Alterations and Its Mechanisms of Wnt Signal Pathway in Human High-matastatatic Large Cell Lung Cancer Cell Line L9981 by Transfecting with Nm23-H1 Gene

Backgroud and Objective Tumor metastasis is not only the malignant marker and characteristics of lung cancer, but also the main cause of failure to cure and lose their life of the

Influence of Nm23-H1 Gene Site Mutagenesis on Invasive And Metastatic Phenotype in Human High-Metastatic Large Cell Lung Cancer Cell Line L9981

Background and Objective Invasion and metastasis is not only the malignant phenotypes of lung cancer but also the main cause of death. To study and elucidate the molecular mechanism

Inhibitory effect of a new gossypol derivative apogossypolone (ApoG2) on xenograft of human prostate cancer cell line PC-3

Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were established via subcutaneous injection of PC-3 cells and the tumor-transplanted mice were divided into 4 groups: control group and three ApoG2 treatment groups, with 10 mice in each group. Volumes of the tumor were estimated every 2 d and the morphology of tumor tissues was observed. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and caspase-8 in tumor tissues. Results: ApoG2 (2.5 mg/kg-10 mg/kg) given intraperitoneally once a day can obviously inhibit the growth of subcutaneous prostatic carcinoma implant. The tumor volume decreased obviously when the treatment dosage was bigger than 5.0 mg/kg (P<0.01). Meanwhile, ApoG2 decreased the expression of PCNA and CD31, and enhanced the expression of caspases-3, caspase-8 in tumor tissues. Conclusion: ApoG2 exert an inhibitory effect on prostatic carcinoma possibly by inducing apoptosis and inhibiting tumor angiogenesis.

The Difference of the Copy Number Variation and Loss of Heterozygosity of Human Lung Large Cell Cancer Cell Line with Different Metastatic Potential

Background and Objective It has been proven that copy number gain/or loss (copy number variation CNV) in uences gene expression and result in phenotypic variation by

Effects of Methylseleninic Acid on the Proliferation and Cell Cycle in Human High Metastatic Large Cell Lung Cancer Cell Line L9981 and Its Molecular Mechanism

Background and Objective Lung cancer is the rst killer of human being in the whole world. Recently, although many treatment strategies have been developed, the anti-cancer effects

Effects of 4-(3-Chloro-Benzyl)-6,7-Dimethoxy-Quinazoline on Kinetics of P120-Catenin and Periplakin in Human Buccal Mucosa Squamous Carcinoma Cell Line

In order to detect molecular markers for the epidermal growth factor inhibitor 4-(3-chloro-benzyl)- 6,7-dimethoxy-quinazoline (tyrphostin), we investigated the kinetics of p120-catenin and periplakin in the human buccal mucosa squamous cancer cell line BICR 10 treated with 3 nM tyrphostin. Growth of BICR 10 cells was inhibited by treatment with tyrphostin. Although changes were not observed in the expression of EGFR and p120-catenin, expression of Akt, Src and periplakin in BICR 10 treated with 3 nM tyrphostin tended to decrease. In addition, phosphorylation of EGFR, Akt and Src was inhibited by treatment with tyrphostin. On immunocytochemical staining, immunoreactions with phosphorylated EGFR, phosphorylated Akt and phosphorylated p120-catenin were weak in BICR 10 treated with tyrphostin. There was a slight immunocy to chemical reaction to periplakin in BICR 10 cells induced by tyrphostin. In conclusion, the decrease in phosphorylation in EGFR and p120-catenin by tyrphostin, following the decrease in Src or Akt phosphorylation, may inhibit expression of several growth factors associated with the proliferation and migration of cancer cells.

