(-)Epigallocatechin gallate and quercetin enhance survival signaling in response to oxidant-induced human endothelial apoptosis

We reported recently that (-)epigallocatechin gallate and quercetin inhibited H2O2-induced apoptosis through modulation of the expression of apoptosis-related Bcl-2 and Bax in endothelial cells. This study attempted to identify possible regulatory sites and mechanisms of antiapoptotic flavonoids, focusing on ROS-mediated signaling in HUVEC. The effects of apigenin on the signaling pathway downstream were compared. Submillimolar H2O2 caused >30% cell killing with intracellular oxidant generation. H2O2-induced oxidant generation markedly decreased total intracellular glutathione (GSH) levels. Micromolar (-)epigallocatechin gallate and quercetin partially eliminated the dichlorodihydrofluorescein (DCF) and phospho-p53 staining, suggesting that these flavonoids inhibited the accumulation of intracellular oxidants and nuclear transactivation of p53 in H2O2-exposed cells. In contrast, cells treated with apigenin remained DCF and phospho-p53 staining positive in response to H2O2. (-)Epigallocatechin gallate significantly raised the total GSH level that had been depleted by H2O2. Caspase-3 activity was enhanced by H2O2, and this increase was inhibited by (-)epigallocatechin gallate and quercetin. Additionally, the upregulation of caspase-3 activation was reversed by these flavonoids at > or =10 micromol/L; these inhibitory effects were dose dependent. Western blot data revealed that H2O2 upregulated phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which was rapidly reversed by quercetin within 30 min; H2O2 activation of c-Jun was downregulated. (-)Epigallocatechin gallate inhibited H2O2-induced phosphorylation of JNK and p38 MAPK after 60 min. These results reveal that quercetin blocks JNK- and p38 MAPK-related signaling triggered by the oxidant and may regulate expression of apoptotic downstream genes, preventing apoptosis and promoting cell survival. (-)Epigallocatechin gallate may function as an antiapoptotic agent through other antiapoptotic pathways.     

Differential inhibition of oxidized LDL-induced apoptosis in human endothelial cells treated with different flavonoids

High plasma level of cholesterol is a well-known risk factor for atherosclerotic diseases. Oxidized LDL induces cellular and nuclear damage that leads to apoptotic cell death. We tested the hypothesis that flavonoids may function as antioxidants with regard to LDL incubated with 5 microm-Cu(2+) alone or in combination with human umbilical vein endothelial cells (HUVEC). Cytotoxicity and formation of thiobarbituric acid-reactive substances induced by Cu(2+)-oxidized LDL were examined in the presence of various subtypes of flavonoid. Flavanols, flavonols and flavanones at a non-toxic dose of 50 microm markedly inhibited LDL oxidation by inhibiting the formation of peroxidative products. In contrast, the flavones luteolin and apigenin had no such effect, with >30 % of cells killed after exposure to 0.1 mg LDL/ml. Protective flavonoids, especially (-)-epigallocatechin gallate, quercetin, rutin and hesperetin, inhibited HUVEC nuclear condensation and fragmentation induced by Cu(2+)-oxidized LDL. In addition, immunochemical staining and Western blot analysis revealed that anti-apoptotic Bcl-2 expression was enhanced following treatment with these protective flavonoids. However, Bax expression and caspase-3 cleavage stimulated by 18 h incubation with oxidized LDL were reduced following treatment with these protective flavonoids. The down-regulation of Bcl-2 and up-regulation of caspase-3 activation were reversed by the cytoprotective flavonoids, (-)-epigallocatechin gallate, quercetin and hesperetin, at >/=10 microm. These results suggest that flavonoids may differentially prevent Cu(2+)-oxidized LDL-induced apoptosis and promote cell survival as potent antioxidants. Survival potentials of certain flavonoids against cytotoxic oxidized LDL appeared to stem from their disparate chemical structure. Furthermore, dietary flavonoids may have therapeutic potential for protecting the endothelium from oxidative stress and oxidized LDL-triggered atherogenesis.     

