Electrospun polyvinyl alcohol fibres incorporating an antimicrobial gel for enzymatically controlled reactive oxygen species release

Wounds pose a risk to the skin,our body's primary defence against infections.The rise of antibiotic resistance has prompted the development of novel therapies.RO-101^(■)is an antimicrobial gel that delivers therapeutic levels of hydrogen peroxide(H_(2)O_(2)),a reactive oxygen species,directly to the wound bed.In this study,electrospinning was used to incorporate RO-101^(■)into a polyvinyl alcohol(PVA)sub-micron fibrous mesh that can act as a delivery agent,achieve a sustained release profile,and provide a barrier against infection.Adequate incorporation of this gel into sub-micron fibres was confirmed via nuclear magnetic resonance spectroscopy.Furthermore,scanning electron microscopy exhibited smooth and uniform meshes with diameters in the 200-500 nm range.PVA/RO-101 electrospun meshes generated H_(2)O_(2) in concentrations exceeding 1 m M/(g·m L)(1 m M=1 mmol/L)after 24 h,and the role of sterilisation on H_(2)O_(2) release was evaluated.PVA/RO-101meshes exhibited antimicrobial activity against both Gram-positive Staphylococcus aureus(S.aureus)and Gram-negative Pseudomonas aeruginosa(P.aeruginosa)bacteria,achieving viable count reductions of up to 1 log unit CFU/mm^(2)(CFU:colony-forming units).Moreover,these meshes were capable of disrupting biofilm formation,even against multidrug-resistant organisms such as methicillin-resistant S.aureus(MRSA).Furthermore,increasing the RO-101^(■)concentration resulted in higher H_(2)O_(2) production and an enhanced antimicrobial effect,while fibroblast cell viability and proliferation tests showed a concentration-dependent response with high cytocompatibility at low RO-101^(■)concentrations.This study therefore demonstrates the potential of highly absorbent PVA/RO-101 meshes as potential antimicrobial wound dressings.

The role of reactive oxygen species in gastric cancer

Gastric cancer(GC)ranks fifth in cancer incidence and fourth in cancer-related mortality worldwide.Reactive oxygen species(ROS)are highly oxidative oxygen-derived products that have crucial roles in cell signaling regulation and maintaining internal balance.ROS are closely associated with the occurrence,development,and treatment of GC.This review summarizes recent findings on the sources of ROS and the bidirectional regulatory effects on GC and discusses various treatment modalities for GC that are related to ROS induction.In addition,the regulation of ROS by natural small molecule compounds with the highest potential for development and applications in anti-GC research is summarized.The aim of the review is to accelerate the clinical application of modulating ROS levels as a therapeutic strategy for GC.

Overexpression of PbrGA2ox1 enhances pear drought tolerance through the regulation of GA_(3)-inhibited reactive oxygen species detoxification and abscisic acid signaling

Drought stress is a devastating natural disaster driven by the continuing intensification of global warming,which seriously threatens the productivity and quality of several horticultural crops,including pear.Gibberellins(GAs)play crucial roles in plant growth,development,and responses to drought stress.Previous studies have shown significant reductions of GA levels in plants under drought stress;however,our understanding of the intrinsic regulation mechanisms of GA-mediated drought stress in pear remains very limited.Here,we show that drought stress can impair the accumulation of bioactive GAs(BGAs),and subsequently identified PbrGA2ox1 as a chloroplast-localized GA deactivation gene.This gene was significantly induced by drought stress and abscisic acid(ABA)treatment,but was suppressed by GA_(3)treatment.PbrGA2ox1-overexpressing transgenic tobacco plants(Nicotiana benthamiana)exhibited enhanced tolerance to dehydration and drought stresses,whereas knock-down of PbrGA2ox1 in pear(Pyrus betulaefolia)by virus-induced gene silencing led to elevated drought sensitivity.Transgenic plants were hypersensitive to ABA,and had a lower BGAs content,enhanced reactive oxygen species(ROS)scavenging ability,and augmented ABA accumulation and signaling under drought stress compared to wild-type plants.However,the opposite effects were observed with PbrGA2ox1 silencing in pear.Moreover,exogenous GA_(3)treatment aggravated the ROS toxic effect and restrained ABA synthesis and signaling,resulting in the compromised drought tolerance of pear.In summary,our results shed light on the mechanism by which BGAs are eliminated in pear leaves under drought stress,providing further insights into the mechanism regulating the effects of GA on the drought tolerance of plants.

