Neuroprotective effects of neolignans isolated from Magnoliae Cortex against glutamate-induced apoptotic stimuli in HT22 cells

In the course of screening for neuroprotective natural products, Magnoliae Cortex showed potent inhibition of hippocampal neuronal HT22 cell death. Obovatol, honokiol, and magnolol were isolated from the ethanolic extract of Magnoliae Cortex. Isolated compounds obovatol, honokiol, and magnolol were protective against 5 mM glutamate-induced cell death. When cells were stressed using glutamate, cell viability decreased to 16.98 ± 4.58% over the control (100.00 ± 10.15%). In contrast, 10 μM obovatol, 10 μM honokiol, and 50 μM magnolol increased cell viability to 91.80 ± 1.70%, 93.59 ± 1.93%, and 85.36 ± 7.40%, respectively. The neuroprotective effects of obovatol and honokiol were attributable to the inhibition of intracellular reactive oxygen species production, followed by protection of the mitochondrial membrane potential (ΔΨ_m), recovery of Bcl-2 and Bid levels, inhibition of apoptosis-inducing factor expression, and phosphorylation of mitogen-activated protein kinases such as p38 kinases, extracellular signal-regulated kinases, and c-Jun N-terminal kinases. On the contrary, magnolol did not show any significant effect on the ΔΨ_m and apoptotic factors. Among three compounds, obovatol most strongly scavenged 2,2-diphenyl-1-picrylhydrazyl radicals and inhibited the elevation of intracellular reactive oxygen species levels in glutamate-stressed HT22 cells. These data suggest that obovatol and honokiol may have clinical applications for preventing neurodegenerative disorders.     

Inhibition of soluble and microsomal epoxide hydrolase by zinc and other metals.

Inhibition of xenobiotic-metabolizing enzymes by metals may represent an important mechanism in regulating enzyme activity. Fourteen cations were evaluated for inhibition of microsomal epoxide hydrolase (mEH) (mouse, rat, and human liver), soluble epoxide hydrolase (sEH) (mouse, rat, and human liver), and recombinant potato sEH. Of the metals tested, Hg2+ and Zn2+ were the strongest inhibitors of mEH, while Cd2+ and Cu2+ were also strong inhibitors of sEH (I50 for all approximately 20 microM). Nickel (divalent) and Pb2+ were moderate inhibitors, but Al2+, Ba2+, Ca2+, Co2+, Fe2+, Fe3+, Mg2+, and Mn2+ were weak inhibitors of both mEH and sEH (less than 50% inhibition by 1 mM metal). Six anions (acetate, bromide, chloride, nitrate, perchlorate, and sulfate) were tested and found to have no effect on the inhibition of sEH or mEH by cations. The kinetics and type of inhibition for zinc inhibition of sEH and mEH were examined for mouse, rat, human, and potato. Zinc inhibits mEH in a competitive manner. Inhibition of human and potato sEH was noncompetitive, but interestingly, zinc inhibition of mouse sEH was very strong and uncompetitive. Inhibition by zinc could be reversed by adding EDTA to the incubation buffer. Additionally, mouse liver microsomes and cytosol were incubated with these chelators. Following incubation at 4 degrees C, samples were dialyzed to remove chelator. Both mEH and sEH activity recovered was greater in samples treated with chelator than in control incubations. Similar treatment with the protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) did not affect enzyme activity recovered. During systemic inflammation, hepatic metallothionien is induced, and liver metal concentrations increase while serum metal concentrations are decreased. The inhibition of microsomal and soluble epoxide hydrolase by metals may represent a mechanism of down-regulation of enzyme activity during inflammation.     

Effect of honokiol on erythrocytes

Honokiol ((3,5-di-(2-propenyl)-1,1-biphenyl-2,2-diol), a component of Magnolia officinalis, stimulates apoptosis and is thus considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal death characterized by cell shrinkage and by breakdown of cell membrane phosphatidylserine asymmetry with phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be triggered following increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored, whether honokiol elicits eryptosis. Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, hemolysis from hemoglobin release, [Ca²⁺]_i from Fluo3-fluorescence, and ceramide from fluorescent antibodies. As a result, a 48 h exposure to honokiol was followed by a slight but significant increase of [Ca²⁺]_i (15 μM), significant decrease of forward scatter (5 μM), significant increase of annexin-V-binding (5 μM) and significant increase of ceramide formation (15 μM). Honokiol further induced slight, but significant hemolysis. Honokiol (15 μM) induced annexin-V-binding was significantly blunted but not abrogated in the nominal absence of extracellular Ca²⁺. In conclusion, honokiol triggers suicidal erythrocyte death or eryptosis, an effect at least in part due to stimulation of Ca²⁺ entry and ceramide formation.     

