Luminex液相芯片技术测定氨甲喋呤对映体耐药A549细胞株中磷酸化酪氨酸激酶受体

目的 探讨氨甲喋呤(MTX)对映体诱导的耐药A549细胞株中磷酸化酪氨酸激酶受体(RTKs)的表达差异及受体间的相关性.方法 用MTX对映体浓度递增结合低剂量持续诱导肺腺癌A549细胞株,用Luminex液相芯片技术检测L-(+)-MTX/A549细胞、D-(-)-MTX/A549细胞和亲本A549细胞3组细胞中7种磷...     

MTX对映体耐药A549细胞株中EGFR基因mRNA的表达分析

【摘要】目的探讨表皮生长因子受体（epidermalgrowthfactorreceptor，EGFR）在氨甲喋呤（methotrexate，MTX）对映体L-（＋）-MTX和D-（-）-MTX耐药A549细胞株中的表达。方法采用RT-PCR方法测定肺癌A549敏感细胞株和15p,moUL、35p,mol／L、55p,mol／L的L-（＋）-MTX和D-（-）-MTXA549耐药细胞株中EGFRmRNA表达情况。结果15μmol／L、35μmol／L、55μmol／LL-（＋）-MTXA549耐药细胞株中EGFR基因表达的mRNA相对含量分别为肺癌A549敏感细胞株的（1．55±0．26）倍、（1．86±0．13）倍、（1．20~0．05）倍。RT—PCR结果显示，A549敏感细胞株和L-（＋）-MTX各浓度A549耐药株均扩增出315bp条带，有EGFR基因表达，而3种浓度的D-（-）-MTX耐药细胞株中EGFR基因不表达。结论MTX诱导耐药后A549细胞株中EGFRmRNA发生变化，且在3种浓度两种对映体细胞株间具有明显的手性差异，MTX可能通过改变EGFR基因启动子的甲基化状态，影响EGFR的表达。     

氨甲蝶呤对映体获得性耐药A549细胞株二氢叶酸还原酶基因表达分析

摘要：目的：研究氨甲蝶呤(MTX)对映体［L-(＋)-MTX和D-(-)-MTX］耐药与二氢叶酸还原酶(DHFR)基因表达的关系。 方法：用浓度递增结合低剂量持续诱导法获得A549细胞对不同构型及不同浓度的MTX对映体的耐药细胞株,荧光定量PCR检测耐药细胞株中DHFR基因的相对含量。 结果：对两种不同对映体的获得性耐药存在差异,D型耐药细胞耐药指数高于L型;对映体各浓度耐药细胞间耐药指数也有差异。15 μmol/L L型、D型MTX首次诱导耐药细胞的DHFR相对含量低于亲本细胞,对该浓度对映体耐药的各细胞组间没有差别（P＞0.05）。35~45 μmol/L浓度耐药细胞的DHFR相对含量增加,45 μmol/L D型耐药细胞DHFR相对含量高于对应浓度L型耐药细胞,差异有显著性(P<0.05)。 结论：首次诱导MTX耐药以抑制DHFR基因为主。DHFR基因表达具有手性差异。DHFR基因的检测有望作为肿瘤治疗监测的指标。     

抑制Smac基因表达对胰腺癌裸鼠移植瘤生长影响的研究

[目的]探讨Smac基因对人胰腺癌裸鼠移植瘤生长的抑制作用.[方法]于裸鼠腋下接种200 μL 107/mL的PANC-1细胞悬液,建立人胰腺癌裸鼠移植瘤模型,将其分为三组：注射PBS液（A组）、转染空载体（B组）、转染Smac基因（C组）,比较三组移植瘤的大小、移植瘤细胞的凋亡率,移植瘤中Smac蛋白表达和微血管密度（MVD）.[结果]与A、B组比较,C组裸鼠移植瘤体积、质量和MVD显著减小;移植瘤细胞的凋亡指数和Smac蛋白的表达率均明显增高,其差异均有统计学意义（P〈0.05）.[结论]脂质体转染Smac基因能抑制人胰腺癌裸鼠移植瘤的生长,可能与该基因诱导胰腺癌细胞凋亡和抑制肿瘤血管形成有关.     

