Discovery of candidate tumor markers for prostate cancer via proteomic analysis of cell culture-conditioned medium.
OBJECTIVE
Prostate-specific antigen measurement, widely used for early detection of prostate cancer (CaP), suffers from low specificity. Additional tumor markers are needed for the early detection of clinically relevant CaP. Our objective was to perform a qualitative proteomic analysis of conditioned medium (CM) from the CaP cell line PC3(AR)(6).


METHODS
We used a roller bottle culture system to culture the PC3(AR)(6) cell line in chemically defined serum-free medium for 14 days. By using strong anion-exchange chromatography, we fractionated the CM and trypsinized the fractions. The tryptic peptides were further fractionated by reversed-phase C-18 chromatography before being subjected to electrospray ionization tandem mass spectrometry. We used MASCOT software to search the mass spectra generated and organized identified proteins based on their genome ontology classification of cellular location. We used an immunoassay to measure a newly identified secreted protein, Mac-2BP, and kallikreins 5, 6, and 11 in serum samples from CaP patients and healthy men.


RESULTS
We classified 262 proteins according to cellular location; the sample was found to contain a significant proportion of secreted (23%) and membrane (16%) proteins. In a proportion of cancer patients compared with healthy men, we determined by ELISA that serum concentrations of a novel candidate biomarker Mac-2BP were increased.


CONCLUSIONS
These identified proteins, and possibly many others found in the CM, may have utility as novel CaP biomarkers.
Sardana
Girish
G
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
Marshall
John
J
Diamandis
Eleftherios P
EP
D000951:Antigens, Neoplasm / Q000032:analysis; D014408:Biomarkers, Tumor / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D005189:False Positive Reactions; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D007118:Immunoassay; D007610:Kallikreins / Q000032:analysis; D008297:Male; D017430:Prostate-Specific Antigen / Q000032:analysis; D011471:Prostatic Neoplasms / Q000175:diagnosis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D012697:Serine Endopeptidases / Q000032:analysis; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry; D016326:Extracellular Matrix Proteins
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D006023:Glycoproteins; D007610:Kallikreins; D017430:Prostate-Specific Antigen; D020543:Proteome; D012697:Serine Endopeptidases; C081856:LGALS3BP protein, human; D016326:Extracellular Matrix Proteins; C114880:trypsin-like serine protease; C400147:KLK5 protein, human; C103551:KLK6 protein, human