EGFR mutation elevates the expression of hypoxia-inducible factor-1 alpha to upregulate the production of glycolytic enzymes, increasing glycolysis and tumor resistance. The inhibition of glycolysis can be a potential strategy for overcoming EGFR-TKI resistance and enhancing the effectiveness of EGFR-TKIs. In this review, we specifically explored the effectiveness of pyruvate dehydrogenase kinase inhibitors and lactate dehydrogenase A inhibitors in combating EGFR-TKI resistance. The aim was to summarize the effects of these natural products in preclinical NSCLC models to provide a comprehensive understanding of the potential therapeutic effects. The study findings suggest that natural products can be promising inhibitors of glycolytic enzymes for the treatment of EGFR-TKI-resistant NSCLC. Further investigations through preclinical and clinical studies are required to validate the efficacy of natural product-based glycolytic inhibitors as innovative therapeutic modalities for NSCLC.

Cellular and subcellular localization of endogenous phospholipase D6 in seminiferous tubules of mouse testes.

Phospholipase D6 (PLD6) plays pivotal roles in mitochondrial dynamics and spermatogenesis, but the cellular and subcellular localization of endogenous PLD6 in testis germ cells is poorly defined. We examined the distribution and subcellular localization of PLD6 in mouse testes using validated specific anti-PLD6 antibodies. Ectopically expressed PLD6 protein was detected in the mitochondria of PLD6-transfected cells, but endogenous PLD6 expression in mouse testes was localized to the perinuclear region of pachytene spermatocytes, and more prominently, to the round (Golgi and cap phases) and elongating spermatids (acrosomal phase); these results suggest that PLD6 is localized to the Golgi apparatus. The distribution of PLD6 in the round spermatids partially overlapped with that of the cis-Golgi marker GM130, indicating that the PLD6 expression corresponded to the GM130-positive subdomains of the Golgi apparatus. Correlative light and electron microscopy revealed that PLD6 expression in developing spermatids was localized almost exclusively to several flattened cisternae, and these structures might correspond to the medial Golgi subcompartment; neither the trans-Golgi networks nor the developing acrosomal system expressed PLD6. Further, we observed that PLD6 interacted with tesmin, a testis-specific transcript necessary for successful spermatogenesis in mouse testes. To our knowledge, these results provide the first evidence of PLD6 as a Golgi-localized protein of pachytene spermatocytes and developing spermatids and suggest that its subcompartment-specific distribution within the Golgi apparatus may be related to the specific functions of this organelle during spermatogenesis.

MicroRNA-320a and microRNA-4496 attenuate Helicobacter pylori cytotoxin-associated gene A (CagA)-induced cancer-initiating potential and chemoresistance by targeting ß-catenin and ATP-binding cassette, subfamily G, member 2.

Infection with Helicobacter pylori is closely linked to an increased risk of gastric cancer. Although cytotoxin-associated gene A (CagA), a major virulence factor of H. pylori, is known to be a causal factor for gastric carcinogenesis, the molecular link between CagA and gastric cancer-initiating cell (CIC)-like properties remains elusive. Here, we demonstrate that CagA is required for increased expression of ß-catenin and its target CIC markers via downregulation of microRNA (miR)-320a and miR-4496. CagA promoted gastric CIC properties and was responsible for chemoresistance. miR-320a and miR-4496 attenuated the in vitro self-renewal and tumour-initiating capacity of CagA-expressing CICs by targeting ß-catenin. Moreover, miR-320a and miR-4496 decreased CagA-induced chemoresistance by targeting ATP-binding cassette, subfamily G, member 2 (ABCG2) at the transcriptional and post-transcriptional levels, respectively. Combination therapy with 5-fluorouracil and miR-320a/miR-4496 suppressed gastric tumourigenesis and metastatic potential in an orthotopic mouse model, probably via suppression of CagA-induced CIC properties and chemoresistance. Our results provide novel evidence that CIC properties, chemoresistance and tumourigenesis associated with H. pylori are linked to CagA-induced upregulation of ß-catenin and ABCG2. These data provide novel insights into the molecular mechanisms of CagA-induced carcinogenisis and the therapeutic potential of of miR-320a and miR-4496. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Targeting pyruvate dehydrogenase kinase 1 overcomes EGFR C797S mutation-driven osimertinib resistance in non-small cell lung cancer.

