叶黄素和玉米黄素抑制口腔上皮细胞癌增殖的实验研究

为阐明玉米蛋白粉类胡萝卜素提取物叶黄素和玉米黄素对人口腔上皮癌细胞株KB凋亡的诱导作用,探讨其分子生物学机制。本文采用单细胞凝胶电泳、DNA凝胶电泳、流式细胞术检测叶黄素和玉米黄素诱导口腔癌细胞凋亡的情况。结果发现:单细胞凝胶电泳显示玉米蛋白粉叶黄素和玉米黄素能造成KB细胞DNA损伤、DNA凝胶电泳显示能使KB细胞DNA发生明显的降解,DNA周期分析主要阻断口腔癌细胞由G1向S期转变;流式细胞仪检测明显抑制人口腔癌细胞株KB中bcl-2基因的蛋白表达,明显升高p53、Bax基因的蛋白表达;说明玉米蛋白粉类胡萝卜素提取物叶黄素和玉米黄素具有明显抑制口腔上皮癌细胞增殖、并诱导其凋亡的作用。     

藻蓝蛋白对非小细胞肺癌H1299生长、迁移和凋亡的功能影响

藻蓝蛋白(C-phycocyanin,C-PC)是一种从顿顶螺旋藻中提取的天然水溶性无毒的活性蛋白。采用MTT法、克隆形成实验、流式细胞术、划痕实验等探究不同浓度的藻蓝蛋白对人类非小细胞肺癌H1299细胞活力、凋亡、迁移等的影响。结果表明:藻蓝蛋白能显著(p<0.05)降低H1299细胞存活率,通过下调周期蛋白cyclinA、CDK2,上调周期抑制因子P21使细胞周期阻滞在S期;藻蓝蛋白处理使细胞形态发生改变,同时通过调控bad、bax、bcl-xL、bcl-2等凋亡相关基因的表达促进细胞凋亡;体外划痕实验表明藻蓝蛋白通过调控基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)和基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)抑制细胞迁移。研究结果表明藻蓝蛋白能够通过细胞周期S期阻滞,调控细胞周期、凋亡和迁移相关基因的表达而抑制非小细胞肺癌H1299细胞的生长和迁移,进而诱导细胞凋亡,为进一步开发功能食品奠定坚实的理论基础。     

三羟异黄酮对人乳腺癌细胞株MCF-7增殖的影响机制

研究了三羟异黄酮(geinstein，Gen)对乳腺癌细胞株MCF-7增殖的影响机制。结果发现Gen对MCF-7细胞生长有显著抑制作用，使细胞生长阻滞于G2／M期，并使cyclin B蛋白表达增加，且呈剂量．效应关系；对cyclin E蛋白表达无影响。Gen降低了bcl-2蛋白的表达，促进了bax蛋白的表达并且降低了bcl-2／bax比值。     

川皮苷体外抗氧化活性及对肿瘤细胞生长抑制作用

目的：研究川皮苷的抗氧化活性及对肿瘤细胞生长抑制作用。方法：在体外测定川皮苷对1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、羟自由基（•OH）和超氧阴离子自由基（O2－•）的清除能力;通过四甲基偶氮唑蓝（methyl thiazolyl tetrazolium,MTT）法筛选对川皮苷作用敏感的肿瘤细胞株,并用倒置显微镜和透射电镜观察敏感细胞株在川皮苷作用之后细胞结构的变化。结果：川皮苷在体外对DPPH自由基、•OH和O2－•都具有清除能力,其中对•OH的清除能力突出。同时,人胰腺癌细胞株PANC-1对川皮苷作用敏感,即川皮苷可抑制PANC-1细胞的生长;透射电镜观察发现川皮苷可使PANC-1细胞的细胞核膜破裂,胞质溶出,形成凋亡小体。结论：川皮苷具有显著的体外抗氧化活性,并且可以抑制PANC-1细胞的增殖,在功能食品等领域具有良好的应用前景。     

Functional expression of bcl-2 protein family and AIF in bovine mammary tissue in early lactation

