基于二级结构的靶向蛋白激酶C-α mRNA的反义药物设计及构效关系分析(英文)

目的:通过优化靶向蛋白激酶C-αmRNA的反义设计,获得优于阳性药ISIS3521的反义药物。方法:RNAstructure预测靶mRNA二级结构,针对二级结构单元设计。体外评价药物对A549细胞增殖的抑制效应。SPSS进行构效关系(QSAR)分析。结果:29个反义药物中3个IC_(50)值显著低于ISIS3521。靶二级结构单元膨胀环、内环、结点的碱基数、药物结构自由能和反应自由能在QSAR方程中有统计意义。多元回归R=0.68,P=0.0193。2个S-ODN有良好的靶结构与自由能但药效差。结论:计算机辅助药物设计有助于获得体外药效优于ISIS3521的反义药物。靶二级结构单元的不稳定程度和自由能影响药效。影响药效的其它因素则有待进一步研究。     

合用C-Raf和PKC-α mRNA为靶点的反义寡核苷酸增强对肺腺癌A549细胞体外生长的抑制作用

观察了靶向C-raf mRNA（ISIS5132）和PKC-α mRNA（ISIS3521）的反义硫代寡核苷酸合用对肺腺癌A549细胞体外增殖的抑制作用(MTT法). 单用药组药物浓度为500,200和80 nmol·L^-1;合用组A两药浓度分别为250,100及40 nmol·L^-1,即两药药量减半,药物摩尔浓度之和与单用药相同. 合用组B两药浓度分别为500,200及80 nmol·L^-1,但作用时间减半（各3 h）,两药作用时间之和与单用药相同. 结果表明,在合用组A,两药浓度水平为100 nmol·L^-1时,对A549细胞生长的抑制作用大于ISIS3521单用药组的200 nmol·L^-1水平(P<0.01);在合用组B,两药浓度水平为200和80 nmol·L^-1时,对A549细胞生长的抑制作用分别大于ISIS3521单用药组的200 nmol·L^-1和ISIS5132单用药组的80 nmol·L^-1水平（P<0.01）;其余合用组各个药物浓度水平的效应,与相应的单用药组的效应相近. 提示靶向不同癌基因的反义药物合用,可能增强对肿瘤细胞的抑制作用.     

合用C-Raf和PKC-αmRNA为靶点的反义寡核苷酸增强对肺腺癌A549细胞体外生长的抑制作用

观察了靶向 C- raf m RNA ( ISIS51 32 )和PKC- α m RNA( ISIS352 1 )的反义硫代寡核苷酸合用对肺腺癌 A549细胞体外增殖的抑制作用 ( MTT法 ) .单用药组药物浓度为 50 0 ,2 0 0和 80 nmol·L-1;合用组 A两药浓度分别为 2 50 ,1 0 0及 40 nmol· L-1,即两药药量减半 ,药物摩尔浓度之和与单用药相同 .合用组 B两药浓度分别为 50 0 ,2 0 0及 80nmol·L-1,但作用时间减半 (各 3h) ,两药作用时间之和与单用药相同 .结果表明 ,在合用组 A,两药浓度水平为 1 0 0 nmol· L-1时 ,对 A549细胞生长的抑制作用大于 ISIS352 1单用药组的 2 0 0 nmol· L-1水平 ( P<0 .0 1 ) ;在合用组 B,两药浓度水平为 2 0 0和 80 nmol· L-1时 ,对 A549细胞生长的抑制作用分别大于 ISIS352 1单用药组的 2 0 0 nmol· L-1和ISIS51 32单用药组的 80 nmol· L-1水平 ( P<0 .0 1 ) ;其余合用组各个药物浓度水平的效应 ,与相应的单用药组的效应相近 .提示靶向不同癌基因的反义药物合用 ,可能增强对肿瘤细胞的抑制作用 .     

