Abstract:
The invention relates to methods and biomarker for evaluating cancer metastasis, pharmaceutical composition for inhibiting cancer metastasis, and method for analyzing secretome. By combining a hollow fiber cartridge (HFC) culture system with quantitative proteomics technology, cancer metastasis-related secrectomes can be found. Furthermore, this is the first time to use PARK7 as a biomarker for judging the process of non-small cell lung cancer.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    1. Field of the Invention 
         [0002]    The present invention relates a method for evaluating lung cancer metastasis, a biomarker for evaluating lung cancer metastasis, a siRNA compound for inhibiting lung cancer metastasis, and a method for analyzing secretome. 
         [0003]    2. Description of Related Art 
         [0004]    Cancer, also known as malignant neoplasm, is a disease involving failed mechanism of cell growth proliferation. Lung cancer is the most common cause of cancer death in the world, and some remarkable problems that get in the way of treating lung cancer are that there is low abundance of effective prognosis tools, and low abundance of prevention-oriented treatment methods for keeping off metastasis. In terms of the most popular type of occurring cancer by number of patients, non-small cell lung cancer (NSCLC) accounts for approximately 85% of the demographics, and among all the histological types of NSCLC, lung adenocarcinoma is the most common type. Many lung cancer patients are diagnosed with distant metastatic, and most of them die as a result of the metastases instead of due to the original tumors. The survival rate of lung cancer patients within a 5 year time frame during treatment period is 15.2% and only 2.8% for lung cancer patients complicated by distant metastases. Metastasis is clearly a fatal condition adversely affecting malignant tumor development. Unfortunately, current technologies in clinical practice cannot keep up to solve this problem as there is still low abundance of effective diagnostic and prognostic tools for metastasis and other progression predictions. To further increase patients&#39; survival rates, studies related to the mechanisms of metastasis are of immediate importance. 
         [0005]    Metastasis, the ultimate event in a cancer progression, can be described as the complex process in which cancer cells travel from a tumor site and migrate through the bloodstream or lymphatic system to other parts of the body. During this intricate process, numerous proteins are required to assist in the progression of the tumor cells. For example, secretory proteins, which are released from cells via various pathways, including the classical ER-golgi pathway, vesicle release, or a specific channel, are known as the secretome. In previous research, cell secretome was widely used via proteomics technologies in cancer research. There are three main aspects of this research, including discovering clinical biomarker, understanding mechanisms of cancer progression, and planning cancer treatment strategy. Previously, there are many studies reporting that some biomarkers are associated with cancer metastasis and aggression and angiogenesis, such as the proteinase MMP-9 and VEGF respectively. 
         [0006]    However, modern technology and the discovered biomarkers still have their shortcomings for cancer metastasis and cancer development process prediction. Therefore, there is still room for people in the art to develop and improve prediction accuracy as well as technical aspects. 
       SUMMARY OF THE INVENTION 
       [0007]    An object of the present invention is to provide a method for evaluating lung cancer metastasis, comprising: (A) providing a specimen sample of a subject, wherein the specimen sample comprises a normal tissue and a tumor tissue to be tested; (B) testing expressions of a biomarker in the normal tissue and in the tumor tissue that is pending to be tested respectively, wherein the biomarker is a genotype or a protein of Parkinson disease type 7 (PARK7); (C) comparing the expressions of the biomarker in the normal tissue and the tumor tissue that is pending to be tested, wherein when the expression of the biomarker in the tumor tissue that is pending to be tested is less than that in the normal tissue, it indicates that the subject is subjected to a cancer metastasis risk. 
         [0008]    In the step (A), the tumor tissue may be, for example, cell tissue, blood, body fluid or protein, but the tumor tissue for the present invention is not limited thereto. 
         [0009]    Particularly, in the step (B), mRNA expression, protein expression, protein derivative expression, or peptide chain expression of proteins of PARK7 in the normal tissue and the tumor tissue to be tested were tested respectively. 
         [0010]    Preferably, in the step (B), protein expression of PARK7 in the normal tissue and the tumor tissue to be tested were tested respectively. 
