The antineoplastic potential of a stable monomeric Au(II) complex with hematoporphyrin IX (Hp), namely [Au(II)Hp-2H.(H2O)2], was investigated in a panel of tumor cell lines. The complex exhibits strong cytotoxicity, whereby the leukaemia- and lymphoma-derived cell lines are more sensitive, with IC50 values comparable to those of the reference anticancer drug cisplatin. In contrast, the solid tumor models are more sensitive to the platinum drug. A comparative assessment of both agents against the human kidney cell line 293T has shown that [Au(II)Hp-2H.(H2O)2] is less cytotoxic. The gold complex induces oligonucleosomal DNA fragmentation in tumour cells following 24-hour treatment and hence its cytotoxic effect is at least partly mediated by induction of apoptotic cell death. A prominent intracellular gold accumulation was detected after treating tumor cells with [Au(II)Hp-2H.(H2O)2] which shows that its putative pharmacological targets are readily accessible after a short incubation period.
1. Introduction
Cisplatin and the structurally related platinum-based drugs represent one of the most important classes of antineoplastic agents, being especially valuable for the treatment of germ cell cancer and a variety of other solid malignancies [1–3]. Despite their important clinical role, however, the platinum-based
chemotherapeutics possess relatively low selectivity to malignant cells and
hence their application is associated with significant dose-limiting organ
toxicities [1]. Beside their unfavorable
safety profile, the major limitation in the clinical application of the currently
marketed platinum agents is the development of acquired resistance by the tumor
cells [2]. Consequently, a significant
interest is manifested towards the design and synthesis of cisplatin-dissimilar
analogues with modified pharmacological properties capable of bypassing the
cellular resistance mechanisms [4–6].
Considering
the fact that the thermodynamic stability and kinetic behavior of the metal
complexes in biological milieu and hence their biochemical and pharmacological
properties depend greatly on the nature of the adduct-forming metal centers, it
is well appreciated that a change of the metal ion could alter the antineoplastic
activity [6]. Among the nonplatinum metal-based
chemotherapeutics much attention has been paid to gold complexes [7]. Well known for their clinical antiarthritic
application [8], the gold-based drugs
have also attracted interest as potential antineoplastic agents with gold(I)-phosphine
derivatives being among the most active in
vivo against murine tumor
models [9]. Currently, the greatest
interest towards development of gold-based chemotherapeutics is focused on the Au(III) compounds which
being isoelectronic with platinum(II) share the propensity of forming square planar complexes, analogous
to cisplatin [7, 10]. It could be
anticipated that similar to Pt(II) compounds, the gold(III) species are capable of binding on DNA and this is the reason for their
cytotoxicity. Unlike platinum(II), however, the gold(III) complexes are extremely unstable under physiological conditions
which practically preclude the interest towards this class of metal-based drugs.
In addition, the gold(III)
complexes are highly reactive and are able to oxidize a series of biomolecules
such as methionine, glycine, and albumin leading to a quick reduction to gold(I) or even to
elemental gold [10–12]. It has been
proven that the stability of Au(III) compounds can be augmented by bonding with nitrogen donor-containing
bi- and multidentate chelating ligands such as ethylendiamine, cyclam, bipyridine,
and so forth, that lower the redox potential of the metal center [10, 13, 14]. Recently,
a large number of reports on the preparation, structural characterization, and
cytotoxic studies of stable gold(III) complexes and organometallic compounds appeared in the scientific literature [10].
With
regard to redox stability and kinetic behavior, the search for proper cytotoxic gold(II) complexes
is an intriguing and previously unexplored area of anticancer drug design. Nowadays,
the Au(II) oxidation
state can be considered as a common state in gold chemistry. Despite the large
number of stable diamagnetic dinuclear and polynuclear gold(II) complexes, the examples of mononuclear
ones are scarce and most of them are with S-containing ligands [15–17]. Recently, the synthesis and structural characterization of a stable monomeric hematoporphyrin Au(II) complex
with general formula [Au(II)Hp-2H.(H2O)2] (Figure 1) and distorted octahedral
structure has been reported [18]. Au(II) species are
stabilized in the complex through coordination via the four nitrogen atoms of
the porphyrin macrocycle and the two water molecules are in axial position.
