CMPD1 inhibited human gastric cancer cell proliferation by inducing apoptosis and G2/M cell cycle arrest.

BACKGROUND: Gastric cancer occupies the fourth highest morbidity rate of cancers worldwide. Clinical therapies of gastric cancer remain limited because of uncertainty of mechanisms and shortness of effective medicine. Thus, new drug candidates for gastric cancer treatment is urgently needed. RESULTS: In this study, CMPD1 as a wildly used MK2 phosphorylation inhibitor was employed to find its impact on gastric cancer cell proliferation, apoptosis and cell cycle using colony formation assay and flow cytometry analysis. Along with its anti-proliferation effect on gastric cancer cell line MKN-45 and SGC7901, CMPD1 also induced massive apoptosis and significant G2/M phase arrest in a time-dependent and dose-dependent manner in MKN-45 cells respectively. Furthermore, Western blot confirmed that the expression of anti-apoptotic proteins Bcl-2 was decreased while BAX, cytochrome c release and cleaved PARP were increased. In addition, oncogene c-Myc was downregulated in response to CMPD1 treatment. CONCLUSIONS: Our results demonstrated that CMPD1 has anti-tumor effect on human gastric cancer cell line MKN-45 possibly via downregulating oncogene c-Myc expression and CMPD1 could be applied as a potential candidate for treating gastric malignancy. To the best of our knowledge, it is the first report of anti-tumor effect of CMPD-1 on human gastric cancer cells.

Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4.

Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (15 mg/kg), omeprazole (15 mg/kg), and midazolam (10 mg/kg), was given orally to rats treated for 7 d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug-CAP interactions.

Anti-Glioma Effect of Pseudosynanceia melanostigma Venom on Isolated Mitochondria from Glioblastoma Cells

Background: Glioblastoma is the most common primary malignant tumor of the central nervous system that occurs in the spinal cord or brain. Pseudosynanceia melanostigma is a venomous stonefish in the Persian Gulf, which our knowledge about is little. This study's goal is to investigate the toxicity of stonefish crude venom on mitochondria isolated from U87 cells. Methods: In the first stage, we extracted venom stonefish and then isolated mitochondria have exposed to different concentrations of venom. Finally, mitochondrial toxicity parameters (Succinate dehydrogenase (SDH) activity, Reactive oxygen species (ROS), cytochrome c release, Mitochondrial Membrane Potential (MMP), and mitochondrial swelling) have evaluated. Results: To determine mitochondrial parameters, we used 115, 230, and 460 µg/ml concentrations. The results of our study show that the venom of stonefish selectively increases upstream parameters of apoptosis such as mitochondrial swelling, cytochrome c release, MMP collapse and ROS. Conclusion: This study suggests that Pseudosynanceia melanostigma crude venom has selectively caused toxicity by increasing active mitochondrial oxygen radicals. This venom could potentially be a candidate for the treatment of glioblastoma.

[The most important clinical interactions among medicines used in cardiologic practice]. / Najwazniejsze interakcje pomiedzy lekami stosowanymi w kardiologii.

This article presents a review of the most important (from the cardiologic practice point of view) interactions among medicines administered to the patients suffering from cardiovascular diseases, in particular among medicines with cytochrome P450 (and its isoenzymes) dependent metabolism. Cardiovascular patients, often advanced in age, usually affected by concomitant diseases and compelled to take many different medicines are particularly exposed to side effects dependent on these interactions. The article describes both beneficial and unprofitable interactions among medicines administered in cardiology.

Dehydroepiandrosterone (DHEA) treatment stimulates oxidative energy metabolism in the cerebral mitochondria from developing rats.

Effects of treatment with dehydroepiandrosterone (DHEA) (0.2 or 1.0mg/kg body weight for 7 days) on oxidative energy metabolism in cerebral mitochondria from developing and young adult rats were examined. Treatment with DHEA did not change the body weight of developing rats but resulted in increase in the brain weight in 5 week group. In young adult rats the body weight increased following treatment with 1.0mg DHEA. State 3 and state 4 respiration rates with all the substrates increased following DHEA treatment, the effect being more pronounced in the developing rats. State 4 respiration rates were stimulated to variable extents. Contents of cytochromes aa(3) and b increased following DHEA treatment and once again the effect was more pronounced in the developing rats. DHEA treatment marginally changed the content of cytochromes c+c(1). In the developing rats the ATPase activity and the levels of dehydrogenases increased significantly by DHEA treatment. Results of our studies have shown that treatment with exogenous DHEA accelerates the process of maturation of cerebral mitochondria thus emphasizing the role of DHEA in brain development in postnatal life.