Cytochrome P450 epoxygenase metabolite, 14,15-EET,protects against isoproterenol-induced cellular hypertrophy in H9c2 rat cell line

We have previously shown that isoproterenol-induced cardiac hypertrophy causes significant changes to cytochromes P450 (CYPs) and soluble epoxide hydrolase (sEH) gene expression. Therefore, in this study, we examined the effect of isoproterenol in H9c2 cells, and the protective effects of 14,15-EET against isoproterenol-induced cellular hypertrophy. Isoproterenol was incubated with H9c2 cells for 24 and 48 h. To determine the protective effects of 14,15-EET, H9c2 cells were incubated with isoproterenol in the absence and presence of 14,15-EET. Thereafter, the expression of hypertrophic markers and different CYP genes were determined by real time-PCR. Our results demonstrated that isoproterenol significantly increased the expression of hypertrophic marker, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), parallel to a significant increase in cell surface area. Also, isoproterenol increased the mRNA expression of CYP1A1, CYP1B1, CYP2J3, CYP4F4 and CYP4F5, as well as the gene encoding sEH, EPHX2. On other hand, 14,15-EET significantly attenuated the isoproterenol-mediated induction of ANP, BNP, CYP1A1, CYP2J3, CYP4F4, CYP4F5 and EPHX2. Moreover 14,15-EET prevented the isoproterenol-mediated increase in cell surface area. Interestingly, 20-hydroxyeicosatetraenoic acid (20-HETE) treatment caused similar effects to that of isoproterenol treatment and induced cellular hypertrophy in H9c2 cells. In conclusion, isoproterenol induces cellular hypertrophy and modulates the expression of CYPs and EPHX2 in H9c2 cells. Furthermore, 14,15-EET exerts a protective effect against isoproterenol-induced cellular hypertrophy whereas, 20-HETE induced cellular hypertrophy in H9c2 cells.     

Maturin acetate from Psacalium peltatum (Kunth) Cass. (Asteraceae) induces immunostimulatory effects in vitro and in vivo

Maturin acetate (MA) is one of main constituents in Psacalium peltatum. The cytotoxic effects of MA on tumorigenic cells were evaluated using the MTT assay. The in vitro immunostimulatory effects of maturin acetate (MA) were evaluated on the viability of murine splenocytes and macrophages, and human peripheral blood mononuclear cells (PBMC). The effects of MA on the production of nitrous oxide, pinocytosis and lysosomal enzyme activity were assayed in murine macrophages RAW 264.7. The effects of MA on the NK cell activity were also assayed. The in vivo immunostimulatory activities of MA were evaluated on BALB/c mice immunosuppressed with cyclophosphamide (CY). MA lacks cytotoxic activity against human cancer cells (IC₅₀ > 200 μM). In the absence of LPS, MA 10 μM or higher stimulated significantly (P ? 0.05), compared to untreated cells (-LPS), the viability of murine macrophages and splenocytes. In the absence of LPS, MA 10 μM or higher stimulated significantly (P ? 0.05), compared to untreated cells (-LPS), the lysosomal enzyme activity and pinocytosis. In immunosuppressed mice, MA increases significantly (P ? 0.05), compared to CY-treated mice, the production of IL-2 and IL-15 and IFN-γ. In conclusion, MA exerts immunostimulatory activities in vitro and in vivo.     

