Deoxyribonucleotidyl Transferase-mediated dUTP Nick End Research Articles

Mechanism of Action of Natural Dipeptidyl Peptidase-IV Inhibitors (Berberine and Mangiferin) in Experimentally Induced Diabetes with Metabolic Syndrome.

Berberine (BER) and mangiferin are known natural dipeptidyl peptidase (DPP-IV) inhibitors. Hence, the study was designed to elucidate the mechanism of action of natural DPP-IV inhibitors (BER and MNG) in experimentally induced diabetes with metabolic syndrome. The aim of this study was to observe mechanism through which natural DPP-IV inhibitor works in diabetes with metabolic syndrome rat model. Wistar rats were fed high-fat diet for 10 weeks and challenged with streptozotocin (STZ) (40 mg/kg) at the 3rd week (high-fat diabetic control [HF-DC] group). After the confirmation of metabolic syndrome in the setting of diabetes, monotherapy (metformin [MET], vildagliptin [VIL], BER, and MNG) and combination (MET + VIL, MET + BER, and MET + MNG) therapy was orally fed to these rats from the 4th to 10th weeks. Insulin resistance (IR) was seen in the HF-DC group as indicated by raised homeostasis model assessment of IR (HOMA-IR) in HF-DC group as compared with normal control (NC) groups. The treatment groups reduced IR as shown by a decrease in HOMA-IR as compared with HF-DC group rats. The marked reduction (P < 0.001) of beta-cell function was observed in the HF-DC group as a reduced level of HOMA for beta-cell function (HOMA-β) was found as compared with the NC group. Increases in HOMA-β as compared to the HFDC group were observed in the therapy groups. The treatment group significantly reduced cholesterol and atherogenic index. The treatment group showed significant preservation of beta-cell mass as per immunohistochemistry and significant anti-apoptotic activity as per Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling assay report. The treated rats significantly (P < 0.05) reduced high-sensitivity C-reactive protein. Lipid peroxidation (thiobarbituric acid reactive substances) marker (P < 0.001) was significantly reduced in the treatment group. The natural DPP-IV inhibitors BER and MNG treatment showed beneficial effects on various components of metabolic syndrome.

A Novel Allele Encoding 7-Hydroxymethyl Chlorophyll a Reductase Confers Bacterial Blight Resistance in Rice.

Rice spotted leaf mutants are helpful to investigate programmed cell death (PCD) and defense response pathways in plants. Using a map-based cloning strategy, we characterized novel rice spotted leaf mutation splHM143 that encodes a 7-hydroxymethyl chlorophyll a reductase (OsHCAR). The wild-type (WT) allele could rescue the mutant phenotype, as evidenced by complementation analysis. OsHCAR was constitutively expressed at all rice tissues tested and its expression products localized to chloroplasts. The mutant exhibited PCD and leaf senescence with increased H2O2 (hydrogen peroxide) accumulation, increased of ROS (reactive oxygen species) scavenging enzymes activities and TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling) -positive nuclei, upregulation of PCD related genes, decreased chlorophyll (Chl) contents, downregulation of photosynthesis-related genes, and upregulation of senescence-associated genes. Besides, the mutant exhibited enhanced bacterial blight resistance with significant upregulation of defense response genes. Knockout lines of OsHCAR exhibited spotted leaf phenotype, cell death, leaf senescence, and showed increased resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) coupled with upregulation of five pathogenesis-related marker genes. The overexpression of OsHCAR resulted in increased susceptibility to Xoo with decreased expression of pathogenesis-related marker genes. Altogether, our findings revealed that OsHCAR is involved in regulating cell death and defense response against bacterial blight pathogen in rice.

