膀胱不同细胞系中CDC25B蛋白表达及意义

目的探讨膀胱癌不同细胞系中CDC25B蛋白表达及意义。方法采用蛋白印迹法检测CDC25B蛋白在膀胱永生化细胞SV-HUC-1细胞系和膀胱癌BIU-87和T24细胞系中的表达。结果 T24细胞中CDC25B蛋白表达水平高于BIU-87细胞和SV-HUC-1细胞系中的表达水平(P<0.05);且BIU-87细胞中CDC25B蛋白表达水平高于SV-HUC-1细胞中的表达水平(P<0.05)。结论 CDC25B可能成为评价肿瘤恶性程度的新指标。     

Discovery and characterization of novel small molecule inhibitors of human Cdc25B dual specificity phosphatase

Cdc25A and Cdc25B dual-specificity phosphatases are key regulators of cell cycle transition and proliferation. They have oncogenic properties and are overexpressed in many human tumors. Because selective Cdc25 phosphatase inhibitors would be valuable biological tools and possible therapeutic agents, we have assayed a small molecule library for in vitro inhibition of Cdc25. We now report the identification of two new structurally distinct classes of Cdc25 inhibitors with cellular activity. The cyclopentaquinoline 3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-4,8-dicarboxylic acid (5661118) and the naphthofurandione 3-benzoyl-naphtho[1,2-b]furan-4,5-dione (5169131) had in vitro IC50 values of 2.5 to 11 microM against recombinant Cdc25 and were less potent inhibitors of other phosphatases. Unlike 5661118, 5169131 caused reversible inhibition of Cdc25B and displayed competitive inhibitor kinetics. No growth inhibitory activity was seen with 5661118, whereas 10 to 30 microM 5169131 caused G1/S and G2/M arrest. We also found that 5169131 inhibited human PC-3 prostate and MDA-MB-435 breast cancer cell proliferation. Concentration-dependent Tyr15 hyperphosphorylation was seen on cyclin-dependent kinase with a 1-h 5169131 treatment, consistent with Cdc25 inhibition. Cells resistant to DNA toposiomerase II inhibitors were as sensitive to 5169131 as parental cells, indicating that this quinone compound does not inhibit topoisomerase II in vivo. Molecular modeling was used to predict a potential interaction site between the inhibitor and Cdc25B and to provide insights as to the molecular origins of the experimental observations. Based on its kinetic profile and cellular activity, we suggest that 5169131 could be an excellent tool for further studies on the cellular roles of Cdc25.     

LGH00031, a novel ortho-quinonoid inhibitor of cell division cycle 25B,inhibits human cancer cells via ROS generation

Aim:

To discover novel cell division cycle 25 (CDC25) B inhibitors and elucidate the mechanisms of inhibition in cancer cells.

Methods:

Cell growth inhibition was detected by MTT assay, the cell cycle was analyzed by flow cytometry, and protein expression and phosphorylation was examined by Western blot analysis.

Results:

LGH00031 inhibited CDC25B irreversibly in vitro in a dose-dependent manner, and impaired the proliferation of tumor cell lines. In synchronized HeLa cells, LGH00031 delayed the cell cycle progression at the G₂/M phase. LGH00031 increased cyclin-dependent kinase 1 (CDK1) tyrosine 15 phosphorylation and cyclin B1 protein level. The activity of LGH00031 against CDC25B in vitro relied on the existence of 1,4-dithiothreitol (DTT) or dihydrolipoic acid and oxygen. The oxygen free radical scavenger catalase and superoxide dismutase reduced the inactivation of CDC25 by LGH00031, confirming that reactive oxygen species (ROS) mediate the inactivation process in vitro. LGH00031 accelerated cellular ROS production in a dose-dependent manner, and N-acetyl cysteine (NAC) markedly decreased the ROS production induced by LGH00031. Correspondingly, the LGH00031-induced decrease in cell viability and cell cycle arrest, cyclin B1 protein level, and phosphorylation of CDK1 tyrosine 15 were also rescued by NAC that decreased ROS production.

Conclusion:

The activity of LGH00031 at the molecular and cellular level is mediated by ROS.     

