A potent agent with antitumor activity

Article abstract of DOI:10.1021/jm100459k

In this study, we investigated the anticancer properties of methoxy-substituted nickel(II)(salophene) derivatives. We demonstrated that the most active complex [Ni^II(3-OMe-salophene)] is not necrotic in Burkitt-like lymphoma cells (BJAB) and hu

Full text of DOI:10.1021/jm100459k

6064 J. Med. Chem. 2010, 53, 6064–6070
DOI: 10.1021/jm100459k
[NiII(3-OMe-salophene)]: A Potent Agent with Antitumor Activity
†
‡
§
†
†
‡
€
Soo-Young Lee, Annegret Hille, Corazon Frias, Benjamin Kater, Birgit Bonitzki, Stefan Wolfl, Heike Scheffler,
Aram Prokop,†,§ and Ronald Gust*,‡,^
†Department of Pediatric Oncology/Hematology, University Medical Center Chariteꢀ, Berlin, Germany, ‡Institute of Pharmacy, Freie Universita€t
Berlin, Berlin, Germany, §Department of Pediatric Oncology, Children’s Hospital of the City of Cologne, Cologne, Germany, Institute of Phamacy
and Molecular Biotechnology, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany, and ^Department of Pharmaceutical Chemistry, Institute
of Pharmacy, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
Received April 14, 2010
In this study, we investigated the anticancer properties of methoxy-substituted nickel(II)(salophene)
derivatives. We demonstrated that the most active complex [NiII(3-OMe-salophene)] is not necrotic in
Burkitt-like lymphoma cells (BJAB) and human B-cell precursor cells (Nalm-6). [NiII(3-OMe-sal-
ophene)] inhibited proliferation and induced apoptosis in a concentration dependent manner, giving
evidence for the involvement of CD95 receptor-mediated, extrinsic pathway. Furthermore, [NiII(3-
OMe-salophene)] overcame vincristine drug resistance in BJAB and Nalm-6 cells.
Introduction
Despite all these different modes of action, data about cell
selectivity and resistance overcome of nickel(salophene) com-
plexes are missing. Here we describe antiproliferative effects
of methoxy-substituted nickel(II)(salophene) complexes. The
most active compound [NiII(3-OMe-salophene)] was further
investigated for its ability to induce programmed cell death
and to overcome resistance to the common therapeutic drug
vincristine in lymphoma and leukemia cells. Furthermore,
we demonstrated that the [NiII(3-OMe-salophene)]-induced
apoptosis is mediated by the CD95 receptor and is dependent
on the expression of the Fas-associated protein with death
domain (FADDa) and the second mitochondria activator of
caspases (smac), thus implying that the anticancer activity is
involved in the extrinsic apoptotic pathway.
Medicinal applications of inorganic chemistry are varied,
encompassing many aspects of the introduction of metal ions
into the body for therapeutic effect.1,2 The ligands of orga-
nometallic complexes are needed to ensure cellular uptake
culminating in precisely targeted particular tissues or en-
zymes. Additionally, biomolecular interactions of these me-
tal-based drugs are determined by the electrochemical and
kinetic variables of the respective central atom. The charac-
teristics of diverse non-platinum-containing metal com-
pounds and their use and/or putative mode of action in
modern cancer treatment are described in several review
articles (e.g., see refs 3 and 4). Recently we showed that es-
pecially the iron(salophene) complexes have increased anti-
tumor activity in MCF-7 and MDA-MB-231 breast as well as
HT-29 colon cancer cells.5 Furthermore, we demonstrated
the importance of the methoxy group in the salicylidene
moieties of the salophene ligand. The results obtained in a
time-dependent chemosensitivity test clearly confirmed the
correlation between cytotoxicity of the complexes and the
position of the methoxy substituents; the best cell growth
inhibitory effects were obtained with the complexes bearing
the electron donating group in positions ortho and para to the
imine moiety. Recently, we showed that [FeIII(salophene)Cl]
induced mitochondrial apoptosis in a concentration-depen-
dent manner and overcame resistance to the conventional
therapeutics daunorubicine and vincristine.6,7
Results
Synthesis and Characterization of the Test Compounds by
1H NMR Spectroscopy. The methoxy-substituted salophene
ligands were synthesized as already described.5,7 Reaction of
the respective ligand with Ni(II) acetate tetrahydrate (Table 1)
yielded the desired Ni(II)(salophene) complex in sufficient
purity.
