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930
Intracellular Accumulation of Thallium as a Marker of
Cisplatin Cytotoxicity in Nonsmall Cell Lung
Carcinoma
An Application of Inductively Coupled Plasma Mass Spectrometry
Taro Hanada, M.D.1
Hiroshi Isobe, M.D.2
Takeshi Saito, Ph.D.3
Shigeaki Ogura, M.D.1
Hironori Takekawa, M.D.1
Kohichi Yamazaki, M.D.1
Yoshio Tokuchi, M.D.1
Yoshikazu Kawakami, M.D.1
1
First Department of Medicine, School of Medicine, Hokkaido University, Sapporo, Japan.
2
Department of Pulmonary Diseases and Clinical
Research Institute, Sapporo National Hospital,
Sapporo, Japan.
3
Hygene and Preventive Medicine, School of Medicine, Hokkaido University, Sapporo, Japan.
Presented in part at the 37th Annual Meetings of
the Japan Lung Cancer Society, Kobe, Japan, October 31 to November 1, 1996.
The authors thank Dr. Kazuo Saito and the Hygiene
and Preventive Medicine fellows for assisting in
the ICP-MS and for their help in the design of this
study.
Address for reprints: Taro Hanada, M.D., Department of Internal Medicine, Tenshi Hospital, North
12, East 3, Higashi-ku, Sapporo, 065-8611, Japan.
Received 27 October, 1997; revision received February 17, 1998; accepted February 17, 1998.
© 1998 American Cancer Society
BACKGROUND. Thallium-201 (201Tl) scintigraphy has been used to detect malignant
pulmonary disease. The mechanism of Tl influx in tumor cells is believed to be
similar to that of cisplatin (CDDP) mediated by sodium- and potassium-activated
adenosine triphosphatase (Na-K ATPase), and the Na-K ATPase activity may determine the cellular CDDP accumulation and sensitivity to CDDP. The objective of
this study was to determine the accumulation of CDDP and Tl in vitro by using
inductively coupled plasma mass spectrometry (ICP-MS), a new analytic technique
for detecting ultra trace elements, and to evaluate the correlations between cellular
CDDP and Tl accumulation, between CDDP 50% inhibitory concentration (IC50)
values and cellular CDDP accumulation, and between CDDP IC50 values and
cellular Tl accumulation.
METHODS. Eight nonsmall cell lung carcinoma (NSCLC) cell lines were used (five
adenocarcinomas and three squamous cell carcinomas). The cell lines were exposed to CDDP or Tl for 1 hour, and the resulting cellular accumulation of
platinum and Tl was determined by ICP-MS. CDDP IC50 values were determined by
a soluble tetrazolium/formazan assay.
RESULTS. The authors were able to measure cellular CDDP and Tl accumulation
precisely, and heterogeneity in the cellular accumulation of CDDP and Tl existed
among the NSCLC cell lines. A significant inverse correlation was observed between CDDP IC50 values and the cellular accumulation of both CDDP and Tl.
CONCLUSIONS. ICP-MS is suitable for the determination of cellular CDDP and Tl
accumulation in NSCLC cell lines. Cellular Tl accumulation determined by ICP-MS
may reflect CDDP cytotoxicity rather than cellular CDDP accumulation. Cancer
1998;83:930 –5. © 1998 American Cancer Society.
KEYWORDS. cisplatin accumulation, thallium accumulation, inductively coupled
plasma mass spectrometry, Na-K-activated adenosine triphosphatase, thallium-201
scintigraphy.
hallium-201 (201Tl) is used to diagnose myocardial infarction,1
myocardial ischemia,2 and thyroid tumor.3 Recently, 201Tl single
photon emission computed tomography (SPECT) has been used for
the detection of malignant pulmonary lesions4 and has been shown to
be superior to gallium scintigraphy for lung carcinoma detection.5
The mechanism of 201Tl influx in tumor cells is believed to be
active transport through the cell membrane mediated by the sodiumand potassium-activated adenosine triphosphatase (Na-K ATPase)
system.6 – 8 In a similar manner, the Na-K ATPase system may also
contribute to the cellular influx of cisplatin (CDDP).9 On the other
T
Tl Accumulation Determined by ICP-MS/Hanada et al.
