黏着斑激酶及其小分子抑制剂作为抗肿瘤药物的研究进展

抗黏着斑激酶是一种非受体型酪氨酸蛋白激酶,在许多肿瘤的发生和发展过程中均有过表达。研究表明,作为细胞内重要的骨架蛋白和调节多种细胞信号通路的关键分子,黏着斑激酶在肿瘤发生、发展、迁移和侵袭的各个阶段都起着重要作用。因此,以黏着斑激酶作为抗肿瘤靶点开发其抑制剂的研究受到广泛关注。综述黏着斑激酶的结构与功能、它与肿瘤的关联及其小分子抑制剂的研究与开发。     

Raf激酶抑制剂及其耐药机制研究进展

Ras/Raf/MEK/ERK 通路是调节细胞生长与增殖的重要信号传导通路。在Ras/Raf/MEK/ERK 通路中某些成员的突变往往与恶性肿瘤的发生密切相关。B-Raf 激酶是该通路中Raf 家族最重要的亚型,其主要突变形式B-RafV600E 在黑色素瘤等多种肿瘤中高度表达。选择性B-RafV600E 抑制剂vemurafenib 和dabrafenib 的上市使得晚期黑色素瘤的治疗进入新纪元,但是耐药性和副作用依然限制了二者的使用。综述目前Raf 激酶抑制剂耐药性与副作用产生机制以及Raf 激酶抑制剂的最新研究进展。     

蛋白激酶B/Akt抑制剂

磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/Akt)信号通路在细胞生长与存活中起着关键作用,PI3K/Akt通路的过度激活在多种肿瘤中常见。Akt激酶本身以及Akt激酶上游调节分子,例如PTEN和PI3K,在超过50%的人类肿瘤中均有异常变化。因此Akt成为肿瘤预防和肿瘤靶向治疗的热点之一。许多小分子化合物通过不同机制抑制Akt活性,根据小分子抑制剂与激酶的结合部位和化学结构不同,主要分为ATP竞争性抑制剂、Akt变构抑制剂和磷脂酰肌醇类似物抑制剂。本文综述了PI3K/Akt通路与肿瘤的关系和Akt抑制剂的研究现状,为新型抗癌药物的设计研究提供参考。     

M1型丙酮酸激酶在肿瘤中的作用

M型丙酮酸激酶(PKM)包括M1型丙酮酸激酶(PKM1)和M2型丙酮酸激酶(PKM2)。既往的研究认为PKM2与肿瘤存在着密切关系,PKM2通过促进Warburg效应和合成代谢以及发挥蛋白激酶样作用,促进肿瘤细胞增殖,在肿瘤的形成过程中起着关键作用。但近年来的研究表明,PKM1不仅促进线粒体的氧化磷酸化过程,也可促进Warburg效应、生物合成代谢、细胞增殖及肿瘤形成,特别是在肺神经内分泌癌和肝细胞癌的形成和进展中可能发挥着重要作用,有望成为肿瘤治疗的新靶点。     

IA型磷脂酰肌醇3-激酶与肿瘤的关系及其抑制剂分子机制研究进展

磷脂酰肌醇3-激酶(phosphatidylinositol-3 kinase,PI3K)是细胞内重要的信号分子,它具有调节细胞增殖、分化、代谢、凋亡等功能。PI3K的基因易发生突变和扩增,从而导致PI3K被激活,与肿瘤的形成和发展密切相关。IA型的PI3K及其下游的信号分子组成的通路参与调节肿瘤细胞的增殖、存活、黏附、迁移等活动。综述了IA型PI3K——PI3Kα、PI3Kβ和PI3Kδ与肿瘤发生、发展的关系,列举了20个具有代表性的IA型PI3K抑制剂,并讨论了它们的分子抑制机制。     

膀胱ICC细胞的研究现状和展望

膀胱ICC细胞(Interstitial cells of Cajal in bladder)早在上个世纪已被发现,与胃肠道ICC细胞同族,膀胱ICC以自发电活动为特性,源于胞内贮存钙离子释放和钙激活的氯同道开放引起自发短暂去极化。膀胱ICC细胞起初被理解为起搏细胞,其自发的电活动作为起搏器引起下游平滑肌的收缩,这种假说尚缺乏立足的直接证据,目前认为ICC细胞仅仅是膀胱平滑肌收缩活动的调节器,与上皮-传入神经以及神经-平滑肌的信号传递密切相关。病理状态下ICC细胞的作用似乎比生理状态下更为突出,例如膀胱过度活动症,多篇文献报道膀胱过度活动症患者膀胱的ICC细胞数目比正常增多,而且其平滑肌的收缩对酪氨酸激酶受体(tyrosine kinase receptor,c-Kit)拮抗剂格列卫更加敏感。未来膀胱ICC细胞的研究集中在阐明病理及生理状态下ICC的作用机制和信号通路。     