Osimertinib, a selective third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), effectively targets the EGFR T790M mutant in non-small cell lung cancer (NSCLC). However, the newly identified EGFR C797S mutation confers resistance to osimertinib. In this study, we explored the role of pyruvate dehydrogenase kinase 1 (PDK1) in osimertinib resistance. Patients exhibiting osimertinib resistance initially displayed elevated PDK1 expression. Osimertinib-resistant cell lines with the EGFR C797S mutation were established using A549, NCI-H292, PC-9, and NCI-H1975 NSCLC cells for both in vitro and in vivo investigations. These EGFR C797S mutant cells exhibited heightened phosphorylation of EGFR, leading to the activation of downstream oncogenic pathways. The EGFR C797S mutation appeared to increase PDK1-driven glycolysis through the EGFR/AKT/HIF-1α axis. Combining osimertinib with the PDK1 inhibitor leelamine helped successfully overcome osimertinib resistance in allograft models. CRISPR-mediated PDK1 knockout effectively inhibited tumor formation in xenograft models. Our study established a clear link between the EGFR C797S mutation and elevated PDK1 expression, opening new avenues for the discovery of targeted therapies and improving our understanding of the roles of EGFR mutations in cancer progression.

Andrographolide suppresses aerobic glycolysis and induces apoptotic cell death by inhibiting pyruvate dehydrogenase kinase 1 expression.

Metabolic disorder is a major characteristic of cancer cells, and controlling genes involved in metabolic shifts can be an effective strategy for cancer treatment. Andrographolide (AG), a diterpenoid lactone, is widely recognized as a natural anticancer drug due to its ability to inhibit cancer growth. The present study aimed to investigate the mechanism underlying the mitochondrial­mediated anticancer effect of AG by inhibiting pyruvate dehydrogenase kinase 1 (PDK1) expression in lung cancer cells. Cells were treated with AG and PDK1 mRNA and protein expression was determined using reverse transcription­quantitative PCR and western blotting, respectively. As a result, AG significantly inhibited the viability of human lung cancer cells and suppressed aerobic glycolysis by decreasing lactate generation. AG further decreased the PDK1 protein and mRNA levels in a dose­dependent manner. AG­induced cell death was assessed by flow cytometry and fluorescence microscopy. AG induced apoptotic cell death that was associated with the cleavage of poly (ADP ribose) polymerase, activation of caspase­3, and mitochondrial damage, which was associated with an increase in reactive oxygen species and loss of mitochondrial membrane potential. AG­induced cell death was partially suppressed via PDK1 overexpression in lung cancer cells. Therefore, the anticancer effects of AG on human lung cancer cells may negatively regulate the expression of PDK1.

Acrolein sensitizes human renal cancer Caki cells to TRAIL-induced apoptosis via ROS-mediated up-regulation of death receptor-5 (DR5) and down-regulation of Bcl-2.

TRAIL resistance in many cancer cells is one of the major problems in TRAIL-based cancer therapy. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are strictly needed for the improvement of anti-cancer effect of TRAIL. Acrolein is a byproduct of lipid peroxidation, which has been involved in pulmonary, cardiac and neurodegenerative diseases. We investigated whether acrolein, an α,ß-unsaturated aldehyde, can potentiate TRAIL-induced apoptosis in human renal cancer cells. The combined treatment with acrolein and TRAIL significantly induced apoptosis, and stimulated of caspase-3 activity, DNA fragmentation, and cleavage of PARP. We found that acrolein down-regulated the protein level of Bcl-2 and Bcl-2 overexpression inhibited the cell death induced by the combined treatment with acrolein and TRAIL. In addition, acrolein up-regulated C/EBP homologous protein (CHOP) and TRAIL death receptor 5 (DR5) and down-regulation of CHOP or DR5 expression using the respective small interfering RNA significantly attenuated the apoptosis induced by acrolein plus TRAIL. Interestingly, pretreatment with an antioxidant, N-acetylcysteine (NAC), inhibited not only CHOP and DR5 up-regulation but also the cell death induced by acrolein plus TRAIL. Taken together, our results demonstrated that acrolein enhances TRAIL-induced apoptosis in Caki cells through down-regulation of Bcl-2 and ROS dependent up-regulation of DR5.

