KR101247636B1 - Diagnostic marker and kit for squamous cell carcinoma - Google Patents

Abstract

The present invention relates to diagnostic markers specific for squamous cell carcinoma. The present invention also relates to compositions, kits, and methods for diagnosing squamous cell carcinoma using the same, comprising a formulation for measuring the presence of said marker.

Description

Diagnostic marker and kit for squamous cell carcinoma of squamous cell carcinoma

The present invention is a composition for detecting a marker for diagnosing squamous cell carcinoma in lung cancer, comprising a preparation for measuring the level of one or more proteins selected from the group consisting of p-Pak1, p-LIMK1, and p-cophylline, kit comprising the same And it relates to a method for diagnosing squamous cell carcinoma using the same.

Cancer deaths continue to increase worldwide. Despite intensive research on cancer over the years, cancer is still the leading cause of death worldwide, of which lung cancer is a high incidence and mortality rate worldwide. Lung cancer can be largely divided into non-small cell lung cancer and small cell lung cancer.Non-small cell lung cancer is divided into adenocarcinoma (30-40%), squamous cell carcinoma (20-30%), and large cell cancer (10%). Can be. Among these, squamous cell carcinoma (SCC), which occurs mainly in large bronchus and grows into the bronchial lumen, is commonly seen in older men and in large amounts of smokers. 95% occurs in the center of the lungs and 5% occurs around the lung lobes. Symptoms usually appear in the bronchus and sometimes form large masses that form cavities. Tumor markers unique to squamous cell carcinoma that can detect such squamous cell carcinoma at an early stage have not yet been identified.

In addition, adenocarcinoma (also referred to as AC) occurs in the peripheral area of the lungs and is most likely to occur in women and non-smokers, and is thought to be caused by changes in smoking habits, changes in smoking amount, changes in diet, and environmental factors.

Although the squamous cell carcinoma and adenocarcinoma are the major histological types of non-small cell lung cancer, and can be distinguished histologically or clinically, the distinction between them is not easy, and also the exact mechanism of its pre-invasion or invasion. It is not known yet. Accurate early diagnosis of squamous cell carcinoma is a prerequisite for proper treatment. Therefore, in order to facilitate proper therapeutic action by surgical resection, radiation therapy, chemotherapy or other known treatment methods, A quick and simple method of diagnosis is required. X-rays, computed tomography, bronchoscopy, radiographic examinations, and bronchoscopy can be used to diagnose cancer.However, these tests can determine the degree of anatomical progression, but the cellular physiological status and molecular genetics. Accurate assessment and prediction cannot be made.

In addition, since the recurrence often occurs after surgery as a surgical therapy for the treatment of squamous cell carcinoma, it is necessary to undergo regular examination to check for recurrence or metastasis every 3-4 months after surgery. . More than 80% of these recurrences are found within 3 years after surgery, so recurrence within 5 years after surgery is the cure criteria. Therefore, there is also a need for the development of markers that can facilitate the prognosis of recurrence or metastasis or invasion after surgery.

Meanwhile, various attempts have been made to diagnose lung cancer using markers. Korean Patent Application No. 10-1998-0038212 relates to a method for confirming metastasis of lung cancer, which measures the expression of mitogen activated protein kinase phosphatase-1 (MKP-1) in lung cancer tissues. It describes a method for diagnosing local metastatic lung cancer. International patent WO04005891 describes a method for diagnosing lung cancer using genes such as OE372, ATP5D, B4GALT, Ppase, GRP58, GSTM4, P4HB, TPI, and UCHLI as lung cancer diagnostic markers. US patent US6117981 describes a method for the diagnosis and treatment of non-small cell lung cancer and ovarian cancer using monoclonal antibodies targeting LCGA. European patent EP0804451 describes a method for diagnosing and treating lung cancer using lung cancer specific antigen HCAVIII. In addition, US Pat. No. 6,759,508, US Pat. No. 6,746,846, US Pat. No. 6,737,514, European Patent EP0621480 and the like describe lung cancer diagnostic markers.

