![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
-Specific Inhibitor O1-Hexadecyl-
-glutamyl-S-benzylcysteinyl-D-phenylglycine Ethylester
Fourth Department of Internal Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan (T.N., T.T., K.M., A.N., T.H., T.A., J.K., Y.N.); and Teijin Ltd., Tokyo, Japan (K.S., Y.N., H.T.)
Received April 30, 2003; accepted May 29, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
(GSTP1-1) is involved
in resistance to anticancer drugs in cholangiocarcinoma and whether
GSTP1-1-specific inhibitors can overcome this resistance. First,
immunohistochemical examination disclosed strong staining of all our 17
cholangiocarcinoma specimens for GSTP1-1, irrespective of histological type.
Transfection of the GSTP1-1 antisense expression vector into a human
cholangiocarcinoma cell line (HuCCT1) apparently decreased its intracellular
GSTP1-1 concentration, and the sensitivity of transfectants to adriamycin
(ADR), cisplatin, and alkylating agents such as melphalan and
4-hydroxyperoxycyclophosphamide (4-HC) was increased significantly, compared
with that of mock transfectants. We next synthesized GSTP1-1-specific
inhibitors by elongating the carbon chain of the ethylester at the
N-terminal of
-glutamyl-S-benzylcysteinyl-phenylglycyl diethylester and
performed a pharmacokinetic study on them. Of six GSTP1-1 inhibitors tested,
O1-hexadecyl--glutamyl-S-benzylcysteinyl-D-phenylglycine
ethylester (C16C2) showed the smallest volume of central compartment and
smallest volume of distribution at steady state and the second smallest
clearance, being the most effective inhibitor in vivo. The IC50
value of ADR or 4-HC for HuCCT1 cells decreased greater by treatment with
C16C2 in a dose-dependent manner, paralleling the decrease in GSTP1-1
activity, than that of ADR or 4-HC alone. The antitumor activity of ADR or
cyclophosphamide was clearly enhanced by combination therapy with C16C2 in a
xenograft model. In conclusion, our results demonstrated that GSTP1-1 is a
resistance factor for anticancer drugs in cholangiocarcinoma and that C16C2, a
GSTP1-1-specific inhibitor, is a potent agent against the resistance.
;
Isa et al., 2001;
Patel, 2001;
Okuda et al., 2002). However,
the mechanism of chemoresistance of this disease is totally unknown.
It has been reported that there are gene abnormalities in
K-ras, p53, and APC in cholangiocarcinoma
(Tada et al., 1990;
Kiba et al., 1993;
Ohashi et al., 1995;
Tannapfel et al., 2000;
Isa et al., 2002). In
particular, K-ras mutation has been detected in as many as 48 to 80%
of these cases. We have recently reported the close relationship between
K-ras mutation and the expression of
glutathione-S-transferase- (GSTP1-1), a detoxification
enzyme in precancerous lesions as well as in cancer tissue
(Miyanishi et al., 2001). We
also showed that GSTP1-1 is a multidrug-resistant factor for adriamycin (ADR),
cisplatin (CDDP), and alkylating agents such as melphalan
(Ban et al., 1996;
Kuga et al., 1997). Hayes et
al. (1991) examined the
expression of GSTP1-1 in cholangiocarcinoma by immunohistochemical staining
and found that it was positive in eight of eight cases, indicating that
GSTP1-1 may be a viable marker for cholangiocarcinoma, although they did not
refer to its possible role as a resistance factor to anticancer drugs.
In this study, we first examined the expression of GSTP1-1 in
cholangiocarcinoma. Then, to prove that GSTP1-1 is responsible for
chemoresistance, we introduced GSTP1-1 antisense cDNA into a
cholangiocarcinoma cell line, HuCCT1, which expresses GSTP1-1 abundantly, and
revealed that the transfectants became sensitive to ADR, CDDP, melphalan, and
4-hydroxyperoxycyclophosphamide (4-HC), an active form of cyclophosphamide
(CPA) in in vitro experiments. Furthermore, we synthesized a GSTP1-1-specific
inhibitor,
O1-hexadecyl--glutamyl-S-benzylcysteinyl-D-phenylglycine
ethylester, and examined whether the sensitivities to ADR and CPA were
increased by administration of the inhibitor simultaneously with anticancer
drugs in nude mice inoculated subcutaneously with HuCCT1 cells.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Anticancer Drugs. ADR and 5-fluorouracil (5-FU) were purchased from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan), whereas CDDP, vincristine (VCR), CPA, and 4-HC were obtained from Shionogi Co., Ltd. (Tokyo, Japan). Melphalan was purchased from Sigma-Aldrich (St. Louis, MO).
Immunohistochemistry. The paraffin-embedded sections were deparaffinized in three changes of xylene and rehydrated through graded alcohol solutions at room temperature. A 10 mM sodium phosphate buffer containing 0.9% NaCl [phosphate-buffered saline (PBS), pH 7.4] was used for washes between various steps; incubations were performed in a humidified chamber. Sections were treated with 5% normal horse serum (Invitrogen, Carlsbad, CA) in PBS and then incubated with 1:100 dilution of anti-GSTP1-1-specific monoclonal antibody (DAKO, Kyoto, Japan) overnight at 4°C, followed by incubation with biotinylated horse anti-mouse immunoglobulin G at room temperature and detection with the ABC kit (Vector Laboratories, Burlingame, CA).