Compound C sensitizes Caki renal cancer cells to TRAIL-induced apoptosis through reactive oxygen species-mediated down-regulation of c-FLIPL and Mcl-1.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), either alone or in combination with other anticancer drugs, is considered as a new strategy for anticancer therapy. Compound C, a cell-permeable pyrrazolopyrimidine derivative, acts as a potent, selective, reversible ATP-competitive inhibitor of AMP-activated protein kinase (AMPK). In this study, we show that compound C sensitizes Caki human renal cancer cells, but not normal human skin fibroblast cells (HSF) and human mesangial cells, to TRAIL-mediated apoptosis. However, AMPK siRNA failed to affect TRAIL-mediated apoptosis in Caki cells and transduction of dominant negative AMPK rather attenuated TRAIL-induced apoptosis, indicating that the effect of compound C on sensitization of TRAIL-induced apoptosis is independent of AMPK activity. Interestingly, we found that down-regulation of c-FLIP(L) and Mcl-1 contributes to compound C-enhanced TRAIL-induced apoptosis. Reduced expression of c-FLIP(L) and Mcl-1 were caused by the decreased protein stability of c-FLIP(L) and Mcl-1, but not by their transcriptional control, in compound C-treated cells. Pretreatment with N-acetyl-L-cysteine (NAC) significantly inhibited the cell death induced by the combined treatment with compound C and TRAIL as well as recovered the expression levels of c-FLIP(L) and Mcl-1 down-regulated by the combinatory treatment with compound C plus TRAIL, suggesting that compound C-stimulated TRAIL-induced apoptosis appears to be dependent on the generation of reactive oxygen species for down-regulation of c-FLIP(L) and Mcl-1. Taken together, the present study demonstrates that compound C enhances TRAIL-induced apoptosis in human renal cancer cells by ROS-mediated c-FLIP(L) and Mcl-1 down-regulation.

Herbal formulation MIT ameliorates high-fat diet-induced non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases and is caused by obesity, diabetes, high blood pressure, and insulin resistance. Many studies have explored novel candidates to treat NAFLD using herbal medicines owing to their fewer side effects. In this study, we examined the effect of MIT, an herbal formula comprising Ephedra sinica, Panax ginseng, and Alisma orientale, on the murine model of NAFLD. METHODS: To evaluate the effect of MIT on NAFLD, we used the high-fat diet (HFD)-induced NAFLD mice model. The mice were divided into four groups: control, HFD, HFD with metformin administration, and HFD with MIT administration. Freeze-dried MIT was dissolved in phosphate buffered saline and orally administered for 8 weeks to MIT-treated mice (60 mg/kg) after feeding them with HFD for 16 weeks. RESULTS: MIT treatment significantly attenuated fat accumulation, serum glucose levels, and excessive cholesterol. It also reduced the activation of NF-κB, JNK, ERK, mammalian target of rapamycin, and peroxisome proliferator-activated receptor γ in the HFD-induced NAFLD mice. The expression level of enzymes involved in the synthesis and oxidation of fatty acids, acetyl-coA carboxylase and CYP2E1, were clearly reduced by MIT treatment. Reactive oxygen species (ROS) production and subsequent liver damage were effectively reduced by MIT treatment. CONCLUSION: We suggest that MIT is a potent herbal formula that can be used for the prevention and treatment of obesity-related NAFLD via regulating the levels of serum glucose and free fatty acids, inflammation, lipid accumulation, and ROS-mediated liver damage.

The coffee diterpene kahweol induces apoptosis in human leukemia U937 cells through down-regulation of Akt phosphorylation and activation of JNK.

Kahweol, the coffee-specific diterpene, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity. The mechanism by which kahweol initiates apoptosis remains poorly understood. In the present study, we investigated the effect of kahweol on the apoptotic pathway in U937 human promonocytic cells. We show that kahweol induces apoptosis in association with the activation of caspase 3 and cytochrome c release from the mitochondria to the cytosol, as well as down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and XIAP). Kahweol altered the phosphorylation state of members of the MAPKs and Akt. Ectopic expression of Bcl-2 or constitutive active Akt (myr-Akt) in U937 cells attenuates kahweol-induced apoptosis. In addition, we have also shown that JNK and Akt signal pathway plays a crucial role in kahweol-induced apoptosis in U937 cells. Taken together, our results show the activity of kahweol to modulate multiple components in apoptotic response of human leukemia cells and raise the possibility a novel therapeutic strategy in hematological malignancies.

Regulation of heat shock-induced apoptosis by sensitive to apoptosis gene protein.

Heat shock may increase oxidative stress due to increased production of reactive oxygen species and/or the promotion of cellular oxidation events. Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates heat shock-induced apoptosis. When we examined the protective role of SAG against heat shock-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed a significant decrease in the endogenous production of reactive oxygen species and oxidative DNA damage in SAG-overexpressed cells compared to control cells on exposure to heat shock. In addition, transfection of PC3 cells with SAG small interfering RNA markedly decreased the expression of SAG, enhancing the susceptibility of heat shock-induced apoptosis. Taken together, these results indicate that SAG may play an important role in regulating the apoptosis induced by heat shock presumably through maintaining the cellular redox status.