Reactive oxygen species and oxidative stress in acute pancreatitis:Pathogenesis and new therapeutic interventions

Acute pancreatitis(AP)is a common acute gastrointestinal disorder affecting approximately 20%of patients with systemic inflammatory responses that may cause pancreatic and peripancreatic fat necrosis.This condition often progresses to multiple organ failure,significantly increasing morbidity and mortality.Oxidative stress,characterized by an imbalance between the body’s reactive oxygen species(ROS)and antioxidants,activates the inflammatory signaling pathways.Although the pathogenesis of AP is not fully understood,ROS are increasingly recognized as critical in the disease's progression and development.Modulating the oxidative stress pathway has shown efficacy in mitigating the progression of AP.Despite numerous basic studies examining this pathway,comprehensive reviews of recent research remain sparse.This systematic review offers an in-depth examination of the critical role of oxidative stress in the pathogenesis and progression of AP and evaluates the therapeutic potential of antioxidant interventions in its management.

OsbZIP53 Negatively Regulates Immunity Response by Involving in Reactive Oxygen Species and Salicylic Acid Metabolism in Rice

The basic region/leucine zipper(bZIP)transcription factors play important roles in plant development and responses to abiotic and biotic stresses.OsbZIP53 regulates resistance to Magnaporthe oryzae in rice by analyzing APIP5-RNAi transgenic plants.To further investigate the biological functions of OsbZIP53,we generated osbzip53 mutants using CRISPR/Cas9 editing and also constructed OsbZIP53 over-expression transgenic plants.Comprehensive analysis of phenotypical,physiological,and transcriptional data showed that knocking-out OsbZIP53 not only improved disease resistance by inducing a hypersensitivity response in plants,but also regulated the immune response through the salicylic acid pathway.Specifically,disrupting OsbZIP53 increased H2O2 accumulation by promoting reactive oxygen species generation through up-regulation of several respiratory burst oxidase homologs(Osrboh genes)and weakened H2O2 degradation by directly targeting OsMYBS1.In addition,the growth of osbzip53 mutants was seriously impaired,while OsbZIP53 over-expression lines displayed a similar phenotype to the wild type,suggesting that OsbZIP53 has a balancing effect on rice immune response and growth.

Microarrow sensor array with enhanced skin adhesion for transdermal continuous monitoring of glucose and reactive oxygen species

Conventional blood sampling for glucose detection is prone to cause pain and fails to continuously record glucose fluctuations in vivo.Continuous glucose monitoring based on implantable electrodes could induce pain and potential tissue inflammation,and the presence of reactive oxygen species(ROS)due to inflammationmay affect glucose detection.Microneedle technology is less invasive,yet microneedle adhesion with skin tissue is limited.In this work,we developed a microarrow sensor array(MASA),which provided enhanced skin surface adhesion and enabled simultaneous detection of glucose and H_(2)O_(2)(representative of ROS)in interstitial fluid in vivo.The microarrows fabricated via laser micromachining were modified with functional coating and integrated into a patch of a three-dimensional(3D)microneedle array.Due to the arrow tip mechanically interlocking with the tissue,the microarrow array could better adhere to the skin surface after penetration into skin.The MASA was demonstrated to provide continuous in vivo monitoring of glucose and H_(2)O_(2) concentrations,with the detection of H_(2)O_(2) providing a valuable reference for assessing the inflammation state.Finally,the MASA was integrated into a monitoring system using custom circuitry.This work provides a promising tool for the stable and reliable monitoring of blood glucose in diabetic patients.

