ORIGINAL PAPER

The ruthenium(II)–arene compound RAPTA-C induces apoptosisin EAC cells through mitochondrial and p53–JNK pathways

Soumya Chatterjee Æ Subhadip Kundu ÆArindam Bhattacharyya Æ Christian G. Hartinger ÆPaul J. Dyson

Received: 19 March 2008 / Accepted: 9 June 2008 / Published online: 3 July 2008

� SBIC 2008

Abstract An investigation of the molecular mechanism

of the anticancer activity demonstrated by the ruthe-

nium(II)–arene compound [Ru(g6-p-cymene)Cl2(pta)] (pta

is 1,3,5-triaza-7-phosphaadamantane), termed ‘‘RAPTA-

C’’, in Ehrlich ascites carcinoma (EAC) bearing mice is

described. RAPTA-C exhibits effective cell growth inhi-

bition by triggering G2/M phase arrest and apoptosis in

cancer cells. Cell cycle arrest is associated with increased

levels of p21 and reduced amounts of cyclin E. RAPTA-C

treatment also enhances the levels of p53, and its treatment

triggers the mitochondrial apoptotic pathway, as shown by

the change in Bax to Bcl-2 ratios, resulting in cytochrome c

release and caspase-9 activation. c-Jun NH2-terminal

kinase (JNK) is a critical mediator in RAPTA-C-induced

cell growth inhibition. Activation of JNK by RAPTA-C

increases significantly during apoptosis. Overall, these

results suggest a critical role for JNK and p53 in RAPTA-

C-induced G2/M arrest and apoptosis of EAC-bearing

mice. Consequently, RAPTA-C treatment results in a sig-

nificant inhibition in the progression of cancer in an animal

model, which emulates the human disease, and does so

with remarkably low general toxicity; hence, RAPTA-C

has potential for clinical application.

Keywords Anticancer research � Apoptosis �Bioorganometallics � p53 � Ruthenium

Introduction

Cisplatin entered clinical trials in 1971 and, within a rel-

atively short time, became the most widely used anticancer

medicine [1]. However, its application is limited by its

severe toxicity and also by drug resistance [2]. Some

tumours exhibit natural resistance to cisplatin and others

develop resistance after initial treatment [3]. The limita-

tions of cisplatin have motivated extensive investigations

into alternative metal-based cancer therapies. Although

these investigations are yet to produce a drug as widely

used as cisplatin and its derivatives, drug targets have been

identified. Amongst these targets, the tumour suppressor

protein p53 and its regulators, including c-Jun NH2-

terminal kinase (JNK), are considered important as they

can trigger the onset of reversible growth arrest or

apoptosis [4, 5].

Investigations into new anticancer drugs have high-

lighted ruthenium as a potential metal centre for new

therapies [6–8]. Ruthenium complexes have similar ligand

exchange kinetics to those of platinum(II) complexes,

different oxidation states are accessible under physiologi-

cal conditions [9, 10], and ruthenium mimics iron in

binding to important carrier proteins [11–13]. This latter-

most feature is thought to be why some ruthenium

complexes have particularly low general toxicity, allowing

larger administrative doses of the compounds and, there-

fore, more effective anticancer therapies [14].

Several ruthenium complexes have already been shown

to exhibit excellent in vivo antitumor activity [15],

although displaying an in vitro activity approximately

S. Chatterjee � S. Kundu � A. Bhattacharyya (&)

Department of Environmental Science,

University of Kalyani,

Kalyani, India

e-mail: arindam19@yahoo.com

S. Chatterjee

e-mail: chatterjees_1234@yahoo.com

C. G. Hartinger � P. J. Dyson

Institut des Sciences et Ingenierie Chimiques,

Ecole Polytechnique Federale de Lausanne (EPFL),

1015 Lausanne, Switzerland

123

J Biol Inorg Chem (2008) 13:1149–1155

DOI 10.1007/s00775-008-0400-9

10 times lower than that of cisplatin. Ruthenium com-

plexes containing indazole heterocycles coordinated to the

metal centre through nitrogen have been shown to act as

cytostatic and cytotoxic drugs in colorectal tumour cells

both in vivo and in vitro [16–19]. Studies have shown that

the drugs are efficiently taken up into the cells, probably

via interactions with transferrin [20–24], where they induce

apoptosis in a Bcl2-independent fashion [25]. Such

compounds are highly valuable for use in tumours over-

expressing Bcl-2, for example colorectal carcinomas.

