KR20190014785A - Screening method of cancer therapeutic composition using N-acetylglucosamine kinase - Google Patents

Abstract

The present invention relates to a method for screening a composition for treating a cancer, which can be useful as a tool for selecting a candidate substance to be used as a therapeutic agent for a cancer. The method comprises: a step (a) of allowing a cell to be in contact with a candidate substance; a step (b) of measuring an expression level of N-acetylglucosamine kinase (NAGK) of cells in contact with the candidate substance; and a step (c) of selecting the candidate substance inhibiting the expression level of the N-acetylglucosamine kinase.

Description

[0002] Screening methods of cancer therapeutic compositions using N-acetylglucosamine kinase using N-acetylglucosamine kinase [

The present invention relates to a method for screening compositions for treating cancer.

Cancer is one of the greatest threats to the health of human beings, a disease that occurs when cells multiply in an unlimited, uncontrolled way through a series of mutation processes. Although various biochemical mechanisms related to cancer have been identified and the development of advanced detection methods for cancer, mass screening, and therapeutic agents and treatment methods have been continuously developed and applied to clinical practice, Despite the fact that many cancer types still have insufficient response rates for treatment, many patients are exposed to highly toxic anticancer drugs, but the symptoms are less effective or extend their life span. To improve this phenomenon, it is essential to study the development of better anticancer drugs and standard therapies using them.

In this situation, efforts are being made to develop anticancer drugs by identifying various in vivo molecules associated with cancer and screening them for drugs. In the conventional in vivo screening method, a subrenal capsule assay was used. The tumor tissue of the patient was excised, cut into small pieces, inserted underneath the renal capsule of the mouse, treated with an anticancer agent to change the size of the transplanted tumor . However, there is a problem in that it is difficult to apply the effect of the anticancer drug administered to the mouse due to the long time for screening the effect depending on the dose and the cycle, so that it is not effective in screening various kinds of anticancer drugs.

N-acetylglucosamine kinase (GlcNAc kinase; NAGK) was first discovered in 1970, and mRNA and protein of N-acetylglucosamine kinase are expressed in various cell lines and tissues. NAGK is characterized by a V-shaped fold consisting of two domains with the β6βαβαβ sequence and belongs to the sugar kinase, heat shock protein 70 and actin superfamily . As the domain binds to the substrate, a structural change occurs in which the angle of the gap between the two domains decreases.

The function of NAGK is to produce GlcNAc-6 phosphate by phosphorylating GlcNAc in the GlcNAc recycling pathway. GlcNAc-6-phosphate is then used to phosphorylate N- / O-glycans, glycolipids and glycosaminoglycans is known to be used to produce UDP-GlcNAc, which is used in the synthesis of glycosaminoglycans.

Dynein is a cytoskeletal motor protein that moves along microtubules in cells, and is known to carry a variety of cellular materials, provide important forces and displacements in mitosis, and induce pulsation of eukaryotic cell cilia and flagella.

Transforming growth factor-β (TGF _β ) superfamily signal transduction processes are involved in various functions such as cell growth, differentiation, apoptosis, motility and invasion, extracellular matrix (ECM) generation, angiogenesis, and immune response. The initiation and progression of the epithelial mesenchymal transition (EMT) occurs at the transcriptional level, where TGFβ induces EMT via SMAD, and SMAD complex migrates into the nucleus and cooperates with other transcription factors to induce EMT.

Accordingly, the present inventors have made intensive research to discover a method for screening a composition for treating cancer, and as a result, they have confirmed that NAGK and dinne are related to cell division, EMT, and cell migration, thereby completing the present invention.

Korean Patent Publication No. 10-2011-0038778

It is an object of the present invention to provide a method for screening compositions for treating cancer, which measures the expression level of N-acetylglucosamine kinase.

Another object of the present invention is to provide a method for screening a composition for treating cancer which quantifies N-acetylglucosamine kinase complex.

An aspect of the present invention provides a method for producing a cell, comprising: (a) contacting a candidate substance to a cell; (b) measuring the expression level of N-acetylglucosamine kinase in cells contacted with the candidate substance; And (c) selecting a candidate substance that inhibits the expression level of the N-acetylglucosamine kinase, as compared to a negative control not treated with the candidate substance.

The first step (a) of the present invention is to bring the candidate substance into contact with the cell.

According to one embodiment of the present invention, the cell may be a cancer cell.

The cancer cells of the present invention can be used for the treatment of cancer, colon cancer, cervical cancer, pancreatic cancer, non-small cell lung cancer, liver cancer, bone cancer, skin cancer, head cancer, Cancer, endometrioid cancer, thyroid cancer, pituitary cancer, soft tissue sarcoma, soft tissue sarcoma, urethral cancer, penile cancer, uterine cancer, endometrial carcinoma, endometrial carcinoma Cancer cells selected from the group consisting of prostate cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma. Preferably, the cancer cell is selected from the group consisting of lung cancer, non-small cell lung cancer, brain tumor, adrenal cancer, ureter cancer, renal cell carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma May be cells of cancerous disease.

According to one embodiment of the present invention, the cell may be selected from the group consisting of mouse neuronal cell line GT1-7, human kidney cell line HEK293T, human glioma cell line U87, and human lung cancer cell line A549 .

The candidate substance of the present invention refers to a substance which is applied to a cancer patient and can improve or change the symptom of a patient by cancer, and the substance exhibiting the above characteristics such as an anticancer drug and an antitumor antibiotic can be included without limitation.

