KR101192700B1

KR101192700B1 - A Method of Screening for anti-cancer drug using TGase2 and VHL

Info

Publication number: KR101192700B1
Application number: KR20100068557A
Authority: KR
Inventors: 김수열
Original assignee: 국립암센터
Priority date: 2010-07-15
Filing date: 2010-07-15
Publication date: 2012-10-19
Also published as: KR20120007825A; WO2012008806A3; WO2012008806A2

Abstract

In the present invention, TGase2 (Transglutaminse 2) modulates VHL (Von Hippel-Lindau) to induce HIP-1α (Hypoxia-inducible factor-1α), IGF-1α (Insulin-like growth factor receptor) and VEGF (Vascular) during cancer formation. To identify TGase2 inhibitors or promoters by determining the stability of endothelial growth factor (NF-κB) and the activity of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and by measuring VHL protein levels or polymerization of VHL Provide a way to. Also provided are methods of detecting TGase2 inhibitors or promoters by further measuring the protein levels of HIF-1α, IGF-1R or VEGF. In the present invention, the TGase2 inhibitor may be usefully used as an anticancer agent and a TGase2 promoter as a therapeutic agent for a viral infection disease.

Description

A method of screening for anti-cancer drug using TGase2 and VHL}

Hypoxia-inducible factor-1α is an inducible transcription factor whose expression is regulated by hypoxia. Accumulation of HIF-1α in cancer cells leads to angiogenesis and drug resistance (Wartenberg, M. et al. FASEB J 17, 503-5 (2003)). Recent reports indicate that HIF-1α is induced by TGase2 (Jang, GY et al. Oncogene 29, 356-67). TGase2 is a cross-linking enzyme that is involved in a variety of cell pathways, including cancer cell proliferation, migration, angiogenesis, mutation of epithelial cells into mesenchymal cells, and drug resistance (Verma, A. & Mehta, K. Drug Resist Updat 10, 144-51 (2007), Iismaa, SE, Mearns, BM, Lorand, L. & Graham, RM Physiol Rev 89, 991-1023 (2009), Kim, SY Advnace in Enzymology 78 (2010) in press). Induction of TGase2 contributes to structural NF-κB activity through polymerization of inhibitors of NF-κB (I-κBα) (Lee, J. et al. J Biol Chem 279, 53725-35 (2004)), which has been reported to be one of the mechanisms of drug resistance in cancer cells (Mehta, K., Fok, J., Miller, FR, Koul, D. & Sahin, AA Clin Cancer Res 10, 8068-76 (2004). Kim, DS, Park, SS, Nam, BH, Kim, IH & Kim, SY Cancer Res 66, 10936-43 (2006). Park, SS, Kim, JM, Kim, DS, Kim, IH & Kim, SY J Biol Chem 281, 34965-72 (2006), Gangadharan, C., Thoh, M. & Manna, SK J Cell Biochem 107, 203-13 (2009), Kim, DS, Park, KS & Kim, SY Front Biosci 14, 2514-21 (2009), Kim, JM et al. J Mol Biol 384, 756-65 (2008)). Increased TGase2 expression in breast cancer tissues is associated with increased NF-κB activity (Park, KS, Kim, DS, Jeong, KC & Kim, SY Front) Biosci 14, 1945-51 (2009)). Amplification of NF-κB linked signal activity by TGase2 appears to be due to a decrease in peroxisome proliferator-activated receptor-g (PPAR-γ) known as NF-κB inhibitors (Maiuri, L. et al. J Immunol 180, 7697-705 (2008)). In both cases there is a reduced inflammatory transcription factor (Von Hippel-Lindau, VHL) inhibitor.

We propose that TGase2-mediated protein-protein crosslinking results in a unique mechanism of secondary signaling through post-translational modification. We found that expression of TGase2 downregulates inhibitors of HIF-1α in cancer cells.

Von Hippel-Lindau (VHL) tumor suppressor protein is part of the ubiquitin-ligase complex and down-regulates HIF-1α through poly-ubiquitination and proteosomal degradation (Kaelin, WG, Jr.). .Nat Rev Cancer 8, 865-73 (2008). In renal adenocarcinoma cells, VHL is a FLICE inhibitor protein (c-FLIP), survivin, apoptosis-1 inhibitor (inhibitor of apoptosisDownregulates NF-κB activity by inhibiting NF-κB-targeted anti-apoptotic genes such as -1, c-IAP-1) and c-IAP-2, and VHL induces tumor necrosis factor (TNF) Be sensitive to toxicity (Qi, H. & Ohh, M.Cancer Res 63, 7076-80 (2003). The mechanism of NF-κB inhibition by VHL is well known, but it is not known how the reduction of VHL induces NF-κB activity. In addition, VHL regulation by deletion, mutation, or promoter methylation of the VHL gene is well known (Tischoff, I. & Tannapfel, A.Z Gastroenterol 46, 1202-6 (2008). Cowey, C.L. & Rathmell, W.K.Curr Oncol Rep 11, 94-101 (2009). Curtis, C. D. & Goggins, M.Methods Mol Med 103, 123-36 (2005)), the mechanism of VHL regulation at the protein level is not yet described.

Against this background, we have found that TGase2 modulates VHL, resulting in the stability of HIF-1α, VEGF and insulin-like growth factor receptor-1 (IGF-1R) and NF-κB activity during tumor formation. It was confirmed that the present invention was completed.

One object of the present invention is to (a) treat a VHL-expressing cell with a candidate inhibitor or promoter of TGase2 (Transglutaminse 2), (b) induce the expression of TGase2, and (c) VHL protein levels and To provide a method of detecting a TGase2 inhibitor or promoter comprising the step of comparing one or more of the polymerizations of the VHL protein with a control.

Another object of the invention is to (a) treat a isolated VHL as a candidate inhibitor or promoter of TGase2 (Transglutaminse 2), (b) treat the isolated TGase2, and (c) VHL protein levels and VHL. To provide a method of detecting a TGase2 inhibitor or promoter comprising the step of comparing one or more of the polymerizations of the protein with the control.