Chemical structure of the tested gold complex [Au(II)Hp-2H.(H2O)2].
The
rationale design for synthesizing porphyrin-based metal complexes as anticancer
drugs is based on their selective accumulation within malignant tissue together
with participation in augmentation of the cytotoxicity upon light irradiation. Hence,
such complexes are expected to behave like hybrid drugs with combined cytotoxic/phototoxic
properties [4]. Brunner and coworkers
have described large series of planar platinum(II)-porphyrin conjugates whereby the
metal centers are coordinated with the porphyrin residues via the pendant
functionalities [19–21]. Recently, we
have synthesized and characterized three stable octahedral platinum
hematoporphyrin complexes in the unusual oxidation state of platinum 3+. In
these complexes the hematoporphyrin ligand is coordinated as follows: via four
pyrrole N-atoms forming metalloporphyrin type complex; or by asymmetric
coordination through two N-atoms from adjacent pyrrole rings forming SAT-type
complex; or by the side chains propionic COO−groups outside the
porphyrin macrocycle. The complexes displayed significant cytotoxic and
proapoptotic activities against human tumor cell lines [22].
This
study deals with the cytotoxic activity of the newly synthesized stable gold(II) complex Au(II)Hp-2H.(H2O)2 against a spectrum of tumor cell lines. Its effect on the human epidermal
kidney cell line 293T has been studied as well in order to estimate the
selectivity of cytotoxicity.
2. Materials and Methods2.1. Chemicals and Reagents
RPMI-1640 and DMEM growth
media, fetal calf serum, and L-glutamine were purchased from 3. Sigma-Aldrich Co, “St. Louis”, Missouri, USA.
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- Triton
X-100, gold standard, Tris HCl, DMSO, and EDTA were supplied from Merck Co.
The gold(II) complex with hematoporphyrin—[Au(II)Hp-2H.(H2O)2]
was synthesized as previously described [18].
The reference anticancer drug cisplatin was purchased from Sigma. Stock
solutions of both agents were freshly prepared in DMSO and promptly diluted serially
with RPMI-1640 medium to the desired extent. The DMSO concentration never
exceeded 1% in the final dilutions obtained.
2.2. Cell Lines and Culture Conditions
The T-cell leukaemia
SKW-3 (a KE-37 derivative) (DSMZ No.: ACC 53); the nonHodgkin lymphoma DOHH-2 (DSMZ No.: ACC 47); the chronic myeloid leukaemias K-562
(DSMZ No. ACC 10), and LAMA-84 (DSMZ No. ACC 168) as well as the urinary bladder
carcinoma-derived 5637 (DSMZ No.: ACC 35) were obtained from DSMZ GmbH (Braunschweig , Germany). The human urinary bladder
carcinoma cell line MGH-U1 was supplied by American
Type Cell Culture (Rockville, MD, USA). The cells were maintained as
suspension type cultures (leukaemias) or as adherent cultures (5637 and MGH-U1) in controlled
environment: RPMI-1640 medium, supplemented by 10%
heat-inactivated fetal calf serum and 2 mM L-glutamine, at 37°C in a “Heraeus”
incubator with 5% CO2 humidified atmosphere. In order to keep cells
in log phase, the cultures were refed with fresh RPMI-1640 medium two or three
times/week.
2.3. Cytotoxicity Assay
Cell viability was assessed using the standard MTT-dye reduction assay as previously described [23] with minor modifications [24].
