Download Accumulation of simple organic cations correlates with

Leukemia (1997) 11, 1156–1159
 1997 Stockton Press All rights reserved 0887-6924/97 $12.00
Accumulation of simple organic cations correlates with differential cytotoxicity in
multidrug-resistant and -sensitive human and rodent cells
TJ Lampidis1, Y-F Shi1, CL Calderon2, D Kolonias1, H Tapiero3 and N Savaraj4
Department of Cell Biology and Anatomy, 2Department of Immunology and Microbiology, University of Miami, School of Medicine, Miami
FL, USA; 3Department of Pharmacology, School of Pharmacy, Chatenay, France; and 4Veterans Administration Medical Center Miami, FL,
USA
1
Structure/functional studies previously reported showed that in
a series of simple organic cations in which the charge is delocalized, an aromatic ring and a minimal degree of lipophilicity
(log P . 21) were required for recognition by murine cells
which express P-glycoprotein (p-gp)-mediated multidrug resistance (MDR). In the present report we find that 3H-octylpyridinium, the simple aromatic cation which has been shown to be
preferentially toxic to MDR2 as compared to MDR1 cells,
accumulates 4.7-fold greater in the MDR2 cell line. In contrast,
we find that 3H-guanidinium which displays no selective toxicity between MDR1 and MDR2 cells, shows no significant
uptake differences between these two cell types. We also
present data which demonstrate that other organic cations
which contain aromatic rings, a minimal degree of lipophilicity
(log P. 21) and carry a delocalized (Rho 123) or shielded
(triphenylmethyl phosphonium) positive charge, also accumulate to a greater degree in MDR2 vs MDR1 cells. Additionally,
we find that human cells which express p-gp MDR, have similar
requirements for recognition of these simple compounds. In
fact, the sensitivity profiles of these compounds closely correlate between murine and human cell lines. It was also found
that none of the series of simple organic compounds tested
showed modulatory activity in MDR1 cells, as assayed by
monitoring retention of Rho 123. Thus, the requirements for
MDR recognition vs those for MDR modulation are clearly distinguished with these simple structured compounds. In comparison, the calcium channel antagonist, verapamil, and a calcium channel agonist, Bay K 8644, both showed modulatory
activity by increasing Rho 123 retention in MDR1 cells, further
supporting the interpretation that verapamil’s modulation of
MDR is unrelated to its action on calcium flux. Overall, the data
presented here add further information for defining the structural requirements of compounds for their recognition by, or
modulation of, human cells expressing p-gp-mediated MDR
Keywords: MDR; simple organic cations; lipophilicity; uptake
Introduction
One of the unanswered questions associated with the
phenomenon of multidrug resistance (MDR) is how does this
process recognize such a wide variety of compounds? We
have shown previously that cationic rhodamines are much
better recognized by cells expressing the MDR phenotype and
p-glycoprotein (p-gp) MDR genotype than zwitterionic homologs.1 Thus, for this group of compounds chemical charge
appeared to play a role in p-gp-mediated MDR recognition.
It was also shown that anthracyclines with reduced or no
charge are accumulated more equally in sensitive- and multidrug-resistant cell variants.2
Since most of the compounds recognized by MDR are complex, results from structure/functional studies are difficult to
interpret. Therefore, a series of simple aromatic (pyridinium)
and non-aromatic (guanidinium) cations which differ in lipophilicity by addition of single alkyl groups were used to better
study MDR.3 Experiments with these simple compounds
revealed that an aromatic ring and a certain degree of lipophilicity was necessary before MDR+ cells showed resistance
to these agents. Furthermore, it was found that the resistance
ratio between MDR+/MDR− to the alkylpyridiniums increased
as a function of lipophilicity. Although differential cytotoxicity
of these compounds was found in MDR+ vs MDR− cells, drug
uptake studies to determine whether resistance correlated with
decreased drug accumulation were not available. Here, utilizing radioactive analogs of a select number of these simple
guanidinium and pyridinium homologs, as well as other
lipophilic cations, differential drug uptake in MDR+ and
MDR− cells was studied.
Moreover, since previous cytotoxicity studies with these
simple compounds were done in murine cell lines, the question of whether human MDR+ cells had similar requirements
for recognition of these agents was addressed in the present
studies. Additionally, we also used these simple compounds
to compare the chemical requirements necessary for MDR
recognition vs those necessary for MDR modulation.
