Cardiac Troponin I Is Abnormally Expressed In Non-small Cell Lung Cancer Tissues And Human Cancer Cells

BackgroundTroponin (Tn) is the thin filament regulatory complex in the process of contraction of striated muscle. It consists of three different subunits-troponin I (TnI), troponin T (TnT) and troponin C (TnC). Along with the tropomyosin, it participates in the control of calcium-mediated interaction of actin and myosin. Troponin I (TnI) is the inhibitory subunit of the complex, because it inhibits the actomyosin Mg^-ATPase. Mammals have three TnI expressing TnI known as slow twitch (TNNI1), fast twitch (TNNI2) and cardiac (TNNI3). Human TNNI3gene is located at19q13.4, and codes for approximately210amino acids (24kDa) protein. The cardiac TnI isoform (cTnI) protein contains a unique N-terminal extension about30amino acids, compared with the fast and slow skeletal isoforms of TnI. Thus, it has high cardiac specificity. Cardiac troponin I exists in cytosolic and structural pools of myocardial cells with free and binding forms, respectively. Approximately three to eight percent of cTnI is located in cytoplasm as free protein. Most of cTnI exists in the form of a complex with troponin T and C subunits located in myofilaments. Moreover, it has been found in nuclei of cardiomyocytes by experimental evidences recently.The cTnI is considered to be one of the most sensitive and specific biomarkers in diagnosing myocardial injury. In clinical, cTnI measurement can also be used as a valid diagnostic tool for assessment and monitoring of cardiotoxicity induced by radio-, chemo-and immuno-therapy for patients with cancer. The conventional view holds that the cTnI is expressed exclusively in cardiac muscle under physiological conditions. Recently there has been reported that cTnI could be expressed transiently in developing skeletal muscle. To date no relevant study investigated the cTnI expression in non-muscle cells, including cancer cells, under pathological conditions. We aim to test cTnI is abnormally expressed in human cancer tissues and cancer cells, and thus to provide experimental basis for the study of the expression, pathological function and mechanisms of cTnI.AimThe present study was designed to investigate whether the cTnI is abnormally expressed in non-small cell lung cancer (NSCLC) tissues and human cancer cells.Methods1. The epitope identification of mouse anti-human cTnI monoclonal antibodies (mAbs):Three mouse anti-cTnI mAbs, XY15, XY13and XY10, were prepared in our lab by conventional hybridoma method. The purified human cTnI (hcTnI) and synthetic peptides of cTnI were used as antigens to identify mAb epitope and specificity by indirect ELISA and western blot. The same methods were applied to identify the epitope of mouse anti-cTnI mAb2F6.6, which was a generous gift from Dr. Jack H. Ladenson. In addition, the mouse anti-cTnI mAbs19C7(epitope41-49)16A11(epitope86-90) and84(epitope117-126) were purchased from Hytest.2. The detection of cTnI expression on the NSCLC tissues:43cases of NSCLC tissues and6cases of other lung diseases tissues in paraffin-embedded blocks were provided by Departments of Cardiothoracic Surgery and Respiratory Diseases of Jinling Hospital from2007to2008. The specimens were obtained from patients diagnosed with lung adenocarcinoma (AC, n=21), lung squamous cell carcinoma (SCC, n=17), lung adenosquamous carcinoma (ASC, n=3), lung large cell carcinoma (LC, n=2), lung metastatic sarcoma (n=1), lung tuberculosis (TB, n=3), pulmonary sclerosing hemangioma (PSH, n=1), and pulmonary inflammatory pseudotumor (n=1) through pathological evidence. Meanwhile, seven non-cancer-bearing lung tissues obtained away from the tumor were submitted for histology. Patients with NSCLC were staged according to the tumor-node-metastasis (TNM) system. Patients did not receive chemo-, radio-or immuno-therapy prior to specimen collection. We detected the cTnl expression on the pathological tissues by immunohistochemical method with the anti-human monoclonal antibody2F6.6and/or19C7. The cTnI immunopositivity was scored according to staining intensity (0, negative;1, weak staining;2, moderate staining; and3, strong staining).3. The detection of cTnI expression in cancer cell lines:Cultured human lung adenocarcinoma cell line SPCA-1, human gastric cancer cell line BGC823and liver cancer cell lines Huh-7, MHCC-97L and MHCC-97H were used in the experiment. We detected the expression and location of cTnI in human cancer cells by immunofluorescence (IF) staining with seven different anti-human cTnI mAbs.4. Real-Time PCR analysis of cTnI gene expression in human cancer cells:Total RNA was extracted from SPCA-1, BGC823cells and cardiomyocytes using trizol reagent. The housekeeping gene GAPDH was used as an internal control. The fold change of cTnI mRNA (TNNI3) expression in cancer cells relative to that of the cardiomyocytes was analyzed by the2-ΔΔCt method.5. Western blot analysis of cTnI protein expressions in human cancer cells:The collected SPCA-1, BGC823and cardiomyocytes were lysed with ice-cold RIP A lysis buffer. Total protein concentrations of supernatant were quantified using BCA Protein Assay. Western blot analysis was performed to detect the cTnI protein expression in cancer cells.Results1. We tested the epitopes of the mAbs by ELISA assay and western blot. The mAbs, XY15, XY13, XY10and2F6.6could recognize synthetic cTnI peptides56-80,76-110,181-210and13-36, respectively, as well as purified human cTnI by western blot. The mAb XY15belongs to IgG2b, and the XY13and XY10belong to IgGl.2. The results of cTnI immunohistochemical staining in lung tissues were that of the49cases of NSCLC,34(69.4%) demonstrated positive staining with anti-cTnI mAb2F6.6or19C7. There was no significant difference in the overall ratios of positive staining (intensity1-3) between lung adenocarcinoma and lung squamous cell carcinoma (14/21,67%vs.11/17,65%, Pearson chi-square test, P=0.899). Notably, all6cases of the strong staining were lung squamous cell carcinoma. The positive staining could be accumulated, diffused or scattered. However, non-cancer-bearing tissues (9/9,100%) except tuberculosis were negative for cTnI. The mAbs2F6.6and19C7staining overlapped in the tissues from the same patient. But with higher magnification and without counterstaining, it could be distinguished that the cell membrane and the inner local area were stained by2F6.6, and nucleus was stained by19C7. The immunopositivity of cTnI had no connection with TNM stage (P=0.839), lymph node status (P=0.281), tumor size and invasiveness (P=0.721) of NSCLC patients.3. The positive staining pattern within SPCA-1and BGC823by cTnI IF method was dependent on the epitope of the cTnI antibodies. The intensive staining was occurred at cell membrane and nucleolus by using anti-cTnI N-terminal antibodies. The cytoplasmic staining by using anti-cTnI C-terminal antibodies and anti-cTnI middle regions antibodies. Furthermore, we confirmed the cTnI IF staining in human hepatoma cell lines such as Huh-7, MHCC-97L and MHCC-97H cells. The staining pattern was the same as SPCA-1and BGC823cells. Meanwhile, we observed that cTnC expressed in the nuclei of cancer cell lines by using IF staining. We performed the double IF staining with a rabbit anti-cTnI polyclonal antibody and the mouse anti-cTnC mAb in the cultured neonatal rat cardiomyocytes and endothelial cells, in which the two antibodies co-stained positively in cytoplasm of cardiomyocytes, but negatively in endothelial cells.4. The western blot showed that a-25kDa band was detected in SPCA-1, BGC823and cardiomyocyte extracts using mouse anti-cTnI mAb XY15. Real-time PCR showed that the relative gene expressions of TNNI3in BGC823and SPCA-1cells to cardiomyocytes were1.0x10-4and1.8x10-4, respectively (the TNNI3gene expression in cardiomyocytes was regarded as1). We collected the PCR products and performed2%agarose gel electrophoresis. The result showed that there were single bands about250bp in groups BGC823and SPCA-1, similar to that in the cardiomyocytes. There was no band in negative control.Conclusion1. cTnI was abnormally expressed in some non-small cell lung cancer tissues. The positive staining could be accumulated, diffused or scattered.2. cTnI was expressed in a variety of cancer cell lines and distributed in cell membrane, nucleus and cytoplasm. cTnI-cTnC co-existed in nucleus of cancer cells. The cTnI mRNA expression level in cancer cell was about ten thousand times less than that in cardiomyocytes.