Cell proliferation and apoptosis were measured in bovine mammary tissue around the time of peak milk production in three heifers, and compared with changes in the expression of bcl-2-related intracellular signals and apoptosis-inducing factor (AIF). Cell proliferation and apoptosis were relatively constant during the study period with no significant change in the incidence of either event. However, the ratio of apoptotic to proliferating cells tended to change from 0.99 on day 45 of lactation to 1.82 on day 63 (P=0.064), suggesting that in the course of the study, a dynamic balance may have been succeeded by net cell loss. Average milk production recorded 6 d after biopsy was correlated with the estimated number of cells (r=0.898; P<0.01) but not with the apoptosis to proliferation ratio (r=-0.224, P>0.05). Turnover of the cell population was associated with relatively constant expression of anti-apoptotic bcl-2. Competitive PCR also indicated expression of bax, in contrast to observations in lactating rodent mammary tissue. Bax expression was relatively low compared with that of bcl-2, but immunohistochemical staining for bax protein, which was not detectable on day 45, was observed on day 53 and, more intensely, on day 60 when the protein appeared to be membrane-associated. A partial coding sequence for bovine AIF was identified and AIF expression was evaluated by in situ hybridization. The results indicated that AIF was expressed in luminal alveolar cells and that, in concert with a change in bax to bcl-2 ratio, they might contribute to signalling of a change in the dynamic balance of the cell population as lactation progresses.     

Bcl-xl和p53基因在条斑紫菜多糖抑制胃癌BGC-823细胞增殖中的作用

目的:探讨Bcl-xl、p53基因在条斑紫菜多糖抑制BGC-823细胞增殖和诱导凋亡中的作用。方法:将不同浓度条斑紫菜多糖作用于BGC-823细胞,利用MTT法检测细胞增殖抑制率,RT-PCR法检测Bcl-xl、p53基因表达量的变化、流式细胞仪检测细胞周期和细胞凋亡情况。结果:条斑紫菜多糖能抑制BGC-823细胞增殖及诱导细胞凋亡,并上调p53和降低Bcl-xl表达。结论:条斑紫菜多糖抑制人胃癌细胞增殖、促进凋亡,其机制可能与激活p53、抑制Bcl-xl表达有关。     

槐花醇提取物促进肝癌大鼠的细胞凋亡

探究槐花醇提取物对肝癌大鼠细胞凋亡的干预效果及作用机制。选取50只SD大鼠,10只为正常组,其余40只建立有肝癌(分为模型组、药物对照组、低、高浓度干预组)。观察各组大鼠细胞周期分布及增殖、凋亡情况,并检测各组大鼠PTEN、p-Akt、m TOR及Bcl-2、PI3K、Akt表达。研究结果显示,高浓度组大鼠癌组织处于G1期细胞比例、凋亡率、PTEN相对表达量分别为54.72%、45.34%、0.73,均高于模型组、药物对照组、低浓度组(p<0.05);增殖率、p-Akt、m TOR、Bcl-2、PI3K、Akt相对表达量分别为13.33%、1.33、1.34、1.31、1.32、1.35,均低于模型组、药物对照组、低浓度组(p<0.05)。在槐花醇提取物的干预下,肝癌大鼠癌组织细胞增殖、凋亡能力及周期分布受到明显调控,PTEN、p-Akt、m TOR及Bcl-2、PI3K、Akt表达受到明显的调控,说明槐花醇提取物能够阻滞肝癌组织细胞周期分布,抑制肝组织细胞增殖,促进肝癌组织细胞凋亡,其能力呈现浓度依赖,为肝癌症状的临床治疗提供一定参考价值。     

灵芝总皂苷对结肠癌HCT116细胞的抑制作用

为探究灵芝总皂苷提取物对结肠癌HCT116细胞的抑制作用和相关机制，以灵芝子实体为原料，醇提并纯化总皂苷，通过CCK8法测定灵芝总皂苷提取物对结肠癌HCT116细胞的抑制效果，同时采用流式细胞术检测细胞凋亡，荧光分光光度计检测细胞内线粒体膜电位及细胞内活性氧含量，实时荧光定量PCR检测凋亡相关基因p53、noax、bax、bcl-2、caspase-9和caspase-3的表达。结果显示，与空白组相比，灵芝总皂苷质量浓度为40 mg/L以上时，可极显著地抑制HCT116细胞增殖，且抑制效果与时间呈正相关。经灵芝总皂苷干预后，细胞凋亡率显著增加，当加入灵芝总皂苷的质量浓度为150 mg/L时，其凋亡率达到50.2%，且灵芝总皂苷可显著降低细胞内线粒体膜电位，增加细胞内活性氧含量，增加促凋亡基因p53、noax、bax、caspase-9和caspase-3的表达，降低抗凋亡基因bcl-2的表达。综上，灵芝总皂苷对于结肠癌细胞具有显著抑制作用，且其抑制效果可通过线粒体凋亡途径实现，是值得进一步研究的抑结肠癌候选药物和食品添加剂。     

