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

Article abstract of DOI:10.3390/md12073818

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.

Full text of DOI:10.3390/md12073818

Mar. Drugs 2014, 12, 3818-3837; doi:10.3390/md12073818
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Lamellarin O, a Pyrrole Alkaloid from an Australian Marine
Sponge, Ianthella sp., Reverses BCRP Mediated Drug
Resistance in Cancer Cells
Xiao-Cong Huang 1,†, Xue Xiao 1,†, Yun-Kai Zhang 2, Tanaji T. Talele 2, Angela A. Salim 1,
Zhe-Sheng Chen 2,* and Robert J. Capon 1,*
1
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of
Queensland, Brisbane QLD 4072, Australia; E-Mails: xiaocong.huang@uqconnect.edu.au (X.-C.H.);
xue.xiao@imb.uq.edu.au (X.X.); a.salim@imb.uq.edu.au (A.A.S.)
2
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences,
St. John’s University, Queens, NY 11439, USA; E-Mails: helloyunyun@live.com (Y.-K.Z.);
talelet@stjohns.edu (T.T.T.)
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: chenz@stjohns.edu (Z.-S.C.);
r.capon@uq.edu.au (R.J.C.); Tel.: +1-718-990-1432 (Z.-S.C.); +61-7-3346-2979 (R.J.C.);
Fax: +1-718-990-1877 (Z.-S.C.); +61-7-3346-2090 (R.J.C.).
Received: 29 May 2014; in revised form: 13 June 2014 / Accepted: 16 June 2014 /
Published: 27 June 2014
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.

Mar. Drugs 2014, 12
3819
Keywords: ABC transporter; multidrug resistance; cancer; P-glycoprotein; BCRP; MRP1;
lamellarin O; marine natural products
1. Introduction
Cancer is the leading cause of death and represents a large diverse group of diseases, of which some
can spread to other sites of the body and result in systemic metastasis. The only effective therapy for
metastasis is chemotherapy, which all too frequently fails due to innate or acquired multi-drug
resistance (MDR). Historically, the over-expression of three ATP binding cassette (ABC) transporters,
P-glycoprotein (P-gp) [1,2], breast cancer resistant protein (BCRP) [3] and/or multi-drug resistance
protein 1 (MRP1) [4], have been correlated with many occurrences of MDR. Over-expression of these
proteins in cancer cells can lead to premature efflux of chemotherapeutic agents, compromising
treatments. The past three decades have seen numerous efforts to overcome MDR caused by P-gp,
BCRP and MRP1, with direct inhibition believed to be the most promising solution. Unfortunately,
although three generations of P-gp inhibitors have been developed [5], none have proved to be
clinically useful. Despite these disappointing results, the need to discover clinically effective inhibitors
remains as compelling as ever [6], with marine natural products representing an attractive source of
bioactive chemical diversity.
The search for marine natural products as inhibitors of ABC transporters commenced some decades
ago, and includes a 1992 report by Prinsep et al. [7] on tolyporphin from the blue-green alga
Tolypothrix nodosa Bharadwaja, which increased the cytotoxicity of doxorubicin and vinblastine in
P-gp overexpressing SK-VLB cells [7]. Following this discovery, at least a dozen classes of marine
metabolites have been reported with P-gp, BCRP or MRP1 inhibitory activity from sponges [8,9],
bryozoans [10], gorgonians [11], ascidians [12], sea pens [13], antinomycetes [14] as well as algae [15]
and tunicates [16].
Our prior investigations into metabolites of southern Australian and Antarctic marine
invertebrates, algae and microbes, have led to the discovery of a number of very promising P-gp
inhibitor scaffolds, including diketopiperazines from the marine-sediment derived actinomycete
Nocardiopsis sp. (CMB-M0232) [17], alkaloids from tunicates of the genus Didemnum [18], and
bromoterpenes from the red alga Laurencia filiformis [19]. This report describes our evaluation of the
ABC transporter inhibitory properties of a suite of alkaloids (1–12, Figure 1) isolated from a southern
Australian marine sponge, Ianthella sp. (CMB-01245).

Mar. Drugs 2014, 12
Figure 1. Metabolites isolated from Ianthella sp. (CMB-01245).
3820
2. Results and Discussion
2.1. Cytotoxicity of 1–12 against SW620 and SW620 Ad300
Prior to investigating the interaction between 1–12 and P-gp we assessed cytotoxicity against
SW620 and the MDR (P-gp over-expressing) daughter SW620 Ad300 cell line, to establish the
non-cytotoxic concentration required for such studies. This study demonstrated that ianthellidones 1–8
and lamellarins 9–10 and 12 were non-cytotoxic towards SW620 and SW620 Ad300 (IC50 > 30 μM),
while lamellarin O (11) exhibited comparable and moderate cytotoxicity towards both SW620
(IC50 22.0 μM) and SW620 Ad300 (IC50 22.3 μM) (Supplementary Table S1), with the maximal
concentration for >80% survival of SW620 and SW620 Ad300 cells being 15 μM.
2.2. Lamellarin O (11) as a P-gp Inhibitor in SW620 Ad300 Cancer Cells (Calcein AM Assay)
The Calcein AM accumulation assay (96-well plate format, Section 3.3) [19] was used as the
primary screen to assess P-gp inhibitory properties of 1–12, with a compound designated as an
inhibitor if a 20 μM treatment increased calcein fluorescence ≥30% of that exhibited by a 100 μM
treatment with the positive control verapamil. While the majority of metabolites tested did not exhibit
inhibitory activity against P-gp, 11 displayed a moderate response (85% of the positive control)
(Figure 2), an observation confirmed by cell flow cytometry (Sections 2.3 and 2.4) and MDR reversal
(Section 2.5) assays.

