Reversing the polarization of tumor-associated macrophages inhibits tumor metastasis

ObjectiveThe M2 phenotype is dominant in tumor associated macrophages (TAM), and plays a key role in promoting tumor growth, invasion and metastasis. Converting TAM polarization from M2 to M1 may contribute to eliciting anti-tumor-specific immune responses and inhibiting tumor metastasis. In this study, the effect of reversing the polarization of TAM on tumor metastasis was investigated.MethodsPeritoneal macrophages were obtained from BABL/c mice, and M2 polarization was induced by IL-4. In an in vivo experiment, BABL/c mice were transplanted with 4 T1 tumor cells. In vitro and in vivo experimental studies, M2 macrophage polarization was reversed with CpG-DNA or CpG-DNA combined with anti-IL-10R Ab. CD68, MHCII and FRβ molecular expression in macrophages were examined with immunofluorescence staining. The mRNA expression of IL-2, IL-6, IL-13, VEGF and MMP-9 were detected with RT-PCR. VEGF and MMP-9 protein expression of tumors in situ was measured by western blot assay. Lung-metastasis of the tumor was observed and assessed by micro-CT.ResultsCpG-DNA and CpG-DNA combined with anti-IL-10R Ab could promote MHCII, IL-2, IL-6 and IL-13 molecular expression, and suppress the expression of FRβ, MMP-9 and VEGF, in both freshly isolated peritoneal macrophages and M2 macrophages. In the CpG-DNA combined with anti-IL-10R Ab injecting group, the percentage of CD68⁺ MHCII⁺ cells were significantly higher than that of CD68⁺ FRβ⁺ cells (P < 0.05). This was distinct from the result of the control group, which CD68⁺ FRβ⁺ was higher than CD68⁺ MHCII⁺ cells (P < 0.01). Furthermore, VEGF-A and MMP-9 level in primary tumor tissues in the experimental group was significantly lower (P < 0.01), compared to the control group. Moreover, the number of detectable lung-metastasis foci was significantly lower in the experimental group than in the control group (P < 0.05).ConclusionReversing the polarization of TAM from M2 to M1 phenotype can inhibit tumor metastasis.     

Troubleshooting the dichlorofluorescein assay to avoid artifacts in measurement of toxicant-stimulated cellular production of reactive oxidant species

IntroductionThe dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA).MethodsDCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA). MEHP was incubated with carboxy-H₂DCFDA in cell-free solutions of Hank's buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H₂DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells).ResultsMEHP did not increase fluorescence in solutions of carboxy-H₂DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180 μM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS containing 25 μM TBBPA exhibited concentration-dependent increased fluorescence with 5–100 μM carboxy-H₂DCFDA (p < 0.05), but not 1 μM carboxy-H₂DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5–25 μM) (p < 0.05), as we had observed in cell-free buffer.DiscussionMEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H₂DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H₂DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.     

Kynurenic acid inhibits proliferation and migration of human glioblastoma T98G cells

BackgroundKynurenic acid (KYNA), tryptophan metabolite synthesized in the kynurenine pathway, is an endogenous antagonist of α-7 nicotinic receptor and all ionotropic glutamate receptors: N-methyl-d-aspartate (NMDA) receptor, α-amino-3-hydroxy-5-methyl-4-isoxasole propionate (AMPA) receptor and kainate receptor. The antiproliferative activity of KYNA toward colon and renal cancer cells has recently been discovered. The aim of the study was to verify whether human Glioblastoma tumors contain KYNA and if KYNA influences glioma cell proliferation and migration.MethodsKYNA content in Glioblastoma tumor samples was determined using HPLC. Proliferation of human glioblastoma T98G cells was measured by means of MTT and BrdU assays. Wound assay was used to evaluate the effect of KYNA on cancer cell migration.ResultsKYNA was detected in all tested Glioblastoma tumor samples (100.3 ± 17.6 pmol/g wet weight). In a series of experiments the antiproliferative activity of KYNA against T98G cells was revealed (IC₅₀ = 1.3 mM). Moreover, KYNA reversed the stimulatory effect of glutamate on glioma cell proliferation and enhanced antiproliferative effect of glutamate receptor antagonists MK801 and GYKI 52466. Next, KYNA at concentrations much lower than those needed to reduce cell proliferation elicited a prominent inhibitory effect on glioma cell motility. Moreover, co-incubation of temozolomide, a drug commonly used in antiglioblastoma therapy, with KYNA gave a superior effect than each of the substances applied alone.ConclusionsWe demonstrate the antiproliferative and antimigrative potential of KYNA against glioma cells in vitro.     