Effect of selected phytochemicals on cell proliferation in A549 lung cancer cells

Effects of quercetin 3-β-d-glucoside, resveratrol, and curcumin on A549 lung cancer cell proliferation and the mechanism of these phytochemicals in regulating apoptosis and cell cycle arrest were investigated. A549 cells were treated with quercetin 3-β-d-glucoside, resveratrol, or curcumin at 37°C for 96 hr and cell viability was measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Proteins related to apoptosis and cell cycle in A549 cells were quantified with Western blotting assay. Quercetin 3-β-d-glucoside, resveratrol, and curcumin inhibited A549 cell proliferation in a dose-dependent manner (p<0.05). Quercetin 3-β-Dglucoside significantly decrease the expression level of CDK4 at concentrations of 5 mM and above (p<0.05). Curcumin lowered expression level of Bcl-2, CDK4, and cyclin D1 at concentrations of 100, 50, and above, and 50 mM and above, respectively (p<0.05). These results suggest that phytochemicals, which can be found in a normal diet, inhibit lung cancer cell proliferation and regulate the expression of the proteins involved in apoptosis and cell cycle.     

EGCG inhibits protein synthesis,lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells

In the previous studies, (–)‐epigallocatechin‐3‐gallate (EGCG) has been shown to have anticarcinogenic effects via modulation in protein expression of p53. Using p53 positive Hep G2 and p53 negative Hep 3B cells, we found that treatment of EGCG resulted in dose‐dependent inhibition of cellular proliferation, which suggests that the interaction of EGCG with p53 may not fully explain its inhibitory effect on proliferation. Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents. EGCG has multiple beneficial activities similar to those associated with CR. One key enzyme thought to be activated during CR is AMP‐activated kinase (AMPK), a sensor of cellular energy levels. Here, we showed that EGCG activated AMPK in both p53 positive and negative human hepatoma cells. The activation of AMPK suppressed downstream substrates, such as mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E‐binding protein‐1 (4E‐BP1) and a general decrease in mRNA translation. Moreover, EGCG activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl‐CoA carboxylase (ACC). Interestingly, the decision between apoptosis and growth arrest following AMPK activation is greatly influenced by p53 status. In p53 positive Hep G2 cells, EGCG blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up‐regulating p21 expression. However, EGCG inducted apoptosis in p53 negative Hep 3B cells. Based on these results, we have demonstrated that EGCG has a potential to be a chemoprevention and anti‐lipogenesis agent for human hepatoma cells.     

Shallot and licorice constituent isoliquiritigenin arrests cell cycle progression and induces apoptosis through the induction of ATM/p53 and initiation of the mitochondrial system in human cervical carcinoma HeLa cells

This study is the first to investigate the anticancer effect of isoliquiritigenin (ISL) in human cervical carcinoma HeLa cells. The results reveal that ISL inhibits HeLa cells by blocking cell cycle progression in the G2/M phase and inducing apoptosis. Blockade of cell cycle is associated with increased activation of ataxia telangiectasia‐mutated (ATM). Activation of ATM by ISL phosphorylated p53 at Serine15, resulting in increased stability of p53 by decreasing p53 and murine double minute‐2 (MDM2) interaction. In addition, ISL‐mediated G2/M phase arrest was also associated with decreases in the amounts of cyclin B, cyclin A, cdc2, and cdc25C, and increases in the phosphorylation of Chk2, cdc25C, and cdc2. The specific ATM inhibitor caffeine significantly decreased ISL‐mediated G2/M arrest by inhibiting the phosphorylation of p53 (Serine15) and Chk2. ISL induced apoptotic cell death is associated with changes in the expression of Bax and Bak, decreasing levels of Bcl‐2 and Bcl‐X_L, and subsequently triggering mitochondrial apoptotic pathway. In addition, pretreatment of cells with caspase‐9 inhibitor blocked ISL‐induced apoptosis, indicating that caspase‐9 activation is involved in ISL‐mediated HeLa cell apoptosis. These findings suggest that ISL may be a promising chemopreventive agent against human uterine cervical cancer.     