Cell Lines and Cell Culture. Human cholangiocarcinoma cell lines HuCCT1 and HuH28, human cervical carcinoma cell line HeLa, human mammary carcinoma cell line MCF7, human gastric carcinoma cell line TMK-1, human hepatocellular carcinoma cell lines HepG2 and PLC/PRF/5, and human colon carcinoma cell line M7609 were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). HuCCT1, HuH28, and M7609 were cultured in RPMI 1640 medium (Invitrogen) and HeLa, MCF7, TMK-1, HepG2, and PLC/PRF/5 were cultured in Dulbecco's modified Eagle's medium containing 10% FCS (Flow Laboratories, North Ryde, Australia) in tissue culture flasks; incubation was performed at 37°C in an atmosphere of air containing 5% CO2.
GSTP1-1 Quantitation by ELISA. After washing each cell preparation
two times in cold PBS, the cells were adjusted to a concentration of 1 x
106 /ml in the same buffer and were homogenized with a Dounce
homogenizer. The lysates were then centrifuged at 12,000 rpm for 15 min, and
the concentration of GSTP1-1 in each supernatant was measured by sandwich
ELISA established in our laboratory as described previously
(Takahashi et al., 1989;
Kura et al., 1996).
Construction of a GSTP1-1 Antisense Vector. The plasmid pGpi2
(Nakasa et al., 1997)
containing GSTP1-1 cDNA was obtained from the Japanese Cancer Research
Resources Bank. pGpi2 was digested with EcoRI, and a 0.7-kb
EcoRI-EcoRI fragment containing the whole coding region for
GSTP1-1 was recovered. Both ends of this fragment were then blunted with the
Klenow fragment (Takara Shuzo Co., Ltd., Kyoto, Japan). The pLJ vector
described by Korman et al.
(1987) was linearized with
BamHI; the blunting of both terminals was similarly performed using
the Klenow fragment and was dephosphorylated with bacterial alkaline
phosphatase (Takara Shuzo Co., Ltd.). The two processed fragments were ligated
with T4 ligase, and a clone was selected in which the GSTP1-1 cDNA was
inserted in the reverse direction. This clone was named pLJ/anti-GSTP.
Gene Transfer. The transfection of the pLJ/antiGSTP into the HuCCT1 cells was performed by the lipofection method. Briefly, 2.5 x 105 cells were dispersed in a 3.5-cm culture dish and were incubated for 24 h. The attached cells were then washed three times with RPMI 1640 medium (Invitrogen), followed by the addition of 3 ml of the same culture medium to the dish. Next, 100 µl of plasmid lipofectin reagent (Invitrogen) was mixed with 3 µg of pLJ/antiGSTP and incubated at room temperature for 15 min. This mixture was then added to each culture dish, and the dishes were incubated at 37°C for 6 h. RPMI 1640 medium (3 ml) containing 10% FCS was added to each culture dish, and incubation was continued for another 72 h. G418 (Invitrogen) was added to the culture medium in each dish to a concentration of 400 µg/ml, and the cells were cultured for approximately 2 weeks at 37°C in an atmosphere of air containing 5% CO2. The G418-resistant clones were obtained and designated HuCCT1/antiGSTP1 and HuCCT1/antiGSTP2. The pLJ vector without GSTP1-1 antisense cDNA was transfected to HuCCT1 cells to obtain a control transfectant, HuCCT1/pLJ.
GSTP1-1 Inhibitors. GSTP1-1 inhibitors
(Fig. 1) were synthesized by
elongating the carbon chain of the alkylester (R1) at the N terminus of
-glutamyl-S-benzylcysteinyl-phenylglycine, the active form of
the GSTP1-1-specific inhibitor (Morgan et
al., 1996), to increase their stability in circulation. The
inhibitors were synthesized by a conventional method of peptide synthesis as
described by Erickson and Merrifield
(1976). In brief,
D-phenylglycine-OEt HCl was synthesized from
D-phenylglycine and thionyl chloride (SOCl2) in ethanol
at room temperature (1). To a mixture of 1, Boc-Cys(Bzl)-OH,
hydroxybenzotriazole, and N-methylmorpholine in dimethylformamide was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (WSC) at
0°C, and the mixture was stirred at room temperature for 80 min. The
reaction mixture was poured into water, extracted with ethyl acetate, washed
with saturated aqueous sodium chloride, dried with anhydrous magnesium
sulfate, and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography to afford Boc-Cys(Bzl)-D-Phg-OEt
(2). Compound 2 was treated with hydrogen chloride in dioxane and concentrated
under reduced pressure to afford Cys(Bzl)-D-Phg-OEt HCl (3). To a
mixture of Boc-Glu(OBzl)-OH and NaHCO3 in dimethylformamide was
added 1-bromo hexadecane at room temperature. After stirring for 38 h, the
reaction mixture was poured into water, extracted with ethyl acetate, washed
with saturated aqueous sodium chloride, dried with anhydrous magnesium
sulfate, and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography to afford Boc-Glu(OBzl)-O(Hexadecyl) (4).
