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