蜂毒素协同TRAIL诱导人肝细胞癌细胞凋亡的实验研究
摘要
一、研究目的及背景:
肝癌是世界第五大癌症,每年有上百万人死于肝癌,我国为世界上肝癌的高发区,原发性肝癌是最常见的恶性肿瘤之一,占癌症死亡率的第二位。目前对肝癌的主要治疗方法是早期进行手术切除,但在我国,由于多数肝癌的发现均较晚或早期即有肝内播散;肝癌病人多伴较重肝硬化,肝功能异常;年龄或其他疾病的限制等,使临床肝癌手术切除治疗受到很大限制,并且肝癌术后复发率也很高,也极大的影响患者的生存。此外,放疗、介入治疗和肝移植等治疗方法虽然取得一定进展,但是总体疗效仍然不能令人满意,且在应用中受到诸多禁忌症的限制。一方面可能与肝癌细胞的多耐药基因有关,但更重要的原因是因为肝癌的发生发展是一个多步骤多因素的过程,涉及到细胞内的一系列复杂的生物学变化。细胞的生长调控机制紊乱是肿瘤发生的主要原因,即在某些致癌因素的作用下或细胞内基因突变发生突变,导致促进细胞生长和增殖的信号通路异常活化,从而引起分化不成熟的细胞异常增生,而促进细胞有序凋亡和成熟分化的信号通路受到抑制,细胞异常存活,细胞增殖和死亡调控信号失衡,从而导致肿瘤的产生。这是一个多因素共同作用和多步骤致瘤的缓慢过程。其机理涉及原癌基因的活化、抗癌基因的失活、生长增殖信号的持续作用、死亡信号衰减或抑制、细胞周期异常及肿瘤血管内皮细胞增生等多个环节。此外,肝癌的发生还和病毒感染密切相关。由于肝癌的发生和发展涉及到多因素、多途径的共同作用,因此,仅针对肝癌发生机制中某一因素的单一药物治疗效果不够理想。因而,在深入研究肝癌形成、发生、发展的分子机制,在分子水平明确肝癌细胞各种生长调控因素间的动态平衡的基础上,针对肝癌发生发展中多个环节,探索能够特异性抑制肝癌细胞生长的药物或能够联合抑制肝癌细胞增殖、促进凋亡的药物组合并揭示其作用机制是国内外研究的热点之一,对于我国这样一个肝癌的高发国度而言,更是具有重要的理论意义和应用价值,具有潜在的经济效应和社会效应。
TRAIL(TNF-related apoptosis-inducing ligand,人肿瘤坏死因子相关凋亡诱导配体)是肿瘤坏死因子超家族的成员之一,TRAIL蛋白是一种Ⅱ型糖蛋白,分为细胞外C端区域、跨膜区、胞内N端区域三个部分,其C末端的胞外区与TNF家族的其它成员具有较高的同源性,TRAIL通过与特异性受体结合发挥生物学功能,目前发现的TRAIL受体有5种:TRAIL-R1/DR4、TRAIL-R2/DR5、TRAIL-R3/DcR1、TRAIL-R4/DcR2和OPG。按其功能和结构可分为三类:(1)死亡受体:DR4和DR5;(2)诱骗受体:DcR1和DcR2;(3)可溶性受体OPG。其中,介导凋亡的主要是死亡受体DR4和DR5。TRAIL通过与其活化型受体DR4或DR5的胞外段结合,引起受体的三聚作用并使胞内段的死亡结构域(DD)活化,并通过其死亡结构域募集胞浆死亡信号传递蛋白FADD,FADD进而活化caspase-8和caspase-10等凋亡执行分子,导致细胞的损伤,同时,TRAIL信号通过影响Bcl-2家族分子,破坏线粒体膜的完整性,既而引起线粒体内cytochrome c及Smac/DIABLO的释放,凋亡小体的形成,从而引起DNA的断裂。TRAIL除可引起凋亡信号的活化,还可以通过激活TRAF2(TNF受体相关因子2),引起下游RIP、TAK1等分子的活化,最后引起MAPK和NFκB信号通路的活化。放疗或化疗通常是通过P53基因的激活来达到杀伤肿瘤细胞的目的,由于很多人肿瘤细胞包括肝癌细胞中P53基因是缺失或突变的,因而很多肿瘤对于放疗或化疗不敏感。TRAIL诱导的凋亡并不依赖于P53的活化,其能够在体内外诱导多种肿瘤细胞凋亡但对正常组织细胞无明显影响,是常用的凋亡诱导及基因治疗模型,其重组细胞因子已应用于某些肿瘤的临床前实验及临床治疗,具备良好的应用前景25。但是,大多数人肝细胞癌细胞对TRAIL诱导的凋亡并不敏感,限制了其在人肝癌治疗中的应用。因而,寻找能够特异性增强TRAIL凋亡诱导作用的药物是目前国际肿瘤基因治疗研究的热点之一,受到国际学术界相关领域的关注。面对这一状况,我国丰富的中药资源是我们的优势之一,很多动植物中提取的生物活性成分都被证实有抗肝癌作用,并被广泛应用于临床。因此,寻找新的生物活性成分,探索其抗肿瘤机理并研究其与TRAIL的协同诱导凋亡效应是一项具有一定理论意义和应用前景的工作。基于本科室以往工作的基础上,我们选择蜂毒的有效活性成分蜂毒素单体作为了研究对象。
蜂毒素(melittin)是蜂毒的主要活性成分,约占蜂毒干质量的50%。蜂毒素单体是经现代工艺从蜂毒中提取的有效活性成分,纯度可达99%以上。它是由26个氨基酸组成的碱性多肽,其N端和C端分别具有疏水和亲水的生化基团,具有典型的两性分子结构,具有很好的水溶性,能够和细胞膜上的磷脂相互作用,在以往的研究中经常作为细胞膜上脂-蛋白相互作用的模型。蜂毒素是Ca2+-ATP酶、H+K+-ATP酶、Na+K+-ATP酶的抑制剂,可以抑制GTP结合蛋白的内在活性,近年来,随着蜂毒分离、提取、纯化技术的不断提高,对蜂毒素的功能及作用机制的研究也日益广泛。目前的研究主要集中在蜂毒素的抗炎作用上,有研究表明蜂毒素能够通过与NF-kB上游的IkB作用从而抑制NF-kB的活性发挥抗炎作用;也有报道认为蜂毒素能够通过与NF-kB的P50亚基相互作用从而起到抗炎效应;在骨关节炎的软骨细胞中能够通过抑制MMP-3从而减少炎性因子的分泌。有研究显示报道去除蜂毒素C端12-26位的谷氨酰氨残基可以增强它的抗炎活性;蜂毒素对体内外支原体和衣原体引起感染也均有抑制作用;也有研究表明蜂毒素对某些肿瘤细胞具有凋亡诱导效应,如蜂毒素可以通过抑制NF-kB和Akt的活性并上调凋亡相关蛋白的表达从而诱导鼠血管平滑肌细胞凋亡,可以引起Ca2+内流继而导致骨肉瘤细胞MG63凋亡。本科室以往的工作曾对蜂毒素的生物学活性有初步研究,证明携带蜂毒素的重组腺病毒在体外能够肝癌细胞的生长。此外,蜂毒素可通过抑制Rac1依赖的信号通路在体内外抑制肝癌的转移。那么,蜂毒素能否诱导肝癌细胞凋亡?蜂毒素是否与TRAIL具有协同效应?在既往工作的基础上,我们对蜂毒素单体对人肝癌细胞生长增殖的作用进行了初步实验。结果显示蜂毒素单体对人肝癌细胞hepG2的生长有明显的凋亡诱导作用,且这种作用存在明显的时间和浓度依赖性。此外,我们发现,在5ug/ml的蜂毒素协同作用下,低浓度(50ng/ml)的TRAIL即可引起TRAIL不敏感肝癌细胞株hepG2出现明显凋亡,相比于单独的蜂毒素处理组和TRAIL处理组差异显著。因此,我们初步推测蜂毒素能够诱导肝癌细胞凋亡,并与TRAIL具有协同效应,能够增强肝癌细胞对TRAIL的敏感性。本课题拟针对这一设想进行研究,探讨蜂毒素与TRAIL协同诱导肝癌细胞凋亡的分子机制。主要研究目的为:
1、明确蜂毒素对肝癌细胞的凋亡诱导效应及其机制。2、探讨蜂毒素能否增强与肝癌细胞对TRAIL诱导凋亡的敏感性及其分子机制。3、观察蜂毒素与TRAIL协同诱导肝癌细胞凋亡的体内效应。
二、研究方法:
1、流式细胞仪检测蜂毒素诱导hepG2细胞凋亡的效应及其时间剂量关系,western-blot、激酶分析法及报告基因方法检测凋亡及生长增殖相关信号通路关键分子的变化,并使用相应信号通路的抑制剂处理细胞以改变该信号通路的活化状况,观察凋亡效应的变化,明确相应信号通路与蜂毒素诱导的凋亡效应的关系,探讨其分子机制。
2、分别设立不加刺激的对照组、蜂毒素单独处理组、TRAIL单独处理组及协同作用组,作用HepG2细胞,流式细胞仪检测细胞凋亡的差异,观察蜂毒素能否增强肝癌细胞对TRAIL诱导凋亡的敏感性,并检测相关信号通路的改变,干预其活化状态,明确其与蜂毒素与TRAIL可能的协同效应的关系,探讨蜂毒素与TRAIL可能的协同效应的作用环节及机理。3、建立裸鼠荷瘤模型,通过小鼠尾静脉途径给药的方法,分别设立TRAIL单独处理组(10mg/kg)、蜂毒素低剂量组(50ug/kg)、蜂毒素高剂量组(100ug/kg)及协同作用组(TRAIL10mg/kg +蜂毒素50ug/kg),以注射PBS小鼠为实验对照组,观察各组间肿瘤大小的差异,观察蜂毒素与TRAIL的体内抗肿瘤效应。
三、研究结果及结论:
1、蜂毒素能够诱导肝癌细胞HepG2凋亡及其机制:我们分别选用不同浓度(0、5ug/ml、10 ug/ml)的蜂毒素作用于HepG2细胞不同时间点(0、12h、24h、48h),分别通过AnnexinV-PI双标、Rho123标记及DHR123标记,流式细胞仪检测细胞凋亡的变化。结果显示,蜂毒素能够诱导HepG2细胞凋亡,呈明显的时间和浓度依赖性,且凋亡现象伴随线粒体电势差的降低。在其他人肝癌细胞系Hep3B、BEL7402及SMMC7721中也得到了类似的结果。10 ug/ml的蜂毒素作用12h后,HepG2细胞中caspase-9、caspase-3、PARP均有活化,cytochromeC及smac/DIABLO的释放增多,使用相应的caspase抑制剂、钙离子抑制剂BAPTA、ROS抑制剂NAC预处理细胞后,细胞凋亡有明显逆转,此外,BAPTA及NAC预处理能够抑制caspase-9、caspase-3、PARP的活化及cytochrome c及smac/DIABLO的释放。蜂毒素作用0、15min、30min、60min后,能够活化CaMKII-TAK1-MKK-JNK/p38信号通路,相应的抑制剂作用后,能够部分逆转蜂毒素诱导的凋亡。蜂毒素作用还能够抑制IKKβ的激酶活性及TAK1介导的NF-κB的活化。2、蜂毒素能够协同TRAIL诱导肝癌细胞凋亡及其机制:我们分别设立不加刺激的对照组(ctrl)、蜂毒素单独处理组(5ug/ml)、TRAIL单独处理组(50ng/ml)及协同作用组(5ug/ml+50ng/ml),刺激HepG2细胞12h,Annexin V-PI双标,流式细胞仪检测细胞凋亡的差异。结果显示,蜂毒素和TRAIL共同作用后,细胞凋亡明显增加,显著高于蜂毒素单独处理组及TRAIL单独处理组,提示蜂毒素和TRAIL具有协同诱导人肝癌细胞凋亡的作用。在其他对TRAIL不敏感的人肝癌细胞系Hep3B、BEL7402及SMMC7721中也得到了类似的结果,我们同时观察了TRAIL敏感的肿瘤细胞系Hela和Jurkat中,在TRAIL剂量较低时(5ng/ml、10 ng/ml),较之单独的TRAIL作用,蜂毒素和TRAIL共同作用后细胞的凋亡明显增加,当TRAIL剂量较高时则差异不明显。此外,我们设立时间梯度,分别刺激以上各细胞系0、6h、12h、24h、48h,观察各组间凋亡的差异,结果显示,随着作用时间的延长,蜂毒素和TRAIL协同诱导的凋亡逐渐增加,呈现明显的时间依赖性。既而我们检测了蜂毒素和TRAIL共同作用对HepG2细胞信号通路的影响,较之TRAIL单独作用组及蜂毒素单独作用组,蜂毒素(5ug/ml)和TRAIL(10ng/ml、50 ng/ml)共同作用后,P38和JNK的磷酸化程度显著升高,使用相应的抑制剂预处理后,细胞凋亡有部分逆转。同时,蜂毒素(5ug/ml)和TRAIL(10ng/ml、50 ng/ml)共同作用能够促进TAK1的磷酸化并增强TAK1的激酶活性。在HepG2细胞中转染TAK1的显性负突变体(dominant—negative)TAK1K63W后,蜂毒素和TRAIL协同诱导的凋亡有显著的逆转,P38和JNK的活化也受到明显的抑制。此外,我们观察到,相比于TRAIL单独作用引起的NF-κB的活化,蜂毒素和TRAIL共同作用能够明显抑制IKBα的磷酸化、IKKβ的激酶活性及NF-κB的活化,相应的,NF-κB依赖的抗凋亡分子Bcl-xl及c-IAP1的表达也受到抑制。3、蜂毒素能够协同TRAIL在体内抑制肿瘤生长及其机制:为了检测蜂毒素协同TRAIL体内抗肿瘤的效应,我们通过皮下接种HepG2及SMMC7721细胞的方法建立了裸鼠荷瘤模型。经过小鼠尾静脉途径连续给药7天,实验分组为:TRAIL单独处理组(10mg/kg)、蜂毒素低剂量组(50ug/kg)、蜂毒素高剂量组(100ug/kg)及协同作用组(TRAIL10mg/kg +蜂毒素50ug/kg),以注射PBS小鼠为实验对照组,观察各组间肿瘤大小的差异。我们发现,相对于其他各组,蜂毒素和TRAIL协同给药组的肿瘤体积显著减小。同时,为了观察各组间肿瘤细胞凋亡的变化,我们分别选取种瘤后第7天(未给药)、第10天(第3次给药后24小时)、第15天(第7次给药后24小时)瘤体,分离肿瘤组织细胞,ELISA检测caspase-3的活化。结果显示,相对于其他各组,蜂毒素和TRAIL协同给药能够显著活化caspase-3。上述结果说明,蜂毒素和TRAIL协同给药在体内有显著的抗肿瘤作用。
结论:综上所述,我们检测了中药单体蜂毒素对人肝癌细胞的凋亡诱导作用及其机制,发现其与TRAIL存在协同诱导肝癌细胞凋亡的效应并探讨了其协同作用的信号转导途径及分子机理,证明蜂毒素和TRAIL可以通过激活CaMKII-TAK1-MKK-JNK/p38信号通路并抑制IKK—NF-κB信号通路的方式诱导人肝癌细胞的凋亡。我们的工作有助于进一步完善对蜂毒素生物学活性的了解,为肝癌的治疗提供新的靶点,并为临床治疗联合用药方案提供新的思路和参考。
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide. In China, HCC is the second cause of death due to cancer. HCC usually develops in the presence of continuous inflammation and hepatocyte regeneration in the setting of chronic hepatitis and cirrhosis, although the molecular mechanisms linking chronic inflammation to malignant transformation remain to be further defined. Treatment of HCC is complex. Involvement of surgery、chemotherapy and radiotherapy, but up to now there has been no satisfying result. The majority of patients with HCC presented with an advanced stage beyond surgical treatment. In addition, chemotherapy and radiotherapy have limited efficacy in hepatocellular carcinoma of an advanced stage. So it is important to explore effective drugs and combination methods for therapy of this disease.
Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic. It has been reported that melittin has multiple effects, inculding antibacteria, antivirus and anti-inflammation, in various cell types. We and others have shown that melittin can induce cell cycle arrest, growth inhibition and apoptosis in various tumor cells. However, the mechanisms of the anti-cancer effects of melittin have not been fully elucidated.
TNF-related apoptosis-inducing ligand (TRAIL) is a member of tumor necrosis factor (TNF) superfamily. In its soluble form,it is emerging as an attractive anticancer agent because of its cancer cell specificity and potent antitumor activity.TRAIL signals by interacting with its receptors. Thus far, five receptors (TRAIL-R) have been identified, namely, the two agonistic receptors, TRAIL-R1 and TRAIL-R2, and the three antagonistic receptors TRAIL-R3, TRAIL-R4, and osteoprotegerin. Binding of TRAIL to the extracellular domain of agonistic receptors results in the trimerization of the receptors and clustering of the intracellular death domains (DDs), which leads to the recruitment of the adaptor molecule Fas-associated protein with death domain (FADD). Subsequently, FADD recruits and activates initiator caspase-8 and caspase-10, leading to cellular disassembly.
Meanwhile, TRAIL-initiatedapoptotic signaling requires an amplification loop by mitochondrial pathwayengagement through impairment of the mitochondrial membrane permeability regulated by Bcl-2 family members, which sequentially leads to cytochrome c or Smac/DIABLO[second mitochondrial activator of caspases/direct IAP-binding protein with low isoelectric point (pI)] release, apoptosome formation, and the final DNA fragmentation.
Similar to TNF-induced activation of the nuclear factorκB (NFκB) transcription factor and the mitogen-activated protein kinase (MAPK) pathway,TRAIL can also initiate the activation of signaling pathways that involve the adaptor molecules TNF receptor-associated factor 2 (TRAF2), receptor interacting protein(RIP) and transforming growth factor-β(TGF-β)-activated kinase 1 (TAK1), finally leading to the activation of the MAPK pathway (including extravellular signal-related kinase ERK1/2, c-Jun N-terminal kinase JNK1/2, and p38) and the IκBαkinase (IKK)-NFκB pathway.
Unfortunately, human hepatoma-derived cell types exhibit a major resistance to TRAIL-induced cell death. In the current study, we tested the effects of melittin in the induction of apoptosis of HCC cells and explored the mechanisms involved in melittin-induced apoptosis of TRAIL-resistant HepG2 cells. We show that melittin can initiate an apoptotic machinery that depends on calcium influx and activation of Ca2+/calmodulin(CaM)-dependent protein kinase (CaMKⅡ)-TAK1-JNK/p38 signaling pathway. Moreover, we find that melittin can sensitize HCC cells to TRAIL-induced apoptosis by activating CaMKⅡ-TAK1-JNK/p38 but inhibiting IKK- NFκB pathways. The differential roles of melittin in activation of TAK1-JNK/p38 but inhibition of IKK-NFκB in response to TRAIL may hold true for other TNF superfamily members because we also find that melittin can potentiate the TNFα-induced activation of TAK1-JNK/p38 but inhibit TNFα-induced activation of IKK-NFκB. Additionally, we find that melittin can not potentiate the drug-induced cell death of BEL-7402 cells that have been selected under chemotherapeutic drugs adriamycin or 5-fluorouracil to establish multi-drug resistance. Therefore, the sensitization of HCC cells to apoptosis by melittin may only be applicable to a limited set of apoptotic stimuli that employ apoptotic machinery similarly to TRAIL. Whether melittin can potentiate apoptosis by the other cancer treatments, however, may need further investigations.
In conclusion, we have demonstrated in the present study that melittin potentiated the apoptotic effects of TRAIL in human HCC cells by activating CaMKⅡ-TAK1-JNK/p38 pathway but inhibiting IKK-NFκB pathway. Our data suggest that melittin may exhibit anti-tumor activity by sensitizing HCC cells to TRAIL-mediated apoptosis, and that the combination of TRAIL with melittin may have therapeutic potential in the treatment of human HCC.
肝癌是世界第五大癌症,每年有上百万人死于肝癌,我国为世界上肝癌的高发区,原发性肝癌是最常见的恶性肿瘤之一,占癌症死亡率的第二位。目前对肝癌的主要治疗方法是早期进行手术切除,但在我国,由于多数肝癌的发现均较晚或早期即有肝内播散;肝癌病人多伴较重肝硬化,肝功能异常;年龄或其他疾病的限制等,使临床肝癌手术切除治疗受到很大限制,并且肝癌术后复发率也很高,也极大的影响患者的生存。此外,放疗、介入治疗和肝移植等治疗方法虽然取得一定进展,但是总体疗效仍然不能令人满意,且在应用中受到诸多禁忌症的限制。一方面可能与肝癌细胞的多耐药基因有关,但更重要的原因是因为肝癌的发生发展是一个多步骤多因素的过程,涉及到细胞内的一系列复杂的生物学变化。细胞的生长调控机制紊乱是肿瘤发生的主要原因,即在某些致癌因素的作用下或细胞内基因突变发生突变,导致促进细胞生长和增殖的信号通路异常活化,从而引起分化不成熟的细胞异常增生,而促进细胞有序凋亡和成熟分化的信号通路受到抑制,细胞异常存活,细胞增殖和死亡调控信号失衡,从而导致肿瘤的产生。这是一个多因素共同作用和多步骤致瘤的缓慢过程。其机理涉及原癌基因的活化、抗癌基因的失活、生长增殖信号的持续作用、死亡信号衰减或抑制、细胞周期异常及肿瘤血管内皮细胞增生等多个环节。此外,肝癌的发生还和病毒感染密切相关。由于肝癌的发生和发展涉及到多因素、多途径的共同作用,因此,仅针对肝癌发生机制中某一因素的单一药物治疗效果不够理想。因而,在深入研究肝癌形成、发生、发展的分子机制,在分子水平明确肝癌细胞各种生长调控因素间的动态平衡的基础上,针对肝癌发生发展中多个环节,探索能够特异性抑制肝癌细胞生长的药物或能够联合抑制肝癌细胞增殖、促进凋亡的药物组合并揭示其作用机制是国内外研究的热点之一,对于我国这样一个肝癌的高发国度而言,更是具有重要的理论意义和应用价值,具有潜在的经济效应和社会效应。
TRAIL(TNF-related apoptosis-inducing ligand,人肿瘤坏死因子相关凋亡诱导配体)是肿瘤坏死因子超家族的成员之一,TRAIL蛋白是一种Ⅱ型糖蛋白,分为细胞外C端区域、跨膜区、胞内N端区域三个部分,其C末端的胞外区与TNF家族的其它成员具有较高的同源性,TRAIL通过与特异性受体结合发挥生物学功能,目前发现的TRAIL受体有5种:TRAIL-R1/DR4、TRAIL-R2/DR5、TRAIL-R3/DcR1、TRAIL-R4/DcR2和OPG。按其功能和结构可分为三类:(1)死亡受体:DR4和DR5;(2)诱骗受体:DcR1和DcR2;(3)可溶性受体OPG。其中,介导凋亡的主要是死亡受体DR4和DR5。TRAIL通过与其活化型受体DR4或DR5的胞外段结合,引起受体的三聚作用并使胞内段的死亡结构域(DD)活化,并通过其死亡结构域募集胞浆死亡信号传递蛋白FADD,FADD进而活化caspase-8和caspase-10等凋亡执行分子,导致细胞的损伤,同时,TRAIL信号通过影响Bcl-2家族分子,破坏线粒体膜的完整性,既而引起线粒体内cytochrome c及Smac/DIABLO的释放,凋亡小体的形成,从而引起DNA的断裂。TRAIL除可引起凋亡信号的活化,还可以通过激活TRAF2(TNF受体相关因子2),引起下游RIP、TAK1等分子的活化,最后引起MAPK和NFκB信号通路的活化。放疗或化疗通常是通过P53基因的激活来达到杀伤肿瘤细胞的目的,由于很多人肿瘤细胞包括肝癌细胞中P53基因是缺失或突变的,因而很多肿瘤对于放疗或化疗不敏感。TRAIL诱导的凋亡并不依赖于P53的活化,其能够在体内外诱导多种肿瘤细胞凋亡但对正常组织细胞无明显影响,是常用的凋亡诱导及基因治疗模型,其重组细胞因子已应用于某些肿瘤的临床前实验及临床治疗,具备良好的应用前景25。但是,大多数人肝细胞癌细胞对TRAIL诱导的凋亡并不敏感,限制了其在人肝癌治疗中的应用。因而,寻找能够特异性增强TRAIL凋亡诱导作用的药物是目前国际肿瘤基因治疗研究的热点之一,受到国际学术界相关领域的关注。面对这一状况,我国丰富的中药资源是我们的优势之一,很多动植物中提取的生物活性成分都被证实有抗肝癌作用,并被广泛应用于临床。因此,寻找新的生物活性成分,探索其抗肿瘤机理并研究其与TRAIL的协同诱导凋亡效应是一项具有一定理论意义和应用前景的工作。基于本科室以往工作的基础上,我们选择蜂毒的有效活性成分蜂毒素单体作为了研究对象。