Nevertheless, the cellular mechanisms of apoptosis induc-

tion are still largely unknown, although it was postulated

that interactions with DNA are involved [26, 27].

Ruthenium(II)–arene complexes containing a 1,3,5-

triaza-7-phosphaadamantane (PTA) ligand have shown

moderate anticancer activity in various cell lines and

excellent activity with regard to reducing the number and

weight of solid metastases, although not affecting the pri-

mary tumour [28]. While many different [Ru(g6-arene)

Cl2(pta)] (RAPTA) compounds have been developed

[29–32], the prototype compound [Ru(g6-p-cymene)

Cl2(pta)] (RAPTA-C) (Fig. 1) remains the best anticancer

compound of this series that has been characterised.

However, the molecular mechanism and the signalling

pathways remain to be elucidated.

The current investigations attempt to elucidate the

pathways involved in RAPTA-C-induced response, focus-

ing on the expression of p53 and its regulators using

Ehrlich ascites carcinoma (EAC) as a tumour model.

Materials and methods

Materials

RNase-A and general reagents were purchased from Sigma

(St Louis, MO, USA) and Pharmacia Fine Chemicals,

Sweden. CycleTEST PLUS DNA reagent kit and annexin V–

propidium iodide (PI) kit were purchased from Becton

Dickinson Immunocytometry Systems (San Jose, CA,

USA). Polyclonal anti-Bax (14-6704), anti-Bcl-2 (14-6992),

anti-p21 (14-6715), anticyclin E (14-1714), anti-p53 (14-

6045), and anti-JNK (14-6725) antibodies were obtained

from eBioscience, and anticytochrome c (556433, and

antiprocaspase-9 (550438) were procured from BD Pharm-

ingen (San Jose, CA, USA). The remaining chemicals were

purchased from local companies (India) and were of highest-

purity grade. RAPTA-C was prepared using a literature

method [33].

Animal models and treatment

All animal experiments were performed following the

‘‘Principles of laboratory animal care’’ (NIH publication

no. 85-23, revised in 1985) as well as specific Indian laws

on the ‘‘Protection of Animals’’ under the prevision of

authorised investigators. Swiss albino mice (approximately

25 g each; five mice in each group) were randomly divided

into different groups, including (1) an EAC-treated set and

(2) a EAC + RAPTA-C treated set. Animals were given

intraperitoneal injections (i.p.) of different concentrations

of RAPTA-C at regular intervals (40 mg/kg body weight

per week) and were killed on day 28.

Isolation of EAC and cell viability assay

EAC cells were isolated from the peritoneal cavity of

tumour-bearing mice (control or treated). Two to three

millilitres of sterile phosphate-buffered saline (PBS) was

injected into the peritoneal cavity and the fluid containing

the tumour cells was withdrawn, collected in sterile Petri

dishes, and incubated at 37 �C for 2 h. The cells of

macrophage lineage adhered to the bottom of the Petri

dishes to form a confluent monolayer. The non-adherent

population was gently aspirated out and washed repeatedly

with PBS. More than 98% of the non-adherent population

was found to be CD16 (specific surface marker for mac-

rophages) negative by flow cytometry analysis. More than

92% of the CD16-negative cells were morphologically

characterised as EAC by Wright staining and viability was

assessed to be more than 95% by trypan blue dye exclu-

sion. The viable EAC cells were processed for further

experiments.

