新型SMO抑制剂SI-4650对人神经胶质瘤U87MG细胞增殖、凋亡和自噬能力的影响

目的研究新型精胺氧化酶(spermine oxidase, SMO)小分子抑制剂SI-4650对人胶质瘤U87MG细胞增殖、凋亡和自噬的影响及其分子机制。方法 MTT法检测细胞增殖情况;化学发光法检测SMO和乙酰多胺氧化酶(N~1-acetylpolyamine oxidase, APAO)的酶活性;高效液相色谱法(HPLC)检测细胞内多胺含量;Transwell法分析U87MG细胞的迁移能力;PI单染后结合流式细胞术分析细胞周期;PI/FITC-Annexin V双染后结合流式细胞术、Western blot分析细胞凋亡;激光共聚焦显微镜(LSCM)和Western blot分析细胞自噬。结果 SI-4650可明显抑制U87MG细胞内SMO和APAO的酶活性,干扰多胺代谢,并降低U87MG细胞内总多胺含量。用SI-4650处理能抑制U87MG细胞的增殖和迁移能力,使细胞发生G_0/G_1周期阻滞,并诱导U87MG细胞发生凋亡和自噬性死亡。结论 SI-4650具有杀伤神经胶质瘤U87MG细胞的药理活性,其机制可能与干扰多胺代谢和诱导细胞凋亡和自噬相关。     

精胺氧化酶抑制剂的筛选及抗肿瘤活性评价

目的 筛选多胺代谢关键酶精胺氧化酶小分子抑制剂,并评价其对人肺癌A549细胞的精胺氧化酶活性抑制效应、抗肿瘤作用及分子机制。方法 以精胺和精胺氧化酶复合物结构为基础,用计算机辅助药物设计技术,在化学数据库中虚拟筛选获得候选的小分子抑制剂。以A549细胞为肿瘤细胞模型,利用化学发光法、HPLC法分析小分子抑制剂对精胺氧化酶活性抑制及多胺含量的影响;采用MTT法、流式细胞术检测小分子抑制剂对细胞增值、细胞周期、诱导细胞凋亡的影响;在倒置荧光显微镜、透射电子显微镜下观察小分子抑制剂诱导细胞自噬小体的形成;Western blot法分析小分子抑制剂对细胞周期蛋白(Cyclin B1、p21、Cyclin D)、凋亡相关蛋白(Bax、Bcl-2、细胞色素C)和自噬相关蛋白(LC3、P62)表达的影响。结果 筛选出有效抑制精胺氧化酶活性的新型小分子抑制剂SI-1338,对精胺氧化酶的半数抑制浓度为880μmol·L^-1。SI-1338可有效抑制A549细胞中精胺氧化酶活性,干扰多胺代谢,减少总多胺的含量;抑制A549细胞的增殖,周期蛋白Cyclin B1、p21、Cyclin...     

多胺类似物CPENSpm通过干扰多胺代谢抑制肺癌细胞的增殖

目的研究多胺类似物CPENSpm对肺癌细胞株A549增殖和细胞凋亡的影响,以探讨CPENSpm抗肿瘤的作用机制。方法MTS法分析细胞的增殖速度,化学分析法测定多胺代谢酶的活性,HPLC法分析细胞内的多胺含量,亚凋亡峰测定法和DNA片段化分析法鉴定细胞程序性凋亡。结果CPENSpm处理A549肺癌细胞可导致:①癌细胞生长抑制并激发细胞凋亡;②抑制多胺合成关键酶ODC的活性,活化多胺降解代谢关键酶SSAT和SMO的活性;③耗竭细胞内多胺含量。SMO抑制剂MDL72527可拮抗CPENSpm对A549细胞的生长抑制作用。结论CPENSpm通过干扰A549细胞的多胺代谢途径,耗竭肿瘤快速生长必需的多胺成分,诱导产生活性氧H2O2从而抑制癌细胞生长并激发细胞程序性凋亡。     

