肺癌中EGFR分子靶向药物及其疗效与毒性的分子预测指标

表皮生长因子受体(epidermal growth factor receptor,EGFR)信号转导通路是非小细胞肺癌(non-small cell lung cancer,NSCLC)治疗的重要靶点.通过小分子的酪氨酸抑制剂如厄洛替尼、吉非替尼或单克隆抗体如西妥昔单抗可阻止EGFR信号转导通路.     

厄洛替尼与西妥昔单抗联合应用治疗吉非替尼耐药NSCLC的疗效

本研究通过EGFR-TKI和EGFR单克隆抗体联合应用探讨治疗EGFR突变阴性和EGFR T790M突变继发性耐药的NSCLC的疗效。方法:应用EGFR突变阴性和EGFR T790M突变继发性耐药的NSCLL细胞原代培养及药敏技术检验EGFR-TKI和EGFR单克隆抗体联合应用的疗效。结果:检测厄洛替尼和西妥昔单抗联合处理对于15例EGFR突变阴性和8例T790M突变阳性的继发性耐药的NSCLC患者原代细胞的影响,应用浓度分别为50 μg/mL西妥昔单抗和1 μM厄洛替尼作用于EGFR突变阴性的NSCLC患者原代细胞,结果显示这三组间T/C值无显著性差异（P>0.05）,对于T790M突变阳性的继发性耐药的NSCLC原代细胞这三组间T/C值有显著性差异（P<0.05）,联合用药组疗效明显高于单药组。结论:进一步验证了厄洛替尼和西妥昔单抗联合应用对于EGFR突变阴性的NSCLC患者无效,但对于T790M突变阳性的继发性耐药的NSCLC患者有效。      

利妥昔单抗与依鲁替尼对弥漫性大B细胞淋巴瘤细胞增殖和凋亡协同作用研究

目的 近年来发现依鲁替尼是B细胞恶性肿瘤的新型靶向药物,利妥昔单抗联合新药依鲁替尼对弥漫性大B细胞淋巴瘤(diffuse large B cell lymphoma,DLBCL)的研究逐步展开,本研究探讨利妥昔单抗与依鲁替尼对DLBCL细胞系Farage增殖及凋亡影响的协同作用.方法 不同浓度的单药利妥昔单抗和单药依鲁替尼处理Farage细胞24、48和72 h后,采用CCK8法检测细胞增殖抑制率.15 μmol/L依鲁替尼处理Farage细胞48 h后,采用RT-PCR法检测凋亡相关因子mRNA的表达变化.含人补体的培养条件下,1 μg/mL利妥昔单抗联合10 μmol/L依鲁替尼同时处理Farage细胞48 h后,采用CCK8法检测增殖抑制率,采用流式细胞术检测凋亡率.结果 利妥昔单抗在不含人补体的培养基中对Farage细胞增殖抑制作用不明显,在含人补体的培养基中有明显增殖抑制作用,并呈浓度和时间依赖性.依鲁替尼在含或不含人补体的培养条件下对Farage细胞均有明显增殖抑制作用,呈浓度和时间依赖性,2种条件下的增殖抑制率差异无统计学意义(F=0.978,P=0.329),15 μmol/L依鲁替尼作用Farage细胞48 h时,Fas(1.63±0.09)、Caspase-8(1.90±0.11)和Caspase-3(2.20±0.11)mRNA相对表达量均高于空白组(1.00±0.00).在含人补体的培养基中,1 μg/mL利妥昔单抗联合10 μmol/L依鲁替尼同时处理Farage细胞48 h后,联合组增殖抑制率为(57.06±1.48)%,高于依鲁替尼单药组(33.83±3.39)%和利妥昔单抗单药组(26.92±2.74)%,差异有统计学意义,F=87.403,P<0.001.联合组的凋亡和坏死率(44.30±2.20)%高于利妥昔单抗单药组(21.73±2.00)%和依鲁替尼单药组(17.44±1.60)%,且利妥昔单抗单药组和依鲁替尼单药组均高于空白组(2.32±0.21)%, 差异均有统计学意义,F=327.205,P<0.001.结论 利妥昔单抗可通过CDC效应引起DLBCL细胞凋亡坏死及增殖抑制,依鲁替尼可能通过上调Fas、Caspase-3和Caspase-8基因的表达,促使DLBCL细胞凋亡和增殖抑制,利妥昔单抗联合依鲁替尼对DLBCL细胞的增殖抑制和凋亡坏死作用明显强于单药利妥昔单抗或依鲁替尼.     

