中药诱导肿瘤细胞凋亡机制研究进展

中药及其生物活性成分具有毒副作用小、无残留、药理作用独特等特点,在抗肿瘤研究方面,已经成为抗肿瘤药物或其辅助药物,目前研究的热点是中药治疗肿瘤作用机制的研究。论文从单味中药及其有效成分、中药复方诱导肿瘤细胞凋亡机制等方面对中药诱导细胞凋亡在肿瘤的发生、发展和转归中所起的作用进行了综述。中药可以诱导肿瘤细胞凋亡,其可能的作用机制是通过调控原癌基因和抑癌基因的表达、影响细胞凋亡通路的信号传导及阻滞肿瘤细胞增殖周期等抑制肿瘤细胞的永生化。     

“肿瘤消”诱导肿瘤细胞凋亡的作用研究

研究“肿瘤消”的抗肿瘤活性和作用,为临床用药提供依据。通过 MTT 法检测“肿瘤消”对多种肿瘤细胞株和正常细胞存活率的影响;采用 Hoechst 染色法和 DNA 片段化分析对“肿瘤消”诱导 MDCC-MSBl 细胞凋亡的作用进行检测;Western blot 检测“肿瘤消”诱导 MDCC-MSBl 细胞凋亡相关蛋白因子表达情况。MTT 法检测结果显示,“肿瘤消”对 MDCC-MSBl、C6、SP2／0和 A549细胞的增殖具有明显抑制作用,对293A 细胞增殖无显著影响;Hoechst 染色和 DNA 片段化分析均证实“肿瘤消”能诱导 MDCC-MSBl细胞凋亡;Western blot 检测发现“肿瘤消”诱导 MDCC-MSBl 细胞凋亡与凋亡因子 caspase 3、caspase 8和caspase 9的活化有关。证实“肿瘤消”具有诱导肿瘤细胞凋亡的作用。     

肿瘤坏死因子诱导凋亡配体TRAIL的研究进展

肿瘤坏死因子诱导凋亡配体TRAIL作为肿瘤坏死因子超家族成员之一,其能和死亡受体相结合从而选择性诱导肿瘤细胞凋亡,却不会影响正常细胞,其正逐渐成为临床上新型抗肿瘤制剂。本文从肿瘤坏死因子诱导凋亡配体TRAIL何受体诱导凋亡机制入手,探讨其在临床方面的应用。     

凋亡素（apoptin）诱导人白血病细胞的．凋亡

为了观察凋亡素（apoptin)能否诱导白血病细胞发生凋亡，利用脂质体介导将凋亡素基因真核表达质粒转染白血病细胞系。通过琼脂糖胶电泳分析、电子显微镜观察，结果表明，凋亡素能促使白血病细胞发生细胞凋亡。     

细菌感染与抗凋亡

凋亡是细胞对环境刺激的一种正常反应,在多细胞生物中经常发生.在微生物感染中也发现有细胞凋亡的现象,并可能影响疾病的进程和转归.令人不解的是还有许多细菌可减轻、甚至抑制细胞凋亡.本文从细胞凋亡的分子途径来探索细胞发生凋亡的生物学条件.揭示细胞凋亡的诱导和抑制机制.本文侧重阐述各种细菌的抗凋亡活性,并试图阐明哺乳动物细胞与细菌间的接触是如何影响细胞对凋亡的抵抗力,二者的作用分别是什么,以及二者的相互作用与细胞凋亡的分子途径有何关系.     

线粒体在细胞凋亡中的作用

细胞凋亡属机体的生理机制 ,是多细胞生物更新正常细胞和清除异常细胞的重要手段 ,线粒体为细胞各种生命活动提供能量 ,二者紧密相关 ;线粒体参与细胞凋亡 ,并且是细胞凋亡的调控中心。淋巴细胞 ,巨噬细胞 ,神经细胞 ,肿瘤细胞等的凋亡都证实了这一点 ;NO和 Ca2 诱导的细胞凋亡也通过线粒体来完成 ;在线粒体调控细胞凋亡机理的研究上也有大量研究成果 ,如 :半胱天冬酶的诱导机制 ,细胞色素 c引起细胞凋亡的机制 ,胞内氧化还原电势改变引起细胞凋亡 ,Bcl-2家族蛋白调控细胞凋亡等。但线粒体参与调控的凋亡机制并不是唯一的细胞凋亡通路。本文综述了近年来有关线粒体与细胞凋亡关系的研究进展     

