MiR-24反义核酸通过BIM-Smac/Diablo途径促进多柔比星对结肠癌细胞的凋亡诱导效应

目的 探讨miR-24在结肠癌中发挥的作用并研究其是否和多柔比星的体外治疗有关。方法 用RT-qPCR法检测miR-24在结肠癌患者血浆中及结肠癌细胞系中的表达水平。MTT法检测miR-24反义核酸对多柔比星杀伤结肠癌细胞能力的影响。利用生物信息学、定量PCR及western blot法验证miR-24是否调节结肠癌细胞BIM的表达。运用JC-1染色、Annexin V染色及western blot法研究miR-24反义核酸影响多柔比星疗效的信号通路。结果 结肠癌患者血浆及结肠癌细胞系中miR-24表达水平显著升高。miR-24反义核酸可显著增强多柔比星对SW480细胞的杀伤活性。定量PCR及western blot实验表明miR-24的靶基因可能为BIM。miR-24反义核酸联合多柔比星可引起SW480细胞线粒体膜电位的丧失并诱导线粒体内Smac/DIABLO的释放,进而引起caspase-3的活化和凋亡的发生。转染BIM siRNA后miR-24反义核酸联合多柔比星对SW480细胞的凋亡诱导效应显著降低。结论 MiR-24反义核酸通过BIM-Smac/Diablo途径促进多柔比星对结肠癌细胞的凋亡诱导效应。     

上调NDRG1表达降低奥沙利铂耐药结肠癌细胞的存活率

目的观察上调NDRG1表达对结肠癌细胞及奥沙利铂耐药结肠癌细胞的毒性作用并探讨其机制。方法以重组真核表达质粒pEGFP-NDRG1-N3转染HCT 116及奥沙利铂耐药的OHP-HCT 116结肠癌细胞,以实时定量(qRT)-PCR法和Western blot法检测转染效率。MTT法检测奥沙利铂对不同结肠癌细胞的毒性及验证OHP-HCT 116细胞的耐药性。给予奥沙利铂干预转染pEGFP-NDRG1-N3的HCT 116细胞及OHP-HCT 116细胞,MTT法检测细胞存活率,流式细胞术检测细胞凋亡率,Western blot法检测凋亡蛋白Bcl-2及p53蛋白水平变化。结果不同浓度奥沙利铂干预下的3株结肠癌细胞中HCT 116细胞对奥沙利铂最敏感,OHP-HCT 116细胞对奥沙利铂耐药得到验证。以不同浓度梯度奥沙利铂干扰转染pEGFP-NDRG1-N3的HCT 116细胞及OHP-HCT 116细胞,与MOCK及siNC组比较,在HCT 116细胞及OHP-HCT 116细胞,转染pEGFP-NDRG1-N3细胞的存活率下降(P<0.05),凋亡率升高(P<0.05),Bcl-2表达降低(P<0.05),p53表达升高(P<0.05)。结论上调NDRG1表达通过调控p53表达改善结肠癌细胞奥沙利铂耐药,提高化疗敏感性。     

二甲双胍在结肠癌中通过抑制lncRNA-MALAT1的表达发挥抗肿瘤效应

目的 探讨二甲双胍（MET）对结肠癌细胞的抑制作用及其机制。方法 采用CCK-8、流式细胞术及Transwell检测MET对结肠癌细胞SW480和SW620增殖、凋亡及侵袭的影响,qRT-PCR检测5种lncRNA在结肠癌细胞中的表达,Western blotting检测MET和/或MALAT1基因敲减在体外对PI3K/AKT和ERK通路活性的影响。结果 MET在体外对SW480和SW620细胞具有抑制作用,且呈剂量和时间依赖性（P<0.05）。MET可促进SW480和SW620细胞凋亡（P<0.05）。在5种lncRNA中,lncRNA-MALAT1在SW480和SW620细胞中的表达水平最高（P<0.01）。siRNA可抑制lncRNA-MALAT1表达,并进一步增强MET介导的抗结肠癌作用（P<0.05）。MET可下调结肠癌细胞中lncRNA-MALAT1的表达（P<0.05）,并通过PI3K/AKT和ERK信号通路与lncRNAMALAT1敲减协同抑制结肠癌细胞的增殖和侵袭,并促进其凋亡（P<0.01）。结论 MET在结肠癌细胞中通过抑制ln...     

