KR101900142B1 - Method for diagnosing cancer using heat shock protein 60 - Google Patents

Abstract

The present invention relates to a method of providing information for cancer diagnosis using a heat shock protein 60 (HSP60) protein. More specifically, the present invention relates to a method for providing information for cancer diagnosis using a heat shock protein 60 (HSP60) To provide information for cancer diagnosis by confirming the cleavage pattern of the protein, and to a composition for diagnosing cancer including an antibody and a hydroxylamine which specifically bind to HSP60 protein, and a cancer diagnosis kit.
The present invention can identify the functional and structural differences of HSP60 in cancer, and utilize HSP60 as a biomarker capable of grasping the normal state and pathological state of human, thereby making it possible to efficiently and objectively diagnose cancer. It can contribute to cost reduction and efficiency increase in diagnosis of cancer.

Description

[0001] The present invention relates to a method for diagnosing cancer using HSP60 protein,

The present invention relates to a method of providing information for cancer diagnosis using a heat shock protein 60 (HSP60) protein. More specifically, the present invention relates to a method for providing information for cancer diagnosis using a heat shock protein 60 (HSP60) To provide information for cancer diagnosis by confirming the cleavage pattern of the protein, and to a composition for diagnosing cancer including an antibody and a hydroxylamine which specifically bind to HSP60 protein, and a cancer diagnosis kit.

The cells that make up our body contain many proteins called Heat Shock Proteins (HSPs) as chaperones. HSP is induced in the cell when it is exposed to a variety of stresses such as external stimuli such as high fever, infection, free radicals, and heavy metals. In this study, we investigated the role of HSP in the pathogenesis of HSP, and found that HSP is a novel marker for the pathogenesis and diagnosis of HSP. Studies are underway in vaccine development.

Representative HSPs are proteins with molecular weights of 90,000, 70,000 and 60,000, and are named HSP90, HSP70 and HSP60, respectively, with numerical values indicating the molecular weight (unit: thousand). Among them, HSP60 is a protein involved in mitochondrial protein transport and polymer assembly. There is no systematic integration of HSP60 related research contents. Especially, there are many studies on the structure of HSP60.

On the other hand, some studies have been reported to infer the quantitative and functional differences of HSP60 between normal and cancer cells. Previous studies by the present inventors have confirmed the difference in the expression pattern of HSP60 mRNA in cervical cancer tissues and cell lines and confirmed that HSP60 is upregulated through 2D-experiments ( Yonsei Med J , 2009, 50 (3): 399-406). However, there has been no report on the structural differences of HSP60 between cancer cells and normal cells.

Accordingly, the inventors of the present invention found that there is a difference in the pattern of cleavage of HSP60 protein by hydroxylamine in cancer cells and normal cells as a result of intensive efforts to identify the structural difference between cancer cells and normal cells of HSP60 Thus completing the present invention. It is expected to be useful for diagnosis of cancer using the difference in the protein cleavage pattern.

(A) reacting a heat shock protein 60 (HSP60) protein present in the isolated biological sample with a hydroxylamine in a patient suspected of having cancer; And (b) identifying the cleavage pattern of the reacted HSP60 protein.

It is another object of the present invention to provide a cancer diagnostic composition comprising an antibody and a hydroxylamine that specifically binds to the HSP60 protein.

It is still another object of the present invention to provide a cancer diagnostic kit comprising the composition.

(A) reacting a heat shock protein 60 (HSP60) protein present in a biological sample isolated from a patient suspected of having cancer with hydroxylamine step; And (b) identifying a cleavage pattern of the reacted HSP60 protein.

In the present invention, when the HSP60 protein existing in the normal cells and the cancer cells was electrophoresed, the difference in position was observed on the gel, and thus it was confirmed that there was a clear difference in the cleavage pattern when reacted with hydroxylamine. From the above results, it was found that the cut-off pattern for the hydroxylamine of the HSP60 protein isolated from the sample of the cancer-predicting patient can be easily and cost-effectively diagnosed. Accordingly, the present invention can be effectively used as a method for providing information for cancer diagnosis and a composition for cancer diagnosis. Hereinafter, the present invention will be described in detail.

In the present invention, the term " diagnosis " is used to identify the presence or characteristic of a pathologic condition. For the purpose of the present invention, not only the confirmation of the onset of cancer but also the recurrence, metastasis, And the like.