Effects of heavy metals on mitogen-activated protein kinase pathways

The signaling pathways leading to cellular protection or cell death following exposure to heavy metals have not been fully clarified. Mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK transmit extracellular signals into the nucleus, and have been shown to participate in a diverse array of cellular functions such as cell growth, differentiation and apoptosis. Treatment with cadmium, inorganic mercury or tributyltin can activate ERK, JNK and p38 MAPK, and induces the expression of c-fos and c-jun genes prior to the development of apoptosis. However, the members of the MAPK family appear to be differentially activated depending on the heavy metal and the cell type exposed. Consequently, various cellular responses may be caused by the distinct pattern of MAPKs activation. MAPKs may be one of the important cellular signal transduction pathways affected by various environmental pollutants, including heavy metals.     

Low dose beta-carotene supplementation of ferrets attenuates smoke-induced lung phosphorylation of JNK, p38 MAPK, and p53 proteins

We demonstrated previously that smoke exposure and/or high-dose beta-carotene supplementation decreases levels of retinoic acid and retinoic acid receptor beta (RARbeta) protein, but increase levels of c-Jun and proliferating cellular nuclear antigen protein in the lungs of ferrets. In contrast, low-dose beta-carotene can prevent the decreased lung retinoic acid and the smoke-induced lung lesions. In the present study, we investigated whether smoke exposure and/or beta-carotene supplementation could affect Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and p53 in the lungs of ferrets. Ferrets were subjected to cigarette smoke exposure and either a high or low dose of beta-carotene (2 x 3 factorial design) for 6 mo. There were greater protein levels of phosphorylated JNK, p38, and c-Jun, but lower levels of MAPK phophatase-1 (MKP-1) in groups exposed to smoke and/or high dose beta-carotene. Both phosphorylated-p53 and total p53 were substantially increased in the lungs of these groups. In contrast, low-dose beta-carotene greatly attenuated the smoke-induced phosphorylation of JNK, p38, c-Jun, p53, and total p53, accompanied by upregulated MKP-1. Smoke exposure increased MAPK kinase-4 (MKK4) phosphorylation regardless of beta-carotene supplementation. These data indicate that restoration of retinoic acid and MKP-1 by low-dose beta-carotene in the lungs of ferrets may prevent the smoke-induced activation of the JNK-dependent signaling pathway, p38 MAPK, and the associated phosphorylation of p53, thereby lowering the risk of the smoke-related lung lesions. These data provide supportive evidence that the beneficial vs. detrimental effects of beta-carotene supplementation are related to the dosage of beta-carotene administered.     

c-Jun氨基末端激酶和细胞外调节蛋白激酶信号通路对苯并(a)芘诱导人胚肺成纤维细胞c-Jun活化的调控

目的探讨丝裂原活化蛋白激酶（MAPKs）信号通路在调控苯并（a）芘[B（a）P]诱导人胚肺成纤维细胞（HELF）c—Jun活化中的作用。方法2．0μmol/LB（a）P处理HELF0、3、6、12、24h后或加入不同浓度B（a）P（0．0、0．5、1．0、2．0μmol/L）处理12h后，通过免疫印迹法检测B（a）P对细胞c-Jun活性的影响；利用p38、c—Jun氨基末端激酶（JNK）以及细胞外调节蛋白激酶（ERK）的显性失活突变体分别阻断p38、JNK和ERK活性后，观察3种MAPKs信号分子与B（a）P诱导c-Jun活化之间的关系。结果在所观察的B（a）P作用时间和浓度范围内，c—Jun蛋白表达量无明显变化；B（a）P可诱导细胞内磷酸化c—Jun（Ser63／Ser73）水平增高，并随着作用时间的延长，细胞内c—Jun的磷酸化水平也逐渐增强，至12h达到峰值（1．61±0．12，1．82±0．18），c-Jun磷酸化Ser63、Ser73与actin灰度比值分别是对照组的20．1倍、15．2倍，作用24h时细胞内c-Jun磷酸化水平呈现下降趋势，而且随着B（a）P浓度的增加，细胞内c-Jun的磷酸化水平也逐渐升高，呈现剂量-反应关系；使用显性失活突变体分别阻断JNK和ERK活性均可明显抑制B（a）P诱导细胞c—Jun磷酸化水平增加，但是阻断p38活性对B（a）P诱导细胞c—Jun磷酸化水平升高无明显影响。结论JNK和ERK信号通路调控B（a）P诱导的HELF细胞c—Jun活化，B（a）P促进c-Jun磷酸化的过程与p38信号通路无关。     