姜黄素体内外逆转结肠癌的5-氟尿嘧啶耐药研究及机制探讨

摘要：目的 探究姜黄素在体内外对结肠癌5-氟尿嘧啶（5-FU）耐药的逆转作用及其相关机制。 方法 以SW480结肠癌细胞为亲代细胞,构建5-FU耐药细胞株SW480R,采用MTT法检测SW480R细胞的耐药指数以及不同浓度的姜黄素对SW480R细胞增殖能力的影响;流式细胞术检测姜黄素对SW480R细胞周期和凋亡的影响;Western blot检测姜黄素对SW480R细胞上皮-间充质转化（epithelial-mesenchymal transition, EMT）相关蛋白以及Wnt信号通路相关蛋白的影响;采用裸鼠移植瘤模型检测肿瘤体积变化情况,计算姜黄素的体内抑瘤率,Western blot检测肿瘤组织中相关蛋白变化。结果 SW480R的耐药指数为12.16,姜黄素能够剂量依赖性地抑制SW480R细胞的增殖,阻滞SW480R细胞周期于G0/G1期,以及诱导SW480R细胞凋亡;Western blot结果表明姜黄素能够在体内外抑制EMT和Wnt信号通路的活性;体内实验表明姜黄素能够有效抑制裸鼠移植瘤的生长。结论 姜黄素能够在体内外逆转结肠癌的5-FU耐药,其机制可能是通过调控Wnt信号通路从而抑制EMT的发生。     

顺铂处理卵巢癌细胞系OVCAR3引起Survivin表达差异的研究

的 探讨顺铂(DDP)处理卵巢癌细胞系时Sur-vivin表达是否有变化,进而探讨Survivin表达与肿瘤细胞耐药之间的关系及其在肿瘤细胞获得性耐药及细胞凋亡耐受所起的重要作用。方法 以卵巢癌细胞系OVCAR3为材料,用MTT比色法检测细胞活性,流式细胞术检测细胞凋亡。RT-PCR检测Survivin mRNA表达差异,Western blot检测Survivin蛋白表达变化。结果 顺铂抑制卵巢癌细胞生长,具有时间和浓度依赖效应,即随时间延长和浓度增加,细胞生长抑制越强,细胞凋亡率也有同样的趋势,最高达31.6％。1mg·L~(-1)DDP处理卵巢癌细胞,Survivin mRNA及蛋白表达随作用时间的增长,分别在8 h和12 h达最高,随后又降低。结论 抗凋亡蛋白Survivin在卵巢癌细胞系中呈高表达,顺铂处理卵巢癌细胞,Survivin mRNA表达随作用时间延长而增加,这可能是卵巢癌细胞对化疗药物产生耐药性的重要因素之一。     

欧前胡素对顺铂的骨肉瘤细胞抗肿瘤作用的增效作用

目的 研究欧前胡素是否能提高人骨肉瘤细胞(human osteosarcoma cells,HOS细胞)对顺铂的敏感性。方法 HOS细胞用欧前胡素和顺铂处理,MTT法检测体外细胞活力,Western blot检测Bcl-2 蛋白家族成员(Mcl-1、Bcl-2、Bcl-xl、Bad和Bax)的表达水平,流式细胞术检测细胞凋亡水平和线粒体膜电位的变化情况。构建Mcl-1真核表达载体,MTT法检测Mcl-1表达载体转染对欧前胡素联合顺铂治疗骨肉瘤疗效的影响。结果 欧前胡素在体外可显著提高顺铂对骨肉瘤细胞系HOS的杀伤活性。欧前胡素可显著降低HOS细胞Mcl-1的表达,而顺铂对Mcl-1的表达水平无影响。相比于欧前胡素或顺铂单治疗组,两者联合可显著诱导HOS细胞发生凋亡并降低其线粒体膜电位。体外转染Mcl-1真核表达载体显著降低顺铂联合欧前胡素对HOS细胞的杀伤活性。结论 欧前胡素通过靶向于Mcl-1增强顺铂对骨肉瘤细胞的杀伤活性。     

