Cathepsins D, B, and L in transformed human breast epithelial cells

Abstract

To investigate the regulation of lysosomal enzymes during carcinogenesis, we measured cathepsins (Cats) D, B, and L in MCF-10F, which is a human breast epithelial cell line, and cells evolved after treatment with carcinogen and transfected with c-Ha-ras oncogene. The clones used in this study, MCF-10FTras, D3, D3-1, and D3-1Tras, expressed no estrogen receptors and gradually increased invasive potential, while oncogene-transfected lines were also tumorigenic in SCID mice [16, 19]. Cats D, B, and L were determined in the cells and in cell media using enzyme-linked immunosorbent assay (ELISA), specific enzyme activity measurements, and immunocytochemistry. The major intra- and extracellular lysosomal proteinase in these cells was Cat D (30-180 pm/mg), followed by Cat B (2-10 pm/mg) and Cat L (1-5 pm/mg). An inverse relationship between intracellular Cat D levels and invasive potential of carcinogen-treated and c-Ha-ras oncogene-transfected cell lines was observed. No significant changes in extracellular concentration of Cat D precursor in this series of cell lines was observed. Intracellular levels of Cats B and L were unchanged or slightly lower in carcinogen-treated D3 and D3-1 cells, as well as in MCF-10FTras. On the other hand, in D3-1Tras cell line, evolving from c-Ha-ras transfected D3-1 line, 3.5 fold and 4.4 fold increases in Cat B and Cat L, respectively, but a 2 fold decrease in Cat D, were observed compared to the parental cell line. Immunocytochemical staining showed a granular, polarized perinuclear and cytoplasmic staining of cathepsins in all cell lines. Cysteine proteinases stained more frequently and more intensely in D3-1Tras compared to other lines, confirming the immunochemical assays. We hypothesize that several molecular events, caused by a carcinogen and an oncogene such as c-Ha-ras, are needed to increase Cat B and Cat L, but not Cat D, expression. Therefore, the cysteine and aspartic lysosomal proteinases are differentially expressed in the breast cell lines with more invasive phenotype.

Invasiveness of Transformed Human Breast Epithelial Cell Lines Is Related to Cathepsin B and Inhibited by Cysteine Proteinase Inhibitors

“…This was observed at mRNA, protein and activity levels, suggesting that CatB is unregulated at the transcription level, as shown previously (Yan et al, 2000). Since CatB expression was not induced by carcinogen-treatment alone, it was concluded that induction of CatB was a consequence of biochemical changes induced specifically by the c-Ha-ras oncogene activation (Lah et al, 1996). Furthermore, the subcellular localization of the enzyme changed significantly upon transfection: in the parental cell line MCF10A, the localization of CatB was mostly perinuclear, whereas in the transfected cell line, more intense staining at the cell periphery was observed.…”

“…CatB expression paralleled the invasiveness, but not the tumorigenicity, of the cell lines. An association between the ras oncogene and CatB expression has also been observed in the MCF10F cell line (a non-adherent variant of the MCF10A cell line) after ras-transfection and treatment with a carcinogen (Lah et al, 1996). In the c-Ha-ras transfected cell line MCF10AT, increased CatB protein concentrations and elevated activity levels were observed, although a significantly altered CatB mRNA expression was not (Sloane et al, 1994;Sameni et al, 2000).…”

Antitumor and antimetastatic activity of fucoidan, a sulfated polysaccharide isolated from the Okhotsk sea Fucus evanescens brown alga

“…Lysosomal proteases, along with other cell proteases (serine proteases, plasminogen activators and inhibitors, matrix metalloproteases) play an important role in regulation of tumor growth and are involved in the realization of tumor cell apoptotic or necrotic death [2,[5][6][7][8]. Three injections of fucoidan in a dose of 10 mg/kg alone or in combination with CP significantly reduced cathepsin L activity in tumor tissue, but did not modify Note.…”

Cathepsin L targeting in cancer treatment

“…However, Gottesman and colleagues observed a strong association between CTSL synthesis and malignancy of Kirsten virus transformed NIH 3T3 cells (Gottesman & Sobel, 1980). Subsequent investigations performed in several different transformation models (different viral oncogenes and chemical carcinogens) reported consistent CTSL over-expression irrespective of the mode of transformation (Doherty, et al, 1985; Gottesman & Sobel, 1980; Lah, et al, 1996; Rabin, Doherty, & Gottesman, 1986). In addition, tumor secreted cytokines that have been widely implicated in malignant progression including VEGF, FGF, PDGF, EGF, NGF, INFγ and IL-6 have also been shown to significantly enhance CTSL promoter activity and synthesis (Asanuma, Shirato, Ishidoh, Kominami, & Tomino, 2002; Frick, Doherty, Gottesman, & Scher, 1985; Gallardo, de Andres, & Illa, 2001; Gerber, Wille, Welte, Ansorge, & Buhling, 2001; Keerthivasan, Keerthivasan, Mittal, & Chauhan, 2007; Lah, Hawley, Rock, & Goldberg, 1995; Nilsen-Hamilton, Hamilton, Allen, & Massoglia, 1981; Scher, Dick, Whipple, & Locatell, 1983).…”