Synthesis and evaluation of esterified Hsp70 agonists in cellular models of protein aggregation and folding.

Over-expression of the Hsp70 molecular chaperone prevents protein aggregation and ameliorates neurodegenerative disease phenotypes in model systems. We identified an Hsp70 activator, MAL1-271, that reduces α-synuclein aggregation in a Parkinson's Disease model. We now report that MAL1-271 directly increases the ATPase activity of a eukaryotic Hsp70. Next, twelve MAL1-271 derivatives were synthesized and examined in a refined α-synuclein aggregation model as well as in an assay that monitors maturation of a disease-causing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutant, which is also linked to Hsp70 function. Compared to the control, MAL1-271 significantly increased the number of cells lacking α-synuclein inclusions and increased the steady-state levels of the CFTR mutant. We also found that a nitrile-containing MAL1-271 analog exhibited similar effects in both assays. None of the derivatives exhibited cellular toxicity at concentrations up to 100 µm, nor were cellular stress response pathways induced. These data serve as a gateway for the continued development of a new class of Hsp70 agonists with efficacy in these and potentially other disease models.

Small molecule IVQ, as a prodrug of gluconeogenesis inhibitor QVO, efficiently ameliorates glucose homeostasis in type 2 diabetic mice.

Gluconeogenesis is a major source of hyperglycemia in patients with type 2 diabetes mellitus (T2DM), thus targeting gluconeogenesis to suppress glucose production is a promising strategy for anti-T2DM drug discovery. In our preliminary in vitro studies, we found that a small-molecule (E)-3-(2-(quinoline-4-yl)vinyl)-1H-indol-6-ol (QVO) inhibited the hepatic glucose production (HGP) in primary hepatocytes. We further revealed that QVO suppressed hepatic gluconeogenesis involving calmodulin-dependent protein kinase kinase ß- and liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathways as well as AMPK-independent mitochondrial function-related signaling pathway. To evaluate QVO's anti-T2DM activity in vivo, which was impeded by the complicated synthesis route of QVO with a low yield, we designed and synthesized 4-[2-(1H-indol-3-yl)vinyl]quinoline (IVQ) as a prodrug with easier synthesis route and higher yield. IVQ did not inhibit the HGP in primary hepatocytes in vitro. Pharmacokinetic studies demonstrated that IVQ was quickly converted to QVO in mice and rats following administration. In both db/db and ob/ob mice, oral administration of IVQ hydrochloride (IVQ-HCl) (23 and 46 mg/kg every day, for 5 weeks) ameliorated hyperglycemia, and suppressed hepatic gluconeogenesis and activated AMPK signaling pathway in the liver tissues. Furthermore, IVQ caused neither cardiovascular system dysfunction nor genotoxicity. The good druggability of IVQ has highlighted its potential in the treatment of T2DM and the prodrug design for anti-T2DM drug development.

Low dose ouabain stimulates NaK ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells.

Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of NaK ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of NaK ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1µg/kg body wt./day) for 9days and increased angiotensin II levels in cell culture media after 24h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10pM stimulated NKA-mediated 86Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension.

Utilization of the Zucker Diabetic Fatty (ZDF) Rat Model for Investigating Hypoglycemia-related Toxicities.

Glucokinase (GK) catalyzes the initial step in glycolysis and is a key regulator of glucose homeostasis. Therefore, glucokinase activators (GKa) have potential benefit in treating type 2 diabetes. Administration of a Bristol-Myers Squibb GKa (BMS-820132) to healthy euglycemic Sprague-Dawley (SD) rats and beagle dogs in 1 mo toxicology studies resulted in marked and extended hypoglycemia with associated clinical signs of toxicity and degenerative histopathological changes in the stomach, sciatic nerve, myocardium, and skeletal muscles at exposures comparable to those expected at therapeutic clinical exposures. To investigate whether these adverse effects were secondary to exaggerated pharmacology (prolonged hypoglycemia), BMS-820132 was administered daily to male Zucker diabetic fatty (ZDF) rats for 1 mo. ZDF rats are markedly hyperglycemic and insulin resistant. BMS-820132 did not induce hypoglycemia, clinical signs of hypoglycemia, or any of the histopathologic adverse effects observed in the 1 mo toxicology studies at exposures that exceeded those observed in SD rats and dogs. This indicates that the toxicity observed in euglycemic animals was secondary to the exaggerated pharmacology of potent GK activation. This study indicates that ZDF rats, with conventional toxicity studies, are a useful disease model for testing antidiabetic agents and determining toxicities that are independent of prolonged hypoglycemia.