Variants of glutathione s-transferase pi 1 exhibit differential enzymatic activity and inhibition by heavy metals

Nonsynonymous single nucleotide polymorphisms in glutathione s-transferase pi 1 (GSTP1; Ile/Val 105, Ala/Val 114) have been associated with altered toxicant metabolism in epidemiological cohorts. We explored the impact of GSTP1 genotype on enzyme kinetics and heavy metal inhibition in vitro. Four GSTP1 allozymes (105/114: Ile/Ala, Val/Ala, Ile/Val, Val/Val) were expressed in and purified from Escherichia coli. Enzyme activity assays quantifying the rate of glutathione conjugation with 1-chloro-2,4-dinitrobenzene (CDNB) revealed significant differences in kinetic parameters depending on genotype (p < 0.01). Allozymes with Ile105 had better catalytic efficiency and greater affinity for CDNB (mean ± SEM: Ile105 Ala114 K_m = 0.33 ± 0.07 mM vs. Val105 Ala114 K_m = 1.15 ± 0.07 mM). Inhibition of GSTP1 activity by heavy metals was assessed following treatment with mercury (inorganic-HgCl₂, methylmercury-MeHg), selenium, cadmium, lead, arsenic, and manganese. All allozymes were inhibited by HgCl₂ (IC₅₀ range: 24.1–172 μM), MeHg (93.9–480 μM), and selenium (43.7–62.8 μM). Genotype significantly influenced the potency of mercury with GSTP1 Ile105 Val114 the least sensitive and Val105 Ala114 the most sensitive to inhibition by HgCl₂ and MeHg. Overall, genotype of two nonsynonymous polymorphisms in GSTP1 influenced enzyme kinetics pertaining to an electrophilic substrate and inhibition by two mercury species.     

The influence of amitriptyline and carbamazepine on levomepromazine metabolism in human liver: An in vitro study

BackgroundJoint administration of phenothiazine neuroleptics and an antidepressant or carbamazepine is applied in the therapy of many complex psychiatric disorders. The aim of the present study was to investigate possible effects of the tricyclic antidepressant drug amitriptyline and the anticonvulsant drug carbamazepine on the metabolism of the aliphatic-type phenothiazine neuroleptic levomepromazine in human liver.MethodsThe experiment was performed in vitro using human liver microsomes. The rates of levomepromazine 5-sulfoxidation and N-demethylation (levomepromazine concentrations: 5, 10, 25 and 50 μM) were assessed in the absence and presence of amitriptyline or carbamazepine added in vitro (drug concentrations: 1, 2.5, 5, 10, 25 μM).ResultsA kinetic analysis of levomepromazine metabolism carried out in the absence or presence of carbamazepine showed that the anticonvulsant drug potently inhibited levomepromazine 5-sulfoxidation (K_i = 7.6 μM, non-competitive inhibition), and moderately decreased the rate of levomepromazine N-demethylation (K_i = 15.4 μM, mixed inhibition) at therapeutic drug concentrations. On the other hand, amitriptyline weakly diminished the rate of levomepromazine 5-sulfoxidation (K_i = 63 μM, mixed inhibition) and N-demethylation (K_i = 47.7 μM, mixed inhibition).ConclusionRegarding the central and peripheral effects of levomepromazine and some of its metabolites, the observed metabolic interaction between this neuroleptic and carbamazepine may be of pharmacological and clinical importance.     

Evaluation of a predictive in vitro Leydig cell assay for anti-androgenicity of phthalate esters in the rat

An in vitro assay using the rat Leydig cell line R2C was evaluated for its ability to quantitatively predict inhibition of testosterone synthesis. Results obtained for endocrine active phthalates (MEHP, MBP), and inactive phthalates (MMP and MEP) were highly consistent with in vivo results based on tissue and media concentrations. Statistically significant inhibition of testosterone synthesis (p < 0.05, 1-way ANOVA) was observed at 1 μM MBP and 3 μM MEHP, while MEP and MMP did not affect inhibition of testosterone synthesis until much higher concentrations (?100 μM). Concentrations causing 50% inhibition of testosterone synthesis for MBP and MEHP (3 and 6 μM respectively), were similar to in vivo values (3 and 7 μM). The R2C assay was used to determine the relative potency of 14 structurally diverse monoesters and oxidative metabolites of MEHP. Monoesters with alkyl chains 4–5 carbons in length had the highest potency for testosterone inhibition, while 0–2 carbon alkyl chains were least potent. Phase I metabolism did not completely inactivate MEHP, underscoring the need for metabolism data when interpreting in vitro pharmacodynamic data. This steroid inhibition assay provides a predictive in vitro alternative to expensive and timeconsuming developmental rat studies for phthalate-induced antiandrogenicity.     

