Identification and expression of troponin T, a new marker on the surface of cultured tumor endothelial cells by aptamer ligand

Abstract

The identification of a specific biomarker involves the development of new clinical diagnostic tools, and an in-depth understanding of the disease at the molecular level. When new blood vessels form in tumor cells, endothelial cell production is induced, a process that plays a key role in disease progression and metastasis to distinct organs for solid tumor types. The present study reports on the identification of a new biomarker on primary cultured mouse tumor endothelial cells (mTECs) using our recently developed high-affinity DNA aptamer AraHH001 (Kd = 43 nmol/L) assisted proteomics approach. We applied a strategy involving aptamer-facilitated biomarker discovery. Biotin-tagged AraHH001 was incubated with lysates of mTECs and the aptamer-proteins were then conjugated with streptavidin magnetic beads. Finally, the bound proteins were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. We identified troponin T via matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, the molecular target of aptamer AraHH001, and its presence was confirmed by measuring mRNA, protein levels, western blot, immunostaining, a gel shift assay of AraHH001 with troponin T. We first report here on the discovery of troponin T on mTECs, a promising and interesting diagnostic tool in the development of antiangiogenic therapy techniques the involves the targeting of the tumor vasculature.

Keywords: Biomarker; DNA aptamer; MALDI-TOF-mass spectroscopy; mTECs; troponin T.

Figure 1

A new DNA aptamer AraHH001 binds to mTECs, and isolated target protein troponin T via aptamer protein pulls down from mTECs. (A) Flow cytometry-binding assay of the selected FITC-tagged AraHH001 DNA aptamer with mTECs. The purple, red, blue, and green curve represents untreated cells, treated cells with a 200 pmol zero cycle ssDNAs pool, 200 pmol and 1000 pmol FITC-tagged AraHH001, respectively. (B) SDS-PAGE analysis of the aptamer AraHH001-based purification of the target protein. Lane 1 represents the protein marker (7–175 kDa), lane 2 represents proteins treated with magnetic beads (MB) with a whole cell lysates and lanes 3 and 4 represents aptamer-bound target proteins treated with MB without and with a linker. Lanes 5, 6, and 7 represents unbound fractions from a whole cell lysates, aptamer without linker and aptamer with linker, respectively.

Figure 2

Confirmation of the aptamer AraHH001 target protein troponin T. (A) Troponin T expressed on mTECs. Flow cytometry assay of a FITC-conjugated cardiac troponin T (cTnT) antibody with mTECs and skin-ECs. The blue and red curves represent nontreated and treated mTECs with a FITC-conjugated cTnT antibody. The green and red curves represent nontreated and treated skin-ECs with the FITC-conjugated cTnT antibody. (B) Relative expression of troponin T mRNA level in mTECs and skin-ECs. RT-PCR analyses of the relative expression of troponin T in mTECs as well as in skin-ECs, compared with standard GAPDH. (C) Electrophoretic mobility shift assay (EMSA) of an AraHH001 aptamer with its target protein troponin T. EMSA result represents the binding of 10 nmol/L AraHH001 with 0, 5.33, 16, 80, 400, and 2000 nmol/L cTnT.

Figure 3

A flow cytometry assay of troponin T expression on trypsin pretreated mTECs and trypsin untreated mTECs. In flow cytometry binding assay, red and green curve represents the binding of the anti-troponin T antibody with mTECs pretreated with or without 0.1× trypsin, respectively.

Figure 4

Western blot analysis of detecting troponin T protein from mTECs cellular extracts contains membrane proteins. In WB analysis, cardiac TnT in first lane recognized by anticardiac troponin T (cTnT) antibody. In the second and third lane, nonrecognized prestained protein marker and recognized troponin T from mTECs cellular lysates by anti-cTnT antibody.

Figure 5

Expression of troponin T on human tumor blood vessel. In immunostaining staining experiment, cardiac troponin T as green fluorescence signal expressed in human tumor blood vessels. Positive control, CD31 with red signal was shown as red. Both green and red signal showing as merge.