蜂毒素(melittin)是蜂毒的主要活性成分,约占蜂毒干质量的50%。蜂毒素单体是经现代工艺从蜂毒中提取的有效活性成分,纯度可达99%以上。它是由26个氨基酸组成的碱性多肽,其N端和C端分别具有疏水和亲水的生化基团,具有典型的两性分子结构,具有很好的水溶性,能够和细胞膜上的磷脂相互作用,在以往的研究中经常作为细胞膜上脂-蛋白相互作用的模型。蜂毒素是Ca2+-ATP酶、H+K+-ATP酶、Na+K+-ATP酶的抑制剂,可以抑制GTP结合蛋白的内在活性,近年来,随着蜂毒分离、提取、纯化技术的不断提高,对蜂毒素的功能及作用机制的研究也日益广泛。目前的研究主要集中在蜂毒素的抗炎作用上,有研究表明蜂毒素能够通过与NF-kB上游的IkB作用从而抑制NF-kB的活性发挥抗炎作用;也有报道认为蜂毒素能够通过与NF-kB的P50亚基相互作用从而起到抗炎效应;在骨关节炎的软骨细胞中能够通过抑制MMP-3从而减少炎性因子的分泌。有研究显示报道去除蜂毒素C端12-26位的谷氨酰氨残基可以增强它的抗炎活性;蜂毒素对体内外支原体和衣原体引起感染也均有抑制作用;也有研究表明蜂毒素对某些肿瘤细胞具有凋亡诱导效应,如蜂毒素可以通过抑制NF-kB和Akt的活性并上调凋亡相关蛋白的表达从而诱导鼠血管平滑肌细胞凋亡,可以引起Ca2+内流继而导致骨肉瘤细胞MG63凋亡。本科室以往的工作曾对蜂毒素的生物学活性有初步研究,证明携带蜂毒素的重组腺病毒在体外能够肝癌细胞的生长。此外,蜂毒素可通过抑制Rac1依赖的信号通路在体内外抑制肝癌的转移。那么,蜂毒素能否诱导肝癌细胞凋亡?蜂毒素是否与TRAIL具有协同效应?在既往工作的基础上,我们对蜂毒素单体对人肝癌细胞生长增殖的作用进行了初步实验。结果显示蜂毒素单体对人肝癌细胞hepG2的生长有明显的凋亡诱导作用,且这种作用存在明显的时间和浓度依赖性。此外,我们发现,在5ug/ml的蜂毒素协同作用下,低浓度(50ng/ml)的TRAIL即可引起TRAIL不敏感肝癌细胞株hepG2出现明显凋亡,相比于单独的蜂毒素处理组和TRAIL处理组差异显著。因此,我们初步推测蜂毒素能够诱导肝癌细胞凋亡,并与TRAIL具有协同效应,能够增强肝癌细胞对TRAIL的敏感性。本课题拟针对这一设想进行研究,探讨蜂毒素与TRAIL协同诱导肝癌细胞凋亡的分子机制。主要研究目的为:
1、明确蜂毒素对肝癌细胞的凋亡诱导效应及其机制。2、探讨蜂毒素能否增强与肝癌细胞对TRAIL诱导凋亡的敏感性及其分子机制。3、观察蜂毒素与TRAIL协同诱导肝癌细胞凋亡的体内效应。
二、研究方法:
1、流式细胞仪检测蜂毒素诱导hepG2细胞凋亡的效应及其时间剂量关系,western-blot、激酶分析法及报告基因方法检测凋亡及生长增殖相关信号通路关键分子的变化,并使用相应信号通路的抑制剂处理细胞以改变该信号通路的活化状况,观察凋亡效应的变化,明确相应信号通路与蜂毒素诱导的凋亡效应的关系,探讨其分子机制。
2、分别设立不加刺激的对照组、蜂毒素单独处理组、TRAIL单独处理组及协同作用组,作用HepG2细胞,流式细胞仪检测细胞凋亡的差异,观察蜂毒素能否增强肝癌细胞对TRAIL诱导凋亡的敏感性,并检测相关信号通路的改变,干预其活化状态,明确其与蜂毒素与TRAIL可能的协同效应的关系,探讨蜂毒素与TRAIL可能的协同效应的作用环节及机理。3、建立裸鼠荷瘤模型,通过小鼠尾静脉途径给药的方法,分别设立TRAIL单独处理组(10mg/kg)、蜂毒素低剂量组(50ug/kg)、蜂毒素高剂量组(100ug/kg)及协同作用组(TRAIL10mg/kg +蜂毒素50ug/kg),以注射PBS小鼠为实验对照组,观察各组间肿瘤大小的差异,观察蜂毒素与TRAIL的体内抗肿瘤效应。
三、研究结果及结论:
1、蜂毒素能够诱导肝癌细胞HepG2凋亡及其机制:我们分别选用不同浓度(0、5ug/ml、10 ug/ml)的蜂毒素作用于HepG2细胞不同时间点(0、12h、24h、48h),分别通过AnnexinV-PI双标、Rho123标记及DHR123标记,流式细胞仪检测细胞凋亡的变化。结果显示,蜂毒素能够诱导HepG2细胞凋亡,呈明显的时间和浓度依赖性,且凋亡现象伴随线粒体电势差的降低。在其他人肝癌细胞系Hep3B、BEL7402及SMMC7721中也得到了类似的结果。10 ug/ml的蜂毒素作用12h后,HepG2细胞中caspase-9、caspase-3、PARP均有活化,cytochromeC及smac/DIABLO的释放增多,使用相应的caspase抑制剂、钙离子抑制剂BAPTA、ROS抑制剂NAC预处理细胞后,细胞凋亡有明显逆转,此外,BAPTA及NAC预处理能够抑制caspase-9、caspase-3、PARP的活化及cytochrome c及smac/DIABLO的释放。蜂毒素作用0、15min、30min、60min后,能够活化CaMKII-TAK1-MKK-JNK/p38信号通路,相应的抑制剂作用后,能够部分逆转蜂毒素诱导的凋亡。蜂毒素作用还能够抑制IKKβ的激酶活性及TAK1介导的NF-κB的活化。2、蜂毒素能够协同TRAIL诱导肝癌细胞凋亡及其机制:我们分别设立不加刺激的对照组(ctrl)、蜂毒素单独处理组(5ug/ml)、TRAIL单独处理组(50ng/ml)及协同作用组(5ug/ml+50ng/ml),刺激HepG2细胞12h,Annexin V-PI双标,流式细胞仪检测细胞凋亡的差异。结果显示,蜂毒素和TRAIL共同作用后,细胞凋亡明显增加,显著高于蜂毒素单独处理组及TRAIL单独处理组,提示蜂毒素和TRAIL具有协同诱导人肝癌细胞凋亡的作用。在其他对TRAIL不敏感的人肝癌细胞系Hep3B、BEL7402及SMMC7721中也得到了类似的结果,我们同时观察了TRAIL敏感的肿瘤细胞系Hela和Jurkat中,在TRAIL剂量较低时(5ng/ml、10 ng/ml),较之单独的TRAIL作用,蜂毒素和TRAIL共同作用后细胞的凋亡明显增加,当TRAIL剂量较高时则差异不明显。此外,我们设立时间梯度,分别刺激以上各细胞系0、6h、12h、24h、48h,观察各组间凋亡的差异,结果显示,随着作用时间的延长,蜂毒素和TRAIL协同诱导的凋亡逐渐增加,呈现明显的时间依赖性。既而我们检测了蜂毒素和TRAIL共同作用对HepG2细胞信号通路的影响,较之TRAIL单独作用组及蜂毒素单独作用组,蜂毒素(5ug/ml)和TRAIL(10ng/ml、50 ng/ml)共同作用后,P38和JNK的磷酸化程度显著升高,使用相应的抑制剂预处理后,细胞凋亡有部分逆转。同时,蜂毒素(5ug/ml)和TRAIL(10ng/ml、50 ng/ml)共同作用能够促进TAK1的磷酸化并增强TAK1的激酶活性。在HepG2细胞中转染TAK1的显性负突变体(dominant—negative)TAK1K63W后,蜂毒素和TRAIL协同诱导的凋亡有显著的逆转,P38和JNK的活化也受到明显的抑制。此外,我们观察到,相比于TRAIL单独作用引起的NF-κB的活化,蜂毒素和TRAIL共同作用能够明显抑制IKBα的磷酸化、IKKβ的激酶活性及NF-κB的活化,相应的,NF-κB依赖的抗凋亡分子Bcl-xl及c-IAP1的表达也受到抑制。3、蜂毒素能够协同TRAIL在体内抑制肿瘤生长及其机制:为了检测蜂毒素协同TRAIL体内抗肿瘤的效应,我们通过皮下接种HepG2及SMMC7721细胞的方法建立了裸鼠荷瘤模型。经过小鼠尾静脉途径连续给药7天,实验分组为:TRAIL单独处理组(10mg/kg)、蜂毒素低剂量组(50ug/kg)、蜂毒素高剂量组(100ug/kg)及协同作用组(TRAIL10mg/kg +蜂毒素50ug/kg),以注射PBS小鼠为实验对照组,观察各组间肿瘤大小的差异。我们发现,相对于其他各组,蜂毒素和TRAIL协同给药组的肿瘤体积显著减小。同时,为了观察各组间肿瘤细胞凋亡的变化,我们分别选取种瘤后第7天(未给药)、第10天(第3次给药后24小时)、第15天(第7次给药后24小时)瘤体,分离肿瘤组织细胞,ELISA检测caspase-3的活化。结果显示,相对于其他各组,蜂毒素和TRAIL协同给药能够显著活化caspase-3。上述结果说明,蜂毒素和TRAIL协同给药在体内有显著的抗肿瘤作用。
结论:综上所述,我们检测了中药单体蜂毒素对人肝癌细胞的凋亡诱导作用及其机制,发现其与TRAIL存在协同诱导肝癌细胞凋亡的效应并探讨了其协同作用的信号转导途径及分子机理,证明蜂毒素和TRAIL可以通过激活CaMKII-TAK1-MKK-JNK/p38信号通路并抑制IKK—NF-κB信号通路的方式诱导人肝癌细胞的凋亡。我们的工作有助于进一步完善对蜂毒素生物学活性的了解,为肝癌的治疗提供新的靶点,并为临床治疗联合用药方案提供新的思路和参考。
Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide. In China, HCC is the second cause of death due to cancer. HCC usually develops in the presence of continuous inflammation and hepatocyte regeneration in the setting of chronic hepatitis and cirrhosis, although the molecular mechanisms linking chronic inflammation to malignant transformation remain to be further defined. Treatment of HCC is complex. Involvement of surgery、chemotherapy and radiotherapy, but up to now there has been no satisfying result. The majority of patients with HCC presented with an advanced stage beyond surgical treatment. In addition, chemotherapy and radiotherapy have limited efficacy in hepatocellular carcinoma of an advanced stage. So it is important to explore effective drugs and combination methods for therapy of this disease.
Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic. It has been reported that melittin has multiple effects, inculding antibacteria, antivirus and anti-inflammation, in various cell types. We and others have shown that melittin can induce cell cycle arrest, growth inhibition and apoptosis in various tumor cells. However, the mechanisms of the anti-cancer effects of melittin have not been fully elucidated.
TNF-related apoptosis-inducing ligand (TRAIL) is a member of tumor necrosis factor (TNF) superfamily. In its soluble form,it is emerging as an attractive anticancer agent because of its cancer cell specificity and potent antitumor activity.TRAIL signals by interacting with its receptors. Thus far, five receptors (TRAIL-R) have been identified, namely, the two agonistic receptors, TRAIL-R1 and TRAIL-R2, and the three antagonistic receptors TRAIL-R3, TRAIL-R4, and osteoprotegerin. Binding of TRAIL to the extracellular domain of agonistic receptors results in the trimerization of the receptors and clustering of the intracellular death domains (DDs), which leads to the recruitment of the adaptor molecule Fas-associated protein with death domain (FADD). Subsequently, FADD recruits and activates initiator caspase-8 and caspase-10, leading to cellular disassembly.
Meanwhile, TRAIL-initiatedapoptotic signaling requires an amplification loop by mitochondrial pathwayengagement through impairment of the mitochondrial membrane permeability regulated by Bcl-2 family members, which sequentially leads to cytochrome c or Smac/DIABLO[second mitochondrial activator of caspases/direct IAP-binding protein with low isoelectric point (pI)] release, apoptosome formation, and the final DNA fragmentation.
Similar to TNF-induced activation of the nuclear factorκB (NFκB) transcription factor and the mitogen-activated protein kinase (MAPK) pathway,TRAIL can also initiate the activation of signaling pathways that involve the adaptor molecules TNF receptor-associated factor 2 (TRAF2), receptor interacting protein(RIP) and transforming growth factor-β(TGF-β)-activated kinase 1 (TAK1), finally leading to the activation of the MAPK pathway (including extravellular signal-related kinase ERK1/2, c-Jun N-terminal kinase JNK1/2, and p38) and the IκBαkinase (IKK)-NFκB pathway.
