槲皮素对胃癌细胞SGC-7901上皮间质转化的抑制作用

目的探讨槲皮素对人胃癌细胞SGC-7901上皮间质转化的影响及机制。方法胃癌细胞SGC-7901分为对照组(普通培养液)、LY294002组(培养液+LY294002)、槲皮素组(培养液+槲皮素);采用MTT法检测槲皮素对胃癌细胞SGC-7901增殖的影响,依据细胞生长抑制率计算槲皮素对胃癌细胞SGC-7901的半数抑制浓度(median inhibitory concentration,IC_(50))值,参照IC_(50)值进行后续实验;采用划痕试验检测3组胃癌细胞SGC-7901增殖、迁移能力;采用Western blot法检测胃癌细胞AKT、p-AKT、Snail、Vimentin及E-钙黏附素(E-cadherin)蛋白表达情况;采用荧光定量PCR法检测3组胃癌细胞上皮间质转化相关因子Snail、Vimentin及E-cadherinmRNA的表达情况。结果槲皮素对胃癌细胞SGC-7901有明显的增殖抑制作用,其IC_(50)为(112.9±2.05)μmol/L;LY294002组胃癌细胞迁移距离[(0.16±0.03)mm]、槲皮素组胃癌细胞迁移距离[(0.15±0.02)mm]均低于对照组[(0.44±0.04)mm](P0.05),LY294002组与槲皮素组比较差异无统计学意义(P0.05);Snail、Vimentin、p-AKT蛋白表达水平在LY294002组(0.760±0.003,0.750±0.006,0.71±0.03)、槲皮素组(0.750±0.006,0.690±0.004,0.68±0.02)均明显低于对照组(1,1,1),E-cadherin表达在LY294002组(2.89±0.19)与槲皮素组(3.66±0.18)均高于对照组(1)(P0.05);LY294002组、槲皮素组、对照组AKT蛋白表达水平(1.03±0.02,1.02±0.01,1)比较差异无统计学意义(P0.05);Snail、Vimentin mRNA表达水平在LY294002组(0.72±0.02,0.78±0.03)、槲皮素组(0.71±0.01,0.79±0.03)均明显低于对照组(1,1),E-cadherin mRNA表达水平在LY294002组(2.51±0.08)、槲皮素组(2.77±0.16)均高于对照组(1)(P0.05);LY294002组Snail、Vimentin、E-cadherin mRNA及Snail、Vimentin、E-cadherin、p-AKT蛋白表达水平与槲皮素组比较差异均无统计学意义(P0.05)。结论槲皮素对人胃癌细胞SGC-7901上皮间质转化有抑制作用,其作用机制可能是抑制AKT信号通路。     

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     

OTUB2表达下调对胃癌细胞增殖、迁移、侵袭能力及PI3K/AKT信号通路的影响

目的探讨含OTU结构域的泛素醛结合蛋白2(OTUB2)表达下调对胃癌细胞增殖、迁移、侵袭能力及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路的影响。方法采用Lipofectmine 2000转染试剂向MGC-803细胞中转染OTUB2小干扰RNA(si-OTUB2)序列(si-OTUB2组)和si-NC序列(si-NC组),以不进行任何转染的胃癌MGC-803细胞作为空白对照组。分别采用实时荧光定量PCR和蛋白质印迹法(Western blot)检测细胞中OTUB2mRNA及蛋白的表达水平。采用CCK-8法检测细胞增殖能力,Transwell小室检测细胞迁移和侵袭能力,Western blot检测增殖细胞核抗原(PCNA)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、AKT、磷酸化蛋白激酶B(p-AKT)蛋白表达水平。结果与空白对照组和si-NC组比较,siOTUB2组细胞中OTUB2mRNA及蛋白表达水平降低(P0.05),细胞增殖能力减弱(P0.05),细胞迁移数和侵袭数减少(P0.05),细胞中PCNA、N-cadherin和p-AKT蛋白表达水平降低(P0.05),E-cadherin蛋白表达水平升高(P0.05)。结论下调OTUB2可以抑制胃癌细胞的增殖、迁移、侵袭,其作用机制可能与抑制PI3K/AKT信号通路有关。     