Boc-Glu(OBzl)-Oet (5), Boc-Glu(OBzl)-O(Octyl) (6), Boc-Glu(OBzl)-O(Dodecyl)
(7), Boc-Glu(OBzl)-O(Tetradecyl) (8), Boc-Glu(OBzl)O(Octadecyl) (9) were
synthesized similarly. A mixture of 4 and catalytic amount of 10% palladium
carbon in dioxane was stirred under an atmosphere of hydrogen at room
temperature for 19 h. After filtration through celite pad, the filtrate was
concentrated under reduced pressure. Dimethylformamide,
3-hydroxybenzotriazole, N-methylmorpholine, and WSC were added, and
the mixture was stirred at room temperature for 2 h. The reaction mixture was
poured into water, extracted with ethyl acetate, washed with saturated aqueous
sodium chloride, dried with anhydrous magnesium sulfate, and concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography to afford Boc-Glu(Cys-
(Bzl)-D-Phg-OEt)-O(Hexadecyl).
Boc-Glu(Cys(Bzl)-D-Phg-OEt)-O(Hexadecyl) was treated with 4 N HCl
in dioxane at room temperature, and the reaction mixture was concentrated
under reduced pressure. Diethyl ether was added and the precipitated
Glu(Cys(Bzl)-D-Phg-OEt)-O(Hexadecyl) HCl (C16C2) was collected by
filtration. Similarly, Glu(Cys(Bzl)-D-Phg-OEt)-OEt HCl (C2C2),
Glu(Cys- (Bzl)-D-Phg-OEt)-O(Octyl) HCl (C8C2),
Glu(Cys(Bzl)-D-Phg-OEt)O(Dodecyl) HCl (C12C2),
Glu(Cys(Bzl)-D-Phg-OEt)-O(Tetradecyl) HCl (C14C2), and
Glu(Cys(Bzl)-D-Phg-OEt)-O(Octadecyl) HCl (C18C2) were synthesized
from compounds 5, 6, 7, 8, and 9, respectively.
|
Pharmacokinetic Study of GSTP1-1 Inhibitors. GSTP1-1 inhibitors were
administered in vivo by formulation with lipid microspheres (LMs) to
facilitate their solubility in plasma. In brief, they were dissolved in a
mixture of benzyl alcohol and ethanol (1:4) at 40 mg/ml and then added to a
fat emulsion (Intralipos; Nihonseiyaku Inc., Tokyo, Japan) at a ratio of
1:0.075, mixed well by vortexing, and subsequently passed through a 1.2-µm
pore-sized filter. The mean particle size of the emulsified LMs thus
synthesized was 243 ± 86 nm (data not shown), which is suitable for in
vivo administration (Yamaguchi and
Mizushima, 1994). LM solution containing GSTP1-1 inhibitors was
intravenously injected into a rabbit at 3 to 30 ml/kg. Blood was sampled 5,
10, 15, 30, 60, 120, and 240 min after injection, and the plasma concentration
of inhibitors was determined by high-performance liquid chromatography
(HPLC).
HPLC. A reverse phase HPLC was performed on the HPLC column (YMC-Pack Pro C18, 4.6 x 25 cm; YMC, Inc., Tokyo, Japan) with an LC-10A equipped with a UV detector (Shimadzu, Kyoto, Japan).
In Vitro Deesterification of C16C2 by Esterase. C16C2 (200 µg/ml)
was incubated with rabbit liver esterase (134 units/ml) in PBS containing
bovine serum albumin at 37°C for 24 h to hydrolyze the C16C2 ester bonds.
The resultant solution was applied to the HPLC column to analyze the presence
of C16C2 and its active form,
-glutamyl-S-benzylcysteinyl-phenylglycine. As an internal
standard, parahydroxymethyl benzoate (10 µg/ml) was added to the solution
before application to the reverse phase HPLC column. The solution of the
active form itself (10 µg/ml) containing parahydroxymethyl benzoate (10
µg/ml) was also applied to the HPLC column as a control.
Cytotoxicity Assays. The sensitivities of each cultured cell line to
the anticancer drugs ADR, melphalan, 4-HC, VCR, CDDP, and 5-FU were determined
by the dye-uptake method. Briefly, 1 x 104 cells in 100 µl
were dispensed in 96-well culture plates, and GSTP1-1 inhibitors were added at
various concentrations. After incubation for 24 h at 37°C, anticancer
drugs were added to each well at various concentrations, and the cells were
incubated for another 48 h at 37°C. Next, 25 µl of a 25% glutaraldehyde
solution was added to each well to fix the cells, and the plates were then
washed with water, dried, stained with a 0.05% methylene blue solution, and
eluted with 0.33 N HCl. The absorbance at 665 nm was measured with an ELISA
reader (MS-3096F; SLT-LAB Instruments Co., Salzburg, Austria). The cell
survival rates were deduced from the relative absorbance values of the samples
to control. Because C16C2 was practically insoluble in water, it was treated
as follows before use. First, 10 mg of L-
-lecithin was
dissolved in chloroform and air-dried. Ethanol containing C16C2 at 30 mg/ml
was then added to the air-dried lecithin. After further addition of 10 ml of
RPMI 1640 medium, the mixture was sonicated for 10 min and then added to the
culture solution (Parsaee et al.,
2002).
