Selection of insulin-producing rat insulinoma (RINm) cells with improved resistance to oxidative stress

The defense system against reactive oxygen species is believed to be crucial for the survival of insulin-producing cells after various injuries. The aim of our study was to select a subpopulation of insulin-producing RINm cells with higher resistance to oxidative stress. The cells resistant to hydrogen peroxide (RINmHP) were obtained by repeated exposure of parental RINm cells to 100 and 200 microM hydrogen peroxide (HP). The increased resistance of RINmHP cells to HP was confirmed by three different cytotoxicity assays. In addition, the selected cells also were resistant to the cytotoxic effect of activated rat splenocytes compared to parental cells. The half-life of HP in the RINmHP cell culture medium was about 2.5 times lower than that of the parental cells, corresponding to the increased level of catalase expression and activity in selected cells. The increased defense property of the selected cells was not associated with any significant changes in insulin content and insulin response to a mixture of glucose with isobutyl methyl xanthine or potassium chloride. In conclusion, repeated exposure to HP induces selection of RINm cells with improved resistance to oxidative stress. This improved defense characteristic probably is due to an increased level of catalase expression and activity in the selected cells.     

AKR1B10 induces cell resistance to daunorubicin and idarubicin by reducing C13 ketonic group

Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at V_max of 837.42 ± 81.39 nmol/mg/min, K_m of 9.317 ± 2.25 mM and k_cat/K_m of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with V_max at 460.23 ± 28.12 nmol/mg/min, K_m at 0.461 ± 0.09 mM and k_cat/K_m at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50 nM) and idarubicin (30 nM) to corresponding alcohols. Within 24 h, approximately 20 ± 2.7% of daunorubicin (1 μM) or 23 ± 2.3% of idarubicin (1 μM) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7 ± 0.9% and 5 ± 1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggest that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin.     

Exposure of human skin to benzo[a]pyrene: Role of CYP1A1 and aryl hydrocarbon receptor in oxidative stress generation

Exposure to benzo[a]pyrene (BaP) can induce inflammatory skin diseases and skin cancer, which are both associated to oxidative stress. BaP is known to bind with high specificity to the aryl hydrocarbon receptor (AhR), modifying the expression of CYP1A1, involved both in cancer and inflammation.     

Positive allosteric modulators as an approach to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations

Neuronal nicotinic acetylcholine receptors (nAChR), recognized targets for drug development in cognitive and neuro-degenerative disorders, are allosteric proteins with dynamic interconversions between multiple functional states. Activation of the nAChR ion channel is primarily controlled by the binding of ligands (agonists, partial agonists, competitive antagonists) at conventional agonist binding sites, but is also regulated in either negative or positive ways by the binding of ligands to other modulatory sites. In this review, we discuss models for the activation and desensitization of nAChR, and the discovery of multiple types of ligands that influence those processes in both heteromeric nAChR, such as the high-affinity nicotine receptors of the brain, and homomeric α7-type receptors. In recent years, α7 nAChRs have been identified as a potential target for therapeutic indications leading to the development of α7-selective agonists and partial agonists. However, unique properties of α7 nAChR, including low probability of channel opening and rapid desensitization, may limit the therapeutic usefulness of ligands binding exclusively to conventional agonist binding sites. New enthusiasm for the therapeutic targeting of α7 has come from the identification of α7-selective positive allosteric modulators (PAMs) that work effectively on the intrinsic factors that limit α7 ion channel activation. While these new drugs appear promising for therapeutic development, we also consider potential caveats and possible limitations for their use, including PAM-insensitive forms of desensitization and cytotoxicity issues.     

Baicalein inhibition of oxidative-stress-induced apoptosis via modulation of ERKs activation and induction of HO-1 gene expression in rat glioma cells C6