高活性ODC微生物菌株的筛选

鸟氨酸脱羧酶(ornithine decarboxylase,ODC)是影响细胞内多胺生物合成及代谢的酶,细胞增殖中多胺是重要的调控因素之一。ODC催化L-鸟氨酸脱羧转化为腐胺,而腐胺则是多胺生物合成途径中的限速酶,多胺能够促进正常细胞的生长于分化。ODC极为广泛的存在于各种生物体细胞内。大量的实验和临床研究证明,在胚胎、再生组织以及恶性肿瘤等生长迅     

MEK抑制剂CQN系列化合物对A549细胞体外抗肿瘤作用

目的观察丝裂原激活蛋白激酶的激酶(MEK)抑制剂CQN系列化合物对A549细胞体外抗肿瘤作用。方法采用非小细胞肺癌A549细胞(K-ras突变)体外抗肿瘤增殖抑制实验对9个CQN系列MEK抑制剂进行筛选,得到具有显著抗肿瘤活性的化合物。通过流式细胞术检测化合物对细胞周期和凋亡的影响;并通过ELISA法检测化合物对肿瘤发生关键蛋白ERK1/2磷酸化的抑制作用;通过细胞划痕实验检测化合物对细胞迁移能力的影响。结果细胞增殖实验筛选出高活性化合物CQN-4和CQN-5,两者的体外抗A549细胞增殖抑制活性与对照药曲美替尼(trametinib)相当。CQN-4和CQN-5可将A549细胞阻滞于G1期,与对照相比可使细胞凋亡率明显增加(P<0.01),且呈浓度依赖性。与对照相比,CQN-4和CQN-5对A549细胞ERK1/2磷酸化均有抑制作用(P<0.01),且具有浓度依赖性。细胞划痕愈合实验显示CQN-4和CQN-5对A549细胞迁移有显著的抑制作用,且具有浓度依赖性。结论 MEK抑制剂化合物CQN-4和CQN-5对A549细胞具有显著的体外抗肿瘤活性。     

氨甲喋呤对映体药物对A549人肺腺癌细胞株增殖抑制作用及诱导凋亡作用

目的 探讨氨甲喋呤对映体[(+)MTX,(-)MTX]对A549细胞的增殖抑制作用及诱导凋亡作用。方法 采用培养的A549细胞,应用MTT比色法分析其活性;用光学显微镜和荧光显微镜观察细胞的形态学变化;碘化丙啶(PI)单染流式细胞术检测细胞周期;DNA梯度电泳检测凋亡。结果 在0.1~150 μmol·L^-1范围内,(+)MTX和(-)MTX作用于A549细胞24,48,72 h,均抑制细胞A549增值,但抑制强度为(+)MTX>(-)MTX,倒置显微镜和荧光显微镜观察不同浓度(+)MTX和(-)MTX作用A549细胞不同时间后,出现细胞不同程度的形态学改变;用10 μmol·L^-1的(+)MTX和(-)MTX作用A549细胞48 h后,PI单染流式细胞术检测A549细胞周期的影响,表明氨甲喋呤对映体干扰A549细胞DNA合成;DNA梯度电泳检测结果发现MTX作用组有凋亡条带出现,其中(+)MTX最为明显。结论 (+)MTX和(-)MTX对A549细胞的抗增殖作用具有化学结构的立体选择性,(+)MTX的抗A549细胞增殖作用明显强于(-)MTX。     

花旗松素对人肺癌细胞A549增殖的抑制作用及其机制研究

目的研究花旗松素对人肺癌细胞A549增殖的抑制作用,并探讨其诱导细胞凋亡的可能机制。方法 WST-1法检测不同质量浓度(0~200μg/m L)花旗松素对A549细胞增殖的影响;Annexin-V/PI双染法检测花旗松素对A549细胞凋亡的影响;细胞免疫荧光法观察花旗松素处理后A549细胞中凋亡蛋白Bax的表达;Western blotting法检测Bcl-2、Akt、P53蛋白表达的变化。结果 WST-1法检测到花旗松素能有效抑制A549细胞的增殖,呈现时间–剂量–效应关系。通过流式细胞仪分析,花旗松素诱导细胞凋亡,上调了促凋亡蛋白Bax的表达,下调抗凋亡蛋白Bcl-2的表达,抑制Akt的过表达,并且促进P53的表达。结论花旗松素对A549细胞的增殖具有显著的抑制作用,其诱导的凋亡机制可能与Bcl-2、Akt相关信号通路的激活有关。     

