JP5721628B2 - Protein kinase Cα as a marker for cancer diagnosis - Google Patents

Description

  The present invention relates to protein kinase Cα (hereinafter sometimes referred to as PKCα) as a cancer marker. Specifically, it relates to extracellular PKCα as a biomarker for cancer diagnosis of a plurality of cancer types.

  Over the past 30 years, the discovery of biomarkers for cancer diagnosis has mainly involved the comparison between cancer tissues and normal tissues (see Non-Patent Documents 1 to 3). In recent years, the search for biomarkers in blood, urine, or saliva has attracted much interest because it is easier to collect and handle samples compared to analysis of tissue samples (see Non-Patent Documents 4 to 7). For example, serum prostate specific antigen (PSA) can be assayed extensively to diagnose prostate cancer or to monitor recurrence of prostate cancer after surgical resection or radiation treatment (Non-Patent Document 3 and 8).

Living cells contain a vast number of intracellular signaling pathways that control or regulate gene expression in response to extracellular signals. Phosphorylation by protein kinase activates target proteins in these intracellular signal transduction pathways and plays an important role in cell proliferation (see Non-Patent Documents 9 to 11). Protein kinase C (PKC), one of the phospholipid-dependent serine / threonine kinases, is one of the most important kinases and is classified into three subfamilies based on composition and activation characteristics. That is, conventional or classical 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) (non- (See Patent Documents 12 to 14).
Among PKC isozymes, PKCα is known to exhibit very little activity in normal tissues and is overexpressed or highly activated in most cancer cells (eg, liver cancer, breast cancer and melanoma) ( Non-patent documents 15 to 19).

Sidransky, D. (2002) Emerging molecular markers of cancer. Nat. Rev. Cancer 2, 210-219. Harris, L., Fritsche, H., Mennel, R., Norton, L., Ravdin, P., Taube, S., Somerfield, MR, Hayes, DF, and Bast Jr., RC (2007) American society of clinical oncology 2007 update of recommendations for the use of tumor markers in breast cancer.J. Clin. Oncol. 25, 5287-5312. Chatterjee, S. K., and Zetter, B. R. (2005) Cancer biomarkers: knowing the present and predicting the future. Future Oncol. 1, 37-50. Roos, P. H., Golka, K., and Hengstler, J. G. (2008) Predictive biomarkers and signature in urinary bladder cancer. Cuur. Opin. Mol. Ther. 10, 243-250. Schrohl, A.- S. Wurtz, S., Kohn, E., Banks, RE, Nielsen, HJ, Sweep, FCGJ, and Brunner, N. (2008) Banking of biological fluids for studies of disease-associated protein biomarkers. Mol Cell Proteomics 7, 2061-2066. Decramer, S., de Peredo, AG, Breuil, B., Mischak, H., Monsarrat, B., Bascands, J.- L., and Schanstra, JP (2008) Urine in clinical proteomics. Mol. Cell Proteomics 7 , 1850-1862. Wong, D. T. (2006) Salivary diagnostics powered by nanotechnologies, proteomics and genomics.JADA 137, 313-321. Damber, J.- E., and Aus, G. (2008) Prostate cancer. Lancet 371, 1710-1721. Adjei, A. A., and Hidalgo, M. (2005) Intracellular signal transduction pathway proteins as targets for cancer therapy.J. Clin. Oncol. 23, 5386-5403. 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 .

Bode, A. M., 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. Newton, A. C. (1997) Regulation of protein kinase C. Curr. Opin. Cell Biol. 9, 161-167. Blobe, G. C., Obeid, L. M., and Hannun, Y. A. (1994) Regulation of protein kinase C and role in cancer biology. Cancer Metastasis Rev. 13, 411-431. Webb, B. L. J., Hirst, S. J., and Giembycz, M. A. (2000) Protein kinase C isoenzymes: a review of their structure, regulation and role in regulating airways smooth muscle tone and mitogenesis.Br. J. Pharmacol. 130, 1433-1452. Oka, M., and Kikkawa, U. (2005) Protein kinase C in melanoma. Cancer Metast. Rev. 24, 287-300. Mackay, H. J., and Twelves, C. J. (2003) Protein kinase C: a target for anticancer drugs? Endocrin. Relat. Cancer 10, 389-396. Hofmann, J. (2004) Protein kinase C isozymes as potential targets for anticancer therapy. Curr. Cancer Drug Targets 4, 125-146. Goekjian, P. G., and Jirousek, M. R. (2001) Protein kinase C inhibitors as novel anticancer drugs. Expert. Opin. Investig. Drugs 10, 2117-2140. 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.

  Under such circumstances, development of a novel biomarker for cancer diagnosis that is simple and highly reliable has been desired.

The present invention has been made in view of the above situation, and includes the following cancer diagnostic markers, cancer diagnostic pharmaceutical compositions, cancer diagnostic kits, methods for evaluating cancer status, cancer preventive drugs and / or The present invention provides a method for screening cancer drugs and the like.
(1) A marker for cancer diagnosis, comprising protein kinase Cα.
Examples of the marker of the present invention include blood markers.
Examples of the marker of the present invention include those used for diagnosis of early cancer or recurrent cancer, and those used for diagnosis of at least two or more types of cancer, and further all types of cancer.

(2) A pharmaceutical composition for cancer diagnosis, comprising a substrate peptide of protein kinase Cα or an antibody against protein kinase Cα.
(3) A kit for diagnosing cancer, comprising the pharmaceutical composition of (2) above.
(4) A method in which a substrate peptide of protein kinase Cα and a biological sample are reacted to detect the phosphorylated peptide, and the cancer state is evaluated using the obtained detection result as an index.
(5) A method of detecting protein kinase Cα by reacting an antibody against protein kinase Cα with a biological sample, and evaluating the cancer state using the obtained detection result as an index.
In the methods (4) and (5), examples of the biological sample include blood.

(6) A method for screening for preventive and / or therapeutic agents for cancer,
A step of administering a candidate substance to a cancer-bearing model non-human mammal, a step of detecting protein kinase Cα in the blood of the non-human mammal, and a target cancer preventive drug and / or cancer treatment using the obtained detection result as an index Said method comprising the step of selecting a drug.

According to the present invention, a novel biomarker for cancer diagnosis that is simple and highly reliable can be provided.
The cancer diagnostic marker of the present invention is a marker in the blood and is easy to handle, can be used for diagnosis of multiple cancer types, and is particularly suitable for diagnosis of early cancer and recurrent cancer, Very useful.