The characterization of salophene ligands and Ni(II)
complexes were performed by using 1H NMR spectroscopy
(Figures 1). Nickel is one of the late row transition metals
forming with salophene-like ligands square planar diamag-
netic Schiff base complexes suitable for NMR investigation.
As depicted in Figure 1A, the ligands 5 and 9 showed the
resonance peaks of Ha and Hc at nearly the same frequency
with an upfield shift, indicating high electron density at these
Taking into account that some nickel (Schiff base) com-
plexes reveal their antitumor activity via DNA adduct forma-
tion and double strand breaks, promoting selective oxidation
of Z-DNA, depleting glutathione and protein sulfhydryl
groups, we focused our attention on the biological properties
of methoxy-substituted nickel(salophene) complexes, too.8-12
aAbbreviations: ALL, acute lymphoblastic leukemia; DMEM, Dul-
becco’s modified Eagle’s medium; FACS, fluorescence activated cell
sorting; FADD, Fas-associated protein with death domain; INT, 2-[4-
iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride; JC-1, 5,50,
6,60-tetrachloro-1,1,3,30-tetraethylbenzimidazolylcarbocyanine iodide;
LDH, lactate dehydrogenase; PBS, phosphate buffered saline; smac,
second mitochondria activator of caspases; Vcr, vincristine.
*To whomcorrespondence should beaddressed. Address:Department
of Pharmaceutical Chemistry, Institute of Pharmacy, University of
Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria. Phone: þ43 512
507 5245. Fax: þ43 512 507 2940. E-mail: ronald.gust@uibk.ac.at.
r
pubs.acs.org/jmc
Published on Web 07/29/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6065
Table 1. Synthesized Compounds with e, f, and g as protons
a
b
c
d
compd
no.
IC50 (μM)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
salophene
[NiIIsalophene]
3-OMe-salophene
1
2
7.98
0.36
34.2
>50
8.85
OMe
OMe
H
3
[NiII(3-OMe-salophene)]
4-OMe-salophene
4
OMe
OMe
H
5
H
[NiII(4-OMe-salophene)]
5-OMe-salophene
6
H
OMe
OMe
H
7
H
H
[NiII(5-OMe-salophene)]
6-OMe-salophene
8
H
H
OMe
OMe
9
10
H
H
H
[NiII(6-OMe-salophene)]
positions. Additionally, these signals of 5 were separated
as a double-doublet (Hc) and doublet (Ha) while those of
9 were superimposed to a quasi triplet. After coordination
(compound 10, Figure 1B), Ha and Hc were well separated
because of a strong high field shift of Ha, whereas the signals
of Ha and Hc of compound 6 remained nearly unchanged.
The resonance signals assigned to Hf and Hg of the ligands
appeared at 7.4-7.5 ppm, irrespective of the substitution
pattern, indicating that the electron density distribution
within the phenylene moiety was not significantly affected
by the position of the OCH3 substituent in the salicylidene
ring. After coordination of the ligands, however, Hf was
characteristically shifted to lower field (from about 7.4 to
8-8.2 ppm). Interestingly, both resonance signals occurred
at the same frequencies of 8.16 ppm (Hf) and 7.35 ppm (Hg)
in the spectra of the complexes 2, 4, and 8.
In Vitro Chemosensitivity Assay. Anticancer activity of
the nickel(II) complexes 2, 4, 6, 8, and 10 against MCF-7
mammary carcinoma cells was evaluated in a time-depen-
dent (see Figure S1 of Supporting Information) and concen-
tration-dependent (IC50 determination; see Table 1) crystal
violet assay. Whereas all ligands were inactive (data not
shown), the position of the methoxy substituent determined
the potency of the respective nickel complex.
membrane damage and the early release of lactate dehydro-
genase (LDH). In order to exclude such an unspecific
cytotoxic effect, we determined the amount of extracellular
LDH in Burkitt-like lymphoma cells (BJAB) by incubating
the cells with various concentrations (0-20 μM) of the
complex [NiII(3-OMe-salophene)] for 3 h and measuring
the LDH release into the culture medium via ELISA detec-
tion. As depicted in Figure 2, no significant LDH release
could be detected after 3 h of incubation with the agent. It is
assumed that necrosis does not have a significant impact on
the potency of [NiII(3-OMe-salophene)].