Fermented Herbal Decoction Selectively Targeting Human Cancer Cell Line and Human Pathogenic Microorganism

Introduction: Prolonged immuno-suppressed status promised to induce internal growth of malignant cell and infectious agent, yet, only a small part of affected individuals seek medical attention or berried by commercially over-flowed fake information. Several studies have described complementary and alternative medicine as effective strategies for improving anti-infectious agent including malignant cell. The purpose of this study was to investigate the effect of a fermented herbal decoction (FHD) both in vitro and in vivo to malignant cells and microorganism by regulating leukocyte subset proportioning FHD as dietary material. Methods: In this approach of alternative study, selective anti-cancer effect by fermented decoction was tried to show first in vitro system both, cancer cell and virus strain. The fermented herbal decoction consisting of 80 sorts of herbs and fruits. The selective toxicity was set up and then for immunological factors in animal and human. The most important factor is to reduce side effect for a normal cell. Results: First, FHD was proved as safe by animal test. FHD regulated also the proportion of granulocyte and lymphocyte ratio both animal and human. In vitro culture showed selective toxicity by FHD against human melanoma and leukemia cell line but reduced toxicity was showed by normal cell line. As for the anti-virus activity, anti-virus effect was tested on the feeder layer of human fibroblast cell, after 9 days of culture. Second, FHD inhibits colon cancer growth in 3-methylholanthrene induced cancer in rat. Conclusion: The present results suggest that our fermented herbal decoction showed selective anti-cancer activities and anti-virus activities, together with the regulative effect on the immune system.

Far Infrared Ray Radiation Inhibits the Proliferation of A549, HSC3 and Sa3 Cancer Cells through Enhancing the Expression of ATF3 Gene

Far-infrared ray (FIR) is electromagnetic wave between 4 and 1000 μm. FIR causes heating, but how it affects cells is not well understood. In this study, we developed a culture incubator that can continuously irradiate cells with FIR and examined the effects of FIR on five human cancer cell lines, namely A431 (vulva), A549 (lung), HSC3 (tongue), MCF7 (breast) and Sa3 (gingiva). We found that FIR inhibits cell proliferation and induces cell hypertrophy without apoptosis in A549, HSC3 and Sa3 cells. Flow cytometry revealed that the inhibition of proliferation was due to G2/M arrest. Contrary, FIR did not inhibit cell proliferation and cause cell hypertrophy in A431 or MCF7 cells. Microarray analysis revealed that FIR suppressed the expression of cell proliferation-related and stress-responsive genes in FIR-sensitive cell lines (A549, HSC3 and Sa3). ATF3 in particular was identified as a key mediator of the FIR effect. Over-expression of ATF3 inhibited cell proliferation and knockdown of ATF3 mRNA using an antisense oligonucleotide suppressed FIR-induced growth arrest. These results indicate that a body temperature range of FIR radiation suppresses the proliferation of A549, HSC3, Sa3 cells and it appears that ATF3 play important roles in this effect.

Inhibition Mechanism of Novel Pyrazolo[1,5-a]pyrazin-4(5H)-one Derivatives Against Proliferation of A549 and H322 Cancer Cells

Objective To explore the inhibition mechanism and safety of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives against proliferation of human lung cancer A549 cells, H322 cells, and human umbilical vein endothelial cell(HUVEC). Methods Cells were treated with 40 μmol/L of the ppo3 a, ppo3 b, ppo3 i, and 0.1% DMSO(control) for 48 hours, respectively. Apoptosis was determined by Hoechst 33258 staining assay in H322 and A549 cells. Cell cycle distribution was determined by flow cytometry analysis in A549 cell. LC3-II, p53, and heat shock protein(HSP) 70 protein levels were detected by Western blotting in A549 cells treated with ppo3 b for 48 hours. The morphology and viability of HUVEC were observed by inverted microscope and sulforhodamine B(SRB) assay. Results Ppo3 a, ppo3 b, and ppo3 i significantly induced apoptosis in H322 and A549 cells. A strong G1-phase arrest was concomitant with the growth inhibitory effect on A549 cells. Ppo3 b effectively elevated the p53 protein level, but significantly reduced the HSP70 protein level. There were no significantly inhibitory effect on the morphology and viability of HUVEC when treated with ppo3 a, ppo3 b, and ppo3 i. Conclusions ppo3 a, ppo3 b, and ppo3 i could inhibit H322 proliferation through apoptosis and inhibit A549 through apoptosis and G1-phase arrest. The protein p53 and HSP70 might involve in the inhibition effects. These derivatives might be a clue to find effective and safe drug for lung cancers.