Materials and methods
Cell lines
The murine MDR+ cells used were developed from parental
MDR-Friend leukemia cells (FLC) by exposure to stepwise
increases of Adriamycin as previously described.4 They were
shown to have the following typical MDR properties: broadspectrum resistance to cytotoxic drugs (.2000-fold resistance
to Adriamycin as compared to MDR− cells), high levels of
MDR1 gene expression, lowered plasma transmembrane
potentials, reduced accumulation of Adriamycin, Rho 123 and
other lipophilic cations, compared to their sensitive, MDR−,
counterparts.2 Lower drug accumulation in these MDR+ cells
is reversed by the widely used MDR modulator, verapamil,
with concomitant reversal of resistance to these agents.5 The
human MDR+ cell line was a gift from Dr Victor Ling, Ontario
Cancer Institute, Toronto, Ontario, Canada and was generated
by stepwise exposure of the parental CEM MDR− cell line
to vinblastine.6
Compounds
Correspondence: TJ Lampidis, Department of Cell Biology and Anatomy, University of Miami, School of Medicine (R-124), PO Box
016960, Miami, FL 33101, USA
Received 13 September 1996; accepted 22 November 1996
Alkylguanidiniums, alkylpyridiniums and triphenylymethyl
phosphonium were synthesized as previously described.7,8
Verapamil was purchased from Sigma (St Louis, MO, USA).
Simple organic compounds and MDR
TJ Lampidis et al
Growth inhibition assays
Growth inhibition assays were performed by seeding exponentially growing human or murine MDR− and MDR+ cells
at 1.0 × 105 cells/ml in Eagle’s minimal essential medium supplemented with 10% fetal bovine serum at 37°C in 10% CO2
+ air and treating each continuously with the indicated doses
of guanidinium, pyridinium analogs or triphenylmethyl phosphonium. At 72 h, cells excluding trypan blue were counted,
and ID50s were derived as previously described.5
Assay of drug accumulation
Cells were collected by centrifugation and concentrated to
40 × 106/ml, placed in a beaker with 2 ml of normal media
and magnetically stirred at 37°C in 5% CO2. The appropriate
radioactive analog was added and incubated with the cells for
30 min. To a microcentrifuge tube layered with 25% sucrose
(s.g. 1.10) on the bottom (100 ml) and 20% sucrose at the top
(100 ml) in blue dextran, 200 ml of cells were gently added.
The cells were centrifuged at 15 000 r.p.m. immediately upon
being placed in the tube. Sixty microliters of the top solution
were removed from the solution, (labeled sample A), added
to 4 ml scintillation solution and read in a liquid scintillation
counter. The rest of the fluid was removed and the cell pellet
was washed twice with cold isotonic solution. One hundred
microliter of 10% HClO4 was added to the washed pellet
which was centrifuged for 5 min at 15 000 r.p.m. Sixty microliters of the cell pellet fluid (labeled sample B) were placed in
4 ml of scintillation fluid and counted. In order to standardize
measurement of different radioactive analogs used intracellular drug accumulation was calculated as a ratio as follows:
drug accumulation = (c.p.m. sample B/(c.p.m. sample A) × 2.
Assay of MDR modulation
Previously we have shown that MDR+ Friend leukemic cells
are cross-resistant to Rho 123.4 Since this compound is fluorescent and enters/exits the cell rapidly it has become a quick
and easy screen for determining whether a cell has the MDR
phenotype. Using a fluorescence microscope, cells are examined after they have been treated with 10 mg/ml of Rho 123
for 15 min, rinsed in and placed in Rho 123-free medium.
Those cells expressing the MDR+ p−gp-mediated phenotype
will show either less brightly stained mitochondria than equivalent MDR− cells or no visibly fluorescent mitochondria at
all. In addition, verapamil has been shown to modulate this
phenomenon5 therefore a cell that is MDR+ will retain Rho
123 in its mitochondria in the presence of 10 mM verapamil
for a substantial period of time after Rho 123 has been rinsed
off. In order to test other compounds for their MDR modulation potency, MDR+ cells were loaded with 10 mg/ml Rho
123 for 15 min in the presence or absence of the test modulator (10 mM) and rinsed free of Rho 123. In the case where
the test compound was coincubated with Rho 123 the same
concentration of the test compound was maintained in the
Rho 123-free rinse solution (Eagle’s minimal essential medium
supplemented with 10% fetal bovine serum). If the test compound modulates MDR then the mitochondria remain as
brightly stained as parental MDR− cells (FLC) whereas if it
does not, the cell does not retain the dye as well as MDR−
cells, or not at all, in which case the mitochondria are not
visible under UV
emission 520 l).