糖原合酶激酶3β抑制剂对前列腺癌细胞裸鼠移植瘤生长的抑制作用

目的研究糖原合酶激酶3β（glycogen synthase kinase-3β,GSK-3β）抑制剂抗前列腺癌细胞裸鼠移植瘤生长的效用。方法建立前列腺癌PC-3和LNCaP／Bcl-xl细胞株裸鼠移植瘤模型,观测GSK-3β抑制剂氯化锂（LiCl）及SB216763腹腔内给药后移植瘤重量及体积的变化。同时通过BrdU掺入的免疫组化染色探讨移植瘤的增殖状态,TUNEL染色比较对移植瘤细胞凋亡的影响。结果LiCl及SB216763均可明显抑制移植瘤生长（P〈0．05）。与对照组相比,LiCl给药组细胞BrdU标记明显降低（P〈0．05）,TUNEL染色差异无统计学意义。结论抑制GSK-3β活性可抑制前列腺癌裸鼠移植瘤细胞在体内的生长,其中LiCl可能通过干扰癌细胞DNA复制发挥作用。     

血管紧张素Ⅱ受体1在人胃癌细胞中的表达及其拮抗剂对胃癌荷瘤裸鼠生长的抑制作用

目的：检测血管紧张素Ⅱ受体1（AT1R）在人胃癌细胞株中的表达及其拮抗剂对人胃癌荷瘤裸鼠生长的抑制作用。方法：采用实时定量PCR和蛋白印迹分析法检测AT1R在胃癌细胞株中的表达。建立人胃癌裸鼠移植瘤模型,分别以2mg/kg和5mg/kgTCV-116（AT1R拮抗剂）每日给荷瘤裸鼠灌胃,观察肿瘤体积的变化并绘制肿瘤生长曲线;以免疫组织化学染色观察胃癌裸鼠移植瘤的微血管密度（MVD）和血管内皮生长因子（VEGF）表达。结果：AT1R在人胃癌细胞株中有较高水平的表达。经TCV-116处理的胃癌荷瘤裸鼠移植瘤体积较对照组显著减小,且TCV-116处理组的MVD和VEGF表达水平也较对照组显著降低。结论：选择性AT1R拮抗剂对人胃癌裸鼠移植瘤的生长具抑制作用,其可能是通过抑制肿瘤血管生成的途径产生抑制作用;故阻断血管紧张素Ⅱ（AngⅡ）可能是治疗胃癌的一种新方法。     

拉帕替尼对人卵巢癌SKOV3裸鼠移植瘤的影响

【目的】探讨新型靶向治疗药物拉帕替尼对人卵巢癌SKOV3裸鼠移植瘤生长的影响。【方法】将人SKVO3细胞株接种于裸小鼠胸壁皮下建立卵巢癌裸小鼠移植瘤模型。随机将其分为空白对照组（A组）和实验组（B组）,实验组根据拉帕替尼药物浓度的不同分为两个亚组：100 mg/kg （B1）和200 mg/kg （B2）,检测各组拉帕替尼用药前后荷瘤体积及裸鼠体质量的变化。【结果】拉帕替尼可显著抑制SKOV3裸小鼠移植瘤的生长,呈剂量依赖性,B2组抑瘤率显著高于B1组,其差异有统计学意义（P＜0．05）,但小鼠体质量用药前后比较无显著性差异（P＞0．05）。【结论】拉帕替尼可显著缩小人卵巢癌细胞株SKVO3裸鼠移植瘤的体积,为卵巢癌新型靶向治疗提供了理论基础。     

