Composition for diagnosing cancer using potassium channel proteins

Abstract

This disclosure relates to a composition for diagnosing cancer by using potassium channel proteins; to a kit for diagnosing cancer comprising the composition; and to an information providing method for diagnosing cancer. Specifically, the composition or kit for diagnosing cancer provided in this disclosure may be used to diagnose the onset of cancer regardless of its type, by measuring the expression levels of potassium channels, KCa3.1 channel and KCa2.3 channel, or a regulator thereof from vascular endothelial cells treated with a sample of a subject, or from red blood cells isolated from the subject, and thus can be widely utilized in determining the stages of progression (growth, metastasis, prognosis, and recurrence) of various cancers.

Claims

1. A method for diagnosing cancer, the method comprising: (a) measuring an expression level of a KCa3.1 channel protein or the mRNA expressed from a gene encoding the KCa3.1 channel protein (i) in the red blood cells of a subject suspected of having cancer, or (ii) in vascular endothelial cells exposed to the serum of a subject suspected of having cancer; (b) comparing the expression level of the protein or mRNA measured in the step (a) with an expression level measured in a normal control sample; (c) measuring an expression level of at least one protein selected from the group consisting of clathrin, caveolin1, EEA and Rab5C, or the mRNA expressed from a gene encoding the at least one protein of the subject in the step (a); and (d) comparing the expression level of the protein or mRNA measured in the step (c) with an expression level measured in a normal control sample.

2. The method of claim 1, further comprising: (e) calculating a ratio of the expression level measured in the step (a) and the expression level measured in the step (c), and (f) comparing the ratio calculated in step (e) with a ratio of the expression levels measured in the normal control sample.

3. The method of claim 1, wherein the KCa 3.1 channel protein expression level is measured using an antibody or an aptamer capable specifically binding to the KCa 3.1 channel protein.

4. The method of claim 1, wherein the mRNA expression levels in steps (a) and (c) are measured by a primer, a probe, or an antisense oligonucleotide capable of specifically binding to the gene.

5. The method of claim 1, wherein the cancer is one selected from the group consisting of liver cancer, lung cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, uterine cancer, cervical cancer, brain cancer, oral cancer, colon cancer, biliary cancer, bone cancer, and skin cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 is an image of western blot analysis illustrating the result of comparing expression levels of potassium channels, KCa3.1 channel and KCa2.3 channel, measured in blood sample(serum)-treated vascular endothelial cells of a patient with liver cancer to that of a control group, and a graph illustrating the quantitated results of the expression levels of the potassium channels.

[0061] FIG. 2a is an image of western blot analysis illustrating the result of comparing the expression level of the potassium channel, KCa3.1 channel, measured in red blood cells of a patient with liver cancer to that of a control group, and a graph illustrating the quantitated result of the expression level of the potassium channel.

[0062] FIG. 2b is an image of western blot analysis illustrating the result of comparing the expression level of the potassium channel, KCa3.1 channel, measured in red blood cells of a patient with liver cirrhosis to that of a control group, and a graph illustrating the quantitated result of the expression level of the potassium channel.

[0063] FIG. 2c is an image of western blot analysis illustrating the result of comparing the expression level of a regulatory factor of the potassium channel, clathrin, measured in red blood cells of a patient with liver cancer and a patient with liver cirrhosis, to that of a control group, and a graph illustrating the quantitated result of the expression level of the clathrin.

[0064] FIG. 3 is an image of western blot analysis illustrating the result of comparing the expression level of a KCa3.1 channel and a regulatory factor thereof, clathrin, measured in red blood cells of a patient with pancreatic cancer to that of a control group, and graphs illustrating the quantitated results of the expression levels of the KCa3.1 channel and the clathrin.

[0065] FIG. 4 is images of western blot analysis illustrating the results of comparing the expression levels of the potassium channels, KCa3.1 channel and KCa2.3 channel, and regulatory factors thereof, caveolin 1 and EEA1, measured in blood sample(serum)-treated vascular endothelial cells of a patient with liver cancer in which the blood sample(serum) of the patient with liver cancer is first diluted.

[0066] FIG. 5a is a graph illustrating the results of comparing the expression level ratios of KCa3.1 channel and clathrin measured in red blood cells of a patient with liver cancer and a patient with liver cirrhosis.

[0067] FIG. 5b is a graph illustrating the results of comparing the expression level ratio of KCa3.1 channel and clathrin measured in red blood cells of a patient with pancreatic cancer.

[0068] FIG. 6 is an image of western blot analysis illustrating the result of comparing the expression levels of the KCa3.1 channel or the KCa2.3 channel expressed in liver tissue cells of a liver cancer model mouse (CerS2), in which a gene encoding ceramide synthase 2 is deleted to induce liver cancer, to that of a control group, and a graph illustrating the quantitated result of the expression level of KCa3.1 channel.

[0069] Hereinafter, this disclosure will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of this disclosure is not limited to these examples.

Example 1

Effect of Blood Samples of a Patient with Liver Cancer on Potassium Channels of Vascular Endothelial Cells

[0070] Vascular endothelial cells were treated with a blood sample of a patient with liver cancer to determine whether expression levels of a KCa3.1 channel and a KCa2.3 channel are changed or not.

