Comparison of Mitochondrial and Antineoplastic Effects of Amiodarone and Desethylamiodarone in MDA-MB-231 Cancer Line.

Previously, we have demonstrated that amiodarone (AM), a widely used antiarrhythmic drug, and its major metabolite desethylamiodarone (DEA) both affect several mitochondrial processes in isolated heart and liver mitochondria. Also, we have established DEA's antitumor properties in various cancer cell lines and in a rodent metastasis model. In the present study, we compared AM's and DEA's mitochondrial and antineoplastic effects in a human triple-negative breast cancer (TNBC) cell line. Both compounds reduced viability in monolayer and sphere cultures and the invasive growth of the MDA-MB-231 TNBC line by inducing apoptosis. They lowered mitochondrial trans-membrane potential, increased Ca2+ influx, induced mitochondrial permeability transition, and promoted mitochondrial fragmentation. In accordance with their mitochondrial effects, both substances massively decreased overall, and even to a greater extent, mitochondrial ATP production decreased, as determined using a Seahorse live cell respirometer. In all these effects, DEA was more effective than AM, indicating that DEA may have higher potential in the therapy of TNBC than its parent compound.

Clinical Study of Metabolic Parameters, Leptin and the SGLT2 Inhibitor Empagliflozin among Patients with Obesity and Type 2 Diabetes.

Obesity is a major public health problem worldwide, and it is associated with many diseases and abnormalities, most importantly, type 2 diabetes. The visceral adipose tissue produces an immense variety of adipokines. Leptin is the first identified adipokine which plays a crucial role in the regulation of food intake and metabolism. Sodium glucose co-transport 2 inhibitors are potent antihyperglycemic drugs with various beneficial systemic effects. We aimed to investigate the metabolic state and leptin level among patients with obesity and type 2 diabetes mellitus, and the effect of empagliflozin upon these parameters. We recruited 102 patients into our clinical study, then we performed anthropometric, laboratory, and immunoassay tests. Body mass index, body fat, visceral fat, urea nitrogen, creatinine, and leptin levels were significantly lower in the empagliflozin treated group when compared to obese and diabetic patients receiving conventional antidiabetic treatments. Interestingly, leptin was increased not only among obese patients but in type 2 diabetic patients as well. Body mass index, body fat, and visceral fat percentages were lower, and renal function was preserved in patients receiving empagliflozin treatment. In addition to the known beneficial effects of empagliflozin regarding the cardio-metabolic and renal systems, it may also influence leptin resistance.

Involvement of Mitochondrial Mechanisms and Cyclooxygenase-2 Activation in the Effect of Desethylamiodarone on 4T1 Triple-Negative Breast Cancer Line.

Novel compounds significantly interfering with the mitochondrial energy production may have therapeutic value in triple-negative breast cancer (TNBC). This criterion is clearly fulfilled by desethylamiodarone (DEA), which is a major metabolite of amiodarone, a widely used antiarrhythmic drug, since the DEA previously demonstrated anti-neoplastic, anti-metastasizing, and direct mitochondrial effects in B16F10 melanoma cells. Additionally, the more than fifty years of clinical experience with amiodarone should answer most of the safety concerns about DEA. Accordingly, in the present study, we investigated DEA's potential in TNBC by using a TN and a hormone receptor positive (HR+) BC cell line. DEA reduced the viability, colony formation, and invasive growth of the 4T1 cell line and led to a higher extent of the MCF-7 cell line. It lowered mitochondrial transmembrane potential and induced mitochondrial fragmentation. On the other hand, DEA failed to significantly affect various parameters of the cellular energy metabolism as determined by a Seahorse live cell respirometer. Cyclooxygenase 2 (COX-2), which was upregulated by DEA in the TNBC cell line only, accounted for most of 4T1's DEA resistance, which was counteracted by the selective COX-2 inhibitor celecoxib. All these data indicate that DEA may have potentiality in the therapy of TNBC.

Cytostatic Effect of a Novel Mitochondria-Targeted Pyrroline Nitroxide in Human Breast Cancer Lines.