hand, Morikage et al.10 found that the expected CDDP
accumulation was reduced consistently in a number
of cell lines that had CDDP resistance. From these
findings, we speculated that a lung tumor that has a
high degree of 201Tl uptake may also accumulate a
high degree of CDDP and, therefore, may have a high
CDDP sensitivity. In fact, in our previous study on
small cell lung carcinoma (SCLC) patients who had
undergone 201Tl SPECT, we found that 201Tl accumulation correlated to patient response to chemotherapy
of platinum compounds.11 Based on this clinical
study, we hypothesized that a significant correlation
exists between the in vitro accumulations of CDDP
and Tl. To evaluate this hypothesis, we investigated
the cellular accumulation of CDDP and Tl in nonsmall
cell lung carcinoma (NSCLC) cell lines by using inductively coupled plasma mass spectrometry (ICP-MS).
ICP-MS is a powerful analytical technique for
the detection of ultratrace elements in biological
materials12 and has been applied to the analysis of
the trace element composition of blood,13 multielement analysis of human tissue,14 and metabolic
studies of minerals in humans.15 In a previous
study, we were able to measure cellular CDDP accumulation by using ICP-MS.16
The objective of the present study was to analyze
the cellular accumulation of CDDP and Tl by using
ICP-MS and to determine the correlation between cellular accumulation of CDDP or Tl and the chemosensitivity to CDDP in NSCLC cell lines.
MATERIALS AND METHODS
931
lung adenocarcinoma cell lines and EBC-1, LK-2 and
PC-10 squamous lung carcinoma cell lines. Cell lines
were obtained from the Japanese Cancer Research
Resources Bank (Tokyo, Japan). All cell lines were cultivated in tissue culture flasks (Falcon; Beckton Dickinson Labware, Franklin Lakes, NJ) in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine
serum, 100 units/mL penicillin, and 100 mg/mL streptomycin.
Chemosensitivity Test
Chemosensitivity to CDDP was determined by using the
2,3-bis(2-methoxy-4-nitro-5-sulphphenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide, inner
salt, sodium salt (XTT) assay, as described previously.18
Briefly, single-cell suspensions were harvested during
the exponential growth phase and seeded into 96-well
microplates. The cells were preincubated at 37°C overnight, after which, various concentrations of the required drugs were added. The cells were then incubated
at 37°C for 96 hours. After incubation, 50 mg of XTT
(Sigma Chemical Company) with 0.38 mg of phenazine
methosulfate (PMS; Sigma Chemical Company) were
added to each well and incubated at 37°C for 4 hours.
The plates were agitated on a plate shaker for 5 minutes
to solubilize the formazan crystals. Their absorbance
was measured at 450 by nm using an enzyme-linked
immunosorbent assay (ELISA) reader. The 50% inhibitory concentration (IC50) value was defined by determining the drug concentration that reduced absorbance by
50%. This value was derived graphically from the concentration-response curve.
Instrumentation
The ICP-MS apparatus used was an SPQ6500 (Seiko
Instruments, Co., Shizuoka, Japan), with a nebulizer
and a spray chamber made of borosilicate glass. Samples are introduced into a nebulizer, and positively
charged ions are then produced by a high-temperature, inductively coupled plasma. The ions pass
through a sampling cone interface into a high-vacuum, quadruple mass spectrometer, which has the
ability to carry out multielement analysis.17
Drugs and Chemicals
CDDP was purchased from Sigma Chemical Company
(St. Louis, MO). Thallium chloride, standard solutions
of platinum (Pt), and Tl were purchased from Wako
Pure Chemical Company (Osaka, Japan). RPMI 1640
was purchased from Nissui Pharmaceutical Company
(Tokyo, Japan).