Unfortunately, human hepatoma-derived cell types exhibit a major resistance to TRAIL-induced cell death. In the current study, we tested the effects of melittin in the induction of apoptosis of HCC cells and explored the mechanisms involved in melittin-induced apoptosis of TRAIL-resistant HepG2 cells. We show that melittin can initiate an apoptotic machinery that depends on calcium influx and activation of Ca2+/calmodulin(CaM)-dependent protein kinase (CaMKⅡ)-TAK1-JNK/p38 signaling pathway. Moreover, we find that melittin can sensitize HCC cells to TRAIL-induced apoptosis by activating CaMKⅡ-TAK1-JNK/p38 but inhibiting IKK- NFκB pathways. The differential roles of melittin in activation of TAK1-JNK/p38 but inhibition of IKK-NFκB in response to TRAIL may hold true for other TNF superfamily members because we also find that melittin can potentiate the TNFα-induced activation of TAK1-JNK/p38 but inhibit TNFα-induced activation of IKK-NFκB. Additionally, we find that melittin can not potentiate the drug-induced cell death of BEL-7402 cells that have been selected under chemotherapeutic drugs adriamycin or 5-fluorouracil to establish multi-drug resistance. Therefore, the sensitization of HCC cells to apoptosis by melittin may only be applicable to a limited set of apoptotic stimuli that employ apoptotic machinery similarly to TRAIL. Whether melittin can potentiate apoptosis by the other cancer treatments, however, may need further investigations.
In conclusion, we have demonstrated in the present study that melittin potentiated the apoptotic effects of TRAIL in human HCC cells by activating CaMKⅡ-TAK1-JNK/p38 pathway but inhibiting IKK-NFκB pathway. Our data suggest that melittin may exhibit anti-tumor activity by sensitizing HCC cells to TRAIL-mediated apoptosis, and that the combination of TRAIL with melittin may have therapeutic potential in the treatment of human HCC.
引文
[1] El-Serag HB, Marrero JA, Rudolph L, Reddy KR. Diagnosis and treatment of hepatocellular carcinoma. Gastroenterology, 2008 ,134(6):1752-63.
[2] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics.2002.CA Cancer J Clin,2005,55(2):74-108.
[3] El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology, 2007 ,132(7):2557-76.
[4] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell, 2000, 100(1):57-70.
[5] Lee HC. Systemic chemotherapy of hepatocellular carcinoma--Korean experience. Oncology, 2008,75:114-8.
[6] Hunter T. Oncoprotein networks. Cell, 1997, 88(3):333-46.
[7] Chau BN, Wang JY. Coordinated regulation of life and death by RB. Nat Rev Cancer, 2003, 3(2):130-8.
[8] Mangues R, Symmans WF, Lu S, Schwartz S, Pellicer A. Activated N-ras oncogene and N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis. Oncogene, 1996, 13(5):1053-63.
[9] Graham SM, Cox AD, Drivas G, Rush MG, D'Eustachio P, Der CJ. Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation. Mol Cell Biol, 1994, 14(6):4108-15.
[10] Kennedy NJ, Davis RJ. Role of JNK in tumor development. Cell Cycle, 2003, 2(3):199-201.
[11] Olashaw N, Pledger WJ. Paradigms of growth control: relation to Cdk activation. Sci STKE, 2002, 134:RE7.
[12] Marrero CR, Marrero JA. Viral hepatitis and hepatocellular carcinoma. Arch Med Res, 2007 ,38(6):612-20.
[13] Heathcote EJ. Treatment of hepatitis B: the next five years. Clin Med, 2007 ,7(5):472-7.
[14] Mason WS, Litwin S, Xu C, Jilbert AR. Hepatocyte turnover in transient and chronic hepadnavirus infections. J Viral Hepat, 2007,14 (1):22-8.
[15] Llovet JM,Burroughs A,Bruix J.Hepatocellular carcinoma.Lancet,2003,362 (9399):1907-17.
[16] Llovet JM ,Bruix J.Systematic review of randomized trials for unresectable hepatocellular carcinoma:chemoembolization improves survival.Hepatology,2003,37(2):429-42.
[17] Bruix J,Sherman M.Management of hepatocellular carcinoma. Hepatology, 2005,42(5):1208-36.
[18]滕红丽.原发性肝癌中医药临床研究进展.中国中医药信息杂志,2005, 12(9): 103-5.
[19]凌昌全,刘庆,李东涛,岳小强.原发性肝癌常见中医基本证候定性诊断规范的研究.中西医结合学报,2005,3(2):95-8.
[20]凌昌全.中医药防治原发性肝癌的研究及其进展.第二军医大学学报,2001,22(7):603-5.
[21]凌昌全.肿瘤治疗存在的问题及中西医结合的研究重点.中西医结合学报,2003,1(3):168-70.
[22] Alisi A, Balsano C. Enhancing the efficacy of hepatocellular carcinoma chemotherapeutics with natural anticancer agents.Nutr Rev,2007 ,65(12):550-3.
[23] Wang S. The promise of cancer therapeutics targeting the TNF-related apoptosis-inducing ligand and TRAIL receptor pathway. Oncogene, 2008,27(48):6207-15.
[24] Aggarwal BB.Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol, 2003, 3(9):745-56.
[25] Carlo-Stella C, Lavazza C, Locatelli A, ViganòL, Gianni AM, Gianni L. Targeting TRAIL agonistic receptors for cancer therapy. Clin Cancer Res, 2007 ,13(8):2313-7.
[26] Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science,1997 , 277(5327):815-8.
[27] Suliman A, Lam A, Datta R, Srivastava RK. Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene,2001,20(17):2122-33.
[28] Pang RW, Poon RT. From molecular biology to targeted therapies for hepatocellular carcinoma: the future is now.Oncology,2007,72 (1):30-44.
[29] Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep,2007, 27(4-5):189-223.
[30] Batenburg AM, Hibbeln JC, de Kruijff B. Lipid specific penetration of melittin into phospholipid model membranes. Biochim Biophys Acta,1987,903(1):155–65.
[31] Cserh?ti T, Sz?gyi M. Interaction of phospholipids with proteins and peptides. New advances IV. Int J Biochem,1994,26:1–18.
[32] Dawson CR, Drake AF, Helliwell J, Hider RC.The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta,1978,510(1):75–86.
[33] Baker KJ, East JM, Lee AG. Mechanism of inhibition of the Ca(2+)-ATPase by melittin. Biochemistry,1995,34(11):3596-604.
[34] Cuppoletti J, Blumenthal KM, MalinowskaDH . Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin. Arch Biochem Biophys,1989,275(1):263–70.
[35] Cuppoletti J, Abbott AJ. Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change. Arch Biochem Biophys ,1990,283(2):249–57.
[36] Fukushima N, Kohno M, Kato T, Kawamoto S, Okuda K, Misu Y, Ueda H. Melittin, a metabostatic peptide inhibiting Gs activity. Peptides ,1998,19(5):811–19.
[37] Park HJ, Son DJ, Lee CW, Choi MS, Lee US, Song HS, Lee JM, Hong JT. Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase. Biochem Pharmacol,2007,73(2):237-47.
[38] Park HJ,Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon DY, Hong JT. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum, 2004 ,50(11):3504-15.
[39] Nah SS,Ha E, Lee HJ, Chung JH. Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes. Toxicon,2007,49(6):881-5.
[40] Sun X,Chen S,Li S, Yan H,Fan Y,Mi H. Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity. Peptides,2005 ,26(3):369-75.
[41] Lazarev VN,Parfenova TM,Gularyan SK, Misyurina OY, Akopian TA, Govorun VM.Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line. Int J Antimicrob Agents, 2002 ,19(2):133-7.
[42] Lazarev VN,Shkarupeta MM,Titova GA,Kostrjukova ES,Akopian TA,Govorun VM. Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo. Biochem Biophys Res Commun,2005,338(2):946-50.
[43] Son DJ,Ha SJ,Song HS,Lim Y,Yun YP,Lee JW,Moon DC,Park YH,Park BS,Song MJ,Hong JT. Melittin inhibits vascular smooth muscle cell proliferation through induction of apoptosis via suppression of nuclear factor-kappaB and Akt activation and enhancement of apoptotic protein expression. J Pharmacol Exp Ther,2006,317(2):627-34.
[44] Chu ST, Cheng HH, Huang CJ, Chang HC, Chi CC, Su HH, Hsu SS, Wang JL, Chen IS, Liu SI, Lu YC, Huang JK, Ho CM, Jan CR.Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells. Life Sci,2007,80(4):364-9.
[45] Liu SJ, Yu M, He Y, Xiao L, Wang F, Song C, Sun SH, Ling CQ, Xu ZH. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology ,2008,47(6), 1964-73.
[46] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ. Growth Arrest and Apoptosis of the Human Hepatocellular Carcinoma Cell Line Bel-7402 Induced by Melittin. Onkologie,2006,29(8-9):367-71.
[47] Ling CQ,Li B, Zhang C, Zhu DZ, Huang XQ, Gu W,Li SX.Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma. Annals of Oncology,2005, 16(1):109-15.
[1] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell, 2000, 100(1):57-70.
[2] Hunter T. Oncoprotein networks. Cell, 1997, 88(3):333-46.
[3] Chau BN, Wang JY. Coordinated regulation of life and death by RB.Nat Rev Cancer,2003,3(2):130-138.
[4] Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature, 2006,441(7092):424-30.
[5] Michor F, Iwasa Y, Nowak MA. Dynamics of cancer progression. Nat Rev Cancer, 2004,4(3):197-205.
[6] Mangues R, Symmans WF, Lu S, Schwartz S, Pellicer A. Activated N-ras oncogene and N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis. Oncogene,1996, 13(5):1053-63.
[7] Graham SM, Cox AD, Drivas G, Rush MG, D'Eustachio P, Der CJ. Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation. Mol Cell Biol, 1994, 14(6):4108-15.
[8] Prochownik EV. c-Myc: linking transformation and genomic instability. Curr Mol Med , 2008 , 8(6):446-58.
[9] Hainaut P, Hollstein M. p53 and human cancer: the first ten thousand mutations. Adv Cancer Res,2000, 77:81-137.
[10] Minamoto T, Buschmann T, Habelhah H, Matusevich E, Tahara H, Boerresen-Dale AL, Harris C, Sidransky D, Ronai Z. Distinct pattern of p53 phosphorylation in human tumors. Oncogene, 2001, 20(26):3341-7.
[11] Wang SP, Zhou HJ, Chen XP, Ren GY, Ruan XX, Zhang Y, Zhang RL, Chen J. Loss of expression of Kruppel-like factor 6 in primary hepatocellular carcinoma and hepatoma cell lines. J Exp Clin Cancer Res, 2007,26(1):117-24.
[12] Sakemi R, Yano H, Ogasawara S, Akiba J, Nakashima O, Fukahori S, Sata M, Kojiro M.X-linked inhibitor of apoptosis (XIAP) and XIAP-associated factor-1 expressions and their relationship to apoptosis in human hepatocellular carcinoma and non-cancerous liver tissues.Oncol Rep,2007,18(1):65-70.
[13] Harbour JW, Dean DC. Rb function in cell-cycle regulation and apoptosis. Nat Cell Biol,2000, 2(4):E65-7.
[14] Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C. PI3K/Akt and apoptosis: size matters. Oncogene,2003, 22(56):8983-98.
[15] Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol, 2001, 17:615-75.
[16] Lahti JM, Li H, Kidd VJ. Elimination of cyclin D1 in vertebrate cells leads to an altered cell cycle phenotype, which is rescued by overexpression of murine cyclins D1, D2, or D3 but not by a mutant cyclin D1. J Biol Chem,1997, 272(16):10859-69.
[17] Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG, Calin GA, Giovannini C, Ferrazzi E, Grazi GL, Croce CM, Bolondi L, Negrini M.Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma.Cancer Res, 2007,67(13):6092-9.
[18] Marrero CR, Marrero JA. Viral hepatitis and hepatocellular carcinoma. Arch Med Res, 2007 ,38(6):612-20.
[19] Heathcote EJ. Treatment of hepatitis B: the next five years.Clin Med, 2007,7(5):472-7.
[20] Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology, 2008,48(4):1312-27.
[21] Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep,2007, 27(4-5):189-223.
[22] Batenburg AM, Hibbeln JC, de Kruijff B. Lipid specific penetration of melittin into phospholipid model membranes. Biochim Biophys Acta,1987,903:155–65.
[23] Cserh?ti T, Sz?gyi M.Interaction of phospholipids with proteins and peptides. New advances IV. Int J Biochem,1994,26(1):1–18.
[24] Dawson CR, Drake AF, Helliwell J, Hider RC.The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta,1978,510(1):75–86.
[25] Baker KJ, East JM, Lee AG. Mechanism of inhibition of the Ca(2+)-ATPase by melittin. Biochemistry,1995,34(11):3596-604.
[26] Cuppoletti J, Blumenthal KM, MalinowskaDH . Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin. Arch Biochem Biophys,1989,275(1):263–70.
[27] Cuppoletti J, Abbott AJ. Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change. Arch Biochem Biophys,1990,283(2):249–57.
[28] Fukushima N, Kohno M, Kato T, Kawamoto S, Okuda K, Misu Y, Ueda H. Melittin,a metabostatic peptide inhibiting Gs activity. Peptides ,1998,19(5):811–19.
[29] Asthana N, Yadav SP, Ghosh JK. Dissection of antibacterial and toxic activity of melittin: a leucine zipper motif plays a crucial role in determining its hemolytic activity but not antibacterial activity. J Biol Chem,2004,279(53):55042-50.