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.     

The effect of Phloretin on human γδ T cells killing colon cancer SW-1116 cells

ObjectiveTo explore the effect and mechanism of Phloretin on human γδ T cells killing colon cancer SW-1116 cells.Methodsγδ T cells were amplified in vitro from human peripheral blood mononuclear cells through isopentenyl pyrophosphate method (IPP). After cocultured different concentrations of Phloretin with γδ T cells or SW-1116 cells for 48 h respectively, MTT assay was used to test the growth curve of these two cells; Flow cytometry to test the expression of Granzyme B (GraB), perforin (PFP), CD107a and IFN-γ of γδ T cells; Lactate dehydrogenase (LDH) release assay to test the cytotoxic activity of the γδ T cells on SW-1116 cells; and Western blot to test the Wnt3a expression of the γδ T cells.ResultsAfter cultured with IPP for ten days, the percentage of γδ T cells increased from 3.31 ± 3.00% to 78.40 ± 10.30%. Compared to the control group, when the concentration of Phloretin increased from 2.35 μg/ml to 18.75 μg/ml, it could significantly proliferate the γδ T cell growth (P < 0.05) and inhibit the growth of SW-1116 cells in dose–response, and the expression of GraB, PFP, CD107a and Wnt3a significantly increased (P < 0.05). Significant positive relationships were observed among CD107a and PFP, GraB, cytotoxicity (P < 0.05). The percentage of IFN-γ producing γδ T cells treated with Phloretin was significantly higher than control group.ConclusionPhloretin can enhance the killing effect of γδ T cells on SW-1116 cells; the mechanism may be that Phloretin could proliferate the γδ T cell growth, increase the expression of PFP and GraB, activate the Wnt signaling pathway, and produce higher level of IFN-γ. Indeed CD107a expression probably correlates quite well with antitumor activity.     

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.     

Inhibitory effects and mechanism of 25-OH-PPD on glomerular mesangial cell proliferation induced by high glucose

ObjectiveTo investigate the protective effects and potential mechanism of the compound 25-OH-PPD (PPD) on the glomerular mesangial cells (GMC) under high glucose condition.MethodsThe hypertrophic GMC cells were established by DMEM containing glucose and randomly divided into five groups, including the normal control group (Control), the high glucose model group (HG, 25 mmol L⁻¹), the PPD low dose group (1 μmol L⁻¹, PPD-L), the PPD middle dose group (5 μmol L⁻¹, PPD −M) and the PPD high dose group (10 μmol L⁻¹, UCN-H). The GMC were incubated for 48 h under different treatment factors. Total protein content was determined by Lowry method. The diameter of the single GMC and volume were measured by computer photograph analysis system. The GMC cell viability was analyzed by MTT assay. The level of malondialdehyde (MDA), the content of glutathione (GSH) and superoxide dismutase (SOD) activity were measured by ELISA. [Ca²⁺]_і transient was measured by Till image system and by cell-loading Fura-2/AM. The expression of COX-1 and COX-2 were also determined using ELISA method.ResultsThe viability of GMC and the total protein content were decreased in HG group, different dosage PPD group could increase these indexes (P < 0.05). The level of MDA was increased, the content of GSH and SOD was decreased in HG group, while PPD could reduce the MDA and enhance GSH and SOD (P < 0.05). Following treatment with different dosage (PPD-L, PPD-M or PPD-H), the [Ca²⁺]_і transient was reduced (P < 0.05 or P < 0.01). Moreover, the expression of COX-1 was decreased while COX-2 expression was increased in different dosage PPD groups.ConclusionThe protective effects of PPD on GMC from HG-induced hypertrophy may be associated with the inhibition of [Ca²⁺]_і transient and decreasing expression of COX-1 via the oxidative-stress injure pathway.     

