Patent Application
20110124006
The present invention relates to a screening method for a therapeutic agent for cancer. More particularly, the present invention relates to a screening method for a therapeutic agent for cancer, which uses the interaction between NLRR1 (neuronal leucine-rich repeat protein 1) and EGFR (epidermal growth factor receptor) as an indicator.
NLRR family genes are expressed in the relatively normal nervous system, and, from the analysis of the expression in the development process of mouse, are thought to play an important role in processes for regulating proliferation, differentiation and cell death of cells in the formation of the nervous system and organs in fetal life (Non Patent Literature 1).
The present inventors have isolated about 5400 genes from neuroblastoma cDNA library (Patent Literature 1). The present inventors have identified human NLRR1 from these genes as a gene whose expression amount is different between neuroblastoma with a good prognosis and neuroblastoma with a poor prognosis (Non Patent Literature 2).
The present inventors have clarified that the expression of NLRR1 is significantly high in neuroblastoma that has a poor prognosis and is refractory (Non Patent Literature 2). Furthermore, the present inventors have found that transcription of NLRR1 is directly induced by Myc (N-Myc and c-Myc transcription factors) that is a typical oncogene. Thus, the relation between NLRR1 and cancer has been strongly suggested, but the function of NLRR1 has not been clear.
Clarification of the function of NLRR1 leads to the elucidation of the relation between NLRR1 and cancer, and, to the development of a therapeutic agent for cancer based on a new mechanism. Therefore, one object of the present invention is to clarify the function of NLRR1. Another object of the present invention is to provide a screening method for a therapeutic agent for cancer based on a new mechanism from the elucidated findings.
As shown below, the present inventors have clarified in the present invention that NLRR1 and EGFR interact with each other to form a complex. Furthermore, the present inventors have clarified that this interaction remarkably increases the cell proliferation signal from the EGFR. Furthermore, the present inventors have clarified that the NLRR1 is involved in the malignant alteration of a variety of cancer types including refractory neuroblastoma, especially, many refractory cancers in which amplification or expression increase of N-Myc or c-Myc was found. Therefore, a substance that suppresses the interaction between NLRR1 and EGFR could be a therapeutic agent for cancer.
The present invention provides a screening method for a therapeutic agent for cancer, which includes a step of measuring an interaction between NLRR1 and EGFR under each condition of being in the presence of a test substance and in the absence of a test substance; and a step of determining that the test substance is a therapeutic agent for cancer when the interaction between NLRR1 and EGFR in the presence of the test substance is weaker than the interaction between NLRR1 and EGFR in the absence of the test substance. The screening method is based on a molecular mechanism in which NLRR1 and EGFR directly interact with each other to form a complex and to increase an intracellular proliferation signal from EGFR remarkably. This molecular mechanism has been found by the present inventors in the present invention, which provides a screening method for a therapeutic agent for cancer based on a new mechanism.
The present invention also provides a screening method for a therapeutic agent for cancer, which includes a step of culturing cells expressing NLRR1 and EGFR under each condition of being in the presence of a test substance and in the absence of a test substance; a step of measuring an interaction between NLRR1 and EGFR in each cultured cells; and a step of determining that the test substance is a therapeutic agent for cancer when the interaction between NLRR1 and EGFR in the cells cultured in the presence of the test substance is weaker than the interaction between NLRR1 and EGFR in the cells cultured in the absence of the test substance. According to this screening method, screening of a therapeutic agent for cancer can be carried out based on a new mechanism.
The present invention provides a kit for measuring an interaction between NLRR1 and EGFR, which includes an antibody recognizing NLRR1 and an antibody recognizing EGFR. The use of such a kit makes it possible to carry out a step of measuring the interaction between NLRR1 and EGFR in the above-mentioned screening method in a simple and easy manner.
The present invention also provides an antibody that suppress an interaction between NLRR1 and EGFR. Such an antibody can be applied to a therapeutic agent for cancer.
According to the present invention, a screening method for a therapeutic agent for cancer based on a new mechanism is provided.