定量构效关系方程对靶向蛋白激酶C-α mRNA的反义药物的活性预测

以往研究对34个针对蛋白激酶C-α mRNA二级结构设计的反义药物（AS-ODNs）进行了定量构效关系（QSAR）分析并得到QSAR方程. 本研究再次设计10个AS-ODNs验证QSAR方程对药物活性预测的可靠性．结果显示其中4个AS-ODNs IC₅₀值明显低于阳性对照ISIS3521（P<0.05）. 8个AS-ODNs与ISIS3521的实测IC₅₀与QSAR方程预测相符,实测与预测IC₅₀之间相关系数为0.76(P<0.05）. 两个AS-ODNs,AP1261(20)和AP0186(20)的实测IC₅₀与预测不符. 结果提 示QSAR方程一定程度上反映了AS ODNs活性与靶结构的关系,对预测反义药物生物活性有益. 但QSAR方程难以解释的其他因素需进一步研究以优化反义药物设计.     

反义寡脱氧核苷酸抑制人胶质瘤细胞的血管内皮生长因子(英文)

目的:了解反义VEGF寡脱氧核苷酸(ODN)对人胶质细胞瘤系(A172细胞)VEGF表达的抑制作用。方法:应用半定量PCR、免疫组化方法分别了解细胞内VEGF mRNA和蛋白的变化,并利用ELISA检测培养上清液中VEGF蛋白的含量。结果:A172细胞经反义VEGF ODN作用后VEGF mRNA的表达量明显减少,且随其浓度的增加而明显减少,但其表达量不受正义和错义VEGF ODN的影响。当A172细胞经50μmol·L~(-1)反义VEGF ODN作用后,细胞内及上清液内VEGF蛋白水平较对照显著减少。结论:反义VEGF ODN特异地抑制A172细胞VEGF mRNA和蛋白的表达。     

核因子-kappa B反义核苷酸对小鼠移植心脏存活时间的影响

目的;设计特定的反义硫代寡聚脱氧核苷酸(ODN)来特异性地抑制小鼠核因子-kappa B(NF-кB)的mRNA,并观察其对小鼠移植心脏存活时间的影响。方法;将30只小鼠随机分成NF-кB反义ODN给药组、碱基错配ODN对照组和未给药组,小鼠心脏移植血管重建后经腹腔缓释泵分别以12.5mg·kg~(-1)·d~(-1)的剂量全身给予NF-кB反义ODN和碱基错配ODN,持续给药14d,而未给药组不给药物。观察受体小鼠移植心脏存活时间;术后d7各组分别处死5只小鼠,测定受体小鼠脾细胞的混合淋巴细胞培养(MLC)和细胞毒T细胞(CTL)活性。结果:给药组小鼠的移植心脏存活时间较对照组和未给药组明显延长 (P<0.05);MLC显示给药组小鼠脾细胞对供体抗原刺激的增殖反应受到抑制(P<0.05);给药组受体小鼠的脾细胞的CTL活性也明显减弱(P<0.05)。结论:NF-кB反义ODN能有效抑制受体对小鼠移植物的细胞免疫反应,使同种异体的心脏移植存活时间延长。     

反义磷脂酰肌醇3-激酶寡脱氧核苷酸抑制白介素-1诱导的NF-κB活化

目的　研究反义磷脂酰肌醇 3 激酶 (PI 3 激酶 )寡脱氧核苷酸 (oligodeoxynucleotides,ODN)对核因子 κB(NF κB)活化的影响。方法　Lipofectin介导反义PI 3 激酶ODN转染HepG2细胞 ,以逆转录PCR法检测PI 3 激酶mRNA表达水平 ,用SandwichELISA法检测NF κB的活化。结果　(1)反义PI 3 激酶ODN抑制PI 3 激酶mRNA表达。 (2 )反义PI 3 激酶ODN呈剂量 (1～ 8μg)和时间 (5～ 2 4h)依赖性地抑制NF κB活化 ,2 μg反义PI 3 激酶ODN与细胞共孵育 8h时抑制效果最好。结论　反义PI 3 激酶ODN通过阻断PI 3 激酶表达抑制NF κB活化 ,预示反义PI 3 激酶ODN可潜在抑制IL 1的炎症活性     

