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Introduction
Multidrug resistance (MDR) restricts successful tumor
chemotherapy. The overexpression of P-glycoprotein
(P-gp) is one of the prominent mechanisms that contribute
to the MDR phenotype[1]. In fact, P-gp is a 170 kDa
membrane-associated glycoprotein that may actively extrude
various substrates such as cytotoxic drugs from the cell
cytoplasm to the outside of the plasma membrane, thus
lowering the effective drug concentrations within the cells and
leading to MDR. To restore the MDR cell sensitivity, a
combination of a conventional chemotherapeutic regimen and
an MDR modulator is a promising
strategy[2]. Recently, drug carriers such as microspheres, liposomes, or nanoparticles
were found to improve the intracellular accumulation of
anticancer drugs in MDR cells in vitro and
in vivo[3]. Barraud et
al[4] reported that a higher antitumor efficacy on
hepatocellular carcinoma appeared in doxorubicin (Dox)-loaded
polyisohexylcyano-acrylate nanoparticles than in free Dox
both in vitro and in vivo. The nanoparticle, acting as a drug
carrier, could improve solubility for hydrophobic drugs and
may increase drug influx into MDR cells through endocytic
and/or pinocytic activity, thus avoiding P-gp efflux and
increasing intracellular drug
accumulation[5].
Polymeric micelles are particles with diameters typically
smaller than 100 nm formed by amphiphilic polymers
dispersed in aqueous media[6,7]. Polymeric micelles solubilize
insoluble drugs by incorporating them into their
hydrophobic cores, thus allowing for an increased bioavailability.
Meanwhile, their hydrophilic shells can restrict their
recognition and uptake by the mononuclear phagocyte system
and allow for extended circulation time. On the other hand,
the hyperpermeability of tumor vasculature and the lack of
an effective lymphatic drainage of tumor tissue lead to an
accumulation of particles in tumor tissue. This passive
targeting phenomenon was termed enhanced permeability and
retention effect[8]. In vitro,
IC50 indicated that both cisplatin-loaded polymeric micellar
poly(e-caprolactone)/poly[2-(N,N-dimethylamino)ethyl methacrylate]/Cisplatin
(PCL/PDMA/CDDP) and PCL/PEG/CDDP had greatly enhanced
cytotoxicity to the tumor cells compared with free
cisplatin[9]. Camptothecin (CPT) loaded in polymeric micelles showed
enhanced tumor inhibitory activity. The micelles prolonged
the drug's blood circulation time and increased
accumulation in tumors compared with free
CPT[10].
FG020326, an imidazole derivative, is a highly potent,
efficacious MDR modulator in vitro and in
vivo. It can inhibit the function of P-gp and increase the accumulation of
anticancer drugs in MDR tumor cells[11], but FG020326 is insoluble
in water. This work was intended to evaluate the
pharmacodynamics of FG020326-loaded nanoparticles with hydrophilic
PEG and hydrophobic PDLLA diblock copolymer (PEDLLA)
termed as PEDLLA-FG020326 on reversing MDR in
vitro and in vivo.
Materials and methods
Materials Dox and vincristine (VCR) were purchased
from Hisun Pharmaceutical (Taizhou, Zhejiang, China).
Rhodamine 123 (Rh123) and tetrazolium (MTT) were
purchased from Sigma Chemical Co (St Louis, MO, USA).
RPMI-1640 was purchased from Gibco BRL (Grand Island, NY,
USA). FG020326 was synthesized and obtained by chromatography, the isolated powder with a purity of more
than 98%. The FG020326 solution was freshly made
with 0.1% DMSO which did not exert any cytotoxicity to the cells.
PEDLLA-FG020326 was obtained from the School of
Chemistry and Chemical Engineering of Sun Yat-Sen University
(Guangzhou, China).
Animals Athymic nude mice (BALB/c-nu-nu) were used
for the KBv200 cell xenografts. The mice were obtained from
and brought up in the Center of Experimental Animals, Sun
Yat-Sen University (China). The animals were fed specific
pathogen free (SPF)-grade sterilized food and water. Female
and male mice used in the experiment were 6_8 weeks old,
weighing 18_24 g.
Cell lines and cell culture KB and KBv200 are human
epidermoid carcinoma cell lines. KBv200, an MDR cell line
with high expression of P-gp, is about 99-fold resistant to
VCR compared with its drug-sensitive parental KB cells. The
KBv200 cells were cloned from parental drug-sensitive KB
cells by stepwise exposure to increasing doses of VCR and
ethylmethane sulfonate mutagenesis. The KBv200 cells and
parental sensitive KB cells were obtained from Prof Liu YS
(Chinese Academy of Medical Sciences, Beijing, China). The
KBv200 cells and KB cells were cultured with RPMI-1640
culture medium with 10% fetal blood serum (FBS) at 37 °C in
a humidified atmosphere of 5%
CO2[12].