The method of providing information for cancer diagnosis of the present invention can provide information on the incidence of cancer in the individual by confirming the cleavage pattern of the HSP60 protein by hydroxylamine. Specifically, the method can be used for the treatment and prophylactic treatment of hepatocellular carcinoma, lung cancer, breast cancer, thyroid cancer, testicular cancer, bone marrow dysplasia, oral cancer, bacterial sarcoma, acute myelogenous leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian cancer, ovarian germ cell tumor, Cholangiocarcinoma, non-small cell lung cancer, small cell lung cancer, pediatric brain tumor, pediatric lymphoma, vulvar cancer, primary malignant cancer, gastric lymphoma, malignant melanoma, myeloma, gallbladder cancer, biliary cancer, colon cancer, chronic myelogenous leukemia, retinoblastoma, choroidal melanoma, Cancer of the uterus, endometrial cancer, uterine sarcoma, prostate cancer, metastatic brain tumor, rectal carcinoid tumor, vaginal cancer, pancreatic cancer, pancreatic cancer, uterine cancer, endometrial cancer, uterine sarcoma, gastric cancer, gastric cancer, stomach cancer, Spinal cord cancer, neuroendocrine carcinoma, pancreatic cancer, salivary gland cancer, tonsil cancer, squamous cell carcinoma, adenocarcinoma, large cell carcinoma, skin cancer and laryngeal cancer, It can be used in the diagnosis of cancer, but are not limited thereto.

Using the fact that the HSP60 protein is structurally different in the normal cells and the cancer cells, the method of providing information for cancer diagnosis of the present invention is a method in which the HSP60 protein isolated from the patient to be diagnosed is simply reacted with hydroxylamine After confirming the cut pattern of the reacted protein, it is possible to easily diagnose the onset of cancer.

The method of providing information for the diagnosis comprises: (a) reacting a HSP60 protein present in a biological sample isolated from a patient suspected of having cancer with a hydroxylamine.

As used herein, the term " biological sample " refers to a tissue or cell isolated from a patient suspected of having cancer. The present invention relates to a method of treating a cancer selected from the group consisting of hepatocellular carcinoma, lung cancer, breast cancer, thyroid cancer, testicular cancer, bone marrow dysplasia, oral cancer, bacterial sarcoma, acute myelogenous leukemia, acute lymphocytic composition, basal cell carcinoma, ovarian cancer, ovarian germ cell tumor, Lymphoma, vulvar cancer, primary malignant cancer, gastric lymphoma, stomach cancer, non-small cell lung cancer, small cell lung cancer, pediatric brain tumor, pancreatic cancer, cholangiocarcinoma, chronic myelogenous leukemia, retinoblastoma, choroidal melanoma, bladder cancer, Cancer of the uterus, uterine sarcoma, uterine sarcoma, prostate cancer, metastatic brain tumor, rectal carcinoid, vaginal cancer, spinal cord cancer, gastrointestinal stromal tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, breast tumor, breast cancer, And may be a tissue or a cell of a site expected to be a neuroendocrine tumor, a neuroendocrine tumor, a pancreatic cancer, an salivary gland cancer, a tonsil cancer, a squamous cell carcinoma, adenocarcinoma, large cell cancer, skin cancer or laryngeal cancer, Or it may be a part of the tissue or cells are expected to cervical cancer, but is not limited thereto.

In the present invention, the term " HSP60 (heat shock protein 60) " refers to a mitochondrial chaperonin that functions in transportation and refolding from a cytoplasm to a mitochondrial matrix. HSP60 has a size of about 60 kDa.

The term " hydroxylamine " in the present invention means an inorganic compound having a composition of NH ₂ OH. Hydroxylamines are mainly used to add oxime functional groups and are used as intermediates for biological nitrification. It is also used to very selectively cleave asparagine-glycine peptide bonds in peptides and proteins. In the present invention, the amino acid sequence of the HSP60 protein was analyzed by a peptide cutter. As a result, it was confirmed that only the 177th position from the N-terminal of the HSP60 protein could be cleaved during the hydroxylamine treatment, Respectively.

In the present invention, the reaction is a reaction of a HSP60 protein isolated from a biological sample of a patient suspected of having cancer with a hydroxylamine, which comprises contacting the separated total protein comprising the HSP60 protein or the isolated HSP60 protein with a hydroxylamine And the reaction is carried out. Isolation of the total protein and separation of the HSP60 protein can be performed by any known method, and in particular, the isolation of the HSP60 protein may be performed by immunoprecipitation, but is not limited thereto.