Salidroside induces apoptosis and autophagy in gastric cancer cells via regulation of mitogen-activated protein kinases signaling pathway

Background: Salidroside, an active ingredient of Rhodiola rosea, exhibits antiproliferative effect in gastric cancer (GC) cells. However, the involvement of salidroside in apoptosis and autophagy of GC cells has not been elucidated. Materials and Methods: Cell viability of BGC-823 cells was assessed by CCK-8 assay, and apoptosis was analyzed with terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling assay and flow cytometry. The apoptosis- and autophagy-associated proteins and mitogen-activated protein kinases (MAPKs) were determined by Western blot analysis. Autophagy was evaluated by green fluorescent protein-fused LC3 punctate formation. Results: Salidroside inhibited proliferation and promoted apoptosis and autophagy of BGC-823 cells in a dose-dependent manner. In addition, salidroside enhanced the phosphorylation of p38 MAPK, ERK1/2, and JNK. Furthermore, inhibition of MAPKs significantly abolished the effects of salidroside in BGC-823 cells. Conclusion: Salidroside induced apoptosis and autophagy of BGC-823 cells via activation of MAPK signaling pathway.

HIF-1α inhibits mitochondria-mediated apoptosis and improves the survival of human adipose-derived stem cells in ischemic microenvironments

Human adipose mesenchymal stem cells (hADSCs) show poor survival after transplantation, limiting their clinical application. Tissue regeneration resulting from stem cell treatment may be caused by attenuation of hypoxia-inducible factor-1α (HIF-1α). In this study, we constructed hADSCs stably expressing HIF-1α and investigated the potential effects of HIF-1α expression in the ischemic microenvironment on mitochondrial apoptosis and survival of hADSCs, and studied the mechanisms involved. Apoptosis was induced by an ischemic microenvironment in vitro. ADSCs with stable HIF-1α expression were established. Cell survival and apoptosis were observed by CCK-8 assay, western blotting, flow cytometry, and fluorescence staining. ADSCs were subcutaneously transplanted into nude mice in the location where a hypoxia ischemic microenvironment was simulated in vivo. After 1, 3, and 7 d, mitochondrial apoptotic proteins were evaluated by immunohistochemistry and immunofluorescence staining. Exogenous HIF-1α downregulated mitochondrial reactive oxygen species, cytochrome c, caspase-9, and caspase-3, but inhibited mitochondrial membrane potential depolarization and increased the Bcl-2/bax ratio. HIF-1α prevented apoptosis and promoted vascular endothelial growth factor (VEGF) secretion as demonstrated by enzyme-linked immunosorbent assay (ELISA), terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and flow cytometry analysis. HIF-1α enhanced the survival of transplanted ADSCs in nude mice. We have shown that through inhibition of the mitochondria-mediated apoptotic pathway and promotion of VEGF secretion in hADSCs in an ischemic microenvironment, HIF-1α may potentially be applied in clinical therapy and as an alternative strategy for improving hADSC therapy.

A circular RNA from NFIX facilitates oxidative stress-induced H9c2 cells apoptosis.

Myocardial infarction is the leading cause of death worldwide, and cardiomyocyte apoptosis during myocardial infarction and reperfusion is a significant factor of poor prognosis. As important regulatory molecules, biofunctions of circRNAs in the pathogenesis of myocardial infarction remain elusive. To confirm the expression level and biological function of circNFIX in cardiomyocytes upon oxidative stress. Divergent polymerase chain reaction and Sanger sequencing were performed to verify the circular structure. The stability of circNFIX was confirmed by RNase R treatment and actinomycin D assay. In order to simulate oxidative stress during myocardial infarction, H9c2 cells were subjected to hydrogen peroxide and hypoxia stimulation. In vivo, mouse models of myocardial ischemia were established. The biological function of circNFIX in cardiomyocytes was investigated through loss- and gain-of-function assays, and cardiomyocyte apoptosis level was detected by the terminal deoxyribonucleotidyl transferase-mediated TdT-mediated dUTP nick end labeling assay and Western blot. CircNFIX is abundant, conserved, and stable in H9c2 cells. The expression of circNFIX was significantly downregulated in cardiomyocytes subjected to oxidative stress. Enforced CircNFIX promotes H9c2 cells apoptosis induced by hydrogen peroxide, in sharp contrast to circNFIX knockdown. In this study, we found that circNFIX served as a pro-apoptosis factor in cardiomyocyte apoptosis. CircNFIX possesses potential to be the biomarker and therapeutic target in myocardial infarction.