The application of the comet assay to assess the genotoxicity of environmental pollutants in the nematode Caenorhabditis elegans

This study aimed to establish a protocol for cell dissociation from the nematode Caenorhabditis elegans (C. elegans) to assess the genotoxicity of the environmental pollutant benzo[a]pyrene (BaP) using the alkaline version of the single cell electrophoresis assay (comet assay). BaP genotoxicity was assessed in C. elegans (wild-type [WT]; N2, Bristol) after 48 h exposure (0–40 μM). Induction of comets by BaP was concentration-dependent up to 20 μM; comet% tail DNA was ∼30% at 20 μM BaP and ∼10% in controls. Similarly, BaP-induced DNA damage was evaluated in C. elegans mutant strains deficient in DNA repair. In xpa-1 and apn-1 mutants BaP-induced comet formation was diminished to WT background levels suggesting that the damage formed by BaP that is detected in the comet assay is not recognised in cells deficient in nucleotide and base excision repair, respectively. In summary, our study provides a protocol to evaluate DNA damage of environmental pollutants in whole nematodes using the comet assay.     

Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells

IntroductionPrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity.Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts.MethodsThe anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1–200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol — a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB).ResultsAt higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC₅₀ values compared to other modes of measurement, however, the statistically significant differences in EC₅₀ values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract > resveratrol > walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay.DiscussionIn conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.     

Cytotoxicity and genotoxicity of phenazine in two human cell lines

Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9–123 μM) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2′-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC₅₀: 11 μM; 48 h IC₅₀: 7.8 μM) observed as low as 1.9 μM. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC₅₀: 47 μM; 48 h IC₅₀: 17 μM). IC₅₀ values for HepG2 proliferation and viability were 54–77% lower compared to T24 cells. In both cell lines, IC₅₀ values for proliferation were 66–90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9–30.8 μM) experienced no significant genotoxic effects, while T24 cells (7.7–123 μM) experienced significant genotoxicity at ⩾61.5 μM. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study.     

Protection of S-adenosyl methionine against the toxicity of clivorine on hepatocytes

In this study, we investigated the protective effects of S-adenosyl-l-methionine (SAM), which is a precursor of cellular reduced glutathione (GSH), against the hepatotoxicity of pyrrolizidine alkaloid clivorine. MTT assay showed that SAM (5 μM) prevented the cytotoxicity of clivorine on human normal liver L-02 cells. DNA fragmentation assay showed that SAM (5 μM) improved clivorine-induced L-02 cell apoptosis, and the results of Western blot showed that SAM (5 μM) decreased clivorine-induced caspase-3 activation. Cellular GSH analysis showed that when L-02 cells were exposed to different concentrations (0, 3, 10, 30, 50 and 100 μM) of clivorine for 48 h, cellular GSH was decreased in a concentration-dependent manner, while SAM (5 μM) enhanced 50 μM clivorine decreased cellular GSH. Further MTT assay showed that 5 mM GSH and 5 mM N-acetyl-l-cysteine (NAC) both had protective effects against clivorine-induced hepatotoxicity. Our results suggest that SAM has protective effects against the hepatotoxicity of clivorine possibly by enhancing cellular GSH level and increasing cellular defensive ability against clivorine-induced cytotoxicity.     

The role of MAPK in the biphasic dose-response phenomenon induced by cadmium and mercury in HEK293 cells

Some studies show that Cd²⁺ and Hg²⁺ may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells. However, mechanisms underlying this phenomenon are still in puzzle. In this study, we aim at detecting the biphasic effects of Cd²⁺ and Hg²⁺ on proliferation and apoptosis of human embryonic kidney 293 (HEK293) cells, analyzing the change of the mitogen-activated protein kinase (MAPK) pathways, and discussing the relationship between them. The results demonstrate that Cd²⁺ and Hg²⁺ can stimulate cell proliferation at lower concentrations (0.05 and 0.5 μM) but inhibit it at higher concentrations (50 and 500 μM). Apoptosis increases at higher concentrations (50 and 500 μM) of Cd²⁺ and Hg²⁺. While 0.5 μM Cd²⁺ and Hg²⁺ decrease the JNK phosphorylation, 50 μM Cd²⁺ and Hg²⁺ increase the JNK and P38 phosphorylation. When HEK293 cells are treated with 20 μM JNK inhibitor or 100 μM ERK1/2 inhibitor, the cell proliferation do not increase significantly at low concentrations (0.05 and 0.5 μM), but still decrease at high concentrations (50 and 500 μM). When HEK293 cells are treated with 20 μM P38 inhibitor, the tendency of cell proliferation is not affected. Data in our study suggests that activation of MAPK pathway may be involved in the biphasic effect induced by Cd²⁺ and Hg²⁺.     

