Methods for diagnosing cancer, cancer diagnostic compositions, cancer diagnostic kits, methods for evaluating cancer status, and methods for screening cancer preventive and / or therapeutic drugs.

Abstract

This invention discloses a method for diagnosing cancer, which includes the step of detecting active protein kinase Cα in urine.

Description

Technical Field

[0001] This invention relates to methods for diagnosing cancer, cancer diagnostic compositions, cancer diagnostic kits, methods for evaluating cancer status, and methods for screening cancer preventive and / or therapeutic drugs. Background Technology

[0002] Living cells contain a vast number of intracellular signal transduction pathways that respond to extracellular signals and regulate or modulate gene expression. Phosphorylation induced by protein kinases activates target proteins in these intracellular signal transduction pathways, playing a crucial role in cell proliferation (see Non-Patent Literature 1–3). Protein kinase C (PKC), a phospholipid-dependent serine / threonine kinase, is one of the most important kinases and can be classified into three subfamilies based on their composition and activation characteristics: conventional or classic PKC (cPKC; α, βI, βII, and γ), novel or non-classical PKC (nPKC; δ, ε, η, and θ), and atypical PKC (ζ, ι, and λ) (see Non-Patent Literature 4–6).

[0003] Among PKC isoenzymes, PKCα is known to exhibit minimal activity in normal tissues, but is overexpressed or highly activated in most cancer cells (e.g., liver cancer, breast cancer, and melanoma) (see Non-Patent Literature 7–10).

[0004] Existing technical documents

[0005] Non-patent literature

[0006] Non-patent literature 1: Adjei, AA, and Hidalgo, M. (2005) Intracellular signal transduction pathway proteins as targets for cancer therapy. J. Clin. Oncol. 23, 5386-5403.

[0007] Non-patent literature 2: Cross, TG, Scheel-Toellner, D., Henriquez, NV, Deacon, E., Salmon, M., and Lord, JM (2000) Serine / threonine protein kinases and apoptosis. Exp. Cell Res. 256, 34-41.

[0008] Non-patent literature 3: Bode, AM, and Dong, Z. (2005) Signal transduction pathways in cancer development and as targets for cancer prevention. Prog. Nucleic Acid Res. Mol. Biol. 79, 237-297.

[0009] Non-patent literature 4: Newton, AC (1997) Regulation of protein kinase C. Curr. Opin. Cell Biol. 9, 161-167.

[0010] Non-patent literature 5: Blobe, GC, Obeid, LM, and Hannun, YA (1994) Regulation of protein kinase C and role in cancer biology. Cancer Metastasis Rev. 13, 411-431.

[0011] Non-patent document 6: Webb, BLJ, Hirst, SJ, and Giembycz, MA (2000) Proteinkinase C isoenzymes: a review of their structure, regulation and role inregulating airways smooth muscle tone and mitogenesis. Br.J.Pharmacol. 130, 1433-1452.

[0012] Non-patent literature 7: Oka, M., and Kikkawa, U. (2005) Protein kinase C in melanoma. Cancer Metast. Rev. 24, 287-300.

[0013] Non-patent literature 8: Mackay, HJ, and Twelves, CJ (2003) Protein kinase C: a target for anticancer drugs? Endocrin. Relat. Cancer 10, 389-396.

[0014] Non-patent literature 9: Hofmann, J. (2004) Protein kinase C isozymes as potential targets for anticancer therapy. Curr. Cancer Drug Targets 4, 125-146.

[0015] Non-patent literature 10: Goekjian, PG, and Jirousek, MR (2001) Protein kinase Cinhibitors as novel anticancer drugs. Expert. Opin. Investig. Drugs 10, 2117-2140.

[0016] Non-patent literature 11: O'Brian, CA, Chu, F., Bornmann, WG, and Maxwell, DS (2006) Protein kinase Cα and ε small-molecule targeted therapeutics: a new roadmap to two holy grails in drug discovery? Expert.Rev.Anticancer Ther.6,175-186. Summary of the Invention

[0017] The problem that the invention aims to solve

[0018] The purpose of this invention is to provide novel biomarkers for cancer.

[0019] Methods for solving problems

[0020] As mentioned above, PKCα has been reported to be highly expressed or highly activated in most cancer cells. On the other hand, based on the function of the glomerulus in the renal corpuscle, it is predicted that it will not be excreted in the urine. This is because PKCα is a negatively charged molecule with a molecular weight of 82 kDa, while the glomerulus in the renal corpuscle has two functions: a pore size barrier effect that prevents protein molecules larger than 60 kDa from passing through, and a charge barrier that prevents negatively charged substances from passing through due to electrorepulsion.

[0021] Contrary to this prediction, the inventors of this application discovered that active protein kinase Cα (active PKCα) can be detected in the urine of cancer patients, thus completing this invention.

[0022] That is, the present invention includes, for example, the following [1] to [9].

[0023] [1] A method for diagnosing cancer, which includes the step of detecting active protein kinase Cα (active PKCα) in urine.

[0024] [2] As described in [1], wherein the cancers mentioned above include urinary system cancers.

[0025] [3] The method as described in [1] or [2], wherein the cancer described above includes urothelial carcinoma.

[0026] [4] The method of any one of [1] to [3], wherein the cancer comprises non-muscle-invasive cancer.

[0027] [5] A cancer diagnostic composition, characterized in that it contains an antibody or substrate peptide for detecting active PKCα, and the diagnosis is performed using the urine of a subject.

[0028] [6] A cancer diagnostic kit, characterized in that it contains an antibody or substrate peptide for detecting active PKCα, and the diagnosis is performed using the urine of the subject.

[0029] [7] The method involves reacting an antibody against active PKCα with the urine of a subject to detect active PKCα, and using the results as an indicator to evaluate the cancer status.

[0030] [8] The method involves reacting the substrate peptide of active PKCα with the urine of the subject, detecting the phosphorylated peptide, and using the obtained detection results as an indicator to evaluate the cancer status.

[0031] [9] A method for screening cancer preventive and / or therapeutic drugs, comprising the following steps:

[0032] Procedures for administering candidate substances to non-human mammalian models of urinary tract cancer;

[0033] The steps for detecting active PKCα in the urine of the aforementioned non-human mammals; and

[0034] The steps for selecting cancer prevention and / or treatment drugs based on the obtained results.

[0035] Invention Effects

[0036] According to the present invention, methods for diagnosing cancer, cancer diagnostic compositions, cancer diagnostic kits, methods for evaluating cancer status, and methods for screening cancer preventive and / or therapeutic drugs are provided, utilizing active PKCα in urine as a biomarker. The present invention is simple and non-invasive because it uses a biomarker in urine. Furthermore, as described below, it also exhibits high specificity and sensitivity for early-stage cancer. Attached Figure Description

[0037] [ Figure 1The image shown is a MALDI-TOF MS spectrum, illustrating the results obtained from analyzing the phosphorylation of substrate peptides in cultured cells (KU-1, KU-7, T24, TCCSUP, UMUC-3) derived from bladder cancer using MALDI-TOF MS.