Synthesis of a tetronic acid library focused on inhibitors of tyrosine and dual-specificity protein phosphatases and its evaluation regarding VHR and cdc25B inhibition

Selective inhibitors of protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs) are expected to be useful tools for clarifying the biological functions of the PTPs themselves and also to be candidates for novel therapeutics. We planned a library approach for the identification of PTP/DSP inhibitors in which 3-acyltetronic acid is used as a "core" phosphate mimic. A series of novel tetronic acid derivatives were synthesized and evaluated as inhibitors of the dual-specificity protein phosphatases VHR and cdc25B. Several compounds are found to be potent inhibitors of cdc25B, which is a key enzyme for cell-cycle progression. The promising results described herein strongly indicated that this tetronic acid library is potent as a library focused on the PTP/DSP-selective inhibitor.     

Monoamine oxidase inhibitors reduce conditioned fear stress-induced freezing behavior in rats

The present study examined the acute anxiolytic effects of monoamine oxidase inhibitors on freezing behavior, a putative index of anxiety induced by conditioned fear stress. The selective serotonin 1A receptor agonist tandospirone (0.1-10 mg/kg) inhibited freezing dose dependently. The irreversible, non-selective monoamine oxidase inhibitors tranylcypromine (3 and 15 mg/kg) and phenelzine (30 and 80 mg/kg) reduced freezing significantly. Clorgyline (10 mg/kg, irreversible selective monoamine oxidase A inhibitor), N-(2-aminoethyl)-5-(m-fluorophenyl)-4-thiazole carboxamide (Ro 41-1049) (30 mg/kg, reversible selective monoamine oxidase A inhibitor), selegiline (3 mg/kg, irreversible selective monoamine oxidase B inhibitor) and lazabemide (10 mg/kg, reversible selective monoamine oxidase B inhibitor) had no effect on freezing behavior. However, combined administration of clorgyline (10 mg/kg) and selegiline (3 mg/kg) reduced freezing significantly, as well as combined administration of clorgyline (10 mg/kg) and lazabemide (10 mg/kg), Ro 41-1049 (30 mg/kg) and selegiline (3 mg/kg), or Ro 41-1049 (30 mg/kg) and lazabemide (10 mg/kg). These effects of monoamine oxidase inhibitors on freezing were not due to non-specific motor effects. These results suggest that acute inhibition of both monoamine oxidase A and B reduced anxiety or fear, while inhibition of monoamine oxidase A or B alone failed to reduce anxiety or fear.     

Two inhibitors of angiotensin-converting enzyme, enalapril and captopril, increase salt appetite of rats

Male rats were orally administered an inhibitor of angiotensin-converting enzyme (ACE), N-[(S)-1-(ethoxycarbonyl)-3-phenylpropyl]-1-ala-1-pro maleate (enalapril, MK-0421) at dosage levels of 10, 30, and 90 mg/kg X d. After 2-6 wk of dosing, the rats receiving 30 and 90 mg/kg X d produced large numbers of seminal plugs and had lacerated penises due to licking in an attempt to recover urine. Providing 0.9% saline as the source of drinking water prevented this behavior and subsequent lesions. There were no adverse effects on reproductive performance. A subsequent study showed that enalapril at 5 mg/kg X d po and captopril (another ACE inhibitor) at 25 mg/kg X d po increased NaCl intake in rats. Our results with captopril confirm those of Fregly (1980) and Evered and Robinson (1983) and show that both converting-enzyme inhibitors (enalapril and captopril) increase salt appetite in rats.     

