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Evaluation of Cisplatin Concentration in Response to
Tumor Hypoxia in Esophageal Squamous Cell
Carcinoma
M. Sakai a) , N. Tanaka a) , H. Kimura a) , T. Inose a) , M. Sohda a) , M. Nakajima a) , H. Kato a) ,
T. Asao a) , H. Kuwano a) , M. Oikawa b) , T. Satoh c) and T. Kamiya c)
a) Graduate School of Medicine, Gunma University, b) Fundamental Technology Center, National
Institute of Radiological Science, c) Department of Advanced Radiation Technology, TARRI, JAEA
Chemotherapy and radiotherapy can provide effective
treatments for Esophageal Squamous Cell Carcinoma
(ESCC). Cis-diaminedichloroplatinum (II) (CDDP) or
cisplatin, a DNA-damaging agent, is widely used as a
chemotherapeutic drug for the treatment of various human
malignancies, including ESCC. To date, tumor hypoxia
appears to be strongly associated with tumor propagation,
malignant progression, and resistance to therapy, and it has
thus become a central issue in tumor physiology and cancer
treatment. The aim of our study is to evaluate cisplatin
concentration in response to oxygenation state in a single
tumor cell (TE-2: human ESCC cell line) using In-Air
Micro-PIXE, the newly-developed device that enable to
visualize trace element distribution in a single cell.
Cell sample preparation was performed as follows.
Exponentially growing TE-2 cells were labeled with 100 μL
(per 10-cm dish) bromodeoxyuridine (BrdU; BD
Biosciences, San Jose, CA, USA) for 24 h. The 4-μm thick
Mylar films were stretched between Pyrex glass and Viton
rings, followed by immersion in 5 mol sulfuric acid for 2 h
and washing with distilled water three times. Twenty-four
hours after the BrdU labeling procedure, cells were
trypsinized and counted using a hemocytometer, then 1 ×
10 6 cells were seeded on a Mylar film in a culture dish for
24 h under normoxia (O2 : 21%) or hypoxia (O2 : 1%).
Then, the original medium on Mylar film was replaced with
CDDP containing medium (0.5-mmol CDDP). After
2 hours of CDDP containing medium exposure, this medium
was removed and cells were washed five times with a
tris-hydroxymethylaminomethane-HNO 3 solution (pH 7.4).
The cells were then cryofixed by soaking the samples in
isopentane at liquid nitrogen temperature. The frozen
samples were dried for 24 h in a freeze-drier and kept in a
desiccator until PIXE analysis. The In-Air Micro-PIXE
analysis was performed at TIARA as described previously 1) .
We used the calibration curve constructed by Sakurai et al. 1)
for the quantitative evaluation of cisplatin.
Figure 1 shows the comparison of intracellular and
nuclear CDDP concentration in TE-2 cultured under
normoxia and hypoxia. Intracellular CDDP concentration
in TE-2 cultured under hypoxia was significantly lower than
that of under normoxia (p=0.0365). Although nuclear
CDDP concentration in TE-2 had no significant deference
between cultured under hypoxia and normoxia (p=0.1259),
nuclear CDDP concentration in TE-2 cultured under
JAEA-Review 2010-065
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hypoxia had a tendency to be lower than that of under
normoxia. Our results show that tumor hypoxia may affect
intracellular and nuclear CDDP concentration.
2)
In the previous study , we investigated several factors
that affected CDDP concentration in two human ESCC cell
lines (TE-2 and TE-13. TE-2 cells were more sensitive to
cisplatin than TE-13 cells). Multidrug resistance protein 2
(MRP2), which belong to the ATP-binding cassette cell
3)
membrane transporter family , is one of those factors.
4)
MRP2 is reported to be involved in resistance to cisplatin .
We compared MRP2 expression in cisplatin-treated TE-2
cells and TE-13 cells as a chemoresistance marker for
cisplatin-based therapy, using real-time reverse
transcriptase-polymerase chain reaction (RT-PCR). MRP2
expression in TE-13 cells was significantly higher than that
in TE-2 cells. It therefore seems likely that cell membrane
transporters play an important role in affecting intracellular
cisplatin concentration and its sensitivity. In our present
study, the mechanism of the effect of tumor hypoxia to
intracellular CDDP concentration was not fully investigated.
It is clear that further studies are needed to elucidate the
expression of the factors which was involved in resistance to
cisplatin, such as MRP2, in ESCC under hypoxia.
References
1) H. Sakurai et al., Cancer Sci. 99 (2008) 901-904.
2) N. Tanaka et al., Cancer Sci. (2010) Feb 25.
3) M. Michael et al., Nat. Rev. Cancer 2 (2002) 48-58.
4) R. G. Deeley et al., Physiol. Rev. 86 (2006) 849-99.
CDDP concentration (×10-18 mol/μm2 CDDP concentration (×10 )
-18 mol/μm2 )
16
14
12
10
8
6
4
2
intracellular
p= 0.0365
20
17.5
15
12.5
10
7.5
2.5
0
Normoxia Hypoxia
0
(n=5) (n=6)
Normoxia
(n=2)
Hypoxia
(n=3)
Fig. 1 Comparison of intracellular and nuclear CDDP
concentration in TE-2.
5
nuclear
p= 0.1259