Assay for GSTP1-1 Activity in Cultured Cells. HuCCT1 cells were
seeded at a concentration of 2 x 105/2 ml in a 12-well
culture dish and cultured in RPMI 1640 medium containing 10% FCS for 24 h.
C16C2 was added to each well at various concentrations, and the cells were
cultured for another 24 h. They were harvested using cell scrapers, incubated
in hypotonic buffer (pH 7.4) (10 mM Tris, 1.5 mM MgCl2) for 20 min
at 4°C, homogenized using Dounce homogenizers, and centrifuged at
10,000g for 30 min at 4°C to collect cytosolic proteins. GSTP1-1
activities were measured using 1-chloro-2, 4-dinitrobenzene (CDNB) as a
substrate, according to the method of Habig et al.
(1974). In brief, protein
samples (10-50 µl) were added to 1 ml of 0.1 M sodium phosphate buffer (pH
6.5) containing 1.3 mM CDNB and 2.5 mM reduced glutathione (Sigma-Aldrich),
and the absorbance at 343 nm was measured at 25°C.
Xenotransplantation. The experiments were performed in accordance
with the recommendations of the Guide for the Care and Use of Laboratory
Animals of Sapporo Medical University School of Medicine. Cells (2 x
106) in 100 µl of RPMI 1640 medium were inoculated
subcutaneously into the back of each nude mouse (5 weeks old; Sankyo Labo
Service Co., Ltd, Tokyo, Japan). When tumor sizes reached 7 mm in diameter, 36
mice were randomized into six groups. To three groups, C16C2 was administered
into the tail vein at a dose of 20 mg/kg on days 1, 2, 3, 8, 9, and 10. Of
these three groups, two groups received intraperitoneal administrations of
either 4 mg/kg ADR or 200 mg/kg CPA on days 2 and 9. Of the remaining three
groups, two groups received intraperitoneal administrations of 4 mg/kg ADR or
200 mg/kg CPA on days 2 and 9. The tumor size was measured with a sliding
caliper every 4 days. Tumor volume (V) was calculated with the
following formula: V = length x (width)2 x
0.5, in accordance with the protocol of Geran et al.
(1972).
To measure the GSTP1-1 activities in tumor tissues of xenografts, tumors were resected on day 3, minced in hypotonic buffer, and the cytosol fractions were extracted. Then, GST activities were measured as described above.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
Intracellular GSTP1-1 Concentration in Various Cell Lines. We measured the concentrations of GSTP1-1 in HeLa cells from human cervical carcinoma, MCF7 cells from human mammary carcinoma, TMK-1 cells from human gastric carcinoma, HepG2 and PLC/PRF/5 cells from human hepatocellular carcinoma, M7609 cells from human colon carcinoma, and HuCCT1 and HuH28 cells from human cholangiocarcinoma using ELISA (Table 2). The GSTP1-1 concentrations in the two cell lines (HuCCT1 and HuH28) derived from cholangiocarcinoma were clearly higher than the others, and the HuCCT1 cell line, which had the highest GSTP1-1 concentration, was used for the following examination.
|
Anticancer Drug Sensitivities of HuCCT1/anti-GSTP1 and HuCCT1/antiGSTP2 Cells. To examine whether GSTP1-1 expressed in HuCCT1 cells was involved in the resistance to anticancer drugs, we introduced GSTP1-1 antisense cDNA into HuCCT1 cells and investigated changes in their drug sensitivity. The intracellular GSTP1-1 concentrations in the two clones, HuCCT1/antiGSTP1 and HuCCT1/antiGSTP2, were decreased to about half of those of the parental cell line and the control cells, HuCCT1/PLJ. Both the HuCCT1/antiGSTP1 and the HuCCT1/antiGSTP2 cells showed significantly elevated sensitivities to ADR, melphalan, 4-HC, and CDDP compared with that of the HuCCT1/PLJ cells. Neither HuCCT1/antiGSTP1 nor HuCCT1/antiGSTP2 cells showed statistically significant changes in their sensitivities to VCR or 5-FU (Table 3).
|
Pharmacokinetic Study of Synthesized GSTP1-1 Inhibitors in Rabbit. Because the plasma esterase activity is similar in rabbits and humans, which is low compared with the activity in rats or mice, we used rabbits to examine the pharmacokinetics of GSTP1-1 inhibitors (Fig. 3). The plasma concentration of C2C2 was already less than 0.1 µg/ml at 10 min after administration and decreased sharply within 30 min. As the number of the carbons in the alkylester at the N terminus increased from eight (C8C2) to 18 (C18C2), the plasma concentration of GSTP1-1 inhibitors gradually increased. The maximal plasma concentration of C16C2 was 17.2 µg/ml at 5 min. There was no apparent difference between the maximal plasma concentrations of C16C2 and C18C2.