Diallyl trisulfide inhibits angiogenic features of human umbilical vein endothelial cells by causing Akt inactivation and down-regulation of VEGF and VEGF-R2

We have shown recently that diallyl trisulfide (DATS), a cancer-chemopreventive constituent of garlic, inactivates Akt to trigger mitochondrial translocation of proapoptotic protein BAD in human prostate cancer cells. Because Akt activation is implicated in the promotion of endothelial cell survival and angiogenesis, we hypothesized that DATS may inhibit angiogenesis. In the present study, we tested this hypothesis using human umbilical vein endothelial cells (HUVECs) as a model. Survival of HUVECs was reduced significantly in the presence of DATS in a concentration-dependent manner, with an IC50 of approximately 4 microM. The DATS-mediated suppression of HUVEC survival was associated with apoptosis induction characterized by accumulation of subdiploid cells, cytoplasmic histone-associated DNA fragmentation, and cleavage of caspase-3 and poly-(ADP-ribose)-polymerase. The DATS-induced DNA fragmentation was significantly attenuated in the presence of pan-caspase inhibitor zVAD-fmk and specific inhibitors of caspase-9 (zLEHD-fmk) and caspase-8 (zIETD-fmk). DATS treatment inhibited the formation of capillary-like tube structure and migration by HUVECs in association with suppression of vascular endothelial growth factor (VEGF) secretion and VEGF receptor-2 protein level and inactivation of Akt kinase. DATS treatment also caused activation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) or p38 mitogen-activated protein kinase (p38MAPK).DATS-mediatedapoptosis induction and inhibition of HUVEC tube formation was partially but statistically significantly attenuated by pharmacologic inhibition of ERK1/2 but not JNK or p38MAPK. The present study demonstrates, for the first time, that DATS has the ability to inhibit angiogenic features of human endothelial cells.     

Neuroprotective effect of cocoa flavonids on in vitro oxidative stress

Background  Cocoa is a rich source of flavonoids that, among other functions, can act as antioxidants. In living systems, the production of reactive oxygen species (ROS) activate an array of intracellular cascades, including mitogen-activated protein kinases (MAPK), that are closely associated with cell death or survival pathways. Aim of the study  To ascertain the role of a cocoa extract and its main flavonoid, (-)-epicatechin, in an in vitro model of oxidative stress induced in a neuronal cell line. Methods  We analyzed ROS production by fluorometry (dichlorofluorescein assay), and activation of MAPK pathways including extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38, by Western blot analysis. Results  Cells incubated with cocoa extract or (-)-epicatechin, reduced ROS production in a dose-dependent manner, reaching 35% inhibition. pJNK and p38, involved in apoptosis, were down-modulated by cocoa extract and (-)-epicatechin with p38 inhibition reaching up to 70%. Conclusions  Our results show that cocoa extract and (-)-epicatechin may exert a neuroprotective action by reducing ROS production and modulating MAPK activation. An erratum to this article can be found under doi:. An erratum to this article can be found at     

Effects of pentachlorophenol and tetrachlorohydroquinone on mitogen-activated protein kinase pathways in Jurkat T cells

When Jurkat human T cells were incubated with 20 microM of pentachlorophenol (PCP) or its metabolite, tetrachlorohydroquinone (TCHQ), for 10 hr, flow cytometric analyses revealed marked increase in the number of apoptotic cells. DNA fragmentation was also observed in these cells. TCHQ was more potent than PCP in causing apoptosis. After incubation with 20 microM TCHQ for 1 hr, all mitogen-activated protein kinases (MAPKs) examined [i.e., extracellular signal-regulated protein kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK)] were phosphorylated, whereas no clear phosphorylation was induced by PCP. TCHQ-induced apoptosis was markedly suppressed by treatment with a p38 inhibitor (SB203580) and mildly (but significantly) suppressed by treatment with a MAPK/ERK kinase inhibitor (U0126). When cells were treated with both inhibitors at the same time, TCHQ-induced apoptosis disappeared almost completely. PCP-induced apoptosis was also suppressed by SB203580 and/or U0126. Nevertheless, treatment with LL-Z1640-2, which inhibits JNK phosphorylation, did not suppress the apoptosis caused by either TCHQ or PCP. Thus, p38 and ERK appear to be important signal transduction pathways leading to apoptosis in a human T-cell line exposed to a ubiquitous pollutant or its metabolite in the general and occupational environment.     