&lt;p&gt;&lt;strong&gt;Antileishmanial apoptotic activity of &lt;em&gt;Nigella sativa&lt;/em&gt; L. essential oil and thymoquinone triggers on &lt;em&gt;Leishmania tropica&lt;/em&gt;&lt;/strong&gt;&lt;/p&gt;

Nigella sativa L., commonly called Black cumin, is well-known in folk medicine and numerous studies have shown its various pharmacological activities. In this study, we estimated the cytotoxic effects of N. sativa essential oil (NEO) and its major bioactive component Thymoquinone (TQ) on Leishmania tropica promastigotes that cause cutaneous leishmanias, and also observed the programmed cell death features. The extraction of NEO was done by hydro-distillation and analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Antileishmanial activity of NEO and TQ was determined using 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) assay and the obtained results are expressed as 50% inhibitory concentration (IC 50 ). The leishmanicidal activity of NEO and TQ was mediated via apoptosis as evidenced by in situ labelling of DNA fragments using terminal deoxyribonucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell cycle arrest at sub G 0 /G 1 phase. The IC 50 values were 5 µg/mL and 1.3 µg/mL, respectively. We noted a significant increase in DNA fragmentation in treated parasites with IC 50 of both NEO and TQ as well as a cell cycle arrest. These results revealed that NEO and TQ possess potential antileishmanial activity that mediated high possibility by programmed cell death.

Effects of sorafenib on fibroblast-like synoviocyte apoptosis in rats with adjuvant arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by synovial inflammation and hyperplasia resulting from an imbalance between the proliferation and apoptosis of fibroblast-like synoviocytes (FLSs). Our previous study found that sorafenib had inhibitory effects in rats with adjuvant arthritis (AA). The present study investigated the role of sorafenib in the induction of AA FLS apoptosis in vitro. FLSs obtained from AA rats were cultured in vitro and identified. Cell apoptosis was detected using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. Real-time PCR and Western blotting assays were used to quantify the expression levels of Fas, Caspase-3, Mcl-1, NF-κB and C-jun gene products in AA FLSs. Our data revealed that sorafenib (4 μmol/L) induced apoptosis in AA FLSs, and flow cytometry analysis showed that AA FLSs treated with sorafenib (4 μmol/L) in vitro accumulated in early and late apoptosis. There were significant increases in the expression levels of Fas, Caspase-3 and Mcl-1, and significant decreases in NF-κB and C-jun expression in AA FLSs treated with sorafenib. In summary, these results demonstrate that sorafenib promotes AA FLS apoptosis, which may be related to the upregulation of Fas and Caspase-3 and downregulation of NF-κB and C-jun. All of these findings suggest that sorafenib exerts an inhibitory effect on AA rats in vivo via AA FLS apoptotic induction, which has potential therapeutic implications for RA.

Anti-EGFR-iRGD recombinant protein modified biomimetic nanoparticles loaded with gambogic acid to enhance targeting and antitumor ability in colorectal cancer treatment.