抗EGFR单抗联合EGFR酪氨酸激酶抑制剂对人肺腺癌细胞凋亡的影响及其机制

目的:目前抗表皮生长因子受体(epidermal growth factor receptor, EGFR)的小分子酪氨酸激酶抑制剂吉非替尼(gefitinib)和抗EGFR单克隆抗体西妥昔单抗(cetuximab)在肺癌的临床应用中颇为广泛.鉴于这两种药物均针对EGFR分子靶点,因此本研究旨在就上述两种药物联合用药对人肺腺癌细胞凋亡的影响及其分子机制进行探讨.方法:吉非替尼和西妥昔单抗单独或联合用药作用于人肺腺癌细胞株A549和SPC-A-1后,用碘化丙啶标记,采用流式细胞术观察细胞凋亡情况,活细胞计数试剂盒测定各组细胞增殖抑制情况,Western印迹法检测两种药物对EGFR下游信号通路蛋白[磷酸化蛋白激酶B(phosphorylated Akt, p-Akt)、磷酸化EGFR(phosphorylated EGFR,p-EGFR)和磷酸化丝裂原激活蛋白激酶(phosphorylated mitogen-activated protein kinase, p-MAPK)]在蛋白水平表达的影响.结果:吉非替尼或西妥昔单抗单独作用后,A549和SPC-A-1细胞均明显凋亡,同时细胞增殖受到不同程度的抑制.Western印迹法检测p-Akt、p-EGFR和p-MAPK蛋白表达量均较不用药对照组下降.吉非替尼和西妥昔单抗联合作用后,肺腺癌细胞的凋亡、增殖以及在EGFR分子层次表现出较单一用药更为显著的作用.结论:吉非替尼和西妥昔单抗两种药物之间具有良好的协同作用,联合用药可能在临床治疗非小细胞肺癌中具有较大的应用潜力.     

美国临床肿瘤学会Ⅳ期非小细胞肺癌化疗的临床实践指南更新

本文旨在为Ⅳ期非小细胞肺癌患者的治疗提供更新版推荐.本文资料检索源自2002年以来公布的相关随机试验文献.此指南范围限于化疗与生物治疗.更新委员会对这些文献进行了总结并提供了推荐更新.162篇文献符合标准被纳入参考.本推荐基于可改善总生存期的治疗方法.仅改善无进展生存期的治疗方法推动了对毒性及生存质量的监测.对于体力状态评分为0分或1分患者的一线治疗,可推荐以铂类为基础的细胞毒性药物的两药联用.对铂类治疗有禁忌的患者,可采用非铂类细胞毒性两药联合.对于体力状态评分为2分的患者,单一细胞毒性药物即可.对于疾病进展或经过4个周期的治疗仍对治疗无反应的患者,应停止一线细胞毒性化疗.即使在6个周期后患者对治疗仍有反应,亦应停止两药细胞毒性化疗.对于伴有明确的表皮生长因子受体(epidermal growth factor receptor,EGFR)突变的患者,可推荐一线采用吉非替尼治疗;对于EGFR突变为阴性或不明确的患者,细胞毒性化疗更佳.除具有特定临床特征的患者外,可推荐贝伐单抗与卡铂-紫杉醇联用.对于通过免疫组化证实EGFR阳性的肿瘤患者,可推荐西妥昔单抗与顺铂-长春瑞滨联用.多西紫杉醇、厄洛替尼、吉非替尼或培美曲塞被推荐作为二线治疗.对于未曾接受过厄洛替尼或吉非替尼治疗的患者,可推荐厄洛替尼作为三线治疗.现有数据不足以推荐常规三线采用细胞毒性药物.已有的证据也不足以推荐常规应用分子标记物选择化疗.     