A screening method for a therapeutic agent for cancer of the present invention includes a step of measuring an interaction between NLRR1 and EGFR under each condition of being in the presence of a test substance and in the absence of a test substance; and a step of determining that the test substance is a therapeutic agent for cancer when the interaction between NLRR1 and EGFR in the presence of the test substance is weaker than the interaction between NLRR1 and EGFR in the absence of the test substance. The interaction between NLRR1 and EGFR specifically denotes binding therebetween, that is, a formation of a complex. For the measurement of the interaction, measurement systems for measuring the interaction between proteins, which are well known to a person skilled in the art, can be used, and examples of the measurement systems include a yeast two-hybrid method, a PCA (protein fragment complementation assay) method, an immunoprecipitation method, an analysis by BIAcore (trade name), a gel shift method, and the like. The yeast two-hybrid method and the PCA method are briefly described below.
Firstly, the yeast two-hybrid method is described. Yeast Ga14 is a transcriptional regulating factor composed of an N-terminal DNA-binding domain (DBD) and a C-terminal transcriptional activation domain (AD). Basically, both domains autonomously function and DBD can bind to DNA by itself but cannot activate transcription. AD is the converse. The yeast two-hybrid method was developed by applying this property. That is to say, when a fusion protein (bait) of the protein P of interest with DBD of Ga14 and a fusion protein (prey) of another protein Q with AD of Ga14 are introduced into yeast cells, if the proteins P and Q interact with each other in the nucleus, a transcriptional regulation complex is reconstituted in the yeast cell, resulting in transcriptional activation dependent on a binding site for Ga14. By using reporter genes, the interaction between the proteins P and Q can be evaluated easily by detecting this activity. Examples of the reporter genes can include HIS3, lacZ, and URA3. In addition to yeast Ga14, a system using SRF or LexA is also available.
The screening method of the present invention may be carried out by a system using NLRR1 as the protein P and EGFR as the protein Q (the converse is possible) in the yeast two-hybrid method. That is to say, yeasts are allowed to express any one of NLRR1 and EGFR as a bait and the other of NLRR1 and EGFR as a prey, and the yeasts are cultured under each condition of being in the presence of a test substance and in the absence of a test substance; transcriptional activities (which represent the intensity of interaction between NLRR1 and EGFR) of reporter genes of the cultured yeasts are measured; and when the transcription activity in the presence of the test substance is increased from the transcription activity in the absence of the test substance, it can be determined that the test substance is a therapeutic agent for cancer.
Next, the PCA method is described. In the PCA method, one functional protein A (for example, enzyme, transcriptional factor) is divided into two fragments A1 and A2, which are then fused to the proteins P and Q of interest, respectively, to prepare fusion proteins A1-P and A2-Q. This method is based on the principle in which if the proteins P and Q of interest bind to each other, the functional protein A recovers its function, and, by detecting the activity thereof, the interaction between the proteins P and Q is determined. Examples of the functional protein may include β-lactamase. Hereinafter, the PCA method using β-lactamase is described.
The β-lactamase is a β-lactam ring-cleaving enzyme derived from bacteria. The β-lactamase is divided into the N-terminal α197 fragment (25 to 197 residues) and the C-terminal ω198 fragment (198 to 288 residues), which are respectively expressed as fusion proteins with proteins of interest. Only when they are bound to each other, the β-lactamase protein recovers its three-dimensional structure and exhibits the activity. The β-lactamase activity is detected with a cell-permeable fluorescent probe CCF2/AM (CCF2/acetoxymethyl ester). The CCF2/AM has a structure in which two different fluorescent substances, coumarin and fluorescein, are bound to both ends of a cephalosporin molecule, and exhibits intramolecular FRET (fluorescence resonance energy transfer) using coumarin as a donor and fluorescein as an acceptor. That is to say, the excitation of coumarin with light of 409 nm results in the emission of fluorescence derived from fluorescein at 520 nm. However, if CCF2 is degraded by the β-lactamase activity, since the two fluorescent substances dissociate from each other, and FRET is not observed, so that coumarin emits its original fluorescence at 447 nm by the excitation with light of 409 nm. The measurement of the fluorescence intensity at 447 nm allows the measurement of the β-lactamase activity, that is, the intensity of interaction between the proteins of interest.