内皮细胞的M2型丙酮酸激酶有助于维持血管完整性

丙酮酸激酶(pyruvate kinase,PK)是细胞内催化糖酵解的关键酶,其中M2型丙酮酸激酶(PKM2)在增殖能力强的细胞特别是癌细胞中高表达,因此成为临床上肿瘤治疗的一个新靶点。然而,全身应用PKM2抑制剂对于其他细胞如血管内皮细胞是否存在副作用尚不清楚。     

MEK激酶及其抑制剂的研究进展

丝裂原活化的细胞外信号调节激酶(mitogen-activated extracellular signal-regulated kinase,MEK)是一种可磷酸化靶蛋白上丝氨酸/苏氨酸和酪氨酸残基的双特异性激酶,也是RASRAF-MEK-ERK信号转导通路的主要组分。该信号通路参与了细胞凋亡,细胞周期进行,细胞迁移、分化、代谢和细胞增殖等众多过程的调节。大量研究表明,MEK结构及其表达水平的改变与肿瘤等多种疾病的发生密切相关。因此,对MEK特异性抑制剂的筛选成了当前国际上关于肿瘤治疗研究的热点。目前,已有多种MEK抑制剂被发现,部分已用于肿瘤等疾病的治疗,并显示出较好的临床疗效。该文将对MEK的结构、功能及MEK抑制剂的临床应用等方面的研究进展作一综述。     

以分子开关Ras蛋白为靶点的抗肿瘤药物研究进展

Ras原癌基因编码的蛋白是细胞信号转导中不可或缺的分子开关,在细胞增殖、分化、凋亡等生理过程中起着重要的作用。Ras基因的功能获得性突变是肿瘤发生和发展的重要驱动因素,因此多年来人们一直致力于靶向Ras蛋白的抗肿瘤药物研究。简介Ras的结构与功能及其与肿瘤的相关性,着重综述近年来靶向Ras的小分子抑制剂的研究进展。     

M2型丙酮酸激酶与宫颈癌关系的研究进展

M2型丙酮酸激酶是近年来发现的一种器官非特异性肿瘤标记物,在多种恶性肿瘤,尤其是在宫颈癌的发生发展中发挥着重要作用.本文综述了M2型丙酮酸激酶的生物学特征、及其在宫颈癌中的表达情况、致癌机制以及应用前景等.     

c-Kit--a hematopoietic cell essential receptor tyrosine kinase

The receptor tyrosine kinase c-Kit is expressed in hematopoietic stem and progenitor cells and in several non-hematopoietic tissues. In the hematopoietic system, c-Kit is critical for proliferation, survival and differentiation. During recent years exploration of the signalling pathways downstream of this receptor has yielded significant new insights in the field. In this review, we will summarise the c-Kit background, structure, downstream signalling and medical significance with particular focus on its role in hematopoietic progenitor cells and mast cells.     

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation

The receptor tyrosine kinase c-Kit, also known as the stem cell factor receptor, plays a key role in several developmental processes. Activating mutations in c-Kit lead to alteration of these cellular processes and have been implicated in many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia, testicular seminomas and mastocytosis. Regulation of the catalytic activity of several kinases is known to be governed by phosphorylation of tyrosine residues in the activation loop of the kinase domain. However, in the case of c-Kit phosphorylation of Tyr-823 has been demonstrated to be a late event that is not required for kinase activation. However, because phosphorylation of Tyr-823 is a ligand-activated event, we sought to investigate the functional consequences of Tyr-823 phosphorylation. By using a tyrosine-to-phenylalanine mutant of tyrosine 823, we investigated the impact of Tyr-823 on c-Kit signaling. We demonstrate here that Tyr-823 is crucial for cell survival and proliferation and that mutation of Tyr-823 to phenylalanine leads to decreased sustained phosphorylation and ubiquitination of c-Kit as compared with the wild-type receptor. Furthermore, the mutated receptor was, upon ligand-stimulation, quickly internalized and degraded. Phosphorylation of the E3 ubiquitin ligase Cbl was transient, followed by a substantial reduction in phosphorylation of downstream signaling molecules such as Akt, Erk, p38, Shc, and Gab2. Thus, we propose that activation loop tyrosine 823 is crucial for activation of both the MAPK and PI3K pathways and that its disruption leads to a destabilization of the c-Kit receptor and decreased survival of cells.     

Grb2 mediates negative regulation of stem cell factor receptor/c-Kit signaling by recruitment of Cbl

Aberrant activation of c-Kit is involved in a number of human diseases including cancers and leukemias. Certain receptor tyrosine kinases, such as epidermal growth factor receptor, have been shown to indirectly recruit Cbl through the adapter protein Grb2, leading to receptor ubiquitination and degradation. In order to study the role of Grb2 in c-Kit degradation, a series of mutations of the Grb2 binding sites in c-Kit were generated (Y703F, Y936F, and Y703F/Y936F). Since other signal transduction molecules are also known to bind Y703 and Y936, the more selective asparagine-to-alanine (N-to-A) mutants N705A, N938A, and N705A/N938A were generated. We could clearly demonstrate that binding of Grb2 was dependent on intact phosphorylation sites Y703 and Y936. Furthermore, we could demonstrate the presence of Cbl in a complex with Grb2 and c-Kit. Thus, Grb2 is able to indirectly recruit Cbl to c-Kit. In the N-to-A mutants, Cbl phosphorylation was strongly reduced, which correlated with reduced ubiquitination of c-Kit as well as decreased internalization and degradation of the receptor. Taken together, we have demonstrated that, in addition to its role in positive signaling via the Ras/Erk pathway, Grb2 mediates c-Kit degradation through recruitment of Cbl to c-Kit, leading to ubiquitination of c-Kit followed by internalization and degradation.     

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45(low) c-Kit(+) cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45(low) c-Kit(-) cells that showed a granulocyte morphology; CD45(high) c-Kit(low/-) that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45(low) c-Kit(+) cells from the AGM culture had the abilities to reproduce CD45(low) c-Kit(+) cells and differentiate into CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) cells, whereas CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) did not produce CD45(low) c-Kit(+) cells. Furthermore, CD45(low) c-Kit(+) cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45(low) c-Kit(+) cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.     

Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk

We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.     

The direct association of the multiple PDZ domain containing proteins (MUPP-1) with the human c-Kit C-terminus is regulated by tyrosine kinase activity

We have identified the multiple PDZ domain containing protein (MUPP-1 or MPDZ) as a novel binding partner of the human c-Kit. c-Kit binds specifically to the 10th PDZ domain of MUPP-1 via its C-terminal sequence. Furthermore, a kinase negative-mutant receptor interacted more strongly with MUPP-1 than the wild-type c-Kit. Strikingly, a constitutively activated c-Kit (D816V-Kit) did not bind to MUPP-1, although this oncogenic form retains the PDZ binding motif 'HDDV' at the C-terminal end. Deletion of V967 of c-Kit abolished binding to MUPP-1 and drastically reduced its tyrosine kinase activity, suggesting that the structure of the C-terminal tail of c-Kit influences its enzymatic activity.     

Expression of c-Kit receptor tyrosine kinase and effect on beta-cell development in the human fetal pancreas

The receptor, c-Kit, and its ligand, stem cell factor (SCF), are critical for hematopoietic stem cell differentiation and have been implicated in the development, function, and survival of rodent islets. Previously, we reported that exogenous SCF treatments of cultured human fetal (14-16 wk fetal age) islet-epithelial clusters enhanced islet cell differentiation and proliferation (Li J, Goodyer CG, Fellows F, Wang R. Int J Biochem Cell Biol 38: 961-972, 2006). In the present study, we examined the expression pattern of c-Kit in early to midgestation human fetal pancreata and the relevance of c-Kit receptor tyrosine kinase for insulin gene expression and beta-cell survival. c-Kit is expressed in the intact pancreas in a cell-specific manner, with a significant decrease in immunoreactivity in the duct regions from 8 to 21 wk fetal age, paralleled by a significant increase in expression within endocrine regions. These c-Kit-positive cells are highly proliferative and show frequent coexpression with insulin and glucagon. Treatment of islet-epithelial clusters with anti-ACK45 antibody stimulates c-Kit phosphorylation paralleled by a significant increase in PDX-1 and insulin expression, increased cell proliferation, and reduced beta-cell death. In contrast, transient transfection with c-Kit siRNA results in a three- to fourfold decrease in c-Kit, PDX-1, and insulin expression and decreased cell proliferation. This study describes important changes in the distribution and dynamics of c-Kit-expressing cells during human fetal pancreatic neogenesis, suggesting that c-Kit may be a marker for human pancreatic islet progenitor cells. Functional analysis of the c-Kit receptor tyrosine kinase provides evidence that phosphorylation of c-Kit receptor may be involved in mediating early beta-cell differentiation and survival.     

Effect of STI-571 (imatinib mesylate) in combination with retinoic acid and gamma-irradiation on viability of neuroblastoma cells

Neuroblastoma (NB) expresses the tyrosine kinase receptors c-Kit, PDGFR-alpha and -beta-targets for STI-571.We investigated a possible combination therapy of STI-571 with retinoic acid (RA) and gamma-irradiation on NB cell viability in vitro. Expression of tyrosine kinase receptors and their ligands was examined in 6 NB cell lines by RT-PCR and FACS. The effect on cell viability was determined by MTT assay. Cell viability of all 6 NB cell lines was significantly inhibited after treatment with 20 microM STI-571 for 72h, two cell lines responding already to 10 microM. Cell lines responded irrespective of their mRNA status or cell surface expression of c-Kit, PDGFR-alpha and -beta. Co-incubation with 9-cis RA sensitized cells to the inhibitory effects of STI-571. However, pre-treatment with 9-cis RA resulted in resistance of NB cell lines to STI-571 and gamma-irradiation. Treatment of NB with STI-571 in combination with 9-cis RA might be a therapeutic strategy for patients in consolidation therapy who have completed gamma-irradiation therapy.     

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa.Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.     

Tyrosine kinase inhibition: Ligand binding and conformational change in c-Kit and c-Abl

The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamily often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.