吉非替尼联合西妥昔单抗对人肠癌Lovo细胞作用的研究

目的 通过研究吉非替尼与西妥昔单抗的不同给药方案对人肠癌Lovo细胞的杀伤作用,探讨两药单药或联合使用对肠癌的作用结果。方法 通过PCR扩增后产物直接测序的方法筛选肠癌细胞,以药物联合效应测定方法,评价吉非替尼和西妥昔单抗不同时间、浓度、给药顺序对K-ras基因没有发生突变的肠癌Lovo细胞的抑制作用,流式细胞仪测定最佳方案对Lovo细胞周期分布及凋亡率的影响。结果 证实药物诱导细胞凋亡存在,吉非替尼联合西妥昔单抗对Lovo细胞的抑制作用优于单药的分别作用,而不同浓度、时间、给药顺序没有显著差异(P＞0.05)。结论 吉非替尼与西妥昔单抗联合应用对人肠癌Lovo细胞的增殖存在抑制作用。     

厄洛替尼单药治疗EGFR突变的晚期NSCLC患者试验汇合分析

背景与目的 厄洛替尼被全球多个国家批准用于晚期非小细胞肺癌(non-small cell lung cancer,NSCLC)患者的二、三线治疗.有报道显示EGFR突变的患者应用厄洛替尼治疗疗效更好.本文拟探讨厄洛替尼单药治疗EGFR突变晚期NSCLC患者的疗效.方法 计算机检索MEDLINE(2004-2009)、CBMdjc(2004-2008)、CNKI(2004-2008),互联网检索以及纳入试验的参考文献.纳入相关临床试验,提取并汇总试验结果.结果 共纳入研究7篇,包括晚期非小细胞肺癌患者463例.厄洛替尼单药治疗EGFR突变的晚期非小细胞肺癌患者,有效率达到66%,1年生存率达到73%,2年生存率达到5396,中位生存时间23个月以上,无进展生存时间8.6个月以上.结论 在当前研究的基础上,厄洛替尼单药一线和二线治疗EGFR突变的晚期NSCLC有效率高,无进展生存期和总生存期长,可以推荐作为EGFR突变的晚期NSCLC的首选治疗.尚需要更详尽的证据评价厄洛替尼单药方案或厄洛替尼与化疗联合方案与标准含铂化疗方案在治疗晚期NSCLC中的地位.     

肿瘤分子标志物与个体化治疗

 个体化治疗的关键是根据肿瘤的分子标志物来选择最佳药物,目前已有研究证实某些分子标志物可以预测药物的有效性,如西妥昔单抗可以使K-ras基因野生型的结直肠癌患者获益;吉非替尼和厄洛替尼可使表皮生长因子受体（EGFR）基因突变的非小细胞肺癌（NSCLC）患者获益;伊马替尼可使C-KIT基因表达或突变阳性的胃肠道间质瘤患者获益。而在安全性方面,尿苷二磷酸葡萄糖醛酸基转移酶1A1(UGT1A1)基因启动子区序列的多态性可以预测伊立替康的不良反应。     

西妥昔单抗治疗非小细胞肺癌耐药机制的研究进展

目的:总结国内外关于西妥昔单抗治疗非小细胞肺癌中耐药机制的研究进展.方法:应用Medline、PubMed、EMBASE及中国生物医学文献数据库检索系统,以"西妥昔单抗、非小细胞肺癌、表皮生长因子受体及耐药机制"为关键词,检索1990-01-2010-02的相关文献.纳入标准: 1)EGFR旁路信号传导通路与西妥昔单抗抵抗的关系;2)非小细胞肺癌患者EGFR基因突变与西妥昔单抗耐药;3)EGFR下游信号转导通路的活化与西妥昔单抗抵抗的关系.根据纳入标准分析34篇文献.结果:由于表皮生长因子受体(EGFR)调节多种细胞功能,EGFR抑制剂西妥昔单抗耐药现象可能与多个传导通路紊乱有关,包括旁路信号途径的激活、受体突变、配体自分泌/旁分泌的产生以及下游信号蛋白的组成性活化.结论:目前对西妥昔单抗产生耐药的确切机制尚不明确,要想解决其耐药问题,进一步提高西妥昔单抗治疗非小细胞肺癌的疗效,尚需进行更多项进一步的临床和基础研究来加以证实.     