[30] Park HJ, Son DJ, Lee CW, Choi MS, Lee US, Song HS, Lee JM, Hong JT. Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase. Biochem Pharmacol,2007,73(2):237-47.
[31] Park HJ,Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon DY, Hong JT. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum, 2004 ,50(11):3504-15.
[32] Nah SS,Ha E, Lee HJ, Chung JH. Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes. Toxicon,2007,49(6):881-5.
[33] Sun X,Chen S,Li S, Yan H,Fan Y,Mi H. Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity. Peptides,2005 ,26(3):369-75.
[34] Lazarev VN,Parfenova TM,Gularyan SK, Misyurina OY, Akopian TA, Govorun VM.Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line. Int J Antimicrob Agents, 2002 ,19(2):133-7.
[35] Lazarev VN,Shkarupeta MM,Titova GA,Kostrjukova ES,Akopian TA,Govorun VM. Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo. Biochem Biophys Res Commun,2005,338(2):946-50.
[36] Son DJ,Ha SJ,Song HS,Lim Y,Yun YP,Lee JW,Moon DC,Park YH,Park BS,Song MJ,Hong JT. Melittin inhibits vascular smooth muscle cell proliferation through induction of apoptosis via suppression of nuclear factor-kappaB and Akt activation and enhancement of apoptotic protein expression. J Pharmacol Exp Ther,2006,317(2):627-34.
[37] Chu ST, Cheng HH, Huang CJ, Chang HC, Chi CC, Su HH, Hsu SS, Wang JL, Chen IS, Liu SI, Lu YC, Huang JK, Ho CM, Jan CR.Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcomacells. Life Sci,2007,80(4):364-9.
[38] Liu SJ, Yu M, He Y, Xiao L, Wang F, Song C, Sun SH, Ling CQ, Xu ZH. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology ,2008,47(6), 1964-73.
[39] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ. Growth Arrest and Apoptosis of the Human Hepatocellular Carcinoma Cell Line Bel-7402 Induced by Melittin. Onkologie,2006,29(8-9):367-71.
[40] Ling CQ,Li B, Zhang C, Zhu DZ, Huang XQ, Gu W,Li SX.Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma. Annals of Oncology,2005, 16(1):109-15.
[41] Li H, Wang X, Li N, Qiu J, Zhang Y, Cao X. hPEBP4 resists TRAIL-induced apoptosis of human prostate cancer cells by activating Akt and deactivating ERK1/2 pathways. J Biol Chem,2007,282(7):4943-50.
[42] Bouchier-Hayes L, Conroy H, Egan H, Adrain C, Creagh EM, MacFarlane M, Martin SJ. CARDINAL, a novel caspase recruitment domain protein, is an inhibitor of multiple NF-kappa B activation pathways. J Biol Chem, 2001, 276(47):44069-77.
[43] Chen T, Guo J, Yang M, Han C, Zhang M, Chen W, Liu Q, Wang J, Cao X. Cyclosporin A impairs dendritic cell migration by regulating chemokine receptor expression and inhibiting cyclooxygenase-2 expression. Blood,2004,103(2):413-21.
[44] Wang Y, Chen T, Han C, He D, Liu H, An H, Cai Z, Cao X. Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4. Blood ,2007, 110(3):962-71.
[45] Duke RC, Witter RZ, Nash PB, Young JD, Ojcius DM. Cytolysis mediated by ionophores and pore-forming agents: role of intracellular calcium in apoptosis. FASEB J,1994, 8(2):237-46.
[46] Mix LL, Dinerstein RJ, Villereal ML. Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem Biophys Res Commun,1984,119(1):69-75.
[47] Okamoto T, Isoda H, Kubota N, Takahata K, Takahashi T, Kishi T, Nakamura TY, Muromachi Y, Matsui Y, Goshima K. Melittin cardiotoxicity in cultured mouse cardiac myocytes and its correlation with calcium overload. Toxicol Appl Pharmacol,1995,133(1):150-63.
[48] Saini SS, Chopra AK, Peterson JW. Melittin activates endogenous phospholipase Dduring cytolysis of human monocytic leukemia cells.Toxicon, 1999 ,37(11):1605-19.
[49] Bullough DA, Ceccarelli EA, Roise D, Allison WS. Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides. Biochim Biophys Acta,1989,975(3):377-83.
[50] Zhang C, Li B, Lu SQ, Li Y, Su YH, Ling CQ. Effects of melittin on expressions of mitochondria membrane protein 7A6, cell apoptosis-related gene products Fas and Fas ligand in hepatocarcinoma cells. Zhong Xi Yi Jie He Xue Bao,2007,5(5):559-63.
[51] Hudmon A, Schulman H. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II. Biochem J, 2002 ,364(Pt 3):593-611.
[52] Ishitani T, Kishida S, Hyodo-Miura J, Ueno N, Yasuda J, Waterman M, Shibuya H, Moon RT, Ninomiya-Tsuji J, Matsumoto K. The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol, 2003 ,23(1):131-9.
[53] Delaney JR, Mlodzik M. TGF-beta activated kinase-1: new insights into the diverse roles of TAK1 in development and immunity. Cell Cycle, 2006,5(24):2852-5.
[54] Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature,2001,412(6844):346-51.
[55] Zhang D, Gaussin V, Taffet GE, Belaguli NS, Yamada M, Schwartz RJ, Michael LH, Overbeek PA, Schneider MD. TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice. Nat Med,2000,6(5):556-63.
[56] Arsura M, Panta GR, Bilyeu JD, Cavin LG, Sovak MA, Oliver AA, Factor V, Heuchel R, Mercurio F, Thorgeirsson SS, Sonenshein GE. Transient activation of NF-kappaB through a TAK1/IKK kinase pathway by TGF-beta1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation. Oncogene,2003,22(3):412-25.
[57] Thiefes A, Wolter S, Mushinski JF, Hoffmann E, Dittrich-Breiholz O, Graue N, D?rrie A, Schneider H, Wirth D, Luckow B, Resch K, Kracht M. Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes. J Biol Chem,2005,280(30):27728-41.
[58] Sayama K, Hanakawa Y, Nagai H, Shirakata Y, Dai X, Hirakawa S, Tokumaru S, Tohyama M, Yang L, Sato S, Shizuo A, Hashimoto K. Transforming growthfactor-beta-activated kinase 1 is essential for differentiation and the prevention of apoptosis in epidermis. J Biol Chem,2006,281(31):22013-20.
[59] Kim YS, Schwabe RF, Qian T, Lemasters JJ, Brenner DA. TRAIL-mediated apoptosis requires NF-kappaB inhibition and the mitochondrial permeability transition in human hepatoma cells. Hepatology,2002,36(6):1498-508.
[60] Yamaguchi Y, Shiraki K, Fuke H, Inoue T, Miyashita K, Yamanaka Y, Saitou Y, Sugimoto K, Nakano T. Targeting of X-linked inhibitor of apoptosis protein or survivin by short interfering RNAs sensitize hepatoma cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic agent-induced cell death. Oncol Rep,2005,14(5):1311-6.
[61] He SQ, Rehman H, Gong MG, Zhao YZ, Huang ZY, Li CH, Zhang WG, Chen XP. Inhibiting survivin expression enhances TRAIL-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest. Cancer Biol Ther,2007,6(8):1247-57.
[62] Schuchmann M, Schulze-Bergkamen H, Fleischer B, Schattenberg JM, Siebler J, Weinmann A, Teufel A, W?rns M, Fischer T, Strand S, Lohse AW, Galle PR. Histone deacetylase inhibition by valproic acid down-regulates c-FLIP/CASH and sensitizes hepatoma cells towards CD95- and TRAIL receptor-mediated apoptosis and chemotherapy. Oncol Rep,2006,15(1):227-30.
[63] Walczak H, Bouchon A, Stahl H, Krammer PH. Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells. Cancer Res,2000,60(11):3051-7.
[64] Hinz S, Trauzold A, Boenicke L, Sandberg C, Beckmann S, Bayer E, Walczak H, Kalthoff H, Ungefroren H. Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis. Oncogene,2000,19(48):5477-86.
[65] Stuhlmeier KM. Apis mellifera venom and melittin block neither NF-kappa B-p50-DNA interactions nor the activation of NF-kappa B, instead they activate the transcription of proinflammatory genes and the release of reactive oxygen intermediates.J Immunol,2007,179(1):655-64.
[66] Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol,2003,3(9):745-56.
[67] Carlo-Stella C, Lavazza C, Locatelli A, ViganòL, Gianni AM, Gianni L. TargetingTRAIL agonistic receptors for cancer therapy. Clin Cancer Res,2007,13(8):2313-7.
[68] Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science,1997 , 277(5327):815-8.
[69] Pan G, Ni J, Yu G, Wei YF, Dixit VM. TRUNDD, a new member of the TRAIL receptor family that antagonizes TRAIL signalling. FEBS Lett,1998,424(1-2):41-5.
[70] Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Baldwin D, Yuan J, Gurney A, Goddard AD, Godowski P, Ashkenazi A. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr Biol,1997,7(12):1003-6.
[71] Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science,1997,277(5327):818-21.
[72] Suliman A, Lam A, Datta R, Srivastava RK. Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene,2001,20(17):2122-33.
[73] Wajant H, Haas E, Schwenzer R, Muhlenbeck F, Kreuz S, Schubert G, Grell M, Smith C, Scheurich P. Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD). J Biol Chem,2000,275(32):24357-66.
[74] Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway. Immunity, 1997,7(6):821-30.
[75] Sprick MR, Weigand MA, Rieser E, Rauch CT, Juo P, Blenis J, Krammer PH, Walczak H. FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity, 2000,12(6):599-609.
[76] Lin Y, Devin A, Cook A, Keane MM, Kelliher M, Lipkowitz S, Liu ZG. The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of IkappaB kinase and c-Jun N-terminal kinase. Mol Cell Biol,2000,20(18):6638-45.
[77] Choo MK, Kawasaki N, Singhirunnusorn P, Koizumi K, Sato S, Akira S, Saiki I, Sakurai H. Blockade of transforming growth factor-beta-activated kinase 1 activity enhances TRAIL-induced apoptosis through activation of a caspase cascade. Mol Cancer Ther, 2006,5(12):2970-6.
[78] Shigeno M, Nakao K, Ichikawa T, Suzuki K, Kawakami A, Abiru S, Miyazoe S, Nakagawa Y, Ishikawa H, Hamasaki K, Nakata K, Ishii N, Eguchi K. Interferon-alpha sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-kappa B inactivation. Oncogene,2003,22(11):1653-62.
[79] Yamanaka T, Shiraki K, Sugimoto K, Ito T, Fujikawa K, Ito M, Takase K, Moriyama M, Nakano T, Suzuki A. Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines. Hepatology,2000,32(3):482-90.
[80] Wirth T, Kühnel F, Fleischmann-Mundt B, Woller N, Djojosubroto M, Rudolph KL, Manns M, Zender L, Kubicka S. Telomerase-dependent virotherapy overcomes resistance of hepatocellular carcinomas against chemotherapy and tumor necrosis factor-related apoptosis-inducing ligand by elimination of Mcl-1. Cancer Res,2005,65(16):7393-402.
[81] Ganten TM, Koschny R, Haas TL, Sykora J, Li-Weber M, Herzer K, Walczak H. Proteasome inhibition sensitizes hepatocellular carcinoma cells, but not human hepatocytes, to TRAIL. Hepatology,2005,42(3):588-97.
[82] Pathil A, Armeanu S, Venturelli S, Mascagni P, Weiss TS, Gregor M, Lauer UM, Bitzer M. HDAC inhibitor treatment of hepatoma cells induces both TRAIL-independent apoptosis and restoration of sensitivity to TRAIL. Hepatology,2006,43(3):425-34.
[83] Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med,1999,5(2):157-63.
[1] Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep,2007, 27(4-5):189-223.
[2] Batenburg AM, Hibbeln JC, de Kruijff B. Lipid specific penetration of melittin into phospholipid model membranes. Biochim Biophys Acta,1987,903:155–65.
[3] Cserh?ti T, Sz?gyi M.Interaction of phospholipids with proteins and peptides. New advances IV. Int J Biochem,1994,26(1):1–18.
[4] Dawson CR, Drake AF, Helliwell J, Hider RC.The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta,1978,510(1):75–86.
[5] Baker KJ, East JM, Lee AG. Mechanism of inhibition of the Ca(2+)-ATPase by melittin. Biochemistry,1995,34(11):3596-604.
[6] Cuppoletti J, Blumenthal KM, MalinowskaDH . Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin. Arch Biochem Biophys,1989,275(1):263–70.
[7] Cuppoletti J, Abbott AJ. Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change. Arch Biochem Biophys,1990,283(2):249–57.
[8] Fukushima N, Kohno M, Kato T, Kawamoto S, Okuda K, Misu Y, Ueda H. Melittin, a metabostatic peptide inhibiting Gs activity. Peptides ,1998,19(5):811–9.