However, there have been no studies or disclosures on markers for specifically diagnosing squamous cell carcinoma among non-small cell lung cancers. In addition, squamous cell carcinoma could not be diagnosed more accurately because there was no useful marker that can easily distinguish between squamous cell carcinoma and adenocarcinoma.

Accordingly, the present inventors have made intensive efforts to develop molecular markers for the specific diagnosis of squamous cell carcinoma. Compared to normal lung tissue or adenocarcinoma, the present invention was completed by elucidating specificity in squamous cell carcinoma tissues and cells, and confirming whether squamous cell carcinoma is highly reliable when used as a marker.

Disclosure of Invention An object of the present invention is to provide a composition for detecting a marker for diagnosing squamous cell carcinoma in lung cancer, comprising an agent for measuring the level of at least one protein selected from the group consisting of p-Pak1, p-LIMK1, and p-cophylline. To provide.

Another object of the present invention to provide a kit for diagnosing squamous cell cancer among lung cancer comprising the composition.

Still another object of the present invention is to provide a method for diagnosing squamous cell cancer among lung cancers.

It is another object of the present invention to provide that the markers of the present invention can be used to predict recurrence and prognosis of lung cancer resection surgery.

As one aspect for achieving the above object, the diagnosis of squamous cell carcinoma of lung cancer, comprising an agent for measuring the level of at least one protein selected from the group consisting of p-Pak1, p-LIMK1, and p-cophylline It relates to a composition for detecting a marker for.

Preferably, the composition may be a composition comprising an agent that measures the levels of p-Pak1, p-LIMK1, and p-cophylline proteins.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether squamous cell carcinoma develops.

In the present invention, the term "squamous cell carcinoma" is also called squamous cell carcinoma, or squamous cell carcinoma, and refers to a type of non-small cell lung cancer in which the carcinoma develops in the squamous cell in the lung. The squamous cell carcinoma mainly occurs in the large bronchus and grows into the bronchial lumen, and the regional bronchus, lobe bronchus, and the main bronchus 95% occur in the center of the lung, and about 5% of the lung lobe occurs.

As used herein, the term “diagnostic marker, diagnostic marker, or diagnostic marker” is a substance capable of diagnosing squamous cell carcinoma cells from normal cells, and has squamous cell carcinoma compared to normal cells. Organic biomolecules such as polypeptides or nucleic acids (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), which show an increasing pattern in cells. For purposes of the present invention, the marker for diagnosing squamous cell carcinoma is p-Pak1, p-LIMK1, and p-cophylline, which show a specific high level of expression in squamous epithelial cells compared to cells of normal lung tissue. One or more proteins selected from the group consisting of, preferably p-Pak1, p-LIMK1, and p-cophylline proteins.

Cofilin is a widely distributed intracellular actin-regulating protein that binds to and depolymerizes fibrous F-actin and inhibits polymerization of monomeric G-actin in a pH-dependent manner. Cophylline is involved in the transfer of actin-cophylline conjugates from the cytoplasm to the nucleus. Cophylline is a small actin-binding protein that regulates actin polymerization and depolymerization and separates actin filaments and is widely distributed in various tissues. The activity of copilin is regulated through a variety of mechanisms, including phosphorylation. Cophylline is an important protein for cell motility that acts to increase actin dynamics in the lamellopodia associated with cell motility. In the present invention, p-cophylline means phosphorylated cofilin.

In the present invention, the phosphorylation level of Pak1 / LIMK1-mediated cophylline was found to be closely related to the diagnosis of squamous cell carcinoma of lung cancer. In addition, the phosphorylation level of Pak1 / LIMK1-mediated cophylline was found to be a determinant factor for the migration and invasion of squamous cell carcinoma of lung cancer, a factor that distinguishes between squamous cell carcinoma and adenocarcinoma type among non-small cell lung cancer subtypes. Was identified.

In the present invention, Pak1 (p21-associated kinase 1) refers to p21-associated kinase 1. Pak1 is an important regulator of actin cytoskeleton and motility, and it has been reported that Pak1 gene amplification and increased Pak1 protein levels are associated with uterine cancer and breast cancer. In the present invention, the expression level of phosphorylated Pak1 is closely related to the diagnosis of squamous cell carcinoma and it can be a diagnostic marker.