Exponentially growing cells were seeded in 96-well flat-bottomed microplates
(100 μL/well) at a density of 1×105 cells per mL and after 24-hour incubation at 37°C; they were exposed to
various concentrations of the tested complexes for 72 hours. For each concentration at least 8 wells were
used. After the incubation with the test compounds MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) solution
(10 mg/mL in PBS) was added (10 μL/well). Microplates were further incubated
for 4 hours at 37°C and the quantity of formazan product obtained was
determined spectrophotometrically using a microprocessor-controlled multiplate
reader (Labexim LMR-1) at 580 nm. The cell survival fractions were calculated as percentage of the
untreated control (untreated control = 100%). The experimental data were
transformed to sigmoidal dose-response curves using nonlinear regression
analysis (GraphPad Prizm), which enabled the calculation of the corresponding IC50 values.
2.4. DNA Fragmentation Analysis
The
characteristic for apoptosis mono- and oligonucleosomal fragmentation of
genomic DNA was detected using “Cell Death Detection” ELISA kit (Roche Diagnostics, Germany). The exponentially growing MGH-U1, K-562,
HD-MY-Z, and SKW-3 cells were plated in sterile petry dishes and exposed to
equipotent concentrations for 24 hours. Cytosolic
fractions of 1×104 cells per group (treated or
untreated) served as antigen source in a sandwich ELISA utilizing primary
antihistone antibody-coated microplate and a secondary peroxidase-conjugated
anti-DNA antibody. The photometric immunoassay for histone-associated DNA fragments was executed in accordance with the
manufacturer's instructions at 405 nm using ELISA reader (Labexim LMR-1). The
results were expressed as the oligonucleosome enrichment factor representing the
ratio between the absorption in the treated versus the untreated control
samples.
2.5. Cellular Accumulation Kinetics
Aliquots of 2×107 K-562 cells and HD-MY-Z cells
(in 2 mL RPMI 1640) were placed in sterile petry dishes and exposed to
different concentrations of the gold complex: (12.5, 25, 50, and 100 μM) for 30 or 60 minutes. After the exposure
period, the cells were spun at 2000 rpm for 5 minutes and the drug-containing medium was
discarded. The cells were then washed thrice with phosphate buffered saline and aliquots were taken
for counting and cell viability determination (trypan blue dye exclusion
assay). Thereafter, the cells were digested in 50 μL 10% Triton X100 (EDTA) for 5 minutes, and 950 μL mixture of concentrated hydrochloric acid and
concentrated nitric acid (3 : 1) was added in order to allow complete
decomposition of the cells, and dissolution of the accumulated gold complex for
1 hour. Following the complete decomposition of the cells, the volume of the
samples was adjusted to 1 mL and the gold concentration was determined. The gold concentrations in the sample solutions
higher than 0.5 μg/mL were determined using flame atomic
absorption spectrometry (FAAS) with an air-acetylene flame (PYE UNICAM SP
1950). Lower gold concentrations were determined by electrothermal AAS using a
Perkin-Elmer Zeeman 3030 spectrometer with an HGA-600 graphite furnace. The
light source was a hollow cathode lamp for Au. The spectral bandpass was 0.7 nm. Standard uncoated graphite tubes were used as atomizer. Only peak areas
were used for quantification. The results
were expressed as nmol gold/106 cells.
2.6. Data Processing and Statistics
The cytotoxicity assays were carried
out in eight separate experiments, whereas the apoptosis induction evaluation
was conducted in quadruplicate. The data processing exploited MS Excel and
GraphPad Prizm software for PC. Student’s t-test was performed with P≤.05
taken as significance level.
3. Results3.1. Cytotoxicity Against Tumor Cell Lines
The
cytotoxic potential of the novel Au(II) complex was studied in a panel of malignant cell lines,
originating from leukaemias, lymphomas, and solid tumors. The results of the
chemosensitivity screening program, following 72-hour treatment, are
summarized in Table 1. Throughout the screening investigation cisplatin was
used as a positive control.
IC50 values of Au(II)Hp-2H.(H2O)2 and cisplatin against a panel of tumor cell lines assessed after
72-hour exposure (MTT-dye reduction assay).