Cytotoxin-induced NADPH oxides activation: roles in regulation of cell death.

Numerous studies have shown that a variety of cytotoxic agents can activate the NADPH oxidase system and induce redox-dependent regulation of cellular functions. Cytotoxin-induced NADPH oxidase activation may either exert cytoprotective actions (e.g., survival, proliferation, and stress tolerance) or cause cell death. Here we summarize the experimental evidence showing the context-dependent dichotomous effects of NADPH oxidase on cell fate under cytotoxic stress conditions and the potential redox signaling mechanisms underlying this phenomenon. Clearly, it is difficult to create a unified paradigm on the toxicological implications of NADPH oxidase activation in response to cytotoxic stimuli. We suggest that interventional strategies targeting the NADPH oxidase system to prevent the adverse impacts of cytotoxins need to be contemplated in a stimuli- and cell type-specific manner.

Doxorubicin causes transient activation of protein poly(ADP-ribosyl)ation in H9c2 cardiomyocytes.

Possible involvement of the system of protein poly(ADP-ribosyl)ation in the mechanisms of cardiotoxicity of doxorubicin, one of the most frequently used anticancer drug, was studied in cultures of cardiomyocytes H9c2. The treatment of H9c2 cells with doxorubicin (1 µM) led to a transient (after 6 h of incubation) increase in the nuclear level of poly(ADP-ribosyl)ated proteins. The observed data indirectly indicate the development of genotoxic stress in the doxorubicin-treated cells, probably caused by the stimulatory effects of doxorubicin and its metabolites on the production of reactive oxygen and nitrogen species.

Biochemical alterations induced by acute oral doses of iron oxide nanoparticles in Wistar rats.

Magnetic iron oxide nanoparticles with appropriate surface chemistry have been widely used with potential new applications in biomedical industry. Therefore, the aim of this study was to assess the size-, dose-, and time-dependent effects, after acute oral exposure to iron oxide-30 NP (Fe(2)O(3)-30), on various biochemical enzyme activities of clinical significances in a female Wistar rat model. Rats were exposed to three different doses (500, 1,000, and 2,000 mg/kg) of Fe(2)O(3)-30 and Fe(2)O(3)-Bulk along with control. Fe(2)O(3)-30 had no effect on growth, behavior, and nutritional performance of animals. Fe(2)O(3)-30 caused significant inhibition of acetylcholinestrase in red blood cells as well as in brains of treated rats. Further, more than 50% inhibition of total, Na(+)-K(+), Mg(2+), and Ca(2+)-ATPases activities, as observed in brains of exposed female rats, may be the result of disturbances in cellular physiology and the iono-regulatory process. Activation of the hepatotoxicity marker enzymes, aspartate aminotransferase and alanine aminotransferase, was recorded in serum and liver, whereas inhibition was observed in kidney. Similarly, enhancement of lactate dehydrogenase activity was observed in serum and liver; however, a decrease in enzyme levels was observed in kidneys of Fe(2)O(3)-30-treated rats. On the other hand, Fe(2)O(3)-Bulk did not depict any significant changes in these biochemical parameters, and alterations were near to control. Therefore, this study suggests that exposure to nanosize particles at acute doses may cause adverse changes in animal biochemical profiles. The use of the rat model signifies the correlation with the human system.

The role of DNA damage and caspase activation in cytotoxicity and genotoxicity of macrophages induced by bisphenol-A-glycidyldimethacrylate.