夏枯草乙醇提取物体外诱导肺癌细胞A549凋亡的研究

目的探讨夏枯草乙醇提取物对人肺癌细胞系A549细胞的增殖、迁移、细胞周期及凋亡的影响。方法不同质量浓度夏枯草提取物作用于人肺癌A549细胞,采用MTT比色法、细胞划痕实验等检测其对肿瘤细胞的生长及迁移的抑制作用,应用流式细胞术检测细胞增殖周期及凋亡率的变化情况,并使用Annexin-V FITC和PI双染试剂盒进行凋亡分析。结果一定质量浓度的夏枯草提取物可明显抑制肺癌细胞的增殖和迁移,并呈量效和时效关系,可诱导细胞发生G0/G1期或G2/M期细胞周期阻滞,且Sub G1峰比例明显上升,在低、中质量浓度(1.0和2.0mg·L-1)时以晚期凋亡为主,在高质量浓度(4.0mg·L-1)时主要发生早期凋亡。结论夏枯草提取物可抑制肺癌A549细胞的增殖和迁移,改变细胞周期,诱导肿瘤细胞凋亡。     

鱼藤素对非小细胞肺癌细胞体内外增殖的影响

目的探究鱼藤素(deguelin)对非小细胞肺癌细胞A549与裸鼠移植瘤增殖的影响。方法采用CCK-8法测定鱼藤素对A549细胞增殖的抑制作用;通过赫斯特染色和AnnexinV-FITC/PI双染实验探究鱼藤素对细胞凋亡形态以及细胞凋亡率的影响;流式细胞术测定鱼藤素对A549细胞周期的影响;此外,通过裸鼠移植瘤模型的体内实验和HE染色探究鱼藤素对裸鼠移植瘤的影响。结果鱼藤素呈浓度和时间依赖性抑制A549细胞增殖;赫斯特染色和AnnexinV-FITC/PI双染实验进一步证实了鱼藤素能诱导A549细胞凋亡;鱼藤素呈浓度依赖性阻滞A549细胞周期于G 2/M期;裸鼠移植瘤模型实验和HE染色实验发现,鱼藤素对A549裸鼠移植瘤的生长有明显抑制作用(P<0.01)。结论鱼藤素对肺癌细胞A549具有显著抑制作用,为鱼藤素的抗肿瘤活性提供新的基础。     

内源性大麻素联用顺铂对人肺腺癌A549细胞的诱导凋亡作用

目的观察内源性大麻素（AEA）、顺铂（DDP）单用或联用对人肺癌A549细胞增殖抑制和诱导凋亡的作用。方法采用四甲基偶氮唑蓝（MTT）法检测AEA和DDP对肺癌A549细胞的增殖抑制作用,以流式细胞仪（FCM）PI单染检测AEA联用DDP对细胞周期的影响,以Annexinv／PI双染法检测AEA联用DDP对细胞凋亡的影响。结果AEA对肺癌细胞增殖有明显的抑制作用,且呈剂量、时间依赖性。10,20μmol／L的AEA和1,2mg／L的DDP联合作用24,48,72h后,细胞增殖抑制率显著高于单用组;两药联用后诱导细胞凋亡的作用显著增强,AEA可使A549细胞阻滞于G2／M期。结论AEA及DDP对肺癌A549细胞的增殖具有显著的抑制作用,并可诱导细胞凋亡,在一定范围内呈量效关系,且两者联合应用具有相加或协同作用。     

Molecular basis for the antiproliferative effect of agmatine in tumor cells of colonic, hepatic, and neuronal origin