Cell line
Origin/Cell type
IC50(μM)
cisplatin
Au(II)Hp-2H.(H2O)2
LAMA-84
chronic myeloid leukaemia
65.1
20.3
K-562
chronic myeloid leukaemia
161.2
32.0
SKW-3(a)
T-cell leukaemia
7.6
11.7
DOHH-2
nonHodgkin lymphoma
50.1
35.0
HD-MY-Z
Hodgkin’s lymphoma
43.1
12.2
MGH-U1(b)
urinary bladder cancer
56.4
5.9
MCF-7
breast cancer
166.8
33.4
SAOS-2
osteogenic sarcoma
>200
15.2
Neuro-2a
murine neuroblastoma
>200
>200
(a) KE-37 derivative;
(b) Formerly designated as EJ.
The cytotoxic effects of the Au(II) complex were
evaluated using the concentration-response curves presented in Figure 2. The
cellular viability was reduced significantly causing 50% inhibition at
micromolar concentrations in the majority of the cell lines tested.
Concentration-response curves of [Au(II)Hp-2H.(H2O)2] (▪) and cisplatin (▴) against a panel of tumor cell lines as assessed by the MTT-dye reduction assay after 72-hour exposure. Each data point represents the arithmetic mean ± sd of eight separate experiments.
Generally, the leukaemia and
lymphoma-derived cell lines were more sensitive to the Au(II) complex and the IC50 values obtained were comparable to those of the referent anticancer drug cisplatin.
Among them the most responsive tumor model was the T-cell leukaemia SKW-3
(KE-37 derivative) (Table 1, Figure 2). Against this cell line the relative
potency of [Au(II)Hp-2H.(H2O)2] even surpassed that of cisplatin. In contrast, the
solid-tumor-derived cell lines showed far more pronounced sensitivity to
cisplatin as compared to the novel gold(II) complex. The murine neuroblastoma Neuro2A was
resistant to both metal complexes.
3.2. In Vitro Cytotoxicity Study of [Au(II)Hp-2H.(H2O)2] on
Human Kidney Cells in Comparison to Cisplatin
The nephrotoxicity
of the novel compound in an in vitro test system versus the established nephrotoxic drug cisplatin was estimated. The human embryonic
kidney 293T cells were exposed for 72 hours to either cisplatin or [Au(II)Hp-2H.(H2O)2] and thereafter, their viability was detected with MTT-dye reduction assay (Figure 3). Throughout the tested range of concentrations the gold(II) complex proved to be only
marginally cytotoxic and failed to cause 50% reduction of cell viability. In
contrast, the referent drug cisplatin exhibited more pronounced cytotoxicity
upon kidney cells with an IC50 value of 3.87 μM.
Cytotoxic effects of [Au(II)Hp-2H.(H2O)2] (▪) and cisplatin (▴) against the human embryonic kidney cell line 293T as assessed by the MTT-dye reduction assay after 72 hours exposure. Each data point represents the arithmetic mean ± sd of eight separate experiments.
3.3. Induction of Apoptosis Following [Au(II)Hp-2H.(H2O)2] Treatment
Despite their
principle modes of action, the majority of anticancer drugs share the
distinction of being capable of recruiting the apoptotic cell death signaling
pathways in malignant cells. Therefore, the ability of [Au(II)Hp-2H.(H2O)2] to evoke genomic DNA-fragmentation which is a key hallmark of programmed cell
death was investigated. For this purpose, the exponentially growing SKW-3, K-562,
HD-MY-Z, and MGH-U1 cells were exposed to equieffective concentrations of the gold(II) complex or
cisplatin for 24 hours and thereafter, the levels of oligonucleosomal DNA
fragmentation were assessed using a commercially available ELISA kit (Figure 4).
Internucleosomal DNA
fragmentation in SKW-3, K-562, HD-MY-Z, and MGH-U1 cells after 24-hour exposure to
equipotent concentrations of [Au(II)Hp-2H.(H2O)2] (white columns) or cisplatin (gray
columns). The level of DNA fragmentation expressed as the corresponding enrichment factor
(ef=1 in untreated control) was determined using “Cell Death Detection”
ELISA (Roche Diagnostics).
