Type V collagen induces apoptosis of 8701-BC breast cancer cells and enhances m-calpain expression

Abstract

We previously reported that ductal infiltrating carcinomas (DIC) of the human breast profound modifications of the stromal architecture, associated with anomalous collagen composition. The major alterations observed the interstitial collagen were an abnormal ratio between type I and type III collagens, the appearence of an onco-foetal form of collagen (OF/LB) and a relative increase of type V collagen content. Biological assays performed by culturing a DIC-derived cell line (8701-BC) onto type V collagen substrate demonstrated that the latter was able to restrain cell growth and to inhibit cell motility and invasion in vitro, differently from what found with other collagen species tested. To search for molecular mechanisms underlying the observed inhibitory effect of collagen type V on breast cancer cells. As a reference model, we used culture substrates prepared with type IV collagen, which represents the physiological substrate for cells of epithelial origin. Apoptosis was studied by both fluorescence microscopical analyses of cell viability and DNA fragmentation assays preparations of 8701-BC cells grown onto either type V and type IV collagen. Differences gene expression following cell adhesion onto the two substrates were analyzed by a differential display PCR (DD-PCR) technique and Western blot. In this paper we demonstrate that the inhibitory effect exerted by type V collagen is consistently associated with the switching-on of a death program by a significant proportion of the cell culture, concomitant with the formation of cohesive cell islands displaying a progressive decrease of cell spreading. DD-PCR and Western blot assays demonstrated a consistent association of type V collagen-promoted apoptosis with the up-regulation of the large subunit of m-calpain (L-mC) at both mRNA and protein level. Cell exposure to calpain inibitor I decreased the amount of DNA fragmentation by 30%. The present data substantiate our previous postulates that cases of breast DIC the zonal increase of type V collagen contribute to the assembly of a non-permissive micro-environment for tumor cells, antagonist to other local permissive substrates. It is therefore conceivable that the spatio-temporal derangement of stromal components may actively modulate neoplastic cell behavior and metastatic propensity, thus contributing to the selection and development of more or less malignant tumor phenotypes.