Transcriptional Inhibition of Type I Collagen Gene Expression in Scleroderma Fibroblasts by the Antineoplastic Drug Ecteinascidin 743

Abstract

We previously showed that COL1A1 expression is upregulated at the transcriptional level in systemic sclerosis (SSc) fibroblasts and that the CCAAT-binding factor (CBF) is involved in this increased expression. Ecteinascidin 743 (ET-743) is a chemotherapeutic agent that binds with sequence specificity to the minor groove of DNA and inhibits CBF-mediated transcriptional activation of numerous genes. Therefore, we examined the effects of ET-743 on the increased COL1A1 expression in SSc fibroblasts. The drug caused a potent and dose-dependent inhibition of type I collagen biosynthesis, which reached 70 -90% at 700 pM without affecting cell viability. The same drug concentration caused 60 -80% reduction in COL1A1 mRNA levels. The stability of the corresponding transcripts was not affected. In vitro nuclear transcription assays demonstrated a 54% downregulation of COL1A1 transcription. Transient transfections with COL1A1 promoter constructs containing the specific CBF binding sequence into SSc cells previously treated with 700 pM ET-743 failed to show an effect on COL1A1 promoter activity. Furthermore, ET-743 did not affect the binding of CBF or Sp1 transcription factors to their cognate COL1A1 elements. However, treatment with 700 pM ET-743 of stably transfected NIH 3T3 cells expressing a human type II procollagen gene under the control of the human COL1A1 promoter caused a greater than 50% reduction in the production of type II procollagen and a similar decrease in the corresponding type II procollagen transcripts. These results indicate that ET-743 is a potent inhibitor of COL1A1 transcription. However, this effect cannot be explained by a direct effect on CBF binding to the COL1A1 promoter. Although the exact mechanisms responsible for the transcriptional inhibition of COL1A1 by ET-743 are not apparent, our observations suggest that the drug may be an effective agent to decrease collagen overproduction in SSc and other fibrotic diseases.

Tumor-Associated Macrophages as Incessant Builders and Destroyers of the Cancer Stroma

“…Trabectedin also affects gene transcription with a peculiar selectivity; some inflammatory cytokines and chemokines are reduced by the drug, such as IL-6, CCL2, CXCL8, VEGF, while TNF is not inhibited [158]. Another affected gene is collagen [159]. We therefore tested whether other ECM-related genes produced by macrophages were reduced by the drug.…”

Selective Effects of the Anticancer Drug Yondelis (ET-743) on Cell-Cycle Promoters

“…Likewise, NF-Y is also unlikely to be an intended target, despite the fact that several CCAAT-containing promoters are inhibited, including HSP40, HSP70, MDR1, collagen (Jin et al, 2000;Minuzzo et al, 2000;Friedman et al, 2002;Louneva et al, 2003), cyclin B1/B2, cyclin A, TK, and DHFR, and some of the CCAAT-less, including metallothionein and SV40, are not. In fact, several findings are inconsistent with this interpretation: 1) the E2F1 promoter, which heavily depends on NF-Y binding (28), is up-regulated (Figs.…”

“…It is important to note that no inhibitory effect of the drug on the basal level of transcription was reported (Jin et al, 2000;Minuzzo et al, 2000;Friedman et al, 2002). Another gene shown to be sensitive to pharmacological doses of the drug is type I collagen (Louneva et al, 2003). Collagen, HSP70, and MDR1 have different architectures, and a common feature, that is, the presence of a CCAAT box on which NF-Y acts.…”

Scleroderma, fibroblasts, signaling, and excessive extracellular matrix

“…It is also reported that 5-fluorouracil blocks COL1A2 expression through a JNK/AP-1 activation [61]. Antineoplastic drug ecteinascidin 743 was reported to inhibit the type I collagen gene expression in SSc fibroblasts [62].…”