AIM: To evaluate the potential toxicological implications of BisGMA on murine macrophage cell line RAW264.7. METHODOLOGY: Lactate dehydrogenase release, flow cytometry, Western blot and fluorometric assays were used to detect cell viability, mode of cell death and caspase activities, respectively. In addition, alkaline single-cell gel electrophoresis and cytokinesis-block micronucleus assays were applied to detect genotoxicity. Statistical analyses were performed using anova followed by the Bonferroni's t-test for multi-group comparisons test. RESULTS: BisGMA demonstrated a cytotoxic effect on RAW264.7 cells in a dose-dependent and a time-dependent manner (P < 0.05). BisGMA was found to induce two modes of cell death. The mode of cell death changed from apoptosis to necrosis as the concentrations of BisGMA elevated. Caspase-3, caspase-8 and caspase-9 activities were significantly induced by BisGMA in a dose-dependent manner (P < 0.05). Moreover, BisGMA exhibited genotoxicity via a dose-related increase in the numbers of micronucleus and DNA strand breaks (P < 0.05). CONCLUSIONS: Cytotoxicity and genotoxicity induced by BisGMA are mediated by DNA damage and caspase activation.

Activation of NAD(P)H:quinone oxidoreductase 1 prevents arterial restenosis by suppressing vascular smooth muscle cell proliferation.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are important pathogenic mechanisms in atherosclerosis and restenosis after vascular injury. In this study, we investigated the effects of beta-lapachone (betaL) (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), which is a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase (NQO)1 activity, on neointimal formation in animals given vascular injury and on the proliferation of VSMCs cultured in vitro. betaL significantly reduced the neointimal formation induced by balloon injury. betaL also dose-dependently inhibited the FCS- or platelet-derived growth factor-induced proliferation of VSMCs by inhibiting G(1)/S phase transition. betaL increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase 1 in rat and human VSMCs. Chemical inhibitors of AMPK or dominant-negative AMPK blocked the betaL-induced suppression of cell proliferation and the G(1) cell cycle arrest, in vitro and in vivo. The activation of AMPK in VSMCs by betaL is mediated by LKB1 in the presence of NQO1. Taken together, these results show that betaL inhibits VSMCs proliferation via the NQO1 and LKB1-dependent activation of AMPK. These observations provide the molecular basis that pharmacological stimulation of NQO1 activity is a new therapy for the treatment of vascular restenosis and/or atherosclerosis which are caused by proliferation of VSMCs.

Analogs of the Dopamine Metabolite 5,6-Dihydroxyindole Bind Directly to and Activate the Nuclear Receptor Nurr1.

The nuclear receptor-related 1 protein, Nurr1, is a transcription factor critical for the development and maintenance of dopamine-producing neurons in the substantia nigra pars compacta, a cell population that progressively loses the ability to make dopamine and degenerates in Parkinson's disease. Recently, we demonstrated that Nurr1 binds directly to and is regulated by the endogenous dopamine metabolite 5,6-dihydroxyindole (DHI). Unfortunately, DHI is an unstable compound, and thus a poor tool for studying Nurr1 function. Here, we report that 5-chloroindole, an unreactive analog of DHI, binds directly to the Nurr1 ligand binding domain with micromolar affinity and stimulates the activity of Nurr1, including the transcription of genes governing the synthesis and packaging of dopamine.

A novel glucokinase activator TMG-123 causes long-lasting hypoglycemia and impairs spermatogenesis irreversibly in rats.

The importance of glucose is well known as an energy source in testes. In order to evaluate the effects of long-lasting hypoglycemia on testes, a novel glucokinase activator, TMG-123, was dosed to rats at 5, 20 and 100 mg/kg for 13 weeks. As a result, plasma glucose levels decreased for several hours with increasing doses over the dose range of 5 to 100 mg/kg. No toxicological findings attributable to the test article were observed in clinical observation, measurements of body weight and food consumption, necropsy, and organ weight measurement. Histopathology showed scattered degeneration of seminiferous tubules in testes, and exfoliation of germ cells related to the degeneration of seminiferous tubules was observed in the lumen of both epididymides in the same animals at the end of the dosing period. Similar histopathological findings were noted at the end of the recovery period. In addition, a fertility study was conducted at the same doses for 13 weeks for males and 5 weeks for females. Sperm analysis showed decreases in the sperm concentration and the motility index and an increase in the incidences of sperm malformations. However, there were no abnormalities in the copulation or fertility rate. These results suggest that long-lasting hypoglycemia in rats is harmful to spermatogenesis and the testicular damage does not recover.

Role of aromatase activation on sodium arsenite-induced proliferation, migration, and invasion of MDA-MB-231 and MDA-MB-453 breast cancer cell lines.