The results obtained indicate
that [Au(II)Hp-2H.(H2O)2] is a potent apoptosis inductor causing
similar level of DNA fragmentation as the referent drug cisplatin if applied at
equipotent concentrations. The effect of the gold complex in MGH-U1 and HD-MY-Z
was characterized by a straightforward concentration dependence, whereby the
proportion of apoptotic cells arise increasing the concentration. In a contrast,
the level of oligonucleosomal DNA-fragmentation was found to decrease at the
highest concentration as compared to the lower ones in SKW-3. This result could
be ascribed to the relative increase of the proportion of necrotic cells
undetectable in our experimental setting. The gold complex failed to induce
prominent DNA-fragmentation in K-562 cells throughout the concentration range
used. The level of apoptosis was significantly higher than the control only
after exposure of cells to the highest concentration (twice the IC50 value).
3.4. Intracellular Accumulation of [Au(II)Hp-2H.(H2O)2]
The
determination of the intracellular levels of gold attained after 30 minutes or
60 minutes of treatment of either K-562 cells or HD-MY-Z with the Au(II) complex is
depicted on Figure 5. A prominent time-
and concentration-dependent pattern of gold accumulation is evident, whereby
the intracellular levels in the K-562 cells are substantially higher as
compared to those in HD-MY-Z cells. These data indicate that putative
pharmacological targets of the tested compounds are readily accessible after a
short incubation period.
Intracellular accumulation of gold following
[Au(II)Hp-2H.(H2O)2] treatment of HD-MY-Z or K-562 cells for 30 minutes (white columns) or 60 minutes
(gray columns), means of 4 independent
experiments.
4. Discussion
To our best knowledge, the present study is the first one addressing
the cytotoxic potential of stable monomeric octahedral Au(II) complexes. The antiproliferative
activity of the novel compound was evaluated in a wide spectrum of cell lines,
representative for some important types of human cancer. The results of the
MTT-dye reduction assay unambiguously indicate that [Au(II)Hp-2H.(H2O)2] exerts potent cytotoxic/antiproliferative effect which in some cases is
comparable to that of the referent cytotoxic drug cisplatin. Among the cell
lines under evaluation the human T-cell leukaemia SKW-3 proved to be
the most sensitive to Au(II)-complex treatment, actually the IC50 value in these cells was lower than cisplatin.
In the other leukaemia models, the relative potency of [Au(II)Hp-2H.(H2O)2] was somewhat lower but more or less comparable to that of cisplatin. Our
experimental data indicated that cisplatin was prominently superior against the
solid tumor-derived cell lines.
The clinically used platinum drugs as well as the gold antirheumatic
agents are characterized by significant nephrotoxicity which is recognized as
major dose-limiting factor. Hence, we sought to determine the nephrotoxic
potential of [Au(II)Hp-2H.(H2O)2] versus the established nephrotoxin cisplatin. The transformed cell lines appear
to be an attractive model among the in
vitro test systems used for assessment of nephrotoxicity. They are more
versatile than the primary cells on the one hand and retain most of the
biochemical features of the normal kidney tissue on the other hand [25]. In the
present study, 293T cells were used which have been recently characterized as a
suitable model for in vitro assessment
of nephrotoxicity [26–28]. The newly
synthesized complex [Au(II)Hp-2H.(H2O)2] proves
to be far less cytotoxic against kidney cells and in contrast to cisplatin, it
fails to induce 50% inhibition of cellular viability. In contrast cisplatin was
found to exert prominent cytotoxic effects with the IC50 value
similar to those obtained in the cancer cell lines.
Another important objective of the present investigation was to
determine the intracellular penetration of the gold complex. Although the
specific mechanisms of the cytotoxicity of gold species are not fully
elucidated, there is a general
consensus that they interact with intracellular targets, a feature which is
common for most of the anticancer drugs [7, 10]. Thus, the cellular accumulation
of the drug appears to be a crucial prerequisite for optimal cytotoxic
activity. The novel gold(II) complex is characterised by a significant intracellular accumulation which is
more pronounced in the leukemic model K-562 than in HD-MY-Z Hodgkin’s lymphoma.