细菌感染与抗凋亡

凋亡是细胞对环境刺激的一种正常反应，在多细胞生物中经常发生。在微生物感染中也发现有细胞凋亡的现象，并可能影响疾病的进程和转归。令人不解的是还有许多细菌可减轻、甚至抑制细胞凋亡。本文从细胞凋亡的分子途径来探索细胞发生凋亡的生物学条件。揭示细胞凋亡的诱导和抑制机制。本文侧重阐述各种细菌的抗凋亡活性，并试图阐明哺乳动物细胞与细菌间的接触是如何影响细胞对凋亡的抵抗力，二者的作用分别是什么，以及二者的相互作用与细胞调亡的分子途径有何关系。     

大蒜素诱导肿瘤细胞凋亡研究进展

近年来许多体外研究表明大蒜素能够诱导包括白血病、肝癌、卵巢癌、胃癌等多种肿瘤细胞的凋亡,并取得了很好的效果,其诱导肿瘤细胞凋亡的途径可能是通过影响细胞生长周期、相关凋亡基因、caspase蛋白表达、细胞端粒酶活性等方式来实现的。大蒜素作为大蒜的主要活性成分,其抗癌效果备受人们的关注。大蒜素诱导肿瘤细胞凋亡是一个比较复杂的生物学过程,论文对大蒜等诱导肿瘤细胞凋亡的作用机制进行了综述。     

三氧化二砷诱导鸡MD肿瘤细胞凋亡及对细胞内钙离子浓度的影响

为探讨As2O3诱导MD肿瘤细胞株凋亡的机制,采用MTT法检测As2O3对MDCC-MSB1的抑制作用.采用荧光显微镜观察细胞形态学变化,DNA Ladder法检测细胞凋亡,Fura-2/AM负载双波长荧光分光光度计检测细胞内游离钙离子浓度([Ca2 ]i)变化,观察A2O3对鸡MD肿瘤细胞株MDCC-MSB1的影响.结果显示:As2O3能抑制鸡MD肿瘤细胞株MDCC-MSB1的生长,呈剂量依赖关系(P<0.05或P<0.01);在荧光显微镜下可见As2O3作用后MDCC-MSB1细胞呈现典型的凋亡特征,并出现典型的DNA Ladder,双波长荧光分光光度计检测结果显示:As2O3作用后的MDCC-MSB1细胞内[Ca2 ]i升高(P<0.05或P<0.01),呈剂量依赖关系.As2O3能明显抑制鸡MD肿瘤细胞株MDCC-MSB1细胞的生长,并诱导其发生凋亡,细胞内[Ca2 ]i高可能是其诱导细胞凋亡的机制之一.     

细胞凋亡的特征及其检测方法

细胞凋亡是动物机体组织清除受损、衰老或多余细胞的一种自杀性过程,它对于保持机体各组织器官正常生长、发育、结构、功能动态平衡和维持内环境相对稳定等都具有重要意义。发生凋亡的细胞大多数是生物个体中多余或老化的细胞,有些也是发生肿瘤时的细胞或受到病毒或细菌感染的细胞。根据不同的检测方法来检测不同类型的细胞凋亡,进而来研究细胞凋亡的现象和发生机制,为揭示机体与细胞凋亡的关系和疾病与凋亡的关系提供理论依据。文章对凋亡细胞的特征、细胞凋亡各种检测方法的检测原理和结构判定,分别进行了概述。     