BackgroundRed blood cell membrane-coated nanoparticle (RBCm-NP) platform, which consist of natural RBCm and synthetic polymeric core, can extend circulation time in vivo with an improved biocompatibility and stability of this biomimetic nanocarrier. To achieve better bioavailability of antitumor drugs that were loaded in RBCm-NPs, the functionalization of coated RBCm with specific targeting ability is essential. Bispecific recombinant protein anti-EGFR-iRGD, containing both tumor penetrating peptide (internalizing RGD peptide) and EGFR single-domain antibody (sdAb), seems to be an optimal targeting ligand for RBCm-NPs in the treatment of multiple tumors, especially colorectal cancer with high EGFR expression.Materials and methodsWe modified the anti-EGFR-iRGD recombinant protein on the surface of RBCm-NPs by lipid insertion method to construct iE–RBCm–PLGA NPs and confirmed the presentation of active tumor-targeting ability in colorectal cancer models with high EGFR expression when compared with RBCm–PLGA NPs. In addition, potential anti-tumor drug gambogic acid (GA) was loaded into the NPs to endow the antitumor efficiency of iE–RBCm–GA/PLGA NPs. It was simultaneously evaluated whether GA can reach better biocompatibility benefiting from the improved antitumor efficiency of iE–RBCm–GA/PLGA NPs in colorectal cancer models.ResultsWe successfully modified anti-EGFR-iRGD proteins on the surface of biomimetic NPs with integrated and stable “shell–core” structure. iE–RBCm–PLGA NPs showed its improved targeting ability in vitro (multicellular spheroids [MCS]) and in vivo (nude mice bearing tumors). Besides, no matter on short-term cell apoptosis at tumor site (terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]) and long-term tumor inhibition, iE–RBCm–GA/PLGA NPs achieved better antitumor efficacy than free GA in spite of the similar effects of cytotoxicity and apoptosis to GA in vitro.ConclusionWe expect that the bispecific biomimetic nanocarrier can extend the clinical application of many other potential antitumor drugs similar to GA and become a novel drug carrier in the colorectal cancer treatment.

Bioactivity-guided isolation of ginsenosides from Korean Red Ginseng with cytotoxic activity against human lung adenocarcinoma cells

BackgroundLung cancer is the leading cause of cancer-related death worldwide. In this study, we used a bioactivity-guided isolation technique to identify constituents of Korean Red Ginseng (KRG) with antiproliferative activity against human lung adenocarcinoma cells. MethodsBioactivity-guided fractionation and preparative/semipreparative HPLC purification were used with LC/MS analysis to separate the bioactive constituents. Cell viability and apoptosis in human lung cancer cell lines (A549, H1264, H1299, and Calu-6) after treatment with KRG extract fractions and constituents thereof were assessed using the water-soluble tetrazolium salt (WST-1) assay and terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining, respectively. Caspase activation was assessed by detecting its surrogate marker, cleaved poly adenosine diphosphate (ADP-ribose) polymerase, using an immunoblot assay. The expression and subcellular localization of apoptosis-inducing factor were assessed using immunoblotting and immunofluorescence, respectively. Results and conclusionBioactivity-guided fractionation of the KRG extract revealed that its ethyl acetate–soluble fraction exerts significant cytotoxic activity against all human lung cancer cell lines tested by inducing apoptosis. Chemical investigation of the ethyl acetatesoluble fraction led to the isolation of six ginsenosides, including ginsenoside Rb1 (1), ginsenoside Rb2 (2), ginsenoside Rc (3), ginsenoside Rd (4), ginsenoside Rg1 (5), and ginsenoside Rg3 (6). Among the isolated ginsenosides, ginsenoside Rg3 exhibited the most cytotoxic activity against all human lung cancer cell lines examined, with IC50 values ranging from 161.1 μM to 264.6 μM. The cytotoxicity of ginsenoside Rg3 was found to be mediated by induction of apoptosis in a caspase-independent manner. These findings provide experimental evidence for a novel biological activity of ginsenoside Rg3 against human lung cancer cells.

