Cathepsin B (CB) is involved in invasion and
metastasis of a variety of solid organ
tumors, including human
prostate cancer. The tertiary structures of the
proenzyme and mature forms of CB are related closely, as revealed by crystallographic studies. However, the cellular distributions of the CB forms have not been defined in human prostate and its
tumors. Our objective was to investigate the distribution and codistribution of CB and
procathepsin B (proCB) in human prostate
tumors. Human prostate tissue samples that were obtained from 21
prostatectomy and/or
cystectomy patients were collected immediately after surgery and processed for this study. We used a rabbit antihuman liver CB
immunoglobulin G (
IgG) that recognizes both mature CB and proCB and a mouse antipropeptide
monoclonal antibody IgG that recognizes only proCB.
Fluorescein isothiocyanate (
FITC)-conjugated donkey antirabbit
IgG and indocarbocyanine (Cy3;
rhodamine)-conjugated donkey antimouse
IgG were used to differentiate localization of the
enzyme forms. Immunofluorescence of
FITC and Cy3 was examined in prostate sections by using epifluorescence and confocal
laser-scanning microscopy. Because fluorescence is dependent on section thickness, time needed for study and photography, and the antigenic sites of proCB and mature CB localized by
antibodies and by fluorescent markers (Cy3 vs.
FITC), the cellular distributions and the relative intensity of fluorescence on cryostat sections were assessed qualitatively. Immunofluorescence of Cy3 for localizing proCB and of
FITC for localizing mature CB were observed in prostatic epithelial cells and their
tumors and in stromal connective tissue cells. By using confocal microscopy, colocalization of the
enzyme forms in the same cells was indicated by yellow fluorescence. In stromal cells (such as smooth muscles, fibroblast, and macrophages), the distribution of proCB and relative fluorescence intensity was moderate to predominant in human prostate and its
tumors. In neoplastic prostate, the cellular distributions of CB ranged from low to predominant levels. In some neoplastic glands, Cy3 fluorescence for proCB was absent, whereas the mature form of CB localized in
cancer cells and in the subjacent extracellular matrix. Confocal microscopy showed a close association of CB with extracellular matrix surrounding neoplastic acini and invasive cells, indicating that the
enzyme form was probably involved in degradation of the matrix
proteins. The negative control study showed no specific immunofluorescence for proCB or CB in
prostate cancer cases. We have shown a differential distribution of
proenzyme and mature forms of CB in normal prostate,
benign prostatic hyperplasia, and neoplastic prostate. The
enzyme forms were assessed by determining the cellular distributions of CB and proCB. Our study indicates that the differential distribution of proCB and CB might provide clues into aggressiveness of
prostate cancers within Gleason grades. However, we emphasize that our observation should be evaluated in a larger series of prostate samples before a definitive conclusion can be reached. This is the first report to show codistribution of
proenzyme and mature forms of CB by using confocal microscopy.