The aim of the present study was to challenge potential mechanisms of action underlying the inhibition of tumor cell proliferation by agmatine. Agmatine inhibited proliferation of the human hepatoma cells HepG2, the human adenocarcinoma cells HT29, the rat hepatoma cells McRH7777, and the rat pheochromocytoma cells PC-12. Inhibition of proliferation of HepG2 cells was associated with an abolition of expression of ornithine decarboxylase (ODC) protein and a doubling of mRNA content encoding ODC. In HepG2 cells, silencing of ODC-antizyme-1, but not of antizyme inhibitor, by RNA interference resulted in an increase of agmatine's antiproliferative effect. Thus, the distinct decrease in intracellular polyamine content by agmatine was due to a reduced translation of the synthesizing protein ODC but was not essentially mediated by induction of ODC-antizyme or blockade of antizyme inhibitor. In interaction experiments 1 mM L-arginine, 1 mM D-arginine, 1 mM citrulline, 100 microM N(omega)-nitro-L-arginine methyl ester, 1 and 10 microM sodium nitroprusside, and 1 microM N1-guanyl-1,7-diaminoheptane failed to alter agmatine's antiproliferative effect. Hence, the antiproliferative effect of agmatine in HT29 and HepG2 cells is due to an interaction with neither the NO synthases, the hypusination of eIF5A, nor an agmatine-induced reduction in availability of intracellular L-arginine. L-Arginine and citrulline, but not d-arginine, inhibited tumor cell proliferation by themselves. Their inhibitory effect was abolished after silencing of arginine decarboxylase (ADC) expression by RNA interference indicating the conversion to agmatine by ADC. Finally, in the four cell lines under study, agmatine-induced inhibition of cell proliferation was paralleled by an increase in intracellular caspase-3 activity, indicating a promotion of apoptosis.     

去甲泽拉木醛通过调控细胞周期和自噬诱导的细胞凋亡抑制K562细胞生长的机制

目的研究去甲泽拉木醛(demethylzeylasteral,ZST93)在体外抑制慢性髓系白血病(chronic myeloid leukemia,CML)细胞的增殖作用,并初步探讨其可能的作用机制。方法以K562细胞为研究对象,采用CCK-8、细胞生长曲线和倒置显微镜检测ZST93对K562细胞增殖抑制作用;细胞转染和Western blot分析细胞自噬;PI染色、Annexin V-FITC/PI和流式细胞术检测细胞周期和细胞凋亡。结果ZST93对K562细胞株的生长呈剂量和时间依赖性的抑制作用,IC₅₀值为2.59μmol·L^-1,使细胞周期阻滞于G₁期。自噬检测中发现ZST93可诱导GFP-LC3积累、LC3-Ⅰ转化为LC3-Ⅱ以及p62表达水平降低。ZST93通过调控自噬激活caspase-8,诱导外部凋亡信号通路,促使caspase-9、caspase-3和PARP剪切而被激活,针对caspase-3的抑制剂Z-DEVD-FMK能够降低ZST93诱导的K562细胞凋亡。结论ZST93可有效抑制K562细胞增殖,促进细胞周期阻滞、细胞凋亡和自噬激活,可能与自噬激活/caspase-8/caspase-3凋亡信号通路有关。     

De novo ceramide biosynthesis is associated with resveratrol-induced inhibition of ornithine decarboxylase activity