Sensitization of Radio-Resistant Lung Cancer Cells with a B Subunit of Bacterial Cytolethal Distending Toxin from Aggregatibacter actinomycetemcomitans

Background: Combination cancer therapy is a promising strategy which employs multiple therapeutic agents with different mechanisms of action along with minimal intolerable side effects. For example, a combination of radiotherapy with gene therapy can overcome the development of resistance to therapeutic doses of irradiation (IR) and normal tissue damages caused by high-dose radiation. Recent studies have revealed radio-resistance in non-small cell lung cancer (NSCLC) cells. In this study, for the first time, subunit B of cytolethal distending toxin (cdtB)-expressing plasmid was introduced as a sensitizer of the cells to IR with a high efficacy. Methods: A vector expressing cdtB suicide gene of human periodontal bacterium Aggregatibacter actinomycetemcomitans was constructed and then transfected into A549 cell line. In the next step, cells transfected with pcDNA3.1/cdtB were irradiated and its growth inhibitory effect was evaluated in NSCLC cancer in vitro by MTT (3-(4, 5-methylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide) assay. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were carried out in order to examine the apoptosis induction by a combination of IR with cdtB. Results: Our data indicated significant cell death in NSCLC cells in comparison with controls with an increase from 5% in response to IR up to 73.27% for combination of IR with cdtB. Moreover, the result of TUNEL assay showed significant differences in the number of apoptotic cells among the different affected groups. Conclusions: Our results confirmed that cdtB-expressing plasmid sensitizes NSCLC cells to IR and significantly increases the efficacy of radiotherapy and therefore, combining toxin with IR has a synergistic effect on NSCLC.

Resveratrol decreases FoXO protein expression through PI3K-Akt-dependent pathway inhibition in H₂O₂-treated synoviocytes.

The aim of this study was to investigate the effects of resveratrol (Res) on hydrogen peroxide (H₂O₂)-treated fibroblast-like synoviocytes (FLSs) in vitro. We studied the phosphoinositide 3-kinase (PI3K)-Akt pathway inhibition-mediated effects of Res on forkhead box O (FoXO) mRNA expression levels. FLS viability was determined by Cell Counting Kit-8 (CCK-8) assay, and FLS apoptosis was measured by terminal deoxyribo nucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining and flow cytometry. FoXO1, FoXO3 and FoXO4 mRNA expression levels in FLSs were determined by RT-PCR, and p-Akt, Akt, p-FoXO1, FoXO1, p-FoXO3, FoXO3, Bcl-2 and Bax protein expression levels were determined by western blotting. Our results showed that low H₂O₂ concentrations (20 μM) can promote FLS growth and that Res significantly inhibited FLS activity. Moreover, Res significantly increased the number of apoptotic cells and the ratio of Bax/Bcl-2 protein expression in the group treated with Res compared with the group treated with H₂O₂ and LY294002, a PI3K inhibitor. Res also decreased FoXO1, FoXO3 and FoXO4 mRNA expression levels and p-Akt/Akt, p-FoXO1/FoXO1, p-FoXO3/FoXO3 protein expression levels. Taken together, these findings indicate that Res can induce apoptosis in H₂O₂-treated FLSs in part by inhibiting the PI3K-Akt signaling pathway.

Genistein suppresses the mitochondrial apoptotic pathway in hippocampal neurons in rats with Alzheimer's disease.

Genistein is effective against amyloid-β toxicity, but the underlying mechanisms are unclear. We hypothesized that genistein may protect neurons by inhibiting the mitochondrial apoptotic pathway, and thereby play a role in the prevention of Alzheimer's disease. A rat model of Alzheimer's disease was established by intraperitoneal injection of D-galactose and intracerebral injection of amyloid-β peptide (25-35). In the genistein treatment groups, a 7-day pretreatment with genistein (10, 30, 90 mg/kg) was given prior to establishing Alzheimer's disease model, for 49 consecutive days. Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated a reduction in apoptosis in the hippocampus of rats treated with genistein. Western blot analysis showed that expression levels of capase-3, Bax and cytochrome c were decreased compared with the model group. Furthermore, immunohistochemical staining revealed reductions in cytochrome c and Bax immunoreactivity in these rats. Morris water maze revealed a substantial shortening of escape latency by genistein in Alzheimer's disease rats. These findings suggest that genistein decreases neuronal loss in the hippocampus, and improves learning and memory ability. The neuroprotective effects of genistein are associated with the inhibition of the mitochondrial apoptotic pathway, as shown by its ability to reduce levels of caspase-3, Bax and cytochrome c.

