Serum factors that suppress cytotoxic effect of methotrexate
To study the phenomenon that human erythroid leukemia K-562 cells are more sensitive to cytotoxic effect of antimetabolites when cultured in a serum-free medium than in a conventional medium containing fetal calf serum (FCS). Methods: Cytotoxic effects of methotrexate, azaserine and 5-fluorouracil w...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2009
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| Цитувати: | Serum factors that suppress cytotoxic effect of methotrexate / T. Kojima, Y. Hashimoto, Y. Inamura, T. Koide, H. Nagata, N. Ito, K. Suzumura // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 209-213. — Бібліогр.: 8 назв. — англ. |
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irk-123456789-1382042018-06-19T03:03:56Z Serum factors that suppress cytotoxic effect of methotrexate Kojima, T. Hashimoto, Y. Inamura, Y. Koide, T. Nagata, H. Ito, N. Suzumura, K. Original contributions To study the phenomenon that human erythroid leukemia K-562 cells are more sensitive to cytotoxic effect of antimetabolites when cultured in a serum-free medium than in a conventional medium containing fetal calf serum (FCS). Methods: Cytotoxic effects of methotrexate, azaserine and 5-fluorouracil were estimated by accessing the lactate dehydrogenase (LDH) activity of viable tumor cells. Proteins of FCS were separated using two-dimensional electrophoresis followed by mass spectrometry analysis. Results: Addition of 10% FCS attenuated anti-tumor activity of methotrexate and azaserine against K-562 cells compared with serum-free medium. Such an activity of FCS was different for each serum lot. Comparison of the proteins in active serum lot with those in not active one using two-dimensional electrophoresis showed that in the active serum there were proteins 150 kDa, which were absent in the not active serum lot. Mass spectrometry indicated that all those proteins had the amino acid sequence of albumin. Sera of one healthy volunteer and two patients with thyroid cancer also attenuated the activity of the agent. Conclusion: Several lots of FCS and human serum demonstrated the ability to attenuate the cytotoxic effect of methotrexate in vitro, possibly due to the formation of albumin dimers/MTX complexes. 2009 Article Serum factors that suppress cytotoxic effect of methotrexate / T. Kojima, Y. Hashimoto, Y. Inamura, T. Koide, H. Nagata, N. Ito, K. Suzumura // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 209-213. — Бібліогр.: 8 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138204 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
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Original contributions Original contributions |
| spellingShingle |
Original contributions Original contributions Kojima, T. Hashimoto, Y. Inamura, Y. Koide, T. Nagata, H. Ito, N. Suzumura, K. Serum factors that suppress cytotoxic effect of methotrexate Experimental Oncology |
| description |
To study the phenomenon that human erythroid leukemia K-562 cells are more sensitive to cytotoxic effect of antimetabolites when cultured in a serum-free medium than in a conventional medium containing fetal calf serum (FCS). Methods: Cytotoxic effects of methotrexate, azaserine and 5-fluorouracil were estimated by accessing the lactate dehydrogenase (LDH) activity of viable tumor cells. Proteins of FCS were separated using two-dimensional electrophoresis followed by mass spectrometry analysis. Results: Addition of 10% FCS attenuated anti-tumor activity of methotrexate and azaserine against K-562 cells compared with serum-free medium. Such an activity of FCS was different for each serum lot. Comparison of the proteins in active serum lot with those in not active one using two-dimensional electrophoresis showed that in the active serum there were proteins 150 kDa, which were absent in the not active serum lot. Mass spectrometry indicated that all those proteins had the amino acid sequence of albumin. Sera of one healthy volunteer and two patients with thyroid cancer also attenuated the activity of the agent. Conclusion: Several lots of FCS and human serum demonstrated the ability to attenuate the cytotoxic effect of methotrexate in vitro, possibly due to the formation of albumin dimers/MTX complexes. |