西妥昔单抗疗效预测指标的研究进展

西妥昔单抗(cetuximal)是一种抗表皮因子受体(epidermal growth factor receptor, EGFR)人/鼠嵌合型IgG1单克隆抗体.能够阻碍内源EGFR配体的结合,进而阻断受体的二聚化、酪氨酸激酶磷酸化及其信号转导从而抑制受体的功能.此外,西妥昔单抗还可以介导抗体依赖的细胞介导的细胞毒作用(Antibody-dependent cellular cytotoxicity;ADCC)靶向杀伤表达EGFR的肿瘤细胞.西妥昔单抗对许多种肿瘤均有效,并能够提高放化疗的敏感性.EGFR免疫组化阳性表达是最初进行西妥昔单抗治疗的患者入选标准,然而,部分患者并未从西妥昔单抗的治疗中获益.一些临床研究结果也证实EGFR免疫组化结果与EGFR单抗治疗疗效并不甚相关.因此这就需要进一步探讨西妥昔单抗治疗有效性的标记物.     

Tripartite motif-containing 3 (TRIM3) inhibits tumor growth and metastasis of liver cancer

Background:Reduced expression of tripartite motif-containing 3 (TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma (HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines (SK-Hep1,Hep3B,Huh7,HepG2,Bel-7402) and one normal liver cell line (L02) were detected with Western blotting.HepG2 and Bel-7402 cells with IowTRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3 (LV-TRIM3),whereas Huh7 and Hep3B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA (siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect of TRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed thatTRIM3 overexpression induced G0/G1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3B cells by siTRIM3 led to significantly decreased percentages of both cells in the G0/G1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed thatTRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G0/G1 phase.     

Erlotinib is a viable treatment for tumors with acquired resistance to cetuximab

The epidermal growth factor receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase (RTK) and is recognized as a key mediator of tumorigenesis in many human tumors. Currently there are five EGFR inhibitors used in oncology, two monoclonal antibodies (panitumumab, and cetuximab) and three tyrosine kinase inhibitors (erlotinib, gefitinib, and lapatinib). Both strategies of EGFR inhibition have demonstrated clinical successes, however many tumors remain non-responsive or acquire resistance during therapy. To explore potential molecular mechanisms of acquired resistance to cetuximab we previously established a series of cetuximab-resistant clones by chronically exposing the NCI-H226 NSCLC cell line to escalating doses of cetuximab. Cetuximab-resistant clones exhibited a dramatic increase in steady-state expression of EGFR, HER2, and HER3 receptors as well as increased signaling through the MAPK and AKT pathways. RNAi studies demonstrated dependence of cetuximab-resistant clones on the EGFR signaling network. These findings prompted investigation on whether or not cells with acquired resistance to cetuximab would be sensitive to the EGFR targeted TKI erlotinib. In vitro, erlotinib was able to decrease signaling through the EGFR axis, decrease cellular proliferation, and induce apoptosis. To determine if erlotinib could have therapeutic benefit in vivo, we established cetuximab-resistant NCI-H226 mouse xenografts, and subsequently treated them with erlotinib. Mice harboring cetuximab-resistant tumors treated with erlotinib exhibited either a tumor regression or growth delay as compared to vehicle controls. Analysis of the erlotinib treated tumors demonstrated a decrease in cell proliferation and increase rates of apoptosis. The work presented herein suggests that 1) cells with acquired resistance to cetuximab maintain their dependence on EGFR and 2) tumors developing resistance to cetuximab can benefit from subsequent treatment with erlotinib, providing rationale for its use in the setting of cetuximab resistance.     

