I. Introduction
Malignant fibrous histiocytoma (MFH) is one of the
most common high-grade sarcomas in late adult life that develops in bone and
soft tissue. MFH has a predilection for extremities, and the prognosis of the
disease is reported to be poor due to its chemo-resistance (Papai et al, 2000; Reichardt, 2002; Spira and Ettinger,
2002).
Recent studies introduced combination adjuvant chemotherapy protocols
consisting of doxorubicin, cisplatin, and high-dose methotrexate (HD-MTX) for
skeletal osteosarcomas, and the successful clinical results have been reported (Bacci et al, 2001, 2002; Thompson et al, 2002).
Of these, HD-MTX with citrovorum factor (CF) rescue regimen is one of the
frequently used chemotherapy protocol for osteosarcoma (Breithaupt and Kuenzlen, 1983).
In contrast, the effectiveness of adjuvant chemotherapy for spindle-pleomorphic
sarcomas including MFH of soft tissue remains controversial. Although many
chemotherapy protocols currently used for spindle-pleomorphic soft tissue
sarcomas consist of doxorubicin, epirubicin, cisplatin, and ifosfamide (Edmonson et al, 2002; Reichardt, 2002),
there is little consensus concerning the doses and the combinations of these
chemotherapeutic agents. To our knowledge, little has been reported concerning
the clinical use of HD-MTX for soft- tissue MFH. The purpose of the present
study is to experimentally examine the effects of the HD-MTX with CF rescue
regimen on proliferative activity of a MFH cell line, comparing that of an
osteosarcoma cell line.
II.
Materials and methods
A. Cell lines and cell culture
The MFH
cell line TNMY1 that was previously established in our laboratory was used (Nakatani et al, 2001). The osteosarcoma cell line KHOS/NP was
purchased from American Type Culture Collection (CRL-1427, Rockville, MD, USA).
Each cell line was maintained as a monolayer culture in a humidified 5% CO2
incubator at 37¡C. The culture medium consisted of EagleÕs minimum essential
medium (MEM) containing 0.292 g/L L-glutamine (Sigma Chemical Co., St. Louis,
MO, USA) supplemented with 10% fetal bovine serum (Sigma Chemical Co.), 100
U/ml penicillin G (Sigma Chemical Co.), and 100 U/ml streptomycin (Sigma
Chemical Co.).
B.
Chemicals
Methotrexate and CF were kindly provided by Lederle Laboratories (Pearl River, NY, USA).
C. The
effect of MTX on the cell proliferation
The cells were suspended in the culture medium at a
concentration of 5.0x103/ml. A hundred microliter aliquot of the
cell suspension was dispersed to each well in 96-well cell culture plates
(Costar, Cambridge, Massachusetts, U.S.A), and the cells were kept quiescent
for 48 hours. The culture medium of each well was replaced by 100 ml of fresh medium, and MTX was pulsed to each well at a
final concentration of 1.0 x 10-2 to 1.0 x 10-8mol/L. An
equivalent volume of fresh medium, instead of MTX, was added to certain wells
to serve as controls. At this time, the serum concentration in the culture
medium was reduced to 1%. After the cells were incubated for 24 hours, the
culture medium was replaced by a new medium following cell rinse with phosphate
buffer saline (PBS). Finally, the cells were incubated another 0, 48, or 96
hours.
The cell viability was determined using a modified MTS
tetrazolium assay (Malich et al, 1997). The medium in each well was replaced by 100 ml of EagleÕs MEM with 0.292 g/L L-glutamine without
FBS, and 20 ml
of MTS reagent (CellTiter 96Ⓡ AQueous
Non-Radioactive Cell Proliferation Assay, Promega Co., Madison, WI, USA) was
pulsed to each well. After 2 hours of further incubation at 37¡C in a
humidified 5% CO2 incubator, the optical density (OD) value of each
well was measured at 490nm using an automatic microplate reader (Microplate
Reader, Bio Rad, USA). The growth inhibition (Gi) rate of the cells in each
well was determined by the following equation (Reinecke
et al, 2000).
Gi = {1- (ODS -ODB)/ (ODC-ODB)}x100
Gi; growth inhibition rate (%)
ODS; OD value of each sample
ODC; OD value of control
ODB; OD value of blank well
The data were shown in mean ± SD from six replicate
wells for each MTX concentration and three replicate experiments for each cell
lines.