Previous studies could demonstrate, that the naturally occuring polyphenol resveratrol inhibits cell growth of colon carcinoma cells at least in part by inhibition of protooncogene ornithine decarboxylase (ODC). The objective of this study was to provide several lines of evidence suggesting that the induction of ceramide synthesis is involved in this regulatory mechanisms. Cell growth was determined by BrdU incorporation and crystal violet staining. Ceramide concentrations were detected by HPLC-coupled mass-spectrometry. Protein levels were examined by Western blot analysis. ODC activity was assayed radiometrically measuring [(14)CO(2)]-liberation. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Antiproliferative effects of resveratrol closely correlate with a dose-dependent increase of endogenous ceramides (p<0.001). Compared to controls the cell-permeable ceramide analogues C2- and C6-ceramide significantly inhibit ODC-activity (p<0.001) in colorectal cancer cells. C6-ceramide further diminished protein levels of protooncogenes c-myc (p<0.05) and ODC (p<0.01), which is strictly related to the ability of ceramides to inhibit cell growth in a time- and dose-dependent manner. These results were further confirmed using inhibitors of sphingolipid metabolism, where only co-incubation with a serine palmitoyltransferase (SPT) inhibitor could significantly counteract resveratrol-mediated actions. These data suggest that the induction of ceramide de novo biosynthesis but not hydrolysis of sphingomyelin is involved in resveratrol-mediated inhibition of ODC. In contrast to the regulation of catabolic spermidine/spermine acetyltransferase by resveratrol, inhibitory effects on ODC occur PPARgamma-independently, indicating independent pathways of resveratrol-action. Due to our findings resveratrol could show great chemopreventive and therapeutic potential in the treatment of colorectal cancers.     

紫花牡荆素诱导人肺腺癌A549细胞凋亡及其机制的探讨

目的探讨紫花牡荆素（CAS）诱导人肺腺癌A549细胞凋亡及其机制。方法平皿集落法测定CAS对A549细胞生长的抑制作用;碘化丙啶（PI）染色流式细胞术（FCM）分析细胞凋亡率;丫啶橙／溴乙啶（AO／EB）荧光染色观察细胞凋亡形态;Westernblot检测FoxMl（forkhead box protein M1）表达水平。结果CAS能抑制人肺腺癌A549细胞生长,呈浓度依赖性,半数抑制浓度（IC50）值为7．26p．mol·L^-1;CAS处理后A549细胞呈典型凋亡细胞形态学改变,诱导细胞凋亡呈浓度依赖性;CAS诱导A549细胞FoxM1蛋白表达下调,呈浓度和时间依赖性。结论CAS抑制人肺腺癌A549细胞的生长并诱导其凋亡,其作用机制可能与FoxM1表达下调有关。     

氨甲喋呤对映体药物对A549人肺腺癌细胞株增殖抑制作用及诱导凋亡作用

目的探讨氨甲喋呤对映体[（＋）MTX,（-）MTX]对A549细胞的增殖抑制作用及诱导凋亡作用。方法采用培养的A549细胞,应用MTT比色法分析其活性;用光学显微镜和荧光显微镜观察细胞的形态学变化;碘化丙啶（PI）单染流式细胞术检测细胞周期;DNA梯度电泳检测凋亡。结果在0.1～150μmol·L-1范围内,（＋）MTX和（-）MTX作用于A549细胞24,48,72h,均抑制细胞A549增值,但抑制强度为（＋）MTX〉（-）MTX,倒置显微镜和荧光显微镜观察不同浓度（＋）MTX和（-）MTX作用A549细胞不同时间后,出现细胞不同程度的形态学改变;用10μmol·L-1的（＋）MTX和（-）MTX作用A549细胞48h后,PI单染流式细胞术检测A549细胞周期的影响,表明氨甲喋呤对映体干扰A549细胞DNA合成;DNA梯度电泳检测结果发现MTX作用组有凋亡条带出现,其中（＋）MTX最为明显。结论 （＋）MTX和（-）MTX对A549细胞的抗增殖作用具有化学结构的立体选择性,（＋）MTX的抗A549细胞增殖作用明显强于（-）MTX。     

Impact of siRNA targeting pirh2 on proliferation and cell cycle control of the lung adenocarcinoma cell line A549

The aim of this study was to investigate the role of pirh2 (p53-induced RING-H2) protein in the proliferation, apoptosis and cell cycle control of the lung cancer cell line A549. Pirh2 expression was detected by immunofluorescence, Western blot analysis and real-time polymerase chain reaction (PCR). Cell proliferation was assessed by cell counting kit-8 (CCK-8). Cell cycle control and apoptosis were analyzed by flow cytometry. The results showed that pirh2 was expressed in the cytoplasm of A549 cells. The inhibition of pirh2 expression by siRNA (psiRNA-pirh2) resulted in reduced cell proliferation and increased apoptosis. In addition, the number of G₀/G₁ phase cells was increased but G₂/M cells were not affected significantly. Taken together, the inhibition of pirh2 expression in the lung adenocarcinoma cell line A549 resulted in reduced tumor cell growth via the inhibition of cell proliferation, the activation of apoptosis and the interruption of cell cycle transition.     