Identification and characterization of diarylimidazoles as hybrid inhibitors of butyrylcholinesterase and amyloid beta fibril formation

In this contribution, a chemical collection of aromatic compounds was screened for inhibition on butyrylcholinesterase (BChE)’s hydrolase activity using Ellman’s reaction. A set of diarylimidazoles was identified as highly selective inhibitors of BChE hydrolase activity and amyloid β (Aβ) fibril formation. New derivatives were synthesized resulting in several additional hits, from which the most active was 6c, 4-(3-ethylthiophenyl)-2-(3-thienyl)-1H-imidazole, an uncompetitive inhibitor of BChE hydrolase activity (IC₅₀ BChE = 0.10 μM; K_i = 0.073 ± 0.011 μM) acting also on Aβ fibril formation (IC₅₀ = 5.8 μM). With the aid of structure–activity relationship (SAR) studies, chemical motifs influencing the BChE inhibitory activity of these imidazoles were proposed. These bifunctional inhibitors represent good tools in basic studies of BChE and/or promising lead molecules for AD therapy.     

Effect of selected antioxidants in β-cyfluthrin-induced oxidative stress in human erythrocytes in vitro

β-Cyfluthrin is one of the most widely used type II pyrethroid in agriculture. The aim of this study was to examine (1) the possibility of β-cyfluthrin to induce oxidative stress in human erythrocytes in vitro and its effect on catalase (CAT) and superoxide dismutase (SOD) activities as well as (2) the role of melatonin (MEL; 2 mM), its precursor – N-acetylserotonin (NAS; 1 mM), quercetin (Q; 80 μM) and rutin (R; 80 μM) in alleviating the cytotoxic effects of β-cyfluthrin. Erythrocytes were divided into portions. The first portion was incubated for 4 h at 37 °C with different concentrations (0, 43, 215, 1075 ppm) of β-cyfluthrin. The other portions were preincubated with selected antioxidant, respectively for 30 min and followed by β-cyfluthrin incubation for 4 h. Malondialdehyde (MDA) concentrations, CAT and SOD activities, as well as haemolysis percentage (H) were measured in all treatment portions of erythrocytes.It could be concluded that the in vitro toxicity of β-cyfluthrin may be associated with oxidative stress. Significant reduction in the activities of CAT was observed at all β-cyfluthrin concentrations, while SOD activities were significantly decreased only in erythrocytes incubated with the highest β-cyfluthrin concentration. SOD activity of the non-pretreated erythrocytes exposed to the lowest dose of β-cyfluthrin was significantly greater when compared to comparably β-cyfluthrin-exposed antioxidant pretreated cells. The highest concentration of β-cyfluthrin has caused over 35% haemolysis, and the lowest concentration about 15%. MEL pretreatment had no effect on H and MDA induction by β-cyfluthrin. NAS, Q and R reduced H and MDA level, but could not prevent induction of these parameters.Compared to other antioxidants NAS appeared to maintain better the CAT activity at control levels for all doses of β-cyfluthrin. Pretreatment with Q was found to protect against the decrease in SOD activity induced by β-cyfluthrin.     

Interactions between dietary flavonoids apigenin or luteolin and chemotherapeutic drugs to potentiate anti-proliferative effect on human pancreatic cancer cells,in vitro

The objectives were to assess the potential of dietary flavonoids apigenin (Api) and luteolin (Lut) to enhance the anti-proliferative effects of chemotherapeutic drugs on BxPC-3 human pancreatic cancer cells and to investigate the potential molecular mechanism of action. Simultaneous treatment or pretreatment (0, 6, 24 and 42 h) of flavonoids and chemotherapeutic drugs at various concentrations (0–50 μM) were assessed using the MTS cell proliferation assay. Simultaneous treatment with either flavonoid (13, 25 or 50 μM) and chemotherapeutic drugs 5-fluorouracil (5-FU, 50 μM) or gemcitabine (Gem, 10 μM) for 60 h resulted in mostly less-than-additive effects (p < 0.05). Pretreatment for 24 h with 13 μM of either Api or Lut, followed by Gem for 36 h was optimal to inhibit cell proliferation. Pretreatment of cells with 11–19 μM of either flavonoid for 24 h resulted in 59–73% growth inhibition when followed by Gem (10 μM, 36 h). Lut (15 μM, 24 h) pretreatment followed by Gem (10 μM, 36 h), significantly decreased protein expression of nuclear GSK-3β and NF-κB p65 and increased pro-apoptotic cytosolic cytochrome c. Pretreatment of BxPC-3 human pancreatic cancer cells with low concentrations of Api or Lut effectively aid in the anti-proliferative activity of chemotherapeutic drugs.     