Thus, the screening method of the present invention using the PCA method may be carried out by the following procedures. Cells are allowed to express fusion proteins of two functional protein fragments A1 and A2 fused with NLRR1 on one side, and EGFR on the other side, and cultured under each condition of being in the presence of a test substance or in the absence of a test substance; the respective functional protein activities in the cultured cells are measured; and it can be determined that the test substance is a therapeutic agent for cancer when the activity of the functional protein in the cells cultured in the presence of the test substance is lower than the activity of the functional protein in the cells cultured in the absence of the test substance.
Furthermore, the screening method for a therapeutic agent for cancer of the present invention includes a step of culturing cells expressing NLRR1 and EGFR under each condition of being in the presence of a test substance and in the absence of a test substance; a step of measuring an interaction between NLRR1 and EGFR in each cultured cells; and a step of determining that the test substance is a therapeutic agent for cancer when the interaction between NLRR1 and EGFR in the cells cultured in the presence of the test substance is weaker than the interaction between NLRR1 and EGFR in the cells cultured in the absence of the test substance. The measurement of the interaction can be carried out by using systems for measuring the interaction between proteins, which are well known to a person skilled in the art, and, for example, an immunoprecipitation method can be used. Hereinafter, the screening method using the immunoprecipitation method is described.
Firstly, cells expressing NLRR1 and EGFR are prepared. The cells can be prepared by introducing an expression plasmid of NLRR1 and EGFR into, for example, 293 cells. For facilitating the analysis, NLRR1 and EGFR genes to be introduced may be linked to genes encoding labeling peptide such as a HA tag, a FLAG tag, and a Myc tag. Then, cells expressing NLRR1 and EGFR are cultured under each condition of being in the presence of a test substance and in the absence of a test substance.
Next, the interactions between NLRR1 and EGFR in each cultured cells are measured. To measure the interaction, the cultured cells are firstly homogenized to prepare cell lysates. Before homogenization of cells, EGF may be added to the medium so as to stimulate EGFR. The stimulation is preferably carried out by adding, for example, EGF at the concentration of 10 ng/ml to the medium and culturing for three minutes. Furthermore, before cells are homogenized, a protein cross-linker such as DSP (dithiobis[succinimidyl propionate]) may be reacted. Although cell lysates from the whole cells may be used, it is preferred that cell lysates from the cell membrane fractions are used because the interaction between NLRR1 and EGFR occurs on the cell membrane. Immunoprecipitation is carried out by adding an antibody against either of NLRR1 or EGFR to the prepared cell lysates. Alternatively, immunoprecipitation may be carried out by using an antibody against labeling peptide linked to these proteins. Then, the obtained precipitate (containing a complex of NLRR1 and EGFR) is subjected to an immunological approach (for example, immunoblot) using an antibody against the other molecule, by which the interaction between NLRR1 and EGFR can be measured by detecting and quantifying the complex of NLRR1 and EGFR.
As a result of the measurement, when the interaction between NLRR1 and EGFR in the cells cultured in the presence of the test substance is weaker than the interaction between NLRR1 and EGFR in the cells cultured in the absence of the test substance (when the formation amount of protein complex is larger), it can be determined that the test substance is a therapeutic agent for cancer.
Examples of the above-mentioned test substances may include a low molecular weight compound, an antibody, an antibody fragment, an antibody derivative, lectin and aptamer. As a supply source of the low molecular weight compound, for example, a commercially available compound library can be used. The preferable test substance can further include an antibody, an antibody fragment, an antibody derivative and aptamer whose antigen is NLRR1 and EGFR, as well as lecithin, and the like. These test substances are easily available, and techniques for mass production of therapeutic agents for cancer have been established.
As described above, the expression of NLRR1 is significantly high in neuroblastoma that has a poor prognosis and is refractory. Furthermore, as mentioned below, enhancement of a proliferation signal by NLRR1 is found not only in an EGFR but also in an IGF (insulin-like growth factor) receptor (IGFR) that is the other membrane receptor for sending a proliferation signal. Therefore, it is thought that NLRR1 is involved not only in refractory neuroblastoma but also in various cancers. Furthermore, as mentioned above, NLRR1 is directly induced to transcription by Myc (N-Myc and c-Myc transcription factors) that is a typical oncogene. Thus, it is thought that NLRR1 is deeply involved in the malignant alteration of many refractory cancers in which amplification or expression increase of N-Myc and c-Myc is observed.