罗米地辛对卵巢癌细胞顺铂耐药及PD-L1表达的影响

目的 探究组蛋白去乙酰化酶抑制剂罗米地辛调控卵巢癌细胞顺铂耐药及PD-L1表达的作用。方法 体外培养顺铂耐药卵巢癌细胞系COC1/DDP,用CCK-8实验检测细胞活力和顺铂半数抑制质量浓度(IC_(50)),用流式细胞术检测细胞凋亡和PD-L1的表达水平,用实时荧光PCR实验检测细胞PD-L1和STAT3的表达水平,用Western blot检测组蛋白乙酰化水平。结果 与Control组相比,Romidepsin组的卵巢癌细胞活力显著下降(P<0.05),IC_(50)值降低,细胞早期凋亡率和晚期凋亡率显著升高(P=0.004、P=0.003),PD-L1 mRNA的表达水平和蛋白表达水平显著升高(P=0.012 7),组蛋白H3乙酰化水平显著升高(P<0.000 1),STAT3的表达水平显著降低(P=0.027 4)。结论 Romidepsin可能通过调控组蛋白乙酰化水平和STAT3的表达水平逆转卵巢癌细胞对顺铂的耐药性,促进顺铂诱导细胞凋亡,提高PD-L1的表达水平。     

沉默ciRS-7通过靶向miR-944逆转胃癌细胞顺铂耐药性机制

目的 探讨沉默环状RNA(circRNA)ciRS-7对胃癌细胞顺铂耐药性的影响及相关机制。方法 构建顺铂耐药胃癌细胞HGC-27/DDP,沉默ciRS-7并用顺铂处理HGC-27/DDP细胞后，用实时荧光定量PCR(qRT-PCR)检测ciRS-7、微小RNA-944(miR-944)表达水平，细胞计数试剂盒-8(CCK-8)检测细胞存活率、克隆形成实验检测克隆形成数，蛋白免疫印迹(Western blot)检测细胞周期蛋白D1(cyclinD1)、增殖细胞核抗原(PCNA)、B淋巴细胞瘤-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、裂解的天冬氨酸特异性半胱氨酸蛋白酶3(cleaved-caspase-3)蛋白表达水平，流式细胞术检测细胞凋亡率，原位末端标记法(TUNEL)检测细胞凋亡指数，双荧光素酶实验验证ciRS-7与miR-944的靶向关系。结果 ciRS-7在顺铂耐药胃癌组织和细胞中高表达，miR-944在顺铂耐药胃癌组织和细胞中低表达，沉默ciRS-7明显降低顺铂处理的HGC-27/DDP细胞存活率、克隆形成数、cyclinD1、PCNA、Bcl-2蛋白表达水平...     

雷公藤红素促进RIP1蛋白的去泛素化增强TNF-α对结肠癌细胞的凋亡诱导活性的研究

目的 探讨中药活性成分雷公藤红素对TNF-α体外抗结肠癌活性的影响并研究其机制。方法 用雷公藤红素联合TNF-α体外治疗结肠癌细胞系SW480,MTT法检测SW480细胞的细胞活力。SW480细胞用雷公藤红素联合TNF-α治疗后,Annexin V/PI染色法检测细胞的凋亡,Western blot法检测细胞caspase-8、caspase-9和caspase-3的活化和对CYLD蛋白表达的影响,并用免疫共沉淀法检测SW480细胞RIP1蛋白的泛素化水平。结果 雷公藤红素联合TNF-α对结肠癌细胞系SW480的细胞活力抑制率和凋亡诱导活性均显著高于雷公藤红素及TNF-α单治疗组。雷公藤红素联合TNF-α对SW480细胞caspase-8、caspase-9和caspase-3的活化显著高于雷公藤红素及TNF-α单治疗组,且两者联合治疗后,caspase-8的活化时间显著早于caspase-9和caspase-3。SW480细胞用雷公藤红素联合TNF-α治疗后,其RIP1蛋白的泛素化水平显著低于雷公藤红素及TNF-α单治疗组。进一步研究发现,雷公藤红素能显著诱导SW480细胞CYLD蛋白的表达,而TNF-α对CYLD的表达水平无影响,当用小干扰RNA沉默CYLD的表达后,雷公藤红素对TNF-α的协同效应丧失。结论 雷公藤红素通过促进RIP1蛋白的去泛素化增强TNF-α对结肠癌细胞的凋亡诱导活性。     