|
The plasma inhibitor concentration was analyzed as a function of time by
the nonlinear least-squares program MULTI, according to a two-compartment
model (Metzler, 1971;
Zuideveld et al., 2002)
(Table 4). The volume of
central compartment (Vc) and volume of distribution at
steady state (Vss) were the smallest for C16C2, whereas
the total body clearance (CL) was the smallest for C18C2 among the synthesized
inhibitors (Table 4). Thus,
C16C2 most effectively of all inhibitors maintained a sustained high blood
concentration. In addition, we have confirmed that C16C2 showed high
stability, even in rats and mice (data not shown).
|
Deesterification of C16C2 in Vitro. C16C2 was incubated in vitro
with an esterase for 24 h to examine whether an active form of C16C2 could be
indeed generated during the esterase treatment. The control active form of a
GSTP1-1-specific inhibitor,
-glutamyl-S-benzylcysteinyl-phenylglycine, was eluted at the
16-min site on a reverse phase HPLC (Fig.
4B). An analysis of C16C2 before the esterase treatment
demonstrated no peak at the 16-min site
(Fig. 4C). After C16C2 was
incubated with an esterase for 24 h, an analysis of the resultant solution by
reverse phase HPLC revealed a small peak at the 16 min site which represented
the elution of the active form of C16C2
(Fig. 4D).
|
Inhibitory Effects of C16C2 on GSTP1-1 Activity in HuCCT1 Cells and Its Effects on Sensitivities to Anticancer Drugs in Comparison with Those of C2C2. We examined whether GSTP1-1 activity in HuCCT1 cells could be indeed inhibited by C16C2. As shown in Fig. 5A, GSTP1-1 activity in HuCCT1 cells was dose dependently inhibited by treatment with both C16C2 and C2C2, although the inhibition rate of C16C2 at each dose was slightly lower than that of C2C2 with no significant difference. Incidentally, there was no significant cytotoxicity of C16C2 to HuCCT1 cells when it was used at the concentration of 0 to 200 µM (data not shown). We next evaluated the ability of C16C2 to potentiate the killing effect of ADR or 4-HC on HuCCT1 cells. The IC50 value for both ADR (Fig. 5B) and 4-HC (Fig. 5C) decreased by treatment with C16C2 and C2C2 in a dose-dependent manner, paralleling the decrease of GSTP1-1 activity (Fig. 5A). The decrement rates of IC50 and GSTP1-1 activity with C16C2 was also lower than that of C2C2 with no significant difference.
|
Effects of C16C2 on Sensitivities to Anticancer Drugs in Xenograft Models. We examined whether the antitumor activity of ADR or 4-HC against HuCCT1 tumors transplanted into nude mice was enhanced by C16C2. C16C2 itself did not exert any effect on tumor growth (Fig. 6) and exhibited no apparent adverse effects such as weight loss, abnormal liver function, or dysfunction of hematopoiesis (data not shown). When C16C2-treated mice were examined histologically, liver, kidney, heart, lung, stomach, and intestine showed no apparent abnormalities. Although either ADR or CPA alone showed some inhibitory activity on tumor growth, when they were combined with C16C2, the inhibitory activity became more evident. Moreover, the tumor disappeared completely in one of the six mice in which CPA was administered in combination with C16C2.
|
GST Activities in Tumor Tissues of the Mice Receiving C16C2. To verify that the augmented antitumor effect of anticancer drugs combined with C16C2 is due to the inhibition of GSTP1-1 activity by the latter agent, we measured GSTP1-1 activity in tumors resected from mice. The mean GSTP1-1 activity in tumor tissues of the control mice was 13.6 ± 1.9 µM/min/mg of protein, whereas that of the mice administered with C16C2 was 6.1 ± 1.1 µM/min/mg of protein. The result indicated that the GSTP1-1 activity was inhibited to 45% of the control level by administration of C16C2.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
;
Penketh et al., 1996;
Kuga et al., 1997;
Niitsu et al., 1998;
Goto et al., 2001).
There have been several studies to overcome the anticancer drug resistance
using GST inhibitors. Tew et al.
(1988) reported that the
sensitivity to chlorambucil increased in cells of the Walker 256 rat mammary
tumor cell line and a human colon cancer cell line treated with pyriplost, an
analog of prostaglandin I, and ethacrynic acid, a diuretic compound as well as
a substrate for GST. However, ethacrynic acid has a very problematic side
effect in addition to its ability to induce the metabolic abnormality and its
strong diuretic activity because it suppresses the bone marrow function
markedly when used together with anticancer drugs. Hall et al.
(1989) reported that a
nonsteroidal anti-inflammatory drug (NSAID), indomethacin, known to bind to
GSTP1-1, is useful in overcoming chlorambucil resistance. We ourselves found
that ketoprofen, one of the NSAIDs that inhibited the GST activity, clearly
overcame the ADR resistance (Niitsu et
al., 1990). In these reports, however, the effectiveness of NSAIDs
in vivo has not yet been clarified. Maeda et al.