Analysis of cell cycle by flowcytometry

EAC cells were fixed with formaldehyde and permeabi-

lised with Triton X-100, and the nuclear DNA was labelled

with PI using a Cycle TEST PLUS DNA reagent kit. Cell

cycle phase distribution of nuclear DNA was determined

using a fluorescence-activated cell sorter (FACS) having a

fluorescence detector equipped with a 488 nm argon laser

light source and a 623 nm band pass filter (linear scale).

A total of 10,000 events were acquired and flow cytometry

Ru

ClCl

N

NP

N

Fig. 1 Structural formula of [Ru(g6-p-cymene)Cl2(pta)] (pta is 1,3,5-

triaza-7-phosphaadamantane) (RAPTA-C)

1150 J Biol Inorg Chem (2008) 13:1149–1155

123

data were analysed using Cell Quest software (Becton

Dickinson). The DNA content (x-axis, PI fluorescence)

versus counts (y-axis) was plotted as a histogram.

Detection of apoptosis by annexin V binding assay

A double-labelling system was used to distinguish between

apoptosis and necrosis. Cells (1 9 106 in each case),

treated or untreated, were harvested and PI, annexin-

V-FLUOS (Boehringer Mannheim) were added directly to

the culture medium or to the cell suspension. The mixture

was incubated for 15 min at 37 �C. Excess fluorophore was

washed off, and the cells were fixed and then analysed

using a Calibur fluorescence-activated cell sorter (equipped

with a 488 nm argon laser light source; 515 nm band pass

filter, FL1-H, and a 623 nm band pass filter, FL2-H).

Electronic compensation of the instrument was performed

to exclude overlapping of the emission spectra. A total of

10,000 events were acquired. The cells were properly gated

and a dual-parameter dot plot of FL1-H (x-axis; FLUOS

fluorescence) versus FL2-H (y-axis; PI fluorescence) was

shown to be logarithmic in fluorescence intensity.

Preparation of cytosol

After treatment, EAC cells were homogenised in 5 mM

N-(2-hydroxyethyl)piperazine-N0-ethanesulfonic acid buf-

fer containing 0.25 M sucrose and 1 mM EDTA, pH 7.2.

The homogenate was centrifuged at 500g to pellet nuclei

and the resulting supernatant was centrifuged at 100,000g

(29,000 rpm) for 60 min at 4 �C in a 50.2 Ti rotor. The

cytosolic supernatant was collected, disbursed into 50-lL

aliquots, and frozen by immersion in liquid nitrogen (and

stored at -80 �C until required).

Western blot analysis

For western blot analysis of p53, Bcl-2, Bax, procaspase-9,

cytochrome c, p21, cyclin E, and JNK, the cell lysate

was loaded onto a 10–15% sodium dodecyl sulfate poly-

acrylamide gel. After electrophoresis, the protein was

transferred to a nitrocellulose membrane and blocked with

non-fat dry milk in PBS containing Tween-20 prior to

antibody treatment. The protein of interest was visualised

by chemiluminescence.

Statistical analysis

Values are shown as the standard error of the mean, except

where otherwise indicated. Data were analysed and, where

appropriate, the significance of the differences between the

mean values were determined using a Student’s t test.

Results were considered significant at p \ 0.05.

Results

Effect of RAPTA-C on the growth inhibition of tumour

cells in vivo

The LD50 values were determined at different doses of

RAPTA-C by administering the drug for a period of

28 days once a week at a concentration of mg/kg body

weight to EAC mice. The tumour-inoculated mice dem-

onstrated a dose-dependent reduction in tumour volume,

with decreased rate of alopecia and vomiting during the

treatment programme. About 50% of the EAC cell popu-

lation was reduced (Fig. 2) using a dose of 40 mg/kg body

weight. At higher doses, for example 50 mg/kg body

weight, approximately 75% of the cell population died.

Thus, 40 mg/kg body weight was chosen as the treatment

dose for further studies (five mice in each experimental

group).