评价新型3,5-二取代吡唑啉衍生物抗人肺癌细胞株(A549)的潜能

目的 评估新型3,5-二取代吡唑啉衍生物抗人肺癌细胞株(A549)的潜能。方法 制备新型吡唑啉衍生物,评价这种衍生物在体外对抗人非小细胞肺癌(NSCLC)细胞株A549的效能。利用MTT法与SRB法筛查全部最终衍生物的抗癌潜力,测定其存在的细胞毒性。结果 本文条件下制备最终化合物用时10d,在表达抗A549中存在细胞毒性潜能,占比约为71.24%,对照药物为阿奇霉素,阿奇霉素所具有的细胞毒性为80.55%,两组相较并无差异(P>0.05)。所制备的新型吡唑啉衍生物的抗癌活性SRB检查结果与MTT法检查所得的细胞毒性结果类似,其成分为具有甲硫基、氟基团、甲氧基官能团的化合物,10d对抗A549的GI50水平为11.41μg/mL,对照药物ADR的TGI水平>100μm,化合物10d为46.76μm。结论 新型吡唑啉衍生物抗NSCLC的A549细胞株的潜力理想,细胞毒性水平较为合理。

HER3-targeted therapeutic antibodies and antibody-drug conjugates in non-small cell lung cancer refractory to EGFR-tyrosine kinase inhibitors

Human epidermal growth factor receptor 3(HER3)is a unique member of the human epidermal growth factor receptor(HER/EGFR)family,since it has negligible kinase activity.Therefore,HER3 must interact with a kinase-proficient receptor to form a heterodimer,leading to the activation of signaling cascades.Overexpression of HER3 is observed in various human cancers,including non-small cell lung cancer(NSCLC),and correlates with poor clinical outcomes in patients.Studies on the underlying mechanism demonstrate that HER3-initiated signaling promotes tumor metastasis and causes treatment failure in human cancers.Upregulation of HER3 is frequently observed in EGFR-mutant NSCLC treated with EGFR-tyrosine kinase inhibitors(TKIs).Increased expression of HER3 triggers the so-called EGFR-independent mechanism via interactions with other receptors to activate“by-pass signaling pathways”,thereby resulting in resistance to EGFR-TKIs.To date,no HER3-targeted therapy has been approved for cancer treatment.In both preclinical and clinical studies,targeting HER3 with a blocking an-tibody(Ab)is the only strategy being examined.Recent evaluations of an anti-HER3 Ab-drug conjugate(ADC)show promising results in patients with EGFR-TKI-resistant NSCLC.Herein,we summarize our understanding of the unique biology of HER3 in NSCLC refractory to EGFR-TKIs,with a focus on its dimerization partners and subsequent activation of signaling pathways.We also discuss the latest development of the therapeutic Abs and ADCs targeting HER3 to abrogate EGFR-TKI resistance in NSCLC.

nm23-H_1基因对人肺癌细胞中细胞外信号调节激酶活性的影响

目的 探讨肿瘤转移抑制基因nm2 3 H1 对人高转移大细胞肺癌细胞株L9981中细胞外信号调节激酶ERK1/2活性的影响。方法 应用特异性识别总ERK1/2 ( p44 /4 2MAPkinase)和双磷酸化ERK1/2( phospho p44 /4 2MAPkinase)的抗体及蛋白印迹法 (Westernblot) ,检测L9981(缺失nm2 3 H1 基因的原代肺癌细胞株 )、L9981 nm 2 3 H1 (转染了nm 2 3 H1 基因的L9981细胞株 )、L9981 PLXSN (转染了空载体的L9981细胞株 )中总ERK1/2和磷酸化ERK1/2的水平。磷酸化ERK 1/2活性应用非放射性免疫沉淀和Westernblot法以及 p44 /4 2MAPkinase分析试剂盒予以检测。 结果 L9981 nm2 3 H1 细胞株中磷酸化ERK 1/2的水平 ,以及ERK1/2活性均显著低于L9981细胞株和L9981 PLXSN细胞株 (P <0 .0 1) ,L9981和L9981 PLXSN细胞株间磷酸化ERK 1/2水平和ERK 1/2活性比较均无显著性差异 (P >0 .0 5 )。三个细胞株间总ERK1/2水平比较均无显著性差异 (P >0 .0 5 )。结论 nm2 3 H1 基因可明显靶向地抑制人高转移肺癌细胞株L9981中ERK 1/2的转录表达和ERK 1/2的活性。推测nm2 3 H1 基因的作用机制可能与其抑制了MAPK/ERK信号传导通路有关。

