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Summary of the thesis “Dinuclear platinum complexes as potential anticancer
drugs” by Ganna Kalayda, Leiden University
This thesis deals with various aspects of intracellular behavior of antitumor-active
dinuclear platinum complexes, i.e. their anticancer activity, cellular distribution and
nephrotoxicity. This final chapter presents a summary of the most important results described
in the thesis and gives an overview of the future perspectives in this research field.
A major part of this thesis is focused on the investigation of cellular processing of
dinuclear platinum anticancer drugs using fluorescence microscopy. Besides, design of new
dinuclear platinum anticancer compounds is described, the relationships between the
structure and nephrotoxicity of dinuclear complexes are discussed, and application of
dinuclear platinum complexes in drug targeting is evaluated.
Chapter 1 presents an introduction to the research topics described in this thesis and
gives an overview of the relevant literature. First, the basic concepts of platinum antitumor
chemistry and biochemistry are introduced, and then the relevant research areas are reviewed
in detail.
New antitumor-active azine-bridged dinuclear platinum complexes are described in
Chapter 2. These complexes contain 2,5-dimethylpyrazine, quinazoline and phthalazine as
bridging ligands. They are structurally similar to the recently described cytotoxic complexes
with pyrazine, pyrimidine and pyridazine,1 and thus, extend this new class of dinuclear
platinum anticancer drugs. The complexes described in Chapter 2 show only moderate
cytotoxicity in various cancer cell lines. However, they induce apoptosis and partly overcome
cisplatin resistance in mouse leukemia cells. The structure-activity relationship for azinebridged dinuclear platinum complexes is established. The complexes that possess additional
groups in the azine ring, which induce steric hindrance and hamper binding of the complex to
nuclear DNA, are less cytotoxic than the complexes with unsubstituted azines. The
substituents on the ligand, which can provide additional interaction with DNA such as
intercalation, significantly improve the antitumor properties of the complex.
Chapters 3 – 5 deal with investigation of intracellular distribution of dinuclear platinum
anticancer drugs using fluorescence microscopy. Fluorescence microscopy is a powerful
method, which allows to study drug processing in living cells. However, most of platinum
drugs are not fluorescent. Two different approaches have been considered to enable
fluorescence microscopy studies of platinum complexes. One approach has been the design
of new platinum antitumor drugs with fluorescent ligands. This method combines the
development of new drugs and investigation of their intracellular behavior. Another approach
has been the labeling of promising platinum anticancer complexes with a fluorescent
reporter. The first approach has been applied in Chapters 3 and 4. These chapters report
cellular processing of new dinuclear platinum complexes with fluorescent
diaminoanthraquinones and the respective free ligands in different cancer cell lines. Both the
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anthraquinones and their dinuclear complexes were earlier shown to exhibit high cytotoxicity
in A2780 human ovarian carcinoma cells.2 Their cellular processing in A2780 and cisplatinresistant A2780cisR cell lines is described in Chapter 3. The ligands are processed similarly
by sensitive and resistant cells, which is in agreement with the cytotoxicity data showing that
the compounds overcome cisplatin resistance in A2780cisR cells. In contrast, the
intracellular distribution of the dinuclear platinum complexes with diaminoanthraquinones in
the resistant and sensitive cell lines is very different. In A2780cisR cells, the complexes are
sequestered in acidic vesicles in the cytosol, which prevents them from binding to nuclear
DNA. This phenomenon was not observed in the sensitive A2780 cell line. The cytotoxicity
tests showed that the dinuclear complexes with anthraquinones are cross-resistant with
cisplatin in A2780cisR. Thus, sequestration of the complexes in lysosomal vesicles explains
their decreased activity in this cell line. However, this mechanism was found to be not related
to the deactivation of platinum complexes by glutathione, which plays an important role in
the resistance profile of A2780cisR cell line. Therefore, encapsulation of the complexes and
their inactivation by intracellular GSH are two different resistance mechanisms, which
appear to operate independently. Chapter 3 presents the first example of sequestration in
cellular organelles as a mechanism of cisplatin resistance.
Chapter 4 is closely related to Chapter 3. It deals with cellular processing of the abovementioned dinuclear platinum complexes with diaminoanthraquinones and the respective free
ligands in U2-OS human osteosarcoma cell line and its cisplatin-resistant derivative,
U2-OS/Pt subline. Cellular distribution of the compounds in U2-OS sensitive/resistant pair of
cell lines is different from their distribution in A2780 sensitive/resistant cell line pair.
Cellular processing of the platinum complexes, as well as the anthraquinones, is similar in
U2-OS and U2-OS/Pt cells. This finding is consistent with the results of the cytotoxicity
tests, as all the compounds overcome resistance in the U2-OS/Pt cell line. Furthermore, no
sequestration of the platinum complexes in this cell line has been observed. Higher activity
and different intracellular distribution of the dinuclear complexes in U2-OS/Pt cells
compared to A2780cisR cells results from different resistant profiles of these cell lines.
Intracellular GSH content in A2780cisR is much higher than in U2-OS/Pt cells,3,4 which
partly accounts for cross-resistance of the dinuclear complexes with cisplatin. In contrast to
U2-OS/Pt cells, A2780cisR cells sequester the complexes in lysosomes, thereby preventing
them form binding to nuclear DNA. Sequestration of the platinum compounds in A2780cisR
cell line has been found to result from alkalinization of lysosomes. Failure to maintain
normal lysosomal pH leads to a general defect in endocytosis, which appears to facilitate
sequestration.
