Investigations on Distribution and Quantity of Zinc in
Prostate Cancer Using Synchrotron Radiation Microbeams
Takuo Kawakami a, Ari Ide-Ektessabi b†, Kazurou Sugimura c, Yuri Kitamura c,
Akinobu Gotoh d, and Toshiro Shirakawa d
a
Department of Precision Engineering, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
b
International Innovation Center, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
c
Department of Radiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe
650-0017, Japan
d
Department of Urology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 6500017, Japan
Abstract. Synchrotron radiation (SR) micro beams were used to investigate ultra-trace elements in a single cell. X-ray
fluorescence (XRF) spectroscopy using SR was applied to determine the distribution and density of the ultra-trace
elements in incubated prostate cancer cells. Metallic elements such as zinc, iron and copper play an important role in the
metabolism and regulatory dynamics of cells. In this study, we focused on zinc because the prostate tissues contain the
highest levels of zinc compared to any other tissues in the body and the level of zinc is a factor that influences the
progress of malignancy. We measured two types of cells (LNCaP and C4-2) that were incubated in the normal culture
medium, zinc-contained one or testosterone-contained one. The distribution and density of zinc, calcium, copper and
iron have close relations to the existence of zinc and androgen in the culture medium.
prostate tissue. Inhibition of phosphatidylinositol-3
kinase induces cellular apoptosis. Androgen inhibits
this pathway, i.e. androgen inhibits apoptosis [5]. Zn2+
induces stimulation of phosphoinositide 3-kinase [6].
Thereby zinc inhibits apoptosis. Apoptosis is a
mechanism of programmed death of cell, and the
deficient of apoptosis are related with the progression
of malignancy.
It has been of great interest to understand how the
fluctuation of the density of zinc can be related to the
process of progression of prostate malignancy.
Previous studies using a homogenization method were
successful in measuring the density of zinc in the
tissues, but it was not possible to obtain the
distributions of zinc at the single cell level. The
synchrotron radiation x-ray fluorescence (SRXRF)
technique makes it possible to determine trace metallic
elements non-destructively [7].
In this study, we applied X-ray fluorescence (XRF)
analysis to LNCaP and C4-2 cells (human malignant
prostate cell lines). The aim was to investigate the
INTRODUCTION
Metallic elements and their organic compounds
have dynamic regulatory functions in cells. The roll of
zinc and its possible causal effects in prostate cancer
has attracted attention in recent years. A human
prostate gland has a unique feature to possess highlevel accumulation of zinc compared to other organs
[1]. In prostate cancer tissue the unique zinc
metabolism is altered, resulting in the decrease of zinc
level in the epithelial cells [1,2]. Zinc accumulation in
glandular cells is relevant to the citrate metabolism in
the citric acid cycle. High level of zinc may involve
the regulation of citrate oxidation in a prostate gland.
In a prostate cancer tissue, citrate remarkably
decreases as well as the level of zinc. The inability to
accumulate necessary amounts of zinc results in
increased citrate oxidation and coupled ATP
production, which is essential for the progression of
malignancy [3,4]. On the other hand, a hormone is
closely related to the zinc level and malignancy in a
†
Corresponding author: Ari Ide-Ektessabi. Email: [email protected]
CP680, Application of Accelerators in Research and Industry: 17th Int'l. Conference, edited by J. L. Duggan and I. L. Morgan
© 2003 American Institute of Physics 0-7354-0149-7/03/$20.00
526
TABLE 1. The characteristics of the prostate cancerous
cell lines.
Androgen
Metastatic Site
LNCaP
Dependent
Lymph node
C4-2
Independent
Lymph node
Quantification of the Density of the
Elements
Quantification was performed in the procedure as
follows. The peak areas were calculated using a
computer code developed by Ektessabi et al. [9] for
quantification of very low content elements in
biomedical samples. In this program, the background
is estimated from the untreated spectra, and the peak is
obtained using Gaussian curve fitting and the least
squares method. The absorption of fluorescent x-rays
by the sample and the consequent excitation of other
elements were negligibly small and hence were not
accounted for.
differences in the densities and the distribution of zinc
and other elements, and to reveal that LNCaP and C42 cells up-take and accumulate zinc from the medium.
EXPERIMENTAL
Materials
The specimens used in this study are the human
malignant cell lines (LNCaP and C4-2). The features
of these cell lines are shown in Table 1. LNCaP is
sensitive to androgen, while C4-2, which is LNCaPderived subline, is independent of androgen. Each cell
was incubated in three kinds of medium (normal
medium, zinc-added medium and testosterone-added
medium).