Mitochondria have emerged as a prospective target to overcome drug resistance that limits triple-negative breast cancer therapy. A novel mitochondria-targeted compound, HO-5114, demonstrated higher cytotoxicity against human breast cancer lines than its component-derivative, Mito-CP. In this study, we examined HO-5114's anti-neoplastic properties and its effects on mitochondrial functions in MCF7 and MDA-MB-231 human breast cancer cell lines. At a 10 µM concentration and within 24 h, the drug markedly reduced viability and elevated apoptosis in both cell lines. After seven days of exposure, even at a 75 nM concentration, HO-5114 significantly reduced invasive growth and colony formation. A 4 h treatment with 2.5 µM HO-5114 caused a massive loss of mitochondrial membrane potential, a decrease in basal and maximal respiration, and mitochondrial and glycolytic ATP production. However, reactive oxygen species production was only moderately elevated by HO-5114, indicating that oxidative stress did not significantly contribute to the drug's anti-neoplastic effect. These data indicate that HO-5114 may have potential for use in the therapy of triple-negative breast cancer; however, the in vivo toxicity and anti-neoplastic effectiveness of the drug must be determined to confirm its potential.

Involvement of Mitochondrial Mechanisms in the Cytostatic Effect of Desethylamiodarone in B16F10 Melanoma Cells.

Previously, we showed that desethylamiodarone (DEA), a major metabolite of the widely used antiarrhythmic drug amiodarone, has direct mitochondrial effects. We hypothesized that these effects account for its observed cytotoxic properties and ability to limit in vivo metastasis. Accordingly, we examined DEA's rapid (3-12 h) cytotoxicity and its early (3-6 h) effects on various mitochondrial processes in B16F10 melanoma cells. DEA did not affect cellular oxygen radical formation, as determined using two fluorescent dyes. However, it did decrease the mitochondrial transmembrane potential, as assessed by JC-1 dye and fluorescence microscopy. It also induced mitochondrial fragmentation, as visualized by confocal fluorescence microscopy. DEA decreased maximal respiration, ATP production, coupling efficiency, glycolysis, and non-mitochondrial oxygen consumption measured by a Seahorse cellular energy metabolism analyzer. In addition, it induced a cyclosporine A-independent mitochondrial permeability transition, as determined by Co2+-mediated calcein fluorescence quenching measured using a high-content imaging system. DEA also caused outer mitochondrial membrane permeabilization, as assessed by the immunoblot analysis of cytochrome C, apoptosis inducing factor, Akt, phospho-Akt, Bad, and phospho-Bad. All of these data supported our initial hypothesis.

Amiodarone's major metabolite, desethylamiodarone inhibits proliferation of B16-F10 melanoma cells and limits lung metastasis formation in an in vivo experimental model.

Previously, we demonstrated the in vitro anti-tumor effects of desethylamiodarone (DEA) in bladder and cervix cancer cell lines. In the present study, we intended to establish its potentiality in B16-F10 metastatic melanoma cells in vitro and in vivo. We assessed cell proliferation, apoptosis and cell cycle by using sulforhodamine B assay, Muse™ Annexin V & Dead Cell and Muse® Cell Cycle assays, respectively. We determined colony formation after crystal violet staining. For studying mechanistic aspects, immunoblotting analysis was performed. We used a C57BL/6 experimental lung metastasis model for demonstrating in vivo anti-metastatic potential of DEA. DEA inhibited in vitro proliferation and colony formation, and in vivo lung metastasizing properties of B16-F10 cells. It arrested the cells in G0/G1 phase of their cycle likely via p21 in a p53-dependent fashion, and induced caspase mediated apoptosis likely via inversely regulating Bcl-2 and Bax levels, and reducing Akt and ERK1/2 activation. In this study, we provided in vitro and in vivo experimental evidences for DEA's potentiality in the therapy of metastatic melanomas. Since DEA is the major metabolite of amiodarone, a worldwide used antiarrhythmic drug, safety concerns could be resolved more easily for it than for a novel pharmacological agent.

PARP inhibition induces Akt-mediated cytoprotective effects through the formation of a mitochondria-targeted phospho-ATM-NEMO-Akt-mTOR signalosome.