It is a figure showing the typical MALDI-TOF MS spectrum obtained from the phosphorylation reaction of the substrate peptide and the plasma extract | collected from the U87 mouse | mouth. The phosphorylated peptide was confirmed from the increase of 80 Da. Substrate peptide, 5 types of tumor-bearing mice (U87, A549, A431, HuH-7 and B16 melanoma; diameter 1.1 cm or 1.2 cm) and 2 normal mice (control mice; BALB / c and BALD / cN nu / It is a graph which shows the result of the phosphorylation reaction with the plasma sample extract | collected from (nu). After the phosphorylation reaction, each sample was analyzed by MALDI-TOF MS. *: P <0.01; **: P <0.05 It is a graph which shows the influence of the peptide concentration with respect to a phosphorylation rate. Blood samples were collected from B16 melanoma mice. After the phosphorylation reaction, each sample was analyzed by MALDI-TOF MS. *: P <0.05 (A) Normal mouse (BALB / cN nu / nu) or U87 mouse, (B) Normal mouse (BALB / cN nu / nu) or A549 mouse, and (C) Normal mouse (BALB / c nu / nu) or B16 It is a figure which shows the result of the Western blot analysis of the plasma sample extract | collected from the melanoma mouse. Plasma (50 μg protein per sample) was immunoblotted with anti-phosphorylated PKCα (Ser657) antibody. N: Plasma sample collected from normal mice; T: Plasma sample collected from cancer-bearing mice.

It is a graph which shows the influence of the kinase inhibitor with respect to the phosphorylation rate of a substrate peptide. At concentrations of 0, 50, 500 or 5000 nM for Ro-31-7549, 20 μM for lotrelin, and 10 μM for H-89, the inhibitors Ro-31-7549, rottrelin and H-89 were Added to the oxidation reaction. After phosphorylation of the substrate peptide and plasma collected from B16 melanoma mice, the phosphorylation rate was measured by MALDI-TOF MS analysis. It is a graph which shows the relationship between tumor size and phosphorylation rate. Three tumor sizes (0.6, 0.9 and 1.2 cm) were prepared from U87 or B16 melanoma mice. After phosphorylation of the substrate peptide and plasma, each sample was analyzed by MALDI-TOF MS. It is a graph which shows the change of the level of the activated PKC (alpha) in the blood by the recurrence of the cancer after the surgical removal of the tumor. After phosphorylation of the substrate peptide and plasma collected at 0, 7, 14, 21 and 28 days after tumor resection, each sample was analyzed by MALDI-TOF MS.

  Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention. In addition, this specification includes the entirety of Japanese Patent Application No. 2009-171144 (filed on July 22, 2009), which is the basis for claiming priority of the present application. In addition, all publications cited in the present specification, for example, prior art documents, and publications, patent publications and other patent documents are incorporated herein by reference.

1. Summary of the Invention Protein kinase Cα (PKCα) plays a key role in differentiation, proliferation and apoptosis of cancer cells, and its activity is known to be higher in cancer cells than in normal cells.
The inventor has identified a substrate peptide (Alphatomega) specific for PKCα from a library containing over 1,700 candidate peptides (Kang, J.-H. et al. (2008) Proteomics 8, 2006-2011. This peptide showed a higher phosphorylation rate in cancer cells and tissues than in normal tissues (Kang, J.-H. et al. (2008) Proteomics 8, 2006-2011. (Supra); Kang, J .- H. et al. (2008) J. Am. Chem. Soc., 130, 14906-14907. As such, alpha tomega can be used to detect PKCα activity.

The present inventor investigated whether or not activated PKCα was present in the blood, which was not conventionally known, using alpha-mega. Specifically, blood samples prepared from normal mice (control mice) and cancer xenograft mouse models (cancer-bearing mice) were analyzed by Western blot analysis and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). ).
As a result, compared with the blood sample derived from the control mouse, a high activated PKCα level and a high phosphorylation rate of alpha tomega were observed in the blood sample derived from the tumor-bearing mouse. Specifically, the level of activated PKCα is higher in the blood of tumor-bearing mice (U87, A549, A431, HuH-7 and B16 melanoma) than the level observed in control mice, and the alpha tomega phosphorylation rate is , Increased in proportion to tumor size. Furthermore, the addition of Ro-31-7549, a highly specific inhibitor of PKCα, reduces the phosphorylation rate in a dose-dependent manner, while the addition of non-PKCα inhibitors rottlerin and H-89. However, the phosphorylation rate was not significantly affected. In addition, the level of activated PKCα decreased after cancer resection, but increased when the cancer recurred.

  From the above results, the present inventors have found that 1) activated PKCα in blood can be a novel biomarker for cancer diagnosis, particularly a novel biomarker useful for diagnosis of multiple types of cancers, and 2) activated PKCα. It was found that the presence or absence of recurrent cancer (for example, recurrent cancer after surgical excision) or early cancer can be evaluated by monitoring the level of the cancer. Thus, the present inventor has been able to demonstrate for the first time that the existence of activated PKCα in blood and the discovery of a novel biomarker for cancer diagnosis based on this existence.

2. Marker for cancer diagnosis The marker for cancer diagnosis of the present invention contains PKCα as described above. The amount of PKCα in a biological sample (especially blood, specifically plasma or serum) of a test animal is PKCα in a biological sample (especially blood, particularly plasma or serum) of a normal animal (control animal not showing cancer pathology). If the amount of the test animal is larger than the amount of the test animal, it can be determined that the subject animal has developed or is highly likely to develop cancer. Since the detection target sample of PKCα that is substantially a cancer diagnostic marker is blood, among biological samples, the cancer diagnostic marker of the present invention is preferably a blood marker (particularly a plasma or serum marker). .

  The cancer (cancer type) to be diagnosed or evaluated in the present invention is not particularly limited, and two or more types of cancer can be the target of diagnosis or evaluation, and in some cases, preferably all cancer types Can also be targeted for diagnosis and evaluation. Specifically, the cancer diagnostic marker of the present invention is capable of diagnosing (detecting cancer) when any cancer develops or is highly likely to develop regardless of the type of cancer. For example, it is useful in that it can be used for the first widespread diagnosis and detection of cancer at the stage before identifying the cancer type. In addition, since the concentration of PKCα in the blood is particularly high at the time of cancer recurrence or at an early stage, the preferred use of the marker for cancer diagnosis of the present invention includes diagnosis of recurrent cancer and early cancer. In addition, although it changes with cancer types, an early stage cancer basically means the cancer of the stage to the stage I only or the stage II.

3. 3. Pharmaceutical composition for diagnosis 3-1. Pharmaceutical Composition Containing Substrate Peptide The pharmaceutical composition of the present invention is a pharmaceutical composition for diagnosing cancer characterized by containing a PKCα substrate peptide (hereinafter, PKCα substrate peptide) as described above.
The present invention includes a method for diagnosing cancer characterized by using a PKCα substrate peptide, and the use of the substrate peptide for producing a drug for cancer diagnosis.

(1) PKCα substrate peptide
The PKCα substrate peptide only needs to be a peptide that can be specifically phosphorylated by PKCα, and is not limited, for example, 10 to 30 amino acid residues (preferably 10 to 20 amino acid residues, more preferably 10 to 15 amino acids) Substrate peptides consisting of residues). Specific examples of the peptide include a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 (FKKQGSFAKKK), a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 (FKKQGTFAKKK), and the like. The peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is referred to as “alpha tomega” in the present specification.
PKCα substrate peptides can be synthesized, for example, using an automated peptide synthesizer and following standard Fmoc chemistry procedures. After the synthesis, it can be purified by a known purification method using various chromatography or the like.