In the present study, we examined the protective mechanism of baicalein (BE) and its glycoside, baicalin (BI), on hydrogen-peroxide (H(2)O(2))-induced cell death in rat glioma C6 cells. Results of the MTT assay, LDH release assay, and morphological observation showed that H(2)O(2) addition reduced the viability of C6 cells, and this was prevented by the addition of BE but not BI. Incubation of C6 cells with BE significantly decreased the intracellular peroxide level induced by H(2)O(2) according to flow cytometric analysis using DCHF-DA as a fluorescent substrate. Suppression of H(2)O(2)-induced apoptotic events including DNA ladders, hypodiploid cells, and activation of caspases 3, 8, and, 9 by BE but not BI was identified in C6 cells. The cytotoxicity and phosphorylation of ERK proteins induced by H(2)O(2) were blocked by the ERK inhibitor PD98059. Catalase addition prevented H(2)O(2)-induced ROS production, ERKs protein phosphorylation, and cell death, and BE dose-dependently inhibited H(2)O(2)-induced ERK protein phosphorylation in C6 cells. These data suggest that ROS-scavenging activity is involved in BE prevention of H(2)O(2)-induced cell death via blocking ERKs activation. Additionally, BE but not BI induced heat shock protein 32 (HSP32; HO-1) protein expression in both time- and dose-dependent manners, but not heme oxygenase 2 (HO-2), heat shock protein 70 (HSP70), or heat shock protein 90 (HSP90) protein expression. In the absence of H(2)O(2), BE induces ERKs protein phosphorylation, and HO-1 protein expression induced by BE was blocked by the addition of cycloheximide, actinomycin D, and the ERK inhibitor PD98059. The addition of the HO inhibitor ZnPP inhibited the protective effect of BE against H(2)O(2)-induced cytotoxicity in C6 cells according to the MTT assay and apoptotic morphology under microscopic observation, accompanied by blocking the ROS-scavenging activity of BE in C6 cells. However, BE treatment was unable to protect C6 cells from C2-ceramide-induced cell death. These data indicate that BE possesses abilities to inhibit ROS-mediated cytotoxic effects through modulation of ERKs activation and induction of HO-1 protein expression. The role of HO-1 in ROS-scavenging activity of BE is proposed.     

A comparative study on the effect of algal and fish oil on viability and cell proliferation of Caco-2 cells.

Polyunsaturated fatty acid (PUFA) rich micro-algal oil was tested in vitro and compared with fish oil for antiproliferative properties on cancer cells in vitro. Oils derived from Crypthecodinium cohnii, Schizochytrium sp. and Nitzschia laevis, three commercial algal oil capsules, and menhaden fish oil were used in cell viability and proliferation tests with human colon adenocarcinoma Caco-2 cells. With these tests no difference was found between algal oil and fish oil. The nonhydrolysed algal oils and fish oil showed a much lower toxic effect on cell viability, and cell proliferation in Caco-2 cells than the hydrolysed oils and the free fatty acids (FFAs). Eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3) were used as samples for comparison with the tested hydrolysed and nonhydrolysed oils. The hydrolysed samples showed comparative toxicity as the free fatty acids and no difference between algal and fish oil. Oxidative stress was shown to play a role in the antiproliferative properties of EPA and DHA, as alpha-tocopherol could partially reverse the EPA/DHA-induced effects. The results of the present study support a similar mode of action of algal oil and fish oil on cancer cells in vitro, in spite of their different PUFA content.     

Quercetin, but not rutin and quercitrin, prevention of H2O2-induced apoptosis via anti-oxidant activity and heme oxygenase 1 gene expression in macrophages

In the present study, we examine the protective mechanism of quercetin (QE) on oxidative stress-induced cytotoxic effect in RAW264.7 macrophages. Results of Western blotting show that QE but not its glycoside rutin (RUT) and quicitrin-induced HO-1 protein expression in a time- and dose-dependent manner, and HO-1 protein induced by QE was blocked by an addition of cycloheximide or actinomycin D. Induction of HO-1 gene expression by QE was accompanied by inducing ERKs, but not JNKs or p38, proteins phosphorylation. Addition of PD98059, but not SB203580 or SP600125, significantly attenuates QE-induced HO-1 protein and mRNA expression associated with blocking the expression of phosphorylated ERKs proteins. H(2)O(2) addition reduces the viability of cells by MTT assay, and appearance of DNA ladders, hypodiploid cells, and an increase in intracellular peroxide level was detected. Addition of QE, but not QI or RUT, significantly reduced the cytotoxic effect induced by H(2)O(2) associated with blocking the production of intracellular peroxide, DNA ladders, and hypodiploid cells. QE protection of cells from H(2)O(2)-induced apoptosis was significantly suppressed by adding HO inhibitor SnPP or ERKs inhibitor PD98059. Additionally, QE protects cells from H(2)O(2)-induced a decrease in the mitochondrial membrane potential and a release of cytochrome c from mitochondria to cytosol by DiOC6 and Western blotting assay, respectively. Activation of apoptotic proteins including the caspase 3, caspase 9, PARP, D4-GDI proteins was identified in H(2)O(2)-treated cells by Western blotting and enzyme activity assay, and that was significantly blocked by an addition of QE, but not RUT and QI. Furthermore, HO-1 catalytic metabolites carbon monoxide (CO), but not Fe(2+), Fe(3+), biliverdin or bilirubin, performed protective effect on cells from H(2)O(2)-induced cell death with an increase in HO-1 protein expression and ERKs protein phosphorylation. These data suggest that induction of HO-1 protein may participate in the protective mechanism of QE on oxidative stress (H(2)O(2))-induced apoptosis, and reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events were involved. Differential anti-apoptotic effect between QE and its glycosides RUT and QI via distinct HO-1 protein induction was also delineated.     