Decreased osteogenesis of adult mesenchymal stem cel s by reactive oxygen species under cyclic stretch: a possible mechanism of age related osteoporosis

Age related defect of the osteogenic differentiation of mesenchymal stem cells（MSCs） plays a key role in osteoporosis. Mechanical loading is one of the most important physical stimuli for osteoblast differentiation.Here, we compared the osteogenic potential of MSCs from young and adult rats under three rounds of 2 h of cyclic stretch of 2.5% elongation at 1 Hz on 3 consecutive days. Cyclic stretch induced a significant osteogenic differentiation of MSCs from young rats, while a compromised osteogenesis in MSCs from the adult rats.Accordingly, there were much more reactive oxygen species（ROS） production in adult MSCs under cyclic stretch compared to young MSCs. Moreover, ROS scavenger N-acetylcysteine rescued the osteogenic differentiation of adult MSCs under cyclic stretch. Gene expression analysis revealed that superoxide dismutase 1（SOD1） was significantly downregulated in those MSCs from adult rats. In summary, our data suggest that reduced SOD1 may result in excessive ROS production in adult MSCs under cyclic stretch, and thus manipulation of the MSCs from the adult donors with antioxidant would improve their osteogenic ability.

Autophagy plays a protective role in advanced glycation end products-induced apoptosis of chondrocytes via regulation of tumor necrosis factor-α,nuclear factor-κ B and reactive oxygen species

Objective: To study the adverse effects of advanced glycation end products(AGEs) on chondrocytes and the role of autophagy in this process. Methods: Chondrocytes were harvested from the human articular cartilage tissues in surgery. AGEs were administered during chondrocytes culture. The rapamycin was used to induce autophagy. The cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide(MTT) assay.The expression of tumor necrosis factor-α(TNF-α) and nuclear factor-κ B(NF-κ B) was detected by quantitative real-time polymerase chain reaction. The reactive oxygen species(ROS) production and apoptosis of the chondrocytes were determined by fluorescent probe and flow cytometer, respectively. Results: The chondrocytes viability was significantly reduced after 12 h incubation with AGEs(P<0.01)). In contrast, rapamycin pretreatment increased the chondrocytes viability through autophagy. AGEs increased TNF-α and NF-κ B mRNA expression of chondrocytes and autophagy receded or proceeded the change. AGEs increased intracellular ROS accumulation and autophagy reversed the change. AGEs accelerated chondrocytes apoptosis and autophagy suspended apoptosis. Conclusions: Accumulation of AGEs may have an adverse role for chondrocytes by increasing TNF-α and NF-κB expression, ROS accumulation and apoptosis; meanwhile, autophagy ameliorates the AGEsinduced adverse effects.

Anti- MSP-1_(19) antibody (IgG) and reactive oxygen species (ROS) response against malaria infection in pregnancy in South Western Nigeria

Objective:Although immunity to malaria is reduced in pregnancy,the maternal immune system still continues to respond to malaria infection by the production of antibodies.IgG has been reported to play significant role in immune response against P.falciparum.Anti-MSP-1_(19) antibody and reactive oxygen species have been shown to be protective against malaria infection in children.This work assessed the response of anti-MSP-1_(19) antibody(a promising blood stage vaccine candidate antigen) and oxidative stress in 250 pregnant women.Methods: Blood samples were collected in dry and wet seasons.Plasmodium falciparum infection was determined by microscopy, anti-MSP-1_(19) IgG level was investigated using ELISA.Malondiadelhyde(MDA) and reduced glutathione (GSH) were used as indicators of oxidative stress and they were quantified spectrophotometrically.Results: Parasitaemia was significantly higher(P<0.05) in wet than dry season and its level decreased with gravidity.There was a significant increase(P<0.05) in anti-MSP-1_(19) IgG and MDA levels in the dry than wet season.Anti-MSP-1_(19) IgG and MDA levels were significantly higher in P.falciparum positive primigravidae than P.falciparum negative primigravidae in both wet and dry seasons.In wet season anti-MSP-1_(19) IgG level was significantly increased(P<0.05) in P.falciparum positive multigravidae than P.falciparum negative. The anti-MSP-1_(19) IgG and MDA were significant higher in P.falciparum positive multigravidae than primigravidae. Reduced glutathione(GSH) level was significantly reduced(P<0.05) among malaria positive than malaria negative patients in both seasons.Conclusion:This study suggests that IgG and MDA response were positively associated with the presence of malaria infection.