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-3 kinase，P13K)／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、P13K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；Western Blot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&;gt;0．05)；高浓度时(wonmannin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&;lt;0．01)。Western Blot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woftmannin(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等蛋白的磷酸化，抑制了细胞凋亡事件的发生。但是PI3K／AKT途径可能在神经干细胞的分化中的作用甚微。     

加巴喷丁通过PI3K-AKT信号通路减轻大鼠心肌缺血—再灌注损伤北大核心CSCD

目的观察加巴喷丁对心肌缺血-再灌注损伤的影响,并进一步探讨其机制。方法清洁级雄性SD大鼠60只,10周龄,体重250 g~300 g,采用随机数字表法分为5组,每组12只:假手术组(Sham组)、心肌缺血-再灌注组(I/R组)、加巴喷丁组(Gap组)、LY294002组(LY组)和加巴喷丁+LY294002组(Gap+LY组)。采用左冠状动脉前降支结扎30 min和再灌注120 min,制备心肌缺血-再灌注损伤模型。记录心肌缺血前基线(T0)、缺血30 min(T1)和再灌注120 min(T2)时大鼠的心率(Heart rate,HR)、平均动脉压(mean arterial pressure,MAP)和心率血压乘积(Rate pressure product,RPP),评价心肌缺血再灌注期间血流动力学变化;记录室性早搏(Premature ventricular complexes,PVCs)和室速/室颤(ventricular tachycardia/ventricular fi brillation,VT/VF)次数,评价缺血-再灌注期间心律失常。术毕取心肌组织采用2,3,5-氯化三苯基四氮唑(2,3,5-Triph enyltetrazoliumchloride,TTC)染色法,检测心肌梗死面积;免疫印迹法检测心肌组织PI3K和p-AKT蛋白表达水平。结果与I/R组相比,Gap组心肌梗死面积显著下降,PVCs和VT/VF次数显著减少,PI3K蛋白表达水平和p-AKT/AKT比值显著升高(P<0.05)。与Gap组相比,Gap+LY组心肌梗死面积显著增加,PVCs和VT/VF次数显著升高,PI3K蛋白表达水平和p-AKT/AKT比值显著降低(P<0.05)。结论加巴喷丁可以减轻心肌缺血再灌注损伤,与激活PI3K-AKT信号通路有关。     

人参皂苷Rh2对人急性T淋巴细胞白血病Jurkat细胞诱导凋亡的作用及其机制

目的:探讨人参皂苷Rh2诱导人急性T淋巴细胞白血病(T-cell acute lymphoblastic leukemia,T-ALL)Jurkat细胞的凋亡及其机制。方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度的Rh2(0、10、20、40和80μg/ml)对Jurkat细胞增殖活性的影响,并计算48 h下Rh2对Jurkat细胞的半抑制浓度(IC_(50));采用形态学方法及Hoechst 33258荧光染色观察IC_(50)剂量下Rh2共培养48 h的Jurkat细胞凋亡状况。后将细胞实验分为4组:对照组、Rh2(IC_(50))组、PI3K抑制剂LY294002(50μmol/l)组以及Rh2(IC_(50))+LY294002(50μmol/l)组。同步培养48 h后,采用PI单染和Annexin V-FITC/PI双染分别检测Jurkat细胞凋亡及细胞周期变化;采用Western blot检测各组Jurkat细胞凋亡相关蛋白BAX、BCL-2、Cleaved-Caspase 3,细胞周期相关蛋白Cyclin D1以及PI3K/AKT信号通路相关蛋白AKT、p-AKT的表达水平。结果:Rh2(10-80μg/ml)呈剂量-时间依赖性抑制Jurkat细胞增殖(r_(48h)=0.999,P 0.01;r_(80μg/ml)=0.991,P 0.05),并伴有明显的细胞凋亡形态学改变。流式细胞术检测结果显示,与对照组比较,Rh2组和LY294002组细胞凋亡率显著增加,且细胞多数被阻滞在G0/G1期;而与Rh2组和LY294002组比较,Rh2+LY294002组细胞凋亡及细胞阻滞现象更为显著。Western blot结果显示,与对照组比较,Rh2能显著促进BAX、Cleaved-Caspase 3蛋白表达,抑制BCL-2、Cyclin D1及p-AKT的表达,且LY294002对这一效应具有显著的促进作用。结论:Rh2能够呈时间-剂量依赖性地诱导Jurkat细胞的凋亡;且能对Jurkat细胞产生明显的G0/G1期阻滞;这可能与Rh2抑制PI3K/AKT通路进而介导的一系列凋亡信号级联反应密切相关。     

