Inhibition of Tumorigenicity and Metastasis of Human Melanoma Cells by Anti-Cathepsin L Single Chain Variable Fragment

Abstract

We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anticathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.

Cellular Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Cleaves C3b, an Essential Component of the Complement System

“…5). These data agree well with the observations of others (35,36) who evaluated the effect of the distinct but functionally similar cathepsin L protease on the deposition of C3b on cell surfaces. Our findings show that MT1-MMP inhibits complement activation by clearing cell surface-deposited C3b and C4b from a tumor cell with the resultant release of their respective inactive soluble forms into the extracellular milieu.…”

“…Conversely, inhibition of the C3-cleaving cathepsin L-like cysteine proteinase p39 strongly decreased tumorigenicity and the metastatic potential of human melanoma xenografts in nude mice (17). In agreement, transfection with L-cathepsin transformed non-metastatic melanoma cells into highly tumorigenic and metastatic cells (35,36). In a similar fashion, expression of rat Crry (an inhibitor of complement activation) inhibited the in vivo deposition of complement C3 fragments and sharply enhanced the tumorigenicity of MCF7 cells.…”

Intratumoral gene delivery of anti-cathepsin L single-chain variable fragment by lentiviral vector inhibits tumor progression induced by human melanoma cells

“…18 Then, we demonstrated that in vitro stable transfection with the cytomegalovirus promoter (pCMV)/myc/ER-anti-CTSLScFv expression vector of A375SM human melanoma cells, characterized by a high tumorigenic and strong metastatic phenotype, led to: (1) intracellular expression of anti-human CTSL-ScFv; (2) strong inhibition of procathepsin L secretion by transfected cells, without modifying their intracellular amount or processing pattern of cathepsin L forms; and (3) switch their phenotype to very poorly tumorigenic and non-metastatic. 19 In vivo, cells transfected with this anti-CTSLScFv produced less and smaller tumors with a decrease of vascularization (angiogenesis) and an increase of cell apoptosis. 19 Furthermore, our recent demonstrations that overexpression of cathepsin L in human tumor cells is under the control of separate and complex regulatory mechanisms 20,21 support that procathepsin L secretion is a key event on which we should focus to inhibit tumorigenic and metastatic phenotype of human melanoma.…”

“…19 In vivo, cells transfected with this anti-CTSLScFv produced less and smaller tumors with a decrease of vascularization (angiogenesis) and an increase of cell apoptosis. 19 Furthermore, our recent demonstrations that overexpression of cathepsin L in human tumor cells is under the control of separate and complex regulatory mechanisms 20,21 support that procathepsin L secretion is a key event on which we should focus to inhibit tumorigenic and metastatic phenotype of human melanoma. As detailed above, our previous experiments were performed by processing human melanoma cells before their injections in nude mice, with polyclonal anti-CTSL antibodies, 8 cathepsin L cDNA 10 or anti-CTSL-ScFv.…”

“…As detailed above, our previous experiments were performed by processing human melanoma cells before their injections in nude mice, with polyclonal anti-CTSL antibodies, 8 cathepsin L cDNA 10 or anti-CTSL-ScFv. 18,19 Thus, we herein analyzed whether it is possible to inhibit later the growth of human tumors already induced in nude mice. For this purpose, we cloned the anti-CTSLScFv in a lentiviral vector construct, optimized to allow in vitro high-transduction efficiency of human melanoma cells.…”