         [0011]    In the method for evaluating lung cancer metastasis of the present invention, wherein, protein expression of PARK7 in the normal tissue and the tumor tissue to be tested are tested respectively by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunoprecipitation (IP), or mass spectrometry (MS). 
         [0012]    In the method for evaluating lung cancer metastasis according to the present invention, the lung cancer particularly refers to non-small cell lung cancer (NSCLC). 
         [0013]    The present invention also provides a biomarker for evaluating lung cancer metastasis, which may be a nucleotide sequence of PARK7, a complementary strand of the nucleotide sequence of PARK7, a derivative of an oligonucleotide sequence of PARK7, a protein sequence of PARK7, a derivative of the protein sequence of PARK7, a fragment of the protein sequence of PARK7, a variant of the protein sequence of PARK7, or an antibody corresponding to the protein sequence of PARK7. 
         [0014]    Particularly, the biomarker for evaluating lung cancer metastasis according to the present invention is a protein sequence of PARK7, a derivative of the protein sequence of PARK7, a fragment of the protein sequence of PARK7, a variant of the protein sequence of PARK7, and an antibody corresponding to the protein sequence of PARK7. 
         [0015]    The biomarker PARK7 for evaluating lung cancer metastasis according to the present invention may serve as a biomarker for cancer risk assessment, cancer diagnosis, and cancer progression prediction, wherein the nucleotide sequence of PARK7 is a sequence represented by SEQ ID NO: 1. 
         [0016]    In the case of the biomarker for evaluating lung cancer metastasis according to the present invention, the protein sequence of PARK7 is a sequence represented by SEQ ID NO: 2. 
         [0017]    Another object of the present invention is to provide an siRNA compound for inhibiting lung cancer metastasis, comprising a target sequence selected from a gene of PARK7. 
         [0018]    Particularly, the target sequence comprises 20-25 oligonucleotides. 
         [0019]    Particularly, the nucleotide sequence of PARK7 is a sequence represented by SEQ ID NO: 1. 
         [0020]    Another object of the present invention is to provide a method for analyzing secretome, comprising the following steps: (A) providing a hollow fiber cartridge (HFC) culture system comprising one or a plurality of hollow fiber cartridges (HFC), a circulation pump, a culture medium supply unit, and a discharge unit, wherein two opposite ends of the circulation pump are connected to the hollow fiber cartridges and the culture medium supply unit respectively; two opposite ends of the hollow fiber cartridges are connected to the circulation pump and the discharge unit respectively; culture medium in the culture medium supply unit is supplied to the hollow fiber cartridges by the circulation pump, and the culture medium flowing through the hollow fiber cartridges is discharged by the discharge unit; (B) culturing a cell pending to be analyzed in the hollow fiber cartridge (HFC) culture system, wherein the cell to be analyzed is cultured on the hollow fiber cartridges; (C) collecting secretomes secreted by the cell to be analyzed; and (D) purifying the secretomes and analyzing protein types thereof before comparing the obtained protein types against a proteome database. 
         [0021]    The HFC culture system can provide a three-dimensional (3D) space and a circulating environment for cell growth. The hollow fiber cartridge (HFC) culture system is a non-stationary system, it operates to employ the high surface area of the HFC to emulate a three-dimensional intra-body environment to grow cells, lending itself to improve cell growth. This system allows cell metabolite to be discharged, which effects decrease of cell mortality, and obtainment of sufficient amount of the secretomes by way of small-volume sampling, which helps analysis of the secretomes. 
         [0022]    According to the method for analyzing secretome of the present invention, particularly, in the step (D), the secretomes is analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). 
         [0023]    In the method for analyzing secretome of the present invention, in the step (D), purifying the secretomes and discretionally marking the secretomes, to analyze the protein types thereof. 
         [0024]    In the event that the secretomes is not marked, protein in the secretomes is analyzed for the protein&#39;s m/z, charge, and retention time. 
         [0025]    In addition, in the method for analyzing secretome of the present invention, the culture medium for the hollow fiber cartridge (HFC) culture system is a serum-free medium (SFM) or a serum medium. In the interest of avoiding serum contamination or serum replacement from factoring into cell growth, serum-free medium is preferred or serum replacement from factoring into cell growth, serum-free medium is preferred. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0026]      FIG. 1A  shows the micrographs (200×) of the morphologies of the CL1-0 and CL1-5 cells before and after serum-free medium (SFM) adaptation, which were observed by a microscope (200×). 