[0038] [ Figure 2 [Graph showing the phosphorylation rate (%) of substrate peptides in cultured cells derived from bladder cancer (KU-1, KU-7, T24, TCCSUP, UMUC-3).]

[0039] [ Figure 3 This section displays case information for patients with urothelial carcinoma. Gray cells indicate positive results or values ​​above a threshold. Histological type (primary) indicates the histological type (primary) of the cancer, urinalysis (RBC) indicates urine examination (red blood cells), urinalysis (WBC) indicates urine examination (white blood cells), and urine cytology indicates a urine cell diagnosis.

[0040] [ Figure 4 This section displays case information for patients without urothelial carcinoma. Gray cells indicate positive results or values ​​above a threshold. RBC (red blood cell count) represents urine analysis, and WBC (white blood cell count) represents urine analysis.

[0041] [ Figure 5 This is a histogram showing the phosphorylation rate of substrate peptides in urine samples from patients with and without urothelial carcinoma. White dots represent lines from urothelial carcinoma patients, and black dots represent lines from non-urothelial carcinoma patients.

[0042] [ Figure 6 This is a graph representing the relationship between the phosphorylation rate and frequency of substrate peptides in urine samples from patients with and without urothelial carcinoma, expressed using probability density functions. White dots represent lines from patients with urothelial carcinoma, and black dots represent lines from patients without urothelial carcinoma.

[0043] [ Figure 7 This is a graph showing the relationship between the phosphorylation rate and frequency of substrate peptides in urine samples from patients with and without urothelial carcinoma, expressed using the cumulative distribution function. White dots represent lines from urothelial carcinoma patients, and black dots represent lines from non-urothelial carcinoma patients.

[0044] [ Figure 8[Graph showing the phosphorylation rate of substrate peptides in urine samples from patients with low-grade and high-grade urothelial carcinoma (*: P < 0.05). Dashed lines indicate the cutoff value (phosphorylation rate 2%).]

[0045] [ Figure 9 [Image showing receiver operating characteristic (ROC) curves for a diagnosis based on the phosphorylation rate of substrate peptides using urinary samples. The dashed line represents the ROC curve for patients with low-grade urothelial carcinoma, and the solid line represents the ROC curve for patients with high-grade urothelial carcinoma.] Detailed Implementation

[0046] This invention is based on the discovery that active PKCα exists in the urine of cancer patients, and uses active PKCα in urine as a marker of cancer.

[0047] [Active PKCα]

[0048] Active PKCα means PKCα activated by phosphorylation of the threonine residue at position 497, the threonine residue at position 638, and the serine residue at position 657 in human PKCα, or the corresponding threonine and serine residues in PKCα of other animals.

[0049] [The Cancer That Becomes the Object]

[0050] There are no particular limitations on the types of cancer that can be used for diagnosis and evaluation; two or more types of cancer can be used. Cancers that are preferred as subjects of diagnosis and evaluation include urinary system cancers. Examples of urinary system cancers include tumors of the renal parenchyma and the urinary tract formed by the renal pelvis, ureter, bladder, and urethra, as well as tumors of the reproductive organs such as the prostate, seminal vesicles, penis, and testes. Representative urinary system cancers include urothelial carcinoma, prostate cancer, renal cancer, and seminal vesicle cancer.

[0051] The cancers that can be considered include urothelial carcinomas. Examples of urothelial carcinomas include bladder cancer, ureteral cancer, urethral cancer, and renal pelvis cancer. The cancers that can be considered can be selected from the group consisting of bladder cancer, ureteral cancer, urethral cancer, and renal pelvis cancer.

[0052] Urothelial carcinoma is a type of cancer that originates from cells in the mucosal urinary tract, specifically the urothelial (transitional epithelium), which forms part of the urinary tract connecting the renal pelvis to the ureter, bladder, and urethra. One of the main types of urothelial carcinoma is bladder cancer. Bladder cancer is assessed by atypia (tumor grade) in two stages: low atypia (low grade) and high atypia (high grade), or by three stages from G1 to G3. High atypia and G3 tumors exhibit strong cellular atypia and are highly malignant. In addition, the depth of invasion (T stage) is divided as follows: from Ta to T1 is non-muscle-invasive carcinoma, of which Tis is called carcinoma in situ, and T2 or above is muscle-invasive carcinoma (Japanese Urological Society, Japanese Society of Pathology and Japanese Society of Radiology, "Guidelines for the Operation of Renal Pelvis, Ureter and Bladder Cancer (Guidelines for the Operation of Renal Pelvis, Ureter and Bladder Cancer) April 2011 (1st Edition)" (Kinbara Publishing)).

[0053] TX: Unable to evaluate primary tumor

[0054] T0: No primary tumor

[0055] Ta: Papillary noninvasive carcinoma

[0056] Tis: Carcinoma in situ (CIS)

[0057] T1: Tumors infiltrating into the subepithelial connective tissue

[0058] T2a: Infiltrates to the middle of the muscle layer

[0059] T2b: Infiltrates beyond the middle of the muscle layer

[0060] T3a: Imagine microscopic infiltration into the peribladder adipose tissue.

[0061] T3b: Imagines a visible extramural infiltration into the peribladder adipose tissue.

[0062] T4a: Infiltration into the prostate, seminal vesicles, uterus, or vagina.

[0063] T4b: Infiltration into the pelvic or abdominal wall

[0064] Currently, biomarkers for cancer include nuclear matrix protein 22 (NMP22) and the Bladder Tumor Antigen Test (BTA test), but these all suffer from low sensitivity as bladder cancer biomarkers (especially for low-grade cancers). In contrast, this invention utilizes a biomarker of active PKCα in urine, which exhibits high specificity and sensitivity for non-muscle-invasive cancers, particularly low-grade cancers. Therefore, the target cancers can be, for example, non-muscle-invasive cancers, low-grade cancers, or carcinoma in situ.

[0065] [Methods used for cancer diagnosis]

[0066] The method of this embodiment provides an indicator for diagnosing cancer in a subject (recipient) based on the presence of active PKCα in the urine of a cancer patient. The method for diagnosing cancer in this embodiment includes the step of detecting active PKCα in urine.

[0067] The method of this embodiment may further include the step of collecting urine as a test sample (urine sample). Urine may be collected using a urine collection container, for example. Urine may be collected from a subject. The subject may be a human or a non-human mammal. The subject may be an animal suspected of having cancer.

[0068] The method of this embodiment may include a step of appropriately pretreating the urine sample according to the detection method of active PKCα. Examples of such pretreatment include centrifugation, heating, adding surfactants, cell disruption using homogenizers and ultrasonic disruption devices, dilution using sample buffers, and treatment with protease inhibitors.