As used herein, the term "contact " is a generic term for it and means that one or more substances and / or cells are located at a distance which is close enough to affect other substances and / or cells. Contact can occur in vitro. For example, combining cells with one or more substances in a test tube or other container. Also, contact can occur in cells or in situ. For example, the expression of a recombinant polynucleotide encoding a polypeptide in a cell affects the expression of the polypeptide in the cell and / or the cell lysate.

The second step (b) of the present invention is to measure the expression level of N-acetylglucosamine kinase in the cells contacted with the candidate substance.

 The method for measuring the expression level of N-acetylglucosamine kinase can be used without limitation as long as it is generally used in the art for measuring protein expression. However, PCR, reverse transcription polymerase chain reaction (RT- Acetylglucosamine kinase mRNA expression levels were measured by PCR, real-time PCR, RNase protection assay (RPA), microarray or northern blotting Is preferably measured.

The last step (c) of the present invention is to select candidates that inhibit the expression level of the N-acetylglucosamine kinase as compared to the negative control not treated with the candidate substance.

Acetylglucosamine kinase is inhibited in comparison with a negative control group that is not treated with the candidate substance, it can be selected as a candidate substance, but the expression level of N-acetylglucosamine kinase is inhibited by 20% or more Candidate substances are preferable, and candidates that suppress the expression level of N-acetylglucosamine kinase by 40% or more relative to the negative control are most preferable.

As used herein, the term "treatment" refers to the amelioration of a disease or disorder. This includes the treatment of a full spectrum of the disease or disorder and any disease that afflicts the patient, including relief, healing and prevention of symptoms resulting therefrom.

Another aspect of the present invention is a method for producing a cell, comprising: (a) contacting a candidate substance to a cell; (b) quantitatively analyzing N-acetylglucosamine kinase (NAGK) complex formed in cells contacted with the candidate substance; And (c) selecting a candidate substance that inhibits the formation of the complex, as compared to a negative control to which the candidate substance has not been treated.

The first step (a) of the present invention is to bring the candidate substance into contact with the cell.

The term "contact" used in this step is used interchangeably with the meaning of "contact"

The second step (b) of the present invention is to quantitatively analyze the N-acetylglucosamine kinase (NAGK) complex formed in the cells contacted with the candidate substance.

The method for quantitatively analyzing N-acetylglucosamine kinase complex can be used without limitation as long as it is generally used in the art for quantifying protein, but it may be applied by Western blotting, immunocytochemistry, immunohistochemistry, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, immunofluorescence, ouchterlony, It is preferable to quantitate the N-acetylglucosamine kinase complex with a complement fixation assay or a protein chip.

The final step (c) of the present invention is to select candidates that inhibit the formation of the N-acetylglucosamine kinase complex as compared to the negative control without treatment of the candidate substance.

Acetylglucosamine kinase complex formation can be selected as a candidate substance as long as it inhibits the formation of the N-acetylglucosamine kinase complex in comparison with the negative control group which is not treated with the candidate substance, but the formation of N-acetylglucosamine kinase complex is inhibited by 20% Candidate substances are preferable, and candidate substances that inhibit the formation of N-acetylglucosamine kinase complex by 40% or more relative to the negative control are most preferable.

According to one embodiment of the present invention, the complex may include one or more proteins selected from the group consisting of dynein, Lis1, NudE1 and SMAD2.

According to one embodiment of the present invention, the dinine may be a dynein light chain roadblock-type 1 (DYNLRB1).

According to one embodiment of the present invention, (b) can be performed in a kinetochore of metaphase chromosome.

According to one embodiment of the present invention, step (b) can be performed in the cytoplasm of the transformed growth factor-β (TGFβ) -treated cell after step (a).

The cancer screening composition screening method of the present invention can be usefully used as a tool for screening candidates for use as therapeutic agents for cancer.