In addition, LIM domain kinase (LIMK) refers to LIM domain kinases and is the most frequent and predominant cophylline kinase in infiltrating carcinoma cells. LIMK is phosphorylated and activated by Pak1 or ROCK (Rho kinase). In the present invention, the expression level of phosphorylated LIMK is closely related to the diagnosis of squamous cell carcinoma, and can be a diagnostic marker.

On the other hand, the migration of tumor cells is very important for the formation of solid cancer and is essential for metastasis to other tissues. This cancer migration and infiltration process is associated with changes in cytoskeletal signaling pathways, increased directional motility, and increased cell survival. Pak1 plays an important role in regulating actin cytoskeleton and also increases the phosphorylation of LIMK and cophylline under the activity of Pak. However, the expression state of LIMK or cophylline alone is not sufficient to determine the state of migration and infiltration of tumor cells.

The inventors of the present invention have studied the relationship between the Pak1 / LIMK1 / cophylline pathways in SCC and AC and the differences in mechanisms, differences in migration and invasion, and the like, while p-Pak1, p-LIMK1, and p-cophylline are Although it does not appear in normal lung tissue, it was found to be markedly increased in squamous cell carcinoma tissues and cells, and it was confirmed that these proteins could act as markers for diagnosing squamous cell carcinoma among lung cancers. In addition, the expression level of p-cophylline was found to be different between squamous cell carcinoma and adenocarcinoma, and this difference was related to the difference in migration and invasion between squamous cell carcinoma and adenocarcinoma. It was also found to be associated with prognostic predictions such as metastasis.

In a specific embodiment of the present invention, Pak1 / LIMK1 was obtained by collecting tumor samples and adjacent normal lung tissue from a total of 42 patients who had SCC (n = 27) and AC (n = 15) among lung cancer patients. Analysis of the / cophylline pathway protein showed increased expression of p-Pak1, p-LIMK1, p-cophylline in all SCC tumor samples compared to adjacent normal lung tissue (see Figure 2). However, in the case of AC tumor samples, it was found that the expression of p-Pak1 and p-LIMK1 was not shown (see FIG. 3). These results indicate that the Pak1 / LIMK1 / cophylline pathway is not predominant in AC and is flattened from normal tissue and adenocarcinoma by measuring the expression levels of proteins of p-Pak1, p-LIMK1, and p-cophylline It was found that epithelial cell carcinoma can be specifically diagnosed.

In addition, in a specific embodiment of the present invention, SK-MES-1, SK-MES-1 and A549 cell lines in parallel with the lung cancer tissues as a result of comparing the expression of the Pak1 / LIMK1 / cophylline pathway protein, SK-MES-1 The expression of the protein was increased in (SCC type) cells, and wound-healing and migration assays were performed to investigate the difference in cell migration between SCC and AC. It was confirmed that the migration and infiltration in MES-1 cells was significantly high (see FIG. 4).

In the present invention, "measurement of expression level of protein" refers to a process of confirming the presence and degree of expression of a protein expressed from a squamous cell carcinoma marker gene in a biological sample in order to diagnose squamous cell carcinoma. The amount of protein can be determined by using an antibody that specifically binds to the protein. Analytical methods for this purpose include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket Immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Fluorescence Activated Cell Sorter (FACS), protein chip, etc., but are not limited to these. .

In another aspect, the present invention provides a squamous cell carcinoma diagnostic marker composition comprising an antibody specific for at least one protein selected from the group consisting of p-Pak1, p-LIMK1, and p-cophylline.

Preferably, the composition may be a composition comprising an antibody specific for the proteins of p-Pak1, p-LIMK1, and p-cophylline.

In the present invention, " antibody " means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since squamous cell carcinoma marker proteins have been identified as described above, the production of antibodies using them can be readily prepared using techniques well known in the art.

Polyclonal antibodies can be produced by methods well known in the art for injecting the squamous cell carcinoma marker protein antigen described above into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

Monoclonal antibodies are known in the art by the hybridoma method (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519), or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.

