High-resolution crystal structure of apolipoprotein(a) kringle IV type 7: insights into ligand binding

Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV(1) to KIV(10)) followed by sequences similar to the kringle V and protease domains of plasminogen. The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs. KIV(10) represents the high-affinity lysine-binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV(5)-KIV(8) and has been directly demonstrated in KIV(7). The present study describes the first crystal structure of apo(a) KIV(7), refined to a resolution of 1.45 A, representing the highest resolution for a kringle structure determined to date. A critical substitution of Tyr-62 in KIV(7) for the corresponding Phe-62 residue in KIV(10), in conjunction with the presence of Arg-35 in KIV(7), results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg-35, Tyr-62, Asp-54) and a peripheral tyrosine residue (Tyr-40). These interactions restrain the flexibility of key LBS residues (Arg-35, Asp-54) and, in turn, reduce their adaptability in accommodating lysine and its analogs. Steric hindrance involving Tyr-62, as well as the elimination of critical ligand-stabilizing interactions within the LBS are also consequences of this interaction network. Thus, these subtle yet critical structural features are responsible for the weak lysine-binding affinity exhibited by KIV(7) relative to that of KIV(10).     

Cancer/testis antigen cancer-associated gene (CAGE) promotes motility of cancer cells through activation of focal adhesion kinase (FAK)

The cancer-associated gene (CAGE) is a novel cancer/testis antigen. Over-expression of it increased phosphorylation of focal adhesion kinase (FAK) and enhanced motility of SNU387 cells. Focal adhesion, kinase-related non-kinase (FRNK), an endogenous inhibitor of FAK, was significantly suppressed. This suggests that CAGE-promoted motility requires FAK. The inhibition of Rho-Associated coiled-coil-containing protein kinase (ROCK), an activator of FAK, also suppressed CAGE-promoted motility.     

脂蛋白（a）的研究进展──生物化学与分子生物学方面

脂蛋白（ａ）［Ｌｐ（ａ）］是一种与低密度脂蛋白类似的脂蛋白,其特殊处在于多含一种具明显多态性的糖蛋白──载脂蛋白（ａ）［Ａｐｏ（ａ）］。Ａｐｏ（ａ）与血浆纤溶酶原有很大同源性。Ｌｐ（ａ）由肝合成,其分解可能主要经非特异途径。Ａｐｏ（ａ）大小及血浆Ｌｐ（ａ）浓度主要由Ａｐｏ（ａ）基因决定。Ｌｐ（ａ）易沉积于血管壁,并可促进平滑肌细胞生长及抑制纤维蛋白溶解,这可能是其促动脉粥样硬化和血栓形成的机理所在。Ｌｐ（ａ）的生理功能尚不清楚。     

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.     

GHRH antagonist inhibits focal adhesion kinase (FAK) and decreases expression of vascular endothelial growth factor (VEGF) in human lung cancer cells in vitro

Lung cancers which show increased vascularization and high microvessel density are considered highly metastatic and with poor prognosis. Growth hormone releasing hormone (GHRH) antagonists are anticancer agents without adverse events in lung cancer tumor models. In the present study we investigated the in vitro effect of GHRH antagonist, MZ-5-156, on focal adhesion kinase (FAK) activity, on the expression of MMP-2 and MMP-9 metalloproteinases, as well as on vascular endothelial growth factor (VEGF) levels in A549 non-small cell lung (NSCLC) cancer cells and H727 bronchial carcinoid cells. We demonstrate for the first time that GHRH antagonist, MZ-5-156, inhibits FAK signaling in lung cancer cells and decreases the expression of additional factors involved in angiogenesis and invasion. In contrast, GHRH itself counteracted these effects. Our study contributes to the further understanding of the processes which govern the mechanism of action of GHRH and its antagonists in cancers.     

人血浆载脂蛋白（a）的分离及纯化

本文报道从人血浆脂蛋白Ｌｐ（ａ）中,分离纯化载脂蛋白（ａ）。收集富含Ｌｐ（ａ）的混合血浆,超离心,获密度１．０５ｇ／ｍｌ至１．０８ｇ／ｍｌ的粗制Ｌｐ（ａ）,经过Ｂｉｏ－Ｇｅｌ Ａ５ｍ层析后,证明纯化后的Ｌｐ（ａ）仅与ａｐｏ（ａ）抗血清反应,经ＤＴＴ处理过的Ｌｐ（ａ）,在琼脂糖电泳中的泳动率由胶β位移到β位,在印迹免疫反应中,对ａｐｏ（ａ）的抗血清反应依然显示在前β位,ＳＤＳ聚丙烯凝胶电脉的迁移率慢     

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.     