(2) Blending ratio of antibody In the pharmaceutical composition of the present invention, the blending ratio of the PKCα substrate peptide is not particularly limited, but is preferably 1 to 100% by weight, and more preferably 10 to 100% by weight.
(3) Other components The pharmaceutical composition of the present invention may contain other components other than the PKCα substrate peptide as long as the effect of the present invention is not significantly impaired, and is not limited. For example, an antibody against a phosphorylated PKCα substrate peptide can be included.

(4) Detection and Quantification of Phosphorylated PKCα Substrate Peptide The pharmaceutical composition of the present invention comprises a biological sample collected from a subject or a patient (test sample; particularly blood, in particular plasma, the PKCα substrate peptide in the composition). Or serum)), and then the phosphorylated PKCα substrate peptide is detected and quantified, and compared with the amount detected in healthy individuals, the possibility of onset and onset of cancer In addition, the degree of cancer pathology can be determined (diagnosed). In addition, by monitoring the amount of phosphorylated PKCα substrate peptide regularly in the same subject, it is also possible to determine the state of cancer (disease state) and the presence or absence of recurrence.

(i) Detection of phosphorylated PKCα substrate peptide Blood collected as a test sample may be diluted 1 to 100 times with, for example, a sample buffer after being subjected to treatment such as plasma separation. Good. When performing the dilution, for example, phosphate buffered saline can be used as the sample buffer.
The test sample after the above treatment is brought into contact with the pharmaceutical composition of the present invention and used for the phosphorylation reaction with the PKCα substrate peptide. In the phosphorylation reaction, the reaction medium is not particularly limited. For example, a buffer obtained by mixing Tris-HCl and magnesium chloride can be used. For example, adenosine triphosphate (ATP) can be used as a phosphate group-donating substrate. Etc.) can be used by mixing in the buffer.
The reaction between the test sample and the PKCα substrate peptide is performed at 30 to 40 ° C. (preferably 37 ° C.) for 10 to 60 minutes (preferably 30 to 60 minutes).

  The detection of the phosphorylated PKCα substrate peptide is not limited, but can be performed using a known immunoassay according to the conventional method. Immunoassays include labeled immunoassay and immunoturbidimetry (TIA), but the former is preferred.For example, in addition to enzyme immunoassay (EIA) such as ELISA, radioimmunoassay (RIA) And fluorescent immunoassay (FIA) are preferred. Further, as a labeled immunoassay method, a Western blot method (in combination with electrophoresis method) or the like is preferably used as a method utilizing a combination with other separation methods. Among these, ELISA and / or Western blotting are preferable. In addition, one or more types of monoclonal antibodies against phosphorylated PKCα substrate peptides may be used, and detection may be performed by combining two or more types of immunoassay methods. Detection can be performed.

  In the present invention, as a method for detecting a phosphorylated PKCα substrate peptide, a method using a metal colloid such as a metal nanoparticle can also be employed. More specifically, the method involves phosphorylation of a substrate peptide by a protein kinase when the substrate peptide and protein kinase are reacted in the presence of a metal colloid, or when a metal colloid is added after the reaction of the substrate peptide and protein kinase. It is a method based on the principle that the level of oxidation can be evaluated based on the color change of the metal colloid. This color change is caused by the presence of metal colloid in the phosphorylation reaction of the substrate peptide by protein kinase, causing the aggregation of the phosphorylated substrate peptide by the intervention of the metal colloid particles, and the color tone of the metal colloid is based on the degree of this aggregation. It changes. In addition, the color tone of the metal colloid changes depending on the degree of aggregation substantially proportional to the phosphorylation rate of the substrate peptide.

Therefore, in the present invention, as described above, the reaction between the test sample and the PKCα substrate peptide is performed in the presence of the metal colloid, or after the reaction, the metal colloid is added to change the color tone of the sample. The presence or absence of phosphorylation of the PKCα substrate peptide can be detected by comparing with a sample before the reaction or by comparing with a change in color tone in a control (reaction with a sample collected from a healthy person).
In the method using the metal colloid, as the reaction medium, for example, a buffer obtained by mixing HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) and magnesium chloride is used. As the donor substrate, ATP or the like is used as described above.
Examples of metal colloids such as metal nanoparticles include gold colloid, silver colloid, platinum colloid, iron colloid, iron hydroxide colloid, aluminum hydroxide colloid, palladium colloid, and rhodium colloid. Of these metal colloids, gold colloid and silver colloid are preferable, and gold colloid is more preferable. These metal colloids can be produced by methods described in known literature, and commercially available products can also be used. For example, gold nanoparticles can be generally produced by a liquid phase reduction method in which a reducing solution is added to a solution containing gold ions to reduce the gold ions (see, for example, JP-A-2003-253310, JP (See Kaikai 2006-152438).

The particle size of the metal colloid is not particularly limited, but is preferably, for example, about 1 nm to 500 nm, more preferably 3 nm to 200 nm, still more preferably 5 nm to 100 nm.
Although the metal colloid depends on the particle size, the gold colloid usually exhibits a red color, and the silver colloid exhibits a yellow color. On the other hand, for example, when gold colloid is present, the color of the gold colloid changes from red to blue due to phosphorylation and aggregation of the PKCα substrate peptide. Based on such color change, the phosphorylated PKCα substrate peptide can be detected. The color change of the metal colloid is preferably examined by measuring the absorbance in the range of about 600 nm to 800 nm, more preferably in the range of about 600 nm to 750 nm, and still more preferably about 700 nm.

(ii) Quantification of phosphorylated PKCα substrate peptide Although quantification is not limited, for example, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for the PKCα substrate peptide used in the reaction. It can be carried out by determining the phosphorylation rate (the ionic strength ratio of phosphorylated substrate and non-phosphorylated substrate).
In addition, when the phosphorylated PKCα substrate peptide is detected by the above-described method using a metal colloid, it is based on the degree of color tone change of the metal colloid (specifically, the degree of color tone change with an appropriate control). Or by comparison with a calibration curve prepared in advance), the phosphorylated substrate peptide can be substantially quantified.
A commercially available kit can also be used for quantification or detection of the phosphorylated PKCα substrate peptide.

(iii) Evaluation of cancer state In the present invention, the cancer state can be evaluated using the detection result (detection amount) of the phosphorylated PKCα substrate peptide detected and quantified as described above as an index. . That is, the present invention detects a phosphorylated substrate peptide by reacting a PKCα substrate peptide with a biological sample (test sample; particularly blood, specifically plasma or serum), and uses the obtained detection result as an index to detect cancer. It includes a method for evaluating the condition.
Here, “evaluating the state of cancer” means determining the presence / absence, progression, severity, treatment reactivity, prognosis, etc. of cancer (recurrent cancer, early cancer). The evaluation may be performed by combining subjective amounts and the like in addition to the detected amount of phosphorylated PKCα substrate peptide. Although it can be evaluated by measuring the phosphorylated PKCα substrate peptide at a frequency of once or twice a year as in regular medical examinations, the change in the detected amount of the phosphorylated substrate peptide is periodically monitored. It is preferable to comprehensively evaluate the state of cancer by monitoring (including monitoring the presence or absence of cancer recurrence). As such an evaluation method, for example, if the detected amount of the phosphorylated PKCα substrate peptide exceeds a certain value, it is diagnosed that there is a high possibility that some cancer has developed. Diagnosing that the treatment is successful if the detected amount of the peptide decreases, and diagnosing that the case where the detected amount of the phosphorylated substrate peptide exceeds a certain value and changes to a high value cannot be treated and recovered. Is also possible.