| format |
Article |
| author |
Kojima, T. Hashimoto, Y. Inamura, Y. Koide, T. Nagata, H. Ito, N. Suzumura, K. |
| author_facet |
Kojima, T. Hashimoto, Y. Inamura, Y. Koide, T. Nagata, H. Ito, N. Suzumura, K. |
| author_sort |
Kojima, T. |
| title |
Serum factors that suppress cytotoxic effect of methotrexate |
| title_short |
Serum factors that suppress cytotoxic effect of methotrexate |
| title_full |
Serum factors that suppress cytotoxic effect of methotrexate |
| title_fullStr |
Serum factors that suppress cytotoxic effect of methotrexate |
| title_full_unstemmed |
Serum factors that suppress cytotoxic effect of methotrexate |
| title_sort |
serum factors that suppress cytotoxic effect of methotrexate |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2009 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138204 |
| citation_txt |
Serum factors that suppress cytotoxic effect of methotrexate / T. Kojima, Y. Hashimoto, Y. Inamura, T. Koide, H. Nagata, N. Ito, K. Suzumura // Experimental Oncology. — 2009. — Т. 31, № 4. — С. 209-213. — Бібліогр.: 8 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-10T05:20:10Z |
| last_indexed |
2025-07-10T05:20:10Z |
| _version_ |
1837236027891122176 |
| fulltext |
Experimental Oncology 31, 209–213, 2009 (December) 209
Antimetabolites, e. g., methotrexate (MTX), cyto-
sine arabinoside (Ara-C) and 5-fluorouracil (5-FU),
are widely used drugs in anti-tumor therapy. However,
the currently used antimetabolites have not displayed
sa tisfactory results in the clinical treatment of solid
tumors and leukemia. Originally antimetabolites were
thought to inhibit the activities of enzymes for de novo
pathways in nucleic acid biosynthesis. However, in
cancer cells the activities of enzymes for salvage
pathway of nucleic acid biosynthesis are also high.
So, researchers proposed to combine inhibitors of key
enzymes for de novo pathways of nucleic acid biosyn-
thesis with blockers of the salvage pathway [1, 2].
Recently, we have observed the phenomenon that
the anti-tumor activity of azaserine, one of the antime-
tabolites used for in vitro studies of human erythrogenic
leukemia cell line K-562, was altered by different culture
medium. The anti-tumor activity was higher in a serum-
free medium Cosmedium-001 than in another serum-
free medium –ASF 104, or modified minimum essential
medium (MEM) with 10% fetal calf serum (FCS).
MATERIALS AND METHODS
Cells, medium and FCS. A human erythroid
leukemia cell line K-562 was maintained in 10% FCS
modified Eagle’s minimum essential medium (MEM).
Modified MEM was composed of 2 mM L-glutamine,
5 x 10–5 M 2-mercaptoethanol, 25 mM HEPES and
MEM. Two serum-free media, Cosmedium-001 and
ASF 104 were purchased from Cosmo Bio Co., Ltd.
(Tokyo, Japan) and AJINOMOTO Co., Ltd. (Tokyo,
Japan), respectively. Two lots (SFB30-1674 & 1663) of
FCS were purchased from Equitec-Bio, Inc. (TX, USA),
and inactivated by incubation at 56 °C for 30 min. Bo-
vine serum albumin (BSA) was purchased from Sigma
Chemical Co. Ltd. (St. Louis, MO, USA).
Antimetabolites. Azaserine, 5-FU, MTX, Ara-C and
trimethoprim were purchased from Sigma Chemical
Co. Ltd. (St. Louis, MO, USA). Drugs were used at follow-
ing concentrations: 10–40 μM, 500–2000 ng/ml, 0.025–
0.1 μM, 25–100 μg/ml and 0.25–1 mM, respectively.
Human serum albumin. Albumin (25%) Cutter
(D419) and albumin from human serum (ALF0020)
were purchased from Bayer in Japan (Osaka, Japan)
and Wako Pure Chemical Industries Ltd. (Osaka, Ja-
pan), respectively.
