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.

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.

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.

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.

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 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.

Controlled systemic delivery by polymeric implants enhances tissue and plasma curcumin levels compared with oral administration.

Curcumin possesses potent anti-inflammatory and anti-proliferative activities but with poor biopharmaceutical attributes. To overcome these limitations, curcumin implants were developed and tissue (plasma, brain and liver) curcumin concentrations were measured in female ACI rats for 3 months. Biological efficacy of tissue levels achieved was analyzed by modulation of hepatic cytochromes. Curcumin implants exhibited diffusion-mediated biphasic release pattern with ∼2-fold higher in vivo release as compared to in vitro. Plasma curcumin concentration from implants was ∼3.3 ng/ml on day 1, which dropped to ∼0.2 ng/ml after 3 months, whereas only 0.2-0.3 ng/ml concentration was observed from 4-12 days with diet and was undetected subsequently. Almost 10-fold higher curcumin levels were observed in brain on day 1 from implants compared with diet (30.1 ± 7.3 vs 2.7 ± 0.8 ng/g) and were still significant even after 90 days (7.7 ± 3.8 vs 2.2 ± 0.8 ng/g). Although curcumin levels were similar in liver from both the routes (∼25-30 ng/g from day 1-4 and ∼10-15 ng/g at 90 days), implants were more efficacious in altering hepatic CYP1A1 levels and CYP3A4 activity at ∼28-fold lower doses at 90 days. Curcumin implants provided much higher plasma and tissue concentrations and are a viable alternative for delivery of curcumin to various organs like brain.

[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.

DNA damage and altered gene expression of enzymes for metabolism and DNA repair by tamoxifen and toremifene in the female rat liver.

Effects of hepatocarcinogenic TAM and non-hepatocarcinogenic TOR on the formation of hepatic DNA adducts and on the gene expression of hepatic drug-metabolizing enzymes and DNA repair enzymes/proteins were comparatively examined in female Sprague-Dawley rats treated with TAM (20 or 40 mg/kg/day, i.g.) or TOR (40 mg/kg/day, i.g.) for 1, 2 or 8 weeks. Hepatic TAM-DNA adducts were formed even after 1 week of treatment with TAM at either dose, and the adduct levels increased in a dose- and treatment period-dependent manner, whereas no DNA adducts were detected in any of the TOR-treated rats. Conversely, TAM and TOR showed almost the same capacity for increasing the gene expression of drug-metabolizing enzymes responsible for metabolic activation and detoxification, at least up to the 2-week treatment mark. Accordingly, differences in DNA adduct formation between TAM- and TOR-treated rats would not be primarily dependent on the capacity for inducing hepatic drug-metabolizing enzymes. In addition, a drastic increase in the gene expression of cytochrome P4503A2 (CYP3A2), an activation enzyme of TAM, by the 8-week treatment with TAM might have contributed to the increased formation of DNA adducts. Gene expressions of DNA repair enzymes/proteins responsible for a nucleotide excision repair system were not significantly changed in any of the rats treated with either drug. The present findings suggest that the difference between TAM and TOR in hepatocarcinogenic potency is dependent on the capacity to form DNA adducts rather than modulating the expression of drug-metabolizing enzymes and DNA repair enzymes/proteins.

Insulin status differentially affects energy transduction in cardiac mitochondria from male and female rats.

AIM: The incidence of coronary heart diseases (CHD), congestive heart failure (CHF) and myocardial infarction is higher in diabetic patients than in non-diabetic groups, with these incidences being more in women than in the men. Hence, we examined involvement of mitochondrial energy transduction functions. METHODS: Mitochondrial energy metabolism in cardiomyopathy was studied using streptozotocin (STZ)-diabetic male and female rats as the model system. Effects of insulin treatment were also evaluated. RESULTS: The body and heart weights decreased in both male and female diabetic rats. Insulin treatments resulted in significant increase in the body and heart weights in the female rats. Mitochondrial respiration rates with all the substrates tested decreased in diabetic condition in both males and females. Treatment with two dose-regimens of insulin had differential restorative effect on mitochondrial substrate oxidation in the males but caused hyper-stimulation in the females. Diabetic state brought about 19% decrease in the cytochrome aa(3) content in the female rats. Treatment with 0.6 units of insulin significantly increased the cytochrome contents in general in both the sexes whereas higher dose (1.0 unit) caused decrease in the cytochromes content in the females. Diabetic state resulted in decreased dehydrogenases activities; insulin treatments had differential effect on the dehydrogenase activity in the males and the females. The results suggest that insulin treatment-induced hyper-stimulation of respiration in female rats may lead to increased production of reactive oxygen species. Besides, increased formation of advanced glycosylated end products may further lead to increased risk of CHF and CHD. CONCLUSIONS: The results suggest that differential effects of STZ-diabetes and insulin treatments in the female rats than in males may be the underlying cause for increased incidence of diabetic cardiomyopathies in the females.

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.

Treatment with antimalarials adversely affects the oxidative energy metabolism in rat liver mitochondria.

Effects of in vivo treatment with the three antimalarials chloroquine, primaquine and quinine on rat liver mitochondrial energy transduction functions were examined. Treatment with all the three antimalarials decreased the state 3 and state 4 respiration rates drastically. The extent of inhibition was higher with pyruvate + malate as the substrate than with glutamate. The antimalarials also acted as uncouplers. The uncoupling effect was seen on site II and site III of phosphorylation; site I was unaffected. As a consequence the ADP phosphorylation rates also decreased significantly. Following antimalarials treatment the basal and Mg2+ stimulated ATPase activities increased while the DNP-stimulated ATPase activity was reduced by half. Treatment with chloroquine resulted in decreased contents of cytochromes aa3 and b; primaquine and quinine treatments increased the contents of the two cytochromes in 14 day treatment groups.