Role of ornithine decarboxylase suppression and polyamine depletion in the antiproliferative activity of polyamine analogs.

Two transfected cell lines, one carrying a mammalian ornithine decarboxylase (ODC) that is suppressed by polyamines and one carrying a trypanosomal ODC that is not, were used to ask whether ODC suppression is necessary for the antiproliferative activities of two polyamine analogs, N1,N8-bis(ethyl)spermidine (BES) and N1,N14-bis(ethyl)homospermine (BE444). Both analogs accumulated within cells and suppressed S-adenosylmethionine decarboxylase, as well as polyamine-sensitive mouse ODC activity. Neither drug was able to suppress the activity of the polyamine-refractory trypanosome ODC. But, whereas BE444 was able to inhibit growth of both cell lines, BES could inhibit only growth of cells carrying the polyamine-sensitive ODC, under conditions that cause prolonged depletion of endogenous polyamines. We conclude from these studies that the antiproliferative activity of BES, a less potent drug, requires the suppression of ODC. The efficacy of BE444 is enhanced by its ability to suppress ODC. However, it can function without ODC suppression, whereas BES cannot.     

Effect of glutathione combined with cisplatin and oxaliplatin on the proliferation and apoptosis of lung carcinoma cell line

Reduced glutathione (GSH) is generally administered for patients with cancer to reduce the side-effects of anti-cancer drugs. However, whether its protective effects interfere with anti-carcinogenicity is still unclear. The aim of this study was to investigate the effect of exogenous GSH on effects of oxaliplatin (L-OHP) or cisplatin (CDDP) by observing the proliferation and apoptosis of lung carcinoma cell line A549. Cell proliferation was evaluated by sulforhodamine-B assay and morphological changes were observed by an inverted microscope. Cell cycle distribution and apoptosis rate were observed by flow cytometry (FCM). Results showed that GSH did not change the inhibiting effects of L-OHP and CDDP on A549 proliferation, and did not reduce the apoptosis induced by CDDP. The FCM analysis showed the GSH combined with the CDDP group had fewer cells in the S-phase and had an apoptotic peak, which was not significantly different from that of the CDDP alone group (p?>?0.05). These results indicated that GSH does not reduce the effects of L-OHP and CDDP to inhibit A549 growth in vitro, and doesn’t affect the apoptosis induced by CDDP.     

The methionine salvage pathway compound 4-methylthio-2-oxobutanate causes apoptosis independent of down-regulation of ornithine decarboxylase

4-Methylthio-2-oxobutanoic acid (MTOB) is the final compound of the methionine salvage pathway that converts the polyamine byproduct methylthioadenosine to adenine and methionine. Here we find that MTOB inhibits growth of several human cell lines in a dose-dependent manner. Growth inhibition was specific for MTOB as we did not observe any inhibition with other chemically related compounds. MTOB treatment causes apoptosis and reduction of ornithine decarboxylase (ODC) activity but not ODC mRNA. To determine if MTOB exerts its effects primarily via ODC inhibition, we compared the effects of MTOB with the ODC-specific inhibitor difluoromethylornithine (DFMO). We found that MTOB was a more potent inducer of apoptosis than DFMO, lacked activation of caspase 3/7, and was able to induce apoptosis in cells lacking p53. Our results show that MTOB-induced growth inhibition and apoptosis is not simply secondary due to ODC inhibition and implies that MTOB activates apoptosis via other mechanisms.