ERK和JNK/活化蛋白-1通路在苯并(a)芘诱导人胚肺成纤维细胞周期改变中的作用

目的探讨丝裂原活化蛋白激酶(MAPK)/转录因子活化蛋白-1(AP-1)信号通路在调控苯并(a)芘[B(a)P]致人胚肺成纤维细胞(HELF)周期改变中的作用。方法用AP-1荧光素酶报告基因技术检测AP-1荧光素酶活力,流式细胞术测定细胞周期时相分布,免疫印迹法检测MAPK[包括细胞外调节蛋白激酶(ERK)、c-Jun氨基末端激酶(JNK)和p38激酶]总量及磷酸化水平,用MAPK显性失活突变体(DN)(DN-ERK2、DN-JNK1和DN-p38)证明通路的上下游关系。结果2μmol/L B(a)P分别处理细胞0、6、122、4 h,AP-1活力在12 h达峰值,是对照组的2.22倍,差异有统计学意义(P<0.05);ERK1/2、JNK1/2和p38蛋白激酶的磷酸化水平明显提高,分别是对照组的2.5、14.0和2.1倍;B(a)P处理组S期细胞比例(50.2%±4.6%)与对照组(16.7%±8.1%)相比明显增加,差异有统计学意义(P<0.01);ERK2和JNK1显性失活突变体的过表达均可明显降低B(a)P诱导的AP-1活力增强,并且明显降低B(a)P处理组S期细胞比例(分别为33.3%±1.7%,30.8%±3.9%),差异均有统计学意义(P<0.05);p38显性失活突变体的过表达对B(a)P引起的AP-1活力增强及S期细胞比例增加无影响。AP-1化学抑制剂姜黄素(20μmol/L)可明显降低B(a)P引起的S期细胞比例增加(13.6%±2.9%),差异均有统计学意义(P<0.05)。结论ERK和JNK通过活化AP-1介导B(a)P诱导的细胞周期改变;而B(a)P诱导的AP-1活力增强及细胞周期改变与p38无关。     

p53介导蜕膜化受损致复发性流产的研究进展

复发性流产（RSA）病因复杂,约50%的RSA患者病因不明,其中由于蜕膜化受损所致的胚胎反复丢失成为学者关注的焦点。p53通过参与多种信号通路介导蜕膜化受损从而引发RSA。其中p53/p21信号通路过度激活可干扰细胞周期调控因子的表达,使细胞周期进入停滞状态,蜕膜多倍体化障碍,转而促进细胞凋亡,影响胚胎着床。而在雌二醇（E2）/p53-白血病抑制因子（LIF）-信号转导和转录激活因子3（STAT3）信号通路中,p53在雌激素的协同作用下促进植入期子宫内膜腺体分泌LIF,并激活JAK/STAT信号通路,为胚胎着床创造有利条件。研究表明,参与调控p53信号通路的长链非编码RNA（lncRNA）功能异常及p53通路基因多态性均可影响p53的活性,从而影响蜕膜化及妊娠结局。现就近年来国内外p53蛋白家族及其通路参与蜕膜化的相关研究进行综述。     

High-power resistance exercise induces MAPK phosphorylation in weightlifting trained men

