Secreted human apolipoprotein(a) kringle IV-10 and kringle V inhibit angiogenesis and xenografted tumor growth

Abstract Angiogenesis plays an important role in normal physiology of blood vessel growth, but can contribute to the pathogenesis of diseases, such as cancer. A new anti-angiogenic recombinant kringle protein, composed of the fused domains of human apolipoprotein(a) carboxyl-terminal kringle IV-10 and kringle V, was expressed in Pichia pastoris and human colorectal carcinoma (HCT 116) cells to investigate its influence on angiogenesis and tumor growth. The mature recombinant protein exhibited the characteristic features of kringle-containing proteins (glycosylation and disulfide bond formation) and, when added to cultures of human umbilical vein endothelial cell, resulted in a 31% decrease in proliferation relative to untreated controls (p<0.05). The neo-angiogenesis was diminished by 63% in chick embryos treated with 10 mug recombinant protein compared with 7% for phosphate buffer solution-treated embryos (p<0.01). Transfection of a kringle IV-10-kringle V fusion protein construct into HCT 116 cells decreased tumorigenesis and inhibited tumor growth in vivo without affecting tumor cell proliferation. HCT 116 cells that expressed recombinant protein displayed a much lower relative growth ratio of 8% (p<0.01) against the control tumor cells. From these results, we conclude that human apolipoprotein(a) carboxyl-terminal kringle IV-10-kringle V fusion protein is an effective inhibitor of angiogenesis and angiogenesis-dependent tumor growth.     

Production of the kringle fragments of human apolipoprotein(a) by continuous lactose induction strategy

A novel lactose induction strategy for the production of rhLK68, the kringle fragments of human apolipoprotein(a) (apo(a)) as a novel anti-angiogenic protein, was investigated. A scale-up of the production was accompanied by a decrease in expression level, and severe aggregation occurred during the solubilization of rhLK68 from the inclusion body during a conventional single introduction of lactose. To overcome this problem, a continuous induction strategy was applied where lactose was mixed with glycerol and fed continuously in a dissolved oxygen (DO)-stat manner. With the sub-optimal feed medium consisted of 1:50 of lactose/glycerol (w/w), the expression level reached 16% of the total cellular protein, which was 1.6-fold higher than that obtained from the conventional lactose induction. Moreover, the solubilization yield of rhLK68 from the inclusion body increased from 30 +/- 5 to 85 +/- 3% compared to the conventional single introduction of lactose. This result suggests that the continuous lactose induction strategy beneficially influenced the expression level of rhLK68 and the quality of its inclusion body.     

Expression of human apolipoprotein(a) kringles in colon cancer cells suppresses angiogenesis-dependent tumor growth and peritoneal dissemination

BACKGROUND: Anti-angiogenesis therapy has been regarded as a promising treatment of cancer based on the fact that most tumors and their metastasis are angiogenesis-dependent. Gene therapy can potentially expand the horizons of tumor angiogenesis therapy by virtue of its ability to produce high concentrations of therapeutic agents in a local area for a sustained period. The present study was performed to evaluate the therapeutic potential of gene therapy for the treatment of cancer and metastasis. METHODS: The murine colon carcinoma cell line CT26 was manipulated ex vivo to express an anti-angiogenic molecule, LK68, consisting of human apolipoprotein(a) kringle domains, KIV(9)-KIV(10)-KV, using retrovirus-mediated gene transfer. Its effects on colon tumor growth and metastasis were evaluated in experimental animal models established by injecting LK68-expressing and control CT26 cells subcutaneously or into the peritoneal cavity of BALB/c mice, respectively. RESULTS: Expression of LK68 significantly suppressed colon tumor growth in mice, but did not influence the growth of tumor cells in vitro. Immunohistochemical analysis of tumor tissues revealed a significant reduction in microvessel density in LK68-expressing tumors. Thus, the suppression of tumor growth appears to result mainly from inhibition of tumor angiogenesis. This decrease in vessel density is correlated with a notable increase in tumor cell apoptosis in vivo, but has no influence on proliferation. Moreover, expression of LK68 prevents peritoneal dissemination, and consequently improves overall host survival. CONCLUSIONS: These results collectively indicate that a gene therapy strategy using LK68 cDNA is useful for the treatment for both colon tumor growth and peritoneal dissemination.     