Chapter 5 describes new fluorescent-labeled dinuclear platinum complexes designed for
investigation of cellular processing of promising dinuclear platinum antitumor drugs. The
cis- and trans-configured complexes have been modified with a fluorogenic tag. The
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modified compounds have been shown to be good models for the parent dinuclear platinum
anticancer complexes. They interact with a guanine model base similarly to the label-free
complexes. The labeled complexes also mimic some of the pharmacokinetic properties of the
unlabelled compounds. Cellular processing of the new complexes with a fluorescent label in
U2-OS and cisplatin-resistant U2-OS/Pt human osteosarcoma cells has been investigated. A
platinum-free compound with the same label has been used in the control experiment. Both
cis and trans dinuclear platinum complexes have been found to accumulate in the nucleus
one hour after internalization, and to be excreted out of the cell via the Golgi complex.
Platinum-free control compounds showed no specific localization, which confirms that the
observed results are induced by the dinuclear platinum moiety, and not just by the label.
Application of dinuclear platinum complexes in drug targeting is described in
Chapter 6. The dinuclear platinum moiety has been found to be suitable for coupling small
organic drugs to carrier proteins. However, the synthesis of drug-carrier conjugates based on
dinuclear platinum unit is rather complicated, and the overall yield is quite low. For these
reasons, dinuclear platinum complexes are not very likely to find a broad application in drug
targeting.
Chapter 7 discusses the relationships between structure of dinuclear platinum
complexes and their nephrotoxicity. In the case of the dinuclear platinum complexes with
rigid ligands, sterically hindered complexes are less toxic because of their poor uptake and
lower reactivity. Toxicity of the dinuclear complexes with flexible ligands depends on the
geometry of the ligands around platinum: cis-configured compounds are more toxic than
their trans-counterparts.
Nowadays, cancer is one of the most widespread diseases in the world. It can start
growing in any organ of the body and may later have serious consequences for the whole
organism. A number of cancer types exist, and they are all very different from one another.
Each cancer has its specific features, which makes treatment a challenging and complicated
task.
Chapter 4 clearly shows that drug behavior in different tumor types is not the same.
Therefore, investigation of cellular processing of platinum and organic anticancer drugs in
various cancer cell lines is of great importance. Fluorescence microscopy appears a very
useful tool in these studies. As shown in this thesis, platinum complexes with fluorescent
ligands or fluorescent-labeled analogues of platinum drugs can be used for investigation of
cellular distribution of platinum compounds. Resistant cell lines with different resistant
profiles may be studied. It would be also very interesting to compare cellular processing of
platinum drugs in cell lines with intrinsic and acquired resistance to cisplatin. Identification
of proteins involved in intracellular transport of platinum complexes is of great interest. It
has been recently shown that copper homeostasis proteins are involved in uptake and efflux
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of platinum anticancer drugs.5-8 Therefore, the role of copper transporters in intracellular
distribution of platinum complexes deserves detailed investigation.
Platinum drugs are now widely used in cancer chemotherapy. However, toxicity and
tumor resistance significantly limit their clinical use. Therefore, development of new
anticancer agents remains of great importance. Targeting drugs to a tumor appears a very
promising approach, as it may increase the therapeutic efficacy of drugs, decreasing at the
same time undesirable side effects. Drug targeting may also help to overcome resistance. As
shown in this thesis, drugs can be coupled to carrier proteins via a platinum moiety. This
approach presents some advantages over the widely used covalent coupling. It allows
coupling of drugs, which lack chemically reactive groups for covalent binding. Furthermore,
the rate of drug release in the targeting conjugates based on platinum complexes can be
controlled by choosing an appropriate coordination surrounding of platinum.
Worldwide an increasing number of new drug candidates is being designed. Many of
them show high anticancer activity and overcome resistance. However, it is very important to
perform preliminary toxicity tests on new compounds synthesized as potential antitumor
drugs. Defining structure-activity and structure-toxicity relationships for new classes of
compounds is of great interest. It will help in the design of new drugs, which exhibit high
antitumor activity in combination with low toxicity.
References
1. Komeda, S.; Kalayda, G. V.; Lutz, M.; Spek, A. L.; Yamanaka, Y.; Sato, T.; Chikuma,
M.; Reedijk, J. Journal of Medicinal Chemistry 2003, 46, 1210.
2. Jansen, B. A. J.; Wielaard, P.; Kalayda, G. V.; Ferrari, M.; Molenaar, C.; Tanke, H. J.;
Brouwer, J.; Reedijk, J. J. Biol. Inorg. Chem. 2004, 9, 403.
3. Perego, P.; Caserini, C.; Gatti, L.; Carenini, N.; Romanelli, S.; Supino, R.; Colangelo, D.;
Viano, I.; Leone, R.; Spinelli, S.; Pezzoni, G.; Manzotti, C.; Farrell, N.; Zunino, F. Mol.
Pharmacol. 1999, 55, 528.
4. Perez, J. M.; Montero, E. I.; Quiroga, A. G.; Fuertes, M. A.; Alonso, C.; NavarroRanninger, C. Metal-Based Drugs 2001, 8, 29.
5. Lin, X. J.; Okuda, T.; Holzer, A.; Howell, S. B. Mol. Pharmacol. 2002, 62, 1154.
6. Samimi, G.; Safaei, R.; Katano, K.; Holzer, A. K.; Rochdi, M.; Tomioka, M.; Goodman,
M.; Howell, S. B. Clin. Cancer Res. 2004, 10, 4661.
7. Katano, K.; Safaei, R.; Samimi, G.; Holzer, A.; Tomioka, M.; Goodman, M.; Howell, S.
B. Clin. Cancer Res. 2004, 10, 4578.
8. Safaei, R.; Howell, S. B. Critical Reviews in Oncology Hematology 2005, 53, 13.
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