Chicken anemia virus induced apoptosis: underlying molecular mechanisms

In 1990, the chicken anemia virus (CAV) genome was cloned by us and proven to be representative for CAV isolates worldwide. This genome contains unique promoter/enhancer replication elements and genes. Upon infection of its target cells, CAV replicates via a double-stranded (ds) DNA intermediate. From this ds CAV molecule, a single mRNA is transcribed, which encodes for three distinct proteins VP1, VP2, and VP3 or apoptin. Its capsid contains only the VP1 protein. However, for the production of the neutralizing epitope, co-synthesis of VP1 and VP2 are needed. CAV genomes with mutations in the 12 bp insert of the promoter/enhancer region were shown to produce immunogenic functional CAV particles. Mutations in these and other regulatory elements of CAV might also decrease its virus load resulting in a reduced pathogenic effect. CAV causes fatal cytopathogenic effects in e.g. chicken thymocytes via apoptosis. Under in vitro conditions, CAV replicates only in transformed chicken cell lines, which indicates that at least a part of the CAV life-cycle requires transformed-like cellular events. In these transformed cell lines, the synthesis of the apoptin protein alone mimics the CAV-induced apoptosis, whereas the VP2 protein also harbors some apoptotic activity. Extensive studies on apoptin resulted in the characterization of domains essential for its apoptotic activity and nuclear localization, which seems to be related with its ability to induce apoptosis. Therefore, both VP2 and apoptin are of interest in reducing the pathogenicity of CAV infections. A series of biomedical studies on apoptin have been carried out in human cell systems, which are informative about the mechanism of CAV-induced apoptosis in chicken (transformed) cells. Synthesis of apoptin alone induces apoptosis in various human transformed and/or tumorigenic cell lines, but not in normal human diploid cells. A striking difference in the cellular localization of apoptin was observed in human normal diploid cells versus tumor cells. In all tumor cells, apoptin is located mainly in the heterochromatic regions of the nucleus, whereas in normal cells it is present in peri-nuclear structures. Apoptin contains a bipartite nuclear localization signal, and one domain that resemble a nuclear export signal. Elucidation of parts of the apoptin-induced apoptotic pathway revealed unique characteristics: apoptin-induced apoptosis is independent of the tumor suppressor p53. The anti-apoptotic protein Bcl-2 does not inhibit but even accelerates apoptin-induced apoptosis in tumor cells, whereas over expression of Bcl-2 in normal cells has no effect on the apoptin activity. Upstream caspases are not involved, whereas downstream caspase 3 is, but seems not to be essential. A number of novel proteins were shown to interact with apoptin in transformed cells. Future studies of apoptin, VP2 and related cellular proteins in chicken cells will unravel the regulatory aspects of CAV-induced apoptosis.     

Trientine, a copper-chelating agent, induced apoptosis in murine fibrosarcoma cells in vivo and in vitro

Anti-copper treatments have been investigated to determine whether they suppress angiogenesis and tumor development since Cu is widely accepted as being required for angiogenesis. We examined the effects of treatment with trientine, a copper-chelating agent, on tumor development in a murine xenograft model using fibrosarcoma-derived transplantable QRsp-11 cells and C57BL/6 mice and induction of apoptosis in tumor cells and endothelial cells in vivo and in vitro. The tumor volumes increased more slowly in trientine-treated mice than in untreated mice. Tumor volumes in the treated mice were significantly smaller than those in the untreated mice at 24 days postinoculation (d.p.i.) of tumor cells. A cluster of pyknotic tumor cells and morphological abnormalities in capillary endothelial cells were observed in the tumors of trientine-treated mice but not in the tumors of untreated mice. The proportions of apoptotic and necrotic cells in the tumors of treated mice were approximately 3.5-fold higher than those in the tumors of untreated mice at 14 d.p.i. When the cells were treated with trientine in vitro, mouse endothelial cells and bovine primary endothelial cells showed an approximately 10-fold higher sensitivity to trientine than QRsp-11 cells in terms of D37. However, the proportion of apoptotic cells in endothelial cells was significantly lower than that in QRsp-11 cells after treatment with trientine. These results show that apoptosis was induced in tumor cells by treatment with trientine in vivo and in vitro.     

Oncolysis of canine tumor cells by myxoma virus lacking the serp2 gene

Objective-To determine the oncolytic efficacy of an attenuated form of myxoma virus lacking the serp2 gene in canine tumor cells. Sample-Primary cells were isolated from tumors that were surgically removed from dogs and from connective tissue obtained from the cadaver of a dog. Cells of various established cell lines from tumors and nontumorous tissues were obtained. Procedures-Experiments were performed with cells in monolayer culture. Cell cultures were inoculated with wild-type myxoma viruses or myxoma viruses lacking the serp2 gene, and measures of cytopathic effects, viral growth kinetics, and cell death and apoptosis were determined. Results-Myxoma viruses replicated in cells of many of the primary and established canine tumor cell lines. Canine tumor cells in which expression of activated protein kinase B was upregulated were more permissive to myxoma virus infection than were cells in which expression of activated protein kinase B was not upregulated. Myxoma viruses lacking the serp2 gene caused more cytopathic effects in canine tumor cells because of apoptosis than did wild-type myxoma viruses. Conclusions and Clinical Relevance-Results of the present study indicated myxoma viruses lacking the serp2 gene may be useful for treatment of cancer in dogs. Impact for Human Medicine-Results of the present study may be useful for development of novel oncolytic treatments for tumors in humans.     