Solution structure of human apolipoprotein(a) kringle IV type 6.

The structure of apo(a) KIVT6 was investigated by two- and three-dimensional homo- and heteronuclear NMR spectroscopy. The solution structure of apo(a) KIVT6 contains only a small amount of regular secondary structure elements, comprising a short piece of antiparallel beta-sheet formed by residues Trp62-Tyr64 and Trp72-Tyr74, a short piece of parallel beta-sheet formed by the residues Cys1-Tyr2 and Thr78-Gln79, and a small 3(10)-helix within residues Thr38-Tyr40. The backbone as well as the side chains are arranged in a way similar to those of apo(a) KIVT7, apo(a) KIVT10, and plasminogen K4. We determined additionally the K(d) value of 0.31 +/- 0.04 mM for the binding of epsilon-aminocaproic acid (EACA) to apo(a) KIVT6 and mapped the binding region on apo(a) KIVT6 by means of chemical shift perturbation. This lysine binding activity, which was reported to occur within apo(a) KIVT5-8, is functionally different from the lysine binding activity found for apo(a) KIVT10.     

Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases

Apolipoprotein(a) [apo(a)] contains the largest numbers of kringle domains identified to date. Of these, apo(a) kringle V shows significant sequence homology with plasminogen kringle 5, which is reported to be a potent angiogenesis inhibitor. To determine the effects of apo(a) kringle V on angiogenesis, it was expressed as a soluble protein (termed rhLK8) in Pichia pastoris and its in vitro and in vivo anti-angiogenic properties were examined. rhLK8 inhibited the migration of human umbilical vein endothelial cells in vitro in a dose-dependent manner. This function was associated with the down-regulation of the activation of focal adhesion kinase and the inhibition of the consequent formation of actin stress fibers/focal adhesions. rhLK8 also inhibited new capillary formation in vivo, as assessed by the chick chorioallantoic membrane assay and the Matrigel plug assay. These results indicate that rhLK8 may be an effective angiogenesis inhibitor both in vitro and in vivo.     

Inhibition of tumor cell growth by a human B-cell line

An Adenocarcinoma cell line (Breast-M) and an Epstein-Barr virus (EBV)-infected B-cell line (Hairy-BM) were established from breast tumor tissue. The Hairy-BM was CD20⁺, CD25 (Tac)+ and surface immunoglobulin (sIg)+. Hairy-BM suppressed the in vitro proliferation of Breast-M in a time and a dose-dependent manner. The suppression was also found in 5 different human tumor targets showing tumor-Hairy-BM binding, but not; in 2 murine tumor targets showing no significant tumor-Hairy-BM binding. Lytic activity of Hairy-BM was found only against Breast-M.Abbreviations sIg Surface immunoglobulin - CTL Cytotoxic T-cells - NK Natural killer - IL2 Interleukin 2 - LAK Lymphokine activated killer - CSN Culture supernatant - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoolium bromide - PCR Polymerase chain reaction - TIL Tumor-infiltrating lymphocytes - HCL Hairy cell leukemia - TNF Tumor necrosis factor     

Expression and purification of kringle 4-type 2 of human apolipoprotein (a) in Escherichia coli.