1 is a fluorescence photograph showing the expression of NAGK in liver (A), electricity (B), middle (C), late (D), late (E) and cytoplasmic cleavage (F).
FIG. 2 is a fluorescence photograph showing the interaction of NAGK-DYNLRB1 in liver (A), electricity (B), middle (C), late (D), late (E) and cytoplasmic cleavage (F).
3 is a fluorescence photograph showing the interaction of NAGK-Lis1 in the liver (A), the liver (B), the middle (C), the late (D), the liver (E) and the cytoplasmic cleavage (F).
4 is a fluorescence image showing the interaction of NAGK and DYNLRB1 (A) in GT1-7 cells and the interaction of NAGK-Dinein complex with Lis1 (B) or NudE1 (C) in HEK293T cells.
FIG. 5 shows that in the cell division of HEK293T cells, a pair of NAGK-tubulin (A), NAGK-Lis1 (B) and NAGK-NudE1 (C) And fluorescence photographs showing that NAGK and Lis1 (D) are dispersed in the cytoplasm.
FIG. 6 shows that the NAGK-Dnae interaction (Aa) was distributed over a majority of chromosomes (Ab) and the NAGK-Lis1 interaction (Ba) was distributed over more than half of the chromosomes (Bb) in the middle cell division of HEK293T cells Fluorescence photographs and graphs.
Figure 7 is a fluorescence image showing that the NAGK-Dinein complex is located at the same position as Lis1 (A) and NudE1 (B).
FIG. 8 is a fluorescence image showing that NAGK and diner are located at the same place of the cytoplasm in the mid-cell division of HEK293T cells by the ICC (A) and PLA (B) methods.
FIG. 9 shows photographs showing the division of cells (Ab) by the co-transfection of the pDsRed2 vector only transfected cells (Aa) and pDsRed2 vector / NAGK shRNA, and the ratios (Ba and Bb) of the divided cells Graph.
FIG. 10 is a fluorescence image showing that NAGK and SMAD signals are located at the same positions in U87 (A) and A549 (B) cells.
Figure 11 shows that cluster-like SMAD2 production and exogenous NAGK and SMAD2 are located at the same location in the cytoplasm (C) after 5 minutes (A) and 15 minutes (B) of treatment with TGFbeta in A549 cells Fig.
FIG. 12 is a fluorescence photograph showing the interaction of NAGK-SMAD2 and the control group (A) after 5 minutes (B) and 15 minutes (C) of TGFβ treatment of A549 cells.
Fig. 13 is a graph showing the interaction of NAGK-SMAD2 after 5 minutes with TGF? Treatment on A549 cells.
14 is PLA (NAGK-SMAD2), DYNLRB1 ICC and DAPI control staining fluorescence photograph of A549 cells.
Figure 15 shows z-axis serial photodissection images obtained in PLA (NAGK-SMAD2), DYNLRB1 ICC and DAPI contrast dye samples, reconstructed in 3D, and then reconstructed in NAGK- A fluorescence image showing a tripartite complex of SMAD2-Dynein (DYNLRB1).
FIG. 16 is a graph showing the fluorescence of NAGK-SMAD2-Dynein (DYNLRB1) in a fluorescence photograph and a 3D model showing that the NAGK-SMAD complex is located in the early endosome (A) in A549 cells transfected with GFP-labeled EEA1 vector 3 shows a fluorescence image showing the three-way complex (B).
17 is a fluorescence image showing the nuclear SMAD2 signal and NAGK small domain (E) of U87 cell (A / B) and A549 cell (C / D) according to knockdown of NAGK.
Figure 18 shows the expression of the SMAD2 vector and GFP vector (A), Flag labeled SMAD2 vector and GFP labeled wild type (WT) NAGK vector (B and C), GFP labeled WT NAGK vector (D) This is a fluorescence image showing the nuclear SMAD2 signal according to the transfection of the GFP-labeled NAGK small domain vector (E).
FIG. 19 is a fluorescence photograph showing fibronectin signals of A549 cells (A / B) and U87 cells (C / D) according to the knockdown of NAGK.
Figure 20 shows that NAGK-NudC interaction (A), NAGK-DYNLRB1 complex is present at the same position as NudC (B), and NAGK-NudC complex is located at the same position as DYNLRB1 in migrating HEK293T cells (C) and NAGK-DYNLRB1 complex are present at the same position (D) in NudC, leading neurites and nuclear membrane.
FIG. 21 is a fluorescence image showing that PLA signals of the nuclear membrane and the PLA signals of the bifurcate are present at the same positions as the corresponding ICC signals A and B from different angles.
In addition, the interaction of NAGK-NudC (A) and NAGK-DYNLRB1 (B) was confirmed by PLA method in mouse embryo (E-17.5) cerebral cortex.
FIG. 22 is a fluorescence image showing that the interaction between NAGK-NudC (A) and NAGK-DYNLRB1 (B) is in the same position in the nuclear membrane of the dendritic cell and the third protein of the tertiary complex.
23 shows the results of microinjection of the pDsRed2 labeled NAGK vector (A), pDsRed2 vector (B) or NAGK shRNA vector and pDsRed2 vector (C) to the ventricular zone of the E14.5 mouse embryonic brain And a fluorescence image introduced by electroporation.
24 is a graph showing counts of cells injected into the cortical plate (CP), the middle region (IZ), and the ventricular system (VZ).
25 is a fluorescence image showing that A549 cells were transfected with a DsRed2 control vector (A), a DsRed2-labeled NAGK vector (B), a NAGK shRNA vector and a DsRed2 control vector (C) to induce a mesenchymal transition .
26 is a graph showing the number of transfected cells of cells transfected with NAGK vector.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  One. NAGK's  Methods for promoting cell division, cancer development and cell migration effect

≪ 1-1 > Antibody preparation and detection method

Antibodies to be used for the experiment were prepared as shown in Table 1 below.

Primary antibody name Classification of experiments Dilution
ratio Where to buy Remarks chicken polyclonal NAGK ICC 1: 1000 Sigma, USA mouse monoclonal NAGK PLA 1:10 Santa Cruz Biotechnology, USA rabbit polyclonal NAGK PLA 1:50 GeneTex, USA rabbit polyclonal DYNLRB1 / LC7 ICC 1:50 Proteintech Group, USA PLA 1:25 Proteintech Group, USA mouse polyclonal dynein IC1 PLA 1:25 Santa Cruz Biotechnology rabbit polyclonal Lis1 ICC 1: 200 Santa Cruz Biotechnology PLA 1:25 Santa Cruz Biotechnology rabbit polyclonal NudE1 ICC 1:50 Proteintech Group, USA rabbit polyclonal CENP-B ICC 1: 500 Abcam, UK PLA 1: 500 Abcam, UK mouse monoclonal alpha-tubulin ICC 1:10 DSHB at University of Iowa, USA broth preparation PLA 1:10 DSHB at University of Iowa, USA rabbit polyclonal lamina B ICC 1: 1000 Young In Frontier Inc., Korea PLA 1: 1000 Young In Frontier Inc., Korea Rabbit NudC ICC 1:50 Proteintech Group, USA PLA 1:50 Proteintech Group, USA Mouse fibronectin ICC 1:25 DSHB at University of Iowa, USA Mouse anti-Flag ICC 1: 500 Sigma, USA Rabbit SMAD2 ICC 1:50 Proteintech Group, USA PLA 1:50 Proteintech Group, USA

The primary antibodies were detected using fluorescently labeled secondary antibodies Alexa Fluor 488 and 568 (Invitrogen, USA).