3-2. Pharmaceutical Composition Containing Antibody As described above, the pharmaceutical composition of the present invention is a pharmaceutical composition for diagnosing cancer comprising an antibody against PKCα (hereinafter, anti-PKCα antibody).
In addition, this invention includes the use of the anti-PKC (alpha) antibody for manufacturing the cancer diagnostic method characterized by using an anti- PKC (alpha) antibody and the chemical | medical agent for cancer diagnosis.

(1) Anti-PKCα antibody An anti-PKCα antibody can be prepared as follows.
(A) Preparation of polyclonal antibody
(i) Preparation of antigen and its solution In producing an anti-PKCα antibody, it is first necessary to prepare or obtain a protein to be used as an immunogen (antigen).
As the antigen protein, purified PKCα can be used, but is not limited thereto.For example, the PKCα consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of PKCα, and PKCα. A protein having activity can also be used. As PKCα, for example, PKCα derived from mammals, specifically, PKCα derived from human, PKCα derived from mouse, PKCα derived from rat, or the like can be used. Here, the base sequence and amino acid sequence information of PKCα derived from each animal can be obtained from the following accession numbers.

<Human-derived PKCα>
・ GenBank accession number: NM 002737 (base sequence; SEQ ID NO: 3), NP 002728 (amino acid sequence; SEQ ID NO: 4)
GenBank accession number: X52479 (base sequence; SEQ ID NO: 5), CAA36718 (amino acid sequence; SEQ ID NO: 6)
<Mouse-derived PKCα>
・ GenBank accession number: NM 011101 (base sequence; SEQ ID NO: 7), NP 035231 (amino acid sequence; SEQ ID NO: 8)
GenBank accession numbers: M25811 (base sequence; SEQ ID NO: 9), AAA39934 (amino acid sequence; SEQ ID NO: 10)
<Rat-derived PKCα>
・ GenBank accession number: XM 343935 (base sequence; SEQ ID NO: 11), XP 343976 (amino acid sequence; SEQ ID NO: 12)

The method for preparing purified PKCα as an antigen is not limited. For example, PKCα can be produced using transformants of appropriate host cells (human-derived cultured fibroblasts, Chinese hamster ovary cells, yeast, etc.). And general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination. .
Next, the obtained purified PKCα is dissolved in a buffer solution to prepare an antigen solution. In that case, you may add the adjuvant for performing immunization effectively as needed. As the adjuvant, for example, commercially available Freund's complete adjuvant or Freund's incomplete adjuvant can be used. These adjuvants may be used alone or in combination of two or more, and are not limited.

(ii) Immunization and collection of antiserum Immunization is performed by administering a solution containing the purified PKCα to a mammal (eg, mouse, rat, rabbit, etc.). Administration is mainly performed by injecting intravenously, subcutaneously or intraperitoneally.
The dose per one dose of the antigen solution is not limited. For example, the amount of purified PKCα is preferably 2 to 500 μg per animal, and more preferably 10 to 100 μg.
The administration interval is not limited, and is preferably, for example, an interval of several days to several weeks, more preferably an interval of 2 to 3 weeks. Moreover, the frequency | count of administration is 2-10 times, for example.
The collection of serum (antiserum) obtained by the immunization is not limited, but is preferably performed, for example, 1 to 28 days after the last administration day, and more preferably 2 to 14 days later. The antiserum can be collected from the blood of the immunized animal according to a conventional method.

(iii) Selection of target antiserum Out of the antisera collected from each of the immunized animals, the target antiserum, ie, an antiserum containing an anti-PKCα antibody, is screened.
The screening method is not limited, but, for example, collected antiserum and purified PKCα or recombinant PKCα as an antigen (produced by cultured human fibroblasts, Chinese hamster ovary cells, yeast, etc.) or those A known immunoassay can be performed using the mutated enzyme according to the conventional method. Immunoassays include labeled immunoassay and immunoturbidimetry (TIA), but the former is preferred.For example, in addition to enzyme immunoassay (EIA) such as ELISA, radioimmunoassay (RIA) And fluorescent immunoassay (FIA) are preferred. Further, as a labeled immunoassay method, a Western blot method (in combination with electrophoresis method) or the like is preferably used as a method utilizing a combination with other separation methods. In the case of Western blotting, since the protein sample to be detected is denatured with heat and a surfactant and used, the antigen in the screening method becomes a mutant enzyme.

The anti-PKCα antibody contained in the target antiserum by the above screening is usually a polyclonal antibody. When it is necessary to purify the antibody, for example, known purification methods such as ammonium sulfate salting-out method, ion exchange chromatography, affinity chromatography, gel chromatography, etc., are used as appropriate, combining only one type or two or more types. Then you can do it.
The reaction site in PKCα to which the anti-PKCα antibody can specifically bind (can be recognized) is not particularly limited.

(B) Production of monoclonal antibodies
(i) Preparation of antigen and solution thereof Antigen and solution thereof can be prepared in the same manner as in the production of the polyclonal antibody.
(ii) Immunization and collection of antibody-producing cells For the immunization method, the dosage, administration interval, and number of administrations using the antigen solution, the same methods and conditions as in the production of the polyclonal antibody can be employed.
The collection of cells that produce anti-PKCα antibody (antibody-producing cells) obtained by the above immunization is not limited, but is preferably performed, for example, 1 to 14 days after the last administration day, more preferably 2 to 4 days later It is.
Preferred examples of antibody-producing cells include spleen cells, lymph node cells (particularly local lymph node cells), peripheral blood cells, etc. Among them, spleen cells and local lymph node cells (for example, sub-knee lymph node cells) are preferable. Is more preferable.

(iii) Cell fusion A fused cell (hybridoma) can be obtained by performing cell fusion between the collected antibody-producing cells and myeloma cells.
As myeloma cells, for example, cell lines that are generally available in mammals such as mice can be used. Specifically, cell lines that have drug selectivity and cannot grow on HAT selection media (hypoxanthine, aminopterin and thymine-containing media) in an unfused state, but can grow in a state fused with antibody-producing cells. preferable. Specifically, as mouse myeloma cells, for example, PAI, P3X63-Ag.8.U1 (P3U1), NS-I and the like can be used.
The cell fusion is performed, for example, by mixing antibody-producing cells and myeloma cells in a culture medium for animal cell culture such as RPMI-1640 medium without serum. The mixing ratio of antibody-producing cells and myeloma cells is, for example, 5: 1.
In general, the fusion reaction is preferably performed in the presence of a cell fusion promoter, and as the promoter, polyethylene glycol having an average molecular weight of 1000 to 6000 daltons or the like can be used. Alternatively, antibody-producing cells and myeloma cells can be fused using a commercially available cell fusion device utilizing electrical stimulation (for example, electroporation).
The cells after the fusion reaction are cultured using, for example, a HAT selection medium. After the above culture, cells in which growth on the HAT selection medium is observed become fused cells (hybridomas).

