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

Abstract

IntroductionWe previously reported that ductal infiltrating carcinomas (DIC) of the human breast display profound modifications of the stromal architecture, associated with anomalous collagen composition. The major alterations observed in 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. AimsTo 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. MethodsApoptosis was studied by both fluorescence microscopical analyses of cell viability and DNA fragmentation assays in preparations of 8701-BC cells grown onto either type V and type IV collagen. Differences in gene expression following cell adhesion onto the two substrates were analyzed by a "differential display" PCR (DD-PCR) technique and Western blot. ResultsIn 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%. DiscussionThe present data substantiate our previous postulates that in 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 spatiotemporal 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. Keywords apoptosis, breast cancer, calpain, collagen type V, differential display.

Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells

“…In the present study we extend previous analyses reported by Pucci-Minafra et al (2000) and demonstrate that type V collagen is able to impair survival of 8701-BC breast cancer cells by promoting a caspase-dependent apoptotic type of death, providing also some information at gene expression level. In fact, our results report the up-regulation of apoptotic initiators caspase-8 and -9, and of the apotosis-involved caspase-1 and the related caspase-5 and -14, as well as a similar increase of the activity of some of the enzyme products.…”

“…At an overall observation, the morphological appearance of cells on the two different collagen substrates was markedly diversified, as already-described in previous publications (Luparello et al, 1990(Luparello et al, , 1991Pucci-Minafra et al, 2000), with cells cultured onto type V collagen showing a less-spread cell morphology and a ''packed'' organization, and also a minor density of the culture, if compared with the counterpart grown as a flat monolayer onto type IV collagen substrate, despite a 2-fold amount of cells was initially seeded on the former substrate. In addition, as shown in Figure 1, more cells stained positively for apoptosis using the apoptosis-discriminating ApopTag oligo A; the percentage of Apoptag-positive cells was calculated, and the values for the cultures grown onto type IV or type V collagen substrates were 4.1 AE 0.58% and 60.4 AE 4.2% (when using oligo A), and 2.2 AE 0.37% and 25.2 AE 2% (when using oligo B), respectively.…”

“…In addition, as shown in Figure 1, more cells stained positively for apoptosis using the apoptosis-discriminating ApopTag oligo A; the percentage of Apoptag-positive cells was calculated, and the values for the cultures grown onto type IV or type V collagen substrates were 4.1 AE 0.58% and 60.4 AE 4.2% (when using oligo A), and 2.2 AE 0.37% and 25.2 AE 2% (when using oligo B), respectively. In consideration of the already published data (Pucci- Minafra et al, 2000) reporting an effect of type V collagen on cellular uptake rate of ethidium bromide and on the onset of oligonucleosomal DNA fragmentation, taking previous and present data together, the concept that type V collagen, already known as a poor substrate for 8701-BC cell survival and growth, had apoptosispromoting property was strongly supported.…”

“…In fact, collagen type V has been proven to represent an optimal substrate for the attachment and growth of hepatocytes (Takai et al, 2001), Schwann cells (Chernousov et al, 2001), normal smooth muscle cells (Grotendorst et al, 1981;Leushner and Haust, 1985), and of some tumor cell types (Ruggiero et al, 1994); conversely, an anti-adhesive and anti-proliferative role played by this collagen species on neurons (Chernousov et al, 2001), various epithelial and endothelial cell lines (Parekh et al, 1998;Underwood et al, 1998), and breast cancer cells (Luparello et al, 1990(Luparello et al, , 1991(Luparello et al, , 2003aPucci-Minafra et al, 2000) has been described.…”

“…Subsequent ''in vitro'' experiments using the 8701-BC cell line, isolated from a biopsy fragment of primary DIC (Minafra et al, 1989), demonstrated that type V collagen was a poorly-adhesive substrate and that the subpopulation of 8701-BC cells able to attach onto it mainly utilized a non-integrin receptor recognized by an anti-67 kDa-elastin/lamininbinding protein antibody (Luparello et al, 1990(Luparello et al, , 1994Minafra et al, 1992). On the other hand, adhesion of 8701-BC cells onto type V collagen resulted in a prominent slow-down of growth rate and inhibition of motility and invasion ''in vitro'' (Luparello et al, 1990(Luparello et al, , 1991Pucci-Minafra et al, 1995); interestingly, more recent results (Pucci-Minafra et al, 2000) provided evidence that type V collagen was able to trigger oligonucleosomal DNA fragmentation, enhance the cellular uptake of ethidium bromide, a marker of loss of membrane integrity (Luparello et al, 2001), and upregulate m-calpain whose product, a Ca þþ -dependent neutral cysteine protease, has been found to be involved in cytoskeletal reorganization and apoptotic promotion in some model systems (Squier et al, 1994).…”