fluorescent
light
(excitation 488 l,
Results
Growth inhibition of human vs murine MDR+ and
MDR− cell lines by alkylguanidiniums and
alkylpyridiniums and other organic cations
A comparison between human and murine cell line MDR+
and MDR− pairs in their growth inhibitory response to alkylguanidiums, alkylpyridiniums and other organic cations was
made (Table 1). Utilizing the human cell lines, CEM and
CEM/VBL, which have been shown to be MDR− and MDR+,
respectively,6 we found that with continuous exposure to
guanidiniums and pyridiniums varying in lipophilicity the former compounds were not selectively toxic in these cells
(Table 1). This correlates to what we had previously found
with the eight guanidinium homologs tested in the murine
MDR+ and MDR− cell lines.3 Correspondingly, the ID50 for
octylpyridinium was higher in this human MDR+ cell line than
in its MDR− cell counterpart as was found for the murine cell
lines.3 With dodecylpyridinium which has a log P value
greater than that of octylpyridinium the resistance ratio
between MDR− and MDR+ was even higher. Moreover, the
organic cation, triphenylmethyl phosphonium, with a log P
value of 1.20 when tested in both the murine and human cell
lines also showed a high ratio of resistance.
When the human MDR+ cells were co-treated with verapamil each of the ID50s to Rho 123, triphenylmethyl phosphonium, dodecylpyridinium and octylpyridinium decreased
respectively, while the ID50 to the guanidinium homologs
remained unaffected (data not shown). This correlates with the
results we reported for the response of murine MDR+ and
MDR− cell lines to these compounds when co-treated with
verapamil.3 Table 1 illustrates that the MDR− and MDR+
murine and human cell lines show very similar cytotoxicity
profiles to the series of simple cationic compounds used in
these studies as well as to triphenylmethyl phosphonium and
Rho 123.
Differential accumulation of alkylpyridiniums and
guanidiniums in MDR+ and MDR− cells
With 1 h exposure to 3H-octylpyridinium, the MDR+ rodent
cell line ARN 15 accumulated 3.4 times less than its parental
MDR− counterpart, FLC. In contrast, 3H-pyridinium accumulated equivalently in both cell types. To determine further
whether the marked increased cytotoxicity we found with the
organic cation triphenylmethyl phosphonium in MDR− vs
MDR+ cells correlated with increased drug accumulation in
the former cell type, cells were incubated with 3H triphenyl
phosphonium and drug uptake was evaluated. The results
showed greater than 46 times accumulation of this compound
in MDR− vs MDR+ cells (Table 2). In comparison about nine
times more Rho 123 is found in these same MDR− vs MDR+
cells lines. The latter result was obtained from studies directly
measuring the amount of Rho 123 intracellularly using HPLC
as described previously.4
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Simple organic compounds and MDR
TJ Lampidis et al
1158
Table 1
Growth inhibitory doses in murine vs human MDR+ and MDR− cell lines (ID50 mM)
Compound
Murine
Guanidinium
Octylguanidinium
Octylpyridinium
Dodecylpyridinium
Triphenylmethyl
phosphonium (mg/ml)
Rho 123 (mg/ml)
Table 2
Human
MDR+
MDR−
Ratio
MDR+
MDR−
Ratio
4000.0
1.7
85.0
24.0
350.0
2000.0
1.3
2.3
0.06
0.22
2.0
1.3
37.0
400.0
1595.0
2800.0
7.3
198.0
23.0
263.0
5000.0
5.0
28.0
0.37
0.75
0.6
1.5
7.1
62.0
351.0
175.0
0.10
1750.0
300.0
0.30
1000.0
Differential drug accumulation in MDR+ and MDR− cells
Compound
RHO 123a
Octylpyridinium
Guanidinium
Triphenylmethyl
phosphonium
MDR−
390
0.204
0.026
0.371
MDR+
41
0.043
0.025
0.008
MDR−/MDR+
ratio
9.50
4.70
1.04
46.30
a
Measured by HPLC in ng/ml 3 h incubation.
Chemical requirements for MDR recognition vs MDR
modulation
Rho 123 is a substrate for MDR5 and thus retention of this
fluorescent dye has become a standard technique for assaying
whether cells express the p-gp-mediated MDR phenotype.
Rho 123 staining in live cells can also be used to measure
whether a test compound can modulate MDR. Thus, in the
presence of verapamil, the classical blocker of the MDR efflux
pump, Rho 123 fluorescence is retained in MDR+ cells for
extended periods (hours) after cells are rinsed free of the
dye.2,5 Since we have shown that MDR+ cells do not recognize the entire series of cationic guanidiniums but do recognize pyridiniums with alkyl chains .4, it was surprising to
find that neither of these two series of compounds could
modulate the retention of Rho 123 in MDR+ cells (Table 3).