(1993) synthesized a
calmodulin antagonist (Ca2+ antagonist), W-77, which
blocked GSTP1-1 activity, and reported that it enhanced the ADR sensitivity
through inhibition of both GSTP1-1 and p-glycoprotein. However, it
was highly toxic in vivo by itself.
Lyttle et al. (1994)
recently synthesized a glutathione analog
(-glutamyl-S-benzylcysteinyl-phenylglycyl diethylester), which
was designed to block GSTP1-1 activity by directly binding to its glutathione
site after entrance into the cells, where diethyester is hydrolyzed by
esterase to become -glutamyl-S-benzylcysteinyl-phenylglycine,
and they demonstrated that the analog had extremely high and specific
inhibitory activity to GSTP1-1 with a Ki value of 0.42
µM compared with that to GST A1-1 (Ki value of 24.3
µM), GST M1-1 (Ki value of 57.8 µM), and GST M2-2
(Ki value of 184 µM). Morgan et al.
(1996) reported further that
the treatment with this glutathione analog of a colon cancer cell line in
which GSTP1-1 was highly expressed resulted in an increase of sensitivity to
chlorambucil. The half-life of this compound, however, was so short in vivo
that it was difficult to attain an effective blood concentration. The short
half-life was conceivably attributable to the readiness of two ethyl groups to
be hydrolyzed by esterase in the blood. To circumvent this problem, we
therefore elongated the ethyl group at the N terminus of
-glutamyl-S-benzylcysteinyl-phenylglycyl diethylester with
long alkyl carbon chains to form various compounds (C8C2, C12C2, C14C2, C16C2,
and C18C2), which should be resistant to deesterification in the blood. When
we administered the above-mentioned compounds to rabbits, the blood
concentration of compounds with longer chains (C16C2 and C18C2) was indeed
maintained at a higher level. Because C16C2 showed the smallest volume of
central compartment (Vc) and the smallest volume of
distribution at steady state (Vss) by a two-compartment
model analysis, we chose C16C2 for further experiments. To verify the in vivo
observation, we then incubated C16C2 with esterase in vitro for 24 h. The
result that only a small amount of the deesterified form was detected on HPLC
indeed indicated the stable nature of C16C2 against esterase. However, when in
vitro activity of C16C2 on tumor cells
(Fig. 5) was examined, it was
almost as high as that of C2C2, although slightly impaired. Together, it was
surmised that C16C2 is quite stable as an inactive form in circulation despite
the presence of esterase, whereas intracellularly, the agent may readily
convert into the active form due to a high concentration of esterase
(Butterworth et al., 1993;
van Ark-Otte et al., 1998),
and thus it exerts nearly the same potent anti-GSTP1-1 activity as C2C2.
Nevertheless, this property of C16C2 is considered to be quite suitable for in
vivo use. Furthermore, for administration of C16C2, we prepared colloidal
microspheres containing C16C2 to facilitate its transfer into cancerous
tissues. Accordingly, the effect of C16C2 was examined in vivo with
tumor-bearing mice. C16C2 was administered three consecutive days (on the day
of administration of an anticancer drug, and on the days before and after
that) because GSTP1-1 must be inhibited before and during anticancer drug
administration. The applied dose was determined based on the inhibitory
activity of C16C2 on GSTP1-1 in vitro (Fig.
5) and the plasma concentration of C16C2 in vivo
(Fig. 3). We found that the
combination of C16C2 with ADR and CPA evoked significantly higher antitumor
effects than administration of each drug in isolation. We also found that the
tumor disappeared completely in one of the mice in which CPA was administered
in combination with C16C2. These results clearly indicated that C16C2 is
highly potent in overcoming the chemoresistance caused by GSTP1-1.
Incidentally, in this study we presented the results of experiments in which
animals were treated with two courses of combination therapy. The results were
much more favorable than those of one-course therapy (data not shown),
suggesting the possibility that the efficacy of treatment may increase as the
number of therapy course increases. However, multiple therapy courses are not
practically feasible in this model because of damage to the tail vein, where
the drugs are injected. This obstacle will be circumvented when therapy is
applied to humans in the future.
Because -glutamyl-S-benzylcysteinyl-phenylglycine, the
active form of C16C2 has a high selectivity to GSTP1-1, it is not expected to
affect the activity of other GST isozymes, and if it does, the affect should
not be serious. However, further detailed investigations are needed. With
regard to toxicity of C16C2, there was no significant cytotoxicity to HuCCT1
cells when it was used at the concentration of 0 to 200 µM. In the
xenograft experiment, mice administered C16C2 showed no significant decrease
in body weight compared with those administered vehicle alone, and no blood
chemical abnormality was found. Histological examinations of liver, kidney,
heart, lung, stomach and intestine showed no apparent abnormalities. Moreover,
the LD50 value of C16C2 was very high, ranging from 800 to 1,000
mg/kg (data not shown). However, a more detailed examination of C16C2
toxicities should be performed. In conclusion, C16C2 is considered to be
useful for treatment of cholangiocarcinoma in combination with anticancer
drugs to which GSTP1-1 is a resistant factor.