The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In still another aspect of the present invention, there is provided a squamous cell carcinoma diagnostic kit comprising the composition for diagnosing squamous cell carcinoma according to the present invention. Preferably, the squamous cell carcinoma diagnostic kit may further comprise one or more other component compositions, solutions or devices suitable for analytical methods.

Preferably, the kit may be a diagnostic kit comprising the necessary elements necessary to perform Western blot or tissue immunostaining. The kit includes antibodies specific for the marker protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The kit may also include antibodies specific for the control protein.

In still another aspect, the present invention provides a method for providing information for diagnosing squamous cell carcinoma in lung cancer using the composition for diagnosing squamous cell carcinoma or the squamous cell carcinoma diagnostic kit.

In a specific embodiment, the present invention comprises the steps of measuring the level of proteins of p-Pak1, p-LIMK1, and p-cophylline from biological samples isolated from patients with squamous cell carcinoma; And comparing the level of the protein with that of a normal control sample.

Preferably, measuring the protein level comprises contacting an antibody specific for p-Pak1, p-LIMK1, and p-cophylline proteins with a biological sample from a suspected squamous cell carcinoma, to form an antigen-antibody complex. Checking may be made. In addition, the comparing with the protein level may be comparing the increase in the amount of protein formation with the protein level of the normal control sample.

As used herein, the term “biological sample” includes tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differ in expression level of squamous cell carcinoma markers due to the development of squamous cell carcinoma. However, it is not limited thereto.

The separation of proteins from biological samples can be carried out using known processes and protein levels can be measured in a variety of ways.

Through the above assay methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in patients with suspected squamous cell carcinoma can be compared. By determining whether the expression level is increased, it is possible to diagnose whether or not the actual squamous cell carcinoma of the patient suspected squamous cell carcinoma.

As used herein, the term “antigen-antibody complex” means a combination of a squamous cell carcinoma marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed quantitatively through the magnitude of the signal of the detection label. It can be measured by

Such a detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not necessarily limited thereto. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapies, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, β-latamase, and the like, but are not limited thereto. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN) ₈ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. .

Analytical methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, Protein chips and the like, but is not limited thereto.

Preferably, the protein expression level is measured using Western blot using one or more antibodies against the squamous cell carcinoma marker. The whole protein is isolated from the sample, electrophoresed to separate the protein according to size, and then transferred to the nitrocellulose membrane to react with the antibody. By checking the amount of the generated antigen-antibody complexes using labeled antibodies, the amount of protein produced by expression of genes can be confirmed to determine whether squamous cell carcinoma is developed. The detection method comprises a method of examining the expression level of the marker protein in the control group and the expression level of the marker protein in the cells with squamous cell carcinoma. Protein levels can be expressed as absolute (eg μg / ml) or relative (eg relative intensity of signals) differences of the marker proteins described above. Also preferably, immunohistostaining is performed using at least one antibody against the squamous cell carcinoma marker. After collecting and fixing normal lung epithelial tissue and suspected squamous cell carcinoma, paraffin embedding blocks are prepared by methods well known in the art. These are sliced to a thickness of several um and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. The unreacted antibody is then washed and labeled with one of the above-mentioned detection labels to read whether or not the antibody is labeled on a microscope.

In another aspect, the present invention compares the expression level of p-cophylline from the biological sample of a patient who has undergone lung cancer resection surgery with the expression level of normal cells in order to provide information necessary for predicting recurrence and prognosis of lung cancer resection surgery. Provided are methods for detecting p-cophylline.

As used herein, the term "prognostic marker" refers to a substance capable of confirming the progress, survival or cure of lung cancer after lung cancer treatment such as squamous cell carcinoma resection surgery. mRNA, etc.), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like. For purposes of the present invention, the marker for predicting prognostic squamous cell carcinoma of the present invention is p-cophylline protein.