重组腺病毒ADV-TK对肝癌抑制作用的实验研究

目的观察已构建的含胸苷激酶（TK）自杀基因的重组腺病毒（ADV-TK）对肝癌细胞的体外杀伤作用和对肝癌裸鼠移植瘤的治疗效果。方法将ADV-TK体外感染人肝癌细胞株SMMC-7721,噻唑蓝（MTT）法检测受感染的SMMC-7721细胞被不同浓度更昔洛韦（GCV）作用后的细胞存活率情况。构建肝癌SMMC-7721裸鼠移植瘤模型,观察肿瘤注射重组腺病毒ADV-TK结合GCV治疗移植瘤的变化。结果相同滴度的重组腺病毒与不同浓度的GCV作用于肝癌细胞株SMMC-7721后,MTT法检测到细胞的存活率随着GCV浓度的增加而不断降低。动物实验中ADV-TK治疗组肿瘤体积明显小于对照组（ADV-null及NS）（P〈0.01）。结论重组腺病毒ADV-TK对肝癌SMMC-7721细胞的体外增殖和裸鼠体内的移植瘤生长均有明显的抑制作用。     

卡铂与顺铂对宫颈癌移植瘤胸苷磷酸化酶的调节作用

目的：探讨卡铂与顺铂对宫颈癌细胞株裸鼠移植瘤中胸苷磷酸化酶（thymi-dine phosphorylase，TP）的调节作用。方法：36只裸鼠均予皮下接种人宫颈鳞状细胞癌Siha细胞株，生成直径约为6～7mm的移植瘤。随机分为3组，每组12只，分别予腹腔内注射等量的卡铂、顺铂和生理氯化钠，给药10d后处死裸鼠，测量移植瘤体积及重量，计算抑瘤率，并以ELISA法测定移植瘤组织中TP的含量。结果：给药后顺铂组和卡铂组人宫颈癌细胞株裸鼠移植瘤体积均比给药前缩小，且均小于对照组（均为P〈0．05），3组的移植瘤重量由低到高依次为顺铂组、卡铂组、对照组（均为P〈0．05）。卡铂组和顺铂组的抑瘤率分别为1．6％和5．5％，2组的抑瘤率比较差异有统计学意义（P〈0．05）。3组移植瘤组织中TP含量由低至高依次为对照组、卡铂组和顺铂组（P〈0．05-0．01）。结论：卡铂和顺铂对宫颈癌细胞株裸鼠移植瘤中TP的表达具有上调作用，其中以顺铂对TP的上调作用较强。     

小干扰RNA抑制卵巢癌模型裸鼠移植瘤葡萄糖调节蛋白94的表达及意义

背景：一些实验在模型鼠移植瘤中葡萄糖调节蛋白94被敲除后,细胞黏附中断,刺激肝起源细胞增殖,进而促进肝肿瘤的发生,推测葡萄糖调节蛋白94在肝癌中起到保护作用。 目的：应用小干扰RNA技术抑制裸鼠卵巢癌模型皮下移植瘤内质网分子伴侣葡萄糖调节蛋白94的基因表达,探讨移植瘤中葡萄糖调节蛋白94基因及蛋白表达的变化对移植瘤生长的影响。 方法：从GeneBank中选取人类葡萄糖调节蛋白94基因序列,构建受控于人RNA聚合酶Ⅲ启动子U6的真核表达载体psiSTRIKETM/葡萄糖调节蛋白94。建立人卵巢癌HO-8910细胞株裸鼠皮下接种模型,并将真核表达载体转染入裸鼠移植瘤体内,观察肿瘤生长情况。卵巢癌裸鼠模型经过不同给药方案干预后分为特异性小干扰RNA组,非特异性小干扰RNA组和生理盐水对照组, RT-PCR和免疫组化SP法检测葡萄糖调节蛋白94 mRNA和蛋白在肿瘤内的表达情况,并观察模型裸鼠的移植瘤生长情况。 结果与结论：成功构建RNA干扰质粒载体,所有裸鼠模型均接种成功,5 d后即可见皮下肿瘤形成,14 d左右肿瘤直径达7-10 mm。转染质粒完毕后抑瘤率为65.1%,与非特异性小干扰RNA组和生理盐水对照组比较,差异有显著性意义（P＜0.01）。psiSTRIKETM/GRP94治疗后瘤体内葡萄糖调节蛋白94 mRNA和蛋白显著下调（P ＜0.01）。说明靶向葡萄糖调节蛋白94 mRNA的小干扰RNA可显著抑制人卵巢癌裸鼠移植瘤的生长,其机制可能是诱导葡萄糖调节蛋白94 mRNA和蛋白表达下调所致。     