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: GSTP1-1, glutathione S-transferase P1-1; ADR, adriamycin; CDDP, cisplatin; 4-HC, 4-hydroxyperoxycyclophosphamide; CPA, cyclophosphamide; 5-FU, 5-fluorouracil; VCR, vincristine; PBS, phosphate-buffered saline; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; WSC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; LM, lipid microsphere; HPLC, high-performance liquid chromatography; CDNB, 1-chloro-2,4-dinitrobenzene; V, volume; Vc, volume of central compartment; Vss, volume of distribution at steady state; CL, total body clearance; GST, glutathione S-transferase; NSAID, nonsteroidal anti-inflammatory drug.
Address correspondence to: Dr. Yoshiro Niitsu, The 4th Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo, Japan, 060-8543. E-mail: niitsu{at}sapmed.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Ban N, Takahashi Y, Takayama T, Kura T, Katahira T, Sakamaki S, and
Niitsu Y (1996) Transfection of glutathione
S-transferase (GST)- antisense complementary DNA increases the
sensitivity of a colon cancer cell line to adriamycin, cisplatin, melphalan
and etoposide. Cancer Res
56:
3577-3582.
Butterworth M, Upshall DG, and Cohen GM (1993) A novel role for carboxylesterase in the elevation of cellular cysteine by esters of cysteine. Biochem Pharmacol 46: 1131-1137.[CrossRef][Medline]
de Groen PC, Gores GJ, LaRusso NF, Gunderson LL, and Nagorney DM
(1999) Biliary tract cancers. N Engl J of
Med 341:
1368-1378.
Erickson BW and Merrifield RB (1976) The Proteins, 3rd ed., vol 2, pp 255-292, Academic Press, New York.
Geran RI, Greenberg NH, Macdonald MM, Schumacher AM, and Abbott BJ (1972) Protocols for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother Rep Part 3 3: 1-103.
Goto S, Ihara Y, Urata Y, Izumi S, Abe K, Koji T, and Kondo T
(2001) Doxorubicin-induced DNA intercalation and scavenging by
nuclear glutathione S-transferase pi. FASEB J
15:
2702-2714.
Habig WH, Pabst MJ, and Jakoby WB (1974) Glutathione
S-transferases. The first enzymatic step in mercapturic acid
formation. J Biol Chem
249:
7130-7139.
Hall A, Robson CN, Hickson ID, Harris AL, Proctor SJ, and Cattan AR
(1989) Possible role of inhibition of glutathione
S-transferase in the partial reversal of chlorambucil resistance by
indomethacin in a Chinese hamster ovary cell. Cancer
Res 49:
6265-6268.
Hayes PC, May L, Hayes JD, and Harrison DJ (1991)
Glutathione S-transferases in human liver cancer.
Gut 32:
1546-1549.
Isa T, Kusano T, Shimoji H, Takeshima Y, Muto Y, and Furukawa M (2001) Predictive factors for long-term survival in patients with intrahepatic cholangicarcinoma. Am J Surg 181: 507-511.[CrossRef][Medline]
Isa T, Tomita S, Nakachi A, Miyazato H, Shimoji H, Kusano T, and Furukawa M (2002) Analysis of microsatellite instability, K-ras gene mutation and p53 protein overexpression in intrahepatic cholangiocarcinoma. Hepatogastroenterology 49: 604-608.[Medline]
Kiba T, Tsuda H, Pairojkul C, Inoue S, Sugimura T, and Hirohashi S (1993) Mutation of the p53 tumor suppressor gene and the ras gene family in intrahepatic cholangiocarcinomas in Japan and Thailand. Mol Carcinog 8: 312-318.[Medline]
Korman AJ, Frantz JD, Strominger JL, and Mulligan RC
(1987) Expression of human class II major histocompatibility
complex antigens using retrovirus vectors. Proc Natl Acad Sci
USA 84:
2150-2154.
Kuga T, Sakamaki S, Matsunaga T, Hirayama Y, Kuroda H, Takahashi Y, and Niitsu Y (1997) Fibronectin fragment facilitated retroviral transfer of the glutathione S-transferase pi gene into CD34+ cells to protect them against alkylating agents. Hum Gene Ther 8: 1901-1910.[Medline]
Kura T, Takahashi Y, Takayama T, Ban N, Saito T, Kuga T, and Niitsu
Y (1996) Glutathione S-transferase is secreted as a
monomer into human plasma by platelets and tumor cells. Biochim
Biophys Acta 1209:
317-323.
Lyttle MH, Hocker MD, Hui HC, Caldwell CG, Aaron DT, Engqvist-Goldstein A, Flatgaard JE, and Bauer KE (1994) Isozyme-specific glutathione-S-transferase inhibitors: design and synthesis. J Med Chem 37: 189-194.[CrossRef][Medline]
Maeda O, Terasawa M, Ishikawa T, Oguchi H, Mizuno K, Kawai M,
Kikkawa F, Tomoda Y, and Hidaka H (1993) A newly synthesized
bifunctional inhibitor, W-77, enhances adriamycin activity against human
ovarian carcinoma cells. Cancer Res
53:
2051-2056.