Flow cytometry analysis of RAPTA-C-induced EAC

cell cycle phase distribution of nuclear DNA

The cell cycle phase distribution of EAC cells in response

to RAPTA-C was studied to further elucidate the mecha-

nism of drug activity. After the 28th day of treatment with

a RAPTA-C dose of 40 mg/kg body weight per week, the

content of hypoploid DNA (less than 2n DNA) was

increased in the RAPTA-C treated group compared with

the untreated cells (Fig. 3a). In contrast, the DNA content

in S phases decreased from 6.2 to 3.6%. As a result, counts

of the G2/M phases also decreased significantly (Fig. 3a),

Fig. 2 Effect of RAPTA-C on the survival of Ehrlich ascites

carcinoma (EAC) cells. The results are representative of an average

of five (n = 5) independent experiments in triplicate. The

mean ± standard error of the mean is plotted; statistical significance

p \ 0.05, compared with the control set

J Biol Inorg Chem (2008) 13:1149–1155 1151

123

suggesting that RAPTA-C-induced DNA damage is

followed by suppressed EAC proliferation.

The mode of cell death of the EAC cells was investi-

gated using an annexin V binding assay. Surprisingly, it

was found that the number of annexin V+/PI-, i.e.

apoptotic, cells, increases significantly (Fig. 3b) compared

with the untreated mice (4%), whereas there was no such

fluorescence in the annexin V-/PI+ and annexin V+/PI+

quadrant, confirming a dose of approximately 40 mg/kg

body weight RAPTA-C induces significant apoptosis in

EAC cells. Both of these data suggest that RAPTA-C

induces apoptosis to produce DNA damage.

Involvement of p53 protein and JNK

in RAPTA-C-induced EAC apoptosis

Key proteins involved in apoptosis or permanent growth

arrest in response to cellular stresses include p53 and JNK

[34]; however, from various in vitro reports it is evident

that in ruthenium-induced cytotoxicity p53 play a major

role [35, 36]. Therefore, to evaluate the cellular basis of

EAC response in correlation to RAPTA-C-induced stress,

the expression of p53 or JNK was analysed using western

blotting. A dose of 40 mg/kg body weight RAPTA-C

markedly induced the expression of p53 (Fig. 4). Further-

more, the expression of the upstream regulator of p53,

JNK, was significantly higher than in the untreated cells.

Effect of RAPTA-C on cell cycle regulatory proteins

activity

In our previous flow cytometry analysis it was found that

RAPTA-C causes arrest of the cell cycle phases, which was

also evident from the works of the other groups [35].

Therefore, to provide more information on the cell cycle

regulation, the expressions of p21, a probable downstream

mediator of p53 [35], and cyclin E were studied. It was found

that in response to RAPTA-C treatment the expression of p21

significantly increased in the EAC-bearing host (Fig. 5),

with a simultaneous reduction in the expression of cyclin E,

which is necessary to drive the cells towards the M phase.

Effect of RAPTA-C on Bcl-2 and Bax expression

in EAC

It is known that interactions of p53 with mitochondrial

target antiapoptotic factors, including Bcl-2, and proapop-

totic factors, such as Bax, are not only an important

regulator of apoptosis, but also play a crucial role in

ruthenium-induced cytotoxicity [34–37]. We also have

previously seen that in response to RAPTA-C there was an

Fig. 3 Flow cytometry analysis

of the EAC cell cycle phase

distribution and apoptosis in

response to RAPTA-C. aHistogram display of DNA

content (x-axis; propidium

iodide, PI, fluorescence) versus

counts (y-axis). b Dot plot

display of annexin V

fluorescence (x-axis; linear

scale) versus PI fluorescence

(y-axis; logarithmic scale)

Fig. 4 Effect of RAPTA-C on p53 and c-Jun NH2-terminal kinase

(JNK) expression in EAC cells. One representative result from three

independent western blot experiments using anti-p53 and anti-JNK

antibodies visualised by chemiluminesence

1152 J Biol Inorg Chem (2008) 13:1149–1155

123

increased expression of p53 and its upstream regulator JNK

(Fig. 2). Therefore, to address the question of how the p53

expression and apoptosis is related, we studied the

expression of the antiapoptotic and proapoptotic factors

Bcl-2 and Bax, respectively. RAPTA-C treatment results in

decreased expression of antiapoptotic Bcl-2 and increased

expression of proapoptotic Bax protein (Fig. 6), compared

with the case in untreated mice. Therefore, the balance

between these positive and negative regulators of apoptosis

clearly shifts towards apoptosis.