Cumulative and irreversible cardiac mitochondrial dysfunction induced by doxorubicin.

Interference with mitochondrial calcium regulation is proposed to be a primary causative event in the mechanism of doxorubicin-induced cardiotoxicity. We previously reported disruption of mitochondrial calcium homeostasis after chronic doxorubicin administration (Solen et al. Toxicol. Appl. Pharmacol, 129: 214-222, 1994). The present study was designed to characterize the dose-dependent and cumulative interference with mitochondrial calcium regulation and to assess the reversibility of this functional lesion. Sprague Dawley rats were treated with 2 mg/kg/week doxorubicin s.c. for 4-8 weeks. With succinate as substrate, cardiac mitochondria isolated from rats after 4 weeks of treatment with doxorubicin expressed a lower calcium loading capacity compared with control. This suppression of calcium loading capacity increased with successive doses to 8 weeks of treatment (P < 0.05) and persisted for 5 weeks after the last doxorubicin injection, and was corroborated by dose-dependent and irreversible histopathological changes. Preincubation of mitochondria with tamoxifen, DTT, or monobromobimane did not reverse the diminished calcium loading capacity caused by doxorubicin. In contrast, incubation with cyclosporin A abolished any discernible difference in mitochondrial calcium loading capacity between doxorubicin-treated and saline-treated rats. The decrease in cardiac mitochondrial calcium loading capacity was not attributable to bioenergetic changes in the electron transport chain, because the mitochondrial coupling efficiency was not altered by doxorubicin treatment. However, the ADP/ATP translocase content was significantly lower in mitochondria from rats that received 8 weeks of doxorubicin treatment. These data indicate that doxorubicin treatment in vivo causes a dose-dependent and irreversible decrease in mitochondrial calcium loading capacity. Suppression of adenine nucleotide translocase content may be a key factor altering the calcium-dependent regulation of the mitochondrial permeability transition pore, which may account for the cumulative and irreversible loss of myocardial function in patients receiving doxorubicin chemotherapy.

Increased myocardial vulnerability to ischemia-reperfusion injury in the presence of left ventricular hypertrophy.

OBJECTIVE: Despite its high prevalence among patients suffering myocardial infarction, the significance of left ventricle hypertrophy for infarct size is not known. We asked whether infarct size might be increased by this condition, and whether any such increase might be associated with an increased mitochondrial damage following coronary occlusion. METHODS: Occlusion of the left descending artery in isolated, perfused hearts of SHR-SP (spontaneously hypertensive rat stroke-prone) (left ventricular hypertrophy) or Wistar-Kyoto (WKY) (control) rats was used, followed by reperfusion with or without exendin-4 (Exe-4), a glucagon-like peptide-1 receptor agonist. Infarct size relative to area-at-risk was determined. Separately, mitochondria were isolated after global ischemia. Activities of complexes III and IV and amounts of selected complex subunits and cytochromes a, b, c, and c1 were determined. RESULTS: Infarct size (ischemia 35  min and 120  min reperfusion) was 65.8% (±3.3%) and 37.1% (±3.4%) in the SHR-SP and WKY hearts, respectively (P < 0.05). Exe-4 significantly decreased infarct size and hypercontracture in WKY, but not in SHR-SP, hearts. After ischemia 15  min in SHR-SP hearts, Exe-4 reduced the infarct (26.6%, ±3.8% to 9.3% ± 1.5%; P < 0.05). Mitochondria from postischemic SHR-SP hearts showed a reduction of complex III (368.1 ± 37.5 to 175.8 ± 23.0  nmoles/min × mg; P < 0.05) and complex IV (14.4 ± 0.22 to 5.8 ± 0.8 1/s × mg; P < 0.05) activities and decreased amounts of cytochromes a, b, and c. CONCLUSION: Hearts from hypertensive (SHR-SP) rats with left ventricle hypertrophy appeared more vulnerable to ischemia-reperfusion injury, as supported by a more profound infarct development and an earlier loss of postconditioning by Exe-4. Mitochondrial complexes III and IV were identified among possible loci of this increased, hypertrophy-associated vulnerability.

Apoptotic pathway activation from mitochondria and death receptors without caspase-3 cleavage in failing human myocardium: fragile balance of myocyte survival?