[0071] A serum sample was obtained from the blood of a patient with liver cancer. The serum sample was treated with human vascular endothelial cells, and then cultured for 24 hours. After completion of the culture, expression levels of the KCa3.1 channel and the KCa2.3 channel expressed in the vascular endothelial cells were measured by western blot analysis and compared (FIG. 1). Vascular endothelial cells treated with a normal serum sample were used as a control group. The expression level of the KCa3.1 channel protein was measured using an antibody having an amino acid sequence of RQVRLKHRKLREQV (SEQ ID NO: 1), and the expression level of the KCa2.3 channel protein was measured by using an antibody having an amino acid sequence of LHSSPTAFRAPPSSNSTAILHPSSRQGSQLNLNDHLLGHSPSSTA (SEQ ID NO: 2) and particularly binding to the KCa2.3 channel protein. GAPDH was used as an internal control.

[0072] As shown in FIG. 1, the expression levels of the KCa3.1 channel and the KCa2.3 channel were increased in the serum sample-treated vascular endothelial cells of the patient with liver cancer, unlike the normal serum sample-treated vascular endothelial cells.

Example 2

Analysis of Expression Levels of Potassium Channels and Regulatory Factors Thereof Expressed in Red Blood Cells in a Patient With Liver Cancer and a Patient With Liver Cirrhosis

[0073] From the results of Example 1, it was confirmed that the expression levels of the KCa3.1 channel and the KCa2.3 channel were increased in the serum sample-treated vascular endothelial cells of the patient with liver cancer. Therefore, expression levels of potassium channels and regulatory factors thereof were analyzed in red blood cells of the patient with liver cancer.

Example 2-1

Analysis pf Expression Levels of Potassium Channels Expressed in Red Blood Cells of a Patient With Liver Cancer and a Patient With Liver Cirrhosis

[0074] It was confirmed in western blot analysis that the expression level of the KCa3.1 channel was increased in red blood cells of a patient with liver cancer (FIG. 2a). Here, normal human red blood cells were used as a control group and GAPDH was used as an internal control. As shown in FIG. 2a, it was confirmed that the expression of the KCa3.1 channel in the red blood cells of the patient with liver cancer was higher than that of the normal red blood cells.

[0075] It was further confirmed in western blot analysis that the expression of the KCa3.1 channel in red blood cells of a patient with liver cirrhosis, instead of the patient with liver cancer, was higher than that of the normal red blood cells (FIG. 2b). Here, normal human red blood cells were used as a control group and GAPDH was used as an internal control.

[0076] As shown in FIG. 2b, it was confirmed that the expression of the KCa3.1 channel in the red blood cells of the patient with liver cirrhosis was higher than that of the normal red blood cells.

Example 2-2

Analysis of Expression Levels of Regulatory Factors of the Potassium Channel Expressed in Red Blood Cells of a Patient With Liver Cancer and a Patient With Liver Cirrhosis

[0077] The expression levels of clathrin, which is known as a regulatory factor of KCa3.1 channel and KCa2.3 channel, in the red blood cells obtained from the blood samples of the patient with liver cancer and the patient with liver cirrhosis used in Example 2-1 were measured by western blot analysis using an antibody having an amino acid sequence of PQLMLTAGPSVAVPPQAPFGYGYTAPPYGQPQPGFGYS (SEQ ID NO: 3) and compared (FIG. 2c). Here, normal human red blood cells were used as a control group and GAPDH was used as an internal control.

[0078] As shown in FIG. 2c, it was confirmed that the expression levels of clathrin, a regulatory factor of the potassium channel, were decreased in red blood cells of the patient with liver cancer and the patient with liver cirrhosis in which the expression level of the KCa3.1 channel is increased.

Example 3

Analysis of Expression Levels of Potassium Channels and Regulatory Factors Thereof Expressed in Red Blood Cells in a Patient With Pancreatic Cancer

[0079] From the results of Example 2, it was confirmed that the expression level of the KCa3.1 channel protein was increased in the red blood cells of the patient with liver cancer and the expression level of the clathrin, the regulatory factor of the KCa3.1 channel protein, was decreased. Thus, it was tested to determine whether the same result would be obtained from a patient with pancreatic cancer.

[0080] Red blood cells were obtained from the blood of a patient with pancreatic cancer, and then expression levels of the KCa3.1 channel and the clathrin expressed from the red blood cells were measured by western blot analysis and compared (FIG. 3). Here, normal human red blood cells were used as a control group and GAPDH was used as an internal control.

[0081] As shown in FIG. 3, it was confirmed that the expression of the KCa3.1 channel in the red blood cells of the patient with pancreatic cancer was increased and the expression of clathrin was decreased as shown in the red blood cells of the patient with liver cancer.