Polyphenolic extract of Sorghum bicolor grains enhances reactive oxygen species detoxification in N-nitrosodiethylamine-treated rats

Reactive oxygen species detoxification potentials of Sorghum bicolor polyphenolic extract was investigated in the liver of N-nitrosodiethylaminetreated rats.Male rats,weighing(135±5.5)g were completely randomized into 7 groups(A–G)of five rats each.Rats in C,D,E and F were administered orally once daily at 24-h interval for 7 d with 500,125,250 and 500 mg/kg body weight of polyphenolic extract of S.bicolor,respectively.Group G was given 100 mg/kg body weight of vitamin C.On the sixth day,groups B,D,E,F and G were administered with 100 mg/kg body weight N-nitrosodiethylamine(NDEA).Group A,which served as the control was treated like the test groups except,that the animals received distilled water only.Reactive oxygen species detoxifying enzymes(superoxide dismutase,catalase,glutathione peroxidase,glutathione reductase and glucose 6-phosphate dehydrogenase)activities were significantly(P<0.05)induced by S.bicolor.These inductions significantly(P<0.05)attenuated the NDEA-mediated decrease in reactive oxygen species detoxifying enzymes and compared favourably with vitamin C.NDEA-mediated elevation in the concentrations of oxidative stress biomarkers;malondialdehyde,conjugated dienes,lipid hydroperoxides,protein carbonyl and percentage DNA fragmentation were significantly(P<0.05)lowered by S.bicolor polyphenolic extract.Overall,the results obtained from this study revealed that the polyphenolic extract of S.bicolor grains enhanced the detoxification of reactive oxygen species in NDEA-treated rats.The polyphenols also prevented the peroxidation of lipid,oxidation of proteins as well as fragmentation of DNA component in the liver of rats and hence gave the evidence of possible prophylactic potentials of S.bicolor grains.©2013 Beijing Academy of Food Sciences.Production and hosting by Elsevier B.V.All rights reserved.

The production and cytotoxicity of the reactive oxygen species (ROS) induced by diallyl trisulfide (DATS) in human myeloid leukemia HL-60 cells

Objective To explore the production and cytotoxicity of the reactive oxygen species(ROS)induced by diallyl trisulfid(DATS)in HL-60 cells.Methods HL-60 cells were either treated with various doses of DATS alone,or DATS combination with Apocynin,a specific NADPH oxidase inhibitor,or with antioxidant N-acetyl-L-cysteine(NAC)for 0,1,3,6,12 and 24 hours,respectively.The intracellular ROS level was measured by flow cytometry.The activity of NADPH oxidase was evaluated by NBT reduction experiment.The content of both malondialdehyde(MDA)and the protein carbonyl was analyzed by spectrophotometer.Results The results from flow cytometry indicated that DATS significantly increased the intracellular ROS level in HL-60 cells(P<0.05),which is a dose-and time-dependent.The fluorescence intensities of ROS reached at maximuam when HL-60 cells were incubated with 150 μmol·L-1 DATS for 3 hours.The NBT reduction experiment showed that DATS activated NADPH oxidase which had highest activity when cell were exposed to 150 μmol·L-1 DATS for 3 hours.Results DATS induced MDA and protein carbonyl production in HL-60 cells.Furrthermore,both MDA and protein carbonyl in the cells exposed to 150 μmol·L-1 DATS for 3 hours reached the highest level.Apocynin and NAC could attenuate the production of MDA and protein carbonyl,which suggested that ROS induced by DATS was involved in the toxicity to cells.Conclusions DATS induce ROS production through activating NADPH oxidase in HL-60 cells.ROS induced by DATS increase the oxidation of the membrane lipid and the protein of HL-60 cell.