Reversal of galectin-1 gene silencing on resistance to cisplatin in human lung adenocarcinoma A549 cells

This study aims to investigate reversal of Galectin-1 gene silencing on resistance to cisplatin in human lung adenocarcinoma A549 (or A549/DDP) in vivo and in vitro. The stably transfected lentivirus vector was used to silence Galectin-1 in human lung adenocarcinoma cell line A549 and A549/DDP cells and the cell lines were cultured and passaged. RT-PCR and western blot assay were used to test A549, A549/DDP cells, silenced Galectin-1A549 (A549/I) cells, Galectin-1 mRNA and protein expression levels, respectively, in A549/DDP (A549/DDP/I) cells. CCK8 assay was used to measure median inhibitory concentration (IC50) in each group and resistant index of A549/DDP cells and A549/DDP/I cells. Tumor model in nude mice was established by armpit injection of A549, A549/DDP, A549/I, A549/DDP/I cells. Cisplatin was injected intraperitoneally in tumor models and growth of tumor was observed in vivo model. Four weeks later, nude mice were killed and tumor weight and diameter was measured. mRNA and protein expression of Galectin-1 in A549/DDP cells was higher than that in A549 cells. mRNA and protein expression of Galectin-1 in A549/DDP/I cells was lower than that in A549/DDP cells. Moreover, IC50 values ​​and resistance index in A549/DDP cells was higher than that in A549 cells group and IC50 values ​​and resistance index A549/DDP/I cell group were lower than that in A549/DDP cells. Additionally, tumor weight and volume in A549/DDP/I cell group were lower than that in A549/DDP. In conclusion, Galectin-1 gene silencing would improve the sensitivity of A549/DDP cells to cisplatin in vivo and in vitro.     

重组人血管内皮抑制素的抗肿瘤活性研究

目的观察重组人内皮抑素的抗肿瘤活性及对血管内皮细胞增殖的抑制作用。方法以重组人内皮抑素作用于小鼠移植性肿瘤模型及人癌裸鼠移植性肿瘤,观察其对肿瘤生长的抑制效果,并与传统细胞毒性药物进行比较;同时,以MTT法观察其对血管内皮细胞ECV细胞和结缔组织类成纤维细胞L929细胞的生长抑制作用。结果重组人内皮抑素对小鼠移植性肿瘤Lewis肺癌及人癌裸鼠移植性肿瘤QGY肝癌、A549肺癌、Bcap37乳腺癌均有明显的肿瘤抑制作用;MTT实验显示重组人内皮抑素对血管内皮细胞及结缔组织类成纤维细胞具有生长抑制作用。结论重组人内皮抑素具有一定的抗肿瘤效果,其作用机理可能在于抑制肿瘤血管内皮细胞的增殖。     