[0069] The method of this embodiment can provide, for example, an indicator for diagnosing cancer based on the amount of active PKCα in urine. Examples of indicators for diagnosing cancer include those indicating the presence or high probability of cancer, and conversely, those indicating the presence or low probability of cancer. For example, a higher amount of active PKCα in a subject's urine than a baseline value can indicate the presence or high probability of cancer. The baseline value can be, for example, a value set based on the amount of active PKCα in the urine of a healthy person, a value set based on the amount of active PKCα in the urine of a cancer patient, or a value set by comparing the amount of active PKCα in the urine of a healthy person with the amount of active PKCα in the urine of a cancer patient. This baseline value can be, for example, a value used for directly comparing the amount of active PKCα in the subject, or a value set as a multiple (e.g., 10 times) of the amount of active PKCα compared to the amount in the urine of a healthy person. The baseline value can be a value set based on multiple samples, such as an average value. Here, "healthy person" can be replaced with a person who has not yet developed cancer (non-cancer patient). For example, a non-cancer patient could be someone who has been diagnosed with a specific cancer (e.g., urinary tract cancer) but has not yet developed cancer.

[0070] Furthermore, the method of this embodiment can also be used to determine the state (disease) of cancer by periodically monitoring the amount of active PKCα in the same subject. For example, an increasing trend in the amount of active PKCα in the subject's urine indicates that cancer has developed or is highly likely to develop.

[0071] There are no particular limitations on the method for detecting active PKCα in the urine of a subject, and known methods can be used. For example, since active PKCα has phosphorylation activity, its substrate peptide can be reacted with the urine of the subject to detect the phosphorylated peptide. Alternatively, for example, active PKCα can be directly detected by reacting an antibody against the urine. Therefore, the steps for detecting active PKCα in urine may include, for example, reacting the substrate peptide of active PKCα with the urine to detect the phosphorylated peptide, or reacting an antibody against the urine to detect PKCα.

[0072] [Substrate peptide detection was used]

[0073] The steps for detecting active PKCα in urine include reacting the substrate peptide of active PKCα with urine to detect phosphorylated PKCα. If the detected amount of phosphorylated PKCα substrate peptide is higher than a baseline value, it indicates that the subject has cancer. The baseline value can be, for example, a value set based on the detected amount of phosphorylated PKCα substrate peptide in the urine of a healthy person, a value set based on the detected amount of phosphorylated PKCα substrate peptide in the urine of a cancer patient, or a value set by comparing the detected amount of phosphorylated PKCα substrate peptide in the urine of a healthy person with that in the urine of a cancer patient. This baseline value can be, for example, a value used for directly comparing the detected amount of phosphorylated PKCα substrate peptide in the subject, or a value set as a factor (e.g., 10-fold) when comparing the detected amount of phosphorylated PKCα substrate peptide in the urine of a healthy person. The baseline value can be a value set based on multiple samples, such as an average value. As described later, when quantifying the phosphorylated PKCα substrate peptide by determining the phosphorylation rate of the PKCα substrate peptide used in the reaction, the phosphorylation rate can be used as a reference value. The phosphorylation rate used as the reference value can be set, for example, in the range of 1–7%, 1.5%–5%, 1.5%–4%, or 2%–3%.

[0074] The substrate peptide for active PKCα is not limited to any peptide that can be specifically phosphorylated by PKCα. Examples include substrate peptides formed from 10 to 30 amino acid residues (preferably 10 to 20 amino acid residues, more preferably 10 to 15 amino acid residues). Specific examples of such peptides include peptides formed from the amino acid sequence shown in Serial No. 1 (FKKQGSFAKKK) and peptides formed from the amino acid sequence shown in Serial No. 2 (FKKQGTFAKKK). It should be noted that the peptide formed from the amino acid sequence shown in Serial No. 1 is also referred to as "Alphatomega" in this specification.

[0075] PKCα substrate peptides can be synthesized, for example, using an automated peptide synthesizer following standard Fmoc chemical procedures. After synthesis, they can be purified using known purification methods employing various chromatographic techniques, etc.

[0076] In the method of this embodiment, the amount of PKCα substrate peptide used can be appropriately set by those skilled in the art, for example, it can be 1 to 1000 μM (preferably 1 to 500 μM, more preferably 1 to 50 μM).

[0077] (i) Detection of phosphorylated PKCα substrate peptides

[0078] Urine samples collected for testing may undergo pretreatment such as centrifugation, heating, addition of surfactants, cell disruption using homogenizers and ultrasonic disruption devices, dilution with sample buffers, or treatment with protease inhibitors. When performing such dilution, sample buffers may include, for example, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer or phosphate-buffered saline.

[0079] After pretreatment as needed, when the test sample is contacted with the PKCα substrate peptide, if the test sample contains active PKCα, the PKCα substrate peptide is phosphorylated through phosphorylation. The reaction medium is not particularly limited in this phosphorylation reaction; for example, a buffer solution composed of Tris-HCl and magnesium chloride can be used as the substrate to supply the phosphate group. Adenosine triphosphate (ATP) can also be mixed into this buffer solution.

[0080] The reaction between the test sample and the PKCα substrate peptide is carried out at 30–40°C (preferably 37°C) for 10–60 minutes (preferably 30–60 minutes).

[0081] The detection of phosphorylated PKCα substrate peptides is not limited and can be performed using known immunoassays according to conventional methods. Examples of immunoassays include labeled immunoassays and turbidimetric assays (TIAs), such as enzyme-linked immunosorbent assays (EIA) like ELISA, as well as radioimmunoassays (RIA) and fluorescence immunoassays (FIA). Furthermore, as labeled immunoassays, those utilizing combinations with other separation methods include Western blotting (combined with electrophoresis). Among these, ELISA and / or Western blotting are preferred. Additionally, one or more monoclonal antibodies targeting phosphorylated PKCα substrate peptides can be used in combination with two or more immunoassays for detection. This allows for superior detection with higher sensitivity and reliability.

[0082] As a method for detecting phosphorylated PKCα substrate peptides, a method using metal colloids such as metal nanoparticles can also be employed. Specifically, this method is based on the principle that, in the case of reacting the substrate peptide with a protein kinase in the presence of a metal colloid, or in the case of adding a metal colloid after the reaction of the substrate peptide with a protein kinase, the level of phosphorylation of the protein kinase-based substrate peptide can be evaluated based on the color change of the metal colloid. This color change refers to the change in the color of the metal colloid based on the degree of aggregation of the phosphorylated substrate peptide caused by the presence of metal colloids during the phosphorylation reaction of the protein kinase-based substrate peptide. Furthermore, the color change of the metal colloid varies substantially proportionally to the degree of aggregation, which is proportional to the phosphorylation ratio of the substrate peptide.

[0083] Therefore, as described above, by reacting the test sample with the PKCα substrate peptide in the presence of metal colloids, or by adding metal colloids after the reaction, and comparing the color change of the sample with that of the sample before the reaction, or with the color change of the control (the reaction with a sample collected from a healthy person), the phosphorylation of the PKCα substrate peptide can be detected.

[0084] In the above-described method using metal colloids, the reaction medium can be, for example, a buffer solution made by mixing HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) with magnesium chloride, etc., as the substrate for supplying phosphate groups, and ATP can be used in the same way as described above.