(iv) Selection of target hybridoma and cloning thereof The target hybridoma, that is, a hybridoma producing an anti-PKCα antibody, is screened from the hybridoma obtained by the above culture. Specifically, a culture supernatant containing a target anti-PKCα antibody is screened.
Although the screening method is not limited, for example, a part of the culture supernatant is collected and purified PKCα or recombinant PKCα (antigen-produced human-derived cultured fibroblasts, Chinese hamster ovary cells, yeast, etc.) ) Or a mutant enzyme thereof, and a known immunoassay can be performed according to the conventional method. Preferred examples of the immunoassay include enzyme immunoassay (EIA) such as ELISA, radioimmunoassay (RIA), and fluorescent immunoassay (FIA). In addition, Western blotting (in combination with electrophoresis) and the like are also preferred. In the case of Western blotting, since the protein sample to be detected is denatured with heat and a surfactant and used, the antigen in the screening method becomes a mutant enzyme.

The anti-PKCα antibody contained in the culture supernatant of the target hybridoma after the screening is an antibody obtained before cloning of the hybridoma, but may be an antibody consisting of a single molecule (monoclonal antibody), There is no limitation.
The reaction site in PKCα to which the anti-PKCα antibody can specifically bind (can be recognized) is not particularly limited.
Cloning of the target hybridoma by the above screening, that is, establishment of a monoclonal antibody-producing cell line can be generally performed by selecting colonies derived from one cell by culturing using a limiting dilution method or the like.

(v) Collection of monoclonal antibody The method of collecting the monoclonal antibody from the hybridoma obtained by the above cloning is not limited, but generally a cell culture method or ascites formation method can be employed.
In the cell culture method, the hybridoma obtained by cloning is cultured, for example, in an animal cell culture medium under conditions of 37 ° C. and 5% CO ₂ for 7 to 14 days. Monoclonal antibodies can be collected.
In the ascites formation method, for example, about 10 ⁶ hybridomas obtained by cloning are administered into the abdominal cavity of a mammal of the same kind as the mammal from which the myeloma cells used for cell fusion are derived. Then, the target monoclonal antibody can be collected from ascites or serum collected 7 to 14 days after the hybridoma is proliferated and administered.
In any of the cell culture method and ascites formation method, when purification of the antibody is required at the time of collecting or after collecting the monoclonal antibody, ammonium sulfate salting out method, ion exchange chromatography, affinity chromatography, gel chromatography, etc. Known purification methods can be adopted and implemented as appropriate by combining only one type or a combination of two or more types.

(2) Blending ratio of antibody In the pharmaceutical composition of the present invention, the blending ratio of the anti-PKCα antibody is not particularly limited, but is preferably 1 to 100% by weight, and more preferably 10 to 100% by weight.
(3) Other components The pharmaceutical composition of the present invention may contain other components other than the anti-PKCα antibody as long as the effect of the present invention is not significantly impaired, and is not limited. For example, it can contain a primary antibody detection reagent, a chromogenic substrate, and the like.

(4) Detection and quantification of PKCα The pharmaceutical composition of the present invention is used for detecting and quantifying PKCα in a collected biological sample of a subject or patient (test sample; particularly blood, specifically plasma or serum), The presence or absence of cancer, the possibility of its onset, and the degree of the disease state can be determined (diagnosed) by comparison with the detected amount in healthy individuals. Moreover, the state (disease state) of cancer can also be judged by monitoring the amount of PKCα of the same subject regularly.
(i) Detection of PKCα Blood or the like collected as a test sample is generally diluted 1 to 100 times with a sample buffer or the like after being subjected to treatment such as serum separation. As the sample buffer, for example, phosphate buffered saline can be used.
The test sample after the treatment is brought into contact with the pharmaceutical composition of the present invention and used for a reaction with an anti-PKCα antibody (a monoclonal antibody is generally preferred).
The reaction between the test sample and the anti-PKCα antibody is performed at 20 to 40 ° C. (preferably 30 to 37 ° C.) for 30 to 720 minutes (preferably 30 to 90 minutes).

  Detection of PKCα can be performed using a known immunoassay according to the conventional method. Immunoassays include labeled immunoassay and immunoturbidimetry (TIA), but the former is preferred.For example, in addition to enzyme immunoassay (EIA) such as ELISA, radioimmunoassay (RIA) And fluorescent immunoassay (FIA) are preferred. Further, as a labeled immunoassay method, a Western blot method (in combination with electrophoresis method) or the like is preferably used as a method utilizing a combination with other separation methods. Among these, ELISA and / or Western blotting are preferable. In addition, one or two or more types of anti-PKCα monoclonal antibodies may be used, and detection may be performed by combining two or more types of immunoassays, and excellent detection with higher sensitivity and reliability can be performed. .

(ii) Quantification of PKCα Although quantification is not limited, it is preferable to quantify PKCα contained in the test sample from a calibration curve based on the relationship between the amount of antigen (antigen concentration: PKCα concentration) and the detected value. The calibration curve is prepared in advance based on the detection value (detection data) obtained using purified PKCα as an antigen and using the above-described anti-PKCα antibody. Specifically, information on the detected value with respect to the antigen concentration is obtained by a method similar to the immunoassay method (ELISA, Western blotting method, etc.) described as the detection method, and prepared based on this. By comparing the calibration curve with the actual detection value (actual measurement value), the amount of PKCα in the test sample can be obtained.
A commercially available kit can also be used for quantification or detection of PKCα.

(iii) Evaluation of cancer state In the present invention, the state of cancer can be evaluated using the PKCα value determined or detected as described above as an index. That is, the present invention includes a method of detecting PKCα by reacting an anti-PKCα antibody with a biological sample (test sample), and evaluating the cancer state using the obtained detection result as an index.
Here, “evaluating the state of cancer” means determining the presence / absence, progression, severity, treatment reactivity, prognosis, etc. of cancer (recurrent cancer, early cancer). Evaluation may be performed in combination with subjective symptoms in addition to the amount of PKCα. Although it can be evaluated by measuring PKCα at a frequency of once or twice a year as in regular medical examinations, the cancer status is comprehensively evaluated by periodically monitoring the transition of the amount of PKCα. (Including monitoring for the recurrence of cancer). As such an evaluation method, for example, if the amount of PKCα exceeds a certain value, it is diagnosed that there is a high possibility that some cancer has developed. If the amount of PKCα is reduced in a cancer case that is already being treated, the treatment is successful. It is also possible to diagnose that the case where the amount of PKCα exceeds a certain value and changes at a high value cannot be treated and recovered.