血小板凝血酶蛋白1对犬乳腺肿瘤细胞体外生物学特性的影响分析

本研究旨在深入探讨血小板凝血酶蛋白1(THBS1)对犬乳腺肿瘤细胞CHMp的影响,并阐明其影响犬乳腺肿瘤发生发展的作用机制。通过构建THBS1慢病毒稳定过表达和THBS1沉默表达的犬乳腺肿瘤细胞CHMp细胞系,采用CCK-8试验、划痕试验、Transwell试验、原位荧光检测和流式细胞术检测THBS1对CHMp细胞增殖、迁移、侵袭、细胞凋亡和细胞周期情况的影响;通过qRT-PCR和Western blot检测THBS1对CHMp细胞凋亡相关因子(p53、Bcl-2、Bax)表达情况的影响,验证THBS1对CHMp细胞凋亡通路的影响。结果显示,过表达THBS1能有效增强犬乳腺肿瘤细胞CHMp的增殖、迁移和侵袭能力,并且减少CHMp细胞的凋亡数量,而沉默THBS1后结果与之相反;流式细胞术得出THBS1能够影响CHMp细胞的细胞周期分布;经qRT-PCR和Western blot检测发现,在THBS1过表达后,p53和Bcl-2的表达量均明显增高,Bax的表达量明显减少,在沉默THBS1后结果与之相反。结果表明,THBS1的异常表达能够影响犬乳腺肿瘤细胞CHMp的增殖、迁移、侵袭以及细胞周期;此外,THBS1能够通过影响细胞凋亡因子p53、Bcl-2和Bax的表达来影响CHMp细胞凋亡相关通路,进而影响犬乳腺肿瘤的发生发展。     

Mitochondrial transmembrane potential and reactive oxygen species generation regulate the enhanced effect of CCCP on TRAIL-induced SNU-638 cell apoptosis

TRAIL is a member of the tumor necrosis factor family and engages apoptosis via recruitment and rapid activation of caspase-8. This study investigated the effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP), a classic uncoupler of oxidative phosphorylation, on TRAIL-induced apoptosis in SNU-638 cells derived from human gastric cancer cells. It was found that treatment with CCCP followed by incubation with TRAIL markedly enhanced apoptosis by 2 fold compared with treatment with TRAIL alone. This effect was accompanied by reduction in mitochondrial transmembrane potential and generation of reactive oxygen species. This sensitization was inhibited by N-acetyl-l-cysteine, which restored the mitochondrial transmembrane potential and reduced reactive oxygen species generation. Treatment with N-acetyl-L-cysteine also inhibited expression of apoptotic proteins such as Bax and Smac and abrogated caspase-8 activation. Moreover, treatment with N-acetyl-L-cysteine prior to induction with TRAIL increased expression of the anti-apoptotic Bcl-2 protein. These data indicate that CCCP enhanced TRAIL-induced apoptosis by dissipation of mitochondrial transmembrane potential and reactive oxygen species, suggesting that treatment with CCCP combined with that with TRAIL can be an efficient method to induce death of tumor cells, particularly cells that are resistant to TRAIL-induced apoptosis.     

The bone biologic effects of zoledronate in healthy dogs and dogs with malignant osteolysis