Levosimendan and its metabolite OR-1896 elicit KATP channel-dependent dilation in resistance arteries in vivo

BackgroundLevosimendan and its long-lived metabolite OR-1896 produce vasodilation in different types of vessels by activating ATP-sensitive (K_ATP) and other potassium channels.MethodsIn the present study we applied intravital videomicroscopy to investigate the in situ effects of levosimendan and OR-1896 on the diameters of real resistance arterioles (rat cremaster muscle arterioles with diameters of ~ 20 μm).ResultsLevosimendan and OR-1896 induced concentration-dependent (1 nM – 100 μM) dilations to similar extents in these arterioles (maximal dilation from 23 ± 2 to 33 ± 2 μm and from 22 ± 1 to 32 ± 1 μm, respectively). The arteriolar dilations induced by the selective K_ATP channel opener pinacidil (1 nM – 10 μM) (maximal dilation from 22 ± 4 μm to 35 ± 3 μm) were diminished in the presence of the selective K_ATP channel blocker – glibenclamide (5 μM) (maximal diameter attained: 22 ± 1 μm). Glibenclamide also counteracted the maximal dilations in response to levosimendan or OR-1896 (to 23 ± 3 μm or 22 ± 5 μm, respectively).ConclusionsIn conclusion, this is the first demonstration that levosimendan and OR-1896 elicit arteriolar dilation in vivo, via activation of K_ATP channels in real resistance vessels in the rat.     

Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells

The aims of this study were to examine the effects of CdCl₂ on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 μM CdCl₂ was sub-cytotoxic at all time-points. Exposure to 1 and 12 μM CdCl₂ for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 μM CdCl₂ in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl₂ caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd^2 + also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1–12 μM Cd^2 + is attainable in vivo, our findings are consistent with the possibility that Cd^2 + induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.     

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.     

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.     

Cytotoxicity and apoptosis induction on RTG-2 cells of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209)

Recently, the environmental residues of polybrominated diphenyl ethers (PBDEs) have markedly increased. In particular, the levels of certain PBDE congeners in fish have raised concern regarding potential risks associated with dietary PBDEs exposures. However, little is known regarding PBDE-mediated cell injury in relevant in vitro fish cell models. In this study, the cytotoxic effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and decabrominated diphenyl ether (BDE-209) on RTG-2 cells were investigated. RTG-2 cells were incubated with different concentrations of BDE-47 and BDE-209 (1–100 μM) for 72 h, and a set of bioassays were conducted to measure: cell viability (evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and neutral red (NR) uptake), lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) formation and cell apoptosis. The results showed that BDE-47 and BDE-209 inhibited the cells viability, increased LDH leakage, and induced cell apoptosis in time and concentration-dependent manner. All significant effects were observed at concentrations of 12.5 μM and above for BDE-47 and 25 μM and above for BDE-209 (P < 0.05). At the concentration of 100 μM BDE-47 and BDE-209, the cell viability of the exposed cells dropped to about 40% and 50% of the control, and the apoptotic rates were 52.6% and 34.6%, respectively. After 12 h exposure, a concentration-dependent increases of BDE-47 and BDE-209 (12.5–100 μM) in ROS formation were observed. Collectively, the results of cell viability, LDH leakage, cell apoptosis and ROS formation demonstrated that the toxic mechanism of PBDEs on RTG-2 might be mediated by oxidative stress.     

