EGCG对人结肠癌HT-29细胞凋亡的影响以及对MMP-2,RECK的调节作用

目的：观察表没食子儿茶素没食子酸酯（Epigallaocatechin-3-gallate,EGCG）对人结肠癌HT-29细胞增殖和凋亡的影响,并探讨其对MMP-2,RECK的调节作用。方法：体外培养人结肠癌HT-29细胞,MTT比色法检测EGCG对HT-29细胞的生长抑制作用;Histone/DNA ELISA检测细胞凋亡;FITC标记Annexin-V/PI双染流式细胞术分析凋亡细胞百分率;Western Blot和RT-PCR方法检测EGCG对MMP-2,RECK蛋白和mRNA表达的影响。结果：EGCG呈浓度和时间依赖性抑制HT-29细胞的增殖,并且增加HT-29细胞Histone/DNA碎片的渗漏;EGCG诱导HT-29细胞凋亡百分率增高;EGCG抑制MMP-2蛋白和mRNA的表达,促进RECK蛋白和mRNA的表达。结论：EGCG抑制人结肠癌HT-29细胞的增殖,促进其凋亡,并且呈浓度和时间依赖性;其作用机制可能与其下调MMP-2蛋白和mRNA的表达、上调RECK蛋白和mRNA的表达有关。     

Antioxidants modulate the IL-6 induced inhibition of negative acute-phase protein secretion in HepG2 cells

Despite increasing evidence on the potential of dietary antioxidants in modulating the etiology of certain chronic diseases such as cancer and cardiovascular diseases, little is known about their beneficial role in acute-phase responses and inflammatory diseases. From this viewpoint the aim of this study was to investigate the effect of selected dietary antioxidants in modulating the secretion of negative acute-phase proteins caused by interleukin-6 (IL-6) in HepG2 cells. Cells were first stimulated with a fixed dose of IL-6 for 24 h then incubated for a further 8 h with varying concentrations of eight antioxidants, alpha-lipoic acid (LA), (-)-epicatechin (EC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), alpha-tocopherol (TOC), ascorbic acid (AA) and N-acetylcysteine (NAC). The culture supernatants were assayed for transthyretin (TTR) and retinol binding protein (RBP) using ELISA. The data revealed that IL-6 significantly reduced TTR and RBP secretion compared with the basal production. All tested antioxidants attenuate the reduction in TTR and RPB levels. The strongest effects were achieved with the highest concentration of each antioxidant. The order of effect were LA > EGCG > ECG > TOC > EGC > EC > NAC > AA. In conclusion, these results provide evidence that the dietary antioxidants can play a fundamental role in inflammatory processes.     

EGCG 对人结肠癌HT-29 细胞凋亡的影响以及对MMP-2，RECK 的 调节作用

目的:观察表没食子儿茶素没食子酸酯(Epigallaocatechin-3-gallate,EGCG)对人结肠癌HT-29细胞增殖和凋亡的影响,并探讨其对MMP-2,RECK的调节作用。方法:体外培养人结肠癌HT-29细胞,MTT比色法检测EGCG对HT-29细胞的生长抑制作用;Histone/DNA ELISA检测细胞凋亡;FITC标记Annexin-V/PI双染流式细胞术分析凋亡细胞百分率;Western Blot和RT-PCR方法检测EGCG对MMP-2,RECK蛋白和mRNA表达的影响。结果:EGCG呈浓度和时间依赖性抑制HT-29细胞的增殖,并且增加HT-29细胞Histone/DNA碎片的渗漏;EGCG诱导HT-29细胞凋亡百分率增高;EGCG抑制MMP-2蛋白和mRNA的表达,促进RECK蛋白和mRNA的表达。结论:EGCG抑制人结肠癌HT-29细胞的增殖,促进其凋亡,并且呈浓度和时间依赖性;其作用机制可能与其下调MMP-2蛋白和mRNA的表达、上调RECK蛋白和mRNA的表达有关。     

(?)-Epigallocatechin-3-Gallate Induces Non-Apoptotic Cell Death in Human Cancer Cells via ROS-Mediated Lysosomal Membrane Permeabilization