A possible explanation is the discrepancy between the cultures type in these
cell lines. While the K-562 cells are suspended in the medium, the HD-MY-Z cells
tend to attach to the bottom of the cultivation vessel forming monolayers.
Conversely, the exposure area in K-562 cells is greater in comparison to that
of the semiadherent HD-MY-Z.
In order to elucidate the mechanisms underlying the observed cytotoxicity
of [Au(II)Hp-2H.(H2O)2], the level of
DNA-fragmentation has been quantified. The induction of programmed cell death
appears to be a common feature mediating the cytotoxic effects of anticancer
agents and in particular of metal-based drugs [1, 4]. The results reported here
confirm the general character of this phenomenon: [Au(II)Hp-2H.(H2O)2] was found to induce apoptotic cell death in SKW-3, MGH-U1, K-562, and HD-MY-Z
cells after 24-hour exposure. The low responsiveness of K-562 cells to the
proapoptotic effects of [Au(II)Hp-2H.(H2O)2] is the most probable explanation for the discrepancy between its low
sensitivity to the gold agent and its excellent intracellular accumulation
patterns.
The present data for the Au(II) metalloporphyrin complex are in accordance with the
effects established with structurally similar, octahedral Pt(III) complexes which are
characterized by a significant cytotoxicity too [22]. The complex [Au(II)Hp-2H.(H2O)2]
has an analogous structure with one of these Pt(III) complexes, both having octahedral
structure with the metal center being coordinated in the porphyrin ring via the
four pyrrolic nitrogens. The juxtaposition of their cytotoxicity shows that
while the platinum complex is far more active against the chronic myeloid leukaemia LAMA-84 [18], the Au(II) complex studied exerts
superior activity against the T-cell leukaemia SKW3 (KE-37). These data
correlate well with the estimated specific inhibiting effect of gold species
upon immune cells and T-cells in particular [7].
The selective uptake of porphyrins in malignant tissues/cells is due
to complex mechanisms, among them the most important being the LDL-receptor
mediated endocytosis of porphyrin/lipoprotein complexes formed in the
circulation. Hence, porphyrins are employed as targeting moieties to ensure
selective accumulation of cytotoxic agents into the solid tumor
microenvironment [19–21]. The novel
complex used in the present study demonstrated significant intracellular
accumulation presumably mediated by formation of FCS-lipoprotein complexes and
subsequent endocytosis. Due to the phototoxic properties of the ligand, a
light-borne augmentation of the cytotoxicity of [Au(II)Hp-2H.(H2O)2] could not be ruled out and would be addressed in a further, more detailed
evaluation of this compound.
All experimental
data presented in this study indicate that [Au(II)Hp-2H.(H2O)2] is a biologically active compound with well-pronounced cytotoxic and proapoptotic
properties against malignant cells. As compared to cisplatin, it is less
cytotoxic for human kidney cells and this feature may prove to be advantageous.
Acknowledgment
The present study was financially supported by the National Science Fund of the Bulgarian Ministry of Education and Science through Grant no. WU-06/05.