D. The reversal effect of CF on the cytotoxicity of MTX
The monolayered cell culture of TNMY1 cell line was prepared in a plastic 96-well plate in the same way as was described above. After the cells were exposed to subsequent doses of MTX for 24 hours, the culture supernatant was replaced by refresh culture medium containing 1% FBS, 100U/ml penicillin G, and 100U/ml streptomycin. Then, the cells were cultured for 96 hours in absence or presence of CF at the concentration of 10-7 mol/L. The cell growth inhibition rate of each well was determined by MTS assay.
E.
Statistical analysis
The data was analyzed using One-way repeated measures ANOVA for comparing the Gi rates in each group, and Two-way repeated measures ANOVA for comparison between each cell line or each incubation time. The p-values less than 0.05 were considered statistically significant.
III. Results
A. The effect of MTX on the cell
proliferation
The Gi rates of the KHOS/NP cells after 24 hours
exposure to MTX and additional 0 hour incubation are shown in Table 1A and Figure 1, and those in the TNMY1 cell line are shown in Table 1B and Figure 2, respectively. One-way repeated measures ANOVA showed that MTX
significantly inhibited the cell growth of the KHOS/NP cell line at the
concentrations of 10-8 to 10-2 M dose-dependently, but no
significant effect was observed on the TNMY1 cell line.
The Gi rates of the cells after 24 hours exposure to
MTX and additional 48-hour incubation in the KHOS/NP cell line are shown in Table
2A and Figure 1, and those in the TNMY1 cell line are shown in Table
2B and Figure 2, respectively. One-way repeated measures ANOVA showed
that MTX significantly inhibited the cell growth at the concentrations of 10-8
to 10-2 M in the KHOS/NP cell line, while MTX inhibited the cell
growth at the concentrations of 10-6 to 10-2 M in the
TNMY1 cell line.
The Gi rates of the cells after 24 hours exposure to
MTX and additional 48-hour incubation in the KHOS/NP cell line are shown in Table
3A and Figure 1, and those in the TNMY1 cell line are shown in Table
3B and Figure 2, respectively. One-way repeated measures ANOVA showed
that MTX significantly inhibited the cell growth at the concentrations of 10-8
to 10-2 M in the KHOS/NP cell line, while MTX inhibited the cell
growth at the concentrations of 10-7 to 10-2 M in the
TNMY1 cell line.
Two-way repeated measures ANOVA revealed that the
growth inhibitory effect of MTX on both KHOS/NP and TNMY1 cell lines
significantly increased by incubating for 48 and 96 hours after the cells were
exposed to MTX.
Table
1A. Gi rates of the KHOS/NP cells
after 24 hours exposure to MTX and additional 0 hour incubation
|
Gi rate (%) KHOS/NP cell line |
MTX concentration (M) |
|
35.0±15.4 |
10-8 |
|
49.8±6.2 |
10-7 |
|
50.1±6.7 |
10-6 |
|
51.0±7.9 |
10-5 |
|
50.3±6.7 |
10-4 |
|
51.4±6.7 |
10-3 |
|
60.5±4.6 |
10-2 |
Table
1B. Gi rates of the TNMY1 cell line
after 24 hours exposure to MTX and additional 0 hour incubation
|
Gi rate (%) TNMY1 cell line |
MTX concentration (M) |
|
1.4±2.2 |
10-8 |
|
2.5±2.6 |
10-7 |
|
4.5±1.7 |
10-6 |
|
4.7±2.9 |
10-5 |
|
6.9±5.9 |
10-4 |
|
7.6±5.5 |
10-3 |
|
4.0±7.9 |
10-2 |
Table
2A. Gi rates of the KHOS/NP cells
after 24 hours exposure to MTX and additional 48-hour incubation
|
Gi rate (%) KHOS/NP cell line |
MTX concentration (M) |
|
67.4±3.1 |
10-8 |
|
69.2±2.6 |
10-7 |
|
69.2±2.8 |
10-6 |
|
69.9±2.8 |
10-5 |
|
70.5±2.6 |
10-4 |
|
72.1±3.3 |
10-3 |
|
0.6±1.8 |
10-2 |
Table
2B. Gi rates of the TNMY1 cell line
after 24 hours exposure to MTX and additional 48-hour incubation.