(−)-Epigallocatechin-3-gallate (EGCG) is the most extensive studied tea polyphenol for its anti-cancer function. In this study, we report a novel mechanism of action for EGCG-mediated cell death by identifying the critical role of lysosomal membrane permeabilization (LMP). First, EGCG-induced cell death in human cancer cells (both HepG2 and HeLa) was found to be caspase-independent and accompanied by evident cytosolic vacuolization, only observable when cells were treated in serum-free medium. The cytosolic vacuolization observed in EGCG-treated cells was most probably caused by lysosomal dilation. Interestingly, EGCG was able to disrupt autophagic flux at the degradation stage by impairment of lysosomal function, and EGCG-induced cell death was independent of Atg5 or autophagy. The key finding of this study is that EGCG is able to trigger LMP, as evidenced by Lyso-Tracker Red staining, cathepsin D cytosolic translocation and cytosolic acidification. Consistently, a lysosomotropic agent, chloroquine, effectively rescues the cell death via suppressing LMP-caused cytosolic acidification. Lastly, we found that EGCG promotes production of intracellular ROS upstream of LMP and cell death, as evidenced by increased level of ROS in cells treated with EGCG and the protective effects of antioxidant N-acetylcysteine (NAC) against EGCG-mediated LMP and cell death. Taken together, data from our study reveal a novel mechanism underlying EGCG-induced cell death involving ROS and LMP. Therefore, understanding this lysosome-associated cell death pathway shed new lights on the anti-cancer effects of EGCG.     

Butein blocks tumor necrosis factor alpha-induced interleukin 8 and matrix metalloproteinase 7 production by inhibiting p38 kinase and osteopontin mediated signaling events in HT-29 cells

In this study, we evaluated whether butein can inhibit the effects of tumor necrosis factor alpha (TNF-alpha), an inflammatory mediator, in intestinal epithelial HT-29 cells. Butein significantly inhibited TNF-alpha-induced interleukin 8 (IL-8) secretion and mRNA expression. Moreover, butein suppressed the expression of matrix metalloproteinase 7 (MMP-7) mRNA and extracellular pro-MMP-7 secretion. The signal transduction study revealed that butein significantly attenuates p38 phosphorylation and inhibits osteopontin (OPN) mediated inhibitory factor kappaBalpha (I-kappaBalpha) phosphorylation in TNF-alpha-stimulated HT-29 cells. Using specific kinase inhibitors, we also found that blocking the p38 pathway is critical for, and blocking of OPN-mediated I-kappaBalpha phosphorylation pathway is at least for, the inhibitory effect by butein on TNF-alpha-induced IL-8 and MMP-7 expression. Furthermore, using an MMP inhibitor, we showed that IL-8 lies upstream of MMP-7 in the TNF-alpha-induced signaling process in HT-29 cells. Collectively, these results suggest that butein may be an effective agent for the treatment of intestinal inflammation.     

N-acetylcysteine stimulates osteoblastic differentiation of mouse calvarial cells

Estrogen deficiency causes osteoporosis via increased generation of reactive oxygen species (ROS), and thus, antioxidants may prove to be the effective therapeutic candidates. We examined the effects of the antioxidant N-acetylcysteine (NAC) on osteoblastic differentiation in mouse calvarial cells. NAC (10-30 mM) enhanced alkaline phosphatase activity, mRNA expression of osteoblast differentiation-associated genes and mineralized nodule formation. It also increased expression of bone morphogenetic proteins-2, -4, and -7. The osteogenic activity of NAC was partially reduced by inhibition of glutathione synthesis. Since caffeic acid phenethyl ester did not stimulate osteoblast differentiation, it is unlikely that ROS scavenging activity of NAC is sufficient for osteogenic activity. We observed that NAC suppressed small GTPase RhoA activity and activation of RhoA by Pasteurella multocida toxin suppressed the osteogenic activity of NAC. These results suggest that NAC might exert its osteogenic activity via increased glutathione synthesis and inhibition of RhoA activation.     

Nobiletin, a citrus flavonoid, down-regulates matrix metalloproteinase-7 (matrilysin) expression in HT-29 human colorectal cancer cells

Overexpression of matrix metalloproteinases (MMPs) is associated with cancer metastasis. We assessed mRNA expression of MMPs in six human colorectal cancer cell lines and found a considerable level of MMP-7 expression in HT-29 cells. Next, we searched for natural and synthetic compounds that cause a reduction in the production of proMMP-7 protein, and found that nobiletin (NOB), quercetin, valeryl salicylate, and sulindac sulfone demonstrated marked inhibition. Importantly, NOB attenuated proMMP-7 protein and its mRNA expression both concentration- and time-dependently via a reduction of activator protein-1 (AP-1) DNA binding activity, suggesting it as a promising agent for suppression of cancer cell invasion and metastasis through MMP-7 gene repression.     