Mar. Drugs 2014, 12
Figure 2. Effect of 1–12 on the accumulation of calcein AM. SW620 Ad300 cells in
3821
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.
200
*
*
160
120
80
40
0
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.
verapamil
lamellarin O (11)
lamellarin O1 (9)
lamellarin O2 (10)
lamellarin Q (12)
PBS
2
3
4
5
0
10
10
10
10
Fluorescent intensity

Mar. Drugs 2014, 12
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2.3. Lamellarin O (11) as a P-gp Inhibitor in SW620 Ad300 Cells (Calcein AM by Cell Flow Cytometry)
Cell flow cytometry is an established methodology that when coupled with the Calcein AM assay
provides a reliable and accurate means to quantify P-gp inhibitors [20]. Calcein AM assay coupled with
cell flow cytometry (Section 3.4) yielded results that were in accord with those detailed above (96-well
plate format, Section 2.2) and confirmed that 11 (20 μM) exhibited a moderate (5.1-fold) inhibitory
effect on the accumulation of calcein AM from SW620 Ad300 cells (Figure 3 and Supplementary
Table S2), with the remaining co-metabolites exhibiting no inhibitory activity (<1.0 fold).
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.
2.4. Lamellarin O (11) as a P-gp Inhibitor in SW620 Ad300 Cells (Hoechst 33342
Accumulation/Efflux)
To further validate the P-gp inhibitory properties of 11 we used cell flow cytometry to quantify the
accumulation and efflux of Hoechst 33342 from P-gp over-expressing SW620 Ad300 cells (Section 3.4).
In the accumulation phase treatment with 11 (20 μM) increased intracellular Hoechst 33342 fluorescence
levels (2.5-fold) comparable to that achieved by the positive control verapamil (2.3-fold) (Figure 4A),
whereas in the efflux phase treatment with 11 resulted in an increase in intracellular Hoechst 33342

Mar. Drugs 2014, 12
3823
fluorescence levels (1.4-fold) below than that achieved by verapamil (3.8-fold increase (Figure 4B).
These results are consistent with the Calcein AM accumulation (96-well plate format, Section 2.2) and
cell flow cytometry (Section 2.3) results detailed above, and confirm that 11 is a moderate inhibitor of
P-gp mediated efflux.
2.5. Lamellarin O (11) Reverses P-gp Mediated Doxorubicin Resistance in SW620 Ad300 Cells
P-gp over-expressing colon cancer SW620 Ad300 cells treated with 5.0, 10 and 15 μM aliquots
of 11 (Table 1) exhibited 1.5-, 3.1- and 4.8-fold increases in sensitivity towards doxorubicin,
a well-known cancer chemotherapeutic and P-gp substrate. By contrast, treatment with 2.5 μM
verapamil resulted in a 8.8-fold increase in sensitivity. These results are consistent with calcein AM
accumulation (Sections 2.2 and 2.3), and Hoechst 33342 accumulation/efflux (Section 2.4) assays,
establishing 11 as a moderate P-gp inhibitor capable of reversing P-gp mediated doxorubicin resistance
in SW620 Ad300 cells.
Table 1. Effect of lamellarin O (11) on P-gp mediated doxorubicin resistance in SW620
Ad300 cells a.
Treatment
IC50 a (nM)
4440 ± 210
2910 ± 350
1430 ± 160
930 ± 120
500 ± 230
DMF b
doxorubicin
+ lamellarin O (11) 5.0 μM
+ lamellarin O (11) 10 μM
+ lamellarin O (11) 15 μM
+ verapamil 2.5 μM
1.5
3.1
4.8
8.8
a
Cell survival was determined by MTT assay (Section 3.5). Data are mean values ± SEM of at least three
b
independent experiments performed in duplicate; DMF: Dose-modifying factor was the ratio of IC50 value
of doxorubicin against SW620 Ad300 without an inhibitor to IC50 value of doxorubicin against SW620
Ad300 with an inhibitor.
2.6. Lamellarin O (11) Reverses BCRP Mediated Efflux in NCI-H460/MX20 Cells
The flow cytometry based mitoxantrone efflux assay (Section 3.4) [19] was used to screen the
BCRP inhibitory properties of 1–12 against the mitoxantrone resistant human lung cancer NCI-H460/
MX20 cells, with a compound designated an inhibitor if a 20 μM treatment increased intracellular
mitoxantrone fluorescence ≥50% of that exhibited by a 10 μM treatment of the positive control
FTC [21,22]. While the majority of metabolites tested did not exhibit inhibitory activity against BCRP,
the increase in intracellular mitoxantrone exhibited by 11 (3.05-fold, 94.5%) was comparable to that of
FTC (3.17-fold, 100%), indicating that 11 was a promising BCRP inhibitor (Table 2).

Mar. Drugs 2014, 12
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Table 2. Effect of 1–12 on BCRP mediated efflux in NCI-H460/MX20 cells.
Treatment
lamellarin O (11)
FAR a
3.05
0.97
1.07
1.12
1.15
1.14
1.21
1.09
1.13
1.23
1.11
1.41
1.00
3.17
% FTC b
94.5
<1.00
3.13
5.60
6.66
6.20
9.79
4.06
6.06
10.6
5.13
18.6
0.00
100
ianthellidone A (1)
ianthellidone B (2)
ianthellidone C (3)
ianthellidone D (4)
ianthellidone E (5)
ianthellidone F (6)
ianthellidone G (7)
ianthellidone H (8)
lamellarin O1 (9)
lamellarin O2 (10)
lamellarin Q (12)
PBS
FTC 10 μM
a
FAR (fluorescence arbitrary ratio) = mitoxantrone fluorescence intensity (geometric mean) in the presence
of metabolites from Ianthella sp. (CMB-01245) at 20 μM/mitoxantrone fluorescence intensity (geometric
mean) in the presence of PBS, expressed as a ratio. Positive control is FTC at 10 μM which FAR = 3.17;
b
activities for inhibition on mitoxantrone efflux were normalized to 10 μM FTC response according to the
following equation: % FTC = (RFU test sample − RFU negative)/(RFU FTC − RFU negative) × 100%; RFU test sample is
relative intracellular mitoxantrone fluorescent unit (geometric mean) in the presence of compound (20 μM),
RFU
is relative intracellular mitoxantrone fluorescent unit (geometric mean) in the presence of 10 μM
FTC
FTC (positive control) and RFU negative is relative intracellular mitoxantrone fluorescent unit (geometric mean)
in the absence of sample (PBS only). A sample was determined to inhibit BCRP when % maximum
was >50% [21,22].
2.7. Lamellarin O (11) Reverses Efflux of [3H]-Mitoxantrone in NCI-H460/MX20 Cells
The BCRP inhibitory activity exhibited by 11 was further verified by evaluating its effect on the
intracellular accumulation and efflux of [3H]-mitoxantrone. Briefly, in accumulation phase, parental
cells (NCI-H460) and BCRP over-expressing daughter cells (NCI-H460/MX20) were cultured with
[3H]-mitoxantrone (0.10 μM) in the presence or absence of FTC (5.0 μM), or 11 (10 or 30 μM).
After 1 h incubation, the intracellular [3H]-mitoxantrone was measured using a liquid scintillation
analyzer, quantifying the ability of 11 to increase the uptake of the BCRP substrate [3H]-mitoxantrone.
In the efflux phase, cells from the accumulation phase were washed to remove extracellular
[3H]-mitoxantrone and incubated with additional FTC (5.0 μM), or 11 (10 or 30 μM) in media without
[3H]-mitoxantrone. By measuring intracellular and media levels of [3H]-mitoxantrone it was possible
to quantify the effect of FTC and 11 on BCRP mediated efflux of a [3H]-mitoxantrone.
In the accumulation phase neither 11 (10 or 30 μM) nor FTC (5.0 μM) significantly changed
intracellular [3H]-mitoxantrone levels in NCI-H460 cells, however, by contrast 11 increased
[3H]-mitoxantrone accumulation in NCI-H460/MX20 cells (1.3- and 2.3-fold by 10 and 30 μM,
respectively) comparable to the increase observed for FTC (2.2-fold) (Figure 5).

Mar. Drugs 2014, 12
Figure 5. Effect of 11 on the accumulation of [3H]-mitoxantrone. The accumulation of
3825
[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.
control
lamellarin O (11) 10 µM
lamellarin O (11) 30 µM
FTC 5 µM
1.0
0.8
*
*
0.6
*
0.4
0.2
0.0
NCI-H460
NCI-H460/MX20
In the efflux phase the intracellular level of [3H]-mitoxantrone in BCRP over-expressing NCI-H460/
MX20 cells (Figure 6B) was significantly less than that in parental cells (Figure 6A), however, when
treated with 11 (10 and 30 μM) NCI-H460/MX20 cells displayed a concentration dependent increase
in intracellular [3H]-mitoxantrone levels, comparable with treatment with FTC (5.0 μM). Neither 11
nor FTC had a significant effect on the intracellular levels of [3H]-mitoxantrone in parental cells
(Figure 6). This study indicates that 11 is a promising BCRP inhibitor, with the potential to increase
uptake and decrease efflux of cancer chemotherapeutics in BCRP over-expressing cells.
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.

Mar. Drugs 2014, 12
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2.8. Effect of Lamellarin O (11) on BCRP Expression in NCI-H460/MX20 Cells
In addition to possibly acting as a small molecule inhibitor of BCRP functionality, an alternative
mechanism by which 11 may decrease BCRP mediated cellular efflux of [3H]-mitoxantrone is via
decreasing cellular levels of BCRP. In order to test this alternative mechanism we incubated NCI-H460
MX20 cancer cells in the absence (0 h) and in the presence of 30 μM 11 (24, 48 and 72 h), and
determined BCRP expression using Western blotting analysis. As illustrated (Figure 7A) BCRP was
expressed significantly in NCI-H460/MX20 cells, but slightly in NCI-H460 cells. The BCRP levels in
NCI-H460/MX20 cells were not significantly changed after 24, 48 or 78 h (Figure 7B,C), confirming
that 11 does not alter BCRP expression.
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.
2.9. Structure Activity Relationship (SAR) Studies on Lamellarin O (11)
The results as outlined above established 11 as a significantly more potent BCRP inhibitor than the
biosynthetically related co-metabolites 1–10 and 12, indicative of a structure-activity relationship (SAR)
in which the methoxy-acetophenone moiety (highlighted in Figure 8) may be a critical determinant.
In light of this we reasoned that further SAR studies, including an evaluation of methylated analogues
incorporating the core methoxy-acetophenone moiety, would be highly informative. To support SAR

Mar. Drugs 2014, 12
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studies we sourced the synthetic analogues 13–16 from an in-house compound library, and prepared
the mono and dimethyl analogues 17–19. All these analogues incorporate the methoxy-acetophenone
moiety unique to 11, but absent from the closely related and BCRP inactive 9–10.
Figure 8. SAR studies on lamellarin O (11) with the methoxy-acetophenone moiety highlighted.
Evaluation of 13–16 using the flow cytometry based mitoxantrone efflux assay (Section 3.4)
showed that none of these compounds inhibited BCRP (<10% of 10 μM FTC, Supplementary Table S3).
The BCRP inhibitory properties of 17+18 (mixture) and 19 were also evaluated over a range of
concentrations (0.1 to 100 μM), and compared to 11, using the flow cytometry based mitoxantrone
efflux assay (Table 3, Figure 9). This analysis confirmed the IC50 and maximal responses (%FTC) for
11 (4.7 μM and 113%) were far superior to those for 17+18 (20 μM and 66%) and 19 (100 μM
and 46%).
Table 3. The effect of lamellarin O (11) and methylated derivatives 17–19 on BCRP
mediated efflux of mitoxantrone in BCRP over-expressing NCI-H460/MX20 cells.
IC50 (μM) a
4.7 ± 0.6
20.2 ± 2.1
>100
Maximal Activity (% of FTC)
11
17+18
19
112.9
65.7
45.4
a IC50 values were calculated from dose-response data using Prism analysis.
While limited, these SAR results nevertheless confirm that replacing the phenyl substituents
pendant to the pyrrole moiety in 11 with bromo groups (e.g., 13–16), or mono/di methylation of
phenolic moieties (e.g., 17–19), leads to a significant reduction in BCRP inhibitory activity. From this
we conclude that while the methoxy-acetophenone moiety in 11 appears essential to delivering BCRP
inhibitory activity, as seen by the loss of activity in 9–10, by itself this moiety is not sufficient to
deliver a BCRP inhibitory effect.

Mar. Drugs 2014, 12
Figure 9. Effect of 11 and its methylated derivatives 17–19 on the BCRP mediated efflux
3828
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.
11
19
150
17 + 18
125
100
75
50
25
0
µM
0.1
0.3
1
3
10
30
100
2.10. Docking Analysis of Lamellarin O (11) and 19 with Human ABCG2 Homology Models
To support SAR analyses we compared docking studies on the BCRP inhibitor 11, with those
on the BCRP inactive analogue 19. In these studies, docking of 11 into the large drug-binding
cavity of human ABCG2 revealed hydrogen bonding between the two phenolic residues, and Arg482
(-O···HN-Arg482, 2.2 Å) and Met515 (-OH···OC-Met515, 1.8 Å), respectively (Figure 10A).
Similarly, the ester and methoxy-acetophenone moieties were seen to engage in hydrogen bonding
with Tyr570 (-COO···HO-Tyr570, 2.2 Å) and Tyr464 (-O···HO-Tyr464, 1.9 Å), respectively,
while 11 was further stabilized in the hydrophobic pocket by several hydrophobic and aromatic
interactions. Importantly, hydrogen bonding to the phenol residues provides an insight into the likely
mechanism behind the loss of BCRP inhibitory activity observed in the dimethylated analogue 19 as
shown in Figure 10B.
2.11. The Effect of Lamellarin O (11) on the MRP1 Mediated Efflux of Calcein in 2008/MRP1 Cells
Inspired by its P-gp and BCRP inhibitory properties, we used the Calcein AM assay supported by
cell flow cytometry (Sections 3.3 and 3.4) to examine the effect of 11 on the MRP1 mediated efflux of
calcein AM. Treatment of 2008/MRP1 cells with 20 μM of 11 revealed only a modest increase in
intracellular calcein fluorescence (1.4-fold), below than that exhibited by treatment with 50 μM of the
positive control MK571 (3.2-fold), confirming that 11 is a weak inhibitor of MRP1 (Figure 11).

Mar. Drugs 2014, 12
Figure 10. XP-Glide predicted binding mode of lamellarin O (11) with homology modeled
3829
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.

Mar. Drugs 2014, 12
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3. Experimental Section
3.1. Chemical Materials
Doxorubicin was a gift from BioAustralis Fine Chemicals (Smithfield, NSW, Australia). MK571
was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Fumitremorgin C (FTC),
originally prepared by Thomas McCloud (Developmental Therapeutics Program, Natural Products
Extraction Laboratory, NIH, Bethesda, MD, USA), was provided by Susan E. Bates and
Robert W. Robey. [3H]-mitoxantrone (2.5 Ci/mmol) was supplied from Moravek Biochemicals Inc.
(Brea, CA, USA). Monoclonal antibody BXP-21 (against ABCG2) was purchased from Signet
Laboratories Inc. (Dedham, MA, USA). Calcein AM and Hoechst 33342 were purchased from
Enzo Life Inc. (Plymouth Meeting, PA, USA). Dimethylsulfoxide (DMSO) was acquired from
Chem-Supply Pty. Ltd. (Gillman, SA, Australia). G418 was supplied from Invitrogen (Carlsbad, CA,
USA). Pheophorbide A (PhA) was purchased from Frontier Scientific, Inc. (Logan, UT, USA).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), mitoxantrone, verapamil, propidium
iodide, and other chemicals were obtained from Sigma-Aldrich Pty. Ltd. (Castle Hill, NSW, Australia).
The marine natural products 1–12, reported in 2012 by Zhang et al. [23] from a southern Australian
marine sponge, Ianthella sp. (CMB-01245), and the structurally related synthetic compounds 13–16
were available from one of the authors (Robert J. Capon). The analogues 17–19 were prepared from
the natural product 11 as described below. All compounds (1–19) were subjected to HPLC-DAD-MS
and 1H NMR analysis prior to detailed biological experiments.
3.2. Cell Lines and Cell Culture
Three human cancer cell lines (SW620, NCI-H460 and 2008) and their ABC transporter
over-expressing counterparts (SW620 Ad300, NCI-H460/MX20 and 2008/MRP1) were examined in
this study. The drug-resistant P-gp over-expressing daughter cell line SW620 Ad300 was established
by stepwise selection of the parental human colon cancer cell line SW620 in increasing concentrations
of doxorubicin, and was maintained in RPMI1640 (Invitrogen, Carlsbad, CA, USA) containing
doxorubicin (300 ng/mL). The drug-resistant BCRP over-expressing daughter cell line NCI-H460/MX20
was established by stepwise selection of the parental human non-small cell lung cancer cell NCI-H460
in increasing concentrations of mitoxantrone, and was maintained in RPMI1640 containing mitoxantrone
(20 nM). These four cell lines were kindly provided by Susan E. Bates and Robert W. Robey of the
National Cancer Institute, Bethesda, MD, USA [24]. In order to confirm their drug resistance
characteristics, the drug-selected MDR cell lines (SW620 Ad300 and NCI-H460/MX20 were
transferred to drug-free medium until two weeks before assays. The human ovarian carcinoma cell line
2008 and its ABCC1 transduced sub-line 2008/MRP1 [25] were generously provided by Johan Allen
(The University of Sydney) with permission from Piet Borst (the National Cancer Institute,
Amsterdam, The Netherlands). All cell lines were grown in flasks in a humidified incubator at 37 °C
with 5% CO2 with RPMI 1640, which was supplemented with 10% fetal bovine serum, L-glutamine
(2 mM), penicillin (100 unit/mL) and streptomycin (100 μg/mL) (Invitrogen, Carlsbad, CA, USA).