High glucose-induced inhibition of 2-deoxyglucose uptake is mediated by cAMP, protein kinase C, oxidative stress and mitogen-activated protein kinases in mouse embryonic stem cells

Abnormally high glucose levels may play an important role in early embryo development and function. In the present study, we investigated the effect of high glucose on 2-deoxyglucose (2-DG) uptake and its related signalling pathway in mouse embryonic stem (ES) cells. 2. 2-Deoxyglucose uptake was maximally inhibited by 25 mmol/L glucose after 24 h treatment. However, 25 mmol/L mannitol and dextran did not affect 2-DG uptake. Indeed, 25 mmol/L glucose decreased GLUT-1 mRNA and protein levels. The glucose (25 mmol/L)-induced inhibition of 2-DG uptake was blocked by pertussis toxin (a G(i)-protein inhibitor; 2 ng/mL), SQ 22,536 (an adenylate cyclase inhibitor; 10(-6) mol/L) and the protein kinase (PK) A inhibitor myristoylated PKI amide-(14-22) (10(-6) mol/L). Indeed, 25 mmol/L glucose increased intracellular cAMP content. 3. Furthermore, 25 mmol/L glucose-induced inhibition of 2-DG uptake was prevented by 10(-4) mol/L neomycin or 10(-6) mol/L U 73,122 (phospholipase C (PLC) inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase (PK) C inhibitors). At 25 mmol/L, glucose increased translocation of PKC from the cytoplasmic fraction to the membrane fraction. The 25 mmol/L glucose-induced inhibition of 2-DG uptake and GLUT-1 protein levels was blocked by SQ 22,536, bisindolylmaleimide I or combined treatment. In addition, 25 mmol/L glucose increased cellular reactive oxygen species and the glucose-induced inhibition of 2-DG uptake were blocked by the anti-oxidants N-acetylcysteine (NAC; 10(-5) mol/L) or taurine (2 yen 10(-3) mol/L). 4. Glucose (25 mmol/L) activated p38 mitogen-activated protein kinase (MAPK) and p44/42 MAPK. Staurosporine (10(-6) mol/L), NAC (10(-5) mol/L) and PD 98059 (10(-7) mol/L) attenuated the phosphorylation of p44/42 MAPK. Both SB 203580 (a p38 MAPK inhibitor; 10(-7) mol/L) and PD 98059 (a p44/42 MAPK inhibitor; 10(-7) mol/L) blocked 25 mmol/L glucose-induced inhibition of 2-DG uptake. 5. In conclusion, high glucose inhibits 2-DG uptake through cAMP, PLC/PKC, oxidative stress or MAPK in mouse ES cells.     

MAPK-specific tyrosine phosphatases: new targets for drug discovery?

Protein tyrosine phosphatases (PTPs) have key roles in a diverse range of cellular processes, and their dysregulation is associated with several human diseases. Many PTPs are recognized as potential drug targets; however, inhibitor development has focused only on a small number of enzymes, most notably PTP1B for type II diabetes and obesity, and MKP1 and CDC25 for cancer. The future challenge of selective-inhibitor development for PTPs will be significantly facilitated by the recent rapid progress in the structural biology of the 'PTPome'. In this article, we focus on the family of mitogen-activated protein kinase (MAPK)-specific tyrosine phosphatases--PTPN5 [also called striatal-enriched phosphatase (STEP)], PTPN7 (also called hematopoietic PTP) and PTPRR (also called PC12 PTP or STEP-like PTP)--and discuss approaches for achieving selectivity for the MAPK-PTPs at the molecular level using recently determined high-resolution X-ray crystal structures. We believe that the development of specific inhibitors would provide a valuable set of experimental pharmacological tools for investigating the physiological role of these phosphatases and exploring their emerging role in human disease.     

INHIBITION OF RAT LUNG MIXED-FUNCTION OXIDASE ACTIVITY FOLLOWING REPEATED LOW-LEVEL TOLUENE INHALATION: POSSIBLE ROLE OF TOLUENE METABOLITES