A novel cyclo-oxygenase-2 inhibitor modulates catabolic and antiinflammatory mediators in osteoarthritis

ITB (6-(p-bromophenyl)amino-7-(p-chlorophenyl)indazolo[2',3':1,5]-1,2,4-triazolo[4,3-a]-1,3,5-benzotriazepine) is a novel inhibitor of cyclo-oxygenase-2 (COX-2) with antiinflammatory activity in animal models. In the present study, we investigated the effect of this compound on the production of catabolic or antiinflammatory mediators in osteoarthritis (OA) cartilage. In OA cartilage explants, ITB inhibited the production of prostaglandin E(2) (PGE(2)), tumour necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-13 (MMP-13) in a concentration-dependent manner, whereas nitrite was partially reduced. On the contrary, ITB increased the production of interleukin (IL)-10 and the expression of heme oxygenase-1 (HO-1). ITB inhibited the production of catabolic mediators at concentrations able to increase IL-10 and HO-1 in OA cartilage, suggesting that this compound may be useful in the prevention of cartilage degradation.     

Oxathiolene oxides: a novel family of compounds that induce ferritin,glutathione S-transferase,and other proteins of the phase II response

Compounds that induce the synthesis of cytoprotective phase II enzymes have shown promise as cancer chemopreventive agents. Although chemically diverse, phase II enzyme inducers are capable of participating in Michael reaction chemistry. We have synthesized a novel class of organosulfur compounds, termed oxathiolene oxides (OTEOs). Based on their chemical properties, we hypothesized that these compounds could function as phase II enzyme inducers. Northern blot analysis showed that oxathiolene oxides induce the phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1 (NQO1), and ferritin H and L mRNA in a concentration-dependent fashion in a normal embryonic mouse liver cell line, BNLCL.2. OTEO-562 (3-cyclohexenyl-4-methyl-1,2-oxathiol-3-ene-2-oxide) was the strongest inducer. Western blot analysis demonstrated that GST-alpha and ferritin H protein levels were also induced in cells treated with OTEO-562, as was total GST and NQO1 enzyme activity. Further, induction of NQO1 activity by OTEO-562 was equivalent in aromatic hydrocarbon (Ah) receptor wild-type and Ah receptor mutant cell lines, suggesting that oxathiolene oxides activate phase II enzymes by an Ah receptor-independent mechanism. Consistent with this observation, OTEO-562 failed to induce cytochrome P450 1A1 mRNA. These results suggest that oxathiolene oxides may merit further investigation as candidate chemopreventive agents.     

Assessment of skin absorption and irritation potential of arachidonic acid and glyceryl arachidonate using in vitro diffusion cell techniques.

Arachidonic acid (AA), a precursor of pro-inflammatory mediators, and its glycerin ester, glyceryl arachidonate (GA), are reportedly used in cosmetic products. In vitro skin penetration of AA and GA and GA's ester hydrolysis was determined in flow-through diffusion cells. AA penetration with human and rat skin was 19.5% and 52.3% of the applied dose respectively, a substantial amount of which remained in the skin at 24h. Similar penetration results were obtained with GA in human skin. However, GA penetration through cultured skin (EpiDerm) was 51% of the applied dose, almost all of which appeared in the receptor fluid. At least 27.8% of GA penetrating skin was hydrolyzed to AA. In vitro methods were used to assess skin irritation in diffusion cells. Skin irritation of AA, sodium lauryl sulfate (SLS), and Tween 80 was determined by changes in transepidermal water loss (TEWL), skin viability (3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide, MTT, formation), and cytokine release (IL-1alpha). SLS irritation was much less pronounced in an emulsion versus an aqueous vehicle. No significant irritation was observed in vitro from AA in an emulsion. This work predicts that AA would penetrate human skin in vivo and that it could be formed in skin from topically applied GA.     

DIESEL particulate exposed macrophages alter endothelial cell expression of eNOS, iNOS, MCP1, and glutathione synthesis genes

There is considerable debate regarding inhaled diesel exhaust particulate (DEP) causing impairments in vascular reactivity. Although there is evidence that inhaled particles can translocate from the lung into the systemic circulation, it has been suggested that inflammatory factors produced in the lung following macrophage particle engulfment also pass into the circulation. To investigate these differing hypotheses, we used in vitro systems to model each exposure. By using a direct exposure system and a macrophage-endothelial cell co-culture model, we compared the effects of direct DEP exposure and exposure to inflammatory factors produced by DEP-treated macrophages, on endothelial cell mRNA levels for eNOS, iNOS, endothelin-1, and endothelin-converting-enzyme-1. As markers of oxidative stress, we measured the effects of DEP treatment on glutathione (GSH) synthesis genes and on total GSH. In addition, we analyzed the effect of DEP treatment on monocyte chemo-attractant protein-1. Direct DEP exposure increased endothelial GCLC and GCLM as well as total GSH in addition to increased eNOS, iNOS, and Mcp1 mRNA. Alternatively, inflammatory factors released from DEP-exposed macrophages markedly up-regulated endothelial iNOS and Mcp1 while modestly down-regulating eNOS. These data support both direct exposure to DEP and the release of inflammatory cytokines as explanations for DEP-induced impairments in vascular reactivity.     

