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UNIVERSIDADE DE LISBOAFACULDADE DE CIÊNCIAS
DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTIONOF ORGANOMETALLIC COMPLEXES
Daniel Mosebo Fernandes Bandarra
Mestrado em Bioquímica
Área de especialização em Bioquímica Médica
2010
UNIVERSIDADE DE LISBOAFACULDADE DE CIÊNCIAS
DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTIONOF ORGANOMETALLIC COMPLEXES
Daniel Mosebo Fernandes Bandarra
Dissertação orientada por:Doutora Margarida MeirelesDoutora Mª José Calhorda
Mestrado em Bioquímica
Área de especialização em Bioquímica Médica
2010
Este trabalho foi realizado na Faculdade de Ciências da Universidade de Lisboa
Parte dos resultados incluídos no presente trabalho fazem parte do artigo científico já publicado [73].No cumprimento do disposto no nº2 do artigo 8º do Decreto-lei 388/07, declaro que participei naconcepção e na execução do trabalho que esteve na base do artigo, bem como na interpretação eredacção dos resultados.
Dedico este trabalho a todos os que procuram fazer a diferença neste Mundo, colaborando com assuas ideias e experiências de modo a contribuir para a sua evolução.
Jeg dedikerer dette arbejde til alle dem, der søger at gøre en forskel i denne verden, og somsamarbejder med deres ideer og erfaringer til at bidrage til dens udvikling.
I dedicate this work to all those who try to make a difference in this world, collaborating with theirideas and experiences in order to contribute to its evolution.
How wonderful that we have met with a paradox, now we have some hope of making progress
Niels Bohr
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | xi
RESUMO
O cancro é uma das doenças com maior mortalidade a nível mundial e, de acordo com a Organização
Mundial de Saúde, prevê-se um aumento do número de mortes, podendo chegar a 12 milhões em
2030. Apesar desta tendência, muitos estudos têm sido feitos para a contrariar, nomeadamente ao
nível da quimioterapia. Actualmente a quimioterapia e a radioterapia constituem as opções mais
recorrentes para o tratamento de cancro, sendo exemplos de tratamentos que exploram a
sensibilidade das células tumorais a danos no DNA.
O uso de metais na medicina remota à Antiga China com o uso de ouro. Outros metais como a
cisplatina têm sido usados desde a segunda metade do século XX. De facto a cisplatina e os seus
análogos são os fármacos actualmente mais usados no tratamento de tumores sólidos, devido à sua
elevada eficácia. No entanto, apresentam diversas limitações, como o desenvolvimento de resistência
do organismo ou a sua elevada toxicidade. Por essa razão, novos complexos com outros centros
metálicos, como o ruténio, o vanádio ou o molibdénio, têm sido testados.
Vários estudos de agentes antitumorais com compostos com molibdénio têm sido feitos, tendo já
sido comprovadas as suas propriedades citotóxicas. No entanto o seu mecanismo de acção encontra-
se por se esclarecer.
O presente trabalho teve como objectivo o estudo das propriedades antitumorais de dois complexos
organometálicos com molibdénio, [Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)], B1, e [Mo(3-
C3H5)CF3SO3(CO)2(2,2’-bipiridil)], T2 (Figura 1), e o estudo do mecanismo de acção citotóxica
destes complexos em linhas tumorais.
A actividade citotóxica de B1 e T2 foi estudada em três linhas tumorais: células HeLa (cancro do
colo do útero), células MCF-7 (cancro da mama) e RPE (epitélio pigmentado da retina imortalizada
pela expressão da proteína telomerase humana), através do ensaio do MTT (brometo de 3-(4,5-
dimetiltiazon-2-il)-2,5-difeniltetrazólio), um ensaio metabólico universalmente usado. Para o
complexo B1 foram obtidos valores de IC50 (inibição da viabilidade celular em 50%) entre 1 e 9 M
e para T2 entre 13 e 46 M para as três linhas celulares estudadas (Tabela 1). Estes valores
encontram-se na gama de valores considerados muito bons ao nível da citotoxicidade em linhas
tumorais, em partícular B1, que apresenta valores de IC50 semelhantes aos da cisplatina.
Figura 1 – Estrutura esquemática de B1 e T2.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | xi
RESUMO
O cancro é uma das doenças com maior mortalidade a nível mundial e, de acordo com a Organização
Mundial de Saúde, prevê-se um aumento do número de mortes, podendo chegar a 12 milhões em
2030. Apesar desta tendência, muitos estudos têm sido feitos para a contrariar, nomeadamente ao
nível da quimioterapia. Actualmente a quimioterapia e a radioterapia constituem as opções mais
recorrentes para o tratamento de cancro, sendo exemplos de tratamentos que exploram a
sensibilidade das células tumorais a danos no DNA.
O uso de metais na medicina remota à Antiga China com o uso de ouro. Outros metais como a
cisplatina têm sido usados desde a segunda metade do século XX. De facto a cisplatina e os seus
análogos são os fármacos actualmente mais usados no tratamento de tumores sólidos, devido à sua
elevada eficácia. No entanto, apresentam diversas limitações, como o desenvolvimento de resistência
do organismo ou a sua elevada toxicidade. Por essa razão, novos complexos com outros centros
metálicos, como o ruténio, o vanádio ou o molibdénio, têm sido testados.
Vários estudos de agentes antitumorais com compostos com molibdénio têm sido feitos, tendo já
sido comprovadas as suas propriedades citotóxicas. No entanto o seu mecanismo de acção encontra-
se por se esclarecer.
O presente trabalho teve como objectivo o estudo das propriedades antitumorais de dois complexos
organometálicos com molibdénio, [Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)], B1, e [Mo(3-
C3H5)CF3SO3(CO)2(2,2’-bipiridil)], T2 (Figura 1), e o estudo do mecanismo de acção citotóxica
destes complexos em linhas tumorais.
A actividade citotóxica de B1 e T2 foi estudada em três linhas tumorais: células HeLa (cancro do
colo do útero), células MCF-7 (cancro da mama) e RPE (epitélio pigmentado da retina imortalizada
pela expressão da proteína telomerase humana), através do ensaio do MTT (brometo de 3-(4,5-
dimetiltiazon-2-il)-2,5-difeniltetrazólio), um ensaio metabólico universalmente usado. Para o
complexo B1 foram obtidos valores de IC50 (inibição da viabilidade celular em 50%) entre 1 e 9 M
e para T2 entre 13 e 46 M para as três linhas celulares estudadas (Tabela 1). Estes valores
encontram-se na gama de valores considerados muito bons ao nível da citotoxicidade em linhas
tumorais, em partícular B1, que apresenta valores de IC50 semelhantes aos da cisplatina.
Figura 1 – Estrutura esquemática de B1 e T2.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | xi
RESUMO
O cancro é uma das doenças com maior mortalidade a nível mundial e, de acordo com a Organização
Mundial de Saúde, prevê-se um aumento do número de mortes, podendo chegar a 12 milhões em
2030. Apesar desta tendência, muitos estudos têm sido feitos para a contrariar, nomeadamente ao
nível da quimioterapia. Actualmente a quimioterapia e a radioterapia constituem as opções mais
recorrentes para o tratamento de cancro, sendo exemplos de tratamentos que exploram a
sensibilidade das células tumorais a danos no DNA.
O uso de metais na medicina remota à Antiga China com o uso de ouro. Outros metais como a
cisplatina têm sido usados desde a segunda metade do século XX. De facto a cisplatina e os seus
análogos são os fármacos actualmente mais usados no tratamento de tumores sólidos, devido à sua
elevada eficácia. No entanto, apresentam diversas limitações, como o desenvolvimento de resistência
do organismo ou a sua elevada toxicidade. Por essa razão, novos complexos com outros centros
metálicos, como o ruténio, o vanádio ou o molibdénio, têm sido testados.
Vários estudos de agentes antitumorais com compostos com molibdénio têm sido feitos, tendo já
sido comprovadas as suas propriedades citotóxicas. No entanto o seu mecanismo de acção encontra-
se por se esclarecer.
O presente trabalho teve como objectivo o estudo das propriedades antitumorais de dois complexos
organometálicos com molibdénio, [Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)], B1, e [Mo(3-
C3H5)CF3SO3(CO)2(2,2’-bipiridil)], T2 (Figura 1), e o estudo do mecanismo de acção citotóxica
destes complexos em linhas tumorais.
A actividade citotóxica de B1 e T2 foi estudada em três linhas tumorais: células HeLa (cancro do
colo do útero), células MCF-7 (cancro da mama) e RPE (epitélio pigmentado da retina imortalizada
pela expressão da proteína telomerase humana), através do ensaio do MTT (brometo de 3-(4,5-
dimetiltiazon-2-il)-2,5-difeniltetrazólio), um ensaio metabólico universalmente usado. Para o
complexo B1 foram obtidos valores de IC50 (inibição da viabilidade celular em 50%) entre 1 e 9 M
e para T2 entre 13 e 46 M para as três linhas celulares estudadas (Tabela 1). Estes valores
encontram-se na gama de valores considerados muito bons ao nível da citotoxicidade em linhas
tumorais, em partícular B1, que apresenta valores de IC50 semelhantes aos da cisplatina.
Figura 1 – Estrutura esquemática de B1 e T2.
xii | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
CompostosIC50 (M)
HeLa MCF-7 RPE
T2 23,7 ± 0,01 44,5 ± 0,7 13,1 ± 2,4
B1 5,1 ± 1,0 8,9 ± 0,5 0,7 ± 0,1
Para compreender o mecanismo da acção citotóxica destes complexos em linhas tumorais, começou-
se por estudar a interacção dos complexos com a membrana plasmática através da determinação do
coeficiente de partição octanol/água dos complexos em solução. Ambos os complexos apresentam
um comportamento hidrofóbico em solução, sendo B1 mais hidrofóbico que T2. De forma a
perceber melhor a interacção composto-membrana plasmática, procedeu-se à quantificação de
molibdénio em células tumurais HeLa após a incubação destas com os compostos, durante 48 horas e
com diversas concentrações. Para além disso, nas mesmas condições, procedeu-se à quantificação de
molibdénio em fracções citosólicas e nucleares. Relativamente às células controlo (sem composto),
observou-se um aumento dos níveis de molibdénio quer no citosol, quer no núcleo, sendo este
aumento dependente da concentração de composto. Estes resultados parecem indicar que B1 e T2
possuem propriedades que lhes permitem entrar na célula e fundamentalmente no núcleo.
Face aos resultados obtidos, colocou-se a hipótese de que os complexos pudessem exercer o seu
efeito citotóxico através da interacção com o DNA, inibindo assim o crescimento das células
tumorais.
Procedeu-se a uma série de ensaios visando a detecção da interacção entre os compostos e o DNA.
Começou-se por estudar a interacção dos complexos com DNA plasmídico, através da realização de
electroforeses, em gel de agarose, de soluções de DNA plasmídico após incubação com os
compostos. Observou-se que ambos os complexos levam a alterações na mobilidade do DNA
palsmídico, sendo este efeito mais evidente para B1. Estes resultados sugerem que há interacções dos
compostos com o DNA.
Efectuaram-se também estudos de titulação de calf thymus DNA (ctDNA) por espectrofotometria
UV-Vis e por dicroísmo circular. Nos ensaios por espectrofotometria UV-Vis mediram-se variações
de absorvência após a adição de quantidades crescentes de ctDNA, fixando-se a concentração de
composto, com os respectivos controlos. Nos ensaios de dicroísmo circular fixou-se a concentração
de DNA e adicionaram-se quantidades crescentes de composto. Os resultados obtidos sugerem que a
interacção entre os compostos e o DNA seja maioritariamente por intercalação, não podendo no
entanto ser excluídos outros tipos de interacções. A partir da titulação determinaram-se as constantes
Quadro 1 – Estudos in vitro da actividade citotóxica dos complexos demolibdénio T2 e B1 em células Hela, MCF-7 [74] e RPE (os valorescorrespondem à média ± desvio padrão de três replicados)
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | xiii
de ligação entre ctDNA e os complexos, tendo-se obtido valores de 2,08 (±0,98) × 105 e 3,68 (±2,01)
× 105 M-1 para B1 e T2, respectivamente. Os valores das constantes de ligação DNA-composto
encontram-se na mesma gama de valores obtidos para o brometo de etídeo, um intercalador clássico,
confirmando assim a natureza intercalativa do tipo de interacção entre os complexos e o DNA.
A microscopia de força atómica (AFM) de soluções de DNA plasmídico com e sem B1 permitiu
estudar possíveis transições estruturais do DNA face a interacções do complexo. Obtiveram-se
imagens topográficas onde se observaram alterações a nível da estrutura terciária do DNA
plasmídico aquando da presença de B1. Estes resultados são comparativos com os obtidos
anteriormente, onde é demonstrado a interacção dos complexos com o DNA. Infelizmente, devido a
limitações experimentais, não foi possível efectuar os estudos de AFM para o complexo T2.
Em suma, a realização deste trabalho permitiu, a partir dos estudos de actividade citotóxica,
identificar dois compostos como potenciais agentes antitumorais e esclarecer um dos possíveis
mecanismos responsáveis pela sua acção citotóxica em células tumorais. Tendo em conta os
resultados obtidos, conclui-se que ambos os compostos apresentam propriedades que lhes permitam
entrar na célula e chegar ao núcleo. Neste compartimento, os complexos levam à inibição do
crescimento celular através da sua interacção, maioritariamente por intercalação, com o DNA,
levando, possivelmente, a alterações ao nível da sua conformação e ultimamente à morte celular.
O presente trabalho mostra a potencialidade do uso de B1 e T2 na quimioterapia, podendo trazer
novas perspectivas nesta área, de modo a ultrapassar as limitações existentes actualmente, uma vez
que se trata de compostos metálicos com diferentes propriedades químicas relativamente aos
actualmente usados. A utilização destes ligandos e a possibilidade de combinar outros novos,
constituem uma mais valia no tratamento do cancro.
É de referir ainda a oportunidade que este trabalho proporcionou a publicação de um artigo científico
com o título: “Mo(II) complexes: A new family of cytotoxic agents?”, na revista Jounal of Inorganic
Biochemistry [73] (doi:10.1016/j.jinorgbio.2010.07.006).
xiv | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Abstract
Antitumor properties of two molybdenum complexes, [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)]
(B1) and [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) have been tested in vitro against human
cervical cancer cell line (HeLa), human breast cancer cell line (MCF-7), and human telomerase
reverse transcriptase – retinal epithelial cells (RPE) using a metabolic activity test, MTT (3-(4,5-
dimethylthiazon-2-yl)-2,5-diphenyltetrazolium bromide), with IC50 values ranged from 1 to 9 M,
and from 13 to 46 M, approximately, for B1 and T2, respectively. In order to understand the
mechanism of action against cancer cell lines several studies have been made. Cellular uptake of
molybdenum and octanol/water partition assays revealed that both B1 and T2 exhibit a selective
uptake by cells with the ability to reach the nucleus, and a hydrophobic behavior in solution, B1
being more hydrophobic than T2. The interaction of the complexes with DNA was also studied.
According to gel electrophoresis studies, both complexes seem to interact with plasmid DNA. The
binding constants of B1 and T2 with calf thymus DNA (ctDNA), as determined by absorption
titration, are 2.08 (±0.98) × 105 and 3.68 (±2.01) × 105 M-1, respectively. These results together with
data obtained from circular dichroism suggest that the complexes interact with DNA, mainly by
intercalation, changing its conformation and possibly inducing cell death. Preliminary studies of
structural transitions in the tertiary structure of plasmid DNA using atomic force microscopy showed
that B1 seems to induce structural changes on the plasmid, plectonemic supercoiling being the
predominant form adopted by the plasmid. These results show that in future both complexes may
provide a valuable tool in cancer chemotherapy.
Keywords:
Molybdenum, antitumor activity, interaction with DNA, chemotherapy
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 1
Index
1. Index of figures ............................................................................................................................3
2. Index of tables ..............................................................................................................................5
3. Abbreviations list.........................................................................................................................6
4. Introduction .................................................................................................................................7
4.1. The current state of cancer research ......................................................................................7
4.2. The hallmarks of chemotherapy ............................................................................................7
4.3. The limit to chemotherapy.....................................................................................................9
4.4. Medicinal inorganic chemistry ............................................................................................11
4.5. Mode of action of metal anticancer compounds..................................................................12
4.6. Experimental strategies .......................................................................................................13
4.7. Electronic absorption titration .............................................................................................13
4.8. Circular dichroism...............................................................................................................14
4.9. Atomic force microscopy (AFM) ........................................................................................14
5. Aim..............................................................................................................................................17
6. Experimental..............................................................................................................................19
6.1. Instrumentation and materials .............................................................................................19
6.2. Synthesis of molybdenum(II) complexes ............................................................................19
6.2.1. [Mo(3-C3H5)(CF3SO3)(CO)2(2,2’-bpy)] (T2) ............................................................19
6.2.2. [Mo(3-C3H5)(Br)(CO)2(1,10-phenanthroline)] (B1)..................................................20
6.3. Cell cultures.........................................................................................................................20
6.4. Subculture of cells ...............................................................................................................20
6.5. Cell quantification ..............................................................................................................20
6.6. Cryopreservation of cells.....................................................................................................21
6.7. Resuscitation of frozen cells................................................................................................21
6.8. Cytotoxic activity assay in vitro ..........................................................................................21
6.9. Octanol/water partition coefficient ......................................................................................22
6.10. Conductimetry .................................................................................................................22
6.11. Cellular molybdenum uptake...........................................................................................22
6.12. DNA binding studies .......................................................................................................23
6.12.1. Electronic absorption titration .....................................................................................23
6.12.2. Circular dichroism.......................................................................................................23
6.12.3. Gel electrophoresis studies ..........................................................................................23
6.12.4. Atomic force microscopy ............................................................................................24
7. Results and discussion...............................................................................................................25
7.1. Cytotoxic activity assay in vitro ..........................................................................................25
2 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
7.2. Octanol/water partition coefficient ......................................................................................29
7.3. Conductimetry .....................................................................................................................30
7.4. Cellular molybdenum uptake ..............................................................................................31
7.5. DNA Binding Studies..........................................................................................................32
7.5.1. Electronic absorption titration .....................................................................................32
7.5.2. Circular dichroism.......................................................................................................34
7.5.3. Gel electrophoresis studies ..........................................................................................35
7.5.4. Atomic force microscopy ............................................................................................35
8. Conclusions ................................................................................................................................37
9. Acknowledgements ....................................................................................................................39
10. References ..................................................................................................................................41
11. Annex..........................................................................................................................................47
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 3
1. Index of figures
Figure 1 - Cisplatin and its analogues (Adapted from [17]). ............................................................................. 8Figure 2 - Dose-response relationships and proposed resistance mechanisms (Adapted from [24])............... 10Figure 3 – Drawing schemes of the transitions in plasmid DNA tertiary structure in response to an intercalatoragent: (a) predominantly relaxed; (b) toroidally supercoiled; (c) mixed toroidal and plectonemic supercoils; (d)complete plectonemic supercoiling (adapted from [59])............................................................................... 15Figure 4 - Schematic illustration of the overview of the work plan. Mo(II) complexes were tested and acted aspotent cytotoxic drugs, interacting with DNA in vitro. Do they enter the cell and directly damage DNA toinhibit cell growth? ...................................................................................................................................... 18Figure 5 – Haemocytometer (adapted from [62]) ......................................................................................... 21Figure 6 - Schematic structure of B1 and T2.................................................................................................. 25Figure 7 - In vitro cytotoxic assays for T2 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with T2 (right). ............................................................................. 25Figure 8 - In vitro cytotoxic assays for B1 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with B1 (right). ............................................................................. 26Figure 9 - In vitro cytotoxic assays for B1 against RPE cells (left). Dose-response curve obtained by nonlinearregression analysis for RPE cells treated with B1 (right)................................................................................ 26Figure 10 - In vitro cytotoxic assays for T2 against RPE cells (left). Dose-response curve obtained by nonlinearregression analysis for RPE cells treated with T2 (right). ............................................................................... 27Figure 11 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with B1 for1, 2, 24, and 48 hours (left). In vitro cytotoxic activity for B1 against HeLa cells at 1, 2, 24, and 48 hours (right)..................................................................................................................................................................... 28Figure 12 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with T2 for 1,2, 24, and 48 hours (left). In vitro cytotoxic activity for T2 against HeLa cells at 1, 2, 24, and 48 hours (right).29Figure 13 - Mean log octanol/water partition coefficients (Log P) of the molybdenum compounds. .............. 29Figure 14 - Specific conductivity of B1 in 0,5% DMSO in water solution......................................................... 30Figure 15 - Specific conductivity of T2 in water solution................................................................................ 30Figure 16 - Comparison of the intracellular molybdenum concentration in HeLa cells after 48 h of exposure tocompounds T2 and B1. ................................................................................................................................ 31Figure 17 - Molybdenum concentration in cytosolic (a) and nuclear (b) extracts of HeLa cells after 48 h ofexposure to compounds T2 and B1. ............................................................................................................. 32Figure 18 - Left: UV-Vis absorption spectra of T2 (20 M) in Tris buffer in the presence of increasing amountsof ctDNA. [DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNAconcentration; right: plot of D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at299 nm. ....................................................................................................................................................... 33Figure 19 - Left: UV-Vis absorption spectra of B1 (20 M) in Tris buffer in the presence of increasing amountsof ctDNA. [DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNAconcentration; right: plot of D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at272 nm. ....................................................................................................................................................... 34Figure 20 – Circular Dichroism of ctDNA incubated with B1 (left) and T2 (right). [Complex] = 0, 10, 25, 50, 75,100, 250 M. The arrow indicates the signal changes upon increasing complex concentration. .................... 35Figure 21 - Electrophoretic mobility pattern of pYES2 plasmid DNA (C) incubated with complex B1 (right) andT2 (left) at 10, 50 and 100 M...................................................................................................................... 35Figure 22 - A Selection of topographic images recorded of plasmid pYES2 (a) and plasmid pYES2 incubatedwith T2 (b) adsorbed to AP-mica. The scale bars correspond to 250 nm........................................................ 36Figure 23 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO with no incubation time (0 hours)...................................................................................................... 47Figure 24 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO after 2 hours of incubation time at 37 ºC. .......................................................................................... 48
4 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 25 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO after 24 hours of incubation time at 37 ºC. ........................................................................................ 49Figure 26 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved inDMSO after 48 hours of incubation time at 37 ºC. ........................................................................................ 50Figure 27 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO with no incubation time (0 hours)...................................................................................................... 51Figure 28 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO after 2 hours of incubation time at 37 ºC. .......................................................................................... 52Figure 29 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO after 24 hours of incubation time at 37 ºC. ........................................................................................ 53Figure 30 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved inDMSO after 48 hours of incubation time at 37 ºC. ........................................................................................ 54Figure 31 - Absorption spectra of B1 ([Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)]). .......................................... 55Figure 32 - Absorption spectra of T2 ([Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipiridil)])............................................. 55
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 5
2. Index of tables
Table 1 - Drugs of Choice for Different Types of Cancer (Adapted from [5])..................................................... 9Table 2 - In vitro cytotoxicity assays for molybdenum complexes T2 and B1 against HeLa, MCF-7 [74], and RPEcells (data are mean ±SD of three replicates each)........................................................................................ 27
6 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
3. Abbreviations list
AFM – Atomic force microscopy
ALL – Acute lymphoblastic leukaemia
AML – Acute myelogenous leukemia
B1 – [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)]
BC – Before Christ
CD – Circular dichroism
ctDNA – calf thymus DNA
DCR – Dose-response curve
DMSO – Dimethyl sulfoxide
DNA – Desoxyribonucleotic acid
DTT – Dithiothreitol
EDTA – Ethylenediamine tetraacetic acid
FBS – Fetal bovine serum
FDA – Food and drug administration
HeLa – Human cervical cancer cell line
HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
IC50 – Inhibitory concentration (dose causing 50% inhibition of cell growth)
ICP-MS – Inductively coupled plasma mass spectrometry
MCF-7 – Human breast cancer cell line
MDR – Multidrug resistance
MOPP – Mustargen-oncovin-procarbazine-prednisone
MTT – 3-(4,5-dimethylthiazon-2-yl)-2,5-diphenyltetrazolium bromide
NCI – American national cancer institute
PBS – Phosphate buffered saline
RNA – Ribonucleic acid
ROS – Reactive oxygen species
RPE – Human telomerase reverse transcriptase – retinal epithelial cells
RPMI – Roswell park memorial institute medium
T2 – [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)]
TAE buffer – Buffer solution containing a mixture of tris base, acetic acid and EDTA
TE buffer – Buffer solution containing tris and EDTA
WHO – World health organization
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 7
4. Introduction
4.1. The current state of cancer research
Cancer is one of the major causes of death worldwide [1]. According to World Health Organization
(WHO) cancer is the world’s second biggest killer, despite cancer being one of the research fields
where most money has been invested. As a result, many developments have been achieved regarding
the understanding of the pathogenesis of cancer [2]. Furthermore, properties such as unlimited
proliferation capacity, self-sufficiency in growth signals, insensitivity to growth-inhibitory signals,
evasion of programmed cell death (apoptosis), sustained angiogenesis, tissue invasion and metastasis
are considered to be a set of characteristics acquired during tumor development [3,4]. Even though
remarkable progress is being made in understanding the molecular, biochemical and cellular basis of
cancer, such knowledge has not been sufficient to cure the disease efficiently [5,6]. Improvements in
cancer treatment are however being achieved, namely on chemotherapy, which is the treatment of
choice for patients diagnosed in the late stages of locally and advanced widespread metastatic
cancers [4,5,7].
4.2. The hallmarks of chemotherapy
Chemotherapy emerged in the late 1940s and 1950s with the classical alkylating agents, nitrogen
mustard. First used as sulfur mustard gas during the First World War, it was applied to treat patients
with non-Hodgkin’s lymphoma, whose remission only lasted few weeks [5,8,9]. Cyclophosphamide
and chlorambucil were later developed to treat patients with lymphomas, leukaemias and solid
tumors; however these alkylating agents quickly proved inefficient, as tumors became resistant to
these drugs [8,9,10]. After the Second World War, new approaches arose with the synthesis of
antimetabolites, like methotrexate, a folate analogue which, as single agent, exhibited antitumor
activity against epithelial cancer, along with breast, ovarian, bladder, head and neck cancers. Folate
analogues also became the first drugs to induce successfully remission in patients with acute
lymphoblastic leukaemia (ALL), although the effect did not last long [5,8,10,11]. Methotrexate is
still mainly used on patients with ALL as well as other lymphomas. The application and research
done with these compounds have provided an important model for understanding mechanisms of
resistance for other agents. Further research led to the development of new drugs, such as vincristine,
a Vinca alkaloid, which was found to inhibit cell division [8,12,13]. In the 1960s several experiments
have shown that killing cancer cells might be cell cycle dependent, whereas some DNA synthesis
inhibitors (methotrexate) were more effective against rapidly dividing cells. On the other hand,
alkylating drugs, such as cyclophosphamide, that physically damage DNA, could kill cells in any
stage of the cell cycle. Furthermore, it was shown that cytotoxic activity was dose dependent, and it
was also important to use several therapies together to prevent drug resistance [8]. In the late 1960s
nitrogen mustard, vincristine, procarbazine and prednisone – known as the MOPP regimen – was
8 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
successfully used as combination chemotherapy to treat Hodgkin’s and non-Hodgkin’s lymphoma,
proving that drugs were most efficient when used as combination therapy and against tumors of
small volume [8,10,13, 14]. Barnett Rosenberg, a Michigan State University researcher, discovered
in 1964 that cisdiamminedichloroplatinum(II), known as cisplatin, could be used as a successful
agent in the therapy of testicular and ovarian cancer, after having received approval for clinical use
by Food and Drug Administration (FDA) [5,9,15,16] (Figure 1).
Figure 1 - Cisplatin and its analogues (Adapted from [17]).
Despite several successful reported cases using chemotherapy, cancer drug discoveries gained a
reputation of low efficacy and high toxicity risk, leading FDA to approve for marketing only 10%
(29 of 280) of new agents into Phase I (dose-finding) clinical trials in patients in the period of 1975
to 1994 [18]. During those years several new drugs were used in chemotherapy, such as
anthracyclines and epipodophyllotoxins, which are related to the topoisomerase II inhibition, an
enzyme essential for DNA replication, transcription and repair. In the early 1980s chemotherapy
progression decreased, which led the American National Cancer Institute (NCI), division of cancer
treatment, to create an innovative screening system, where panels of 60 human tumor cell lines were
used to test new drugs. This methodology is now widely used by industry, including a rapid
colorimetric cell viability assay, the MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) assay, and high-throughput automated screening [8]. Recently, a new era arose with
targeted-therapy, whereas targets such as growths factors, signaling molecules, cell-cycle proteins,
apoptotic signals and molecules related to angiogenesis became a priority in chemotherapy (Table 1)
[4].
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 9
Table 1 - Drugs of Choice for Different Types of Cancer (Adapted from [5])
Drug Class Principal mechanism of action Example(s) Types of tumors commonlyused for
Alkylating agents To form covalent bonds with DNAand prevent DNA replication
CyclophosphamideCarmustineCisplatin
Non-Hodgkin’s lymphomaBrain gliobastomaOvarian, head, neck, lung andtesticular
Antimetabolites
Folate antagonist
Pyrimidine analogues
Purine analogues
To block or subvert pathways inDNA synthesisTo inhibit dihydrofolate reductase,preventing generation oftetrahydrofolate, thereby interferingwith thymidylate synthesisTo convert into nucleotides andinhibit thymidylate synthesis6-thiol analogues of the endogenous6-OH purine bases that becomeconverted into nucleotides
Methotrexate
Fluorouracil
MercaptopurineThioguanine
Acute lymphocytic leukemia(ALL)
Colorectal and gastric
Acute myelogenous leukemia(AML)
Antitumor antibiotics To interfere with topoisomerase IIaction, inhibiting DNA and RNAsynthesisTo cause fragmentation of DNAchainsTo intercalate in DNA, interfere withRNA polymerase and inhibittranscriptionTo act as an alkylating agent
Doxorubicin
Bleomycin
Dactinomycin
Mitomycin
Osteogenic sarcoma,Hodgkin’s disease, CML, softtissue sarcomaCervical
Wilms’ tumor
LungPlant alkaloidsVinca alkaloids
Podophyllotoxins
Taxoids
Camptothecins
To inhibit mitosis at metaphase bybinding to tubulinTo inhibit DNA synthesis byinterfering with topoisomerase II, andalso mitochondrial functionTo promote the polymerization oftubulin and inhibit the disassembly ofmicrotubulesTo inhibit topoisomerase I and DNAand RNA synthetases, and alsomicrotubule formation
Vincristine,vinblastineEtoposide
Taxol
Irinotecan, topotecan
Lung, Non-Hodgkin’slymphomaLung, Kaposi’s sarcoma
Ovarian, breast and lung
Refractory colorectal andadvanced ovarian
Nowadays better drugs have been developed and knowledge regarding cancer has grown, so that the
new challenge is to design new strategies where targeted-therapy and cytotoxics can be combined in
the most effective manner [8].
4.3. The limit to chemotherapy
Cytotoxic agents of chemotherapy are conditioned by several factors. Drug metabolizing enzymes
and drug transporters are essential in the understanding of the overall process where antitumor
compounds are involved [19,20]. It is also important to understand the side effects of these agents in
order to make them less toxic to the organism without significant cytotoxic activity loss [21]. Thus it
would be crucial to achieve a balance between drug sensitivity and resistance displayed by target
tumor cells to maximize the efficacy and minimize the toxicity of treatment [21,20]. Although the
use of chemotherapy has improved quality of life and prolonged survival for many cancer patients,
10 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
its potential to induce toxicity is also predictable. Several side effects arise, the most common being
gastrointestinal toxicity, alopecia (such as hair loss) and myelosuppression [21]. Such malignancies
are due to the fact that the target of most chemotherapeutic agents consists of rapidly dividing cells,
leading adverse effects into normal cells with a high growth proliferation, such as hair cells. Patients
whose tumor does not respond to the therapeutic agents seem to possess resistance mechanisms,
which are considered to be the major limitation for successful cancer treatment [22,72].
Chemotherapeutic resistance can be classified as ‘acquired’ or ‘intrinsic’. Intrinsic resistance limits
the usefulness of therapeutic agents, while acquired resistance might be related to a resistance
achieved after an initial successful progress, which somehow became powerless [22,23,72]. These
mechanisms of resistance can also be reflected by dose-response relationships: if the dose-response
curve (DCR) has a “shoulder” it will be classified as ‘active’, otherwise ‘passive’ results in a DRC
terminal plateau (saturable passive) or in a decreased DRC slope (non-saturable passive) (Figure 2,
[24]).
Figure 2 - Dose-response relationships and proposed resistance mechanisms (Adapted from [24]).
Examples of active resistance would be efflux pumps, DNA repair systems, anti-apoptotic factors,
etc. Passive resistance would include drug uptake or activating systems, proapoptotic factors or
factors that are part of the apoptotic cascade, cells in a sensitive phase of the cell cycle, etc.
Furthermore, several factors may contribute to resistance: blood flow and drug delivery, extracellular
environment, drug efflux, drug uptake, drug detoxification, drug binding, DNA repair, decreased
DNA mismatch repair, reduced apoptotic response, apoptosis inhibitors, etc [22,24,25]. Although
these mechanisms can act individually, they can also act synergistically, resulting in multidrug
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 11
resistance (MDR) [26,72]. To diminish side-effects and reduce propensity to induce drug resistance,
novel antitumor compounds have been designed in order to overpass chemotherapy limitations.
4.4. Medicinal inorganic chemistry
Metals are essential to many life-related processes and their use as therapeutic agents can be traced
back to 2500 BC by the ancient Chinese, who have attributed medicinal proprieties to gold.
Paracelsus (1493-1541), considered to be the ‘true father of modern metallotherapy’, used several
heavy metals to treat patients with different malignancies, such as cancer [9,27,28]. Since then
several reports have described the use of metals in medicine. As already mentioned, the First World
War has changed the era of chemotherapy with the cytotoxic action of nitrogen mustards established
years after, and further investigation led to the development of efficient antitumor drugs, such as
cisplatin and its derivatives [8,9,15]. Nowadays they continue to be the most frequently used
cytostatic drugs worldwide, constituting the main component of combination chemotherapy for the
treatment of solid carcinomas [29]. This knowledge led to the development of other metal
compounds with cytostatic activity, the groups 13 to 15 of the periodic table and certain transition
metals of groups 4 to 11 being very active [9]. Metals can bind to organic fragments affording
organometallic compounds, whose chemistry offers a rich field in medicine with a high potential to
develop effective antitumor agents [30]. Furthermore, the limited selectivity and toxicity of metals
alone can be overcome by the formation of organometallic compounds. Features such as the metal
coordination numbers, the coordination geometries, the redox potential and the thermodynamic and
kinetic properties have to be considered on the design of organometallic agents suitable for
successful interaction with biological molecules [15,29]. This variety of options provides unlimited
possibilities of combination that results in an extensive spectrum of mechanisms open to
organometallic compounds.
Although platinum complexes are widely used for the treatment of cancer, the appearance of drug
resistance among the drug toxicity induced and its side-effects have led to the development of other
non-platinum drugs, metal-based compounds, in order to overcome platinum compounds limitations,
namely ruthenium, vanadium and molybdenum compounds [9,15,29,30].
Molybdenum is an essential trace element commonly used in the cell as cofactor by important
enzymes (e.g. xanthine oxidase/dehydrogenase). In 1979, Köpf and Köpf-Maier reported the
antitumor action of several metal-based complexes with Mo [31]. Since then a number of
molybdenum containing molecules have been described to display cancerostatic activity. These
include Na2MoO4, heteropolyacid Mo salts, polyoxomolybdenum complexes, Mo complexes bound
to small carborane ligands, and chiral octahedral complexes [32,33,34,35]. Portuguese investigators
in 2005 studied several molybdenum(II) compounds, concluding that they were very efficient
cytotoxic agents against six cell lines, and filed a patent [36]. The mechanisms of action of most of
the organometallic complexes of molybdenum are far from being understood. Since cell growth is
12 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
slowed down by those compounds, it seems conceivable that the inhibitory activity might in some
way be related to DNA damage. This can be due to a direct action on the DNA molecule (eg.
intercalation in the double helix) or by the oxidative action of oxygen free radicals generated by the
chemical agents (chapter 4.5).
4.5. Mode of action of metal anticancer compounds
Metal compounds have a high propensity to bind and to interact with many important biological
molecules, e.g. DNA, which is considered to be the primary intracellular target of the antitumor
agents [17,27,28,37]. On the other hand, organometallic compounds can act as enzyme inhibitors or
modulators blocking substrate interaction [38]. Other possible mechanism can be the generation of
endogenous reactive oxygen species (ROS), which can directly or indirectly induce oxidative DNA
damage [38,39]. Therefore covalent and noncovalent interactions can be considered in the interaction
of the organometallic compounds with DNA. Thus, covalent interactions lead to complete inhibition
of DNA processes and they usually are irreversible, contrarily to noncovalent bindings which are
reversible [40,41]. One example of a covalent binder is the chemotherapeutic agent cisplatin and its
analogues (Figure 1), which form covalent bonds between platinum and the nitrogen atom of some
of the DNA base pairs [17]. Additionally three major noncovalent interactions can be found: minor
groove binding, major groove binding and intercalation. However, one complex can participate in
more than one mode of interaction, as actinomycin D, a well-known transcription inhibitor [41,42].
These metal compounds-DNA interactions, ultimately, trigger programmed cell death [27,37].
A classification of metal anticancer compounds based on their mode of action has been suggested
[43]. This classification relies on the metal compounds, rather than the nature of the compound
targets (e.g. proteins, DNA, enzymes, transporters, cellular transduction pathways, etc.), since the
knowledge about their interaction is poorly understood and unreliable. Thus five classes were
created:
1) The metal as a functional role
2) The metal as a structural role
3) The metal as a carrier of ligands
4) The metal as a catalyst
5) The metal as a photosensitizer
One of the most successful metals antitumor compounds, cisplatin, with an antitumor activity
associated with interaction with DNA, forming intrastand crosslinks to adjacent guanosine residues,
leading to cell apoptosis [17,28,37], belongs to the first class. The cytotoxic activity of compounds
belonging to this class depends essentially of the thermodynamic and kinetic parameters of the metal
center and the nature of the ligand. The main disadvantage of these compounds is related to the high
toxicity caused by uncontrolled reactivity with molecules other than their targets. The second class
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 13
compounds should not bind directly to the biological molecules (e.g. non-covalent interactions with
DNA) and be less toxic than functional compounds [43]. One of the most successful drugs belonging
to this class is the antimalarial ferroquine. Several other platinum compounds can intercalate into
DNA rather than forming covalent adducts as cisplatin does. Compounds of the third class carry a
ligand, which is delivered in vivo. The ligand is protected by the metal until its delivery. The metal as
a catalyst is associated to the generation of ROS that cause cell damage [28,39]. Several metal
compounds need to be primarily induced to be antitumor active (e.g. ruthenium complexes with
polypyridine ligands) [17,43]. Although several classes have been created, all the compounds used in
clinical practice belong to the first class, and no compounds, from any of the others classes, are
expected to enter in clinical use hereafter, even though a lot of research in this field is being made in
order to develop new drugs [43].
4.6. Experimental strategies
Several approaches can be used in order to study possible mechanisms by which antitumor metal
compounds inhibit cell proliferation, particularly by interactions with DNA [44,45]. Gel
electrophoresis studies constitute a rapidly and economical mode to evaluate the effect of metal
complexes on DNA tertiary structure. Absorption titration and circular dichroism are universally
used to examine the binding mode of DNA-complexes [42,46]. The analysis of the transitions in the
tertiary structure of plasmid DNA induced by metals complex constitutes a complementary approach
to the DNA-complex interaction study and it can be achieved by using atomic force microscopy
(AFM) [47].
4.7. Electronic absorption titration
Electronic absorption titration spectroscopy is one of the most useful techniques and widely used in
DNA binding studies [44]. The mode of interaction can be evaluated by the changes on the
absorption spectra. Thus, if the complex intercalates in DNA, a red-shift (bathochromism) along with
a decrease in the intensity of the complex spectral band (hypochromism) can be observed. The
existence of a bathochromism is also indicative of the stabilization of DNA duplex [48,49]. On the
other hand, the increase in the intensity of the complex absorption band (hyperchromism) is related
to the damage of the secondary structure of DNA. Then, hyperchromism and hypochromism are
related to spectral features of the double helix structure of DNA, where hypochromism means that
the DNA-binding mode of complex may be due to electrostatic effect or intercalation and
hyperchromism is related to the loss of secondary structure of DNA.
The complex-DNA binding strength can be evaluated by the intrinsic binding constant, K, which
represents the binding constant per DNA base pair [50]. In order to determine the binding constant,
the neighbor exclusion model was primarily used by Benesi and Hildebrand [76]. It was limited to
molecules that occupy only one binding site, and does not correspond to most biological ligands
14 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
which interact with more than one base pair in the DNA [41]. Schemechel and Crothers [77] adapted
the previous model and Wolf et al applied it successfully to determine K according to the following
equation (Eq 1) [48,50,52]:
[DNA]( a f) = [DNA]( b f)+ ( b f) (Eq 1)
where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficients a, f
and b correspond to Absorbance observed/[complex], the extinction coefficient for the free complex
and the extinction coefficient for the free complex in the fully bound form, respectively. K is given
by the ratio of slope to intercept in plots of [DNA]/(a-f) versus [DNA].
4.8. Circular dichroism
Circular dichroism (CD) results from the difference between left- and right-handed polarized
components of the incident light absorbed by chiral molecules. Those components are absorbed
differently by the sample, leading to a difference in the absorption coefficients: = left-right (M-1
cm-1). The signal is measured in units of cm2g-1 although it is widely used as ellipticity () [53]. CD
can be used to follow rapidly and efficiently DNA conformational transitions, which constitute an
important tool to evaluate with high selectively the behavior of complexes that recognize DNA
structures or sequences [49,53]. For these reasons CD represents a further source of useful
information in the study of the interactions of metals complexes with DNA. The most common DNA
conformation observed is the B-form, which is characterized by positive bands at about 260-280 nm
and a negative band at around 245 nm. Since the base pairs of DNA are perpendicular to the double-
helix axis in the B-form, the peak intensities are relatively small, considering the weak chirality of
the molecule [49,54,55]. Nevertheless, CD being an extremely sensitive method, small
conformational changes (e.g. due to the interaction with metal complexes) can be measured
efficiently [53]. The intercalation of the complex in DNA enhances the intensities of both the bands
stabilizing the DNA conformation, as is commonly observed for the classical intercalators [46]. On
the other hand, simple groove binding and electrostatic interactions would lead to no perturbations or
minimal perturbations in both bands of B-DNA, because these binding modes do not influence the
secondary structure of DNA [45,49,55].
4.9. Atomic force microscopy (AFM)
Atomic force microscopy has become a successful technique to investigate DNA structure and
dynamics at very high resolution [56]. AFM is an ultra-high resolution microscopic technique that
does not require complicated sample preparation or any other complex treatment [47]. It can be used
to study DNA tertiary structure as well as the influence of factors that induce conformational
transitions, such as intercalator complexes (e.g. ethidium bromide) [57]. Recent publications have
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 15
shown the potential of AFM to investigate the interactions of DNA with anti-tumor agents of metal
complexes (e.g. cisplatin), where conformational structure changes were evaluated [58]. Furthermore
studies carried out in order to study the effect of ethidium bromide, a classical DNA intercalator on
DNA plasmid has led to the definition of some schematic structures (Figure 3) [59]. Four structures
were considered: predominantly relaxed, toroidally supercoiled, mixed toroidal and plectonemic
supercoils, and complete plectonemic supercoiling. Hence, with the interaction of metal complexes
intercalators the DNA plasmid would become increasingly supercoiled.
Figure 3 – Drawing schemes of the transitions in plasmid DNA tertiary structure in
response to an intercalator agent: (a) predominantly relaxed; (b) toroidally
supercoiled; (c) mixed toroidal and plectonemic supercoils; (d) complete plectonemic
supercoiling (adapted from [59]).
16 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 17
5. Aims
Several metal-based complexes with molybdenum were reported as having antitumor and
cancerostatic activity [31,36]. The mechanisms of action of most of the organometallic complexes of
molybdenum are far from being understood, although they might in some way be related to DNA
damage [43].
The present study aimed at evaluating the antitumor activity of two molybdenum complexes, B1
([Mo(3-C3H5)Br(CO)2(1,10-phen)]) and T2 ([Mo(3-C3H5)CF3SO3(CO)2(2,2’-bpy)]), and study
their mechanism of action against cancer cell lines.
To evaluate the cytotoxic activity of the complexes a metabolic activity test (MTT) was used.
Octanol/water partition assays and the determination of intracellular molybdenum were carried out in
order to understand how easily the complexes could pass through the membrane and where may start
the process that triggers cell death. Several other studies regarding the interactions between the
complexes and DNA were also performed, namely absorption titration and circular dichroism.
Complementary approaches, such as gel electrophoresis studies and atomic force microscopy, were
used to contribute to the study of the binding mode of DNA-complexes.
Understanding the mechanism of action of B1 and T2 will potentially constitute a valuable tool in
cancer chemotherapy in order to reduce drug resistance of the chemotherapeutic agents used
nowadays and to overcome some chemotherapy limitations. An overview of the work plan is
illustrated in Figure 4.
18 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 4 - Schematic illustration of the overview of the work plan. Mo(II) complexes were tested and acted as potentcytotoxic drugs, interacting with DNA in vitro. Do they enter the cell and directly damage DNA to inhibit cell growth?
18 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 4 - Schematic illustration of the overview of the work plan. Mo(II) complexes were tested and acted as potentcytotoxic drugs, interacting with DNA in vitro. Do they enter the cell and directly damage DNA to inhibit cell growth?
18 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 4 - Schematic illustration of the overview of the work plan. Mo(II) complexes were tested and acted as potentcytotoxic drugs, interacting with DNA in vitro. Do they enter the cell and directly damage DNA to inhibit cell growth?
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 19
6. Experimental
6.1. Instrumentation and materials
Commercially available reagents and all solvents were purchased from standard chemical suppliers.
Octanol was purchased from Riedel-de Haën, Germany. The RPMI 1640 cell culture medium, fetal
bovine serum (FBS) were purchased from LONZA Co. MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) was purchased from Sigma Chemical Co, USA. Calf thymus DNA
(ctDNA) was purchased from Sigma Chemical Co. Ltd. and a stock solution was prepared by
dilution in a buffer solution (50 mM NaCl/5 mM Tris-HCl, pH 7.1) followed by stirring at 4 ºC for
two days. This solution was stored at 4 ºC. The stock solution of ctDNA gave a ratio of UV
absorbance at 260 and 280 nm (A260/A280) > 1.8, indicating that the DNA was sufficiently free of
protein contamination [60]. The DNA concentration was determined by the UV absorbance at 260
nm after 1:10 dilution using = 6600 M-1 cm-1 [61]. MTT was dissolved (5 mg/ml) in phosphate
buffer saline pH 7.2.
Infrared spectra were measured on a Mattson 7000 FT spectrometer. Samples were run as KBr
pellets. NMR spectra were recorded on a Bruker Avance-400 spectrometer in CDCl3 or deuterated
DMSO. Elemental analyses were carried out at the University of Vigo, Spain. UV-Vis spectra were
recorded on a Shimadzu UV-2450 equipped with a Peltier cell for temperature control.
6.2. Synthesis of molybdenum(II) complexes
6.2.1. [Mo(3-C3H5)(CF3SO3)(CO)2(2,2’-bpy)] (T2)
Thallium triflate (TlCF3SO3) (0.353 g, 1 mmol) was added to a solution of [MoBr(3-
C3H5)(CO)2(2,2’-bipyridyl)] (0.429 g, 1 mmol) in acetonitrile (20 ml), and the mixture was refluxed
for 5 hours. A white solid of TlBr was formed and filtered with celite. The solid was washed 3 times
with acetonitrile. The filtrate was evaporated and the solid residue dissolved in dichloromethane.
Addition of n-hexane resulted in the formation of red crystals after a few days [73].
Yield: 72% (0.359 g)
IR (KBr disc) (cm-1): 3436; 3069; 1947; 1863; 1602; 1573; 1495; 1474; 1441; 1389; 1312; 1302;
1287; 1237; 1219; 1174; 1158; 1127; 1109; 1077; 1034; 930; 795; 764; 734; 657; 650; 630; 577;
570; 516; 504; 439; 418.
1H NMR (400 MHz, DMSO-d6): 1.63 (d, Hanti); 3.76 (d, Hsyn); 4.06 (m, Hmeso); 7.69 (t, H3/H6); 8.08
(t, H2/H7); 8.16 (d, H4/H5); 9.2 (s, H1/H8).
20 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
6.2.2. [Mo(3-C3H5)(Br)(CO)2(1,10-phenanthroline)] (B1)
A solution of 1,10-phenantroline (2 mmol, 0.3965 g) was added to a stirring solution of (3-
C3H5)(CO)2(MeCN)2] (0.7100 g, 2 mmol) in dicholoromethane (20 ml) and the mixture was kept
stirring overnight. The red precipitate formed was washed with dichloromethane (10 ml), diethyl
ether (10 ml) and dried under vacuum.
Yield: 87% (0.7875g)
IR (KBr disc) (cm-1): 1926; 1863; 1832; 1626; 1463, 1426;
1H NMR (400 MHz, CDCl3): 1.51 (d, Hanti); 2.86 (m, Hmeso); 3.14 (d, Hsyn); 4.15 (m Hmeso); 7.82 (m,
H7); 7.96 (s, H4/H5); 8.01 (s, H2); 8.5 (m, H3/H6); 9.23 (t, H8); 9.4 (s, H1);
13C NMR (400 MHz, CDCl3): 53 (Csyn); 54 (Canti); 62 (Cmeso); 124 (C7); 127 (C2/C4/C5); 136 (C3/C6); 151 (C8).
6.3. Cell cultures
Different cell lines were used in order to study intracellular processes, as the cytotoxic activity of the
molybdenum complexes synthesized (Chapter 6.2), as well as their mechanism of action. Thus
HeLa (cervical carcinoma), MCF-7 (breast carcinoma) and hTERT-RPE1 (human telomerase reverse
transcriptase – retinal carcinoma) were maintained in RPMI 1640 supplemented with 10% fetal
bovine serum (FBS), 200 U/ml penicillin, 100 µg/ml streptomycin and 0.3 g/ml L-glutamine in a
humidified atmosphere of 95% air /5% CO2 at 37º C. All cells culture procedures were carried out in
a culture cabinet under sterile conditions, as well as all the material used sterilely.
6.4. Subculture of cells
In order to maintain the cells in a healthy and viable state they were subcultured, process also known
as passaging. Thus the adherent cells were harvested enzymatically from culture dishes occupied 80
to 90% of the surface with cells (confluent state). Thus, trypsin was used to detach the monolayer of
adherent cells and, after diluted in phosphate buffered saline (PBS), it was inactivated by the addition
of medium containing serum. The suspension of cells was then distributed to new culture dishes and
incubated in a humidified atmosphere of 95% air /5% CO2 at 37º C.
6.5. Cell quantification
Seed with the appropriate seeding density is crucial to reach the optimum growth. One way to
quantify cells is by using a haemocytometer, which is both simple and cheap. It contains 9 large
squares and inside it has 16 small squares. Each large square measures 1 mm x 1 mm and is 0.1 mm
deep (Figure 5).
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 21
Figure 5 – Haemocytometer (adapted from [62])
Furthermore each square has a volume of 0.1 mm3 and from this it is possible to determine the
concentration of cells and the total number of cells per cubic centimeter. Then cells are counted in
each large square and the average of cells is calculated to increase the accuracy. The count can be
converted to the number of cells per mL of suspension according to the following equation (Eq 2).
Cells (cells/mL) = Average of the number of cells counted × 10 (Eq 2)
6.6. Cryopreservation of cells
One way to preserve cells is by freezing stocks in liquid nitrogen. This process, known as
cryopreservation, grants a renewable source of cells for later use. To avoid the formation of ice
crystals inside the cell and changes in pH, DMSO is used to lower the freezing point. In order to
accomplish a successful cell cryopreservation a freezing chamber (‘Mr Frosty’) was used to cool
down slowly from room temperature to -80 ºC at a rate of 1-3 ºC per minute. Cells were then
harvested and resuspended in a solution of 60% medium and 40% of serum, from which was added
DMSO (10%) and stored in vials in a ‘Mr Frosty’ cryo freezing container. After that it was placed in
a -80 ºC freezer at overnight and hereafter transferred to liquid nitrogen.
6.7. Resuscitation of frozen cells
In order to revive frozen stocks from cryogenic vials stored in liquid nitrogen it is necessary to warm
up the vials at 37 ºC for 1-2 min, avoiding that cells warm up to 37 ºC, otherwise they may rapidly
die. The cell suspension is then added to fresh growth medium pre-warmed and placed in a culture
flask, which is incubated in a humidified atmosphere of 95% air /5% CO2 at 37º C.
6.8. Cytotoxic activity assay in vitro
In order to determine the cell viability towards molybdenum complexes, cytotoxic activity assay was
performed by the MTT (3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method
22 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
previously described with some modifications [63]. Exponentially growing cells were seeded at a
density of approximately 4x105 cells/ml, in a 96-well flat-bottomed microplate, and incubated for 48
h in an atmosphere of 5% CO2/95% air at 37ºC. Then the cells were treated with the complexes and
incubated for 48 h. Compounds were dissolved in the culture medium with 0.5% DMSO and tested
in concentrations ranging from 1 to 1000 M. Control wells contained supplemented media with
0.5% DMSO. After incubation time MTT solution (100 L, 0,5 mg/mL) was added into each well,
and incubated for 2 hours at 37ºC h in an atmosphere of 5% CO2/95% air. After medium removal,
100 L DMSO was added to dissolve the formazan crystals. The optical density was measured at
570 nm using a 96-well multiscanner autoreader. The IC50 values (concentration that caused 50%
growth inhibition) were calculated by non-linear regression analysis. For the kinetic studies, cell
cultures were exposed to different concentrations of compounds for different periods of time (1, 2,
24, and 48 h), after which the medium with the drug was removed and was replaced by fresh
medium. Each experiment included ten replicates for the different concentrations of complexes and
results represent at least 3 independent experiments. UV-Vis and mass spectrometry spectra of
solutions with appropriate concentrations were measured during this period and showed no
decomposition of the complexes being studied (Annex).
6.9. Octanol/water partition coefficient
Water-saturated octanol and octanol-saturated water were prepared by shaking equal volumes of
octanol and water for 5 hours and allowing the mixture to separate into the respective phases for 24
hours. Solutions of molybdenum complexes (20 M) were prepared in water-saturated octanol and
their absorbance was analyzed by UV spectrophotometry. Three and six milliliters of complex
solution were then added to 40 ml of octanol-saturated water. These solutions were shaken
vigorously for 2 hours. The aqueous phase was separated ensuring that there was no contamination
from the octanol phase, and each of these solutions was analyzed by UV spectrophotometry to obtain
the absorbance of the compounds.
6.10. Conductimetry
The specific conductivity of molybdenum complexes solutions was measured at 25ºC using a
Radiometer Copenhagen – Meterlab CMD 230 conductimeter. The relative uncertainty in
determining the specific conductivity of the compounds solution was within 0.5%. The repeatability
of the conductivity measurement, estimated from two successive runs, was about ± 3. The
conductance reading was checked every 20 s until it reached a steady value.
6.11. Cellular molybdenum uptake
The protocol used was as previously described with some modifications [64]. HeLa cells were
seeded in 100 mm dishes at 4x105 cells/ml and incubated at 37 ºC in an atmosphere of 5% CO2/95%
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 23
air for 48 h. The culture medium was removed and replaced with medium containing the
molybdenum complex at a concentration of 0, 10, 50 and 100 M for 48 h. Following treatment, the
cell monolayer was scraped off the culture dishes and the molybdenum content was determined by
inductively coupled plasma mass spectrometry (ICP-MS) by standard protocol at the University of
Vigo, Spain.
Molybdenum content of cytosolic and nuclear extracts was also analyzed. Cells were washed twice
with PBS buffer and lysed with cytosolic lysis buffer (HEPES 50 mM, pH 7.2, EDTA 2 mM, NaCl
10 mM, sucrose 250 mM, DTT 2 mM). Cells were then scraped up and centrifuged (3000 g, 4 min,
4ºC) (supernatant - cytosolic fraction). The pellet was washed with cytosolic buffer and
ressuspended with nuclear lysis buffer (HEPES 50 mM, pH 7.2, EDTA 2 mM, NaCl 400 mM,
glycerol 20% (v/v), DTT 2 mM) (pellet - nuclear fraction). The molybdenum content of both
fractions was determined by ICP-MS at the University of Vigo, Spain.
6.12. DNA binding studies
6.12.1.Electronic absorption titration
Calf thymus DNA (ct DNA) solutions of various concentrations (0 – 100 M) were added to 20 M
buffered solutions (5 mM Tris, 50 mM NaCl, pH 7.2) of the metal complexes. Absorption spectra
were recorded after equilibration at 37.0 ºC for 10 min. The intrinsic binding constant, K, was
determined according to Eq 1 (Chapter 4.7).
6.12.2.Circular dichroism
The CD spectra of ctDNA (500 M) in the absence and presence of molybdenum complexes at
various concentrations (0, 10, 25, 50, 75, 100, 250 M) were recorded on a Jasco J810
spectropolarimeter at 37ºC (Julabo F25 temperature control unit). The region of wavelength between
220-360 nm was scanned for each sample using a 1 mm path quartz cell, and the result was displayed
in millidegreed (deg).
6.12.3.Gel electrophoresis studies
E. coli. Bacteria were transformed with pYES2 DNA plasmid (5856 nucleotides, multiple cloning
site, ampicillin resistance gene) by prior treatment with Ca2+ at 4ºC in order to become competent.
DNA was added to the suspension of competent cells and taken up during a brief increase in
temperature (heat shock). After a brief incubation to allow expression of the antibiotic resistance
genes the cells were plated onto medium containing the antibiotic, ampicillin. The plasmid was then
isolated from the bacteria using a kit from GE Healthcare and stored at 4ºC. The pYES2 was
incubated with various concentrations (0, 10, 50 and 100 M) of molybdenum complexes (B1 and
T2) in TE buffer (Tris HCl 10 mM, EDTA 1 mM, pH 8.0). DNA digested with Hind III and plasmid
24 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
without complexes were used as marker and control, respectively. Samples were incubated for 10
min. After incubation samples were run on an agarose gel (1% in TEA Buffer [Tris acetate 40 mM,
EDTA 1 mM, pH 8.0]) stained with ethidium bromide, and imaged with a common transilluminator.
6.12.4.Atomic force microscopy
Squares of mica were stuck to steel discs ready for mounting samples onto the AFM instrument. The
mica squares were cleaved with adhesive tape immediately prior to use. A method involving divalent
cations to bridge between the negatively charged mica substrate and DNA backbone was used. The
plasmid, pYES2, was diluted in a solution of Tris-HCl (20 mM, pH 7.5), MgCl2 (5 mM), and B1 (20
M). Solution of plasmid diluted in Tris-HCl and MgCl2 was used as control. The solution was then
spotted directly onto freshly cleaved mica and, following a 2 min incubation period, the mica was
gently rinsed with H2O and blown dry with compressed N2.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 25
Figure 7 - In vitro cytotoxic assays for T2 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with T2 (right).
7. Results and discussion
7.1. Cytotoxic activity assay in vitro
To evaluate the potential antiproliferative activity of the molybdenum complexes T1 and B2 (Figure
6), human cervical cancer cell line (HeLa), human breast cancer cell line (MCF-7) [74], and human
telomerase reverse transcriptase – retinal epithelial cells (RPE) were incubated for 48 hours with
varying concentrations of compounds and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was used .
The well known MTT assay measures cell viability in terms of metabolic turnover, as indicated by
the oxidation of MTT to purple formazan by mitochondria. The relation between cell viability and
compound concentration obtained for HeLa and RPE cells treated with compound T2 and B1 was
determined (Figure 7 - 10, left).
0
50
100
0 1 10 25 50 100
% C
ell V
iabi
lity
[T2] ( M)
Figure 6 - Schematic structure of B1 and T2.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 25
Figure 7 - In vitro cytotoxic assays for T2 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with T2 (right).
7. Results and discussion
7.1. Cytotoxic activity assay in vitro
To evaluate the potential antiproliferative activity of the molybdenum complexes T1 and B2 (Figure
6), human cervical cancer cell line (HeLa), human breast cancer cell line (MCF-7) [74], and human
telomerase reverse transcriptase – retinal epithelial cells (RPE) were incubated for 48 hours with
varying concentrations of compounds and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was used .
The well known MTT assay measures cell viability in terms of metabolic turnover, as indicated by
the oxidation of MTT to purple formazan by mitochondria. The relation between cell viability and
compound concentration obtained for HeLa and RPE cells treated with compound T2 and B1 was
determined (Figure 7 - 10, left).
100 250 500 1000[T2] ( M)
Figure 6 - Schematic structure of B1 and T2.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 25
Figure 7 - In vitro cytotoxic assays for T2 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with T2 (right).
7. Results and discussion
7.1. Cytotoxic activity assay in vitro
To evaluate the potential antiproliferative activity of the molybdenum complexes T1 and B2 (Figure
6), human cervical cancer cell line (HeLa), human breast cancer cell line (MCF-7) [74], and human
telomerase reverse transcriptase – retinal epithelial cells (RPE) were incubated for 48 hours with
varying concentrations of compounds and the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was used .
The well known MTT assay measures cell viability in terms of metabolic turnover, as indicated by
the oxidation of MTT to purple formazan by mitochondria. The relation between cell viability and
compound concentration obtained for HeLa and RPE cells treated with compound T2 and B1 was
determined (Figure 7 - 10, left).
Figure 6 - Schematic structure of B1 and T2.
26 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 8 - In vitro cytotoxic assays for B1 against HeLa cells (left). Dose-response curve obtained by nonlinearregression analysis for HeLa cells treated with B1 (right).
Figure 9 - In vitro cytotoxic assays for B1 against RPE cells (left). Dose-response curve obtained by nonlinear regressionanalysis for RPE cells treated with B1 (right).
From the relation between cell viability and compound concentration, dose-response curves obtained
by non linear regression analysis for HeLa and RPE cell lines treated with the complexes were done
in order to determine the IC50 value (final concentration ≤0.5% DMSO), which corresponds to the
concentration of compound required to inhibit cell proliferation by 50% (Figure 7-10, right).
0
50
100
0 1 10 25 50 100 250 500 1000
% C
ell V
iabi
lity
[B1] ( M)
0
50
100
0 1 2.5 5 10 20 30 50 100
% C
ell V
iabi
lity
[B1] ( M)
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 27
Figure 10 - In vitro cytotoxic assays for T2 against RPE cells (left). Dose-response curve obtained by nonlinearregression analysis for RPE cells treated with T2 (right).
The IC50 values of B1 and T2 against HeLa, MCF-7 [74], and RPE were then calculated from the
dose-response curves (Table 2).
Table 2 - In vitro cytotoxicity assays for molybdenum complexes T2 and B1against HeLa, MCF-7 [74], and RPE cells (data are mean ±SD of three replicateseach).
CompoundsIC50 (M)
HeLa MCF-7 RPE
T2 23.7 ± 0.01 44.5 ± 0.7 13.1 ± 2.4
B1 5.1 ± 1.0 8.9 ± 0.5 0.7 ± 0.1
As demonstrated by the IC50 values (Table 2), the molybdenum complexes showed to be very
effective as cytotoxic agents against the in vitro growth of various cancer cell lines. B1 exhibited
activities with IC50 values ranging from 1 to 9 M, approximately, and for T2 ranging from 13 to 46
M, approximately, for the cell lines studied. The complexes exhibited significant potency against
RPE cells, but less toxic toward MCF-7. Thus, it is clear that the RPE cells are the most sensitive,
whereas MCF-7 cells turned out to be the most resistant against the cytotoxic agents. It is also
evident that B1 is more effective than T2. This interesting behavior may be related to the ligand,
1,10-phenanthroline, which already has been shown to be very effective as cytotoxic agent [44].
Furthermore, both the complexes achieved almost total inhibition (<10% cell viability) at the
maximum concentration tested (100 M) for all cell lines. In fact, B1 values are comparable to
0
50
100
0 1 2.5 5 10 20 30 50 100
% C
ell V
iabi
lity
[T2] ( M)
28 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 11 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with B1 for 1, 2, 24,and 48 hours (left). In vitro cytotoxic activity for B1 against HeLa cells at 1, 2, 24, and 48 hours (right).
cisplatin, which has IC50 values below 10 M with a range of cell lines [65,66]. For further
comparison between these two complexes and the two ligands, complementary studies have been
performed.
The effect of the incubation time of cells with the compounds was also investigated. Thus dose-
response curves (Figure 11-12, left) and cytotoxic activity (Figure 11-12, right) for the treatment of
HeLa cells with the compounds for 1, 2, 24, and 48 hours were determined.
These results indicate that the molybdenum complexes inhibit cell proliferation in a duration-
dependent manner. The IC50 value of both complexes decrease as the exposure time increases,
although the difference between 24 h and 48 h does not seem relevant. Indeed, it seems that some
time is needed in order to achieve the maximum cytotoxic effect, which may be related to the
formation of new species from the molybdenum complexes. In fact, observing the MS spectra for
both the complexes (Annex, Figure 23-30), a cationic complex is formed
([(Mo)(C12H8N2)(C3H5)(O)2]+ , for B1 and [(Mo)(C10H8N2)(O)2]+ for T2). For B1 it is formed after
two hours of incubation time and for T2 is formed immediately. Thus, it is reasonable to admit that
this cationic form may be related to the cytotoxic effect of both the complexes, since it appears after
24 hours with a significative relative abundance, as the cytotoxic activity starts to achieve the
maximum effect. This relation can be seen clearly with B1, which seems to inflict more cytotoxic
effect as the cationic form is created. T2 seems to form more of this cationic complex at 24 hours,
the maximum effect; after that, no relative abundance increase is observed. This may be related to
the stabilizing landing observed for the cytotoxic effect after 24 hours (Figure 12). The increase of
the cationic form may be the key to achieve the maximum cytotoxic effect.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 29
Figure 13 - Mean log octanol/water partition coefficients (Log P) ofthe molybdenum compounds.
Figure 12 - Dose-response curve obtained by nonlinear regression analysis for HeLa cells treated with T2 for 1, 2, 24,and 48 hours (left). In vitro cytotoxic activity for T2 against HeLa cells at 1, 2, 24, and 48 hours (right).
7.2. Octanol/water partition coefficient
Partition coefficients, P, determinations were carried out to estimate how easily the compounds T2
and B1 are able to pass through a biological membrane. The P measurements are based on the
difference in solubility that a given compound exhibits in an aqueous versus a hydrophobic medium
[67]. Complex B1 presented a
hydrophobic behavior (log P = 0.760 ±
0.039). For complex T2 it was not
possible to determine a conclusive value
of log P, nevertheless T2 seems to be
hydrophobic; however, less than B1
(Figure 13). In order to study further the
behavior of the complexes in solution,
the determination of the conductance
were carried out (Chapter 4.3).0
1/2
1
T2 B1
LogP
30 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 15 - Specific conductivity of T2 in water solution
7.3. Conductimetry
Conductimetry is the measurement of the conductance of a solution, where conductance is the ability
of a material to conduct electric current. It is expressed in siemens (S), although since conductivity
values are affected by electrode dimensions, it is commonly expressed in specific conductivity units
(S.cm-1).
This assay contributes to the understanding of the
metal complexes capability for ionizing in
solution, and it functions as a complement of the
octanol/water partition coefficient determination,
telling to interpret the results observed. The
conductance was determined by the measurement
of the current through the solution subject to a
potential applied between two plates. The
measurements made for both complexes were
done in different conditions; for B1 was used
water with 0.5% of DMSO (amount necessary to
complete solubilization of the complex), and for T2 was used only water (at low concentrations it
can be dissolved only in water).
The conductance measured for the water solution was significantly lower than the conductance
measured for the T2 solution, which means that T2 leads to the formation of ionic specimens that
makes the T2 solution more conductive than the
water solution (Figure 15). In fact T2 has a weak
bond to triflate ion, which may lead to T2 loss of
triflate when in contact with oxygen (from water)
originating probably the cationic
[(Mo)(C10H8N2)(O)2]+. As a result, the absorbance
of T2 fraction in octanol solution becomes lower
when ionic specimens are created (Figure 13),
which means that the log P determined does not
correspond effectively to a log P, but a log D (of
distribution). Relatively to B1, and opposite to
the observed for T2, the conductance measured
for the water solution with 0.5% of DMSO was significantly higher than the conductance measured
for the B1 solution (Figure 14). This indicates that B1 has a low tendency to form ionic specimens,
as suspected from inert Mo-Br bond. This effect is consistent with the result obtained for B1 partition
coefficient (Figure 13). Thus, the hypothesis that B1 has a higher hydrophobic behavior than T2 can
now be confirmed.
*P<0.001
0
2
4
Water T2
Spec
ific
Con
dutiv
ity(
S.cm
-1)
*P<0.001
0
13
25
Water B1
Spec
ific
Cond
utiv
ity (m
S.cm
-1)
Figure 14 - Specific conductivity of B1 in 0,5% DMSO inwater solution.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 31
Figure 16 - Comparison of the intracellular molybdenum concentrationin HeLa cells after 48 h of exposure to compounds T2 and B1.
7.4. Cellular molybdenum uptake
In order to understand the beginning of the processes that trigger cell death, cellular molybdenum
content was determined after treatment of HeLa cells with T2 and B1 (Figure 16). The correlation of
the Mo cell uptake with the cytotoxicity previously determined (Chapter 7.1) provides useful
information regarding the intracellular cytotoxic potential and mechanism of action of the
complexes. Each compound was tested
at three different concentrations (10,
50 and 100 M), based on the
cytotoxicity data reported previously.
The intracellular molybdenum levels
of the cells treated with B1 revealed a
significant increase and a dose
dependent relation, contrasting to the
cells treated with T2. However, this
single analysis does not seem to be
enough to conclude about the relation
between the intracellular molybdenum
and T2 concentration, so more studies
are needed in order to avoid
significative experimental errors.
To understand better if the complexes can enter the cell and reach the nucleus, nuclear and cytosolic
fractions of cells pre-incubated with compounds for 48 hours were analyzed (Figure 17). Noted that
during the cell fractionation the plasma membrane is released as vesicles [75], and the molybdenum
content from the plasmatic membrane will be measured in the cytosolic fraction, leaving the nuclear
fraction with low plasmatic membrane contamination.
0
25
50
Control 10 50 100
Intr
acel
lula
r M
o×
104
(m
ol/m
illio
n ce
lls)
Concentration of Mo complex ( M)
B1
T2
32 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
As observed in Figure 17, the molybdenum levels of the cytosolic and nuclear extracts treated with
compounds B1 and T2 showed a significant increase and also a dose dependent relation. B1 has
clearly higher values than T2 in both the nucleus and cytosol fractions, which is consistent with the
octanol-water partition coefficient determined (chapter 7.2), as B1 having an extended hydrophobic
behavior would explain the ease with which it interacts with the membrane, contrarily to T2. These
results may also explain the cytotoxic activity of both the complexes, as B1 showed a stronger
cytotoxicity effect than T2 (Chapter 7.1, Table 2). The presence of both the complexes in the
nucleus supports the hypothesis that the complexes reach the nucleus, may interact with DNA and
take part in the process that leads to cell death (Chapter 7.5).
7.5. DNA Binding Studies
Extensive investigations have established DNA as a primary intracellular target of antitumor agents
[27]. Metal complexes can bind to DNA via covalent or/and noncovalent interactions. Whereas in
covalent binding a ligand is replaced by a nitrogen in a base of DNA, noncovalent binding is related
to the interactions of the complexes outside the DNA helix, by intercalative mode for example [48].
Absorption titration and circular dichroism were used to test the hypothesis that B1 and T2 inhibit
cancer cell proliferation by interacting with DNA. The binding mode of DNA-complexes was also
examined.
7.5.1. Electronic absorption titration
Electronic absorption spectroscopy is universally employed to study the binding mode of DNA to
complexes [44]. The absorption spectra changed with the addition of the complexes (Figure 18-19).
The addition of ctDNA (0-50 M bp-1) to T2 led to spectral changes with hyperchromism of the 299
0
20
40
Control 10 50 100
Nuc
lear
Mo
×10
10(m
mol
/mill
ion
cells
)
Concentration of Mo complex ( M)
B1T2
b)
0
5
10
Control 10 50 100
Cyt
osol
ic M
o×
1010
(mm
ol/m
illio
n ce
lls)
Concentration of Mo complex ( M)
B1
T2
a)
Figure 17 - Molybdenum concentration in cytosolic (a) and nuclear (b) extracts of HeLa cells after 48 h of exposure tocompounds T2 and B1.
(a) (b)
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 33
0
8
15
0 25 50
D/
ap×
1010
(M2
cm)
D ( M)
Figure 18 - Left: UV-Vis absorption spectra of T2 (20 M) in Tris buffer in the presence of increasing amounts of ctDNA.[DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNA concentration; right: plotof D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at 299 nm.
nm band, although no obvious red shift can be observed. Generally, hyperchromism is related to the
loss of secondary structure of DNA [48,49]. For the absorbance data at 299 nm, a plot of D/ap vs.
D was done (Figure 18, right), in order to determine the binding constant value (Chapter 4.7, K). It
shows a linear relationship with a K value of 2.08 (±0.98) × 105 M-1.
The binding of B1 to ctDNA also led to similar spectral changes (Figure 19), with hyperchromism
of the 272 nm absorption band. The K value for B1 was calculated to be 3.68 (±2.01) × 105 M-1. The
results indicate that the complex T2 binds more strongly than B1. Both K values are similar to those
of a typical classical intercalator, ethidium bromide, with K values 1.23 (±0.07) × 105 M-1 [68].
These results suggest that both complexes can bind to ctDNA, probably by electrostatic interaction
leading to the stabilization of DNA duplex. Nevertheless, both complexes have aromatic ligands with
extended systems, which means that intercalation could be one of the binding patterns, since the
extend systems can intercalate in DNA. Other approaches, such as circular dichroism, need to be
pursued in order to confirm this result (Chapter 7.5.2).
0
1
2
240 295 350
Abs
orba
nce
Wavelength (nm)
34 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
7.5.2. Circular dichroism
CD can be used to follow rapidly and efficiently DNA conformational transitions [53]. Generally, in
noncovalent interactions, such as electrostatic interaction, no spectral chances are observed in the
DNA spectra, since these modes of binding do not affect the secondary structure of DNA. On the
other hand, intercalation mode leads to changes in the DNA spectra [55]. DNA show a positive band
at 275 nm and a negative band at 245 nm, which is consistent with the characteristic B conformation
of DNA (Figure 20) [54]. With the addition of B1, the intensity of the positive band increased and
no significant changes was observed for the negative band. T2 also led to changes in the ctDNA
spectra, with the decrease of positive and negative bands. These changes in the CD spectrum of ct
DNA indicate strong interaction between the complexes and DNA mainly by intercalation mode
[55]. This effect can be seen clearly with complex T2. Despite the existence of intercalation
interactions, electrostatics and groove binding may also occur, mainly with B1 where the alterations
in the DNA spectra are not so obvious. Thus, as far as the binding mode of complexes is concerned it
seems to take place mainly via intercalation.
0
1
2
240 295 350
Abs
orba
nce
Wavelength (nm)
0
15
30
0 25 50D
/ap
×10
10(M
2cm
)D ( M)
Figure 19 - Left: UV-Vis absorption spectra of B1 (20 M) in Tris buffer in the presence of increasing amounts of ctDNA.[DNA] = 0, 10, 20, 30, 40, 50 M. The arrow indicates the absorbance changes upon increasing DNA concentration; right: plotof D/ap vs. D for the titration of DNA to complex. Absorbance was monitored at 272 nm.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 35
Figure 20 – Circular Dichroism of ctDNA incubated with B1 (left) and T2 (right). [Complex] = 0, 10, 25, 50, 75, 100,250 M. The arrow indicates the signal changes upon increasing complex concentration.
Figure 21 - Electrophoretic mobility pattern of pYES2 plasmid DNA (C) incubated with complex B1 (right) and T2 (left)at 10, 50 and 100 M.
7.5.3. Gel electrophoresis studies
Gel electrophoresis studies were performed in order to evaluate the effect of molybdenum complexes
on DNA tertiary structure. Thus, the effect of T1 and B2 on plasmid DNA, pYES2, was studied
(Figure 21).
Plasmid DNA is visualized as supercoiled (high mobility form), linear (intermediate mobility form)
and circular (low mobility form). With the increasing of B1 concentration, a mobility decrease can
be observed. This effect can be observed more clearly with circular and linear forms. The effect of
B1 is similar to the one observed for intercalator agents, such as cisplatin, which means that
interaction of the complex with DNA occurs and probably by intercalation [70]. This pattern, where
plasmid forms retardation are observed, can be visualized for T2, although more slightly and only in
the supercoiled form, the only form visualized.
7.5.4. Atomic force microscopy
AFM can be used to study the influence of factors, such as intercalators, that induce conformational
transitions on the DNA tertiary structure [57]. Thus, preliminary studies of structural transitions in
the tertiary structure of plasmid DNA were performed. AFM images of the free plasmid pYES2 and
-8
-4
0
4
8
230 270x
103
(deg
)
(nm)
T2B1
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 35
-5
-3
0
3
5
230 270x
103
(deg
)
(nm)
Figure 20 – Circular Dichroism of ctDNA incubated with B1 (left) and T2 (right). [Complex] = 0, 10, 25, 50, 75, 100,250 M. The arrow indicates the signal changes upon increasing complex concentration.
Figure 21 - Electrophoretic mobility pattern of pYES2 plasmid DNA (C) incubated with complex B1 (right) and T2 (left)at 10, 50 and 100 M.
7.5.3. Gel electrophoresis studies
Gel electrophoresis studies were performed in order to evaluate the effect of molybdenum complexes
on DNA tertiary structure. Thus, the effect of T1 and B2 on plasmid DNA, pYES2, was studied
(Figure 21).
Plasmid DNA is visualized as supercoiled (high mobility form), linear (intermediate mobility form)
and circular (low mobility form). With the increasing of B1 concentration, a mobility decrease can
be observed. This effect can be observed more clearly with circular and linear forms. The effect of
B1 is similar to the one observed for intercalator agents, such as cisplatin, which means that
interaction of the complex with DNA occurs and probably by intercalation [70]. This pattern, where
plasmid forms retardation are observed, can be visualized for T2, although more slightly and only in
the supercoiled form, the only form visualized.
7.5.4. Atomic force microscopy
AFM can be used to study the influence of factors, such as intercalators, that induce conformational
transitions on the DNA tertiary structure [57]. Thus, preliminary studies of structural transitions in
the tertiary structure of plasmid DNA were performed. AFM images of the free plasmid pYES2 and
310
T2B1
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 35
270 310
(nm)
Figure 20 – Circular Dichroism of ctDNA incubated with B1 (left) and T2 (right). [Complex] = 0, 10, 25, 50, 75, 100,250 M. The arrow indicates the signal changes upon increasing complex concentration.
Figure 21 - Electrophoretic mobility pattern of pYES2 plasmid DNA (C) incubated with complex B1 (right) and T2 (left)at 10, 50 and 100 M.
7.5.3. Gel electrophoresis studies
Gel electrophoresis studies were performed in order to evaluate the effect of molybdenum complexes
on DNA tertiary structure. Thus, the effect of T1 and B2 on plasmid DNA, pYES2, was studied
(Figure 21).
Plasmid DNA is visualized as supercoiled (high mobility form), linear (intermediate mobility form)
and circular (low mobility form). With the increasing of B1 concentration, a mobility decrease can
be observed. This effect can be observed more clearly with circular and linear forms. The effect of
B1 is similar to the one observed for intercalator agents, such as cisplatin, which means that
interaction of the complex with DNA occurs and probably by intercalation [70]. This pattern, where
plasmid forms retardation are observed, can be visualized for T2, although more slightly and only in
the supercoiled form, the only form visualized.
7.5.4. Atomic force microscopy
AFM can be used to study the influence of factors, such as intercalators, that induce conformational
transitions on the DNA tertiary structure [57]. Thus, preliminary studies of structural transitions in
the tertiary structure of plasmid DNA were performed. AFM images of the free plasmid pYES2 and
T2B1
36 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
pYES2 incubated with T2 were taken (Figure 22). Images were obtained after a very short time of
incubation (2 min) in order to allow the observation of the changes produced on DNA forms. At
longer times it was not possible to observe the images properly, probably due to the excessive
amount of DNA adhered to the mica. These results showed that T2 induce changes in the DNA
structure as a consequence of its interaction with DNA chains. T2 seems to originate mixed toroidal
and plectonemic supercoiling forms (see Figure 3, Chapter 4.9). As observed in previous studies,
intercalation of aromatic rings between base pairs of DNA seems to be the most probable way of
interaction. The image obtained is similar to others obtained for intercalators [70,71]. Unfortunately,
the study of structural transitions in the tertiary structure of plasmid pYES2 using B1 was not
possible owing to experimental limitations.
Figure 22 - A Selection of topographic images recorded of plasmid pYES2 (a) and plasmidpYES2 incubated with T2 (b) adsorbed to AP-mica. The scale bars correspond to 250 nm.
36 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
pYES2 incubated with T2 were taken (Figure 22). Images were obtained after a very short time of
incubation (2 min) in order to allow the observation of the changes produced on DNA forms. At
longer times it was not possible to observe the images properly, probably due to the excessive
amount of DNA adhered to the mica. These results showed that T2 induce changes in the DNA
structure as a consequence of its interaction with DNA chains. T2 seems to originate mixed toroidal
and plectonemic supercoiling forms (see Figure 3, Chapter 4.9). As observed in previous studies,
intercalation of aromatic rings between base pairs of DNA seems to be the most probable way of
interaction. The image obtained is similar to others obtained for intercalators [70,71]. Unfortunately,
the study of structural transitions in the tertiary structure of plasmid pYES2 using B1 was not
possible owing to experimental limitations.
Figure 22 - A Selection of topographic images recorded of plasmid pYES2 (a) and plasmidpYES2 incubated with T2 (b) adsorbed to AP-mica. The scale bars correspond to 250 nm.
36 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
pYES2 incubated with T2 were taken (Figure 22). Images were obtained after a very short time of
incubation (2 min) in order to allow the observation of the changes produced on DNA forms. At
longer times it was not possible to observe the images properly, probably due to the excessive
amount of DNA adhered to the mica. These results showed that T2 induce changes in the DNA
structure as a consequence of its interaction with DNA chains. T2 seems to originate mixed toroidal
and plectonemic supercoiling forms (see Figure 3, Chapter 4.9). As observed in previous studies,
intercalation of aromatic rings between base pairs of DNA seems to be the most probable way of
interaction. The image obtained is similar to others obtained for intercalators [70,71]. Unfortunately,
the study of structural transitions in the tertiary structure of plasmid pYES2 using B1 was not
possible owing to experimental limitations.
Figure 22 - A Selection of topographic images recorded of plasmid pYES2 (a) and plasmidpYES2 incubated with T2 (b) adsorbed to AP-mica. The scale bars correspond to 250 nm.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 37
8. Conclusions
Organometallic compounds offer a rich chemistry field in medicine with a high potential to develop
effective antitumor drugs [30]. This variety of options provides unlimited possibilities of
combination that result in an extensive spectrum of mechanisms open to organometallic compounds.
Two organometallic compounds [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)], B1, and [Mo(3-
C3H5)CF3SO3(CO)2(2,2’-bipyridyl)], T2, have been studied in the search for innovative and valuable
tools in cancer chemotherapy.
Complexes T2 and B1 exhibited a high cytotoxic activity toward several human cancer cell lines:
HeLa, MCF-7, and RPE. MCF-7 showed to be the most resistant cell line, whereas RPE was the
most sensitive against the complexes. Although both the complexes showed very significant IC50
values, B1 showed to be a little more effective than T2, which may be related to the 1,10-
phenanthroline ligand. Furthermore, B1 presents a range of IC50 values comparable to cisplatin,
which makes it a good candidate as a substitute of cisplatin, avoiding some of the cisplatin
limitations, such as acquired resistance. The kinetic studies revealed that a cationic form of
molybdenum compound may be related to the maximum cytotoxic effect. According to the cellular
uptake of molybdenum and octanol/water partition assays, both complexes seem to enter the cells in
a selective and dose dependent manner and reach the nucleus. These results may be related to the
hydrophobic behavior of the complexes in solution, which showed that B1 is more hydrophobic than
T2. B1 might then be able to pass more easily through a biological membrane than T2. As far as
complex-DNA interaction is concerned, both the complexes showed interaction with DNA (DNA
plasmid and ctDNA). B1 and T2 bind to ctDNA with binding constants of 2.08 (±0.98) × 105 and
3.68 (±2.01) × 105 M-1, respectively. The data from absorption, circular dichroism and atomic force
microscopy support a specific binding mode of both the complexes with DNA, via intercalation,
although other modes of binding can also occur. The interaction of the complexes with DNA, mainly
by intercalation, changing its conformation, may be one pathway responsible for cell death.
It has been shown that both complexes are present in the nucleus and that they interact with DNA in
vitro, so it becomes clear that this process may also occur in vivo.
As both the complexes showed promising results, they may be able to become a successful
alternative to the existing platinum chemotherapeutic agents, the only compounds with clinical use in
chemotherapy currently, and they might exhibit unique properties, such as the reactivity of the
ligands or differences in coordination geometry, that can be used in order to overcome chemotherapy
current limitations.
38 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
However, compounds with high cytotoxicity against cancer cell lines do not necessarily mean that all
the criteria for anticancer drugs are fulfilled, and despite the fact that these compounds presented a
high cytotoxic potency, further studies involving in vivo experiments need to be done in order to
raise these compounds to a proper therapeutic window.
Future work will address the intracellular localization of molybdenum, for example by the use of
fluorescents ligands, and the study underlying the metabolism responsible for DNA damage repair,
which would be useful to understand the global picture related to the mechanism of action of these
molybdenum complexes. Long-term work would be related to the use of molybdenum compounds in
in vivo studies.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 39
9. Acknowledgements
É com enorme gratidão que venho agradecer à Professora Margarida Meireles e à Professora Maria
José Calhorda, as orientadoras responsáveis pelo meu projecto de mestrado, por me receberem no
laboratório e por me integrarem no grupo de Química Inorgânica e Teórica. Foi para mim uma honra
partilhar ideias e trabalhos com diversas pessoas experientes e de diferentes áreas. Também queria
agradecer a oportunidade de integrar neste projecto onde pude aprender e aquirir imensa prática
laboratorial. O facto de haver sempre disponibilidade de todos os membros do grupo contribuiu para
o sucesso do trabalho, que culminou com a publicação do artigo científico. Manifesto assim a minha
gratidão a todo o grupo e, em particular, às minhas orientadoras.
Deixo um agradecimento aos diversos grupos de investigação que colaboraram neste projecto e que,
com toda a disponibilidade e empenho, tornaram possível a realização das várias experiências:
Ao Dr. Alexandre Quintas e Dr. Luís Oliveira pela colaboração nos ensaios de dicroísmo circular no
Instituto Superior de Ciências da Saúde Egas Moniz;
Ao grupo de enzimologia da FCUL com especial ênfase para a Dra. Marta Silva pela colaboração
nos ensaios com DNA plasmídico;
À Dra. Filomena Martins e à Dra. Cristina Ventura pela colaboração nos ensaios de partição
octanol/água;
À Dra. M. Soledade Santos pela colaboração nos ensaios das condutividades;
À Marta Saraiva e à Dra. Carla Nunes pelo apoio na síntese química dos complexos;
E à Faculdade de Ciências, nomeadamente ao Departamento de Quimíca e Bioquímica, pela
oportunidade da concretização deste projecto e por tornar possível a minha formação quer científica,
quer pessoal, oferecendo todas as condições necessárias.
Um sincero agradecimento ao CQB pela oportunidade que me deram de apresentar um seminário,
juntamente com a minha colega de grupo Marta Saraiva, com o título “Synthesis and cytotoxic
characteristics of a family of molybdenum(II) complexes”.
Queria também agradecer ao meu colega de laboratório Miguel Lopes, cuja presença tornou este
trabalho mais interessante e desafiador. Com ele pude contar sempre em todos os momentos, quer na
realização dos vários ensaios, quer na vida pessoal. Aqui fica um profundo e sincero agradecimento
de uma amizade que já dura 17 anos.
A todas as pessoas com quem fui partilhando experiências ao longo da vida e que me influenciaram
de algum modo, a todos os meus amigos, a todos os meus colegas de licenciatura, com especial
ênfase para Ana Filipa Ribeiro, Armando Cruz, Bruno Moraes, Carlos Neves, Maria João Lima,
40 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Mariana Oliveira, Miguel Lopes, Sara Carvalhal e Soraia Martins, aqui fica um forte abraço.
Obrigado pelas companhias nos almoços, pelas visitas motivadoras ao laboratório, pelo
conhecimentos partilhados ao longo da licenciatura e do mestrado e, acima de tudo, pela diversão
que tão bem sabemos desfrutar! Um especial agradecimento para a Sara, “a rapariga do laboratório
ao lado”, cuja companhia muito me ajudou nesta experiência.
Por fim, deixo um sincero e especial agradecimento aos meus pais, irmãs e madrinha pelo suporte
sólido, com quem posso sempre contar para o que quer que seja, em qualquer altura.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 41
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role of coligands on DNA binding and cleavage and anticancer activity. Inorg chem, 48(4): 1309-22,
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11.Annex
Figure 23 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO with no incubation time (0 hours).
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 47
11.Annex
Figure 23 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO with no incubation time (0 hours).
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 47
11.Annex
Figure 23 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO with no incubation time (0 hours).
48 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 24 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.
48 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 24 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.
48 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 24 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 49
Figure 25 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 24 hours of incubation time at 37 ºC.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 49
Figure 25 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 24 hours of incubation time at 37 ºC.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 49
Figure 25 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 24 hours of incubation time at 37 ºC.
50 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 26 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 48 hours of incubation time at 37 ºC.
50 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 26 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 48 hours of incubation time at 37 ºC.
50 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 26 - ESI+ mass spectrum of the [Mo(3-C3H5)Br(CO)2(1,10-phenanthroline)] (B1) complex dissolved in DMSO after 48 hours of incubation time at 37 ºC.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 51
Figure 27 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO with no incubation time (0 hours).
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Figure 27 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO with no incubation time (0 hours).
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 51
Figure 27 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO with no incubation time (0 hours).
52 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 28 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.
52 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 28 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.
52 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 28 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 2 hours of incubation time at 37 ºC.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 53
Figure 29 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 24 hours of incubation time at 37 ºC.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 53
Figure 29 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 24 hours of incubation time at 37 ºC.
CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES | 53
Figure 29 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 24 hours of incubation time at 37 ºC.
54 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 30 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 48 hours of incubation time at 37 ºC.
54 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 30 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 48 hours of incubation time at 37 ºC.
54 | CYTOTOXIC ACTIVITY AND MECHANISM OF ACTION OF ORGANOMETALLIC COMPLEXES
Figure 30 - ESI+ mass spectrum of the [Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipyridyl)] (T2) complex dissolved in DMSO after 48 hours of incubation time at 37 ºC.
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Figure 31 - Absorption spectra of B1 ([Mo(3-C3H5)Br(CO)2(1,10-fenantrolina)]).
Figure 32 - Absorption spectra of T2 ([Mo(3-C3H5)CF3SO3(CO)2(2,2’-bipiridil)]).
0.00
0.25
0.50
240 420 600
Abs
orba
nce
(nm)
0 h
1 h
2 h
24 h
48 h
B1
0.00
0.25
0.50
270 435 600
Abs
orba
nce
(nm)
0 h
1 h
2 h
24 h
48 h
T2