nm23-H_1基因转染能上调人肺癌细胞株L9981中GSK-3β激酶活性

目的 探讨转移抑制基因nm2 3 H1对人高转移大细胞肺癌细胞株L9981中Wnt信号传导激酶GSK 3 β的表达量和活性的影响 ,为全面阐明nm2 3 H1基因调控肺癌转移相关信号传导通路的分子机制提供实验依据。方法 将稳定转染nm 2 3 H1基因的人高转移大细胞肺癌细胞株L9981 nm2 3 H1、原代细胞株L9981和空载体转染细胞株L9981 pLXSN培养传代 ,应用Westernblot分别检测应用 2 0mmol/LLiCl处理前后三个肺癌细胞株胞浆、胞核中GSK 3 β的表达水平 ,应用免疫共沉淀同位素闪烁计数法测定上述肺癌细胞株胞浆和胞核中的GSK 3 β激酶活性。 结果 ( 1)L9981 nm2 3 H1胞浆和胞核中GSK 3 β蛋白表达量分别为( 63 41± 5 41)和 ( 4 3 5 6± 490 )IOD ,L9981 pLXSN分别为 ( 3 613± 3 83 )和 ( 70 5± 75 )IOD ,L9981分别为 ( 373 6± 2 98)和 ( 65 7± 5 7)IOD。三个细胞株间胞浆和胞核中GSK 3 β表达量均有非常显著差异 (P <0 .0 1) ;两两比较 :L9981 nm2 3 H1胞浆和胞核GSK 3 β表达水平均显著高于L9981 pLXSN和L9981(P <0 .0 1) ,而后两者之间比较均无显著性差异 (P >0 .0 5 )。 ( 2 )L9981 nm2 3 H1胞浆和胞核GSK 3 β激酶活性分别为 ( 2 895 5±2 5 0 9)和 ( 92 47± 92 4)CPM ,L9981 pLXSN分别为 ( 112 41±

常规化疗药物连续刺激对人肺腺癌细胞株表达erbB-2,p53和MDR1的影响

目的:分析常规化疗药物连续刺激对肿瘤细胞株表达erbB-2,p53和MDR1的影响,为联合应用生物治疗提供实验基础。方法:人肺癌细胞株经ADM,VP-16,DDP单药及联合用药连续刺激,每种药物分2个剂量梯度。应用流式细胞术分别检测erbB-2,p53和MDR1的阳性细胞数及平均荧光强度,以此推算出不同药物在不同浓度下连续2—3次作用后细胞株以上各蛋白表达的细胞数、平均表达量、总表达量,同时设对照组。结果:随着培养时间的延长各项检测指标其阳性细胞的百分数、平均荧光强度及总荧光强度均呈下降趋势。高剂量各组erbB-2和MDR1以上各项指标基本上均随刺激次数的增加呈下降趋势,而低剂量各组的检测指标则有升有降;p53的表达无一定规律,但第三次刺激后药物组的表达均高于对照组。结论:化疗药物连续刺激后,特别是小剂量化疗后,erbB-2,p53和NDR1的表达可不同程度增高,提示临床上针对这些分子对病人实施生物治疗时应考虑化疗药物的影响。

42℃温热联合顺铂对人卵巢癌细胞SKOV3毒性的影响

目的探讨42℃温热联合化疗药物顺铂对人卵巢癌细胞株SKOV-3作用的规律及其机制。方法应用四氮唑蓝快速比色法测定42℃热疗组、单纯化疗组以及以不同序贯方式结合的热化疗组对人卵巢癌细胞SKOV-3的生长抑制率;流式细胞仪测定各组细胞周期的变化;金氏公式分析相互作用指数,评价温热化疗两因素联合的作用。结果 42℃单纯热疗和单纯化疗均对卵巢癌癌细胞有抑制和杀伤作用;42℃热疗与化疗药物以不同方式联合,抑制作用均强于单纯化疗组和单纯热疗组(P<0.05),尤以热化疗同时作用组效果最佳(P<0.001);细胞周期分析发现:42℃热疗降低了S期细胞所占比例;与单纯化疗组相比,先化疗后热疗组、先热疗后化疗组和热化疗同时组的S期细胞所占比例减少,G1期细胞增多,其中热化疗同时组增减的幅度最大。结论 42℃温热可以明显增强化疗药顺铂的毒性,结合方式上以热化疗同时进行最佳,其作用机制可能与干扰细胞周期有关。

