Desenvolvimento de novos complexos de prata(I) contendo ligantes tiossemicarbazonas com potencial antitumoral contra células de câncer de mama triplo negativo
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Data
2020-11-06
Autores
Silva, Débora Eduarda Soares
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Universidade Estadual Paulista (Unesp)
Resumo
O câncer de mama é o câncer mais incidente em mulheres em todo o mundo. Entre os diferentes subtipos moleculares do câncer de mama, o câncer de mama triplo negativo (TNBC) representa aproximadamente 20% de todos os casos. Possui um prognóstico ruim e, atualmente, não existem terapias direcionadas e eficazes para seu tratamento. Desse modo, o desenvolvimento de novos fármacos contra tumores TNBC é urgente. Seis novos complexos de prata (I) contendo ligantes de interesse biológico foram sintetizados, de fórmula geral [Ag(phen)(L1-L3)](NO3) (1–3) e [AgCl(PPh3)2(L4-L5)] (4–6), onde phen = 1,10-fenantrolina; PPh3 = trifenilfosfina; L1 = 2-formilpiridina tiossemicarbazona; L2 = 2-formilpiridina-N(4)-metil-tiossemicarbazona; L3 = 2-formilpiridina-N(4)-etil-tiossemicarbazona; L4 = 3-metoxi-4-hidroxibenzaldeído tiossemicarbazona; L5 = 3-metoxi-4-[2-(1-morfolinil)etóxi]benzadeíldo tiossemicarbazona; L6 = 3-metoxi-4-[2-(1-piperidinil)etóxi]benzadeíldo tiossemicarbazona. Os compostos foram caracterizados por análise elementar, condutividade molar, espectrometria de massas, espectroscopia de absorção na região do infravermelho e resssonância magnética nuclear de 1H, 13C e 31P. Os complexos [AgCl(PPh3)2(L5)] e [AgCl(PPh3)2(L6)] foram caracterizados por difração de raios X de monocristal. A citotoxicidade in vitro dos compostos foi determinada frente as linhagens tumorais A549 (pulmão), MCF-7 (mama - hormônio sensitiva) e MDA-MB-231 (mama triplo negativo), e linhagem não tumoral MCF-10A (mama). Os complexos foram ativos contra todas as células tumorais testadas, com valores de IC50 para 48 horas de incubação na faixa de 1,5 – 20 μM. Particularmente, os compostos apresentaram promissora atividade citotóxica frente a linhagem tumoral MDA-MB-231. Além disso, os compostos foram menos citotóxicos frente as células MCF-10A. Estudos de quantificação celular de prata mostraram que os complexos de prata derivados da trifenilfosfina são internalizados de forma mais eficiente do que os complexos derivados da 1,10 fenantrolina nas células MDA-MB-231. Ensaios de morte celular demonstraram que os complexos [Ag(phen)(L1)](NO3) e [AgCl(PPh3)2(L4)] induzem alterações morfológicas características de apoptose, como encolhimento e arredondamento celular, aumento da porcentagem de células na fase sub-G1 do ciclo celular e aumento da porcentagem de células apoptóticas marcadas com Anexina V, que evidenciam a morte das células MDA-MB-231 por apoptose. Ambos os compostos também induziram a despolarização do potencial de membrana mitocondrial (Δψm) e aumento dos níveis de espécies reativas de oxigênio (ROS) intracelular, eventos que podem estar envolvidos na indução de apoptose das células MDA-MB-231. Ensaios de interação com o ctDNA por titulação espectroscópica, dicroísmo circular e ensaios de ligação competitiva evidenciaram que os compostos [Ag(phen)(L1)](NO3) e [AgCl(PPh3)2(L4)] podem interagir com o ctDNA sem modificar a estrutura do DNA (forma B), possivelmente com o sulco menor. A ligação dos complexos de prata com o ctDNA é fraca em comparação com o Hoechst 33258. Nossos resultados sugerem que outros alvos intracelulares ao invés do DNA sejam responsáveis pelos efeitos citótoxicos observados nas células MDA-MB-231, possivelmente as mitocôndrias.