Ultrafiltration. FCS or medium was filtered with
the use of Amicon centrifugal filter devices, YM-30
(nominal molecular weight: > 30 kDa) and YM-100
(nominal molecular weight: > 100 kDa) (Millipore Cor-
poration, Bedford, MA, USA). Preliminary experiments
showed that retentates of ultrafiltration with YM-30
and -100 contained molecules with molecular weight
30 kDa and 100 kDa, respectively. Both filtrate and re-
tentate were reconstituted with the starting medium.
Estimation of anti-tumor activity. Human leuke-
mic cells, mainly K-562 cells (5 x 103/well) were incu-
bated with several factors in 96-well round-bottomed
microtiter plates, each well containing 200 μl of medium
at 37 °C for 48 h. Then, the number of viable cells was
estimated by measuring the activity of released LDH
from viable cells using a kit, CytoTox 96® Non-Radioac-
tive Cytotoxicity Assay (Promega Corporation, USA).
Two-dimensional electrophoresis and mass
spectrometric analysis of proteins. In order to de-
SERUM FACTORS THAT SUPPRESS CYTOTOXIC EFFECT
OF METHOTREXATE
T. Kojima1, *, Y. Hashimoto2,Y. Inamura3, T. Koide1, H. Nagata4, N. Ito4, K. Suzumura4
1Department of Surgery, Aichi-Gakuin University School of Dentistry, Nagoya-City, Aichi 464-8651, Japan
2Department of Biochemistry, Aichi-Gakuin University School of Dentistry, Nagoya-City, Aichi 464-8650, Japan
3Department of Surgery, Yamaguchi Hospital, Seto-City, Aichi 489-0866, Japan
4Department of Digestive Surgery, Aichi Medical University, Aichi 480-1103, Japan
Aim: To study the phenomenon that human erythroid leukemia K-562 cells are more sensitive to cytotoxic effect of antimetabolites
when cultured in a serum-free medium than in a conventional medium containing fetal calf serum (FCS). Methods: Cytotoxic effects of
methotrexate, azaserine and 5-fluorouracil were estimated by accessing the lactate dehydrogenase (LDH) activity of viable tumor cells.
Proteins of FCS were separated using two-dimensional electrophoresis followed by mass spectrometry analysis. Results: Addition of
10% FCS attenuated anti-tumor activity of methotrexate and azaserine against K-562 cells compared with serum-free medium. Such
an activity of FCS was different for each serum lot. Comparison of the proteins in active serum lot with those in not active one using
two-dimensional electrophoresis showed that in the active serum there were proteins 150 kDa, which were absent in the not active serum
lot. Mass spectrometry indicated that all those proteins had the amino acid sequence of albumin. Sera of one healthy volunteer and two
patients with thyroid cancer also attenuated the activity of the agent. Conclusion: Several lots of FCS and human serum demonstrated the
ability to attenuate the cytotoxic effect of methotrexate in vitro, possibly due to the formation of albumin dimers/MTX complexes.
Key Words: serum, methotrexate, azaserine, K-562, anti-tumor activity.
Received: October 12, 2009.
*Correspondence: E-mail: taku@dpc.aichi-gakuin.ac.jp
Abbreviations used: 5-FU — 5-fluorouracil; ACN — acetonitrile;
Ara-C — cytosine arabinoside; BSA — bovine serum albumin;
FCS — fetal calf serum; HSA — human serum albumin; LDH — lac-
tate dehydrogenase; MEM — minimum essential medium; MTX —
methotrexate; TFA — trifluoroacetic acid.
Exp Oncol 2009
31, 4, 209–213
210 Experimental Oncology 31, 209–213, 2009 (December)
tect factors, responsible for the effect of FCS, we have
used the technical services of Towa Kankyo Kagaku
Co. Ltd. (Hiroshima, Japan). Two lots of FCS (active
and no activat) were separated by 2-dimenshonal
electrophoresis, and analyzed by mass spectrometry.
Isoelectric focusing (IEF) was performed using Im-
mobiline dry strip pH 4–7 (GE Healthcare UK Ltd.) and
Ampholine pH 3.5–10. Solubilized protein (100 μg) was
loaded onto a strip. SDS-PAGE was performed using
9–18% gradient gels. Protein spots were detected by
SYPRO Ruby staining (Invitrogen, CA, USA).