|
Gi rate (%) TNMY1 cell line |
MTX concentration (M) |
|
2.6±3.9 |
10-8 |
|
7.0±4.3 |
10-7 |
|
20.2±5.9 |
10-6 |
|
35.8±4.5 |
10-5 |
|
34.7±4.8 |
10-4 |
|
34.2±3.9 |
10-3 |
|
35.1±5.5 |
10-2 |
Table
3A. Gi rates of the KHOS/NP cells
after 24 hours exposure to MTX and additional 96-hour incubation
|
Gi rate (%) KHOS/NP cell line |
MTX concentration (M) |
|
34.7±13.8 |
10-8 |
|
71.9±12.2 |
10-7 |
|
74.8±9.9 |
10-6 |
|
75.6±9.4 |
10-5 |
|
75.1±9.3 |
10-4 |
|
74.3±8.7 |
10-3 |
|
77.3±6.2 |
10-2 |
Table
3B. Gi rates of the TNMY1 cell line
after 24 hours exposure to MTX and additional 96- hour incubation
|
Gi rate (%) TNMY1 cell line |
MTX concentration (M) |
|
0.9±2.1 |
10-8 |
|
15.1±4.5 |
10-7 |
|
43.5±16.6 |
10-6 |
|
63.4±5.0 |
10-5 |
|
62.9±4.2 |
10-4 |
|
63.1±6.0 |
10-3 |
|
60.3±9.4 |
10-2 |
Figure
1: The effect of MTX on the cell
proliferation of the osteosarcoma cell line KHOS/NP. Each value represents the
mean ± standard deviation from MTS assays of six replicate wells per microtiter
plate and three replicate experiments per cell line (n=18). An asterisk beside
each error bar indicates statistical significance (p<0.05) compared to the
control specimen. MTX: methotrexate
Figure
2: The effect of MTX on the cell
proliferation of the MFH cell line TNMY1. Each value represents the mean ±
standard deviation from MTS assays of six replicate wells per microtiter plate
and three replicate experiments per cell line (n=18). An asterisk beside each
error bar indicates statistical significance (p<0.05) compared to the
control specimen. MTX: methotrexate
B. The
reversal effect of CF on the cytotoxicity of MTX
The Gi rates of the TNMY1 cells after 24 hours exposure to MTX and subsequent 96-hour incubation in the absence of CF at the MTX concentrations of 10-7, 10-6, 10-5, 10-4, and 10-3 M, are shown in Table 4A and Figure 3, and those in the presence of 10-7 M CF are shown in Table 4B and Figure 3, respectively. Two-way repeated measures ANOVA showed no significant decrease in the Gi rates in the presence of 1.0 x 10-7 mol/L CF. One-way repeated measures ANOVA revealed no significant decrease in the Gi rates at any concentrations of MTX in the presence of CF except for at 1x10-6 M.
Table
4A. Gi rates of the TNMY1 cell line
after 24 hours exposure to MTX and additional 96- hour incubation in the
absence of CF.
|
Gi rate (%) TNMY1 cell line |
MTX concentration (M) |
|
6.1±11.1 |
10-7 |
|
36.1±15.0 |
10-6 |
|
63.1±5.7 |
10-5 |
|
61.7±4.2 |
10-4 |
|
59.7±2.7 |
10-3 |
Table
4B. Gi rates of the TNMY1 cell line
after 24 hours exposure to MTX and additional 96- hour incubation in the
presence of 10-7 M CF.
|
Gi rate (%) TNMY1 cell line |
MTX concentration (M) |
|
6.0±11.6 |
10-7 |
|
20.9±9.4 |
10-6 |
|
57.4±7.4 |
10-5 |
|
60.7±3.6 |
10-4 |
|
60.8±3.0 |
10-3 |
Figure 3: The reversal effect of CF on the cytotoxicity of MTX. Each value represents the mean±standard deviation from MTS assays of six replicate wells per microtiter plate and three replicate experiments per cell line (n=18). An asterisk beside each error bar indicates statistical significance (p<0.05). MTX: methotrexate, CF: Citrovorum factor.
