Ecteinascidin-743 (ET-743), a natural marine compound, with a unique mechanism of action

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Abstract

The mode of action of Ecteinascidin-743 (ET-743), a marine tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, which has shown very potent antitumour activity in preclinical systems and encouraging results in Phase I clinical trials was investigated at a cellular level. Both SW620 and LoVo human intestinal carcinoma cell lines exposed for 1 h to ET-743 progress through S phase more slowly than control cells and then accumulate in the G2M phase. The sensitivity to ET-743 of G1 synchronised cells was much higher than that of cells synchronised in S phase and even higher than that of cells synchronised in G2M. ET-743 concentrations up to four times higher than the IC50 value caused no detectable DNA breaks or DNA–protein cross-links as assessed by alkaline elution techniques. ET-743 induced a significant increase in p53 levels in cell lines expressing wild-type (wt) (p53). However, the p53 status does not appear to be related to the ET-743 cytotoxic activity as demonstrated by comparing the drug sensitivity in p53 (−/−) or (+/+) mouse embryo fibroblasts and in A2780 ovarian cancer cells or the A2780/CX3 sub-line transfected with a dominant-negative mutant TP53. The cytotoxic potency of ET-743 was comparatively evaluated in CHO cell lines proficient or deficient in nucleotide excision repair (NER), and it was found that ET-743 was approximately 7–8 times less active in ERCC3/XPB and ERCC1-deficient cells than control cells. The findings that G1 phase cells are hypersensitive and that NER-deficient cells are resistant to ET-743 indicate that the mode of action of ET-743 is unique and different from that of other DNA-interacting drugs.

Introduction

Ecteinascidin-743 (ET-743) is a marine tetrahydroisoquinoline alkaloid isolated from Ectenascidia turbinata, a tunicate that grows on mangrove roots throughout the Caribbean sea 1, 2, 3. At nanomolar concentrations, ET-743 is active against a variety of solid tumour cell lines, including melanoma, non-small cell lung carcinoma (NSCLC), ovarian and colon cell lines [4] and against a variety of surgically derived human tumour specimens growing in primary cultures [5]. Furthermore, ET-743 is very active in vivo against several types of human solid tumour xenografts [6]. On the basis of these preclinical data, the drug was selected for clinical development. The results obtained so far in phase I clinical trials in Europe and the USA are quite encouraging in view of the many objective responses that have been observed at tolerable drug doses in a variety of human tumours including soft tissue sarcomas, osteosarcoma, melanoma and breast cancers 7, 8.

The mechanism of action of ET-743 has yet to be fully elucidated. However, it has been shown that ET-743 binds to the minor groove of DNA forming covalent adducts at the N2 position of guanine 9, 10. At relatively high concentrations, ET-743 causes disorganisation of microtubule assembly and is a topoisomerase I poison 11, 12.

Most mechanistic studies performed so far have been done using ET-743 concentrations much higher than the optimal cytotoxic concentrations and, therefore, possibly irrelevant for explaining the antitumour activity of this compound.

The present study was performed to investigate the cell cycle perturbations and the mechanism of cytotoxicity after a short exposure with relatively low drug concentrations.

Several factors such as cell cycle phase, p53 and nucleotide excision repair (NER) status have been examined and the overall picture that is emerging from these studies is that the primary mechanism of action for ET-743 is distinct from the other known DNA-interacting drugs.

Section snippets

Cells and culture conditions

The human colon adenocarcinoma LoVo cell line was grown in a monolayer in Ham's F12 medium supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) l-glutamine (200 mM), 1% (v/v) vitamins (BME vitamin solution; 100×; Gibco Europe, Paisley, UK) at 37°C in a humidified 5% CO2 atmosphere in T25 cm2 tissue flasks (IWAKI, Bibby Sterilin, Staffordshire, UK). The human colon adenocarcinoma SW620 cell line was grown in monolayer in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented

Results

Fig. 2 shows the clonogenic inhibitory effect of 1 h treatment with ET-743 on LoVo and SW620 cells. One hour exposure caused a significant inhibition in the clonogenicity that was higher in SW620 than in LoVo cells.

Fig. 3, Fig. 4 show the cell cycle phase perturbations induced by ET-743. In these figures the upper panel shows the biparametric BrdUrd/DNA cytograms of control and ET-743-treated cells, while the lower panel shows the percentage of cells in the different cell cycle phases evaluated

Discussion

Many anticancer agents currently used therapeutically act by causing DNA damage either directly by reacting with DNA or indirectly by poisoning DNA-processing enzymes as in the case of the inhibitors of DNA-topoisomerase enzymes. The basis of selectivity is largely unknown, but it may not rely only on the type of DNA damage, but on many other cellular mechanisms involved in the recognition of the damage, DNA repair as well as several kinds of responses which follow the drug-induced cellular

Acknowledgements

This work was partially supported by Fondazione Nerina e Mario Mattioli. The generous contribution of the Italian Association for Cancer Research is gratefully acknowledged. We thank Drs Heiko Mueller and Tullia Casini, European Institute of Oncology, Milan, Italy, for kindly providing synchronised cells by centrifugal elutriation.

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