Patent Application
20130236913
Embodiments relate to serological markers for detecting colorectal cancer and a method for using the serological markers, especially to a serological marker for detecting colorectal cancer and a method for using the serological marker with high sensitivity and specificity. Further, embodiments relate to the capability of colorectal cancer inhibition by neutralizing the function of selected serological markers.
Cancers are shown as the major causes of death. Earlier detecting of cancer, inhibiting the development and spread of cancer cells, and even researching a method for preventing cancer cells are major issues focused by researchers and the public.
Colorectal cancer (CRC) has been the third common cancer among all cancers. Every year, there are a million new cases and 500,000 people death due to the colorectal cancer. Researches reveal earlier detection and diagnosis will help to decrease the mortality and morbidity of colorectal cancer.
Screening methods currently available for detecting the colorectal cancer include digital rectal examination, fecal occult blood test (FOBT), sigmoidoscopy, and colonoscopy. Although above-mentioned methods have improved the detection efficiency of the colorectal cancer, however, their diagnostic value as a general screening tool is limited because of poor sensitivity and a high false positive rate (FOBT), costs, risks, and inconvenience (colonoscopy). To overcome these problems, the development of novel biomarkers that can allow for the early detection of CRC is crucial.
The advancement of molecular biology, proteomic and preventive medicine provide more useful information or tools to increase the proceeding of develop a novel detection or therapy for colorectal cancer. A biomarker revealing over-expression in tumor tissues (i.e. a tumor tissue or a tissue biopsy) by using one of above-mentioned methods might not release to the circulation of the blood, show a large amount of expression in the blood, or has a long half-life to be detected. A carcinoembryonic antigen (CEA), a clinically established serological marker, is a marker currently widely used in colorectal cancer detection but is always suggested to be used in estimating the prognosis of CRC patients due to the limited sensitivity and specificity.
Accordingly, using different biotechnological methods to search and develop a biomarker to earlier diagnosis the colorectal cancer and to improve the therapeutic efficiency is very important
According to one aspect of an embodiment of the invention, a serological marker for detecting a colorectal cancer with high sensitivity and specificity at least comprises a phospholipid scramblase 1 (PLSCR1) protein. The PLCSR1 is highly expressed in colorectal cancer patients. Therefore, the colorectal cancer is easy to be determined the possibility and risk by collecting and assaying the PLSCR1 expression in a blood sample instead of invasive obtaining a biopsy. The PLSCR1 protein is increased in the blood at the earlier stage of the colorectal cancer so that the PLSCR1 protein could be applied in the early detection of the colorectal cancer. In an embodiment, the PLSCR1 protein is combined with other proteins, such as SEC61β or STOML2 protein, that are sensitive and specific with the colorectal cancer to improve the detection efficiency.
According to another aspect of an embodiment of the invention, a method for detecting the colorectal cancer comprises steps of establishing a database, collecting a blood sample, detecting a serological marker in the blood sample and comparing the serological maker expression with the database.
According to yet another aspect of an embodiment of the invention, a detection device comprises a sample container, at least a PLSCR1 protein antibody and a substrate. A blood sample is collected and applied in the detection device to test the expression of a serological marker, which is compared with a database to determine the possibility of the colorectal cancer.
Due to the highly sensitivity and specificity to the colorectal cancer, an antigen of the PLSCR1 is used to as a serological marker in detecting the colorectal cancer, and the PLSCR1 or a recombinant protein of PLSCR1 is applied in a detection device for detecting the colorectal cancer. Otherwise, an antibody neutralizing the function of selected serological marker has capability to inhibiting the cell growth of the colorectal cancer.
The above objects and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings in which:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The embodiment selects a phospholipid scramblase 1 (PLSCR1) as a serological marker for detecting the colorectal cancer. The PLSCR1 is over-expressed in the colorectal cancer tissue and is tested the efficiency as a serological marker to detect the colorectal cancer.
28 colorectal cancer patients are enrolled. A colorectal cancer tissue (tumor tissue) and a normal tissue away from the colorectal cancer tissue for at least 10 centimeters collected from each patient is respectively analyzed the proteomic profile. All tissues are cut into small pieces, washed by using 0.9% sodium chloride to remove blood, homogenized in STM solution (5 ml STM solution/g tissue, 0.25 M sucrose, 10 mM Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) and 1 mM magnesium chloride) to form a homogenous solution.
The homogenous solution is centrifuged at 260×g for 5 minutes to remove the precipitated nuclei and tissue debris. A separated supernatant solution is further centrifuged at 1500×g for 10 minutes to precipitate the crude membrane proteins. The crude membrane proteins are homogenized in two-thirds volume of the original homogenate volume (contains 0.25M STM solution and protease inhibitor) and centrifuged at 16000×g for an hour to purify the membrane proteins. The membrane pellet is washed by 0.1M sodium carbonate for overnight and is centrifuged at 16000×g for an hour to re-collect the membrane proteins. The purified membrane proteins are homogenized in 90% formic acid and stored in −80° C.