帕米膦酸二钠抑制人肝癌细胞增殖及诱导凋亡机制研究

目的 肝癌是常见的恶性肿瘤之一,寻找其低毒有效的化疗药物具有重要的临床价值.本研究旨在探讨一种临床常用的骨吸收抑制剂帕米膦酸二钠对人肝癌HepG2及Bel-7402细胞体外增殖和凋亡的影响及其机制.方法 采用MTS法检测不同浓度的帕米膦酸二钠对HepG2和Bel-7402细胞体外增殖的影响;以TUNEL末端标记法和Annexin-Ⅴ-FITC双标记流式法检测帕米膦酸二钠对细胞凋亡的影响采用蛋白质印迹法检测帕米膦酸二钠对凋亡相关蛋白:聚腺苷二磷酸-核糖聚合酶(poly ADP-ribose polymerase,PARP)、Caspase-9、Caspase-3、active-Caspase-3、Bax及Bcl-2表达的影响.结果 MTS检测显示,帕米膦酸二钠对HepG2及Bel-7402细胞均具有明显抑制作用,并呈剂量依赖性,药物的半数抑制浓度(50％inhibitory concentration,IC50)分别为46.12和51.35 μmol/L.流式细胞术检测发现,帕米膦酸二钠可诱导肝癌细胞剂量依赖性凋亡,20、40和60 μmol/L作用HepG2细胞24 h凋亡率分别为(25.70±6.00)％、(43.53±4.06)％和(74.73±2.90)％,与对照组的(3.43土1.07)％相比差异有统计学意义,F=175.5,P＜0.001;20、40和60 μmol/L作用Bel-7402细胞24 h凋亡率分别为(25.50±3.00)％、(48.30±1.02)％和(68.97±4.90)％,与对照组(3.33±0.05)％相比差异有统计学意义,F=81.97,P＜0.001.蛋白质印迹法检测结果显示,帕米磷酸二钠可诱导细胞内凋亡相关蛋白Caspase-9、Caspase-3及PARP的活化,促凋亡蛋白Bax的上调,抑凋亡蛋白Bcl-2的下调.结论 帕米膦酸二钠可以明显抑制人肝癌HepG2及Bel-7402细胞的体外增殖,其机制可能与线粒体凋亡途径的激活而诱导癌细胞发生凋亡相关,帕米膦酸二钠可能成为潜在的肝癌治疗药物.     

The combination of epidermal growth factor receptor inhibitors with gemcitabine and radiation in pancreatic cancer

PURPOSE: Gemcitabine-radiotherapy is a standard treatment for locally advanced pancreatic cancer. Clinical data have shown that gemcitabine plus erlotinib is superior to gemcitabine alone for advanced pancreatic cancer. Therefore, we investigated the effects of the combination of epidermal growth factor receptor inhibitors with gemcitabine and radiation on a pancreatic cancer model. EXPERIMENTAL DESIGN: EGFR signaling was analyzed by measuring phosphorylated EGFR (pEGFR(Y845, (Y1173)) and AKT (pAKT(S473)) protein levels in pancreatic cancer cell lines and tumors. The effects of scheduling on gemcitabine-mediated cytotoxicity and radiosensitization combined with erlotinib were determined by clonogenic survival. In vivo, the effects of cetuximab or erlotinib in combination with gemcitabine-radiation on the growth of BxPC-3 tumor xenografts were measured. RESULTS: We found in vitro that gemcitabine induced phosphorylation of EGFR at Y845 and Y1173 that was blocked by erlotinib. Treatment of BxPC-3 cells with gemcitabine before erlotinib enhanced gemcitabine-mediated cytotoxicity without abrogating radiosensitization. In vivo, cetuximab or erlotinib in combination with gemcitabine-radiation inhibited growth compared with gemcitabine-radiation (time to tumor doubling: gemcitabine + radiation, 19 +/- 3 days; cetuximab + gemcitabine + radiation, 30 +/- 3 days; P < 0.05, erlotinib + gemcitabine + radiation 28 +/- 3 days; P < 0.1). Cetuximab or erlotinib in combination with gemcitabine-radiation resulted in significant inhibition of pEGFR(Y1173) and pAKT(S473) early in treatment, and pEGFR(Y845), pEGFR(Y1173), and pAKT(S473) by the end of treatment. This study shows a novel difference pEGFR(Y845) and pEGFR(Y1173) in response to EGFR inhibition. CONCLUSIONS: These results show that the EGFR inhibitors cetuximab and erlotinib increase the efficacy of gemcitabine-radiation. This work supports the integration of EGFR inhibitors with gemcitabine-radiation in clinical trials for pancreatic cancer.     