[0085] In addition, metal colloids such as metal nanoparticles can be used, for example, gold colloids, silver colloids, platinum colloids, iron colloids, ferric hydroxide colloids, aluminum hydroxide colloids, palladium colloids, and rhodium colloids. Among the above-mentioned metal colloids, gold colloids and silver colloids are preferred, and gold colloids are more preferred. These metal colloids can be prepared using methods described in known literature, and commercially available products can also be used. For example, gold nanoparticles can usually be prepared by liquid-phase reduction method, which involves adding a reducing solution to a solution containing gold ions to reduce the gold ions (see, for example, Japanese Patent Application Publication No. 2003-253310 and Japanese Patent Application Publication No. 2006-152438).

[0086] The particle size of the metal colloid is not particularly limited, but is preferably about 1 nm to 500 nm, more preferably 3 nm to 200 nm, and even more preferably 5 nm to 100 nm.

[0087] For metal colloids, although the color varies depending on the particle size, gold colloids typically appear red, while silver colloids appear yellow. In contrast, for example, in the presence of gold colloids, the PKCα substrate peptide is phosphorylated and aggregated, causing the hue of the gold colloid to change from red to blue. This hue change can be used to detect the phosphorylated PKCα substrate peptide. It should be noted that the hue change of the metal colloid is preferably examined by measuring absorbance in the range of approximately 600 nm to 800 nm, more preferably in the range of approximately 600 nm to 750 nm, and even more preferably around 700 nm.

[0088] (ii) Quantification of phosphorylated PKCα substrate peptides

[0089] Quantification is not limited; for example, it can be performed by using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine the phosphorylation rate (the ratio of the ionic strength of the phosphorylated substrate to the non-phosphorylated substrate) of the PKCα substrate peptide used in the reaction.

[0090] Furthermore, when detecting phosphorylated PKCα substrate peptides using the aforementioned metal colloid method, the phosphorylated substrate peptides can be substantially quantified based on the degree of hue change of the metal colloid (specifically, by comparing the degree of hue change with that of a suitable control and a pre-prepared calibration curve).

[0091] The quantification or detection of phosphorylated PKCα substrate peptides can also be performed using commercially available kits.

[0092] [Antibody-based detection]

[0093] The steps for detecting active PKCα in urine include reacting an antibody against the active PKCα with the urine to detect PKCα. If the detected level of active PKCα is higher than a baseline value, it indicates that the subject has cancer. The baseline value can be, for example, a value set based on the detected level of active PKCα in the urine of a healthy person, a value set based on the detected level of active PKCα in the urine of a cancer patient, or a value set by comparing the detected level of active PKCα in the urine of a healthy person with that in the urine of a cancer patient. This baseline value can be, for example, a value used for directly comparing the detected level of active PKCα in the subject, or a value set as a factor (e.g., 10-fold) when comparing the detected level of active PKCα in the urine of a healthy person. The baseline value can be a value set based on multiple samples, such as the average value.

[0094] Antibodies against active PKCα (anti-PKCα antibodies) can be prepared in the following manner.

[0095] (A) Preparation of polyclonal antibodies

[0096] (i) Preparation of antigen and its solution

[0097] To produce anti-PKCα antibodies, the first step is to prepare or obtain a protein that will be used as an immunogen (antigen).

[0098] Purified PKCα can be used as the antigen protein, but it is not limited to this. For example, proteins with PKCα activity can also be used, formed by the deletion, substitution, or addition of one or more amino acids in the amino acid sequence of PKCα. It should be noted that, for example, PKCα derived from mammals can be used, specifically, PKCα derived from humans, mice, or rats. The base and amino acid sequence information of PKCα derived from each animal can be obtained from the following accession numbers.

[0099] <Originating from human PKCα>

[0100] • GenBank accession numbers: NM 002737 (base sequence; sequence number 3), NP 002728 (amino acid sequence; sequence number 4)

[0101] • GenBank accession numbers: X52479 (base sequence; sequence number 5), CAA36718 (amino acid sequence; sequence number 6)

[0102] <PKCα derived from mice>

[0103] • GenBank accession numbers: NM 011101 (base sequence; sequence number 7), NP 035231 (amino acid sequence; sequence number 8)

[0104] • GenBank accession numbers: M25811 (base sequence; sequence number 9), AAA39934 (amino acid sequence; sequence number 10)

[0105] <PKCα derived from rats>

[0106] • GenBank accession numbers: NM 001105713.1 (base sequence; sequence number 11), XM 343975 (base sequence; sequence number 12)

[0107] There are no limitations on the method for preparing purified PKCα as an antigen. Examples include: generating PKCα using a transformant derived from a suitable host cell (such as cultured fibroblasts from humans, Chinese hamster ovary cells, and yeast); or preparing PKCα using conventional biochemical methods used in protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, and affinity chromatography, either alone or in appropriate combinations.

[0108] Next, the purified PKCα is dissolved in buffer solution to prepare an antigen solution. At this point, adjuvants for effective immunization can be added as needed. Examples of adjuvants include commercially available Freund's complete adjuvant and Freund's incomplete adjuvant. There are no restrictions on the use of one or more adjuvants.

[0109] (ii) Collection of immune and antiserum

[0110] Immunization can be performed by administering a solution containing the purified PKCα described above to mammals (e.g., mice, rats, and rabbits). Administration is primarily carried out via intravenous, subcutaneous, or intraperitoneal injection.

[0111] There is no limitation on the amount of antigen solution administered per dose. For example, for one animal, it is preferred to use 2 to 500 μg of purified PKCα, and more preferably 10 to 100 μg.

[0112] There is no limit to the interval between applications; for example, an interval of several days to several weeks is preferred, and an interval of 2 to 3 weeks is more preferred. In addition, the number of applications is, for example, 2 to 10.

[0113] The collection of serum (antiserum) obtained through the above immunization is not limited, but it is preferred to be carried out 1 to 28 days after the final administration, more preferably 2 to 14 days later. The antiserum can be collected from the blood of the immunized animal using conventional methods.

[0114] (iii) Screening of target antiserum

[0115] The target antiserum, i.e. the antiserum containing anti-PKCα antibody, is screened from antiserum collected from various immunized animals.

[0116] The screening method is not limited. For example, it can be performed using collected antiserum, purified PKCα as an antigen, recombinant PKCα (derived from cultured human fibroblasts, Chinese hamster ovary cells, yeast, etc.), or their mutant enzymes, using a known immunoassay method according to its conventional procedures. As immunoassays, there are labeled immunoassays and immunoturbidimetric assays (TIAs), with the former being preferred. Examples include enzyme-linked immunosorbent assays (EIA) such as ELISA, radioimmunoassays (RIA), and fluorescence immunoassays (FIA). Furthermore, regarding labeled immunoassays, as assays utilizing combinations with other separation methods, Western blotting (combined with electrophoresis) is also a preferred example. It should be noted that in the case of Western blotting, since the protein sample to be tested is denatured using heat and surfactants, the antigen in the screening method becomes a mutant enzyme.

[0117] The anti-PKCα antibodies contained in the target antiserum screened above are usually polyclonal antibodies. If purification of this antibody is required, it can be carried out using, for example, one or a combination of two or more known purification methods such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, or gel chromatography.

