Patent Application
20120071405
1. Field of the Invention
The present invention relates to a method for inhibiting angiogenesis, and particularly relates to inhibition of angiogenesis and/or angiogenesis-associated diseases with LECT2 protein.
2. Description of the Related Art
Leukocyte cell-derived chemotaxin 2 (hereinafter LECT2), a 16-kDa protein, is a novel neutrophil chemotactic factor which has already been purified from the culture of human T-cell leukemia SWK3 cell induced by phytohemagglutinin (Biochim Biophys Acta 1396:105-13 (1998)). In addition, its mRNA has been known to specifically express in adult or fetal liver and human liver cancer cell strains by isolation of cDNA that encodes LECT2 (Biochem Biophys Res Commun 237:116-20 (1997)). LECT2 is a chondromodulin-II (a cartilage regulatory factor) which is a bovine protein stimulating the proliferation of chondrocytes and osteoblasts (J Bio Chem 125:436-42 (1999)). Currently, it has been reported that the expression of murine LECT2 is transiently reduced in hepatitis induced by concanavaline A (Con A). Such hepatitis induced by Con A is an animal model of human autoimmune hepatitis that can be induced by immunocyte (such as CD4+ T cells or macrophages) associated cytohormones or cytotoxic molecules (J Clin Invest 90:196 (1992)). To date, however, it has not been revealed in any literature that LECT2 protein can inhibit angiogenesis.
Angiogenesis refers to the generation of new vessels to repair injuries and resume the blood supply of tissues, which occurs when a healthy individual suffers injuries. The process of angiogenesis is strictly regulated by various positive and negative regulatory factors. However, in many disease conditions, the angiogenesis of patients is out of control.
Angiogenesis is an important process to the growth of newborns, and is also very important to injury repair. It, however, is also a pathologic feature of many clinical diseases including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, degeneration of macula caused by retinal angiogenesis, and the like. These clinical manifestations associated with angiogenesis are also known as angiogenic diseases. Angiogenic diseases generally include cancer (including solid and liquid tumors), cardiovascular diseases (such as arteriosclerosis), chronic inflammation (such as rheumatoid arthritis and Crohn's disease), diabetes (such as diabetic retinopathy), psoriasis, endometriosis and obesity.
Therefore, there is an urgent need in medical field for a method and composition to effectively inhibit angiogenesis to treat or prevent the aforementioned diseases.
The present invention provides a method for inhibiting pathologic angiogenesis, comprising administering an effective amount of LECT2 protein or analogue thereof, and a pharmaceutically acceptable carrier to an individual.
The present invention also provides a composition that inhibits angiogenesis, comprising an effective amount of LECT2 protein or analogue thereof, and a pharmaceutically acceptable carrier.
The present invention further provides a method for treatment and/or inhibition of cell proliferative disorders, comprising administering an effective amount of LECT2 protein or analogue thereof, and a pharmaceutically acceptable carrier to an individual.
The present invention further provides a composition that treats and/or inhibits cell proliferative disorders, comprising an effective amount of LECT2 protein or analogue thereof, and a pharmaceutically acceptable carrier.
The present invention further provides a method for inhibiting pathologic angiogenesis, comprising administering an effective amount of LECT2 protein or analogue thereof, and a pharmaceutically acceptable carrier to an individual.
In order to render such and other purposes, aspects, and advantages of the present invention obvious and clear, preferred embodiments are specified in the following and illustrated below in detail in conjunction with the attached figures.
The present invention provides a composition that inhibits pathologic angiogenesis and/or cell proliferative disorders. Such composition comprises an effective amount of LECT2 protein and analogue thereof, and a pharmaceutically acceptable carrier.
“LECT2” or “LECT2 protein” as described herein refers to all forms of leukocyte-derived chemotaxin 2 protein including active or inactive forms. Any analogue/fragment of LECT2 protein that has biological activity is within the scope of the present invention. Analogue/fragment can be conjugated to carrier molecules such as immunoproteins for various applications, including increasing valence of protein binding sites. Furthermore, the LECT2 protein analogue of the present invention can also be obtained by replacement, deletion, addition or insertion of one or more amino acids in the original protein and still retains at least one active component of the original protein.
“LECT2 mutants” or “mutant proteins” as described herein include proteins that share at least 60% sequence identity with LECT2 protein, wherein sequence similarity is defined by the amino acid sequences of the proteins to maximize the common components and minimize sequence difference.
The LECT2 proteins of the present invention also include modified LECT2 proteins which are analogous to the native LECT2 protein. A person skilled in the art can modify peptide or DNA sequences with common and conventional techniques. For example, modification of protein sequences may include alteration, replacement, substitution, insertion, or deletion of one amino acid in the coding sequence. The methods employed in alteration, replacement, substitution, insertion, or deletion of the sequences are commonly known techniques. The sequences preferably retain the activity of LECT2 protein after the alteration, replacement, substitution, insertion or deletion.