Example 4

Effect of Dilution of Blood Samples of a Patient With Liver Cancer on Potassium Channels of Vascular Endothelial Cells

[0082] A serum sample was obtained from the blood of a patient with liver cancer. The serum sample was diluted with a culture solution, treated with vascular endothelial cells, and then cultured. After completion of the culture, expression levels of the KCa3.1 channel and the KCa2.3 channel, which are potassium channels, and caveolin1 and EEA1, which are regulatory factors of the potassium channel, expressed in the vascular endothelial cells were measured by western blot analysis and compared. The expression levels of caveolin1 and EEA1 were measured using an antibody having the amino acid sequence of MADELSEKQVYDAHTKEID (SEQ ID NO: 4) and an antibody having the amino acid sequence of FCAECSAKNALTPSSKKPVR (SEQ ID NO: 5), respectively. GAPDH was used as an internal control.

[0083] As shown in FIG. 4, the expression levels of the KCa3.1 channel and the KCa2.3 channel were increased in the serum-treated vascular endothelial cells of the patient with liver cancer, but the expression levels of caveolin-1 and EEA1 were decreased at the same time.

Example 5

Analysis of Ratios of Expression Levels of Potassium Channels and Regulatory Factors Thereof in Blood Sample-Treated Vascular Endothelial Cells

[0084] Blood samples (red blood cells or serums) of the patient with liver cancer, the patient with liver cirrhosis and the patient with pancreatic cancer, who were used in Example 1 to Example 4, were treated with vascular endothelial cells and cultured. After completion of the culture, expression levels of the KCa3.1 channel, which is a potassium channel, and clathrin, which is a regulatory factor of the potassium channel, expressed from the vascular endothelial cells were measured. The measured values were applied to the following equation to calculate a ratio of the measured value of the expression level of the channel protein to the measured value of the expression level of the regulatory factor thereof. The calculated ratio was compared to the ratio of the measurements calculated in a normal control (FIG. 5a and FIG. 5b). Here, normal blood sample-treated vascular endothelial cells were used as the control.

Ratio of Measured Values=Measured Expression Level of KCa3.1 Channel/Measured Expression Level of a Regulatory Factor of the Potassium Channel

[0085] As shown in FIG. 5a and FIG. 5b, the calculated ratio of the measured values of the blood sample-treated (red blood cell-treated or serum sample-treated) vascular endothelial cells was 2.0 or higher, while that of the normal control group was about 1.0.

[0086] It is noted that when only the expression levels of potassium channels or regulatory factors of the potassium channel are measured and compared and the measured expression levels are similar in a patient with cancer and a normal subject, any error may occur in the diagnosis result. On the other hand, when both the expression levels of potassium channels and regulatory factors of the potassium channel are measured and the ratios thereof are calculated and the measured values are similar, the ratios thereof calculated from a patient with cancer and a normal subject are clearly distinguished from each other so that the likelihood of errors may be significantly reduced in the diagnosis result.

[0087] Accordingly, it confirms that the ratio of expression levels between the potassium channels and regulatory factors of the potassium channel may be usefully utilized to determine progression levels of cancers.

Example 6

Analysis of Expression Levels of Potassium Channels in Lever Tissue of a Liver Cancer Model Mouse

[0088] Since it was confirmed that the expression level of the KCa3.1 channel or the KCa2.3 channel was increased in red blood cells of the patient with liver cancer or the patient with pancreatic cancer in Examples above, the expression level of the potassium channel was measured in a liver tissue of a liver cancer model mouse.

[0089] A liver tissue was obtained from a liver cancer model mouse (CerS2) in which the liver cancer was induced by deleting a gene encoding ceramide synthase 2 and expression level of the KCa3.1 channel or the KCa2.3 channel expressed in the obtained liver tissue was measured and quantitated by western blot analysis (FIG. 6). Here, a normal mouse liver tissue was used as a control group and alpha-tubulin was used as an internal control group.

[0090] As shown in FIG. 6, it was confirmed that the expression levels of KCa3.1 channel and KCa2.3 channel in the liver tissue of the liver cancer model mouse were increased.

[0091] Collectively, the results for Examples described above suggest that cancer patients can be distinguished from normal subjects by utilizing expression levels of the potassium channel proteins in blood sample-treated vascular endothelial cells or red blood cells of cancer patients, expression levels of regulatory factors of the channel proteins, or each ratio of the expression levels.

[0092] Similar results were obtained in patients with liver cirrhosis of whom liver cancer was not started but who were more likely to develop liver cancer.

[0093] From the above description, it is noted that it is possible not only to diagnose the onset of cancer in patients but also to diagnose the probability of cancer early before the cancer occurs by using the expression level of the protein of the potassium channel or regulatory factors thereof measured in the blood sample-treated vascular endothelial cells of a patient suspected of having cancer or the red blood cells of the patient.

[0094] Throughout the description of the present disclosure, when describing a certain technology is determined as that the point of the present disclosure can be fully understood by those who are skilled in the art, the pertinent detailed description has been omitted. While this disclosure includes specific examples, it will be apparent after an understanding of the disclosure of this application that various changes in form and details may be made in these examples without departing from the spirit and scope of the claims and their equivalents. Therefore, the scope of the disclosure is defined not by the detailed description, but by the claims and their equivalents, and all variations within the scope of the claims and their equivalents are to be construed as being included in the disclosure.