Induction of Apoptosis by [NiII(3-OMe-salophene)]. [NiII-
(3-OMe-salophene)] showed efficient antiproliferative effects
in BJAB cells. As gauged from Figure 3, cell proliferation
could be significantly decreased in a concentration-dependent
manner. To further quantify the [NiII(3-OMe-salophene)]-
induced induction of apoptosis, we exposed BJAB cells to the
drug at different concentrations for 72 h and analyzed DNA
fragmentation by flow cytometric determination via fluores-
cence activated cell sorting (FACS). The data, shown in
Figure 4, verify specific dose-dependent induction of apopto-
sis with a half-maximum concentration of about 10 μM.
Involvement of the Mitochondrial Pathway in the Induction
of Apoptosis. In order to determine the involvement of the
mitochondrial pathway in the induction of apoptosis, the
mitochondrial activation was measured by performing a JC-
1 (5,50,6,60-tetrachloro-1,1,3,30-tetraethylbenzimidazolylcar-
bocyanine iodide) assay.13 For this purpose BJAB cells were
incubated with [NiII(3-OMe-salophene)] for 48 h, followed
by staining the treated cells with the fluorescence dye JC-1
and quantification of the mitochondrial permeability via
flow cytometric determination of cells with declined fluores-
cence. Our investigations revealed a minimal loss of the
mitochondrial membrane potential at higher concentrations,
thus indicating that the mitochondrial pathway does not
play a very important role in [NiII(3-OMe-salophene)]-in-
duced apoptosis (Figure 5).
The unsubstituted (2) and the 6-OCH3 derivative (10)
caused low growth inhibitory effects (50-60% reduction)
at 1 μM. A shift of the OCH3 group from position 6 to 5 or 4
terminated the activity at this concentration, while a shift to
position 3 led to an analogue (4, [NiII(3-OMe-salophene)])
with cytocidal properties (see Figure S1). It already blocked
cell growth after an incubation time of 60 h.
For a better comparison of the antitumor potency, the
IC50 values were calculated after an incubation time of 72 h
(Table 1). At that time all complexes reached their maximal
growth inhibitory effects (see Figure S1), which increased in
the the following order: 8 (5-OCH3, IC50>50 μM)<6 (4-
OCH3, IC50=34.2 μM)<10 (6-OCH3, IC50=8.85 μM) ∼ 2
(H, IC50=7.98 μM) , 4 (3-OCH3, IC50=0.36 μM).
Induction of Apoptosis by [NiII(3-OMe-salophene)] in Vin-
cristine-Resistant Leukemia and Lymphoma Cell Lines. The
development of resistance to various conventional chemother-
apeutic drugs remains a major component in the failure of
chemotherapy. Therefore, the effect of [NiII(3-OMe-salophene)]
Because of its outstanding cytotoxicity, [NiII(3-OMe-sal-
ophene)] was selected for further biological investigations.
Exclusion of Necrotic Effects. Many cytotoxic substances
cause undesired necrotic cell death which is characterized by

6066 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Lee et al.
Figure 1. (A) 1H NMR of the ligands 1, 3, 5, 7, and 9. (B) 1H NMR of the nickel(II) complexes 2, 4, 6, 8, 10.
on vincristine (Vcr) resistant leukemia and lymphoma cell
lines was tested. After treatment of regular leukemia cells
(Nalm-6) and lymphoma cells (BJAB) and a Vcr modifica-
tion of both cell lines for 72 h, the apoptosis induction was
determined by flow cytometric measurements. As depicted in
Figure 6, [NiII(3-OMe-salophene)] induced higher apoptosis
in the resistant cells compared to the control cells. Thus, it can
be assumed that the agent was capable of overcoming drug
resistance in Vcr-resistant leukemia and lymphoma cells.