Toxicity assessment on trophoblast cells for some environment polluting chemicals and 17β-estradiol

The identification of reproductive toxicants is a major scientific challenge for human health. We investigated the effects of a selected group of environmental polluting chemicals mostly provided with estrogenic activity on the human trophoblast cell lines BeWo and HTR-8/SVneo. Cells were exposed for 24 h to various concentrations (from 0.1 pM to 1 mM) of atrazine (ATR), diethylstilbestrol (DES), para-nonylphenol (p-NP), resveratrol (RES) and 17 β-estradiol (E2) and assayed for cell viability and human beta-Chorionic Gonadotropin (β-hCG) secretion.Decrease of cell viability as respect to control, vehicle-treated, cultures was obtained for all chemicals in the concentration range of 1 μM–1 mM in both cell types.A parallel decrease of β-hCG secretion was observed in BeWo cells, at 1 μM–1 mM concentrations, with the only exception of ATR which caused an increase at concentrations up to 1 mM. β-hCG release was also unexpectedly inhibited by ATR, DES, p-NP and RES at non-toxic (pM–nM) concentrations.These findings raise concern about the negative, potential effects of various environmental polluting chemicals on pregnancy success and fetal health.     

Apoptogenic and necrogenic effects of mercuric acetate on the chromatin structure of K562 human erythroleukemia cells

Time lapse video photography was used to follow the movement of individual cells after in vitro treatment with Hg(II) acetate. Cellular changes of mercuric ions were characterized by their properties of causing reduced cellular mobility (10–50 μM), and complete lack of cellular movement at higher concentrations (100–1000 μM). Results show that after mercury treatment at subtoxic levels (⩽1 μM): (a) chromatin changes were the earliest signs of cytotoxicity, (b) two major parts in nuclear material of K562 erythroleukemia cells could be distinguished, highly condensed supercoiled and decondensed veil-like chromatin, (c) decondensed chromosomes were rejected as clustered puffs and (d) often the nuclear material was broken down to apoptotic bodies. Nuclear changes caused by Hg(II) acetate in the concentration range between 10 and 50 μM were characterized by apoptosis seen as broken nuclei and apoptotic bodies. High concentration of Hg²⁺ ions (100 μM) initiated necrotic nuclear changes, with enlarged leaky or opened nuclei.     

Anti-allergic activity of emodin on IgE-mediated activation in RBL-2H3 cells

BackgroundEmodin (1,3,8-trihydroxy-6-methylanthraquinone) is a Chinese herbal anthraquinone derivative from the rhizome of rhubarb (Rheum palmatum L.) that exhibits numerous biological activities, such as antitumor, antibacterial, antiinflammatory, and immunosuppressive. In the present studies, the anti-allergic activities of emodin were investigated to elucidate the underlying active mechanisms.MethodsThe inhibitory effects of emodin on the IgE-mediated allergic response in rat basophilic leukemia (RBL-2H3) cells were evaluated by measuring the release of granules and cytokines. The Ca²⁺ mobilization in RBL-2H3 cells loaded with the Ca²⁺-reactive fluorescent probe Fluo-4 AM was also measured by laser scanning confocal microscope.ResultsEmodin inhibited the release of β-hexosaminidase (β-HEX; IC₅₀ = 5.5 μM) and tumor necrosis factor (TNF)-α (IC₅₀ = 11.5 μM) from RBL-2H3 cells induced by 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) and displayed stronger inhibition of β-HEX release than ketotifen fumarate salt (IC₅₀ = 63.8 μM). Emodin at a concentration of 12.5 μM also inhibited the DNP-BSA-induced influx of extracellular Ca²⁺ in RBL-2H3 cells.ConclusionsThese results suggested that emodin likely exhibits anti-allergic activities via increasing the stability of the cell membrane and inhibiting extracellular Ca²⁺ influx.     