“A recombinant protein” as described herein refers to a protein comprising one or more recombinant components. Site-specific recombinases are a group of enzymes existing in various viruses and bacteria, which have both endonuclease and ligase activity. Such recombinases can recognize specific DNA sequences and alter DNA fragments.
In the present invention, LECT2 protein, LECT2 protein analogues/fragments or recombinant LECT2 proteins can be expressed in animal cells, low eukaryotes or prokaryotes. Animal cells may include monkey COS cells, CHO cells, human renal 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cells, normal diploid cells, in vitro cultured primary tissue-derived cell strains, primary colonies, Hela cells, murine L cells, BHK, HL-60, U937, HaK or Jurkat cells. Low eukaryotes include yeast, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida or any yeast that can express heterogeneous proteins. Prokaryotes may include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterium that can express heterogeneous proteins. If yeasts or bacteria are used to express a protein, the expressed protein can be further modified, for example, by phosphorylation or glycosylation at approrpiate sites to impart function to the protein.
In the present invention, a composition is applied to a tissue having pathologic angiogenesis, wherein the composition comprises an effective amount of LECT2 protein or a nucleotide vector expressing active LECT2 protein. The tissue can be any tissue or organ undergoing angiogenesis, including brain, skin, muscle, stomach, connective tissues, joints, bones, and the like. Pathologic angiogenesis can be induced by a growth factor, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF).
In addition, the LECT2 protein, LECT2 protein analogues/fragments or LECT2 recombinant proteins can also inhibit the generation of tube-like structures of nascent vessels, migration and proliferation of endothelial cells, vascular invasion of tumors, or other angiogenesis-associated diseases. Angiogenesis-associated diseases include, but are not limited to, inflammatory diseases, chronic rheumatoid arthritis and psoriasis, diseases associated with abnormal vascular invasion, for example, diabetic retinopathy, neovascular glaucoma, restenosis of vessels, and cell proliferative disorders, such as tumor or tumor-associated diseases (for example, solid tumors, solid tumor metastasia, liver cancer, lung cancer, breast cancer, colorectal cancer, stomach cancer and melanoma).
“Cell proliferative disorders” as described herein refer to damages to a multiple-cell tissue caused by abnormal proliferation of one or more cell populations in the multiple-cell tissue. Cell proliferative disorders can occur in different animals and in human. Cell proliferative disorders include, but are not limited to, cancer, vessel hyperplasia and fibrosis.
The LECT2 protein and/or the pharmaceutical composition comprising LECT2 protein of the present invention can be administered orally, parenterally, by inhalation, rectally, vaginally, intradermally, transdermally or topically. A dosage unit can include a conventional nontoxic carrier, adjuvant or vehicle that is pharmaceutically acceptable, and can be administered by peristaltic means.
The LECT2 protein and/or the pharmaceutical composition comprising LECT2 protein of the present invention can be administered once, administered multiple times within 24 hours or administered consecutively. When the injection is consecutively administered, an appropriate well-known way can be selected, including but not limited to, intravenous infusion, intravenous injection pump, imbedded injection pump or topical administration. The time of treatment can be properly adjusted according to different circumstances, for example, the progression and severity of angiogenesis. LECT2 protein is administered alone or combined with other agents of the present invention until the termination of pruritus or the treatment is throughout the lifetime.
In another example, the present invention provides a pharmaceutical composition that inhibits pathologic angiogenesis and/or cell proliferative disorder. Such pharmaceutical composition comprises an effective amount of LECT2 protein or analogue thereof, and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include solvents, dispering agents, coatings, antibiotics/antifungal agents, isotonic agents, absorption delaying agents and the like.
In another example, the present invention further provides a method for inhibiting pathologic angiogenesis, including administering an effective amount of LECT2 protein or analogue thereof, and a pharmaceutically acceptable carrier to an individual.
First, the biomarkers of endothelial cells and the expression of LECT2 protein in 14 human liver cancer (HCC) tissues provided by National Taiwan University Hospital were analyzed by immunohistochemical staining. Briefly, primary liver cancer tissue were obtained from patients having liver cancer, fixed with formalin and embedded in paraffin, followed by immunostaining. After deparaffinage and dehydration treatment, they are washed 3 times with PBST and treated with 1:500 dilution of CD34 antibody followed by biotin-labeled anti-mouse IgG secondary antibody (Vector Laboratories, Inc., Burlingame, Calif.) for 1 hour at room temperature, and the bound antibody was probed with ABC kit (Vector Laboratories Inc.). The sections were stained with diaminobenzidine, washed, and finally counterstained with Delafield's hematoxylin before analysis and counting. Microvascular density and extent of angiogenesis were assayed by staining the endothelial cell antigen. The measurement of microvascular density (MVD) was presented as the count of CD34 antibody-stained endothelial cells. For each tumor, three highly-vascularized regions were analyzed in high magnitude. Count the total number of microvessels in each region, and the average number of microvessels in each tumor.