Power is critical to muscle performance, specifically in athletic populations. Mitogen-activated protein kinase (MAPK) pathways (extracellular signal-regulated protein kinase (ERK 1/2), p38, and c-Jun NH(2)-terminal kinase (JNK)) are intracellular signal transduction mechanisms that partially regulate exercise-induced skeletal muscle alterations. These pathways are highly responsive to exercise, but their reaction to high power, multi-joint resistance exercise is yet to be examined. Nine weightlifting-trained men performed 15 sets of three repetitions of a dynamic clean pull exercise at 85% of their one repetition maximum. Vastus lateralis biopsies were obtained prior to (pre) and after the 8th (mid) and 15th set (post) of exercise. Three subjects returned to serve as non-exercising controls for a similar sequence of biopsies (CON). The ratio of phosphorylated MAPK to total MAPK increased significantly for p38 (3.0 fold, p??0.05), and thus the biopsy procedure itself did not account for the entire increase in MAPK phosphorylation during EX. These data indicate MAPK pathways are activated early and remain elevated throughout the duration of high power resistance exercise. These findings help describe the mechanisms partially responsible for chronic adaptations in response to high intensity, high power resistance training in humans.     

Latex of Euphorbia antiquorum induces apoptosis in human cervical cancer cells via c-jun n-terminal kinase activation and reactive oxygen species production

Latex of Euphorbia antiquorum (EA) has inhibitory effects on several different cancer cell lines. However, the molecular mechanism of EA inhibitory effects on human cervical cancer HeLa cell growth has not been explored. EA induced apoptosis, which was characterized by morphological change, DNA fragmentation, increased sub-G1 population, and alterations in levels of apoptosis-associated proteins. Treatment with EA increased cell death and expression levels of caspase-8, -9, and -3. EA suppressed expression of Bcl-2, increased Bax, and reduced cleavage of Bid and the translocation of tBid to the mitochondria and the release of cytochrome c from mitochondria. EA caused a loss of mitochondrial membrane potential (ΔΨm) and an increase in cellular reactive oxygen species (ROS). EA-induced ROS formation was suppressed by cyclosporine A (an inhibitor of the ΔΨm) or allopurinol (an effective scavenger of ROS). EA also increased expression of Fas, FasL, and c-Jun N-terminal kinase (JNK), p38, and mitogen-activated protein kinase (MAPK) and decreased expression of extracellular signal-regulated kinase (ERK) 1/2-p. Co-treatment with the JNK inhibitor SP600125 inhibited EA-induced apoptosis and the activation of caspase-8, -9, and -3. Results of this study provide support for the hypothesis that EA causes cell death via apoptotic pathways in human cervical adenocarcinoma HeLa cells.     

Latex of Euphorbia Antiquorum Induces Apoptosis in Human Cervical Cancer Cells via c-Jun N-terminal Kinase Activation and Reactive Oxygen Species Production

Latex of Euphorbia antiquorum (EA) has inhibitory effects on several different cancer cell lines. However, the molecular mechanism of EA inhibitory effects on human cervical cancer HeLa cell growth has not been explored. EA induced apoptosis, which was characterized by morphological change, DNA fragmentation, increased sub-G1 population, and alterations in levels of apoptosis-associated proteins. Treatment with EA increased cell death and expression levels of caspase-8, -9, and -3. EA suppressed expression of Bcl-2, increased Bax, and reduced cleavage of Bid and the translocation of tBid to the mitochondria and the release of cytochrome c from mitochondria. EA caused a loss of mitochondrial membrane potential (ΔΨm) and an increase in cellular reactive oxygen species (ROS). EA-induced ROS formation was suppressed by cyclosporine A (an inhibitor of the ΔΨm) or allopurinol (an effective scavenger of ROS). EA also increased expression of Fas, FasL, and c-Jun N-terminal kinase (JNK), p38, and mitogen-activated protein kinase (MAPK) and decreased expression of extracellular signal-regulated kinase (ERK) 1/2-p. Co-treatment with the JNK inhibitor SP600125 inhibited EA-induced apoptosis and the activation of caspase-8, -9, and -3. Results of this study provide support for the hypothesis that EA causes cell death via apoptotic pathways in human cervical adenocarcinoma HeLa cells.     