PURPOSE: The cytoprotective effect of poly(ADP-ribose) polymerase 1 (PARP1) inhibition is well documented in various cell types subjected to oxidative stress. Previously, we have demonstrated that PARP1 inhibition activates Akt, and showed that this response plays a critical role in the maintenance of mitochondrial integrity and in cell survival. However, it has not yet been defined how nuclear PARP1 signals to cytoplasmic Akt. METHODS: WRL 68, HeLa and MCF7 cells were grown in culture. Oxidative stress was induced with hydrogen peroxide. PARP was inhibited with the PARP inhibitor PJ34. ATM, mTOR- and NEMO were silenced using specific siRNAs. Cell viability assays were based on the MTT assay. PARP-ATM pulldown experiments were conducted; each protein was visualized by Western blotting. Immunoprecipitation of ATM, phospho-ATM and NEMO was performed from cytoplasmic and mitochondrial cell fractions and proteins were detected by Western blotting. In some experiments, a continually active Akt construct was introduced. Nuclear to cytoplasmic and mitochondrial translocation of phospho-Akt was visualized by confocal microscopy. RESULTS: Here we present evidence for a PARP1 mediated, PARylation-dependent interaction between ATM and NEMO, which is responsible for the cytoplasmic transport of phosphorylated (thus, activated) ATM kinase. In turn, the cytoplasmic p-ATM and NEMO forms complex with mTOR and Akt, yielding the phospho-ATM-NEMO-Akt-mTOR signalosome, which is responsible for the PARP-inhibition induced Akt activation. The phospho-ATM-NEMO-Akt-mTOR signalosome localizes to the mitochondria and is essential for the PARP-inhibition-mediated cytoprotective effects in oxidatively stressed cells. When the formation of the signalosome is prevented, the cytoprotective effects diminish, but cells can be rescued by constantly active Akt1, further confirming the critical role of Akt activation in cytoprotection. CONCLUSIONS: Taken together, the data presented in the current paper are consistent with the hypothesis that PARP inhibition suppresses the PARylation of ATM, which, in turn, forms an ATM-NEMO complex, which exits the nucleus, and combines in the cytosol with mTOR and Act, resulting in Act phosphorylation (i.e. activation), which, in turn, produces the cytoprotective action via the induction of Akt-mediated survival pathways. This mechanism can be important in the protective effect of PARP inhibitor in various diseases associated with oxidative stress. Moreover, disruption of the formation or action of the phospho-ATM-NEMO-Akt-mTOR signalosome may offer potential future experimental therapeutic checkpoints.

Amiodarone's major metabolite, desethylamiodarone, induces apoptosis in human cervical cancer cells.

Previously, we found that desethylamiodarone (DEA) may have therapeutic potentiality in bladder cancer. In this study, we determined its effects on human cervical cancer cells (HeLa). Cell viability was evaluated by Muse Cell Count & Viability Assay; cell apoptosis was detected by Muse Annexin V & Dead Cell Assay. Cell cycle was flow cytometrically determined by Muse Cell Cycle Kit and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33342 staining. The changes in the expression levels of apoptosis-related proteins in the HeLa cells were assessed by immunoblot. Our results showed that DEA significantly inhibited the proliferation and viability of HeLa cells and induced apoptosis in vitro in dose-dependent and also in cell cycle-dependent manner because DEA induced G0/G1 phase arrest in the HeLa cell line. We found that DEA treatment downregulated the expression of phospho-Akt and phospho-Bad. In addition, DEA could downregulate expression of Bcl-2, upregulate Bax, and induce cytochrome c release. Our results indicate that DEA might have significance as an anti-tumor agent against human cervical cancer.

Desethylamiodarone-A metabolite of amiodarone-Induces apoptosis on T24 human bladder cancer cells via multiple pathways.

Bladder cancer (BC) is a common malignancy of the urinary tract that has a higher frequency in men than in women. Cytostatic resistance and metastasis formation are significant risk factors in BC therapy; therefore, there is great interest in overcoming drug resistance and in initiating research for novel chemotherapeutic approaches. Here, we suggest that desethylamiodarone (DEA)-a metabolite of amiodarone-may have cytostatic potential. DEA activates the collapse of mitochondrial membrane potential (detected by JC-1 fluorescence), and induces cell death in T24 human transitional-cell bladder carcinoma cell line at physiologically achievable concentrations. DEA induces cell cycle arrest in the G0/G1 phase, which may contribute to the inhibition of cell proliferation, and shifts the Bax/Bcl-2 ratio to initiate apoptosis, induce AIF nuclear translocation, and activate PARP-1 cleavage and caspase-3 activation. The major cytoprotective kinases-ERK and Akt-are inhibited by DEA, which may contribute to its cell death-inducing effects. DEA also inhibits the expression of B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1) and reduces colony formation of T24 bladder carcinoma cells, indicating its possible inhibitory effect on metastatic potential. These data show that DEA is a novel anti-cancer candidate of multiple cell death-inducing effects and metastatic potential. Our findings recommend further evaluation of its effects in clinical studies.