[9] Hider RC. Honeybee venom: a rich source of pharmacologically active peptides.Endeavour,1988,12(2): 60?5.
[10] Park HJ,Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon DY, Hong JT. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum, 2004 ,50(11):3504-15.
[11] Park HJ, Son DJ, Lee CW, Choi MS, Lee US, Song HS, Lee JM, Hong JT. Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase. Biochem Pharmacol,2007,73(2):237-47.
[12] Stuhlmeier KM. Apis mellifera venom and melittin block neither NF-kappa B-p50-DNA interactions nor the activation of NF-kappa B, instead they activate the transcription of proinflammatory genes and the release of reactive oxygen intermediates.J Immunol,2007,179(1):655-64.
[13] Nah SS,Ha E, Lee HJ, Chung JH. Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes. Toxicon,2007,49(6):881-5.
[14] Sun X,Chen S,Li S, Yan H,Fan Y,Mi H. Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity. Peptides,2005 ,26(3):369-75.
[15] Sharma SV. Melittin resistance: a counterselection for ras transformation. Oncogene, 1992,7(2):193-201.
[16] Sharma SV. Melittin-induced hyperactivation of phospholipase A2 activity and calcium influx in ras-transformed cells.Oncogene ,1993,8(4):939-47.
[17] Hait WN, Grais L, Benz C, Cadman EC. Inhibition of growth of leukemic cells by inhibitors of calmodulin: phenothiazines and melittin. Cancer Chemother Pharmacol,1985,14(3):202?5.
[18] Hait WN, Lee GL. Characteristics of the cytotoxic effects of the phenothiazine class of calmodulin antagonists. Biochem Pharmacol, 1985,34(22):3973-8.
[19] Lee GL, Hait WN. Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin. Life Sci,1985,36(4):347-54.
[20] Lazo JS, Hait WN, Kennedy KA, Braun ID, Meandzija B. Enhanced bleomycin-induced DNA damage and cytotoxicity with calmodulin antagonists. Mol Pharmacol ,1985,27(3):387?93.
[21] Lazo JS, Chen DL, Gallicchio VS, Hait WN. Increased lethality of calmodulin antagonists and bleomycin to human bone marrow and bleomycin-resistant malignant cells. Cancer Res,1986, 46(5):2236?40.
[22] Killion JJ,Dunn JD. Differential cytolysis of murine spleen,bone-marrow and leukemia cells by melittin reveals differences in membrane topography. Biochem Biophys Res Commun,1986,139(1): 222?7.
[23] Zhu HG, Tayeh I, Israel L, Castagna M. Different susceptibility of lung cell lines to inhibitors of tumor promotion and inducers of differentiation. J Biol Regul Homeost Agents,1991, 5(2): 52?8.
[24] Arora AS, de Groen PC, Croall DE, Emori Y, Gores GJ. Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation. J Cell Physiol,1996,167(3):434?42.
[25] Wu YL, Jiang XR, Newland AC, Kelsey SM. Failure to activate cytosolicphospholipase A2 causes TNF resistance in human leukemic cells. J Immunol,1998,160(12):5929?35.
[26] Saini SS, Chopra AK, Peterson JW. Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells.Toxicon, 1999 ,37(11):1605-19.
[27] Chu ST, Cheng HH, Huang CJ, Chang HC, Chi CC, Su HH, Hsu SS, Wang JL, Chen IS, Liu SI, Lu YC, Huang JK, Ho CM, Jan CR.Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells. Life Sci,2007,80(4):364-9.
[28] Moon DO, Park SY, Choi YH, Kim ND, Lee C, Kim GY.Melittin induces Bcl-2 and caspase-3-dependent apoptosis through downregulation of Akt phosphorylation in human leukemic U937 cells.Toxicon,2008,51(1):112-20.
[29] Chen YQ, Zhu ZA, Hao YQ, Dai KR, Zhang C. Effect of melittin on apoptosis and necrosis of U2 OS cells. Zhong Xi Yi Jie He Xue Bao,2004, 2(3):208-9.
[30] Holle L, Song W, Holle E, Wei Y, Wagner T, Yu X. A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo. Int J Oncol,2003,22(1): 93?8.
[31] Wang C, Chen TY, Zhang N, Yang MJ, Li B, LüX, Cao XT, Ling CQ. Melittin, a major component of bee venom, sensitizes human hepatocellular carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by activating CaMKII-TAK1-JNK/p38 and inhibiting IkappaBalpha kinase-NFkappaB.J Biol Chem,2009,284(6):3804–13.
[32] Liu SJ, Yu M, He Y, Xiao L, Wang F, Song C, Sun SH, Ling CQ, Xu ZH. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology ,2008,47(6): 1964-73.
[33] Winder D, Günzburg WH, Erfle V, Salmons B. Expression of antimicrobial peptides has an antitumour effect in human cells. Biochem Biophys Res Commun ,1998,242(3):608?12.
[34] Ling CQ, Li B, Zhang C, Gu W, Li SX, Huang XQ, Zhang YN. Anti-hepatocarcinoma effect of recombinant adenovirus carrying melittin gene. Zhonghua Gan Zang Bing Za Zhi,2004,12(12):741?4.
[35] Ling CQ,Li B, Zhang C, Zhu DZ, Huang XQ, Gu W,Li SX.Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma. Annals of Oncology,2005, 16(1):109-15.
[36] Li B, Ling CQ, Zhang C, Gu W, Li SX, Huang XQ, Zhang YN, Yu CQ. The induced apoptosis of recombinant adenovirus carrying melittin gene for hepatocellular carcinoma cell. Zhonghua Gan Zang Bing Za Zhi,2004,12(8):453?5.
[37] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ. Growth Arrest and Apoptosis of the Human Hepatocellular Carcinoma Cell Line Bel-7402 Induced by Melittin. Onkologie,2006,29(8-9):367-71.
[38] Lazarev VN,Parfenova TM,Gularyan SK, Misyurina OY, Akopian TA, Govorun VM.Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line. Int J Antimicrob Agents, 2002 ,19(2):133-7.
[39] Lazarev VN,Shkarupeta MM,Titova GA,Kostrjukova ES,Akopian TA,Govorun VM. Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo. Biochem Biophys Res Commun,2005,338(2):946-50.
[2] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics.2002.CA Cancer J Clin,2005,55(2):74-108.
[3] El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology, 2007 ,132(7):2557-76.
[4] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell, 2000, 100(1):57-70.
[5] Lee HC. Systemic chemotherapy of hepatocellular carcinoma--Korean experience. Oncology, 2008,75:114-8.
[6] Hunter T. Oncoprotein networks. Cell, 1997, 88(3):333-46.
[7] Chau BN, Wang JY. Coordinated regulation of life and death by RB. Nat Rev Cancer, 2003, 3(2):130-8.
[8] Mangues R, Symmans WF, Lu S, Schwartz S, Pellicer A. Activated N-ras oncogene and N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis. Oncogene, 1996, 13(5):1053-63.
[9] Graham SM, Cox AD, Drivas G, Rush MG, D'Eustachio P, Der CJ. Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation. Mol Cell Biol, 1994, 14(6):4108-15.
[10] Kennedy NJ, Davis RJ. Role of JNK in tumor development. Cell Cycle, 2003, 2(3):199-201.
[11] Olashaw N, Pledger WJ. Paradigms of growth control: relation to Cdk activation. Sci STKE, 2002, 134:RE7.
[12] Marrero CR, Marrero JA. Viral hepatitis and hepatocellular carcinoma. Arch Med Res, 2007 ,38(6):612-20.
[13] Heathcote EJ. Treatment of hepatitis B: the next five years. Clin Med, 2007 ,7(5):472-7.
[14] Mason WS, Litwin S, Xu C, Jilbert AR. Hepatocyte turnover in transient and chronic hepadnavirus infections. J Viral Hepat, 2007,14 (1):22-8.
[15] Llovet JM,Burroughs A,Bruix J.Hepatocellular carcinoma.Lancet,2003,362 (9399):1907-17.
[16] Llovet JM ,Bruix J.Systematic review of randomized trials for unresectable hepatocellular carcinoma:chemoembolization improves survival.Hepatology,2003,37(2):429-42.
[17] Bruix J,Sherman M.Management of hepatocellular carcinoma. Hepatology, 2005,42(5):1208-36.
[18]滕红丽.原发性肝癌中医药临床研究进展.中国中医药信息杂志,2005, 12(9): 103-5.
[19]凌昌全,刘庆,李东涛,岳小强.原发性肝癌常见中医基本证候定性诊断规范的研究.中西医结合学报,2005,3(2):95-8.
[20]凌昌全.中医药防治原发性肝癌的研究及其进展.第二军医大学学报,2001,22(7):603-5.
[21]凌昌全.肿瘤治疗存在的问题及中西医结合的研究重点.中西医结合学报,2003,1(3):168-70.
[22] Alisi A, Balsano C. Enhancing the efficacy of hepatocellular carcinoma chemotherapeutics with natural anticancer agents.Nutr Rev,2007 ,65(12):550-3.
[23] Wang S. The promise of cancer therapeutics targeting the TNF-related apoptosis-inducing ligand and TRAIL receptor pathway. Oncogene, 2008,27(48):6207-15.
[24] Aggarwal BB.Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol, 2003, 3(9):745-56.
[25] Carlo-Stella C, Lavazza C, Locatelli A, ViganòL, Gianni AM, Gianni L. Targeting TRAIL agonistic receptors for cancer therapy. Clin Cancer Res, 2007 ,13(8):2313-7.
[26] Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science,1997 , 277(5327):815-8.
[27] Suliman A, Lam A, Datta R, Srivastava RK. Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene,2001,20(17):2122-33.
[28] Pang RW, Poon RT. From molecular biology to targeted therapies for hepatocellular carcinoma: the future is now.Oncology,2007,72 (1):30-44.
[29] Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep,2007, 27(4-5):189-223.
[30] Batenburg AM, Hibbeln JC, de Kruijff B. Lipid specific penetration of melittin into phospholipid model membranes. Biochim Biophys Acta,1987,903(1):155–65.
[31] Cserh?ti T, Sz?gyi M. Interaction of phospholipids with proteins and peptides. New advances IV. Int J Biochem,1994,26:1–18.
[32] Dawson CR, Drake AF, Helliwell J, Hider RC.The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta,1978,510(1):75–86.
[33] Baker KJ, East JM, Lee AG. Mechanism of inhibition of the Ca(2+)-ATPase by melittin. Biochemistry,1995,34(11):3596-604.
[34] Cuppoletti J, Blumenthal KM, MalinowskaDH . Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin. Arch Biochem Biophys,1989,275(1):263–70.
[35] Cuppoletti J, Abbott AJ. Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change. Arch Biochem Biophys ,1990,283(2):249–57.
[36] Fukushima N, Kohno M, Kato T, Kawamoto S, Okuda K, Misu Y, Ueda H. Melittin, a metabostatic peptide inhibiting Gs activity. Peptides ,1998,19(5):811–19.
[37] Park HJ, Son DJ, Lee CW, Choi MS, Lee US, Song HS, Lee JM, Hong JT. Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase. Biochem Pharmacol,2007,73(2):237-47.
[38] Park HJ,Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon DY, Hong JT. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum, 2004 ,50(11):3504-15.
[39] Nah SS,Ha E, Lee HJ, Chung JH. Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes. Toxicon,2007,49(6):881-5.
[40] Sun X,Chen S,Li S, Yan H,Fan Y,Mi H. Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity. Peptides,2005 ,26(3):369-75.
[41] Lazarev VN,Parfenova TM,Gularyan SK, Misyurina OY, Akopian TA, Govorun VM.Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line. Int J Antimicrob Agents, 2002 ,19(2):133-7.
[42] Lazarev VN,Shkarupeta MM,Titova GA,Kostrjukova ES,Akopian TA,Govorun VM. Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo. Biochem Biophys Res Commun,2005,338(2):946-50.
[43] Son DJ,Ha SJ,Song HS,Lim Y,Yun YP,Lee JW,Moon DC,Park YH,Park BS,Song MJ,Hong JT. Melittin inhibits vascular smooth muscle cell proliferation through induction of apoptosis via suppression of nuclear factor-kappaB and Akt activation and enhancement of apoptotic protein expression. J Pharmacol Exp Ther,2006,317(2):627-34.
[44] Chu ST, Cheng HH, Huang CJ, Chang HC, Chi CC, Su HH, Hsu SS, Wang JL, Chen IS, Liu SI, Lu YC, Huang JK, Ho CM, Jan CR.Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells. Life Sci,2007,80(4):364-9.
[45] Liu SJ, Yu M, He Y, Xiao L, Wang F, Song C, Sun SH, Ling CQ, Xu ZH. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology ,2008,47(6), 1964-73.
[46] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ. Growth Arrest and Apoptosis of the Human Hepatocellular Carcinoma Cell Line Bel-7402 Induced by Melittin. Onkologie,2006,29(8-9):367-71.