Sphingosine-1-phosphate inhibition of apoptosis requires mitogen-activated protein kinase phosphatase-1 in mouse fibroblast C3H10T 1/2 cells

The roles of extracellular regulated kinase (ERK) activation and mitogen-activated protein kinase phosphatase-1 (MKP-1) were examined in sphingosine-1-phosphate (S1P)-mediated inhibition of apoptosis in C3H10T 1/2 fibroblast cells. Apoptosis induced by the ceramide analog, C8-ceramine, was inhibited by S1P (ceramine/S1P). Stress activated protein kinase or c-Jun N-terminal kinase (SAPK/JNK) activation was significantly higher after ceramine and ceramine/S1P treatments. Ceramine/S1P treatment also significantly increased ERK activation and MKP-1 protein levels. ERK activation was required for the inhibition of apoptosis by S1P as shown using the mitogen-activated protein kinase kinase inhibitor, PD98059. Transfection with a dominant negative mutant construct of the MKP-1 gene prevented S1P inhibition of apoptosis and resulted in sustained SAPK/JNK activity. The MKP-1 mutant did not affect ERK activity, indicating that MKP-1 preferentially down-regulates SAPK/JNK in C3H10T 1/2 cells. Finally, the S1P activation of ERK and inhibition of apoptosis were reduced by pertussis toxin treatment, suggesting that G-protein-coupled receptors, such as the endothelial differentiation gene (EDG) receptor, play a role. Thus, both ERK activation and MKP-1, which down-regulates SAPK/JNK, are required for S1P-mediated inhibition of apoptosis.     

Role of cell signaling in poxvirus-mediated foreign gene expression in mammalian cells

Poxviruses have been extensively used as a promising vehicle to efficiently deliver a variety of antigens in mammalian hosts to induce immune responses against infectious diseases and cancer. Using recombinant vaccinia virus (VV) and canarypox virus (ALVAC) expressing enhanced green fluorescent protein (EGFP) or multiple HIV-1 gene products, we studied the role of four cellular signaling pathways, the phosphoinositide-3-OH kinase (PI3K), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK), in poxvirus-mediated foreign gene expression in mammalian cells. In nonpermissive infection (human monocytes), activation of PI3K, ERK, p38 MAPK, and JNK was observed in both VV and ALVAC and blocking PI3K, p38 MAKP, and JNK pathways with their specific inhibitors significantly reduced viral and vaccine antigen gene expression. Whereas, blocking the ERK pathway had no significant effect. Among these cellular signaling pathways studied, PI3K was the most critical pathway involved in gene expression by VV- or ALVAC-infected monocytes. The important role of PI3K in poxvirus-mediated gene expression was further confirmed in mouse epidermal cells stably transfected with dominant-negative PI3K mutant, as poxvirus-mediated targeted gene expression was significantly decreased in these cells when compared with their parental cells. Signaling pathway activation influenced gene expression at the mRNA level rather than virus binding. In permissive mammalian cells, however, VV DNA copies were also significantly decreased in the absence of normal function of the PI3K pathway. Poxvirus-triggered activation of PI3K pathway could be completely abolished by atazanavir, a new generation of antiretroviral protease inhibitors (PIs). As a consequence, ALVAC-mediated EGFP or HIV-1 gag gene expression in infected primary human monocytes was significantly reduced in the presence of atazanavir. These findings implicate that antiretroviral therapy (ART), also known as highly active antiretroviral therapy (HAART), may negatively impact the efficacy of live poxvirus vector-based vaccines and should be carefully considered when administering such live vaccines to individuals on ART.     

Red wine polyphenols inhibit the growth of colon carcinoma cells and modulate the activation pattern of mitogen-activated protein kinases