           [0027]      FIG. 1B  shows the proliferation of the CL1-0 cells before and after serum-free medium (SFM) adaptation, which was investigated via MTT assay. 
           [0028]      FIG. 1C  shows the proliferation of the CL1-5 cells before and after serum-free medium (SFM) adaptation, which was investigated via MTT assay. 
           [0029]      FIG. 2  shows the experimental workflow of secretomes analyses for lung adenocarcinoma metastasis according to an example of the present invention. 
           [0030]      FIG. 3  shows different expression levels of the housekeeping protein G3PDH with and without exosome purification in an identical amount of CM. 
           [0031]      FIG. 4  shows the comparison of candidate protein expressions in CL1-0 and CL1-5 via Western blot according to an example of the present invention. 
           [0032]      FIG. 5A  shows the PARK7 protein expressions in cells treated with PARK7 siRNA or the plasmids transfected with PARK7 DNA according to an example of the present invention. 
           [0033]      FIG. 5B  shows the proliferation of the CL1-5 cell line treated with PARK7 siRNA according to an example of the present invention. 
           [0034]      FIG. 5C  shows the proliferation of the A549 cell line treated with PARK7 siRNA according to an example of the present invention. 
           [0035]      FIG. 5D  shows the proliferation of the CL1-0 cell line treated with PARK7 siRNA according to an example of the present invention. 
           [0036]      FIG. 5E  shows the proliferation of the CL1-0 cell line treated with the plasmids transfected with PARK7 DNA according to an example of the present invention. 
           [0037]      FIG. 5F  shows the amount of the migrated CL1-5, A549 and CL1-0 cells respectively in each region according to an example of the present invention. 
           [0038]      FIG. 6A  shows the expression levels of PARK7 between adjacent normal tissue specimens and cancerous tissues with overall TNM stages I, II, and III, wherein the statistical difference therebetween was obtained. 
           [0039]      FIG. 6B  shows the receiver operating characteristic (ROC) curve according to tissue specimens of the present invention, wherein TNM stages I and II are clearly distinguished. 
           [0040]      FIG. 6C  shows the Kaplan Meier estimator of the survival rates according to the plasma samples of the present invention. 
           [0041]      FIG. 6D  shows the Kaplan Meier estimator of the progression-free survival rates according to the plasma samples of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0042]    Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various forms. The following embodiments are described in order to enable those of ordinary skill in the art to embody and practice the present invention, and those skilled in the art will appreciate that various modifications, additions and substitutions are possible without departing from the scope and spirit of the invention as disclosed in the accompanying claims. 
         [0043]    Hereinafter, the lung adenocarcinoma cell line will be used as an example to describe a specific embodiment of the present invention in detail, and reference to the flow chart ( FIG. 2 ) is required so the present invention can be more easily understood and applied. 
         [0044]    Harvesting Conditioned Media from CL1 Cancer Cell Lines Using a Hollow Fiber Cartridge (HFC) Culture System 
         [0045]    In this example, the lung adenocarcinoma cell lines CL1-0 (lowly invasive) and CL1-5 (highly invasive) derived from the same parental cell line with low and high invasive abilities respectively were provided and cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS). The volume of serum medium was slowly reduced and replaced with serum-free medium consisting of RPMI 1640 with 15% CDM-HD serum replacement and 1% antibiotics. Following 2-3 passages, the morphologies of cell lines CL1-0 and CL1-5 ( FIG. 1A ) were observed with a microscope (200×), and the cell growth status was tested by MTT technology, referring to  FIGS. 1B ,  1 C. Our results demonstrated that the morphologies and growing conditions for the CL1-0 and CL1-5 cells did not change significantly after SFM adaptation, compared to cells grown in serum medium. After that, the adapted cells were transferred to hollow fiber cartridge (HFC) culture system. 