ABT-737增强人卵巢癌细胞顺铂敏感性并诱导线粒体分裂

目的观察Bcl-2抑制剂ABT-737对人卵巢癌顺铂耐药(SKOV3/DDP)细胞株顺铂敏感性和细胞线粒体形态的影响,为降低人卵巢癌细胞顺铂耐药性的进一步研究提供依据。方法将处于对数生长期的SKOV3/DDP细胞随机分为对照组、顺铂组、ABT-737组和顺铂联合ABT-737组(联合组)。MTT法检测各组SKOV3/DDP细胞生存率,流式细胞术(flow cytometry,FCM)检测各组细胞凋亡率,MitoTracker Red荧光探针法共聚焦显微镜下观察线粒体形态变化,免疫荧光法、蛋白印记法(Western blot)检测细胞中线粒体动力相关蛋白Drp-1和线粒体外膜分子Fis1的表达。结果 MTT结果发现与对照组比较,顺铂组和联合组SKOV3/DDP细胞生存率明显降低(P0.05),且联合组细胞存活率明显低于顺铂组(P0.05)。流式细胞术检测结果显示顺铂组细胞凋亡率明显高于对照组,联合组细胞凋亡率高于顺铂组。MitoTracker Red荧光探针法检测发现顺铂组细胞胞浆线粒体较对照组细胞胞浆内阳性着色颗粒感增强,联合组趋势强于顺铂组。蛋白免疫印记法检测Drp-1和Fis1表达水平,结果显示与顺铂组比较,联合组细胞中Drp1和Fis1蛋白表达水平明显增强。免疫荧光法结果呈现相同趋势。结论 ABT-737能够增强SKOV3/DDP细胞的顺铂敏感性,且对线粒体分裂存在明显诱导作用。     

Modification of the sensitivity to cisplatin with c-myc over-expression or down-regulation in colon cancer cells.

Human colon cancer SW480 cells express the c-myc gene. On the other hand, SW480DDP cell lines resistant to cisplatin exhibited decreased c-myc gene expression, but their cell growth rates remained similar to those of their parental cells. Antisense oligonucleotides to c-myc inhibited c-myc expression and induced increased resistance to cisplatin in SW480 cells. In contrast, SW480DDP cells showed increased c-myc expression and reversed sensitivity to cisplatin when these cells were transfected with c-myc cDNA from the pLNCX plasmid. Moreover, SW480DDP cells transfected with c-myc cDNA induced apoptosis when exposed to cisplatin, but not SW480 cells transfected with an antisense sequence for c-myc. Transfection either with a c-myc antisense sequence or c-myc cDNA to these cells did not change their growth rates. Thus enforced expression of c-myc in SW480 and SW620 lines sensitizes cells to cisplatin-induced apoptosis, whereas the down-regulation of c-myc in SW480DDP and SW620DDP increases cisplatin resistance when using antisense strategy.     

百里醌抑制大肠癌生长的实验研究

目的:探讨百里醌抑制体内外大肠癌生长的影响及机制。方法:不同浓度百里醌作用人大肠癌细胞株SW480后,CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡;Western blotting检测大肠癌细胞中NF-κB、Bcl-2和Survivin的表达;建立裸鼠大肠癌皮下移植瘤模型,随机分为对照组和实验组(n=10),第3周开始分别经灌胃给予溶媒(1%乙醇)和百里醌(3 mg/只),每周3次,共两周,术后第8周处死裸鼠,测量肿瘤瘤重并计算抑瘤率;免疫组织化学法检测肿瘤组织的NF-κB、Bcl-2和Survivin的表达。结果:与对照组相比,百里醌可显著抑制大肠癌SW480细胞生长,并诱导细胞凋亡;百里醌可明显抑制NF-κB、Bcl-2和Survivin在SW480细胞中表达;与对照组相比较,实验组裸鼠皮下移植瘤生长被显著抑制,肿瘤组织中NF-κB、Bcl-2和Survivin表达下调。结论:百里醌具有抑制体内外大肠癌生长的作用,可能是通过抑制大肠癌中NF-κB及其调控蛋白Bcl-2及Survivin的表达而实现。     