MTT cytotoxicity assay The cells were harvested in a
logarithmic growth phase and seeded in 96-well plates
at 3.0×103 cells each well for KBv200 and KB cells
in a final volume of 170 µL. After incubation for
24 h, 20 µL 20 µmol/L FG020326, PEDLLA-FG020326, or drug-unloaded micelle
PEDLLA, and 10 µL cytotoxic agent VCR or Dox were added
to triplicate wells and incubated for 72 h. Then 10 µL
10
mg/mL MTT solution was added to each well. DMSO (100
µL) was added to each well 4 h later.
IC50 was calculated from the cytotoxicity curves (Bliss's software,
Bliss Co, CA, USA). The degree of resistance was calculated by dividing
the IC50 for the MDR cells by that for the parental sensitive
cells. The reversal fold of MDR was calculated by dividing
the IC50 for cells to the anticancer drug in the absence of the
modulator by that in the presence of the
modulator[13].
Dox accumulation and efflux The intracellular Dox
accumulation was examined as described by Fu et
al[13]. In detail, the logarithmically growing KB cells and KBv200 cells
were harvested and resuspended at a concentration of
1×106 cells/mL. The cells were treated with FG020326 (1, 2, or
4 µmol/L), PEDLLA-FG020326 (1, 2, or 4 µmol/L), PEDLLA
(4 µmol/L), or vehicle at 37 °C for 2 h in the RPMI-1640 medium.
Then Dox was added to a final concentration of 10
µmol/L. The cells were incubated for another 3 h at 37 °C, then
collected, centrifuged, washed 3 times with cold phosphate
buffered solution (PBS), and resuspended in 0.3 mol/L HCl
in 60% ethanol. After centrifugation at 13
201×g for 15 min, the supernatant was removed and assayed
spectrofluoro-metrically at lex=482 nm and
lem=593 nm. PEDLLA-FG020326, FG020326, and PEDLLA did not affect the absorbance or
emission spectra of Dox. The accumulation of Dox was
calculated by the standard curve of Dox. The fold of the Dox
accumulation was calculated by dividing the value in the
presence of the modulator by that without the modulator.
To measure drug efflux, the KBv200 or KB cells were
incubated in energy-supplied buffer and incubated with 10
µmol/L Dox for 3 h at 37 °C. After that, each dish was washed
once with PBS, then FG020326 (4 µmol/L),
PEDLLA-FG020326 (4 µmol/L), or PEDLLA (4
µmol/L) was added. The cells were incubated for the indicated times at 37 °C and harvested,
then quantified as described
earlier[13].
Rh123 accumulation studies The KBv200 cells and KB
cells were exposed to FG020326 (1, 2, or 4 µmol/L),
PEDLLA-FG020326 (1, 2, or 4 µmol/L), or PEDLLA
(4 µmol/L) for 2 h. The cells were collected and washed once and then
resuspended in 1 mL RPMI-1640 at the concentration of
2×105 cells/mL. Then the cells were loaded with 5
µg/mL Rh123 for 30 min at 37 °C. After washing with RPMI-1640 once, the
cells were allowed to efflux the dye for 10 min in dye-free
RPMI-1640 at 37 °C. The cells were then washed and
resuspended in 1 mL RPMI-1640 containing 10 ng/mL verapamil,
after which fluorescence analysis was carried out on a
FACScan flow cytometer (Becton and Dickinson, Mountain
View, CA, USA)[13].
Reversal of MDR in the KBv200 cell xenografts
The KBv200 cell xenograft model was established as described
by Liang et al[14]. Briefly, the KBv200 cells were harvested,
resuspended at a concentration of
1.5×107 cells/mL, and implanted subcutaneously with 0.2 mL cell suspension under
the right armpits of nude mice. When the tumors had reached
a diameter of 5 mm, the animals were divided into 5 groups
according to their body weight, and treated with various
regimens. The groups included a saline group and groups
receiving VCR alone (0.2 mg/kg ip), every 2 d (q2d),
PEDLLA-FG020326 alone (50 mg/kg ip) q2d, VCR (0.2 mg/kg) plus
FG020326 (50 mg/kg ip) q2d, or VCR (0.2 mg/kg) plus
PEDLLA-FG020326 (50 mg/kg ip) q2d. The body weight of the animals
was measured every 2 d for the modulation of the drug
dosage. The tumor volume was measured in 2 perpendicular
diameters (A and B) every 2 d, and the tumor volume
(V) was estimated according to the following
formula[12,13]:
The curve of tumor growth was drawn according to
tumor volulme and time of implantation. The mice were
ethically killed when the mean tumor weights were over 1 g in the
control group. Tumor tissue was excised from the mice and
weighed. The rate of inhibition (IR) was calculated
according to the formula[12,13]:
Statistical analysis All data were repeated at least 3
times in independent experiments and differences were
determined by Student's t-test. Significance was determined
at P<0.05.