[0118] The reaction site in the (identifiable) PKCα that this anti-PKCα antibody can specifically bind to is not particularly limited.

[0119] (B) Production of monoclonal antibodies

[0120] (i) Preparation of antigen and its solution

[0121] The preparation of antigens and their solutions can be carried out in the same manner as the preparation of polyclonal antibodies described above.

[0122] (ii) Collection of immune and antibody-producing cells

[0123] The immunization method, the amount of the antigen solution used, the interval between administrations, and the number of administrations can be the same as those used in the preparation of the polyclonal antibodies.

[0124] There are no limitations on the collection of cells (antibody-producing cells) that produce anti-PKCα antibodies obtained through the above immunization. For example, it is preferable to collect them 1 to 14 days after the final administration, more preferably 2 to 4 days after the administration.

[0125] Antibody-producing cells may preferably include spleen cells, lymph node cells (especially local lymph node cells) and peripheral blood cells, with spleen cells and local lymph node cells (e.g., below-knee lymph node cells) being more preferred.

[0126] (iii) Cell fusion

[0127] By fusing collected antibody-producing cells with myeloma cells, fused cells (hybridomas) can be obtained.

[0128] As myeloma cells, lineages commonly obtained from mammals such as mice can be used, for example. Specifically, lineages that are drug-selective and cannot grow in an unfused state in HAT selective medium (a medium containing hypoxanthine, aminopterin, and thymine) but can grow in a state fused with antibody-producing cells are preferred. Specifically, as mouse myeloma cells, PAI, P3X63-Ag.8.U1 (P3U1), and NS-I can be used, for example.

[0129] The aforementioned cell fusion is performed, for example, by mixing antibody-producing cells with myeloma cells in an animal cell culture medium such as serum-free RPMI-1640 medium and allowing a fusion reaction to occur. The mixing ratio of antibody-producing cells to myeloma cells is, for example, 5:1.

[0130] Fusion reactions are typically carried out preferably in the presence of a cell fusion promoter, such as polyethylene glycol with an average molecular weight of 1000–6000 Daltons. Alternatively, commercially available cell fusion devices utilizing electrical stimulation (e.g., electroporation) can be used to fuse antibody-producing cells with myeloma cells.

[0131] The cells after the fusion reaction are cultured, for example, in HAT selective medium. After this culture, cells that proliferate in the HAT selective medium are observed to be fused cells (hybridomas).

[0132] (iv) Screening and cloning of target hybridomas

[0133] The target hybridoma, namely the hybridoma that produces anti-PKCα antibody, is screened from the hybridomas obtained through the above culture. Specifically, hybridomas containing anti-PKCα antibody are screened from the culture supernatant.

[0134] The screening method is not limited. For example, a portion of the culture supernatant can be collected, and purified PKCα, recombinant PKCα (derived from cultured human fibroblasts, Chinese hamster ovary cells, and yeast, etc.), or their mutant enzymes can be used as antigens. The screening can then be performed using a known immunoassay method, following standard procedures. Examples of preferred immunoassay methods include enzyme-linked immunosorbent assays (EIA) such as ELISA, radioimmunoassays (RIA), and fluorescence immunoassays (FIA). Additionally, Western blotting (combined with electrophoresis) is also preferred. It should be noted that in the case of Western blotting, since the protein sample to be tested is denatured using heat and surfactants, the antigen in the screening method becomes a mutant enzyme.

[0135] The anti-PKCα antibody contained in the culture supernatant of the target hybridoma after the above screening can be an antibody obtained before the hybridoma is cloned, or it can be an antibody formed from a single molecule (monoclonal antibody), there is no limitation.

[0136] The specific binding sites of the anti-PKCα antibody to which it can bind are not particularly limited. For example, antibodies that specifically recognize phosphorylated sites of PKCα (e.g., Thr497, Thr638, and Ser657 sites) can be used. By using antibodies that specifically recognize phosphorylated sites of PKCα, improved accuracy in the detection of active PKCα can be expected.

[0137] The establishment of target hybridoma clones, i.e., monoclonal antibody-producing cell lines, based on the above screening is usually carried out by selecting colonies derived from a single cell using culture methods employing limiting dilution.

[0138] (v) Collection of monoclonal antibodies

[0139] There are no restrictions on the method for collecting monoclonal antibodies from hybridomas obtained through the above cloning process; cell culture or ascites formation methods are commonly used.

[0140] In the cell culture method, the hybridoma obtained by cloning is cultured in a medium for animal cell culture, for example, at 37°C and 5% CO2 for 7 to 14 days, and the target monoclonal antibody can be collected from the culture supernatant afterward.

[0141] In ascites formation methods, for example, hybridomas obtained through cloning are distributed at 10 per animal. 6Approximately one dose is administered into the peritoneal cavity of an animal of the same lineage as the myeloma cells used for cell fusion, causing the hybridoma to proliferate rapidly. The target monoclonal antibody can be collected from ascites or serum 7–14 days after administration.

[0142] In either cell culture or ascites formation methods, if purification of the monoclonal antibody is required during or after collection, known purification methods such as ammonium sulfate precipitation, ion exchange chromatography, affinity chromatography, and gel chromatography can be appropriately employed, either alone or in combination with two or more of them.

[0143] In the method of this embodiment, the amount of anti-PKCα antibody used can be appropriately set by those skilled in the art. For example, based on 1 μg, it can be 1 μg to 10 μg, 1 μg to 100 μg, or 1 μg to 1000 μg.

[0144] (C) Detection and quantification of active PKCα

[0145] (i) Detection of active PKCα

[0146] Urine samples collected for testing can be pretreated by methods such as centrifugation, heating, addition of surfactants, cell disruption using homogenizers and ultrasonic disruption devices, dilution with sample buffers, and treatment with protease inhibitors. For example, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer and phosphate-buffered saline can be used as sample buffers.

[0147] After pretreatment as required, when the test sample is reacted with an anti-PKCα antibody (usually a monoclonal antibody is preferred), an antigen-antibody reaction will occur if the test sample contains active PKCα.

[0148] The reaction between the test sample and the anti-PKCα antibody is carried out at 20–40°C (preferably 30–37°C) for 30–720 minutes (preferably 30–90 minutes).

[0149] The detection of active PKCα can be performed using known immunoassays according to conventional methods. Examples of immunoassays include labeled immunoassays and turbidimetric assays (TIAs), with the former being preferred. Examples include enzyme-linked immunosorbent assays (EIA) such as ELISA, radioimmunoassays (RIA), and fluorescence immunoassays (FIA). Furthermore, regarding labeled immunoassays, Western blotting (combined with electrophoresis) is a preferred method that utilizes a combination of other separation methods. Of these, ELISA and / or Western blotting are preferred. Additionally, one or more anti-PKCα monoclonal antibodies can be used in combination with two or more immunoassays for detection. This allows for superior detection with higher sensitivity and reliability.