缓激肽介导的治疗心血管和糖尿病药物致血管性水肿的研究进展

目的 本研究旨在调查慢性射血分数降低的心力衰竭(HFrEF)患者神经内分泌抑制剂-血管紧张素转换酶抑制剂(ACEI)或血管紧张素II受体阻滞剂(ARB)、β受体阻滞剂(BB)和醛固酮受体拮抗剂(MRA)使用现状及其与指南的差距。方法 搜集并分析2018年01月到12月HFrEF住院患者基本资料和三类神经内分泌抑制剂使用情况。结果 共纳入301例慢性HFrEF患者,平均年龄为71.3±11岁,男性占56.5%,平均EF值32.1±7.2%,51.8%合并有高血压,心衰最常见的病因是心脏瓣膜病。ACEI/ARB、BB和MRA使用率分别为77.4%,60.5%和94.0%,剂量达标率分别为44.2%、22.5%和100%。48.8%患者采用指南推荐的联合用药,包括1.3%两药联合(ACEI/ARB+BB)和47.4%三药联合(ACEI/ARB+ BB+MRA)。结论 该院慢性HFrEF患者神经内分泌抑制剂应用现状与指南仍有差异,ACEI/ARB和BB使用率和剂量达标率不足,而MRA使用过度,需进一步提高医生对指南依从性。     

Virosecurinine induces apoptosis by affecting Bcl-2 and Bax expression in human colon cancer SW480 cells

Virosecurinine, the major alkaloid isolated from Securinega suffruticosa Pall Rehd was found to exhibit growth inhibition and cytotoxicity against huaman colon cancer SW480 cells via the microculture tetrazolium (MTT) assay. Due to its greater cytotoxic potency and selectivity towards SW480 cells, flow cytometry was used to analyze the cell cycle distribution of control and treated SW480 cells whereas Annexin V-FITC/PI flow cytometry analysis was carried out to confirm apoptosis induced by virosecurinine in SW480 cells. Apoptotic regulatory genes were determined by RT-PCR analysis. Virosecurinine was found to induce G1/S cell cycle arrest which led to predominantly apoptotic mode of cell death. Mechanistically, virosecurinine was found to up-regulated the Bax gene expression and down-regulated the Bcl-2 expression in SW480, The ratio of Bcl-2 to Bax was significantly decreased. Hence, we suggest that virosecurinine induced apoptosis in SW480 cells by affecting the expression of bcl-2 and bax.     

The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells

In the current paper, berberine, an isoquinoline alkaloid, was tested for its chemopreventive potential in colon cancer (SW480) cells. Berberine inhibited proliferation of colon cancer cells in a dose- and time-dependent manner. Interestingly, this compound exhibited minimum toxicity in normal cells at 200 μM. Berberine arrested SW480 cell cycle at G2/M phase, which was supported by induction of p21 expression. Induction of a series of biochemical events, including loss of mitochondrial membrane potential, release of cytochrome-c to cytosol, induction of Bcl-2 family proteins, caspases and cleavage of poly (ADP-ribose) polymerase (PARP), by berberine suggests its ability to induce apoptosis. In addition, berberine also inhibited inflammation, as evidenced by induction of expression of NFκB and Cox(2). Furthermore, berberine inhibited caspase-8 mediated angiogenesis, as confirmed through expression of tumor necrotic factor related apoptosis-inducing ligand (TRAIL), vascular endothelial growth factor (VEGF) and survivin. The results of the current study demonstrated that berberine has the ability to cause cell cycle arrest, induce apoptosis and inhibit inflammation in colon cancer cells. The magnitude of the effects observed suggests that berberine may be worth considering for further studies of its potential applications for improving health, either as a preventative or a potential treatment.     