The most frequently occurring kringle 4 domain of human apolipoprotein (a), Kringle 4-subtype 2 (K4(2)), was expressed as a fusion protein with the maltose binding protein in Escherichia coli using the "tac" promoter. Although the fusion protein was expressed without a signal sequence, 25% was secreted into the periplasmic space; the remainder was found associated with the soluble cytosolic fraction. The fusion protein was readily isolated from whole cell lysate by amylose agarose affinity chromatography. Although a factor Xa cleavage site was engineered into the fusion protein, it was found that release of the K4(2) protein was most conveniently achieved by proteolysis with subtilisin A. The cleavage product produced in this way was shown to be intact K4(2) with only the first three amino acid residues of the leading flanking peptide missing, as judged by N-terminal sequence analysis. K4(2) was isolated from the hydrolysate by FPLC on a Mono-Q column with a yield of 170 +/- 30 micrograms/g wet cells. The resulting protein was monomeric in phosphate-buffered saline as judged by size-exclusion chromatography and appeared to be folded as shown by spectroscopic and immunological assays. The recombinant K4(2) did not bind to either lysine- or proline-Sepharose, suggesting that the ligand binding activities of lipoprotein (a) may reside in the other kringle domains of apolipoprotein (a).     

Lysine-phosphatidylcholine adducts in kringle V impart unique immunological and potential pro-inflammatory properties to human apolipoprotein(a)

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.     

A novel peptide derived from human apolipoprotein E is an inhibitor of tumor growth and ocular angiogenesis

Angiogenesis is a hallmark of tumor development and metastasis and now a validated target for cancer treatment. We previously reported that a novel dimer peptide (apoEdp) derived from the receptor binding region of human apolipoprotein E (apoE) inhibits virus-induced angiogenesis. However, its role in tumor anti-angiogenesis is unknown. This study demonstrates that apoEdp has anti-angiogenic property in vivo through reduction of tumor growth in a mouse model and ocular angiogenesis in a rabbit eye model. Our in vitro studies show that apoEdp inhibits human umbilical vein endothelial cell proliferation, migration, invasion and capillary tube formation. We document that apoEdp inhibits vascular endothelial growth factor-induced Flk-1 activation as well as downstream signaling pathways that involve c-Src, Akt, eNOS, FAK, and ERK1/2. These in vitro data suggest potential sites of the apoE dipeptide inhibition that could occur in vivo.This is the first evidence that a synthetic dimer peptide mimicking human apoE has anti-angiogenesis functions and could be an anti-tumor drug candidate.     

Inhibition of tumor specific angiogenesis by amentoflavone

The formation of new capillaries from existing blood vessels is critical for tumor growth and metastasis. In this study we report that amentoflavone, a biflavonoid from Biophytum sensitivum, could inhibit the process of angiogenesis. Amentoflavone at nontoxic concentrations (0.05–0.2 μg/ml) showed significant inhibition in the proliferation, migration, and tube formation of endothelial cells, which are key events in the process of angiogenesis. In vivo studies in C57BL/6 mice using amentoflavone showed remarkable inhibition (52.9%) of tumor directed capillary formation. Amentoflavone showed inhibitory effect on the production of various endogenous factors such as IL-1β, IL-6, TNF-α, GM-CSF, and VEGF that control the process of angiogenesis. Amentoflavone treatment could increase the production of IL-2 and TIMP-1, which could successfully shift the equilibrium towards an angiostatic condition. The antiangiogenic activity of amentoflavone was supported by its remarkable suppression in sprouting of microvessels from rat aorta. Our results also show that amentoflavone could inhibit the production of VEGF mRNA in B16–F10 cells. These findings indicate that amentoflavone inhibits angiogenesis by disrupting the integrity of endothelial cells and by altering the endogenous factors that are required for the process of neovascularization. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 2, pp. 258–269.     