肺腺癌细胞总RNA转染树突状细胞诱导特异性CTLs的体内抗肿瘤效应研究

目的探讨人肺腺癌细胞总RNA转染的树突状细胞(DCs)诱导抗原特异性的细胞毒T细胞(CTLs)对移植瘤裸鼠的抗肿瘤作用。方法 1)从外周血单个核细胞(PBMCs)中诱导DCs,提取人肺癌细胞A549总RNA,用于电转染DCs,转染后的DCs与自体T细胞混合培养,诱导抗原特异性的CTLs;2)实验分为转染RNA的DCs组、转染PBS的DCs组和未转染DCs组,流式细胞术(FCM)鉴定T细胞表型,3H-TdR掺入检测T细胞增殖能力;3)建立肺癌细胞株A549荷瘤裸鼠模型,过继回输CTLs,比较肿瘤体积,计算抑瘤率,免疫组化法检测移植瘤组织中Bax、Bcl-2、COX-2和VEGF的表达,并用全自动图像分析系统分析其平均光密度值(MOD)。结果 1)转染RNA的DCs和自体T细胞共育诱导的CTLs的CD8+T细胞大幅度上调,表达率(79.29±2.18)%明显高于转染PBS的DCs组CD8+T细胞(26.10±1.76)%和未转染DCs组CD8+T细胞(25.58±2.73)%(P0.05);2)转染RNA的DCs显著刺激了自体T细胞增殖,3H-TdR渗入所得的cpm值:阳性对照组为17 105±130,转染RNA的DCs组为7 759±493,转染PBS的DCs组为2 611±161,未转染DCs组为2 248±332(P0.05);3)成功建立肺癌裸鼠移植瘤模型,实验结束时,转染RNA组裸鼠荷瘤体积(540±99)mm3明显小于转染PBS组(1 572±147)mm3和未转染组(2 043±395)mm3(P0.05);4)转染RNA组和转染PBS组的抑瘤率分别为68.53%和8.62%;5)转染RNA的DCs诱导的CTLs能显著上调Bax的表达(转染RNA组MOD值为335±105),显著下调Bcl-2、COX-2和VEGF的表达(转染RNA组MOD值分别为146±47、122±33和128±58)。结论人肺腺癌细胞总RNA转染DCs诱导的抗原特异性CTLs对肺腺癌裸鼠移植瘤的生长产生抑制作用。     

Inhibitory effects of hyperoside on lung cancer by inducing apoptosis and suppressing inflammatory response via caspase-3 and NF-κB signaling pathway

Lung cancer is one of the most common malignancies in the world and the most threatening cancer to human health. Effective therapies based on non-cytotoxic induction in cell inflammation- and apoptosis-responsive pathways are thought to represent a novel advance in treating lung cancer. However, many studies are still required for effective pharmaceutical to induce cancer cell death. Hyperoside (Hyp) is the chief component of some Chinese herbs with anticancer effect. Here, we investigated the role of hyperoside on the lung cancer cell migration, invasion, inflammation and apoptosis in A549 cells in vitro and xenografts of nude mice in vivo. A549 cells were injected in nude mice for establishing tumors. Our results showed that hyperoside suppressed the proliferation, migration and invasion. Additionally, apoptosis was induced by hyperoside via Bcl-2/Bax-regulated Caspase3 activation, suggesting that hyperoside might inhibit lung cancer progression through apoptotic induction. And also, hyperoside could prevent progression and development of lung cancer through inactivating NF-κB signaling pathway. Subsequently, inflammatory cytokines, including TNF-α, IL-6, IL-1β and IL-18, were down-regulated significantly. And animal experiments also illustrated that the tumor volume and weight were reduced after hyperoside administration, which was also through apoptosis induction and prevention of inflammation response by Caspase3 activation and NF-κB inactivation. To our knowledge, it was the first time to evaluate the effects of hyperoside on preventing progression and development of lung cancer in vivo and in vitro to assess the possible therapies of hyperoside as a future approach for preventing lung cancer progression and development.     

Activation of nonsteroidal anti-inflammatory drug-activated gene-1 via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase revealed a isochaihulactone-triggered apoptotic pathway in human lung cancer A549 cells