4). Cancer Diagnostic Kit A cancer diagnostic kit can be used for diagnosing cancer, and includes, for example, the pharmaceutical composition for cancer diagnosis described in the above sections 3-1 and 3-2. Kits can be mentioned.
In the kit, when the PKCα substrate peptide is included, the substrate peptide is in a powder state or a solution state (pure water, buffer, physiological saline, etc.) in consideration of stability (storage stability) and ease of use. It is preferably stored under conditions of refrigeration or freezing (-30 ° C to -80 ° C).
When the kit contains an anti-PKCα antibody, the antibody is preferably provided in a dissolved state in consideration of stability (storage stability) and ease of use.

The kit can contain other components in addition to the above PKCα substrate peptide or anti-PKCα antibody. Examples of other components include a primary antibody detection reagent and a chromogenic substrate that can be used for ELISA and Western blotting.
The kit only needs to have at least the above PKCα substrate peptide or anti-PKCα antibody as a component. Therefore, all the components essential for the diagnosis of cancer may be provided together with the PKCα substrate peptide or the anti-PKCα antibody or not, and there is no limitation.

5). Screening Method As described above, the method of screening for a cancer preventive and / or therapeutic agent of the present invention is a method comprising the following steps (a) to (c). The screening method of the present invention may include other steps as necessary.
(a) A step of administering a candidate substance to a cancer-bearing model non-human mammal (administration step)
(b) A step of detecting PKCα in the blood of the above cancer-bearing model non-human mammal (detection step)
(c) A process of selecting a target cancer preventive drug and / or cancer therapeutic drug using the obtained detection result as an index (evaluation process)

Below, each said process is demonstrated.
(a) Administration process The tumor-bearing model non-human mammal to be the test animal and the control animal are not particularly limited, but usually the same kind of non-human mammal is used in that an accurate comparative experiment can be performed. Preferably, non-human mammals of the same litter are used, more preferably non-human mammals of the same sex and the same age are used. Moreover, it is preferable that the test animals and the control animals have the same breeding conditions other than the amount of feed intake.
As the test animal, a cancer-bearing model non-human mammal in which tumor is developed by transplanting a tumor tissue or tumor cell to a normal non-human mammal by a known method can be used. Furthermore, in the present invention, a non-human mammal obtained by surgically excising a tumor tissue or tumor cell from the above cancer-bearing model non-human mammal can also be used. When the non-human mammal after the excision is used as a test animal, for example, a screening method for a preventive drug for recurrent cancer can be performed. In the present invention, the non-human mammal after the excision is also included in the cancer-bearing model non-human mammal.
The control animal is not limited as long as it is suitable for use as a control for comparison with a test animal. For example, a normal non-human mammal not transplanted with a tumor tissue or the like is used, and a candidate substance is used. Non-administered cancer-bearing model non-human mammals or test animals prior to administration of candidate substances can also be used as control animals.

Candidate substances to be administered to a test animal are not limited, but various naturally or artificially synthesized peptides, proteins (including enzymes and antibodies), nucleic acids (polynucleotide (DNA, RNA), oligonucleotides (siRNA, etc.) ), Peptide nucleic acids (PNA), etc.), low molecular or high molecular organic compounds, and the like.
The candidate substance can be administered orally or parenterally, and is not limited. In any case, known administration methods, administration conditions, and the like can be adopted. The dose can be appropriately set in consideration of the type and condition of the test animal, the type of candidate substance, and the like.

(b) Detection step In this step, PKCα is detected from the blood (blood sample) of the test animal to which the candidate substance has been administered. A well-known method can be applied as a blood sample collection method. The number of detections, the detection period, and the like of PKCα are not particularly limited, and can be appropriately set in consideration of the efficacy of the target cancer preventive drug or cancer therapeutic drug.
The method for detecting and quantifying PKCα in blood is not limited, but in Sections 3-1. And 3-2 (especially 3-1. (4) and 3-2. (4)). The described method can be adopted as appropriate.

(c) Evaluation process When screening for a cancer preventive drug and / or a cancer therapeutic drug, for example, the amount of PKCα detected in the blood (detection of the test animal to which the candidate substance has been administered and the control animal to which the candidate substance has not been administered) Level). Based on this evaluation, it is preferable to select whether the candidate substance is a target cancer preventive drug and / or cancer therapeutic drug.
Specifically, for a cancer-bearing model non-human mammal having the same level of blood PKCα, when one is a test animal to which a candidate substance is administered and the other is a control animal to which no candidate substance is administered, When the blood PKCα level is lower than the blood PKCα level in the control animal, the candidate substance can be selected as a target cancer therapeutic agent. On the other hand, when the blood PKCα level in the test animal maintains the same level as or higher than the blood PKCα level in the control animal, the candidate substance may not be selected as the target cancer therapeutic agent. Can not. In this case, the control animal can be the test animal itself before administration of the candidate substance.

  In addition, a cancer-bearing model non-human mammal obtained by surgically excising tumor tissue or the like, or a cancer-bearing model non-human mammal once cancer treatment has been completed by a method other than surgical excision (medical method, etc.) When the normal non-human mammal is used as a control animal and the blood PKCα level in the test animal is continuously maintained at the same level as the blood PKCα level in the control animal, the candidate substance Can be selected as a cancer preventive drug (specifically, a preventive drug for recurrent cancer). On the other hand, when the blood PKCα level in the test animal is higher than the blood PKCα level in the control animal, the candidate substance cannot be selected as the target cancer preventive drug. In this case, the control animal can be the test animal itself immediately before administration of the candidate substance (before recurrence).

Since cancer screening based on the amount of PKCα in the blood can be screened for a plurality of cancer types (preferably all cancer types), a cancer-bearing model non-human mammal As described above, when a cancer-bearing model non-human mammal of a specific cancer type is prepared and used, a preventive and / or therapeutic drug for the specific cancer type can be screened.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

"Extracellular PKCα as a diagnostic biomarker for multiple cancers"
<Materials and methods>
Peptide synthesis An automated peptide synthesizer was used to synthesize alpha tomega (FKKQGSFAKKK (SEQ ID NO: 1)) as a substrate peptide according to standard Fmoc chemistry procedures. After treatment with trifluoroacetic acid (TFA), the peptide was subjected to BioCAD perfusion chromatography (Ikemoto Rika Kogyo Co., Ltd.) on an Inertsil ODS-3 column (250 × 20 mm, 3.5 μm; GL Sciences, Tokyo, Japan). And an AB linear gradient (eluent A is water containing 0.1% TFA, eluent B is acetonitrile containing 0.1% TFA) at a flow rate of 8 ml / min.

MALDI-TOF MS analysis α-cyano-4-hydroxycinnamic acid (CHCA) matrix (10 mg / ml) was prepared in 50% water / acetonitrile and 0.1% TFA. The matrix and sample were mixed in a 20: 1 ratio. A total volume of 1 μl of the sample / matrix mixture was applied to the sample plate and dried to allow crystallization. In this example, the Voyager DE RP biospectrometry workstation (Applied Biosystems) was used using a positive or negative ion reflectron mode. The acceleration voltage was 20 kV and the extraction delay time was 100 ns. Typically, 100 laser shots were averaged to improve the signal / noise ratio. All spectra were analyzed using Data Explore software (Applied Biosystems). The phosphorylation rate was defined as the ionic strength ratio of phosphorylated to non-phosphorylated and calculated according to existing explanations (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.).