Arsenic is an endocrine disruptor that promotes breast cancer (BCa) development. Estrogen synthesis, through aromatase activation, is essential for BCa promotion and progression through activating the G-coupled estrogen receptor 1 (GPER1), regulating rapid nongenomic effects involved in cell proliferation and migration of BCa cells. Herein, was studied the role of aromatase activation and the GPER1 pathway on sodium arsenite-induced promotion and progression of MDA-MB-231 and MDA-MB-453 BCa cell lines. Our results demonstrated that 0.1 µM of sodium arsenite induces cell proliferation, migration, invasion, and stimulates aromatase activity of BCa cell lines MDA-MB-231, MDA-MB-453, MCF-7, but not in a nontumorigenic breast epithelial cell line (MCF-12A). Using letrozole (an aromatase inhibitor) and G-15 (a GPER1-selective antagonist), we demonstrated that sodium arsenite-induced proliferation and migration is mediated by induction of aromatase enzyme and, at least in part, by GPER1 activation in MDA-MB-231 and MDA-MB-453 cells. Sodium arsenite induced phosphorylation of Src that participated in sodium arsenite-induced aromatase activity, and -cell proliferation of MDA-MB-231 cell line. Overall, data suggests that sodium arsenite induces a positive-feedback loop, resulting in the promotion and progression of BCa cells, through induction of aromatase activity, E2 production, GPER1 stimulation, and Src activation.

The effect of cotinine on telomerase activity in human vascular smooth muscle cells.

AIM: Cotinine, the main stable metabolite of nicotine, has been shown to have a biological half-life approximately 10 times longer than nicotine. It has also been demonstrated to have a powerful effect on vascular smooth muscle cell (VSMC) proliferation. Telomerase activation is known to play an important role in cell viability and proliferation. The purpose of our experiment was to evaluate the effect of cotinine on proliferative potential of vascular smooth muscle cells via its effects on telomerase activity. METHODS: Primary cultures of human VSMC obtained from greater saphenous veins were used in this experiment from 3(rd) to 5(th) passage. Cotinine was added in doses equivalent to plasma levels of cotinine in an active smoker by dissolving, 0.0, 2.88x10(-6), 5.76x10(-6), and 1.44x10(-5) mol/L of cotinine in the media. The number of viable cells was assessed by trypan blue exclusion. The Telomeric Repeat Amplification Protocol (TRAP) was used to detect telomerase activity. TRAP products were detected by ELISA. RESULTS: The mitogenic effect of cotinine in VSMC was observed at 48 hours after treatment. The viable cell numbers were significantly increased (4.0x10(7)) at lower doses of cotinine exposure as compared to untreated cultures (2.5x10(5)). At the concentration of 1.44x10(-5) mol/L, cotinine was cytotoxic to VSMCs. Telomerase activity was detected in all sets of VSMC cultures treated with cotinine (P<0.01). CONCLUSIONS: Cotinine causes abnormal cell proliferation as demonstrated by increased cell numbers and reactivation of telomerase in a dose dependent manner. This study demonstrated cotinine's stimulatory effect on human SMC proliferation in vitro at low doses while high doses of cotinine had a toxic effect. These data correlate with the results of other studies concerning the mitogenic effect of cotinine and telomerase activation during cellular proliferative response.

Xanthine Oxidase Activation Modulates the Endothelial (Vascular) Dysfunction Related to HgCl2 Exposure Plus Myocardial Infarction in Rats.

Heavy metal exposure is associated with cardiovascular diseases such as myocardial infarction (MI). Vascular dysfunction is related to both the causes and the consequences of MI. We investigated whether chronic exposure to low doses of mercury chloride (HgCl2) worsens MI-induced endothelial dysfunction 7 days after MI. Male Wistar rats were divided into four groups: Control (vehicle), HgCl2 (4 weeks of exposure), surgically induced MI and combined HgCl2-MI. Morphological and hemodynamic measurements were used to characterize the MI model 7 days after the insult. Vascular reactivity was evaluated in aortic rings. Chronic HgCl2 exposure did not cause more heart injury than MI alone in terms of the morphological or hemodynamic parameters. Vascular reactivity increased in all groups, but the combination of HgCl2-MI increased the vasorelaxation induced by ACh compared with the HgCl2 and MI groups. Results showed reduced endothelial nitric oxide synthase (eNOS) protein expression in the MI group; increased iNOS activity in the HgCl2-MI group, although without enough magnitude to reverse the reduction in NO bioavailability; and increased phenylephrine response in the HgCl2-MI group due to an increase in ROS production, notably via xanthine oxidase (XO). Results suggest that the combination of 1 month pre-exposure of HgCl2 before MI changed the endothelial generation of oxidative stress induced by mercury exposure from NADPH oxidase pathway to XO (xanthine oxidase)-dependent ROS production.