RNA interference-mediated silencing of focal adhesion kinase inhibits growth of human malignant glioma xenograft in nude mice

FAK (focal adhesion kinase), which plays a pivotal role in mediating cell proliferation, survival and migration, is frequently overexpressed in human malignant glioma. The expression of FAK increases with the advance of tumour grade and stage. Based on these observations, we hypothesized that attenuation of FAK expression may have inhibitory effects on the growth of malignant glioma. In the present study, human glioma cell line U251 was transfected with plasmids containing U6 promoter-driven shRNAs (small-hairpin RNAs) against human FAK using cationic liposome. The effects of FAK knockdown in U251 cells in vitro were analysed by using flow cytometry and PI (propidium iodide)-staining assays. Based on the encouraging in vitro results with FAK silencing, plasmids encoding FAK-targeted shRNA were encapsulated by DOTAP (dioleoyltrimethylammonium propane):Chol (cholesterol) cationic liposome and injected via tail vein to evaluate its therapeutic efficiency on suppressing tumour growth in a human glioma xenograft model. PCNA (proliferating-cell nuclear antigen), CD34 immunostaining and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay were used to assess the changes in tumour angiogenesis, apoptosis and proliferation respectively. The results indicated that DOTAP:Chol cationic liposome could deliver therapeutic plasmids systemically to tumour xenografts, resulting in suppression of tumour growth. Treatment with plasmid encoding FAK-targeted shRNA reduced mean tumour volume by approx. 70% compared with control groups (P<0.05), accompanied with angiogenesis inhibition (P<0.05), tumour cell proliferation suppression (P<0.05) and apoptosis induction (P<0.05). Taken together, our results demonstrated that shRNA-mediated silencing of FAK might be a potential therapeutic approach against human malignant glioma.     

Comparing mechano-transduction in fibroblasts deficient of focal adhesion proteins

Mechano-transduction was studied in wildtype and focal adhesion (FA) protein-deficient mouse embryonic fibroblasts (MEFs). Using a cell stretcher, we determined the effect of stretch on cell morphology, apoptosis, and phosphorylation of ERK^1/2. After 20% cyclic, uniaxial stretch, FA-deficient MEFs showed morphological changes and levels of apoptosis of the order: focal adhesion kinase > p130Cas > vinculin compared to wildtype cells. ERK^1/2 phosphorylation peaked in wildtype cells at around 10 min, and in all FA-deficient cells at around 5 min. The relative change in strain energy of FA-deficient cells compared to wildtype cells was of the order: vinculin > FAK > p130Cas. Taken together, FAK and p130Cas are more important in the stretch-mediated downstream signaling and cell survival pathway, while vinculin is more critical in maintaining cell contractility.     

Dynamin II interacts with syndecan-4, a regulator of focal adhesion and stress-fiber formation

Dynamin is a large mechanochemical GTPase that has been implicated in vesicle formation in multiple cellular compartments. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. To identify potential intracellular proteins that interact with the PH domain of dynamin II, we carried out a yeast two-hybrid screen in which the PH domain of dynamin II was used as bait. The cell surface heparan sulfate proteoglycan syndecan-4 that acts in conjunction with integrins to promote the formation of actin stress fibers and focal adhesions was isolated as a binding partner for the PH domain of dynamin II. In vitro binding assays, immunoprecipitation, and confocal microscopy analysis confirmed the association of dynamin II with syndecan-4. Most dramatic finding of our study is that the cytoplasmic distribution of dynamin II and syndecan-4 changes in fibroblasts that have been stimulated to form the focal adhesions and stress fibers with LPA. In quiescent cells, dynamin II is evenly distributed in the cytoplasm and colocalizes with syndecan-4 near the nucleus. Upon treatment with LPA to induce focal adhesions and stress-fiber formation, dynamin II becomes markedly associated with syndecan-4 at focal adhesion sites. We further established the colocalization of syndecan-4 and dynamin with paxillin and actin as marker proteins for focal adhesions and stress fibers, respectively. All of these results suggest that the interaction between dynamin II and syndecan-4 is important in mediating focal adhesion and stress-fiber formation.     

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12 h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.     

K-252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells

Abstract: The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.     

A novel inhibitor, 16-hydroxy-cleroda-3,13-dien-16,15-olide,blocks the autophosphorylation site of focal adhesion kinase (Y397) by molecular docking

Background

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine plays an important role in a number of cell signaling pathways, including cell migration, proliferation, and cell survival. This study was aimed to identify novel and specific inhibitors from natural compounds via molecular docking of FAK (Y397).

