Anti-hepatoma cells function of luteolin through inducing apoptosis and cell cycle arrest

The aim of this study is to explore the apoptotic induction and cell cycle arrest function of luteolin on the liver cancer cells and the related mechanism. The liver cancer cell line SMMC-7721, BEL-7402, and normal liver cells HL-7702 were treated with different concentrations of luteolin. Cell proliferation ability was tested. Morphological changes of the apoptotic cells were observed under inverted fluorescence microscope after Hoechst33342 staining. We investigated the effect of luteolin on cell cycling and apoptosis with flow cytometry. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Caspases-3 and Bcl-2 family proteins expression were analyzed by real-time PCR. Cell proliferation of SMMC-7721 and BEL-7402 were inhibited by luteolin, and the inhibition was dose–time-dependent. Luteolin could arrest the cells at G1/S stage, reduce mitochondrial membrane potential, and induce higher apoptosis rate and the typical apoptotic morphological changes of the liver carcinoma cells. Q-RT-PCR results also showed that luteolin increased Bax and caspase-3 expression significantly and upregulated Bcl-2 expression in a dose-dependent manner in liver carcinoma cells. However, the normal liver cells HL-7702 was almost not affected by luteolin treatment. Luteolin can inhibit SMMC-7721 and BEL-7402 cell proliferation in a time- and dose-dependent manner. And the mechanism maybe through arresting cell cycle at phase G1/S, enhancing Bax level, reducing anti-apoptotic protein Bcl-2 level, resulting in activating caspase-3 enzyme and decrease of mitochondrial membrane potential, and finally leading to cell apoptosis.     

Luteolin Induced-growth Inhibition and Apoptosis of Human Esophageal Squamous Carcinoma Cell Line Eca109 Cells in vitro

Luteolin is a plant flavonoid which exhibits anti-oxidative, anti-inflammatory and anti-tumor effects. However,the antiproliferative potential of luteolin is not fully understood. In this study, we investigated the effect of luteolinon cell cycling and apoptosis in human esophageal squamous carcinoma cell line Eca109 cells. MTT assays showedthat luteolin had obvious cytotoxicity on Eca109 with an IC50 of 70.7±1.72μM at 24h. Luteolin arrested cell cycleprogression in the G0/G1 phase and prevented entry into S phase in a dose- and time-dependent manner. asassessed by FCM. Luteolin induced apoptosis of Eca109 cells was demonstrated by AO/EB staining assay andannexin V-FITC/PI staining. Moreover, luteolin downregulated the expression of cyclin D1, survivin and c-myc,and it also upregulated the expression of p53, in line with the fact that luteolin was able to inhibit Eca109 cellproliferation.     

姜黄素抗血管生成分子机制的探讨

目的：研究姜黄素抑制血管生成的分子机制。方法：采用MTT法、流式细胞术（FCM）观察姜黄素对人脐静脉内皮细胞（HUVECs）的抑制作用及促凋亡作用。采用RT-PCR法及Western-blot法检测姜黄素作用人肺腺癌A2细胞不同时间后血管生成素（Ang-1、Ang-2）、血小板反应素（TSP）mRNA的表达水平和血管内皮生长因子（VEGF）,基质金属蛋白酶（MMP-9）的蛋白表达水平。结果：姜黄素能抑制HUVECs的增殖,呈时间及浓度依赖性,且能诱导HUVECs凋亡,凋亡率明显高于对照组（P〈0.01）。A2细胞经100μmol/L姜黄素作用不同时间后Ang-1、Ang-2、TSPmRNA的表达率明显下降;VEGF与MMP-9的蛋白表达也明显减少。结论：姜黄素可能通过下列途径抑制肿瘤的血管生成：1）直接抑制血管内皮细胞增殖并促进其凋亡;2）抑制血管生成促进因子（VEGF,Ang-1,Ang-2）的表达;3）促进血管生成抑制因子TSP的表达;4）降低基质金属蛋白酶MMP-9的活性从而抑制细胞外基质降解。     

Luteolin Suppresses Teratoma Cell Growth and Induces Cell Apoptosis via Inhibiting Bcl-2

Luteolin, which is found in plant foods, has a range of therapeutic applications. In order to examine the potential roles of luteolin in ovarian teratocarcinoma, the human ovarian teratocarcinoma cell line PA-1 was selected for functional experiments in vitro and in vivo. We demonstrated that luteolin inhibited the proliferation and colony formation of PA-1 cells in vitro. The flow cytometry results suggested that luteolin induced apoptosis of PA-1 cells in a dose-dependent manner. Immunofluorescence and qRT-PCR results showed that the expression of B-cell lymphoma-2 (Bcl-2) was decreased in luteolin-treated cells, whereas the expression of Bcl-2- associated X (Bax) was increased compared with that in the control group. In addition, luteolin inhibited the tumor growth of ovarian teratocarcinoma cells in a xenograft model. All the results suggested that luteolin induced cell apoptosis and inhibited tumor growth of PA-1 cells.     