As one aspect for achieving the above object, the present invention comprises the steps of (a) treating a VHL-expressing cell candidate inhibitor or promoter of TGase2 (Transglutaminse 2), (b) inducing expression of TGase2, and (c) comparing the at least one of the VHL protein level and the polymerization of the VHL protein with a control, the method of detecting a TGase2 inhibitor or promoter.

The mechanism by which TGase2 modulates VHL in tumorigenesis has not been determined to date. The present inventors confirmed that VHL is inhibited by expression of TGase2 in cancer cells, and conversely, when TGase2 is inhibited by using cystamine or TGase2 variants, the level of VHL is restored (FIG. 1), and TGase2 cross-links VHL. It was confirmed that catalyzing the depletion of the VHL monomer (monomer) and increasing the VHL polymer (Fig. 2), it was confirmed that downregulation of VHL by TGase2 is important for IGF-1R protein expression (Fig. 3). In addition, TGase2 inhibited VHL in TGase2-induced transgenic mice, indicating that NF-κB activity and IGF-1R expression were induced in vivo (FIG. 4), and between VHL and TGase2 in human kidney cancer tissue samples. The inverse relationship was reconfirmed (FIG. 5).

Based on this understanding, substances that modulate the activity of TGase2 can be detected by measuring VHL protein levels or polymerization of VHL proteins. Since TGase2 is a calcium-dependent enzyme and its activity changes sensitively even with small changes in calcium, it is more preferable to measure VHL protein level or polymerization of VHL protein than to measure TGase2 protein level. Hereinafter, each step of the present invention will be described in more detail.

The first step of the present invention is the step of (a) treating a VHL-expressing cell with a candidate inhibitor or promoter of TGase2 (Transglutaminse 2).

As used herein, the term "von Hippel-Lindau" (VHL) is a tumor suppressor protein that is part of the ubiquitin-ligase complex and downregulates HIF-1α through poly-ubiquitination and proteosomal degradation. In addition, VHL enhances apoptosis through the transcriptional activity of p53 and inhibits the cell cycle, thus functioning as a cancer suppressor. In renal cancer cells, VHL also downregulates NF-κB by inhibiting nf-κB targeted anti-apoptotic genes such as c-FLIP, subvavin, c-IAP-1, c-IAP-2. In the present invention, it was first identified that TGase2 downregulates VHL.

As used herein, the term "TGase2" refers to transglutaminase 2 (EC 2.3.2.13, protein-glutamine γrglutamyltransferase; Transglutaminase 2; TGase2) between N ε- (γ-L-glutamyl) -L-lysine between lysine and glutamine. iso refers to Ca ⁺ ² dependent enzymes to promote peptide bond formation. Nε- (γ-L-glutamyl) -L-lysine crosslinks are known to form protective barrier function, cell death, extracellular matrix in stabilizing epithelial cells and to stabilize proteins present in and outside of cells. TGase2 is normally expressed at low levels in various tissues and is abnormally activated in various pathological conditions. In particular, TGase2 expression levels are known to increase in inflammatory diseases.

As used herein, the term "inhibitor" refers to a substance that functions to reduce the expression or decrease the activity of TGase2.

As used herein, the term "activator" refers to a substance that acts to increase the expression or increase the activity of TGase2.

As used herein, the term "candidate inhibitor" or "candidate promoter" means a candidate substance expected to be an inhibitor or promoter of TGase2. Such candidate materials may include a single compound such as an organic or inorganic compound, a complex of a plurality of compounds, a polymer compound such as a protein, a carbohydrate, a nucleic acid molecule (RNA, DNA, etc.) and a lipid, and the like.

As used herein, the term "treatment" refers not only to the direct contact of a candidate substance, ie, a TGase2 candidate inhibitor or candidate promoter, with TGase2, but also that the substance affects the cell membrane and the signal generated from the cell membrane contacts or affects TGase2. It also includes cases that affect. Therefore, in the present invention, the candidate substance includes not only a substance which is permeable to the cell membrane, but also a substance which is impermeable to the cell membrane. At this time, the candidate substance should be treated within an effective amount range. The term “effective amount” means an amount sufficient to induce a reaction, and below the effective amount range, accurate results cannot be obtained. Investigate the function.

Detection of the inhibitor or promoter of TGase2 of the present invention includes all cells of animal origin, such as human or bovine, goat, pig, mouse, rabbit, hamster, rat, guinea pig, which express TGase2, and are primary, secondary and immortalized. Prepared cells and the like can be used. In addition, a recombinant vector into which TGase2 has been introduced may be used to use cells that have been engineered to stably or temporarily overexpress TGase2 in cells. Specifically, the human multi-drug resistant breast cancer cell line NCI / ADR-RES, the breast cancer cell line MDA-MB-231, the human embryonic kidney cell line HEK293, and the human breast cancer adenocarcinoma cell line MCF-7 were used.

Detection of inhibitors or promoters of TGase2 can be performed in vivo using experimental animals such as mice, rabbits, hamsters, rats, guinea pigs, as well as at the cellular level.

The second step of the invention is (b) inducing expression of TGase2.

In this step, THLase-induced expression of TGase2 is induced after a period of time after treatment of VHL-expressing cells with a candidate inhibitor or promoter of TGase2, or treatment of VHL-expressing cells with a candidate inhibitor or promoter of TGase and simultaneous expression of TGase2. Can be induced. In some cases, expression of TGase2 may be first induced and treated with candidate inhibitors or promoters of TGase2.

TGase2 expression is an element known to induce the expression of TGase2, such as ultraviolet light, glutamate, calcium inophore, maitotoxin, retinoic acid, inflammation-induced cytokines, glutamate, and the like. Can be induced through treatment, oxidative environment, viral infection, and the like, and TGase2 can be cloned into a recombinant vector and introduced into the cell transiently. The substance or method for inducing TGase2 is not particularly limited.

The third step of the invention is the step of detecting a TGase2 inhibitor or promoter by comparing (c) one or more of VHL protein levels and polymerization of VHL protein with a control.

The extent to which TGase2 is inhibited or promoted by candidate treatment can be significantly confirmed by confirming at least one of VHL protein levels and polymerization of VHL protein with the control.

As used herein, the term "control" refers to a cell in which VHL protein levels or polymerization of VHL protein are not artificially regulated by treating an inhibitor or promoter of TGase2.