<1-2> Cell culture

GT1-7 cells, HEK293T cells, U87 cells and A549 cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and 1% penicillin streptomycin on polylysine coated glass cover slips, respectively.

&Lt; 1-3 > Transfusion ( transfection ) Way

According to the manufacturer's instructions it was infected with Lipofectamine ^® 2000 by using the reagent (Invitrogen) transfection vector injected into the cells of Examples 1-2 at all times.

<1-4> Immunocytochemistry Immunocytochemistry ; ICC)

The cells of Example 1-2 were routinely cultured in PBS (20 mM sodium phosphate buffer, pH 7.4 and 0.9% NaCl) containing 4% paraformaldehyde at room temperature for 10 minutes and then cultured in methanol at -20 ° C for 20 minutes To fix the cells.

The immobilized cells were cultured for 4 hours in preblocking buffer (5% normal chlorine plasma, 0.05% Triton X-100, PBS and pH 7.4) for 6 hours or longer, treated with the antibody of Example 1-1, &Lt; / RTI &gt; was fixed on a glass slide.

<1-5> Proximity Ligation Assay PLA )

The cells of Example 1-2 were immersed in a preblocking buffer (PBS containing 5% normal goat serum and 0.05% Triton X-100, pH 7.4) containing the primary antibody of Example 1-1, At 4 ° C overnight and then washed three times for 20 minutes with preblocking buffer at room temperature to perform in situ PLA using a Duolink assay kit (Olink Bioscience, Sweden). Then, PLA probe anti-mouse MINUS and PLA probe anti-rabbit PLUS were treated with the secondary antibody of Example 1-1, in which the oligonucleotide diluted with preblocking buffer was conjugated, and the thermostatic chamber at 37 ° C for 2 hours.

For the combination of PLA and ICC, the PLA step was performed first and then the primary antibody of Example 1-1 was added and kept overnight at 4 ° C. Subsequently, the secondary antibody of Example 1-1, to which the fluorescent substance was conjugated, was added according to Example 1-4, and the primary antibody was detected by storing at room temperature for 1 hour.

<1-6> NAGK's  Knocked-down method

The HEK293T cells of Example 1-2 were transfected with a vector expressing NAGK small hairpin (sh) RNA according to the above Example 1-3, and the NAGK was knocked-down. A vector expressing the red fluorescent protein pDsRed2 (Fig. 9Aa) was co-transfected with the control and NAGK shRNA vectors (Fig. 9Ab). Then, the cells were cultured for 48 hours to express the protein.

<1-7> Image analysis

Fluorescence (epifluorescence) images were taken using an Olympus microscope BX53 equipped with UNA, BNA and GW filter sets. Images were acquired at high resolution using CellSens Imaging Software (Olympus) and a digital camera DP72 (Olympus, Japan).

<1-8> 3D model analysis

A z-axis serial photodissection image obtained by LAS X software combined with a default 3-dimensional deconvolution algorithm was taken with a super sensitive high resolution confocal laser scanning microscope (Leica TCS SP8) ) Were reconstructed in 3D, and 3D model analysis was performed by acquiring images viewed from different angles of the 3D model.

<1-9> Statistical analysis

Cells (n = 500) obtained from 2 to 3 different positions on each coverslip at 48 hours and 72 hours after infection of the cells were counted and the number of transfected cells was determined as the ratio (%).

<1-10> in utero electroporation ( IEU ) Way

14.5 days pregnant mice were anesthetized with isoflurane, and they were implanted with 2-3 μg of DNA into the ventricle of the fetus by laparotomy. After 5 electric shocks (45V, 50ms long pulses, 950ms intervals) were applied using a square-wave pulse generator (ECM 830; BTX, USA), the brains were separated and immunohistochemically stained after 24 hours. For immunohistochemical staining, embryonic brain was treated with 4% paraformaldehyde at 4 ° C for 2 hours and stored at 4 ° C overnight in phosphate buffered saline (PBS) containing 30% sucrose. After cryosections (10 μM) were placed in Tissue-Tek OCT (Sakura Finetek, USA), the brains were attached to MAS-coated glass slides (Matsunami glass, Japan) and citrate buffer (10 mM, pH 6.0) , Heat-treated at 95 ° C for 5 minutes, and treated overnight in PBS containing 5% normal donkey serum. The following day, the primary antibody (anit-GFP; Abcam; 1: 1000) was added, treated overnight, and the Alexa Fluor 594-labeled secondary antibody was added (Jackson ImmunoResearch Laboratories; 1: 400) for reaction at room temperature for 2 hours. Images were then obtained by observation with a fluorescence microscope, which was washed 2-3 times with PBS.

Example  2. NAGK's  Cell division promoting function analysis

<2-1> Before the cell division, NAGK's  Confirmation of expression

In order to confirm the expression pattern of NAGK in cell division, ICC analysis and DAPI staining were carried out according to Example 1-4 using anti-NAGK and anti-tubulin antibody at each cell division stage of HEK293T cells Respectively.

As a result, the expression of NAGK was observed in the mesenchymal cells, and the cytoplasmic NAGK signal was mainly located in the microtubule (FIG. 1A). In the electricity where the chromosomes started to condense, some NAGK signals were located at the same location as the microtubules of the nuclear membrane (FIG. 1B). During the metaphase, in which spindle microtubules are sequentially attached to the condensed chromosomal kinetochores, the NAGK signal is transmitted through thin MT spindle shafts and positive ends of chromosomes attached to chromosomes ) (Fig. 1C). A similar distribution pattern was observed during anaphase (FIG. 1D) and telophase (FIG. 1E). As they entered the cytoplasmic breakdown stage, NAGK was redistributed throughout the cells, including the nucleus (Fig. 1F).