The novel lignan isochaihulactone inhibits cell proliferation and is an effective inducer of apoptosis in a variety of carcinoma cell lines. To determine the mechanisms underlying these effects, we examined isochaihulactone-induced changes in gene expression using oligodeoxynucleotide-based microarray screening of a human lung carcinoma cell line, A549. Isochaihulactone-inducible genes included the early growth response gene-1 (EGR-1) and nonsteroidal anti-inflammatory drug-activated gene (NAG-1). Isochaihulactone increased EGR-1 and then NAG-1 mRNA and protein expression. Pure isochaihulactone induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Isochaihulactone-induced increases in EGR-1 and NAG-1 expression were reduced by the mitogen-activated protein kinase kinase 1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059), and this effect was not blocked by the phosphatidylinositol 3-kinase/protein kinase B pathway inhibitor 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002). Inhibition of isochaihulactone-induced NAG-1 expression by EGR-1 small interfering RNA blocked isochaihulactone-induced apoptosis in A549 cells, suggesting that induction of EGR-1 expression decreases survival of A549 cells. RNA interference using double-stranded RNA specific for NAG-1 also inhibited isochaihulactone-induced apoptosis, and cells transfected to increased NAG-1 expression had a greater apoptotic response to isochaihulactone and reduced colony formation efficiency. In addition, treatment of nude mice with isochaihulactone increased the in vivo NAG-1 expression as examined by immunohistochemistry from tumor biopsy. Isochaihulactone treatment increased the luciferase activity of NAG-1 in A549 cells transfected with the NAG-1 promoter construct. This induction increased expression of NAG-1 that was p53-independent and Sp1-dependent. Our findings suggest that NAG-1 expression is up-regulated by isochaihulactone through an ERK-dependent pathway involving the activation of EGR-1.     

Production and characterization of rat monoclonal antibodies with an apparent specificity for certain lung cancers and their potential clinical application

This paper describes two rat monoclonal antibodies (MAbs) (1A6 and 2H7, both IgG2a) directed against a human adenocarcinoma of the lung (A549). Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry (Avidin-Biotin-complex method) as the screening of the hybridoma cells revealed that 1A6 and 2H7 MAb mainly reacted with adenocarcinoma of the lung, breast and pancreas, and 2H7 MAb also recognized large cell carcinoma, epithelia of the lung, trachea, tongue or small cell carcinoma of the lung. The antigen molecules recognized by 1A6 MAb was not detected by immunoprecipitation but the molecular size of the antigen by 2H7 was ca. 40Kd. The antibody-producing heterohybridoma cells could be transplanted into peritoneal cavities of nude mice to form ascites. We found these monoclonals useful for immunohistodiagnosis of surgical specimens from patients with lung cancer. Furthermore, we utilized 125I-labeled 2H7 MAb, aiming at localizing A549 tumor mass transplanted into nude mice subcutaneously. This antibody clearly localized the tumor mass. It is suggested that they may be useful as both diagnostic and therapeutic agents.     

Epidermal proliferation of nude mouse skin, pig skin, and pig skin grafts. Failure of nude mouse skin to respond to the tumor promoter 12- O-tetradecanoyl phorbol 13-acetate

Human skin transplanted to nude mice offers a possible experimental system for the study of normal epidermal proliferation and differentiation, and for their pathological counterparts. Crucial to the development of such a system is the demonstration that such grafts retain the responsive features of donor skin. To document that donor proliferative characteristics are maintained in the grafts, a comparative analysis of agents that induce proliferation was made on skin of mice homozygous and heterozygous for nude, on pig skin, and on pig skin transplanted onto nude mice. A wave of epidermal proliferation could be induced in pig skin and pig skin grafted onto nude mice, but not in nude mouse skin after the topical application of 10 ng 12-O- tetradecanoyl phorbol 13-acetate (TPA). A 10-fold greater concentration of TPA or 5% croton oil induced proliferation in all species of epidermis studied. Mice, heterozygous for nude, showed a normal response to 10 ng TPA, suggesting that the ability to respond to TPA may be related, in part, to a recessive genetic trait. Nude mouse skin transplanted to a heterozygous littermate capable of responding to 10 ng TPA does not respond. These observations argue that: the graft retains its donor proliferative characteristics when transplanted to the nude, and the inability of the nude mouse to respond to lower doses of TPA may be related to absorption, the nude gene(s), or an inherent threshold to response. The lack of response to the promoter TPA provides a plausible explanation for the decreased incidence of tumors arising in nude mice during two-stage carcinogenesis experiments.     