PYDDT,a novel phase 2 enzymes inducer,activates Keap1–Nrf2 pathway via depleting the cellular level of glutathione

Keap1–Nrf2 pathway has emerged as a regulator for the endogenous antioxidant response, which is critical in defending cells against carcinogenesis. Herein, we demonstrated that depleting the cellular level of glutathione (GSH) by a novel electrophilic agent 2-(pro-1-ynyl)-5-(5,6-dihydroxypenta-1,3-diynyl) thiophene (PYDDT) could activate Keap1–Nrf2 pathway. In above process, it was found that Keap1 was modified by S-glutathionylation, an important post-translational modification of protein cysteines with critical roles in oxidative stress and signal transduction. We concluded from our findings that conjugation with intracellular GSH by PYDDT might lead to Keap1 S-glutathionylation and was a key event involved in its Nrf2 inducing activity.     

Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes

A series of seven platinum(II) cyclobutane-1,1-dicarboxylato (cbdc) complexes {[Pt(cbdc)(L_n)₂], 1-7}, derived from carboplatin by a substitution of two NH₃ molecules for two 2,6,9-trisubstituted 6-benzylaminopurine-based N-donor ligands (L_n), was studied by the MTT assay for their in vitro cytotoxic activity against seven human cancer cell lines, i.e. lung carcinoma (A549), cervix epithelioid carcinoma (HeLa), osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma (A2780) and its cisplatin-resistant analogue (A2780cis), and against two primary cultures of human hepatocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in some cases even more than cisplatin. The best results were achieved for complexes 1 (IC₅₀ = 17.4 ± 2.0 μM) and 2 (IC₅₀ = 14.8 ± 2.1 μΜ) against HOS cells, 1 (IC₅₀ = 15.1 ± 6.8 μM), 2 (IC₅₀ = 13.6 ± 5.2 μM) and 6 (IC₅₀ = 19.0 ± 6.6 μM) against MCF7, 6 (IC₅₀ = 6.4 ± 0.1 μM) against A2780, and 1-6 (IC₅₀ = 15.6 ± 4.0, 12.9 ± 3.7, 15.8 ± 3.8, 16.6 ± 5.5, 22.1 ± 2.5, and 5.6 ± 1.7 μM, respectively) against A2780cis. Viability of human hepatocytes was not declined by the tested complexes up to the concentration of 50 μM (for 1, 3-7) and 20 μM (for 2; caused by lower solubility of this complex).     

R-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphtalenylmethanone (WIN-2) ameliorates experimental autoimmune encephalomyelitis and induces encephalitogenic T cell apoptosis: partial involvement of the CB(2) receptor

Many reports have shown that cannabinoids might be beneficial in the symptomatic treatment of multiple sclerosis (MS). We have investigated the therapeutic properties of the non-selective cannabinoid receptor agonist WIN-2 as a suppressive drug in the experimental autoimmune encephalomyelitis (EAE) model of MS. In the passive variety of EAE, induced in Lewis rats by adoptive transfer of myelin-reactive T cells, WIN-2 ameliorates the clinical signs and diminishes the cell infiltration of the spinal cord. Due to the involvement of cannabinoids in the regulation of cell death and survival, we investigated the effects of WIN-2 on the encephalitogenic T cell population. WIN-2 induced a profound increase of apoptosis in a dose- and time-dependent manner. The potential involvement of cannabinoid receptors (CB) was investigated by encephalitogenic T cell stimulation in the presence of the CB(1) (SR141716A) and CB(2) (SR144528) antagonists, pertussis toxin (PTX) and the inactive enantiomer WIN-3. WIN-2-induced apoptosis was partially blocked by SR144528 and PTX, whereas, WIN-3 only exerted a mild effect on cell viability. These results point to the partial involvement of CB(2) receptor together with other receptor-independent mechanism or by yet unknown cannabinoid receptors. Moreover, WIN-2 induced the extrinsic pathway of apoptosis, as shown by caspase-10 and -3 activation. These results suggest that cannabinoid-induced apoptosis of encephalitogenic T cells may cooperate in their anti-inflammatory action in EAE models. The partial involvement of CB(2) receptors in WIN-2 action may open new therapeutic doors in the management of MS by non-psychoactive selective cannabinoid agonists.