金雀异黄素合成品诱导人胃癌细胞SGC-7901凋亡的体外研究

目的:探讨金雀异黄素合成品诱导人胃癌细胞SGC-7901凋亡的作用机制。方法:采用2.5mg·L~(-1)、5.0mg·L~(-1)、10.0mg·L~(-1)和20.0mg·L~(-1)的金雀异黄素处理人胃癌细胞SGC-7901后,用流式细胞仪检测细胞凋亡率,电镜下观察细胞形态变化,RT-PCR法检测凋亡相关基因表达。结果:10.0mg·L~(-1),20.0mg·L~(-1)金雀异黄素能诱导胃癌细胞SGC-7901凋亡,凋亡率与剂量正相关(相关系数r=0.9830),10.0mg·L~(-1)金雀异黄素诱导胃癌细胞SGC-7901发生凋亡的形态学改变,2.5mg·L~(-1)、5.0mg·L~(-1)、10.0mg·L~(-1)和20.0mg·L~(-1)金雀异黄素使Bcl-2mRNA表达下调,Fas mRNA表达上调。结论:金雀异黄素能诱导胃癌细胞SGC-7901凋亡,降低Bcl-2 mRNA表达,增加Fas mRNA表达为其诱发SGC-7901细胞凋亡的机制之一。     

金雀异黄素抑制IL-1α刺激破骨样细胞的组织蛋白酶K表达

从人骨巨细胞瘤组织中纯化破骨样细胞,用不同浓度的金雀异黄素温育48h,观察IL-1α刺激后1h后组织蛋白酶K表达。结果发现:与阴性对照组相比,IL-1α明显刺激破骨样细胞表达组织蛋白酶K(P<0.01);而金雀异黄素抑制IL-1α刺激后组织蛋白酶K转录及表达,且呈剂量依赖关系(P<0.01);加用雌激素受体拮抗剂ICI182.780后,金雀异黄素作用被部分拮抗。金雀异黄素通过雌激素受体部分抑制IL-1α刺激破骨样细胞的组织蛋白酶K表达。     

苏云金芽孢杆菌Bt9875晶体蛋白诱导HL-60细胞凋亡

[目的]研究苏云金芽孢杆菌(Bacillus thuringiensis,Bt)Bt9875菌株晶体蛋白对人急性髓细胞性白血病细胞HL-60的影响.[方法]采用MTT比色、荧光显微观察、DNA凝胶电泳、流式细胞术等方法来检测不同浓度的Bt9875晶体蛋白处理后HL-60细胞的凋亡特征.[结果]Bt9875晶体蛋白对HL-60细胞的生长具有明显的抑制作用,且随着蛋白质浓度的增加对HL-60细胞生长抑制愈加明显,而对正常人外周血单个核细胞(PBMC)无作用;荧光显微镜下观察发现经该蛋白作用后HL-60细胞核的形态呈现凋亡特征;流式细胞术分析表明,HL-60细胞经100 μg/mL晶体蛋白作用后,凋亡率达到52%;琼脂糖凝胶电泳显示细胞DNA呈梯状降解.[结论]初步证明了Bt9875晶体蛋白在体外能够明显抑制HL-60细胞的增长,并诱导其凋亡,这为苏云金芽抱杆菌晶体蛋白的应用开创了新的思路.     

微波辐射对肾小管上皮细胞的影响以及金雀异黄素对其保护作用

目的：观察微波辐照对人近端肾小管上皮细胞（HKC）的影响及金雀异黄素对其的保护作用。方法：HKC分为对照组、微波辐照组、金雀异黄素组（n=6）。金雀异黄素组在辐照前2 h用含30μmol/L金雀异黄素的DMEM培养基进行预培养。辐照后24 h留取上清进行乳酸脱氢酶（LDH）、β-N-乙酰氨基葡萄糖苷酶（NAG）活性及丙二醛（MDA）、超氧化物歧化酶（SOD）活性检测。Hoechst 33258染色观察细胞凋亡情况。结果：与对照组比较,微波辐照组上清NAG、LDH活性明显增加（P < 0.01）,金雀异黄素预处理组则较微波辐照组明显下降（P < 0.01）;微波辐照组上清活性也较对照组明显增加（P < 0.01）。Hoechst 33258染色显示,微波辐照可导致较多量的细胞凋亡,而应用金雀异黄素预处理后细胞凋亡的比例均大大减少。微波辐照可大大提高HKC细胞中的MDA含量,SOD活性降低（P< 0.01）,应用金雀异黄素预处理后MDA的含量无明显降低,SOD的活性明显增大（P < 0.01）。结论：微波辐照可导致人近端肾小管上皮细胞出现功能损伤,金雀异黄素对其具有一定的保护作用,可能与其减轻氧化应激损伤、减少细胞凋亡有关。     

黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响

为研究黄泥螺提取物对小鼠黑色素瘤细胞B16生长的影响,以及初步研究其作用机制,采用磺酰罗丹明B(SRB)法测黄泥螺提取物对B16细胞的生长抑制作用,得到48 h后半数抑制浓度(IC50)为68.56 ug/ml.当黄泥螺提取物浓度为70ug/ml时,台盼蓝排斥试验显示有部分细胞死亡.经Hoechest 33258染色并用荧光显微镜观察,发现培养的细胞中出现凋亡小体,细胞核发生固缩;DNA琼脂糖凝胶电泳呈现出DNA大片段;用流式细胞仪进一步检测表明,细胞生长周期发生变化并检测到凋亡峰.结果表明,体外培养的B16细胞经过黄泥螺提取物处理后,B16细胞增殖受到抑制,细胞周期被阻滞,B16细胞受到诱导后存在凋亡.因此,黄泥螺提取物对小鼠黑色素瘤细胞B16生长有明显的影响.     

羊栖菜多糖诱导HL-60细胞凋亡的研究

用MTT法观察羊栖莱多糖(SFPS)在体外抗人白血病HL-60细胞增殖作用;扫描电镜、透射电镜、DNA电泳和流式细胞仪检测HL-60细胞凋亡。结果表明SFPS对HL-60细胞具有显著生长抑制作用,并呈量效和时效关系,药物作用24,36,48,72h的IC50分别为390,362,402,421mg／L;药物浓度为300mg／L和500mg／L作用HL-60细胞后,琼脂糖凝胶电泳显示有凋亡细胞特有的DNA梯状条带,细胞微绒毛减少、染色质固缩、边集,凋亡小体形成;DNA直方图出现亚G1峰。在一定浓度范围内,SFPS诱导细胞凋亡的作用呈现浓度和时间依赖性,同时G2／M期细胞比例增多。因此,SFPS抗肿瘤作用与诱导细胞凋亡和G2／M期细胞阻滞有关。     

羊栖菜多糖诱导HL-60细胞凋亡的研究

用MTT法观察羊栖菜多糖(SFPS)在体外抗人白血病HL-60细胞增殖作用;扫描电镜、透射电镜、DNA电泳和流式细胞仪检测HL-60细胞凋亡。结果表明SFPS对HL-60细胞具有显著生长抑制作用,并呈量效和时效关系,药物作用24,36,48,72h的IC_(50)分别为390,362,402,421mg/L;药物浓度为300mg/L和500mg/L作用HL-60细胞后,琼脂糖凝胶电泳显示有凋亡细胞特有的DNA梯状条带,细胞微绒毛减少、染色质固缩、过集,凋亡小体形成;DNA直方图出现亚G_1峰。在一定浓度范围内,SFPS诱导细胞凋亡的作用呈现浓度和时间依赖性,同时G_2/M期细胞比例增多。因此,SFPS抗肿瘤作用与诱导细胞凋亡和G_2/M期细胞阻滞有关。     

高良姜素对人食管鳞癌KYSE-510细胞的抑制作用

探讨在体外高良姜素对人食管鳞癌KYSE-510细胞的抑制作用以及可能的作用机制.MTT结果表明,高良姜素对人食管鳞癌KYSE-510细胞具有很强的生长抑制作用.光学显微镜、激光共聚焦显微镜以及流式细胞仪的分析结果表明,高良姜素可诱导KYSE-510细胞分化.荧光定量RT-PCR和Western印迹分析结果表明,高良姜素诱导P21^waf1、抑制细胞周期蛋白B1和细胞周期蛋白D1的表达,推测上述基因可能是高良姜素实现细胞分化诱导作用的靶基因.     