[0150] (ii) Quantitative analysis of active PKCα

[0151] Quantification is not limited, but it is preferable to quantify the active PKCα contained in the test sample based on a calibration curve relating antigen concentration (antigen concentration: PKCα concentration) to its detection value. This calibration curve is pre-prepared using purified PKCα as the antigen and based on detection values ​​(detection data) obtained using the aforementioned anti-PKCα antibody. Specifically, it is prepared based on information obtained from detection values ​​for antigen concentration using the same method as the immunoassay methods (ELISA, Western blotting, etc.) described as detection methods. The amount of active PKCα in the test sample can be determined by comparing this calibration curve with the actual detection values ​​(measured values). PKCα quantification or detection can also be performed using commercially available kits.

[0152] [Compositions for Cancer Diagnosis]

[0153] The cancer diagnostic composition of this embodiment is characterized by containing an antibody or substrate peptide for detecting active PKCα, and using the urine of a subject for diagnosis.

[0154] The antibodies or substrate peptides used to detect active PKCα can be the substances described above. The cancer diagnostic composition of this embodiment can be used to detect and quantify active PKCα in the urine of a subject, and can be used in the aforementioned diagnostic procedures. The methods for detecting active PKCα are also the same as those described above.

[0155] The proportion of the antibodies in the cancer diagnostic composition is not particularly limited. For example, based on the total weight of the composition, it can be 1-100 wt%, 5-100 wt%, 10-100 wt%, 3-99 wt%, 5-99 wt%, 10-99 wt%, 10-95 wt%, or 20-95 wt%. Without significantly impairing the effects of the present invention, the cancer diagnostic composition in this embodiment may contain other components besides the anti-PKCα antibody, and there are no limitations. For example, it may contain reagents for primary antibody detection, chromogenic substrates, etc.

[0156] The proportion of the PKCα substrate peptide in the cancer diagnostic composition is not particularly limited. For example, based on the total weight of the above composition, it can be 1-100% by weight, 5-100% by weight, 10-100% by weight, 3-99% by weight, 5-99% by weight, 10-99% by weight, 10-95% by weight, or 20-95% by weight. Without significantly impairing the effects of the present invention, the cancer diagnostic composition in this embodiment may contain other components besides the PKCα substrate peptide, and there are no limitations. For example, it may contain antibodies against the phosphorylated PKCα substrate peptide.

[0157] [Cancer Diagnostic Kit]

[0158] The cancer diagnostic kit of this embodiment is characterized by containing an antibody or substrate peptide for detecting active PKCα, and using the subject's urine for diagnosis.

[0159] The antibodies or substrate peptides used to detect active PKCα can be the substances described above. The cancer diagnostic kit in this embodiment is used to detect and quantify active PKCα in the urine of a subject, and can be used in the diagnostic procedures described above. The methods for detecting active PKCα are also the same as described above.

[0160] The cancer diagnostic kit of this embodiment may contain the above-described cancer diagnostic composition.

[0161] When a cancer diagnostic kit contains an anti-PKCα antibody, it is preferable that the antibody be in a dissolved state, taking into account factors such as stability (storageability) and ease of use.

[0162] When a cancer diagnostic kit contains a PKCα substrate peptide, considering stability (storageability) and ease of use, it is preferable to store the substrate peptide in powder or solution form (dissolved in pure water, buffer solution, physiological saline, etc.) under refrigerated or frozen conditions (-30°C to -80°C).

[0163] In addition to the aforementioned PKCα substrate peptide or anti-PKCα antibody, this kit may contain other components. Examples of other components include reagents and chromogenic substrates used in ELISA and Western blotting for primary antibody detection.

[0164] This kit only needs to contain at least the aforementioned PKCα substrate peptide or anti-PKCα antibody as a component. Therefore, it may or may not contain all the components necessary for cancer diagnosis, and there are no restrictions.

[0165] In addition, the kit may include means of collecting urine, such as a urine collection container.

[0166] In addition, the kit may come with instructions for using the kit to detect active PKCα in the urine of subjects and for cancer diagnosis.

[0167] Methods for assessing cancer status

[0168] The method for evaluating cancer status in this embodiment involves reacting an antibody against the active PKCα with the subject's urine to detect the active PKCα, and using the obtained detection result as an indicator to evaluate the cancer status. Alternatively, another embodiment of the method for evaluating cancer status involves reacting a substrate peptide of the active PKCα with the subject's urine to detect the phosphorylated peptide, and using the obtained detection result as an indicator to evaluate the cancer status.

[0169] The aforementioned substances can be used as antibodies or substrate peptides for detecting active PKCα. Furthermore, the method for detecting the reaction with the subject's urine is the same as described above.

[0170] The method for evaluating cancer status according to this embodiment uses the detection result (detection amount) of the detected and quantified active PKCα or phosphorylated substrate peptide as an indicator to evaluate the cancer status. That is, active PKCα is detected by reacting an antibody against the subject's urine, or phosphorylated substrate peptide is detected by reacting a PKCα substrate peptide with urine, and the cancer status is evaluated using the obtained detection result as an indicator.

[0171] Here, "evaluating the cancer status" means determining whether the cancer has developed, its progression, severity, treatment responsiveness, and prognosis. The evaluation can be conducted by combining the detection levels of active PKCα or phosphorylated PKCα substrate peptides, as well as self-reported symptoms. For example, evaluation can be performed by measuring active PKCα or phosphorylated PKCα substrate peptides once or twice a year, similar to regular physical examinations. Preferably, the cancer status (including monitoring for recurrence) is comprehensively evaluated by regularly monitoring changes in the detection levels of active PKCα or the phosphorylated substrate peptide. As an example, if the detection levels of active PKCα or phosphorylated PKCα substrate peptides exceed a certain value, a high probability of developing a certain type of cancer can be diagnosed; if the detection levels of active PKCα or the phosphorylated substrate peptides decrease in cancer cases already under treatment, treatment effectiveness can be diagnosed; if the detection levels of active PKCα or the phosphorylated substrate peptides change to high values ​​exceeding a certain threshold, the case can be diagnosed as untreatable and unrecoverable.

[0172] Methods for screening cancer prevention and / or treatment drugs

[0173] The method for screening cancer preventive and / or therapeutic drugs according to this embodiment includes the following steps:

[0174] (a) The procedure for administering the candidate substance to a non-human mammalian model of urinary tract cancer (the administration procedure);

[0175] (b) The steps for detecting active PKCα in the urine of the aforementioned non-human mammals (detection steps); and

[0176] (c) The steps of selecting cancer prevention and / or treatment drugs as targets based on the results obtained (evaluation steps).

[0177] It should be noted that the screening method in this embodiment may include other steps as needed.

[0178] The following explains each of the above steps.

[0179] (a) Application steps

[0180] There are no particular limitations on the non-human mammals used as test animals for urinary system cancer models and control animals. However, for the purpose of conducting accurate comparative experiments, it is generally preferred to use non-human mammals of the same species, more preferably non-human mammals of the same mother, and even more preferably non-human mammals of the same sex and age. In addition, it is preferable that the test animals and control animals have the same rearing conditions except for the amount of feed intake.