[47] Ling CQ,Li B, Zhang C, Zhu DZ, Huang XQ, Gu W,Li SX.Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma. Annals of Oncology,2005, 16(1):109-15.
[1] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell, 2000, 100(1):57-70.
[2] Hunter T. Oncoprotein networks. Cell, 1997, 88(3):333-46.
[3] Chau BN, Wang JY. Coordinated regulation of life and death by RB.Nat Rev Cancer,2003,3(2):130-138.
[4] Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature, 2006,441(7092):424-30.
[5] Michor F, Iwasa Y, Nowak MA. Dynamics of cancer progression. Nat Rev Cancer, 2004,4(3):197-205.
[6] Mangues R, Symmans WF, Lu S, Schwartz S, Pellicer A. Activated N-ras oncogene and N-ras proto-oncogene act through the same pathway for in vivo tumorigenesis. Oncogene,1996, 13(5):1053-63.
[7] Graham SM, Cox AD, Drivas G, Rush MG, D'Eustachio P, Der CJ. Aberrant function of the Ras-related protein TC21/R-Ras2 triggers malignant transformation. Mol Cell Biol, 1994, 14(6):4108-15.
[8] Prochownik EV. c-Myc: linking transformation and genomic instability. Curr Mol Med , 2008 , 8(6):446-58.
[9] Hainaut P, Hollstein M. p53 and human cancer: the first ten thousand mutations. Adv Cancer Res,2000, 77:81-137.
[10] Minamoto T, Buschmann T, Habelhah H, Matusevich E, Tahara H, Boerresen-Dale AL, Harris C, Sidransky D, Ronai Z. Distinct pattern of p53 phosphorylation in human tumors. Oncogene, 2001, 20(26):3341-7.
[11] Wang SP, Zhou HJ, Chen XP, Ren GY, Ruan XX, Zhang Y, Zhang RL, Chen J. Loss of expression of Kruppel-like factor 6 in primary hepatocellular carcinoma and hepatoma cell lines. J Exp Clin Cancer Res, 2007,26(1):117-24.
[12] Sakemi R, Yano H, Ogasawara S, Akiba J, Nakashima O, Fukahori S, Sata M, Kojiro M.X-linked inhibitor of apoptosis (XIAP) and XIAP-associated factor-1 expressions and their relationship to apoptosis in human hepatocellular carcinoma and non-cancerous liver tissues.Oncol Rep,2007,18(1):65-70.
[13] Harbour JW, Dean DC. Rb function in cell-cycle regulation and apoptosis. Nat Cell Biol,2000, 2(4):E65-7.
[14] Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C. PI3K/Akt and apoptosis: size matters. Oncogene,2003, 22(56):8983-98.
[15] Katso R, Okkenhaug K, Ahmadi K, White S, Timms J, Waterfield MD. Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer. Annu Rev Cell Dev Biol, 2001, 17:615-75.
[16] Lahti JM, Li H, Kidd VJ. Elimination of cyclin D1 in vertebrate cells leads to an altered cell cycle phenotype, which is rescued by overexpression of murine cyclins D1, D2, or D3 but not by a mutant cyclin D1. J Biol Chem,1997, 272(16):10859-69.
[17] Gramantieri L, Ferracin M, Fornari F, Veronese A, Sabbioni S, Liu CG, Calin GA, Giovannini C, Ferrazzi E, Grazi GL, Croce CM, Bolondi L, Negrini M.Cyclin G1 is a target of miR-122a, a microRNA frequently down-regulated in human hepatocellular carcinoma.Cancer Res, 2007,67(13):6092-9.
[18] Marrero CR, Marrero JA. Viral hepatitis and hepatocellular carcinoma. Arch Med Res, 2007 ,38(6):612-20.
[19] Heathcote EJ. Treatment of hepatitis B: the next five years.Clin Med, 2007,7(5):472-7.
[20] Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology, 2008,48(4):1312-27.
[21] Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep,2007, 27(4-5):189-223.
[22] Batenburg AM, Hibbeln JC, de Kruijff B. Lipid specific penetration of melittin into phospholipid model membranes. Biochim Biophys Acta,1987,903:155–65.
[23] Cserh?ti T, Sz?gyi M.Interaction of phospholipids with proteins and peptides. New advances IV. Int J Biochem,1994,26(1):1–18.
[24] Dawson CR, Drake AF, Helliwell J, Hider RC.The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta,1978,510(1):75–86.
[25] Baker KJ, East JM, Lee AG. Mechanism of inhibition of the Ca(2+)-ATPase by melittin. Biochemistry,1995,34(11):3596-604.
[26] Cuppoletti J, Blumenthal KM, MalinowskaDH . Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin. Arch Biochem Biophys,1989,275(1):263–70.
[27] Cuppoletti J, Abbott AJ. Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change. Arch Biochem Biophys,1990,283(2):249–57.
[28] Fukushima N, Kohno M, Kato T, Kawamoto S, Okuda K, Misu Y, Ueda H. Melittin,a metabostatic peptide inhibiting Gs activity. Peptides ,1998,19(5):811–19.
[29] Asthana N, Yadav SP, Ghosh JK. Dissection of antibacterial and toxic activity of melittin: a leucine zipper motif plays a crucial role in determining its hemolytic activity but not antibacterial activity. J Biol Chem,2004,279(53):55042-50.
[30] Park HJ, Son DJ, Lee CW, Choi MS, Lee US, Song HS, Lee JM, Hong JT. Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase. Biochem Pharmacol,2007,73(2):237-47.
[31] Park HJ,Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon DY, Hong JT. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum, 2004 ,50(11):3504-15.
[32] Nah SS,Ha E, Lee HJ, Chung JH. Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes. Toxicon,2007,49(6):881-5.
[33] Sun X,Chen S,Li S, Yan H,Fan Y,Mi H. Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity. Peptides,2005 ,26(3):369-75.
[34] Lazarev VN,Parfenova TM,Gularyan SK, Misyurina OY, Akopian TA, Govorun VM.Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line. Int J Antimicrob Agents, 2002 ,19(2):133-7.
[35] Lazarev VN,Shkarupeta MM,Titova GA,Kostrjukova ES,Akopian TA,Govorun VM. Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo. Biochem Biophys Res Commun,2005,338(2):946-50.
[36] Son DJ,Ha SJ,Song HS,Lim Y,Yun YP,Lee JW,Moon DC,Park YH,Park BS,Song MJ,Hong JT. Melittin inhibits vascular smooth muscle cell proliferation through induction of apoptosis via suppression of nuclear factor-kappaB and Akt activation and enhancement of apoptotic protein expression. J Pharmacol Exp Ther,2006,317(2):627-34.
[37] Chu ST, Cheng HH, Huang CJ, Chang HC, Chi CC, Su HH, Hsu SS, Wang JL, Chen IS, Liu SI, Lu YC, Huang JK, Ho CM, Jan CR.Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcomacells. Life Sci,2007,80(4):364-9.
[38] Liu SJ, Yu M, He Y, Xiao L, Wang F, Song C, Sun SH, Ling CQ, Xu ZH. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology ,2008,47(6), 1964-73.
[39] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ. Growth Arrest and Apoptosis of the Human Hepatocellular Carcinoma Cell Line Bel-7402 Induced by Melittin. Onkologie,2006,29(8-9):367-71.
[40] Ling CQ,Li B, Zhang C, Zhu DZ, Huang XQ, Gu W,Li SX.Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma. Annals of Oncology,2005, 16(1):109-15.
[41] Li H, Wang X, Li N, Qiu J, Zhang Y, Cao X. hPEBP4 resists TRAIL-induced apoptosis of human prostate cancer cells by activating Akt and deactivating ERK1/2 pathways. J Biol Chem,2007,282(7):4943-50.
[42] Bouchier-Hayes L, Conroy H, Egan H, Adrain C, Creagh EM, MacFarlane M, Martin SJ. CARDINAL, a novel caspase recruitment domain protein, is an inhibitor of multiple NF-kappa B activation pathways. J Biol Chem, 2001, 276(47):44069-77.
[43] Chen T, Guo J, Yang M, Han C, Zhang M, Chen W, Liu Q, Wang J, Cao X. Cyclosporin A impairs dendritic cell migration by regulating chemokine receptor expression and inhibiting cyclooxygenase-2 expression. Blood,2004,103(2):413-21.
[44] Wang Y, Chen T, Han C, He D, Liu H, An H, Cai Z, Cao X. Lysosome-associated small Rab GTPase Rab7b negatively regulates TLR4 signaling in macrophages by promoting lysosomal degradation of TLR4. Blood ,2007, 110(3):962-71.
[45] Duke RC, Witter RZ, Nash PB, Young JD, Ojcius DM. Cytolysis mediated by ionophores and pore-forming agents: role of intracellular calcium in apoptosis. FASEB J,1994, 8(2):237-46.
[46] Mix LL, Dinerstein RJ, Villereal ML. Mitogens and melittin stimulate an increase in intracellular free calcium concentration in human fibroblasts. Biochem Biophys Res Commun,1984,119(1):69-75.
[47] Okamoto T, Isoda H, Kubota N, Takahata K, Takahashi T, Kishi T, Nakamura TY, Muromachi Y, Matsui Y, Goshima K. Melittin cardiotoxicity in cultured mouse cardiac myocytes and its correlation with calcium overload. Toxicol Appl Pharmacol,1995,133(1):150-63.
[48] Saini SS, Chopra AK, Peterson JW. Melittin activates endogenous phospholipase Dduring cytolysis of human monocytic leukemia cells.Toxicon, 1999 ,37(11):1605-19.
[49] Bullough DA, Ceccarelli EA, Roise D, Allison WS. Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides. Biochim Biophys Acta,1989,975(3):377-83.
[50] Zhang C, Li B, Lu SQ, Li Y, Su YH, Ling CQ. Effects of melittin on expressions of mitochondria membrane protein 7A6, cell apoptosis-related gene products Fas and Fas ligand in hepatocarcinoma cells. Zhong Xi Yi Jie He Xue Bao,2007,5(5):559-63.
[51] Hudmon A, Schulman H. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II. Biochem J, 2002 ,364(Pt 3):593-611.
[52] Ishitani T, Kishida S, Hyodo-Miura J, Ueno N, Yasuda J, Waterman M, Shibuya H, Moon RT, Ninomiya-Tsuji J, Matsumoto K. The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca(2+) pathway to antagonize Wnt/beta-catenin signaling. Mol Cell Biol, 2003 ,23(1):131-9.
[53] Delaney JR, Mlodzik M. TGF-beta activated kinase-1: new insights into the diverse roles of TAK1 in development and immunity. Cell Cycle, 2006,5(24):2852-5.
[54] Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature,2001,412(6844):346-51.
[55] Zhang D, Gaussin V, Taffet GE, Belaguli NS, Yamada M, Schwartz RJ, Michael LH, Overbeek PA, Schneider MD. TAK1 is activated in the myocardium after pressure overload and is sufficient to provoke heart failure in transgenic mice. Nat Med,2000,6(5):556-63.
[56] Arsura M, Panta GR, Bilyeu JD, Cavin LG, Sovak MA, Oliver AA, Factor V, Heuchel R, Mercurio F, Thorgeirsson SS, Sonenshein GE. Transient activation of NF-kappaB through a TAK1/IKK kinase pathway by TGF-beta1 inhibits AP-1/SMAD signaling and apoptosis: implications in liver tumor formation. Oncogene,2003,22(3):412-25.
[57] Thiefes A, Wolter S, Mushinski JF, Hoffmann E, Dittrich-Breiholz O, Graue N, D?rrie A, Schneider H, Wirth D, Luckow B, Resch K, Kracht M. Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes. J Biol Chem,2005,280(30):27728-41.
[58] Sayama K, Hanakawa Y, Nagai H, Shirakata Y, Dai X, Hirakawa S, Tokumaru S, Tohyama M, Yang L, Sato S, Shizuo A, Hashimoto K. Transforming growthfactor-beta-activated kinase 1 is essential for differentiation and the prevention of apoptosis in epidermis. J Biol Chem,2006,281(31):22013-20.
[59] Kim YS, Schwabe RF, Qian T, Lemasters JJ, Brenner DA. TRAIL-mediated apoptosis requires NF-kappaB inhibition and the mitochondrial permeability transition in human hepatoma cells. Hepatology,2002,36(6):1498-508.
[60] Yamaguchi Y, Shiraki K, Fuke H, Inoue T, Miyashita K, Yamanaka Y, Saitou Y, Sugimoto K, Nakano T. Targeting of X-linked inhibitor of apoptosis protein or survivin by short interfering RNAs sensitize hepatoma cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic agent-induced cell death. Oncol Rep,2005,14(5):1311-6.
[61] He SQ, Rehman H, Gong MG, Zhao YZ, Huang ZY, Li CH, Zhang WG, Chen XP. Inhibiting survivin expression enhances TRAIL-induced tumoricidal activity in human hepatocellular carcinoma via cell cycle arrest. Cancer Biol Ther,2007,6(8):1247-57.
