Codistribution of procathepsin B and mature cathepsin B forms in human prostate tumors detected by confocal and immunofluorescence microscopy.

Abstract

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.