Neferine from Nelumbo nucifera induces autophagy through the inhibition of PI3K/Akt/mTOR pathway and ROS hyper generation in A549 cells

Previously we have reported that neferine from the medicinal plant Nelumbo nucifera, inhibited cancer cell proliferation by inducing apoptosis. The present study was focused on the action mechanism of neferine in inducing autophagy in lung cancer cells. Neferine markedly inhibited A549 cell proliferation in a dose dependent manner. Acidic vesicular accumulation was observed in neferine treated cells as an indication of autophagy. Neferine could induce the conversion of LC3B-I to LC3B-II without affecting the expression levels of PI3KCIII and Beclin1. It has been observed that neferine mediated autophagy is dependent on inhibition of PI3K/Akt/mTOR signaling by neferine. Neferine treatment could also lead to the ROS hypergeneration and depletion of cellular antioxidant, GSH. The results demonstrate that neferine-induced autophagy is mediated through ROS hypergeneration and mTOR inhibition. Taken together, the present study unveils a novel mechanism of action of neferine on lung cancer cells in the induction of autophagy.     

Coumestrol induces senescence through protein kinase CKII inhibition-mediated reactive oxygen species production in human breast cancer and colon cancer cells

An inhibitor of the protein kinase CKII (CKII) was purified from leaves of Glycine max (L.) Merrill and was identified as coumestrol by structural analysis. Coumestrol inhibited the phosphotransferase activity of CKII toward β-casein, with an IC₅₀ of about 5 μM. It acted as a competitive inhibitor with respect to ATP as a substrate, with an apparent K_i value of 7.67 μM. Coumestrol at 50 μM resulted in 50% and 30% growth inhibition of human breast cancer MCF-7 and colorectal cancer HCT116 cells, respectively. Coumestrol promoted senescence through the p53-p21^Cip1/WAF1 pathway by inducing reactive oxygen species (ROS) production in MCF-7 and HCT116 cells. The ROS scavenger N-acetyl-l-cysteine (NAC), NADPH oxidase inhibitor apocynin and p22^phox siRNA almost completely abolished this event. Overexpression of CKIIα antagonised cellular senescence mediated by coumestrol, indicating that this compound induced senescence via a CKII-dependent pathway. Since senescence is an important tumour suppression process in vivo, these results suggest that coumestrol can function by inhibiting oncogenic disease, at least in part, through CKII inhibition-mediated cellular senescence.     

Antioxidant behaviour of carotenoids highly accumulated in HepG2 cells

The antioxidant behaviour of major dietary carotenoids accumulated at high concentrations in human hepatoma HepG2 cells was evaluated, in comparison with α-tocopherol. The cells that accumulated carotenoids and α-tocopherol at levels higher than the values reported in the human liver were exposed to mild oxidative stress with tert-butylhydroperoxide. β-Carotene (>2.6 nmol/mg protein) and astaxanthin (>1.8 nmol/mg protein) significantly suppressed lipid peroxidation, while β-cryptoxanthin and lutein did not. α-Tocopherol remarkably suppressed lipid peroxidation with an IC₅₀ value of 0.16 nmol/mg protein. Neither α-tocopherol nor any of the carotenoids except for lycopene showed pro-oxidant action even at high cellular concentrations. The antioxidant behaviours of carotenoids in a cellular milieu were quite different from those previously found in liposomes and homogeneous solutions. Further studies are required to assess the implications of the antioxidant behaviours found in the cultured cells on human health.     

过氧化氢诱导人肝癌细胞产生氧化应激最佳作用浓度的研究

不同浓度过氧化氢作用人肝癌细胞(HepG2)4 h后,用单细胞凝胶电泳技术测定DNA损伤状况,分光光度法测定MDA含量、SOD及GSH-Px活性,研究过氧化氢的最佳作用浓度,构建体外氧化应激细胞模型。实验结果表明,过氧化氢的最佳作用浓度为100μmol/L,作用细胞的时间选择4 h,可以成功构建以过氧化氢为氧化应激诱导剂的HepG2细胞体外氧化应激模型。     

Investigation of the cytotoxicity of food-grade nanoemulsions in Caco-2 cell monolayers and HepG2 cells

Nanoemulsions represent one of the emerging formulations for nutraceutical delivery. However, the possible toxicity associated with the small droplet size (diameter <200 nm) is still unknown. In this study, three nanoemulsions emulsified by modified starch, Tween 20 and whey protein isolate, respectively, were prepared and their cytotoxicity was examined by comparing with the corresponding micron-sized emulsions. Caco-2 cell monolayers were used to mimic the small intestine epithelium. Integrity of the cell membrane and tight junctions was tested by measuring the lactate dehydrogenase leakage and transepithelial electrical resistance, respectively. All three nanoemulsions did not reveal significant difference from their micron-sized counterparts, suggesting no apparent toxicity of the nanoemulsions on the small intestine. Meanwhile, the possible hepatic toxicity was investigated using MTT assay on HepG2 cells. It was found that nanoemulsions made with modified starch and whey protein isolate, but not Tween 20, affected the cell viability/proliferation more than did the micron-sized emulsions. Further in vivo investigation is required to examine the possible hepatic toxicity of nanoemulsions.     

Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway

Ovarian cancer is a significant malignancy for women in the western world, and its death rate has remained unchanged over the past 50 years, leaving room for proper chemoprevention. Kaempferol is a natural flavonoid widely distributed in fruits and vegetables, and epidemiological studies have found a negative correlation between kaempferol consumption and ovarian cancer risk. To understand the mechanism behind this negative correlation, we investigated kaempferol's ability to induce apoptosis in A2780/CP70, A2780/wt, and OVCAR-3 ovarian cancer cell lines. Kaempferol inhibited cell proliferation but did not cause necrosis in all 3 cell lines. For the apoptosis, caspase 3/7 levels were induced in a concentration-dependent manner by kaempferol treatment, with A2780/wt cells being the most responsive. This induction can be diminished by pre-treatment with a caspase-9 inhibitor, indicating an intrinsic apoptosis pathway. Western blot analysis revealed that protein levels of Bcl-x(L) were decreased in ovarian cancer cells, while p53, Bad, and Bax proteins were up-regulated by kaempferol treatment. Our data indicate that kaempferol induces apoptosis in ovarian cancer cells through regulating pro-apoptotic and anti-apoptotic protein expressions in the intrinsic apoptosis pathways, and is a good candidate for the chemoprevention of ovarian cancers in humans. Further studies in animal models and clinical trials are therefore warranted.     

Generation of reactive oxygen species mediated by humic-like substances in atmospheric aerosols

Particulate matter (PM)-mediated reactive oxygen species (ROS) generation has been implicated in health effects posed by PM. Humic-like substances (HULIS) are an unresolved mixture of water-extracted organic compounds from atmospheric aerosol particles or isolated from fog/cloudwater samples. In this study, we use a cell-free dithiothreitol (DTT) assay to measure ROS production mediated by HULIS. The HULIS samples are isolated from aerosols collected at a rural location and a suburban location in the Pearl River Delta, China. In our experiments, ROS activities by residue metal ions in the HULIS fraction are suppressed by including a strong chelating agent in the DTT assay. Under conditions of DTT consumption not exceeding 90%, the HULIS-catalyzed oxidation of DTT follows the zero-order kinetics with respect to DTT concentration, and the rate of DTT oxidation is proportional to the dose of HULIS. The ROS activity of the aerosol HULIS, on a per unit mass basis is 2% of the ROS activity by a reference quinone compound, 1,4-naphthoquinone and exceeds that of two aquatic fulvic acids. The HULIS fraction in the ambient samples tested exhibits comparable ROS activities to the organic solvent extractable fraction, which would contain compounds such as quinones, a known organic compound class capable of catalyzing generation of ROS in cells. HULIS was found to be the major redox active constituent of the water-extractable organic fraction in PM. It is plausible that HULIS contains reversible redox sites, thereby serving as electron carriers to catalyze the formation of ROS. Our work suggests that HULIS could be an active PM component in generating ROS and further work is warranted to characterize its redox properties.     

Induction of apoptosis by Myagropsis myagroides extracts is associated with a caspase dependent pathway and reactive oxygen species generation in human cancer cells

The purpose of this study was to elucidate the mechanisms underlying apoptosis induced by an ethanol extracts from Myagropsis myagroides (ME) in HeLa, U937, and PC-3 cells. ME treatment for 24 h significantly inhibited cell viability in a dose-dependent manner, and induced apoptosis. Moreover, ME treatment triggered the cleavage of caspase-8, ?9, ?3, and poly(ADP-ribose) polymerase (PARP). A general caspase inhibitor (z-VAD-fmk) inhibited ME-induced activation of caspase-3, PARP cleavage, and cell death. ME treatment also triggered the release of cytochrome c from the mitochondria to the cytosol and stimulated the cleavage of Bid, up-regulation of Bax, and down-regulation of Bcl-2. Furthermore, ME treatment caused reactive oxygen species (ROS) generation. An antioxidant N-acetylcysteine (NAC) blocked MEinduced activation of caspase-3, PARP cleavage, and cell death. Overall, these results suggest that ME-induced apoptosis is mediated by a caspase dependent pathway and ROS generation in HeLa, U937, and PC-3 cells.     

白花蛇舌草对HepG2人肝癌细胞Bcl-2和Caspase-3表达的影响

目的：研究白花蛇舌草对HepG2人肝癌细胞Bcl-2和Caspase-3表达的影响。方法：MTT检测白花蛇舌草提取液（EHD）对HeDG2细胞增殖的抑制作用和免疫组化SP法检测Bcl-2和Caspase-3蛋白表达。结果：EHD在一定浓度和时间内能抑制HepG2细胞的生长,并具有剂量和时间依赖性;EHD（160mg／m;_）作用HepG2细胞48h后,Bcl-2的表达水平下降,而CaspaseJ的表达水平升高。结论：EHD（160mg／mL）对HepG2细胞具有抑制增殖和诱导凋亡作用,婪机制可能与相关基因BcL2、 Casnase-3的袁主太调控有美．     

Induction of apoptosis by cinnamaldehyde from indigenous cinnamon Cinnamomum osmophloeum Kaneh through reactive oxygen species production,glutathione depletion,and caspase activation in human leukemia K562 cells

The compositions of essential oils from leaves of two Cinnamomum osmophloeum clones (A and B) commercially cultivated by Taiwan Cinnamon Biotech Co. Ltd., in Taiwan were investigated. GC and GC–MS analyses showed that Cinnamomum osmophloeum clones A and B contain trans-cinnamaldehyde (91.15%) and cinnamyl acetate (46.39%), respectively, as the major component. This study demonstrated that cinnamaldehyde was able to induce apoptosis in a concentration-dependent manner. Cinnamaldehyde-induced cell death was characterized with changes in nuclear morphology, DNA fragmentation, and cell morphology. Furthermore, treatment with cinnamaldehyde caused a rapid loss of mitochondrial transmembrane potential, stimulation of reactive oxygen species (ROS) production, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 and procaspase-3 processing. Taken together, these results suggest that ROS production and depletion of the glutathione that committed to cinnamaldehyde-induced apoptosis in K562 cells.     

The interaction of sesamol with DNA and cytotoxicity,apoptosis, and localization in HepG2 cells

Sesamol, a nutritional antioxidant phenolic compound present in sesame seed, has a potential therapeutic molecule effect against cancers. In this study, the interaction between sesamol and DNA was investigated by employing ultraviolet/visible (UV/Vis), fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), and molecular modeling. The fluorescence analysis indicated that the fluorescence quenching mechanism of sesamol by calf thymus DNA (ctDNA) occurred through static quenching. The UV/Vis, CD, FT-IR spectra and molecular docking results implied that the primary binding mode was minor groove binding. Furthermore, the intracellular interaction of sesamol with DNA and its bioactivity effect were explored. The cell activity results demonstrated that sesamol induced hepatic cell line (HepG2) death. The acridine orange (AO)/ethidium bromide (EB) staining assay and DNA fragmentation confirmed that sesamol could efficiently induce the apoptosis of HepG2 cells. Moreover, addition of sesamol to HepG2 cells resulted in nuclear localization, as visualized by confocal microscopy.     

Antioxidant properties of rare sugar D-allose: Effects on mitochondrial reactive oxygen species production in Neuro2A cells

The anti-oxidative activity of the rare sugar D-allose has recently been reported, but the mechanism is largely unclear. In this study, we evaluated the reactive oxygen species (ROS) scavenging activities of D-allose and then examined the effects of D-allose on ROS production in mitochondria to clarify the antioxidant properties of D-allose. While D-allose did not scavenge hydrogen peroxide and superoxide anions, it eliminated hydroxyl radicals to the same extent as D-glucose. Rotenone, an uncoupler of mitochondrial respiratory complex I, induces ROS production in mouse neuroblastoma Neuro2A cells in the presence of D-glucose. However, in the presence of D-allose, there was no change in the ROS levels in Neuro2A cells following rotenone treatment. Furthermore, treatment with D-allose attenuated the D-glucose-dependent ROS generation induced by rotenone. Whereas treatment with D-glucose enhanced ATP synthesis in Neuro2A cells, D-allose was less effective in producing intracellular ATP than D-glucose. Treatment with D-allose inhibited the ATP synthesis stimulated by D-glucose. These results suggest that D-allose suppresses ROS production in the mitochondria due to competition with D-glucose at the cellular level.     

The potential of extracts of Caryocar villosum pulp to scavenge reactive oxygen and nitrogen species

Caryocar villosum (piquiá) is a native fruit from the Amazonian region, considered to be an interesting source of bioactive compounds. In this paper, five extracts of C. villosum pulp were obtained, using solvents with different polarities and their in vitro scavenging capacity against reactive oxygen species (ROS) and reactive nitrogen species (RNS) was determined. Additionally, the phenolic compounds and carotenoids in each extract were identified and quantified by a high performance liquid chromatography coupled to diode array and mass spectrometer detectors (HPLC-DAD-MS/MS). The ethanol/water and water extracts, which presented the highest phenolic contents (5163 and 1745μg/g extract, respectively), with ellagic acid as the major phenolic compound, proved to have the highest ROS and RNS scavenging potential. Nevertheless, in general, ellagic acid was less effective in scavenging ROS (IC(50) from 1.7 to 108μg/ml) and RNS (IC(50) from 0.05 to 0.59μg/ml), when compared to gallic acid (IC(50) from 0.4 to 226μg/ml for ROS and IC(50) from 0.04 to 0.12μg/ml for RNS). The results obtained in the present study clearly demonstrated that the in vitro antioxidant efficiency of C. villosum extracts was closely related to their contents of phenolic compounds.     

知母水溶性小分子提取物对胰岛素抵抗HepG2细胞体外糖代谢的影响

目的 研究知母水溶性小分子提取物的制备及其对胰岛素抵抗HepG2 (IR-HepG2)细胞糖代谢的作用.方法 采用水提取,膜分离方式制备知母提取物;采用高浓度胰岛素持续作用于HepG2细胞24 h建立胰岛素抵抗模型,加入知母水溶性小分子提取物保温24 h后,测定对照组和加药组细胞内葡萄糖消耗量、肝糖原含量及己糖激酶(hexokinase,HK)、丙酮酸激酶(pyruvate kinase,PK)的活性.结果 知母水溶性小分子提取物明显增加细胞的葡萄糖消耗、提高IR-HepG2细胞HK、PK活性,增加肝糖原含量.结论 知母水溶性小分子提取物可明显改善胰岛素抵抗状态下HepG2细胞对葡萄糖的利用,提高肝HK、PK活性,促进葡萄糖进入肝细胞,增加肝糖原合成,促进糖代谢.     

Antioxidant activity of blueberry anthocyanin extracts and their protective effects against acrylamide‐induced toxicity in HepG2 cells

In this study, the antioxidant activities of blueberry anthocyanin extracts from ten blueberry varieties were evaluated based on the methods of scavenging activities for DPPH radicals, ABTS radicals, hydroxyl radicals and ferric reducing antioxidant power (FRAP) assay. Among the ten blueberry varieties, Polaris had the highest antioxidant abilities and the largest amounts of anthocyanins identified by HPLC‐MS. The protective effects of anthocyanin extracts from Polaris (AEP) against acrylamide (AA)‐induced toxicity in HepG2 cell models were also evaluated due to the neurotoxic, genotoxic and potentially carcinogenic effects of AA. The protective effects of AEP on the damage of HepG2 cells were explored from the aspects of cell viability, T‐SOD and CAT activity and MDA level. The AEP (5, 10, 20 μg mL^?1) could significantly increase cell viability (P < 0.01) and inhibit AA‐induced cytotoxicity. Polaris also markedly promoted the activity of SOD, CAT and inhibited MDA level. The results showed that AEP had strong antioxidant activities, presenting high protective effects against AA‐induced cell damage in HepG2 cell models.     

黄酮对AAPH诱导的HepG2细胞氧化应激的作用

研究部分黄酮对AAPH诱导的HepG2细胞氧化应激的作用。建立AAPH诱导HepG2细胞氧化应激的模型,比较对照组和实验组细胞内的氧化自由基ROS的含量。结果表明,用不同浓度的黄酮处理HepG2细胞1h后,经过相同浓度的氧化剂作用后,细胞内自由基ROS的含量相对空白对照组均发生变化。这些选择性的黄酮对于AAPH诱导的HepG2细胞氧化应激具有促进或者抑制作用,其作用机制可能清除HepG2细胞内ROS以及与细胞内氧化酶和抗氧化酶系统的作用有关。     

Protective effects of protein hydrolysate from marine microalgae Navicula incerta on ethanol-induced toxicity in HepG2/CYP2E1 cells

Ethanol is independently known to cause tissue damage through various mechanisms. This study was designed to evaluate the protective effect of marine microalgae, Navicula incerta protein enzymatic hydrolysates (NEHs) against ethanol-induced hepatotoxicity in HepG2 cells transfected with human CYP2E1. Induction of CYP2E1 by ethanol is one of the central pathways by which ethanol generates a state of oxidative stress in hepatocytes. When the alcohol-induced cells were treated with NEHs at various concentrations, there was a dose-dependent decrease of gamma-glutamyl transpeptidase (GGT) activity in the culture media and loss of cell viability. Among the NEHs constituents the hydrolysates which were obtained by papain (P-NEH), pronase-E (PR-NEH) and α-chymotrypsin (A-NEH) activity attenuated the ethanol cytotoxicity effectively, respectively. The activity appeared to be a GGT inhibitor. Therefore, the cytoprotective effects at alcohol-induced HepG2/CYP2E1 cells could be attributed to the inhibition of GGT activity by NEHs. This study suggests that NEHs have enough potential to be considered as highly active compounds against ethanol toxicity which leads NEHs to be significant nutraceuticals.     

Protective effects of triterpenoids from Ganoderma resinaceum on H2O2-induced toxicity in HepG2 cells

Ganoderma resinaceum Boud. (Polyporeseae) has long been used for antioxidant, immunoregulation and liver protection. From the fruiting bodies of G. resinaceum, eight new lanostanoids, lucidones D–G (1–4), 7-oxo-ganoderic acid Z₂ (5), 7-oxo-ganoderic acid Z₃ (6), ganoderesin A (7), and ganoderesin B (8), together with six known lanostanoids (9–14) were isolated. The structures of new compounds were elucidated through extensive spectroscopic analysis. In an in vitro model, ganoderesin B (8), ganoderol B (10) and lucidone A (11) showed inhibitory effects against the increase of ALT and AST levels in HepG2 cells induced by H₂O₂ compared to a control group in the range of their maximum non-toxic concentration (MNTC). However, compounds 8, 10 and 11 displayed no anti-oxidant activities by DPPH assay. Meanwhile, activation for PXR (Pregnane X Receptor) of ganoderesin B (8), ganoderol B (10) and lucidone A (11) was evaluated; ganoderol (10) exhibited a vital activation for PXR-induced CYP3A4 expression. These results suggested that GTs (Ganoderma triterpenoids) exhibited hepatoprotective activities by lowering ALT and AST levels.     

Cell death induced by serum deprivation in luteal cells involves the intrinsic pathway of apoptosis

The corpus luteum is a transient endocrine gland specializing in the production of progesterone. The regression of the corpus luteum involves an abrupt decline in its capacity for producing progesterone followed by its structural involution, which is associated with apoptosis of the luteal cells. An in vitro experimental approach is needed to study the molecular mechanisms underlying hormonal regulation of luteal cell death under defined experimental conditions. In this study, we investigated simian virus-40-transformed luteal cells to determine whether they can be driven to apoptosis and, if so, to define the intracellular pathway involved. Luteal cells were cultured in the presence or absence of fetal bovine serum for 24 or 48 h. Under serum starvation conditions, the luteal cells underwent growth arrest accompanied by cell death as evaluated by dye exclusion, and confirmed by two-color fluorescence cell viability/cytotoxicity assay. We next studied whether serum starvation-induced death of luteal cells occurred by apoptosis. Morphologic features of apoptosis were observed in cells stained with hematoxylin after being subjected to serum starvation for 48 h. The apoptotic nature was further confirmed by in situ 3'-end labeling and fragmentation of genomic DNA. Apoptosis of serum-deprived luteal cells was dependent upon caspase activation. Serum starvation induced cleavage of poly (ADP-ribose) polymerase (PARP), suggesting that caspase-3 had been activated under the stress of withdrawal of growth factors. This was confirmed by cleavage of full-length procaspase-3. Finally, the fact that serum starvation promoted the cleavage of full-length procaspase-9 and the decrease in the expression of endogenous Bid, a BH-3-only proapoptotic protein of the Bcl-2 family, indicates that the intrinsic (i.e., mitochondrial) pathway of apoptosis was activated. In summary, we have characterized an in vitro experimental model of luteal cell death that can be utilized to evaluate the role of hormones in apoptosis of luteal cells under defined culture conditions, and to study the mechanism of luteal regression.