Effect of RAPTA-C on cytochrome c release

and procaspase-9 activity

To map the further downstream events of apoptosis, we

investigated the release of cytochrome c [38]. After

RAPTA-C treatment, cytosolic cytochrome c protein levels

are increased (Fig. 7). Various in vitro studies have shown

that apoptosis involves activation of caspases [38, 39]. On

the other hand, it is known that cytochrome c causes

activation of caspases by cleaving the procaspases.

Therefore, less procaspase-9 detected in the cell cytosol

(Fig. 7) suggests that RAPTA-C increases cytochrome c

expression, which causes cytochrome c mediated cleavage/

activation of caspases to be elevated.

Discussion

Current platinum-based anticancer therapies have been

very successful in the clinic to combat a range of cancers,

but they are not universally applicable. Active research in

developing new therapies has led to extensive investiga-

tions into novel complexes with alternative metal centres.

Some ruthenium complexes have been demonstrated to

play a major role in the regression of cancer [40], and in

addition, some have a surprisingly low general toxicity.

RAPTA-C is one complex with both attributes [41].

RAPTA-C has been demonstrated to be an effective

treatment for solid metastatic tumours. The current study

aimed to elucidate the mechanism of this activity using

EAC cells grown in the peritoneal cavity of Swiss albino

mice.

The tumour suppressor gene p53 is central to the

induction of cell cycle arrest and apoptosis in response to

DNA damage or cellular stress in human cells [42]. The

results of the current study show that treatment of EAC-

bearing hosts with RAPTA-C results in the accumulation of

p53, suggesting that RAPTA-C-induced apoptosis and cell

cycle arrest involve p53.

Cell cycle arrest mediated by p53 involves transactiva-

tion of cell-cycle-related factors, such as p21 [43].

Upregulated p21 expression is associated with cell cycle

inhibition, differentiation, and cellular senescence [44]. In

addition, p21 can block DNA synthesis by binding to

proliferating cell nuclear antigens [45]. As such, activation

of p21 causes subsequent arrest in the G1/G0 or G2/M

phase of the cell cycle by interacting with the cyclin–

Fig. 5 Effect of RAPTA-C on p21 and cyclin E expression in EAC

cells. Representative western blot result using anti-p21 and anticyclin

E antibodies and visualised by chemiluminesence

Fig. 6 Effect of RAPTA-C on Bax and Bcl-2 expression in EAC

cells. Representative western blot result using anti-Bax and anti-Bcl-2

antibodies and visualised by chemiluminesence

Fig. 7 Effect of RAPTA-C on procaspase-9 and cytochrome cexpression in EAC cells. Representative western blot data using

procaspase-9 and anticytochrome c antibodies and visualised by

chemiluminesence

J Biol Inorg Chem (2008) 13:1149–1155 1153

123

cyclin-dependent kinase complex [43, 46]. RAPTA-C

increased the expression of p21 and arrests the cell cycle in

the G2/M phase, which is followed by apoptosis in EAC

cells.

The upregulation of p21 by p53 induction has been

shown to reduce cell death in quercetin-treated A549 cells

[47]. Furthermore, p21 has been shown to influence the

outcome of the p53 response to DNA damage and play a

protective role against apoptosis [47, 48]. These different

observations may be due to the cell type, the cell content,

and the specificity of apoptosis inducers or their subsequent

signalling transduction pathways. Although our results

indicate that RAPTA-C induces apoptosis in EAC cells in a

background of elevated p21, the actual role of p21 in the

relation of G2/M arrest and apoptosis requires further

investigation.