In the present invention, the reaction may be carried out at 30 to 50 ° C and at a pH of 6.0 to 10.0, and more specifically, at 35 to 45 ° C and at a pH of 7.0 to 9.0. If the tissue-derived HSP60 protein is a condition capable of selectively cleaving by hydroxylamine at the 177th position, those skilled in the art can appropriately select and perform the condition.

In the present invention, " cleavage " refers to cleavage of a specific position of a protein resulting in detection of 2 to several proteins, and " cleavage " refers to cleavage of the protein to a level at which the protein is chopped. The reaction can be carried out for 1 to 5 hours, more specifically for 2 to 4 hours, but is not particularly limited thereto. Although the degree of cleavage or degradation of the HSP60 protein may vary depending on the reaction time, the present invention provides information for diagnosis by confirming whether or not there is a difference in cleavage pattern or whether or not there is a cleavage pattern. The degree of degradation may be different, but the results of the diagnosis are not different.

In a specific example of the present invention, when HSP60 protein isolated from normal tissues, cancer tissues, and cancer cell lines was confirmed by Western blotting, it was confirmed that the positions of HSP60 proteins were different on the gel during electrophoresis (FIGS. 1 and 2 ). Specifically, HSP60 protein derived from cancer tissue or cancer cell line showed a band somewhat lower position on the gel than that derived from normal cells. The position of the protein band on the Western blotting gel may be influenced by the size, structure, and the like of the protein. As a result, the size or the structure of the HSP60 protein may be different in the cancerous tissue and the normal tissue.

In another specific example of the present invention, in order to clarify the cause of the difference of the HSP60 protein band position, HSP60 protein was reacted with hydroxylamine to confirm the cleavage pattern (FIGS. 3 and 4). As a result of analyzing the normal protein by peptide cutter, the HSP60 protein derived from normal tissues was expected to appear in a pattern in which only the 177th position was cleaved from the N-terminal when reacted with hydroxylamine, and the 177th position was cleaved through the actual reaction It was confirmed that a protein fragment of about 41 kDa size was produced. However, when the cancer tissue-derived HSP60 protein was reacted with hydroxylamine at 37 DEG C and pH 7.0 for 2 hours, a band of about 60 kDa of the HSP60 protein having a size of about 60 kDa was faintly detected as compared with the control without hydroxylamine treatment And a band of the HSP60 fragment cut only at position 177 was scarcely observed (FIG. 3). Further, it was confirmed that HSP60 protein was not detected at all when the conditions were changed to react with hydroxylamine at 41 ° C and pH 9.0 for 4 hours (FIG. 4).

From the above results, it was found that the HSP60 protein derived from cancer tissue was completely decomposed by the hydroxylamine, unlike the normal tissue-derived HSP60 protein showing a pattern of cleavage by hydroxylamine only at the 177th position. This means that the HSP60 protein has a structural variation specifically for cancer tissue.

Next, (b) identifying the cleavage pattern of the reacted HSP60 protein may provide information for cancer diagnosis.

The term " cleavage pattern " in the present invention means a form in which cleavage of a protein occurs by reacting HSP60 protein with hydroxylamine. The step of confirming the cleavage pattern may further include a step of confirming whether the reacted HSP60 protein is degraded. As described above, the HSP60 protein from normal tissues is cleaved by the hydroxylamine to cleave the 177th amino acid position from the N-terminal, that is, only the position is cleaved to generate two peptide fragments. Specifically, the HSP60 protein of about 60 kDa can be divided into two fragments of about 41 kDa and 19 kDa by the cleavage. However, in the case of cancer tissue-derived HSP60, it can be completely degraded by hydroxylamine.

The step of confirming the cleavage pattern of the HSP60 protein can be performed, for example, through Western blotting, but it is not limited thereto and those skilled in the art can appropriately perform the cleavage pattern of the HSP60 protein by known methods. In particular, whether or not the HSP60 protein derived from cancer tissue is degraded by hydroxylamine, that is, whether the protein is degraded refers to a state in which the protein is completely decomposed and can not be detected, or the protein is degraded Concentration in the sample.

In summary, a method of providing information for cancer diagnosis of the present invention comprises reacting a HSP60 protein separated from a biological sample of a patient suspected of having cancer with a hydroxylamine to form a cleavage pattern of the protein with hydroxylamine By confirming, if the protein is not only cleaved at the 177th position from the N-terminus but is degraded, it can be diagnosed as having cancer. This can provide information for cancer diagnosis in a relatively inexpensive and easy way.