         [0046]    Next, CL1-0 and CL1-5 cells (˜5×10 7 ) were suspended in serum-free medium then inoculated into the extra-capillary space (ECS) of the hollow fiber cartridge. 15 mL of secretome samples in conditioned media (CM) from the extra-capillary space (ECS) of the hollow fiber cartridge (HFC) culture system were collected every 24 hrs. The media used for maintaining cell growth was refreshed every day. In addition, glucose and lactate concentrations were also measured daily to monitor cell growth in the hollow fiber cartridge (HFC) culture system. 
         [0047]    CM harvested from the ECS of the hollow fiber cartridge (HFC) culture system was ultracentrifuged at 10000×g for an hour to remove cell debris. Then, the protein concentrations of the secretome samples were determined using the Bradford assay (Bio-Rad, Hercules, Calif., USA). 
         [0048]    In order to confirm that the intracellular proteins were come from the cell secretion rather than via cell lysis, a total of 100 m of CM sample was equally separated into two parts. One of the CM parts was used for exosome purification, to be compared against the housekeeping protein G3PDH between the CM samples (50 μg) applied to exosome purification and another CM part (50 μg). Referring to  FIG. 3 , the G3PDH protein appeared in similar amounts between the CM sample (50 μg) and exosome fraction. These results confirm that intracellular proteins were released into the extracellular space via exosome secretion. 
         [0049]    Sample Purification and Analysis 
         [0050]    The stacking gel-aided purification method was previously established in the conventional knowledge for use in secretome sample clean-up, wherein the CM samples were mixed with the sample dye and 0.5 M DTT before being added into a self-poured stacking gel that contained 50% running gel and a 4% stacking gel. Then, SDS-PAGE was performed at 55 V for 30 min. The protein samples were stacked to the border between the stacking and running gel. 
         [0051]    In this example, the CM samples during the second to tenth day of cell growth were collected, and then the protein in the CM sample of CL1-0 and CL1-5 CM cells were recovered using the stacking gel-aided purification method for uses in subsequent experiments of staining and hydrolyzing colloid into peptide fragments. 
         [0052]    Coomassie Brilliant Blue R-250 was used to stain the gel. All bands were excised and digested in-gel with trypsin. The gel pieces were reduced with 0.5 M DTT (56° C.) and alkylated with saturated iodoacetamide at room temperature, with each step requiring 1 hr. 
         [0053]    20 μL of 0.1 μg/μL of modified trypsin was added to the gel pieces, and they were incubated overnight at 37° C. The digested peptide samples were purified with a C18 tip followed by mass spectrometry analyses. 
         [0054]    Mass Spectrometry Analyses 
         [0055]    In the present example, the MS samples of CL1-0 and CL1-5 CM cells were analyzed using a LTQ-Orbitrap hybrid mass spectrometer with a nanoelectrospray ion source (Nanoelectrospray, branded under ThermoElectron, San Jose Calif.) coupled to a nano flow HPLC (Nanoflow HPLC, branded under Agilent Technologies 1200 series). 
         [0056]    Protein Identification and Quantification 
         [0057]    In the protein identification of the present example, searching with the Mascot database search was performed, and a protein was selected if it had at least two unique peptide sequences that could be quantified, where in the two peptide sequence defined as RANK 1 are the candidate protein of the present invention. A total of 412 and 531 proteins were identified in CL1-0 and CL1-5 cells, respectively. Subsequently, identified proteins were quantified via IDEAL-Q software. 
         [0058]    Next, protein quantification was performed in terms of the candidate protein&#39;s m/z, charge, and retention time in the LC-MS/MS analysis, using IDEAL-Q software. By means of this method, a total of 50 candidate proteins were identified with different levels between CL1-0 and CL1-5 cell lines. Among these proteins, 25 and 25 proteins exhibited high levels in the CL1-0 and CL1-5 cells, respectively. 
         [0059]    Selection of Protein Candidates Via Interactome Analysis 
         [0060]    Next, interactions between the 50 candidate proteins were analyzed using the STRING 9.0 database and 7 proteins were selected based on these results. The proteins ACTN4, FN1, PARK7, PRDX4 and GRP78 within the highly invasive CL 1-5 cells were expressed at high levels, and proteins MYO6 and GSR within the lowly invasive CL1-0 cells were expressed at high levels. 