In addition, treatment of EAC cells with RAPTA-C

decreased the expression of cyclin E, a key regulator in the

G1-S/G2-M [49, 50] transition. Therefore, in addition to

inducing apoptosis, RAPTA-C slows down cell division.

The mitochondrial apoptotic pathway appears to be

central to the signalling of cell death in mammalian cells

[51]. The activity of proapoptotic and antiapoptotic Bcl-2

family members, such as Bim and Bcl-2 itself, has been

highlighted as important for the mitochondrial apoptotic

pathway [48, 52, 53]. Following the treatment of EAC cells

with RAPTA-C, a significant increase of Bax and decrease

of Bcl-2 was observed. This altered ratio of proapoptotic

and antiapoptotic factors favours the promotion of apop-

tosis. In addition, RAPTA-C reduced the ability of Bcl-2 to

bind to Bax and enhanced the translocation of Bax from

cytosol to mitochondria, further increasing the suscepti-

bility of the cells to apoptosis [54, 55]. Thus, in response to

RAPTA-C treatment, at least one of the mechanisms of

apoptosis in tumour cells involves the mitochondrial

pathway.

RAPTA-C treatment also elevates the amount of cytosolic

cytochrome c and decreases the amount of procaspase-9,

suggesting procaspase-9 activation. Various studies have

linked activation of caspases to apoptosis; hence, the data

suggest an additional molecular mechanism for RAPTA-C-

induced apoptosis.

The mechanism of apoptosis induced by several DNA-

damaging agents has been suggested to involve the acti-

vation of JNK, which in turn activates further proapoptotic

factors by phosphorylation [56]. The treatment of EAC

cells with RAPTA-C resulted in the accumulation of

phospho-JNK and its substrate, i.e. the transcription factor

phospho-c-Jun, suggesting that RAPTA-C partly mediates

its effect by modulating the activity of this regulatory

pathway. The JNK targets are not clearly defined, but in

addition to c-Jun, p53 is a likely candidate. The half-life

and transcriptional activity of p53 are increased in response

to phosphorylation in the N-terminal domain [52], which

fits with the observed effects of RAPTA-C treatment.

Conclusions

The present study demonstrates that in vivo experimental

models of EAC cells are highly sensitive to growth inhi-

bition by RAPTA-C:

1. Increased survival of mice after exposure to RAPTA-C

is associated with G2/M phase cell cycle arrest and

apoptosis induction.

2. RAPTA-C can inhibit cell cycle progression at the G2/

M phase by increasing p21 expression in a p53-

dependent manner and by decreasing the expression of

cyclin E.

3. Increased cytochrome c levels induced by RAPTA-C

treatment activate procaspase-9, promoting apoptosis.

4. RAPTA-C-induced cell growth inhibition in the EAC

cells is mediated by activation of JNK stabilising p53.

This study has highlighted changes in expression and

activity of key proteins known to be involved in the reg-

ulation of the cell cycle and apoptosis. Each interaction

promotes cell cycle arrest or apoptosis, suggesting that the

effect of RAPTA-C is mediated via different molecular

pathways, reducing the likelihood of developing some

types of drug resistance. The increased survival of the mice

is in part due to the low toxicity of RAPTA-C. Taken

together, these results suggest a critical role for JNK and

p53 in RAPTA-C-induced G2/M arrest and apoptosis of

EAC cells and further demonstrate the potential of RAP-

TA-C as a candidate for further evaluation as an anticancer

agent. Moreover, the fact that multiple pathways are

involved in RAPTA-C-induced apoptosis means that it is

less likely to suffer from acquired resistance during

chemotherapy.

Acknowledgments The authors are indebted to the Department of

Science and Technology, India, and the Indo Swiss Bilateral Research

Initiative (EPFL) for funding this research. The support of the the

Austrian Science Fund (C.G.H.; Schrodinger Fellowship J2613-N19)

and of COST D39 is gratefully acknowledged.