Therefore, a therapeutic agent for cancer obtained by a screening method of the present invention may be applied to many refractory cancers in which amplification or increased expression of N-Myc or c-Myc is observed, more preferably, may be applied to neuroblastoma, and further preferably, may be applied to neuroblastoma that has a poor prognosis and is refractory.
Furthermore, a screening method of the present invention may further include a step of measuring a proliferation rate of cells expressing NLRR1 and EGFR under each condition of being in the presence of a test substance and in the absence of a test substance.
A therapeutic agent for cancer obtained by a screening method of the present invention is used as a lead compound, and subjected to chemical modification, and thereby it is possible to obtain pharmaceutical preparation having high activity and improved physical property, pharmacokinetics, and toxicity.
The present invention further provides a kit for measuring an interaction between NLRR1 and EGFR, which includes an antibody recognizing NLRR1 and an antibody recognizing EGFR. The use of such a kit allows a step of measuring the interaction between NLRR1 and EGFR in the above-mentioned screening method to be carried out in an easy and simple manner by, for example, an immunoprecipitation method. These antibodies may be prepared by immunizing animals such as a mouse, a rabbit, or the like, with, for example, entire or a part of NLRR1 and EGFR protein as an antigen, or may be selected from the phage display library of antibodies.
The above-mentioned kit may further include expression plasmids of NLRR1 and EGFR. These expression plasmids are inserted into cultured cells such as 293 cells, and thereby cells expressing NLRR1 and EGFR can be obtained. The cells can be used for the above-mentioned screening method for a therapeutic agent for cancer. Furthermore, the above-mentioned expression plasmids of NLRR1 and EGFR may have a structure in which NLRR1 and EGFR proteins are expressed as fused proteins with a labeled peptide such as a myc tag or a HA tag. In this case, an antibody included in the kit may recognize a labeled peptide linked to NLRR1 and EGFR instead of recognizing NLRR1 and EGFR.
The above-mentioned kit may further include a cell line that highly expresses NLRR1 and EGFR. This cell can be used for the above-mentioned screening method for a therapeutic agent for cancer.
The present invention also provides an antibody suppressing the interaction between NLRR1 and EGFR. Such an antibody can be applied for a therapeutic agent for cancer. This antibody may be an antibody recognizing a binding site of NLRR1 and EGFR. In this case, the antibody is bound to the above-mentioned binding site, and thereby the binding between NLRR1 and EGFR is inhibited.
The above-mentioned antibody can be selected from antibodies that are bound to NLRR1 and EGFR.
Hereinafter, the present invention is described more specifically with reference to Examples of the present invention. However, the present invention is not intended to be limited to these Examples, and a variety of modifications can be carried out within the scopes of technical idea of the present invention.
(Materials)
MTT assay kit (Cell counting kit) was purchased from Wako Pure Chemical Industries, Ltd. In vitro Transcription/Translation System (L1170) was purchased from Promega. pcDNA3.1 was purchased from Invitrogen. U0126 and LY294002 were purchased from Cell Signaling Technology. AG1478, FTI277, SP600125 and SB203580 were purchased from Calbiochem. EGF (epidermal growth factor), G418 and Tunicamycine were purchased from Sigma. An isotope-labeled 125I-EGF was purchased from GE Healthcare. IGF was purchased from Sigma. DAPI (4′6-diamino-2-phenylindole) and PI (propidium iodide) were purchased from Molecular Probes. DSP was purchased from PIERCE. An anti-myc antibody (9B11), an anti-phosphorylated p38MAPK antibody, an anti-p38MAPK antibody, an anti-phosphorylated-EGFR antibody, an anti-EGFR antibody, an anti-phosphorylated-ERK antibody, an anti-ERK antibody, an anti-phosphorylated-Akt antibody, an anti-Akt antibody, and an anti-phosphorylated p90RSK antibody were purchased from Cell Signaling Technology. An anti-phosphorylated tyrosine antibody (PY20) was purchased from BD Science. An anti-actin antibody and an anti-HA antibody were purchased from Sigma. An anti-MYCN antibody was purchased from Oncogene Research Products.