OBJECTIVES: Activation of the caspase cascade through the mitochondrial and/or death receptor pathway was investigated in the failing human myocardium, in which the mode and extent of the cascade activation are unknown. BACKGROUND: In terminal heart failure, a loss of cardiomyocytes by overload-induced apoptosis is an attractive mechanism, explaining the progressive character of the disease. However, its relevance is unclear, because the specificity of probes for apoptotic deoxyribonucleic acid damage is under debate. METHODS: Left ventricular specimens from 36 explanted failing and 21 nonfailing donor hearts were used for messenger ribonucleic acid detection by semiquantitative reverse-transcription polymerase chain reaction. From these groups, immunoblot analysis was performed in samples from nine failing and six nonfailing donor hearts. RESULTS: In terminally failing hearts, there was a significant accumulation of cytochrome c in the cytosol, which was associated with activation of caspase-9 and downregulation of its inhibitor, caspase-9S. Similarly, the death receptor-induced pathway revealed activation of caspase-8, combined with downregulation of its inhibitors, flice-like inhibitory protein-L (FLIP(L)) and FLIP(S). The unspecific caspase inhibitors, XIAP, hIAP-1 and hIAP-2, were also downregulated. However, the terminal effector caspase-3 was not activated, and its substrate gelsolin, acting in its uncleaved form as a feedback inhibitor of caspase-3, was not cleaved. CONCLUSIONS: In the terminally failing human myocardium, the caspase cascade is partially activated in the presence of a consistent phenotype shift toward enhanced susceptibility to apoptosis. Although the system is still under a fragile control, the partial initiation of the apoptotic program may be of functional relevance also for the surviving cardiomyocytes.

The concomitant expression and availability of conventional and alternative, cyanide-insensitive, respiratory pathways in Candida albicans.

The opportunistic oral pathogen Candida albicans expresses a cyanide-insensitive alternative oxidase (AOX) upon exposure to respiratory inhibitors that act downstream from coenzyme Q, and upon ageing of cells. To investigate whether the conventional pathway is retained when the alternative pathway is induced, cells were grown in the presence of sodium cyanide, a reversible inhibitor of cytochrome oxidase. AOX expression was monitored by Western blotting and the presence of cytochromes associated with complexes III and IV of the conventional pathway was monitored by recording spectra between 500 and 650 nm at 77K. The activities of complexes III and IV were determined in polarographic and enzyme-kinetic experiments using specific respiratory substrates and inhibitors. Results indicated that complexes III and IV are constitutively expressed and are functional in cells expressing AOX. Furthermore, the enzymatic activities of complexes III and IV were similar in mitochondrial preparations from cells grown with or without cyanide. We next investigated whether both pathways are simultaneously available for electron transfer from the Q pool to molecular oxygen. Respiration was virtually completely inhibited by the combination of cyanide and salicyl hydroxamic acid (SHAM) or antimycin A and SHAM, but only partly inhibited by either of these inhibitors alone. This indicates that electrons can in principle flow either through the conventional or the alternative respiratory pathway. The availability of two electron pathways in C. albicans and the potential use of either pathway endows this pleomorphic fungus with another level at which it can rapidly adjust to altered environmental conditions.

Purple rice bran extract attenuates the aflatoxin B1-induced initiation stage of hepatocarcinogenesis by alteration of xenobiotic metabolizing enzymes.

Pigmented rice bran has been suggested to be a valuable source of beneficial phytochemicals. We investigated genotoxic and anti-genotoxic effects of purple rice bran extract (PRBE) in rats using a liver micronucleus assay. Purple rice bran was extracted with methanol, obtaining large amounts of phenolic compounds, including anthocyanins and small amounts of gamma-oryzanol. The experimental protocols were divided into two sets. Male rats were divided into three groups. Group 1 was a negative control, while Groups 2 and 3 were fed with 100 and 500 mg/kg bw of PRBE, respectively, for 28 days. PRBE had no effect on micronucleus formation or xenobiotic metabolizing enzymes in rat liver. Experiments concerning the effect of PRBE on AFB1 showed that PRBE significantly lessened the amount of micronucleated hepatocytes in AFB1 treated rats. Furthermore, it modulated metabolic activation of AFB1 metabolism in the liver by suppressing activity and protein expression of CYP1A2, CYP3A and CYP 450 reductase, and enhancing phase II enzymes including GST and UGT. Overall, purple rice bran extract was not genotoxic in rats. It exhibited anti-genotoxicity by modulation some xenobiotic enzymes active in AFB1 metabolism.

Effects of cobalt on haem proteins of erythropoietin-producing HepG2 cells in multicellular spheroid culture.