Toluene is a commonly used solvent that has been shown to alter mixed-function oxidase (MFO) activity, in an organ- and isozyme-specific pattern, following intraperitoneal administration. The purpose of this study was to determine whether similar changes occurred following repeated, low-level inhalation exposure, and to investigate the role of toluene metabolites in these alterations. Exposure to 375 ppm toluene, 6 h/d for up to 5 d, resulted in significant inhibition of the activity of pulmonary arylhydrocarbon hydroxylase (AHH), cytochrome P-4502B1 (CYP2B1), and CYP4B1, but not CYP1A1. After exposure to lower toluene levels (125 ppm, 6 h/ d, 3 d), the activities of lung AHH, CYP2B1, and CYP4B1 were also significantly decreased, but in a dose-related manner. MFO activity was not consistently altered in liver. Control pulmonary or liver microsomes were incubated with various concentrations (0.01-10 m M) of toluene or its metabolites and CYP2B1, CYP1A1, and/or CYP4B1 activities were subsequently determined. Benzaldehyde produced a significant dose-related inhibition in the activity of all three lung P-450s examined (IC50 10-3 M). Toluene was found to be a more potent inhibitor of lung CYP2B1 and CYP1A1 (IC50, 10-4 M) than benzaldehyde, but neither toluene nor benzyl alcohol was an effective inhibitor of lung CYP4B1. Toluene and its metabolites were weaker inhibitors of CYP1A1 than of CYP2B1. For CYP2B1 and CYP1A1, the order of inhibitory potency was toluene &gt; benzaldehyde &gt; benzyl alcohol and suggests that both the parent molecule and its metabolites may act in concert to inhibit catalytic activity of these cytochromes. The MFO inhibition seen after repeated low-level toluene inhalation exposure could result in altered metabolic profiles of other xenobiotics in an organ-specific fashion.     

Endothelium-dependent hyperpolarization to acetylcholine in carotid artery of guinea pig: role of lipoxygenase

This study was designed to determine whether lipoxygenase-dependent metabolites of arachidonic acid are involved in the endothelium-dependent hyperpolarization of the guinea pig carotid artery. The membrane potential of vascular smooth muscle cells was measured with intracellular microelectrodes and potassium channels were studied on freshly isolated cells with the patch-clamp technique. Acetylcholine-induced hyperpolarizations were not affected by arachidonyl trifluoromethyl ketone (AACOCF3), quinacrine (phospholipase A inhibitors), or eicosatetraenoic acid (nonspecific inhibitor of lipoxygenase, cytochrome P450, and cyclooxygenase). In contrast, cinnamyl-3,4 dihydroxy-alpha-cyanocinnamate (CDC) and AA861 (lipoxygenase inhibitors) as well as 1-(6-(17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U-73122) (phospholipase C inhibitor) produced a significant inhibition of the hyperpolarization. An opener of intermediate conductance calcium-activated potassium channels, 1-ethyl-2-benzamidazolinone (1-EBIO), induced a hyperpolarization that was unaffected by AACOCF3, CDC, AA861, or U-73122 but was inhibited by charybdotoxin. (+/-)12-hydroxy-eicosatetraenoic acid (12-HETE) and 12(S)-hydroperoxy-eicosatetraenoic acid (12(S)-HpETE) did not induce any significant changes in membrane potential. CDC inhibited the voltage-gated potassium current and increased the large conductance calcium-activated potassium current whereas AA861 inhibited both potassium currents. These results confirm that, in the isolated carotid artery of the guinea pig, stimulation of endothelial muscarinic receptors involves phospholipase C activation and indicate that the activation of phospholipase A2 and the release of lipoxygenase metabolites is unlikely to explain endothelium-dependent hyperpolarization.     

Discovery of new inhibitors of Cdc25B dual specificity phosphatases by structure-based virtual screening

Cell division cycle 25 (Cdc25) proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases and represent attractive drug targets for anticancer therapies. To discover more potent and diverse inhibitors of Cdc25 biological activity, virtual screening was performed by docking 2.1 million compounds into the Cdc25B active site. An initial subset of top-ranked compounds was selected and assayed, and 15 were found to have enzyme inhibition activity at micromolar concentration. Among these, four structurally diverse inhibitors with a different inhibition profile were found to inhibit human MCF-7, PC-3, and K562 cancer cell proliferation and significantly affect the cell cycle progression. A subsequent hierarchical similarity search with the most active reversible Cdc25B inhibitor found led to the identification of an additional set of 19 ligands, three of which were confirmed as Cdc25B inhibitors with IC(50) values of 7.9, 4.2, and 9.9 μM, respectively.     