激活FXR抑制结肠癌细胞浸润转移及MMP-7的表达

目的:探讨法尼酯X受体(FXR)特异性激动剂GW4064抑制结肠癌细胞浸润转移的机制。方法:在体外培养人结肠癌细胞HT-29,应用GW4064作用于结肠癌细胞,以四唑氮蓝还原法(MTT)检测细胞活性的变化。用transwell小室研究结肠癌细胞的迁移及浸润。用RT-PCR检测FXRm RNA及MMP-7mRNA表达的变化,用western blot检测FXR及MMP-7蛋白表达的变化。结果:MTT结果显示GW4064作用于人结直肠HT-29细胞的生长抑制率呈浓度依赖性;transwell小室结果显示GW4064抑制结肠癌细胞的浸润转移,与对照组相比,差异具有统计学意义(P0.05),RT-PCR及Western blot显示GW4064促进FXR m RNA及蛋白表达,抑制MMP-7mRNA及蛋白的表达,与对照组相比差异有统计学意义(P0.05)。结论:GW4064抑制结肠癌细胞的生长及转移,上调HT-29细胞FXR m RNA及蛋白的表达,下调HT-29细胞MMP-7 m RNA及蛋白的表达。FXR被激活后抑制结肠癌细胞转移,MMP-7可能是其作用通路之一。     

Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells

(-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells.     

Phospholipase C, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astroglioma cells.

We show that epigallocatechin-3 gallate (EGCG), a major component of green tea, stimulates phospholipase D (PLD) activity in U87 human astroglioma cells. EGCG-induced PLD activation was abolished by the phospholipase C (PLC) inhibitor and a lipase inactive PLC-gamma1 mutant, which is dependent on intracellular or extracellular Ca(2+), with the possible involvement of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). EGCG induced translocation of PLC-gamma1 from the cytosol to the membrane and PLC-gamma1 interaction with PLD1. EGCG regulates the activity of PLD by modulating the redox state of the cells, and antioxidants reverse this effect. Moreover, EGCG-induced PLD activation was reduced by PKC inhibitors or down-regulation of PKC. Taken together, these results show that, in human astroglioma cells, EGCG regulates PLD activity via a signaling pathway involving changes in the redox state that stimulates a PLC-gamma1 [Ins(1,4,5)P(3)-Ca(2+)]-CaM kinase II-PLD pathway and a PLC-gamma1 (diacylglycerol)-PKC-PLD pathway.     

Epigallocatechin-3-gallate induces cell apoptosis of human chondrosarcoma cells through apoptosis signal-regulating kinase 1 pathway

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. (-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit tumorigenesis and cancer cell growth in animal models. The aim of this study was to elucidate the mechanism of EGCG-induced apoptosis of human chondrosarcoma cells. EGCG induced cell apoptosis in human chondrosarcoma cell lines but not primary chondrocytes. EGCG induced upregulation of Bax and Bak, downregulation of Bcl-2 and Bcl-XL, and dysfunction of mitochondria in chondrosarcoma. We also found that the accumulation of reactive oxygen species (ROS) is a critical mediator in EGCG-induced cell death. EGCG induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of chondrosarcoma cells with EGCG induced p38 and c-jun-NH2-kinase (JNK) phosphorylation. Transfection with ASK1 siRNA or p38 and JNK mutant antagonized the EGCG-induced cell apoptosis. Therefore, EGCG triggered ROS and activated the ASK1-p38/JNK pathway, resulting chondrosarcoma cell death. Importantly, animal studies revealed a dramatic reduction in tumor volume after 24 days of treatment. Thus, EGCG may be a novel anti-cancer agent for the treatment of chondrosarcoma.     

N-Acetylcysteine enhances the lung cancer inhibitory effect of epigallocatechin-3-gallate and forms a new adduct

The major tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), inhibits carcinogenesis in many in vivo models. Many potential mechanisms of action have been proposed based on cell line studies, including prooxidant activity. In the present study, we studied the effect of N-acetylcysteine (NAC) on the inhibitory effects of EGCG on lung cancer cell growth. We found that NAC (0-2 mM) dose dependently enhanced the growth inhibitory activity of EGCG against murine and human lung cancer cells. The combination of NAC and EGCG caused an 8.8-fold increase in apoptosis in CL13 mouse lung cancer cells compared to treatment with either agent alone. Addition of 2 mM NAC increased the stability of EGCG in the presence of CL13 cells (t 1/2=8.5 h vs 22.7 h). Intracellular levels of EGCG were increased 5.5-fold by the addition of 2 mM NAC. HPLC and LC-MS analyses of cell culture medium from CL13 cells treated with EGCG and NAC for 24 h revealed that EGCG-2'-NAC was time dependently formed. This adduct was not formed in the absence of NAC. The present results show that under cell culture conditions, EGCG and NAC interact to form a previously unreported adduct, EGCG-2'-NAC, which may contribute to enhancement of EGCG-mediated cell killing.     