Metzler CM (1971) Usefulness of the two-compartment open model in pharmacokinetics. J Am Stat Assoc 66: 49-53.[CrossRef]
Miyanishi K, Takayama T, Ohi M, Hayashi T, Nobuoka A, Nakajima T,
and Niitsu Y (2001) Glutathione S-transferase-
overexpression is closely associated with K-ras mutation during human colon
carcinogenesis. Gastroenterology
121:
865-874.[CrossRef][Medline]
Morgan AS, Ciaccio PJ, Tew KD, and Kauvar LM (1996) Isozyme-specific glutathione S-transferase inhibitors potentiate drug sensitivity in cultured human tumor cell lines. Cancer Chemother Pharmacol 37: 363-370.[CrossRef][Medline]
Nakasa H, Mera N, Ohmori S, Itahashi K, Igarashi T, Ishii I, and Kitada M (1997) Nucleotide sequence of pi class glutathione S-transferase in human fetal liver. Res Commun Mol Pathol Pharmacol 97: 67-78.[Medline]
Niitsu Y, Ishigaki S, and Takahashi Y (1990) GST-pi assay for serodiagnosis of malignancy, in Glutathione S-Transferase and Drug Resistance (Heyes JD, Pickett LB, and Mantle TJ eds) pp 409-417, Taylor & Francis Ltd., London.
Niitsu Y, Takahashi Y, Ban N, Takayama T, Saito T, Katahira T,
Umetsu Y, and Nakajima T (1998) A proof of glutathione
S-transferase--related multidrug resistance by transfer of
antisense gene to cancer cells and sense gene to bone marrow stem cells.
Chem Biol Interact 24:
325-332.
Ohashi K, Nakajima Y, Kanehiro H, Tsutsumi M, Taki J, Aomatsu Y, and Yagura K (1995) Ki-ras mutations and p53 protein expressions in intrahepatic cholangiocarcinomas: relation to gross tumor morphology. Gastroenterology 109: 1612-1617.[CrossRef][Medline]
Okuda N, Nakanuma Y, and Miyazaki M (2002) Cholangiocarcinoma: recent progress. Part 1:epidemiology and etiology. J Gastroenterol Hepatol 17: 1049-1055.[CrossRef][Medline]
Parsaee S, Sarbolouki MN, and Parnianpour M (2002) In vitro release of diclofenac diethylammonium from lipid-based formulations. Int J Pharm 241: 185-190.[CrossRef][Medline]
Patel T (2001) Increasing incidence and mortality of primary intrahepatic cholangiocarcinoma in united states. Hepatology 33: 1353-1357.[CrossRef][Medline]
Penketh PG, Shyam K, and Sartorelli AC (1996) Mechanisms of resistance to alkylating agents. Cancer Treat Res 87: 65-81.[Medline]
Tada M, Omata M, and Ohto M (1990) Analysis of ras
gene mutations in human hepatic malignant tumors by polymerase chain reaction
and direct sequencing. Cancer Res
50:
1121-1124.
Takahashi Y, Hirata Y, Saito T, Yamashina T, Niitsu Y, Suzuki E,
and Hosoda K (1989) Enzyme immunoassay for human serum
glutathione S-transferase using monoclonal and polyclonal
antibodies. Cancer J 2:
225-229.
Tannapfel A, Benicke M, Katalinic A, Uhlmann D, Kockerling F, Hauss
J, and Wittekind C (2000) Frequency of p16(INK4A) alterations and
K-ras mutations in intrahepatic cholangiocarcinoma of the liver.
Gut 47:
721-727.
Tew KD, Bomber AM, and Hoffman SJ (1988) Ethacrynic
acid and piriprost as enhancers of cytotoxicity in drug resistant and
sensitive cell lines. Cancer Res
48:
3622-3625.
van Ark-Otte J, Kedde MA, van der Vijgh WJ, Dingemans AM, Jansen WJ, Pinedo HM, and Boven E (1998) Determinants of CPT-11 and SN-38 activities in human lung cancer cells. Br J Cancer 77: 2171-2176.[Medline]
Yamaguchi T and Mizushima Y (1994) Lipid microspheres for drug delivery from the pharmaceutical viewpoint. Crit Rev Ther Drug Carrier Syst 11: 215-229.[Medline]
Zuideveld KP, Rusic-Pavletic J, Maas HJ, Peletier LA, van der Graaf
PH, and Danhof M (2002) Pharmacokinetic-pharmacodynamic modeling
of buspirone and its metabolite 1-(2-pyrimidinyl)-piperazine in rats.
J Pharmacol Exp Ther
303:
1130-1137.
This article has been cited by other articles:
|
|
 |
|
  S. Hohaus, A. Di Ruscio, A. Di Febo, G. Massini, F. D'Alo', F. Guidi, G. Mansueto, M. T. Voso, and G. Leone Glutathione S-transferase P1 Genotype and Prognosis in Hodgkin's Lymphoma Clin. Cancer Res., March 15, 2005; 11(6): 2175 - 2179. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