An expression plasmid encoding EGFR and an expression plasmid encoding NLRR1 tagged with an HA tag were introduced into 293 cells to force the expression. The cells were cultured for three minutes in a medium containing 10 ng/ml EGF. The cells were washed with phosphate-buffered saline (PBS), then subjected to cross-linking with DSP, and the cells were collected and subjected to immunoprecipitation with an anti-EGFR antibody. Next, samples were subjected to SDS-polyacrylamide gel electrophoresis, and transferred to a membrane, followed by an analysis of binding between EGFR and NLRR1 by an immunoblot method using an anti-HA antibody and an anti-EGFR antibody.
An expression plasmid encoding NLRR1 was introduced into MCF-7 cells to force the expression. As a control, cells into which an empty vector plasmid was introduced were used. These cells were cultured in a medium containing 1 nM isotope-labeled 125I-EGF on ice for 60 minutes. The cells were washed with PBS that had been cooled to 4° C., and the cells were collected by using 1M NaOH, followed by measuring the radioactivity detected from the collected cells with the use of a gamma-ray measuring device. The measurement result was corrected by the amount of protein contained in a sample.
An expression plasmid encoding NLRR1 tagged with a HA tag was introduced into MCF-7 cells to force the expression. As a control, cells into which an empty vector plasmid was introduced were used. These cells were cultured in media containing IGF at the concentrations of 0, 0.1, 1, and 10 ng/ml, and cells were collected after 10 minutes. These cells were subjected to immunoblot using an anti-phosphorylated-ERK antibody, an anti-ERK antibody, an anti-phosphorylated-Akt antibody and an anti-Akt antibody.
For the purpose of confirming the presence or absence of sugar chain modification, a plasmid encoding NLRR1 tagged with a myc tag was introduced into COS7 cells, the cells were cultured for 24 hours in the presence and absence of 2 μg/ml tunicamycin as a sugar chain synthesis inhibitor. After the cells were collected, the expression of NLRR1 was detected by an immunoblot method to the myc tag.
A plasmid encoding NLRR1 tagged with a myc tag was introduced into COS7 cells and cultured for 48 hours. The cells were collected and were subjected to immunoprecipitation using an anti-myc antibody. Next, samples were subjected to an SDS-polyacrylamide gel electrophoresis, and transferred to a membrane, followed by the detection by an immunoblot method using an anti-myc antibody and an anti-phosphorylated tyrosine antibody.
A plasmid encoding NLRR1 tagged with a myc tag was introduced into COST cells, and the cells were cultured for 48 hours in the condition containing 10% fetal bovine serum (FBS) and in the serum free condition. The cells were collected and were subjected to immunoprecipitation using an anti-myc antibody. Furthermore, as a control, immunoprecipitation using a standard mouse IgG was also carried out. Next, samples were subjected to an SDS-polyacrylamide gel electrophoresis, and transferred to a membrane, followed by detection by an immunoblot method using an anti-phosphorylated tyrosine antibody. Next, the membrane was subjected to reprobing and detected by an immunoblot method using an anti-myc antibody.
A plasmid encoding NLRR1 tagged with a myc tag was introduced into SK-N-BE cells derived from neuroblastoma, and the cells were forced to express. The proliferation of cells was measured by using a MTT assay kit. As a control, SK-N-BE cells into which an empty vector plasmid was introduced were used and the same measurement was carried out (which corresponds to “mock” in
SK-N-BE cells into which NLRR1 tagged with a myc tag was introduced and SK-N-BE cells into which an empty vector plasmid was introduced (which corresponds to “mock” in
To SK-N-BE cells derived from neuroblastoma, siRNA against NLRR1 was introduced so as to suppress the expression of NLRR1. As the siRNA, RNA fragment of the base sequences shown SEQ ID NOs: 3 and 4 were annealed and used.