Ammonia triggers the promotion of oxidative stress in the aquatic macrophyte Myriophyllum mattogrossense.

The effect of increased ammonia content on sub-acute biochemical responses was assessed in the rooted submersed aquatic macrophyte Myriophyllum mattogrossense (common name: "Brazil Milfoil" or "Matogrosso Milfoil"), in a seven day aquarium experiment. The pH and temperature were monitored in order to determine the proportions of both ionized (NH4+) and un-ionized (NH3) forms of ammonia. Specific activities of several enzymes such as catalase (CAT), guaiacol peroxidase (POD), glutathione peroxidase (GPx) and glutathione S-transferase (GST's) were measured as well as the content of the soluble antioxidant glutathione and lipid peroxidation were determined as these parameters are considered as indicators of cell-level disorder. The results showed that ammonia is able to generate oxidative stress, expressed through an elevated GSH content and the enhancement of CAT, POD, GPx and GST's activities in treatments with elevated ammonia content. As the toxic mechanism of ammonia is a complex phenomenon, this work adds an additional point of view to explain in parts the oxidative stress generating effect of ammonia promoting oxidative stress. Additionally the different modes of action proposed by other research groups are discussed, thus trying to combine the various points of view.

Aluminium sulfate exposure: A set of effects on hydrolases from brain, muscle and digestive tract of juvenile Nile tilapia (Oreochromis niloticus).

Aluminium is a major pollutant due to its constant disposal in aquatic environments through anthropogenic activities. The physiological effects of this metal in fish are still scarce in the literature. This study investigated the in vivo and in vitro effects of aluminium sulfate on the activity of enzymes from Nile tilapia (Oreochromis niloticus): brain acetylcholinesterase (AChE), muscle cholinesterases (AChE-like and BChE-like activities), pepsin, trypsin, chymotrypsin and amylase. Fish were in vivo exposed during 14days when the following experimental groups were assayed: control group (CG), exposure to Al2(SO4)3 at 1µg·mL-1 (G1) and 3µg·mL-1 (G3) (concentrations compatible with the use of aluminium sulfate as coagulant in water treatment). In vitro exposure was performed using animals of CG treatment. Both in vivo and in vitro exposure increased cholinesterase activity in relation to controls. The highest cholinesterase activity was observed for muscle BChE-like enzyme in G3. In contrast, the digestive enzymes showed decreased activity in both in vivo and in vitro exposures. The highest inhibitory effect was observed for pepsin activity. The inhibition of serine proteases was also quantitatively analyzed in zymograms using pixel optical densitometry as area under the peaks (AUP) and integrated density (ID). These results suggest that the inhibition of digestive enzymes in combination with activation of cholinesterases in O. niloticus is a set of biochemical effects that evidence the presence of aluminium in the aquatic environment. Moreover, these enzymatic alterations may support further studies on physiological changes in this species with implications for its neurological and digestive metabolisms.

Characterization of bile salt-induced apoptosis in colon cancer cell lines.