Results
Comparison of PEDLLA-FG020326 and FG020326 in reversing MDR
in vitro The KBv200 cells were approximately 99-fold resistant to VCR and 16-fold resistant to Dox
in comparison with the KB cells. PEDLLA-FG020326,
FG020326, and PEDLLA did not appear to have any
cytotoxicity to KBv200 cells and KB cells until 50 µmol/L; PEDLLA
did not reverse MDR action. PEDLLA-FG020326 exhibited
stronger activity than free FG020326 on reversing MDR in
the KBv200 cells. However, both PEDLLA-FG020326 and
FG020326 had no effect on the enhancement of drug
cytotoxicity in drug sensitive KB cells (Table 1; Figure 1).
PEDLLA-FG020326 induced more intracellular Dox
accumulation and less Dox efflux than FG020326 in KBv200
cells The intracellular accumulation of
Dox in the KB cells was 12-fold of that of the KBv200 cells. PEDLLA-FG020326
and FG020326 increased concentration-dependent
intracellular Dox accumulation in the KBv200 cells, but not in the KB
cells. Importantly, PEDLLA-FG020326 was more effective
for the enhancement of intracellular Dox accumulation than
FG020326 (Figure 2).
A higher percentage of Dox extrusion appeared in the
KBv200 cells than that in the KB cells. At 60 min, more than
70% of the accumulated Dox was extruded from the KBv200
cells, while only 40% was extruded from the KB cells. PEDLLA
did not affect the Dox efflux in the KBv200 and KB cells.
PEDLLA-FG020326 was stronger in inhibiting the efflux of
Dox than FG020326 in the KBv200 cells, but both had no
effect on the KB cells (Figure 3).
PEDLLA-FG020326 was stronger in the inhibition of
P-gp function than FG020326 Rh123 is a substrate of P-gp
so the intracellular Rh123 accumulation acts as an index of
P-gp function. PEDLLA-FG020326 and FG020326 increased
concentration-dependently intracellular Rh123 accumulation
in the KBv200 cells, but not in the KB cells. Importantly, the
Rh123 accumulation in the KBv200 cells treated with
PEDLLA-FG020326 was much more than that of the KBv200 cells treated
with FG020326 at the same concentration (Figure 4).
PEDLLA-FG020326 improved the pharmacodynamics
of FG020326 in reversing MDR in vivo PEDLLA-FG020326
had stronger activity in reversing MDR in
vitro than FG020326. To determine whether PEDLLA-FG020326 would
improve the pharmacodynamics of FG020326 in reversing
MDR in vivo, a tumor growth suppression experiment was
conducted in nude mice bearing a KBv200 cell solid tumor.
In vivo experiments showed that neither PEDLLA-FG020326
alone nor VCR alone resulted in the inhibition of tumor
growth. However, the combination of VCR and FG020326 or
PEDLLA-FG020326 elicited marked inhibition of tumor
growth. The tumors were much smaller in the group of VCR
plus PEDLLA-FG020326 than VCR plus FG020326; the
inhibition ratio of tumor growth was 50.5% and 31.2%,
respectively (Table 2; Figure 5).
Discussion
MDR is the main obstacle to successful chemotherapy.
To deal with this issue, a number of MDR reversal agents
with P-gp inhibitory activity, also known as "modulators",
were identified[1]. FG020326 is a recently developed
modulator[11]. In comparison to the early generation of modulators
(eg cyclosporin-A, verapamil) that are not optimized for MDR
reversal[1,2], FG020326 has a higher potency for P-gp and
lower cellular toxicity[11].
Drug delivery systems such as microspheres, liposomes,
or polymeric nanoparticles may improve the effectiveness
and decrease the side effects of cancer chemotherapeutics.
Several in vivo studies have shown that polymeric micelles
are able to improve the efficiency of anticancer drugs against
leukemia[19,20]and solid
tumors[21,22]. There are some mechanisms for the increased pharmaceutical activity of
drug-loaded polymeric micelles such as specific targeting in
tumor tissues, increased permeability of the drug through the
biological membrane, and prolonged circulation time due to
slow drug release[7].