Sp1反义寡核苷酸抑制Jurkat T细胞hTERT表达而抑制端粒酶活性(英文)

目的:研究转录因子Sp1反义寡核苷酸(ODN)对Jurkat T细胞端粒酶活性和端粒酶催化亚基hTERT表达的影响。方法:设计Sp1反义ODN并用脂质体转染细胞,用端粒酶PCR-ELISA方法检测端粒酶活性;用逆转录PCR方法检测Sp1和hTERT mRNA水平,用Western blot检测蛋白水平。结果:sp1反义ODN(1μmol/L)明显抑制Jurkat T细胞Sp1 mRNA和蛋白表达,抑制率分别为44.8％(P<0.05)和57％(P<0.01),并抑制hTERT mRNA表达,抑制率为43.7％(P<0.01);当浓度从0.25到2.0μmol/L,Sp1反义ODN对端粒酶活性抑制率从27.1％到64.6％,呈明显的剂量依赖性关系。结论:Sp1反义寡核苷酸通过抑制hTERT mRNA表达而抑制端粒酶活性。     

bcl-2反义药物的设计及其对白血病细胞生长的影响

目的　通过计算机模拟bcl 2基因的mRNA二级结构 ,优化反义药物设计。方法　用计算机程序Mfold软件模拟mRNA的二级结构后 ,筛选出不稳定的二级结构区域 ,进行反义药物设计 ;用实验评价所设计反义药物的生物效应 ,筛选出的bcl 2反义寡核苷酸对白血病细胞株HL 60、K5 62生物学活性的影响。结果　有 2个位点的反义寡核苷酸在计量为 10 μmol·L-1以上时 ,能明显抑制细胞的生长活性。结论　bcl 2mRNA二级结构的模拟是设计反义药物的一条新的途径     

埃坡霉素B对A549人肺腺癌细胞的体内外抗肿瘤药效学研究

目的:观察埃坡霉素B在体内外对A549人肺腺癌细胞生长的抑制作用。方法:用MTT法观察埃坡霉素B体外作用72h对A549人肺腺癌细胞增殖的抑制作用;用A549人肺腺癌裸鼠移植瘤模型评价埃坡霉素B在1.0、0.5、0.25mg/kg剂量下,给药2~3次后的体内抗肿瘤活性。结果:实验结果显示,埃坡霉素B在体外对人肺腺癌细胞A549抑制作用的IC50为(0.53±0.11)nmol/L,实际检测的最大抑制率为68.6%。体内实验表明受试物对A549人肺腺癌裸鼠移植瘤具有较强的抑制作用,并呈现出良好的剂量依赖性。给予1.0、0.5、0.25mg/kg受试物,第一次实验,其对A549裸鼠移植瘤的抑制率分别为78.6%、58.8%和48.3%;第二次实验,其对A549裸鼠移植瘤的抑制率分别为84.2%、76%和68.2%。阳性药物紫杉醇15mg/kg多次给药的抑瘤率分别为56.0%和85.7%。结论:埃坡霉素B在体内外对A549人肺腺癌具有较强的抑制作用。     

定量构效关系方程对靶向蛋白激酶 C-α m RNA的反义药物的活性预测(英文)