The purified membrane proteins are resuspended in 6M urea, 5 mM ethylene diamine tetraacetic acid (EDTA), and 2% sodium dodecyl sulfate (SDS) in 0.1M triethylammonium bicarbonate (TEABC) and sonicated at 4° C. for 10 minutes. A bovine serum albumin (BSA) is used as an internal standard (0.1% BSA in the purified membrane proteins). The purified membrane proteins were reduced by 5 mM Tris(2-carboxyethyl)-phosphine (TCEP) and alkylated by 2 mM methyl methanethiosulfonate (MMTS) at room temperature for 30 mM. A 40% acrylamide solution (acrylamide: bisacrylamide is 29:1, v/v), 10% (w/v) ammonium persulfate (APS) and 100% N,N,N,N-Tetramethylethylenediamine (TEMED) are added to the purified membrane protein mixture to polymerize as a gel.
The gel is cut into small pieces and subjected to tryptic digestion in 25 mM TEABC solution at 37° C. to form peptides. The peptides are extracted from the gel using sequential extraction with 25 mM TEABC, 0.1% (v/v) trifluoroacetic acid (TFA) in water, 0.1% TFA in acetonitrile (ACN), and 100% ACN. The extracted peptides are concentrated, desalted and analyzed by a liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Information collected from LC-MS/MS is searched by Mascot v2.2 against International Protein Index (IPI) human database from the European Bioinformatics Institute. The selection constraints: (a) only tryptic peptides with up to two missed cleavage sites were allowed; and (b) 0.3-Da mass tolerances for MS and 0.1-Da mass tolerances for MS/MS fragment ions. Only unique peptides with scores ≧35 (p<0.05) were confidently assigned. The amino acid sequence of enrolled peptide is further compared to identify the corresponding protein.
Results from the LC-MS/MS analysis are used to compare the expression of a specific protein in the colorectal cancer tissue (tumor tissue) and the normal tissue. For example, peak area of protein A in the colorectal cancer tissue and the normal tissue is R1 and R2 respectively. The peak area of the internal standard in the colorectal cancer tissue and the normal tissue is S1 and S2 respectively. Comparing the ratio of the protein A and the internal standard in the colorectal cancer tissue and the normal tissue, a difference in abundance of 1.5-fold to indicate a statistically significance of higher or lower expression, respectively.
PLSCR 1 is selected from the LC-MS/MS analysis due to the significant overexpression in the colorectal cancer tissue than the matching normal tissue, and is further tested the expression in the western blotting assay and the immunohistochemistry (IHC) assay.
Western Blotting Analysis
The colorectal cancer tissue and the matching normal tissue are suspended in a lysis buffer (0.25 M sucrose, 10 mM Tris-HCl, pH 7.6, 1 mM MgCl2, 1% SDS) with protease inhibitor mixture (20 μg/μL aprotinin, 20 μg/μL leupeptin, and 1 mmol/L phenylmethanesulfonyl fluoride (PMSF); protein:protein inhibitor was 100:1, v/v) and homogenized on ice to form a protein suspension. 40 μg of protein suspension was subjected to the western blot analysis.
With reference to Table 1, Table 1 illustrates the expression of the PLSCR1 in the colorectal cancer tissue compared with their normal counterpart. 18 of 28 paired tissues show at least 1.5 folds overexpression in the colorectal cancer tissue compared with their normal counterpart.
The PLSCR1 of the embodiment has an UniProt accession number 015162 with sequence shows as following (SEQ ID NO: 1):
However, one of ordinary skill in the art will realize that any sequence has more than 90% similarity with above-mentioned sequence is capable to apply in the present invention.
In order to understand whether the PLSCR 1 is significant presented in a blood specimen or has capability to be a serological marker, blood samples respectively from colorectal patients and healthy controls are collected and electrophoresised to separate proteins in blood samples by SDS-PAGE. Proteins in the SDS-PAGE are transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane is blocked with 5% skim milk in Tris-buffered saline-Tween buffer (25 mmol/L Tris, 190 mmol/L NaCl, and 0.5% [v/v] Tween 20, pH 7.5) and then incubated with primary rabbit antihuman PLSCR1 polyclonal antibody (1:1000 dilution) at 4° C., overnight. After being washed, the membranes were incubated at 25° C. for 1 h with peroxidase-conjugated mouse antirabbit IgG antibody (1:5000 dilution), and then were developed with a chemiluminescence reagent kit and photographed. Immunoblot images were analyzed by an Imagemaster analyser, and the band intensities are presented in arbitrary units (AU).
With reference to
One of ordinary skill in the art realizes the western blot assay for detecting the PLSCR1 expression might be replaced by an immunoassay such as an enzyme linked immunosorbent assay (ELISA).
One of ordinary skill in the art also obviously understands when the PLSCR1 is highly expressed in the colorectal cancer, a corresponding antibody (autoantibody) induced by the PLSCR1 will be increased. Therefore, the autoantibody of PLSCR1 is able to be a serological marker for detecting the colorectal cancer.