Involvement of the Extrinsic Pathway in [NiII(3-OMe-
salophene)]-Induced Apoptosis. As described before, the mi-
tochondrial pathway does not seem to play an important role
in the induction of apoptosis, since there was no significant
loss of the mitochondrial membrane potential observed. To
emphasize the suggestion that the [NiII(3-OMe-salophene)]-
induced apoptosis mainly follows the extrinsic pathway, we
used Jurkat cells (human T cell leukemia cell line) over-
expressing smac, a proapoptotic mitochondrial protein that
is released during the intrinsic apoptotic pathway. As de-
picted in Figure 7, the [NiII(3-OMe-salophene)]-induced cell
death turned out to be independent of the expression of the
proapoptotic intrinsic factor smac.
Additionally, the Fas-associated death domain dependence
was investigated to further prove if the alternative extrinsic
pathway takes part. In the extrinsic pathway binding of the Fas
ligand to Fas/CD95 activates the apoptotic program by recruit-
ing a FADD adaptor, finally leading to DNA fragmentation.14
Using BJAB cells overexpressing a dominant-negative FADD
(FADD-dn) mutant, we could show that these cells underwent
apoptosis to a lower level than the control cell line (BJAB
mock), revealing that the DNA fragmentation was significantly
influenced by FADD-dn overexpression, which in turn indi-
cates that [NiII(3-OMe-salophene)]-induced cell death proceeds
dependently of CD95/Fas signaling (Figure 8).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6067
Figure 2. Quantification of lactate dehydrogenase (LDH) release.
To evaluate necrotic properties of [NiII(3-OMe-salophene)], BJAB
cells were treated with different concentrations of [NiII(3-OMe-
salophene)] for 3 h. Viability of the cells was determined by LDH
release through photometrical measurement via ELISA reader.
LDH activity of lysed cells was used as a 100% control. Values
are given as % of control ( SD (n = 3).
Figure 5. Influence of [NiII(3-OMe-salophene)] on the mitochon-
drial membrane potential. After 48 h of incubation of BJAB cells
with [NiII(3-OMe-salophene)] the mitochondrial permeability tran-
sition was measured by flow cytometric analysis. Values of the
mitochondrial permeability transition are given as the fraction of
cells with decreased membrane potential (ΔΨm) in % ( SD (n = 3).
Figure 3. Antiproliferative effect of [NiII(3-OMe-salophene)] in
BJAB cells. Inhibition of cell proliferation in a concentration-
dependent manner after treatment with [NiII(3-OMe-salophene)]
for 24 h as measured by a Casy cell counter system. Bars indicate the
number of cells after 24 h of incubation in units of 105 cells mL-1
(
SD (n = 3). The line chart indicates the inhibition of cell prolifera-
tion as % of the control.
Figure 6. Apoptosis induced by [NiII(3-OMe-salophene)] in vin-
cristine-resistant leukemia (Nalm-6) and lymphoma (BJAB) cells.
Apoptosis was determined by DNA fragmentation in regular
leukemia and lymphoma cells as well as in (A) Vcr-resistant
Nalm-6 cells and (B) Vcr-resistant BJAB cells after an incubation
period of 72 h with various concentrations of the indicated drug.
Data are given as percentage hypodiploidy (subG1) ( SD (n = 3),
which reflects the number of apoptotic cells.
Figure 4. Apoptosis induction in BJAB cells after treatment with
different concentrations of [NiII(3-OMe-salophene)]. Nuclear DNA
fragmentation was measured after 72 h of incubation via flow
cytometric anaylysis by determining the hypodiploid DNA. Data
are given as % hypodiploidy (subG1) ( SD (n = 3), which reflects
the number of apoptotic cells.
induction of apoptosis in tumor cells are known to be the
major therapeutic mechanisms of anticancer drugs, since a
deregulation of both processes causes malignant diseases.15
We already demonstrated the cytotoxic potential of the
Schiff base iron complex [FeIII(salophene)Cl] in vitro and ex
vivo and illustrated its excellent ability to overcome multiple
drug resistance. Treatment of BJAB lymphoma cells with
[FeIII(salophene)Cl] led to a concentration-dependent inhibi-
tion of proliferation up to 100%. A significant loss of the
Discussion
Despite the improved outcome of patients with relapsed
acute lymphoblastic leukemia (ALL), chemotherapy failure
due to cellular drug resistance remains a leading problem in
pediatric oncology. The inhibition of proliferation and the

6068 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Lee et al.
tance of the transition metal on the biological properties of the
respective complex and the involvement of the metal in the
mode of action.