Long-term use of proton pump inhibitors and vitamin B12 status in elderly individuals

Background  Some studies have shown that short-term use of proton pump inhibitors decreases the absorption of vitamin B12, but the results of studies into long-term proton pump inhibitor use and vitamin B12 deficiency are inconsistent.
Aim  To investigate whether long-term proton pump inhibitor use is associated with an abnormal vitamin B12 status in elderly individuals.
Methods  One hundred and twenty-five long-term (>3, years) proton pump inhibitor users aged 65, years and above were recruited from general practices. Their 125 partners (who did not use proton pump inhibitors) served as the reference group. Vitamin B12 status was determined by serum levels of vitamin B12 and homocysteine, and mean corpuscular volume.
Results  No differences in mean vitamin B12 levels were observed between the long-term proton pump inhibitor users and their partners [345 (s.d. 126), p m vs. 339 (s.d. 133), p m , P, = , 0.73], even after adjustment for age, gender, Helicobacter pylori status and C-reactive protein levels ( P, = , 0.87). Four proton pump inhibitor users and three partners had vitamin B12 levels <150, p m (3% vs. 2%, P, = , 1.00). No differences between the groups were observed in homocysteine levels and mean corpuscular volume.
Conclusions  No association between long-term proton pump inhibitor use and vitamin B12 status was observed. Regular testing for low vitamin B12 levels in elderly patients on long-term treatment with proton pump inhibitors is therefore not recommended.     

CDC25 phosphatase inhibitors: an update

The cell division cycle 25 (CDC25) family of proteins is a group of highly conserved dual-specificity phosphatases. They are key regulators of normal cell division and the cell response to DNA damage, and play a fundamental role in transitions between cell cycle phases during normal cell division, via the activation of CdK/cyclin complexes. Their abnormal expression, detected in a number of tumors, often correlated with a poor clinical prognosis, implies that their dysregulation is involved in malignant transformation. Thus, inhibition of these proteins represents an attractive therapeutic target in oncology, as evidenced from many patents and papers published on the subject in recent years. Hence, this review aims to provide an overview of recent developments in the field of CDC25 phosphatase inhibitor design since 2008.     

Inhibition of cytochrome P450s enhances (+)‐usnic acid cytotoxicity in primary cultured rat hepatocytes

(+)‐Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non‐isoenzyme‐selective inhibitor SKF‐525A (20 μM), or the CYP1A inhibitor alpha‐naphthoflavone (10 μM), or the CYP3A inhibitor ketoconazole (25 μM), the cytotoxicity of UA at 3 ~ 6 μM after 3 ~ 20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA‐induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 μM, showed no effects in parallel experiments. These data demonstrate that UA is bio‐transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Induced fit binding of aldose reductase inhibitors to AKR1B10

AKR1B10 is a recently identified NADP+ dependent aldo–keto reductase. It is strongly over expressed in lung and hepatic carcinomas as well as in colorectal and uterine cancers. AKR1B10 has 71% sequence identity with aldose reductase, the latter plays an important role in diabetic complications. The enzyme also exhibits substrate-specificity and inhibitor-sensitivity similar to aldose reductase. Various aldose reductase inhibitors show induced fit phenomenon in aldose reductase. It is reported that the selective ALR2 inhibitor, zopolrestat, also inhibits wild type AKR1B10. In this study, we have performed the induced fit docking of a few aldose reductase inhibitors in crystal structure of AKR1B10 (i.e., 1ZUA). This study elucidates the binding mode of various aldose reductase inhibitors in AKR1B10 and provides insights for the design of more selective and specific inhibitors.     

Comparison of a vasopeptidase inhibitor with neutral endopeptidase and angiotensin-converting enzyme inhibitors on bradykinin metabolism in the rat coronary bed