         [0061]    In this example, as shown in  FIG. 4 , the 7 protein levels were further analyzed via western blot (the housekeeping protein tubulin as a control). The findings produced by the western blot analysis and the mass spectrometry data were compared against each other to analyze for consistency. 
         [0062]    In this case, the results of PARK7 from the western blot analysis and from the mass spectrometry PARK7 were consistent with each other, this confirms that PARK7 has high levels of expression in CL1-5 cells. 
         [0063]    PARK7&#39;s Significant Impact on Cell Proliferation, Migration/Invasiveness in Lung Adenocarcinoma Cells 
         [0064]    To further verify the PARK7&#39;s potential related to metastasis-associated functions and service as a biological marker, in the present example, the A549 cells were additionally employed for test. As shown in  FIG. 5 , first, CL1-5, A549 and CL1-0 cells were treated with PARK1 siRNA respectively. The siRNA of PARK7 was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). The sequence of the siRNA was the sc-37080A sense: CUCCACUUGUUCUUAAAGATT (SEQ ID NO: 3) and antisense: UCUUUAAGAACAAGUGGAGTT (SEQ ID NO: 4), sc-37080B sense: CGACGAUCACUUAGAGAAATT (SEQ ID NO: 5), and antisense: UUUCUCUAAGUGAUCGUCGTT (SEQ ID NO: 6), sc-37080C sense: GGAAGUAUGGAAGUCACAATT (SEQ ID NO: 7), and antisense sc-37080C:UUGUGACUUCCAUACUUCCTT (SEQ ID NO: 8), and compared against scrambled siRNA which served as the control. As shown in  FIG. 5A , first, the level of the PARK7 protein expression in the cell lines having plasmids treated with PARK7 siRNA or transfected with PARK7 DNA was confirmed, to ensure that siRNA successfully inhibits PARK7 performance in the cells, or the level of the PARK7 protein expression was increased due to the plasmids transfected with PARK7 DNA. 
         [0065]    The results of MTT assay indicated that with the reduced synthesis and secretion of PARK7 in CL1-5 ( FIG. 5B ) and A549 ( FIG. 5C ), cell proliferation was significantly reduced. 
         [0066]    According to the results of MTT assay, the proliferation of CL1-0 treated with PARK7 siRNA was also influenced and was reduced slightly ( FIG. 5D ). Further, CL1-0 was transfected respectively into empty plasmids (control group) and plasmids carrying PARK7 DNA. The results of MTT assay shown that CL1-0 cell transfected with the plasmids carrying PARK7 DNA CL1-0 grew better compared to the control ( FIG. 5E ). 
         [0067]    In addition,  FIG. 5F  shown that the migration of CL1-5 and A549 cell lines were greatly reduced due to the treatment with PARK7 siRNA. In the case of lowly invasive CL1-0, it was also found that the cell growth and migration were reduced when the expression of PARK7 protein decreased. Conversely, the growth and migration of lowly invasive CL1-0 cell line were promoted when PARK7 was over-expressed. 
         [0068]    Clinical Expression of PARK7 in Tissue Specimen and Plasma 
         [0069]    Next, the clinical expression of PARK7 was confirmed by tissue specimen and plasma. The tissue microarray including 64 cancerous tissues and 31 adjacent normal tissues was used. By international standards, the number of the cancer diagnosis phases is defined in terms of the TNM system embodying different notations including the tumor size (T), regional Lymph Nodes (N), distant Metastasis (M), wherein the level of the PARK7 expression had a high degree of correlation with TNM stage and lymph node metastasis. In addition, as shown in  FIG. 6A , the levels of the PARK7 expression in cancerous tissues were significantly higher than that in normal tissues. According to the result of receiver operating characteristic (ROC) curve analysis, the stage I and stage II of the patients were distinguished clearly by the different levels of the PARK7 expression ( FIG. 6B ). In clinical expression, PARK7 levels within the plasma samples were significantly higher than that in the normal tissue. According to the Kaplan-Meier of  FIGS. 6C and 6D , when the cut-off value of PARK7&#39;s expression level was within the first quartile, patients with levels within this quartile had 3-year survival and progression-free rates; however, when the cut-off value was below the first quartile, patients with such a level had lower survival rates than patients with levels above this quartile.