Inhibition of prostate tumor angiogenesis by the tumor suppressor CEACAM1

We have previously shown that CEACAM1, a cell-adhesion molecule, acts as a tumor suppressor in prostate carcinoma. Expression of CEACAM1 in prostate cancer cells suppresses their growth in vivo. However, CEACAM1 has no effect on the growth of prostate cancer cells in vitro. This difference suggests that the antitumor effect of CEACAM1 may be due to inhibition of tumor angiogenesis, perhaps by increased secretion of antiangiogenic molecules from the cells. In this study, we have demonstrated that expression of CEACAM1 in DU145 prostate cancer cells induced the production of a factor or factors that specifically blocked the growth of endothelial but not epithelial cells. Conditioned medium from the CEACAM1-expressing cells but not control luciferase-expressing cells inhibited endothelial cell migration up a gradient of stimulatory vascular endothelial growth factor in vitro and inhibited corneal neovascularization induced by basic fibroblast growth factor in vivo. Moreover, conditioned medium from CEACAM1-expressing cells induced endothelial cell apoptosis in vitro. Only medium conditioned by CEACAM1 mutants that were able to suppress tumor growth in vivo could cause endothelial cell apoptosis. These observations suggest that CEACAM1-mediated tumor suppression in vivo is, at least in part, due to the ability of CEACAM1 to inhibit tumor angiogenesis.     

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V)

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.     

Inhibition of tumor angiogenesis by a natural fragment of a tRNA synthetase

Human tyrosyl- and tryptophanyl-tRNA synthetases (TyrRS and TrpRS, respectively) link protein synthesis to signal-transduction pathways, including angiogenesis. Fragments of TyrRS stimulate angiogenesis, whereas those of TrpRS (T2-TrpRS) inhibit angiogenesis. Thus, these two synthetases acquired opposing activities during evolution, possibly as a coordinated mechanism for regulating angiogenesis. The recent identification of the cellular target of T2-TrpRS sheds light into the mechanism of angiogenesis inhibition. This mechanism provides a molecular basis for the lack of effect of T2-TrpRS on the normal vasculature. With these features, we suggest that this fragment of a tRNA synthetase might safely be used to arrest neovascularization of tumors. In particular, an anti-angiogenesis agent that stops the growth of tumor vessels without affecting normal vessels might serve as an adjunct to cytotoxic therapy.     

Direct interaction of the extracellular matrix protein DANCE with apolipoprotein(a) mediated by the kringle IV-type 2 domain

Lipoprotein(a) [Lp(a)] consists of LDL and apolipoprotein(a), and has been shown to be a major, independent, risk factor for arteriosclerosis and thrombosis in humans. To further elucidate the (patho)physiological function(s) of Lp(a)/apo(a), we searched for new protein ligands, using the yeast two-hybrid system and employing the highly repetitive kringle IV type 2 (KIV-2) sequence from apo(a) as bait. The extracellular matrix protein DANCE [developmental arteries and neural crest epidermal growth factor (EGF)-like] recently implicated in atherogenesis was identified as an interactor. A direct physical interaction between DANCE and apo(a) was confirmed by co-purification of both recombinant proteins derived from culture supernatants of transiently transfected COS-1 cells. Furthermore, binding of human plasma-derived Lp(a) to recombinant DANCE was also observed. Finally, when monoclonal anti-apo(a) and polyclonal anti-DANCE antibodies were applied to tissue slices of atherosclerotic carotid artery, the two proteins were found to be co-localized in endothelial and smooth muscle cells, suggesting that they occur together in the arterial wall. However, as yet, the in vivo relevance and the possible functional role of this newly identified DANCE:Lp(a)/apo(a) interaction remains speculative.     

Recombinant kringle IV-10 modules of human apolipoprotein(a): structure, ligand binding modes, and biological relevance

The kringle modules of apolipoprotein(a) [apo(a)] of lipoprotein(a) [Lp(a)] are highly homologous with kringle 4 of plasminogen (75-94%) and like the latter are autonomous structural and functional units. Apo(a) contains 14-37 kringle 4 (KIV) repeats distributed into 10 classes (1-10). Lp(a) binds lysine-Sepharose via a lysine binding site (LBS) located in KIV-10 (88% homology with plasminogen K4). However, the W72R substitution that occurs in rhesus monkeys and occasionally in humans leads to impaired lysine binding capacity of KIV-10 and Lp(a). The foregoing has been investigated by determining the structures of KIV-10/M66 (M66 variant) in its unliganded and ligand [epsilon-aminocaproic acid (EACA)] bound modes and the structure of recombinant KIV-10/M66R72 (the W72R mutant). In addition, the EACA liganded structure of a sequence polymorph (M66T in about 42-50% of the human population) was reexamined (KIV-10/T66/EACA). The KIV-10/M66, KIV-10/M66/EACA, and KIV-10/T66/EACA molecular structures are highly isostructural, indicating that the LBS of the kringles is preformed anticipating ligand binding. A displacement of three water molecules from the EACA binding groove and a movement of R35 bringing the guanidinium group close to the carboxylate of EACA to assist R71 in stabilizing the anionic group of the ligand are the only changes accompanying ligand binding. Both EACA structures were in the embedded binding mode utilizing all three binding centers (anionic, hydrophobic, cationic) like plasminogen kringles 1 and 4. The KIV-10/T66/EACA structure determined in this work differs from one previously reported [Mikol, V., Lo Grasso, P. V. and, Boettcher, B. R. (1996) J. Mol. Biol. 256, 751-761], which crystallized in a different crystal system and displayed an unbound binding mode, where only the amino group of EACA interacted with the anionic center of the LBS. The remainder of the ligand extended into solvent perpendicular to the kringle surface, leaving the hydrophobic pocket and the cationic center of the LBS unoccupied. The structure of recombinant KIV-10/M66R72 shows that R72 extends along the ligand binding groove parallel to the expected position of EACA toward the anionic center (D55/D57) and makes a salt bridge with D57. Thus, the R72 side chain mimics ligand binding, and loss of binding ability is the result of steric blockage of the LBS by R72 physically occupying part of the site. The rhesus monkey lysine binding impairment is compared with that of chimpanzee where KIV-10 has been shown to have a D57N mutation instead.     

Human-mouse hybrids carrying fragments of single human chromosomes selected by tumor growth

Fusion of human EJ bladder carcinoma cells to mouse C127 cells, with direct selection for tumor growth, gave rise to hybrid cells in which the human chromosome complement had been reduced dramatically, while selectively retaining the activated HRAS1 at chromosome band 11p15. A single-component hybrid retaining only part of human chromosome 11 is described in detail. Our results suggest a novel and general approach for investigating the chromosomal basis of neoplastic change and for subchromosomal mapping of and enrichment cloning for the human genome.     

A truncated kringle domain of human apolipoprotein(a) inhibits the activation of extracellular signal-regulated kinase 1 and 2 through a tyrosine phosphatase-dependent pathway

Most proangiogenic factors exert their biological effects primarily by activating extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3-K)/Akt signaling pathways. These pathways appear to play a critical role in endothelial cell migration, because selective inhibition of either ERK or PI3-K/Akt signaling almost completely prevented endothelial cell migration. Recently, we demonstrated that a truncated kringle domain of human apolipoprotein(a), termed rhLK68, inhibits endothelial cell migration in vitro. However, its mechanism of action was not well defined. In this study, we determined the effects of rhLK68 on ERK1/2 and PI3-K/Akt signaling pathways to explore the molecular mechanism of rhLK68-mediated inhibition of endothelial cell migration. Treatment with rhLK68 inhibited ERK1/2 phosphorylation but did not influence Akt activation. Interestingly, an inhibitor of protein-tyrosine phosphatase, sodium orthovanadate, dose-dependently reversed both rhLK68-induced dephosphorylation of ERK1/2 and decreased migration of endothelial cells, whereas rhLK68 showed no significant effects on MEKs phosphorylation. In conclusion, these results indicate that inhibition of endothelial cell migration by rhLK68 may be achieved by interfering with ERK1/2 activation via a protein-tyrosine phosphatase-dependent pathway.     

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.