氧化苦参碱对K562肿瘤细胞增殖的影响

目的:研究氧化苦参碱(OM)对人白血痛细胞系K562生长增殖的影响.方法:运用MTT比色法、活细胞计数法、集落形成法以及透射电镜观察检测OM对人白血病细胞系K562增殖抑制作用.结果:MTT实验、生长曲线及集落形成实验显示OM能明显抑制K562细胞的增殖.随着OM浓度的增加,K562细胞存活细胞显著降低,呈现明显的刺量依赖性,经相关分析,细胞抑制率与OM浓度呈正相关(r=0.9010),其半数抑制浓度(IC50)为0.33 mg/ml.透射电镜下显示在低浓度即有明显的诱导细胞凋亡的作用,出现核固缩、核碎裂、凋亡小体等典型的凋亡形态.结论:OM具有抑制K562白血病细胞增殖诱导肿瘤细胞凋亡的作用.     

iNOS抑制剂1400W通过p53途径增强顺铂对宫颈癌SiHa细胞的化疗效果

该文探讨了iNOS抑制剂1400W联合顺铂对宫颈癌SiHa细胞株生长抑制作用及其机制。应用MTT法检测1400W及顺铂对宫颈癌Si Ha细胞生长的抑制作用;流式细胞仪检测1400W及顺铂对宫颈癌Si Ha细胞凋亡的影响;quantitative RT-PCR和Western blot检测1400W及顺铂对宫颈癌Si Ha细胞p53、鼠双微基因2(murine double minut 2,MDM2)、DNA依赖蛋白激酶(DNA-dependent protein kinase,DNA-PK)蛋白及m RNA表达的影响。结果显示,通过i NOS抑制剂1400W联合顺铂作用于宫颈癌Si Ha细胞株发现,1400W联合顺铂能明显抑制Si Ha细胞生长,其抑制作用与剂量呈正相关。1400W可以增强顺铂诱导的细胞凋亡作用。进一步研究证实,1400W可以抑制顺铂诱导的p53积聚,增加鼠双微基因2及DNA依赖蛋白激酶的表达。该实验结果为治疗宫颈癌患者提供了新的治疗策略,即通过联合使用i NOS抑制剂,可以增强顺铂对宫颈癌细胞的杀伤作用,提高顺铂对宫颈癌患者的治疗作用。     

Effects of genistein and daidzein on the cell growth,cell cycle,and differentiation of human and murine melanoma cells(1)

Genistein and daidzein are two major isoflavonoids in dietary soybean that have inhibition effect on the cell growth of different tumor cell lines. We previously reported the anti-tumor activities of genistein and daidzein in human co1on tumor (HCT) cells and their different ability to enhance the activation of murine lymphocytes. In the present study, the effect of genistein and daidzein on the cell growth, cell cycle progression, and differentiation of murine K1735M2 and human WM451 cel1s was investigated. It was found that genistein could inhibit the cell growth of two metastatic melanoma cell lines, murine Kl735M2 and human WM45l in a dose-dependent manner. Flow cytometry showed that genistein could cause arrest of both Kl735M2 and WM45l at G(2)/M phase, while daidzein increased the cell numbers at S phase, decreased the cell numbers at G(1) phase. Detection of melanin and morphological observation showed that genistein can induce Kl735M2 and WM45l to produce dendrite-like structure and produce more melanin by 80%. In contrast, daidzein only retarded the growth of K1735M2 and did not induce differentiation in either K1735M2 or WM451. These results suggest that genistein and daidzein in soybean can inhibit certain malignant phenotype of melanoma via different mechanisms and be potential medical candidates for melanoma cancer therapy.     

Dissociation of hemoglobin accumulation and commitment during murine erythroleukemia cell differentiation by treatment with imidazole

The effect of imidazole on DMSO-induced murine erythroleukemia (MEL) cell differentiation has been examined. While imidazole does inhibit heme, globin mRNA, and hemoglobin accumulation in DMSO-induced MEL cells, it does not affect the commitment of MEL cells to the specific limitation of proliferative capacity associated with the in vitro differentiation program. Furthermore, imidazole treatment does not affect DMSO-induced changes in cell volume, in the relative proportion of nuclear protein IP25, and in the specific activity of the enzyme cytidine deaminase. A clonal analysis in the presence of imidazole indicated that the drug prevents heme accumulation even in MEL cells already committed to terminal differentiation. These observations suggest that imidazole effectively dissociates two aspects of the erythroid differentiation program of MEL cells: globin gene expression and commitment to loss of proliferative capacity.     