Phosphorylation of substrate peptides in plasma samples All animal experiments were performed according to the animal experiment rules presented by Kyushu University. Cancer cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U / ml), streptomycin (100 μg / ml) and amphotericin B (0.25 μg / ml; all Invitrogen). The cancer cells were placed in a humidified atmosphere at 37 ° C. containing 5% CO ₂ and 95% air. For B16 melanoma, 5-week-old BALB / c male mice were used, and for human cancers (A549, U87, HuH-7 and A431), BALB / c nude mice (weight approximately 20 g) were used. For B16 melanoma cells, 100 μl Hank's balanced salt solution (Invitrogen), for human cancer (A549, U87, HuH-7 and A431), 1 × 10 ⁷ cells in 100 μl matrigel (BD Biosciences) were injected by subcutaneous dorsal injection. Mice were inoculated. The cancer was grown until the average diameter was about 1.2 cm. In order to monitor the recurrence of cancer after surgical resection, 90% resection was performed for each cancer (1.1 or 1.2 cm in diameter). For blood collection, heparin at a dose of 3 U / body weight (g) was injected intraperitoneally. After 15 minutes, the mice were sacrificed and blood samples were collected from the posterior vena cava. The blood sample was centrifuged at 2,000 rpm, 4 ° C. for 15 minutes, and the supernatant (plasma) was used for phosphorylation with the substrate peptide. The phosphorylation of the substrate peptide was performed in 30 μl buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 100 μM ATP and 2 mg / ml plasma) containing 30 μM synthetic peptide. After incubation at 37 ° C for 60 minutes, the samples were analyzed by MALDI-TOF MS.

Western blot analysis Plasma (50 μg) was immunoblotted with anti-phosphorylated PKCα (Ser657) antibody (Nippon Millipore). The immune response was visualized by chemiluminescence. In order to activate PKCα, the Ser657 site of PKCα must be phosphorylated, but the anti-phosphorylated PKCα (Ser657) antibody is a monoclonal antibody that specifically recognizes the phosphorylated Ser657 site. .

Statistical analysis results are shown with standard deviation as the average of 3 or 4 samples. All measurements were performed twice. Statistical analysis was performed by significance test of difference in mean value using Student t-test.

<Result>
MALDI-TOF MS was used to confirm the state of phosphorylation of PKCα-specific substrate peptide alpha tomega (FKKQGSFAKKK (SEQ ID NO: 1)). Previous studies have reported that when CHCA is used as a matrix, contaminants and biomolecules (e.g., salts, proteins and lipids) prevent crystal formation and ionization, and the inventor also In the face of similar problems, a dilution method was employed to dilute the sample with CHCA at a 20: 1 CHCA to sample volume ratio to overcome these obstacles. Using MALDI-TOF MS, a wide range of peptide concentrations (10 ^-4 M (100 pmol / μl) to 10 ^-8 M (10 fmol / μl)) could be analyzed. In this example, the concentration of peptide (1.5 μM at 20: 1 dilution) was well within this range. The increase of 80 Da in m / z value confirmed the phosphorylation of the substrate peptide (FIG. 1).
To investigate whether PKCα-specific substrate peptides can be phosphorylated by blood samples, control mice, as well as five different cancerous mice with tumors 1.1-1.2 cm in size (U87, A549, A431). , HuH-7 and B16 melanoma). After phosphorylation between the substrate peptide and plasma, each sample was analyzed by MALDI-TOF MS. In five blood samples collected from a cancer xenograft mouse model, a higher phosphate rate of the substrate peptide was confirmed than that of normal mouse blood (FIG. 2).

Next, whether or not the phosphorylation rate was affected by the substrate peptide concentration (15 to 100 μM) was examined using plasma derived from B16 melanoma mice. The phosphorylation rate decreased as the substrate peptide concentration increased from 30 μM to 100 μM, but there was no difference in phosphorylation rate between 15 μM and 30 μM. A significant reduction in phosphorylation rate (P <0.05) was observed at 100 μM peptide concentration compared to 15 μM and 30 μM peptide concentrations (FIG. 3).
Western blot analysis was employed to confirm whether activated PKCα was present in the blood of tumor-bearing mice. A higher level of activated PKCα was observed in the blood of cancer-bearing (U87, A549 or B16 melanoma) mice than in normal mice (FIG. 4). Next, the effect of inhibitors on the phosphorylation rate was investigated using three protein kinase inhibitors (Ro-31-7549, Lotrelin and H-89). Addition of Ro-31-7549 to a phosphorylation reaction mixture containing plasma collected from B16 melanoma mice resulted in a dose-dependent decrease in phosphorylation, with a 100% decrease at 5 μM. However, no significant decrease in phosphorylation rate was observed with the addition of lottorelin or H-89 (FIG. 5). These results strongly indicated that the peptide was phosphorylated by activated PKCα present in the blood of cancer-bearing mice.

Next, the relationship between tumor size and phosphorylation rate was investigated. Plasma from mice with 0.6 cm, 0.9 cm and 1.2 cm B16 melanoma or U87 tumors was prepared. The phosphorylation rate increased with increasing tumor size. Compared with control mice, mice with tumor size ≧ 0.9 cm showed a significant increase in phosphorylation rate (P <0.05) (FIG. 6). These data indicated that the level of activated PKCα in the blood was related to tumor size.
Finally, it was investigated whether the level of activated PKCα in the blood could be applied to monitor cancer recurrence after surgical resection of the tumor. Although the phosphorylation rate decreased significantly in blood samples taken after surgical resection of B16 melanoma tumors, the phosphorylation rate increased with tumor recurrence 21 days later (FIG. 7). These results indicated that plasma PKCα can be applied to monitor cancer recurrence.

<Discussion>
Phosphorylated peptides have been detected by a variety of techniques including MS-based analysis, radioactive assays using [ ³² P] ATP chip technology, and homogeneous assays. Of these assays, the use of MS-based analysis has increased significantly over the past decade. The most widely used MS technique is MALDI-TOF MS, which is highly sensitive and fast in identification. Accordingly, MALDI-TOF MS was also used in the phosphorylated peptide assay in this example. Dilution methods were incorporated and samples were diluted in CHCA to avoid hindering crystal formation and ionization by contaminants or biomolecules. As a result, phosphorylated peptides can be sufficiently detected (FIG. 1).
PKCα is widely expressed in many tissues and plays a key role in cancer cell differentiation, proliferation and apoptosis. Several reports have shown that PKCα activity is higher in tumor cells than in normal cells. In addition, the concentrations of PKCα activators [diacylglycerol (DAG)] and cofactors [phosphatidylserine (PS) and Ca ²⁺ ] and the migration of PKCα from the cytosol to the membrane were increased in cancer cells. Thus, PKCα has attracted attention as a potential target for anticancer therapy.