In other words, it was confirmed that NAGK plays a role in fixing the spindle to the chromosome of the nuclear membrane and the nuclear fission machine.

<2-2> NAGK  And Of DYNLRB1  Confirm interaction

After in situ localization of NAGK and dinine interactions at various cell division stages by performing PLA according to Example 1-5 on NAGK and dynein light chain roadblock type 1 (DYNLRB1) in HEK293T cells, Tubulin ICC and DAPI counter-staining were performed according to Examples 1-4 above.

As a result, PLA signals were also observed in other z-axial layers of the cells, but mainly in the quatorial nuclear layer. NAGK-DYNLRB1 interactions (PLA analysis) in interphase cells were found in the cytoplasmic region with many PLA signals around and around the nuclear membrane surface (Fig. 2A). Also, when the chromosomes started to condense, NAGK-dynein interactions were observed in the eukaryotic membrane (Fig. 2B).

These results suggest that NAGK-DYNLRB1 interactions may play an important role in the nuclear envelope breakdown (NEB) before nuclear nuclear breakdown (NEB) And that it is a component of the dynein-adapter protein complexes that guide the embedding.

In the middle stage, the chromosomes were aligned in the equatorial plane, and NAGK-dynein interaction appeared in the mitotic axis, chromosomes and peripheral cytoplasmic areas (Fig. 2C) and NAGK-DYNLRB1 binding (PLA signal ) Were observed at similar locations during late, terminal and cytoplasmic cleavage (Figures 2D-2F).

In other words, it was confirmed that NAGK plays a role in regulating dinine function in mitotic spindle and chromosomal kinetochore (KT).

<2-3> In the nuclear membrane during cell division NAGK  And Of Lis1  Confirm interaction

In order to investigate the possibility of interaction between NAGK and dinne-Lys complexes, NAGK-Lis1 PLA was performed on HEK293T cells in each cell division stage according to the above Example 1-5, Tubulin ICC and DAPI control staining were performed.

As a result, NAGK interacted with the cytoplasmic Lis1 and the nuclear outer membrane of the liver (FIG. 3A), and the NAGK-Lis1 complex was also observed in the nuclear membrane of the electric cell (FIG. 3B). In the middle stage, the NAGK-Lis1 PLA signal was present in chromosomal alignment and in the same position as the spindle, and some signals were also observed in astral sites (FIG. 3C). Similar NAGK-Lis1 interactions have been found in mitotic spindles, chromosomes and astral cytoplasmic areas during late and late (FIGS. 3D and 3E). During cytoskeleton, NAGK-Lis1 PLA signal was also observed at the chromosomal and other cytoplasmic sites (Fig. 3F).

Namely, through the above-mentioned results, it can be seen that the NAGK-Lis1 interaction (PLA signal) and the position of the NAGK-dinine complex during cell division are similar in the electric and mid- .

<2-4> In the nuclear envelope during nuclear impingement NAGK  And Dineen - Lis1 - NudE1  Confirmation of complex interaction

In order to investigate NAGK-Dinein interaction during electrical NEB, NAGK-DYNLRB1 PLA was performed in the hypothalamic neuron cell line GT1-7 according to the above Example 1-5, and lamin-B ICC and DAPI control staining was performed.

In order to examine the interaction with dynein-Lis1-NudE1 complex and the role of NAGK in NEB, NAGK-DYNLRB1 PLA was performed on HEK293T cells according to Example 1-5, followed by Lis1 ICC and DAPI control staining counter-staining.

As a result, the NAGK-dynein PLA signal was observed around the mitotic eukaryotic membrane, and when the nuclear lamina was degraded and enriched in the recessed region during the onset of electrical NEB, the strong in situ lamin B signal (Fig. 4A) And the NAGK-dynein complex was found at the same position as the Lis1 ICC spot (puncta) (Fig. 4B) in the nuclear membrane during the electrophoresis.

In a similar experiment on HEK293T cells, it was also observed that NAGK-Dynein interaction was present at the same position as the NudE1 signal (Fig. 4C).

That is, the interaction of NAGK and dynein-Lis1-NudE1 complex was confirmed in the electrophoresis of NEB through the above results.

<2-5> Microtubule  After nuclear destruction Nu1E1 - NAGK  Complex and Lis1 - NAGK  Confirmation of co-existence of complex

HEK293T cells of Example 1-2 were routinely treated with 70 ng / ml nocodazole (Sigma, USA) for 18.5 hours in the same medium to inhibit cell division in G2 / Tubulin and anti-NAGK antibodies were used to perform tubulin ICC and DAPI control staining according to Example 1-4 above.

As a result, after the treatment with nocodazole, the proportion of the electric cells increased. Microtubules were destroyed by ICC, and tubulin staining was mostly present in the centrioles. The NAGK signal was also concentrated at this location, overlapping the signal of tubulin (Fig. 5A).

NAGK / Lis1 (FIG. 5B) and NAGK / NudE1 (FIG. 5C) were in the same position, and cytoplasmic signals for NAGK and Lis1 were observed in the absence of nocodazole (Fig. 5D).

That is, it was confirmed through the above results that NAGK and dynein-Lis1-NudE1 complex interactions and NAGK are involved in nuclear membrane dynein-mediated NEB during the G2 / divider stage.