After siRNA was introduced, the cells were cultured, and the cell proliferation was quantified by the MTT assay over time. As a control, cells into which Silencer (registered trademark) Negative Control #1 siRNA (Ambion) was introduced were used.
A plasmid encoding NLRR1 tagged with a myc tag was introduced into SK-N-AS cells. The SK-N-AS cells are a cell line which is derived from neuroblastoma and in which a MYCN oncogene is not amplified. After introduction of the gene, drug selection was carried out in a medium containing G418, and survival cells were cloned, and thereby a cell line continuously expressing NLRR1 was obtained. As a control cell line, cells subjected to the same operation by using an empty vector plasmid were used.
To SHEP21N cells that were forced to express MYCN in the absence of tetracycline, siRNA against NLRR1 was introduced, and the cell proliferation was quantified by the MTT assay over time.
SK-N-BE cells, SK-N-BE cells into which siRNA against NLRR1 was introduced, and SK-N-BE cells into which Silencer (registered trademark) Negative Control #1 siRNA was introduced were cultured for 10 hours in the presence of 10 ng/ml EGF, and the cells were collected. Next, these cells were subjected to an immunoblot method using an anti-MYCN antibody, an anti-NLRR1 rabbit polyclonal antibody and an anti-actin antibody, and expression of MYCN and NLRR1 in the protein level was detected.
It is known that in the SK-N-AS neuroblastoma cell line, cell death is induced when serum is removed, and that the number of sub-G1 cells is increased from the flow cytometry analysis. SK-N-AS cells into which an empty vector plasmid was introduced (corresponding to “mock” on the left in
A neuroblastoma cell line SK-N-BE was cultured for 10 hours in media containing EGF at a variety of concentrations, by using an anti-phosphorylated EGFR antibody, an anti-EGFR antibody, an anti-MYCN antibody and an anti-actin antibody, the phosphorylation of an EGF receptor and the expression of MYCN were analyzed by an immunoblot method.
An expression plasmid (pcDNA3.1myc.Extra.NLRR1) encoding only an extracellular domain of NLRR1 and an expression plasmid (pcDNA3.1Cyt.NLRR1.myc) encoding only an intracellular domain were prepared. Both plasmids were tagged with a myc tag. To HeLa cells, 0.5 μg and 1.0 μg of pcDNA3.1myc.Extra.NLRR1 were introduced, respectively, followed by detection by an immunoblot method using an anti-Myc antibody. As a control, HeLa cells into which 1.0 μg of empty vector plasmid was introduced were used.
5 μg and 10 μg of pcDNA3.1Cyt.NLRR1.myc were allowed to express, respectively. For expression, In vitro Transcription/Translation System was used. As a control, 5 μg and 10 μg of empty vector plasmids were allowed to express.
SK-N-BE cells that were forced to express NLRR1 were cultured in the presence and absence of ERK1/2 (ERK1 and ERK2) inhibitor, U0126. Furthermore, as a control, SK-N-BE cells into which a plasmid encoding NLRR1 was not introduced were cultured in the absence of U0126. The proliferation of these cells was quantified by an MTT assay over time.
A breast cancer cell line MCF-7 was forced to express NLRR1, which was cultured in media containing 0, 0.1, 1 and 10 ng/ml EGF for 10 minutes. Next, cells were collected and subjected to an immunoblot method using an anti-phosphorylated ERK antibody, an anti-ERK antibody, and an anti-myc antibody.
A combination of a plasmid encoding only NLRR1, a plasmid encoding only EGFR, as well as a plasmid encoding NLRR1 and EGFR were introduced into MCF-7 cells and were expressed. As a control, MCF-7 cells into which genes were not introduced were used. These cells were cultured in a medium containing 1 ng/ml EGF for 10 minutes. Next, the cells were collected and subjected to an immunoblot method using an anti-phosphorylated-Akt antibody, an anti-Akt antibody and an anti-actin antibody.