RNA干扰技术下调CEACAMl基因表达对胶质瘤血管生成的影响

目的：利用RNA干扰技术下调胶质瘤细胞中癌胚抗原相关细胞黏附分子1（ CEACAM1）基因的表达情况,探讨胶质瘤细胞CEACAM1表达变化后其培养上清液诱导人脐静脉内皮细胞（ HUVEC）增殖、血管生成的情况及其可能机制。方法设计并化学合成针对CEACAM1的3对siRNA,脂质体法瞬时转染SHG44细胞,收集细胞培养上清液作为条件培养基。分别采用RT－PCR检测RNA干扰后细胞中CEACAM1及血管内皮生长因子（ VEGF） mRNA表达的变化情况,ELISA法检测上清液中VEGF蛋白的含量。 MTT比色法及Transwell侵袭实验检测共培养刺激后HUVEC增殖及迁移能力的变化,体外血管生成实验检测体外血管生成能力。结果与正常对照组和阴性对照组相比,转染CEACAM1－siRNA后胶质瘤细胞中CEACAM1 mRNA的表达水平下调（P＜0．01）,以CEACAM1－siRNA3组最为显著。 RNA干扰后SHG44细胞VEGF mRNA及上清液中的VEGF蛋白含量均减少,HUVEC的增殖受到抑制,迁移及体外血管生成能力下降。结论 CEACAM1－siRNA能够下调SHG44细胞中CEACAM1 mRNA的表达,并通过减少VEGF分泌,从而影响HUVEC的增殖、迁移和体外血管生成能力。     

Luteolin induces apoptosis via death receptor 5 upregulation in human malignant tumor cells

Luteolin, a naturally occurring flavonoid, induces apoptosis in various cancer cells. Little is known however concerning the underlying molecular mechanisms responsible for this activity. In this report, we reveal a novel mechanism by which luteolin-induced apoptosis occurs, and show for the first time that the apoptosis by luteolin is mediated through death receptor 5 (DR5) upregulation. Luteolin markedly induced the expression of DR5, along with Bcl-2-interacting domain cleavage and the activation of caspase-8, -10, -9 and -3. In addition, suppression of DR5 expression with siRNA efficiently reduced luteolin-induced caspase activation and apoptosis. Human recombinant DR5/Fc also inhibited luteolin-induced apoptosis. On the other hand, luteolin induced neither DR5 protein expression nor apoptosis in normal human peripheral blood mononuclear cells. These results suggest that DR5 induced by luteolin plays a role in luteolin-induced apoptosis, and raises the possibility that treatment with luteolin might be promising as a new therapy against cancer.     

Mechanism of antiproliferative activity of luteolin against stilbene-estrogen stimulation of proliferation of hamster renal epithelial cells

In the present study, we have investigated the influence of a naturally occurring plant flavone luteolin on cell proliferation, nuclear tyrosine kinase activity, and expression of insulin-like growth factor-I (IGF-I) receptor in Syrian hamster renal epithelial cells. Diethylstilbestrol (DES)-induced cell proliferation was inhibited in a dose-dependent manner by cotreatment with luteolin. We also tested the influence of luteolin on nuclear protein tyrosine kinase activity and the expression of IGF-I receptor. The nuclear tyrosine kinase activity was inhibited in a dose-dependent manner by both genistein and luteolin. Luteolin was five times more effective in inhibiting nuclear tyrosine kinase activity than genistein. Data of kinetic constants suggest that luteolin is a noncompetitive inhibitor of nuclear protein tyrosine kinases. Co-treatment of luteolin significantly inhibited DES-induce IGF-I receptor expression. These findings indicate that blocking effects of DES-induced cell proliferation by luteolin may be through the inhibition of nuclear tyrosine kinase activity and of IGF-I receptor expression.     