(a)
Experimental Set Up
Synchrotron radiation XRF (x-ray fluorescence)
analyses were performed at Photon Factory, KEK.
Synchrotron radiation from the storage ring (2.5 GeV,
maximum current 400mA) was monochromated by a
multilayer film monochromator. The incident x-ray
energy was 14.3 keV. Incident x-rays were focused
using Kirkpatrick-Baez optics. The incident beam size
was about 6 x 5 µm2. The incident and transmitted
photon flux was monitored with an ion chamber, while
the fluorescent x-rays were collected by a solid state
detector (SSD). Measurements were performed in air.
(b)
XRF Imaging and Spectra
XRF imaging technique was applied in order to
investigate the distributions of zinc and other elements
in the cells. X-y step pulse motors moved the sample
stage. The measurement areas were divided into
matrices of 40 x 40 pixels. At each pixel, XRF yields
of zinc were integrated by single channel analyzers.
The measurement time was 6 seconds for each pixel.
Point spectra were measured for quantification
analyses at certain points in the samples. The spectra
were obtained by using a multi-channel analyzer. The
measurement time was 200 seconds for each spectrum.
22.50 -- 24.00
21.00 -- 22.50
19.50 -- 21.00
18.00 -- 19.50
16.50 -- 18.00
15.00 -- 16.50
13.50 -- 15.00
12.00 -- 13.50
10.50 -- 12.00
9.000 -- 10.50
7.500 -- 9.000
6.000 -- 7.500
4.500 -- 6.000
3.000 -- 4.500
1.500 -- 3.000
0 -- 1.500
42.19 -- 45.00
39.38 -- 42.19
36.56 -- 39.38
33.75 -- 36.56
30.94 -- 33.75
28.13 -- 30.94
25.31 -- 28.13
22.50 -- 25.31
19.69 -- 22.50
16.88 -- 19.69
14.06 -- 16.88
11.25 -- 14.06
8.438 -- 11.25
5.625 -- 8.438
2.813 -- 5.625
0 -- 2.813
FIGURE 1. Typical XRF imaging of zinc in the incubated
cells of prostate cancer: (a) in the LNCaP that were
incubated in zinc-added medium, (b) in the C4-2 that were
incubated in zinc-added medium.
527
(a)
(a)
1000
1000
K
Ca Ti
Fe
Ca
Zn(-)
Zn(+)
Ar
Cu
100
Cu
100
Zn
Counts
Zn
Counts
T(-)
T(+)
Ar
10
10
1
2
3
4
5
6
7
8
9
1
10
2
3
4
5
6
7
8
9
10
Energy(keV)
Energy(keV)
(b)
(b)
1000
1000
Ar
Zn(-)
Zn(+)
Ca
T(-)
T(+)
Ar
100
Fe
Cu
Ca
Counts
Counts
Zn
Cu
100
Zn
Fe
10
10
1
2
3
4
5
6
7
8
9
10
1
Energy(keV)
2
3
4
5
6
7
8
9
10
Energy(keV)
FIGURE 2. Typical XRF spectra of the incubated cells of
prostate cancer: (a) in the LNCaP which were incubated in
zinc-free medium [Zn(-)] and zinc-added medium [Zn(+)],
(b) in the C4-2 which were incubated in zinc-free medium
and zinc-added medium.
FIGURE 3. Typical XRF spectra of the incubated cells of
prostate cancer: (a) in the LNCaP which were incubated in
testosterone-free medium [T(-)], and testosterone-added
medium [T(+)], (b) in the C4-2 which were incubated in
testosterone-free medium and testosterone-added medium.
medium is higher than the one in zinc-free medium.
Fig. 3 shows the typical spectra of the LNCaP and C42 cells which were incubated in testosterone-added,
zinc-free medium or testosterone-free, zinc-free
medium.
Quantification analyses of elements contained in
the cells were performed. Table 2 shows the density of
the elements in the LNCaP and C4-2. The density of
the sample was assumed to be 1.0 g cm-3.