Konjac glucomannan enhances 5-FU-induced cytotoxicity of hepatocellular carcinoma cells via TLR4/PERK/CHOP signaling to induce endoplasmic reticulum stress

5-Fluorouracil (5-FU) is a commonly used chemotherapeutic agent for various cancers. However, the drug resistance developed by tumor cells hinders the therapeutic effect. Konjac glucomannan (KGM) is indicated to sensitize 5-FU-resistant hepatocellular carcinoma (HCC) cells to 5-FU. In our study, we found that KGM or 5-FU treatment alone did not affect the malignant cell behaviors and endoplasmic reticulum (ER) stress of 5-FU-resistant HCC cells or HepG2/5-FU and Bel-7402/5-FU cells, while cotreatment with KGM and 5-FU significantly facilitated HCC cell apoptosis and ER stress and suppressed cell proliferation potential and migration abilities. Moreover, we explored the underlying mechanism by which KGM induces 5-FU cytotoxicity in HCC cells. We found that Toll-like receptor 4 (TLR4) was downregulated in KGM- and 5-FU-treated HCC cells. TLR4 overexpression reversed the KGM and 5-FU cotreatment-induced inhibition of the malignant behaviors of 5-FU-resistant HCC cells. Furthermore, KGM enhanced 5-FU-induced ER stress by inhibiting TLR4 to activate PERK/ATF4/CHOP signaling. Xenograft mouse models were established using HepG2/5-FU cells, and KGM was demonstrated to reverse 5-FU resistance in HCC tumors in vivo by suppressing TLR4 to enhance ER stress and activate PERK/ATF4/CHOP signaling. In conclusion, KGM combined with 5-FU treatment significantly promoted apoptosis and reduced cell proliferation, migration and ER stress in 5-FU-resistant HCC cells compared with KGM or 5-FU treatment alone by downregulating TLR4 to activate PERK/ATF4/CHOP signaling.     

Dual-agent molecular targeting of the epidermal growth factor receptor (EGFR): combining anti-EGFR antibody with tyrosine kinase inhibitor

Molecular inhibition of epidermal growth factor receptor (EGFR/HER1) signaling is under active investigation as a promising cancer treatment strategy. We examined the potency of EGFR inhibition achieved by combining anti-EGFR monoclonal antibody and tyrosine kinase inhibitor, which target extracellular and intracellular domains of the receptor, respectively. We specifically studied the combination of cetuximab (Erbitux, C225; ImClone Systems, New York, NY) with either gefitinib (Iressa, ZD1839; AstraZeneca, Macclesfield, UK) or erlotinib (Tarceva, OSI-774; Genentech, South San Francisco, CA) across a variety of human cancer cells. The combination of cetuximab plus gefitinib or erlotinib enhanced growth inhibition over that observed with either agent alone. As measured by immunostaining, inhibition of EGFR phosphorylation with the combination of cetuximab plus gefitinib or erlotinib was augmented over that obtained with single-agent therapy in head and neck (H&N) cancer cell lines. Phosphorylation inhibition of downstream effector molecules [mitogen-activated protein kinase (MAPK) and AKT] also was enhanced in tumor cells treated with the combination of cetuximab plus gefitinib or erlotinib. Flow cytometry and immunoblot analysis demonstrated that treatment of H&N tumor cells with cetuximab in combination with either gefitinib or erlotinib amplified the induction of apoptosis. Following establishment of cetuximab-resistant cell lines, we observed that gefitinib or erlotinib retained the capacity to inhibit growth of lung and H&N tumor cells that were highly resistant to cetuximab. Treatment with gefitinib or erlotinib, but not cetuximab, also could further inhibit the activation of downstream effectors of EGFR signaling in cetuximab-resistant cells, including MAPK and AKT. These data suggest that tyrosine kinase inhibitors may further modulate intracellular signaling that is not fully blocked by extracellular anti-EGFR antibody treatment. Finally, animal studies confirmed that single EGFR inhibitor treatment resulted in partial and transient tumor regression in human lung cancer xenografts. In contrast, more profound tumor regression and regrowth delay were observed in mice treated with the combination of cetuximab and gefitinib or erlotinib. Immunohistochemical staining, which demonstrated significant reduction of the proliferative marker proliferating cell nuclear antigen in mice treated with dual EGFR inhibitors, further supported this in vivo observation. Together, these data suggest that combined treatment with distinct EGFR inhibitory agents can augment the potency of EGFR signaling inhibition. This approach suggests potential new strategies to maximize effective target inhibition, which may improve the therapeutic ratio for anti-EGFR-targeted therapies in developing clinical trials.     