The in vitro effects of omapatrilat, a dual vasopeptidase inhibitor that simultaneously inhibits neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), on exogenous bradykinin metabolism after a single passage through the coronary bed were compared with that of a NEP inhibitor (retrothiorphan, 25 nM), an ACE inhibitor (enalaprilat, 130 nM), and omapatrilat (25 nM). Bradykinin and inhibitors were infused into isolated Langendorff rat hearts perfused at 1 ml/min followed by reperfusion at 10 ml/min. Residual bradykinin was quantified in the coronary effluent by enzyme-linked immunosorbent assay to calculate bradykinin recovery and its kinetic parameters (Vmax/Km). Bradykinin degradation rate at 1 ml/min was 4.56 +/- 0.39 1/min per gram without inhibitors and was significantly reduced to 2.57 +/- 0.19 1/min per gram in the presence of enalaprilat, to 2.97 +/- 0.38 1/min per gram with retrothiorphan, to 1.82 +/- 0.17 1/min per gram with both enalaprilat and retrothiorphan, and to 1.14 +/- 0.35 1/min per gram with omapatrilat. In a second set of experiments, the effect of a 14-day treatment of rats with either ACE inhibitors (enalapril, quinapril, and ramipril), a NEP inhibitor (candoxatril), or omapatrilat on exogenous bradykinin metabolism was studied in Langendorff perfused hearts isolated from these long-term treated rats. In untreated rats, bradykinin degradation at a coronary perfusion of 1 ml/min was 4.35 +/- 0.41 1/min per gram. This value was reduced by 30% for the NEP inhibitor, by 50% for all ACE inhibitors, and by 75% for omapatrilat. All inhibitors administered either short term or long term significantly reduced bradykinin degradation during a single passage through the coronary bed. However, omapatrilat administration resulted in the greatest protection from bradykinin breakdown than ACE or NEP inhibitors alone.     

Differential effects of UCN-01, staurosporine and CGP 41 251 on cell cycle progression and CDC2/cyclin B1 regulation in A431 cells synchronized at M phase by nocodazole

UCN-01 (7-hydroxystaurosporine) and CGP 41 251 (4'-N-benzoyl staurosporine), both of which were discovered as protein kinase C selective inhibitors, have entered in phase 1 clinical trials as anti-cancer drugs. In this study, we have directly compared the effects of these drugs as well as staurosporine (STP) on cell cycle progression of A431 human epidermoid carcinoma cells synchronized at M phase by treatment with nocodazole. The nocodazole-synchronized cells progressed from M to G1 phase in the absence of the drug, which was accompanied by a decrease of cyclin B1 protein expression, disappearance of the complex formation of CDC2 with cyclin B1 and reduction of the kinase activity. Treatments of the M phase cells with UCN-01, STP and CGP 41 251 at 80% growth-inhibitory concentrations (IC80S) resulted in specific G1 block, G2M block and polyploidy, respectively. Decreases of cyclin B1 protein expression was partially prevented by treatments with STP and CGP 41 251 but not with UCN-01 at IC80S. Reductions of active complex and kinase activity of CDC2/cyclin B1 were also observed in the presence of the three drugs. In addition, augmentation of CDC2 protein tyrosine phosphorylation was induced only when the cells were treated with STP. These observations demonstrated that higher concentrations of UCN-01, STP and CGP 41 251 showed different effects on cell cycle progression as well as CDC2/cyclin B1 regulation in A431 cells synchronized at M phase. The data suggest that UCN-01 and CGP 41 251 may act at quite different points on the cell cycle.     

Evaluation of the neuronal apoptotic pathways involved in cytoskeletal disruption-induced apoptosis

The cytoskeleton is critical to neuronal functioning and survival. Cytoskeletal alterations are involved in several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. We studied the possible pathways involved in colchicine-induced apoptosis in cerebellar granule neurons (CGNs). Although colchicine evoked an increase in caspase-3, caspase-6 and caspase-9 activation, selective caspase inhibitors did not attenuate apoptosis. Inhibitors of other cysteine proteases such as PD150606 (a calpain-specific inhibitor), Z-Phe-Ala fluoromethyl ketone (a cathepsins-inhibitors) and N(alpha)-p-tosyl-l-lysine chloromethyl ketone (serine-proteases inhibitor) also had no effect on cell death/apoptosis induced by colchicine. However, BAPTA-AM 10 microM (intracellular calcium chelator) prevented apoptosis mediated by cytoskeletal alteration. These data indicate that calcium modulates colchicine-induced apoptosis in CGNs. PARP-1 inhibitors did not prevent apoptosis mediated by colchicine. Finally, colchicine-induced apoptosis in CGNs was attenuated by kenpaullone, a cdk5 inhibitor. Kenpaullone and indirubin also prevented cdk5/p25 activation mediated by colchicine. These findings indicate that cytoskeletal alteration can compromise cdk5 activation, regulating p25 formation and suggest that cdk5 inhibitors attenuate apoptosis mediated by cytoskeletal alteration. The present data indicate the potential therapeutic value of drugs that prevent the formation of p25 for the treatment of neurodegenerative disorders.     

