Characterization of human kallikreins 6 and 10 in ascites fluid from ovarian cancer patients.
OBJECTIVE
Human kallikreins 6 (hK6) and 10 (hK10) are secreted serine proteases. We previously found that hK6 and hK10 are highly overexpressed in epithelial ovarian tumors and demonstrated that serum levels of hK6 and hK10 are valuable biomarkers for ovarian cancer diagnosis and prognosis. Our aim is to purify and characterize these two kallikreins from ascites fluid of ovarian cancer patients.


METHODS
Protein concentrations of hK6 and hK10 in ovarian cancer ascites fluids were measured with ELISA-type immunoassays. hK6 and hK10 were purified from the ascites fluids with immunoaffinity columns, followed by reverse-phase high performance liquid chromatography. Purified hK6 and hK10 were then subjected to N-terminal sequencing. Enzymatic analyses were performed with synthetic fluorogenic peptides.


RESULTS
hK6 and hK10 were present in ovarian cancer ascites fluid at concentrations ranging from 0.2-571 and 0.7-220 microg/l, respectively. The majority of hK6 and hK10 in the ascites fluids were present in the free (uncomplexed) form. Both hK6 and hK10 purified from the ascites fluid were zymogens with a molecular mass of 30 kDa. Purified hK6 exhibited trypsin-like enzymatic activity, whereas no enzymatic activity was observed for purified hK10. The enzymatic activity of hK6 could be suppressed by a neutralizing monoclonal antibody.


CONCLUSIONS
The majority of hK6 secreted by the ovarian tumor cells into the ascites fluid are present in the uncomplexed, zymogen form, possessing weak trypsin-like enzymatic activity. All hK10 present in ovarian cancer ascites fluids are in the uncomplexed, zymogen form and have no detectable enzymatic activity.
Luo
Liu-Ying
LY
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada.
Soosaipillai
Antoninus
A
Grass
Linda
L
Diamandis
Eleftherios P
EP
D000595:Amino Acid Sequence; D001202:Ascitic Fluid / Q000378:metabolism*; D002853:Chromatography, Liquid; D004792:Enzyme Precursors / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015200:Fluoroimmunoassay; D006801:Humans; D007610:Kallikreins / Q000032:analysis / Q000737:chemistry*; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000378:metabolism*
D004792:Enzyme Precursors; C497170:KLK12 protein, human; C103551:KLK6 protein, human; D007610:Kallikreins