nm23-H_1转染对肺癌细胞整合素分子表达的调控

目的 通过基因转染方法将nm 2 3 H1基因转入nm 2 3 H1基因缺失的人高转移大细胞肺癌细胞株L9981,探讨nm2 3 H1基因对肺癌细胞整合素 (integrin)分子表达的调控作用。方法 应用阳离子脂质体介导的基因转染技术 ,将nm 2 3 H1基因导入人高转移大细胞肺癌细胞株L9981;应用半定量RT PCR技术检测nm2 3 H1基因转染前后integrinβ1、integrinβ3基因的mRNA表达 ;利用流式细胞仪技术检测nm2 3 H 1基因转染前后integrinβ1、integrinβ3基因的蛋白表达。 结果 ( 1)转染后获得稳定、高效表达nm 2 3 H1基因的转基因肺癌细胞株L9981 nm2 3 H1,该细胞株的integrinβ1、integrinβ3mRNA表达水平显著低于原代细胞株L9981。 ( 2 )转基因肺癌细胞株L9981 nm2 3 H1中integrinβ1、integrinβ3蛋白表达水平显著低于原代细胞株L9981(P <0 .0 1)。结论 nm2 3 H1基因转染能显著下调人高转移大细胞肺癌细胞株L9981中integrinβ1和integrinβ3mRNA和蛋白表达 ,表明nm2 3 H1基因可通过调控细胞integrin表达逆转人高转移大细胞肺癌L9981细胞株的转移潜能。

nm23-H_1基因转染前后人高转移大细胞肺癌细胞株L9981 PKA活性变化的实验研究

目的 探讨nm2 3 H1基因转染和forskolin对人高转移大细胞肺癌细胞株L9981PKA活性的影响。方法 应用PKA通路特异激动剂forskolin对人高转移大细胞肺癌原代细胞株L9981、转基因细胞株L9981 nm 2 3 H1 pLXSN和空载体转染细胞株L9981 pLXSN共同培养 ,应用放免法检测forskolin处理后不同时间点三个细胞株PKA的活性变化。结果 ( 1)forskolin处理前L9981、L9981 pLXSN和L9981 nm2 3 H1 pLXSN的PKA活性经F检验有显著性差异 (P <0 .0 1) ;两两比较 :L9981 nm2 3 H1 pLXSN的PKA活性显著高于L9981和L9981 pLXSN(P <0 .0 1) ,而L9981与L9981 pLXSN之间比较无显著性差异 (P >0 .0 5 )。 ( 2 )在同一作用时间 ( 3 0min)下 ,三个细胞株经不同浓度forskolin处理后PKA活性均显著升高 (P <0 .0 1) ,在forskolin浓度为 10 0 μmol/L时PKA活性最高 ,呈剂量依赖关系。 ( 3 )三个细胞株在同一浓度forskolin( 10 0μmol/L)处理下 ,PKA活性随作用时间升高 ,在forskolin作用时间为 3 0min时PKA的活性最高 ,呈时间依赖关系。结论 ( 1)nm2 3 H1基因转染能显著上调人高转移大细胞肺癌细胞株L9981的PKA活性 ,nm2 3 H1作为一种肿瘤转移抑制基因的作用机理与PKA信号通路可能有一定联系 ;( 2 )forskolin能显著上调L9981细胞?