Ethanol induces peroxynitrite-mediated toxicity through inactivation of NADP+-dependent isocitrate dehydrogenase and superoxide dismutase.

It has been reported that chronic alcohol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. Several studies have shown the importance of superoxide dismutase (SOD) in protecting cells against ethanol-induced oxidative stress. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. In this report, we demonstrate that ethanol induces the peroxynitrite-mediated cytotoxicity in HepG2 cells through inactivation of antioxidant enzymes such as ICDH and SOD. Upon exposure to 100mM ethanol for 3days to HepG2 cells, a significant decrease in the viability and activities of ICDH and SOD was observed. The ethanol-induced inactivation of antioxidant enzymes resulted in the cellular oxidative damage and modulation of redox status as well as mitochondrial dysfunction in HepG2 cells. The cytoxicity of ethanol and inactivation of antioxidant enzymes were effectively protected by manganeses(III) tetrakis(N-methyl-2-pyridyl) porphyrin, a manganese SOD mimetic, and N'-monomethyl-l-arginine, a nitric oxide synthase inhibitor. These results indicate that ethanol toxicity is mediated by peroxynitrite and the peroxynitrite-mediated damage to ICDH and SOD may be resulted in the perturbation of the cellular antioxidant defense systems and subsequently lead to a pro-oxidant condition.

Epigallocatechin gallate, a constituent of green tea, regulates high glucose-induced apoptosis.

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidative stress. Therefore, compounds that scavenge reactive oxygen species may confer regulatory effects on high glucose-induced apoptosis. Epigallocatechin gallate (EGCG), the major polyphenolic of green tea, is reported to have an antioxidant activity. We investigated the effect of EGCG on high glucose-induced apoptosis in U937 cells. Upon exposure to 35 mM glucose for 2 days, there was a distinct difference between untreated cells and cells pre-treated with 1 microM EGCG for 2 h in regard to cellular redox status and oxidative DNA damage to cells. EGCG pre-treated cells showed significant suppression of apoptotic features such as DNA fragmentation, damage to mitochondrial function, and modulation of apoptotic marker proteins upon exposure to high glucose. This study indicates that EGCG may play an important role in regulating the apoptosis induced by high glucose presumably through scavenging of reactive oxygen species.

Regulation of nitric oxide-induced apoptosis by sensitive to apoptosis gene protein.

Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. Nitric oxide (NO), a radical species produced by many types of cells, is known to play a critical role in many regulatory processes, yet it may also participate in collateral reactions at higher concentrations, leading to cellular oxidative stress. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates NO-induced apoptosis. When we examined the protective role of SAG against NO-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed the significant decrease in the endogenous production of ROS and oxidative DNA damage in SAG-overexpressed cells compared to control cells upon exposure to NO. These results suggest that SAG plays an important protective role in NO-induced apoptosis, presumably, through regulating the cellular redox status.

Protection of peroxynitrite-induced DNA damage by dietary antioxidants.

The present study was undertaken to test the hypothesis that dietary antioxidants protect DNA damage induced by peroxynitrite, a potent physiological inorganic toxin. The present study showed that dietary antioxidants such as (-)-epigallocatechin gallate, quercerin, rutin, resveratrol, and ursolic acid inhibit single strand breaks in supercoiled plasmid DNA induced by 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion. The formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in calf thymus DNA by SIN-1 was also inhibited by dietary antioxidants. When U937 cells were incubated with 1 mM SIN-1 bolus, a significant increase of 8-OH-dG level was observed. However, oxidative DNA damage was significantly lower in the cells pre-treated with dietary antioxidants when cells were exposed to SIN-1.

Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells.

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP+ -dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide.

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. NO donors such as S-nitrosothiols, diethylamine NONOate, spermine NONOate, and 3-morpholinosydnomine N-ethylcarbamide (SIN-1)/superoxide dismutase inactivated ICDH in a dose- and time-dependent manner. The inhibition of ICDH by S-nitrosothiol was partially reversed by thiol, such as dithiothreitol or 2-mercaptoethanol. Loss of enzyme activity was associated with the depletion of the cysteine-reactive 5,5'-dithiobis-(2-nitrobenzoate) and the loss of fluorescent probe N,N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine accessible thiol groups. Using electrospray ionization mass spectrometry with tryptic digestion of protein, we found that nitric oxide forms S-nitrosothiol adducts on Cys305 and Cys387. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by NO. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and intrinsic tryptophan fluorescence. When U937 cells were incubated with 200 microM SNAP for 1 h, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Furthermore, stimulation with lipopolysaccharide significantly decreased intracellular ICDH activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N(omega)-methyl-L-arginine. This result indicates that ICDH was also inactivated by endogenous NO. The NO-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.