Protein spots were excised from the 2-D gels,
washed twice with Milli-Q water, and dehydrated in
100% acetonitrile (ACN) until they turned opaque white.
The spots were then dried in a vacuum centrifuge, and
subsequently rehydrated in 10 μl of digestion solution
consisting of 50 mM NH4HCO3, 5 mM CaCl2, 0.01 μg/μl
modified sequence-grade trypsin (Promega). After 16 h
incubation at 37 °C the digestion was terminated by
ad ding 10 μl of 5% trifluoroacetic acid (TFA). Peptides
were extracted 3 times for 20 min with 50 μl of 5% TFA,
50% ACN, and the extracts were pooled and dried in
a vacuum centrifuge. The dried materials were resus-
pended in 10 μl of 0.1% TFA. To remove excess salts
from the extracts, solid-phase extraction was performed
using C18 ZipTip (Millipore) according to the manufac-
turer’s instruction. Peptides were eluted from ZipTip by
3 μl of 50% ACN, 0.1% TFA and 1 μl of the eluants were
spotted onto a target plate. Then, the spots on the target
plate were immediately mixed with 0.5 μl of a matrix so-
lution containing 0.3 mg/ml α-cyano-hydroxycinnamic
acid, 33% acetone, 66% ethanol, and were completely
air-dried at room temperature. MS and MS/MS spectra
were obtained using an Ultraflex TOF/TOF mass spec-
trometer (Bruker Daltonics). An external peptide mixture
was used to calibrate the instrument. Identification of
proteins was carried out using the MASCOT software
(Matrix Science) and de novo sequencing.
Albumin depletion. Depletion of albumin from
FCS was performed with Vivapure® Anti-HSA Kit for
human albumin depletion (Sartorius Biotech GmbH,
Goettingen, Germany), which is based on an antibody
to human serum albumin (HSA) or MontageTM Albumin
Deplete Kit (Millipore Corporation, Bedford, MA, USA),
which is based on an affinity resin, respectively. In case
of MontageTM Albumin Deplete Kit, albumin depletion
(more than 50%) was confirmed using SDS-PAGE.
Both methods require buffer to deplete albumin from
FCS. So the buffer was removed using a centrifugal
concentrator, Apollo® 7 ml (Orbital Biosciences, LLC,
Topsfield, MA, USA), and the obtained albumin-
depleted FCS was reconstituted with PBS.
Statistics. Difference between 2 groups was
evaluated using two-tailed Student’s t-test or Welch’s
test. P values of 5 or less% were considered statisti-
cally significant.
RESULTS
Cytotoxic effect of antimetabolites on
K-562 cells. Fig. 1, a, b shows that K-562 cells were
more sensitive to azaserine and MTX when cultured
in Cosmedium-001 than in ASF 104 or 10% FCS
modified MEM. Anti-tumor activity of other analyzed
antimetabolites (5-FU, Ara-C and trimethoprim) was
not affected by culture media (Fig. 1, c–e).
Effect of FCS on anti-tumor activities of anti-
metabolites against K-562 cells cultured in Cos-
medium-001. In order to examine why anti-tumor
activities of MTX and azaserine were attenuated when
K-562 cells were cultured in 10% FCS modified MEM,
cells were cultured in Cosmedium-001 with 10% FCS.
It was shown that anti-tumor activity of 5-FU was not
altered by this medium. However, the anti-tumor activi-
ties of MTX and azaserine were attenuated.
Suppressive effect of different FCS lots on the
anti-tumor activity of MTX. Fig. 3 shows that 10% FCS
of lot No.1674, but not lot No.1663 attenuated the anti-
tumor activity of MTX. Lot No.1674 showed the highest
activity among other tested lots of FCS, and the effect
of lot No.1664 was minimal (data not shown).