  In this example, it was confirmed that PKCα is present in the blood. Western blot analysis detected higher levels of activated PKCα in the blood of cancer-bearing mice than in normal mice (FIG. 4). Furthermore, blood derived from tumor-bearing mice had a higher phosphorylation rate for alpha tomega than blood derived from normal mice (FIG. 2). The inventor further examined the rate of peptide phosphorylation using protein kinase inhibitors Ro-31-7549, Lotrelin and H-89. Lotrelin (20µM)> 80% protein kinase A (PKA), mitogen activated protein kinase activated protein kinase 2 (MAPKAP-K2), p38 regulated / activated kinase (PRAK) and glycogen synthase kinase 3 (GSK3) Although it can be inhibited, PKCα was relatively unaffected by this kinase inhibitor (<10% inhibition). On the other hand, MAPKAP-K1b, PKA, mitogen and stress activated protein kinase 1 (MSK1), protein kinase Bα (PKBα), plasma and glucocorticoid-inducible kinase (SGK), p70 ribosomal protein S6 by H-89 (10 μM) Inhibition of kinase (S6K1), Rho-related protein kinase-II (ROCK-II), checkpoint kinase 1 (CHK1), and AMP-activated protein kinase (AMPK) was> 80%, but for PKCα <30%. On the other hand, Ro-31-7549 is a highly specific inhibitor of PKCα (> 90% at 5 μM) and Ro-31-7549 resulted in a dose-dependent decrease in phosphorylation rate, while rottrelin Alternatively, no significant decrease was observed with H-89 (FIG. 5). These results indicate that the substrate peptide is phosphorylated by activated PKCα, and the activated PKCα in blood can be used as a biomarker for cancer diagnosis.

The mechanism of PKCα release into the bloodstream has not been clarified. However, it has been shown so far that cancer cells can secrete PKA into the extracellular space, and N-terminal myristoylation of PKA is essential for secretion. N-myristoylation is an acylation process that attaches myristic acid to the N-terminal glycine of proteins. Protein N-myristoylation results in fragile and reversible protein-membrane interactions. These observations indicate that PKA interaction with the cell membrane plays a decisive role in PKA secretion. Activation of PKCα requires the transfer of PKCα from the cytosol to the membrane and interaction with activators (DGA) and cofactors (PS and Ca ²⁺ ). Thus, active PKCα present in the extracellular space can be derived from the secretion of PKCα docked to the membrane.
Without vascularization, the growth of cancerous tumors is limited to 0.2-0.3 cm due to the limited diffusion range of oxygen and nutrients. For further growth, angiogenesis to obtain a blood supply is essential. Angiogenesis involves angiogenesis-promoting factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor (TNF) and interleukin 8 (IL-8), and interferon (IFN). And is regulated by anti-angiogenic factors such as angiostatin. Increased blood vessel density increases the supply of oxygen and nutrients to cancer cells, leading to cancer growth. In this Example, a significant increase in activated PKCα was confirmed in tumor-bearing mice having tumors with a diameter ≧ 0.9 cm as compared to growing mice (FIG. 6). This indicated that the level of activated PKCα in the blood was closely related to the blood vessel density essential for cancer growth.

Several biomarkers for the diagnosis of specific tumors such as PSA for prostate cancer and human epidermal growth factor receptor 2 (HER2) for breast cancer have been discovered and used clinically. However, few biomarkers have been discovered that can be used for early stage tumor diagnosis and extensive diagnostic screening of one or more cancers. In this example, it was discovered that the level of activated PKCα was higher in the blood of five types of cancer mice than in the blood of normal mice (FIG. 2). These results indicated that plasma activated PKCα could be useful as a diagnostic marker for multiple cancers.
Blood biomarkers are also used to monitor cancer recurrence after surgical resection or to monitor radiotherapy or anticancer drug therapy. The levels of some blood biomarkers have been shown to decrease dramatically after surgical resection (extraction), chemotherapy or radiation treatment of the tumor, but increase with cancer recurrence. Similarly, the inventor confirmed that PKCα levels were low for 14 days after cancer resection but increased after 21 days (FIG. 7). Thus, it was found that plasma PKCα levels can be used as an indicator for monitoring cancer recurrence.

“Detection of PKCα activity in human serum samples”
<Materials and methods>
Peptide Synthesis Alpha tomega (Ac-FKKQGSFAKKK-NH ₂ (SEQ ID NO: 1)) as a substrate peptide was synthesized by the same method as in Example 1.
MALDI-TOF MS analysis All spectra were analyzed by the same method as in Example 1, and the phosphorylation rate of the substrate peptide was calculated.
Phosphorylation of substrate peptides in human serum samples
The serum of lung cancer patients 14 patients were collected, the phosphorylation of the substrate peptide was performed using this as a sample. The final concentration of 30μM substrate peptide (alpha preparative mega), 2.5-fold diluted human serum samples (Samples 1 to 14; see Table 1), 0.3 mM ATP, 1 mM Mg 2+, a solution containing 10 mM HEPES The substrate peptide was phosphorylated for 1 hour at 37 ° C.
Thereafter, the phosphorylation rate was determined by MALDI-TOF-MS.

<Results and discussion>
In eight of the serum samples of 14 patients (case 1, 3, 4, 5, 6, 8, 9, 13), phosphorylation of the substrate peptide was confirmed (see Table 1). Especially in serum samples of Case 9 and Case 13, existing tumor markers (ProGRP, CEA, CYFRA) the reactions were positive observed, phosphorylation of a substrate peptide of the present invention (alpha preparative mega) is confirmed, A positive reaction was observed.

  According to the present invention, a novel biomarker for cancer diagnosis that is simple and highly reliable can be provided. The cancer diagnostic marker of the present invention is a marker in the blood and is easy to handle, can be used for diagnosis of multiple cancer types, and is particularly suitable for diagnosis of early cancer and recurrent cancer, It is extremely useful.

SEQ ID NO: 1: Synthetic peptide SEQ ID NO: 2: Synthetic peptide

Claims (9)

Look including protein kinase C alpha, and characterized in that it is a blood marker, a marker for cancer diagnosis. And it is used in the diagnosis of early cancer or recurrent cancer, marker of claim 1 Symbol placement. The marker according to claim 1 or 2 , which is used for diagnosis of at least two types of cancer. The marker according to claim 3 , which is used for diagnosis of all cancer types. It includes substrate peptide of protein kinase C alpha, or those used to react with the blood as a biological sample, or, an antibody only contains for protein kinase C alpha, detects the protein kinase C alpha in the blood as a biological sample A pharmaceutical composition for cancer diagnosis, which is used for quantification . A cancer diagnostic kit comprising the composition according to claim 5 . And substrate peptide of protein kinase C alpha, methods by reacting the blood as a biological sample, detecting the peptide that is phosphorylated to assess the status of the cancer detection results obtained as an indicator. An antibody against protein kinase C alpha, methods by reacting the blood as a biological sample to detect the protein kinase C alpha, to assess the status of the cancer detection results obtained as an indicator. A method for screening for a preventive and / or therapeutic agent for cancer, comprising:
A step of administering a candidate substance to a cancer-bearing model non-human mammal, a step of detecting protein kinase Cα in the blood of the non-human mammal, and a target cancer preventive drug and / or cancer treatment using the obtained detection result as an index Said method comprising the step of selecting a drug.