Pterocarpanquinone LQB-118 induces apoptosis in Leishmania (Viannia) braziliensis and controls lesions in infected hamsters.

Previous results demonstrate that the hybrid synthetic pterocarpanquinone LQB-118 presents antileishmanial activity against Leishmania amazonensis in a mouse model. The aim of the present study was to use a hamster model to investigate whether LQB-118 presents antileishmanial activity against Leishmania (Viannia) braziliensis, which is the major Leishmania species related to American tegumentary leishmaniasis. The in vitro antileishmanial activity of LQB-118 on L. braziliensis was tested on the promastigote and intracellular amastigote forms. The cell death induced by LQB-118 in the L. braziliensis promastigotes was analyzed using an annexin V-FITC/PI kit, the oxidative stress was evaluated by 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and the ATP content by luminescence. In situ labeling of DNA fragments by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to investigate apoptosis in the intracellular amastigotes. L. braziliensis-infected hamsters were treated from the seventh day of infection with LQB-118 administered intralesionally (26 µg/kg/day, three times a week) or orally (4,3 mg/kg/day, five times a week) for eight weeks. LQB-118 was active against the L. braziliensis promastigotes and intracellular amastigotes, producing IC50 (50% inhibitory concentration) values of 3,4±0,1 and 7,5±0,8 µM, respectively. LQB-118 induced promastigote phosphatidylserine externalization accompanied by increased reactive oxygen species production and ATP depletion. Intracellular amastigote DNA fragmentation was also observed, without affecting the viability of macrophages. The treatment of L. braziliensis-infected hamsters with LQB-118, either orally or intralesionally, was effective in the control of lesion size, parasite load and increase intradermal reaction to parasite antigen. Taken together, these results show that the antileishmanial effect of LQB-118 extends to L. braziliensis in the hamster model, involves the induction of parasite apoptosis and shows promising therapeutic option by oral or local routes in leishmaniasis.

Adiponectin inhibits murine pancreatic cancer growth.

Adiponectin is an adipose tissue-derived secretory hormone whose plasma concentrations are lower in obese individuals. Obesity is a risk factor for the development and growth of pancreatic cancer, and hypoadiponectinemia was suggested to be involved in the growth of Pan02 murine pancreatic cancer cells that were inoculated into the flanks of congenitally obese mice. The aim of this study was to clarify the role of adiponectin in the growth of pancreatic cancer cells. We examined the effect of adiponectin on the growth of Pan02 cells using recombinant adiponectin and adiponectin knockout mice. The in vitro treatment of Pan02 cells with adiponectin inhibited cellular proliferation that was accompanied by increased apoptosis and caspase-3 and caspase-7 activities. Transplantation of Pan02 cells into the pancreas of knockout mice resulted in a larger tumor volume with fewer terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells compared with wild-type mice. The results indicate that adiponectin directly suppresses the proliferation of Pan02 cells.

Spreading area and shape regulate apoptosis and differentiation of osteoblasts

The in vivo observations have indicated that at the remodeling sites of bone, the spreading area or shape of preosteoblasts is confined by the mineralized matrix. But it remains unknown whether this spreading confinement regulates the differentiation or apoptosis of osteoblasts. In the present study, osteoblast-like cells (MC3T3-E1) were seeded on micropatterned islands with different area and shape. The expression of three osteogenic differentiation markers was measured by immunofluorescence staining and apoptotic cells were detected using a terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling assay kit. The membrane fluorescence staining results showed that the actual spreading area of micropatterned osteoblasts coincided with the designed value. When the area of a micropatterned cell was confined as 314 or 615 µm2, which was lower than that of freely spreading osteoblasts, the circular shape promoted the expression of osteogenic differentiation markers and the percentage of apoptotic osteoblasts compared with the branched shape. This shape-regulated differentiation and apoptosis of osteoblasts with confined spreading area were abolished when actin polymerization was inhibited by cytochalasin D. The present study gives an insight into the roles of spreading morphology on osteoblastic differentiation and apoptosis.