Liu et al[17] and Li et
al[18] have reported that the co-encapsulation of anticancer drugs and modulators by
polymeric particles can enhance MDR reversal by the
simultaneous delivery approach. Moreover, lower normal tissue
drug toxicity and fewer drug-drug interactions have
generally been observed in the combinative treatments. However,
so far, there has not been a consensus as to which strategy
provides the optimal treatment outcome.
In this study, we compared the reversal activity of
PEDLLA-FG020326 with FG020326 in vitro and
in vivo. The in vitro results showed that PEDLLA-FG020326 was more
effective than FG020326 in reversing MDR in KBv200 cells.
P-gp-positive MDR cells demonstrated that the emergence
of MDR in these cells was linked to a marked decrease in the
intracellular accumulation of the various cytotoxic drugs.
Most MDR modulators could restore drug accumulation in
MDR tumor cells[15]. In this study, PEDLLA-FG020326 and
FG020326 were tested for their effect on intracellular drug
accumulation. At the same concentration, PEDLLA-FG020326
caused a marked increase of Dox accumulation compared
with FG020326 in the KBv200 cells but not in the sensitive
KB cells. Rh123 is a specific fluorescent substrate of P-gp
which functions as a drug transporter and mediates drug
efflux[16]. Intracellular Rh123 accumulation could be a good
indicator of P-gp function. Chen et
al[11] found that FG020326 can inhibit the function of P-gp and increase Rh123
accumulation in MDR cells. To evaluate whether the increased
accumulation of Dox in the KBv200 cells was due to the
inhibition of Dox efflux, the extrusion of Dox and P-gp function
were examined. PEDLLA-FG020326 caused a slower
extrusion of Dox and more increased Rh123 accumulation in the
KBv200 cells than FG020326. These results suggest that
PEDLLA-FG020326 can inhibit the function of P-gp more
effectively than FG020326. Moreover, PEDLLA-FG020326 can
increase intracellular drug accumulation compared with
FG020326 in MDR KBv200 cells.
The KBv200 cell xenografts in nude mice were
established and proved to be extremely resistant to VCR, and
retained the characteristics of the MDR
phenotype[14]. The combination of PEDLLA-FG020326 and VCR exhibited a more
potent inhibitory effect on the growth of the tumors than the
co-administration of FG020326 and VCR. Neither
PEDLLA-FG020326 alone nor VCR alone resulted in the inhibition of
tumor growth. The results demonstrate that
PEDLLA-FG020326 has stronger reversal activity than FG020326
in vivo. More importantly, the difference of body weight
between the PEDLLA-FG020326 plus VCR-treated group and
the control or VCR-treated groups is not significant, so
co-administration with PEDLLA-FG020326 does not increase
the toxicity of VCR obviously.
In summary, we observed an improvement in MDR
reversal activity in vitro and in
vivo when FG020326 or PEDLLA-FG020326 was co-administered with a conventional
anticancer drug. Importantly, PEDLLA-FG020326 was more
effective in the reversal of MDR than FG020326 in
vitro and in vivo, which was associated with inhibiting the function of
P-gp and enhancing chemotherapeutic drug accumulation
in MDR cells. These results suggest that the
pharmacodynamics of FG020326 is improved by incorporating into
micellar nanoparticles formed with PEG-block-PDLLA diblock
copolymers.
Acknowledgements
We would like to thank Prof Y S LIU (Chinese Academy
of Medical Sciences, Beijng, China) for providing us with
the KB and KBv200 cell lines.
References
1 Endicott JA, Ling V. The biochemistry of
P-glycoprotein-mediated multidrug resistance. Annu Rev Biochem 1989; 58:
137_71.
2 Kellen JA. The reversal of multidrug resistance in cancer.
Anticancer Res 1993; 13: 959_61.
3 Brigger I, Dubernet C, Couvreur P. Nanoparticles in cancer
therapy and diagnosis. Adv Drug Deliv Rev 2002; 54: 631_51.
4 Barraud L, Merle P, Soma E, Lefrancois L, Guerret S, Chevallier
M, et al. Increase of doxorubicin sensitivity by
doxorubicin-loading into nanoparticles for hepatocellular carcinoma cells
in vitro and in vivo. J Hepatol 2005; 42: 736_43.
5 Koziara JM, Whisman TR, Tseng MT, Mumper RJ.
In-vivo efficacy of novel paclitaxel nanoparticles in paclitaxel-resistant
human colorectal tumors. J Control Release 2006; 112: 312_9.