Lipid peroxidation-mediated cytotoxicity and DNA damage in U937 cells.

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. In the present study, we evaluated lipid peroxidation-mediated cytotoxicity and oxidative DNA damage in U937 cells. Upon exposure of U937 cells to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the cells exhibited a reduction in viability and an increase in the endogenous production of reactive oxygen species (ROS), as measured by the oxidation of 2',7'-dichlorodihydrofluorescein. In addition, a significant decrease in the intracellular GSH level and the activities of major antioxidant enzymes were observed. We also observed lipid peroxidation-mediated oxidative DNA damage, reflected by an increase in 8-OH-dG level and loss of the ability of DNA to renature. When the cells were pretreated with the antioxidant N-acetylcysteine (NAC) or the spin trap alpha-phenyl-N-t-butylnitrone (PBN), lipid peroxidation-mediated cytotoxicity in U937 cells was protected. This effect seems to be due to the ability of NAC and PBN to reduce ROS generation induced by lipid peroxidation. These results suggest that lipid peroxidation resulted in a pro-oxidant condition of U937 cells by the depletion of GSH and inactivation of antioxidant enzymes, which consequently leads to a decrease in survival and oxidative damage to DNA. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in oxidative stress-induced cellular damage.

Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products.

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP+-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a prooxidant condition.

Expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in the rat dental pulp and trigeminal ganglion following inflammation.

BACKGROUND: There is increasing evidence that peripheral glutamate signaling mechanism is involved in the nociceptive transmission during pathological conditions. However, little is known about the glutamate signaling mechanism and related specific type of vesicular glutamate transporter (VGLUT) in the dental pulp following inflammation. To address this issue, we investigated expression and protein levels of VGLUT1 and VGLUT2 in the dental pulp and trigeminal ganglion (TG) following complete Freund's adjuvant (CFA) application to the rat dental pulp by light microscopic immunohistochemistry and Western blot analysis. RESULTS: The density of VGLUT2- immunopositive (+) axons in the dental pulp and the number of VGLUT2+ soma in the TG increased significantly in the CFA-treated group, compared to control group. The protein levels of VGLUT2 in the dental pulp and TG were also significantly higher in the CFA-treated group than control group by Western blot analysis. The density of VGLUT1+ axons in the dental pulp and soma in the TG remained unchanged in the CFA-treated group. CONCLUSIONS: These findings suggest that glutamate signaling that is mediated by VGLUT2 in the pulpal axons may be enhanced in the inflamed dental pulp, which may contribute to pulpal axon sensitization leading to hyperalgesia following inflammation.

Ultrastructure and synaptic connectivity of main and accessory olfactory bulb efferent projections terminating in the rat anterior piriform cortex and medial amygdala.

Neurons in the main olfactory bulb relay peripheral odorant signals to the anterior piriform cortex (aPir), whereas neurons of the accessory olfactory bulb relay pheromone signals to the medial amygdala (MeA), suggesting that they belong to two functionally distinct systems. To help understand how odorant and pheromone signals are further processed in the brain, we investigated the synaptic connectivity of identified axon terminals of these neurons in layer Ia of the aPir and posterodorsal part of the MeA, using anterograde tracing with horseradish peroxidase, quantitative ultrastructural analysis of serial thin sections, and immunogold staining. All identified boutons contained round vesicles and some also contained many large dense core vesicles. The number of postsynaptic dendrites per labeled bouton was significantly higher in the aPir than in the MeA, suggesting higher synaptic divergence at a single bouton level. While a large fraction of identified boutons (29%) in the aPir contacted 2-4 postsynaptic dendrites, only 7% of the identified boutons in the MeA contacted multiple postsynaptic dendrites. In addition, the majority of the identified boutons in the aPir (95%) contacted dendritic spines, whereas most identified boutons in the MeA (64%) contacted dendritic shafts. Identified boutons and many of the postsynaptic dendrites showed glutamate immunoreactivity. These findings suggest that odorant and pheromone signals are processed differently in the brain centers of the main and accessory olfactory systems.