Thymic Atrophy and Apoptosis of CD4+CD8+ Thymocytes in the Cuprizone Model of Multiple Sclerosis.

Previous studies on the degenerative animal model of multiple sclerosis suggested that the copper-chelator cuprizone might directly suppress T-cell functions. Peripheral T-cell function in the cuprizone model has already been explored; therefore, in the present study, we investigated, for the first time, how cuprizone feeding affects the thymus, the organ of T-cell maturation and selection. We found that even one week of cuprizone treatment induced significant thymic atrophy, affecting the cortex over the medulla. Fluorescent microscopy and flow-cytometric analyses of thymi from cuprizone- and vehicle-treated mice indicated that eradication of the cluster of the differentiation-4 (CD4)-CD8 double-positive T-cell subset was behind the substantial cell loss. This result was confirmed with CD3-CD4-CD8 triple-staining experiments. Ultrastructurally, we observed degraded as well as enlarged mitochondria, myelin-bodies, large lipid droplets, and large lysosomes in the thymi of cuprizone-treated mice. Some of these features were similar to those in physiological and steroid-induced accelerated aging. According to our results, apoptosis was mainly of mitochondrial origin mediated by both caspase-3- and apoptosis inducing factor-mediated mechanisms. Additionally, mitogen activated protein kinase activation and increased pro-apoptotic B cell lymphoma-2 family protein expression were the major underlying processes. Our results do not indicate a functional relationship between cuprizone-induced thymus involution and the absence of inflammatory responses or the selective demyelination observed in the cuprizone model. On the other hand, due to the reversible nature of cuprizone's deleterious effects, the cuprizone model could be valuable in studying thymus regeneration as well as remyelination processes.

Health related quality of life in patients with bladder cancer: a cross-sectional survey and validation study of the Hungarian version of the Bladder Cancer Index.

Health-related quality of life (HRQoL) is an important outcome in oncology care although an underexplored area in bladder cancer (BC). Our aims were to assess HRQoL of patients with BC, analyse relationships between diverse HRQoL measures and validate the Hungarian version of the Bladder Cancer Index (BCI) questionnaire. A cross-sectional survey was performed among patients with BC (N = 151). Validated Hungarian versions of the FACT-Bl, SF-36 and EQ-5D were applied and SF-6D was derived. Psychometric analysis of the Hungarian BCI was performed. Pearson correlations between the five measures were analysed. Deterioration in SF-36 Physical Functioning was detected among patients aged 45-64 years. The EQ-5D score did not differ significantly from the age-matched population norm. Correlations between the FACT-Bl, EQ-5D and SF-6D utility measures were strong (r > 0.6). Cronbach alpha coefficients of the Hungarian BCI ranged from 0.75 to 0.97 and factor analysis confirmed that data fit to the six predefined subdomains. Test-retest correlations (reliability, N = 50) ranged from 0.67 to 0.87 and interscale correlations between urinary, bowel and sexual BCI domains were weak or moderate (r = 0.29 to 0.49). Convergent validity revealed a stronger correlation with FACT-Bl (r = 0.126 to 0.719) than with generic health state scores (r = 0.096 to 0.584). Results of divergent validity of the Hungarian BCI by treatment groups by Kruskal Wallis test were promising although limited by low sample sizes in cystectomy subgroups. Generic health state measures have limited capacity to capture HRQoL impact of BC. Validity tests yielded favourable results for the Hungarian BCI. Mapping studies to estimate utility scores from FACT-Bl are encouraged but less recommendable with the BCI.

Effects of PACAP on mitochondrial apoptotic pathways and cytokine expression in rats subjected to renal ischemia/reperfusion.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly efficient cytoprotective actions. Its neuroprotective effects are well-known, but PACAP is able to exert similar actions in non-neuronal cells. Recently, we have shown that PACAP prolongs renal ischemic time, decreases mortality, and attenuates tubular degeneration in a rat model of renal ischemia/reperfusion, but the mechanism of renoprotection is not yet known. Therefore, the aim of the present study was to obtain further insight into the renoprotective effects of PACAP by examining its direct effects of PACAP on mitochondrial permeability transition in vitro and on the expression of the anti-apoptotic Bcl-2 and cytokines/chemokines in kidney tissues following 45 and 60 min renal ischemia and reperfusion in vivo. We found that PACAP did not have any direct effect on mitochondrial permeability transition. Cytokine array revealed that the expression of a few cytokines/chemokines was strongly increased after ischemia/reperfusion, which was ameliorated by PACAP treatment. Furthermore, in rats subjected to renal ischemia, PACAP treatment counteracted the ischemia/reperfusion-induced decrease of the anti-apoptotic Bcl-2, both after 45 and 60 min ischemia, as analyzed by Western blot. In summary, we showed that PACAP could attenuate tissue injury involving both anti-inflammatory and anti-apoptotic effects, but not directly acting on mitochondrial permeability transition.