Molecular weights of the FCS molecules that
suppress the anti-tumor activity of MTX. Ultra-
filtration showed that FCS molecules 100 kDa from
lot No.1674 attenuated the cytotoxic effect of MTX
(Fig. 4). However, a preliminary experiments revealed
that this fraction actually contained molecules with ap-
proximate weight of 64.2 kDa (data not shown).
Two-dimensional electrophoresis of both lots
of FCS. Fig. 5, a, b shows that the FCS (lot No.1674)
contained proteins with molecular weight of 150 kDa
(a), which were absent in the FCS (lot No.1663) (b).
Mass spectrometry showed that those proteins had
the same amino acid sequence as albumin.
Effect of albumin depletion on the suppressive
activity of the FCS. Fig. 6 shows that depleting albu-
min from FCS (lot No.1674) with 2 different methods
(antibodies and affinity resin) attenuated its suppres-
sive effect on anti-tumor activity of MTX.
Effect of human sera on the anti-tumor activity
of MTX. Fig. 7 shows that sera of one female healthy
volunteer and two female patients with thyroid cancer
at a concentration of 5% completely abrogated the
cytotoxic effect of MTX against K-562 cells cultured
in Cosmedium-001 and that the activity of individual
serum was different at concentrations of 0.05–0.5%.
Effects of HSA on the anti-tumor activity of MTX.
It is well-known that the concentration of human serum
albumin (HSA) in serum is 30~40 mg/ml. Five percent of
human serum (1.5~2 mg/ml HSA) attenuated the anti-
tumor activity of MTX (Fig. 7). So, we decided to test the
ability of two HSA preparations at the concentration of
5 mg/ml, produced by different manufacturers, to sup-
press the anti-tumor activity of MTX (Fig. 8, a). One of the
HSA preparations (Wako), lost its activity at a concentration
of 10 mg/ml. A bovine serum albumin did not exert such
an activity (data not shown). SDS-PAGE shows that not the
BSA solution but the HSA (Wako) contained proteins with
molecular weight of approximately 200 kDa (Fig. 8, b).
Experimental Oncology 31, 209–213, 2009 (December) 211
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 10 20 40
Azaserine (μM)
O
D4
92
0
0.5
1
1.5
2
0 0.025 0.05 0.1
Methotrexate (μM)
O
D4
92
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 500 1000 2000
5-FU (ng/ml)
O
D4
92
0
0.5
1
1.5
2
0 25 50 100
Ara-C (μg/ml)
O
D4
92
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.25 0.5 1
Trimethoprim (mM)
O
D4
92
a
b
c
d
e
Fig. 1. Cytotoxic effect of antimetabolites on K-562 cells cul-
tured in 3 different media. K-562 cells were incubated with the
indicated concentration of antimetabolite, azaserine (a), MTX (b),
5-FU (c), Ara-C (d) and trimethoprim (e) in Cosmedium-001 ( ),
ASF 104 ( ) or 10% FCS modified MEM ( ) at 37 °C for 48 h. Verti-
cal bars represent standard deviation (SD) of 4 determinants
0
0.5
1
1.5
2
2.5
FCS (- ) FCS (+) FCS (- ) FCS (+) FCS (- ) FCS (+)
MTX Azaserine 5-FU
O
D4
92
Fig. 2. Effect of FCS on anti-tumor activities of antimetabolites
against K-562 cells cultured in Cosmedium-001. K-562 cells
were incubated with ( ) or without ( ) 0.1 μM MTX, 40 μM aza-
serine or 2000 ng/ml 5-FU in Cosmedium-001 containing 10%
FCS < FCS (+) > or without FCS < FCS (−) > at 37 °C for 48 h.