新型棉酚衍生物ApoG2对人前列腺癌PC-3移植瘤生长的抑制

目的:探讨棉酚衍生物ApoG2对前列腺癌的抑制作用,并了解其作用机理。方法:建立人前列腺癌细胞的裸鼠皮下移植瘤模型,在ApoG2作用下观察其对皮下移植瘤生长的抑制作用,通过免疫组化方法检测肿瘤组织中bcl-2、PCNA及caspase-3、-8的表达。结果:ApoG2可明显抑制前列腺癌皮下移植瘤生长速度,肿瘤体积明显减小,ApoG2能增加肿瘤组织中caspase-3、8的表达,减少PCNA表达。结论:ApoG2对人前列腺癌有显著的生长抑制作用。

ω-3多不饱和脂肪酸对人胃腺癌细胞系AGS的作用

目的探讨ω-3多不饱和脂肪酸对人胃腺癌AGS细胞生长增殖的作用和可能的机制。方法以不同浓度梯度的DHA、EPA作用于体外培养的AGS细胞和人微血管内皮细胞HMEC-1,采用四甲基偶氮唑蓝(MTT)观察细胞增殖抑制率,使用流式细胞仪检测细胞周期变化,采用Western blot分析细胞线粒体呼吸链膜蛋白复合体Ⅰ、Ⅱ、Ⅴ表达变化。结果 DHA和EPA对胃腺癌AGS细胞增殖具有明显抑制作用,该抑制作用呈现明显的剂量时间依赖效应的特点(P<0.05),在相同的浓度梯度下,DHA的抑制作用强于EPA(P<0.05)。倒置显微镜下DHA作用后观察到AGS细胞明显皱缩,细胞的贴壁能力明显减弱。流式细胞仪检测显示,DHA、EPA干预后AGS细胞的DNA合成前期(G0/G1期)细胞分布比例明显增加,DNA合成期(S期)细胞分布比例明显减少(P<0.05)。Western blot分析可见,DHA干预AGS细胞24h和48h后,与对照组比较,AGS线粒体呼吸链膜蛋白复合体Ⅰ、Ⅱ、Ⅴ表达灰度值显著下降,而且随着作用时间延长,复合体表达灰度值下降愈明显(P<0.05)。DHA对人微血管内皮细胞的生长增殖、细胞形态和线粒体呼吸链膜蛋白复合体无明显影响(P>0.05)。结论ω-3多不饱和脂肪酸可选择性有效地抑制人胃腺癌AGS细胞的生长增殖。该作用可能是通过阻滞AGS细胞于G0/G1期和抑制AGS细胞能量代谢来实现的。

转染nm23-H_1基因靶向阻断人大细胞肺癌细胞株L9981 Wnt信号传导通路

目的 探讨nm2 3 H1 基因转染靶向阻断人高转移大细胞肺癌细胞株L9981Wnt信号传导通路的可行性 ,为阐明nm2 3 H1 基因调控肺癌转移抑制的分子机制和靶向治疗提供实验依据。方法 以转染nm 2 3 H1 基因的L9981 nm 2 3 H1 、原代L9981、空载L9981 pLXSN三株人高转移大细胞肺癌细胞为研究对象 ,应用Westernblot检测比较各株肺癌细胞胞浆、胞核中Wnt信号传导通路关键激酶GSK 3 β和 β 连环蛋白表达的变化。结果 ①GSK 3 β在L9981 nm 2 3 H1 肺癌细胞胞浆中表达量IOD( 63 41± 5 41)显著高于L9981( 373 6± 2 98)和L9981 pLXSN( 3 613± 3 83 ) (P <0 .0 0 1) ;②GSK 3 β在L9981 nm2 3 H1 肺癌细胞胞核中表达量IOD( 4 3 5 6± 490 )显著高于L9981( 65 7± 5 7)和L9981 pLXSN ( 70 5± 75 ) (P <0 .0 0 1) ;③β 连环蛋白在L9981 nm 2 3 H1 肺癌细胞胞浆中表达量IOD( 3 64 9± 118)显著高于L9981( 14 0 1± 3 1)和L9981 pLXSN ( 13 5 0± 5 5 )(P <0 .0 0 1) ;④β 连环蛋白在L9981 nm 2 3 H1 肺癌细胞胞核中表达量IOD( 2 945± 68)与L9981( 2 60 4± 2 3 )和L9981 pLXSN( 2 65 2± 5 3 )比较无显著性差异 (P >0 .0 5 ) ;⑤L9981与L9981 pLXSN两者间GSK 3 β和β 连环蛋白在肺癌细胞胞浆和胞核的?