Comparison of Mechanisms of N-Nitrosation and N-Nitration of Ammonia and Dimethylamine by Reactive Nitrogen Oxygen Species:A Theoretical Study

Reactive nitrogen oxygen species（RNOS） implicate damage in biological systems,especially leading to inflammation,neurodegenerative and cardiovascular diseases,and cancer by altering the functions of biomolecules through the N-nitrosation and N-nitration reactions.The mechanisms of N-nitrosation and N-nitration reactions of ammonia and dimethylamine by RNOS,i.e.,N2O3,N2O4,N2O5 and ONOOH,were investigated at the CBS-QB3 level of theory.The computational results indicate that the N-nitrosation reaction prefers a concerted mechanism,in which a H-abstraction and ON-addition occur simultaneously,whereas a stepwise mechanism（also called a free radical mechanism） is more favorable for most nitrating agents in the N-nitration reaction,where NO2 first abstracts a hydrogen atom from the nitrogen of amines and then the induced intermediate reacts with NO2 once more to form the nitration products.However,the concerted pathway is still a feasible process for some nitrating agents such as N2O5.In addition,the relationship between the structures of different RNOS and their nitrosating or nitrating abilities was also investigated.

Novel Zn-doped CuO nanocomposites inhibit tumor growth in vitro and in vivo：role of reactive oxygen species-dependent apoptosis and autophagy cross-linked by NF-kappaB pathway

OBJECTIVE Zn-doped CuO nanocomposites(nZn-CuO NPs) are novel nanoparticles synthesized by sonochemical method.This study aimed to further investigate the antitumor effects and mechanism of nZn-CuO NPs,as well as the exact mechanism of reactive oxygen species(ROS) on nZn-CuO NPs-induced death using N-acetylcysteine(NAC).METHODS The antitumor effects of nZn-CuO NPs were evaluated by MTS assay and orthotopic transplantation tumor model in nude mice.The effects of nZn-CuO NPs with or without NAC on ROS production,DNA damage,apoptosis,mitochondrial damage,autophagy,lysosome impairment,and ER and Golgi stress were determined.Also,western blot was used to detect apoptosis and autophagy related proteins,as well as NF-κB pathway related proteins.RESULTS nZn-CuO NPs significantly inhibit tumor growth both in vitro and in vivo.nZn-CuO NPs were able to cause cytotoxicity,ROS production,DAN damage mitochondrial damage,apoptosis,and autophagy,and NAC can attenuate them.Further studies showed that nZn-CuO NPs induced changes of apoptosis,autophagy and NF-κB pathway related proteins,and NAC can restore them.CONCLUSION Overall,our data demonstrated that nZn-CuO NPs could inhibit tumor growth both in vitro and in vivo by ROS-dependent regulation of apoptosis and autophagy,which might be cross-linked by NF-κB pathways.

Evaluation of Reactive Oxygen Species (ROS) Generated on the Surface of Copper Using Chemiluminesence

The antibacterial activity of copper is well-known from an ancient civilization, however, its biocidal mechanism has not been necessarily elucidated. Notwithstanding up to now, mainly 4 processes have been proposed. Among them, it is cleared that 4 kinds of reactive oxygen species (ROS): hydroxyl radical ·OH, hydrogen per oxide H2O2, superoxide anion ·O-2 and singlet oxygen 1O2, play an important role for contact-killing of bacteria, viruses and fungi. In this paper, generation of ROS on the surfaces of copper plates heated from room temperature to 673 K for 4.2 × 102 s in air, was investigated using the chemiluminescence. ROS have been evaluated by selecting the most suitable scavengers, such as 2-propanol for ·OH, sodium pyruvate for H2O2, nitro blue tetrazolium for ·O-2, and sodium azide NaN3 for 1O2. At the same time the outermost surface of copper, on which thin film of cuprous oxide Cu2O was first formed and then cupric oxide CuO was laminated on Cu2O, was examined by thin-film XRD and TEM analysis to estimate the amounts and kinds of copper oxides. It was found that the most amounts of ROS were obtained for the 573 K-heated Cu plate and they were composed of ·OH, H2O2, and ·O-2..