[0181] As test animals, non-human mammals used as models of urinary system cancer can be used, obtained by transplanting tumor tissue and tumor cells into normal non-human mammals to induce cancer using known methods. Alternatively, non-human mammals obtained by surgically removing tumor tissue and tumor cells from the aforementioned non-human mammal models of urinary system cancer can also be used. When these resected non-human mammals are used as test animals, screening methods for drugs to prevent recurrent cancer can be implemented, for example.

[0182] As a control animal, there are no limitations as long as it is suitable for comparison with the test animal. For example, normal non-human mammals without transplanted tumor tissue can be used, as well as non-human mammals with urinary system cancer models that have not been given the candidate substance or test animals before the administration of the candidate substance can be used as control animals.

[0183] Candidate substances for administration to test animals are not limited and can include various naturally or artificially synthesized peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotides (DNA, RNA), oligonucleotides (siRNA, etc.), peptide nucleic acids (PNA), etc.), low- or high-molecular-weight organic compounds, etc.

[0184] Candidate substances can be administered orally or non-orally, without limitation, and any known administration methods and conditions can be used. The dosage can also be appropriately set considering the species and condition of the test animal, as well as the type of candidate substance.

[0185] (b) Detection steps

[0186] In this step, the active form of PKCα is detected in the urine (urine sample) of test animals that have been administered the candidate substance. Any known method can be used to collect the urine sample. There are no particular limitations on the number of tests or the testing time for active PKCα; these can be appropriately set considering factors such as the efficacy of the target cancer prophylaxis or treatment.

[0187] There are no restrictions on the detection and quantification methods for PKCα in urine; the methods described above may be used as appropriate.

[0188] (c) Evaluation steps

[0189] In the screening of cancer prevention and / or cancer treatment drugs, for example, the detection level of active PKCα in urine is compared and evaluated in test animals administered the candidate substance and control animals not administered the candidate substance. Preferably, this evaluation is used to determine whether the candidate substance is a target cancer prevention and / or cancer treatment drug.

[0190] Specifically, for non-human mammalian models of urinary tract cancer with the same level of active PKCα in urine, where one group serves as test animals administered the candidate substance and the other as control animals not administered the candidate substance, the candidate substance can be selected as the target cancer treatment if the level of active PKCα in the urine of the test animals is lower than that in the control animals. Conversely, the candidate substance cannot be selected if the level of active PKCα in the urine of the test animals is maintained at the same level as or higher than that in the control animals. In this case, the control animals can also be the test animals themselves before the administration of the candidate substance.

[0191] In addition, when non-human mammals with urinary tract cancer models obtained through surgical removal of tumor tissue, or non-human mammals with urinary tract cancer models that have temporarily undergone cancer treatment using methods other than surgical removal (medical methods, etc.), are used as test animals to administer the candidate substance, and normal non-human mammals are used as control animals, if the level of active PKCα in the urine of the test animals is consistently maintained at the same level as that in the control animals, the candidate substance can be selected as a cancer prophylactic drug (more specifically, a prophylactic drug for recurrent cancer). In contrast, if the level of PKCα in the urine of the test animals is higher than that in the urine of the control animals, the candidate substance cannot be selected as a target cancer prophylactic drug. In this case, the control animal can also be the test animal itself before the administration of the candidate substance (before recurrence).

[0192] It should be noted that the screening of cancer prevention and / or treatment drugs based on the amount of active PKCα in urine can be carried out for multiple cancer types (preferably all cancer types). Therefore, in the case of non-human mammals that have been used as models of urinary system cancer for a specific cancer type, it is possible to screen for prevention and / or treatment drugs for that specific cancer type.

[0193] Treatment methods for cancer

[0194] The cancer treatment method of this embodiment includes the following steps:

[0195] This includes diagnostic procedures for cancer detection, such as detecting active PKCα in the urine of test subjects; and

[0196] The steps for administering therapeutic drugs to subjects diagnosed with cancer.

[0197] The diagnostic procedure for cancer can be based on the indicators provided in the methods described above for cancer diagnosis, to diagnose whether a subject has cancer. Alternatively, it can be based on the methods described above for evaluating the state of cancer, to diagnose whether a subject has cancer.

[0198] In the procedure of administering therapeutic drugs to subjects diagnosed with cancer, the therapeutic drugs administered to the subjects can be appropriately selected by those skilled in the art based on the type of cancer. For example, in the case of urinary system cancer, anthracycline-type anticancer antibiotics, mitomycin C, and BCG are commonly used as preventative drugs for recurrence after transurethral surgery, while cisplatin and gemcitabine are commonly used as therapeutic drugs. Therapeutic drugs selected using the methods described above for screening cancer preventative and / or therapeutic drugs can also be used. Furthermore, administration can be performed orally or non-orally without limitation, and known administration methods and conditions can be used in any situation. The dosage can also be appropriately set considering the type of cancer, the subject's condition, and the type of therapeutic drug.

[0199] Furthermore, the cancer treatment method of this embodiment may also include a step of determining whether the treatment based on the therapeutic drug is effective by monitoring the detection level of active PKCα or the phosphorylated substrate peptide using the above-described method for evaluating the cancer status after the administration of the therapeutic drug. For example, it may include: if the detection level of active PKCα or the phosphorylated substrate peptide decreases after the administration of the therapeutic drug, it is determined that the treatment is effective; and / or if the detection level of active PKCα or the phosphorylated substrate peptide changes to a high value exceeding a certain value, it is diagnosed as a case where the treatment is ineffective.

[0200] Example

[0201] The following examples illustrate the invention in more detail, but the invention is not limited thereto.

[0202] [Example 1: Phosphorylation of substrate peptides in cancer cell samples]

[0203] (Synthesis of substrate peptides)

[0204] Alphatomega (FKKQGSFAKKK (serial number 1)) was synthesized as the substrate peptide using an automated peptide synthesizer following standard Fmoc chemistry procedures. After treatment with trifluoroacetic acid (TFA), the peptide was purified using an Inertsil ODS-3 column (250 × 20 mm, 3.5 μm; GL Sciences, Tokyo, Japan) with a BioCAD perfusion chromatography system (Ikemoto Rika Co., Ltd.) and an AB linear gradient at a flow rate of 8 mL / min (elution buffer A was water containing 0.1% TFA, and eluent B was acetonitrile containing 0.1% TFA).

[0205] In the following experiments, this peptide was used as a PKCα-specific substrate peptide. It should be noted that the molecular weight of this substrate peptide, FKKQGSFAKKK-NH2, is 1338 Da, but due to PKCα phosphorylation, it increased by 80 Da to 1418 Da.