Cell Lines
The human lung cancer cell lines used were A549,
PC-3, RERF-LC-MS, RERF-LC-OK, and VMRC-LCD
Drug Treatment
The cells were incubated in 10-cm tissue culture plates
(Falcon 3003; Beckton Dickinson Labware) at 5 3 106
cells in the 10-mL culture medium with 200 mL of 0.5
mg/mL CDDP (10 mg/mL) or 200 mL of 1.0 mg/mL TlCl
(20 mg/mL). After 1 hour of incubation at 37°C, cells
were harvested by scraping, transferred to plastic
tubes, and washed, using centrifugation and resuspension, two times with cold phosphate-buffered saline. The cell pellets were resuspended in 1 mL of 0.9%
sodium chloride and were taken up into Teflon resolution vessels (Flon Industry Company, Tokyo, Japan).
The vessels were placed in a vacuum oven and heated
at 120 °C for 2 hours. After cooling, the samples were
diluted to 10 mL with Millipore MilliQ water (Millipore
Corp., Bedford, MA). A CDDP concentration of 10
mg/mL, a TlCl concentration of 20 mg/mL, and an
incubation time of 1 hour were employed, because the
reproducibility of cellular CDDP and Tl accumulation
were good under these experimental conditions using
ICP-MS.
932
CANCER September 1, 1998 / Volume 83 / Number 5
TABLE 1
Resistance of Nonsmall Cell Lung Carcinoma Cell Lines to Cisplatin
Cell lines
Adenocarcinoma
A549
PC-3
RERF-LC-MS
RERF-LC-OK
VMRC-LCD
Squamous cell carcinoma
EBC-1
LK-2
PC-10
a
IC50 values (mM)
21.9 6 3.1a
8.3 6 1.5
2.8 6 1.4
7.8 6 0.8
20.6 6 9.3
17.0 6 1.2
17.1 6 4.0
4.2 6 0.6
Mean 6 S.D. (n $ 3). IC50: 50% inhibitory concentration.
Inductively Coupled Plasma Mass Spectrometry
The digested samples were nebulized into ICP-MS
without further dilution. Quantification of all samples
was performed by using external standard solutions
made up in 1% nitric acid. The standard solutions
used were 10, 50, and 100 mg/mL Pt or Tl, and the
blank solution was 0.1% nitric acid.
Protein Assay
Protein was measured by using the bicinchoninic
acid protein assay (Pierce Chemical Company,
Rockford, IL).
Statistics
A calibration curve and a correlation coefficient between the CDDP IC50 values and cellular CDDP accumulation and between the CDDP IC50 values and cellular Tl accumulation were determined to test for the
significance of the regression. Only P values ,0.05
were considered significant.
RESULTS
Cisplatin Sensitivity
Chemosensitivity to CDDP in NSCLC cell lines was
evaluated (Table 1). IC50 values among the NSCLC cell
lines ranged from 2.8 mM to 21.9 mM by XTT assay.
Calibration Curve
The calibration curve was obtained by using four standard Pt and Tl solutions (Fig. 1). The correlation coefficient of the regression line was 0.999 among 0, 10, 50,
and 100 mg/mL of Pt and Tl.
Accumulation of CDDP and Tl in NSCLC Cell Lines
By using ICP-MS, the cellular accumulation of CDDP
and Tl could be determined after 1 hour of exposure to
CDDP and Tl. Cellular accumulations of CDDP ranged
from 5.4 3 1023 ng/mg protein to 2.6 3 1022 ng/mg
protein, and Tl ranged from 1.7 3 1021 ng/mg protein
to 8.8 3 1021 ng/mg protein (Fig. 2). The cellular
accumulation of CDDP and Tl were heterogeneous
among NSCLC cell lines.