LQB-118, an orally active pterocarpanquinone, induces selective oxidative stress and apoptosis in Leishmania amazonensis

The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing. Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2',7'-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨm) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨm after 24 h of incubation with 2.5 μM (18.7%), 5 μM (63.7%) or 10 μM (70.7%) LQB-118. A sub-G0/G1 cell cycle phenotype was observed in 21%-83% of the promastigotes incubated with 1.25-10 μM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5-10 μM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 μM treatment. Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.

Evaluation of reactive oxygen species (ROS) and DNA integrity assessment in cases of idiopathic male infertility

Background Studies suggest that reactive oxygen species (ROS) attack the integrity of DNA in the sperm nucleus by causing base modifications, DNA strand breaks, and chromatin cross-linking. Sperm DNA damage analysis may reveal hidden sperm DNA abnormalities in infertile men with normal standard semen analysis values who were diagnosed with idiopathic infertility. Aim of the work We explored the levels of ROS and their correlation with sperm DNA damage in patients with idiopathic male infertility. Patients and methods A total of 93 men were included in this study. Among them, 68 presented to our Andrology outpatient clinic with idiopathic infertility and were selected, and 25 were healthy fertile men, who were assigned to the control group. Both groups were subjected to the following laboratory investigations: semen analysis including peroxidase test, measurement of ROS levels by chemiluminescence assay, and sperm DNA damage assessment by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Results There were no significant differences in the semen parameters between idiopathic infertile men and controls. However, ROS and sperm DNA damage levels were significantly higher in idiopathic infertile men compared with controls ( P ͳ 0.001). In addition, the results showed a strong positive correlation between ROS levels and the percentage of sperm DNA damage ( P ͳ 0.001). Conclusion Traditional semen analysis does not reveal seminal defects at the molecular level that might be induced by ROS. Therefore, our results suggest that standard semen analysis should be coupled with measurement of ROS and assessment of DNA integrity in cases of idiopathic male infertility as even sperms with normal morphology and motility may harbor DNA damage.

Blockade of acid-sensing ion channels protects articular chondrocytes from acid-induced apoptotic injury

Acid-sensing ion channels (ASICs) are members of the degenerin/epithelial sodium channel (DEG/ENaC) protein superfamily and play a critical role in acid-induced cell injury. In this study, we examined whether drugs such as amiloride that block ASICs could attenuate acid-induced apoptotic injury to articular chondrocytes. Articular chondrocytes were isolated from Sprague-Dawley rats, and their phenotype was determined by toluidine blue and immunocytochemical staining. Articular chondrocyte viability assay was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Apoptosis of chondrocytes was observed by the terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling method as well as propidium iodide labeling methods. Intracellular calcium ([Ca(2+)](i)) was analyzed by a Ca(2+)-imaging method. In addition, the expression levels of calpain and calcineurin in articular chondrocytes were examined by real-time PCR and immunocytochemical staining. The activity of caspase-3 was evaluated by spectrophotometric assays. Positive staining for glycosaminoglycan and collagen II was seen in articular chondrocytes. Blocking acid-sensing ion channels significantly decreased the cell death percentage and increased cell viability following acid exposure. After pretreated with amiloride, acid-induced [Ca(2+)](i) rises were reduced. Amiloride also inhibited calpain and calcineurin expression levels in acid-induced chondrocytes, and inhibited caspase-3 activity. The data presented in this study provided some experimental evidence that blocking ASICs could protect acid-induced apoptotic injury to chondrocytes.