肠三叶因子介导PI3K/Akt信号通路保护胃黏膜上皮细胞紧密连接

目的：探讨肠三叶因子对胃黏膜上皮细胞紧密连接的保护作用,并研究PI3K/Akt信号通路在其中的作用机制。方法：体外培养GES-1细胞,分别设正常对照组,LPS组,ITF组,LPS＋ITF组,LPS＋ITF＋LY294002组,ITF＋LY294002组。正常对照组：正常培养;LPS组：加入浓度为10mg/L的LPS;ITF组：加入浓度为100μg/L的ITF;LPS＋ITF组：加入浓度为10mg/L的LPS,同时加入100μg/L的ITF;LPS＋ITF＋LY294002组：加入浓度为10mg/L的LPS、100μg/L的ITF,同时加入PI3K/Akt信号通路的抑制剂LY294002（15μM）;ITF＋LY294002组：加入100μg/L的ITF,同时加入15μM的LY294002。培养48h,采用Western blot检测ITF对PI3K/Akt信号通路的作用,采用免疫荧光和Western blot检测细胞紧密连接蛋白Occludin和ZO-1的变化情况。结果：Western blot检测结果说明,与对照组相比,ITF提高了pAkt蛋白的表达水平,而LY294002抑制了ITF激活的pAkt蛋白的表达,说明ITF可以通过激活PI3K/Akt信号通路来调控GES-1细胞的生理活动。免疫荧光和Western blot结果显示LPS导致GES-1细胞的紧密连接遭到破坏,降低紧密连接蛋白Occludin和ZO-1的表达水平,而ITF可以通过激活PI3K/Akt信号通路来保护GES-1细胞的紧密连接的完整性。结论：ITF保护胃黏膜上皮细胞紧密连接的完整性,提高紧密连接蛋白的表达水平,其发挥作用的主要分子机制是通过激活PI3K/Akt信号通路来实现的。     

Gab2对结肠癌细胞细胞外黏附作用的影响

;目的探讨Gab2对结肠癌细胞外黏附作用的影响。方法选取SW620和HCT116两种结肠癌细胞系,建立Gab2表达降低和Gab2表达升高的稳定细胞株。将实验NOD/SCID小鼠分为3组,每组10只。(1)对照组;接种scr/MGC803体结肠癌细胞;(2)Gab2降低组;接种Gab2降低的siGab2/MGC803结肠癌细胞;(3)Gab2升高组;接种Gab2升高的Gab2/MGC803结肠癌细胞。检测细胞迁移能力,测量肿瘤的大小和侵袭范围及细胞凋亡情况。结果处理后3组细胞迁移数计算结果显示,Gab2降低组的细胞迁移数显著低于Gab2升高组和对照组(P<0.05)。各组NOD/SCID小鼠均在接种40 d后处死,比较接种前与接种后小鼠体质量,Gab2降低组显著低于Gab2升高组、对照组,对照组低于Gab2升高组。测量瘤体积,Gab2升高组高于Gab2降低组、对照组。Gab2降低组瘤体积抑制率显著高于对照组(P<0.05)。相关分析发现,MMP-2、MMP-9蛋白的表达与Gab2蛋白表达呈正相关。结论 Gab2可有效调控体外细胞迁移数,显著抑制小鼠结肠癌细胞侵袭及转移,通过调节MMP-2、MMP-9蛋白表达,从而抑制结肠癌生长及转移。     