Involvement of PKA,PKC, CAMK-II and MEK1/2 in the acute antidepressant-like effect of creatine in mice

BackgroundThe aim of this study was to investigate the involvement of signaling pathways on the creatine antidepressant-like effect in the tail suspension test (TST) in mice.MethodsThe TST was used to assess the antidepressant-like properties of creatine.ResultsThe anti-immobility effect of creatine (1 mg/kg, p.o.) in the TST was blocked by i.c.v. pretreatment with H-89 (1 μg/site, PKA inhibitor), KN-62 (1 μg/site, CAMK-II inhibitor), chelerythrine (1 μg/site, PKC inhibitor), U0126 (5 μg/site, MEK1/2 inhibitor) or PD09058 (5 μg/site, MEK1/2 inhibitor).ConclusionThese results suggest that the antidepressant-like effect of creatine is dependent on PKA, CaMK-II, PKC and MEK 1/2 activation.     

Oleanolic acid and ursolic acid induce apoptosis in four human liver cancer cell lines

Apoptotic effects of oleanolic acid (OA) and ursolic acid (UA) on human liver cancer HepG2, Hep3B, Huh7 and HA22T cell lines were examined. OA or UA at 2, 4, 8 μmol/L were used and their effects on cell viability, DNA fragmentation, mitochondrial membrane potential (MMP), activity of Na⁺–K⁺-ATPase, caspase-3 and caspase-8, cell adhesion, level of intercellular adhesion molecule (ICAM)-1 and vascular endothelial growth factor (VEGF) in these cell lines were determined. OA or UA treatments concentration-dependently decreased cell viability and increased DNA fragmentation in HepG2 and Hep3B cell lines (P < 0.05). However, these two compounds reduced viability and increased DNA fragmentation in Huh7 cell only at 4 and 8 μmol/L (P < 0.05). OA or UA treatments concentration-dependently lowered MMP in HepG2, Hep3B and HA22T cell lines (P < 0.05). These two compounds also concentration-dependently diminished Na⁺–K⁺-ATPase activity and VEGF level in four test cell lines (P < 0.05). Besides Huh7 cell, OA or UA treatments concentration-dependently elevated caspase-3 and caspase-8 activities in other three cell lines (P < 0.05). Besides HA22T cell, these two compounds concentration-dependently inhibited cell adhesion and decreased ICAM-1 level in other three cell lines (P < 0.05). These findings support that OA and UA are potent anti-cancer agents to cause apoptosis in these liver cancer cell lines.     

EP3 receptor-mediated contraction of human pulmonary arteries and inhibition of neurogenic tachycardia in pithed rats

BackgroundThe aim of our study was (1) the pharmacological characterization of EP₃ receptors in human pulmonary arteries and (2) the examination of the potential involvement of these receptors in the regulation of neurogenic tachycardia in pithed rats. L-826266 served as the EP3 receptor antagonist.MethodsExperiments were performed on isolated human pulmonary arteries and pithed rats.ResultsThe prostanoid EP₁/EP₃ receptor agonist sulprostone (1 nM – 100 μM) concentration-dependently contracted isolated human pulmonary arteries (pEC₅₀, 6.88 ± 0.10). The EP₁ receptor antagonist SC 19920 (100 μM) did not affect the vasoconstriction induced by sulprostone, the TP receptor antagonist sulotroban (10 μM) only slightly attenuated the effects elicited by sulprostone > 3 μM, whereas L-826266 (10 μM) shifted its concentration-response curve to the right (apparent pA₂ value 6.18; incubation time 0.5 h). In rings exposed to L-826266 (0.1,1 or 10 μM) for 3 h, a concentration-dependent inhibitory effect against the sulprostone-induced vasoconstriction was obtained, yielding a Schild plot-based pA₂ value of 7.39. In pithed rats, sulprostone (10 – 1,000 nmol/kg), but not the IP/EP₁ receptor agonist iloprost (1-100 nmol/kg), inhibited the electrically evoked increase in heart rate (HR) dosedependently, maximally by at least 80%. L-826266 (3 μmol/kg) did not affect basal HR and diastolic blood pressure, but reduced the inhibitory effect of sulprostone 1,000 nmol/kg by about 20%.ConclusionEP₃ receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibers supplying the heart of pithed rats strongly inhibit the neurogenic tachycardia.