白杨素对人肝癌BEL-7402细胞表面超微结构和蛋白质磷酸化及相关通路的影响

背景：白杨素是保健食品和中草药中常见的具有防癌抗癌活性的食源黄酮类天然产物。白杨素是否有助于防治肝癌？其作用机制为何？是否促进肝癌干细胞的分化？对此尚缺乏系统研究。目的：以人肝癌细胞为对象,揭示白杨素的亚细胞和分子作用机制。方法：应用酸性磷酸酶实验、扫描电镜、免疫印迹技术探讨白杨素对BEL-7402细胞的作用及其机制。结果与结论：白杨素显著抑制BEL-7402细胞的体外增殖,半数抑制浓度IC50为24.9mol/L或6.3mg/L。白杨素与PI3K-AKT途径特异性抑制剂LY294002联用具有显著的协同抗癌效应。但白杨素本身并不改变AKT、磷酸化pAKT、AKT下游分子GSK-3和磷酸化pGSK-3的表达水平,虽然高剂量下β-catenin略有下调。白杨素导致细胞表面微绒毛样突起密度明显增加并形成纳米膜粒;细胞回缩,间隙加宽;出现形态上不同于凋亡的死亡细胞。白杨素显著改变多种蛋白质的丝/苏氨酸磷酸化水平;对酪氨酸磷酸化亦有广谱下调作用,接近于表柔比星对照。白杨素对CDK1、磷酸化pCDK1和pCDK2、CDC25A、CDC25B和CD133均无明显影响,但可剂量依赖性诱导PARP-1降解。总之,白杨素对BEL-7402细胞的抑制作用并非起因于PI3K-AKT和Wnt/β-catenin通路阻断或细胞周期核心调控因子阻断,而更可能起因于生物膜功能障碍所致细胞表面超微结构和蛋白质磷酸化的大范围改变,并以非经典方式诱导肝癌细胞死亡。     

PI3K/AKT/FKHRL1信号通路对CXLCl6诱导的平滑肌细胞增殖的影响

目的 探讨PI3K/AKT/FKHRL1信号转导通路在CXCL16诱导的血管平滑肌增殖中的作用。方法 运用细胞计数法及MTT法检测CXCL16对于平滑肌细胞增殖的影响;免疫组化法观察CXCL16对PI3K/AKT/FKHRL1信号转导通路的作用,同时观察PI3K/AKT抑制剂LY294002对CXCL16诱导的上述变化的影响。结果 CXCL16可明显诱导平滑肌细胞增殖,作用高峰时间在第4天,并且可增加磷酸化AKT及磷酸化FKHRL1的表达。PI3K/AKT阻断剂LY294002可明显抑制CXCL16诱导的平滑肌增殖,同时下调磷酸化AKT及磷酸化FKHRL1的表达。结论 CXCL16可能是通过激活平滑肌细胞的AKT通路,诱导AKT及FKHRL1的磷酸化,从而影响平滑肌细胞的增殖。LY294002可以阻断PI3K/AKT/FKHRL1通路而抑制CXCL16诱导的平滑肌细胞增殖。     

蛋白激酶B活化诱导骨桥蛋白表达与肝癌细胞骨转移间的关系

目的:研究蛋白激酶B(AKT)活化诱导骨桥蛋白(OPN)表达在肝细胞肝癌(HCC)骨转移中的作用及其机制。方法:收集40例HCC骨转移灶及其原发灶标本,并另选取22例无转移的HCC组织标本作为对照,行免疫组化染色检测AKT、OPN表达水平;同时用miR-21表达载体转染人HCC细胞系BEL-7402,诱导AKT磷酸化激活,同时用AKT特异性抑制剂(MK-2206)抑制AKT磷酸化激活,分别观察AKT磷酸化水平(p-AKT)与OPN表达间的关系;采用OPN特异性小干扰RNA(OPN-siRNA)沉默BEL-7402细胞OPN表达,并用MTT法检测其对细胞增殖的影响。结果:在发生HCC骨转移灶中,p-AKT和OPN阳性率分别为85.00%(34/40)和77.50%(31/40),明显高于其相应的HCC原发灶组织(p-AKT为60.00%;OPN为55.00%,P<0.05)及无转移的HCC组织(p-AKT为54.55%;OPN为36.36%,P<0.01);BEL-7402细胞转染miR-21诱导p-AKT上调后,OPN表达升高并促进肿瘤细胞增殖加速;反之,MK-2206抑制AKT磷酸化后,BEL-7402细胞OPN表达下降,肿瘤细胞增殖速率降低;采用特异siRNA沉默OPN表达,同样可抑制肿瘤细胞增殖。结论:HCC细胞OPN高表达与AKT信号通路的激活有关,AKT磷酸化水平的升高能诱导OPN表达,进而促进HCC细胞的增殖和转移,提示检测p-AKT与OPN的表达水平对于HCC恶性生物学行为的判断具有潜在的应用价值。     