Bay K8644, a calcium agonist, and reserpine, a calcium antagonist, showed MDR modulatory activity when tested for
increased Rho 123 retention in MDR+ cells (Table 3).
Discussion
The number of compounds reported to be recognized by cells
expressing the MDR phenotype continues to increase.4,9–13
Most of these compounds are complex in nature and contain
many structural differences.4,9–13 Thus, the use of a series of
simple cationic compounds which differ in lipophilicity by
stepwise addition of alkyl groups offered an advantage in
studying the chemical requirements for MDR recognition.3
Our results here with decreased accumulation of the radioactive analogs of octylpyridinium and triphenylmethyl phosphonium in MDR+ vs MDR− cells as well as with Rho 123,
correlates with their reported reduced toxicity in these same
MDR+ as compared to MDR− cell lines as shown here and
reported previously.3 Although the resistance ratio for Rho
123 was found to be somewhat higher than for triphenyl-
Table 3
Modulation of Rho 123 retention in MDR+ cells by various compounds
Compound
Verapamil
Bay K 8644
Reserpine
Guanidinium
C2-guanidinium
C3-guanidinium
C4-guanidinium
C5-guanidinium
C6-guanidinium
C8-guanidinium
C1-pyridinium
C4-pyridinium
C5-pyridinium
C6-pyridinium
C7-pyridinium
C8-pyridinium
C10-pyridinium
C12-pyridinium
Triphenylmethyl phosphonium
Modulates MDR
(increases Rho 123 retentiion in
MDR+ cells)
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
methyl phosphonium while the accumulation ratio was higher
for the latter compound, these differences may be explained
by the short incubation times for accumulation studies vs the
longer times necessary to assay growth inhibition as well as
by the differences that may occur in their mechanisms for
inhibiting growth. Clearly however, the fact that 3H-guanidinium which demonstrates equipotent toxicity in MDR+ and
MDR− cells3 showed no selective accumulation when tested
in both cell types confirms the fact that guanidiniums are not
recognized by the p-gp MDR pump. Thus, interpretation of
our previous cytotoxicity data with these simple compounds
which suggested that an aromatic ring and a certain degree
of lipophilicity are necessary for their recognition by MDR is
supported by the uptake data presented here.
Furthermore, it is interesting to note that Rho 123 and triphenylmethyl phosphonium which are much more complex
compounds than the pyridiniums we have shown to be recognized by MDR+ cells, have in common with these compounds
at least one aromatic moiety and lipophilicity . log P−1. The
log P for Rho 123 is 2.73 while that for triphenyl methyl phosphonium is 1.20 as determined by partition coefficient assays
previously described.3 Our data therefore suggest, that in general, compounds that are recognized by MDR contain at least
one aromatic ring and have a partition coefficient greater than
log P −1.
Simple organic compounds and MDR
TJ Lampidis et al
Although the MDR gene is found in many different species,
phenotypic drug sensitivity profiles have been reported to differ both within the same and across different species.6,14,15
Here we have shown that human cell lines show similar patterns of sensitivities to these simple compounds as compared
to those found in murine cell lines.3 Thus, the understanding
of the minimal requirements of compound composition for
recognition by murine p-gp-mediated MDR is extended to
human MDR. This information should therefore be useful for
drug design aimed at overcoming human multidrug resistance.
Since the discovery of verapamil as an agent which has
blocking activity on the p-gp MDR mechanism there have
been a number of structure/functional studies reporting on the
chemical requirements for modulation of MDR.16–21 Cationic
charge, aromaticity and a minimal degree of lipophilicity have
been suggested as parameters necessary for MDR modulatory
activity.16–21 The data we present here show that none of the
pyridiniums or guanidiniums tested modulate Rho 123 retention in MDR+ cells which clearly demonstrates that there are
different chemical requirements for MDR recognition vs those
for modulation of MDR. Thus, our results indicate that within
the series of simple organic cations tested, additional chemical modifications are necessary for blockage or modulation of
the MDR efflux pump.
The finding that Bay K 8644 which is a calcium channel
agonist and verapamil the calcium channel antagonist work
as modulators of MDR is in agreement with previous reports
which suggest that the effects of verapamil on calcium flux is
distinct from its activity as a modulator of MDR. Thus our
results further indicate that regardless of the direction of perturbation on calcium flux, MDR drug efflux can be modulated.
Acknowledgements
This work was supported in part by an NCl grant No. CA
37109 and a National VA grant.
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