Red wine is a rich source of polyphenols, which exhibit a number of biological effects in different in vitro and in vivo systems. The bioavailability of polyphenols is poor and the plasma concentrations of major red wine polyphenols are usually low after consumption of dietary relevant amounts of red wine. In contrast to most organ systems, the gastrointestinal tract (particularly the epithelial cells of this organ system) is exposed to high concentrations of polyphenols. Here, we show that the total polyphenol pool isolated from a red wine (varity Lemberger, vintage 1998) at micromolar concentrations inhibited the proliferation of transformed colon epithelial cells HT 29 clone 19A induced by epidermal growth factor (EGF). Inhibition of proliferation was also associated with modulation of activation of mitogen-activated protein kinases (MAPK). Stress activated c-Jun N-terminal kinases 1/2 (JNK) and p38 MAPK were significantly activated by red wine polyphenols (6 mmol/L). Maximum phosphorylation of both MAPK was observed after a 1-h treatment with red wine polyphenols. In contrast, activation of extracellular signal regulated kinase (ERK) 1/2 by EGF (1 nmol/L) was significantly inhibited by red wine polyphenols (6 mmol/L). This signaling pattern, activation of JNK 1/2 and p38 MAPK and inhibition of ERK 1/2, is typical for antiproliferative compounds, indicating that red wine polyphenols may inhibit the proliferation of colon carcinoma cells by modulating MAPK intracellular signal transduction pathways.     

尼古丁对PDLFs细胞MAPKs信号通路影响

目的探讨尼古丁诱导人牙周膜成纤维细胞(PDLFs)凋亡过程中丝裂素活化蛋白激酶(MAPK)信号转导通路的作用及作用机制。方法采用不同浓度的尼古丁(1、10和100μg/mL)作用于PDLFs细胞24 h后,使用逆转录聚合酶链反应(RT-PCR)测定JAK 1、JAK 2、MAPKK、MAPK、JNK、p 38和ERK的基因表达水平;Western blot检测caspase-3蛋白的表达水平。结果浓度分别为1、10和100μg/mL的尼古丁作用于PDLFs细胞24 h后,JAK 1转录水平表达量分别为(65.17±8.45)、(106.17±22.22)和(115.50±8.12),JAK 2转录水平表达量分别为(103.00±13.13)、(118.17±13.17)、(159.00±13.74),MAPK转录水平表达量分别为(126.69±18.20)、(143.02±13.97)、(172.64±26.43),MAPKK转录水平表达量分别为(79.17±4.49)、(115.67±7.66)、(122.33±8.41),p 38转录水平表达量分别为(110.64±11.14)、(128.54±11.72)、(132.90±11.99),JNK转录水平表达量分别为(106.16±26.89)、(123.17±13.09)、(132.68±11.97),Caspase 3转录水平表达量分别为(131.52±19.85)、(144.76±13.87)、(153.59±13.85),Caspase 3蛋白翻译水平表达量分别为(128.33±13.50)、(144.00±12.53)、(153.00±12.77),与正常对照组比较,差异均有统计学意义(P<0.05或P<0.01);随着尼古丁剂量的增加,这些基因表达均呈逐渐增高趋势;而ERK的表达水平则无明显变化。结论尼古丁通过介导p 38-MAPK和JNK-MAPK通路诱导人PDLFs细胞凋亡,并导致凋亡执行蛋白Caspase 3的表达增加。     

Epicatechin Gallate Induces Cell Death via p53 Activation and Stimulation of p38 and JNK in Human Colon Cancer SW480 Cells

The tea flavonoid epicatechin gallate (ECG) exhibits a wide range of biological activities. In this study, the in vitro anticancer effects of ECG on SW480 colon cancer cell line was investigated by analyzing the cell cycle, apoptosis, key proteins involved in cellular survival/proliferation, namely AKT/phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs), and the role of p53 in these processes. ECG induced cell cycle arrest at the G0/G1-S phase border associated with the stimulation of p21, p-p53, and p53 and the suppression of cyclins D1 and B1. Exposure of SW480 cells to ECG also led to apoptosis as determined by time-dependent changes in caspase-3 activity, MAPKs [extracellular regulated kinase (ERK), p38, and c-jun amino-terminal kinase (JNK)], p21 and p53 activation, and AKT inhibition. The presence of pifithrin, an inhibitor of p53 function, blocked ECG-induced apoptosis as was manifested by restored cell viability and caspase-3 activity to control values and reestablished the balance among Bcl-2 anti- and proapoptotic protein levels. Interestingly, ECG also inhibited p53 protein and RNA degradation, contributing to the stabilization of p53. In addition, JNK and p38 have been identified as necessary for ECG-induced apoptosis, upon activation by p53. The results suggest that the activation of the p53-p38/JNK cascade is required for ECG-induced cell death in SW480 cells.