Another embodiment is a cancer diagnostic composition comprising an antibody and a hydroxylamine that specifically binds to a HSP60 (heat shock protein 60) protein.

The diagnosis of HSP60 protein, hydroxylamine and cancer has been described above.

As used herein, the term " antibody " includes immunoglobulin molecules immunologically reactive with specific antigens, and may include both polyclonal antibodies and monoclonal antibodies. &Quot; Monoclonal antibody " refers to highly specific antibodies directed against a single antigenic site (epitope) as a single molecule composition of an antibody molecule obtained in a substantially identical population of antibodies, and such monoclonal antibodies typically have different Unlike polyclonal antibodies that contain different antibodies directed against epitopes, they are directed against a single epitope on the antigen.

It will also be readily understood by those skilled in the art that the monoclonal antibodies can be converted into chimeric, humanized and human monoclonal antibodies that have reduced immunogenicity for application to the human body. Such chimeric antibodies are those in which the variable region of a monoclonal antibody is recombined with the constant region of a human antibody and the humanized antibody comprises complementarity determining regions (CDRs) or complementarity determining regions (CDRs) that directly bind to the antigen in the variable region of the monoclonal antibody Among the crystal regions, only the specificity determining residues (SDRs) involved in antigen binding specificity are transplanted into human antibodies.

The humanized antibody may be obtained by replacing the heavy chain variable region or the light chain variable region in the variable region of the monoclonal antibody with a heavy chain variable region or a light chain variable region of a human antibody and thereby producing a hybrid (mouse heavy chain / human light chain or The mouse heavy chain variable region or the light chain variable region is selected from the mouse light chain / human heavy chain antibody and then ligated to a human antibody constant region. The mutant can be easily produced from a monoclonal antibody, Of course. The chimeric antibody, humanized antibody and human monoclonal antibody prepared as described above can be produced in animal cells using a known method.

In addition, the monoclonal antibody in the present invention may be a fragment thereof as long as it has the above-described binding property. That is, the antibody of the present invention can be used not only for the complete form having the full length of two heavy chains and two light chains, but also as a functional fragment of the antibody molecule, for diagnosis and diagnosis of cancer. The functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, and may include Fab, F (ab ') 2, F (ab') 2 and Fv.

In addition, monoclonal antibodies in the present invention can be easily produced by a known monoclonal antibody production technique. For example, by the fusion method well known in the art (Kohler et al., European Journal of Immunology 6; 511-519). This method is similar to the traditional hybridoma production method, but can be obtained by inactivating the immunoglobulin gene of a mouse and preparing a transgenic mouse transplanted with a human immunoglobulin gene locus.

In addition, monoclonal antibodies can be prepared by phage display technology, which is a technique of expressing a human antibody library on the surface of bacteriophages in the form of antibody fragments (Fab, ScRv) to screen antibody clones for specific antigens, The method of producing the antibody is not limited.

Typically, immunoglobulins have a heavy chain and a light chain, and each heavy and light chain comprises a constant region and a variable region (the region also known as a "domain"). The variable regions of the light and heavy chains include three multifunctional regions called "complementarity determining regions" (hereinafter referred to as "CDRs") and four "framework regions". The CDRs mainly serve to bind to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, CDR3 starting from the N-terminus and sequentially identified by the chain in which the particular CDR is located.

The composition of the present invention comprises a hydroxylamine for the purpose of cleaving a protein separated from a biological sample of a patient, particularly HSP60 protein, and for the purpose of confirming the cleavage pattern or decomposition of HSP60 protein in the above-mentioned reacted product And an antibody that specifically binds to the HSP60 protein.

Yet another aspect is a cancer diagnostic kit comprising the composition.

It is obvious that the composition for cancer diagnosis is as described above and can be prepared in the form of a kit which is commonly used in the art, including the composition of the present invention as a constituent element. For the purpose of the present invention, the cancer which can be diagnosed by confirming the cleavage pattern by the hydroxylamine of the HSP60 protein may include the above-mentioned cancer without limitation.

By identifying the functional and structural differences of HSP60 in cancer and using it as a biomarker to detect human normal state and pathologic status, HSP60 can be used for clinical diagnosis of cancer efficiently and objectively. It can contribute to cost reduction and efficiency increase.