In the case of TGase2 promoters, the levels of VHL protein polymers increased with a decrease in intracellular VHL monomer protein levels compared to the control. On the other hand, the TGase2 inhibitor significantly decreases the level of VHL protein polymer while increasing the level of intracellular VHL monomer protein compared to the control.

VHL proteins or protein polymers can be detected using antibodies specific for VHL. As used herein, the term "antibody" refers to a labeling substance capable of detecting the expression of VHL by treating a candidate inhibitor or promoter of TGase2.

The method for detecting VHL protein level or polymerization of VHL protein in cells treated with candidate inhibitors or promoters of TGase2 using the antibody is Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, oukteroni immunodiffusion method, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, complement fixation assays, FACS or protein chip methods, and the like in which the protein is isolated from the cytoplasm and subjected to SDS-PAGE and specific antibody levels and / or Western blots measured by confirming activity are preferred.

In the detection method, the amount of antigen-antibody complex formation can be quantitatively measured through the magnitude of a signal of a detection label. As used herein, the term "detection label" is to be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Refers to a composition that can be. Such detection markers include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes.

In a preferred embodiment, the detection method of the present invention is carried out in a cell further expressing at least one of IGF-1R, HIF-1α and VEGF and the protein level of at least one of IGF-1R, HIF-1α and VEGF is further added to the control group. Comparing may further comprise.

The term "insulin-like growth factor-1" (IGF-1) means insulin-like growth factor-1 and belongs to a group of hormones called somatomedin which have a structure very similar to insulin. It has many similarities to growth hormone in the way it works and, like growth hormone, promotes protein assimilation. In the present invention, it was confirmed that the expression of IGF-1R may be increased by downregulating VHL by TGase2 (FIGS. 3 and 4).

As used herein, the term "HIFoxia-inducible factor-1α" is a nuclear transcription factor that is induced in a large amount in a hypoxic state. In order to maintain oxygen homeostasis in cells, red blood cell production, angiogenesis and sugar It is involved in metabolism and expresses genes. Since it is known that VHL inhibits HIF-1α, it can be seen that VHL is down-regulated by TGase2 in the present invention, resulting in increased stability of HIF-1α.

The term "vascular endothelial growth factor" (VEGF) is a vascular endothelial growth factor, and is known to play a major role in angiogenesis caused by cancer cells. In the present invention, it was confirmed through RT-PCR that VEGF expression is reduced in cancer cells knocked down TGase2-specific siRNA (FIG. 1A). In addition, since it is known that IGF-1R upregulates VEGF, it can be seen that the expression of IGF-1R increases with VEGF expression by downregulating VHL by TGase2 in the present invention.

In another embodiment, the present invention provides a method of treating a isolated VHL with a candidate inhibitor or promoter of TGase2 (Transglutaminse 2), (b) treating the isolated TGase2, and (c) VHL protein levels and A method of detecting a TGase2 inhibitor or promoter comprising comparing one or more of the polymerizations of VHL proteins to a control.

As used herein, the term “isolated” refers to an isolated protein expressed in a cell, and may be separated by applying a general separation technique known in the art. A method for separating and purifying a protein expressed in a cell is not particularly limited.

The isolated VHL can react directly with the TGase2 candidate inhibitor or promoter in vitro and not in the cell, and after treatment with the isolated VHL with the TGase2 candidate inhibitor or promoter, treatment of TGase2 or expression of TGase2 after a certain period of time. Can be induced first and treated with a TGase2 candidate inhibitor or promoter, and the two steps can be performed simultaneously.

The method of comparing one or more of VHL protein levels and polymerization of VHL protein with a control is as described above. In addition, the method may further include the step of performing any one or more of the isolated IGF-1R, HIF-1α, and VEGF to compare the protein level of any one or more of IGF-1R, HIF-1α, and VEGF with a control.

Methods of detecting TGase2 inhibitors or promoters can detect one or more of VHL protein levels and polymerization of VHL proteins using antibodies specific for VHL. In addition, protein levels of any one or more of IGF-1R, HIF-1α and VEGF can be detected using antibodies specific for that protein.

Expression of VHL, HIF-1α, IGF-1R, and VEGF in cells treated with candidate inhibitors or promoters of TGase2 using the antibody is Western blot, ELISA, radioimmunoassay, radioimmunoproliferation method, oukteroni immunity. Diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS or protein chip method, etc., isolate the protein from the cytoplasm and use it to SDS-PAGE and specific antibody levels And / or western blots measured by confirming activity.

By inhibiting TGase 2 signaling in cells by administering an inhibitor obtained by the above method, diseases associated with increased TGase 2 activity, such as inflammatory diseases and cancer diseases, can be effectively treated or prevented. Inflammatory diseases include autoimmune diseases and neurodegenerative diseases. Autoimmune diseases are closely associated with abnormal activity of T cells and macrophages that cause severe inflammation. Abnormally increased expression of TGase 2 in autoimmune inflammatory myopathy and chronic digestive disorders has been reported (Choi et al., (2000) J. Biol. Chem. 275, 88703-88710; Choi et al (2004) Eur Neurol. 51, 10-14; Bruce et al., (1985) Clin. Sci. 68, 573-579).

In addition, TGase 2 was found to increase in autoimmune diseases as a result of macrophage activity and increased expression of TGase 2 appears to be closely associated with autoantibody formation (Novogrodsky et al., (1978) Proc. Natl. Acad. Sci. USA 75, 1157-1161; Murtaugh et al., PJ (1983) J. Biol. Chem. 258, 11074-11081; Leu et al., (1982) Exp. Cell Res. 141, 191-199 ). Autoimmune diseases associated with overexpression or overactivity of TGase 2 include chronic digestive disorders (Dieterich et al., (1997) Nat. Med. 3, 797-801), dermatitis herpetiformis (Dieterich). , et al .. (1999) J. Investig.Dermatol. 113, 133-136), type 1 diabetes (Lampasona et al., (1999) Diabetologia 42, 1195-1198), Lupus (Sanchez, et al., (2000) J. Autoimmun. 15, 441-449), Rheumatoid Arthritis (Picarelli et al., (2003) Clin. Chem. 49, 2091-2094), and the like. However, it is not limited thereto.