Establishment and characterization of two 5-fluorouracil-resistant hepatocellular carcinoma cell lines

5-Fluorouracil (5-FU) chemotherapy is the first choice treatment for advanced hepatocellular carcinoma (HCC), and resistance is the major obstacle to successful treatment. Recent studies have reported that epithelial-to-mesenchymal transition (EMT) is associated with chemoresistance in cancers. We speculated that EMT and 5-FU metabolism are related to the mechanism of 5-FU resistance. First, two 5-FU-resistant cell lines, HLF-R4 and HLF-R10, were established from the HLF undifferentiated human HCC cell line. Whereas cell growth was similar in the HLF and HLF-R cell lines, HLF-Rs are about 4- and 10-fold more resistant compared with the HLF cells; thus, we named these cell lines HLF-R4 and HLF-R10, respectively. The terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay also showed a dramatically decreased number of apoptotic cells in the HLF-Rs after treatment with 5-FU. We next assessed the characteristics of the HLF, HLF-R4 and HLF-R10 cells. Consistent with our hypothesis, the HLF-Rs had typical morphologic phenotypes of EMT, loss of cell-cell adhesion, spindle-shaped morphology and increased formation of pseudopodia. Real-time quantitative reverse transcriptase polymerase chain reaction data showed downregulated E-cadherin and upregulated Twist-1 and also indicated that EMT changes occurred in the HLF-Rs. We also found decreased ribonucleotide reductase and increased multidrug resistance protein 5 genes in the HLF-R cells. Our results suggested that the metabolism of EMT and 5-FU has important roles in 5-FU chemoresistance in the HLF-R cells, and that the HLF-R cells would be useful in vitro models for understanding the 5-FU-resistant mechanisms in HCC.

Large human sperm vacuoles observed in motile spermatozoa under high magnification: nuclear thumbprints linked to failure of chromatin condensation

An embryo's ability to grow and implant can be improved by selection of a normal spermatozoon with a vacuole-free head. However, large vacuoles in spermatozoa have yet to be fully characterized. The present study aimed to determine whether these vacuoles are of nuclear, membrane and/or acrosomal origin. We studied 15 infertile patients with differing sperm profiles. For each sperm sample, we used high-magnification (×10 000) contrast microscopy to select and assess 30 normal 'top' spermatozoa and 30 spermatozoa with a large sperm-head vacuole (≥ 25% of the head's cross-sectional area). We subsequently analysed the spermatozoa's degree of chromatin condensation (aniline blue staining), DNA fragmentation (terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling assay) and chromosome content (fluorescence in situ hybridization X,Y,18). Atomic force microscopy enabled us to map the plasma sperm membrane in detail. Three-dimensional deconvolution microscopy enabled us to reconstruct images of the nucleus and acrosome in 'top' and 'vacuolated' spermatozoa. We studied a total of 450 'top' spermatozoa and 450 vacuolated spermatozoa. The rate of non-condensed chromatin was higher for 'vacuolated' spermatozoa than for 'top' spermatozoa (36.2 ± 1.9 versus 7.6 ± 1.3%, respectively; P < 0.0001). 'Top' and 'vacuolated' spermatozoa did not differ significantly in terms of DNA fragmentation (0.7 ± 0.4 versus 1.3 ± 0.4% respectively; P = 0.25) or aneuploidy (1.1 ± 0.5 versus 2.2 ± 0.7% respectively; P = 0.21). The majority of aneuploid spermatozoa (9 out of 15) lacked chromatin condensation. In all vacuolated spermatozoa, the acrosome was intact, the plasma membrane was sunken but intact and the large vacuole was identified as an abnormal, 'thumbprint'-like nuclear concavity covered by acrosomal and plasmic membranes. The large vacuole appears to be a nuclear 'thumbprint' linked to failure of chromatin condensation.