ELA经PI3K/AKT和MARK通路促进MSCs增殖和迁移

目的:观察ELABELA(ELA)对骨髓间充质干细胞(Bone mesenchymal stem cells,MSCs)增殖和迁移能力的影响,并探讨可能调控机制。方法:将体外培养的MSCs分为Control组、ELA处理组(50 n M、500 n M、5μM、10μM)。MTS法检测细胞增殖,Transwell法检测细胞迁移。Western Blot法检测Control组、5μM ELA、5μM ELA+LY294002(PI3K/AKT通路抑制剂)组、5μM ELA+U0126(MARK通路抑制剂)组中AKT、p-AKT、ERK1/2、p-ERK1/2、Cyclin D1的表达情况。结果:与Control组相比,ELA处理组(50 n M、500 n M、5μM)MSCs的增殖及迁移能力显著增加(P0.01),其中以5μM ELA处理MSCs增殖及迁移能力最佳;与Control组相比,5μMELA组p-AKT/AKT、p-ERK/ERK、Cyclin D1的蛋白水平显著增加(均为P0.01),然而此效应可被LY294002及U0126阻断。结论:5μM ELA处理MSCs增值及迁移能力最佳,此效应可能与激活PI3K/AKT通路及MARK通路相关。     

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。     

miR-212在老年前列腺癌患者中的表达及其对癌细胞增殖、侵袭、转移的影响机制研究

目的探讨miR-212在老年前列腺癌患者中的表达及其对癌细胞增殖、侵袭、转移的影响机制。方法选择2017年10月至2019年10月于厦门市第五医院行手术确诊的前列腺癌患者60例,经病理档案室收集其癌组织标本及配对癌旁正常组织标本,采用实时荧光定量PCR检测前列腺组织miR-212的相对表达水平。将购自上海北诺生物科技有限公司的前列腺癌PC-3细胞24株随机分为空白组(不作任何处理)、对照组(转染空白miR-212对照)、miR-212组(转染miR-212 mimics),每组各8株;检测并比较3组转染后前列腺癌细胞增殖率、侵袭能力和转移率;采用荧光素酶报告实验验证miR-212下游靶基因。结果miR-212在前列腺癌组织中呈低表达,在癌旁正常组织中呈高表达,前列腺癌组织中的miR-212相对表达水平低于癌旁正常组织(P<0.05)。转染miR-212后,PC-3细胞增殖、侵袭、转移均受到抑制。miR-212组PC-3细胞增殖率、侵袭能力、转移率低于对照组、空白组(P<0.05);空白组和对照组比较,PC-3细胞增殖率、侵袭能力、转移率差异无统计学意义(P>0.05)。miR-212组与上皮-间质转化(EMT)-WT(野生型)共转染后细胞荧光活性显著低于对照组与EMT-WT共转染后细胞荧光活性(P<0.05);空白组与对照组比较,与EMT-WT共转染后细胞荧光活性差异无统计学意义(P>0.05)。空白组、对照组、miR-212组与EMT-MUT(突变型)共转染后细胞荧光活性比较,差异无统计学意义(P>0.05)。结论miR-212在前列腺癌组织中呈低表达,上调miR-212的相对表达水平可抑制前列腺癌细胞增殖、侵袭、转移能力,其作用与EMT密切相关。     

Combretastatin A4-induced differential cytotoxicity and reduced metastatic ability by inhibition of AKT function in human gastric cancer cells

