CHAPTER 5. Prohibition of sexual violence under international criminal law
B. The European System of Human Rights Protection
O cancro é uma das principais causas de morte em todo o mundo, e entre os vários tipos, o cancro do colo do útero é um dos mais frequentes nas mulheres.2 Deste modo torna-se
necessário desenvolver novas estratégias terapêuticas.
Este trabalho consistiu no estudo do aptamero G4 AT11, ligandos e complexos G4 AT11-ligando como agentes anticancerígenos, para obtenção de uma maior eficácia e seletividade a nível terapêutico.
Uma vez que os derivados de fenantrolina têm demonstrado ser uma nova classe de agentes anti-tumorais12, os compostos propostos neste trabalho eram macrociclos derivados de
fenantrolina. Contudo, a síntese não foi concluída pois não se conseguiu purificar/isolar os macrociclos puros, passando a estudar-se a como ligandos de G4 os derivados de fenantrolina [32]phen2N4 e [16]phenN2 e os derivados de laranja de acridina C3, C5, C8 e C8-NH2.
Os ensaios de dicroísmo circular permitiram concluir que o aptamero G4 AT11 apresenta uma topologia paralela com banda positiva a 260 nm e negativa a 240 nm, a qual é mantida após adição dos ligandos.
Através dos ensaios biofísicos de dicroísmo circular concluiu-se que os ligandos mais promissores eram os derivados de laranja de acridina C8 e C8-NH2 seguidos dos derivados de fenantrolinas
[16]phenN2 e [32]phen2N4 uma vez que os valores de ΔTm para estes ligandos foram de 23,9;
21,0; 18,9 e 18,7ºC, respetivamente. Nos ensaios de espectroscopia de fluorescência, estes ligandos continuam a ser os mais promissores, uma vez que apresentam afinidades moderadas altas com valores de KD de 9,90x10-7; 2,90 x10-7; 3,53 x10-7 e 2,01 x10-7 M-1 respetivamente.
O estudo de proliferação celular mostrou que o aptamero G4 AT11 provoca uma diminuição da viabilidade celular mais acentuada nas células HeLa comparativamente às NHDF. Relativamente aos ligandos em estudo, os que apresentam uma maior citotoxicidade são os derivados de laranja de acridina C8 e C8-NH2 com valores de IC50 de 0,15 µM e 0,31 µM respetivamente, para
as células HeLa, e 0,48 µM e 0,80 µM respetivamente, para as células NHDF.
A formação dos complexos G4 AT11-ligandos (derivados de fenantrolina e de laranja de acridina) parecem ser uma estratégia promissora, uma vez que a formação destes nas células HeLa diminui a viabilidade celular relativamente ao ligando e G4 AT11 livres, e nas NHDF promove um aumento da viabilidade celular comparativamente ao ligando livre.
O estudo da localização intracelular mostrou que o aptamero G4 AT11 só é internalizado ao fim de 24 horas de incubação, enquanto que o ligando derivado de laranja de acridina C8 é
internalizado após a sua incubação em células HeLa, demonstrando que não há nenhum condicionamento por parte da célula. O aptamero G4 AT11 localiza-se a nível citoplasmático, enquanto o ligando C8 se encontra a nível citoplasmático e nucleolar. Porém, quando há
formação do complexo G4 AT11-C8, este localiza-se no citoplasma, indicando que o aptamero
G4 AT11 não consegue atingir o núcleo da célula, mas que promove morte celular.
O mecanismo envolvido no processo de morte celular poderá estar de acordo com o proposto por Reyes-Reyes e colaboradores para o G4 AS141132, no qual o aptamero G4 AT11 poderá
ligar-se especificamente à nucleolina, promovendo a ativação constante da Rac1 e levando a célula para morte celular através de metuose.
Assim, conclui-se que a formação dos complexos aptamero G4 AT11-ligandos (derivados de laranja de acridina e fenantrolinas) parece ser uma estratégia promissora para terapia do cancro do colo do útero.
Perante os resultados obtidos conclui-se ser necessário a realização de mais ensaios de modo a clarificar os mecanismos envolvidos. Como perspetivas futuras propõem-se as seguintes:
Avaliar a interação nucleolina-G4 AT11 e nucleolina-G4 AT11-ligando por ensaios de SPR
Avaliar a resistência que o G4 AT11 e os complexos G4 AT11-ligandos têm para as nucleases séricas
Avaliar a localização intracelular do G4 AT11, do derivado de laranja de acridina C8 e
formação do complexo G4 AT11-C8 na linha celular de fibroblastos da derme humana
normais (NHDF).
A nível de síntese química, é necessário:
Estudar as condições reacionais da síntese do DPPZ(Me)2 e DPPZ(COH)2 de forma a
melhorar os rendimentos obtidos procedendo assim à síntese dos compostos seguintes. Avaliar a interação de todos os compostos intermediários e finais com o G4 AT11.