Firstly, the endogenous level of cellular zinc and
other elements in LNCaP and C4-2 cells and the
accumulation of zinc and other elements in cells
RESULTS
XRF analyses were performed on the prostate
cancer cells. XRF imaging of zinc in LNCaP and C4-2
that were incubated in zinc-contained medium is
shown in Fig. 1 (a) and (b). Typical XRF spectra of
the LNCaP and C4-2 cells that were incubated in the
zinc-free medium or zinc-added medium are shown in
Fig. 2. As shown in these spectra, the level of zinc in
both LNCaP and C4-2 incubated in zinc-added
TALBE 2. Density (ppm) of copper and zinc contained in LNCaP and C4-2 cells. The values in parentheses are the
average of data for n points.
Cell line
Cu
Zn
n
LNCaP
Control
Zn(+)
T(+)
138-209 (172)
82-139 (114)
191-379 (282)
24-53 (38)
84-167 (130)
36-96 (52)
11
5
10
C4-2
Control
Zn(+)
T(+)
206-383 (327)
200-321 (270)
279-549 (455)
53-90 (73)
110-434 (264)
41-69 (60)
7
10
5
528
The present studies demonstrate that testosterone
regulates accumulation in LNCaP and C4-2 cells. In
LNCaP cells, zinc accumulation was increased, but in
C4-2, zinc accumulation was decreased. The absence
of an effect of testosterone on zinc level of C4-2 cells
might be expected because these cells do not contain
androgen receptor. On the other hand, the copper
accumulation was increased in both LNCaP and C4-2.
Because synchrotron radiation makes it possible to
determine several elements simultaneously, we can
discuss the balance and the equilibrium of elements in
the cells as a new approach to cell microbiology. This
studies provide the basis for further studies of the
characterization of the zinc transport and other
elements in malignant prostate cells, the mechanism of
testosterone regulation of zinc and other elements
transport, the relationship of zinc accumulation in the
pathogenesis and progression of prostate malignancy.
exposed to the zinc-added medium were established.
The endogenous level is defined as the concentration
in the cells grown and maintained in zinc-free medium.
The mean endogenous level of zinc in LNCaP cells
(38 ppm) was 52% lower than the level in C4-2 cells
(73 ppm). When the harvested cells were incubated in
zinc-added medium, the cellular level of zinc in
LNCaP increased 242% to 130 ppm; and C4-2 zinc
level increased 261% to 264 ppm. Thus, both LNCaP
and C4-2 cells took up and accumulated zinc from the
medium. However, C4-2 cells maintained significantly
higher (103%) zinc level than LNCaP cells. The
endogenous level of copper in LNCaP cells (172 ppm)
was 47% lower than the level in C4-2 cells (327 ppm).
When the harvested cells were incubated in zinc-added
medium, the cellular level of zinc in LNCaP decreased
34% to 114 ppm; and C4-2 zinc level decreased 18%
to 270 ppm. Consequently it is clear that LNCaP and
C4-2 discharge copper when they are incubated in
zinc-added medium.
We determined the effects of testosterone treatment
of the cells on zinc accumulation. The harvested cells
were incubated in testosterone-added, zinc-free
medium. In LNCaP cells, zinc accumulation was
increased 36% to 52 ppm. On the other hand, in C4-2
cells, zinc accumulation was decreased 18% to 60 ppm.
When the harvested cells were incubated in
testosterone-added medium, the cellular level of
copper in LNCaP increased 64% to 282 ppm; and C42 copper level increased 39% to 455 ppm.
ACKNOWLEDGMENTS
The microbeam XRF analyses were performed at
the Photon Factory in the High Energy Accelerator
Research Organization, Tsukuba, Japan (project
2001G179). The authors express their thanks to
Professor A. Iida of the Photon Factory.
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DISCUSSION AND CONCLUSION
The present studies demonstrate that the human
malignant prostate cells LNCaP and C4-2 possess the
ability to accumulate high zinc levels, and both
LNCaP and C4-2 cells took up and accumulated zinc
from the medium. However the zinc level in C4-2 is
higher than that in LNCaP. LNCaP is androgendependent while C4-2, which is derived from LNCaP,
is androgen-independent. Moreover, it is reported that,
when exposed to extracellular zinc, the accumulation
of zinc by PC-3, which is the prostate cancer cell,
results in the inhibition of their ability to oxidize
citrate thereby causing in citrate accumulation [8].
This shows that the accumulated zinc includes a
mobile reactive component that enters the
mitochondria and inhibits mitochondria aconitase
activity. To protect against the toxic effects of zinc,
human cells generally employ defensive mechanisms
that prevent the accumulation of mobile reactive zinc.
In contrast, and consistent with their unique function,
prostate secretory epithelial cells employ mechanisms
that facilitate the uptake and accumulation of zinc.
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