Bile salts have been shown to be involved in the etiology of colorectal cancer. Although there is a large body of evidence for bile salts as a cocarcinogen in azoxymethane-induced colorectal cancer, bile salt-induced apoptosis of colorectal cancer cells has not yet been studied in detail. Therefore, we investigated the effects of different bile salts on apoptosis and apoptotic signaling in colon cancer cell lines. Incubation of colorectal cancer cell lines with physiological concentrations of deoxycholic acid led to a dramatic induction of apoptosis. Caspase cleavage and caspase activation occurred as early as 30 min after the addition of deoxycholate. Caspase-2 (Ich-1, Nedd2), caspase-3 (CPP-32, YAMA, Apopain), caspase-7 (Mch-3, ICE-LAP-3), and caspase-8 (FLICE, Mach-1, Mch5) are activated in HT-29, whereas caspase-1 (ICE) remained intact. Caspase activation and cellular apoptosis induced by bile salts were reversed by broad spectrum and selective caspase inhibitors. As opposed to hepatocyte death mediated by bile acids, CD95 was not involved in deoxycholate-induced apoptosis. The cytoprotective effect of ursodeoxycholic acid in hepatocytes or other tumor cell lines, which is mediated by inhibiting the mitochondrial permeability transition, was not observed in colon cancer cell lines as well. This points to distinct intracellular functions of ursodeoxycholate in different cancer cell types. Here we describe the specificity of bile salt-induced apoptosis in colon cancer cell lines. Differences from hepatocytes are shown. Bile acid-specific caspase activation is part of the apoptotic pathway induced by bile salts in colon cancer cell lines. Furthermore, a lack of cytoprotective function of ursodeoxycholate in these cells is demonstrated. Our data raise questions as to the role of bile salts in colorectal carcinogenesis.

Apoptotic response of HL-60 human leukemia cells to the antitumor drug TAS-103.

TAS-103 is a DNA intercalating indeno-quinoline derivative that stimulates DNA cleavage by topoisomerases. This synthetic drug has a broad spectrum of antitumor activity against many human solid tumor xenografts and is currently undergoing clinical trials. We investigated the induction of apoptosis in human promyelocytic leukemia cells treated with TAS-103. The treatment of proliferating human leukemia cells for 24 h with various concentrations of the drug induces significant variations in the mitochondrial transmembrane potential (delta(psi)mt) measured by flow cytometry using the fluorochromes 3,3-dihexyloxacarbocyanine iodide, Mitotracker Red, and tetrachloro-tetraethylbenzimidazolcarbocyanine iodide. The collapse of delta(psi)mt is accompanied by a marked decrease of the intracellular pH. Cleavage experiments with the substrates N-acetyl-Asp-Glu-Val-Asp-pNA, poly(ADP-ribose) polymerase, and pro-caspase-3 reveal unambiguously that caspase-3 is a key mediator of the apoptotic pathway induced by TAS-103. Caspase-8 is also cleaved, and the bcl-2 oncoprotein is underexpressed. Drug-induced internucleosomal DNA fragmentation and the externalization of phosphatidylserine residues in the outer leaflet of the plasma membrane were also characterized. The cell cycle perturbations produced by TAS-103 can be connected with the changes in deltapsi(mt). At low concentrations (2-25 nM), the drug induces a marked G2 arrest and concomitantly provokes an increase in the potential of mitochondrial membranes. In contrast, treatment of the HL-60 cells with higher drug concentrations (50 nM to 1 microM) triggers massive apoptosis and a collapse of deltaP(mt) that is a signature for the opening of the mitochondrial permeability transition pores. The discovery of a correlation between the G2 arrest and changes in mitochondrial membrane potential provides an important mechanistic insight into the action of TAS-103.

Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200µg/L) at different time points after fertilization. Results showed that 200µgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos.

TAT cell-penetrating peptide modulates inflammatory response and apoptosis in human lung epithelial cells.

Cell-penetrating peptides (CPPs) are commonly used as delivery vehicles for the introduction of a variety of macromolecules into cells. Trans-activator of transcription (TAT) is the most commonly used CPP and, as a delivery vehicle, is assumed to be biologically inert. In this study, we pretreated human lung epithelial cells with TAT prior to stimulation with phorbol 12,13-dibutyrate (PDBu), a protein kinase C (PKC) activator. Surprisingly, TAT alone inhibited the production of multiple cytokines induced by PKC activation. Furthermore, PKC activation-induced IκBα degradation was partially reduced by TAT. Moreover, TAT treatment alone induced apoptosis in a dose-dependent manner, influenced expression of several B cell lymphoma 2 (Bcl-2) family members and increased caspase 3 cleavage at a high dose. These findings suggest that TAT as a delivery vehicle should be used cautiously, as it may affect the inflammatory response, as well as signals related to apoptosis.