Specificity and Sensitivity of the PLSCR1 for Detecting the Colorectal Cancer
With reference to
aCutoff value ≧ 5 ng/mL.
bCutoff value ≧ 13.9 AU.
The PLSCR1 alone has better detection efficiency while applying in detecting the early stage and advanced stage colorectal of cancer than the CEA does. The overall detection rate of the PLSCR1 is at least 2 folds than the CEA so that the PLSCR1 has great capability for detecting the colorectal cancer. Moreover, combination of the PLSCR1 and the CEA will increase the efficiency in CRC detection.
Relationship Between the PLSCR1 Expression and the Overall Survival Rate
With reference to
Further, an anti-PLSCR1 antibody that is produced from the PLSCR1 of the embodiment is used to be identified the capability of CRC inhibition in a mouse model. The anti-PLSCR1 antibody is directly contacted with a colorectal cancer HT29 cells. With reference to
With reference to
Table 3 shows selected proteins by using the above mentioned methods which might has potential to be colorectal cancer detection markers. The markers are overexpressed in at least 20 tissue pairs from a total of 28 colorectal tissue pairs.
athe location is annontated by Gene Onotology and Ingenuity Pathway Analysis Knowledge Base. PM indicates plasma membrane and OM indicates organelle membrane
bthe expression of proteins in colorectal cancer tissues are obtained in Human Protein Reference Database
cthe data are obtained from Ingenuity Pathway Analysis
Accordingly, the PLSCR1 is able to be applied in manufacturing a detection device for detecting the colorectal cancer. The detection device comprises a sample container, at least an anti-PLSCR1 antibody and a substrate. Blood samples are collected and applied in the sample container, and reacts with the anti-PLSCR1 antibody. The substrate might a chromophoric reagent or a fluorescence reagent that is combined with another anti-PLSCR1 antibody and develop a signal related to the binding between the PLSCR1 and the anti-PLSCR1 antibody, which is compared with a database or a cutoff value. The database collects the values of PLSCR1 expression from healthy controls. The cutoff value is calculated by estimating the values of PLSCR1 expression from healthy controls.
This embodiment uses a SEC61β protein listed in the Table 3 or an autoantibody induced from the SEC61β protein as a marker to detect the colorectal cancer.
In this embodiment, a comparison of SEC61β expression between the colorectal cancer tissue and the matching normal tissue is established by using western blot assay. Differences between the embodiment 1 and 2 shows as following:
The colorectal cancer tissue and the matching normal tissue is respectively mixed with an electrophoresis buffer contains 2% SDS and 5% 2-mercaptoethanol and heated at 100° C. for 5 minutes. Proteins in the tissues are fractioned by a 12% denatured polyacrylamide gel and then are transferred to the PVDF membrane. The PVDF membrane is blocked with 5% skim milk. Proteins in the PVDF membrane react with a rabbit anti-human SEC61β polyclonal antibody at room temperature for 2 hours and then react with a peroxide-conjugated second antibody at room temperature for 1 hour.
The SEC61β of the embodiment has an UniProt accession number P60468 with sequence shows as following (SEQ ID NO: 2):
However, one of ordinary skill in the art will realize that any sequence has more than 90% similarity with above-mentioned sequence is capable to apply in the present invention.
With reference to
With reference to
With reference to
With reference to Table 5, shows the detection efficiency of the SEC6β autoantibody and the current used CEA in detecting CRC patients with different stages. The SEC61β autoantibody has great capability to detect the colorectal cancer in early stage and has detection efficiency more than 75%.
This embodiment indicates the SEC61β autoantibody has high capability to be a serological marker to detect the colorectal cancer; especially in the early stage, the SEC61β autoantibody has the detection efficiency more than 75%, which is helpful to promote the early detection and the prognosis evaluation.
A STOML2 protein is selected and listed in the Table 3 to be identified the capability to detect the colorectal cancer.
This embodiment has a difference with the embodiment 2, that is, this embodiment uses an ELISA method to detect the expression of the STOML2 protein in blood samples of colorectal cancer patients or healthy controls.
The STOML2 of the embodiment has an UniProt accession number Q9UJZ1 with sequence shows as following (SEQ ID NO: 3):
However, one of ordinary skill in the art will realize that any sequence has more than 90% similarity with above-mentioned sequence is capable to apply in the present invention.
With reference to
With reference to
In this embodiment, the sensitivity and the specificity of different antigens is calculated by a backward elimination method with inultivariate logistic regression model. All antigens have excellent specificity and are able to be applied alone or combined each other to improve the efficiency of the colorectal cancer detection.
1AUC: area under ROC curve
2p-value is calculated by t-test
This application is a Divisional of co-pending application Ser. No. 13/252,976, filed on 4 Oct. 2011, for which priority is claimed under 35 U.S.C. §120, the entire contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13252976 | Oct 2011 | US |
| Child | 13892011 | US |