PTEN/PI3K/AKT信号通路与儿童急性淋巴细胞白血病原代细胞凋亡耐药的相关性

目的:探讨儿童急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)原代细胞中PTEN/PI3K/AKT信号通路蛋白表达与细胞凋亡和柔红霉素(daunorubicin,DNR)耐药的关系。方法:分离45例初诊未治儿童急性B系ALL(B-ALL)骨髓单个核细胞进行原代细胞培养,用终浓度0.5 mg/L的DNR干预24 h后使用细胞凋亡试剂盒检测凋亡率;于培养开始时和药物干预完成后取标本应用Western blot方法检测PTEN、PI3K、AKT蛋白表达水平,并进行各检测指标间的相关性分析。结果:DNR干预后,PTEN低表达组凋亡率较PTEN高表达组低(P0.05),显示凋亡率与干预前PTEN表达呈强正相关;PI3K-AKT低表达组凋亡率较PI3K-AKT高表达组高(P0.01);DNR干预后PI3K-AKT蛋白表达降低(P0.01)。结论:儿童B-ALL原代细胞中PTEN表达存在差异,DNR干预后PTEN表达变化不显著,提示PTEN表达与DNR耐药相关。DNR可通过下调PI3K-AKT信号通路蛋白表达,诱导儿童B-ALL原代细胞凋亡。     

信号通路抑制剂XL765对人白血病KG-1细胞株的抑制效应研究

目的:探讨PI3K/mTOR信号通路抑制剂XL765(SAR245409, Voxtalisib)对人急性髓系白血病细胞株KG-1细胞的抑制效应及可能的作用机制。方法:应用CCK-8法检测XL765对KG-1细胞增殖的影响;平板集落形成实验检测XL765对KG-1细胞集落形成的抑制情况;Annexin V/PI双染色流式细胞术检测XL765对细胞凋亡的影响;实时荧光定量PCR检测细胞凋亡相关基因的表达;Western blot法检测凋亡相关蛋白的表达水平及信号通路分子磷酸化水平的变化。结果:XL765能有效抑制KG-1细胞的增殖,抑制率呈剂量依赖性增加;与DMSO处理组相比,加入XL765后,KG-1细胞的集落形成能力显著下降(P=0.0002);XL765能有效诱导细胞凋亡(P0.001);XL765作用KG-1细胞48 h后,细胞的凋亡相关基因BCL-2表达下调,BAX及Caspase3表达上调,其差异均有统计学意义(P0.05);与DMSO组对比,实验组BAX及Caspase3活化蛋白表达上调,同时BCL-2蛋白表达下调,信号蛋白PI3K、AKT及S6K磷酸化表达下调,其差异均有统计学意义(P值均0.005)。结论:XL765能有效抑制KG-1细胞增殖及集落形成,并诱导细胞凋亡,其机制可能与调节凋亡蛋白BCL2、BAX及Caspase3水平及抑制PI3K、AKT及S6K磷酸化水平相关。     

