Human desmoid fibroblasts: matrix metalloproteinases, their inhibitors and modulation by Toremifene

Abstract

Background: Desmoid tumour is a benign, non metastasising neoplasm characterised by an elevated deposition of organic macromolecules in the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of ECM macromolecules. The MMPs and their natural inhibitors (TIMPs) have been implicated in tumour growth, invasion and metastasis. In this study we provide evidence that the in vitro cultured cell line from desmoid tumour accumulates more collagen fibres in the ECM than healthy fibroblasts.

Methods: We investigated collagen accumulation by 3H-thymidine incorporation, MMP expression by substrate gel zymography and TIMP expression by Western blot analysis.

Results: Desmoid fibroblasts showed a reduction in MMP activity and an increase of type I and III collagen and TIMPs compared to normal fibroblasts.

Conclusion: The increase in collagen in desmoid fibroblasts was due to inhibited collagen degradation (reduction of MMP activity) rather than to increased collagen synthesis. Adding toremifene, an anti-estrogen triphenylethylene derivate, to desmoid fibroblasts reduced collagen accumulation by decreasing mRNA expression and increasing collagen degradation.

Figure 1

Expression of procollagen α₁ (I) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA in normal and desmoid fibroblasts. Panel A: NF: normal fibroblasts; DF: desmoid fibroblasts; DFT: desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. In panel B the absolute counts, obtained by densitometric analysis, were converted to percentages of control value, assuming the untreated level of normal fibroblasts as 100%.

Figure 2

Western blot analysis of type I and III collagen secreted into the medium by normal and desmoid fibroblasts. Panel A. The samples are as follow: NF normal fibroblasts; DF desmoid fibroblasts; DFT desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel B. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%.

Figure 3

Collagenase activity in media derived from normal, desmoid fibroblasts and desmoid fibroblasts plus toremifene. Panel A. NF: normal fibroblasts; DF: desmoid fibroblasts; DFT: desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. In panel B the quantity of 3/4 and 1/4 fragments of digested collagen was determined by densitometric analysis. The absolute counts were converted to percentages assuming the level of normal fibroblasts as 100%.

Figure 4

Zymogram of media from normal fibroblasts, desmoid fibroblasts, desmoid fibroblasts plus toremifene. Collagen zymogram. One set of samples was treated with APMA to activate the proenzymatic forms. Panel A. NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene. In the same zymogram an aliquot of samples was activated with APMA: NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene, C: trypsin. Similar results were obtained in four separate experiments. Panel B. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%. Gelatin zymogram. One set of samples was treated with APMA to activate the proenzymatic forms. Panel C. NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene. In the same zymogram an aliquot of samples was activated with APMA: NF: normal fibroblasts, DF: desmoid fibroblasts, DFT: desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel D. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%.

Figure 5

Western blot analysis of MMP-1, MMP-2, MMP-9 secreted into the medium. Panel A: MMP-1; panel B: MMP-2; panel C: MMP-9. The samples are as follows: NF, normal fibroblasts; DF, desmoid fibroblasts; DFT, desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel D. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%. ND = not determined.

Figure 6

Western blot analysis of TIMP-1 and TIMP-2 secreted into the medium. Panel A; TIMP-1. Panel B; TIMP-2. The samples are as follows: NF, normal fibroblasts; DF, desmoid fibroblasts; DFT, desmoid fibroblasts plus toremifene. Similar results were obtained in four separate experiments. Panel C. The absolute counts, obtained by densitometric analysis, were converted to percentages assuming the level of normal fibroblasts as 100%.