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Stabilization of modified AS1411 G-quadruplex aptamer by acridine orange
derivative for cervical cancer therapy
Márcia Ribeiro
1, Maria P. C. Campello
2, João A. Queiroz
1, António Paulo
2, Carla Cruz
1*
1CICS-UBI – Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506
Covilhã, Portugal
2Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Estrada Nacional 10 (km 139,7),
2695-066 Bobadela LRS, Portugal. *e-mail: [email protected]
e-mail: [email protected]
Introduction
G-quadruplex (G4) AS1411 aptamer has been recently employed as a drug delivery system with efficient cellular internalization compared to non-G4 DNA sequences, and it exhibits anticancer activity by interfering with nucelolin oncogenic functions. [1] Clinical trials of AS1411 have indicated that it is well tolerated with therapeutic activity but improved pharmacology and potency are required for optimal efficacy. [2]
Since G4 AS1411 aptamer has high affinity for nucleolin and it is able to enter more easily into malignant cells that can be used for diagnosis (when associated with imaging agents) and for treatment of cancer (by ligands association) [3] The synthetized ligands used in this study are derivatives of acridine orange [4]. Acridines are a class of heterocyclic compounds that have a wide range of biological and pharmaceutical properties. [5]
In order to have greater entrance in the cell, greater stability and better pharmacological properties, AS1411 has been structurally modified to be used as therapeutic agent.
The aim of this study is to evaluate the AS1411 properties with and without modification and ligands in cancer cell lines.
Materials and Methods
Circular dichroism is performed to evaluate the structure of the AS1411 aptamer modified and G-quadruplex maintenance/interconversion after acridine orange binding. The binding affinity of G-quadruplex AS1411 derivative aptamer with and without acridine orange ligand to nucleolin is assessed by surface plasmon resonance (SPR) biosensor Cell viability assays are performed to evaluate the cytotoxic effect of G-quadruplex AS1411 derivative aptamer on cervical cancer cells.
Results and Discussion
The G-quadruplex AS1411 derivative aptamer showed high stabilization with the acridine orange derivative by circular dichroism. The binding G-quadruplex AS1411 derivative aptamer with and without ligand assessed by SPR biosensor showed high affinity to nucleolin. Cytotoxic studies revealed that the ligand is cytotoxic for cervical cancer cells with IC50 around 2.4-8.9 µM.
Conclusions
Overall, the G-quadruplex AS1411 derivative showed high stability in the presence of acridine orange ligand. The binding AS1411 aptamer modified with and without ligand presented high affinity to nucleolin and cytotoxic studies revealed that the ligand is cytotoxic for cervical cancer. The G-quadruplex AS1411 derivative is an effective targeting agent that can be used to deliver cargoes to cancer cells.
Acknowledgments: This work was supported by FCT project “Projeto de Investigação Exploratória” reference IF/00959/2015 Nucleolin targeting by G-quadruplex aptamers for cervical cancer therapy” financed by Fundo Social Europeu e ao Programa Operacional Potencial Humano.
Stabilization of modified AS1411 G-quadruplex aptamer by acridine orange
and phenantrolines derivatives for cervical cancer therapy.
Márcia Ribeiroa, Josué Carvalhoa, Maria P. C. Campellob, João A. Queiroza, António Paulob, Carla Cruza*.
aCICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã,
Portugal
b Centro de Ciências e Tecnologias nucleares, Instituto superior Técnico, Universidade de Lisboa, estrada nacional 10 (km 139,7), 2695-
066 Bobadela LRS, Portugal
Email: [email protected] | *Email: [email protected]
Introduction
G-quadruplex (G4) AS1411 aptamer is a high-order structure that occur in G-rich sequences and has been recently employed as a drug delivery system because it exhibits anticancer activity by interfering with nucelolin oncogenic functions. [1]
G4 AS1411 aptamer has high affinity for nucleolin and it is able to enter more easily into malignant cells. This can be used for diagnosis and for treatment of cancer (by ligands association) [2]
The synthetized ligands used in this study are derivatives of acridine orange [3] and phenantrolines.
The aim of this study is to evaluate the modified AS1411 properties with and without ligands in cancer cell lines, since it was shown that the AS1411features needed to be optimized [4]
Materials and Methods
Circular dichroism is performed to evaluate the structure of the AS1411 aptamer modified and G4 maintenance/interconversion after ligands binding
Affinity of ligand to the aptamer was measured by fluorescence
Cell viability assays are performed to evaluate the cytotoxic effect of G-quadruplex AS1411 derivative aptamer on cervical cancer cells and normal cells.