苯丁酸氮芥抗套细胞淋巴瘤细胞株Jeko-1作用机制的研究

目的:探讨苯丁酸氮芥抗套细胞淋巴瘤细胞株Jeko-1作用机制。方法:应用MTT法检测苯丁酸氮芥对Jeko-1细胞增殖的影响,Hoechst染色及Annexin V-FITC双染检测苯丁酸氮芥对Jeko-1细胞凋亡的影响,Western blot检测苯丁酸氮芥对Jeko-1细胞凋亡相关蛋白BAX,BCL-2,procaspase 3,procaspase 8,procaspase 9表达水平及PI3K/AKT信号通路的激活情况。结果:苯丁酸氮芥0、5、10和20μmol/L作用Jeko-1细胞24、48、72 h后,细胞增殖受到抑制,并呈时间及剂量依赖性(r=0.873,r=0.932)。0,5,10,20μmol/L苯丁酸氮芥作用Jeko-1细胞72 h后,细胞凋亡率显著提高,BAX,procaspase 3,procaspase 8及procaspase 9蛋白表达水平显著上调,BCL-2表达水平显著下调,PI3K蛋白及AKT磷酸化水平显著降低。结论:苯丁酸氮芥能显著诱导套细胞淋巴瘤细胞株Jeko-1凋亡,其作用机制与调节PI3K/AKT信号通路有关。     

雷公藤甲素诱导急性髓细胞白血病干细胞凋亡及其机制

目的探讨雷公藤甲素诱导急性髓细胞白血病干细胞凋亡的分子机制。方法采用MTT法检测雷公藤甲素对KG-1细胞的增殖抑制作用,计算出IC50值,按照浓度梯度用雷公藤甲素处理KG-1细胞48 h,流式细胞仪检测细胞凋亡变化。Western blot法检测Bcl-2、β-catenin、p-AKT和AKT等蛋白的变化。结果雷公藤甲素对KG-1细胞的增殖抑制呈剂量依赖性,IC50值约为4 nmmol/L,其还可诱导KG-1细胞凋亡。雷公藤甲素能够下调Bcl-2、β-catenin,p-AKT等蛋白的表达水平。结论雷公藤甲素诱导KG-1细胞的分子机制可能与Bcl-2、β-catenin、p-AKT的下调有关。     

外源性PDGF-DD蛋白对肝癌细胞株BEL-7402的体外作用

目的:探讨外源性PDGF-DD蛋白对PDGF-D和β-PDGFR阳性表达的人肝癌细胞株BEL-7402增殖和转移相关基因VEGF及CXCR4表达的影响.方法:浓度为0 ～ 100 pg/mL PDGF-DD蛋白作用于肝癌细胞株BEL-7402 48 h,MTT检测细胞活性;流式细胞仪检测细胞周期;RT-PCR检测VEGF mRNA和CXCR4 mRNA表达.结果:外源性PDGF-DD蛋白干预细胞后,可促进细胞增殖,且在0 ～ 100 pg/mL范围内呈剂量依赖性;周期分布显示G0/G1期细胞比例下降,S期细胞比例增多;且上调VEGF mRNA和CXCR4 mRNA的表达.结论:外源性PDGF-DD能促进BEL-7402细胞增殖,上调VEGF、CXCR4 mRNA的表达.提示PDGF-D及其信号传导系统在肝癌的发生、发展及转移中可能发挥重要的作用,可作为肝癌预后预测指标和治疗靶点.     