In this structure-activity relationship study we could show
that methoxy groups at the salicylidene moiety of [NiII-
salophene] modified the biological properties more efficiently.
On the one hand, the introduction at the 4- or 5-position
terminated the effects at the MCF-7 cell line nearly comple-
tely. The methoxy substituent at position 6 did not change the
activity compared to the unsubstituted [NiIIsalophene] com-
plex. On the other hand, a substitution at position 3 led to a
compound with outstanding cytotoxicity. At the MCF-7 cell
line, IC50=0.34 μM was calculated, which is even lower than that
of the reference cisplatin (IC50 =2.2 μM). Therefore, [NiII(3-
OMe-salophene)] was selected for detailed biological studies.
This complex induced, in contrast to [FeIII(salophene)Cl], apop-
tosis in a FADD- and smac-dependent manner and was accom-
panied by a minor loss of the mitochondrial membrane potential,
thus indicating that [NiII(3-OMe-salophene)] efficiently triggers
the extrinsic pathway of apoptosis. Furthermore, [NiII(3-OMe-
salophene)] overcomes vincristine resistance in leukemia and
lymphoma cells.
Figure 7. Impairment of overexpression of proapoptotic smac in
human T cell leukemia cells (Jurkat) on [NiII(3-OMe-salophene)]-
induced DNA fragmentation. By use of the cell line Jurkat smac,
overexpressing the proapoptotic intrinsic factor smac, DNA frag-
mentation was measured by flow cytometric analysis after 72 h of
incubation with [NiII(3-OMe-salophene)]. Data are given as per-
centage hypodiploidy (sub-G1) ( SD (n = 3), which reflects the
number of apoptotic cells. As a control, vector-transfected Jurkat
cells (Jurkat neo) were used.
Conclusions
We demonstrated in this paper that [NiII(3-OMe-salophene)]
constitutes a novel lead structure with remarkably high levels of
apoptosis induction in leukemia and lymphoma cells and
promising anticancer activities in drug resistant cell lines.
Experimental Section
General. Chemicals were obtained from Sigma Aldrich
(Germany) and used without further purification. The following
instrumentation was used: 1H NMR, Bruker ADX 400 spectro-
meter at 400 MHz (internal standard, TMS); EI-MS spectra, CH-
7A Varian (70 eV); IR spectra (KBr pellets), Perkin-Elmer model
580 A. Elemental C, H, N analyses werecarried out with a Perkin-
Elmer 240 B and 240 C elemental analyzer. The purity of all
synthesized substances was confirmed by elemental analysis. All
complexes reported in the manuscript have a purity of >95%.
Synthesis. Salophene (1), [NiIIsalophene] (2), and the meth-
oxy-substituted salophene ligands 3, 5, 7, and 9 were prepared as
described elsewhere.5,7
Figure 8. Impairment of overexpression of dominant-negative
FADD in BJAB cells on [NiII(3-OMe-salophene)]-induced DNA
fragmentation. After exposure of vector-transfected or FADD dn-
transfected BJAB cells to [NiII(3-OMe-salophene)] for 72 h, DNA
fragmentation was measured. Data are given as percentages of cells
with hypodiploid DNA ( SD (n = 3).
General Procedure for the Synthesis of the Complexes 4, 6, 8,
10. The respective ligand was dissolved in ethanol (10 mL) and
heated to reflux in the presence of 1.5 equiv of Ni(OAc)2 4H2O
mitochondrial membrane potential in lymphoma cells indi-
cated the involvement of the intrinsic mitochondrial path-
way. Specific apoptotic cell death was detected for leukemia
(Nalm-6) and lymphoma cells (BJAB) via DNA fragmenta-
tion. We further confirmed the up- and down-regulation of
various apoptosis relevant genes via real-time PCR and an
overcoming of drug resistance against vincristine- and dau-
norubicine-resistant Nalm-6 cells.