Combretastatin A4 (CA4) is a drug that targets tumor vasculature to inhibit angiogenesis. Whether CA4 has a direct effect on gastric cancer is not known. We herein investigated the effect of CA4 on growth and metastasis of gastric cancer cells at clinically achievable concentration and explored the associated antitumor mechanisms. Nine human gastric cancer cell lines, including two metastatic gastric cancer cell lines (AGS-GFPM1/2), constitutively expressing green fluorescence protein (GFP) were used. These metastatic AGS-GFPM1/2 cells expressed a higher level of phosphorylated serine 473 on AKT (p-AKT). Our results showed that CA4 (0.02-20 microM) has significant in vitro effects on reducing cell attachment, migration, invasiveness, as well as cell cycle G2/M disturbance on p-AKT-positive gastric cancer cells. In addition, a phosphoinositide 3-kinase inhibitor, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], a specific AKT inhibitor, and 0.2 to 20 microM CA4 displayed a similar response profile on p-AKT-positive cells, suggesting that CA4-induced effect was mediated by inhibition of the PI3 kinase/AKT pathway. The results from in vivo GFP monitoring system indicated that CA4 phosphate (CA4-P; 200 mg/kg) significantly inhibited the s.c. and intra-abdominal growth of xenotransplanted AGS-GFPM2 cells in nude mice. Furthermore, CA4-P treatment showed a remarkable ability to inhibit gastric tumor metastasis as well as attenuate p-AKT expression. In conclusion, our study is the first to find that CA4 inhibited AKT activity in human gastric cancer cells. The decreased AKT activity correlated well with the CA4 antitumor growth response and decrease of metastasis. Further investigation on drugs targeting the PI3 kinase-AKT pathway may provide a new approach for the treatment of human gastric cancer.     

Role of phosphatidylinositol 3-kinase/AKT as a survival pathway against CYP2E1-dependent toxicity

The objective of this work was to evaluate the possible role of PI3-kinase/AKT as a survival pathway against CYP2E1-dependent toxicity. E47 cells (HepG2 cells transfected with human CYP2E1 cDNA) exposed to 25 microM iron-nitrilotriacetate+5 microM arachidonic acid (AA+Fe) developed higher toxicity than C34 cells (HepG2 cells transfected with empty plasmid). Toxicity was associated with increased oxidative stress and activation of calcium-dependent hydrolases calpain and phospholipase A2. Treatment of E47, but not C34 cells, with arachidonic acid and iron (AA+Fe) led to a decrease in the phosphorylation state of AKT. 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002), a specific inhibitor of PI3-kinase, produced a further decrease of phosphorylated AKT in AA+Fe-treated E47 cells. LY294002 and down-regulation of endogenous AKT with small interference RNAs increased the toxicity of AA+Fe in E47 cells. Toxicity of AA+Fe in rat hepatocytes was also increased by LY294002. LY294002 did not affect phospholipase A2 or calpain activation, CYP2E1 activity, or lipid peroxidation elicited by AA+Fe. alpha-Tocopherol prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of phosphorylated AKT. LY294002 potentiated AA+Fe-induced loss of mitochondrial membrane potential and ATP, whereas overexpression of constitutively active AKT partially prevented mitochondrial impairment and toxicity. Mitochondrial permeability transition inhibitors prevented both AA+Fe and AA+Fe+LY294002-induced toxicity and decrease of mitochondrial membrane potential. These results suggest that: i) AA+Fe+CYP2E1-induced oxidative stress decreases AKT activation; ii) AKT inactivation induces mitochondrial impairment associated with opening of the permeability transition pore but is not dependent on the activation state of bad, glycogen synthase kinase-3beta, mammalian target of rapamycin, or bcl-xL; and iii) PI3-kinase/AKT may serve as a survival pathway against CYP2E1-dependent toxicity.     

RACK1 promotes the proliferation,migration and invasion capacity of mouse hepatocellular carcinoma cell line in vitro probably by PI3K/Rac1 signaling pathway

Hca-P and Hca-F is a pair of synogenetic mouse hepatocarcinoma ascites cell lines, possessing different capacity of lymphatic metastasis. Receptor of activated C-kinase 1 (Rack1), together with Jnk1 and gelsolin (Gsn) were previously identified as differentially expressed proteins for lymphatic metastatic potential between the two cell lines. As an intracellular scaffold protein, Rack1 could recruit such signaling molecules as integrins, Src, PKC which are involved in many important biological processes and play key roles in cancer progression. In our present studies, pCDNA3.1(+)-Rack1, a eukaryotic expression plasmid, was constructed and stably transfected into Hca-P cells with a low metastatic potential. CCK8 assay and transwell system were used to evaluate the effects of Rack1 on proliferation, migration and invasion of Hca-P cells in vitro. Then, LY294002, an inhibitor of PI3K, was added into the culture medium of pCDNA3.1(+)-Rack1-Hca-P cells and their biological behaviors observed further. Moreover, the expression of Jnk1, Rac1 and Gsn of pCDNA3.1(+)-Rack1-Hca-P cells were detected by western blot after pretreated with various doses of LY294002. As a result, the proliferation, migration and invasion of pCDNA3.1(+)-Rack1-Hca-P cells were significantly enhanced and could be inhibited by LY294002. In addition, the expression of Gsn, Rac1 and Jnk1 of pCDNA3.1(+)-Rack1-Hca-P cells also decreased after pretreated with LY294002. The expression of Gsn can be inhibited by NSC33766 (an inhibitor of Rac1). Taken together, Rack1/PI3K/Rac1 signaling pathway may play a crucial role in malignant biological behaviors of mouse hepatocarcinoma cells with lymphatic metastasis potential. It may be a potential target for therapy of cancer lymphatic metastasis.     

