片仔癀对人卵巢癌OVCAR-3细胞增殖、凋亡及周期的影响

目的：研究片仔癀对人卵巢癌细胞株OVCAR-3增殖抑制作用,及其对细胞周期、细胞凋亡的影响。方法：采用MTT法观察片仔癀对人卵巢癌细胞株OVCAR-3细胞的增殖抑制率,流式细胞仪检测细胞凋亡及细胞周期,westem—blot检测相关蛋白的表达。结果：片仔癀以剂量依赖式抑制人卵巢癌细胞株OVCAR-3细胞增殖,片仔癀250、500、1000μg·mL-1作用于OVCAR-3细胞24h后,其早期凋亡率分别为6．6％、30．9％、43．2％,而对照组为0％,其诱导凋亡作用呈现剂量依赖性;细胞积聚在G0／G1期,同时S期细胞比例减少;Akt、PARP、CDK6表达下调。结论：片仔癀可以抑制OVCAR-3细胞增殖及诱导细胞凋亡作用,并能阻滞细胞于G0／G1期,有望成为卵巢癌治疗药。     

乙酰胆碱对三种人垂体腺瘤细胞增殖及凋亡的影响

为了解乙酰胆碱（acetylcholine,ACh)在人垂体腺瘤发生、发展中的作用,本研究首先观察了人垂体腺瘤细胞内是否存在合成ACh必需的胆碱乙酰转移酶,并应用MTT实验、[^3H]TdR掺入实验、细胞周期分析和TUNEL法测定了ACh对体外培养的人垂体无功能瘤、催乳素瘤和生长激素瘤等三种瘤细胞增殖和凋亡的影响。结果发现：（1）三种垂体腺瘤细胞中均有胆碱酯酶的表达,但明显少于正常垂体;（2）ACh对三种类型的人垂体腺瘤细胞增殖代谢的影响相类似,不同浓度的ACh能明显抑制体外培养的三种人垂体腺瘤细胞的增殖,呈明显的剂量效应关系,同时ACh能减少垂体腺瘤细胞进入S、G2期的细胞比例,而使处于G1期的细胞比例增加;（3）ACh的这种作用可被阿托品阻断,但不受筒箭毒的影响;（4）ACh对体外培养的三种人垂体腺瘤细胞凋亡无明显影响。该结果提示,ACh可能以旁分泌或自分泌的方式作用于垂体前叶细胞,对垂体腺瘤细胞增殖分化有调控作用,而且是通过ACh M受体来实现的。     

木黄酮对人催乳素瘤细胞增殖及其凋亡影响的实验研究

目的：观察木黄酮（genistein,GST)对培养的人垂体催乳素瘤细胞增殖和凋亡的影响。方法：将CST、β-雌二醇（E2）作用于体外培养的人催乳素瘤细胞,测定MTT值及^3H-TdR掺入量,流式细胞仪测定细胞周期,并用TUNEL法观察细胞凋亡情况。结果：不同浓度的GST可抑制催乳素瘤细胞的增殖,并存在着剂量效应,10^-5mol.L^-1GST可使G1期的细胞比例从对照组的55.3%上升为90.3%;不同浓度的E2以剂量依赖方式刺激催乳素瘤细胞的增殖,并使G2期的细胞比例从对照组的15.6%上升为41.8%,GST和E2的共同作用仍可抑制催乳素瘤细胞的增殖,但抑制程度降低,不同浓度的GST均明显促进催乳素瘤细胞的凋亡,E2对GST的促凋亡作用无明显影响。结论：GST在体外能明显抑制培养人催乳素瘤细胞的增殖,并促进其凋亡,E2可部分拮抗GST的抑制增殖作用,但对其促凋亡作用无明显影响。     

木黄酮对人催乳素瘤细胞增殖及其凋亡影响的实验研究

目的观察木黄酮(genistein,GST)对培养的人垂体催乳素瘤细胞增殖和凋亡的影响。方法将GST、β-雌二醇(E2)作用于体外培养的人催乳素瘤细胞,测定MTT值及3H-TdR掺入量,流式细胞仪测定细胞周期,并用TUNEL法观察细胞凋亡情况。结果不同浓度的GST可抑制催乳素瘤细胞的增殖,并存在着剂量效应,10-5mol*L-1GST可使G1期的细胞比例从对照组的55.3%上升为90.3%;不同浓度的E2以剂量依赖方式刺激催乳素瘤细胞的增殖,并使G2期的细胞比例从对照组的15.6%上升为41.8%。GST和E2的共同作用仍可抑制催乳素瘤细胞的增殖,但抑制程度降低。不同浓度的GST均明显促进催乳素瘤细胞的凋亡,E2对GST的促凋亡作用无明显影响。结论GST在体外能明显抑制培养人催乳素瘤细胞的增殖,并促进其凋亡。E2可部分拮抗GST的抑制增殖作用,但对其促凋亡作用无明显影响。     