Vertical bars represent SD of 4 determinants
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
No FCS 1663 1674
FCS lot
O
D4
92
c, d
a, b
d
c
a
b
Fig. 3. Difference between two lots of FCS in their suppressive
effect on the anti-tumor activity of MTX. K-562 cells were incu-
bated without ( ), or with 0.05 μM ( ) or 0.1 μM ( ) of MTX in
Cosmedium-001 in the presence of 10% FCS (lot No.1663 & 1674)
or with no FSC at 37 °C for 48 h. Vertical bars represent SD of
4 determinants. aNot significant (NS), bp < 0.05, c, dp < 0.001.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
None 100 000 < 100 000–30 000
Nominal molecular weight (Da)
O
D4
92
a
a
Fig. 4. The molecular weights of the molecules in the FCS that
suppressed the anti-tumor activity of MTX. K-562 cells were
incubated with ( ) or without ( ) 0.1μM MTX in 180 μl of Cos-
medium-001 and 20 μl of the indicated FCS fraction at 37 °C for
48 h. Vertical bars represent SD of 4 determinants. ap < 0.01.
Effect of pre- or simultaneous treatment of
K-562 cells with the FCS on the cytotoxicity of
MTX. Fig. 9 shows that pre-incubation of K-562 cells
with 10% FCS for 48 h did not attenuate their sensi-
tivity to MTX, but only the simultaneous incubation of
K-562 cells with 10% FCS and MTX had such effect.
Interaction between MTX and the suppres-
sive molecules in the FCS. MTX (0.2 μM) and FCS
(lot No.1674) (20%) were incubated at 37 °C for 24 h.
Their concentrations were 2 times higher than those in
212 Experimental Oncology 31, 209–213, 2009 (December)
the other experiments since they were finally diluted
to 0.1 μM (MTX) and 10% (FCS). Upon filtration by
YM-100 filter, the anti-tumor activities of both filtrates
and retentates were examined. Fig. 10 shows that the
filtrate of MTX without the FCS exerted its anti-tumor
activity. On the other hand, the filtrate of MTX incubated
with the FCS did not exert its anti-tumor activity. Each
retentate of MTX incubated with or without the FCS
exerted weak anti-tumor activity to the same degree.
DISCUSSION
Many authors reported that albumin functions as
a carrier for a variety of molecules. Albumin is the
principle carrier of fatty acids [3–5], bilirubin [4, 5],
metals including zinc [5, 6], hormones [7] and several
important classes of drugs, e. g., calcium blockers,
diuretics and non-steroidal anti-inflammatory drugs
kDa
225
150
100
75
50
35
25
15
10
4 5 6 7 pl
kDa
225
150
100
75
50
35
25
15
10
4 5 6 7 pl
a
b
Fig. 5. Two-dimensional electrophoresis of two lots of FCS. The
active FCS (No. 1674) (a) and the not active FCS (No. 1663) (b).
Spots indicated by circles were specific in each FCS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Albumin-depletedIntact FCS No FCS
O
D4
92
a, b
a a
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Albumin-depletedIntact FCS No FCS
O
D4
92
c, d
c d
a
b
Fig. 6. Effect of albumin depletion on the suppressive activity
of the FCS. K-562 cells were incubated with indicated fractions
(10%) in the presence ( ) or absence ( ) of 0.1 μM MTX in
Cosmedium-001 at 37 °C for 48 h. Albumin depletion: Vivapure
®Anti-HSA Kit (a), Montage Albumin Depletion Kit (b). Vertical
bars represent SD of 4 determinants. ap < 0.01, bp < 0.001,
cp < 0.05 (one-tailed),dp < 0.05.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 0.05 0.5 5 0 0.05 0.5 5 0 0.05 0.5 5
TS TT MM
Serum (%)
O
D4
92
Fig. 7. Effect of human sera on the anti-tumor activity of MTX.
K-562 cells were incubated with ( ) or without ( ) 0.1 μM MTX
in Cosmedium-001 containing the indicated concentrations
of individual human serum at 37 °C for 48 h. TS, TT and MM
represent initials of volunteers. Vertical bars represent SD of
4 determinants.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 5 10 0 5 10
Wako Bayer
HSA (mg/ml)
O
D4
92
a
a
b
b
c
Origin —
204 kDa —
121 kDa —
82 kDa —
50.2 kDa —
34.2 kDa —
28.1 kDa —
19.3 kDa —
Std H B
— *
a
b
Fig. 8. Effect of HSA on the anti-tumor activity of MTX. K-562 cells
were incubated with the indicated concentrations of HSA produced
by two manufacturers, Wako and Bayer, in the presence ( ) or ab-
sence ( ) of 0.1 μM MTX in Cosmedium-001 at 37 °C for 48 h (a).
Vertical bars represent SD of 4 determinants. ap < 0.05, bp < 0.001,
cp < 0.05. Wako’s HSA (H) and BSA (B) were analyzed by SDS-
PAGE (b). *Shows that HSA had indicated band, but BSA did not.
Experimental Oncology 31, 209–213, 2009 (December) 213
[5]. All these authors consider albumin as a carrier
molecule, especially Kratz [4] and his colleagues [8].
The present study suggested that proteins with high
molecular weight derived from albumin (possibly its
dimers or trimers) could bind MTX, and suppress its
anti-tumor activity.
K-562 cells cultured in Cosmedium-001 were more
sensitive to cytotoxic effect of azaserine and MTX than
cells cultured in ASF 104 and 10% FCS modified MEM.
The cytotoxic effect of other antimetabolites, 5-FU,
Ara-C and trimethoprim against K-562 cells was not
altered by culture media. When some lots of FCS were
added to Cosmedium-001 at a concentration of 10%,
MTX and azaserine exerted low anti-tumor activities to
K-562 cells as compared with Cosmedium-001 alone.
This finding suggests that some factors in the FCS
were responsible for suppressing the anti-tumor activi-
ties of both drugs. Lower anti-tumor activities of MTX
and azaserine to K-562 cells cultured in ASF 104 than
in Cosmedium-001, remain to be clarified.
The two-dimensional electrophoresis showed
that the active FCS contained proteins with molecular
weights 150 kDa, which were absent in the not active
FCS lot. Mass spectrometry analysis revealed that those
proteins had the same amino acid sequence as albumin.
It seems that these proteins with high molecular weight
are complexes of albumin. When albumin was depleted
from the active FCS by means of 2 different methods
(albumin binding affinity resin and anti-human serum al-
bumin antibody), its suppressive activity was clearly at-
tenuated. All tested human sera (one healthy volunteer
and two patients with thyroid cancer) suppressed the
anti-tumor activity of MTX against K-562 cells cultured
in Cosmedium-001. Most of HSA samples are expected
to exert such an activity. In fact, 2 tested commercially
available HSA preparations exerted the activity. In any
case, the activity of a larger number of human sera
remains to be examined.
We hypothesized that the FCS did not resolve
MTX chemically, but bound it. We suppose that pos-
sibly such MTX-binding molecules are complexes of
albumin.
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Copyright © Experimental Oncology, 2009
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Pre- Simultaneous
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D4
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Fig. 9. Effect of pre- or simultaneous treatment of K-562 cells with
the FCS on the anti-tumor activity of MTX. K-562 cells (2 x 105/flask)
were incubated with (10%) or without FCS in 3 ml of Cosme-
dium-001 at 37 °C for 48 h. After washing the cells twice with 3 ml
of PBS, they were incubated (5 x 103/well) with ( ) or without ( )
0.1 μM MTX in 200 μl Cosmedium-001 at 37 °C for 48 h (Pre-). Alter-
natively, K-562 cells were incubated with ( ) or without ( ) 0.1 μM
MTX in the presence (10%) or absence of FCS at 37 °C for 48 h
(simultaneous). Vertical bars represent SD of 4 determinants
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Retentate Elute
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D4
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Fig. 10. Interaction between MTX and the suppressive molecules in
the FCS. MTX (0.2 μM) was incubated with ( ) or without ( ) 20%
FCS in 500 μl Cosmedium-001 at 37 °C for 24 h. The media were
ultrafiltrated with a YM-100 filter, and the filtrate and the retentate
were collected followed by reconstitution with Cosmedium-001.
K-562 cells were incubated in 100 μl Cosmedium-001 with 100 μl of
the filtrate or the retentate at 37 °C for 48 h. Vertical bars represent
SD of 4 determinants
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