简介：Objective:ActivatingKRASmutationsarethemostcommondriversinthedevelopmentofnon-smallcelllungcancer(NSCLC).However,unsuccessoftreatmentbydirectinhibitionofKRAShasbeenproven.DeregulationofPI3KsignalingplaysanimportantroleintumorigenesisanddrugresistanceinNSCLC.TheactivityofPI3Kα-selectiveinhibitionagainstKRAS-mutatedNSCLCremainslargelyunknown.Methods:CellproliferationwasdetectedbysulforhodamineBassay.Cellcycledistributionandapoptosisweremeasuredbyflowcytometry.CellsignalingwasassessedbyWesternblotandimmunohistochemistry.RNAinterferencewasusedtodown-regulatetheexpressionofcyclinD1.HumanNSCLCxenograftswereemployedtodetecttherapeuticefficacyinvivo.Results:CYH33possessedvariableactivityagainstapanelofKRAS-mutatedNSCLCcelllines.AlthoughCYH33blockedAKTphosphorylationinalltestedcells,RbphosphorylationdecreasedinCYH33-sensitive,butnotinCYH33-resistantcells,whichwasconsistentwithG1phasearrestinsensitivecells.CombinedtreatmentwiththeCDK4/6inhibitor,PD0332991,andCYH33displayedsynergisticactivityagainsttheproliferationofbothCYH33-sensitiveandCYH33-resistantcells,whichwasaccompaniedbyenhancedG1-phasearrest.Moreover,down-regulationofcyclinD1sensitizedNSCLCcellstoCYH33.Reciprocally,CYH33abrogatedthePD0332991-inducedup-regulationofcyclinD1andphosphorylationofAKTinA549cells.Co-treatmentwiththesetwodrugsdemonstratedsynergisticactivityagainstA549andH23xenografts,withenhancedinhibitionofRbphosphorylation.Conclusions:SimultaneousinhibitionofPI3KαandCDK4/6displayedsynergisticactivityagainstKRAS-mutatedNSCLC.ThesedataprovideamechanisticrationaleforthecombinationofaPI3KαinhibitorandaCDK4/6inhibitorforthetreatmentofKRASmutatedNSCLC.

简介：目的探讨磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(PKB,也称AKT)信号通路相关蛋白在食管癌组织中的表达情况,并分析其与食管癌淋巴结转移和淋巴管生成的关系。方法选取120例食管癌患者,收集经组织病理学检查证实的食管癌组织和癌旁正常黏膜组织。采用免疫组织化学法检测PI3K和AKT蛋白在食管癌组织及其相应癌旁正常黏膜组织中的表达情况;采用D2-40蛋白标记淋巴管,并采用免疫组织化学法检测微淋巴管密度(LMVD),分析PI3K、AKT蛋白的表达与食管癌患者临床特征及微淋巴管密度(LMVD)的关系。结果食管癌组织中PI3K和AKT蛋白的阳性表达率分别为85.0%和86.7%,均明显高于癌旁食管正常黏膜组织的4.2%和5.8%,差异均有统计学意义(P﹤0.01)。不同性别、组织分化程度食管癌患者食管癌组织中PI3K、AKT蛋白的阳性表达率比较,差异均无统计学意义(P﹥0.05)。有淋巴结转移、TNM分期为Ⅲ+Ⅳ期的食管癌患者食管癌组织中PI3K、AKT蛋白的阳性表达率均高于无淋巴结转移、TNM分期为Ⅰ+Ⅱ期的食管癌患者,差异均有统计学意义(P﹤0.05)。PI3K蛋白阳性表达食管癌组织中LMVD的数量为(13.92±3.02),明显高于PI3K蛋白阴性表达食管癌组织的(6.54±1.05),差异有统计学意义(P﹤0.01);AKT蛋白阳性表达食管癌组织中LMVD的数量为(13.84±3.08),明显高于AKT蛋白阴性表达食管癌组织的(6.14±0.89),差异有统计学意义(P﹤0.01)。结论PI3K/AKT信号通路相关蛋白在食管癌组织中的表达水平较高,且能够促进微淋巴管的生成和淋巴结转移,其表达情况与患者的临床特征也具有一定的关系。阻断该通路可作为治疗和预防食管癌及淋巴结转移的新的研究方向。

简介：目的:研究心肌细胞条件培养液(cardiomyocyteconditionedmedium,CMCM)对K562细胞及NCI-H2452细胞增殖的影响,探索CMCM抑癌作用是否有肿瘤特异性,并探究其理化性质。方法:原代培养Wistar乳大鼠心肌细胞,以顺铂(DDP)作为阳性对照,CCK8法分析CMCM对K562细胞及人间皮瘤细胞NCI-H2452以及正常肝细胞HL7702体外增殖的影响。随后将CMCM采取不同比例稀释,探究稀释浓度与抑瘤作用间的关系。将CMCM采用胰酶消化,予以不同温度处理,以探究CMCM的理化性质。结果:K562细胞及NCI-H2452细胞分别与CMCM共同培养后,细胞存活率明显下降,与对照组相比差异均具有统计学意义(P<0.05),而对正常细胞存活率无明显影响(P>0.05)。经胰酶消化及热处理后的CMCM活性仍然存在,CMCM对K562细胞的抑制作用具有时间及浓度依赖性。结论:CMCM可抑制K562细胞及NCI-H2452细胞的增殖,CMCM不能被蛋白酶所破坏,对热也稳定,抑制作用具有时间及浓度依赖性。