Inhibition of human hepatocellular carcinoma by L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13) in vitro and in vivo

A new anticancer tripeptide, L-proline-m-bis (2-chloroethyl) amino-L-phenylalanyl-L-norvaline ethyl ester hydrochloride (MF13), was investigated for its activity and mechanism in human hepatocellular carcinoma (HCC) cell lines. MF13 showed antiproliferative activities in the panel of 7 human HCC cell lines with IC50 in the range of 0.08-2.32 microM. A significant blockade in the S-phase occurred in tumor cells 12 h after their exposure to MF13. The inactivated Rb (phosphorylated Rb, pRb), which is present in the S-phase, was increased within 6 h of treatment. Bcl-2 expression was without change in hepatocarcinoma cells treated with MF13; however, a significant increase of bax was observed, resulting in a decreased ratio of bcl-2/bax. Increased activity of caspase-9, -8 and -3 was detected in the MF13 treated cells, indicating an activated pathway of apoptosis by MF13. Morphological examination as well as DNA gel electrophoresis demonstrated a nuclear fragmentation and DNA degradation in the form of multiple-unit DNA ladder in MF13 treated tumor cells. MF13 alone at 10 mg/kg (i.p.) inhibited HepG2 tumor in nude mice by more than 94% in volume. Bel-7402 tumor originated from a Chinese patient with HCC exhibited a sensitivity to MF13 similar to HepG2 in vivo. Antitumor effect of MF13 in the nude mice bearing human hepatocarcinoma (Bel-7402 or HepG2) was stronger than mitomycin C as well as its precursor m-sarcolysin (p<0.01), and comparable with cyclophosphamide. We believe MF13 merits consideration for further investigation as an agent against human hepatocellular carcinoma.     

23,24-二氢葫芦素B抑制Nrf2-ARE通路逆转肝癌耐药的实验研究

目的探讨23,24-二氢葫芦素B(DHCB)抑制核因子E2相关因子2(Nrf2)/抗氧化反应元件(ARE)通路逆转肝癌细胞耐药的作用机制。方法采用阿霉素(ADM)浓度梯度递增法建立肝癌耐药细胞株Bel-7402/ADM,分为空白对照组、ADM组、DHCB组、ADM+DHCB组、DHCB+TBHQ组和DHCB+N-乙酰基-L-半胱氨酸(NAC)组。MTT法和平板克隆实验检测DHCB对Bel-7402/ADM细胞增殖活性的抑制作用;流式细胞术检测DHCB对Bel-7402/ADM细胞活性氧(ROS)含量和细胞凋亡。Western blotting法检测DHCB对Bel-7402/ADM细胞中Nrf2、醌氧化还原酶1(NQO1)、谷胱甘肽S转移酶-π(GST-π)、多药耐药相关蛋白1(MRP1)、cleaved caspase-3蛋白表达的影响。结果Bel-7402/ADM细胞对ADM的耐药指数RI为6.56;而经DHCB(4μmol/L)协同作用后,Bel-7402/ADM细胞对ADM的耐药指数RI为2.21。与空白对照组比较,ADM+DHCB组细胞克隆形成率明显降低(P<0.05);MRP1、Nrf2、NQO1和GST-π蛋白表达量下调,cleaved caspase-3蛋白表达量升高(P<0.05)。与Nrf2激活剂TBHQ合用后,DHCB抑制Nrf2/ARE通路的作用被逆转。与空白对照组比较,DHCB可上调Bel-7402/ADM细胞ROS表达水平;与DHCB组比较,DHCB+NAC组细胞ROS表达水平降低。DHCB可明显促进Bel-7402/ADM细胞凋亡。结论DHCB可增强Bel-7402/ADM细胞对ADM的敏感性,其作用机制可能为抑制Nrf2/ARE信号通路,进而促进肿瘤细胞发生氧化应激损伤,诱导细胞凋亡。     

