Role of NADPH oxidase-mediated generation of reactive oxygen species in the mechanism of apoptosis induced by phenolic acids in HepG2 human hepatoma cells

Although plant-derived phenolic acids have been reported to have anti-cancer activity, the exact mechanism is not completely understood. In this study, we investigated the role for reactive oxygen species (ROS) as a mediator of the apoptosis induced by caffeic acid (CA) and ferulic acid (FA), common phenolic acids in plants, in HepG2 human hepatoma cells. CA and FA reduced cell viability, and induced apoptotic cell death in a dose-dependent manner. In addition, they evoked a dose-related elevation of intracellular ROS. Treatment with various inhibitors of NADPH oxidase (diphenylene iodonium, apocynin, neopterine) significantly blunted both the generation of ROS and the induction of apoptosis induced by CA and FA. These results suggest that ROS generated through activation of NADPH oxidase may play an essential role in the apoptosis induced by CA and FA in HepG2 cells. These results further suggest that CA and FA may be valuable for the therapeutic management of human hepatomas.     

The essential oils from Zanthoxylum schinifolium pericarp induce apoptosis of HepG2 human hepatoma cells through increased production of reactive oxygen species

The volatile extract from dried pericarp of Zanthoxylum schinifolium that was obtained by simultaneous distillation with dichloromethane and water was composed of 29.9% geranyl acetate, 15.8% citronella, 15.4% sabinene and the minor volatile components included beta-myrcene, linalool, (-)-isopulegol, citronellyl acetate, 1,4-dimethyl pyrazole, alpha-terpinene, 3-methyl-6-(1-methylethyl)-2-cyclo-hexene-1-o1 and trans-geraniol. The volatile extract decreased the cell viability and induced apoptotic death in HepG2 human hepatoma cells in a concentration- and time-related manner. In addition, the volatile extract increased the production of reactive oxygen species in a dose-dependent manner. Pretreatment of the cells with Trolox, a well-known antioxidant, significantly suppressed the generation of reactive oxygen species and cell death induced by the extract. However, caspase-3 activity was not changed in the extract-treated cells, suggesting that the extract-induced apoptosis of HepG2 cells is caspase-3 independent. Furthermore, in nude mice inoculated with Huh-7 human hepatoma cells, the extract significantly inhibited tumor development. These results suggest that the volatile extract from Zanthoxylum schinifolium pericarpium is a good candidate for hepatocellular carcinoma (HCC) therapy and that reactive oxygen species are the key signaling molecules in the volatile extract-induced cell death in HepG2 cells.     

Gypenosides induce apoptosis in human hepatoma Huh-7 cells through a calcium/reactive oxygen species-dependent mitochondrial pathway

We have previously reported that gypenosides induce apoptosis in human hepatocarcinoma Huh-7 cells through a mitochondria-dependent caspase-9 activation cascade. In order to further explore the critical events leading to apoptosis in gypenosides-treated cells, the following effects of gypenosides on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration of the mitochondrial membrane potential (MPT), and the subcellular distribution of Bcl-2 and Bax. We show that gypenosides-induced apoptosis was accompanied by the generation of intracellular ROS, disruption of MPT, and inactivation of ERK, as well as an increase in mitochondrial Bax and a decrease of mitochondrial Bcl-2 levels. Ectopic expression of Bcl-2 or treatment with furosemide attenuated gypenosides-triggered apoptosis. Treatment with ATA caused a drastic prevention of apoptosis and the gypenosides-mediated mitochondrial Bcl-2 decrease and Bax increase, but failed to inhibit ROS generation and MPT dysfunction. Incubation with antioxidants significantly inhibited gypenosides-mediated ROS generation, ERK inactivation, MPT and apoptosis. Moreover, an increase of the intracellular calcium ion (Ca(2+)) concentration rapidly occurred in gypenosides-treated Huh-7 cells. Buffering of the intracellular Ca(2+) increase with a Ca(2+) chelator BAMTA/AM blocked the gypenosides-elicited ERK inactivation, ROS generation, Bcl-2/Bax redistribution, mitochondrial dysfunction, and apoptosis. Based on these results, we propose that the rise in intracellular Ca(2+) concentration plays a pivotal role in the initiation of gypenosides-triggered apoptotic death.     