Correlations between Cellular CDDP and Tl Accumulation,
between CDDP IC50 Values and Cellular CDDP
Accumulation, and between CDDP IC50 Values and
Cellular Tl Accumulation
A significant inverse correlation between the CDDP
IC50 values and its cellular accumulation was observed
in the NSCLC cell lines (Fig. 3). A similar inverse correlation was obtained between the CDDP IC50 values
and Tl cellular accumulation (Fig. 4). No significant
correlation was observed between the cellular CDDP
accumulation and Tl accumulation (data not shown).
DISCUSSION
Na-K ATPase has a number of features in addition to
its contribution to the intracellular influx system of Tl.
Kier19 reported that Na-K ATPase activities were
higher in plasma membranes from metastatic cells
than in those from primary tumor cells and proposed
that this Na-K ATPase increase was related to the cell
surface fluidity and metastatic ability of metastatic
cells. On the other hand, Ohmori et al.9 reported that
CDDP accumulation, Na-K ATPase activity, and Na-K
ATPase mRNA expression were all lower in CDDPresistant NSCLC cell lines than in their parental cell
lines. Accordingly, the authors speculated that Na-K
ATPase plays a crucial role in the transport of CDDP
and that decreased intracellular accumulation of
CDDP is the primary factor of CDDP resistance.
Based on these findings, we hypothesized and
demonstrated previously that 201Tl accumulation
within a tumor is closely related to that tumor’s lymph
node metastasis20 or response to chemotherapy with
CDDP. These results suggested that 201Tl SPECT might
be useful as an indicator of metastasis in adenocarcinoma of the lung and as a predictor of chemotherapeutic responses to platinum compounds in SCLC
patients.
CDDP is an important anticancer drug for the
treatment of many solid tumors, but intrinsic or acquired resistance to CDDP is a significant clinical
problem, leading to treatment failure in NSCLC patients. If we could predict the chemosensitivity of the
NSCLC patient to CDDP by using 201Tl scintigraphy,
then we could avoid giving chemotherapy to a nonresponder. Because the patients with limited disease
were treated by operation, and, for extensive disease,
it was difficult to reach complete remission or partial
remission in NSCLC patients, we were unable to ex-
Tl Accumulation Determined by ICP-MS/Hanada et al.
933
FIGURE 1. Calibration curve for platinum (Pt; A) and thallium (Tl; B) determined by inductively coupled plasma
mass spectrometry. The correlation coefficient of the regression line was 0.999
among 0, 10, 50, and 100 mg/mL of Pt
and Tl. Because the standard deviations
were less than the size of the symbol,
error bars do not appear for dependent
variables. conc., concentration.
FIGURE 2.
Cellular accumulation of
cisplatin (CDDP; A) and thallium (Tl; B)
after a 1 hour exposure to CDDP and Tl
using inductively coupled plasma mass
spectrometry. Heterogeneity in the cellular accumulation of CDDP and Tl was
present among nonsmall cell lung carcinoma (NSCLC) cell lines. Each point
represents the mean value from three or
more independent experiments. Error
bars indicate standard deviation.
FIGURE 3. Correlation between accumulation of cisplatin (CDDP) and chemosensitivity to CDDP. A significant inverse
correlation between the CDDP 50% inhibitory concentration (IC50) values and
its cellular accumulation was observed
in nonsmall cell lung carcinoma cell
lines. Each point represents the mean
value from three or more independent
experiments. Error bars indicate standard deviation.
amine the relation between 201Tl accumulation and
responses to chemotherapy with platinum compounds in NSCLC patients. In this paper, we therefore
investigated whether there was a significant correlation between CDDP IC50 and Tl accumulation in
NSCLC cell lines.
Although the most widely used techniques for
the evaluation of cellular accumulation of CDDP and
Tl in vitro are atomic absorption spectrometry (AAS),
radiolabeled CDDP ([195mPt]-CDDP), and radiolabeled
thallium chloride (201TlCl),8 –10,21,22 we employed
ICP-MS to evaluated the cellular accumulation of
CDDP and Tl in this study. The accuracy of ICP-MS
was superior to AAS in the analysis of reference materials,14 and the sensitivity of ICP-MS was 100-fold
greater than that of AAS.17
The accuracy and the sensitivity of radiolabeling
methodology are not known. However, radiolabeled
tracers are not widely available and involve safety
hazards. Because ICP-MS offers a wide linear range
(six orders of magnitude) and a low detection limit,17,23
the linearity of the calibration curve obtained by using
934
CANCER September 1, 1998 / Volume 83 / Number 5
FIGURE 4. Correlation between accumulation of thallium (Tl) and chemosensitivity to cisplatin (CDDP). A significant
inverse correlation between the CDDP
50% inhibitory concentration (IC50) values and cellular accumulation of Tl was
observed with NSCLC cell lines. Each
point represents the mean value from
three or more independent experiments.
Error bars indicate standard deviation.
four standard Pt and Tl solutions was excellent. Moreover, once the calibration curve was obtained, the
cellular accumulation of CDDP and Tl in several
NSCLC cell lines could be analyzed at one time despite
CDDP or Tl heterogeneity. In fact, it took less than 1
minute to analyze each sample in the present study.
Because the NSCLC cell lines that were not exposed to
CDDP or Tl scarcely contained Pt and Tl, we could
determine their cellular CDDP and Tl accumulation
precisely. Therefore, ICP-MS not only is suitable for
the determination of cellular CDDP and Tl in tumor
cells but also is applicable for determining the cellular
accumulation of Tl in thyroid cells or myocardial cells
in vitro.
In a study using radiolabeled CDDP, Morikage et
al.10 observed an inverse correlation between CDDP
IC50 values and CDDP cellular accumulation in NSCLC
cell lines and proposed that defective CDDP accumulation may play an important role in the natural resistance of NSCLC cells to CDDP. Our previous study
using ICP-MS did not show a significant inverse correlation between the CDDP IC50 values and CDDP
cellular accumulation.16 We speculated that this was
due to the presence of amphotericin B in the culture
medium, because it has been shown to increase CDDP
cytotoxity by increasing cellular CDDP accumulation
in several NSCLC cell lines.10 However, in the present
study, we used a culture medium without amphotericin B, and, in good agreement with Morikage et al.,10
we found a significant inverse correlation between
CDDP IC50 values and CDDP accumulation within
NSCLC cell lines.
Our present and past11 results suggest that intracellular accumulation of Tl could be a suitable marker
of CDDP cytotoxity. On the other hand, no significant
correlation was observed between the cellular accumulation of CDDP and Tl. Although the influx systems
of both CDDP and Tl are mediated by Na-K ATPase,
the intracellular metabolic system of CDDP is different from that of Tl. The mechanism of Tl metabolism has been thought to involve an affinity to sulfhydryl groups7,24,25 and a tendency to combine with
riboflavin.25 In contrast, the metabolism of CDDP
seems to be related to the following mechanisms:
enhanced inactivation by cellular detoxification systems, such as glutathione (nonprotein-bound sulfhydryl groups) and metallothionein (protein-bound sulfhydryl groups), and decreased DNA damage and/or
increased repair.26 These differences between the
CDDP and Tl metabolic systems may contribute to the
lack of correlation between CDDP and Tl accumulation.
Although this is the first report demonstrating a
significant inverse correlation between CDDP IC50 values and cellular Tl accumulation, the correlation was
fairly weak. The weakness of the correlation was due
to the fact that the CDDP IC50 values of the eight cell
lines used were close to each other, so more experience is needed using a variety of cell lines that have
heterogenous CDDP IC50 values. If a good correlation
is obtained from a variety of NSCLC cell lines, then
201
Tl scintigraphy may be useful not only for the detection of malignant tumors but also for predicting the
response of NSCLC patients to chemotherapy with
platinum compounds and the detection of acquired
resistance to platinum compounds in the process of
chemotherapy.
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