体外神经干细胞培养过程中蛋白激酶磷脂酰肌醇-3激酶/AKT信号途径的作用

目的:探讨磷脂酰肌醇-3激酶(phosphatidylinositol-3kinase,PI3K)/AKT信号途径在体外培养神经干细胞存活、分化中的作用。方法:实验于2001-11/2004-04在重庆医科大学神经病学研究所完成。分为对照组、PI3K特异性抑制剂wortmannin组和LY294002组;采用间接免疫荧光法检测nestin,NF-200和GFAP的表达;TUNEL评价细胞凋亡率;WesternBlot法检测磷酸化的AKT,caspase-9和bad的表达。结果:随着wortmannin和LY294002浓度增大,神经干细胞的形态变化逐渐明显,而且细胞凋亡率也逐渐增加。低浓度时(wortmannin浓度为5nmol/L,LY294002浓度为2μmol/L),神经干细胞凋亡率与对照组无显著性意义(P>0.05);高浓度时(wortmannin浓度为50,100nmol/L,LY294002浓度为50,100μmol/L),神经干细胞凋亡率与对照组、低浓度组有非常显著性意义(P<0.01)。WesternBlot结果提示,随着PI3K特异性抑制剂浓度增大,磷酸化AKT的表达逐渐减弱。当高浓度wort-mannin(20nmol/L)和LY294002(10μmol/L)抑制了AKT磷酸化后,磷酸化的caspase-9,Bad表达减弱。此外,将wortmannin组和LY294002组存活的细胞接种后,分化后细胞的NF-200,GFAP表达无显著性意义。结论:神经干细胞存活依赖于PI3K/AKT途径的活化,其机制可能是PI3K/AKT活化后,促进了Bad和caspase-9等蛋     

微小RNA在肝癌耐药Bel-7402/5-FU细胞中的表达分析

目的:通过对比肝癌亲本Bel-7402细胞及其5-FU耐药Bel-7402/5-FU细胞中微小RNA(microRNA, miRNA)的表达差异,探讨miRNA在肝癌细胞多药耐药中的作用。方法:体外培养Bel-7402细胞及其耐药株Bel-7402/5-FU细胞,MTT法检测Bel-7402/5-FU细胞对不同化疗药物的敏感性,miRNA表达芯片检测Bel-7402细胞和Bel-7402/5-FU细胞miRNA的差异表达,qPCR验证差异表达的miRNA。结果:Bel-7402/5-FU细胞对不同的化疗药物均具有耐药性;miRNA芯片检测结果发现,耐药株Bel-7402/5-FU细胞与亲本Bel-7402细胞中,差异表达的miRNA有29个,其中上调14个,下调15个;qPCR验证结果发现,和亲本Bel-7402细胞比较,耐药株Bel-7402/5-FU细胞中miR-10a-5p、-371a-3p、-371a-5p、-372,-373-3p、-146-5p、-6723-5p显著下调,miR-4497、-3148显著上调。结论:肝癌多药耐药的发生与miRNA表达异常有关。     

Chrysin inhibits expression of hypoxia-inducible factor-1alpha through reducing hypoxia-inducible factor-1alpha stability and inhibiting its protein synthesis

Chrysin is a natural flavonoid and has been shown recently to have anticancer effects. However, the mechanisms that chrysin inhibits cancers are not well known. In this study, we investigated the effects of chrysin on expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor in human prostate cancer DU145 cells. Chrysin inhibited insulin-induced expression of HIF-1alpha by reducing its stability. Chrysin increases ubiquitination and degradation of HIF-1alpha by increasing its prolyl hydroxylation. In addition, chrysin interfered with interaction between HIF-1alpha and heat shock protein 90. Chrysin was also found to inhibit HIF-1alpha expression through AKT signaling. Inhibition of HIF-1alpha by chrysin resulted in abrogation of vascular endothelial growth factor expression. Finally, we showed that chrysin inhibited DU145 xenograft-induced angiogenesis in nude mice. Taken together, these results suggest that chrysin is a potent inhibitor of HIF-1alpha and provide a new sight into the mechanisms of chrysin against cancers.     

热化疗逆转人肝癌细胞系BEL-7402/ADM多药耐药性的实验研究

目的　研究热化疗对多药耐药人肝癌细胞系BEL- 74 0 2 /ADM耐药性的影响并探讨相关的机制。方法　通过MTT法测定热化疗对多药耐药人肝癌细胞系BEL -74 0 2 /ADM耐药性影响;利用流式细胞仪检测多药耐药相关基因(MDR)的表达产物P糖蛋白(P- gp)、多药耐药相关蛋白(MRR)、谷胱甘肽转移酶(GSH/GST)的表达。结果　热化疗使多药耐药人肝癌细胞系BEL- 74 0 2 /ADM耐药性降低;P -gp、MRP和GSH/GST的表达均有所下降。结论　热化疗具有逆转多药耐药人肝癌细胞系BEL -74 0 2 /ADM耐药性的作用,可能与多药耐药相关基因表达水平下调有关。     

RPMI-1640和DMEM培养基中肝癌细胞系BEL-7402与HepG-2的生长状态比较

背景：DMEM和RPMI-1640是两种最常用的商品化培养基,二苔对肝癌细胞生长的影响尚未见直接的列比研究．目的：比较两种常用培养基对人肝癌BEL-7402与HepG-2细胞系体外生长和增殖效果的影响,从中选择更适合的培养肇．方法：分别应用高糖DMEM与RPMI-1640完全培养液培养BEL-7402和HepG-2细胞,于培养的0,24,48,72,96,120h用酸性磷酸酶检测法测定细胞生长和增殖速率,并于倒置显微镜下观察细胞的形态。结果与结论：结果发现BEL-7402和HepG-2细胞在RPMI-1640培养基中的生长增殖速率均明显高RDMEM培养碡（P〈0.01）。镜下观察证实细胞在RPMI-1640培养基中的黏附和伸展状态更好。因此,建议阿选RPMI-1640培养基进行肿瘤细胞体外培养。     

乙酰肝素酶反义寡核苷酸对人肝癌细胞株乙酰肝素酶表达的影响

目的:探讨乙酰肝素酶(Hpa)反义寡脱氧核苷酸对人肝癌细胞株Hpa mRNA和蛋白表达的影响。方法:设计合成互补于Hpa mRNA起始密码区的硫代反义寡脱氧核苷酸AS-ODN及相应的无义对照寡脱氧核苷酸NS-ODN,以阳离子脂质体Oligofectamine~(TM) Reagent包埋后转染SMMC-7721、BEL-7402细胞,以RT-PCR和Western-blot检测转染前后细胞Hpa mRNA以及蛋白表达的变化。结果:与正常对照组和NS-ODN相应浓度组相比,转染AS-ODN组肝癌细胞Hp amRNA和蛋白的表达均下降。结论:Hpa反义寡脱氧核苷酸下调Hpa mRNA和蛋白的表达,可能抑制肝癌的侵袭转移。     

小柴胡汤逆转人肝癌细胞BEL-7402／5-FU 的多药耐药作用

目的:研究小柴胡汤对BEL-7402/5-FU细胞多药耐药的逆转作用,并初步探讨其作用机制。方法:采用四甲基偶氮唑蓝(MTT)法检测不同化疗药物以及小柴胡汤对BEL-7402/5-FU细胞的半数抑制浓度(IC50),计算耐药指数和小柴胡汤的逆转倍数;流式细胞仪检测细胞内阿霉素的荧光强度;RT-PCR检测mdr1基因表达;western blot检测p-gp蛋白表达。结果:BEL-7402/5-FU耐药株对5-FU和阿霉素顺铂均具有耐药性,耐药指数分别是6 501.99和31.73;0.5 mg/ml小柴胡汤和5-FU、阿霉素联合应用,其逆转倍数分别是1.66、1.50;1.0 mg/ml小柴胡汤和5-FU、阿霉素联合应用,其逆转倍数分别是3.44、5.58;小柴胡汤增强阿霉素的荧光强度;小柴胡汤降低mdr1基因表达和p-gp蛋白表达,各个浓度均具有统计学意义。结论:小柴胡汤可以抑制mdr1基因编码蛋白产物p-gp的表达,增加细胞内化疗药物的浓度,从而逆转了肝癌细胞的多药耐药性。     

Chrysin,a flavonoid attenuates histological changes of hyperammonemic rats: A dose dependent study