Apoptosis induction by 4-nerolidylcatechol in melanoma cell lines

Pothomorphe umbellata, a native Brazilian plant, is popularly known to be effective in the treatment of skin lesions. This benefit is attributed to 4-nerolidylcatechol (4-NC), a compound extracted from P. umbellata. Since melanomas show prominent resistance to apoptosis and exhibit extreme chemoresistance to multiple forms of therapy, novel compounds addressing induction of cell death are worth investigating. Here, we evaluated effects on cell cycle progression and possible cytotoxic activity of 4-NC in melanoma cell lines as well as human dermal fibroblasts. Inhibitory effects on cell invasion and MMP activity were also investigated. 4-NC showed cytotoxic activity for all melanoma cell lines tested (IC₅₀ = 20–40 μM, 24 h for tumoral cell lines; IC₅₀ = 50 μM for fibroblast cell line) associated with its capacity to induce apoptosis. Furthermore, this is the first time that 4-NC is described as an inhibitor of cell invasiveness, due mainly to a G1 cell cycle arrest and inhibition of MMP-2 activity in melanoma cell lines.     

Epoxide hydrolases in the rat epididymis: possible roles in xenobiotic and endogenous fatty acid metabolism.

Epoxide hydrolases play an important role in detoxifying epoxides that arise from the metabolism of xenobiotic and endogenous compounds. Both the soluble and microsomal forms of epoxide hydrolase (sEH and mEH, respectively) have been detected in the rat testis. Because of the important role the epididymis plays in sperm maturation and protection, the present study evaluated the presence and activity of these two epoxide hydrolases in the rat epididymis. Using Western blotting, protein bands consistent in size with both mEH and sEH were detected in the caput, corpus, and cauda of the epididymis. The mEH immunoreactive bands in the epididymis ( approximately 50 kDa) were consistent with mEH detected in the liver and kidney. The sEH immunoreactive bands in the epididymis ( approximately 65 kDa) were consistent with a recombinant sEH standard and sEH detected in the liver, kidney, and testis. The presence of mEH and sEH in the epididymis was supported by observations from substrate-based enzyme assays. Results indicated that epididymal mEH can hydrolyze [(3)H]-cis-stilbene oxide to the corresponding diol at levels approximately 9% of the kidney. Epididymal sEH hydrolyzed the substrate [(3)H]-trans-diphenylpropene oxide to the corresponding diol and this activity was inhibited by cyclohexyl-dodecyl urea. Arachidonic acid epoxygenase activity was detected in epididymal S9 fractions, suggesting that fatty acid metabolism by epididymal cytochrome P450s can form epoxides that subsequently become substrates for epididymal sEH. Results from the present study indicate that the epididymis contains at least two active forms of epoxide hydrolase. The role of these enzymes in the detoxification of xenobiotic epoxides is well known, although it is unclear what cellular role they may play in the formation of biologically active metabolites in the epididymis.     

Cytotoxic activity of sanguinarine and dihydrosanguinarine in human promyelocytic leukemia HL-60 cells

The benzo[c]phenanthridine alkaloid sanguinarine has been studied for its antiproliferative activity in many cell types. Almost nothing however, is known about the cytotoxic effects of dihydrosanguinarine, a metabolite of sanguinarine. We compared the cytotoxicity of sanguinarine and dihydrosanguinarine in human leukemia HL-60 cells. Sanguinarine produced a dose-dependent decline in cell viability with IC₅₀ (inhibitor concentration required for 50% inhibition of cell viability) of 0.9 μM as determined by MTT assay after 4 h exposure. Dihydrosanguinarine showed much less cytotoxicity than sanguinarine: at the highest concentration tested (20 μM) and 24 h exposure, dihydrosanguinarine decreased viability only to 52%. Cytotoxic effects of both alkaloids were accompanied by activation of the intrinsic apoptotic pathway since we observed the dissipation of mitochondrial membrane potential, induction of caspase-9 and -3 activities, the appearance of sub-G₁ DNA and loss of plasma membrane asymmetry. This aside, sanguinarine also increased the activity of caspase-8. As shown by flow cytometry using annexin V/propidium iodide staining, 0.5 μM sanguinarine induced apoptosis while 1–4 μM sanguinarine caused necrotic cell death. In contrast, dihydrosanguinarine at concentrations from 5 μM induced primarily necrosis, whereas apoptosis occurred at 10 μM and above. We conclude that both alkaloids may cause, depending on the alkaloid concentration, both necrosis and apoptosis of HL-60 cells.     