In a specific embodiment of the present invention, immunohistochemical analysis of tumor cells taken from 43 non-small cell lung cancer patients and follow-up of patients who underwent surgery for 3 years showed that 40.7% (n = 12) of SCC patients had local infiltration. (n = 10) or distant metastasis to the liver, brain, and bone (n = 3), while only 6.7% of AC patients showed relapse. The difference between them was found to be closely related to the difference in expression of pcophylline (see FIG. 5). Therefore, it can be seen that the expression level of p-cophylline can be measured and used as a marker for predicting prognosis such as recurrence, metastasis and invasion of patients undergoing lung cancer resection.

As described in detail above, the phosphorylation of Pak1 and LIMK1, which are enzymes upstream of cophylline and its upstream in the cytoskeletal signal, is specific in squamous cell carcinoma tissues and cells as compared to normal lung tissue or adenocarcinoma. It can be usefully used as a squamous cell carcinoma marker.

1 shows representative images of H & E staining and p-cophylline immunostaining in lung incision tissues obtained from patients with SCC or AC. (A) is a comparison of H & E staining in SCC and AC, and (B) shows immunohistochemical analysis of SCC and AC samples using anti-p-cophylline antibodies (Scale bars = 100 μm).
2 shows the expression of Pak1 / LIMK1 / cophylline signaling pathway proteins in ten representative human normal lung tissue (N) and SCC (T) pairs. ROCK1, p-Pak1, total Pak1, p-LIMK1, total LIMK1, p-cophylline, and total cophylline were examined by Western blot analysis. β-actin was used as loading control. Samples were separated on 10% SDS-polyacrylamide gels and transblotted proteins were probed with each antibody.
3 shows expression of Pak1 / LIMK1 / cophylline signaling pathway proteins in nine representative human normal lung tissue (N) and AC (T) pairs. ROCK1, p-Pak1, total Pak1, p-LIMK1, total LIMK1, p-cophylline, and total cophylline were examined by Western blot analysis. β-actin was used as loading control. Samples were separated on 10% SDS-polyacrylamide gels and transblotted proteins were probed with each antibody.
4 shows the expression results of Pak1 / LIMK1 / cophylline signal protein and their migration and invasion in SK-MES-1 and A549 cells. vitro Assay is shown. 4A is a Western blot analysis of the Pak1 / LIMK1 / cophylline signaling pathway in SCC (SK-MES-1) and AC (A549) cell lines, and FIG. 4B shows the migration of human lung cancer cells in a two-dimensional cell migration assay system. As shown, the top shows a phase-control image of the wound site 0, 9 and 24 hours after the wound of the SK-MES-1 and A549 monolayers, and the bottom shows a wound-healing percentage. Each bar represents three independent experimental means ± SEM, with an asterisk indicating significant difference from initial value ( P <0.05). 4C shows the migration of lung cancer cells in a three-dimensional cell migration assay system, the top of which shows a representative microscopic image of the migration cells, in which transwell culture inserts SK-MES-1 and A549 cells (15,000 cells / well) Seeded and incubated at 37 ° C. for 6 hours, the bottom showing the moving percentage. Each bar represents four independent experimental mean ± SEM, with asterisks indicating significant difference from A549 cells ( P <0.05). 4D also shows SK-MES-1 and A549 cell infiltration assays. Infiltrated cells were stained at the bottom of the membrane. Each bar represents three independent experimental mean ± SEM, with asterisks indicating significant difference from A549 cells ( P <0.05).
5 shows a representative image of p-cophylline immunostaining in pulmonary incision tissue obtained from a patient with SCC. (A) shows the expression of p-cophylline in SCC at SCC stages I, II, III, and IV; Higher results. Tumor recurrence was manifested as local infiltration and distant metastasis (Scale bars = 200 μm).

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Materials and Experiments

Patient and tissue sample

All patients continued to the National Gyeongsang National University Hospital (Jinju, Korea) and were operated from the same surgeon (Jang I) between May 2005 and June 2009. Tumor samples were obtained from a total of 42 surgical patients with SCC (n = 27) and AC (n = 15), frozen and stored at -80 ° C. Diagnosis was based on pathological examination of tissue samples. The characteristics of the tumor samples are summarized in Table 1 below. The patients ranged in age from 38 to 69 years, with 7 women and 35 men. For Western blot analysis, tumor tissues and adjacent normal lung tissues were randomly harvested from selected SCC (n = 10) and AC (n = 9) samples. The use of human tissue samples and the experimental procedures described below were all supervised and approved by the Ethics Committee of Gyeongsang National University Hospital.