[0206] (MALDI-TOF MS analysis)

[0207] An α-cyano-4-hydroxycinnamic acid (CHCA) matrix (10 mg / ml) was prepared in 50% water / acetonitrile and 0.1% TFA. This matrix was mixed with the sample at a ratio of 20:1. A total volume of 1 μl of the sample / matrix mixture was spread onto a sample plate, dried, and allowed to crystallize. In this example, Bruker's Auto FlexSpeed ​​was used and run in both positive and negative modes. The accelerating voltage was set to 20 kV, and the extraction delay time was set to 100 ns. Typically, 100 laser shots were averaged to improve the signal-to-noise ratio. All spectra were analyzed using Flex Analysis software (Bruker). Phosphorylation rate is defined as the ratio of the ionic strength of the phosphorylated substance to that of the non-phosphorylated substance, and is calculated according to existing specifications (Kang, J.-H. et al., (2007) J.Am.Soc.Mass Spectrom.18, 106-112.; Kang J.-H. et al., (2007) J.Am.Soc.Mass Spectrom.18, 1925-1931.).

[0208] To investigate whether PKCα-specific substrate peptides can be phosphorylated by bladder cancer, MALDI-TOF MS was used to analyze cultured cells (KU-1, KU-7, T24, TCCSUP, and UMUC-3) derived from five different bladder cancers. KU-1, KU-7, TCCSUP, and UMUC-3 were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, penicillin (100 U / ml), streptomycin (100 μg / ml), and amphotericin B (0.25 μg / ml; all Invitrogen). T24 was maintained in RPMI-1640 medium. The cancer cells were placed in a humidified atmosphere at 37°C containing 5% CO2 and 95% air. The cultured cells were excised and centrifuged at 1,500 rpm for 5 minutes. The precipitate (cells) was used for phosphorylation of the substrate peptide. Phosphorylation of the substrate peptide was performed in 30 μl buffer containing 30 μM of the synthetic peptide (10 mM HEPES (pH 7.5), 10 mM MgCl2, 100 μM ATP, and 0.2 mg / ml cells). After incubation at 37°C for 60 min, samples were analyzed using MALDI-TOF MS.

[0209] The results are shown in Figure 1 and 2 “Untreated” means no cells. An 80 Da increase in m / z value confirmed phosphorylation of the substrate peptide, with high phosphorylation rates confirmed in all samples prepared from cultured cells derived from bladder cancer.

[0210] [Example 2: Detection of PKCα activity in human urine samples]

[0211] Urine samples were collected from 28 patients with urothelial carcinoma and used for the phosphorylation of substrate peptides. Urine samples from 24 patients without urothelial carcinoma served as a control group. Case information for both urothelial carcinoma and non-urothelial carcinoma patients is presented separately. Figure 3 and Figure 4 .

[0212] 20 ml of urine sample was collected from the patient and centrifuged (5000 g, 15 min), and the precipitate was recovered. 160 μl of HEPES buffer (containing 10% sucrose) and 40 μl of protease inhibitor cocktail were added to resuspend the cells. Cells were lysed on ice for 10 seconds using a probe-type ultrasonic disruptor (SONIFIER 250, BRANSON). After centrifugation (5000 g, 15 min), the supernatant was recovered, and protein quantification was performed to determine the protein concentration. Substrate peptide phosphorylation was performed with a protein concentration of 0.2 mg / ml, 100 μM ATP, 10 mM MgCl2, and 30 μM substrate peptide. After incubation at 37°C for 60 min, the samples were analyzed using MALDI-TOF MS. The analysis of the substrate peptide and MALDI-TOF MS was performed in the same manner as in Example 1.

[0213] Phosphorylation rates were measured separately in patients with and without urothelial carcinoma, and plotted as histograms. Figure 5 ), probability density function ( Figure 6 ), cumulative distribution function Figure 7 The figure indicates that patients with urothelial carcinoma showed higher phosphorylation compared to those without urothelial carcinoma, and a phosphorylation rate of 2% with a large difference was set as the threshold.

[0214] in addition, Figure 8 The figure shows the phosphorylation rate of substrate peptides in urine samples from 9 patients with low-grade urothelial carcinoma and 19 patients with high-grade urothelial carcinoma. Both low-grade and high-grade patients showed higher phosphorylation compared to non-urothelial carcinoma patients.

[0215] [Example 3: Comparison with other urothelial carcinoma markers]

[0216] Based on the phosphorylation rate of PKCα in urothelial carcinoma patients and non-urothelial carcinoma patients calculated in Example 2, a positive reaction threshold was set at 2%, and comparisons were made with other existing urothelial carcinoma markers (urine cell diagnostics, NMP22 (Alere), BTA (FUJIREBIO), and CK18-8 (iDL Biotech)). Existing urothelial carcinoma markers were used according to the appendix document.

[0217] The threshold for PKCα in urothelial carcinoma patients was set at 2%, the threshold for CK18-8 (another urothelial carcinoma marker) was set at 16 ng / mgCr, and the threshold for NMP22 was set at 12 U / ml. The sensitivity, specificity, and accuracy of each assay method were investigated. For urine cell diagnosis and BTA, since these are qualitative methods, a positive and negative criterion was used in the study. PKCα showed a sensitivity of 89.3%, a specificity of 83.3%, and an accuracy of 86.5%, all indicating high levels of results. On the other hand, NMP22 showed a sensitivity of 50.0%, a specificity of 91.7%, and an accuracy of 69.2%; BTA showed a sensitivity of 39.4%, a specificity of 75.0%, and an accuracy of 55.8%; and CK18-8 showed a sensitivity of 75.0%, a specificity of 58.3%, and an accuracy of 67.3%, indicating high specificity but low sensitivity (Table 1).

[0218] [Table 1]

[0219]

[0220] in addition, Figure 9 The diagram shows receiver operating characteristic (ROC) curves for diagnosis based on the phosphorylation rate of substrate peptides, using urinary samples from 9 patients with low-grade urothelial carcinoma and 19 patients with high-grade urothelial carcinoma. Active PKCα in urine showed high detection sensitivity and specificity not only in high-grade urothelial carcinoma but also in low-grade urothelial carcinoma.

Claims

1. The use of reagents for detecting active protein kinase Cα in urine in the manufacture of kits for diagnosing urothelial carcinoma. in, The diagnosis was made using the subject's urine.

2. The use as described in claim 1, wherein, The urothelial carcinoma includes carcinoma in situ.

3. The use as described in claim 1 or 2, characterized in that, The kit contains an antibody or substrate peptide for detecting active protein kinase Cα.

4. The use of reagents for detecting active protein kinase Cα in urine in the manufacture of kits for evaluating the status of urothelial carcinoma, wherein, The antibody targeting active protein kinase Cα reacts with the subject's urine to detect active protein kinase Cα, and the results are used as an indicator to evaluate the status of urothelial carcinoma.

5. The use of reagents for detecting active protein kinase Cα in urine in the manufacture of kits for evaluating the status of urothelial carcinoma, wherein, The substrate peptide of active protein kinase Cα reacts with the urine of the subject, and the phosphorylated peptide is detected. The results are used as an indicator to evaluate the status of urothelial carcinoma.

6. A method for screening cancer preventive and / or therapeutic drugs, comprising the following steps: Procedures for administering candidate substances to non-human mammalian models of urothelial carcinoma; The steps for detecting active protein kinase Cα in the urine of the aforementioned non-human mammals; and The steps for selecting cancer prevention and / or treatment drugs based on the obtained results.

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