BoulikasT.VougioukaM.Cisplatin and platinum drugs at the molecular level200310616631682DesoizeB.bernard.desoize@univ-reims.frMadouletC.Particular aspects of platinum compounds used at present in cancer treatment200242331732510.1016/S1040-8428(01)00219-0KellandL.lkelland@cancertechnology.comThe resurgence of platinum-based cancer chemotherapy20077857358410.1038/nrc2167ZhangC. X.LippardS. J.lippard@lippard.mit.eduNew metal complexes as potential therapeutics20037448148910.1016/S1367-5931(03)00081-4GalanskiM.markus.galanski@univie.ac.atJakupecM. A.KepplerB. K.Update of the preclinical situation of anticancer platinum complexes: novel design strategies and innovative analytical approaches200512182075209410.2174/0929867054637626DesoizeB.bernard.desoize@univ-reims.frMetals and metal compounds in cancer treatment200424315291544ShawC. F.IIIGold-based therapeutic agents19999992589260010.1021/cr980431oDavisP.Auranofin198410369383MirabelliC. K.JohnsonR. K.SungC. M.FaucetteL.MuirheadK.CrookeS. T.Evaluation of the in vivo antitumor activity and in vitro cytotoxic properties of auranofin, a coordinated gold compound, in murine tumor models19854513239GabbianiC.CasiniA.MessoriL.Gold(III) compounds as anticancer drugs20074017381EspósitoB. P.breno@vms.huji.ac.ilNajjarR.Interactions of antitumoral platinum-group metallodrugs with albumin20022321-213714910.1016/S0010-8545(02)00049-8CalamaiP.CarottiS.GuerriA.Biological properties of two gold(III) complexes: AuCl3 (Hpm) and AuCl2 (pm)199766210310910.1016/S0162-0134(96)00190-0MessoriL.AbbateF.MarconG.Gold(III) complexes as potential antitumor agents: solution chemistry and cytotoxic properties of some selected gold(III) compounds200043193541354810.1021/jm990492uMarconG.CarottiS.CoronnelloM.Gold(III) complexes with bipyridyl ligands: solution chemistry, cytotoxicity, and DNA binding properties20024581672167710.1021/jm010997wLagunaA.LagunaM.Coordination chemistry of gold(II) complexes1999193–19583785610.1016/S0010-8545(99)00141-1ThorntonP.The noble metals200410027530110.1039/b311793fShawJ. L.WolowskaJ.CollisonD.Redox non-innocence of thioether macrocycles: elucidation of the electronic structures of mononuclear complexes of gold(II) and silver(II)200612842138271383910.1021/ja0636439GenchevaG.ggencheva@chem.uni-sofia.bgTsekovaD.GochevG.MehandjievD.BontchevP. R.Monomeric Au(II) complex with hematoporphyrin IX20036332532810.1016/S1387-7003(02)00747-5LottnerC.BartK.-C.BernhardtG.BrunnerH.Soluble tetraarylporphyrin-platinum conjugates as cytotoxic and phototoxic antitumor agents200245102079208910.1021/jm0110690LottnerC.BartK.-C.BernhardtG.BrunnerH.Hematoporphyrin-derived soluble porphyrin-platinum conjugates with combined cytotoxic and phototoxic antitumor activity200245102064207810.1021/jm0110688LottnerC.christian.lottner@exfor.uni-regensburg.deKnuechelR.BernhardtG.BrunnerH.Combined chemotherapeutic and photodynamic treatment on human bladder cells by hematoporphyrin-platinum(II) conjugates2004203217118010.1016/j.canlet.2003.09.001GenchevaG.TsekovaD.GochevG.Synthesis, structural characterization, and cytotoxic activity of novel paramagnetic platinum hematoporphyrin IX complexes: potent antitumor agents20072007136737610.1155/2007/67376MosmannT.Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays1983651-2556310.1016/0022-1759(83)90303-4KonstantinovS. M.ElblH.BergerM. R.m.berger@dkfzheidelberg.deBCR-ABL influences the antileukaemic efficacy of alkylphosphocholines1999107236537410.1046/j.1365-2141.1999.01700.xMorinJ.-P.de BroeM. E.PfallerW.SchmuckG.Nephrotoxicity testing in vitro: the current situation: ECVAM nephrotoxicity task force report 11997255497503HamadaT.SasaguriT.TanimotoA.Apoptosis of human kidney 293 cells is promoted by polymerized cadmium-metallothionein1996219382983410.1006/bbrc.1996.0318MaoW.-P.maoweiping@njnu.edu.cnYeJ.-L.GuanZ.-B.Cadmium induces apoptosis in human embryonic kidney (HEK) 293 cells by caspase-dependent and -independent pathways acting on mitochondria200721334335410.1016/j.tiv.2006.09.004ZhouX.xiazhou@usuhs.milYangG.DavisC. A.Hydrogen peroxide mediates FK506-induced cytotoxicity in renal cells200465113914710.1111/j.1523-1755.2004.00380.x