简介：目的探讨微小RNA-148a-3p(miR-148a-3p)对丝裂原活化蛋白激酶激酶激酶9(MAP3K9)的靶向调控作用及对胃癌细胞增殖和凋亡的影响。方法向对数生长期胃癌细胞株MGC-803转染miR-148a-3p模拟物(mimics组)和阴性对照(NC组),以未转染的MGC-803细胞为对照组;采用实时定量PCR(QPCR)检测各组miR-148a-3p水平以评价转染效率,MTT法检测各组细胞增殖能力,流式细胞术检测各组细胞凋亡情况,分别采用QPCR和Westernblotting检测Bcl-2、Bax、caspase-3及MAP3K9mRNA和蛋白水平,同时采用双荧光素酶报告实验验证miR-148a-3p与MAP3K9的靶向作用关系。结果QPCR结果显示,对照组、NC组和mimics组的miR-148a-3p水平分别为1.021±0.123、1.087±0.196和2.854±0.368,与对照组和NC组比较,mimics组的miR-148a-3p水平升高(P<0.05)。mimics组MGC-803细胞的增殖活力较其余两组减弱(P<0.05)。mimics组MGC-803细胞凋亡率为(15.2±1.6)%,高于对照组的(3.5±0.9%)%和NC组的(4.5±1.1)%,差异具有统计学意义(P<0.05)。与对照组和NC组比较,mimics组的MAP3K9和Bcl-2的mRNA和蛋白水平均下调,而Bax和caspase-3的mRNA和蛋白水平均上调(P<0.05);双荧光素酶报告实验证实MAP3K9是miR-148a-3p的直接作用靶点。结论MiR-148a-3p可抑制胃癌细胞MGC-803的增殖并诱导其凋亡,可能通过靶向MAP3K9来发挥抑癌作用,调控miR-148a-3p/MAP3K9轴在胃癌防治中有一定应用前景。

简介：Objective:ToexaminetheeffectofpSer9-GSK-3βonbreastcancerandtodeterminewhethertheunderlyingmetabolicandimmunologicalmechanismisassociatedwithROS/eIF2Bandnaturalkiller(NK)cells.Methods:WeemployedTWS119toinactivateGSK-3βbyphosphorylatingSer9andexploreditseffectonbreastcancerandNKcells.TheexpressionofGSK-3β,naturalkillergroup2memberD(NKG2D)ligands,eIF2BwasquantifiedbyPCRandWesternblot.Wemeasuredintracellularreactiveoxygenspecies(ROS)andmitochondrialROSusingDCFH-DAandMitoSOXTMprobe,respectively,andconductedquantitativeanalysisofcellularrespirationon4T1cellswithmitochondrialrespiratorychaincomplexⅠ/Ⅲkits.Results:OurinvestigationrevealedthatTWS119downregulatedNKG2Dligands(H60aandRae1),suppressedthecytotoxicityofNKcells,andpromotedthemigrationof4T1murinebreastcancercells.Nevertheless,LY290042,whichattenuatesp-GSK-3βformationbyinhibitingthePI3K/Aktpathway,reversedtheseeffects.WealsofoundthathigherexpressionofpSer9-GSK-3βinducedhigherlevelsofROS,andobservedthatabnormalityofmitochondrialrespiratorychaincomplexⅠ/ⅢfunctioninducedthedysfunctionofGSK-3β-inducedelectrontransportchain,naturallydisturbingtheROSlevel.Inaddition,theexpressionofNOX3andNOX4wassignificantlyup-regulated,whichaffectedthegenerationofROSandassociatedwiththemetastasisofbreastcancer.Furthermore,wefoundthattheexpressionofpSer535-eIF2BpromotedtheexpressionofNKG2Dligands(Mult-1andRae1)followingbyexpressionofpSer9-GSK-3βandgenerationofROS.Conclusions:ThePI3K/Akt/GSK-3β/ROS/eIF2BpathwaycouldregulateNKcellactivityandsensitivityoftumorcellstoNKcells,whichresultedinbreastcancergrowthandlungmetastasis.Thus,GSK-3βisapromisingtargetofanti-tumortherapy.

简介：目的研究扶正固本颗粒联合顺铂对荷瘤小鼠体内抗肿瘤治疗及T细胞免疫调节的影响。方法选取昆明种小鼠120只,雌雄各半,随机分为6组,每组20只,建立小鼠S180腹腔积液瘤模型。空白组,无任何处理;模型组:等体积蒸馏水灌胃;化疗组:顺铂注射液(1mg/kg)腹腔注射;低、中、高剂量组:顺铂注射液(1mg/kg)腹腔注射联合扶正固本颗粒(5.625、11.250、22.500g/kg)灌胃给药,每日给药1次,共10天,观察小鼠一般情况,记录体重变化,计算抑瘤率、脏器指数,流式细胞仪检测小鼠外周血中CD4^+、CD8^+及T淋巴细胞亚群水平。结果化疗组和低、中、高剂量组的抑瘤率分别为65.5%、43.2%、44.0%、57.6%。与化疗组比较,低、中、高剂量组小鼠体重与血液中Th1/Th2水平均提高;中、高剂量组小鼠胸腺指数和血液中CD4^+、Th1、Th17水平均提高,CD8^+水平降低;高剂量组小鼠血液中Th2、Treg水平下降,Th17/Treg水平提高,差异均有统计学意义(P﹤0.05)。结论顺铂联合高剂量扶正固本颗粒治疗较单独化疗能增强荷瘤小鼠T细胞免疫。