Luteolin抑制血管生成的机制研究

目的:探讨luteolin对血管的抑制机制。方法:采用不同浓度luteolin处理人微血管内皮细胞,观察luteolin对内皮细胞生长,乳腺癌细胞MDA-MB 231培养液介导的内皮细胞趋化抑制作用。并探讨luteolin对内皮细胞中IL-8信号激活的抑制作用,及luteolin对血管生成抑制作用机制。结果:Luteolin对人微血管内皮细胞细胞增殖抑制作用明显(P<0.01)。Luteolin可抑制乳腺癌细胞MDA-MB 231培养液介导的内皮细胞趋化作用(P<0.01),并明显抑制IL-8对内皮细胞ERK及AKT的激活。结论:Luteolin可抑制人微血管内皮细胞增殖及乳腺癌细胞MDA-MB 231培养液介导的趋化作用,并可抑制IL-8对人微血管内皮细胞的信号激活作用,luteolin抗血管生成作用在预防恶性肿瘤复发及转移中可能有重要的作用。     

Protein kinase C inhibition and x-linked inhibitor of apoptosis protein degradation contribute to the sensitization effect of luteolin on tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an important member of the TNF superfamily with great potential in cancer therapy. Luteolin is a dietary flavonoid commonly found in some medicinal plants. Here we found that pretreatment with a noncytotoxic concentration of luteolin significantly sensitized TRAIL-induced apoptosis in both TRAIL-sensitive (HeLa) and TRAIL-resistant cancer cells (CNE1, HT29, and HepG2). Such sensitization is achieved through enhanced caspase-8 activation and caspase-3 maturation. Further, the protein level of X-linked inhibitor of apoptosis protein (XIAP) was markedly reduced in cells treated with luteolin and TRAIL, and ectopic expression of XIAP protected against cell death induced by luteolin and TRAIL, showing that luteolin sensitizes TRAIL-induced apoptosis through down-regulation of XIAP. In search of the molecular mechanism responsible for XIAP down-regulation, we found that luteolin and TRAIL promoted XIAP ubiquitination and proteasomal degradation. Next, we showed that protein kinase C (PKC) activation prevented cell death induced by luteolin and TRAIL via suppression of XIAP down-regulation. Moreover, luteolin inhibited PKC activity, and bisindolylmaleimide I, a general PKC inhibitor, simulated luteolin in sensitizing TRAIL-induced apoptosis. Taken together, these results present a novel anticancer effect of luteolin and support its potential application in cancer therapy in combination with TRAIL. In addition, our data reveal a new function of PKC in cell death: PKC activation stabilizes XIAP and thus suppresses TRAIL-induced apoptosis.     

Luteolin inhibits proliferation induced by IGF-1 pathway dependent ERα in human breast cancer MCF-7 cells

The growth of many breast tumors is stimulated by IGF-1, which activates signal transduction pathways inducing cell proliferation. ERα is important in this process. The aim of the study was to investigate relationships in vitro among inhibitory effects of luteolin on the growth of MCF-7 cells, IGF-1 pathway and ERα. Our results showed that luteolin could effectively block IGF-1-stimulated MCF-7 cell proliferation in a dose- and time- dependent manner and block cell cycle progression and induce apoptosis evidenced by the flow cytometric detection of sub-G1DNA content. Luteolin markedly decreased IGF-1-dependent IGF-1R and Akt phosphorylation without affecting Erk1/2 phosphorylation. Further experiments pointed out that ERα was directly involved in IGF-1 induced cell growth inhibitory effects of luteolin, which significantly decreased ERα expression. Knockdown of ERα in MCF-7 cells by an ERα-specific siRNA decreased the IGF-1 induced cell growth inhibitory effects of luteolin. ERα is thus a possible target of luteolin. These findings indicate that the inhibitory effect of luteolin on the growth of MCF-7 cells is via inhibiting IGF-1 mediated PI3K-Akt pathway dependent on ERα expression.     