In brain inflammation, glial cells producing toxic factors such as NO and TNF-α are known to be activated (Minagar et al., (2002) J. Neurol. Sci. 202, 13-23; Catania et al., (1998) Ann. NY Acad. Sci. 856, 62-68). Synthesis and secretion of these elements form innate immunity in which the host can be destroyed by pathogen invasion, but excessive generation and accumulation of NO contributes to neurodegeneration (Liu et al., (2002) Ann. NY Acad Sci. 962, 318-331). In particular, TGase 2 induced in activated astrocytes is known to be involved in the pathogenesis of degenerative neurological disease (Campisi et al., (2003) Brain Res. 978, 24-30; Monsonego et al., (1997) J Biol.Chem. 272, 3724-3732). Neurodegenerative diseases associated with overexpression or overactivity of TGase 2 include Parkinson's disease (Jung, Y. et al. Hypoxia-inducible factor induction by tumour necrosis factor in normoxic cells requires receptor-interacting protein-dependent nuclear factor). kappa B activation. Biochem J 370, 1011-7 (2003)), one including Alzheimer's disease (Alzheimer's disease), neurological AIDS (neuro-AIDS), but is not limited thereto.

A representative target gene induced by NF-κB is COX-2. COX-2 is a gene that is considered to be important not only in the treatment of inflammation but also in the prevention and treatment of cancer. Induction of COX-2 in cancer cells and malignant tumor tissues results in much higher levels of prostaglandins than normal cells (Kargman et al., (1995) Cancer Research, 55: 2556-2559; Ristimaki et al., ( 1997) Cancer Research, 57: 1276-1280). Prostaglandins such as prostaglandin E2 (PGE2) promote angiogenesis and promote cell proliferation, so their overproduction provides a good environment for cancer cell growth. Moreover, excessive expression of COX-2 is known to inhibit apoptosis and increase the metastatic capacity of cancer cells. In addition, increased expression of COX-2 was found in several cancers, while COX inhibitors were found to reduce the incidence of cancer (Noguchi et al., (1995) Prostaglandins, Leukotrienes, and Essential Fatty Acids, (1997) 53 : 325-329; Thompson et al., (1997) Cancer Research, 57: 267-271). Thus, COX-2 selective inhibitors can be used as anti-inflammatory as well as anti-cancer agents.

Therefore, it can be seen that TGase 2 inhibitor acts as a therapeutic agent for diseases caused by abnormal expression of TGase 2, for example, as an anticancer agent. Cancers that can be prevented or treated using TGase inhibitors detected by the present invention include colorectal cancer, small intestine cancer, rectal cancer, anal cancer, esophageal cancer, pancreatic cancer, stomach cancer, kidney cancer, uterine cancer, breast cancer, lung cancer, lymph gland cancer, thyroid cancer , Prostate cancer, leukemia, skin cancer, colon cancer, brain tumors, bladder cancer, ovarian cancer, gallbladder cancer, and the like. In addition, TGase2 inhibitors may act as therapeutic agents for inflammatory diseases or degenerative neurological diseases.

In addition, the TGase2 promoter obtained by the method of the present invention can effectively treat or prevent diseases associated with a decrease in TGase 2 activity, such as a viral infection, by promoting TGase 2 signaling in cells. TGase 2 is known to promote its expression by retinoic acid (Moore et al. (1984) J Biol Chem 259, 12794-12802). RA is also known to help treat diseases by inhibiting viral infections or promoting immunity (Lotan R. (1996) FASEB J. 10, 1031-109). In other words, it can be seen that the activation of NF-κB by TGase 2 acts as a protective mechanism against infections such as viruses. Immune activity depends on the activity of NF-κB, as is well known, and NF-κB is activated by overexpression of TGase 2. Therefore, by administering a promoter detected by the above method and promoting TGase 2 signaling in the cell, infectious diseases such as viruses can be effectively treated or prevented.

Through the method of detecting the TGase2 inhibitor or promoter by measuring the level of protein or protein polymerization of VHL identified as the target of transglutaminase 2 of the present invention, and further the expression level of IGF-1R, HIF-1α or VEGF TGase2 inhibitors or promoters can be effectively detected.

