Download Combined effects of bisphosphonate and radiation on fibrosarcoma

Combined effects of bisphosphonate and radiation on fibrosarcoma cells
+* **Sawai, Y; *Murata, H; * Koto, K; *Sakabe T; *Matsui T; *Horie, N; ***Kimura, S; ****Maekawa, T; **Fushiki, S; *Kubo, T
+*Department of Orthopaedics, Graduate School of Medical Science. Kyoto Prefectural University of Medicine, Kyoto, Japan
**Department of Pathology and Applied Neurobiology, Graduate School of Medical Science. Kyoto Prefectural University of Medicine, Kyoto, Japan
***Division of Hematology, Respiratory Medicine, and Oncology, Department of Internal Medicine, Faculty of Medicine, Saga University, Saga, Japan
****Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, Kyoto, Japan
e-mail: [email protected]
INTRODUCTION:
We reported that third-generation bisphosphonates (BPs),
zoledronic acid (ZOL) significantly inhibit the in vitro growth of human
fibrosarcoma cell lines in a time- and dose-dependent manner by
preventing prenylation of small GTPases and inducing apoptosis (53st
ORS). To investigate the effect of BPs further, we examined the way to
augment the effect of BPs by combination with radiation.
Figure 1: X-axis; a: control, b: 1.5µM of ZOL alone, c: 0.5×IC50 of
anti-cancer drug alone, d: combination b with c, e: 1.0×IC50
of anti-cancer drug, f: combination b with d. Y-axis is cell
counts(×105). A: doxorubicin, B: cisplatin, C: etoposide, D:
5-fluorouracil, E: paclitaxel, F: docetaxel, G: gemcitabine, H:
methotrexate. *p＜0.025, **p＞0.05,
MATERIALS AND METHODS:
We used third-generation BPs, ZOL Cultured cells were irradiated
with 20-800cGy X-Ray using SOFTEX M-150WE (SOFTEX CO.,
LTD., Tokyo, Japan). The irradiation conditions selected were a distance
of 1cm from the focus to the specimen, an irradiation rate of 0.5Gy/min
in air. We used human fibrosarcoma cell lines, HT1080 and human
synovial sarcoma, Syo-1.
Concurrent exposure to ZOL and other anti-cancer drugs
Cell growth and viability was evaluated by the MTT assay method.
The IC50 values were obtained by using the nonlinear regression
program CalcuSyn (Biosoft, Cambridge, United Kingdom).
(1)Growth inhibitory effects of ZOL with anti-cancer drugs
To investigate the combind effect of ZOL with other anticancer agents,
the HT1080 cells were treated with 1.5 µM, which is about IC50
concentration, of ZOL alone, an anti-cancer drug alone and the
ZOL/anti-cancer drug combination in 6-well plates for 72h. The
concentrations of these anticancer drugs were 1.0×IC50 and 0.5×IC50
of each. Proliferation of the cell lines were determined using the trypan
blue dye exclusion method. The results are shown as mean ±SD.
(2) Cell cycle analysis and induction of apoptosis
Untreated HT1080 cells or HT1080 cells treated with ZOL alone, an
anti-cancer drug alone and the ZOL/anti-cancer drug combination for 72
h were analyzed for alterations in the cell cycle by staining with
propidium iodide (PI) (Sigma Aldrich) and made histogram.
Sub-G1(%)
anti-cancer agent
0.5×IC50
1.0×IC50
ZOL1.5uM + 0.5×IC50
ZOL1.5uM + 1.0×IC50
B
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5
a
b c d
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5
a
b c d
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b e f
E
a
b e f
b c d
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5
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a
b e f
b c d
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5
a
b c d
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b e f
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5
a
b e f
G
F
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D
C
5
H
5
a
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b c d
b e f
b c d
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b e f
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5
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a
b c d
A
5.51
7.33
29.23
32.75
B
6
21.09
14.99
21.63
C
7.48
8.69
32.15
39.89
D
1.83
2.33
11.4
15.06
E
9.66
26.64
43.98
50.09
F
2.85
6.85
11.43
24.04
G
2.89
3.18
12.34
24.31
Figure 2: Cell cycle analysis using flow cytometry. An anti-cancer
drug is etoposide. X-axis is DNA count. Y-axis is cell count. a－f :
similar to figure1.
RESULTS:
(1) Cytotoxic interactions from concurrent exposure of cells to ZOL and
other anti-cancer drugs
When combined with concentration of 0.5×IC50 values, and 1.0
× IC50 values of doxorubicin, cisplatin, etoposide, 5fluorouracil,
docetaxel, paclitaxel, and gemcitabine, growth inhibitory effects was
augmented compared to by ZOL alone or by anti-cancer drugs alone
(Figure 1). In contrast, when combined with methotrexate, P-value
exceeded 0.05, indicating that combination with methotrexate produces
antagonistic rather than additive effects. Only MTX, combined effect
was not acquired.
A
(2) Effects of ZOL and other anti-cancer drugs on the cell cycle and
induction of cell death
The Sub-G1 in flow cytometric analysis was also increased
by combined treatment ZOL with other anti-cancer drugs excluded
methotrexate.
In
e
a
c
the histogram, G1,
S and G2/M phase
was not so changed
by
combined
treatment
compared to by
f
d
b
each drugs alone.
(Figure 2, table 1).
A-E: similar to
Fig.1.
b e f
Table 1: The sub-G1 in flow cytometry. A－H : similar to figure 1.
DISCUSSION:
We reported that third-generation BPs, ZOL significantly inhibit
the in vitro growth of human fibrosarcoma. But in clinical practice,
administration of ZOL (4mg in 100ml over 15 minutes) resulting a peak
systemic concentration, and the drug is rapidly cleared from the
circulation within 1-2 hours. It is therefore likely that peripheral tumors
are exposed to low concentration of ZOL for only a few hours. So, when
using ZOL in practice, combined treatment with other agents should be
encouraged, we suppose.
The aim of this study was to determine whether the anti-tumor
effect of BPs can be strengthen when it was combined with other antitumor drugs.
In this present study, we found that combination of ZOL with
etoposide has strong antitumor effect against human fibrosarcoma cells
compared to the use of any of these agent alone. These results may have
therapeutic application, particularly for enhancing the efficacy of a drug
that cannot be administered at higher dosages because of its toxicity.
Therefore, we may be able to combine ZOL with the present
fibrosarcoma chemotherapy regimes. In addition, the anti-growth
activities of doxorubicin, cisplatin, 5-fluorouracil, docetaxel, paclitaxel
and gemcitabine were also augmented by ZOL. Therefore, ZOL may
have therapeutic application in enhancing the efficacy of these drugs
such that their effective doses against other malignant neoplasms, such
as osteosarcoma, lung cancer and ovarian cancer, can be reduced.
Although we did not actually determine whether the combination
Poster No. 1424 • ORS 2011 Annual Meeting
H
1.65
2.96
4.84
4.39
overcomes the multi-drug resistance system, these results appear to offer
benefits to fibrosarcoma patients.
In conclusion, our results indicated that the combination of ZOL
with doxorubicin, cisplatin, etoposide, 5-fluorouracil, docetaxel,
paclitaxel, and gemcitabine may have more effectiveness against human
fibrosarcoma than the use of any of these drugs alone.