Methods

The 3D structure of FAK (PDB ID: 2AL6) was used for docking 109 natural compounds. Based on high affinity and energy interaction, four of ten candidate compounds, 16-hydroxy-cleroda-3,13-dien-16,15-olide (HCD), curcumin, quercetin, and catechin hydrate, were hit, and the inhibitory activity against FAK was validated in these compounds in C6 glioma and N18 neuroblastoma cell lines.

Results

HCD showed a potential effect on cell viability by MTT assay and cell arrest in the G0–G1 phase, and a TUNEL assay confirmed further apoptosis. Treatment with HCD decreased anti-apoptotic proteins and increased pro-apoptotic proteins. Atomic force microscopy data depicted that the formation of filopodia on the intracellular surface decreased in treated cells compared with the control. Zymography showed that HCD inhibited the activity of MMP-2 and MMP-9. The protein levels of FAK, pFAK, Rac1 and Cdc42, which are the key regulators for the formation of filopodia, were decreased. Additionally, HCD regulated the expression of epithelial mesenchymal transition proteins.

Conclusions

HCD effectively interacted at the autophosphorylation site of FAK and interaction analysis indicated an H-bond with the Arg 86 and Arg 125 residues.

General significance

This study suggests that HCD could be a potential inhibitor of FAK and could be used for anti-tumorigenesis and anti-metastasis treatments.     

Inhibitory role of focal adhesion kinase on anoikis in the lung cancer cell A549

Resistance to anoikis is a characteristic of malignant cells with increased tumorigenesis and metastasis. Altered FAK activity has been strongly implicated in the development, growth, progression, and metastasis of human cancers, but the mechanism of FAK in regulating anoikis is unknown. In this study, the resistance anoikis role of FAK and its downstream mediators was evaluated in the human lung cancer cell line A549. It has been shown that down regulation of FAK stimulates the apoptosis of cells and the down-regulation of p-ERK, p-PI3K, p-Src, and p-p38. Furthermore, in detached A549 cells, increased FAK phosphorylations (Tyr397, Tyr861, Tyr925) were detected in a time-dependent manner, and the specific inhibitors of MEK1, PI3K, and Src (PD98059, LY294002, and PP2) partly abolished the resistance to the anoikis characteristic of cancer cells. Altogether, our data suggested that Src is involved in the progress of detachment-induced FAK activation in lung tumor cells. PI3K/AKT, MAPK-ERK, and perhaps MAPK-p38 but not MAPK-JNK, appear to be the key downstream effectors of FAK in mediating cell survival. The increased FAK activity upon cell detachment may contribute to the metastasis potential of malignant tumors.     

Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a)

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.     

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)₄K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)₄K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)₄K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)₄K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Pharmacological inhibition of poly(ADP-ribose) polymerase inhibits angiogenesis

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which plays an important role in regulating cell death and cellular responses to DNA repair. Pharmacological inhibitors of PARP are being considered as treatment for cancer both in monotherapy as well as in combination with chemotherapeutic agents and radiation, and were also reported to be protective against untoward effects exerted by certain anticancer drugs. Here we show that pharmacological inhibition of PARP with 3-aminobenzamide or PJ-34 dose-dependently reduces VEGF-induced proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro. These results suggest that treatment with PARP inhibitors may exert additional benefits in various cancers and retinopathies by decreasing angiogenesis.     

6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.     

Role of c-Src and focal adhesion kinase in progression and metastasis of estrogen receptor-positive breast cancer

The non-receptor tyrosine kinases c-Src and focal adhesion kinase (Fak) mediate signal transduction pathways that regulate cell proliferation, survival, invasion, and metastasis. Here, we investigated whether c-Src and Fak are activated during progression of hormone-dependent breast cancer. Maximally active c-Src was overexpressed in a subset of tamoxifen-resistant variants and in metastases of recurrent hormone-treated breast cancer. Active Fak was also frequently observed in these tumors. We also show that estrogen receptor (ER) can bind to Fak and that estrogen can modulate Fak autophosphorylation supporting a cross-talk between these two pathways. Inhibition of c-Src activity blocked proliferation of all tamoxifen-resistant variants, suggesting that inhibitors of c-Src-Fak activity may delay or prevent progression and metastasis of ER-positive tumors. These studies also raise the possibility that fully active forms of c-Src and Fak in breast tumors may be biomarkers to predict tamoxifen resistance and/or risk of recurrence in ER-positive breast cancer.