Increased expression of IL-6 mRNA in hepatocellular carcinoma cell lines correlates with biological characteristics

Background & aims: IL-6 has been implicated in both virus-associated and diethylnitrosamine-induced hepatocellular carcinomas (HCCs). Generally it is produced by immune cells such as Kupffer cells inthe liver. To understand mechanisms by which IL-6 might participate in the genesis of HCCs, the production of IL-6 by cell lines under different conditions was examined to determine inducing factors Methods: Expression of IL-6 mRNA in both hepatoma cell lines and a normal liver cell line L-02 was measured by quantitative RT-PCR. Biological molecules including liposome, dsRNA and cell debris were used to stimulate IL-6 mRNA expression in HepG2 cells and inhibition was effected by RNAi. Proliferation was assessed by MTT and clone formation and migration was determined by scratch assay. Results: All of the HCC cell lines observed expressed IL-6 mRNA, including HepG2, Bel-7402(7402), MHCC-97H and SMMC-7721.Normal liver cell line L-02 also expressed IL-6 mRNA. SiRNA to IL-6 specifically knockdowned IL-6 mRNA expression in HepG2, and liposome, dsRNA and cell debris increased it. Both proliferation and migration of HepG2 cells were related to the level of IL-6 HepG2 expressed. Conclusion: Both normal liver cell line and HCC cell lines can produce IL-6 so that Kupffer cells are noit the only source of the cytokine in the liver well as other immune cells. That the fact that HCC cells reacted to stimulation of biological molecules such as liposome, dsRNA or cell debris with increasing production of IL-6 indicates that the cytokine might play an important role not only in the period of tumor initiation but progression and recurrence as well.     

半乳糖受体介导的c-myc反义寡核苷酸对人肝癌细胞的靶向投递作用

背景与目的:c-myc反义寡核苷酸(antisenseoligodeoxynucleotide,ASODN)能抑制肝癌细胞的增殖,脂质体和腺病毒介导的c-mycASODN投递虽能显著抑制肝癌细胞的增殖,抑制荷肝癌小鼠肿瘤的生长,但这种投递缺乏靶向性;受体介导的药物投递具有高效性和靶向性的特点,已被用于基因及ASODN的靶向投递。本实验以半乳糖(galactose,Gal)-聚乙烯亚胺(polyethyleneimine,PEI)作为载体,介导c-mycASODN作用于人肝癌细胞Bel-7402,探讨c-mycASODN对Bel-7402细胞的靶向投递作用。方法:用流式细胞仪检测Bel-7402、U937细胞对荧光标记的Gal-PEI-c-myc-ASODN(简称Gal-PEI-ASODN)的摄取率及细胞内平均荧光强度;相差/荧光显微镜观察荧光标记的Gal-PEI-ASODN及c-myc-ASODN进入Bel-7402、U937、Raji细胞的形态;台盼蓝拒染法检测不同浓度Gal-PEI-ASODN对这三种细胞增殖的影响。结果:荧光标记的Gal-PEI-ASODN或ASODN与相应细胞孵育10min到4h,Gal-PEI-ASODN-Bel-7402细胞的荧光摄取率为88.25%～98.66%,细胞内平均荧光强度为38.64%～111.90%,与单纯c-mycASODN-Bel-7402细胞组、Gal-PEI-ASODN-U937细胞组相比,所有时间点的细胞荧光摄取率、胞内平均荧光强度均显著增高,经Poission分布检验,均有显著性差异(P<0.01)。相差/荧光显微镜观察到