<2-6> In the mid-term chromosome NAGK  And dynein - Lis1 - NudE1  Confirmation of complex interaction

In order to analyze the position of PLA signal of NAGK-dynein and NAGK-Lis1 in medium-term cells, Example 1-5 was carried out.

As a result, both NAGK-dynein and NAGK-Lis1 were observed in the spindle and chromosomes (Figs. 6Aa and 6Ba) and most NAGK-dynein and NAGK-Lis1 PLA signals (Fig. 6Ab and Fig. 6Bb) Lt; / RTI &gt;

In other words, it was confirmed from the above results that NAGK transports the chromosome toward the cell cortex (cell edge) in association with dynein and Lis1 in the middle of cell division.

In addition, immunostaining and DAPI counter-staining of Lis1 or NudE1 after NAGK-DYNLRB1 PLA revealed that the NAGK-dynein complex was co-located with Lis1 (Fig. 7A) and NudE1 (Fig. 7B).

That is, it was confirmed that NAGK is present in the same position with the DYNLRB1-Lis1-NudE1 complex on the chromosome of the mid-stage cells.

<2-7> In the medium term, NAGK - dynein  Confirm the existence of complex

As a result of double staining of HEK293T cells of Example 1-2 at all times with an antibody against NAGK and an electrophile-labeled protein CENP-B, it was confirmed that CENP-B and NAGK coexist in the intermediate cell cell (Fig. 8A).

After NAGK-dynein PLA, CENP-B immunostaining and DAPI counter-staining were performed to locate the NAGK-Dinein complex in the mobilis (Fig. 8B).

As a result, the NAGK-dynein complex and CENP-B were present at the same position on the chromosome in the middle stage, and the CENP-B signal was observed in the centrosomes.

That is, it was confirmed from the above results that NAGK and dinine interacted in medium term kinetochores.

<2-8> NAKG's  Confirmation of cell division delay due to knockdown

In order to investigate the role of NAGK in cell division, NAGK was knocked down according to Example 1-6, and the biomedical image of the transfected cells was obtained according to Example 1-7, The same incubation area was imaged after 24 hours.

As a result, most of the cells transfected with the control pDsRed2 vector cleaved (Fig. 9Aa) during the 24 hour period (from 48 hours to 72 hours in culture), whereas in contrast, a number of cells transfected with the NAGK shRNA vector (Fig. 9Ab).

In addition, as a result of the statistical analysis (n = 500) according to the above Example 1-9, it was confirmed that the control cells transfected with pDsRed2 for 24 hours proliferated at the same rate as that of the non-transfected cells, The ratio of transformed cells to NAGK shRNA vector remained almost the same after 72 hours (Fig. 9Ba), but the ratio of transfected cells to total cells in NAGK shRNA vector decreased about half It was confirmed that it was not disrupted at normal speed (Fig. 9Bb).

That is, it was confirmed from the above results that the interaction between NAGK and dynein-Lis1-NudE1 complex was reduced by the knockdown of NAGK, thereby delaying the cell division process.

Example  3. NAGK's Cancer incidence  Check the effect

<3-1> NAGK  And Of SMAD2  Identical location

In order to confirm the expression pattern of NAGK and SMAD2 in cancer development, U87 cells and A549 cells of Example 1-2 were double-stained with anti-NAGK and anti-SMAD2 antibody.

As a result, it was confirmed that the NAGK immuno signal and the SMAD immuno puncta exist at the same position in the cytoplasm (FIG. 10).

<3-2> NAGK  And Of SMAD2  Confirmation of binding and nuclear transport effects

In order to confirm the role of NAGK in nuclear transport of SMAD2, A549 cells of Example 1-2 were treated with TGFβ and double stained with anti-NAGK and anti-SMAD2 antibody.

As a result, a cluster-like SMAD2 signal was generated in the cytoplasm 5 minutes after treatment, and this signal was in the same position as the NAGK signal (Fig. 11A). After 15 minutes, a strong nuclear SMAD2 signal was observed, while the SMAD2 signal in the cytoplasm was slightly reduced, but they were in the same position as the NAGK signal (FIG. 11B).

In addition, A549 cells of Example 1-2 were transfected with GFP-labeled NAGK vector, treated with TGFβ for 5 minutes, and double-stained with anti-NAGK and anti-SMAD2 antibody.

As a result, exogenous NAGK was located at the same position as the SMAD2 immunostaining site in the cytoplasm, and a strong signal was observed particularly in the peripheral region (TGFβ receptor of the cell membrane) (FIG. 11C).

That is, it was confirmed that NAGK is complexed with SMAD2 and involved in nuclear transport of SMAD2.

<3-3> NAGK - SMAD2 - Dynein ( DYNLRB1 ) Samwon  Identification of the tripartite complex

In order to confirm whether NAGK, SMAD2 and dinne form a complex, A549 cells of Example 1-2 were treated with TGFβ, PLA (NAGK / SMAD2) was performed according to Example 1-5, ICC and DAPI control staining of DYNLRB1 according to Examples 1-4 was performed. In order to perform 3D model analysis on the PLA (NAGK-SMAD2), DYNLRB1 ICC, and DAPI contrast dye samples (Fig. 14), images viewed from different angles were obtained according to Examples 1-8.

As a result, NAGK-SMAD2 interaction was significantly increased after 5 minutes of treatment compared to the control (Fig. 12A) (Fig. 12B and Fig. 13). The NAGK-SMAD2 complex was located at the same position as the DYNLRB1 immune signal, and the NAGK-SMAD2-Dynein (DYNLRB1) tripartite complex (Fig. 12C) ) (Fig. 15).