三氧化二砷对HL-60细胞和人白血病裸鼠移植瘤模型的作用

目的:探讨三氧化二砷(As2O3)对HL-60细胞增殖和凋亡的影响及其对HL-60细胞皮下移植瘤裸鼠的抗瘤作用和药物毒性。方法:不同浓度的As2O3处理HL-60细胞24h,采用MTT比色法检测细胞增殖活性,Hochest33342染色、DNA凝胶电泳、流式细胞仪检测细胞凋亡。建立皮下移植瘤裸鼠模型,随机分入对照组和As2O3组,比较两组的抑瘤作用和裸鼠一般状态的变化。结果:As2O3可有效抑制HL-60细胞增殖并诱导其凋亡,其中在5～50!mol/L浓度范围内表现出明显的剂量依赖性抑制效应。同时可显著抑制HL-60细胞皮下移植瘤的生长,并延长荷瘤裸鼠的生存时间,对裸鼠一般状况和体重无不良影响。结论:As2O3在体外和体内均可显著抑制HL-60细胞的增殖并诱导其凋亡,且无明显毒副作用。     

双氢青蒿素对人肺腺癌细胞株A549抑制作用的实验研究

目的观察双氢青蒿素对人肺腺癌细胞株A549的作用。方法采用四唑氮蓝（MTT）法测定不同浓度和时间双氢青蒿素对A549细胞的生长抑制作用;应用流式细胞术检测双氢青蒿素对A549细胞增殖和细胞周期的影响;应用透射电镜分析法检测双氢青蒿素对A549细胞凋亡的影响。结果双氢青蒿素对A549细胞有明显的体外增殖抑制作用,72 h的IC50值为580 nmol/L,且有明显的时间和剂量依赖关系;双氢青蒿素作用后G0/G1期细胞数增加;双氢青蒿素作用A549细胞后可以出现凋亡的形态学改变。结论双氢青蒿素对人肺腺癌A549细胞有生长抑制作用,其机制可能与诱导A549细胞周期阻滞和凋亡有关。     

Exisulind-induced apoptosis in a non-small cell lung cancer orthotopic lung tumor model augments docetaxel treatment and contributes to increased survival

We reported previously a significant increase in survival of nude rats harboring orthotopic A549 human non-small cell lung cancer tumors after treatment with a combination of exisulind (Sulindac Sulfone) and docetaxel (D. C. Chan, Clin. Cancer Res., 8: 904-912, 2002). The purpose of the current study was to determine the biochemical mechanisms responsible for the increased survival by an analysis of the effects of both drugs on A549 orthotopic lung tumors and A549 cells in culture. Orthotopic A549 rat lung tissue sections from drug-treated rats and A549 cell culture responses to exisulind and docetaxel were compared using multiple apoptosis and proliferation analyses [i.e., terminal deoxynucleotidyl transferase-mediated nick end labeling, active caspase 3, the caspase cleavage products cytokeratin 18 and p85 poly(ADP-ribose) polymerase, and Ki-67]. Immunohistochemistry was used to determine cyclic GMP (cGMP) phosphodiesterase (PDE) expression in tumors. The cGMP PDE composition of cultured A549 cells was resolved by DEAE-Trisacryl M chromatography and the pharmacological sensitivity to exisulind, and additional known PDE inhibitors were determined by enzyme activity assays. Exisulind inhibited A549 cell cGMP hydrolysis and induced apoptosis of A549 cells grown in culture. PDE5 and 1 cGMP PDE gene family isoforms identified in cultured cells were highly expressed in orthotopic tumors. The in vivo apoptosis rates within the orthotopic tumors increased 7-8-fold in animals treated with the combination of exisulind and docetaxel. Exisulind increased the in vivo apoptosis rates as a single agent. Docetaxel, but not exisulind, decreased proliferative rates within the tumors. The data indicate that exisulind-induced apoptosis contributed significantly to the increased survival in rats treated with exisulind/docetaxel. The mechanism of exisulind-induced apoptosis involves inhibition of cGMP PDEs, and these results are consistent with a cGMP-regulated apoptosis pathway.