Breast cancer is the most incident cancer in women worwilde. Considering the different molecular subtypes of breast cancer, triple negative breast cancer (TNBC) comprises about 20% of all cases. It has a poor prognosis and, currently, there are no specific targeted therapies for its treatment. Therefore, the development of new drugs against TNBC tumors is crucial. Six new silver (I) complexes containing ligands of biological importance were synthesized, of general formula [Ag(phen)(L1-L3)](NO3) (1–3) and [AgCl(PPh3)2(TSC)] (4–6), where phen = 1,10-phenanthroline; PPh3 = triphenylphosphine; L1 = 2-formylpyridine thiosemicarbazone; L2 = 2-formylpyridine-N(4)-methylthiosemicarbazone; L3 = 2-formylpyridine-N(4)-ethylthiosemicarbazone; L4 = 3-methoxy-4-hydroxybenzaldehyde thiosemicarbazone; L5 = 3-methoxy-4-[2-(morpholine-1- yl)ethoxy]benzaldehyde thiosemicarbazone; L6 =3-methoxy-4-[2-(piperidine-1-yl)ethoxy]benzaldehyde thiosemicarbazone. The compounds were characterized by elemental analysis, molar conductance measurements, mass spectrometry, infrared spectroscopy and 1H, 13C and 31P NMR. The molecular structures of complexes [AgCl(PPh3)2(L5)] and [AgCl(PPh3)2(L6)] were determined by single crystal X-ray diffraction. The in vitro cytotoxicity of compounds were evaluated against the tumor cell lines A549 (lung), MCF-7 (breast-hormone-responsive), MDA-MB-231 (triple negative breast cancer), and non-tumor cell line MCF-10A (breast). The silver compounds were cytotoxic against all tumor cells with IC50 values in 48 h of incubation ranging from 1.5 to 20 M. Particularly, the complexes exhibited appreciable cytotoxic activity against MDA-MB-231 cells. Furthermore, the compounds were less cytotoxic against the non-tumor cells MCF-10A. Cell accumulation studies showed that phosphine compounds were more efficiently internalized than phen compounds in the MDA-MB-231 cells. Cell death studies indicated that compounds [Ag(phen)(L1)](NO3) and [AgCl(PPh3)2(L4)] induce morphological changes typical of apoptosis, as cell shrinkage and rounding, an increase of cells at sub-G1 phase of cell cycle and an increase in apoptotic cells stained with Anexin V, evidencing the cell death of MDA-MB-231 cells by apoptosis. Both compounds induced mitochondrial membrane depolarization and increase of reactive oxygen species (ROS) levels, events that may be involved in the induction of apoptosis in the MDA-MB-231 cells. DNA binding studies by spectroscopic titration, circular dichroism and competitive assays with Hoechst 33258 and Thiazole Orange evidenced that compounds [Ag(phen)(L1)](NO3) and [AgCl(PPh3)2(L4)] can interact with ctDNA and does so without modifying the B-structure of the DNA, possibly bind to the minor groove. The binding of silver complexes with ctDNA is weak compared to Hoechst 33258. Our findings suggest that other intracellular targets rather than DNA are responsible for the cytotoxic effects observed in the MDA-MB-231 cells, potentially the mitochondria.
Breast cancer is the most incident cancer in women worwilde. Considering the different molecular subtypes of breast cancer, triple negative breast cancer (TNBC) comprises about 20% of all cases. It has a poor prognosis and, currently, there are no specific targeted therapies for its treatment. Therefore, the development of new drugs against TNBC tumors is crucial. Six new silver (I) complexes containing ligands of biological importance were synthesized, of general formula [Ag(phen)(L1-L3)](NO3) (1–3) and [AgCl(PPh3)2(TSC)] (4–6), where phen = 1,10-phenanthroline; PPh3 = triphenylphosphine; L1 = 2-formylpyridine thiosemicarbazone; L2 = 2-formylpyridine-N(4)-methylthiosemicarbazone; L3 = 2-formylpyridine-N(4)-ethylthiosemicarbazone; L4 = 3-methoxy-4-hydroxybenzaldehyde thiosemicarbazone; L5 = 3-methoxy-4-[2-(morpholine-1- yl)ethoxy]benzaldehyde thiosemicarbazone; L6 =3-methoxy-4-[2-(piperidine-1-yl)ethoxy]benzaldehyde thiosemicarbazone. The compounds were characterized by elemental analysis, molar conductance measurements, mass spectrometry, infrared spectroscopy and 1H, 13C and 31P NMR. The molecular structures of complexes [AgCl(PPh3)2(L5)] and [AgCl(PPh3)2(L6)] were determined by single crystal X-ray diffraction. The in vitro cytotoxicity of compounds were evaluated against the tumor cell lines A549 (lung), MCF-7 (breast-hormone-responsive), MDA-MB-231 (triple negative breast cancer), and non-tumor cell line MCF-10A (breast). The silver compounds were cytotoxic against all tumor cells with IC50 values in 48 h of incubation ranging from 1.5 to 20 M. Particularly, the complexes exhibited appreciable cytotoxic activity against MDA-MB-231 cells. Furthermore, the compounds were less cytotoxic against the non-tumor cells MCF-10A. Cell accumulation studies showed that phosphine compounds were more efficiently internalized than phen compounds in the MDA-MB-231 cells. Cell death studies indicated that compounds [Ag(phen)(L1)](NO3) and [AgCl(PPh3)2(L4)] induce morphological changes typical of apoptosis, as cell shrinkage and rounding, an increase of cells at sub-G1 phase of cell cycle and an increase in apoptotic cells stained with Anexin V, evidencing the cell death of MDA-MB-231 cells by apoptosis. Both compounds induced mitochondrial membrane depolarization and increase of reactive oxygen species (ROS) levels, events that may be involved in the induction of apoptosis in the MDA-MB-231 cells. DNA binding studies by spectroscopic titration, circular dichroism and competitive assays with Hoechst 33258 and Thiazole Orange evidenced that compounds [Ag(phen)(L1)](NO3) and [AgCl(PPh3)2(L4)] can interact with ctDNA and does so without modifying the B-structure of the DNA, possibly bind to the minor groove. The binding of silver complexes with ctDNA is weak compared to Hoechst 33258. Our findings suggest that other intracellular targets rather than DNA are responsible for the cytotoxic effects observed in the MDA-MB-231 cells, potentially the mitochondria.
Descrição
Palavras-chave
Compostos de prata, Tiossemicarbazonas, Mamas-Câncer, Quimioterapia, Apoptose