[62] Schuchmann M, Schulze-Bergkamen H, Fleischer B, Schattenberg JM, Siebler J, Weinmann A, Teufel A, W?rns M, Fischer T, Strand S, Lohse AW, Galle PR. Histone deacetylase inhibition by valproic acid down-regulates c-FLIP/CASH and sensitizes hepatoma cells towards CD95- and TRAIL receptor-mediated apoptosis and chemotherapy. Oncol Rep,2006,15(1):227-30.
[63] Walczak H, Bouchon A, Stahl H, Krammer PH. Tumor necrosis factor-related apoptosis-inducing ligand retains its apoptosis-inducing capacity on Bcl-2- or Bcl-xL-overexpressing chemotherapy-resistant tumor cells. Cancer Res,2000,60(11):3051-7.
[64] Hinz S, Trauzold A, Boenicke L, Sandberg C, Beckmann S, Bayer E, Walczak H, Kalthoff H, Ungefroren H. Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis. Oncogene,2000,19(48):5477-86.
[65] Stuhlmeier KM. Apis mellifera venom and melittin block neither NF-kappa B-p50-DNA interactions nor the activation of NF-kappa B, instead they activate the transcription of proinflammatory genes and the release of reactive oxygen intermediates.J Immunol,2007,179(1):655-64.
[66] Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol,2003,3(9):745-56.
[67] Carlo-Stella C, Lavazza C, Locatelli A, ViganòL, Gianni AM, Gianni L. TargetingTRAIL agonistic receptors for cancer therapy. Clin Cancer Res,2007,13(8):2313-7.
[68] Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science,1997 , 277(5327):815-8.
[69] Pan G, Ni J, Yu G, Wei YF, Dixit VM. TRUNDD, a new member of the TRAIL receptor family that antagonizes TRAIL signalling. FEBS Lett,1998,424(1-2):41-5.
[70] Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Baldwin D, Yuan J, Gurney A, Goddard AD, Godowski P, Ashkenazi A. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr Biol,1997,7(12):1003-6.
[71] Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science,1997,277(5327):818-21.
[72] Suliman A, Lam A, Datta R, Srivastava RK. Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene,2001,20(17):2122-33.
[73] Wajant H, Haas E, Schwenzer R, Muhlenbeck F, Kreuz S, Schubert G, Grell M, Smith C, Scheurich P. Inhibition of death receptor-mediated gene induction by a cycloheximide-sensitive factor occurs at the level of or upstream of Fas-associated death domain protein (FADD). J Biol Chem,2000,275(32):24357-66.
[74] Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway. Immunity, 1997,7(6):821-30.
[75] Sprick MR, Weigand MA, Rieser E, Rauch CT, Juo P, Blenis J, Krammer PH, Walczak H. FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity, 2000,12(6):599-609.
[76] Lin Y, Devin A, Cook A, Keane MM, Kelliher M, Lipkowitz S, Liu ZG. The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of IkappaB kinase and c-Jun N-terminal kinase. Mol Cell Biol,2000,20(18):6638-45.
[77] Choo MK, Kawasaki N, Singhirunnusorn P, Koizumi K, Sato S, Akira S, Saiki I, Sakurai H. Blockade of transforming growth factor-beta-activated kinase 1 activity enhances TRAIL-induced apoptosis through activation of a caspase cascade. Mol Cancer Ther, 2006,5(12):2970-6.
[78] Shigeno M, Nakao K, Ichikawa T, Suzuki K, Kawakami A, Abiru S, Miyazoe S, Nakagawa Y, Ishikawa H, Hamasaki K, Nakata K, Ishii N, Eguchi K. Interferon-alpha sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-kappa B inactivation. Oncogene,2003,22(11):1653-62.
[79] Yamanaka T, Shiraki K, Sugimoto K, Ito T, Fujikawa K, Ito M, Takase K, Moriyama M, Nakano T, Suzuki A. Chemotherapeutic agents augment TRAIL-induced apoptosis in human hepatocellular carcinoma cell lines. Hepatology,2000,32(3):482-90.
[80] Wirth T, Kühnel F, Fleischmann-Mundt B, Woller N, Djojosubroto M, Rudolph KL, Manns M, Zender L, Kubicka S. Telomerase-dependent virotherapy overcomes resistance of hepatocellular carcinomas against chemotherapy and tumor necrosis factor-related apoptosis-inducing ligand by elimination of Mcl-1. Cancer Res,2005,65(16):7393-402.
[81] Ganten TM, Koschny R, Haas TL, Sykora J, Li-Weber M, Herzer K, Walczak H. Proteasome inhibition sensitizes hepatocellular carcinoma cells, but not human hepatocytes, to TRAIL. Hepatology,2005,42(3):588-97.
[82] Pathil A, Armeanu S, Venturelli S, Mascagni P, Weiss TS, Gregor M, Lauer UM, Bitzer M. HDAC inhibitor treatment of hepatoma cells induces both TRAIL-independent apoptosis and restoration of sensitivity to TRAIL. Hepatology,2006,43(3):425-34.
[83] Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med,1999,5(2):157-63.
[1] Raghuraman H, Chattopadhyay A. Melittin: a membrane-active peptide with diverse functions. Biosci Rep,2007, 27(4-5):189-223.
[2] Batenburg AM, Hibbeln JC, de Kruijff B. Lipid specific penetration of melittin into phospholipid model membranes. Biochim Biophys Acta,1987,903:155–65.
[3] Cserh?ti T, Sz?gyi M.Interaction of phospholipids with proteins and peptides. New advances IV. Int J Biochem,1994,26(1):1–18.
[4] Dawson CR, Drake AF, Helliwell J, Hider RC.The interaction of bee melittin with lipid bilayer membranes. Biochim Biophys Acta,1978,510(1):75–86.
[5] Baker KJ, East JM, Lee AG. Mechanism of inhibition of the Ca(2+)-ATPase by melittin. Biochemistry,1995,34(11):3596-604.
[6] Cuppoletti J, Blumenthal KM, MalinowskaDH . Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin. Arch Biochem Biophys,1989,275(1):263–70.
[7] Cuppoletti J, Abbott AJ. Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change. Arch Biochem Biophys,1990,283(2):249–57.
[8] Fukushima N, Kohno M, Kato T, Kawamoto S, Okuda K, Misu Y, Ueda H. Melittin, a metabostatic peptide inhibiting Gs activity. Peptides ,1998,19(5):811–9.
[9] Hider RC. Honeybee venom: a rich source of pharmacologically active peptides.Endeavour,1988,12(2): 60?5.
[10] Park HJ,Lee SH, Son DJ, Oh KW, Kim KH, Song HS, Kim GJ, Oh GT, Yoon DY, Hong JT. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum, 2004 ,50(11):3504-15.
[11] Park HJ, Son DJ, Lee CW, Choi MS, Lee US, Song HS, Lee JM, Hong JT. Melittin inhibits inflammatory target gene expression and mediator generation via interaction with IkappaB kinase. Biochem Pharmacol,2007,73(2):237-47.
[12] Stuhlmeier KM. Apis mellifera venom and melittin block neither NF-kappa B-p50-DNA interactions nor the activation of NF-kappa B, instead they activate the transcription of proinflammatory genes and the release of reactive oxygen intermediates.J Immunol,2007,179(1):655-64.
[13] Nah SS,Ha E, Lee HJ, Chung JH. Inhibitory effects of melittin on the production of lipopolysaccharide-induced matrix metalloproteinase 3 in human osteoarthritic chondrocytes. Toxicon,2007,49(6):881-5.
[14] Sun X,Chen S,Li S, Yan H,Fan Y,Mi H. Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity. Peptides,2005 ,26(3):369-75.
[15] Sharma SV. Melittin resistance: a counterselection for ras transformation. Oncogene, 1992,7(2):193-201.
[16] Sharma SV. Melittin-induced hyperactivation of phospholipase A2 activity and calcium influx in ras-transformed cells.Oncogene ,1993,8(4):939-47.
[17] Hait WN, Grais L, Benz C, Cadman EC. Inhibition of growth of leukemic cells by inhibitors of calmodulin: phenothiazines and melittin. Cancer Chemother Pharmacol,1985,14(3):202?5.
[18] Hait WN, Lee GL. Characteristics of the cytotoxic effects of the phenothiazine class of calmodulin antagonists. Biochem Pharmacol, 1985,34(22):3973-8.
[19] Lee GL, Hait WN. Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin. Life Sci,1985,36(4):347-54.
[20] Lazo JS, Hait WN, Kennedy KA, Braun ID, Meandzija B. Enhanced bleomycin-induced DNA damage and cytotoxicity with calmodulin antagonists. Mol Pharmacol ,1985,27(3):387?93.
[21] Lazo JS, Chen DL, Gallicchio VS, Hait WN. Increased lethality of calmodulin antagonists and bleomycin to human bone marrow and bleomycin-resistant malignant cells. Cancer Res,1986, 46(5):2236?40.
[22] Killion JJ,Dunn JD. Differential cytolysis of murine spleen,bone-marrow and leukemia cells by melittin reveals differences in membrane topography. Biochem Biophys Res Commun,1986,139(1): 222?7.
[23] Zhu HG, Tayeh I, Israel L, Castagna M. Different susceptibility of lung cell lines to inhibitors of tumor promotion and inducers of differentiation. J Biol Regul Homeost Agents,1991, 5(2): 52?8.
[24] Arora AS, de Groen PC, Croall DE, Emori Y, Gores GJ. Hepatocellular carcinoma cells resist necrosis during anoxia by preventing phospholipase-mediated calpain activation. J Cell Physiol,1996,167(3):434?42.
[25] Wu YL, Jiang XR, Newland AC, Kelsey SM. Failure to activate cytosolicphospholipase A2 causes TNF resistance in human leukemic cells. J Immunol,1998,160(12):5929?35.
[26] Saini SS, Chopra AK, Peterson JW. Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells.Toxicon, 1999 ,37(11):1605-19.
[27] Chu ST, Cheng HH, Huang CJ, Chang HC, Chi CC, Su HH, Hsu SS, Wang JL, Chen IS, Liu SI, Lu YC, Huang JK, Ho CM, Jan CR.Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells. Life Sci,2007,80(4):364-9.
[28] Moon DO, Park SY, Choi YH, Kim ND, Lee C, Kim GY.Melittin induces Bcl-2 and caspase-3-dependent apoptosis through downregulation of Akt phosphorylation in human leukemic U937 cells.Toxicon,2008,51(1):112-20.
[29] Chen YQ, Zhu ZA, Hao YQ, Dai KR, Zhang C. Effect of melittin on apoptosis and necrosis of U2 OS cells. Zhong Xi Yi Jie He Xue Bao,2004, 2(3):208-9.
[30] Holle L, Song W, Holle E, Wei Y, Wagner T, Yu X. A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo. Int J Oncol,2003,22(1): 93?8.
[31] Wang C, Chen TY, Zhang N, Yang MJ, Li B, LüX, Cao XT, Ling CQ. Melittin, a major component of bee venom, sensitizes human hepatocellular carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by activating CaMKII-TAK1-JNK/p38 and inhibiting IkappaBalpha kinase-NFkappaB.J Biol Chem,2009,284(6):3804–13.
[32] Liu SJ, Yu M, He Y, Xiao L, Wang F, Song C, Sun SH, Ling CQ, Xu ZH. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology ,2008,47(6): 1964-73.
[33] Winder D, Günzburg WH, Erfle V, Salmons B. Expression of antimicrobial peptides has an antitumour effect in human cells. Biochem Biophys Res Commun ,1998,242(3):608?12.
[34] Ling CQ, Li B, Zhang C, Gu W, Li SX, Huang XQ, Zhang YN. Anti-hepatocarcinoma effect of recombinant adenovirus carrying melittin gene. Zhonghua Gan Zang Bing Za Zhi,2004,12(12):741?4.
[35] Ling CQ,Li B, Zhang C, Zhu DZ, Huang XQ, Gu W,Li SX.Inhibitory effect of recombinant adenovirus carrying melittin gene on hepatocellular carcinoma. Annals of Oncology,2005, 16(1):109-15.
[36] Li B, Ling CQ, Zhang C, Gu W, Li SX, Huang XQ, Zhang YN, Yu CQ. The induced apoptosis of recombinant adenovirus carrying melittin gene for hepatocellular carcinoma cell. Zhonghua Gan Zang Bing Za Zhi,2004,12(8):453?5.
[37] Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ. Growth Arrest and Apoptosis of the Human Hepatocellular Carcinoma Cell Line Bel-7402 Induced by Melittin. Onkologie,2006,29(8-9):367-71.
[38] Lazarev VN,Parfenova TM,Gularyan SK, Misyurina OY, Akopian TA, Govorun VM.Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line. Int J Antimicrob Agents, 2002 ,19(2):133-7.
[39] Lazarev VN,Shkarupeta MM,Titova GA,Kostrjukova ES,Akopian TA,Govorun VM. Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo. Biochem Biophys Res Commun,2005,338(2):946-50.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |