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Palacký University in Olomouc

Faculty of Science

Department of Biophysics

Factors affecting biological effects of metallodrugs

Mgr. Tereza Muchová

Supervisor: prof. RNDr. Viktor Brabec, DrSc.

Olomouc 2013

Abstract

Antitumor metallodrugs are used in clinical treatment very often. Well

known cisplatin is the most notable and the most successful representative of this

group. There are a lot of drugs prepared on the basis of cisplatin and there are

continuously prepared new compounds which should not have such wide spectra of

side effects and nevertheless it should have the same or higher efficiency in

antitumor treatment, including broader spectrum of human tumors sensitive to

metallodrugs.

Successfulness of new synthesized cytostatics is based on their ability of

both transportation through the cell membrane and activation. Characteristic feature

of many antitumor active drugs, which are prepared on the basis of heavy metals, is

their direct binding to the DNA molecule. During platinum compounds binding, four

types of DNA adducts (cross-links) are formed: monofunctional adduct, intrastrand

and interstrand and interduplex cross-links. These bindings result in nucleic acid

structure changes including bending and unwinding the DNA. Most of these damages

are recognized by DNA-binding proteins and DNA-repairing enzymes.

Polynuclear platinum compounds belong among newly synthesized

antitumor platinum drugs. Structure of these compounds is characterised by two or

three heavy metal atoms connected by more or less flexible organic chains. These

drugs form intramolecular or intermolecule interstrand adducts with higher probability

than mononuclear heavy metal compounds. The characteristics of newly synthesized

compounds are studied in vitro and subsequently their effects on cell lines are tested.

The present work deals with the effects of newly synthesized analogues

of cisplatin (cis-[PtCl2(nHaza)2], nHaza is 7-azaindole-halogeno substituent) on

ovarial carcinoma cell line A2780. The cytotoxicity of compounds was tested by

means of MTT assay [3-(4,5-methylethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide]. It is well known that cisplatin creates intrastrand and interstrand DNA

adducts, so these new analogues of cisplatin were tested with respect to creating of

interstrand cross-links. Interstrand cross-links are studied because they present more

serious damage of DNA.

Cisplatin belongs to the most comprehensively studied therapeutics; it is

known that damage of cells caused by this agent very often leads to the cell death,

such as Programmed Cell Death, i.e. apoptosis. In the present work, the effect of

7-azaindole derivatives of cisplatin on induction of programmed cell death and on

consequences to the cell cycle has been studied.

Next part of the work has been concerned on repair mechanisms of DNA

damaged by antitumor drugs and on ability of these compounds to form interduplex

cross-links. Cells exert several types of DNA repair mechanisms. Damages caused

by compounds on the basis of cisplatin are repaired by nucleotide excision repair

(NER) mechanism.

Interduplex cross-links represent minor type of DNA adducts, which can

be set up mainly by polynuclear compounds. This type of cross-links is created when

the molecules of DNA lie in close proximity. This feature is very common in cell

nucleus during replication and recombination. Under the in vitro conditions, this

environment presented in cell may be simulated by solutions containing high

concentrations of ethanol and salt.

All obtained results should help in future studies of another features

associated with the metabolism and reactivity of antitumor platinum compounds. The

main approach is to improve their pharmacological action such as reduction of

inherent and acquired cancer cell resistance and minimisation of side effects.

Abstrakt

Protinádorová metalofarmaka jsou velmi často využívána v klinické

léčbě. Nejpoužívanějším a nejúspěšnějším zástupcem této skupiny je známá

cisplatina. Na jejím základě se připravuje celá řada léčiv a neustále se vyvíjejí léčiva

nová, která by vykazovala menší spektrum vedlejších účinků a přitom si zachovala

stejnou nebo vyšší účinnost v protinádorové léčbě, včetně širšího spektra lidských

nádorů citlivých k chemoterapii metalofarmaky.

Úspěšnost nově připravovaných cytostatik je dána jejich schopností

procházet přes buněčnou membránu a jejich aktivací. Charakteristickou vlastností

většiny protinádorově účinných léčiv vytvořených na bázi přechodných kovů je jejich

přímá vazba na molekulu DNA. Při vazbě platinového komplexu na DNA se vytvářejí

čtyři typy aduktů, monofunkční adukty, vnitrořetězcové můstky a meziřetězcové

můstky uvnitř molekuly DNA nebo mezi molekulami DNA. Tyto vazby vedou ke

změnám ve struktuře DNA včetně rozvinutí dvojité šroubovice a ohybu její podélné

osy. Většina z těchto poškození je rozlišována DNA vazebnými proteiny a opravnými

enzymy.

Mezi nově připravovaná protinádorová léčiva patří polynukleární

platinové komplexy. Jejich struktura je charakterizována dvěma nebo třemi atomy

platiny spojenými více či méně flexibilními organickými řetězci. Tyto komplexy

vytvářejí meziřetězcové nebo mezimolekulové můstky s pravděpodobností větší, než

je tomu u mononukleárních komplexů platiny. U všech nově připravených komplexů

jsou studovány jejich vlastnosti in vitro a následně jsou testovány jejich účinky na

buněčné linie.

Předkládaná práce pojednává o účincích nově připravených analog

cisplatiny (cis-[PtCl2(nHaza)2], kde nHaza je 7-azaindol-halogenidový substituent) na

buněčnou linii ovariálního karcinomu A2780. Byla testována cytotoxicita těchto

komplexů s využitím testu MTT [3-(4,5-methylethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromid]. Protože je známo, že cisplatina vytváří na DNA

vnitrořetězcové a meziřetězcové můstky, byla nově syntetizovaná analoga cisplatiny

studována i s ohledem na vytváření meziřetězcových můstků. Meziřetězcové můstky

jsou studovány zejména proto, že z hlediska přežití buňky se jedná o závažnější

poškození DNA.

Cisplatina je nejlépe prostudovaným léčivem. Bylo ukázáno, že působení

cisplatny na nádorové buňky vede k jejich smrti – jde zejména o programovanou

buněčnou smrt, tzv. apoptózu. V práci byl studován vliv

7-azaindolových derivátů cisplatiny na navození programované buněčné smrti

a jejich vliv na průběh buněčného cyklu.

Další část práce byla zaměřena na zkoumání mechanizmu opravy

poškození DNA protinádorovými léčivy a na schopnost těchto komplexů vytvářet

mezimolekulové můstky. Existuje několik buněčných mechanizmů opravy poškození

DNA. Poškození způsobená komplexy na bázi cisplatiny jsou opravována především

pomocí nukleotidové excizní opravy (NER).

Mezimolekulové můstky jsou minoritním typem aduktů, které mohou být

vytvořeny především polynukleárními komplexy. Tento typ můstků se vytváří za

takových podmínek, kdy jsou molekuly DNA v těsné blízkosti. K takové situaci

dochází zejména v buněčném jádře při replikaci či rekombinaci. V podmínkách

in vitro je možné takové prostředí simulovat roztokem koncentrovaného etanolu

a soli.

Veškeré získané výsledky mohou pomoci při následném studiu dalších

jevů spojených s metabolizmem a reaktivitou platinových cytostatik. Jde především

o zjištění faktorů, jejichž působení je zodpovědné za zlepšení farmakologických

účinků cytostatik, při čemž by měla být snížena inherentní a tzv. získaná rezistence

nádorových buněk a minimalizovány vedlejší účinky.

Statutory declaration

I hereby declare that this doctoral thesis has been written solely by myself.

All the sources quoted in this work are listed in the Reference section. All published

results included in this work are approved by co-authors.

Olomouc, April 29, 2013 ……………………………..

Acknowledgement

Firstly, I would like to thank my supervisor prof. RNDr. Viktor Brabec, DrSc. for

his patient guidance, encouragement and for priceless advices.

My thanks also deserve my colleagues from the Department of Biophysics at

Palacký University in Olomouc and from Department of Molecular Biophysics and

Pharmacology of the Institute of Biophysics ASCR, v.v.i. in Brno for their suggestions

and creating the friendly atmosphere.

At last, my special thanks belong to my family for their support and patience.

The experiments were founded by Grant PrF 2012 026 and Grant PrF

2013 017.

Table of Contents

1 Introduction ...................................................................................................... 10

1.1 Approved platinum compounds .................................................................... 11

1.2 Mechanism of action of platinum compounds ............................................... 12

1.3 Modifications made by anticancer drugs ...................................................... 13

1.4 Repair of DNA lesions formed by anticancer drugs ...................................... 15

1.5 Metallodrugs and cells .................................................................................. 18

1.5.1 Platinum accumulation and binding in cells .......................................... 20

1.5.2 Cell death .............................................................................................. 21

1.5.2.1 Apoptosis ........................................................................................ 21

1.5.2.2 Necrosis (programmed) .................................................................. 23

1.5.2.3 Autophagy ...................................................................................... 24

1.5.2.4 Other types of cell death................................................................. 24

1.5.3 Mechanisms of resistance .................................................................... 25

2 Aims of the study .............................................................................................. 28

3 Materials and methods ..................................................................................... 29

3.1 Chemicals ..................................................................................................... 29

3.2 Other chemicals and biological material ....................................................... 30

3.3 In cellulo assays ........................................................................................... 31

3.3.1 In vitro growth inhibition assay .............................................................. 31

3.3.2 Detection of apoptosis and necrosis ..................................................... 32

3.3.3 Interaction of platinum compounds with cells in real time ..................... 32

3.3.4 Cell cycle analysis ................................................................................. 33

3.3.5 DNA platination in cells ......................................................................... 33

3.3.6 Cellular uptake of platinum compounds ................................................ 33

3.4 In vitro assays............................................................................................... 34

3.4.1 Platination of DNA in cell-free medium containing ethanol ................... 34

3.4.2 Interstrand DNA cross-links in cell-free medium ................................... 34

3.4.3 Interduplex DNA cross-links in cell-free medium ................................... 35

3.4.4 DNA transcription by RNA polymerase in vitro ...................................... 35

3.4.5 Repair DNA synthesis by mammalian cell-free extract ......................... 36

3.5 Other physical methods ................................................................................ 37

4 Summary of results and discussion .................................................................. 38

4.1 Replacement of thiourea with an amidine group in a monofunctional

platinum-acridine antitumor agent. Effect on DNA interactions, DNA adduct

recognition and repair. (Paper I) ............................................................................ 39

4.2 Formation of interduplex DNA cross-links under molecular crowding

condition (Paper II) ................................................................................................. 41

4.3 How to modify 7-azaindole to form cytotoxic PtII complexes: Highly in vitro

anticancer effective cisplatin derivatives involving halogeno-substituted

7-azaindole (Paper III)............................................................................................ 42

4.4 Insight into the toxic effect of the cis-Pt(II)-dichlorido complexes containing

7-azaindole halogeno-derivatives in tumor cells (Paper IV) ................................... 43

5 Conclusion ....................................................................................................... 46

6 References ....................................................................................................... 47

7 List of abbreviation ........................................................................................... 52

8 List of publications ............................................................................................ 54

9 Curriculum vitae ............................................................................................... 55

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1 INTRODUCTION In 1965, Barnett Rosenberg from Michigan State University accidentally

discovered antitumor effects of cisplatin, the structure of which was already well

known in these days. He was studying the influence of electromagnetic field on cell

growth of E. coli. After application of electromagnetic field using platinum electrodes,

the E. coli colonies started to grow in filamentous organisation instead of normal

short rods. This effect was shown not to be due to the electric field but, rather, to

electrolysis products arising from the platinum electrodes. Detailed chemical analysis

identified two active complexes — cis-[PtII(NH3)2Cl2] (cisplatin, which has been

known as Peyrone’s salt since 1845), and a platinum(IV) analogue,

cis-diamminetetrachloridoplatinum(IV). The trans isomer was much less active

(Kelland, 2007).

In the 1970’s, the activity of cisplatin against limited number of human

tumors was registered (Desoize and Madoulet, 2002). Cisplatin was the first

inorganic compound registered to treat human cancer. But the application to humans

is limited by resistance of tumor cells to this treatment, concerned inborn or acquired

resistance to the cisplatin (Brabec and Kašpárková, 2005).

The effects of cisplatin on tumor cells have been studied on several

levels. It is generally accepted that the desired antitumor effects are achieved when

platinum compounds react with deoxyribonucleic acid (DNA). The DNA is the corner

stone of all living organisms. It maintains the basic information for cell division,

progression and for protein synthesis. DNA is also the only molecule which is

repaired. This reparation is necessary to keep genetic information unharmed by

physical or chemical damage.

DNA consists of sugar-phosphate backbone and heterocyclic nucleoside

on purine or pyrimidine bases. In DNA, the sugar is represented by 2’-D-deoxyribose,

where on carbon 5’ there is a phosphate and on carbon 1’ there is nucleoside. The

bases are represented by heterocyclic molecule of guanine and adenine (purines)

and cytosine and thymine (pyrimidines). DNA molecule is formed by two anti-parallel

strands. The nucleotides from opposite strands bind together via hydrogen bonds.

The basic base pairing is following: guanine is paired with cytosine by three hydrogen

INTRODUCTION

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bonds and adenine and thymine are paired by two hydrogen bonds. DNA forms a

double helix, which is in natural (physiological) conditions in

B-conformation. However, DNA can also hold other conformations. At lower hydration

its conformation holds A-structure, which is shorter and there are differences in major

and minor groove size. In the presence of high salt concentration and when purines

and pyrimidines alternate, the DNA can adopt Z-conformation. This is the only

situation when DNA is levorotary.

1.1 APPROVED PLATINUM COMPOUNDS

First approved inorganic compound used to treat the human cancer was

cisplatin (Brabec, 2002). During years, the research in this branch is carried out to

improve the potential activity and to reduce side effects of used compounds. More

than three thousand platinum derivatives were synthesized and tested against cancer

cells, but only several underwent clinical trials. To date, clinically approved platinum

compounds create a small group of four members. It includes cisplatin, approved in

1978, and carboplatin. Both of them are worldwide used in clinical trials. Then,

oxaliplatin has been approved to be used in few countries. And last but not least,

nedaplatin which is approved only in Japan (Desoize and Madoulet, 2002). The newly approved derivatives of cisplatin should be also mentioned: lobaplatin approved in China, and heptaplatin approved in the South Korea.

Cisplatin (cis-diamminedichloridoplatinum(II)) is widely used for treatment of several types of tumors. It is a relatively unreactive

molecule. But in aqueous environment, the chloride ligands are

displaced by water molecule (Berners-Price and Appleton, 2000) and the compound

is then activated. The cytotoxic potential of this compound has several drawbacks

like renal toxicity, ototoxicity etc. and acquired resistance (Brabec and Kašpárková,

2005; McKeage, 2000).

Carboplatin (cis-diammine-[1,1-cyclobutanedicarboxylato]-platinum(II)) is the cisplatin derivative approved in the United

Kingdom and Canada in 1985 (Pasetto et al., 2006). Its formula

contains cyclobutanedicarboxylato ligand instead of two chlorides in cisplatin

molecule (Brabec and Kašpárková, 2005). The main difference between carboplatin

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and cisplatin is in the stability in body fluids and in the rate of reaction with

biologically relevant macromolecules (McKeage, 2000). Carboplatin is less toxic than

cisplatin, but it is active against the same range of tumors (Brabec and Kašpárková,

2005) and also it retains some drawbacks like thrombocytopenia and gastrointestinal

effects (McKeage, 2000).

Oxaliplatin ((1R,2R-diamminocyclohexane)oxalatoplatium(II)) is another analogue of cisplatin which contains the oxalato leaving

group. It is successfully used in combination with 5-fluoruracil and it has been

approved for clinical use in France and the United States. The cytotoxic action is

potentiated by use in combination with other anticancer agents. In addition,

oxaliplatin does not cause serious ototoxicity or nefrotoxicity, but it still has effects on

gastrointestinal tract and causes peripheral neurotoxicity (Brabec and Kašpárková,

2005; McKeage, 2000; Pasetto et al., 2006)

The development of tumor cell resistance to classical compounds drives

the search for novel platinum complexes, and in general for transition metal

complexes. This has resulted in development of mononuclear, dinuclear and even

trinuclear platinum compounds, which might overcome the imperfections of classical

compounds. Examples of these potential novel compounds are dinuclear BBR3535,

cis-[PtCl2(nHaza)2], [PtCl(en)(ACRAMTU)](NO3)2] (McGregor et al., 2002;

Štarha et al., 2012, Kostrhunova et al., 2011) which were studied within the present

doctoral thesis.

1.2 MECHANISM OF ACTION OF PLATINUM COMPOUNDS

As it was mentioned above, cisplatin is active against testicular and

ovarian cancers and is also widely used for treating bladder, cervical, head and neck

oesophageal and small cell lung cancer. However some tumors like colorectal and

non-small cell lung cancer have exerted resistance to cisplatin, whatever acquired or

inborn (Cepeda et al., 2007). Biochemical mechanism of action covers interaction

with various compounds. But the proper mechanism is still to be elucidated (Brabec

and Kašpárková, 2005; Pasetto et al., 2006).

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Before platinum compound enters the cell, it reacts with phospholipids

and phosphatidylserine in cell membrane (Pasetto et al., 2006). Either passive

diffusion or carrier-mediated transport should be involved in the uptake of platinum

compounds. A specific transporter of platinum cannot be determined, due to not

saturable nor inhibitable accumulation of platinum in cells. There is evidence of Na+

dependent influx which can be altered by adenosine triphosphate depletion, cyclic

adenosine monophosphate elevation, protein kinase C agonists, osmotic strength,

pH, membrane polarization, calmodulin antagonists or ras expression (Andrews,

2000).

Also, it is generally accepted that cisplatin and in general platinum

compounds have the ability to bind to DNA. This is the main event responsible for its

antitumor properties. Specific binding of cisplatin to DNA may inhibit transcription

and/or DNA replication (Cepeda et al., 2007). Thus, binding of drugs to DNA could

activate multiple signalling pathways involving p53, Bcl-2 family, caspases, cyclins,

cyclin-dependent kinases, pRb, protein kinase C, MAPK and PI3K/Akt. And also,

after entering the cell, platinum drugs could interact with non-DNA targets, i.e.

proteins (Pasetto et al., 2006).

1.3 MODIFICATIONS MADE BY ANTICANCER DRUGS

The main target of platinum-based drugs is DNA (Brabec, 2002). The

cisplatin reacts with the N7-atom of guanine or adenine residue. The main body of

the complex is coordinated in major groove of the double helix (Jung and Lippard,

2007). Cisplatin forms several types of adducts on DNA (Fig. 1), approx. 65% of

1,2-d(GpG) intrastrand cross-links (CLs), 25% 1,2-d(ApG) intrastrand CLs, 5-10% of

1,3-d(GpNpG) intrastrand CLs, the rest of the adducts is made up by interstrand and

monofunctional adducts (Ahmad, 2010; Cepeda et al., 2007). On the other hand, the

clinically inactive trans analogue of cisplatin forms mainly interstrand CLs and almost

50% of monofunctional adducts (Brabec, 2002).

The formation of various types of adducts leads to destabilization of DNA

by unwinding and bending of the duplex, and also disturbs stacking interactions in

double helical DNA (Jung and Lippard, 2007; Brabec, 2002). When cisplatin forms

the intrastrand CLs, this adduct cause roll between platinated purines and bend of

INTRODUCTION

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the helix axis toward the major groove (Brabec, 2002), thus the minor groove is

widened and shallow (Jung and Lippard, 2007). The interstrand CLs bends the helix

axis toward major groove and locally unwinds the double helix, in addition the rest of

the cisplatin molecule is situated in minor groove and the DNA is locally in

left-handed Z-DNA form (Brabec, 2002).

Fig. 1: Three main cross-links of cisplatin formed on DNA. From top interstrand CLs, 1,2-interstrand

CLs, 1,3-interstrand CLs, protein-DNA CLs (Adapted from Gonzales et al., 2001).

The minor interest was paid to the possibility of formation platinum

CLs between two neighboring DNA molecules, until Pospíšilová and Kypr (1998)

have shown that UV-light in combination with aqueous ethanol environment enables

the formation of CLs between two adjacent DNA molecules (Pospíšilová and Kypr,

1998). Also, another study shows that platinum compounds with long and flexible

linker are able to form interduplex CLs under molecular crowding conditions

(Muchová et al., 2012).

Several classes of cellular proteins have been identified and the

mechanism how they exert antitumor effect of cisplatin was studied. Especially the

intrastrand adducts are well recognized by nuclear proteins with high-mobility group

(HMG) domains (Brabec, 2002). The proteins HMGB1 and HMGB2 belong to this

protein family of small, nonhistone chromatin-associated proteins. This family of

proteins is involved in gene regulation and chromatin structure maintenance

(Gonzalez et al., 2001). The proteins bind selectively to the 1,2-GG or AG adducts of

INTRODUCTION

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cisplatin. The binding fashion of the proteins is to minor groove of DNA opposite to

the formed adduct (Cepeda et al., 2007, Brabec, 2007). When HMGB1 binds to

1,2-intrastrand CLs it can inhibit the nucleotide excision repair (NER) by shielding the

damage site from recognition by NER repair proteins (Jung and Lippard, 2007).

There are two subgroups of HMGB domain proteins, structure-specific, which

recognize DNA structure like four-way junctions and supercoiled DNA and sequence-

specific subgroup, that includes transcription factors like LEF-1 and TCF-1 and SRY

protein. It should be mentioned here, that HMGB domain proteins show selectivity for

platinum-DNA adducts of cisplatin, but they fail to recognize the transplatin adducts

(Ahmad, 2010).

There are also present non-HMG domain proteins in cells. That group

includes the TATA-binding protein (TBP) which is involved in initiation of transcription

and it is able to recognize the 1,2-intrastrand adduct of cisplatin. Then the proteins of

reparative mechanisms recognize the structure deformities introduced by platinum

agents. In the repair of cisplatin adduct there are mainly the NER proteins involved,

such as XPA and RPA proteins, and mismatch repair (Jung and Lippard, 2007)

whose mechanism of action is described below.

1.4 REPAIR OF DNA LESIONS FORMED BY ANTICANCER DRUGS

The human cells are exposed to various damaging agents like UV-light

and natural or synthetic mutagens. During one day the cell genetic information is

exposed to 50 000 single-strand breaks, 10 000 depurinations, 5 000 alkylations,

2 000 oxidations, 600 deaminations and 10 double-strand breaks events (Kryštof,

oral communication). These disruptors cause damage to cells genetic information.

During evolution, cells have developed sophisticated mechanisms to exclude the

damage from their genomes. There are three main excision pathways of reparation of

damaged DNA: base excision repair, nucleotide excision repair and mismatch repair.

To maintain the genome stability, other mechanisms of reparation are also involved

and this will be described further.

First mentioned Base excision repair (BER) is a principal cellular repair mechanism correcting a wide spectra of DNA lesions. This repair pathway is involved

in repair of damages generated by environmental agents – ionizing radiation,

INTRODUCTION

16

alkylating agents and oxidative reagents, but also by endogenously produced oxygen

radicals and other reactive species (Frosina et al., 1999). The BER is executed in two

ways (i) ‘short patch’ and (ii) ‘long patch’ manner. This system of DNA repair is

activated when one or a few bases are damaged. In the reparation, DNA-

glycosylases are involved which recognize damaged base and cleave it out to create

apurinic or apyrimidinic (AP) site in DNA. The AP site is recognized by endonuclease

that cleaves hydrolytically the phosphodiesteric bond on the 5’-side of the AP site.

Then phosphodiesterase excises generated 5’-deoxyribose phosphate terminus to

leave a single nucleotide gap. Consequently, the gap is filled by DNA polymerase

and nick is sealed by DNA ligase. For short patch BER, five proteins are necessary:

UDG, HAP1, DNA polymerase β, XRCC and DNA ligase I or III. In long patch BER,

there are Dnase IV and PCNA active (Reed, 1998; Frosina et al., 1999).

Highly conserved and most studied reparation mechanism is Nucleotide excision repair (NER) which is a primary process to remove platinum damaged DNA and bulky covalent lesion such as UV dimers, polycyclic aromatic hydrocarbons

(Jung and Lippard, 2007; Martin et al., 2008). There are 16 essential proteins of NER

involved (Reed, 1998). This system comprises damage recognition, unwinding of the

DNA around the site of damage, incision on either site of the lesion, removal of

fragment containing the lesion and DNA synthesis and ligation to form a repair patch

about 30 nucleotide long (Biggerstaff and Wood, 1999) to maintain the DNA molecule

(Martin et al., 2008).

The platinum lesion is recognized by different subpathways of NER,

transcription coupled repair and global genomic repair. The transcription coupled

repair is launched by stalled RNA polymerase II, which acts like recognition signal. In

global genomic repair, the initial signal is performed via XPC-HR23B protein dimer.

After crucial recognition of damage, the transcription coupled and global genomic

repair follow similar path (Jung and Lippard, 2007). After recognition of damage,

another set of repair protein is involved. The TFIIH transcription factor with helicase

activity consists of proteins ERCC2 (XPD) and ERCC3 (XPB). Their helicase activity

requires ATP, together with XPA and RPA. After unwinding of damaged sequence,

the XPC-HR23B complex is released when endonuclease XPG binds to unfolded

DNA (Reed, 1998; Jung and Lippard, 2007). After unwinding, the excision complex

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composed of ERCC1 (excision repair cross-complementation group 1), which has

key role in excision, heterodimerizes with XPF and ensures the double excision of

platinated oligonucleotides (Martin et al., 2008). RPA protein remains on incised DNA

and recruites resynthesis factors like PCNA and RFC to fill the gap (Jung and

Lippard, 2007).

Nucleotide excision repair is very effective tool for repair of DNA damage

by platinum agents, especially intrastrand adducts. Due to this feature, the increased

NER contributes to the cisplatin resistance of malignant cells. Good example is testis

tumor cell lines where NER proteins are present at low levels and the cell line retains

sensitivity to cisplatin in vitro. Another potential factor which should be mentioned

involves the fact that DNA-platinum adducts are recognized by nuclear proteins, like

those belonging to HMGB family, which shield the damage, inhibit nucleotide excision

repair and thus enhance cisplatin sensitivity (Ahmad, 2010).

Strand specific, highly conserved, post-replication repair system which

corrects mispaired and unpaired bases in DNA duplex is called Mismatch repair (MMR). The MMR systems involve proteins called Mut which recognize mismatched or unmatched DNA base pairs, deletions or insertions. The recognition of

mismatches in eukaryotic cells starts by hMutSα which binds to single mismatch or

insertion/deletion loop. Subsequently, after recognition by hMutSα, the MutLα and

PCNA are recruited and repair is done. Also, DNA ligase I is used to fill the gap

created by removing misincorporated base (Jung and Lippard, 2007; Martin et al.,

2008).

MMR is critical in cellular sensitivity to cisplatin because loss of human

homologs of MutS and MutL leads to resistance to cisplatin and carboplatin, but

interestingly it does not cause resistance to oxaliplatin and satraplatin (bis-acetato-

ammine-dichloro-cyclohexylamineplatinum(IV), JM216) which was originally

developed to be an orally active version of carboplatin). Defective MMR leads to

almost 4-fold increase in tolerance of cisplatin treatment, which contributes to failure

of therapy of the cancer (Ahmad, 2010).

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18

The cross-linking of DNA has a consequence for DNA synthesis,

replication and transcription (Jung and Lippard, 2007). The ‘classical’ replication

polymerases α, δ and ε are not able to pass through the lesions made by cisplatin.

Nevertheless, there are several polymerases that can do so. These ‘bypassing’

polymerases are polymerase β, η, ξ and ι (Rabik and Dolan, 2007). They are

specialized for the process called translesion synthesis. The process of bypassing the lesion carried along the possibility of introduction of mutation to the genome

(Jung and Lippard, 2007).

Introduction of platinum adduct to DNA leads also to stall the RNA

synthesis necessary for translation and consequently for proteosynthesis. RNA

polymerase II is most abundant protein in cell and it ensures the transcription of

eukaryotic genes. RNA polymerases are strongly blocked by bifunctional adducts of

cisplatin, but not by the monofunctional platinum agents (Jung and Lippard, 2007).

When the RNA polymerase II is stalled on DNA, the cells are not able to pass the cell

cycle correctly – the cells are arrested in G2/M phase of cell cycle (Jamieson and

Lippard, 1999).

The interstrand CLs could induce the double-strand breaks in DNA. To

repair this damage, the homologous recombination plays a role. The components of

NER the ERCC1 and XPF are involved in homologous recombination coupled repair

(Rabik and Dolan, 2007).

1.5 METALLODRUGS AND CELLS

The novel candidate antitumor complexes of transition metals are

currently synthesized. They are intended to improve the response of cells to this

treatment. Tumorigenesis in humans is a multistep process (Fig. 2). These steps

reflect genetic alterations that drive progressive transformation of normal human cells

into highly malignant derivatives. There is a large body of evidence indicating that the

genomes of tumor cells are invariably altered at multiple sites (Hanahan and

Weinberg, 2000).

INTRODUCTION

19

Fig. 2: The hallmark of cancer. (Adapted from Hanahan and Weinberg, 2000)

There are a lot of types of tumors present in human, but Hanahan and

Weinberg (2000) have suggested, and now it is generally accepted, that the cancer

cell genotype is a manifestation of six essential alterations in cell physiology that

collectively dictate malignant growth. It concerns self-sufficiency in growth signals,

insensitivity to growth-inhibitory signals, evasion of programmed cell death, limitless

replicative potential, sustained angiogenesis and tissue invasion and metastasis

(Hanahan and Weinberg, 2000).

The evidence from animal models and cell cultures is mounting the

acquired resistance toward apoptotic signals. It is a hallmark of almost all types of

cancer (Hanahan and Weinberg, 2000). The main goal of cancer chemotherapy is to

force tumor cells to apoptotic type of cell death. Cisplatin is very potent inorganic

compound to induce apoptosis, but on the other hand, the cancer cells are able to

develop the resistance. The resistance phenomenon is developed by chronic

exposure to drug–acquired resistance, or by inborn predisposition (Siddik, 2003).

INTRODUCTION

20

1.5.1 PLATINUM ACCUMULATION AND BINDING IN CELLS

Many of platinum drugs are administered intravenously. Platinum

compounds are therefore allowed to react with plasma proteins through high affinity

to sulphur containing donors, like thiol groups of amino acids like cysteine. In blood,

there is almost 90% of platinum bound to albumin or other plasma proteins

(Cepeda et al., 2007)

After the compound enters the cell, it undergoes aquation, because

intracellular concentration of Cl– ions is low. Due to this phenomenon the chlorines in

platinum complex molecule are easily displaced by water molecules. This product is

a potent electrophile and can react with any nucleophile present within the cell

(Brabec and Kašpárková, 2005).

A lot of resistant cell lines have reduced platinum accumulation. Cisplatin

resistance might be correlated with the rigidity of cell membranes with high

sphingomyelin and cholesterol moiety; it also could be correlated with altered

ganglioside expression. Cellular uptake of platinum compounds is advanced by

copper transporter Ctr1. This protein undergoes cytoplasmic internalization after it is

exposed to cisplatin. This process leads to inactivation of large number of copper

transporters at the surface and this limits further cisplatin uptake (Stewart, 2007).

After the activation, the platinum is allowed to react with constituents of

lipidic bilayer which contains nitrogen and sulphur atoms. It is also reactive with many

cytoplasm components such as cytoskeletal microfilaments,

thiol-containing peptides and proteins and RNA. The literature refers that only 5–10%

of covalently bound cell-associated cisplatin is found in genomic DNA, while 75–85%

of the drug binds to proteins and other cellular constituents (Cepeda et al., 2007).

Intrastrand and interstrand DNA CLs of cisplatin are responsible for

killing cells. The highest platination in cells occurs in exposition of cells to cisplatin

during G1 phase, while the lowest platination occurs during G2/M phase of cell cycle

(Stewart, 2007).

INTRODUCTION

21

1.5.2 CELL DEATH

The death of cells might be described by their morphological

appearance, enzymological criteria, functional aspects or immunological

characteristics. The dying of cell is reversible until the phase or ‘point of no return’ is

crossed. The point of no return could be characterized by massive caspase

activation, loss of mitochondrial trans-membrane potential, complete permeabilization

of the mitochondrial outer membrane or exposure to phosphatidyl serine residues

which emit signals “eat me” to normal neighboring cells (Kroemer et al., 2009).

The term programmed cell death comprises apoptosis, autophagy and

programmed necrosis and it is proposed for death of cell in any pathological format

(Ouyang et al., 2012).

1.5.2.1 APOPTOSIS

The term “apoptosis” is historically referred for programmed cell death

and is induced by several signals (Kerr, 1972). It is derived from Greek words ‘apo’

stands for from/off/without and ‘ptosis’ stands for falling. This is well ordered and

orchestrated cellular process and it is important in both physiological and pathological

conditions. It also has pivotal role in the pathogenesis of many diseases (Wong,

2011).

From morphological point of view, the apoptotic type of cell death affects

both the nucleus and the cytoplasm. The cells round-up, detach from the surface,

reduce cell volume (pyknosis), then chromatin condenses and nucleus is fragmented

(karyorhexis). However, the cytoplasmic organelles are rarely modified. The plasma

membrane blebbing occurs, but the integrity of membrane is kept until final stage of

apoptosis, then cells rest or whole dead cells are engulfed by phagocytes and

eliminated from tissue (Kroemer et al., 2009).

Briefly, extrinsic pathway (Fig. 3) involves death receptors, death ligands

and the subsequent signalling cascade. Well known receptor is the TNF1 (tumor

necrosis factor 1) receptor and related Fas protein (Wong, 2011). Their common

ligands are TNF and Fas-L (Fas ligand), and their binding leads to multimerisation of

death receptors followed by binding of adaptor protein Fas-associated death domain

(FADD) (Ahmad, 2010). This leads to activation of caspase-8 by DISC, which is

INTRODUCTION

22

aggregate of pro-caspace-8-10 and death-effector domain (Ouyang et al., 2012). The

close proximity of pro-caspases-8 in DISC allows self-activation of them. Activated

caspase-8 could activate caspase-3 and other downstream caspases (Ahmad, 2010).

This basic activation leads to initiation of apoptosis by cleaving other downstream or

executioner caspases (Wong, 2011).

Fig. 3: Intrinsic and extrinsic pathway of apoptosis. (Adapted from Wong, 2011)

Second possible pathway of execution of programmed apoptosis is

intrinsic pathway (Fig. 3). This cascade of cell death stimuli is initiated within the cell.

The inner stimuli for intrinsic cascade are irreparable genetic damage, hypoxia,

extremely high concentration of cytosolic calcium ions and severe oxidative stress

(Wong, 2011). Changes in mitochondrial outer membrane often lead to release of

cytochrome c into cytoplasm. In cytosol cytochrome c is allowed to react with Apaf-1.

This complex subsequently binds pro-caspase-9 and forms the apoptosome, which

catalyses the activation of caspase-9. Activated caspase-9 ensures activation of

effector caspases 3, 6 and 7, which effects proteolytic cascade manifesting in gross

apoptotic changes (Ahmad, 2010). This pathway is regulated by Bcl-2 family of

proteins. It was shown that correct execution of apoptosis is dependent on well

balanced manifestation of pro-apoptotic and anti-apoptotic proteins of Bcl-2 family

(Ouyang et al., 2012; Wong, 2011).

INTRODUCTION

23

The third, less known pathway is intrinsic endoplasmic reticulum

pathway. It is proposed that this pathway is activated by cellular stress like hypoxia,

free radicals or glucose starvation and therefore unfolding of proteins and reduced

protein synthesis occur (Wong, 2011).

1.5.2.2 NECROSIS (PROGRAMMED)

The necrosis cell death or necrosis definition by Nomenclature committee

on Cell Death 2009 describes this cell death as morphologically characterized gain in

cell volume (oncosis), swelling of organelles, plasma membrane rupture and

subsequent loss of intracellular contents. For many years, necrosis was presented as

accidental and uncontrolled process. But the committee admits and describes the

possibility that the necrosis is orchestrated by several proteins connected to

programmed cell death (Kroemer et al., 2009).

There are papers dealing with programmed necrosis. Golstein and

Kroemer review that necrosis can occur during development (e. g. death of

chondrocytes controlling the longitudinal growth of bones) and in adult tissue

homeostasis. Also, activation of specific plasma membrane receptors by their

physiological ligands may activate necrosis. So this receptor activation speculates for

specific pathway of activation of necrosis. Propensity to necrotic death can be

regulated by genetic and epigenetic factors. Another specific points were mentioned,

the inhibition of some enzymes and processes which can prevent necrosis. And last

but not least, inhibition of caspases, often connected to manifestation of apoptosis,

can change the morphological appearance of cell death from apoptosis to autophagy

or necrosis (Golstein and Kroemer, 2006).

Briefly, the molecular necrotic pathway is activated by death ligands like

TNFα, TRAIL (TNF-related apoptosis-inducing ligand) and Fas which bind to their

receptors. This results in assembly of supramolecular platform composed of

caspase-8, FADD and RIP1 (receptor interacting protein serine-threonine kinase 1).

In the case of inactivation of caspases, the death receptor ligation ends in assembly

of complex of caspase-8, FADD, RIP1 and RIP3. The pronecrotic complex

RIP1-RIP3 interacts with metabolic enzymes to enhance metabolism leading to rise

of reactive oxygen species production. Other modulators are involved in mechanism

INTRODUCTION

24

of programmed necrosis, like PARP1, NADPH oxidases and calpains (Ouyang et

al., 2012).

1.5.2.3 AUTOPHAGY

According to Nomenclature committee on Cell Death 2009, the

autophagic cell death is morphologically defined type of cell death that occurs without

chromatin condensation but it is accompanied by massive autophagic vacuolization

of the cytoplasm (Kroemer et al., 2009).

Autophagy is major catabolic mechanism regulated by some autophagy-

related genes (ATGs). It is answer of cell to extra- or intracellular stress and can

result in cell survival under certain conditions. Autophagy is activated under extreme

conditions and leads to degradation of intracellular macromolecules to ensure

energetic needs of cell for maintaining minimal cell function when cell is starving

(Ouyang et al., 2012). Macroautophagy is characterized by sequestration of

cytoplasmic material within autophagosome for degradation in lysosomes.

Autophagosomes are two membraned vesicles containing cytoplasmic organelles or

cytosol. Their fusion with lysosomes leads to creation of autophagolysosomes in

which inner membrane and its luminal content are degraded by acidic lysosomal

hydrolases (Kroemer et al., 2009)

But, on the other hand, autophagy plays a critical role in the early stages

of cancer progression. The pro-tumor role in carcinogenesis is secured by regulating

of number of pathways involving Beclin-1, Bcl-2, PI3K (class III and I), mTOR and

p53 (Ouyang et al., 2012).

1.5.2.4 OTHER TYPES OF CELL DEATH

There have been described several other types of programmed cell

death with fine different mechanism of cell death. Mitotic catastrophe belongs among these types and might be present during or shortly after a dysregulated/failed

mitosis. Also, morphological alterations including micronucleation and multinucleation

could be presented. Apoptosis induced by the loss of the attachment to the substrate

or to other cells is called anoikis.

Next two types are described in nervous system. First one is

excitotoxicity performed via excitatory amino acids, i.e. glutamate, which leads to

INTRODUCTION

25

cytoxolic Ca2+ overload and activation of death signals. Second type is wallerian degeneration where cellular catabolism takes place and this lethal incident affects only a part of neuron or axon, not the whole body.

Paraptosis is triggered by expression of the insulin-like growth factor receptor I and leads to cytoplasmatic vacuolization and mitochondrial swelling without

any other morphological hallmark of apoptosis. The phenomenon of pyroptosis has been described in macrophages infected with Salmonella typhimurium. This includes

apical activation of caspase-1 only. Macrophages undergoing pyroptosis exhibit

morphological features of apoptosis, but also show some traits associated with

necrosis.

In lymphoblasts from patients with Huntington’s disease, a form of

“cellular cannibalism” was shown. It has been described as one cell engulfing its live

neighbours which died within the phagosome. This type of cell death was named

entosis and it is not inhibited by bcl-2 or Z-VAD-fmk. The internalized cell appears normal, later it disappears probably by lysosomal degradation. However engulfed

cells are able to divide or should be released.

Often mentioned terms keratinization or cornified envelope formation

mainly refers to process of cornification. The process is a terminal differentiation program similar to those leading to other anucleated tissue. On molecular level, there

is a specific mechanism of epithelial differentiation and cells express all enzymes and

substrates required for building up the epidermal barrier. The specialised cross-

linking enzymes and proteases, together with synthesis of specific lipids released into

extracellular space are involved in the process of cornification (Kroemer et al., 2009).

1.5.3 MECHANISMS OF RESISTANCE

The major goal of cancer chemotherapy is to lead the cell to apoptotic

cell death after exposure to antitumor agents (Siddik, 2003). But some tumor cells

are resistant. Some of them are inborn resistant and unfortunately some of them

develop acquired resistance. The gain of resistance is believed to be multifactorial

(Stewart, 2007). Most of chemotherapeutics are distributed intravenously, so the

platinum agent must enter to the cell. The reduced drug accumulation is considered

as the first possibility of resistance to chemotherapeutic treatment

INTRODUCTION

26

(Cepeda et al., 2007). The reduced influx and increased efflux of cisplatin was

frequently observed (Fig. 4). This phenomenon should be enhanced by active efflux

of glutathione S-conjugate with cisplatin (Kartalou and Essigmann, 2001).

.

Fig. 4: Cell resistance to cisplatin and platinum-based chemotherapy (Adapted from Kelland, 2007)

Second mechanism contributing to resistance of cells to cisplatin is

inactivation of the drug by S-containing molecules in cytoplasm. When the platinum

agent enters the cell almost 85% of the drug is bound to proteins or other cytoplasm

constituents (Cepeda et al., 2007). It is accepted that increased glutathione level

cause resistance of cell, through binding and consequently inactivating the cisplatin

agent. The cisplatin covalently bound to glutathione cannot enter the cell nucleus and

the aggregate of platinum-glutathione is exported from cell by active transport

(Kartalou and Essigmann, 2001). Also the metallothioneins are considered to

contribute to cisplatin resistance (Stewart, 2007).

The alteration in expression of oncogenes and tumor suppressor genes

should be mentioned as possible contributors to cisplatin resistance as well. Cisplatin

resistant cells express higher levels of the c-Myc, however the cells resistant to the

drug have also high frequently mutated the ras alleles. But taken together, the

alteration of expression of regulatory proteins is probably cell type specific and it is

not the predictive marker of response of cells to the therapy (Kartalou and

Essigmann, 2001).

INTRODUCTION

27

Next contributor to cisplatin resistance of cell is the deregulated DNA

repair. The mammalian cells defective in DNA repair mechanisms are more sensitive

to platinating agents (Stewart, 2007). Platinum damage is predominantly repaired by

NER, as mentioned above. It should be mentioned that deficiency in MMR also

contributes to the resistance to cisplatin but not to oxaliplatin. This is a main reason

why oxaliplatin is active in cells resistant to treatment by cisplatin and carboplatin

(Martin et al., 2008). Also recombination is important in cellular resistance together

with the translesion synthesis, because the replicative bypass increases in drug-

resistant cell lines (Ahmad, 2010).

Also, the cisplatin adducts on DNA activated a robust apopototic

response. But in the resistant cells this initiation of cell death is corrupted, due to

alterations in expression of regulators of apoptosis like p53, HER2 and ras (Kartalou

and Essigmann, 2001; Stewart, 2007; Siddik, 2003).

AIMS OF THE STUDY

28

2 AIMS OF THE STUDY The main aims of this thesis are the following:

• To summarize the level of knowledge and development achieved in the

field related to the topics of this thesis

• To describe selected types of adducts formed in DNA by novel

derivatives of cisplatin

• To determine a significance of damages induced in DNA by platinum

compounds using DNA repair synthesis assay

• To evaluate the effect of novel derivatives of cisplatin on processes in

tumor cells

MATERIALS AND METHODS

29

3 MATERIALS AND METHODS

3.1 CHEMICALS

Solutions of platinum compounds were prepared at concentrations of

5 × 10–4 M in NaClO4 (10 mM) or in DMF (100%). The solutions were stored in dark

and in refrigerator (4 °C). In addition, for the assays employing cell lines, the starting

concentrations of platinum compounds were 5 × 10-2 M in DMF (100%). The

solutions used for in cellulo testing were always freshly prepared. The concentrations

of stock solutions were controlled by flameless atomic absorption spectrometry

(FAAS).

Cisplatin and transplatin (purity ≥ 99.9% based on elemental and ICP trace analysis) were from Sigma (Prague, the Czech Republic)

[PtCl(en)(L)](NO3)2, where en = ethane-1,2-diamine; L = 1-[2-(acridin-9-yl-amino)-ethyl]-1,3-dimethylthiourea, and its second-generation analogue

[PtCl(en)(L’)](NO3)2, where L’ = N-[2-(acridin-9-yl-amino)ethyl]-N-methylpropionamidine (Paper I) were synthesized, characterized and provided by

prof. U. Bierbach, Department of Chemistry, Wake Forest University, United States

BBR3535, with chemical formula [{trans-PtCL(NH3)2}2-µ-{trans-(H2N(CH2)6NH2(CH2)2NH2(CH2)6NH2)}]4+ (Paper II), was synthesized, characterized

and provided by prof. N. P. Farrell, Department of Chemistry, Virginia Commonwealth

University, the United States

cis-[PtCl2(3ClHaza)2], cis-[PtCl2(3IHaza)2] and cis-[PtCl2(3BrHaza)2], where 3ClHaza = 3-chloro-7-azaindole, 3IHaza = 3-iodo-7-azaindole and

3BrHaza = 3-bromo-7-azaindole (Paper III and IV), were synthesized, characterized

and provided by prof. Z. Trávníček, Regional centre of advanced technologies and

materials, Department of Inorganic chemistry, Palacký University in Olomouc, the

Czech Republic


30

3.2 OTHER CHEMICALS AND BIOLOGICAL MATERIAL

Calf thymus (CT)-DNA (42% G+C, mean molecular mass ca.

20 000 kDa) was prepared and characterized as described previously (Brabec and

Palecek, 1976).

The plasmids pUC19 (2686 bp), pBR322 (4361 bp) and pSP73KB

(2455 bp) were isolated according to standard protocols.

N,N’-dimethylformamide (DMF), dimethylsulphoxide (DMSO) propidium

iodide (PI) were from Sigma-Aldrich (Prague, the Czech Republic).

Restriction endonuclease EcoRI and SspI, the Klenow fragment from

DNA polymerase I (KF–) were from New Engeland Biolabs (Beverly, MA).

Proteinase K and ATP were from Boehringer (Mannheim, Germany).

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] were

from Calbiochem (Darmstadt, Germany).

Radioactive products were obtained from MP Biomedicals, LLC (Irvine, CA).

RPMI 1640 medium, fetal bovine serum (FBS), trypsin/EDTA were from

PAA (Pasching, Austria).

Gentamycin, sodium dodecylsulphate (SDS), agarose (research grade)

were from Serva (Heidelberg, Germany).

The cell death detection ELISA plus kit was from Roche Molecular

Biochemical (Mannheim, Germany).

DNazol (DNazol genomic DNA isolation reagent) was from MRC

(Cincinnati, OH).

The cell-free extract (CFE) was prepared from the repair proficient HeLa S3

cell line as reported previously (Reardon, 1999).

Cell lines A2780 and A2780cisR (cisplatin resistant variant of A2780 cells)

were kindly supplied by prof. B. Keppler, University of Vienna (Austria).


31

3.3 IN CELLULO ASSAYS

3.3.1 IN VITRO GROWTH INHIBITION ASSAY

The human ovarian carcinoma cisplatin sensitive A2780 cells and

cisplatin resistant A2780cisR cells (cisplatin resistant variant of A2780 cells) have

been grown in RPMI 1640 medium supplemented with gentamycin (50 µg/ml) and

heat inactivated FBS (10%). The acquired resistance of A2780cisR cells was

maintained by supplementing the medium with cisplatin (1 µM) in every second

passage. The cells culture was incubated in humidified incubator at 37°C in 5% CO2

atmosphere and subcultured 2-3 times a week with appropriate plating densities.

The cells were seeded in 96-well tissue culture plates at density

104 cells/well in 100 µl of growth medium. After overnight incubation, the cells were

treated with compounds at the final concentrations of 0 to 100 µM in a final volume of

200 µl/well. The cell lines were incubated for 72 hours with platinum compounds and

cell death was evaluated using a system based on the tetrazolium compound MTT as

described previously (Bugarcic et al., 2008; Kisova et al., 2011).

Stock solutions of drugs were prepared in DMF. Stock solutions were

freshly prepared just before the testing and diluted step-by-step in DMF, giving the

series of nine dilutions. This series were then diluted 500-fold into culture medium to

give concentrations 2-fold lower than the test concentration. Medium (100 µl)

containing the test substance was added to each well. The final concentration of

DMF in all wells was 0.1%, which was shown not to affect the cell growth.

After 72 hours of incubation, freshly diluted MTT solution (10 µl,

2.5 mg/ml) was added to each well. Plate was then incubated for additional 4 hours in

humidified CO2 incubator. The medium was removed at the end of incubation and

formazan product was dissolved in 100 µl of DMSO. Cell viability was evaluated by

absorbance measurement at wavelength of 570 nm using Absorbance Reader Tecan

INFINITE M2000 (Schoeller).

IC50 values (compound concentrations that produce 50% of cell growth

inhibition) were calculated from curves constructed by plotting cell survival (%)


32

against drug concentration (µM). All experiments were done in triplicate. Cytotoxic

effects were expressed as IC50.

3.3.2 DETECTION OF APOPTOSIS AND NECROSIS

The cell death detection ELISA plus kit (Roche) was used as an indicator

of apoptosis and necrosis (Moser et al., 2007). Internucleosomal DNA fragmentation

was quantitatively assayed by antibody-mediated capture and detection of

cytoplasmic mononucleosome and oligonucleosome associated histone-DNA

complexes.

After centrifugation (200 g) 20 µl of the supernatant was used for ELISA

(enzyme-linked immunosorbent assay) for detection of necrosis. A2780 cells were

then resuspended in lysis buffer (200 µl) and incubated for 30 minutes at room

temperature. The cell nuclei were pelleted by centrifugation, and supernatant (20 µl,

cytoplasmatic fraction) was used for ELISA detection of apoptosis according to

manufacturer’s standard protocol.

Absorbance was determined at wavelength of 405 nm and 490 nm with

microplate reader (Sunrice Tecan Infinite M200, Schoeller) after 20 minutes

incubation with peroxidase substrate. Other details of this assay and data analysis

were performed according to manufacturer’s protocol.

3.3.3 INTERACTION OF PLATINUM COMPOUNDS WITH CELLS IN REAL TIME

Background of the E-plates was determined in 100 µl of medium (RPMI), and

subsequently 50 µl of the A2780 cell suspension was added to the concentration

104 cells/well. E-plates were immediately placed into the Real-time Cell Analyzer

(RTCA) station. Cells have been grown for 24 hours in a humidified incubator (37°C,

5% CO2).

Next day four equal parts of the cells were treated, first part with 50 µl of media,

second part with 50 µl of media containing IC20 concentration of tested complex 1, third part with 50 µl of media containing IC20 concentration of tested complex 2 and fourth part with 50 µl of media containing IC20 concentration of cisplatin. Impedance

was monitored every 15 minutes for first 6 hours and then every 30 minutes. The cell-

sensor impedance is displayed as an arbitrary unit Cell index (CI).


33

CI at each time point is defined as (Rt – Rb)/15, where Rt is defined as

the cell-electrode impedance of the well with cells at different time points and Rb is

the background impedance of the well with the media alone. Normalized CI is

calculated by dividing the cell index at particular time points by the CI at the time of

interest. Each treatment was performed in triplicate.

3.3.4 CELL CYCLE ANALYSIS

The A2780 cells were treated by tested compounds and cisplatin at

concentration 3 µM and 5 µM. After 24 hours of incubation, the floating cells were

collected and attached cells were harvested by trypsinization (Trypsin/EDTA in PBS).

Cells picked up in this way were washed in PBS and fixed in 70% ethanol.

Subsequently, the cell pellets were rinsed by PBS and stained with solution of

Propidium iodide (50 µg/ml) supplemented by RNase A (10 µg/ml) for 30 minutes in

a dark at room temperature. DNA content was measured using flow cytometer

Cell lab quanta TM SC-MLP (Beckman Coulter). The percentages of cells in the

individual cell cycle phases were analysed.

3.3.5 DNA PLATINATION IN CELLS

Cells A2780 were grown near confluence. Then the cells were exposed

to 10 µM concentration of tested complexes or cisplatin for 5 hours or 24 hours.

After incubation period, the cells were trypsinized and washed twice in

ice-cold PBS. Later on, the cells were lysed in DNazol (MRC) supplemented with

RNase A (100 µg/ml). The genomic DNA was precipitated from lysate with ethanol,

dried and resuspended in water. The DNA content in each sample was determined

by UV spectroscopy. To avoid the effect of high DNA concentration on ICP-MS

detection of platinum in the sample, the DNA samples were digested in the presence

of hydrochloric acid (11 M) using high pressure microwave mineralization system

(MARS5, CEM). Experiments were performed in triplicate and the values are

presented as means ± SD.

3.3.6 CELLULAR UPTAKE OF PLATINUM COMPOUNDS

Cellular uptake of tested complexes and cisplatin was measured in

A2780 cells. The cells were seeded in tissue culture dishes at confluence

3 × 104 cells/cm2. Later on, the cells were treated with PtII complex (10 µM) for


34

5 hours and 24 hours. These concentrations were verified by the measurement of

platinum in medium by FAAS.

The adherent cells were harvested by trypsinisation and subsequently

washed by ice-cold PBS. The cell pellet was stored at –80 °C. Consequently, the

pellets were digested by high pressure microwave digestion system (MARS5, CEM)

with HCl to give fully homogenized solution. Final platinum content was determined

by FAAS. The results of cellular platinum uptake were corrected for absorption

effects (Egger et al., 2009). All experiments were performed in triplicate.

3.4 IN VITRO ASSAYS

3.4.1 PLATINATION OF DNA IN CELL-FREE MEDIUM CONTAINING ETHANOL

EcoRI linearized plasmid pUC19 DNA or CT-DNA was incubated with

platinum compounds in 0.2 M sodium acetate, pH 5.5 and 75% ethanol at 37°C in the

dark. After 48 hours, the samples were precipitated and resolved in the medium

required for follow-up agarose gel electrophoresis. An aliquot of samples was used to

determine the rb value (number of molecules of platinum compound bound per

nucleotide residue).

3.4.2 INTERSTRAND DNA CROSS-LINKS IN CELL-FREE MEDIUM

The platinum compounds were incubated for 24 hours with 0.5 µg of

linear 2686 bp fragment of pUC19 plasmid linearized by EcoRI. The linear fragment

was first 3’-end labeled by means of the KF– of DNA polymerase I (NEB) in the

presence of [α32P]dATP. The platinated samples were analysed for DNA

intramolecular interstrand CLs (ICL) by previously published procedures (Farrell et

al., 1990; Brabec and Leng, 1993).

The number of ICL was analysed by electrophoresis under denaturating

conditions on alkaline agarose gel (1%). After the electrophoresis had been

completed, the intensities of the bands corresponding to the single strands of DNA

and ICL duplex were quantified.

The frequency of interstrand cross-links was calculated as

%ICL/Pt = XL/5372rb (the DNA fragment contains 5372 nucleotide residues), where

%ICL/Pt is the number of ICL per adduct multiplied by 100, and XL is the number of


35

ICL per molecule of the linearized DNA duplex which was calculated assuming

a Poisson distribution of the ICLs as XL = –ln A, where A stands for the fraction of

molecules running as a band corresponding to the non-cross-linked DNA.

3.4.3 INTERDUPLEX DNA CROSS-LINKS IN CELL-FREE MEDIUM

DNA interduplex cross-linking was examined using plasmid pUC19 DNA

linearized by EcoRI (which cuts only once within this plasmid) by electrophoresis

through native 1% agarose gels with 40 mM

tris(hydroxymethyl)aminomethane/acetate, 1 mM EDTA pH 7.4 running buffer. The

gels were run at 20°C in the dark with voltages ranging between 30 V and 60 V. The

running time depended on the voltage. The resultant gels were stained with ethidium

bromide in water (0.3 µg/ml). Bands were visualised by UV translumination,

photographed and the electrophoretic bands intensities were quantified by means of

AIDA image analyser program (Raytest, Germany).

The frequency of interduplex cross-links was calculated as follows:

frequency of interduplex CLs = XL/5372rb (pUC19 plasmid contains 5372 nucleotide

residues). XL stands for the number of interduplex CLs per molecule of the linearized

DNA duplex and was calculated assuming a Poisson distribution of the interduplex

CLs as XL = –ln A, where A is the fraction of molecules running as a band

corresponding to the non-cross-linked double-stranded DNA.

3.4.4 DNA TRANSCRIPTION BY RNA POLYMERASE IN VITRO

Double stranded DNA template, plasmid pSP73KB (2455 bp), was

digested by NdeI and HpaI restriction endonucleases. The resulting two fragments

212 bp and 2243 bp long were separated on agarose gel (1%) in the buffer

containing 40 mM Tris-acetate (pH8), 1 mM EDTA and 0.5 mg/ml ethidium bromide.

The 212 bp fragment was isolated from gel and purified by Wizard-SV (Promega) and

PCR Clean-Up System (Machery-Nagel). Then, purified fragment was incubated with

the cis-[PtCl2(3ClHaza)2], cis-[PtCl2(3IHaza)2] and cis-[PtCl2(3BrHaza)2] or cisplatin in

NaClO4 (10 mM) for 24 hours at 37°C in the dark. Samples were precipitated by

ethanol and dissolved in the TE buffer (10 mM Tris-Cl pH 7.4, 1 mM EDTA) after

incubation period. The level of platination (rb) in aliquots was checked by FAAS.


36

The analyses of DNA transcription were performed in the absence of

unbound platinum compounds. Transcription of the 212 bp (NdeI/HpaI) restriction

fragment with DNA-dependent T7 RNA polymerase and electrophoretic analysis of

transcripts were performed according to the protocols recommended by Promega

(Promega Protocols and Applications, 43-49 (1989-90)) and previously described

(Brabec and Leng,1993).

3.4.5 REPAIR DNA SYNTHESIS BY MAMMALIAN CELL-FREE EXTRACT

Repair DNA synthesis was assayed using platinated pUC19 plasmid and

cell-free extracts (CFEs) prepared from the repair proficient HeLa S3 cell line. Each

reaction of 50 µl contained 600 ng of unmodified pBR322 and 600 ng of unmodified

or platinated pUC19, 2 mM ATP, 30 mM KCl, 0.05 mg/ml creatine phosphokinase

(rabbit muscle), 20 mM each dGTP, dATP, dTTP, 8 mM dCTP, 74 kBq of [α32P]dCTP

in the buffer composed of 40 mM HEPES-KOH pH 7.5, 5 mM MgCl2, 0.5 mM

dithiotreitol, 22 mM creatine phosphate, 1.4 mg/ml of BSA and 20 mg of CFE.

Reactions were incubated for 3 hours at 30°C and terminated by

incubating for 20 minutes with 20 mM Na2H2EDTA, 0.6% SDS and 250 mg/ml

proteinase K. The products were extracted once with phenol/chloroform (1:1) and

precipitated by adding 3 M sodium acetate and ethanol. After 30 minutes of

incubation at 45°C and centrifugation at 12 000 g for 30 minutes at 4°C, the pellet

was washed with 0.2 ml of 80% ethanol and dried in a vacuum centrifuge.

The DNA was linearized before electrophoresis on agarose gel (1%). Gel

was stained with EtBr for photodocumentation. Experiments were performed in

quadruplicate.


37

3.5 OTHER PHYSICAL METHODS

Absorption spectra were measured with Beckman 7400 DU

spectrophotometer and quartz cells with a thermoelectrically controlled cell holder

and a path length of 1 cm. FAAS measurements were conducted with a Varian

AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite

tube atomizer. For FAAS analysis, DNA was precipitated with ethanol and dissolved

in 0.1 M HCl. Differential pulse polarography was performed with an EG&G Princeton

Applied Research Corporation model 384B polarographic analyser.

SUMMARY OF RESULTS AND DISCUSSION

38

4 SUMMARY OF RESULTS AND DISCUSSION Presented doctoral thesis is based on four manuscripts published and

one manuscript accepted for publication in international, peer reviewed journals (see

the list below). The copies of published papers are attached to the thesis in the

Appendix. The contribution of author to the published work is stated there as well.

Results obtained are also summarized in this section.


39

4.1 REPLACEMENT OF THIOUREA WITH AN AMIDINE GROUP IN

A MONOFUNCTIONAL PLATINUM-ACRIDINE ANTITUMOR AGENT.

EFFECT ON DNA INTERACTIONS, DNA ADDUCT RECOGNITION AND

REPAIR. (PAPER I)

The goal of this project was to delineate mechanistic differences between

two members of a novel class of platinum–acridine antitumor agents and to compare

their DNA damage mechanisms with that of cisplatin. We wanted to elucidate the

consequences of changing the thiourea into the amidine donor group for the

molecular mechanism of the hybrid agents at the DNA level.

The binding experiments confirm that compound 2 has a major advantage over compound 1 with respect to the kinetics of DNA adduct formation. The experiments carried out with randomly and site-specifically modified DNA also

demonstrate that the substitution of the thiourea donor group with an amidine donor

group has consequences for the local DNA adduct structure and global DNA

conformation beyond the adduct sites. DNA conformational changes produced by

adducts formed by compound 2 are more pronounced than the effects caused by compound 1. The higher degree of duplex unwinding and the CD signatures observed for compound 2 are in agreement with geometry more favourable for classical intercalation of the acridine moiety in adducts of this derivative. The adducts

formed by both derivatives do not significantly affect the thermodynamic stability of

modified DNA due to complete enthalpy–entropy compensation.

An important result of the small extent of bending in DNA modified with

compounds 1 and 2 is the lack of recognition of the damage by HMG domain proteins. This notion is further corroborated by the observation that transcription

factors in the cell-free extracts used in this study are hijacked to cisplatin-induced

cross-links but not to the sites of DNA damage produced by complexes 1 and 2.

These important findings may also apply to other nuclear proteins known

to recognize seriously distorted DNA, for example DNA damage recognition proteins

belonging to the nucleotide excision repair (NER) complex. The monofunctional

adducts produced by complex 1 and 2 should be poor substrates for NER. The repair


40

synthesis assay in randomly modified plasmid carried out in this study demonstrates

that the adducts formed by complex 2 are repaired less efficiently than the damage caused by derivative 1, but to a higher extent than cisplatin-type adducts (Fig. 5).

Fig. 5: Reparation of lesion by complex 1, 2 and cisplatin by mammalian cell-free extract

Critical difference between complexes 1 and 2 arose from their ability to inhibit transcription of DNA by stalling DNA dependent RNA polymerase II.

Complex 2 proves to be a significantly more potent inhibitor of RNA synthesis than either complex 1 or cisplatin. Inhibition of DNA transcription is considered to be a major mediator of cytotoxic effect of cisplatin. Monofunctional intercalative adducts of

complex 2 are able to efficiently stall RNA pol II and this suggests that transcription inhibition may contribute to the high cytotoxicity levels observed for the second-

generation platinum-acridine pharmacophore.

The data acquired in this study will help to establish structure-activity

relationships in this class of compounds with the ultimate goal of providing novel

therapies exhibiting a unique mechanism of action.


41

4.2 FORMATION OF INTERDUPLEX DNA CROSS-LINKS UNDER MOLECULAR

CROWDING CONDITION (PAPER II)

Antitumor platinum drugs are able to form various types of adducts on

DNA molecule, especially monofunctional, intrastrand and interstrand adducts. It is

possible to conceive at least two types of DNA interstrand cross-linking by

bifunctional PtII complexes, depending on whether the platinum complex coordinates

to the bases in one DNA molecule or in two different DNA duplexes.

The research was conducted under molecular crowding conditions

mimicking environmental conditions in the cellular nucleus, namely in medium

containing ethanol, which is a commonly used crowding agent.

We investigated the possibility of formation of interduplex CLs by

dinuclear complex BBR3535 and compared the effect with mononuclear cisplatin and

transplatin compounds. The test of ability was carried out in the molecular crowding

conditions ensured by 75% ethanol. We have shown that dinuclear BBR3535 formed

interduplex CLs more efficiently compared with cisplatin and transplatin even in the

lower rb values compared to the mononuclear ones. The frequency of interduplex

CLs of cisplatin, transplatin and BBR3535 were almost independent on the rb and

BBR3535 is 40-fold or 18-fold effective in forming of interduplex CLs in comparison

with cisplatin or transplatin.

Also, we investigated the effect of ethanol concentration on interduplex

CLs formation. The modification of DNA by cisplatin and BBR3535 was carried out at

various ethanol concentrations. As a result, we obtained no interduplex fraction in

media containing up to 30% of ethanol, but the interduplex CLs became evident in

medium containing 50% ethanol. Their fractions grew concomitantly with increasing

concentration of this molecular crowding agent.

The main result obtained by this research is that bifunctional polynuclear

platinum complexes are suitable to form interduplex CLs on DNA under molecular

crowding conditions more efficiently than mononuclear cisplatin and transplatin. Also,

we have shown that sufficient contact of two different DNA molecules is ensured by

mimicking of environmental conditions present in the cellular nucleus.


42

4.3 HOW TO MODIFY 7-AZAINDOLE TO FORM CYTOTOXIC PTII COMPLEXES:

HIGHLY IN VITRO ANTICANCER EFFECTIVE CISPLATIN DERIVATIVES

INVOLVING HALOGENO-SUBSTITUTED 7-AZAINDOLE (PAPER III)

The aim of this work was characterisation of synthesized platinum(II)

dichlorido and oxalate complexes. The geometry was determined by a single-crystal

X-ray analysis. The complexes were screened for their cytotoxic potential in several

human cancer cell lines. The 7-azaindole platinum complexes displayed significantly

higher biological effect against MCF7 and HOS cell line compared with commercially

used cisplatin.

The mechanism of action of 7-azaindole platinum complexes was studied

by means of transcription inhibition by DNA adducts of these compounds compared

to cisplatin. The method is based on RNA synthesis by T7 RNA polymerase in vitro.

T7 RNA polymerase was chosen because it is well characterized, its promoter is

clearly defined and the purified enzyme is commercially available. Briefly, the

NdeI/HpaI restriction fragment of pSP73KB plasmid DNA was globally modified.

The RNA synthesis on the template modified by the platinum complexes yielded

fragments of defined size, which indicates that RNA synthesis on these templates

was prematurely terminated. These results indicates that 7-azaindole platinum

complexes were able to bind DNA forming adducts capable to stall RNA polymerase.

The sequence analysis unveiled that major bands resulting from termination of RNA

synthesis by the 7-azaindole platinum adducts were similar to those produced by

cisplatin.

The accounted complexes with 7-azaindole halogeno-substituents in

structure displayed high cytotoxicity in vitro in contrast to previously reported platinum

dichlorido complex with unsubstituted 7-azaindole. This slight modification of

7-azaindole molecule by halogeno-substituents improved solubility and bioavailability

of the platinum (II) complex with these ligands in structure.


43

4.4 INSIGHT INTO THE TOXIC EFFECT OF THE CIS-PT(II)-DICHLORIDO

COMPLEXES CONTAINING 7-AZAINDOLE HALOGENO-DERIVATIVES IN

TUMOR CELLS (PAPER IV)

The present work deals with the cytotoxic potential of new 7-azaindole

halogeno-derivatives of cisplatin. We have shown that both compounds

cis-[PtCl2(3ClHaza)2] (complex 1) and cis-[PtCl2(3IHaza)2] (complex 2) are toxic to the ovarian tumor cells. The values of IC50 were moderately better compared to

cisplatin in sensitive cell line A2780, but on contrary they were much lower in

cisplatin-resistant line A2780cisR. The cytotoxicity of complexes 1 and 2 to the ovarian tumor cells is characterized by remarkably low resistance factor; markedly

less than 1, therefore complexes 1 and 2 are capable of circumventing of cisplatin resistance in some types of the cisplatin-resistant lines.

Fig. 6: Tested compounds of 7-azaindole halogeno-derivatives of cisplatin and cisplatin molecule

We investigated potential factors which might be involved in the

mechanism underlying the cytotoxic effects of complexes 1 and 2 (Fig. 6) and compared these factors with the mechanism underlying the cytotoxic effects of the

frequently studied anticancer metallodrug cisplatin.

We quantified the levels of apoptosis and necrosis induced by complex 1 and 2. The results show that both complexes induce cell death by apoptosis in sensitive cell line with considerably higher efficiency than cisplatin, and apoptotic type

of cell death prevailed over necrosis. Also we have studied the ability of complex 1 and 2 to arrest cell cycle. Our studies were performed in the cell line with wt p53 status to show the differences between complex 1 and 2 and cisplatin. These two classes of PtII compounds exhibit difference in type and dynamics of cell cycle

perturbations by these compounds. For cells treated with complexes 1 and 2, the


44

nuclear debris from apoptotic or necrotic cells are observed as a sub-G1 population,

but it is markedly lower for cisplatin-treated cells. Results mentioned above are

consistent with former experiment dealing with cell death type. These observations

are in agreement with previous experiment studying cell death type. Cisplatin blocks

the ovarian cancer cells in the G2-phase already at 3 µM concentration, the

complexes 1 and 2 induced weaker block in A2780 cells only at 5 µM concentration.

The difference in cell cycle arrest induced by complex 1 and 2 and cisplatin is also supported by the results of impedance-based real-time monitoring of

the effects of these PtII drugs on cell growth. This method makes it possible to

register very small and rapid changes in cell count, cell adhesion and cell morphology

due to drug toxicity. The results indicate that complexes 1, 2 and cisplatin decrease impedance, suggesting that the reduced cell viability determined in the colorimetric

assay translates into cell death. That suggests existence of critical differences in the

rate and mechanisms of cell kill caused by complexes 1 and 2 unlike cisplatin.

The amount of platinum bound to cellular DNA of A2780 cells incubated

with complexes 1 and 2 for 5 hours and 24 hours was higher than the amount found in cells treated with cisplatin. The results of DNA binding obtained from experiments

carried out in cell free medium indicated that modification reaction resulting in the

irreversible coordination of complexes 1 and 2. Also, determination of monofunctional adducts and interstrand cross-linking efficiency of complexes 1 and 2 suggests that several aspects of the DNA binding mode of complexes 1 and 2 are similar to those of parental cisplatin. It is also possible that adducts on DNA by complex 1 and 2, which are identical to those adducts caused by cisplatin, could distort DNA

conformation differently and could be processed by cellular components differently.

Results describing irreversible binding of GSH to complex 1, 2 and cisplatin showed that the reaction of cisplatin with GSH was 1.2-fold higher than the

one obtained for complex 1 and 2.

Proteins of nucleotide excision repair can most efficiently recognize and

remove DNA adducts that seriously distort and destabilize double-helical DNA.

The adducts produced by complexes 1 and 2 should be poorer substrates for nucleotide excision repair than the adducts of cisplatin. The relative resistance to


45

DNA repair would explain why complexes 1 and 2 show major pharmacological advantages over cisplatin in ovarian cancer cell lines. The presumably most cytotoxic

and major adducts formed by complexes 1 and 2 are repaired considerably less efficiently than the damage caused by cisplatin, this may potentiate toxic effects of

this class of PtII compounds in tumor cells.

CONCLUSION

46

5 CONCLUSION The main goals of the present thesis were to examine the ability of novel

platinum compounds to form cross-links, especially interduplex CLs, on DNA. In the

published paper (Paper II), we have shown that the selected dinuclear and

bifunctional antitumor platinum compounds are efficient interduplex cross-linkers

under suitable conditions mimicking environmental conditions in the cellular nucleus.

For our purposes, the molecular crowding conditions were ensured by aqueous

ethanol solutions supplemented by salt.

Another potential factor of resistance to chemotherapeutics based on

transition metal complexes is repair of lesions formed in DNA. Cross-links formed by

cisplatin are effectively removed by nucleotide excision repair. If the lesion is

successfully repaired, the treatment is inefficient. We have shown that DNA adducts

of new monofunctional platinum-acridine antitumor agent are repaired more efficiently

than those of bifunctional cisplatin.

An attention was also paid to the mechanism underlying action of newly

synthetized 7-azaindole halogeno-derivatives of cisplatin. We have studied the

antitumor effects of these complexes on ovarian cancer cell line and we have shown

that tested compounds were quite toxic to them, also we have shown that these

compounds to accumulation in cells increase, which might be caused by the bulkier

ligand on platinum atom. Also, the possibility of binding of these compounds to DNA

was tested in vitro together with platination of cellular DNA. As a next step, we have

shown (Paper III, Paper IV) that these compounds are able to stop the cell cycle and

induce the apoptotic cell death rather than necrosis.

The obtained results contribute to the understanding of mechanisms

underlying action and resistance of selected chemotherapeutics based on platinum

complexes. The slight change of molecule of cisplatin shows promising possibilities

for novel cisplatin derivatives, which could overcome the resistance of cell to the

cisplatin treatment.

REFERENCES

47

6 REFERENCES Ahmad S. (2010): Platinum-DNA interactions and subsequent cellular processes

controlling sensitivity to anticancer platinum complexes. Chem Biodivers 7:

543-566.

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