Figure 1 is the Western blot results for HSP60 in cervical cancer cell line (MCF7) and human cancer tissues.
Figure 2 shows the Western blot results for HSP60 in breast cancer, cervical cancer and normal tissues.
FIG. 3 shows the result of reacting HSP60 with hydroxylamine at 37 DEG C and pH 7.0 for 2 hours.
FIG. 4 shows the result of reacting HPS60 with hydroxylamine at 41.degree. C., pH 9.0 for 4 hours.

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

Example  1: Experimental material

The tissue diagnosed as cancer from patients who visited the medical center of Gachon University was purchased with IRB approval, and the experiment was conducted with HSP60 protein extracted from cervical cancer tissue and breast cancer tissue.

Example  2: protein extraction

First, tissues were exchanged on ice using a homogenizer. Then, it was centrifuged at 14000 rpm for 10 minutes at 4 DEG C, and the supernatant was collected in a new tube. It was stored at -20 ° C for use on the day of extraction and at -80 ° C for long-term storage.

Example  3: Immunoprecipitation method ( Immunoprecipitation ; IP)

Experiments were performed using an immunoprecipitation kit from Novex (Invitrogen). It is the principle that separates only the target protein after attaching the antibody to the magnetic bead containing Protein G. First, 100 - 1000 μl of the protein extracted from the tissue together with the HSP60 antibody (LK1) was added to the beads, followed by reaction at room temperature for 10 minutes. After the reaction, it was inserted into a magnetic rack to separate the supernatant and beads, and the supernatant was transferred to another tube. The beads were then washed three times with 200 μL of wash buffer and then placed in a magnetic rack to discard the supernatant. Proteins in the beads were then separated using elution buffer.

Example  4: Hydroxylamine  ( Hydroxylamine ) process

To determine the structural differences of the HSP60 protein in normal cells (or tissues) and cancer cells (or tissues), peptide sequence analysis was performed first. As a result, it was confirmed that the partition between the 177th asparagine (N) and the 178th glycine (G) of the HSP60 protein was specifically cleaved by hydroxylamine (http: //web.expasy .org / peptide_cutter /).

Thus, 0.4 to 4 M hydroxylamine (pH 4, 7 or 8) was mixed 1: 1 with the protein (HSP60 isolated from normal or cancer) and incubated at 37 ° C for 2 hours or at 41 ° C for 4 hours Lt; / RTI > After the reaction, Western blot was used to confirm the difference in cleavage pattern or degradation of HSP60 between normal cells and cancer cells.

Example  5: Western blot  (Western blotting)

HSP60 isolated from normal tissues and cancer tissues was divided into two groups before and after the hydroxylamine reaction. Each sample was mixed with 10 μl of the sample buffer, reacted at 95 ° C for 5 minutes, and then reacted on ice for 5 minutes. The samples were then slowly loaded and electrophoresed at 50 V for 15 minutes and 90 V for 1 hour and 30 minutes to 4 hours. Then, it was transferred to the nitrocellulose membrane, blocked for 1 hour, and reacted with the primary antibody overnight and with the secondary antibody for 2 hours. The primary antibody was HSP60 monoclonal antibody (LK-1) and the secondary antibody was goat anti-mouse IgG-HRP. Experimental results were obtained using 4 ~ 12% gradient gel and 10% gel.

Experimental Example  One: Cancer cells  In normal cells HSP60's  Identify structural differences

To investigate the structural differences of HSP60 protein between normal and cancer tissues, the following experiment was performed. (BC), breast tissue (BN), cervical cancer tissue (CC) and cervical cancer tissue normal tissue (CN) in the same human cancer tissue and normal tissue As a result of Western blot analysis of HSP60 protein, HSP60 of normal tissues appeared to be slightly above the gel. As a control, the protein isolated from breast cancer cell line MCF7 was used and protein was detected at the same position as HSP60 isolated from cancer tissue of cancer patients. This suggests that the HSP60 protein differs in size or structure in cancer tissues and normal tissues (Fig. 1).

The protein extracted from normal and cancer tissues is a total protein, and thus contains many other proteins besides HSP60. Then, electrophoresis was performed on SDS-PAGE for 4 to 5 hours for a long time and Western blot results for HSP60 protein were confirmed.

As a result, a difference in the position of HSP60 protein between breast cancer tissue and cervical cancer tissue and normal tissue was observed more clearly than the result observed in FIG. In cancer tissues, a small amount of protein was observed and a large protein band was observed in normal tissue (Fig. 2). These results have not been reported so far and have been confirmed for the first time by the present inventors.