Facilitation of mitochondrial outer and inner membrane permeabilization and cell death in oxidative stress by a novel Bcl-2 homology 3 domain protein.

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x(L), which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.

Potentiation of paclitaxel-induced apoptosis by galectin-13 overexpression via activation of Ask-1-p38-MAP kinase and JNK/SAPK pathways and suppression of Akt and ERK1/2 activation in U-937 human macrophage cells.

Galectin-13 transcripts have been identified in several normal and malignant tissues, but the physiological function of galectin-13 is still poorly understood. Here, we present evidence for its possible role in promoting cell death in the U-937 human macrophage cell line. Transfection of U-937 human macrophages by a galectin-13 cDNA-containing mammalian expression vector increased the galectin-13 level and sensitized the cells to stress stimuli. Galectin-13 overexpression facilitated paclitaxel-induced cell death and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease-G without inducing mitochondrial cytochrome-c release or caspase-3 activation. Immunoblot and immunofluorescence data showed that overexpression of galectin-13 induced long-term activation of c-Jun N-terminal kinase (JNK) and p38-mitogen-activated protein kinase (MAPK) pathways, as well as activation of apoptosis signal-regulating kinase-1 (Ask-1) kinase while it suppressed paclitaxel-induced long-term activation of the phosphatidilylositol-3-kinase (PI-3K)-Akt and extracellular signal-regulated kinase (ERK1/2) cytoprotective pathways. In addition, pharmacological inhibition of JNK and p38-MAPK pathways protected the cells from paclitaxel-induced cell death. All this data indicate that galectin-13 overexpression promoted apoptosis presumably by activating the Ask-1 kinase-JNK and p38-MAPK pro-apoptotic pathways and by suppressing the PI-3K-Akt and ERK1/2 cytoprotective pathways.

PARP-1 inhibition-induced activation of PI-3-kinase-Akt pathway promotes resistance to taxol.

PARP inhibitors combined with DNA-damage inducing cytostatic agents can lead to effective tumor therapy. However, inhibition of poly(ADP-ribose) polymerase (PARP-1; EC 2.4.2.30) induces the activation of PI-3-kinase-Akt pathway, which can counteract the effectiveness of this therapy. To understand the role of Akt activation in the combined use of cytostatic agent and PARP inhibition, we used taxol (paclitaxel) as an antineoplastic agent, which targets microtubules and up-regulates mitochondrial ROS production, together with (i) pharmacological inhibition (PJ-34), (ii) siRNA knock-down and (iii) transdominant expression of the DNA binding domain of PARP-1. In all cases, PARP-1 inhibition leads to suppressed poly-ADP-ribosylation of nuclear proteins, prevention of NAD(+) depletion and significant resistance against taxol induced caspase-3 activation and apoptotic cell death. Paclitaxel induced a moderate increase in Akt activation, which was significantly augmented by PARP inhibition, suggesting that PARP inhibition-induced Akt activation could be responsible for the cytostatic resistance. When activation of the PI-3-kinase-Akt pathway was prevented by LY-294002 or Akt Inhibitor IV, the cytoprotective effect of PARP inhibition was significantly diminished showing that the activation of PI-3-kinase-Akt cascade had significantly contributed to the cytostatic resistance. Our study demonstrates that drug-induced drug resistance can be responsible for the reduced efficacy of antitumor treatment. Although inhibition of PARP-1 can promote cell death in tumor cells by the inhibition of DNA repair, PARP-inhibition promoted activation of the PI-3-kinase-Akt pathway can counteract this facilitating effect, and can cause cytostatic resistance. We suggest augmenting PARP inhibition by the inhibition of the PI-3-kinase-Akt pathway for antitumor therapy.

Critical role of bad phosphorylation by Akt in cytostatic resistance of human bladder cancer cells.