Intramammary inoculation of Panax ginseng extract in cows at drying off enhances early mammary involution

This study was designed to evaluate the effects of a single intramammary infusion of Panax ginseng extract on cell proliferation and death mechanism in bovine mammary gland during early involution. Eight mammary quarters from six non-pregnant cows in late lactation were infused with 10 ml of ginseng solution (3 mg/ml), six quarters were treated with 10 ml of placebo (vehicle alone) and six quarters were maintained as uninoculated controls. Milking was interrupted after infusion. Animals included in the three groups were slaughtered 7 d after inoculation and samples for histological analysis were taken. Morphometric analysis showed a significant increase in percentages of mammary tissue area occupied by stroma in ginseng-treated quarters compared with controls. A significant increase of immunostained area for bax protein and active caspase-3 was observed in ginseng-treated quarters compared with controls, whereas no differences were observed for bcl-2 immunostaining. Expression of bax mRNA was significantly higher in ginseng-treated quarters than in controls. The bax/bcl-2 ratio indicated a significant predominance of bax over bcl-2 mRNA expression in ginseng-treated quarters compared with controls. The rise of epithelial and stromal cell apoptosis in situ by TUNEL was more marked in quarters treated with ginseng than in controls. Ginseng inoculation had no effect on the number of epithelial and stromal proliferating cells labelled with Ki-67 antibody. Ratio of apoptotic to proliferating cells was higher in quarters treated with ginseng compared with controls, indicating a net loss of cells in parenchymal components. Also, the intramammary inoculation of ginseng extract at drying off increased the rate of mammary cell apoptosis without inhibiting cell proliferation. Taken together, these changes are indicative of mammary regression enhancement during early involution.     

The p53-, Bax- and p21-dependent inhibition of colon cancer cell growth by 5-hydroxy polymethoxyflavones

Scope: Previously, we reported that 5‐hydroxy polymethoxyflavones (5OH‐PMFs) isolated from orange, namely 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxyflavone, 5‐hydroxy‐3,6,7,8,3′,4′‐hexamethoxyflavone (5HHMF) and 5‐hydroxy‐6,7,8,4′‐tetramethoxyflavone (5HTMF), potently induced apoptosis and cell‐cycle arrest in multiple human colon cancer cells. Herein, using isogenic variants of HCT116 human colon cancer cells, we investigated the effects of p53, Bax and p21 on the apoptosis and cell‐cycle arrest induced by different 5OH‐PMFs. Methods and results: Annexin V/PI co‐staining assay demonstrated that 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (p53^+/+) cells but not in HCT116 (p53^?/?) cells. Furthermore, 5HHMF and 5HTMF significantly induced apoptosis in HCT116 (Bax^+/?) cells, whereas their pro‐apoptotic effects on HCT116 (Bax^?/?) cells were marginal. All three 5OH‐PMFs increased G0/G1 cell population of HCT116 (p53^+/+) cells, and these effects were abolished in HCT116 (p53^?/?) and HCT116 (p21^?/?) cells. Immunoblotting analysis showed that 5HHMF and 5HTMF increased the levels of cleaved caspase‐3, cleaved PARP in both HCT116 (p53^+/+) and HCT116 (Bax^+/?) cells and these effects were much weaker in HCT116 (p53^?/?) and HCT116 (Bax^?/?) cells. Conclusion: Our results demonstrated that 5OH‐PMFs, especially 5HHMF and 5HTMF, induce apoptosis and cell‐cycle arrest by p53‐, Bax‐ and p21‐dependent mechanism.     

苦荞麦蛋白质对乳腺癌细胞的作用机制

研究了苦荞麦蛋白质TBWSP31对Bcap37乳腺癌细胞的作用机制。采用流式细胞仪分析测定苦荞麦蛋白质TBWSP31对Bcap37细胞作用后,对细胞周期及癌基因和抑癌基因蛋白产生的影响。结果表明,TBWSP31对Bcap37细胞的增殖抑制作用存在时间效应和剂量效应。TB-WSP31作用于Bcap37细胞后,G0/G1期细胞比例增加,DNA合成期-S期细胞减少,说明细胞受阻于G0/G1期,阻滞G0/G1期的细胞向S期转化,细胞的恶性增殖相应减慢。TBWSP31可以上调抑癌基因蛋白Fas的表达,下调癌基因蛋白bcl-2的表达。     

Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: Identification of phenolic acids with cytotoxic potential

Mushrooms are a possible rich source of biologically active compounds with the potential for drug discovery. The aim of this work was to gain further insight into the cytotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterised regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the C. alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI₅₀ concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 μg/ml) for 48 h caused an increase in the levels of wt. p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, evidence was found for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract.     