Cell culture

Lung cancer cell lines (SK-MES-1 and A549) were purchased from American Type Culture Collection (Manassas, VA, USA). SK-MES-1 cells were cultured in EMEM supplemented with 5% -10% FBS and penicillin / streptomycin (Sigma, St. Louis, MO, USA), and A549 cells were cultured with 5% -10% FBS and penicillin / strepto Cultured in DMEM supplemented with mycin (Sigma, St. Louis, MO, USA).

Antibody

The following primary antibodies were used in this example: Rabbit anti-ROCK1 (160 kDa; Cell Signaling Technology, MA, USA), Rabbit anti-phospho (p) -Pak1 (ser144, 61-67 kDa; Cell Signaling Technology), Rabbit anti-Pak1 (70 kDa; Cell Signaling Technology), Rabbit anti-p-LIMK1 (72 kDa; Cell Signaling Technology), Rabbit anti-LIMK1 (70 kDa; Cell Signaling Technology), Rabbit term -p-cofilin (19-21 kDa; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and Goat anti-cofilin (19 kDa; Santa Cruz Biotechnology).

move Assay

For two-dimensional moving wound-healing assays, cells were seeded in 6-well culture dishes at a density of 1 × 10 ⁵ cells / well. After reaching confluence, cells were scratched with a sterile pipette tip to form a 600-μm wide defect in a single membrane. Cells were then washed with PBS and incubated in fresh medium. After incubation for the indicated periods, optical microscopy was taken. Relative cell migration distance (wound closure) was examined by measuring the wound width under a microscope (Axiovert 40C, Carlzeiss Micro Imaging, Gottingen, Germany) and subtracted this value from the initial wound width at 0 h. The data obtained through three scratches in each well was converted to a percentage of wound closure.

Transwell  move Assay

Three-dimensional cell migration was analyzed using the CytoSelect ^™ Cell Migration Assay Kit with polycarbonate membrane inserts (8-μm pore membrane; Cell Biolabs, Inc., CA, USA). Briefly, cell suspensions containing 0.5 × 10 ⁵ cells / ml were prepared in serum-free medium and added to the upper chamber of each insert. The lower chamber contained 10% FBS (chemotactic stimulus) and allowed for cell migration towards the lower face of the transwell culture insert. The cells were incubated at 37 ° C. for 6 hours in a humid 5% CO 2/95% atmosphere. Cells that did not migrate to the inner side of the transwell culture insert were gently rubbed off with a cotton swab. The transferred cells remaining on the bottom surface were stained with 0.5% crystal violet for 10 minutes at room temperature. A micrograph of five individual fields was taken using a microscope (Axiovert 40C) per insert and the number of cells counted to calculate the average number of cells moved. The stained inserts were washed and then incubated in extraction solution (1% Triton X-100) for 10 minutes to dissolve. Each sample was transferred to a 96-well microtiter plate and measured with a flake reader (Infinite® F200, Tecan, Mannedorf, Switzerland) at absorbance 560 nm.

infiltration Assay

Invasion of SK-MES-1 and A549 cells was investigated using a quantitative CytoSelectTM 96-well Cell Invasion Assay (Cell Biolabs) according to the manufacturer's instructions. Experimental instruments and assay protocols are basically similar to those used in the transwell migration assay. Briefly, SK-MES-1 and A549 cells suspended in a serum-free medium at a density of 0.5 × 10 ⁵ cells / ml were seeded on a culture insert (including a coating of a uniform layer of dried basement membrane matrix solution). Was allowed to penetrate the basement membrane membrane for 24 hours. Cells penetrated through the membrane were stained with 0.5% crystal violet and rubbed with a cotton swab to remove uninfiltrated cells on the upper surface of the membrane and observed under a microscope. The invasiveness was quantified by counting the number of infiltrated cells and calculating the average.