双歧杆菌脂磷壁酸对结肠癌细胞CD44v6和MMP-2表达的影响

目的研究双歧杆菌脂磷壁酸(LTA)对结肠癌细胞中CD44v6与基质金属蛋白酶2(MMP-2)表达的影响,探讨其在抑制结肠癌转移中的作用。方法结肠癌LoVo细胞及HT-29细胞用含50 mg/L双歧杆菌LTA的培养液培养24 h后,RT-PCR和免疫细胞化学染色检测CD44v6和MMP-2在结肠癌细胞中的表达变化。结果结肠癌LoVo细胞及HT-29细胞中CD44v6和MMP-2的mRNA和蛋白质均呈高表达,经双歧杆菌LTA处理后,其表达均明显下降,与对照组比较,差异有非常显著性(P0.01)。结论双歧杆菌LTA可能通过下调CD44v6和MMP-2的表达来抑制结肠癌的转移。     

Generation of reactive oxygen species and activation of NF-kappaB by non-Abeta component of Alzheimer's disease amyloid

Non-amyloid beta (Abeta) component of Alzheimer's disease (AD) amyloid (NAC) coexists with Abeta protein in senile plaques. After exposure to NAC fibrils, cortical neurons of rat brain primary culture became apoptotic, while astrocytes were activated with extension of their processes. NAC fibrils decreased the activity of reducing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cortical neurons more markedly (IC(50) = 5.6 microm) than in astrocytes (IC(50) approximately 50 microm). The neuron-specific toxicity of NAC fibrils was indicated also by an increased release of lactate dehydrogenase from the cells. Neuronal apoptosis was suppressed by pre-treatment with the antioxidants, propyl gallate (PG) and N-t-butyl-phenylnitrone (BPN), or overexpression of human Bcl-2. Exposure to NAC fibrils enhanced generation of reactive oxygen species (ROS) in neurons and less efficiently in astrocytes, as demonstrated by oxidation of 2',7'-dichlorofluorescin. The site of ROS generation was shown to be mitochondria by oxidation of chloromethyl-tetramethyl rosamine. Exposure to NAC fibrils increased also the nuclear translocation of nuclear factor kappa B (NF-kappaB) and enhanced its DNA-binding activity, which was inhibited by PG and BPN more efficiently in neurons than in astrocytes. These results suggest that NAC fibrils increase mitochondrial ROS generation and activate NF-kappaB, thereby causing a differential change in gene expression between neurons and astrocytes in the AD brain.     

Syndecan-2 Functions as a Docking Receptor for Pro-matrix Metalloproteinase-7 in Human Colon Cancer Cells

Although elevated syndecan-2 expression is known to be crucial for the tumorigenic activity in colon carcinoma cells, how syndecan-2 regulates colon cancer is unclear. In human colon adenocarcinoma tissue samples, we found that both mRNA and protein expression of syndecan-2 were increased, compared with the neighboring normal epithelium, suggesting that syndecan-2 plays functional roles in human colon cancer cells. Consistent with this notion, syndecan-2-overexpressing HT-29 colon adenocarcinoma cells showed enhanced migration/invasion, anchorage-independent growth, and primary tumor formation in nude mice, paralleling their morphological changes into highly tumorigenic cells. In addition, our experiments revealed that syndecan-2 enhanced both expression and secretion of matrix metalloproteinase-7 (MMP-7), directly interacted with pro-MMP-7, and potentiated the enzymatic activity of pro-MMP-7 by activating its processing into the active MMP-7. Collectively, these data strongly suggest that syndecan-2 functions as a docking receptor for pro-MMP-7 in colon cancer cells.     

Epigallocatechin-3-gallate protects toluene diisocyanate-induced airway inflammation in a murine model of asthma

Epigallocatechin-3-gallate (EGCG), a major form of tea catechin, has anti-allergic properties. To elucidate the anti-allergic mechanisms of EGCG, we investigated its regulation of matrix metalloproteinase (MMP-9) expression in toluene diisocyanate (TDI)-inhalation lung tissues as well as TNF-alpha and Th2 cytokine (IL-5) production in BAL fluid. Compared with untreated asthmatic mice those administrated with EGCG had significantly reduced asthmatic reaction. Also, increased reactive oxygen species (ROS) generation by TDI inhalation was diminished by administration of EGCG in BAL fluid. These results suggest that EGCG regulates inflammatory cell migration possibly by suppressing MMP-9 production and ROS generation, and indicate that EGCG may be useful as an adjuvant therapy for bronchial asthma.     

Phospholipase D isozymes mediate epigallocatechin gallate-induced cyclooxygenase-2 expression in astrocyte cells

Little is known about the effect of epigallocatechin-3 gallate (EGCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2 protein and its product, prostaglandin E(2) (PGE(2)). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and PGE synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38 MAPK), and specific inhibition of p38 MAPK dramatically abolished EGCG-induced PLD activation, COX-2 expression, and PGE(2) formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and PGE(2) accumulation. The same pathways as those obtained (2)in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells.