Targeting multiple signal transduction pathways through inhibition of Hsp90

The multichaperone heat shock protein (Hsp) 90 complex mediates the maturation and stability of a variety of proteins, many of which are crucial in oncogenesis, including epidermal growth factor receptor (EGF-R), Her-2, AKT, Raf, p53, and cdk4. These proteins are referred to as "clients" of Hsp90. Under unstressed conditions these proteins form complexes with Hsp90 and the cochaperones to attain their active conformations or enhance stability. Inhibition of Hsp90 function disrupts the complex and leads to degradation of client proteins in a proteasome-dependent manner. This results in simultaneous interruption of many signal transduction pathways pivotal to tumor progression and survival. Based on the unique role of the Hsp90 complex, extensive effort has been made in identifying Hsp90 inhibitors. Several compounds have been shown to inhibit Hsp90 in vitro and in vivo and the most advanced, 17-allylamino-17-demethoxygeldanamycin (AAG), is in phase I/II clinical trials. Recent findings with 17-AAG indicate that tumor cells utilize Hsp90 quite differently from normal cells, explaining the selectivity of the drug and suggesting a central role of Hsp90 in malignant progression. Thus these small molecule inhibitors have proved not only to be of great value in identifying new Hsp90 client proteins and in understanding the biology of Hsp90 but are also promising therapeutics in a variety of tumors.     

Designed hybrid TPR peptide targeting Hsp90 as a novel anticancer agent

Background  

Despite an ever-improving understanding of the molecular biology of cancer, the treatment of most cancers has not changed dramatically in the past three decades and drugs that do not discriminate between tumor cells and normal tissues remain the mainstays of anticancer therapy. Since Hsp90 is typically involved in cell proliferation and survival, this is thought to play a key role in cancer, and Hsp90 has attracted considerable interest in recent years as a potential therapeutic target.     

Hsp90aa1: a novel target gene of miR-1 in cardiac ischemia / reperfusion injury

<正>The present study aimed to investigate the expression and potential target of microRNA-1(miR-1)in the myocardium of a rat model of ischemia/reperfusion(I/R).The apoptosis of cardiomyocytes in the ischemic rat myocardium increased on day 1,and then decreased on post-I/R day 3 and day 7.While miR-1 was decreased,the reduced heat shock protein 90(Hsp90)aa1 protein,not     

Fibroblast activation protein (FAP) as a possible target of an antitumor strategy

This review was devoted to the use of the universal component of tumoral stroma (fibroblast activation protein, FAP) as a target of the universal tumor therapy. A tumor is a coevolution system, which includes a microenvironment or reactive stroma differing from the normal tissue in its phenotypic and genotypic features. Cancer-associated fibroblasts (CAFs), which contain the typical marker FAP (serine proteinase with the enzymatic activity of dipeptidyl peptidase and endopeptidase), are important elements of the tumor microenvironment. According to the literature, more than 90% of tumors contain FAP-positive activated fibroblasts. FAP is virtually absent in normal tissues, but it is present in the embryonic and tumor tissues, which makes it a selective and universal target. In this work, basic approaches to affecting CAFs using FAP as a target are discussed. The use of FAP as a target provides an important advantage: its proteolytic activity can be used along with the protein-targeted agents. The main areas of development in the therapeutic use of FAP are discussed in this work.     

Mammalian arylsulfatase A functions as a novel component of the extracellular matrix

Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2β1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes.     

A novel use of genipin-fixed gelatin as extracellular matrix for peripheral nerve regeneration

Application of combining herbal medicine and biomedical material science to nerve regeneration is a new approach. In this study, we describe a novel use of purified genipin, which can be extracted from Gardenia jasminoides Ellis, fixing the gelatin to be an extracellular matrix for peripheral nerve regeneration. A 10-mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with either the genipin-fixed gelatin or collagen gel. Silicone rubber tubes filled with saline were used as controls. Six weeks after implantation, regeneration across the nerve gaps occurred in 80 and 90% of the animals from the groups of genipin-fixed gelatin and collagen, respectively, whereas only 30% in the control group. Large numbers of myelinated axons were also seen in the genipin-fixed gelatin (5104 +/- 3278) and the collagen groups (8063 +/- 1807). These findings indicated that the genipin-fixed gelatin could be an acceptable extracellular matrix for nerve regeneration.     

Sialyloligosaccharide chains of laminin as an extracellular matrix target for S fimbriae of Escherichia coli.

S fimbriae purified from recombinant Escherichia coli HB101(pANN801-13) bound strongly to extracellular matrices of cultured endothelial and epithelial cells; only poor binding was seen with the fimbriae purified from the sfaS mutant strain HB101(pANN801-1321). E. coli HB101(pANN801-13) adhered strongly to laminin immobilized on glass; no adhesion was seen to type I, III, IV, or V collagen. Strain HB101(pANN801-1321) failed to adhere to any of the target proteins. Adhesion to laminin of strain HB101(pANN801-13) was inhibited by sialyl-alpha-2,3-lactose as well as by periodate oxidation and neuraminidase treatment of laminin. In Western blotting, the purified S fimbriae recognized more strongly the A chain than the B chains of laminin.     

Antiviral activity and RNA polymerase degradation following Hsp90 inhibition in a range of negative strand viruses

We have analyzed the effectiveness of Hsp90 inhibitors in blocking the replication of negative-strand RNA viruses. In cells infected with the prototype negative strand virus vesicular stomatitis virus (VSV), inhibiting Hsp90 activity reduced viral replication in cells infected at both high and low multiplicities of infection. This inhibition was observed using two Hsp90 inhibitors geldanamycin and radicicol. Silencing of Hsp90 expression using siRNA also reduced viral replication. Hsp90 inhibition changed the half-life of newly synthesized L protein (the large subunit of the VSV polymerase) from >1 h to less than 20 min without affecting the stability of other VSV proteins. Both the inhibition of viral replication and the destabilization of the viral L protein were seen when either geldanamycin or radicicol was added to cells infected with paramyxoviruses SV5, HPIV-2, HPIV-3, or SV41, or to cells infected with the La Crosse bunyavirus. Based on these results, we propose that Hsp90 is a host factor that is important for the replication of many negative strand viruses.     

Toxin-induced calcium oscillations: a novel strategy to affect gene regulation in target cells

Although the mucosal linings continuously are exposed to microbes, the microbes rarely induce disease. This is because mucosal surfaces are protected by a first line of host defence termed the innate immunity system. The innate immune response is an outcome of the complex interplay between microbes and host target cells, and leads to the activation of inflammatory processes. Although inflammation is essential for clearing out infectious agents, it can also be harmful to the host and is therefore subjected to control at multiple levels. We recently discovered that alpha-haemolysin, a toxin secreted by uropathogenic E. coli induces constant, low-frequency Ca2+ oscillations in renal epithelial cells (Uhlén et al., Nature 405, 694-696 (2000)). Ca2+ oscillation at a specific periodicity of 12 min was found to affect gene expression in target epithelial cells, as the proinflammatory cytokine IL-6 and chemokine IL-8 were specifically induced by alpha-haemolysin-induced Ca2+ oscillations. This demonstrates a novel feature of bacterial toxin effects on host target cells: as inducers of second messenger responses which fine-tune gene expression in target epithelial cells into pathways leading to e. g. a pro-inflammatory response.     

Upregulation of bone cell differentiation through immobilization within a synthetic extracellular matrix

There is a need for new therapeutic strategies to treat bone defects caused by trauma, disease or tissue loss. Injectable systems for cell transplantation have the advantage of allowing the use of minimally invasive surgical procedures, and thus for less discomfort to patients. In the present study, it is hypothesized that Arg-Gly-Asp (RGD)-coupled in a binary (low and high molecular weight) injectable alginate composition is able to influence bone cell differentiation in a three-dimensional (3D) structure. Viability, metabolic activity, cytoskeleton organization, ultrastructure and differentiation (alkaline phosphatase (ALP), von Kossa, alizarin red stainings and osteocalcin quantification) of immobilized cells were assessed. Cells within RGD-modified alginate microspheres were able to establish more interactions with the synthetic extracellular matrix as visualized by confocal laser scanning microscope and transmission electron microscopy imaging, and presented a much higher level of differentiation (more intense ALP and mineralization stainings and higher levels of osteocalcin secretion) when compared to cells immobilized within unmodified alginate microspheres. These findings demonstrate that peptides covalently coupled to alginate were efficient in influencing cell behavior within this 3D system, and may provide adequate preparation of osteoblasts for cell transplantation.     

Dermatopontin, a novel player in the biology of the extracellular matrix

Dermatopontin is a widely distributed small molecular weight protein in the extracellular matrix (ECM) and today its homologues are known in five mammals and several invertebrates. The structures of these homologues are relatively well conserved among the species. In the skin, dermatopontin is located mainly on the surface of the collagen fibers. It is found in the conditioned medium and also in the cytoplasm of cultured fibroblasts. Early studies focused on ECM assembly (collagen fibrillogenesis) and interactions (with the proteoglycan decorin). Subsequently, a targeted disruption of dermatopontin resulted in a phenotype similar to Ehlers-Danlos syndrome. In addition, a cell adhesion activity of this protein for dermal fibroblasts and several other cells was found, and this activity might suggest this protein's involvement in wound healing. The expression of dermatopontin around an infarct zone of experimental myocardial infarction may support this possibility. In invertebrates, dermatopontin homologues act mainly as adhesion/agglutination molecules. In addition, we found that transforming growth factor-beta1 interacts with dermatopontin and the function of this cytokine is modified by dermatopontin. Recently, the involvement of this protein in cell proliferation has been indicated. In this review we describe the reported functions of this protein and speculate on the multiple roles of this largely uncharacterized matrix molecule.     

Resolution of inflammation as a novel chemopreventive strategy

Acute inflammation, a physiologic response to protect cells from microbial infection and other noxious stimuli, is automatically terminated by endogenous anti-inflammatory and pro-resolving mediators to restore homeostatic conditions. However, if timely resolution of inflammation is failed, inflammation persists and can progress to a chronic inflammation which has long been thought as a predisposing factor to carcinogenesis. Excessive and pathologic inflammation causes DNA damage, genomic instability, epigenetic dysregulation, and alteration of intracellular signaling, all of which are involved in neoplastic transformation. To prevent chronic inflammation and resulting inflammation-promoted cancer development, understanding the process that resolves inflammation is essential. Resolution of inflammation is an active coordinated process regulated by distinct anti-inflammatory and pro-resolving endogenous lipid mediators, such as resolvins and lipoxins. The role of pro-inflammatory signaling in carcinogenesis has become more and more evident and well characterized, but the potential role of pro-resolving mediators in cancer prevention remains still elusive. In searching for an efficacious way to prevent chronic inflammation-associated cancer, the pro-resolving signal transduction pathways and their regulators should be unraveled.     

Enhancement of complement-induced cell lysis: a novel mechanism for the anticancer effects of Hsp90 inhibitors

Molecular chaperones (heat shock proteins, Hsp-s) play a pleiotropic role in immunological functions. Hsp-s participate in the presentation of peptide antigens, folding of several immunologically important proteins, such as the MHC, and in the maintenance of the activation-competent conformation of key signaling molecules (mostly serine/threonine and tyrosine kinases) of B and T cells activation. The most abundant cytoplasmic chaperone, Hsp90, is in the center of these processes. In recent years Hsp90 inhibitors emerged as very promising anticancer agents. Not surprisingly, Hsp90 inhibitors behave as immunosuppressants, and also cause an induction of superoxide production. Here we extend our previous data by showing the enhancement of complement-induced lysis of several types of tumor cells after Hsp90 inhibition. This novel mechanism may significantly contribute to the anticancer effects of Hsp90 inhibitors in vivo.     

以抑制肿瘤新生血管为靶点的抗肿瘤药物研究进展

恶性肿瘤的生长和转移与肿瘤区域的血管密切相关,肿瘤区域的新生毛细血管是肿瘤赖以生长和生存的物质基础,肿瘤细胞需要新生血管为迅速生长的肿瘤提供营养物质和排出废物。早在七十年代初就有人提出把抑制肿瘤血管形成作为肿瘤治疗的一种途径,以后这种以肿瘤血管为靶标的治疗策     

CrkL is a novel target of Sprouty2 in fibroblast growth factor signaling

Sprouty, an inhibitor of receptor tyrosine kinase signaling, plays an important role in the regulation of a wide variety of biological processes. Although it is established that the Sprouty inhibitory activity is induced by tyrosine phosphorylation in response to stimuli, its action mechanisms have not been fully elucidated. Here, we report identification of a novel target of Sprouty. We find that Sprouty1 and Sprouty2 bind to the adaptor protein CrkL in a stimulus‐dependent manner. Biochemical analyses show that the binding requires tyrosine phosphorylation of Sprouty and that both the SH2 domain and the N‐terminal SH3 domain of CrkL are necessary for the binding. In fibroblast growth factor‐stimulated NIH3T3 cells, CrkL binding to Sprouty2 occurs concomitantly with tyrosine phosphorylation of Sprouty2, which occurs slowly but is sustained. Importantly, our results show that tyrosine‐phosphorylated Sprouty2 suppresses Rap1 activation. These results taken together indicate that Sprouty2 acts as an inhibitor of CrkL‐Rap1 signaling.     

牵张力下mTOR/MMPs信号通路可促进间充质干细胞-成骨细胞系的迁移

背景：目前对于牵张成骨促进间充质干细胞-成骨细胞系迁移的机制尚未明了,亟需基于这一机制的新的治疗方案,以提高牵张成骨临床疗效。 目的：观察牵张应力下间充质干细胞-成骨细胞系mTOR信号通路相关基因的表达以及细胞迁移能力。 方法：将12只SD大鼠随机分为2组：牵张组大鼠(n=6)进行右侧下颌骨牵张成骨,非牵张组大鼠(n=6)行同侧下颌骨截断后安置牵张器但不牵张,术后15 d进行免疫组织化学染色分析新生骨痂中p-mTOR表达变化。另外,对培养的健康SD大鼠下颌骨间充质干细胞进行体外牵张试验,施加(6%,4 h)的静息牵张力,对照组不施加牵张力,实时定量PCR、划痕实验等方法检测间充质干细胞-成骨细胞系mTOR信号通路相关基因的表达及细胞迁移能力的变化。 结果与结论：牵张组大鼠新生骨痂中的p-mTOR表达高于非牵张组(P < 0.05)。与非牵张组(0%,4 h)相比,牵张组(6%,4 h)间充质干细胞-成骨细胞系的 mTOR、Raptor、p70S6K、MMP-2、MMP-9、MMP-13基因表达升高,间充质干细胞-成骨细胞系迁移距离更远(P < 0.05)。结果提示牵张应力可能通过激活间充质干细胞-成骨细胞系的mTOR/MMPs信号通路,促进其迁移。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

姜黄素抑制STAT3磷酸化对乳腺癌细胞增殖及Hsp90α表达的影响

目的探讨姜黄素对乳腺癌细胞增殖和Hsp90α表达的影响及其可能机制。方法以乳腺癌细胞株MCF-7为材料,分别用不同浓度的姜黄素(0、5、10、15、20μmol/L)处理细胞36 h,或以10μmol/L姜黄素分别处理细胞12、24、36、48 h,用MTT法检测细胞生长抑制率,分别采用实时定量PCR和Western blot法检测Hsp90αmRNA、Hsp90α及磷酸化STAT3(p-STAT3)蛋白的表达。结果姜黄素处理MCF-7细胞后,总STAT3蛋白的表达无明显差异,p-STAT3表达明显下调;同时,姜黄素处理组MCF-7细胞代谢MTT的能力降低,细胞生长的抑制率明显上升,Hsp90αmRNA和蛋白质的表达下调,且该效应呈浓度和时间依赖性(P0.05)。结论姜黄素可能通过STAT3信号途径,调控Hsp90α的表达,进而干预乳腺癌细胞的增殖。