抑癌基因PTEN对人神经母细胞瘤细胞组织因子表达调控的研究

目的 研究10号染色体同源缺失的磷酸酶-张力蛋白同源物（phosphatase and tensin homolog deleted on chromosome 10，PTEN）对人神经母细胞瘤细胞组织因子（tissue factor，TF）表达的调控。方法 以人神经母细胞瘤细胞系SK—N-SH和SK-N—MC为对象，用Westernb lot法检测PTEN、TF的表达，用RT—PCR方法检测TF转录水平，以脂质体Lipofectamine2000介导进行PTEN基因表达载体pCMV—PTEN的转染；以特异性抑制剂Ly294002阻断磷脂酰肌醇-3-激酶（PI3K）／AKT的磷酸化；并以Western blot法检测磷酸化水平。结果 SK—N—SH细胞PTEN强表达而TF弱表达，SK—N-MC细胞PTEN基因表达阴性而TF强表达。以PTEN基因表达载体pCMV—PTEN转染SK-N—MC细胞，PTEN表达增强，而TF表达水平降低，并伴有AKT磷酸化的减弱。用PI3K／AKT信号途径特异性抑制剂Ly294002处理后，TF的表达可呈剂量依赖性降低。结论 PTEN基因的表达可能通过抑制PI3K／AKT信号途径而下调人神经母细胞瘤细胞中TF的表达，PTEN基因的缺失可能是TF异常高表达的原因。     

CXCL10/CXCR3 axis promotes the invasion of gastric cancer via PI3K/AKT pathway-dependent MMPs production

CXCR3, a G-protein coupled chemokine receptor, has been found to be overexpressed in many tumors and act as an independent prognostic marker. However, it is still unclear whether CXCR3 is involved in gastric cancer progression. In this study, we found that CXCR3 was markedly expressed in gastric cancer cells and tissues. High CXCR3 expression correlated with advanced tumor stage, vascular invasion, lymph node metastasis and poor survival of gastric cancer patients. Activation of CXCR3 by one of its ligands CXCL10 promoted the invasion and migration of gastric cancer BGC-823 and MGC-803 cells, and increased the secretion and activities of MMP-2 and MMP-9. However, the effects of CXCL10 on gastric cancer cells were attenuated by CXCR3 siRNA transfection. Furthermore, overexpression of CXCR3 enhanced CXCL10-mediated cell invasion and migration of gastric cancer MKN28 cells. In addition, CXCR3 time-dependently induced activation of AKT. PI3K/AKT pathway was required for CXCR3-mediated gastric cancer cell invasion, migration and MMP-2/9 production. Together, our findings suggest that CXCL10/CXCR3 axis promotes gastric cancer cell invasion and migration by upregulating MMP-2 and MMP-9 production via PI3K/AKT pathway. Thus, CXCR3 could be a potential target for the gastric cancer treatment.     