BACKGROUND: Taxol is the most commonly used agent for salvage chemotherapy in transitional cell carcinoma of the urothelium. We examined mechanisms responsible for taxol resistance by using T24 human bladder carcinoma cells. MATERIALS AND METHODS: We used an inhibitor and an activator of the phosphatidylinositol-3 kinase-Akt pathway in cell survival and caspase-3 assays, an HPLC method for determining released cytochrome c and immunoblotting for detecting protein phosphorylation. RESULTS: Activation of Akt increased paclitaxel resistance by increasing Bad phosphorylation, leading to decreased release of mitochondrial cytochrome c and caspase-3-mediated apoptosis. On the other hand, inhibition of Akt prevented paclitaxel resistance by enhancing the effects of paclitaxel on Bad phosphorylation, mitochondrial cytochrome c release and caspase-3-mediated apoptosis, besides diminishing or abolishing the opposing effects of Akt activation. CONCLUSION: Akt-mediated Bad phosphorylation plays an important role in preservation of mitochondrial membrane systems leading to paclitaxel resistance in T24 cells.

Prevalent role of Akt and ERK activation in cardioprotective effect of Ca(2+) channel- and beta-adrenergic receptor blockers.

We studied cardioprotective as well as Akt and extracellular signal-activated kinase (ERK) activating effect of a Ca(2+) antagonist and a beta-adrenergic receptor blocker during ischemia-reperfusion, and compared these properties of the substances with that of a poly(ADP-ribose) polymerase (PARP) inhibitor used as a positive control throughout the experiments. Langendorff-perfused isolated rat hearts were subjected to 25 min global ischemia followed by 45 min reperfusion, and recovery of energy metabolism as well as functional cardiac parameters were monitored. Although to varying extents, all substances improved recovery of creatine phosphate, ATP, intracellular pH, and reutilization of inorganic phosphate. These favorable changes were accompanied by improved recovery of heart function parameters and reduced infarct size. In addition and again to varying extents, all studied substances decreased oxidative damage (lipid peroxidation and protein oxidation), and activated Akt, glycogen synthase kinase (GSK)-3beta, and ERK1/2. Correlation between cardioprotective and kinase activating effectivity of the compounds proved to be statistically significant. Physiological significance of these kinase activations was established by demonstrating that inhibition of Akt by LY294002 and ERK1/2 by PD98059 compromised the cardioprotective effect of all the substances studied. In conclusion, we demonstrated for the first time that activation of phosphatidylinositol-3-kinase (PI-3K)-Akt and ERK2 pathways significantly contributed to cardioprotective effects of a Ca(2+) antagonist and a beta-adrenergic receptor blocker. Furthermore, we found a strong correlation between cardioprotective and kinase-activating potencies of the substances studied (Verapamil, Metoprolol and two PARP inhibitors), which indicated the potentiality of these kinases as drug-targets in the therapy of ischemic heart disease.

Ferulaldehyde, a water-soluble degradation product of polyphenols, inhibits the lipopolysaccharide-induced inflammatory response in mice.

Antiinflammatory properties of polyphenols in natural products, traditional medicines, and healthy foods were recently attributed to highly soluble metabolites produced by the microflora of the intestines rather than the polyphenols themselves. To provide experimental basis for this hypothesis, we measured antiinflammatory properties of ferulaldehyde (FA), a natural intermediate of polyphenol metabolism of intestinal microflora, in a murine lipopolysaccharide (LPS)-induced septic shock model. We found that intraperitoneally administered FA (6 mg/kg) prolonged the lifespan of LPS-treated (40 mg/kg) mice, decreased the inflammatory response detected by T(2)-weighted in vivo MRI, decreased early proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin (IL)-1beta, and increased the antiinflammatory IL-10 in the sera of the mice. Additionally, FA inhibited LPS-induced activation of nuclear factor kappaB transcription factor in the liver of the mice. According to our data, these effects were probably due to attenuating LPS-induced activation of c-Jun N-terminal kinase and Akt. Furthermore, FA decreased free radical and nitrite production in LPS plus interferon-gamma-treated primary mouse hepatocytes, whose effects are expected to contribute to its antiinflammatory property. These data provide direct in vivo evidence, that a water-soluble degradation product of polyphenols could be responsible for, or at least could significantly contribute to, the beneficial antiinflammatory effects of polyphenol-containing healthy foods, natural products, and traditional medicines.

Inhibition of cell death by a novel 16.2 kD heat shock protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation pathway.

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase-Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase-Akt cytoprotective pathway.

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL.

We found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT.