Full Papers
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) sup-
plemented with 10% fetal calf serum (FCS), 50 mgmLÀ1 streptomy-
cin, 50 UmLÀ1 penicillin G, and 2 mm l-glutamine. The doxorubi-
cin-resistant human ovarian carcinoma cell line A2780adr was fur-
ther used for calcein AM assays. This ABCB1-overexpressing cell
line was purchased from the European collection of animal cell cul-
tures (ECACC, 93112520, UK) and cultured in RPMI-1640 medium,
supplemented with 10% FCS, 50 mgmLÀ1 streptomycin, 50 UmLÀ1
penicillin G, and 2 mm l-glutamine. H69AR, the small-cell lung
cancer cell line, expressing ABCC1 (multidrug resistance-associated
protein 1) was obtained from American Type Culture Collection
(ATCC, CRL-11351). These cells were grown in RPMI-1640 medium
with 20% FCS, 50 mgmLÀ1 streptomycin, 50 UmLÀ1 penicillin G,
and 2 mm l-glutamine. Cell lines were incubated in a 5% CO2 hu-
midified atmosphere at 378C and harvested for sub-culturing with
0.05% trypsin and 0.02% EDTA.
tation wavelength of 485 nm and an emission wavelength of
520 nm. For measurement the BMG POLARstar microplate reader
(BMG Labtech, Offenburg, Germany) was used. From the pIC50
values and their standard deviations, IC50 values and standard devi-
ations were calculated according to the equation for log-normal
distributed values.[43,44]
MTT assays for determining cytotoxicity: Intrinsic cytotoxicity of se-
lected compounds was analyzed by MTT colorimetric assay using
MDCK II BCRP and MDCK II wild-type cells.[28,33] Based on mitochon-
drial dehydrogenases of viable cells MTT is reduced to purple for-
mazan, which is quantified spectrophotometrically. The assay was
performed as described previously with minor modifications. Cells
were harvested as described above and seeded into 96-well tissue-
culture treated plates (Starlab GmbH, Hamburg, Germany) at a den-
sity of ~3000 cells per well in a total volume of 180 mL. The plates
were kept under 5% CO2 atmosphere and 378C for 6 h, for attach-
ment of cells. Various concentrations of selected compounds were
prepared in culture medium. Then 20 mL of each dilution was
added to reach the final concentration in a volume of 200 mL. The
highest concentration of DMSO used in the assay was not >0.1%
and of methanol not >4%. Furthermore, two wells per row were
prepared with either 10% (v/v) DMSO (positive control) or pure
culture medium (negative control). For reducing the evaporation
of solvent during the incubation time of 72 h, phosphate-buffered
saline (PBS) was added to the interspaces of the plates. After three
days a solution of MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenylte-
trazolium bromide) in PBS (5 mgmLÀ1) was added to each well in
a volume of 20 mL. During the incubation of 1 h, MTT is reduced to
purple formazan. The liquid was removed to stop the reaction and
the cells were lysed by adding 100 mL DMSO to each well. Absorb-
ance was measured at 570 nm and background correction at
690 nm using a Multiscan Ex microplate photometer (Thermo
Fisher Scientific, Waltham, MA, USA). The obtained data sets were
normalized, and GI50 values calculated by nonlinear regression
analysis, assuming a sigmoidal concentration–response curve with
variable Hill slope (GraphPad Prism, version 5.0, San Diego, CA,
USA).
Hoechst 33342 accumulation assays: To analyze the inhibitory effect
of title compounds on ABCG2, the Hoechst 33342 accumulation
assay was performed as described earlier with small modifica-
tions.[24,30,35] Various dilutions of each compound were prepared in
Krebs-Hepes buffer (KHB). The highest concentrations (10 mm) were
prepared from a 10À2 m stock solution in DMSO, methanol, and
KHB. A volume of 20 mL from each dilution was placed into black
96-well plates (Greiner, Frickenhausen, Germany). For further prep-
aration, cells were harvested after reaching a confluence of 80–
90% by gentle trypsination (0.05% trypsin/0.02% EDTA). Following
the addition of culture medium, cells were transferred into 50 mL
tubes and centrifuged (266 g, 48C; 4 min). Obtained cell pellets
were resuspended in fresh culture medium and cells were counted
using a CASY1 model TT cell counter device (Schaerfe System
GmbH, Reutlingen, Germany). After three washing steps with KHB
by centrifugation, ~30000 cells per well were placed in a volume
of 160 mL. In all assays the highest concentration of DMSO was not
>0.1% and of methanol not >4%. The plates were incubated for
30 min under 5% CO2 at 378C. Furthermore 20 mL of a 10 mm
Hoechst 33342 solution (protected from light) was placed in each
well and the fluorescence was measured at constant time intervals
(60 s) for a period of 120 min at an excitation wavelength of
355 nm and an emission wavelength of 460 nm at 378C. For mea-
surement a BMG POLARstar microplate reader (BMG Labtech, Of-
fenburg, Germany) was used. The average of fluorescence values
in the steady state (100–109 min) was calculated for all concentra-
tions. From these data concentration–response curves were gener-
ated by nonlinear regression using the three- or four-parameter lo-
gistic equation, whichever was statistically preferred (GraphPad
Prism, version 5.0, San Diego, CA, USA). From the pIC50 values and
their standard deviations, IC50 values and standard deviations were
calculated according to the equation for log-normal distributed
values.[43,44]
MTT assay for determining the ability of MDR reversal: Besides the
detection of cytotoxicity, the MTT assay was also used to evaluate
the effect of inhibitors on the reversal of resistance against cyto-
toxic compounds like SN-38 (active metabolite of irinotecan). For
this purpose, the cytotoxic effect of SN-38 in the absence or pres-
ence of various concentrations of selected inhibitors was mea-
sured. MDCK II wild-type and MDCK II BCRP cells were seeded into
96-well tissue-culture treated plates in a total volume of 160 mL
with a density of ~3000 cells per well. Subsequently plates were
incubated under 5% CO2 atmosphere and 378C for 6 h. From pre-
viously prepared dilution series of SN-38 in culture medium, 20 mL
of each concentration was added to the cell containing wells. Af-
terward 20 mL of culture medium (negative control) as well as
1 and 5 mm test compound were added to the wells containing
MDCK II BCRP cells. Thereby was accomplished the final concentra-
tion in a total volume of 200 mL. As positive control, 20 mL of cul-
ture medium was added to the MDCK II wild-type cells. The subse-
quent steps were performed as described above. From the pIC50
values and their standard deviations, IC50 values and standard devi-
ations were calculated according to the equation for log-normal
distributed values.[43,44]
Calcein AM assays: To evaluate the compounds for their selectivity
toward ABCB1 and ABCC1 inhibition, calcein AM assays were per-
formed as described previously with minor modifications.[28,30,32]
The procedure for dilution series and preparation of cell suspen-
sion was identical to the Hoechst 33342 assay. The dilution series
were pipetted into colorless 96-well plates (Greiner, Frickenhausen,
Germany) in a volume of 20 mL. A2780adr cells for ABCB1 and
H69AR cells for ABCC1 were then placed into the plates at a densi-
ty of ~30000 cells per well to reach a total volume of 180 mL. Fol-
lowing a 30 min pre-incubation under 5% CO2 at 378C, 20 mL of
a 3.125 mm calcein AM solution (protected from light) was added
to each well. Immediately, the fluorescence was measured at con-
stant time intervals (60 s) for a period of 60 min at 378C at an exci-
ATPase assays: ATPase activity was measured as described previ-
ously with slight modifications by a sensitive colorimetric detection
of inorganic phosphate.[35] The isolated Sf9 cell membranes were
obtained after three days infection with a recombinant baculovirus
&
ChemMedChem 2016, 11, 1 – 13
10
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!