Experimental Example  2: Cancer cells  In normal cells Hydroxylamine  ( hydroxylamine ) To confirm the cutting pattern

In order to reduce the influence of other proteins other than HSP60, HSP60 protein was identified and tested by IP (Immunoprecipitation) method.

From the results of Experimental Example 1, it was expected that the size or structure of HSP60 protein would be different in cancer cells and normal cells, but it was confirmed that there was no substantial difference in size. Thus, the present inventors sought to identify the structural differences of HSP60 in cancer cells and normal cells.

In order to identify the amino acid sequence causing the structural difference of the HSP60 protein, the HSP60 protein was analyzed with a peptide cutter. As a result, it was confirmed that the HSP60 protein was specifically cleavable by the hydroxylamine only at the 177th position of the HSP60 protein. Experiments were conducted using these characteristics.

This protein was treated with 0.4 M hydroxylamine (pH 7.0) for 2 hours using proteins obtained from normal tissues and cancer tissues, and the partitioning pattern of HSP60 protein was different (FIG. 3). That is, in the normal tissue, the 177th position of the HSP60 protein was cleaved by hydroxylamine to identify two bands with the original size band (about 60 kDa) and the truncated fragment band (about 41 kDa). In the normal tissue, only the band of about 41 kDa was confirmed as the cleaved fragment because the monoclonal antibody used in this Example recognized this portion. As a result, HSP60 from normal tissue was detected by hydroxylamine at position 177 Only two fragments are produced. However, in the cancer tissue, only the original size band was blurred.

In order to confirm this more specifically, the reaction conditions were partially changed and the reaction time was changed to 4 hours. After that, the cleavage pattern of each tissue-derived HSP60 protein was confirmed by Western blotting (FIG. 4).

As a result, it was confirmed that similar results as those shown in FIG. 3 were obtained in normal tissues, and it was confirmed that the concentration of the band of the cut fragments was more enhanced than that of the non-cut bands. This means that as the reaction time is increased, more protein is cleaved by the hydroxylamine. On the other hand, even when the cancer tissue was cut with hydroxylamine, the band which was confirmed in Fig. 3 was not even confirmed. This means that all of the HSP60 isolated from the cancer cells in the hydroxylamine-treated reaction was decomposed.

Taken together, these results indicate that the size of HSP60 protein is not different between cancer cells and normal cells, but it is structurally different. This can be confirmed by a pattern cleaved by hydroxylamine. The normal cell-derived HSP60 protein is cleaved by the hydroxylamine only at the 177th amino acid position, whereas the cancer cell-derived HSP60 protein is treated with hydroxylamine , It can be seen that it is decomposed according to time.

These results suggest that a protein sample isolated from a patient suspected of having cancer may be treated by treating hydroxylamine with a hydroxylamine and then assaying the cleavage pattern of the HSP60 protein for cancer diagnosis.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (12)

(a) reacting a heat shock protein 60 (HSP60) protein present in a biological sample isolated from a patient suspected of having cancer with hydroxylamine; And
(b) identifying a cleavage pattern of the reacted HSP60 protein, the method comprising:
Wherein said cancer is breast or cervical cancer.
2. The method of claim 1, further comprising the step of determining whether the reacted HSP60 protein is degraded.
2. The method of claim 1, wherein the step of reacting with the hydroxylamine is carried out for 1 to 5 hours.
2. The process of claim 1, wherein the step of reacting with the hydroxylamine is carried out at 30 to 50 < 0 > C.
2. The method of claim 1, wherein the step of reacting with the hydroxylamine is performed at a pH of from 6.0 to 10.0.
2. The method of claim 1, wherein identifying the cleavage pattern of the HSP60 protein is performed through Western blot.
delete 3. The method of claim 1, wherein the HSP60 protein reacted with the hydroxylamine is judged to be normal when cleaved at the 177th position from the N-terminus.
3. The method of claim 2, wherein if the HSP60 protein reacted with the hydroxylamine is degraded, the cancer is judged to be cancer.
A composition for diagnosing cancer comprising an antibody specifically binding to a HSP60 (heat shock protein 60) protein and a hydroxylamine,
Wherein the cancer is breast cancer or cervical cancer.
delete A cancer diagnostic kit comprising the composition of claim 10.

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