Effects of Selenium Dioxide on Apoptosis, Bcl-2 and P53 Expression, Intracellular Reactive Oxygen Species and Calcium Level in Three Human Lung Cancer Cell Lines

Objective: To evaluate the anti-tumor effects of SeO2 and its mechanisms on three human lung cancer cell lines. Methods: Three lung cancer cells A549, GLC-82 and PG were treated with 3-30 μmol/L SeO2. Flow cytometry was used to detect apoptosis, and analyze the changes of expression of p53 and Bcl-2, as well as ROS and Ca2+ level within cells. Results:SeO2 markedly inhibited cell proliferation and viability, and prompted apoptosis after 48 h treatment. SeO2 at 10 μmol/L induced 47.8% apoptosis in A549 cells, 40.8% in GLC-82 cells, 18.2% in PG cells. SeO2 at 30 μmol/L induced 37.8% apoposis in PG cells,but did not increase apoptotic raes in other two cells. SeO2 could down-regulate the mean fluorescent intensity of Bcl-2 from 65.8 to 9.6 in A549, but not in GLC-82 and in PG cells, up-regulate wild type p53 level in all three cells. SeO2 decreased the ROS and Ca2+ level markedly within three tested cells. Conclusion: SeO2 showed anti-tumor effect via apoptosis pathway in three lung cancer cell lines. The decrease of ROS and Ca2+ level within cells as well as regulation of Bcl-2 and p53 expression may play important roles in above apoptotic procedure.

Effects of Nitric Oxide on the Germination of Wheat Seeds and Its Reactive Oxygen Species Metabolisms Under Osmotic Stress

Effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on the germination and metabolism of reactive oxygen species were surveyed in wheat (Triticum aestivum L.) seeds. Germination of wheat seeds and even the elongation of radicle and plumule were dramatically promoted by SNP treatments during the germination under osmotic stress. Meanwhile, activities of amylase and EP were enhanced, thus leading to the degradation of storage reserve in seeds. After osmotic stress was removed, higher viability of wheat seeds was also maintained. In addition, the activities of CAT, APX and the content of proline were increased by SNP treatment simultaneously, but activities of LOX were inhibited, and both of which were beneficial for improving the antioxidant capacity during the germination of wheat seeds under osmotic stress. It was also shown that the increase of the activity of amylase induced by SNP in embryoless half-seeds of wheat in the beginning period of germination (6 h) might be indirectly related to GA(3).

The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate,to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry,with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide (PI),indicated that As2O3 (2 μM) could induce apoptosis in NB4 cells prevailingly from G2/M phase,and this efficacy was enhanced upon co-administration of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) (2.5 μM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells' susceptibility to apoptosis by As2O3.

Potential role of reactive oxygen species on testicular pathology associated with infertility

<abstract>Aim: To investigate the level of malondialdehyde (MDA), a direct indicator of lipid peroxidation-induced injury by reactive oxygen species (ROS), in testicular biopsy specimens from infertile patients. Methods: Levels of MDA were measured in testicular biopsy specimens from 29 consequent-randomized infertile men, aged 29.58±4.76 (21-45) years. All patients were evaluated by a complete medical and reproductive history, physical examination, semen analysis (at least two), serum follicle-stimulating hormone and free testosterone levels, testicular biopsy and contact imprint. Scrotal colour Doppler ultrasonography was used to confirm suspected varicocele. The testicular MDA level was measured using the thiobarbituric acid test and the results were expressed per unit tissue weight. Results: As a causal factor in infertility, varicocele was identified in 17 (58.6 %) patients, and idiopathic infertility, testicular failure and obstruction in 4 (13.8 %) patients each. The testicular MDA level was 13.56 (6.01), 49.56 (24.04), 58.53 (48.07), and 32.64 (21.51), 32.72 (13.61), 23.07 (7.82), 42,12 (34.76) pmol/mg tissue in the normal spermatogenesis (control), late maturation arrest, Sertoli cell only (SCO) and hypospermatogenesis (mild, moderete, severe) groups, respectively. The elevation of MDA levels was significant in the testicular tissue from SCO and maturation arrest groups compared with the controls (P<0.05). In addition, the elevation in testicular MDA levels between the SCO and the moderete hypospermatogenesis, and the moderate hypospermatogenesis and the maturation arrest groups was significant (P<0.05). Conclusion: Severe pathologic changes in the testicular tissue are associated with a high level of lipid peroxidation. These findings suggest that overproduction of ROS may play a role in the mechanism of testicular degeneration associated with infertility.

