Nuclear Instruments and Methods in Physics Research B 210 (2003) 364–367

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Nuclear microprobe imaging of gallium nitrate in cancer cells

Richard Ortega a,*, Asami Suda a,b, Guillaume Dev�ees a

a LCNAB, UMR 5084 CNRS, Universit�ee de Bordeaux 1, Le Haut Vigneau, BP 120, 33175 Gradignan, Franceb Department of Quantum Science and Energy Engineering, Tohoku University, Sendai, Japan

Abstract

Gallium nitrate is used in clinical oncology as treatment for hypercalcemia and for cancer that has spread to the bone.

Its mechanism of antitumor action has not been fully elucidated yet. The knowledge of the intracellular distribution of

anticancer drugs is of particular interest in oncology to better understand their cellular pharmacology. In addition, most

metal-based anticancer compounds interact with endogenous trace elements in cells, altering their metabolism. The

purpose of this experiment was to examine, by use of nuclear microprobe analysis, the cellular distribution of gallium

and endogenous trace elements within cancer cells exposed to gallium nitrate. In a majority of cellular analyses, gallium

was found homogeneously distributed in cells following the distribution of carbon. In a smaller number of cells,

however, gallium appeared concentrated together with P, Ca and Fe within round structures of about 2–5 lm diameter

located in the perinuclear region. These intracellular structures are typical of lysosomial material.

� 2003 Elsevier B.V. All rights reserved.

PACS: 82.80.E; 91.65.Nd; 87.64

Keywords: Nuclear microprobe; PIXE; Cancer; Gallium; Iron

1. Introduction

Gallium is the second metal ion, after platinum,

to be used in cancer treatment (for review see[1,2]). Gallium nitrate has shown clinical efficacy in

the treatment of hypercalcemia and bone cancer.

Gallium compounds have also demonstrated

promising antitumor chemotherapeutic activity in

clinical trials for the treatment of non-Hodgkin�slymphoma, metastatic colorectal cancer, carci-

noma of the urothelium, and in combination with

cisplatin against inoperable lung cancer.

* Corresponding author. Tel.: +33-5-57-12-09-07; fax: +33-5-

57-12-09-00.

E-mail address: ortega@cenbg.in2p3.fr (R. Ortega).

0168-583X/$ - see front matter � 2003 Elsevier B.V. All rights reser

doi:10.1016/S0168-583X(03)01052-8

The solution and coordination chemistries of

Ga3þ are very similar to those of Fe3þ. In vivo

studies have shown that nearly all plasma gallium

is tightly bound to the iron-transport proteintransferrin. In cells, gallium is complexed to trans-

ferrin receptors and released from lysosomes. The

mechanism of cell death may result, at least par-

tially, from an intracellular iron deficiency that

triggers apoptosis [3]. In addition, gallium inhibits

the iron-dependant enzyme ribonucleotide reduc-

tase, a key enzyme for DNA synthesis and cell

division. This inhibitory effect could be the resultof a competition of Ga with Fe in M2 subunit of

ribonucleotide reductase. However, the cellular

mechanisms for gallium antitumor activity are still

not fully elucidated. The aim of this study was to

determine the distribution of Ga, and endogenous

ved.

R. Ortega et al. / Nucl. Instr. and Meth. in Phys. Res. B 210 (2003) 364–367 365

trace metals such as Fe, in cancer cells by use of

nuclear microprobe analysis.

Fig. 1. Cell growth inhibition in human ovarian cancer cells

exposed for 48 h to 0, 250, or 500 lM Ga(NO3)3.

2. Material and methods

The biochemical characteristics of human ovar-

ian cancer cells (IGR-OV1), cell culture conditions,

and sample processing for nuclear microprobe

analysis were detailed elsewhere [4]. In brief, IGR-

OV1 cells were maintained in RPMI medium

supplemented with 10% fetal calf serum, plus pen-icillin and streptomycin, 100 IU/ml and 100 mg/

ml, respectively. The cells were incubated at 37 �Cin a humidified atmosphere of air with 5% CO2.

For nuclear microprobe experiments, cells were

cultured as monolayer onto 200 nm thick formvar

foils. After drug exposure, cells were cryofixed at

)164 �C into liquid nitrogen chilled isopentane,

and freeze-dried at )35 �C.Gallium nitrate was obtained from Aldrich.

The certificate of analysis stipulated a purity of

99.999%, and trace concentrations of 3.3 lg/g for

Ca, 0.8 lg/g for Fe. The effect of gallium nitrate on

the proliferation of IGR-OV1 cells was determined

by cell dye exclusion assay with trypan blue [5].

Cells were plated at an initial density of 1� 105

cells/ml in 12-well plates and incubated for 72 hbefore drug exposure. Then, cells were cultured for

48 h with appropriate gallium nitrate concentra-

tions (ranging from 0 to 500 lM). Viable cell

number was determined directly by cell counting

using a hemocytometer. Experiments were carried

out in triplicate.