IV. Discussion
Chemotherapy for locally advanced or metastatic
spindle-pleomorphic soft tissue sarcomas remains highly investigational. Doxorubicin,
epirubicin, and ifosfamide are chemotherapeutic agents commonly used, with a
clinical response rate of approximately 20% (Reichardt, 2002); little has been known concerning the effect of HD-MTX with CF rescue
on spindle-pleomorphic soft tissue sarcomas either in vivo or in vitro. The
present experimental study was designed to provide a conceptual basis for
further optimization of the HD-MTX with CF rescue protocol that may be useful
in the practical use. The concentration ranges of MTX used in this study were
determined to simulate the actual serum levels attained clinically 24 hours
after cessation of intravenous administration of HD-MTX in osteosarcoma
chemotherapy (Breithaupt and Kuenzlen, 1983). The standard dose of CF in the practical regimen in osteosarcoma
chemotherapy is 15 mg/m2 for every six hours, bearing a serum
concentration of 1x10-6 to 1x10-7 M (Diddens
et al, 1987).
In the present study, the data obtained showed that
the response to MTX in the MFH cells was significantly lower than that in the osteosarcoma
cells shortly after the cells were exposed to MTX for 24 hours. However, a
delayed effect of MTX on the MFH cell proliferation was observed; the Gi rates
in the MFH cells significantly increased 48 and 96 hours after 24-hour exposure
to MTX time-dependently, with the maximal Gi rate of approximately 65%.
The cause of such a delayed effect of MTX on the MFH
cell growth is of an important consideration. MTX is a potent inhibitor of
dihydrofolate reductase (DHFR), a key enzyme for intracellular folate
metabolism (Bertino et al, 1996). After transportation into the cell, MTX needs to be polyglutamylated
by folylpolyglutamate synthetase (FPGS) before functioning as DHFR inhibitor.
DHFR inhibition by polyglutamylated MTX results in decreased regeneration of
tetrahydorfolate from dihydrofolate, which is a product of thymidylate
synthase, leading to decrease in DNA biosynthesis (Bertino
et al, 1996). The decreased transport of MTX into the cells, impaired
polyglutamylation of MTX, and increased DHFR enzyme activity of the cells have
been suggested to be the main factors of cell resistance to MTX (Bertino
et al, 1996). Assuming that the MFH cells had increased activities of the latter
two pathways, the growth inhibition rates should have remained low despite the
subsequent incubation after MTX exposure. There is a possibility that a
decreased transport of MTX into the MFH cells could result in a delayed
increase of intracellular concentration of polyglutamylated MTX, leading to the
late response to MTX.
Our data showed that the growth inhibition effects of
MTX on both MFH and osteosarcoma cells increased dose-dependently. The maximal
Gi rate was attained at the concentrations of over 1x10-5 M in the
MFH cells, and at the concentrations of over 10-7 M in the
osteosarcoma cells when the cells were subjected to 96 hours incubation
following 24-hour exposure to MTX. In the clinical HD-MTX chemotherapy, the
serum concentrations of MTX have been reported to be 1x10-5 to 1x10-3
M during 24 hours after administration (Breithaupt and Kuenzlen, 1983). These data indicate that the MTX dosages used in the clinical
chemotherapy for osteosarcomas could sufficiently cover the effective serum
concentration levels for MFH of soft tissue. Although the maximum Gi rate in
MFH cells was lower than that in osteosarcoma cells, the MFH cells are
considered to be sensitive to MTX.
Citrovorum factor is
readily converted to reduced folate independently of DHFR within the cells and
bypasses the block of DHFR by MTX, thereby replenishing cellular pools of
tetrahydrofolate depleted by MTX action (Diddens et al, 1987; Haskell, 1980).
Since a certain neoplastic cell has an insufficient active transporting ability
of CF in its cell membrane, such a protective effect of CF from MTX
cytotoxicity is often more effective in normal tissues than in neoplasms (Haskell, 1980).
The data in the present study showed that 10-7 M CF did not reverse
the cell proliferation of TNMY1 cell line when the cells were pretreated with
over 10-5 M of MTX, although a significant reversal effect of CF was
observed after pretreatment with 10-6 M of MTX. The reversal effect
of CF after pretreatment with the lower dose of MTX could be explained by the fact
that MTX and CF competitively share an active transporting mechanism, hence
high levels of MTX may require large doses of CF to prevent cell death (Haskell, 1980).
These data indicated that the standard concentration of CF administration after
HD-MTX could act as the rescue for normal cells without preventing the
cytotoxicity of MTX for TNMY1 cells.
V.
Conclusions
Our data in the present study have indicated that MFH
of soft tissue is potentially sensitive to MTX. The HD-MTX with CF rescue
chemotherapy protocol that is frequently used for osteosarcoma is possibly
applicable to treatment of MFH of soft tissue, although clinical prospective
studies are required to verify this.
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