In this example, SK-Hep1, a human liver cancer cell strain without endogenous expression of LECT2 protein was used. cDNA of human LECT2 was constructed into pSecTag2A eukaryotic expression vector to obtain an expression vector overexpressing LECT2 protein and having hygromycin B gene downstream to the same promoter. The LECT2 protein expression vector was used to transform liver cancer cell (SK-Hep1) with Lipofectamine 2000 reagent (Invitrogen Life tech.). After screening with hygromycin B, a cell strain overexpressing LECT2 protein (SK-Hep1/LECT2) was obtained. The expression level of LECT2 protein in the LECT2-overexpressing cell strain (SK-Hep1/LECT2) was at least 5 times higher than that in control cell strain (SK-Hep1/Neo). From
Furthermore, the tumor cells overexpressing LECT2 protein were injected into BALB/c mice and analysis was conducted. Other than replacement of SK-Hep1 human liver cancer cells and nude mice with BNL murine liver cancer cells and BALB/c mice, the analysis of this example was the same as Example 2. Mice (BALB/c mice, 8-week old) were subcutaneously injected with BNL murine liver cancer cells stably and highly expressing LECT2 (BNL/LECT2) or control BNL cells (BNL/Neo), with each mouse injected with 1×105 cells. The results of this example were similar to those of Example 2. According to
This example analyzed whether the LECT2 recombinant protein could similarly affect the mice transplanted with HCC tumor and assessed the effect of LECT2 recombinant protein on BALB/c mice injected with BNL/Neo cells. When the tumors of the BNL/Neo group grew to about 150 mm3, the LECT2 recombinant protein (1.25 mg/kg body weight) or 1× PBS buffer (50 μL), was injected into the tumors consecutively for 10 day s, respectively. The results were shown in
In this example, the effect of the conditioned media with SK-Hep1 cells that overexpress LECT2 on angiogenesis was evaluated using chick chorioallantoic membrane assay (CAM assay). This media were from SK-Hep1 cells transfected with LECT2 or the vector. Cancer cells were cultured in DMEM media without bovine fetal serum for 48 hours. The conditioned media of SK-Hep1 cells transfected with LECT2 and the vector were filtered by low speed centrifugation. CAM assay of chicken embryos is a suitable model for studying vascular response to multiple regulatory factors in the process of in vivo angiogenesis. Fertilized eggs were cultured in an incubator with an environment of controlled humidity and aeration at 100° F. for 14 days. At Day 9, a small hole was bored on the top of the eggs, above the major vascular region to separate the chick chorioallantoic membrane with the shell membrane. SK-Hep1/Neo conditioned media, SK-Hep1/LECT2 conditioned media, or SK-Hep1/LECT2 conditioned media pretreated with anti-LECT2 or anti-IgG antibody were used for treatment. Conditioned media which have a total volume of 20 μL were dripped to a small disc of 5 mm diameter, which covered the chick chorioallantoic membrane (3 chicken embryos treated in each group). The hole on the top of the egg was sealed with air permeable tape, and the eggs were put back into the incubator. At the third day (Day 12) and fifth day (Day 14) after the treatment, the eggs were carefully opened up. Angiogenesis in chick chorioallantoic membrane was evaluated by determining the length and density of the vessels. The effect of the LECT2 recombinant protein on angiogenesis was assayed by comparison between the numbers of the vessels in the control and the experimental groups, which were assayed with Image-Pro Plus 4.5 software. According to
Endothelial cells will align on matrigel within 6 hours and form 2-dimensional microtube-like network in vitro. Matrigel assay is an extracellular matrix (ECM) assay which provides a physiological environment useful for analysis of the morphology, biochemical function and gene expression of endothelial cells. Vascular endothelial cells were placed on Matrigel and allowed to differentiate and form microtube-like structures. On Matrigel, formation of microtube-like structures requires cell-matrix interaction, cell-cell communication and cell migration. In order to analyze the in vitro effect of the LECT2 recombinant protein on angiogenesis, vascular endothelial cells were placed in a 24-well culture plate covered with Matrigel, treated with the conditioned media of SK-Hep1 or HCC36 cells which were transfected with LECT2 or Neo, respectively, for 6 hours, and observed for formation of tube-like structures using an inverted phase contrast microscope. Under microscopic observation, 6 tube-like structures randomly selected within the visual field were compared to each other. According to