Results and Discussion
Dicroism circular showed that the G4 AS1411 derivative aptamer has high stabilization with ligands and cytotoxic studies revealed that the ligand is cytotoxic for cervical cancer cells.
Conclusions
Overall, the modified AS1411 G4 aptamer is an effective targeting agent that can be used to deliver ligands to cancer cells.
Acknowledgements: This work was supported by FCT project “Projeto de Investigação Exploratória” reference IF/00959/2015 Nucleolin
targeting by G-quadruplex aptamers for cervical cancer therapy” supported by Fundo Social Europeu and Programa Operacional Potencial Humano.
References:
[1] Carvalho, J., Ferreira, J., Pereira, P., Coutinho, E., Guédin, A., Nottelet, P., … Cruz, C. (2016). Stabilization of novel
immunoglobulin switch regions G-quadruplexes by naphthalene and quinoline-based ligands. Tetrahedron, 72(9), 1229–1237.
[2] Bagheri, Z., Ranjbar, B., Latifi, H., Zibaii, M. I., Moghadam, T. T., & Azizi, A. (2015). Spectral properties and thermal stability of
AS1411 G-quadruplex. International Journal of Biological Macromolecules, 72, 806–811.
[3] Pereira, E., Quental, L., Palma, E., Oliveira, M. C., Mendes, F., Raposinho, P., … Paulo, A. (2017). Evaluation of Acridine
Orange Derivatives as DNA-Targeted Radiopharmaceuticals for Auger Therapy : Influence of the Radionuclide and Distance to DNA. Scientific Reports, 10, 1–16.
[4] Bates, P. J., Reyes-Reyes, E. M., Malik, M. T., Murphy, E. M., O’Toole, M. G., & Trent, J. O. (2017). G-quadruplex
oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochimica et Biophysica Acta - General Subjects,
1861(5), 1414–1428.
Ligands effects on stabilization of DNA G-quadruplex aptamer
M. Ribeiroa, J. Carvalhoa, M. P. C. Campellob, J. A. Queiroza, A. Paulob, C. Cruza*aCICS-UBI - Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã,
Portugal
b Centro de Ciências e Tecnologias nucleares, Instituto superior Técnico, Universidade de Lisboa, estrada nacional 10 (km 139,7),
2695-066 Bobadela LRS, Portugal
Email: [email protected] | *Email: [email protected]
ABSTRACT
Several DNA G-rich sequences can form four-stranded structures called G-quadruplexes (G4s). [1] The G4 AS1411 has been recently employed as an aptamer of nucleolin and a drug delivery system because exhibits anticancer activity by interfering with nucleolin oncogenic functions. [2]
Since G4 AS1411 aptamer has high affinity to nucleolin and is able to enter more easily into malignant cells, acting as agents for diagnosis and treatment [3]
Clinical trials of AS1411 indicated that it is well tolerated with therapeutic activity, but improved pharmacology and potency are required for optimal efficiency. [4] For this purpose, new aptamer derived by AS1411 is developed and its stabilization with ligands was evaluated by circular dichroism and fluorescence spectroscopy.
The ligands are acridine orange derivatives [5] and phenanthroline macrocycles. The cytotoxic effects and cellular uptake of complexes aptamer-ligands were evaluated in cancer cell lines versus fibroblasts
The results showed high stabilization of G4 aptamer by acridine orange ligands and cytotoxic effects in cervical cancer cells. The confocal microscopy experiments confirmed the binding of the aptamer to nucleolin and the nucleus localization of the acridine orange ligands.
Overall, the modified this aptamer is an effective targeting agent that can be used to deliver ligands into cancer cells.
Keywords: G-quadruplex, AS1411, nucleolin. cervical cancer
Acknowledgements: This work was supported by FCT project “Projeto de Investigação Exploratória” reference IF/00959/2015 Nucleolin targeting by G-quadruplex aptamers for cervical cancer therapy” supported by Fundo Social Europeu and Programa Operacional Potencial Humano and Project Fundação Luso-Americana (FLAD) Healthcare 2020 ref 45/2018
References:
[1] Han, H., & Hurley, L. H. (2000). G-quadruplex DNA: A potential target for anti-cancer drug design. Trends in Pharmacological Sciences, 21(4), 136–142.
[2] Carvalho, J., Ferreira, J., Pereira, P., Coutinho, E., Guédin, A., Nottelet, P., … Cruz, C. (2016). Stabilization of novel
immunoglobulin switch regions G-quadruplexes by naphthalene and quinoline-based ligands. Tetrahedron, 72(9), 1229–
1237.
[3] Bagheri, Z., Ranjbar, B., Latifi, H., Zibaii, M. I., Moghadam, T. T., & Azizi, A. (2015). Spectral properties and thermal stability of AS1411 G-quadruplex. International Journal of Biological Macromolecules, 72, 806–811.