纳米银抑制肝癌HepG2细胞活力

目的探讨纳米银(AgNPs)对体外培养的人肝癌细胞株HepG2增殖和凋亡的影响。方法用不同浓度的AgNPs处理肝癌HepG2细胞,采用细胞形态学观察及细胞活力测定(MTT法)来评价AgNPs对HepG2细胞体外增殖的影响,流式细胞仪分析细胞周期分布并检测细胞凋亡率。结果 AgNPs使细胞增殖活性降低,且呈浓度和时间依赖性,细胞呈凋亡形态改变;流式细胞仪检测到细胞凋亡,细胞平均凋亡率呈时间依赖性。AgNPs处理组S期细胞逐渐增多,而G0/G1、G2/M期细胞逐渐减少。结论 AgNPs抑制HepG2细胞生长,使HepG2细胞阻滞于S期,并诱导其凋亡。     

疏肝养血降乳汤含药血清对垂体瘤增殖和凋亡作用的解析

为了探索疏肝养血降乳汤含药血清对垂体瘤(垂体泌乳素腺瘤)细胞增殖和凋亡的影响及其作用机制,采用大鼠垂体瘤细胞系MMQ作为研究对象,通过MTT法检测不同浓度疏肝养血降乳汤含药血清对MMQ细胞活力的影响;流式细胞仪检测不同浓度疏肝养血降乳汤含药血清对MMQ细胞周期和凋亡的影响;蛋白质免疫印迹检测不同浓度疏肝养血降乳汤含药血清对MMQ细胞Caspase-1、Caspase-3蛋白剪切活化的影响。表明,疏肝养血降乳汤含药血清对MMQ细胞增值有很强的抑制作用,通过激活Caspase-1、Caspase-3而促进MMQ细胞的凋亡,含药血清将MMQ细胞阻滞在G2/M期。这些说明疏肝养血降乳汤含药血清能够抑制MMQ细胞增值并促进其凋亡。     

片仔癀对人卵巢癌OVCAR-3细胞增殖、凋亡及周期的影响

目的:研究片仔癀对人卵巢癌细胞株OVCAR-3增殖抑制作用,及其对细胞周期、细胞凋亡的影响。方法:采用MTT法观察片仔癀对人卵巢癌细胞株OVCAR-3细胞的增殖抑制率,流式细胞仪检测细胞凋亡及细胞周期,western-blot检测相关蛋白的表达。结果:片仔癀以剂量依赖式抑制人卵巢癌细胞株OVCAR-3细胞增殖,片仔癀250、500、1000μg·mL-1作用于OVCAR-3细胞24 h后,其早期凋亡率分别为6.6%、30.9%、43.2%,而对照组为0%,其诱导凋亡作用呈现剂量依赖性;细胞积聚在G0/G1期,同时S期细胞比例减少;Akt、PARP、CDK6表达下调。结论:片仔癀可以抑制OVCAR-3细胞增殖及诱导细胞凋亡作用,并能阻滞细胞于G0/G1期,有望成为卵巢癌治疗药。     

连香树精油对人肝癌SMMC-7721细胞抗肿瘤机制的研究

为探讨连香树精油的体外抗肿瘤活性。以人肝癌SMMC-7721细胞为受试细胞株,利用MTT法、流式细胞术、JC-1法和Western blot法评价连香树精油的体外抗肿瘤活性。结果表明:连香树精油处理24 h后可降低SMMC-7721细胞存活率;细胞中G0/G1期的比例下降,G2/M期的比例升高;细胞线粒体膜电位降低;自噬相关蛋白LC3-Ⅱ、Beclin-1表达上升,加入自噬抑制剂氯喹后LC3-Ⅱ蛋白与Bcl-2抗凋亡蛋白表达下降。综上说明连香树精油能抑制SMMC-7721细胞的增殖活性,通过阻滞G2/M期抑制细胞分裂,通过降低线粒体膜电位诱导SMMC-7721细胞凋亡,而自噬在SMMC-7721细胞凋亡过程中作为一种保护机制而存在。     