以往研究对 34个针对蛋白激酶 C- α m RNA二级结构设计的反义药物 (AS- ODNs)进行了定量构效关系 (QSAR)分析并得到 QSAR方程 .本研究再次设计 1 0个 AS- ODNs验证 QSAR方程对药物活性预测的可靠性 .结果显示其中 4个 AS- ODNsIC50 值明显低于阳性对照 ISIS352 1 (P<0 .0 5) . 8个AS- ODNs与 ISIS352 1的实测 IC50 与 QSAR方程预测相符 ,实测与预测 IC50 之间相关系数为 0 .76(P<0 .0 5) .两个 AS- ODNs,AP1 2 61 (2 0 )和 AP0 1 86(2 0 )的实测 IC50 与预测不符 .结果提示 QSAR方程一定程度上反映了 AS- ODNs活性与靶结构的关系 ,对预测反义药物生物活性有益 .但 QSAR方程难以解释的其他因素需进一步研究以优化反义药物设计     

Application of secondary structure prediction in antisense drug design targeting protein kinase C-alpha mRNA and QSAR analysis

AIM: To optimize the design of antisense drug targeting protein kinase C-alpha (PKC-alpha) mRNA and obtain better antisense drugs than ISIS3521 that is undergoing clinical trials. METHODS: RNAstructure (version 3.21, 1999) was utilized to predict the optimal and suboptimal secondary structures of human PKC alpha mRNA (GenBank, X52479), and 29 antisense phosphorothioate oligodeoxynucleotides (S-ODN) targeting the secondary structural elements, 3 partly matched S-ODN and 1 scrambled 3521 were designed. ISIS3521 was set as positive control. Mean (n = 3-5) 50% inhibitory effects on proliferation of A549 cells (IC50) of S-ODN were evaluated. Free energies (delta G degree 37) relating to the target secondary structural elements were calculated according to the nearest neighbor model. The quantitative structure-activity relationship (QSAR) analysis through multiple regression was obtained by SPSS. RESULTS: Three S-ODN; (5'-AGCCCA-GCCGCTTGGCTGGG-3', 5'-AGGAGTGCAGCTGC-GTCAAG-3', 5'-TCAGAGGG-ACTGATGACTTT-3') had lower IC50[(48 +/- 7), (50 +/- 4), (64 +/- 2.7) nmol.L-1, respectively] than that of ISIS3521 [(81 +/- 25) nmol.L-1]. The number of bases comprising the target secondary structural element bulge loop, internal loop, and knot, the free energy of S-ODN (delta G degree 37S), and reaction (delta G degree 37R) were important parameters in QSAR equation. In the multiple regression, R was 0.68, P = 0.0193. Not tally with the equation, two S-ODN (5'-TCAAATGGAGG-CTGCCCGGC-3', 5'-AAAACGTCAGCCATGGTCCC-3') with favorable target structures and delta G degree 37 did not behave good activities. CONCLUSION: Computer aided design was helpful to obtain S-ODN with better in vitro effect than current positive drug. The degree of instability of secondary structural elements and delta G degree 37 were important factors for drug activity. Other important factors needed for further investigation.     

Involvement of endothelin and ET(A) endothelin receptor in mechanical allodynia in mice given orthotopic melanoma inoculation

We investigated whether endothelin (ET) would be involved in skin cancer pain in mice. Orthotopic inoculation of B16-BL6 melanoma cells into the plantar region of the hind paw produced marked mechanical allodynia in C57BL/6 mice. Intraplantar injections of the ET(A)-receptor antagonist BQ-123 (0.3 - 3 nmol/site), but not the ET(B)-receptor antagonist BQ-788 (1 and 3 nmol/site), inhibited mechanical allodynia in mice with grown melanoma. In naive mice, an intraplantar injection of tumor extract (1 and 3 mg/site), which was prepared from the grown melanoma in the paw, produced mechanical allodynia, which was inhibited by BQ-123 and BQ-788 at doses of 3 and 10 nmol/site. An intraplantar injection of ET-1 (1 and 10 pmol/site) elicited licking behavior, which was increased in the melanoma-bearing hind paw. BQ-123 (3 and 10 nmol/site) inhibited licking induced by ET-1 (10 pmol/site). The level of mRNA of ET(A), but not ET(B), receptor, was significantly increased in the dorsal root ganglia on the inoculated side. Cultured B16-BL6 cells contained ET, and the melanoma mass increased the concentration of ET as it grew bigger. These results suggest that ET-1 and ET(A) receptor are at least partly involved in the induction of pain induced by melanoma cell inoculation.     