SK-N-BE cells were cultured in the presence of 10 ng/ml EGF (control, which corresponds to “mock” in
SK-N-BE cells were cultured for six hours in the presence of 10 ng/ml EGF and in the presence of an EGFR inhibitor AG1478, a Ras inhibitor FTI277, an ERK1/2 inhibitor U0126, a PI3Kinase inhibitor LY294002, a INK inhibitor SP600125 or a p38MAPK inhibitor SB203580 in each concentration shown in
SK-N-BE cells, NLF cells, SH-SY5Y cells, SMS-SAN cells, which are cell lines derived from neuroblastoma, and SK-N-BE cells into which siRNA against NLRR1 was introduced were cultured in the presence and absence of 10 ng/ml EGF, and the cell proliferation was quantified by the MTT assay over time.
COST cells were forced to express NLRR1 (tagged with a myc tag) or NLRR2 (tagged with a myc tag) that is one of the NLRR family genes. The cells were subjected to serum starvation for 24 hours; 0.02 ng/ml EGF was then added to the medium; and the cells were collected after 0 minute, 15 minutes, 1 hour, and 4 hours. These cells were subjected to an immunoblot using an anti-myc antibody, an anti-phosphorylated ERK antibody, an anti-ERK antibody, an anti-phosphorylated p38MAPK antibody and an anti-actin antibody.
SK-N-AS cells were forced to express NLRR1 tagged with a myc tag. The cells were subjected to serum starvation for 12 hours; 10% fetal bovine serum (FBS) was then added into a medium; and the cells were collected after 0, 3, 5, 10, 30 and 160 minutes. As a control, SK-N-AS cells into which an empty vector plasmid was introduced was used (which corresponds to “mock” in
From the above-mentioned results, it has been clarified that NLRR1 promotes the phosphorylation of p38MAPK not only in cells derived from neuroblastoma but also in COS7 cells.
An expression plasmid encoding only an intracellular domain of NLRR1 (tagged with a myc tag) or only an extracellular domain (tagged with a myc tag) were introduced into SK-N-BE cells. As a control, cells into which an empty vector plasmid was introduced were used (which corresponds to “mock” in
An expression plasmid of NLRR1 tagged with a myc was introduced into SK-N-BE cells at the concentrations of 0, 100, 200, 500, and 1000 ng/ml. After 48 hours of culturing, the cells were collected and were subjected to an immunoblot method using an anti-myc antibody, an anti-MYCN antibody and an anti-actin antibody.
An expression plasmid of NLRR1 tagged with a myc tag was introduced into SK-N-BE cells at the concentration of 1000 ng/ml. The cells were cultured for 12 hours in the presence of 1 μM of EGFR inhibitor AG1478 or 2 μM of ERK1/2 inhibitor U0126, and then the cells were collected and subjected to an immunoblot method using an anti-myc antibody, an anti-MYCN antibody and an anti-actin antibody.
An expression plasmid encoding only an extracellular domain of NLRR1 tagged with a myc tag was introduced into SK-N-BE cells at the concentrations of 0, 100, 200, 500, and 1000 ng/ml. The cells were cultured for 48 hours, and then the cells were collected and subjected to an immunoblot method using an anti-myc antibody, an anti-MYCN antibody and an anti-actin antibody.
An expression plasmid encoding only an intracellular domain of NLRR1 tagged with a myc tag was introduced into SK-N-BE cells at the concentrations of 0, 100, 200, 500, and 1000 ng/ml. The cells were cultured for 48 hours, and then the cells were collected and subjected to an immunoblot method using an anti-myc antibody, an anti-MYCN antibody and an anti-actin antibody.
From the above-mentioned results, it has been clarified that when either an extracellular domain or an intracellular domain of NLRR1 is absent, the expression of MYCN protein in the downstream cannot be induced.
An expression plasmid encoding only an extracellular domain of NLRR1 (tagged with a myc tag) or only an intracellular domain (tagged with a myc tag) was introduced into SK-N-BE cells. As a control, cells into which an empty vector plasmid was introduced were used (which corresponds to “mock” in
The effect of NLRR1 on EGF signal transduction in cells treated with cyclodextrin (methyl-β-cyclodextrin, mβCD) that inhibits lipid raft was examined.
According to the present invention, a screening method for a therapeutic agent for cancer based on a new mechanism is provided.
Sequence Listing
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-053959 | Mar 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/053699 | 2/27/2009 | WO | 00 | 11/22/2010 |