Western Blot  Analysis method

To extract proteins from tissues, cells transformed from frozen lung tissues were treated with 200 μL of lysis buffer (15 mM HEPES pH 7.9, 0.25 M sucrose, 60 mM KCl, 10 mM NaCl, 1 mM EGTA, 1 mM phenyl methyl sulfonyl). Transfer to a 1.5 mL microcentrifuge tube containing fluoride, and 2 mM NaF). Homogenized tissue was incubated for 10 minutes on ice and sonicated. The sample was then centrifuged at 14,240 × g at 4 ° C. for 10 minutes and the supernatant transferred to a new vial. For protein extraction from cells, cells were washed twice with PBS and then lysed with lysis buffer. Protein concentrations in tissue and cell lysates were quantified using Bio-Rad protein assay (Bio-Rad, USA) and stored at −80 ° C. until the samples were analyzed. For each protein analysis, lung lysates (30 μg per lane) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrophoresis on polyvinylidene difluoride membrane (Millipore, Mass., USA). It was. The membranes were probed with each antibody and visualized using an enhanced chemiluminescence substrate (Pierce, Rockford, IL, USA). The membrane was reprobed with anti-β-actin antibody as a control for equivalent protein loading.

Immunohistochemistry

For immunohistochemical analysis, paraffin-embedded lung tissue was cut into 5 μm thick sections and the sections were attached to gelatin-coated slides. Sections were deparaffinized, rehydrated with grade alcohol and processed using avidin-biotin immunoperoxidase method. Briefly, sample slides were incubated overnight at 4 ° C. with rabbit anti-p-cophylline antibody (diluted 1: 500; Santa Cruz Biotechnology). After washing three times with 0.1 M PBS, the sections were incubated with secondary biotinylated antibody (1: 200) for 1 hour at room temperature. After three additional washes with 0.1 M PBS, the sections were incubated with avidin-biotin-peroxidase complex solution (ABC solution, Vector Laboratories, CA, USA), followed by 0.05% diaminobenzidine with 0.05% H ₂ O ₂ . Devaluated with (DAB, Sigma). Slides were counterstained with hematoxylin, then dihydrated with grade alcohol and cleared with xylene. Then, cover slip-mounted with Permount (Sigma). The sections were visualized using a BX51 light microscope (Olympus, Tokyo, Japan), and digital images were captured and recorded.

statistics

Differences between SCC and AC tumor tissue or cell lines were investigated using two-tailed unpaired Student t- tests. The values are expressed as mean ± standard deviation (SEM). P value <0.05 was considered statistically significant.

Experiment result

The clinicopathological characteristics of 42 patients who underwent surgery for SCC or AC are shown in Table 1 below.

[Table 1]

One. SCC P- Kopilin  Immune staining increased AC Does not increase.

FIG. 1 (A) shows SCC lung tissue showing squamous cell differentiation with keratinized characteristics and AC tissue showing low differentiated adenocarcinoma with secretory gland-like structure. Positive staining for p-cophylline appeared exclusively restricted to tumor cells in tissue sections obtained from SCC and was localized to the cytoplasm; No p-cophylline immunostaining was detected in AC tissues (FIG. 1B).

2. normal lung tissue and SCC In Pak1 Of LIMK1 Of Kopilin  Expression of Pathway Proteins

To assess upstream of cophylline, 10 representative lung tissue samples from 27 matched pairs of surgically dissected lung SCCs were examined to analyze Pak1 / LIMK1 / cophylline pathway proteins in SCC lung tissue (FIG. 2). The expression of Pak1 / LIMK1 / cophylline pathway protein in all tumor samples was found to be increased when compared to samples of corresponding adjacent normal lung tissue. ROCK1 was overexpressed in three of the ten sample pairs (T3, T4, and T10), but overall ROCK1 expression was not significantly different between normal lung tissue and SCC. In addition, there was no difference in p-cophylline expression between different tumor stages.