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3.3. Calcein AM Accumulation Assay (96-Well Plate Format)
The Calcein AM accumulation assay (96-well plate format) was modified based on the original
method [26]. The non-fluorescent reagent calcein AM can diffuse into the cellular cytoplasm and yield
the fluorescent dye calcein after hydrolysis by intracellular esterases. As calcein AM is a P-gp
substrate, in the presence of functioning P-gp it is largely effluxed prior to hydrolysis, resulting in low
levels of intracellular fluorescence. A P-gp inhibitor can block the efflux of calein AM, which then
undergoes hydrolysis to calcein which is not a P-gp substrate, leading to higher levels of intracellular
fluorescence. Briefly, after harvesting with trypsin, 5 × 104 SW620 Ad300 cells (in 100 μL) were
seeded into a well of 96-well flat clear-bottom black-wall microtitre plates (AP, Falcon, 353219, BD,
Franklin Lakes, NJ, USA). Following 48 h incubation at 37 °C with 5% CO2, wells were treated with
an aliquot (25 μL) of test compounds (20 μM), or verapamil (100 μM as a positive control), or 1%
DMSO in PBS (v/v, negative control), for 15 min at 37 °C with 5% CO2. An aliquot (25 μL) of calcein
AM (0.25 μM) was then added to each well, except for background control wells, which were treated
with a 25 μL PBS. Plates were incubated at 37 °C with 5% CO2 for a further 30 min, after which
intracellular calcein fluorescent intensity (arbitrary unit) was measured by a POLARstar Omega plate
reader (BMG LABTECH, Offenburg, Germany) at 490 nm (excitation wavelength) and 510 nm
(emission wavelength). Data (fluorescence unit, FU) were normalized to verapamil treated cells on the
same microtitre plate, and reported as % of 100 μM verapamil activity according to:
% maximum = (FUsample − FUnegative)/(FUverapamil − FUnegative) × 100
(1)
where FUsample is fluorescence in the presence of test compounds (20 μM), FUverapamil is fluorescence in
the presence of verapamil (100 μM) and FUnegative is fluorescence in the absence of sample (negative
control). A sample with P-gp inhibitory activity was determined when % maximum was >30%. All
experiments were performed in duplicate.
3.4. Flow Cytometry Assays
Flow cytometry based assays were used to measure the ability of natural or synthetic compounds to
inhibit P-gp, BCRP or MRP1 mediated accumulation and/or efflux of fluorescent reagents [21].
Briefly, SW620 Ad300 cells (P-gp over-expressing), NCI-H460/MX20 cells (BCRP over-expressing),
or 2008/MRP1 cells (MRP1 over-expressing), were harvested with trypsin and re-suspended in phenol
red-free RPMI 1640 (with 10% FBS) to give a final concentration of 50 × 104 cells/mL. Cells were
then pre-incubated with 20 μM of test compounds, or 20 μM of verapamil (positive control for P-gp),
or 10 μM FTC (positive control for BCRP) or 50 μM MK571 (positive control for MRP1) for 15 min
at 37 °C in 5% CO2. For the comparison of BCRP inhibitory activity of lamellarin O (11) and its
permethylated derivatives (mix of 17+18, or 19), NCI-H460/MX20 cells were treated with compounds
at a series of concentration (0.1, 0.3, 1, 3, 10, 30 and 100 μM). Subsequently, cells were incubated with
reagents (0.25 μM calcein AM or 60 μM Hoechst 33342 for P-gp, 20 μM of mitoxantrone for BCRP,
and 0.25 μM calcein AM for MRP1) for 30 min followed by washing twice with cold medium. For the
calcein AM accumulation assays, cells were directly analyzed by flow cytometry. For the efflux
assays, cells treated with Hoechst 33342 (P-gp), mitoxantrone (BCRP) or calcein AM (MRP1) were

Mar. Drugs 2014, 12
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incubated for 1 h in fluorescence reagent free medium with or without compounds, or corresponding
positive controls (see above), after which cells were washed twice with cold medium.
Intracellular fluorescence intensity was measured on a BD FACSCanto™ II flow cytometer (Becton
Dickinson, San Jose, CA, USA) and data were analyzed through FlowJo (Tree Star, Inc., Ashland, OR,
USA). Calcein was detected with a 480 nm laser and a 530/30 nm band-pass filter, whereas Hoechst
33342 was detected using a 405 nm laser and a 450/50 nm band-pass filter. Mitoxantrone fluorescence
was detected with a 638 nm argon laser and a 660/20 nm band-pass filter. At least 10,000 events were
collected for all of the flow cytometry experiment. By increasing threshold value on forward scatter
versus side scatter, debris was eliminated. Dead cells were excluded based on propidium iodide
staining. Data are means ± SEM of at least three independent experiments performed in duplicate.
3.5. MTT Cytotoxicity Assay
The MTT assay was modified based on that previously described [27], using adherent cell lines
SW620 and NCI-H460, and their P-gp over-expressing daughter cell line SW620 Ad300 and BCRP
over-expressing daughter cell line NCI-H460/MX20, respectively. Briefly, cells were harvested with
trypsin and added into 96-well microtitre assay plates at 2000 cells/well, followed by 18 h incubation
at 37 °C with 5% CO2. Lamellarin O (11) was dissolved in 5% DMSO in PBS (v/v) and aliquots
(20 μL) tested over a series of concentrations. Control wells were treated with 5% aqueous DMSO.
When the assay was used for P-gp MDR reversal activity, SW620 Ad300 cells were co-incubated
with 5, 10 or 15 μM aliquots of lamellarin O (11), or 2.5 μM verapamil, together with doxorubicin at a
series of concentrations. 68 h later, 20 μL MTT (4 mg/mL in PBS) was dispensed into each well (final
concentration 0.40 mg/mL), and plates were incubated for a further 4 h at 37 °C with 5% CO2. Finally
medium was removed and precipitated formazan crystals were dissolved in 100% DMSO. The
absorbance of each well was measured at 580 nm with a PowerWave XS Microplate Reader from
Bio-Tek Instruments Inc. (Vinooski, VT, USA).
IC50 values were calculated using Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA), as the
concentration of analyte required for 50% inhibition of cancer cell growth (compared to negative
controls). All experiments were performed in duplicate.
3.6. Drug Accumulation and Efflux Assay
The effect of lamellarin O (11) on the intracellular accumulation of mitoxantrone in BCRP
over-expressing cells was determined by measuring the intracellular accumulation of [3H]-mitoxantrone
in NCI-H460 and NCI-H460/MX20 cells. Cells were trypsinized and three aliquots (5 × 106 cells)
from each cell line were resuspended in medium and pre-incubated for 1 h at 37 °C with or without 10
or 30 μM lamellarin O (11), or 5 μM FTC. To measure drug accumulation, cells were subsequently
incubated for 2 h at 37 °C with 5% CO2 with 0.1 μM [3H]-mitoxantrone in RPMI 1640 medium, in the
presence or absence of 10, 30 μM lamellarin O (11), or 5 μM FTC. The resulting cells were pelleted
at 4 °C, washed three times with 10 mL ice-cold PBS, placed in lysis buffer, and radioactivity
measured using a Packard TRI-CARB 1900CA liquid scintillation analyzer from Packard Instrument
Company Inc. (Downers Grove, IL, USA) as previously described [28].

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In the efflux study cells were incubated with 0.1 μM [3H]-mitoxantrone as the same in the
accumulation study. After washing three times with cold PBS, the suspended cells were incubated
at 37 °C in fresh medium in the presence or absence of 10 or 30 μM lamellarin O (11), or 5 μM FTC.
After 0, 30, 60, and 120 min aliquots of cells were removed and immediately washed three times
with 10 mL of ice-cold PBS. The resulting cells were pelleted and processed for measurement of
radioactivity (as indicated above).
3.7. Preparation of Total Cell Lysates and Western Blotting
Cells were treated with 30 μM lamellarin O (11) for 0, 24, 48 and 72 h, after which cell lysates were
prepared as previously described [28] and stored at −80 °C until the gel electrophoresis was run.
Protein concentrations were determined by bicinchonic acid (BCA™) based protein assay (Thermo
Scientific, Rockford, IL, USA). Equal amounts of total cell lysates (20 μg protein) were resolved by
SDS-PAGE and electrophoretically transferred onto PVDF membranes. Following 1 h incubation in a
blocking solution in TBST buffer at room temperature, the membranes were immunoblotted for 2 h at
room temperature with primary monoclonal antibodies against actin, at a 1:400 dilution, or ABCG2, at
a 1:100 dilution. The membranes were then further incubated for 1 h at room temperature with
HRP-conjugated secondary antibody (1:2000 dilution). Finally the protein-antibody complex was
detected by enhanced chemiluminescence detection system (Amersham, NJ, USA). In addition, levels
of protein expression were quantified by ImageJ Software (NIH, Bethesda, MD, USA).
3.8. Syntheses of Methylated Lamellarin O Derivatives (17, 18 and 19)
A solution of 11 (7.7 mg, 0.017 mmol) in acetone (2 mL) was treated with a 3-fold excess of MeI
(7.2 mg, 0.051 mmol), and K2CO3 (14.0 mg, 0.10 mmol), and was stirred at ambient temperature
overnight. After filtration the eluent was concentrated in vacuo and the residue purified by
semi-preparative HPLC (Agilent Zorbax Rx-C8, 5 μm, 9.4 × 250 mm column, 15 min gradient elution
at 3.5 mL/min from 90% H2O/MeCN to 100% MeCN with an isocratic 0.01% TFA/H2O modifier) to
afford 4′-methyl-lamellarin O (18); tR 13.52 min, 3.9 mg, 50%), 4″-methyl-lamellarin O (17);
tR 13.7 min, 2.4 mg, 30%) and 4′,4″-dimethyl-lamellarin O (19); tR 15.4 min, 0.6 mg, 8.0%).
A solution of 11 (3 mg, 0.0066 mmol) in acetone (2 mL) was treated with a 10-fold excess of MeI
(9.3 mg, 0.066 mmol), and K2CO3 (9.1 mg, 0.066 mmol), and was stirred at ambient temperature
overnight. Workup and fractionation as detailed above yielded 4′,4″-dimethyl-lamellarin O (19)
(tR 15.4 min, 2.4 mg, 75%).
1
4′-methyl-lamellarin O (18): H NMR (600 MHz, CDCl3): δH = 8.03 (d, J = 8.7 Hz, 2H), 7.15 (d,
J = 8.7 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.92 (s, 1H), 6.82 (d, J = 8.7 Hz,
2H), 6.64 (d, J = 8.7 Hz, 2H), 5.74 (s, 2H), 3.90 (s, 3H), 3.82 (s, 3H), 3.47 (s, 3H); 13C NMR
(150 MHz, CDCl3): δC = 192.1, 164.3, 162.7, 158.5, 154.0, 132.0, 131.3, 130.5, 129.8, 128.1, 128.1,
127.4, 127.3, 124.9, 120.0, 115.2, 114.3, 113.0, 55.7, 55.7, 55.3, 51; UV (MeOH) λmax (log ε)
277 (4.48) nm; HRESI(−)MS m/z 494.1574 [M + Na]+ (calcd for C28H25NO6Na+, 494.1574).
1
4″-methyl-lamellarin O (17): H NMR (600 MHz, CDCl3): δH = 8.03 (d, J = 8.7 Hz, 2H), 7.10 (d,
J = 8.7 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 6.93 (s, 1H), 6.77 (d, J = 8.7 Hz,

Mar. Drugs 2014, 12
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2H), 6.72 (d, J = 8.7 Hz, 2H), 5.74 (s, 2H), 3.90 (s, 3H), 3.75 (s, 3H), 3.46 (s, 3H); 13C NMR (150 MHz,
CDCl3): δC = 192.1, 164.2, 162.5, 158.0, 154.5, 132.2, 131.3, 130.5, 129.6, 128.2, 128.1, 127.3, 127.1,
124.9, 120.0, 114.6, 114.3, 113.7, 55.7, 55.7, 55.3, 51; UV (MeOH) λmax (log ε) 277 (4.46) nm;
HRESI(−)MS m/z 494.1574 [M + Na]+ (calcd for C28H25NO6Na+, 494.1574).
4′,4″-dimethyl-lamellarin O (19): 1H NMR (600 MHz, CDCl3): δH = 8.03 (d, J = 8.7 Hz, 2H), 7.16
(d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 6.93 (s, 1H), 6.82 (d, J = 8.7 Hz,
13
2H), 6.72 (d, J = 8.7 Hz, 2H), 5.74 (s, 2H), 3.90 (s, 3H), 3.82 (s, 3H), 3.75 (s, 3H), 3.47 (s, 3H); C
NMR (150 MHz, CDCl3): δC = 192.0, 164.2, 162.5, 158.4, 158.0, 132.0, 131.3, 130.5, 129.6, 128.1,
128.1, 127.4, 127.1, 124.9, 120.0, 114.3, 113.7, 113.0, 55.7, 55.7, 55.3, 55.3, 51.0; UV (MeOH) λmax
(log ε) 277 (4.68) nm; HRESI(−)MS m/z 508.1732 [M + Na]+ (calcd for C29H27NO6Na+, 508.1731).
3.9. Molecular Modeling
Lamellarin O (11) and 4′,4″-dimethyl-lamellarin O (19) were prepared as ligands for docking
simulations into human ABCG2 following the same protocol as previously described [29]. All grids of
ABCG2 were prepared and generated as per our pervious protocol [30]. Grid-1 generated using
Arg482 had the highest docking score; therefore, docking discussion was based on binding model of
11 and 19 at this site. Glide v6.0 (Schrödinger, LLC, New York, NY, USA, 2013) docking tool was
followed with the default functions. Top scoring conformation was used for graphical analysis.
All computations were carried out on a Dell Precision 490n dual processor with Linux OS
(Ubuntu 12.04 LTS, Canonical Group Limited, London, UK).
3.10. Statistical Analysis
The statistical significance was defined as p < 0.05 by two-tailed Student’s t-test. All experiments
were performed at least three times in duplicate.
4. Conclusions
In this study we assessed the P-gp, BCRP or MRP1 inhibitory properties of a family of marine
alkaloids, ianthellidone A–F (1–9) and lamellarins O1 (9), O2 (10), O (11) and R (12), isolated from
a southern Australian marine sponge Ianthella sp. (CMB-01245), leading to the discovery of 11 as
a potent and selective BCRP inhibitor. The P-gp, MRP1 and BCRP inhibitory properties of 11 were
detected, measured and quantified by calcein AM accumulation and cell flow cytometry based
(accumulation or efflux) assays against human colon cancer (SW620 and SW620 Ad300), ovarian
carcinoma (2008 and 2008/MRP1) and non-small cell lung (NCI-H460 and NCI-460/MX20) cells,
respectively, with further validation of P-gp inhibitory properties measured in Hoechst 33342
accumulation and efflux, and doxorubicin drug resistance reversal assays, and of BCRP inhibitory
properties in [3H]-mitoxantrone drug accumulation and efflux assays. Collectively these studies
demonstrated that, whereas 11 exhibited weak to moderate inhibitory activity against P-gp and MRP1,
it was a selective and potent inhibitor of BCRP (IC50 4.7 ± 0.6 μM). Further studies demonstrated that
11 did not alter (reduce) the expression of BCRP in NCI-460/MX20 cells, suggesting that it was acting
(as predicted) as a small molecule inhibitor of BCRP function. An SAR analysis on the BCRP

Mar. Drugs 2014, 12
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inhibitory properties of 11, drawing on data for the co-metabolites 1–10 and 12, and the synthetic
analogues 12–19, and supported by in silico docking studies on 11 and 19, identified structural
elements that were key to the inhibitory pharmacophore, including the methoxy-acetophenone,
carboxylic ester and phenolic residues. Our discovery and characterization of 11 as a selective inhibitor
of BCRP will hopefully prompt and inform future SAR investigations into this pharmacophore, to
explore and advance its potential to deliver a clinically useful BCRP inhibitor therapeutic.
Acknowledgments
We thank Susan E. Bates and Robert W. Robey (NCE, NIH, Bethesda, MD, USA) for providing
FTC, SW620, SW620 Ad300, NCI-H460, NCI-H460/MX20, and John Allen (USyd) and Piet Borst
(NCI, Amsterdam, The Netherlands) for providing 2008 and 2008/MRP. Xiao-Cong Huang
acknowledges the provision of International Postgraduate Scholarship from The University of
Queensland. This work was partially funded by The Institute for Molecular Bioscience and The
University of Queensland. We also thank Mark F. Rosenberg (University of Manchester, Manchester,
UK) and Zsolt Bikádi (Virtua Drug Ltd., Budapest, Hungary) for providing coordinates of ABCG2
homology model.
Author Contributions
Conceived and designed the experiments: X.-C.H., X.X., Y.-K.Z., T.T.T., Z.-S.C. and R.J.C.
Performed the experiments: X.-C.H., X.X., Y.-K.Z. Analyzed the data: X.-C.H., X.X., Y.-K.Z. Wrote
the paper: X.-C.H., X.X., Y.-K.Z., A.A.S., Z.-S.C. and R.J.C.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Dano, K. Active outward transport of daunomycin in resistant ehrlich ascites tumor-cells.
Biochim. Biophys. Acta 1973, 323, 466–483.
2. Juliano, R.L.; Ling, V. A surface glycoprotein modulating drug permeability in Chinese hamster
ovary cell mutants. Biochim. Biophys. Acta 1976, 455, 152–162.
3. Hegedus, C.; Ozvegy-Laczka, C.; Apati, A.; Magocsi, M.; Nemet, K.; Orfi, L.; Keri, G.;
Katona, M.; Takats, Z.; Varadi, A.; et al. Interaction of nilotinib, dasatinib and bosutinib with
ABCB1 and ABCG2: Implications for altered anti-cancer effects and pharmacological properties.
Br. J. Pharmacol. 2009, 158, 1153–1164.
4. Cole, S.P.C.; Bhardwaj, G.; Gerlach, J.H.; Mackie, J.E.; Grant, C.E.; Almquist, K.C.;
Stewart, A.J.; Kurz, E.U.; Duncan, A.M.V.; Deeley, R.G. Overexpression of a transporter gene in
a multidrug-resistant human lung-cancer cell-line. Science 1992, 258, 1650–1654.
5. Szakacs, G.; Paterson, J.K.; Ludwig, J.A.; Booth-Genthe, C.; Gottesman, M.M. Targeting
multidrug resistance in cancer. Nat. Rev. Drug Discov. 2006, 5, 219–234.

Mar. Drugs 2014, 12
3836
6. Wu, C.P.; Ohnuma, S.; Ambudkar, S.V. Discovering natural product modulators to overcome
multidrug resistance in cancer chemotherapy. Curr. Pharm. Biotechnol. 2011, 12, 609–620.
7. Prinsep, M.R.; Caplan, F.R.; Moore, R.E.; Patterson, G.M.L.; Smith, C.D. Tolyporphin, a novel
multidrug resistance reversing agent from the blue-green-alga tolypothrix-nodosa. J. Am.
Chem. Soc. 1992, 114, 385–387.
8. Aoki, S.; Yoshioka, Y.; Miyamoto, Y.; Higuchi, K.; Setiawan, A.; Murakami, N.; Chen, Z.S.;
Sumizawa, T.; Akiyama, S.; Kobayashi, M. Agosterol a, a novel polyhydroxylated sterol acetate
reversing multidrug resistance from a marine sponge of Spongia sp. Tetrahedron Lett. 1998, 39,
6303–6306.
9. Chen, Z.S.; Aoki, S.; Komatsu, M.; Ueda, K.; Sumizawa, T.; Furukawa, T.; Okumura, H.;
Ren, X.Q.; Belinsky, M.G.; Lee, K.; et al. Reversal of drug resistance mediated by multidrug
resistance protein (MRP) 1 by dual effects of agosterol a on mrp1 function. Int. J. Cancer 2001,
93, 107–113.
10. Spitaler, M.; Utz, I.; Hilbe, W.; Hofmann, J.; Grunicke, H.H. PKC-independent modulation of
multidrug resistance in cells with mutant (v185) but not wild-type (g185) p-glycoprotein by
bryosratin 1. Biochem. Pharmacol. 1998, 56, 861–869.
11. Aoki, S.; Okano, M.; Matsui, K.; Itoh, T.; Satari, R.; Akiyama, S.; Kobayashi, M. Brianthein A, a
novel briarane-type diterpene reversing multidrug resistance in human carcinoma cell line, from
the gorgonian briareum excavatum. Tetrahedron 2001, 57, 8951–8957.
12. Henrich, C.J.; Robey, R.W.; Takada, K.; Bokesch, H.R.; Bates, S.E.; Shukla, S.; Ambudkar, S.V.;
McMahon, J.B.; Gustafson, K.R. Botryllamides: Natural product inhibitors of ABCG2.
ACS Chem. Biol. 2009, 4, 637–647.
13. Tanaka, C.; Yamamoto, Y.; Otsuka, M.; Tanaka, J.; Ichiba, T.; Marriott, G.; Rachmat, R.;
Higa, T. Briarane diterpenes from two species of octocorals, Ellisella sp. and Pteroeides sp.
J. Nat. Prod. 2004, 67, 1368–1373.
14. Fu, P.; Liu, P.P.; Li, X.; Wang, Y.; Wang, S.X.; Hong, K.; Zhu, W.M. Cyclic bipyridine
glycosides from the marine-derived actinomycete actinoalloteichus cyanogriseus WH1-2216-6.
Org. Lett. 2011, 13, 5948–5951.
15. Smith, C.D.; Zilfou, J.T.; Stratmann, K.; Patterson, G.M.L.; Moore, R.E. Welwitindolinone
analogs that reverse p-glycoprotein-mediated multiple-drug resistance. Mol. Pharmacol. 1995, 47,
241–247.
16. Williams, A.B.; Jacobs, R.S. A marine natural product, patellamide-D, reverses
multidrug-resistance in a human leukemic-cell line. Cancer Lett. 1993, 71, 97–102.
17. Raju, R.; Piggott, A.M.; Huang, X.C.; Capon, R.J. Nocardioazines: A novel bridged
diketopiperazine scaffold from a marine-derived bacterium inhibits p-glycoprotein. Org. Lett.
2011, 13, 2770–2773.
18. Plisson, F.; Huang, X.C.; Zhang, H.; Khalil, Z.; Capon, R.J. Lamellarins as inhibitors of
p-glycoprotein-mediated multidrug resistance in a human colon cancer cell line. Chem. Asian J.
2012, 7, 1616–1623.
19. Huang, X.C.; Sun, Y.L.; Salim, A.A.; Chen, Z.S.; Capon, R.J. Parguerenes: Marine red alga
bromoditerpenes as inhibitors of P-glycoprotein (ABCB1) in multidrug resistant human cancer
cells. Biochem. Pharmacol. 2013, 85, 1257–1268.

Mar. Drugs 2014, 12
3837
20. Druley, T.E.; Stein, W.D.; Roninson, I.B. Analysis of MDR1 P-glycoprotein conformational
changes in permeabilized cells using differential immunoreactivity. Biochemistry 2001, 40,
4312–4322.
21. Henrich, C.J.; Bokesch, H.R.; Dean, M.; Bates, S.E.; Robey, R.W.; Goncharova, E.I.;
Wilson, J.A.; McMahon, J.B. A high-throughput cell-based assay for inhibitors of ABCG2
activity. J. Biomol. Screen. 2006, 11, 176–183.
22. Robey, R.W.; Honjo, Y.; van de Laar, A.; Miyake, K.; Regis, J.T.; Litman, T.; Bates, S.E.
A functional assay for detection of the mitoxantrone resistance protein, MXR (ABCG2).
Biochim. Biophys. Acta 2001, 1512, 171–182.
23. Zhang, H.; Conte, M.M.; Huang, X.C.; Khalil, Z.; Capon, R.J. A search for bace inhibitors reveals
new biosynthetically related pyrrolidones, furanones and pyrroles from a Southern Australian
marine sponge, Ianthella sp. Org. Biomol. Chem. 2012, 10, 2656–2663.
24. Robey, R.W.; Steadman, K.; Polgar, O.; Morisaki, K.; Blayney, M.; Mistry, P.; Bates, S.E.
Pheophorbide a is a specific probe for ABCG2 function and inhibition. Cancer Res. 2004, 64,
1242–1246.
25. Hooijberg, J.H.; Broxterman, H.J.; Kool, M.; Assaraf, Y.G.; Peters, G.J.; Noordhuis, P.;
Scheper, R.J.; Borst, P.; Pinedo, M.M.; Jansen, G. Antifolate resistance mediated by the multidrug
resistance proteins MRP1 and MRP2. Cancer Res. 1999, 59, 2532–2535.
26. Tiberghien, F.; Loor, F. Ranking of P-glycoprotein substrates and inhibitors by a calcein-AM
fluorometry screening assay. Anti-Cancer Drugs 1996, 7, 568–578.
27. Carmichael, J.; DeGraff, W.G.; Gazdar, A.F.; Minna, J.D.; Mitchell, J.B. Evaluation of a
tetrazolium-based semiautomated colorimetric assay: Assessment of radiosensitivity. Cancer Res.
1987, 47, 943–946.
28. Kuang, Y.H.; Patel, J.P.; Sodani, K.; Wu, C.P.; Liao, L.Q.; Patel, A.; Tiwari, A.K.; Dai, C.L.;
Chen, X.; Fu, L.W.; et al. OSI-930 analogues as novel reversal agents for ABCG2-mediated
multidrug resistance. Biochem. Pharmacol. 2012, 84, 766–774.
29. Sodani, K.; Tiwari, A.K.; Singh, S.; Patel, A.; Xiao, Z.J.; Chen, J.J.; Sun, Y.L.; Talele, T.T.;
Chen, Z.S. GW583340 and GW2974, human EGFR and HER-2 inhibitors, reverse ABCG2- and
ABCB1-mediated drug resistance. Biochem. Pharmacol. 2012, 83, 1613–1622.
30. Tiwari, A.K.; Sodani, K.; Dai, C.L.; Abuznait, A.H.; Singh, S.; Xiao, Z.J.; Patel, A.; Talele, T.T.;
Fu, L.; Kaddoumi, A.; et al. Nilotinib potentiates anticancer drug sensitivity in murine ABCB1-,
ABCG2-, and ABCC10-multidrug resistance xenograft models. Cancer Lett. 2013, 328, 307–317.
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