6 Kwon GS, Kataoka K. Block copolymer micelles as
longcircu-lating drug vehicles. Adv Drug Deliv 1995; 16: 295_309.
7 Jones M, Leroux J. Polymeric micelles _ a new generation of
colloidal drug carriers. Eur J Pharm Biopharm 1999; 48:
101_11.
8 Maeda H, Fang J, Inutsuka T, Kitamoto Y. Vascular permeability
enhancement in solid tumor: various factors, mechanisms
involved and its implications. Int Immunopharmacol 2003; 3:
319_28.
9 Xu P, Van Kirk EA, Li S, Murdoch WJ, Ren J, Hussain MD,
et al. Highly stable core-surface-crosslinked nanoparticles as cisplatin
carriers for cancer chemotherapy. Colloids Surf B Biointerfaces
2006; 48: 50_7.
10 Kawano K, Watanabe M, Yamamoto T, Yokoyama M, Opanasopit
P, Okano T, et al. Enhanced antitumor effect of camptothecin
loaded in long-circulating polymeric micelles. J Control Release
2006; 112: 329_32.
11 Chen LM, Wu XP, Ruan JW, Liang YJ, Ding Y, Shi Z,
et al. Screening novel, potent multidrug-resistant modulators from
imidazole derivatives. Oncol Res 2004; 14: 355_62.
12 Fu LW, He LR, Liang YJ, Chen LM, Xiong HY, Yang XP,
et al. Experimental chemotherapy against xenografts derived from
multidrug resistant KBv200 cells and parental drug-sensitive KB
cells in nude mice by annonaceous acetogenin 89-2. Acta Pharm
Sin 2003; 38: 565_70.
13 Fu LW, Zhang YM, Liang YJ, Yang XP, Pan QC. The multidrug
resistance of tumour cells was reversed by tetrandrine
in vitro and in xenografts derived from human breast adenocarcinoma
MCF-7/adr cells. Eur J Cancer 2002; 38: 418_26.
14 Liang YJ, Fu LW, Feng HL, He LR, Feng GK, Yang XP,
et al. Establishment of the model of KBv200 nude mice xenograft and
studies on its characterization of multidrug resistance. Chin
Pharmacol Bull 2000; 16: 705_9.
15 Yokoyama M, Miyauchi M, Yamada N, Okano T, Sakurai Y,
Kataoka K, et al. Characterization and anticancer activity of
the micelle-forming polymeric anticancer drug
adriamycin-conjugated poly(ethylene glycol)-poly(aspartic acid)
block copolymer. Cancer Res 1990; 50: 1693_700.
16 Zhang X, Burt HM, Von Hoff D, Dexter D, Mangold G, Degen D,
et al. An investigation of the antitumour activity and
biodistribu-tion of polymeric micellar paclitaxel. Cancer Chemother
Pharmacol 1997; 40: 81_6.
17 Zhang X, Burt HM, Mangold G, Dexter D, Von Hoff D, Mayer L,
et al. Anti-tumor efficacy and biodistribution of intravenous
polymeric micellar paclitaxel. Anticancer Drugs 1997; 8:
696_701.
18 Yokoyama M, Okano T, Sakurai Y, Ekimoto H, Shibazaki C,
Kataoka K. Toxicity and antitumor activity against solid tumors
of micelle-forming polymeric anticancer drug and its extremely
long circulation in blood. Cancer Res 1991; 51: 3229_36.
19 Liu Z, Bendayan R, Wu XY. Triton-X-100-modified polymer
and microspheres for reversal of multidrug resistance. J Pharm
Pharmacol 2001; 53: 1_12.
20 Li X, Ruan GR, Lu WL, Hong HY, Liang GW, Zhang YT,
et al. A novel stealth liposomal topotecan with amlodipine: apoptotic
effect is associated with deletion of intracellular
Ca2+ by amlodipine thus leading to an enhanced antitumor activity in leukemia. J
Control Release 2006; 112: 186_98.
21 Tsuruo T, Naito M, Tomida A, Fujita N, Mashima T, Sakamoto
H, et al. Molecular targeting therapy of cancer: drug resistance,
apoptosis and survival signal. Cancer Sci 2003; 94: 15_21.
22 Croce AC, Supino R, Lanza KS, Locatelli D, Baglioni P, Bottiroli
G. Photosensitizer accumulation in spontaneous multidrug
resistant cells: a comparative study with rhodamine 123, rose bengal
acetate and photofrine. Photochem Photobiol Sci 2002; 1:
71_8. |