The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

The influence of phenobarbital on cytochromes and reactive oxygen species in erythropoietin producing HepG2 cells.

Light absorption photometry of HepG2 cells treated with phenobarbital for enhancing the content of cytochrome P-450 and the synthesis of erythropoietin revealed an influence on all cytochromes detectable in the wavelength range between 400 and 620 nm. No correlation was found between specific changes of cytochrome P-450 absorption and increased EPO synthesis as proposed earlier by Fandrey et al. (Life Sci. (1990) 47, 127-134). In the present study, however, the increased erythropoietin synthesis could be related to a decreased intracellular hydroxyl radical level described as crucial for the oxygen regulated gene expression (Kietzmann et al., Biochem. J. (1998) 335, 425-432; Porwol et al., Eur. J. Biochem. (1998) 256, 16-23).

Spotlight on oxcarbazepine in epilepsy.

Oxcarbazepine (Trileptal, Timox) is structurally related to carbamazepine and has anticonvulsant activity. Studies suggest that the anticonvulsant activity of oxcarbazepine is mediated via the blocking of neuronal ion channels. In patients aged <18 years, the efficacy of oxcarbazepine monotherapy was similar to that of phenytoin in children with partial onset or generalised tonic-clonic seizures in a 48-week trial. Additional supporting findings demonstrated that 43-71% of patients with partial onset, generalised or undetermined epilepsy were seizure free after oxcarbazepine monotherapy (mean dosage 27.7-50 mg/kg/day; duration 1-5 years). In contrast, one small nonblind trial showed more patients treated with oxcarbazepine monotherapy than with carbamazepine monotherapy had recurrent seizures during 16 months of therapy (although the conclusions that can be drawn from this trial are limited). As adjunctive therapy, oxcarbazepine was significantly better than placebo at reducing seizure frequency in children and adolescents with refractory partial onset seizures with or without secondary generalisation: the median percentage change in partial onset seizure frequency was 35% versus 9%, respectively, during 16 weeks of therapy. In noncomparative trials of adjunctive oxcarbazepine (mean dosage of 34.5-56.7 mg/kg/day), 7-11% of patients with partial onset or generalised seizures were seizure free during treatment, and 20-54% had seizure reductions of > or =50%. Oxcarbazepine was generally well tolerated during monotherapy and adjunctive therapy; 2.5% and 10% of patients withdrew from well controlled trials of oxcarbazepine monotherapy and adjunctive therapy. Oxcarbazepine monotherapy was better tolerated than phenytoin and events observed in oxcarbazepine-treated patients were transient. Oxcarbazepine metabolism is largely unaffected by induction of the cytochrome (CYP) P450 system. However, oxcarbazepine can inhibit CYP2C19 and induce CYP3A4 and CYP3A5, thereby interfering with the metabolism of other drugs (e.g. phenytoin). In addition, oxcarbazepine decreases plasma levels of oral contraceptives and alternative contraceptive methods should be used. In conclusion, oxcarbazepine (as both monotherapy and adjunctive therapy) has shown efficacy in the treatment of partial onset seizures in children with epilepsy. Nevertheless, the generally favorable tolerability profile and relatively low potential for drug interactions of oxcarbazepine make it a valuable option in the treatment of childhood epilepsy.

Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli.

The individual and the combined effect of the fnr and arcA regulatory gene products on cytochrome o oxidase and cytochrome d oxidase gene expression in Escherichia coli were evaluated using lacZ reporter fusions to the cyo-ABCDE and cydAB operons. Fnr repressed cyo-lacZ and cyd-lacZ expression during anaerobic growth but not during aerobic growth conditions. ArcA functioned as an anaerobic repressor of cyo-lacZ expression while, in contrast, it activated cydAB expression during both aerobic and anaerobic growth. ArcA and Fnr appear to function independently of each other to control cyo-ABCDE operon expression. In contrast, FNR repression of cydAB expression was dependent on arcA+, as indicated by the inability of fnr+ plasmids to repress cyd-lacZ expression in an arcA strain. Under no conditions tested did Fnr activate cydAB expression. Most, but not all, of the observed aerobic/anaerobic regulation of cyo and cyd was accounted for by the two transcriptional regulators. These data suggest the existence of additional levels of anaerobic gene control in E. coli. Additionally, the expression of the fnr regulatory gene, and regulation of the anaerobic respiratory genes, narGHJI, dmsABC and frdABCD, was found to be independent of ArcA.

Sites of inhibition of mitochondrial electron transport by cadmium.