SH2B1沉默对胃癌SGC-7901细胞增殖、凋亡及PI3K/AKT通路的影响

目的研究沉默SH2B1基因表达对胃癌SGC-7901细胞增殖、凋亡及3-磷酸肌醇激酶(PI3K)/蛋白质丝氨酸苏氨酸蛋白激酶(AKT)通路的影响。方法体外培养胃癌SGC-7901细胞,采用SH2B1 siRNA转染SGC-7901细胞作为研究组,采用国际通用的与所有基因均无同源序列的non-target siRNA转染作为阴性对照组(NC组),以未经处理的胃癌SGC-7901细胞作为空白对照组(BC组),48h后收集各组转染成功细胞,采用荧光定量聚合酶链反应(qRT-PCR)检测SH2B1 mRNA表达情况,采用蛋白质印迹法(Western Blot)检测SH2B1蛋白表达情况;采用细胞计数试剂盒(CKK-8)检测细胞增殖情况,采用流式细胞仪检测细胞凋亡,同时检测Ki67、增殖细胞核抗原(PCNA)、Caspase-9、PI3K、AKT、p-AKT蛋白表达情况。结果研究组SGC-7901细胞SH2B1蛋白及mRNA表达量较BC组和NC组明显降低,差异有统计学意义(P<0.05);转染后,siRNA组SGC-7901细胞增殖明显受到抑制、平板克隆形成率较BC组和NC组明显降低,凋亡率明显升高,差异均有统计学意义(P<0.05);与BC和NC组比较,研究组SGC-7901细胞PI3K、p-AKT蛋白、Ki67及PCNA蛋白呈低表达,差异有统计学意义(P<0.05),Caspase-9蛋白呈高表达,AKT蛋白表达差异无统计学意义(P>0.05)。结论沉默SH2B1基因表达可能通过抑制PI3K/ATK信号通路激活,抑制SGC-7901细胞增殖,促进凋亡。     

ARK5、MMP-2和MMP-9在胃癌中的表达及与侵袭转移的关系

目的:探讨ARK5、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)在胃癌组织中的表达及与胃癌侵袭转移的关系。方法:免疫组织化学方法检测66例胃癌组织及20例正常胃组织中ARK5、MMP-2、MMP-9的表达;脂质体介导法将ARK5-siRNA转染入胃癌细胞株SGC-7901(siARK5/SGC7901细胞组),以SCRsiRNA转染组(scr/SGC7901细胞组)作为对照组,Western blot检测转染后ARK5、MMP-2、MMP-9蛋白表达;Transwell侵袭实验检测细胞体外侵袭能力。结果:ARK5、MMP-2、MMP-9在胃癌组织中的阳性表达率分别为68%、76%、68%,三者之间差异具有统计学意义(P<0.05);ARK5的表达与胃癌的浸润深度、细胞分化程度、TNM分期及淋巴结转移呈正相关(P<0.05),与年龄、性别、肿瘤大小无关(P>0.05);转染后siARK5/SGC7901细胞组表达ARK5、MMP-2和MMP-9蛋白水平降低,体外侵袭能力明显降低(P<0.05)。结论:ARK5、MMP-2和MMP-9在组织中的表达有相关性,ARK5与胃癌的侵袭和转移密切相关,可作为判断预后的指标和化学治疗的靶点。     

Blocking of PI3K/AKT induces apoptosis by its effect on NF-κB activity in gastric carcinoma cell line SGC7901

NF-κB plays an important role in many aspects of tumorigenesis and tumor progression by its antiapoptosis effect. Hence, NF-κB has been regarded as a therapeutic target in cancer, because inhibition of NF-κB not only induces enhancing apoptosis but also causes increasing sensitivity to radiation or chemotherapy in several tumor cells. The activation of NF-κB is presumed to be associated with PI3K/Akt signal pathway in gastric carcinoma, but the underlying molecular mechanism remains unclear. Our work demonstrates that blocking PI3K/Akt by LY294002 inhibits the NF-κB activity with significantly increased apoptosis in gastric cancer cell. Furthermore, when the cells were pretreated with IKK siRNA and/or IκB siRNA then exposed to LY294002, the results suggest that the regulatory significantly increased apoptosis in gastric cancer cell. Furthermore, when the cells were pretreated, effect of PI3K/AKT on NF-κB activity is associated with the influence of PI3K/AKT on IKK/IκB. The apoptosis induced by blocking PI3K/AKT might be ascribed to inhibition of NF-κB activity through IKK/IκB at least in part.