References

1. Reedijk J (1987) Pure Appl Chem 59:181–192

2. Jakupec MA, Galanski M, Arion VB, Hartinger CG, Keppler BK

(2008) Dalton Trans 183–194

3. Wong E, Giandomenico CM (1999) Chem Rev 99:2451–2466

4. Zanzi I, Srivastava SC, Meinken GE, Robeson W, Mausner LF,

Fairchild RG, Margouleff D (1989) Nucl Med Biol 16:397–403

5. Srivastava SC (1996) Semin Nucl Med 26:119–131

6. Rademaker-Lakhai JM, van den Bongard D, Pluim D, Beijnen

JH, Schellens JH (2004) Clin Cancer Res 10:3717–3727

1154 J Biol Inorg Chem (2008) 13:1149–1155

123

7. Hartinger CG, Zorbas-Seifried S, Jakupec MA, Kynast B, Zorbas

H, Keppler BK (2006) J Inorg Biochem 100:891–904

8. Hartinger CG, Jakupec MA, Zorbas-Seifried S, Groessl M, Egger

A, Berger W, Zorbas H, Dyson PJ, Keppler BK (2008) Chem

Biodivers (in press)

9. Schluga P, Hartinger CG, Egger A, Reisner E, Galanski M, Ja-

kupec MA, Keppler BK (2006) Dalton Trans 1796–1802

10. Jakupec MA, Reisner E, Eichinger A, Pongratz M, Arion VB,

Galanski M, Hartinger CG, Keppler BK (2005) J Med Chem

48:2831–2837

11. Allardyce CS, Dyson PJ (2001) Platinum Metals Rev 45:62–69

12. Clarke MJ (2003) Coord Chem Rev 236:209–232

13. Sulyok M, Hann S, Hartinger CG, Keppler BK, Stingeder G,

Koellensperger G (2005) J Anal At Spectrom 20(9):856–863

14. Timerbaev AR, Hartinger CG, Aleksenko SS, Keppler BK (2006)

Chem Rev 106:2224–2248

15. Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H,

Sadler PJ, Jodrell DI (2002) Br J Cancer 86:1652–1657

16. Galeano A, Berger MR, Keppler BK (1992) Arzneimittelfors-

chung 42:821–824

17. Berger MR, Garzon FT, Keppler BK, Schmahl D (1989) Anti-

cancer Res 9:761–765

18. Sava G, Bergamo A (2000) Int J Oncol 17:353–365

19. Seelig MH, Berger MR, Keppler BK (1992) J Cancer Res Clin

Oncol 118:195–200

20. Frasca DR, Gehrig LE, Clarke MJ (2001) J Inorg Biochem

83:139–149

21. Kratz F, Hartmann M, Keppler B, Messori L (1994) J Biol Chem

269:2581–2588

22. Pongratz M, Schluga P, Jakupec MA, Arion VB, Hartinger CG,

Allmaier G, Keppler BK (2004) J Anal At Spectrom 19:46–51

23. Polec-Pawlak K, Abramski JK, Semenova O, Hartinger CG, Ti-

merbaev AR, Keppler BK, Jarosz M (2006) Electrophoresis

27:1128–1135

24. Piccioli F, Sabatini S, Messori L, Orioli P, Hartinger CG, Keppler

BK (2004) J Inorg Biochem 98:1135–1142

25. Kapitza S, Pongratz M, Jakupec MA, Heffeter P, Berger W,

Lackinger L, Keppler BK, Marian B (2005) J Cancer Res Clin

Oncol 131:101–110

26. Cauci S, Alessio E, Mestroni G, Quadrifoglio F (1987) Inorg

Chim Acta 137:19–24

27. Kelly JM, Feeney MM, Tossi AB, Lecomte JP, Kirsch-De