Cadmium is an extremely toxic environmental contaminant having a long half-life in humans. The greatest accumulation occurs in the liver and kidneys. Since mitochondria are the most sensitive targets, the effect of cadmium on the oxygen consumption and on the redox state of electron carriers of rat liver mitochondria has been evaluated. Cadmium markedly inhibits uncoupler-stimulated oxidation on various NADH-linked substrates as well as that of succinate. Experiments on specific segments of the respiratory chain showed that cadmium does not inhibit electron flow through cytochrome oxidase, whereas the inhibition of duroquinol oxidation clearly demonstrates an impairment of electron flow through site 2, the ubiquinone-b-cytochrome c1 complex. On the basis of the ability of N,N,N',N' tetramethyl-p-phenylendiamine and 2,6-dichlorophenolinindophenol bypasses to relieve the cadmium inhibition of succinate oxidation and on the spectroscopic behaviour of the cytochrome b, the inhibition was found to take place before cytochrome b and, more precisely, between ubisemiquinone and cytochrome bT. Furthermore, the finding that cadmium induces a more oxidized state of cytochrome b in state 1 demonstrates the existence of a second point in which it inhibits electron transfer. Spectroscopic evidence demonstrates that cadmium induces an oxidation of NAD(P)H in mitochondria in states 1 and 4 and prevents the reduction of mitochondrial NAD(P)+ by substrates, thus indicating that the site must be localized between NAD-linked substrates and respiratory chain.

Stress-induced changes in ubiquinone concentration and alternative oxidase in plant mitochondria.

We have investigated the influence of stress conditions such as incubation at 4 degrees C and incubation in hyperoxygen atmosphere, on plant tissues. The ubiquinone (Q) content and respiratory activity of purified mitochondria was studied. The rate of respiration of mitochondria isolated from cold-treated green bell peppers (Capsicum annuum L) exceeds that of controls, but this is not so for mitochondria isolated from cold-treated cauliflower (Brassica oleracea L). Treatment with high oxygen does not alter respiration rates of cauliflower mitochondria. Analysis of kinetic data relating oxygen uptake with Q reduction in mitochondria isolated from tissue incubated at 4 degrees C (bell peppers and cauliflowers) and at high oxygen levels (cauliflowers) reveals an increase in the total amount of Q and in the percentage of inoxidizable QH2. The effects are not invariably accompanied by an induction of the alternative oxidase (AOX). In those mitochondria where the AOX is induced (cold-treated bell pepper and cauliflower treated with high oxygen) superoxide production is lower than in the control. The role of reduced Q accumulation and AOX induction in the defense against oxidative damage is discussed.

Effect of long-term in vivo treatment with imipramine on the oxidative energy metabolism in rat brain mitochondria.

The effects of long-term administration of the tricyclic antidepressant imipramine on energy metabolism of rat brain mitochondria were examined. Intraperitoneal administration of the drug resulted in significant stimulation of the state 3 respiration rates with glutamate, pyruvate+malate, beta-hydroxybutyrate and succinate as the substrates. The effect was evident within a week of imipramine administration and was sustained through the second week of the drug treatment. State 4 respiration rates were also found to be increased in general. However, the respiration with ascorbate+TMPD as the electron donor system decreased. The intramitochondrial content of cytochrome b and c+c1 increased in the first week of the drug treatment; that of aa3 cytochrome increased only in the second week.

Effects of cadmium on the energy metabolism of isolated hepatocytes: its relationship with the nonviability of isolated hepatocytes caused by cadmium.

The effects of cadmium on the energy metabolism of mitochondria were studied with isolated hepatocytes and rat liver mitochondria. It was found that cadmium inhibited the respiration of both isolated hepatocytes and mitochondria and decreased the ATP content of isolated hepatocytes. This inhibition of energy metabolism of mitochondria was highly related to the nonviability of isolated hepatocytes caused by cadmium. The site of electron transport of the mitochondrial respiratory chain blocked by cadmium was located between cytochrome b and flavo-proteins. The uncoupling effects of mitochondrial oxidative phosphorylation caused by cadmium may have resulted from changes in the fluidity and permeability of the mitochondrial membrane.

[An evaluation of the toxicity of manganese and iron in their separate and joint body uptake]. / Otsenka toksichnosti margantsa i zheleza pri razdel'nom i sovmestnom postuplenii v organizm.

Different effects for the manganese and iron uptake with a drinking water were received. An antagonism and/or synergism of these ions at levels of 1/50 and 1/10 CL50 were noted.