3. normal lung tissue and AC In Pak1 Of LIMK1 Of Kopilin  Expression of Pathway Proteins

Western blot showed that Pak1 / LIMK1 / cophylline pathway protein was not overexpressed in all AC lung tissues (FIG. 3). p-Pak1 was not expressed in AC tumor tissue except for one sample (T3). No expression of p-LIMK1 was seen in all normal tissue and AC tumor samples. The levels of total LIMK protein and p-cophylline were found to be significantly increased in AC tissues in three and four of nine sample pairs, respectively. These results indicate that the Pak1 / LIMK1 / cophylline pathway is not dominant in AC, and the difference in ROCK1 expression between normal lung tissue and AC tissue is not significant.

4. Compared with A549 cells SK - MES -1 cells Pak1 Of LIMK1 Of Kopilin  Path is upregulated

In parallel with experiments on human lung cancer tissues, we examined the expression of Pak1 / LIMK1 / cophylline pathway proteins in the corresponding SCC and AC cell lines using Western blot analysis (see FIG. 4A). Similar to the results in 17 lung tissues in humans, the expression of Pak1 / LIMK1 / cophylline pathway protein was increased in SK-MES-1 (SCC type) cells as compared to A549 (AC type) cells. As in human SCC and AC tissues, ROCK1 expression was not significantly different between the two cell lines.

5. Than in A549 cells SK - MES Increased migration and infiltration in -1 cells.

To investigate the difference in cell migration between SCC and AC cell types, wound-healing and transwell migration assays were performed (see FIGS. 4B and 4C). As shown in FIG. 4B, at 24 hours the wound closure was significantly stronger in SK-MES-1 cells than in A549 cells (22.8% ± 9.3%) (65.5% ± 8.6%; P <0.05 ). Since wound healing is a complex process involving many cellular activities, including cell migration and proliferation, we sought to confirm the greater migration capacity of SK-MES-1 cells by performing a three-dimensional migration assay. As described in the experimental materials and methods, a transwell assay was performed to monitor cell migration toward chemotactic stimulus (10% FBS). As a result, it was confirmed that the percentage of migrated cells was significantly higher in SK-MES-1 cells (20.3% ± 6.6%) than A549 cells (0.06% ± 0.02%). Invasiveness of SK-MES-1 cells also increased by about 50% compared to A549 cells (see FIG. 4D).

6. Number of patients with local infiltration or distant metastasis AC  Than the organization SCC  Higher in lung tissue.

Immunohistochemical analysis of tumor cells obtained from all 42 NSCLC patients was performed. Western blot analysis showed no significant change in p-cophylline levels, but p-cophylline expression was associated with the histological stage of SCC patients. P-cophylline immunostaining was shown to be stronger than stage I and II in stage III and IV tissues of SCC (see FIG. 5A). In addition, follow-up of 43 patients who were initially diagnosed and operated for three years showed that 40.7% (n = 12) of SCC patients had local infiltration (n = 10) or distant metastasis to liver, brain, and bone (n = 3) appeared. In contrast, only 6.7% of AC patients relapsed, and only one AC patient showed distant metastasis to the brain.

Claims (8)

A composition for detecting a marker for diagnosing squamous cell carcinoma in lung cancer, comprising an agent measuring the level of at least one protein selected from the group consisting of p-Pak1, p-LIMK1, and p-cophylline. The composition of claim 1 comprising an agent for measuring the levels of proteins of p-Pak1, p-LIMK1, and p-cophylline. The composition of claim 1, wherein the agent measuring the level of the protein comprises an antibody specific for the protein. A kit for diagnosing squamous cell carcinoma of lung cancer, comprising the composition according to any one of claims 1 to 3. Measuring the levels of proteins of p-Pak1, p-LIMK1, and p-cophylline from biological samples isolated from suspected squamous cell carcinoma; And
Comparing the level of the protein with that of a normal control sample, Method of providing information for diagnosing squamous cell carcinoma of lung cancer. The method of claim 5, wherein the protein level uses an antibody specific for the protein. The method of claim 5, wherein the method for measuring protein levels is performed by Western blot, ELISA, radioimmunoassay, radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation. Method of providing information for diagnosing squamous cell carcinoma using any one of an assay, FACS or protein chip method. delete

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