单胺氧化酶抑制剂氯吉灵抑制结肠癌的作用及机制研究

目的：研究单胺氧化酶A （monoamine oxidase A,MAO-A）抑制剂氯吉灵（Clorgyline）对结肠癌细胞增殖、转移的作用,以及其对MAO-A的酶活、MAO-A、MMP-2和MMP-9蛋白表达的影响。方法：MTS法检测不同浓度Clorgyline对结肠癌细胞SW480增殖的作用;划痕实验研究Clorgyline对SW480细胞迁移的影响;Transwell实验研究Clorgyline对SW480细胞侵袭的影响;裸鼠移植瘤模型研究Clorgyline对SW480细胞体内增殖的抑制作用;酶活试剂盒检测Clorgyline对裸鼠移植瘤组织中MAO-A的作用;Western blot检测Clorgyline对裸鼠移植瘤组织中的MAO-A、MMP-2和MMP-9蛋白表达的影响。结果：Clorgyline对SW480细胞增殖有抑制作用,且呈现剂量依赖性;Clorgyline 10 μmol·L^-1和20 μmol·L^-1浓度均能够抑制SW480细胞迁移和侵袭能力,与对照组相比具有显著性差异（P<0.01）;Clorgyline 20和40 mg·kg^-1均能够抑制SW480细胞裸鼠移植瘤的生长（P<0.01）,抑制裸鼠移植瘤组织中MAO-A的酶活（P<0.05）,抑制MMP-2和MMP-9蛋白的表达水平,而对MAO-A蛋白的表达水平没有影响。结论：Clorgyline抑制SW480结肠癌细胞的增殖和转移,其机制可能与抑制MAO-A酶活和转移相关蛋白MMP-2、MMP-9的表达有关。     

Increased expression of p27 is associated with the cisplatin resistance in gastric cancer cell line YCC-3

The major obstacle of treating cancer patients is acquisition of chemoresistance, in which treated tumor cells become insensitive after chronic drug exposure. To study the mechanism of acquired cisplatin resistance, we established a cisplatin-resistant human gastric cancer cell line. The cisplatin-resistant cell line (YCC-3/R) was isolated after exposing the gastric cancer cell line, YCC-3, to a constant concentration (0.5 μg/mL) of cisplatin for 12 months. The expression of cell cycle regulatory proteins (p53, Bax, p21, p27) in the YCC-3/R were investigated by western blot analysis. The cisplatin treatment significantly down-regulated the p53 and p21 expression level, while up-regulated the p27 expression in the YCC-3/R cells compared to the parental cells. The Bax expression level was similar in both cells. These results suggest that the p27 dependent-cell cycle arrest may prevent cisplatin-induced apoptosis and give enough time to repair the DNA damage in the YCC-3/R cells.     

Dietary flavanols exert different effects on antioxidant defenses and apoptosis/proliferation in Caco-2 and SW480 colon cancer cells

Flavanols intake has been associated with reduced risk of cancer. In this study, the anticarcinogenic effects of the flavanols epicatechin (EC), epicatechin-gallate (ECG) and procyanidin B2 (PB2) on Caco-2 and SW480 colon cancer cells were investigated. Catechins showed different cytotoxicity depending on the cell line. ECG displayed strong growth inhibitory effects against SW480 cells, but was ineffective on Caco-2 cells. In contrast, PB2 did not affect Caco-2 cells, whereas promoted cell growth in SW480 cells and EC had no obvious effects on any cell line. Exposure of SW480 cells to ECG led to apoptosis as determined by caspase-3 activity, imbalance among Bcl-2 anti- and pro-apoptotic protein levels, ERK activation and AKT inhibition, whereas PB2 treatment enhanced phospho-AKT and phospho-ERK levels. Incubation of Caco-2 cells with ECG increased glutathione levels without affecting the expression of pro- and anti-apoptotic Bcl-2 proteins, AKT or ERK. The results suggest that the different cytotoxicity of flavanols is caused by their different activity and the degree of differentiation of the colon cancer cell line. Thus, ECG induced apoptosis in SW480 cells and contributed to the cytotoxic effect, whereas ECG enhanced the antioxidant potential in Caco-2 cells. PB2 activated cell proliferation and survival/proliferation pathways in SW480 cells.