Particle induced X-ray emission (PIXE) and

Rutherford backscattering spectrometry (RBS)were performed simultaneously using the nuclear

microprobe of Bordeaux Gradignan [6], with a

proton beam of 2.5 MeV incident energy, 2.5 lmdiameter, and 250 pA intensity. PIXE measure-

ments were made with a Si(Li) energy dispersive

detector placed at 45� from the specimen. Trace

element concentrations (ng/cm2) were calculated

with Gupix software [7]. RBS has been simulta-neously carried out for local mass (lg/cm2) deter-

mination of the biological matrix, as described

elsewhere [4]. RBS measurements were performed

using a Si surface barrier detector placed at 135�.

RBS data were processed with RUMPIN code

[8].

3. Results and discussion

To determine the pharmacologically relevant

gallium nitrate concentrations, inhibition of cell

growth has been studied (Fig. 1). Gallium nitrate

inhibited 53� 3% of IGR-OV1 cell growth when

administered at 250 lM for 48 h, and 75� 5% at

500 lM. These two concentrations were chosen fordrug exposure prior to element microanalysis. A

concentration dependant effect of gallium on IGR-

OV1 cell growth was noted. The number of viable

cells decreased by a factor two when drug con-

centration in culture medium increased by a factor

two.

Mean element concentrations in cells were de-

termined from micro-PIXE analysis of large zoneson cellular monolayers, 500 lm� 500 lm scans,

corresponding to the analysis of approximately

400–500 cells. Intracellular gallium concentration

after exposure to 500 lM Ga(NO3)3 was approx-

imately twice the concentration measured after 250

lM exposure (Table 1). This result also indicates a

concentration dependant effect of Ga(NO3)3 on

inhibition of cancer cells growth. Gallium nitratedid not significantly modify the mean cellular

concentration of trace elements such as Mn, Fe,

Cu, or Zn (Table 1). At the cellular scale, however,

Table 1

Quantitative analysis of gallium and trace metals in human ovarian cancer cells exposed in vitro to gallium nitrate during 48 h. Mean

quantitative results were obtained from analysis of large sample zones, 500 lm� 500 lm, containing several hundred cells

Element Control (lg/g� SD) 250 lM Ga(NO3)3 (lg/g�SD) 500 lM Ga(NO3)3 (lg/g� SD)

Mn 13.0� 0.9 9.6� 3.4 9.2� 3.0

Fe 155� 16 114� 35 129� 5

Cu 23.7� 4.7 17.6� 3.6 17.8� 3.0

Zn 258� 18 222� 19 232� 8

Ga – 60.8� 21.7 115� 8

366 R. Ortega et al. / Nucl. Instr. and Meth. in Phys. Res. B 210 (2003) 364–367

changes in trace element distributions were ob-served.

In the majority of cases, about 98% of the cells,

chemical maps of cells exposed to Ga(NO3)3showed an homogeneous intracellular distribu-

tions for P, K, Ca, Fe, as well as Ga, similar to

that of carbon (Fig. 2). Because gallium map fol-

lows the volume distribution of carbon, it can be

concluded that gallium is homogeneously distrib-uted in cells. In about 2% of cells exposed to

Ga(NO3)3, gallium appeared concentrated to-

gether with P, Ca and Fe within round structures

of approximately 2–5 lm diameter located in the

perinuclear region (Fig. 3). These intracellular

structures are typical of lysosomial material.

Lysosomes containing dense granules, rich in

phosphorus and gallium, were already observed byuse of electron X-ray microanalysis in tumor cells

Fig. 2. Chemical mapping of a single human ovarian cancer cell afte

characteristic for a majority of cells showing an homogeneous gallium

2.5 lm spot size, 250 pA. Scan size: 24 lm� 24 lm.

[9]. However, calcium or iron co-localization wasnot mentioned in this report. On the other hand,

the antagonistic effect of gallium on Ca and Fe

metabolism is well established [1,2,10]. The ob-

servation of Ca and Fe, accumulation within

vacuole-like structures in cells exposed to gallium

nitrate suggests a possible role for vacuolar re-

tention of Ca and Fe in inhibition of cell growth.

4. Conclusion

The quantitative and spatially resolved infor-

mation obtained by use of nuclear microprobe

analysis enabled to draw a better picture of gallium

nitrate cellular pharmacology in cancer cells. Gal-

lium is generally homogeneously distributed withincells. In few cases, P, Ca, Fe and Ga accumulated

r exposure to 500 lM Ga(NO3)3 during 48 h. This example is

distribution. Experimental conditions: 2.5 MeV proton beam,

Fig. 3. Chemical mapping of a single human ovarian cancer cell after exposure to 500 lM Ga(NO3)3 during 48 h. Note the

co-localization of P, Ca, Fe and Ga within a round structure of the perinuclear region. Such intracellular structure is typical of

lysosomial material. Experimental conditions: 2.5 MeV proton beam, 2.5 lm spot size, 250 pA. Scan size: 56 lm� 56 lm.

R. Ortega et al. / Nucl. Instr. and Meth. in Phys. Res. B 210 (2003) 364–367 367

in round structures within the cytosol suggesting a

possible role for vacuolar Ca and/or Fe retention in

gallium nitrate anticancer effects.

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