Proteomic identification of anti-cancer proteins in luteolin-treated human hepatoma Huh-7 cells

Luteolin has been shown to exhibit anti-cancer activity against several forms of cancers, including human hepatic cancers. Many in vitro studies have reported anti-oxidant effects of luteolin. Here, we demonstrate using ROS (reactive oxygen species) detection in the human hepatocellular carcinoma cell line, Huh-7, that anti-cancer action of luteolin are mediated through an increasing in intracellular ROS levels. To identify proteins potentially involved in this mechanism, a two-dimensional electrophoresis (2-DE)-based-proteomic approach was employed. Proteomic analysis revealed that several proteins were associated with the anti-cancer effects of luteolin. Interestingly, these proteins included peroxiredoxin 6 (PRDX6) and prohibitin (PHB), which are implicated in ROS metabolism and apoptosis. Western blot analyses confirmed the expression of these proteins in Huh-7 cells following luteolin application. On the basis of these results, we suggest that PRDX6 and PHB are key targets of luteolin that the mechanism of luteolin-induced apoptosis in Huh-7 cells is mediated through effects involving intracellular ROS.     

Potential role for vascular endothelial growth factor‐D as an autocrine factor for human gastric carcinoma cells

Vascular endothelial growth factor (VEGF)‐D induces lymphangiogenesis by activating VEGF receptor (VEGFR)‐3, which is expressed mainly by lymphatic endothelial cells. VEGFR‐3 has also been detected in several types of malignant cells, but the significance of VEGFR‐3 expression by malignant cells remains unclear. We examined the expression and function of VEGF‐D/VEGFR‐3 in human gastric carcinoma cells. Expression of VEGF‐D and VEGFR‐3 was analyzed in three human gastric carcinoma cell lines and 29 surgical specimens. cDNA microarray analysis was used to examine the effect of VEGF‐D on the expression of genes associated with disease progression in VEGFR‐3‐expressing KKLS cells. VEGF‐D‐transfected cells and control cells were transplanted into the gastric wall of nude mice. In 10 of the 29 (34%) gastric carcinoma specimens and two of the three cell lines, cancer cells expressed both VEGF‐D and VEGFR‐3. In vitro treatment of KKLS cells with exogenous VEGF‐D increased expression of cyclin D1 and Bcl‐2 and stimulated cell proliferation. VEGF‐D transfection into KKLS cells resulted in stimulation of angiogenesis, lymphangiogenesis, and cell proliferation, and in inhibition of apoptosis. VEGF‐D may participate in the progression of human gastric carcinoma by acting via autocrine and paracrine mechanisms. (Cancer Sci 2010)     

Luteolin inhibits insulin-like growth factor 1 receptor signaling in prostate cancer cells

Insulin-like growth factor 1 receptor (IGF-1R) activation is required for prostate cell proliferation. Prostate cancer is one of the most commonly diagnosed malignant tumors in Western countries. Overexpression of IGF-1R in prostate cancer is associated with tumor growth. These suggest that IGF-1R inhibitory agents may be of preventive and/or therapeutic value. With evidence accumulating for a chemopreventive role of flavonoids, the effects of luteolin, a bioactive flavonoid, on IGF-1R signaling in prostate cancer cells were examined. Luteolin inhibited insulin-like growth factor 1 (IGF-1) induced activation of IGF-1R and AKT in prostate cancer PC-3 and DU145 cells. Inhibition of AKT by luteolin resulted in decreased phosphorylation of its downstream targets, including p70S6K1, GSK-3beta and FKHR/FKHRL1. Luteolin also inhibited the IGF-1-induced activation of EGFR and MAPK/ERK signaling. Luteolin inhibited expression of cyclin D1 and increased expression of p21. As a result, luteolin suppressed proliferation and induced apoptosis of prostate cancer cells. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of prostate cancer cells. Results of in vivo tumor growth assay indicated that luteolin inhibited PC-3 tumor growth. Immunoblotting of the extracts of tumor tissues showed that luteolin inhibited IGF-1R/AKT signaling. Our results provide a new insight into the mechanisms that luteolin is against cancer cells.     