<3-4> Initial In the early endosomes NAGK - SMAD2  Confirm interaction

In order to confirm the interaction of NAGK and SMAD2 in the early endosome, A549 cells were transfected with GFP-labeled EEA1 (early endosome marker) vector, and after 24 hours, PLA (NAGK-SMAD2) of Example 1-5, anti-GFP ICC of Example 1-4 and DAPI control staining Respectively. In order to perform 3D model analysis on the PLA (NAGK-SMAD2), ICC and DAPI contrast dye samples, images viewed from different angles were obtained according to Examples 1-8.

As a result, it was confirmed that the PLA signal (NAGK-SMAD complex) was located in the initial endosome (Fig. 16A), and a number of tripartite interactions thereof were confirmed in the 3D model (Fig. 16B).

<3-5> NAGK  By knockdown Of SMAD2 Nuclear immobility  Confirmation of inhibition

In order to confirm whether TGFβ inhibits the migration of SMAD2 to the nucleus when NAKG is knocked down, the NAGK shRNA vector and the DsRed2 control vector are co-transfected according to Example 1-6, And the NAGK was knocked down. After 72 hours of transfection, TGFβ was treated for 30 minutes and stained with anti-SMAD2 antibody.

As a result, NAGK shRNA-transfected cells showed no SMAD2 signal in the nucleus (Fig. 17A), but strong SMAD2 signal in nucleus in the cells transfected with the DsRed2 control vector (Fig. 17B).

In addition, in the case of A549 cells of Example 1-2, NAGK knockdown cells showed no expression of SMAD2 in the nucleus (Fig. 17C), whereas in the case of the control group transfected with DsRed2 vector, It was confirmed that strong SMAD2 expression was present in the nucleus as in the non-transfected cells (Fig. 17D). Furthermore, when transfected with a GFP-labeled NAGK small domain vector, there was no SMAD2 signal in the nucleus.

That is, it was confirmed that NAGK is involved in the nuclear internalization of SMAD2, and in particular, the small domain of NAGK plays an important role (FIG. 17E).

<3-6> NAGK  Over-expression Of SMAD2 Nuclear immobility  Confirmation of facilitation effect

In order to confirm whether NADK overexpression promoted nuclear internalization of SMAD2, SMAD2 vector and GFP vector (FIG. 18A), Flag-labeled SMAD2 vector and GFP-labeled wild type (GFP-labeled WT NAGK vector (FIG. 18D) or GFP-labeled NAGK small domain vector (FIG. 18E) and treated with anti-GFP and anti ICC was performed with anti-SMAD2 (Figure 18A, Figure 18B and Figure 18D) or anti-Flag antibody (Figure 18C and Figure 18E).

As a result, exogenous WT NAGK transferred exogenous SMAD2 to the nucleus (Fig. 18B and Fig. 18C). However, when transfected with the WT NAGK vector, the nuclear SMAD2 signal was not induced from the exogenous SMAD2 protein, and SMAD2 protein did not migrate to the nucleus 24 hours after the transfection. In addition, the NAGK small domain showed a dominant negative effect, indicating that there was no nuclear SMAD2 signal due to exogenous SMAD2 in the vector transfected with this vector (Fig. 18E).

That is, it was confirmed from the above results that the SMAD signal pathway was not activated by overexpression of only NAGK or SMAD2 alone.

<3-7> NAGK  By knockdown Liver  implementation( mesenchymal  transition

In order to confirm the effect of inhibiting mesenchymal transition by NAGK knockdown, A549 cells of Example 1-2 were cultured in NAGK shRNA vector and DsRed2 vector (Fig. 19A) or DsRed2 control vector (Fig. 19B) were transfected alone. Twenty-four hours after the transfection, TGFβ was treated and further cultured for 48 hours to induce mesenchymal characteristics and stained with anti-fibronectin antibody.

As a result, fibronectin expression in the cells transfected with the NAGK shRNA vector was very weak, but the cells transfected with the DsRed2 vector showed a very strong signal.

In addition, as a result of performing the same experiment using the U87 cell of Example 1-2, NAGK knockdown cells (FIG. 19C) did not express fibronectin, but cells injected with the DsRed2 vector (FIG. 19D) showed strong fibronectin expression Respectively.

In other words, it was confirmed that NAGK plays an important role in mesenchymal transition through the above results.

Example  4. NAGK's  Identify the role of cell migration

<4-1> During cell migration NAGK - NudC  Confirm interaction

To confirm the interaction of NAGK-NudC during cell migration, NAGK-NudC PLA was performed on migrating HEK293T cells of Example 1-2, followed by staining with anti-DYNLRB1 antibody. The nuclei were stained with DAPI Respectively.

As a result, NAGK-NudC interaction was observed in the nuclear membrane and cell cortex, and NAGK-NudC was located at the same position as the DYNLRB1 immune signal (Fig. 20A).

That is, it was confirmed that NAGK binds with NudC and dynein during cell migration, and is located in the nuclear membrane and the cell membrane layer.

<4-2> During cell migration NAGK - NudC - Of DYNLRB1 Samwon  Confirmation of tripartite interaction

To confirm the interaction of NAGK-NudC-DYNLRB1 during cell migration, NudC ICC and DAPI staining were performed after NAGK-DYNLRB1 PLA.

As a result, the NAGK-DYNLRB1 complex was located at the same position as the leading migratory projections of the cell cortex and the NudC immune signal in the nuclear membrane of the migrating cells (Fig. 20B).

That is, through the above results, it was confirmed that NAGK-NudC-DYNLRB1 tripartite interaction occurs during migration and NAGK regulates the interaction of dynein-NudC in cell migration and nuclear migration.