枸杞多糖促进大鼠肝癌组织细胞的凋亡

为探究枸杞多糖对肝癌模型大鼠癌组织细胞凋亡的干预效果及作用机制。选取45只SD健康雄性大鼠,5只作为正常组,其余40只建立肝癌模型,分为模型组、药物对照组、低、高浓度组,并分别进行干预。观察各组大鼠细胞增殖、凋亡、周期分布情况,检测PTEN、p-Akt、m TOR、Bcl-2、Bax、caspase3表达。研究结果显示,高浓度组G1期细胞比例为57.59%,高于模型组、药物对照组、低浓度组(p<0.05)。高浓度组大鼠增殖率为8.53%,凋亡率为47.68%,均优于模型组、药物对照组和低浓度组。高浓度组大鼠PTEN相对表达量为0.98,p-Akt、m TOR相对表达量分别为1.05、1.11,高浓度组大鼠Bcl-2、caspase3相对表达量分别为1.26、1.19,Bax相对表达量为0.98,干预效果均优于药物对照组和低浓度组(p<0.05)。实验结果表明,在枸杞多糖的干预下,肝癌大鼠癌组织肿瘤细胞增殖、凋亡及周期分布受到明显调控,PTEN、p-Akt、m TOR、Bcl-2、caspase3、Bax表达受到明显的调控,说明枸杞多糖能够阻滞肝癌组织细胞周期分布,抑制肝癌组织细胞增殖,促进细胞凋亡,为肝癌的临床治疗提供一定的理论依据。     

Khz (fusion of ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis in A549 human lung cancer cells by generating reactive oxygen species and decreasing the mitochondrial membrane potential

Khz, a naturally occurring compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia, inhibits the growth of cancer cells. This study aimed to investigate the anti-proliferative effects of Khz on A549 lung cancer cells. Khz cytotoxicity was measured using an MTT assay and the mitochondrial membrane potential (MMP)-related, calcium-induced generation of reactive oxygen species (ROS) in A549 cells was measured by flow cytometry. The expression of p53, Bax, Bcl-2, and actin proteins was analyzed by western blotting. Khz inhibited cell division and induced apoptosis in A549 cells. Flow cytometry analysis showed that the percentage of A549 cells in sub-G1 phase of the cell cycle increased in response to Khz treatment. Khz reduced MMP and Bcl-2 protein levels, but increased ROS generation and p53 and pro-apoptotic proteins. The anti-proliferative and pro-apoptotic effects of Khz suggest that this extract shows great promise as a potential chemotherapeutic agent.     

夏枯草提取物对人食管癌Eca-109细胞增殖和凋亡的影响

目的：观察夏枯草提取物对人食管癌Eca-109细胞增殖和凋亡的影响,探讨夏枯草抗肿瘤作用及机制。方法：不同浓度夏枯草作用于食管癌Eca-109细胞,利用MTr法检测细胞增殖抑制率,免疫组织化学法检测Bcl-2、Bax蛋白表达;Annexin—V—FITC／PI法检测细胞凋亡率。结果：中高剂量夏枯草抑制食管癌Eca-109细胞增殖,并呈量效和时效关系;使细胞凋亡率增高,同时Bcl-2蛋白减少,Bax蛋白表达增加。结论：夏枯草提取物可抑制食管癌Eca-109细胞增殖,使Bcl-2蛋白表达减少,Bax蛋白表达增加,细胞凋亡增加。     

龙须菜藻红蛋白对Hela细胞增殖抑制及其机制的研究

目的:探讨龙须菜藻红蛋白(PE)对人宫颈癌细胞Hela体外的抑制作用及其作用机制。方法:采用MTT法检测不同浓度的藻红蛋白对Hela细胞增殖抑制效果。流式细胞仪和Annexin V-FITC/PI双荧光染色法观察藻红蛋白对Hela细胞周期及细胞凋亡的影响。结果:PE可显著抑制Hela细胞的生长,并呈剂量-效应关系(p<0.01,r=0.84),剂量为20μg/ml,作用48h,其抑制率达70.88%,其48h的IC50值为4.12μg/ml。PE可阻滞Hela细胞从G2/M期进入S期,诱导细胞凋亡。结论:PE对Hela细胞有较强的抑制作用,其作用机制部分与诱导Hela细胞调亡有关。