Lamellarin O, a pyrrole alkaloid from an Australian marine sponge, Ianthella sp., reverses BCRP mediated drug resistance in cancer cells

Abstract

ATP binding cassette (ABC) transporters, such as P-gp, BCRP and MRP1, can increase efflux of clinical chemotherapeutic agents and lead to multi-drug resistance (MDR) in cancer cells. While the discovery and development of clinically useful inhibitors has proved elusive to date, this molecular target nevertheless remains a promising strategy for addressing and potentially overcoming MDR. In a search for new classes of inhibitor, we used fluorescent accumulation and efflux assays supported by cell flow cytometry and MDR reversal assays, against a panel of sensitive and MDR human cancer cell lines, to evaluate the marine sponge co-metabolites 1-12 as inhibitors of P-gp, BCRP or MRP1 initiated MDR. These studies identified and characterized lamellarin O (11) as a selective inhibitor of BCRP mediated drug efflux. A structure-activity relationship analysis inclusive of the natural products 1-12 and the synthetic analogues 13-19, supported by in silico docking studies, revealed key structural requirements for the lamellarin O (11) BCRP inhibitory pharmacophore.

Figure 1

Metabolites isolated from Ianthella sp. (CMB-01245).

Figure 2

Effect of 1–12 on the accumulation of calcein AM. SW620 Ad300 cells in a 96-well micro-titer plate (5 × 10⁴ per well) were cultured at 37 °C in 5% CO₂ for 48 h after which they were treated with either 1–12 (20 μM), or the positive control verapamil (100 μM), and incubated for 15 min. Cells were then treated with calcein AM (0.25 μM) and incubated for a further 30 min. Fluorescence of the calcein AM hydrolysis product calcein was quantified (POLARstar Omega multimode plate reader) and displayed in Figure 2 as the mean value ± SEM of two independent experiments performed in duplicate (* p < 0.05), with the dashed line corresponding to the PBS negative control.

Figure 3

Effect of 9–12 on accumulation of calcein AM in SW620 Ad300 cells using flow cytometry. SW620 Ad300 cells were incubated with calcein AM (0.25 μM) with or without 9–12 (20 μM), or the positive control verapamil (20 μM), after which cells were washed twice with ice-cold PBS and the intracellular calcein fluorescence levels quantified using flow cytometry (Section 3.4). The histogram results (Y-axis represents cell counts with the maximum as 400) for 9–12 and verapamil are illustrated, with data for other metabolites reported in Supplementary Table S2.

Figure 4

Effect of lamellarin O (11) and verapamil on accumulation (A) and efflux (B) of Hoechst 33342 in SW620 Ad300 using flow cytometry. SW620 Ad300 cells were incubated with or without Hoechst 33342 (60 μM), in the absence or presence of 11 (20 μM), or verapamil (20 μM), for 30 min. After incubation cells were washed twice with ice-cold PBS, and intracellular calcein fluorescent intensity measured by flow cytometry (accumulation, Figure A). In an efflux phase assay SW620 Ad300 cells were further incubated in Hoechst 33342-free medium, with or without 11 (20 μM), or verapamil (20 μM) for 1 h. After which cells were washed as indicated above for the accumulation phase, and intracellular calcein fluorescent intensity measured by flow cytometry (Figure B). The accumulation and efflux phase increase in intracellular fluorescence mediated by 11 (thin dashed line) and verapamil (thin solid line), compared with the negative controls of PBS with Hoechst 33342 (heavy solid line), were 2.5-fold and 1.4-fold, and 2.3-fold and 3.8-fold respectively. Heavy dashed line illustrates baseline intracellular fluorescence of SW620 Ad300 cells without Hoechst 33342.

Figure 5

Effect of 11 on the accumulation of [³H]-mitoxantrone. The accumulation of [³H]-mitoxantrone in parental NCI-H460 cells and in BCRP over-expressing NCI-H460/MX20 cells was measured (Section 3.6). Columns are the mean of triplicate determinations. Error bars represent the SD; * p < 0.05 vs. the control group. Experiments were performed in three independent times, and a representative experiment is shown.

Figure 6

Effect of 11 on the efflux of [³H]-mitoxantrone in (A) NCI-H460 cells and (B) NCI-H460/MX20 cells. A time course vs. % of intracellular [³H]-mitoxantrone remaining was plotted; * p < 0.05 vs. control group. Error bars represent the SD. Experiments were carried out in triplicate.

Figure 7

Western blotting analysis of BCRP and effect of 11 on BCRP expression in NCI-H460/MX20 cells. (A) Expression of BCRP in NCI-H460 and NCI- H460/MX20 cells; (B) Effect of 11 (30 μM) on expression level of BCRP in NCI-H460/MX20 cells (0, 24, 48 and 72 h); (C) Quantitative analysis on relative expression levels of ABCG2. Columns are grayscale ratios of BCRP over β-actin. Error bars represent SD. Equal amounts of total cell lysate were used for each Western blotting (Section 3.7). Representative results are shown, with similar results obtained in two other trials.

Figure 8

SAR studies on lamellarin O (11) with the methoxy-acetophenone moiety highlighted.

Figure 9

Effect of 11 and its methylated derivatives 17–19 on the BCRP mediated efflux of mitoxantrone in BCRP over-expressing NCI-H460/MX20 cells. NCI-H460/MX20 cells were incubated with mitoxantrone in the absence or presence of 11, mix of 17+18, or 19 (0.1 to 100 μM), or the positive control FTC (10 μM), after which the cells were washed twice with ice-cold PBS, and incubated without or with test compounds for a further 1 h in mitoxantrone free medium. Mitoxantrone fluorescence was detected with a 638-nm argon laser and a 660/20 nm band-pass filter. Data were collected by cell flow cytometry according (Section 3.4) and normalized to that obtained with 10 μM FTC (signal set as 100%). Data are means ± SEM of at least three independent experiments performed in duplicate.

Figure 10

XP-Glide predicted binding mode of lamellarin O (11) with homology modeled ABCG2. (A) Binding mode of 11 within the drug binding site-1 of human ABCG2. Important residues are depicted as sticks with the atoms colored as carbon–gray, hydrogen–white, nitrogen–blue, oxygen–red, sulfur–yellow whereas 11 is shown as ball and stick model with the same color scheme as above except carbon atoms are represented in orange. Dotted black lines indicate proposed hydrogen bonds; (B) Compound 19 within the drug binding site-1 of human ABCG2.

Figure 11

Effect of lamellarin O (11) on the efflux of MRP1 fluorescent substrate calcein in MRP1 over-expressing 2008/MRP1 cells. Cells were incubated for 30 min with calcein AM in the absence (heavy solid line) or presence (dashed line) of 11 (20 μM), or the positive control MK571 (50 μM) (thin solid light). Subsequently, cells were washed twice with ice-cold PBS and incubated without or with compounds for 1 h in calcein AM-free medium. Intracellular fluorescence of calcein was detected with a 488 nm argon laser and a 530/30 nm band pass filter. Data were analyzed by flow cytometry according (Section 3.4). Representative results from at least two independent experiments were shown.