bFGF和celecoxib对胃癌BGC-823细胞株生长的影响及其机制

目的：探讨bFGF和celecoxib对人胃癌BGC-823细胞增殖及凋亡的作用及对COX-2、NF-κB、VEGF蛋白表达的影响.方法：bFGF和celecoxib作用于BGC-823人胃癌细胞后，MTT比色法测定细胞的增殖，使用流式细胞仪检测细胞凋亡率，荧光显微镜观察细胞凋亡形态，应用Western印迹分析法检测COX-2、NF-κB和VEGF蛋白的表达.结果：25ng/ml bFGF对BGC-823细胞有促增殖作用，能增强COX-2、NF-κB、VEGF蛋白表达，呈时间依赖性（P＜0.01）.不同浓度的celecoxib对BGC-823人胃癌细胞均有增殖抑制作用，并能促进细胞凋亡，使COX-2、NF-κB、VEGF蛋白表达明显减弱，呈浓度依赖性（P＜0.01）.结论：bFGF对BGC-823细胞具有促进增殖和COX-2、NF-κB、VEGF蛋白表达的作用.celecoxib对BGC-823细胞具有促进凋亡和抑制COX-2、NF-κB、VEGF蛋白表达的作用.COX-2、NF-κB、VEGF之间具有相关性（r分别为0.852、0.908、0.930，P＜0.01）.     

Comparative Studies to Evaluate Relative in vitro Potency ofLuteolin in Inducing Cell Cycle Arrest and Apoptosis in HaCaTand A375 Cells

Luteolin is a naturally occurring flavonoid present in many plants with diverse applications in pharmacology.Despite several studies elucidating its significant anti-cancer activity against various cancer cells, the mechanismof action in skin cancer is not well addressed. Hence, we investigated the effects of luteolin in HaCaT (humanimmortalized keratinocytes) and A375 (human melanoma) cells. The radical scavenging abilities of luteolin weredetermined spectrophotometrically, prior to a cytotoxic study (XTT assay). Inhibitory effects were assessedby colony formation assay. Further, the capability of luteolin to induce cell cycle arrest and apoptosis weredemonstrated by flow cytometry and cellular DNA fragmentation ELISA, respectively. The results revealedthat luteolin possesses considerable cytotoxicity against both HaCaT and A375 cells with IC50 values of 37.1μM and 115.1 μM, respectively. Luteolin also inhibited colony formation and induced apoptosis in a dose andtime-dependent manner by disturbing cellular integrity as evident from morphological evaluation by Wright-Giemsa staining. Accumulation of cells in G2/M (0.83-8.14%) phase for HaCaT cells and G0/G1 (60.4-72.6%)phase for A375 cells after 24 h treatment indicated cell cycle arresting potential of this flavonoid. These datasuggest that luteolin inhibits cell proliferation and promotes cell cycle arrest and apoptosis in skin cancer cellswith possible involvement of programmed cell death, providing a substantial basis for it to be developed into apotent chemopreventive template for skin cancer.     

PTEN抑制人脐静脉内皮细胞ECV304的增殖和VEGF的表达

目的:探讨与张力蛋白同源的10号染色体缺失的磷酸酶基因(phosphatase and tensin hemology deleted on chromo-some ten gene,PTEN)对人脐静脉内皮细胞ECV304细胞增殖、凋亡,及血管内皮生长因子(vascular endothelial growth factor,VEGF)及其受体1(VEGF receptor 1,VEGFR1)的影响。方法:将携带有野生型PTEN及绿色荧光蛋白(green fluorescent protein,GFP)基因的腺病毒Ad-PTEN-GFP及空载体腺病毒Ad-GFP感染ECV304细胞,MTT实验、Hoechst3342染色法及流式细胞术检测ECV304细胞的增殖和凋亡。实时荧光定量PCR法检测Ad-PTEN-GFP感染后ECV304细胞中PTEN、VEGF和VEG-FR1 mRNA表达水平,ELISA检测ECV304细胞培养上清中VEGF的水平。鸡胚尿囊膜(chick chorioallantoic membrane,CAM)血管生长实验检测PTEN对鸡胚血管生长的影响。结果:与Ad-GFP相比,Ad-PTEN-GFP感染能明显抑制ECV304细胞的增殖,诱导细胞凋亡,5 d时增殖抑制率可达(50.38±5.42)%、细胞凋亡率达(73.3±5.3)%。当感染复数为100时,Ad-PTEN-GFP组ECV304细胞的VEGF及VEGFR1 mRNA表达水平分别为未感染组的(13.40±1.32)%及(46.12±5.20)%。同时,Ad-PTEN-GFP感染能够明显抑制CAM体内血管生长[血管指数(57.6±3.37)%vs(92.2±4.37)%,P<0.05]。结论:PTEN能显著抑制人脐静脉内皮细胞ECV304的增殖、促进其凋亡,其机制可能与抑制VEGF和VEGFR1表达,抑制血管新生有关。