Mesmaeker A (1990) Anticancer Drug Des 5:69–75

28. Scolaro C, Bergamo A, Brescacin L, Delfino R, Cocchietto M,

Laurenczy G, Geldbach TJ, Sava G, Dyson PJ (2005) J Med

Chem 48:4161–4171

29. Dorcier A, Dyson PJ, Gossens C, Rothlisberger U, Scopelliti R,

Tavernelli I (2005) Organometallics 24:2114–2123

30. Scolaro C, Geldbach TJ, Rochat S, Dorcier A, Gossens A,

Bergamo A, Cocchietto M, Tavernelli I, Sava G, Rothlisberger U,

Dyson PJ (2006) Organometallics 25:756–765

31. Ang WH, Daldini E, Scolaro C, Scopelliti R, Juillerat-Jeannerat

L, Dyson PJ (2006) Inorg Chem 45:9006–9013

32. Scolaro C, Chaplin AB, Hartinger CG, Bergamo A, Cocchietto M,

Keppler BK, Sava G, Dyson PJ (2007) Dalton Trans 5065–5072

33. Allardyce CS, Dyson PJ, Ellis DJ, Heath SL (2001) Chem

Commun 1396–1397

34. Reiber M, Reiber MS (2008) Cancer Biol Ther 7 (in press)

35. Hayward RL, Schornagel QC, Tente R, Macpherson JS, Aird RE,

Guichard S, Habtemariam A, Sadler P, Jodrell DI (2005) Cancer

Chemother Pharmacol 55:577–583

36. Gaiddon C, Jeannequin P, Bischoff P, Pfeffer M, Sirlin C,

Loeffler JP (2005) J Pharmacol Exp Ther 315:1403–1411

37. Perrone G, Vincenzi B, Santini D, Verzı A, Tonini G, Vetrani A,

Rabitti C (2004) Cancer Lett 208:227–234

38. Garrido C, Galluzi L, Brunet M, Puig PE, Didelot C, Kroemer G

(2006) Cell Death Differ 13:1423–1433

39. Montaner B, Perez-Tomas R (2002) Ann N Y Acad Sci 973:246–

249

40. Frasca DR, Ciampa D, Emerson J, Umans RS, Clarke MJ (1996)

Met Based Drugs 3:197–209

41. Dyson PJ, Sava G (2006) Dalton Trans 1929–1933

42. Harris SL, Levine AJ (2005) Oncogene 24:2899–2908

43. Taylor WR, Stark GR (2001) Oncogene 20:1803–1815

44. Chen X, Zhang W, Gao YF, Su XQ, Zhai ZH (2002) Cell Res

12:229–233

45. Jaiswal AS, Bloom LB, Narayan S (2002) Oncogene 21:5912–5922

46. Coqueret O (2003) Trends Cell Biol 13:65–70

47. Seoane J, Pouponnot C, Staller P, Schader M, Eilers M, Massague

J (2001) Nat Cell Biol 3:400–408

48. Kuo PC, Liu HF, Chao JI (2004) J Biol Chem 279:55875–55885

49. Choi J, Chiang A, Taulier N, Gros R, Pirani A, Husain M (2006)

Circ Res 98:1273–1281

50. Li Z, Putzer BM (2008) J Cell Mol Med 9999 (in press)

51. Hengartner MO (2000) Nature 407:770–776

52. Fuchs SY, Adler V, Buschmann T, Yin Z, Wu X, Jones SN,

Ronai Z (1998) Genes Dev 12:2658–2663

53. Yoshida K, Miki Y (2005) Cell Cycle 4:777–779

54. Ishikawa Y, Kusaka E, Enokido Y, Ikeuchi T, Hatanaka H (2003)

Mol Cell Neurosci 24:451–459

55. Zu K, Hawthorn L, Ip C (2005) Mol Cancer Ther 4:43–50

56. Liu J, Lin A (2005) Cell Res 151:36–42

J Biol Inorg Chem (2008) 13:1149–1155 1155

123