OVERCOMING MULTIDRUG RESISTANCE IN HUMAN BREAST CANCER CELLS

Aidan Seeley1, Andrew Irvine1, Tara Schmitz1, Laurent Trembleau2, Wael Houssen2, Andrew McEwan2, Marcel Jaspers2 & Heather M Wallace1

1 Division of Applied Medicine, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD2 Marine Biodiscovery Centre, University of Aberdeen, Meston Building, Aberdeen AB24 3UE

The response to docetaxel wasmeasured in the MDA-MB-231TAX30 cell line by the MTTassay. Cells were seeded at adensity of 2.4 x 104cells/cm2 in96-well plates and grown for48 h in RPMI-1640, 10% foetalbovine serum and 1%penicillin / streptomycin at37oC and 5% CO2. Cells werethen incubated for 48 h in thepresence of docetaxel atincreasing concentrations, andthen with the known P-gpinhibitor, zosuquidar.

A major contributor to the failure of cancer chemotherapy is thedevelopment of multidrug resistance (MDR) which is the primarymechanism by which cancers develop resistance to multiplechemotherapeutic drugs. Heterogenic tumours contain both drug-sensitive and drug-resistant cells, with drug-sensitive cells killed byearly stages of chemotherapy leaving a higher population of drug-resistant cells. Cancers can acquire resistance through increasedexpression of the drug efflux pump, P-glycoprotein (P-gp; Fig. 1).Patellamides have shown promise in the resensitisation of MDRcells1 through inhibition of P-gp but progressed no further followingNCI60 screens2 Biosynthetic manipulation of these patellamides ishoped to yield invaluable therapeutics. This project aimed toestablish the cytotoxic profile of a new patellamide peptide family indrug-resistant breast adenocarcinoma cell line (MDA-MB-231 TAX30)to assess the potential of patellamide-like structures as drugresistance modifiers in cancer cells.

Figure 1. The role of p-glycoprotein in drug resistance. Taken from: http://www.drugdevelopment-technology.com/projects/tesmilifene/tesmilifene2.html

1. WILLIAMS, A.B. and JACOBS, R.S., (1993) A marine natural product, patellamide D, reverses multidrug resistance in a human leukemic cell line. Cancer letters, 71(1-3), pp. 97-102.2. RASHID, M.A., GUSTAFSON, K.R., CARDELLINA, J.H., 2nd and BOYD, M.R., (1995). Patellamide F, A new cytotoxic cyclic peptide from the colonial ascidian Lissoclinum patella. Journal of natural products, 58(4), pp. 594-597.

These results demonstrate that MDA-MB-231 TAX30 are able to besensitised to docetaxel treatment in the presence of zosuquidar and area model cell line for further in vitro studies of novel P-gp inhibitors. Thepatellamide-like compounds exhibit limited cytotoxicity in comparisonto zosuquidar, however the resistance modifying ability is yet to beassessed in vitro. Previous work on patellamide structures1,2 wouldsuggest this limited cytotoxicity may yield invaluable therapeutics intreatment of multidrug resistant cancers through P-gp inhibition.

Figure 4. (a) Effect on the viable cell number after a 48 h incubation with thepatellamide-like compounds; MBC-536, MBC-537 and MBC-541 and (b) shows theeffect on the viable cell number after a 48 h incubation with the patellamide-likecompounds; MBC-538, MBC-539 and MBC-540.

A B

Our aim was to determine if the patellamide-like structures were ableto increase the toxicity of docetaxel in a drug resistant breast cancercell line. The results show that we can use MTT assay as a quickscreen for inhibitors as the dose response curve will be moved to theleft if the test compounds inhibit P-gp. Zosuquidar was used as ourpositive control and did decrease the IC50 value by 25.1%. Weestablished the potential toxicity of the patellamide-like structuresalone and these may be useful in combination with drugs that induceP-gp and drug resistance if shown to be efficacious.

Figure 2. (a) (b) The effect of docetaxel on the viable cell number ofMDA-MB-231 TAX30 cells after a 48 h exposure and (b) 48 h exposure ofMDA-MB-231 TAX30 cells to the P-gp inhibitor, zosuquidar (0-25 µM)

BA

The IC50 values measured by MTT assay for docetaxel andzosuquidar were 112.7 and ~25 µM (Fig. 2A and B). In order to testthe efficacy of zosuquidar in blocking P-gp cells were treated bothwith docetaxel and zosuquidar. Zosuquidar at 12.5 µM moved thedose response curve to the left indicating increased toxicity ofdocetaxel in the presence of zosuquidar, with an IC 50 of 84.4 µM(Fig. 3A). The patellamide–like compounds tested showed varyingtoxicity when tested alone (Fig. 4A and B). However at 12.5 µM(equivalent to the dose of zosuquidar used) none of thepatellamide-like structures exhibited toxicity greater thanzosuquidar. Figure 3. (a) The effect of docetaxel on the viable cell number and the

effect on viable cell number in the presence of docetaxel in combinationwith zosuquidar.

A

The cytotoxic profile of patellamide-like structures were determined byMTT assay after 48 h exposure to the compounds.