1 is a diagram showing that HIF-1α expression is reduced by TGase2 knockdown in normal and cancer cell lines, and that the expression of VHL is inversely related to TGase2 expression. (a) HIF-1α expression was inhibited by TGase2 knockdown in cancer cells. TGase2 was inhibited by treatment with TGase2 specific siRNA, and levels of HIF-1α were analyzed in immunoblot. VEGF expression in cancer cells was inhibited by TGase2 knockdown using TGase2-specific siRNA. VEGF levels were analyzed by RT-PCR. GAPDH mRNA levels were measured as controls. (b) The expression of endogenous VHL was decreased in ovarian carcinoma and breast cancer cells overexpressing TGase2. Cytoplasmic fractions of HEK293, MCF7, NCI / ADR-RES, and MDA-MB-231 cells were analyzed by immunoblot using anti-TGase2 and anti-VHL antibodies. β-actin was analyzed as a loading control. (c) Endogenous VHL protein levels were down regulated by overexpression of TGase2 and recovered by TGase2 inhibitor treatment or expression of TGase2 mutants (C277S). HEK293 and MCF7 cells were transfected stepwise for 24 hours with an expression vector of TGase2 or TGase2 C277S mutant and with or without cystamine for 24 hours (CTM; 0.5 mM). Cytoplasmic fractions of cells were recovered and analyzed by immunoblot using anti-TGase2 and anti-VHL antibodies. (d) Expression of ectopic VHL and TGase2 in HEK-293 and MCF7 cells. Cells were concurrently infected with the expression plasmids presented for 24 hours, and with or without cystamine for 24 hours (CTM; 0.5 mM). TGase2 and VHL levels were analyzed via immunoblot using anti-TGase2 and anti-VHL antibodies. (e) Ectopically expressed VHL was down regulated by dose dependent overexpression of ectopic TGase2. The cells were simultaneously infected with the VHL expression vector and the amount of TGase2 expression vector increased for 48 hours. Cell extracts were prepared and analyzed by immunoblot using anti-TGase2 and VHL antibodies.
FIG. 2 shows that direct physical interaction of TGase2 and VHL results in TGase2-VHL polymer formation and proteasome-dependent degradation. (a) TGase2 induces the accumulation of degraded VHL monomers in the proteasome. Cells were transfected with the plasmids presented for 24 hours, and various inhibitors (calpeptin, calpein inhibitor, 50 μM; leupeptin, lysosomal inhibitor, 50 μM; MG 132, proteasome inhibitor, 5 μM) for 12 hours prior to recovery. Treated and untreated. TGase2 and VHL levels were analyzed by immunoblot using anti-TGase2 and anti-HA antibodies. (b) VHL interacts with TGase2, and this interaction is further increased in the presence of proteasome inhibitors. Cells were simultaneously infected with the indicated expression plasmids for 24 hours, followed by or without MG132 (5 mM) for 12 hours. Whole cell extracts were analyzed by immunoprecipitation using anti-HA antibodies and then immunoblot using anti-TGase2 and anti-VHL antibodies. Simultaneous immunoblot analysis of whole cell extracts with anti-TGase2 and anti-HA antibodies showed that TGase2 and VHL were stably expressed in a proteosome dependent method. (c) TGase2-VHL complexes undergo proteosome-dependent degradation. Cells were simultaneously infected with the indicated expression vectors for 24 hours and MG132 (5 mM) was treated with or without 12 hours. VHL was immunoprecipitated from whole cell extracts with anti-HA antibody (5 times more cell lysate than b) and immune complexes were analyzed by immunoblot with anti-TGase2 and anti-VHL antibodies.
3 is a diagram showing that expression of ectopic VHL with inhibition of TGase2 inhibits NF-κB activity and reduces IGF1R-β levels. (a) NF-κB activity was reduced by ectopic expression of VHL and inhibition of TGase2. Cells were transfected with the indicated expression vectors for 24 hours and either with or without cystamine (CTM; 0.5 mM) (right panel) or TGase2-specific siRNA (left panel). The culture medium was recovered and NF-κB transcriptional activity was measured using SEAP reporter assay. Results are expressed as base error (SEM) of the mean ± 3 sets of experimental means (p <0.05). (b) Nuclear extracts of the cells described in (a) were analyzed by EMSA using a ³² P-labeled NF-kB probe. (c) Cell death was analyzed by annexin V / propidium iodide staining (PI) and immunoblot of cleaved caspase 3 and PARP. Cells were transfected with VHL expressing plasmids and TGase2-specific-siRNA. Annexin V / PI staining was determined by flow cytometry. The percentage of cells recovered in the quadrant corresponding to early and late cell death was shown (top panel). Whole cell extracts were analyzed by immunoblot using anti-cleaved caspase-3, anti-PARP and anti-β-actin antibodies. (d) Inhibition of TGase2 restored VHL protein levels and downregulated endogenous IGF1R-β protein levels. Cells were transfected with the indicated plasmids for 24 hours, and then treated or untreated with cystamine (CTM; 0.5 mM) for 24 hours before recovery (left panel). Alternatively, cells were transfected 24 hours with TGase2-specific siRNA using reversible infection, followed by 24 hours transfection with the indicated expression plasmids (right panel). Whole cell extracts were analyzed by immunoblot with anti-TGase2, anti-IGF1R-β and anti-VHL antibodies.
Figure 4 shows that overexpression of TGase2 induces inhibition of VHL and activity of IGF1R-β and NF-κB in the body. (a) Schematic of the TGase2-pCAGGS genomic construct used to generate TGase2 transgenic mouse species is shown. (b) The potential founder mice to which the TGase2 transplant gene was introduced by PCR were identified. (c) After perfusion, frozen tissues derived from wild-type and TGase2 transgenic mice were homogenized and then TGase2, VHL and IGF1R-β levels were immunized with anti-TGase2, anti-VHL and anti-IGF1R-β antibodies. Relative to blot. (d) Tissues were prepared as described in (a) and nuclear extracts were analyzed by EMSA using ³² P-labeled NF-κB probes. NF-κB DNA binding activity was quantified using a density analyzer. Results are shown as SEM of mean ± three set samples (p <0.05).
Figure 5 shows that TGase2 and VHL expression is in a reversible relationship in human RCC carcinoma samples. In 9 of 11 RCC cell carcinoma samples, TGase2 and VHL expression were inversely related. For samples with high expression of TGase2, VHL expression was low or absent (a and b). For samples with low expression of TGase2, the expression of VHL was relatively high (c and d). The expression of TGase2 was high in invasive progenitor cells (e, arrow), while the expression of VHL was relatively low or absent (f, arrow), providing additional evidence indicating the inverse of TGase2 and VHL expression. All image amplification rates are 100X.
6 shows a model presented for the role of VHL and TGase2 in NF-κB activity. TGase2 is an I-κBα (Kim, DS, Park, SS, Nam, BH, Kim, IH & Kim, SY Reversal of drug resistance in breast cancer cells by transglutaminase 2 inhibition and nuclear factor-kappaB inactivation. Cancer Res 66 , 10936-43 (2006)) and VHL induce or expand the activity of NF-κB. It is a well known major inhibitor of NF-κB, and VHL is a tumor inhibitor that modulates HIF-1α and IGF-1R. Reduction of I-κBα and VHL increases resistance to survival, growth, migration, drug resistance and apoptosis in cancer cells.
7 is a diagram showing that the expression level of VHL is restored by suppressing TGase2 expression by inhibition of cystamine or TGase2-specific siRNA in NCI / ADR-RES and MDA-MB-231 overexpressed TGase2. Cells were transfected with TGase-2 specific siRNA and the plasmids presented above (a. Right panel) or transfected with the plasmids presented above with or without cystamine (b. Left panel). Overexpression of VHL was confirmed by immunoblot analysis using anti-VHL antibodies.
8 shows that localization of ectopic VHL is regulated by TGase2 gene in NCI / ADR-RES and MDA-MB-231 cancer cells. Cells were transfected with TGase2-specific siRNA for 24 hours with invertebrate infection and with the plasmids disclosed above. The cells were recovered and reinoculated on two chamber slides for 16 hours. Cells were analyzed using immuno-chemical analysis using anti-HA primary antibodies followed by fluorescently bound anti-mouse immunoglobulin secondary antibodies for detection of VHL. Staining pattern shows nuclear-cytoplasmic distribution of VHL. Cells were counterstained with 4 ', 6-diamino-2-phenylindole (DAPI) to visualize nuclei. Scale bar, 20 ㎛
9 shows that TGase2 induces a dose dependent polymerization of VHL in vitro. Purified recombinant VHL was polymerized by guinea pig liver TGase2 in vitro. VHL polymers were separated by SDS-PAGE, visualized by Coomassie blue staining (left panel) and analyzed by immunoblot with anti-VHL antibodies (right panel).
Figure 10 shows monomers, dimers and polymers of VHL produced by proteolysis by mass spectrometry of peptides. Purified recombinant VHL was polymerized by TGase2 in vitro. Multiple binding VHL complexes were separated by SDS-PAGE and visualized by Coomassie Blue staining. The quantified bands were collected, reduced and resolved by trypsin treatment. The mass of peptides was determined using MALDI-TOF MS.
Figure 11 shows the results of the purification of the VHL monomer and polymer by MS to identify the cross-linking site in VHL. FIG. 11 shows amino acid sequences of VHL monomers and polymers, in which red letters represent residues identified by MS, and blue letters (in VHL polymer) represent residues blocked after polymerization. This result indicates that glutamine 164 and 203 are associated with TGase2-induced VHL polymerization.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only examples of the present invention and the present invention is not limited thereto.

Example 1: Antibodies and Reagents

Anti-VHL antibodies from BD Pharmingen (USA, CA, San Diego), anti-TGase2 antibodies from NeoMarkers (USA, CA, Fremont, clone CUB 7402), anti-β-actin was Abcam (UK, Cambridge, Head) Quarter, anti-IGF1R-β and anti-HA-probe (F7) antibodies from Santa cruz Biotechnology (Santa Cruz, CA, USA), anti-PARP and anti-cutting caspase-3 (Asp175) antibodies from Cell It was purchased from Signaling Technologies (Beverly, MA, USA). Lipofectamine RNAiMAX, including Lipofectamine 2000, Lipofectamine RNAiMAX Transfection Reagent, and Stealth Negative Control, was purchased from Invitrogen, Calpeptin (Calbiochem), Leupeptin (USA, MO, St. Louis, Sigma-Aldrich). Co), MG132 (Calbiochem) and FITC-hybrid anti-mouse immunoglobulin (US, Pennsylvania, West Grove, Jackson Immunology Laboratory) were purchased commercially.

Example 2: siRNA ( small interfering RNA ) Treatment TGase2 Suppression of silencing )

SiRNA double strands targeting human TGase2 (5′-AAGAGCGAGAUGAUCUGGAACTT-3 ′: SEQ ID NO: 1) were introduced into cells using lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Cells were harvested 48 hours post infection and cytoplasmic fractions or whole cell extracts were prepared for immunoblot analysis. As a negative control, cells were incubated with lipofectamine RNAiMAX alone and with a universal negative siRNA (Invitrogen).

Example 3: Immunoblotting ( Immunoblotting )

Standardized methods (Kim, DS, Park, SS, Nam, BH, Kim, IH & Kim, SY Cancer Res 66 , 10936-43 (2006)) to perform immunoblot analysis.

Example 4: Electrophoresis Change analysis method Electrophoretic mobility shift assay , EMSA )

A double stranded oligonucleotide probe (5'-AGT TGA GGG GAC TTT CCC AGG C-3 ': SEQ ID NO: 2) containing an NF-kB DNA binding site was purchased from Santa Cruz Biotech. EMSA analysis was performed according to the manufacturer's protocol.

Example 5: Apoptosis Assay

Apoptosis analysis by Annexin V binding was performed using the Annexin V-FITC Apoptosis Detection Kit (CA, Mountain View, BD Biosciences) according to the manufacturer's instructions.

Example 6: TGase2 ⁺ Preparation of Transgenic Mice

The mouse transglutaminase 2 gene was inserted into the native EcoRI-BglII site of pCAGGS comprising human CMV enhancer, chicken β-actin promoter-intron and rabbit β-globin polyadenylation sequence. The 5.8 kb DNA fragment product was cut and prepared following the basic method for pronuclei DNA injection. TGase2 transgene was selected by PCR analysis of genomic DNA extracted from a 1 cm tail clip. PCR was performed using the following primers.

5'-CAC CCT TCG TGT TTG CCG AGG TCA-3 '(SEQ ID NO: 3)

5'-TCT CCG AGG TGT CGT TGC CGA TGT-3 '(SEQ ID NO: 4)

Transglutaminase 2 transgene and wild type allele were obtained with 334 basepairs (pb) and 3,056 bp, respectively. In this way, we identified four founder mice with gene copies of the TGase2 transgene.

Example 7: human RCC In the sample TGase2 And VHL Immunochemical staining

Eleven kidney cancer samples obtained by surgical operation at Ilsan Paik Hospital (Korea, Goyang) were selected from pathology records. Use of the recorded organization was approved by the Ethics Association and the Institutional Review Board (IRB number: 1B-2-1003-010). Immunohistochemical staining was performed using the Ultravision LP detection kit and DAB (Lab Vision Corporation Fremont, CA, USA). Paraffinized human RCC tissue was stained. Tissue sections were deparaffinized in xylene and antigen recovery was heat treated at 121 ° C. for 15 minutes in 10 mM Tris buffer at pH 9.0 with 1 mM EDTA. Sections were fixed in 95% ethanol, treated with 0.3% hydrogen peroxide and fixed by irradiating Ultra V for 15 minutes. Sections were reacted for 1 hour at room temperature with anti-TGase2 and anti-VHL primary antibodies in 0.05% TBS-T with 20% FBS. After washing TBS-T, the sections were sequentially reacted with a primary antibody amplifier and then reacted with horseradish peroxidase (HRP) polymers for 30 minutes each. After TBS-T washing, slides were counterstained using DAB and Mayer's hematoxylin (DakoCytomation).

Example 8: immunochemistry

HA-VHL was visualized by immunochemistry and phase contrast microscopy. Cells were seeded in a 2-well slide chamber in the presence of TGase2 specific siRNA, or in the presence of universal negative siRNA the day before transfection as a control. Cells were transfected with a control empty vector (mock) or HA-VHL expression vector for 24 hours and then washed with PBS (Phosphate buffered saline). Immunchemistry was performed according to standard protocols.

Example 9: human VHL Expression and Purification

Human VHL cDNA was subcloned into modified pET32 (Novagen) using BamHI / XhoI restriction enzymes, a 23-residue polyhistidine tag (MHHHHHHGSLVPRSENLYFQGS, 6xHis) at the amino terminus of VHL, and a 10-residue polyhistidine tag at the carboxy terminus (LEHHHHHHHH, 8 × His) was added. Recombinant VHL was purified by established protocol after overexpression in E. coli strain Rosetta 2 (DE3) (Novagen).

Example 10: Guinea Pig liver TGase2 On by VHL Polymerization

Add guinea pig liver TGase2 (5 μl, U / mL) to 30 μl of reaction buffer (50 mM Tris-HCL, pH 7.5, 150 mM NaCl, 15 mM CaCl ₂ ) containing purified human VHL (5 μg). It reacted at 2 degreeC. Proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining or analyzed by immunoblot.

Experiment result

Experimental Example One: VHL And TGase2 of Inverse correlation

In previous studies we have shown that increased TGase2 expression plays a key role in obtaining drug resistance of cancer cells. In particular, HIF-1α was increased in drug resistant cancer cells under normal oxygen conditions (FIG. 1A). Knockdown of the TGase2 gene reduced the expression level of endogenous HIF-1α protein in NCI / ADR-RES and MDA-MB-231 cells (FIG. 1A). The expression levels of VHL in NCI / ADR-RES and MDA-MB-231 breast cancer cells were lower than in HEK-293 and MCF7 cells (FIG. 1B), and endogenous VHL levels in cancer cell lines were inversely related to TGase2 (FIG. 1B). . Endogenous VHL was inhibited by ectopic TGase2 expression in HEK293 and MCF7 breast cancer cells, and this reduction was either by inhibition of TGase2 with cystamine or by expression of catalytically inactivated TGase2 variant (C277S). It was reversed by inhibition of TGAse2 (FIG. 1C). Levels of ectopic VHL were reduced by co-expression of TGase2, and this effect was lost by inhibition of TGase2 using expression of cystamine or catalytically inactivated TGase2 C277S (FIG. 1D). These results indicate that cross-linking catalytic activity of TGase2 is a major cause of VHL reduction.

Experimental Example 2: TGase Suppression in cancer cells VHL Increases stability

To determine whether the TGase2 inhibitor increases the level of VHL by regulating VHL stability, the present invention confirmed the inhibitory effect of endogenous TGase2 activity on VHL protein levels. NCI / ADR-RES and MDA-MB-231 cells expressing high levels of TGase2 were transiently transfected with HA-VHL expression vectors with TGase2-specific siRNA or cystamine. Levels of ectopic HA-VHL increased in response to TGase2 inhibition by cystamine or gene knockdown, compared to control cells expressing only HA-VHL without treatment with TGase2-specific siRNA or cystamine (FIG. 7). It is consistent with the result of FIG. 2A. Immunohistochemical staining of HA-VHL-transfected NCI / ADR-RES and MDA-MB-231 cells with an anti-HA monoclonal antibody showed that VHL was localized in the nucleus. In comparison, cells co-transfected with TGase2-specific siRNA appeared in the cytoplasmic and nuclear regions of HA-VHL (FIG. 8). Thus, TGase2 appears to target VHL in the cytoplasm where TGase2 is most abundant.

Experimental Example 3: VHL Polymer Proteasome Dependent decomposition ( proteasome - dependent degradation )

In order to identify whether the VHL polymer produced via TGase2-catalyzed cross linking is metabolically degraded, we have identified three different proteolytic inhibitors (proteasome inhibitors, lysosomes) for their ability to inhibit VHL degradation. Inhibitors, calpine inhibitors). Reduction of VHL monomers by TGase2 was inhibited by proteasome inhibitor MG132 treatment, but not by calpain or lysosomal inhibitors (FIG. 2A). To determine whether TGase2 and VHL interact directly, cells were co-transfected with expression vectors of HA-VHL and TGase2, with or without MG132 and using anti-HA antibodies for immunoblot analysis. By immunoprecipitation (Fig. 2b). TGase2 was detected in the VHL precipitate, and the amount of precipitated VHL was increased by MG132 treatment (FIG. 2B). These results indicate that VHL interacts with TGase2 and that the VHL complex is degraded via proteasome-dependent mechanisms (FIG. 2A).

VHL polymer was easily detected and the expression level of the polymer was increased in the cells treated with MG132. In addition, polymerized TGase2 was also detected in anti-HA precipitation, indicating that TGase2 acts as a substrate for polymerization with the ability to catalyze polymerization (FIG. 2C). To confirm that VHL is a substrate of TGase2, purified recombinant human VHL was incubated in vitro with purified guinea pig liver TGase. TGase2 induced the polymerization of VHL in a dose dependent manner (FIG. 9). Recovery by high molecular weight gel fraction (arrow head in FIG. 9) and analysis by MS / MS confirmed that VHL was a major component of the polymerized protein complex (FIG. 10). To identify the crosslinking site of VHL, the analyzed gels were purified by MS into VHL monomers and polymers. FIG. 11 shows amino acid sequences of VHL monomers and polymers, the red letters represent residues identified by MS and the blue letters (in VHL polymer) represent residues blocked after polymerization. These results indicate that glutamine 164 and 203 are associated with TGase2-induced VHL polymerization (FIG. 11).

Experimental Example 4: TGase By suppression VHL Manifestation recovery NF results in inhibition of -κB activity

The activity of NF-κB has been reported to be downregulated by VHL. NF-κB activity was reduced by Tgase2 inhibition in VHL expressed cells. As shown in FIG. 3, NF-κB activity in HA-VHL expressing cells treated with TGase2-specific siRNA or Tgase2 inhibitor cystamine was lower than in HA-VHL-only untreated cells (two-fold and 2.5 times) (FIG. 3A). Simultaneously with the SEAP reporter assay, we performed an electrophoretic mobility shift assay (EMSA) to assess NF-κB DNA binding activity. Reduced NF-κB DNA binding activity expressed with ectopic VHL expression alone or with TGase2 inhibition (FIG. 3B). Apoptosis was induced more efficiently in HA-VHL expressing cells treated with TGase2-specific siRNA compared to cells treated with siRNA alone, measured by relative levels of annexin-V positive staining and cleaved PARP (FIG. 3c).

Previous reports have shown that VHL inhibits the expression of IGF-1R. TGase2 inhibition by cystamine or gene knockdown results in the stability of ectopic VHL, which is associated with reduced IGF-1R levels in drug resistant cancer cell lines (FIG. 3D). These results indicate that downregulation of VHL by TGase2 is associated with Indicates that IGF-1R protein expression is essential.

Experimental Example 5: Downregulation of VHL in TGase2 + Transgenic Mice

In order to confirm the relationship with VHL and TGase2 in vivo, we prepared TGase2 + transgenic mice into which the mouse TGase2 + transplant gene was introduced (FIGS. 4A and 4B). The kidneys of TGase2 + transgenic mice showed no histological abnormalities and the appearance was normal. Analysis of VHL protein levels in kidneys of three different wild-type and TGase2 ⁺ transgenic mice via immunoblot showed lower VHL expression levels in TGase2 + transgenic mice than in wild type mice (FIG. 4C). To determine whether VHL reduction by TGase2 + overexpression affected IGF-1R protein levels, we analyzed kidney samples from wild-type and TGase2 + transgenic mice via immunoblot with anti-IGF-1R antibodies. IGF-1R protein levels were expressed higher in TGase2 + transgenic mice than wild type mice as expected (FIG. 4C). As a result of analysis of NF-κB DNA binding activity by EMSA, NF-κB activity was increased almost three-fold in TGase2 + transgenic mice compared to wild type mice (FIG. 4D). Therefore, VHL levels were decreased in the kidneys of TGase2 + transgenic mice compared to wild type mice, which is associated with high expression levels of IGF-1R. These results indicate that TGase2 + inhibits VHL, thereby inducing NF-κB activity and expression of IGF-1R in vivo.

Experimental Example 6: human kidney cells carcinoma In the sample TGase2 + And VHL Protein level station correlation

In order to confirm the validity of the experimental results in cancer cell lines and transgenic TGase2 + mouse models, that is, to confirm the inverse relationship of TGase2 + and VHL expression, the present inventors have performed immunohistochemical analysis of Renal clear cell carcinoma tissue samples. Expression levels of VHL and TGase2 were confirmed in (FIG. 5). In normal kidney tissues, TGase2 was present in glomerular cells and relatively low in tubular cells, whereas VHL was restricted in renal tubular cells. In RCC carcinoma tissues, expression levels of TGase2 generally increased and expression of VHL decreased. Nine out of eleven human RCC carcinomas showed an inverse relationship between TGase2 and VHL levels. As shown in FIGS. 5A-D, almost complete inversion was seen in cancer 6, and in cancer 3, it was shown in positive regions of TGase2 and VHL. In general, for samples with high expression of TGase2, the expression of VHL was low or absent (Figures 5a, b). In the samples with low expression of TGase2, the expression level of VHL was high (Fig. 5C, d). In invasive front cells, TGase2 expression was higher than the rest of the cancer (FIG. 5E) and the expression of VHL was low or absent (FIG. 5F). These results further demonstrate the inverse relationship between TGase2 and VHL expression.

Attach an electronic file to a sequence list

Claims

(a) treating a VHL-expressing cell with a candidate inhibitor or promoter of TGase2 (Transglutaminse 2);
(b) inducing expression of TGase2; And
(c) comparing one or more of VHL protein levels and polymerization of VHL protein with a control, the method of detecting a TGase2 inhibitor or promoter.

The method of claim 1, wherein the protein levels of any one or more of IGF-1R, HIF-1α and VEGF are further compared to the control by performing in cells further expressing any one or more of IGF-1R, HIF-1α and VEGF. Method comprising the steps.

The method of claim 1 wherein steps (a) and (b) are performed simultaneously or sequentially.

According to claim 1, TGase2 expression is characterized in that the expression of lipopolysaccharide (LPS), ultraviolet rays, glutamate (glutamate), calcium inophore (maitotoxin), retinoic acid (RA), inflammation-induced cytokines, glutamate , Using an oxidative environment or viral infection.

The method of claim 1, wherein at least one of VHL protein levels and polymerization of the VHL protein is detected using an antibody specific for VHL.

The method of claim 2, wherein the protein level of any one or more of IGF-1R, HIF-1α and VEGF is detected using an antibody specific for that protein.

The method of claim 5, wherein any one of Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS or protein chip method Method of detection using one.

The method of claim 1, wherein the TGase2 inhibitor is an anticancer agent.

The method of claim 1, wherein the TGase2 promoter is a therapeutic agent for a viral infection disease.

(a) treating the isolated VHL with a candidate inhibitor or promoter of TGase2 (Transglutaminse 2);
(b) treating the isolated TGase2; And
(c) comparing one or more of VHL protein levels and polymerization of VHL protein with a control, the method of detecting a TGase2 inhibitor or promoter.

11. The method of claim 10, further comprising the step of performing at least one of isolated IGF-1R, HIF-1α, and VEGF to further compare the protein levels of any one of IGF-1R, HIF-1α, and VEGF with the control. How to include.

The method of claim 10, wherein steps (a) and (b) are performed simultaneously or sequentially.

The method of claim 10, wherein at least one of VHL protein levels and polymerization of the VHL protein is detected using an antibody specific for VHL.

The method of claim 13, wherein the protein level of any one or more of IGF-1R, HIF-1α, and VEGF is detected using an antibody specific for that protein.

The method of claim 10, wherein any one of Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS or protein chip method Method of detection using one.