Mitochondrial pathway mediated by reactive oxygen species involvement in α-hederin-induced apoptosis in hepatocellular carcinoma cells

AIM To investigate the antitumor activity of α-hederin in hepatocellular carcinoma(HCC) cells and its underlying mechanisms in vitro and in vivo.METHODS SMMC-7721, Hep G-2 and Huh-7 HCC cells were cultured in vitro and treated with α-hederin(0, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L, 25 μmol/L, 30 μmol/L, 35 μmol/L, 40 μmol/L, 45 μmol/L, 50 μmol/L, 55 μmol/L, or 60 μmol/L) for 12 h, 24 h, or 36 h, and cell viability was then detected by the Cell Counting Kit-8. SMMC-7721cells were treated with 0, 5 μmol/L, 10 μmol/L, or 20 μmol/L α-hederin for 24 h with or without DL-buthionineS,R-sulfoximine(2 mmol/L) or N-acetylcysteine(5 mmol/L) pretreatment for 2 h, and additional assays were subsequently performed. Apoptosis was observed after Hoechst staining. Glutathione(GSH) and adenosine triphosphate(ATP) levels were measured using GSH and ATP Assay Kits. Intracellular reactive oxygen species(ROS) levels were determined by measuring the oxidative conversion of 2',7'-dichlorofluorescin diacetate. Disruption of the mitochondrial membrane potential was evaluated using JC-1 staining. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C were detected by western blotting. The antitumor efficacy of α-hederin in vivo was evaluated in a xenograft tumor model.RESULTS The α-hederin treatment induced apoptosis of HCC cells. The apoptosis rates in the control, low-dose α-hederin(5 μmol/L), mid-dose α-hederin(10 μmol/L) and highdose α-hederin(20 μmol/L) groups were 0.90% ± 0.26%, 12% ± 2.0%, 21% ± 2.1% and 37% ± 3.8%, respectively(P < 0.05). The α-hederin treatment reduced intracellular GSH and ATP levels, induced ROS, disrupted the mitochondrial membrane potential, increased the protein levels of Bax, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C, and decreased Bcl-2 expression. The α-hederin treatment also inhibited xenograft tumor growth in vivo. CONCLUSION The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.

HBx-induced reactive oxygen species activates hepatocellular carcinogenesis via dysregulation of PTEN/Akt pathway

AIM:To investigate the role of hepatitis B virus X-protein(HBx)-induced reactive oxygen species(ROS)on liver carcinogenesis in HBx transgenic mice and HepG2-HBx cells.METHODS:Cell growth rate was analyzed,and through western blotting,mitogenic signaling was observed.Endogenous ROS from wild and HBx transgenic mice and HepG2-Mock and HBx cells were assayed by FACS-calibur.Identification of oxidized and reduced phosphatase and tensin homolog(PTEN)was analyzed through N-ethylmaleimide alkylation,nonreducing electrophoresis.RESULTS:We observed that the cell-proliferation-related phosphoinositide 3-kinase/Akt pathway is activated by HBx in vivo and in vitro.Increased ROS were detected by HBx.Tumor suppressor PTEN,via dephosphorylation of Akt,was oxidized and inactivated by increased ROS.Increased oxidized PTEN activated the mitogenic pathway through over-activated Akt.However,treatment with ROS scavenger N-acetyl cysteine can reverse PTEN to a reduced form.Endogenously produced ROS also stimulated HBx expression.CONCLUSION:HBx induced ROS promoted Akt pathways via oxidized inactive PTEN.HBx and ROS maintained a positive regulatory loop,which aggravated carcinogenesis.