Acute and sub-chronic toxicity studies of honokiol microemulsion

The purpose of this study was to investigate the acute and sub-chronic toxicity of honokiol microemulsion. In the acute toxicity tests, the mice were intravenously injected graded doses of honokiol microemulsion and were observed for toxic symptoms and mortality daily for 14 days. In the sub-chronic toxicity study, rats were injected honokiol microemulsion at doses of 100, 500, 2500 μg/kg body weight (BW) for 30 days. After 30 days treatment and 14 days recovery, the rats were sacrificed for hematological, biochemical and histological examination. In the acute toxicity tests, the estimated median lethal dosage (LD₅₀) was 50.5 mg/kg body weight in mice. In the sub-chronic toxicity tests, the non-toxic reaction dose was 500 μg/kg body weight. In each treatment group, degeneration or/and necrosis in vascular endothelial cells and structure change of vessel wall can be observed in the injection site (cauda vein) of a few animals while there were no changes in the vessels of other organs. The overall findings of this study indicate that the honokiol microemulsion is non-toxic up to 500 μg/kg body weight, and it has irritation to the vascular of the injection site which should be paid attention to in clinical medication.     

Inhibitory vs. protective effects of N-acetyl-l-cysteine (NAC) on the electromechanical properties of the spontaneously beating atria of the frog (Rana ridibunda): An ex vivo study

The results of this study have shown that N-acetyl-l-cysteine (NAC), a compound used for protection of tissues or cell cultures against the deleterious effects of various environmental pollutants, has certain unusual effects on the contraction of the spontaneously beating atria of the frog isolated in saline (ex vivo): (1) NAC, 6.0 and 10.0 mM, eliminated, in a concentration-dependent manner, the contractile properties of the atria (force and frequency) within minutes, without affecting its electrical properties; (2) the IC₅₀ of NAC for the force was 5.09 ± 1.01 mM (n = 6) [4.98–5.19 mM, 95% confidence interval (CI)], significantly lower than the IC₅₀ for the frequency, 6.15 ± 1.01 mM, (6.02–6.29 mM, 95% CI), indicating that working atria cells are more sensitive to NAC than autorhythmic cells. The no-observed-effect concentration (NOEC) was 1–2 mM; (3) the pattern of NAC-induced inhibition of electromechanical activity was similar to that of verapamil, an indication that NAC possibly affects L-type voltage-gated calcium channels; (4) NAC at 2 mM protected against cadmium-induced inhibition of atria contraction. The IC₅₀ for cadmium was 17.9 ± 1.1 μM (n = 6) (16.9–19.0 μM, 95% CI), while in the presence of 2 mM NAC, it became 123.3 ± 1.0 μΜ (n = 6) (114.8–132.4 μM, 95% CI). The same concentration of NAC failed to exert any protective effects against rotenone (5 μM)-induced inhibition of atria contraction. The protective effects of NAC are probably due to chelation of cadmium, rather than scavenging of oxidants.     

Inhibition of sphingosine kinase-2 ablates androgen resistant prostate cancer proliferation and survival

BackgroundEndogenous sphingolipid signaling has been shown to play an important role in prostate cancer endocrine resistance.MethodsThe novel SphK2 inhibitor, ABC294640, was used to explore SphK signaling in androgen resistant prostate cancer cell death signaling.ResultsIt dose-dependently decreased PC-3 and LNCaP cell viability, IC₅₀ of 28 ± 6.1 μM (p < 0.05) and 25 ± 4.0 μM (p < 0.05), respectively. ABC294640 was more potent in long-term clonogenic survival assays; IC₅₀ of 14 ± 0.4 μM (p < 0.05) in PC-3 cells and 12 ± 0.9 μM (p < 0.05) in LNCaP cells. Intrinsic apoptotic assays failed to demonstrate increased caspase-9 activity. Ki-67 staining demonstrated decreased proliferation by 50 ± 8.4% (p < 0.01) in PC-3 cells.ConclusionsSphK2 inhibition decreases androgen resistant prostate cancer viability, survival, and proliferation independently of the intrinsic apoptotic pathway. Findings are in contrast to recent observations of ABC29460 acting dependently on the intrinsic pathway in other endocrine resistant cancer cell lines.