BACKGROUND: Malignant osteolysis is a process whereby cancer cells in concert with osteoclasts erode bone matrix. Aminobisphosphonates (NBPs) such as zoledronate induce osteoclast apoptosis and thereby decrease malignant skeletal destruction, severity of bone pain, and frequency of pathologic fracture. HYPOTHESIS: IV-administered zoledronate will reduce homeostatic bone turnover in healthy dogs and pathologic bone resorption in dogs diagnosed with primary and secondary bone tumors. ANIMALS: Six healthy dogs and 20 dogs with naturally occurring primary or metastatic bone tumors were administered zoledronate IV. METHODS: Prospective study: In all dogs, healthy (n = 6) and with malignant osteolysis (n = 20), the bone biologic effects of zoledronate were evaluated by quantifying changes in serum C-telopeptide (CTx) or urine N-telopeptide (NTx) concentrations or both. In dogs with osteosarcoma (OSA) (n = 10), serial changes in tumor relative bone mineral density (rBMD) assessed by dual-energy x-ray absorptiometry were used to characterize zoledronate's antiresorptive effects within the immediate tumor microenvironment. Additionally, the biochemical tolerability of zoledronate was assessed in 9 dogs receiving multiple (> or =2) consecutive treatments. RESULTS: All dogs had significant reductions in serum CTx or urine NTx concentrations or both after zoledronate administration. In a subset of dogs with appendicular OSA, reduced urine NTx concentrations and increased primary tumor rBMD coincided with improved limb usage as reported by pet owners in dogs treated with zoledronate and concurrent oral analgesics. Multiple zoledronate infusions were not associated with biochemical evidence of toxicosis. CONCLUSIONS AND CLINICAL IMPORTANCE: In dogs with skeletal neoplasms, IV-administered zoledronate exerts bone biologic effects, appears safe, and can provide pain relief.     

Effect of the VP3 gene of chicken anemia virus on canine mammary tumor cells

OBJECTIVE: To investigate the antitumor effect of the chicken anemia virus (CAV) VP3 gene in canine mammary tumor (CMT) cells. SAMPLE POPULATIONS: Established primary canine cell lines that originated from epithelial cells of resected CMTs and nonneoplastic mammary gland epithelial (MGE) cells. PROCEDURES: Expression vectors and lentiviral vectors encoding the VP3 gene from a Taiwan-Ilan isolate of CAV were used to deliver the VP3 gene into CMT cells and nonneoplastic MGE cells. Ectopic gene expression and the pro-apoptotic effect of the VP3 gene on CMT and nonneoplastic MGE cells by either transfection or viral infection were evaluated via immunofluorescence microscopy, western blot analysis, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis. RESULTS: Overexpression of the enhanced green fluorescent protein-VP3 fusion protein was detected predominantly in the nuclei of CMT cells. In contrast, the VP3 protein was localized to the cytoplasm of nonneoplastic MGE cells. Among the fusion protein-expressing CMT cells, most underwent characteristic changes of apoptosis, whereas apoptosis was not detected in fusion protein-expressing, nonneoplastic MGE cells. Induction of apoptosis by VP3 gene overexpression in CMT cells was associated with the caspase-9-, but not the caspase-8-, mediated apoptosis pathway. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that the VP3 gene of the CAV induces apoptosis in malignant CMT cells, but not in nonneoplastic canine MGE cells. On the basis of such tumor cell-specific killing, the VP3 gene may be a promising agent for the treatment of malignant mammary gland tumors in dogs.     

颗粒细胞凋亡影响家禽卵泡闭锁的研究进展

卵巢是家禽的重要繁殖器官,会产生大量卵泡,而卵泡在生长发育的各个阶段中都可能因为不同因素的调控而发生闭锁,最终导致繁殖性能衰退。颗粒细胞对卵泡的生长发育有重要调控作用,其凋亡会诱导卵泡发生闭锁。诱导颗粒细胞发生凋亡的因素较多,包括激素、细胞因子、氧化应激、线粒体及其他体外因素。颗粒细胞凋亡主要由线粒体途径导致,其涉及到半胱天冬酶（Caspase）家族参与,当线粒体裂解时会释放细胞色素C （Cyt-C）,随后形成凋亡小体激活Caspase-3和Caspase-8,最终激活Caspase-9导致颗粒细胞凋亡;当颗粒细胞发生凋亡,家禽体内卵泡丧失生物功能并且卵泡细胞之间的调控失衡,促使卵泡内卵母细胞和膜细胞凋亡,最终导致卵泡发生闭锁;颗粒细胞在存活状态下所分泌的生长因子、性腺类固醇、细胞因子能减少卵母细胞氧化损伤,防止细胞内活性氧（ROS）水平过高导致的线粒体DNA损伤,从而避免线粒体功能障碍而造成的颗粒细胞凋亡。作者从颗粒细胞凋亡及其影响因素、颗粒细胞凋亡和卵泡闭锁的关系、颗粒细胞凋亡对卵泡闭锁的影响3个方面进行阐述,以期为减少卵泡闭锁、提高家禽繁殖性能提供理论依据。