A methyl jasmonate derivative,J-7, induces apoptosis in human hepatocarcinoma Hep3B cells in vitro

The pro-apoptotic activity of J-7, a synthetic methyl jasmonate derivative, on the Hep3B human hepatocarcinoma cell line was investigated. Treatment of Hep3B cells with J-7 resulted in growth inhibition and the induction of apoptosis as measured by trypan blue-excluding cells, MTT assay, nuclear staining, DNA fragmentation, and flow cytometry analysis. The increased apoptotic events in Hep3B cells caused by J-7 were associated with the alteration in the ratio of Bax/Bcl-2 protein expression. J-7 treatment induced the expression of death receptor-related proteins such as death receptor 5, which triggered the activation of caspase-8 and the down-regulation of the whole Bid expression. In addition, the apoptosis induction by J-7 was correlated with the activation of caspase-9 and caspase-3, down-regulation IAP family proteins such as XIAP and cIAP-1, and concomitant degradation of poly (ADP-ribose) polymerase. However, the cytotoxic and apoptotic effects induced by J-7 were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. Furthermore, blocking the extracellular signal-regulated protein kinase and c-Jun N-terminal kinase pathways showed increased apoptosis and the activation of caspases in J-7-induced apoptosis. The results indicated that J-7 induces the apoptosis of Hep3B cells through a signaling cascade of death-receptor-mediated extrinsic as well as mitochondria-mediated intrinsic caspase pathways, which are associated with the activation of the mitogen-activated protein kinases signal pathway.     

Role of reactive oxygen species in apoptosis induced by N-ethylmaleimide in HepG2 human hepatoblastoma cells.

We have previously reported that N-ethylmaleimide induces apoptosis through activation of K(+), Cl(-)-cotransport in HepG2 human hepatoblastoma cells. In this study, we investigated the role for reactive oxygen species as a mediator of the apoptosis induced by N-ethylmaleimide. N-ethylmaleimide induced a significant elevation of intracellular level of reactive oxygen species. Treatment with antioxidants (N-acetyl cysteine, N,N'-diphenyl-p-phenylenediamine) which markedly suppressed generation of reactive oxygen species, significantly inhibited the N-ethylmaleimide-induced activation of K(+), Cl(-)-cotransport and apoptosis. Inhibitors of NADPH oxidase (diphenylene iodonium, apocynin, D-(+)-neopterine) also significantly blunted the generation of reactive oxygen species, activation of K(+), Cl(-)-cotransport and apoptosis induced by N-ethylmaleimide. These results suggest that reactive oxygen species generated through activation of NADPH oxidase may play a role in the N-ethylmaleimide-induced stimulation of K(+), Cl(-)-cotransport and apoptosis in HepG2 cells.     

DDTD, an isoflavone derivative, induces cell apoptosis through the reactive oxygen species/apoptosis signal-regulating kinase 1 pathway in human osteosarcoma cells

Osteosarcoma is the most common primary bone tumor associated with childhood and adolescence. In the present study, we investigated the anticancer effect of a new isoflavone derivative, 3',4'-dichloro-3-(3,4-dichlorophenylacetyl)-2,4,6-trihydroxydeoxybenzoin (DDTD) in human osteosarcoma cells. DDTD induced cell apoptosis in human osteosarcoma cell lines (including: U2OS, MG-63, Saos2 and ROS 17/2.8). We found that the accumulation of reactive oxygen species is a critical mediator in DDTD-induced cell death. DDTD induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of osteosarcoma cells with DDTD induced p38 and p53 phosphorylation. Transfection with ASK1, mitogen activated protein kinase (MAPK) kinase (MKK)3/6, and p38 small interfering RNA (siRNA) antagonized the DDTD-induced cell apoptosis. DDTD also triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl2 ratio and Caspase-9 activation. Bax knockdown using a Bax siRNA strategy reduced Bax expression and subsequent cell death. In addition, transfection of cells with ASK1, MKK3/6, and p38 siRNA reduced DDTD-induced p38 activation, p53 phosphorylation and Bax expression. These results suggest that DDTD generates reactive oxygen species and activates the ASK1-MKK3/6-p38-p53-Bax pathway to cause osteosarcoma cell death.     

Berberine-induced apoptosis in human prostate cancer cells is initiated by reactive oxygen species generation

Phytochemicals show promise as potential chemopreventive or chemotherapeutic agents against various cancers. Here we report the chemotherapeutic effects of berberine, a phytochemical, on human prostate cancer cells. The treatment of human prostate cancer cells (PC-3) with berberine induced dose-dependent apoptosis but this effect of berberine was not seen in non-neoplastic human prostate epithelial cells (PWR-1E). Berberine-induced apoptosis was associated with the disruption of the mitochondrial membrane potential, release of apoptogenic molecules (cytochrome c and Smac/DIABLO) from mitochondria and cleavage of caspase-9,-3 and PARP proteins. This effect of berberine on prostate cancer cells was initiated by the generation of reactive oxygen species (ROS) irrespective of their androgen responsiveness, and the generation of ROS was through the increased induction of xanthine oxidase. Treatment of cells with allopurinol, an inhibitor of xanthine oxidase, inhibited berberine-induced oxidative stress in cancer cells. Berberine-induced apoptosis was blocked in the presence of antioxidant, N-acetylcysteine, through the prevention of disruption of mitochondrial membrane potential and subsequently release of cytochrome c and Smac/DIABLO. In conclusion, the present study reveals that the berberine-mediated cell death of human prostate cancer cells is regulated by reactive oxygen species, and therefore suggests that berberine may be considered for further studies as a promising therapeutic candidate for prostate cancer.     

大黄素诱导人肝癌HepG2细胞线粒体凋亡作用研究

目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧（ROS）含量、Ca²⁺浓度、线粒体膜电位（MMP）变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度（IC₅₀）为（77.42±1.25）μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca²⁺浓度明显增加（P<0.05、0.01、0.001）,80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降（P<0.05、0.01、0.001）。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。     

Actinodaphnine induces apoptosis through increased nitric oxide, reactive oxygen species and down-regulation of NF-kappaB signaling in human hepatoma Mahlavu cells.

Actinodaphnine, extracted from Cinnamomum insularimontanum (Lauraceae), possesses cytotoxicity in some cancers, but the mechanism by which actinodaphnine induces apoptosis in human hepatoma cells remains poorly understood. In this study, we investigated the mechanisms of apoptosis induced by actinodaphnine in human hepatoma Mahlavu cells. Treatment with actinodaphnine dose-dependently induced apoptosis in Mahlavu cells that correlated with increased intracellular nitric oxide (NO) and reactive oxygen species (ROS), disruptive mitochondrial transmembrane potential (DeltaPsi(m)), and activation of caspase 3/7. Our data also demonstrated that actinodaphnine down-regulated activity of nuclear factor kappaB (NF-kappaB). The apoptotic response to actinodaphnine was markedly decreased in Mahlavu cells pretreated with dexsamethasone, a NO inhibitor, N-acetylcysteine (NAC), an antioxidant, and Boc-Asp(OMe)-fmk, a broad caspases inhibitor. These results suggested that actinodaphnine-induced apoptosis is initially mediated through the NO and/or ROS increase and caspases-dependent pathway. In conclusion, our results indicate that an increase of ROS and/or NO is the initial essential event that results in the decrease of DeltaPsi(m) and the activation of caspases that commits the cells to the apoptotic pathway in actinodaphnine-treated hepatoma Mahlavu cells.     

Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species

Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect of DOX treatment in cancer patients is the onset of cardiomyopathy and heart failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX cardiotoxicity. Curcumin, a natural compound extracted from Curcuma Longa L., is known for its anti-oxidant properties. It has been identified as increased apoptosis in several cancer cell lines in combination with doxorubicin, but there are few studies about the effect of curcumin and doxorubicin on normal cardiac cells. Therefore, we evaluated the effects of curcumin on apoptosis induced by DOX in cardiac muscle cells. Preteatment with curcumin significantly increased DOX-induced apoptosis of cardiac muscle cells through down regulation of Bcl-2, up-regulation of caspase-8 and caspase-9. The Bax/Bcl-2 ratio increased significantly after 1 h pretreatment with curcumin. As well, curcumin increases ROS generation by DOX. In response to DOX, NF-κB was activated. However, curcumin was able to inhibit NF-κB activation. In conclusion, our results indicated that pretreatment with nontoxic concentrations of curcumin sensitized H9c2 cells to DOX-mediated apoptosis by generation of ROS.     

雷公藤红素通过活性氧诱导结肠癌SW620细胞凋亡

目的:探讨雷公藤红素(celestrol)诱导KRAS驱动的结肠癌SW620细胞产生凋亡的作用和机制。方法:四甲基偶氮唑蓝(MTT)法和台盼蓝拒染法检测细胞增殖;免疫印迹法检测蛋白表达;流式细胞仪和荧光显微镜检测细胞凋亡、细胞周期、线粒体膜电位;荧光显微镜检测细胞内活性氧水平(reactive oxygen species,ROS)。结果:雷公藤红素明显抑制SW620细胞的增殖活性;雷公藤红素下调SW620胞内的p-Akt、NF-κB、Survivin表达,激活caspase-7、caspase-3 和PARP;雷公藤红素增加SW620细胞内的ROS、降低线粒体膜电位、阻滞细胞周期于G₂/M期和诱导凋亡。抗氧化剂N-乙酰半胱氨酸(NAC)抑制雷公藤红素引起的上述作用。结论:通过诱导细胞内ROS的累积导致细胞内线粒体膜电位的下降进而触发细胞发生凋亡是雷公藤红素诱导SW620细胞凋亡的作用机制之一。     

Requirement of reactive oxygen species generation in apoptosis of leukemia cells induced by 2-methoxyestradiol

Aim： To investigate the effects of 2-methoxyestradiol （2-ME） on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms. Methods： Human myeloid leukemia cells HL-60 and U937 were used. Measurement ofmitochondrial membrane potential （Dym） was performed using 5,5′＇,6,6′-Tetrachloro-1, 1′,3,3′- tetraethylbenzimidazolylcarbocyanine iodide （ JC- 1）. Apoptosis and cellular nitric oxide （NO） were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by dihydroethidium （DHE）. Cytotoxicity was analyzed by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl- tetrazolium assay. Results： 2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species （ROS）, including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME. Conclusion： Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application.     

Induction of apoptosis by cordycepin via reactive oxygen species generation in human leukemia cells

Cordycepin (3′-deoxyadenosin), a specific polyadenylation inhibitor, is the main functional component in Cordyceps militaris, one of the top three renowned traditional Chinese medicines. Cordycepin has been shown to possess many pharmacological activities including immunological stimulation, and anti-bacterial, anti-viral, and anti-tumor effects. However, the mechanisms underlying its anti-cancer mechanisms are not yet understood. In this study, the apoptotic effects of cordycepin were investigated in human leukemia cells. Treatment with cordycepin significantly inhibited cell growth in a concentration-dependent manner by inducing apoptosis but not necrosis. This induction was associated with generation of reactive oxygen species (ROS), mitochondrial dysfunction, activation of caspases, and cleavage of poly(ADP-ribose) polymerase protein. However, apoptosis induced by cordycepin was attenuated by caspase inhibitors, indicating an important role for caspases in cordycepin responses. Administration of N-acetyl-l-cysteine, a scavenger of ROS, also significantly inhibited cordycepin-induced apoptosis and activation of caspases. These results support a mechanism whereby cordycepin induces apoptosis of human leukemia cells through a signaling cascade involving a ROS-mediated caspase pathway.     

Small-molecule anthracene-induced cytotoxicity and induction of apoptosis through generation of reactive oxygen species

A series of anthracene derivatives have been synthesized, and their potential individual cytotoxicity was evaluated using Jurkat T cells and peripheral blood mononuclear cells (PBMCs) in vitro. These compounds, except for 2l, showed less cytotoxicity in PBMCs than mitoxantrone. We also analyzed the antiproliferative activity of these derivatives using the annexin V/propidium iodide assay. These synthetic compounds induced apoptosis, thus leading to antitumor effects. Compounds 2b, 2e, 2f, 2g, 2h, 2i, 2j, and mitoxantrone produced dose-dependent cytotoxicity, while the antiproliferative activity of the anthracene pharmacophore was retained in Jurkat T cells base on the detection of DNA degradation and membrane unpacking. These clearly indicate a correlation between cytotoxicity and antitumor activity. Unlike mitoxantrone, cytotoxic properties were observed, as documented by the reactivity of these novel compounds against Jurkat T cells and PBMCs as normal cells, respectively. Various concentrations of 2b, 2e, 2f, 2g, 2h, 2i, and 2j preparations also inhibited Jurkat T cell proliferation and induced apoptosis of Jurkat T cells, potentially confirmed through the detection of DNA degradation and membrane unpacking. In the present report we also investigated the antiinflammatory activity against phorbol-12-myristate-13-acetate induced superoxide anion production, a marker for an inflammatory mediator produced by neutrophils, with IC(50) (microM) values of 2b, 2h, 2l, and 2o of 4.28+/-0.89, 3.31+/-0.88, 4.38+/-0.25, and 5.45+/-1.78, respectively. These results suggest that, in addition to the specific chromosomal aberrations and cell death, elevated apoptosis could also be a marker for exposure to anthracene derivatives.     

Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells

The potential use of low dose chemotherapy has been appealing since lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of Taxol, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic with a capability of activating additional apoptotic signals or inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Esculetin, a well-known lipoxygenase inhibitor, showed an inhibitory effect on the cell cycle progression of HL-60 cells in our previous study. In this report, the effects of a concomitant administration of esculetin and Taxol were investigated in human hepatoma HepG2 cells. Firstly, esculetin alone could exert an antiproliferation effect together with an inhibitory effect on the activation of ERKs and p38 MAPK. As compared to the treatment with Taxol only, a co-administration with esculetin and Taxol could result in a further enhancement of apoptosis as revealed by DNA fragmentation assay and Annexin-V-based assay. Meanwhile, immunoblotting analysis also showed that the co-administration of esculetin and Taxol could increase the expression of Bax and the cytosolic release of cytochrome C and enhance the expression of Fas and Fas ligand while the activation of caspase-8 and caspase-3 was also increased. Finally, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis.     

Induction of apoptosis and autophagy by sodium selenite in A549 human lung carcinoma cells through generation of reactive oxygen species

Selenium in the form of sodium selenite has been reported to exert anti-tumor effects in several cancer cell types by inducing autophagic cell death and apoptosis mediated by reactive oxygen species (ROS). However, the exact molecular pathways underlying these effects have not been fully established. The present study used A549 human lung carcinoma cells for further investigation of the anti-cancer mechanism of sodium selenite. We showed that sodium selenite modulated both the extrinsic and intrinsic apoptotic pathways, which were interconnected by Bid truncation. We used z-VAD-fmk, a pan-caspase inhibitor, to demonstrate that sodium selenite-induced apoptosis was dependent on the activation of caspases. Sodium selenite also increased autophagy, as indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and elevation of autophagic flux. Pretreatment with bafilomycin A1 enhanced sodium selenite-induced apoptosis, indicating that sodium selenite-induced autophagy functioned as a survival mechanism. Sodium selenite treatment also resulted in generation of ROS, which abrogated mitochondrial membrane potential (MMP) and regulated both apoptosis and autophagy. Phospho-nuclear factor erythroid 2-related factor 2 (p-Nrf2) showed a ROS-dependent translocation to the nucleus, which suggested that Nrf2 might increase cell survival by suppressing ROS accumulation and apoptosis mediated by oxidative stress. Sodium selenite treatment of A549 cells therefore appeared to trigger both apoptosis and cytoprotective autophagy, which were both mediated by ROS. The data suggest that regulation of ROS generation and autophagy can be a potential strategy for treating lung cancer that is resistant to pro-apoptotic therapeutics.     

Apoptosis induction by silica nanoparticles mediated through reactive oxygen species in human liver cell line HepG2

Silica nanoparticles are increasingly utilized in various applications including agriculture and medicine. In vivo studies have shown that liver is one of the primary target organ of silica nanoparticles. However, possible mechanisms of hepatotoxicity caused by silica nanoparticles still remain unclear. In this study, we explored the reactive oxygen species (ROS) mediated apoptosis induced by well-characterized 14 nm silica nanoparticles in human liver cell line HepG2. Silica nanoparticles (25-200 μg/ml) induced a dose-dependent cytotoxicity in HepG2 cells. Silica nanoparticles were also found to induce oxidative stress in dose-dependent manner indicated by induction of ROS and lipid peroxidation and depletion of glutathione (GSH). Quantitative real-time PCR and immunoblotting results showed that both the mRNA and protein expressions of cell cycle checkpoint gene p53 and apoptotic genes (bax and caspase-3) were up-regulated while the anti-apoptotic gene bcl-2 was down-regulated in silica nanoparticles treated cells. Moreover, co-treatment of ROS scavenger vitamin C significantly attenuated the modulation of apoptotic markers along with the preservation of cell viability caused by silica nanoparticles. Our data demonstrated that silica nanoparticles induced apoptosis in human liver cells, which is ROS mediated and regulated through p53, bax/bcl-2 and caspase pathways. This study suggests that toxicity mechanisms of silica nanoparticles should be further investigated at in vivo level.     

Hyperglycemia and reactive oxygen species mediate apoptosis in aortic endothelial cells through Janus kinase 2

The generation of reactive oxygen species (ROS) has been implicated in the perturbation of endothelial function and cell death. However, the specific signaling pathways which mediate and modifying this response have not been fully elucidated. Therefore, in this study we tested the hypothesis that activation of JAK2 is involved in the aortic endothelial cell (EC) response to ROS. When ECs were exposed to HG (25 mM) for 6 h or ROS (i.e., H(2)O(2) (100 microM)) for 1 h and returned to normal medium we found a decrease in cell density and morphologic signs of apoptosis. Furthermore, incubation of ECs with HG and H(2)O(2) also resulted in the tyrosine phosphorylation of JAK2. In addition, pretreatment of ECs with AG-490, an inhibitor of JAK2, prevented nuclear fragmentation, whereas inhibitors of Jun kinase (SP 600125), MAP kinase (PD 98059), Src kinase (PP2) or PI-3 kinase (wortmannin) were without effect. Finally, immunoblot analysis of caspase-3 and PARP cleavage confirmed a role for activation of JAK2 in both HG- or ROS-induced apoptosis, based on inhibition by either AG-490 or adenoviral transfection with a dominant-negative JAK2 mutant. In conclusion the activation of JAK2 plays a pivotal role in oxidant stress-induced commitment of ECs to apoptosis, based on studies with HG and H(2)O(2).     

Mechanism of apoptosis induced by apigenin in hepg2 human hepatoma cells: involvement of reactive oxygen species generated by NADPH oxidase

Although plant-derived flavonoids have been reported to have anti-cancer activities, the exact mechanism of these actions is not completely understood. In this study we investigated the role for reactive oxygen species (ROS) as a mediator of the apoptosis induced by apigenin, a widespread flavonoid in plant, in HepG2 human hepatoma cells. Apigenin reduced cell viability, and induced apoptotic cell death in a dose-dependent manner. In addition, it evoked a dose-related elevation of intracellular ROS level. Treatment with various inhibitors of the NADPH oxidase (diphenylene iodonium, apocynin, neopterine) significantly blunted both the generation of ROS and induction of apoptosis induced by apigenin. These results suggest that ROS generated through the activation of the NADPH oxidase may play an essential role in the apoptosis induced by apigenin in HepG2 cells. These results further suggest that apigenin may be valuable for the therapeutic management of human hepatomas.     

Apoptosis induced by a new flavonoid in human hepatoma HepG2 cells involves reactive oxygen species-mediated mitochondrial dysfunction and MAPK activation

Earlier reports suggest that protoapigenone showed remarkable anticancer activities. In the present study, the cytotoxic effect of a new flavonoid, 2-(cis-1, 2-dihydroxy 4-oxo-cyclohex-5-enyl)-5, 7-dihydroxy-chromone (DEDC), which is a protoapigenone analog, was investigated in human hepatoma HepG2 cells. We found that hepatoma cells were highly susceptible to DEDC in contrast with normal cells. The sustainable and rapid generation of reactive oxygen species was observed in DEDC-induced cell death. Following oxidative stress, DEDC sequentially decreased mitochondrial membrane potential (ΔΨm), reduced Bcl-2 expression, increased cytochrome c release, and activated caspase-3, -8, and -9. Phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK) was stimulated by treatment with DEDC. To further investigate the mechanisms of the DEDC-induced cell death, we examined the effects of reactive oxygen species scavenger N-acetyl-L-cysteine (NAC) and selective inhibitors for MAPK pathways on the cell death. The DEDC-induced cell death was significantly inhibited by both NAC and JNK inhibitor SP600125, but promoted by p38 MAPK inhibitor, SB203580. Together, DEDC may have antitumor effects in HepG2 cells through reactive oxygen species production as well as activation of MAPK signaling pathways.