Sea urchin embryos as a model organism for studying anti-mitotic agents and multidrug efflux transporters modulators

A Bouraoui1,2, DR Day2, RS Jacobs2. 1Unité de recherche URSAM, Faculté de Pharmacie, Université de Monastir, Lab. de Pharmacologie, Dept des Sciences Pharmaceutiques 5000, Tunisia, 2University of California, Santa Barbara, Lab. of Marine Pharmacology, Dept of Ecology, Evolution and Marine Biology 93103, USA

The development of multidrug resistance in cancer cells is a major problem in the chemotherapy. As part of our search for a model organism for studying anti-mitotic agents and efflux transporters modulators, the anti-mitotic activity of three compounds were investigated in sea urchin embryos, in absence and in presence of a specific inhibitor of multidrug resistance associated protein1 (MRP1).

Adult purple sea urchins (Strongylocentrotus purpuratus) kept at the laboratory of marine pharmacology (UCSB), in flow-through 13 ± 2°C seawater and were fed kelp weekly. Gametes were collected from two urchins (one male and one female) using 0.5 M KCl solution to induce spawning (white sperm and orange eggs). These gametes were fertilized as described previously (Jacobs and Wilson, 1986). Fertilization success was checked under the microscope. All experiments were conducted at 15°C for approximately 120 min after fertilization. Two marine natural products (Thyrsiferol, dehydrothyrsiferol) and colchicine were used as anti-mitotic agents and MK 571 as specific inhibitor of MRP1. These compounds were dissolved in DMSO and the concentration of this solvent did not exceed 0.5 % of the expected volume.

The present study has established that Thyrsiferol, dehydrothyrsiferol and Colchicine, inhibited the first cleavage of S. purpuratus embryos in a concentration dependent manner with 50% inhibitory concentration (IC50) occurring at approximately at 85 ng/ml, 280 ng/ml and 70 µg/ml, respectively. These three compounds produced 100% inhibition of the first mitosis without lysis or morphological abnormalities. However, when the efflux transporter MRP1 activity of sea urchin embryos is inhibited with 20 µM of MK 571, the effective concentration of the three anti-mitotic agents was decreased. The IC50 of Thyrsiferol, dehydrothyrsiferol and Colchicine were reduced from 85 ng/ml to 55 ng/ml, from 280 ng/ml to 220 ng/ml and from 70 µg/ ml to 7 µg/ml, respectively. We, also, established that in the presence of 25 ng /ml of Thyrsiferol (a concentration which inhibits cell division by approximatively 10 %) the antimitotic activity of colchichine was increased, the IC50 was effectively lowered from 70 µg/ml to 28 µg/ml.

This preliminary study revealed the functional activity of efflux transporter, MRP1 in sea urchin embryos, and the pharmacological inhibition of MRP-mediated efflux activity with MK 571 sensitizes embryos to the anti-mitotic agents. In addition, this study revealed the synergism between the marine natural product, thyrsiferol and colchicine, they act synergistically to inhibit cell proliferation. This synergy could be explained by the action of the two products on different targets. So, sea urchin embryos constitutes a fantastic model organism for in vitro evaluation and identification of potential anticancer agents and ABC transporters reversal agents.

References De Souza et al, 2010; Epel et al, 2006; Semenova et al, 2006; Hamdoun et al, 2004; Hansen et al, 2003; Fernandez et al, 1998; Matsuzama et al, 1994; Jacobs and Wilson, 1986.