Modulation of cyclic AMP levels and differentiation by adenosine analogs in mouse erythroleukemia cells

Friend virus-transformed mouse erythroleukemia (MEL) cells can be induced to undergo erythroid differentiation by a variety of compounds, including dimethyl sulfoxide (DMSO) and the adenosine analog xylosyladenine. The present studies have monitored the effects of the stable adenosine receptor ligand N6-phenylisopropyladenosine (PIA) on induction of MEL cell differentiation. PIA has been previously shown to stimulate adenylate cyclase activity in rat hepatic and mouse Leydig 1-10 cells as well as inhibit adenylate cyclase in adipocytes. In the present study, PIA was ineffective as an inducer of the differentiated MEL cell phenotype. However, the results demonstrate that PIA inhibits the induction of MEL cell differentiation by DMSO and xylosyladenine. The extent of this inhibition as determined by benzidine staining, induction of globin RNA, and loss of self-renewal capacity was dependent on PIA concentration. The results also demonstrate that PIA induces a rapid and sustained increase in cyclic AMP (cAMP) levels. Furthermore, there was a highly significant correlation between cAMP levels and inhibition of xylosyladenine-induced differentiation (r = 0.962, P less than 0.0005). This relationship is further supported by the demonstration that prostaglandins E1 and E2 increase MEL cell cAMP levels and inhibit induction of the differentiated MEL cell phenotype. Moreover, PIA inhibited induction of MEL cell differentiation by butyric acid, diazepam, hypoxanthine, and the aminonucleoside analog of puromycin. These results suggest that cAMP may act as a negative regulatory signal in the induction of MEL cell differentiation.     

Succinylacetone, a potent inhibitor of heme biosynthesis: effect on cell growth, heme content and delta-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells.

4,6-Dioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, δ-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit cell growth of murine erythroleukemia (MEL) cells in culture. SA profoundly inhibited ALA dehydratase in broken cell preparations at concentrations as low as 10^?7 M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.     

Wild type p53 gene causes reorganization of cytoskeleton and,therefore, the impaired deformability and difficult migration of murine erythroleukemia cells

We studied the role of p53 gene in the biophysics and biology in murine erythroleukemia cell line (MEL), with the goal of understanding the influence of this tumor suppressor gene on the deformability and metastasis of tumor cells. Experiments were performed on MEL and p53-transfected MEL (MEL-M with mutant p53 gene and MEL-W with wild-type p53 gene). The cell growth curves indicated that the over-expression of wild-type p53 gene significantly suppressed the growth of MEL, with G(0)-G(1) arrest and apoptosis shown by flow cytometric assays. Confocal laser scanning microscopy revealed that the MEL-W had a more compact organization of the F-Actin cytoskeleton than MEL and MEL-M. Fluorescence polarization measurement indicated a higher membrane fluidity of MEL-W than the other two groups. Fourier transform infrared spectroscopy (FT-IR) showed changes in the composition and/or structure of membrane lipids in MEL-W, with decreases in secondary structures of proteins such as alpha-helix, turns and bends and random coil, in comparison to MEL and MEL-M. The osmotic fragility curves indicated that MEL-W was more fragile and micropipette experiments showed that they had increased elasticity and reduced deformability in comparison to MEL and MEL-M. The adhesion assay with the use of the flow chamber revealed a lower adhesion rate of MEL-W to endothelial cells at high shear stress. The present study on the molecular biology with biophysics of MEL cells contributes to our knowledge on the tumor suppressor gene p53.     