Introduction of OCH3 into the salicylidene moieties modi-
fied the growth inhibitory potency. Ifthe substituent is located
in a position ortho or para to the phenolic oxygen, the acti-
vity will be reduced by half while the attachment at the
meta-position increased the growth inhibitory effects. In each
case, however, a steep concentration-response relation was
observed.
3
in ethanol (5 mL). After 1-2 h, the mixture was allowed to cool
to room temperature and the solid obtained was filtered off,
washed with ethanol, and dried in vacuum.
[NiII(3-OMe-salophene)] 4. Compound 4 was obtained from
N,N0-bis(3-methoxysalicylidene)-1,2-phenylenediamine and 1.5
equiv of Ni(OAc)2 4H2O as a red-black solid. Yield: 237.33 mg
3
(0.57 mmol, 72%). 1H NMR (DMSO-d6): δ=8.91 (s, 2H, He),
8.17-8.14 (AA0XX0, 2H, Hf, 3J=9.6 Hz, 4J=2.9 Hz), 7.35-7.33
(AA0XX0, 2H, Hg, 3J=9.5 Hz, 4J=3.0 Hz), 7.22-7.20 (dd, 2H,
Hb, 3J=6.9 Hz, 4J=1.4 Hz), 6.90-6.89 (dd, 2H, Hd, 3J=7.4 Hz,
4J =1.1 Hz), 6.61-6.57 (dd, 2H, Hc, 3J=7.9 and 7.8 Hz), 3.76
(s, 6H, -OCH3). IR (KBr): ν [cm-1] = 1608 s, 1540 s, 1247 s, 1199
s, 740 m. MS (EI, 100 ꢀC): m/z (%)=432 (100) [Mþ•]. Anal. (C22-
H18N2O4Ni) C, H, N.
Stability of Stock Solution. For stability evaluations in
DMSO-d6 the nickel(II) complexes 2 and 6 were dissolved.
The proton resonance of this solution was measured within a
week of freezing-thawing cycles. Because no changes were
observed in the resulting 1H NMR spectra, the respective stock
solution of the nickel(II) complexes in DMSO could be stored
at -20 ꢀC (data not shown).
In addition to these studies we verified the importance of
the transition metal on controlling cancer cell proliferation of
[FeIII(salophene)Cl]. As soon as the iron central atom was
exchanged by nickel(II), cell growth inhibition and apoptosis
induction were diminished, clearly pointing out the impor-

Article
Biological Methods. Cell Culture Conditions and in Vitro
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6069
staining with propidium iodide. During apoptosis, the phos-
pholipid phosphatidylserine is exposed to the outer leaflet of the
plasma membrane. Annexin-V-FITC then binds to phosphati-
dylserine, leading to an increase in fluorescence.17,18 On the
other hand, propidium iodide is excluded from cells with intact
membranes. Propidium iodide positivity is therefore a sign of
cell necrosis, whereas cells that are annexin-V-FITC positive but
propidium negative are generally defined as apoptotic.19 For the
annexin-V-propidium iodide assay, 1 ꢀ 105 cells were washed
twice with ice cold PBS and then resuspended in a binding buffer
(10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/
NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) at 1 ꢀ 106
cells/mL. Next, 5 mL of annexin-V-FITC (BD Pharmingen,
Heidelberg, Germany) and 10 μL of 50 μg/mL propidium iodide
(Sigma-Aldrich, Taufkirchen, Germany) were added to the cells.
Analyses were performed on a FACScan (Becton Dickinson,
Heidelberg, Germany) using the CellQuest analysis software.
Chemosensitivity Assay. The anticancer activity of all com-
pounds against MCF-7 breast cancer cells was determined as
described previously.5 Prior to use, the respective DMSO stock
solution was diluted with DMEM to obtain the desired con-
centration. The final DMSO concentration amounted to 0.1%.