Antiproliferative activity of cardenolides isolated from Streptocaulon juventas

Sixteen cardenolides, two hemiterpenoids, two phenylpropanoids and a phenylethanoid isolated from the roots of Streptocaulon juventas (LOUR.) MERR. were examined for their antiproliferative activity toward three human-derived (HT-1080 fibrosarcoma, lung A549 adenocarcinoma, cervix HeLa adenocarcinoma) and three murine-derived (colon 26-L5 carcinoma, Lewis lung carcinoma, B16-BL6 melanoma) cell lines. The cardenolides selectively and strongly inhibited proliferation of the HT-1080 (IC(50) values, 0.054-1.6 microM) and A549 (IC(50), 0.016-0.65 microM) cell lines. The characteristic morphological changes and ladder-like DNA fragmentation in those cells treated with the cardenolides indicated the antiproliferative activity was due to the induction of apoptosis.     

反义白介素-1受体相关激酶-1寡核苷酸抑制白介素-1诱导的NF-κB活化

目的 :研究反义白介素 1受体相关激酶 1(IRAK 1)寡核苷酸对核因子 κB(NF κB)活化的影响。方法 :Lipofectin介导反义IRAK 1寡核苷酸转染HepG2细胞 ,以逆转录PCR法检测IRAK 1mRNA表达水平 ,用SandwichELISA法检测NF κB的活化。结果 :反义IRAK 1寡核苷酸抑制IRAK 1mRNA表达 ;反义IRAK 1寡核苷酸呈剂量 (1～ 8μg)和时间 (5～ 2 4h)依赖性地抑制NF κB活化 ,反义IRAK 1寡核苷酸 4μg与细胞共孵育 8h时抑制效果最好。结论 :反义IRAK 1寡核苷酸通过阻断IRAK 1表达抑制NF κB活化 ,预示反义IRAK 1寡核苷酸可潜在抑制IL 1的炎症活性。     

Antitumor activity of the Korean Mistletoe Lectin is attributed to activation of macrophages and NK cells

Inhibitory effect of the lectins (KML-C) isolated from Korean mistletoe (KM; Viscum album coloratum) on tumor metastases produced by murine tumor cells (B16-BL6 melanoma, colon 26-M3.1 carcinoma and L5178Y-ML25 lymphoma cells) was investigated in syngeneic mice. An intravenous (i.v.) administration of KML-C (20-50 ng/mouse) 2 days before tumor inoculation significantly inhibited lung metastases of both B16-BL6 and colon 26-M3.1 cells. The prophylactic effect of 50 ng/mouse of KML-C on lung metastasis was almost the same with that of 100 microg/mouse of KM. Treatment with KML-C 1 day after tumor inoculation induced a significant inhibition of not only the experimental lung metastasis induced by B16-BL6 and colon 26-M3.1 cells but also the liver and spleen metastasis of L5178Y-ML25 cells. Furthermore, multiple administration of KML-C given at 3 day-intervals after tumor inoculation led to a significant reduction of lung metastasis and suppression of the growth of B16-BL6 melanoma cells in a spontaneous metastasis model. In an assay for natural killer (NK) cell activity, i.v. administration of KML-C (50 ng/mouse) significantly augmented NK cytotoxicity against Yac-1 tumor cells 2 days after KML-C treatment. In addition, treatment with KML-C (50 ng/mouse) induced tumoricidal activity of peritoneal macrophages against B16-BL6 and 3LL cells. These results suggest that KML-C has an immunomodulating activity to enhance the host defense system against tumors, and that its prophylactic and therapeutic effect on tumor metastasis is associated with the activation of NK cells and macrophages.