Phytoestrogen genistein as an anti-staphylococcal agent

The soybean-derived isoflavone genistein has been shown to exert beneficial effects on many disorders, including cancer and cardiovascular diseases. The effects of genistein on mammalian cells are mediated by its abilities to inhibit topoisomerase II and protein tyrosine kinase. In order to examine the potential antibacterial activities of genistein, we incubated the bacteria with various concentrations of this compound for different periods of time and assessed the viable counts. Exposure to genistein exhibited an inhibitory effect on all staphylococcal strains tested, including methicillin-resistant strains. Furthermore, the growth of Streptococcus pasteurianus, Bacillus cereus, and Helicobacter pylori was clearly inhibited by genistein, whereas Escherichia coli growth was not suppressed. Daidzein, which is structurally similar to genistein, but deficient in topoisomerase II inhibitory activity, also inhibited the growth of Staphylococcus aureus, albeit with lower potency than genistein. Our results indicate that genistein exerts potent antibacterial properties in vitro, which are possibly mediated by the stabilization of the covalent topoisomerase II-DNA cleavage complex.     

Inhibition of cell proliferation and induction of apoptosis by genistein in experimental hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of cancer related deaths in the world, with increasing incidence in many developed countries. Epidemiological data suggest that consumption of soy products may be associated with a decreased risk of cancer. We investigate the effects of genistein on cell proliferation, apoptosis and caspase-3 in DEN induced (200 mg/kg body weight; by single intraperitoneal injection) and Phenobarbital promoted (0.05% through drinking water for 14 successive weeks) cancer-bearing rats. Immunohistochemistry was employed to detect cell proliferating markers proliferating cell nuclear antigen (PCNA), DNA fragmentation was determined by agarose gel electrophoresis and terminal deoxynucleatide transferase dUTP nick labeling (TUNEL) staining and caspase by enzyme-linked immunosorbent assay. We found inhibition of cell proliferation, induction of apoptosis and activation of caspase-3 in genistein treated animals. From these results, we conclude that genistein inhibit cell proliferation, induced apoptosis. This activation of caspsase-3 in genistein treated liver cancer bearing animals correlated well with its apoptosis inducing effect.     

Book of Abstracts

A widespread occurrence of melatonin (MEL) in plant kingdom has been reported. MEL is a highly conserved molecule occurring in evolutionary distant organisms. Its role in plants seems to be similar to that in animals. Although MEL function in plants is not well known, yet a hypothesis can be put forward that it probably functions as a night signal, coordinating responses to diurnal and photoperiodic environmental cues. It has also been suggested that MEL is an independent plant growth regulator, probably its action is analogous to IAA and it may mediate the actions of other plant growth regulators. Due to its antioxidant properties MEL may also stabilize cell red-ox status and protect them against reactive oxygen species (ROS) and other harmful environmental influence.     

Inhibitory effect of genistein on the invasive potential of human cervical cancer cells via modulation of matrix metalloproteinase-9 and tissue inhibitiors of matrix metalloproteinase-1 expression

BackgroundOne of the most challenging stumbling blocks for the treatment of cancer is the ability of cancer cells to break the natural barriers and spread from its site of origin to non-adjacent regional and distant sites, accounting for high cancer mortality rates. Gamut experimental and epidemiological data advocate the use of pharmacological or nutritional interventions to inhibit or delay various stage(s) of cancer such as invasion and metastasis. Genistein, a promising chemopreventive agent, has gained considerable attention for its powerful anti-carcinogenic, anti-angiogenic and chemosensitizing activities.MethodsIn this study, the cytotoxic potential of genistein on HeLa cells by cell viability assay and the mode of cell death induced by genistein were determined by nuclear morphological examination, DNA laddering assay and cell cycle analysis. Moreover, to establish its inhibitory effect on migration of HeLa cells, scratch wound assay was performed and these results were correlated with the expression of genes involved in invasion and migration (MMP-9 and TIMP-1) by RT-PCR.ResultsThe exposure of HeLa cells to genistein resulted in significant dose- and time-dependent growth inhibition, which was found to be mediated by apoptosis and cell cycle arrest at G₂/M phase. In addition, it induced migration-inhibition in a time-dependent manner by modulating the expression of MMP-9 and TIMP-1.ConclusionOur results signify that genistein may be an effective anti-neoplastic agent to prevent cancer cell growth and invasion and metastasis. Therefore therapeutic strategies utilizing genistein could be developed to substantially reduce cancer morbidity and mortality.