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.

Prediction of differences in in vivo oral clearance of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl] ethylamine monohydrochloride (NE-100) between extensive and poor metabolizers from in vitro metabolic data in human liver microsomes lacking CYP2D6 activity and recombinant CYPs.

1. It has previously been reported that N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100) was predominantly metabolized by cytochrome P450 (CYP) 2D6 in human liver microsomes (HLM). In the present study, the contribution of CYP forms involved in the formation of the major metabolites of NE-100 in human liver lacking CYP2D6 activity (PM-HLM) has been predicted by use of in vitro kinetic data on recombinant CYPs microsomes (rCYPs). 2. In PM-HLM, NE-100 is predicted to be metabolized to N-despropyl-NE-100 (NE-098), p-hydroxy-NE-100 (NE-152) and m-hydroxyl-NE-100 (NE-163), but not to O-demethy-NE-100 (NE-125), which is a major metabolite in pooled human liver microsomes (EM-HLM). The relative activity factor approach assumed that NE-098 formation is predominantly catalysed by CYP3A4 and CYP2C9 and the NE-152+163mix (a mixture of two hydroxylated metabolites, NE-152 and NE-163) formation is only catalysed by CYP3A4. 3. The predicted contribution rates of CYP3A4 and CYP2C9 for NE-098 formation were 58.1 and 34.6%, respectively, in PM-HLM. These predicted results were strongly supported by kinetic and inhibition studies using PM-HLM. The intrinsic clearance of NE-100 predicted from rCYPs (the predicted CLint-HLM-total) corresponded to those observed from EM- and PM-HLM (the observed CLint-HLM). 4. The in vivo oral clearance (CLoral) of NE-100 in extensive metabolizers and poor metabolizers of CYP2D6 was predicted to be 50times higher in extensive metabolizers than poor metabolizers using in vitro-in vivo scaling method based on the dispersion model. These data suggest that polymorphism of CYP2D6 might greatly affect NE-100 metabolism in vivo.

The mitochondrial genome can be altered or lost without lethal effect in the petite-negative yeast Debaryomyces (Schwanniomyces) occidentalis.

The nature of mutations affecting several cytochrome-deficient mutants of Debaryomyces (Schwanniomyces) occidentalis has been characterized. The DR12 mutant, which is deficient in cytochrome b, and the B10Mn mutant, which is deficient in cytochromes b and a, a3, are deleted in the mitochondrial CYB and COX1 genes respectively. The B10 strain, which is partially deficient in cytochrome b, has no detectable change in its mitochondrial DNA and possibly carries nuclear lesion(s). These three mutants, unlike the rho(-) and rho degrees "petite" mutants of Saccharomyces cerevisiae, can still grow on non-fermentable substrates, due to the development of a salicylhydroxamic acid (SHAM)-sensitive alternative pathway linked to phosphorylation at site 1. A gly(-) mutant lacking mtDNA and respiratory capacity has been isolated. For the first time, it is demonstrated that mtDNA can be altered or even lost without lethal consequence in D. occidentalis, although this yeast was classified as a petite-negative species.

Stoichiometry of the photosynthetic apparatus and phycobilisome structure of the cyanobacterium Plectonema boryanum UTEX 485 are regulated by both light and temperature.

The role of growth temperature and growth irradiance on the regulation of the stoichiometry and function of the photosynthetic apparatus was examined in the cyanobacterium Plectonema boryanum UTEX 485 by comparing mid-log phase cultures grown at either 29 degrees C/150 micromol m(-2) s(-1), 29 degrees C/750 micromol m(-2) s(-1), 15 degrees C/150 micromol m(-2) s(-1), or 15 degrees C/10 micromol m(-2) s(-1). Cultures grown at 29 degrees C/750 micromol m(-2) s(-1) were structurally and functionally similar to those grown at 15 degrees C/150 micromol m(-2) s(-1), whereas cultures grown at 29 degrees C/150 micromol m(-2) s(-1) were structurally and functionally similar to those grown at 15 degrees C/10 micromol m(-2) s(-1). The stoichiometry of specific components of the photosynthetic apparatus, such as the ratio of photosystem (PS) I to PSII, phycobilisome size and the relative abundance of the cytochrome b(6)/f complex, the plastoquinone pool size, and the NAD(P)H dehydrogenase complex were regulated by both growth temperature and growth irradiance in a similar manner. This indicates that temperature and irradiance may share a common sensing/signaling pathway to regulate the stoichiometry and function of the photosynthetic apparatus in P. boryanum. In contrast, the accumulation of neither the D1 polypeptide of PSII, the large subunit of Rubisco, nor the CF(1) alpha-subunit appeared to be regulated by the same mechanism. Measurements of P700 photooxidation in vivo in the presence and absence of inhibitors of photosynthetic electron transport coupled with immunoblots of the NAD(P)H dehydrogenase complex in cells grown at either 29 degrees C/750 micromol m(-2) s(-1) or 15 degrees C/150 micromol m(-2) s(-1) are consistent with an increased flow of respiratory electrons into the photosynthetic intersystem electron transport chain maintaining P700 in a reduced state relative to cells grown at either 29 degrees C/150 micromol m(-2) s(-1) or 15 degrees C/10 micromol m(-2) s(-1). These results are discussed in terms of acclimation to excitation pressure imposed by either low growth temperature or high growth irradiance.