Vascular endothelial cells were cultured in M199 medium in a 24-well culture plate (7×104 cells/well). After 24 hours, medium with 5% fetal bovine serum was exchanged to the monolayer of vascular endothelial cells, and cell-free monolayers were obtained by scratching a straight line with blue pipette tips. The HCC conditioned media containing overexpressed LECT2 (SK-Hep1/LECT2 CM) and the HCC conditioned media of the vector control group (SK-Hep1/Neo CM), or the HCC conditioned media with inhibited endogenous LECT2 (Huh-7/shLECT2-2 CM) and the HCC conditioned media of the vector control group (Huh-7/shLuc CM) were cultured under 37 for 14 hours, followed by observing for the migration ability of endothelial cells using inverted phase contrast microscope, and measuring for the degrees of cell migration under 4 separate randomly selected visual fields. The images were obtained via analysis performed by Image Pro-Plus 4.5 software. For each plate, 4 visual fields were observed, and the scratch width after cell removal was measured. According to
Vascular endothelial cells were placed on Matrigel of 24-well plates, treated with VEGF-A (50 ng/mL), PDGF (50 ng/mL), bFGF (30 ng/mL), EGF (50 ng/mL), HGF (40 ng/mL) or LECT2 recombinant protein (200 ng/mL) for 6 hours and assayed for the formation of microtube-like structures. From the results, it can be known that LECT2 recombinant protein effectively inhibited VEGF-A induced neogenesis of vascular tube-like structures, and its inhibition ratio was 52% with respect to the group with VEGF-A (50 ng/mL) treatment, as shown in
The effect of LECT2 recombinant protein on VEGF-A induced angiogenesis was analyzed by CAM assay. Chick chorioallantoic membranes of 9-day old chicken embryo were treated with VEGF-A and different concentrations of LECT2 recombinant proteins (0, 1.25, 2.5 and 5 nM) or Fc-Tag (control) (three chicken embryos were treated in each group): treatments with a total volume of 20 μL were dripped onto discs of 5 mm diameter, the discs were covered on the chick chorioallantoic membranes. The assay was done at third day (i.e., Day 12) or fifth day (i.e., Day 14) after treatment of chick chorioallantoic membranes with VEGF-A and LECT2 recombinant protein. According to
This example analyzed the effect of the LECT2 recombinant protein on vascular endothelial cell migration and angiogenesis. Vascular endothelial cell migration is one of the important factors of angiogenesis, thus the effect of rLECT2 protein on the migration ability of vascular endothelial cell induced by VEGF-A was analyzed. Endothelial cell migration was effected by treatment with 2% FBS (control group), VEGF-A (50 ng/mL), VEGF-A (50 ng/mL) together with different concentrations of LECT2 recombinant protein (0-200 ng/mL), and LECT2 recombinant protein (200 ng/mL) for 14 hours. VEGF-A (50 ng/mL) could improve the migration of vascular endothelial cells. From
This example analyzed the effect of the LECT2 recombinant protein on angiogenesis of different cancer cell strains. Vascular endothelial cells were placed on Matrigel of 24-well plates, treated with the conditioned media of various cancer cell strains for 6 hours or with the conditioned media of cancer cell strains combined with rLECT2 (5 nM) for 6 hours, said cancer cells including liver cancer cells (SK-Hep1 and Huh-7), lung cancer cells (A549 and H1299), breast cancer cells (MB-MDA-231 and MCF7), stomach cancer cells (MKN and N87), rectal cancer cells (HCT 116) and murine melanoma cells (B16F1).
First, murine melanoma cell strain B16F1 which overexpresses LECT2 was constructed. After screening with hygromycin B, the expression level of LECT2 was analyzed, and the effects of LECT2 protein on tumorigenesis and angiogenesis were evaluated using in vivo melanoma xenograft model. B16F1 cells (B16F1/Neo and B16F1/LECT2) were injected subcutaneously into 7-week old female C57BL/6 mice, with 5×105 cells for each mouse. According to
Lung cancer A549 cells were subcutaneously injected into 7-week old female nude mice with each mouse injected with 5×106 cells. At Day 20 after A549 cell injection, D-luciferin (20 mg/mL/kg body weight of mouse) was intraperitoneally injected. The mice were anaesthetized and photographed with Berthold/NightOWL imaging system for 5 minutes. According to
While the present invention has been disclosed with respect to the preferred examples above, it shall not be construed as any limitation to the present invention. Any person skilled in the art can make alteration and modification without departing from the spirits and scope of the present invention. Therefore the scope of the present invention is decided by that defined by the attached claims.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN2009/074086 | 9/22/2009 | WO | 00 | 7/1/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61136635 | Sep 2008 | US |