藻蓝蛋白抑制人结肠癌SW480细胞增殖的初步研究

目的研究藻蓝蛋白对人结肠癌SW480细胞的体外抑瘤作用,为进一步探讨藻蓝蛋白抑制肿瘤的机制提供依据。方法用不同浓度的藻蓝蛋白处理结肠癌SW480细胞后,应用MTT实验来检测藻蓝蛋白对细胞增殖的抑制作用并计算出半数抑制率,利用HE染色法和电镜技术观察凋亡细胞形态结构,采用流式细胞术分析其对SW480细胞周期的影响。结果 MTT试验证明藻蓝蛋白能够抑制SW480细胞的增殖,且呈时间和剂量依赖性;HE染色以及电镜的观察结果显示藻蓝蛋白能够诱导SW480细胞的凋亡,流式细胞术显示SW480细胞被阻滞在G2/M期,G0/G1期细胞比例降低。结论藻蓝蛋白在体外能够抑制结肠癌SW480细胞的增殖,具有可开发人结肠癌治疗光敏剂应用前景。     

曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖和凋亡的作用及其机制

观察曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖及凋亡的作用,并探讨其机制。TSA与p38抑制剂（SB203580,3μmol／L）及JNK抑制剂（SP600125,0．5μmol／L）单独或同时处理143B细胞,分别以MTT、台盼蓝染色、流式细胞术和JC—1（测定线粒体跨膜电位）法检,TSA对143B细胞的增殖、存活、周期以及凋亡的影响。应用RT-PCR、Westernblot检测Bax、Bcl．2、p38／JNK表达。结果显示,TsA能够以时间和剂量依赖方式抑制143B细胞增殖,使细胞周期阻滞于GdG。与G2／M期,并能诱导143B细胞凋亡,引起线粒体膜电位降低,促凋亡蛋白Bax表达上调,抑凋亡蛋白Bcl．2表达下调,同时使p38／ⅢK活化增加。p38／JNK4检测剂则能逆转TSA对Bax／Bcl．2的上调及抑制作用。研究结果揭示,TSA可以时间剂量依赖方式抑制143BN胞增殖,阻滞细胞周期,诱导细胞凋亡;其诱导细胞凋亡的机制可能与活化MAPK通路中p38和JNK的活性从而激发线粒体凋亡通路有关。     

Effects of the tyrosine-kinase inhibitor genistein on DNA synthesis and phospholipid-derived second messenger generation in mouse 10T1/2 fibroblasts and rat liver T51B cells

We have examined the effects of the tyrosine kinase inhibitor genistein on hormone dependent cell proliferation and intracellular signalling in mouse 10T1/2 fibroblasts and rat liver T51B epithelial cells. Genistein inhibits both PDGF and EGF induced mitogenesis with an IC50 of 40 uM and 10 uM respectively. Genistein also inhibits inositol phosphate generation and calcium signalling in response to PDGF, and 1,2-diacylglycerol generation and calpactin II translocation in response to EGF. By contrast genistein does not inhibit inositol phosphate production, Ca2+ signalling or 1,2-diacylglycerol generation in response to ATP or angiotensin II. These data demonstrate that genistein selectively inhibits tyrosine kinase dependent processes without effecting similar responses obtained to hormones which are not dependent upon tyrosine kinase activation.     

Interleukin 1 potentiates agonist-induced secretion of beta-endorphin in anterior pituitary cells

Interleukin 1 (IL-1) has been shown to potentiate the release of beta-endorphin induced by secretagogues, including corticotropin releasing factor (CRF) and phorbol ester (TPA), in the mouse AtT-20 pituitary tumor cell line (Fagarasan et al., PNAS, 1989, 86, 2070-2073). In cultured rat anterior pituitary cells, pretreatment with IL-1 caused only a small increase in beta-endorphin release but significantly potentiated CRF-and vasopressin-stimulated beta-endorphin secretion. Vasopressin stimulates the secretion of beta-endorphin in normal pituitary cells but not in AtT-20 cells. However, treatment of AtT-20 cells with IL-1 induced the expression of vasopressin-mediated beta-endorphin release; this effect of IL-1 was reduced after depletion of protein kinase C by prolonged treatment with TPA. The enhancement of CRF-stimulated beta-endorphin release by IL-1 was also reduced in AtT-20 cells after depletion of protein kinase C, and after treatment with staurosporine. These findings indicate that treatment with IL-1 amplifies receptor-mediated responses to the major physiological secretagogues in normal corticotrophs, and initiates a secretory response to vasopressin in AtT-20 cells.     