The following cell lines were used: BJAB (Burkitt like lympho-
ma cells), mock and FADD transfected cells; Nalm-6 (human B
cell precursor leukemia cells); Jurkat (human T cell leukemia
cells), neo/smac-transfected cells; vincristine-resistant BJAB
and Nalm-6. The cells were subcultured every 3-4 days by
dilution of the cells to 1 ꢀ 105/mL. All experiments were per-
formed in DMEM or RPMI 1640 (GIBCO, Invitrogen) supple-
mented with 10% heat inactivated fetal calf serum, 100 U/mL
penicillin, 100 μg/mL streptomycin, and 0.56 g/L L-glutamine.
Twenty-four hours before the assay was set up, cells were
cultured at a concentration of 3 ꢀ 105/mL to attain standardized
growth conditions. For apoptosis assays, the cells were then
diluted to 1 ꢀ 105/mL immediately before addition of [NiII (3-
OMe-salophene)].
Measurement of Cell Death by LDH-Release Assay. Cyto-
toxicity of the different drugs was measured by the release of
lactate dehydrogenase (LDH). After incubation with different
concentrations of the agents for 3 h, LDH activity released by
BJAB cells was measured in the cell culture supernatants using
the cytotoxicity detection kit from Boehringer Mannheim
(Mannheim, Germany). The supernatants were centrifuged at
1500 rpm for 5 min. An amount of 20 μL of cell-free super-
natants was diluted with 80 μL of phosphate-buffered saline
(PBS), and an amount of 100 μL of reaction mixture containing
2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chlor-
ide (INT), sodium lactate, NADþ, and diaphorase were added.
Then time-dependent formation of the reaction product was
photometrically quantified at 490 nm. The maximum amount of
LDH activity released by the cells was determined after lysis of
the cells using 0.1% Triton X-100 in culture medium and set to
represent 100% cell death.
Determination of Cell Concentration and Cell Viability. Cell
viability was determined by using the “CASY cell counter þ
analyzer system” of Innovatis (Bielefeld, Germany). Settings
were specifically defined for the requirement of the cells used.
With this system the cell concentration can be analyzed simul-
taneously in three different size ranges; thus, cell debris, dead
cells, and viable cells could be determined in one measurement.
Cells were seeded at a density of 1 ꢀ 105cells/mL and treated
with different concentrations of [NiII(3-OMe-salophene)]; non-
treated cells served as control. After a 24 h incubation period,
cells were resuspended completely and 100 μL of each well was
diluted in 10 mL of CASYton (ready-to-use isotonic saline
solution) for immediate automated counting.
Measurement of DNA Fragmentation. Apoptotic cell death
was determined by a modified cell cycle analysis, which detects
DNA fragmentation at the single cell level as described.16 Cells
were seeded at a density of 1ꢀ105 cells/mL and treated with
different concentrations of the agents. After a 72 h incuba-
tion period at 37 ꢀC, cells were collected by centrifugation at
1500 rpm for 5 min, washed with PBS at 4 ꢀC, and fixed in PBS/
2% (v/v) formaldehyde on ice for 30 min. After fixation, cells
were pelleted, incubated with ethanol/PBS (2:1, v/v) for 15 min,
pelleted, and resuspended in PBS containing 40 μg/mL RNase.
RNA was digested for 30 min at 37 ꢀC, after which the cells
were pelleted again and finally resuspended in PBS containing
50 μg/mL propidium iodide. Nuclear DNA fragmentation was
quantified by flow cytometric determination of hypodiploid
DNA. Data were collected and analyzed using a FACScan
(Becton Dickinson, Heidelberg, Germany) equipped with CELL
Quest software. Data are given in percent hypoploidy (subG1),
which reflects the number of apoptotic cells.
Acknowledgment. Financial support by the Deutsche For-
schungsgemeinschaft (DFG-project FOR630) and the Dr.
Kleist Foundation, Berlin, are gratefully acknowledged.
Supporting Information Available: Elemental analysis results
of all complexes; time-dependent growth inhibition of MCF-7
cells; and experimental data of complexes 6, 8, and 10. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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