Chrysin (5,7-dihydroxyflavone) is a major component of some traditional medicinal herbs present in honey, propolis and many plant extracts. The study was aimed to illuminate the effect of chrysin in the pathogenesis of ammonium chloride (NH₄Cl) induced hyperammonemic rat model in a dose dependent manner. Rats were injected with NH₄Cl (100 mg/kg b.w.) by intraperitonially (i.p) thrice a week for 8 consecutive weeks for the induction of experimental hyperammonemia. Hyperammonemic rats were treated with chrysin by orally at a dose of 25, 50 & 100 mg/kg b.w. respectively. Protective effect of chrysin against hyperammonemia was evaluated by performing biochemical estimations and morphopathological investigations of hematoxylin and eosin stained sections of liver, brain and kidney tissues. Supplementation of chrysin reinstated the levels of blood ammonia, plasma urea, uric acid, total bilirubin, creatinine, brain glutamate, glutamine, nitric oxide (NO) and the activities of Na⁺/K⁺-ATPase, and liver marker enzymes. On the other hand increased level of plasma urea was observed in chrysin treated rats as compared with hyperammonemic rats. Chrysin administration caused distortion of hepatic, brain and kidney architecture as shown by histological examination. Chrysin at a dose (100 mg/kg b.w.) showed an utmost decline in the level of all biochemical estimations. Both biochemical and morphological studies clearly revealed that chrysin protects against cell injury induced by ammonia intoxication in a dose-response manner with respect to endogenous antioxidants and hypoammonemic effects.     

Influence of chrysin on D-galactose induced-aging in mice: Up regulation of AMP kinase/liver kinase B1/peroxisome proliferator-activated receptor-γ coactivator 1-α signaling pathway

Adenosine monophosphate kinase/liver kinase B1/peroxisome proliferator-activated receptor-γ coactivator 1-α (AMPK/LKB1/PGC1α) pathway has a vital role in regulating age-related diseases. It controls neurogenesis, cell proliferation, axon outgrowth, and cellular energy homeostasis. AMPK pathway also regulates mitochondrial synthesis. The current study evaluated the effect of chrysin on D-galactose (D-gal) induced-aging, neuron degeneration, mitochondrial dysfunction, oxidative stress, and neuroinflammation in mice. The mice were allocated randomly into four groups (10 each group): Group 1: normal control group, Group 2: D-gal group, Groups 3 and 4: chrysin (125 and 250 mg/kg, respectively). Groups 2–4 were injected with D-gal (200 mg/kg/day; s.c) for 8 weeks to induce aging. Groups 3 and 4 were orally gavaged every day concurrent with D-gal. At the end of experiment, behavioral, brain biochemical and histopathological changes were monitored. Chrysin administration elevated discrimination ratio in object recognition, Y Maze percentage alternation, locomotor activity and brain contents of AMPK, LKB1, PGC1α, NAD (P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), nerve growth factor (NGF) (neurotrophin-3; NT-3), and seretonin as well as reduced brain contents of tumor necrosis factor-alpha (TNF-α), nuclear factor kappa B (NF-κB), advanced glycation end products (AGEs) and glial fibrillary acidic protein (GFAP) compared to D-gal-treated mice. Chrysin also alleviated cerebral cortex and white matter neurons degeneration. Chrysin protects against neurodegeneration, improves mitochondrial autophagy and biogenesis as well as activates antioxidant genes expression. In addition, chrysin ameliorates neuroinflammation and stimulates the release of NGF and serotonin neurotransmitter. So, chrysin has a neuroprotective effect in D-gal induced-aging in mice.     

Combined inhibitory effects of celecoxib and fluvastatin on the growth of human hepatocellular carcinoma xenografts in nude mice

This study was designed to investigate the in vivo growth inhibitory effects of celecoxib, a cyclo-oxygenase-2 inhibitor, and fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on the hepatocellular carcinoma (HCC) cell line, BEL-7402. Athymic nude mice implanted with BEL-7402 cells were given celecoxib and fluvastatin, either alone or in combination, and the effect of treatment on tumour growth was evaluated after 6 weeks. The combination of celecoxib and fluvastatin enhanced inhibition of tumour growth, induction of apoptosis, inhibition of tumour cell proliferation, and inhibition of tumour angiogenesis compared with either treatment alone. The combination of celecoxib and fluvastatin also increased levels of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1), decreased levels of p-Akt, myeloid cell leukaemia-1 (Mcl-1) and survivin protein, but had no effect on Akt protein levels in tumours. These results suggest that celecoxib combined with fluvastatin would be more efficacious for the treatment of HCC than either treatment alone and this combination of therapy warrants further research.