Design and optimize N‐substituted EF24 as effective and low toxicity NF‐κB inhibitor for lung cancer therapy via apoptosis‐to‐pyroptosis switch

As NF‐κB signaling pathway is constitutively activated in lung cancer, targeting NF‐κB has a potential for the treatment. EF24 has been proved to be a NF‐κB inhibitor with good antitumor activity, while whose toxicity possibly became one of the obstacles to enter into clinical application. In order to find high efficiency and low toxicity NF‐κB inhibitors, EF24 was modified and 13d was screened out. It was proved that 13d possessed an effective combination of inhibiting NF‐κB pathway and showing lower cytotoxicity on normal cells as well as less toxicity in acute toxicity experiment compared with the lead compound of EF24. In addition, 13d was found to inhibit cell vitality, arrest cell cycle in G2/M phase, promote cell apoptosis, and suppress the xenograft tumor growth. Furthermore, 13d was elucidated to induce pyroptosis developing from apoptosis, which was associated with the inhibition of NF‐κB. Taken together, it was suggested that 13d was a potent antitumor agent.     

The contribution of cytochrome P-450 isoenzymes to the metabolism of phenothiazine neuroleptics.

The aim of the present study was to determine optimum conditions for studying promazine and perazine metabolism in rat liver microsomes, and to investigate the influence of specific cytochrome P-450 inhibitors on 5-sulfoxidation and N-demethylation of these neuroleptics. Based on the developed method, the metabolism of neuroleptics in liver microsomes was studied at linear dependence of product formation on time, and protein and substrate concentrations (incubation time: 10 min; concentration of microsomal proteins: promazine-0.7 mg ml(-1), perazine-0.5 mg ml(-1); substrate concentrations: promazine-25, 40 and 75 nmol ml(-1), perazine-20, 35, 50 nmol ml(-1)). A Dixon analysis of the metabolism of neuroleptics showed that quinine (a CYP2D1 inhibitor), metyrapone (a CYP2B1/B2 inhibitor) and alpha-naphthoflavone (a CYP1A1/2 inhibitor) affected, whereas erythromycin (a CYP3A inhibitor) and sulfaphenazole (a CYP2C inhibitor) did not change the neuroleptic biotransformation. N-Demethylation of promazine was competitively inhibited by quinine (K(i)=20 microM) and metyrapone (K(i)=83 microM), while that of perazine-by quinine (K(i)=46.5 microM), metyrapone (K(i)=46 microM) and alpha-naphthoflavone (K(i)=78.8 microM). 5-Sulfoxidation of promazine was inhibited only by quinine (K(i)=28.6 microM), whereas that of perazine-by quinine (K(i)=10 microM) and metyrapone (K(i)=96 microM). The results obtained are compared with our previous findings of analogous experiments concerning thioridazine, and with the data on other phenothiazines and species. In summary, it is proposed that N-demethylation of the mentioned phenothiazine neuroleptics in the rat is catalyzed by the isoenzymes CYP2D1, CYP2B2 and CYP1A2 (CYP1A2 does not refer to promazine). 5-Sulfoxidation of these drugs may be mediated by different isoenzymes, e.g. CYP2D1 (promazine and perazine), CYP2B2 (perazine) and CYP1A2 (thioridazine). Isoenzymes belonging to subfamilies CYP2C and CYP3A do not seem to be involved in the metabolism of the investigated neuroleptics in the rat. The results obtained point to the drug structure and species differences in the contribution of cytochrome P-450 isoenzymes to the metabolism of phenothiazines.