Genistein differentially inhibits postreceptor effects of insulin in rat adipocytes without inhibiting the insulin receptor kinase.

Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.     

Genistein Induces G2 Arrest in Malignant B Cells by Decreasing IL-10 Secretion

Chronic B cell malignancies are often chemoresistant and the development of new therapeutic modalities is a high priority. Many B cell malignancies have autocrine production of IL-10, which regulates B cell growth and differentiation. Here we demonstrated that the soy isoflavone genistein, a tyrosine kinase inhibitor, rapidly decreased IL-10 secretion followed by upregulation of IFN-gamma and inhibition of cell proliferation with predominantly G2 arrest. The antiproliferative effects of genistein could be reversed by the addition of exogenous IL-10. Genistein downregulated cdc25C and cdk1 as well as anti-apoptotic proteins survivin and Ian-5. After genistein withdrawal, the G2M arrested cells re-entered the cell cycle and underwent apoptosis, which was significantly augmented by fludarabine. We conclude that genistein can sensitize malignant B cells to the action of other chemotherapeutic agents by modulating the cytokine profile and controlling cell cycle progression.     

Tyrosine kinase-dependent calcium signaling in airway smooth muscle cells

Contractile agonists may stimulate mitogenic responses in airway smooth muscle by mechanisms that involve tyrosine kinases. The role of contractile agonist-evoked activation of tyrosine kinases in contractile signaling is not clear. We addressed this issue using cultured rat airway smooth muscle cells. In these cells, serotonin (5-HT, 1 microM) caused contraction (quantitated by a decrease in cell area), which was blocked by the tyrosine kinase inhibitor genistein (40 microM). Genistein and tyrphostin 23 (40 and 10 microM, respectively) significantly decreased 5-HT-evoked peak Ca(2+) responses, and the effect of genistein could be observed in the absence of extracellular Ca(2+). The specific inhibitor of mitogen-activated protein kinase kinase PD-98059 (30 microM) had no significant effect on peak Ca(2+) levels. Western analysis of cell extracts revealed that 5-HT caused a significant increase in tyrosine phosphorylation of proteins with molecular masses of approximately 70 kDa within 10 s of stimulation but no measurable tyrosine phosphorylation of the gamma isoform of phospholipase C (PLC-gamma). Tyrosine phosphorylation was inhibited by genistein. Furthermore, genistein (40 microM) significantly attenuated 5-HT-induced inositol phosphate production. We conclude that in airway smooth muscle contractile agonists acting on G protein-coupled receptors may activate tyrosine kinase(s), which in turn modulate calcium signaling by affecting, directly or indirectly, PLC-beta activity. It is unlikely that PLC-gamma or the mitogen-activated protein kinase pathway is involved in Ca(2+) signaling to 5-HT.     

Evidence of Tyrosine Kinase Activity in the Protozoan Parasite Trypanosoma brucei

ABSTRACT. Phosphorylation of proteins at tyrosine is an important mechanism for regulating cell growth and proliferation in metazoan organisms. In this report, we have demonstrated that Trypanosoma brucei , a protozoan parasite, possesses a tyrosine kinase that plays a role in regulation of proliferation of this protozoan. Genistein, a tyrosine kinase inhibitor, prevented multiplication of the parasite. An in vitro kinase assay demonstrated the presence of a kinase capable of phosphorylating an exogenous substrate at tyrosine, and genistein was able to reduce trypanosome-mediated phosphorylation of this substrate. An alkali digestion of ³²P-labeled trypanosome proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated several proteins phosphorylated at tyrosine. These results indicate that T. brucei has a tyrosine kinase that is involved in proliferation or growth regulation of the parasite and provide further evidence for the possibility of growth factor regulation and signal transduction in trypanosomes.     

Effect of genistein, a tyrosine kinase inhibitor, on latent EBV activation induced by cross-linkage of membrane IgG in Akata B cells.

The activation of phosphoprotein tyrosine kinases was studied in the regulation of EBV activation in Akata cells after cross-linking membrane IgG with anti-IgG. Protein tyrosine phosphorylation was induced in Akata cells after stimulation with anti-IgG, as determined by immunoblotting with the PY20 anti-phosphotyrosine mAb. The frequency of phosphotyrosine-activated cells was also measured by immunofluorescence with the PY20 antibody. Genistein, an inhibitor of tyrosine kinases, at non-cytotoxic doses blocked EBV activation, as measured in the induction of EBV Ag, EBV immediate early BZLF1 mRNA, and its protein product ZEBRA. Such inhibitions were reversed upon removing genistein from the cultures. Genistein inhibition of early Ag induction depended upon the time of addition of genistein after stimulation with anti-IgG. These findings indicate that activation of tyrosine kinase is required for EBV activation after cross-linking membrane IgG in Akata cells.     

Genistein and erbstatin inhibition of normal mammary epithelial cell proliferation is associated with EGF-receptor down-regulation

Epidermal growth factor (EGF) is a potent mitogen for normal mouse mammary epithelial cells grown in primary culture. EGF activation of the EGF-receptor (EGF-R) induces intrinsic tyrosine kinase activity which results in EGF-R autophosphorylation and tyrosine phosphorylation of other intracellular substrates involved in EGF-R signal transduction. Genistein and erbstatin are anticancer agents which have been shown to be potent tyrosine kinase inhibitors. However, the effects of these compounds in modulating EGF-dependent normal mammary epithelial cell proliferation is presently unknown. Therefore, studies were conducted to determine the effects of genistein and erbstatin on EGF-dependent proliferation, and EGF-R levels and autophosphorylation in normal mouse mammary epithelial cells grown in primary culture and maintained in serum-free media. Chronic treatment with 6.25–100 μM genistein or 1–16 μM erbstatin significantly decreased EGF-dependent mammary epithelial cell proliferation in a dose-responsive manner. However, the highest doses of genistein (100 μM ) and erbstatin (16 μM ) were found to be cytotoxic. Additional studies showed that acute treatment with 6.25–400 μM genistein did not affect EGF-R levels or EGF-induced EGF-R autophosphorylation, while acute treatment with 1–64 μM erbstatin caused a slight reduction in EGF-R levels, but had no effect on EGF-dependent EGF-R autophosphorylation in these cells. In contrast, chronic treatment with similar doses of genistein or erbstatin resulted in a large dose-responsive decrease in EGF-R levels, and a corresponding decrease in total cellular EGF-R autophosphorylation intensity. These results demonstrate that the inhibitory effects of chronic genistein and erbstatin treatment on EGF-dependent mammary epithelial cell proliferation is not due to a direct inhibition of EGF-R tyrosine kinase activity, but results primarily from a down-regulation in EGF-R levels and subsequent decrease in mammary epithelial cell mitogenic-responsiveness to EGF stimulation.     

Growth hormone-releasing factor releases ACTH from an AtT-20 mouse pituitary tumor cell line but not from normal pituitary cells

Corticotropin-releasing factor (CRF) and both human pancreatic growth hormone-releasing factor (hp-GRF) and rat hypothalamic GRF (rh-GRF) stimulated ACTH release from neoplastic AtT-20 mouse pituitary tumor cells in a dose-dependent fashion, with CRF inducing a 10-fold increase and GRF a maximal increment of approximately one-half that of CRF. Neither rh-GRF nor hp-GRF induced ACTH release in normal anterior pituitary cells. Pretreatment with either dexamethasone or somatostatin prior to the addition of rh-GRF inhibited the increase in ACTH release. Both ovine CRF and rh-GRF stimulated adenosine 3,5-monophosphate production in AtT-20 cells. The weak but clearly discernible effect of GRF on ACTH release from AtT-20 cells may be due to an abnormality in the AtT-20 cell receptor.     

Effect of the tyrosine kinase inhibitor, genistein, on interleukin-1 stimulated PGE2 production in mesangial cells

Prostaglandin production and cAMP formation are two signaling pathways identified for IL-1, though neither adequately account for the multitude of effects of IL-1. To investigate the role of tyrosine phosphorylation in IL-1 signaling, we used the tyrosine kinase inhibitor, genistein. At 10-30 micrograms/ml, genistein blocked IL-1 stimulated prostaglandin production and induction of prostaglandin endoperoxide synthase (PES) in glomerular mesangial cells maintained in 10% serum. Addition of genistein hours after IL-1 addition also halted further PGE2 synthesis. Genistein failed to block PES activity in vitro, indicating it was not acting as a PES inhibitor. Overall these data suggest that tyrosine phosphorylation may be a required event for IL-1 stimulation of PGE2 and PES activity, either directly as part of IL-1 signaling, or indirectly as part of a serum/PDGF competence effect on mesangial cells.