<4-3> During cell migration NAGK  And NudC - Dynein  Confirmation of complex interaction

To confirm the interaction of NAGK and NudC-Dynein complex during cell migration, NAGK-NudC PLA was performed on neurons migrating from cultured neurosphere aggregates.

As a result, NAGK-NudC PLA spots were observed in the neuronal cell body and leading neuritic projections of migrating neurons, and the NAGK-NudC complex was located at the same position as the DYNLRB1 spot (Fig. 20C).

In addition, NAGK-DYNLRB1 complex was found in the migrated cells, and the NAGK-DYNLRB1 complex was located at the same position in NudC immunostaining, leading neurites, and nuclear membrane (Fig. 20D).

That is, it was confirmed that NAGK binds to the dynein-NudC complex during cell migration and regulates the migration process.

<4-4> According to 3D model analysis NAGK  And dynein - NudC  Confirmation of complex interaction

In order to confirm the interaction of the NAGK and dynein-NudC complex, a 3D model of the cells stained in Example 4-3 was prepared according to the above Example 1-8 and analyzed.

As a result, the PLA signals (Fig. 21Ab and Fig. 21Bb) of the nuclear membrane and the PLA (Fig. 21Ac and Fig. 21Bc) signals of the dendrites are present at the same positions as the corresponding ICC signals (Figs. 21A and 21B) Respectively.

In addition, the interaction of NAGK-NudC (Fig. 22Aa) and NAGK-DYNLRB1 (Fig. 22Ba) was confirmed by PLA method in mouse embryo (E-17.5) cerebral cortical tissue. When in high resolution, this interaction was at the same location in the nuclear membrane of the dendritic cells and the third protein of the tertiary complex (Fig. 22Ab and Fig. 22Bb).

In other words, it was confirmed that NAGK binds to dynein-NudC complex in migratory cortical neurons and plays an important role in embryonic brain development.

<4-5> in utero in electroporation (IEU)  Following NAGK's  Identify cell migration role

To confirm the role of NAGK in cell migration, (Fig. 23A), pDsRed2 vector (Fig. 23B) or NAGK shRNA vector and pDsRed2 vector (Fig. 23C) were transfected with E14.5 mouse embryonic brain Was introduced into the ventricular zone by microinjection and electroporation according to Example 1-10.

As a result, the knockdown of NAGK by shRNA inhibited the migration of neurons from the cerebral cortex to the cortical plate (Fig. 23c, most of the transplanted neurons dwelt in the brain shield area). On the other hand, NAGK overexpression promoted migration and reached most cortical plates (Fig. 23A).

Further, the cells injected with the transfected cells in the cortical plate (CP), the middle region (IZ) and the brain shunt region (VZ) were counted and displayed as a bar graph (Fig. 24).

As a result, the number of cells reaching the cortical plate during NAGK overexpression was significantly increased (about 50%) than the pDsRed2 control (around 18%). On the other hand, when transfected with the NAGK shRNA vector, the number of cells was significantly reduced (about 5%).

In other words, it was confirmed that NAGK is essential for neuronal migration and brain development.

Example 5. Matrigel &lt; RTI ID = 0.0 &gt;  Transition analysis using metastatis  분석 )

The A549 cells of Example 1-2 were cultivated in a transwell plate and the DsRed2 control vector (Fig. 25A), DsRed2 labeled NAGK vector (Fig. 25B), NAGK shRNA vector and After transfection with the DsRed2 control vector (Fig. 25C) and incubation for 24 hours, TGFβ was treated for 48 hours to induce mesenchymal transitions. This was seeded into matrigel coated transwells, and after 24 hours, the migrated cells were counted.

As a result, NAGK vector-transfected cells showed significant number of transmembrane cells (Fig. 26).

Namely, it was confirmed that the migratory capacity was increased by NAGK.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (9)

(a) contacting the cell with a candidate agent;
(b) measuring the expression level of N-acetylglucosamine kinase (NAGK) in cells contacted with the candidate substance; And
(c) selecting a candidate substance that inhibits the expression level of the N-acetylglucosamine kinase in comparison with the negative control not treated with the candidate substance
Wherein the method comprises the steps of:
(a) contacting the cell with a candidate agent;
(b) quantitatively analyzing N-acetylglucosamine kinase (NAGK) complex formed in cells contacted with the candidate substance; And
(c) selecting a candidate substance that inhibits the formation of the complex as compared with a negative control to which the candidate substance has not been treated
Wherein the method comprises the steps of:
3. The method of screening composition for cancer according to claim 2, wherein the complex comprises at least one protein selected from the group consisting of dynein, Lis1, NudE1 and SMAD2.
4. The method according to claim 3, wherein the dynein is dynein light chain roadblock-type 1 (DYNLRB1).
3. The method according to claim 1 or 2,
wherein the step (b) is performed in a kinetochore of metaphase chromosome.
3. The method according to claim 1 or 2,
wherein step (b) is performed in the cytoplasm of a cell treated with Transforming growth factor-β (TGFβ) after step (a).
The method of screening composition for cancer according to claim 1 or 2, wherein the cell is a cancer cell.
The method of claim 7, wherein the cancer cells are selected from the group consisting of lung cancer, non-small cell lung cancer, brain tumor, adrenal cancer, ureter cancer, renal cell carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma Wherein the cancer is a cell of a selected cancer.
3. The method of screening composition for cancer according to claim 1 or 2, wherein the cell is a cell selected from the group consisting of GT1-7, HEK293T, U87 and A549.

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