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Novas estratégias para combater a resistência a múltiplos fármacos na leucemia mieloide crónica
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Resumo(s)
Os inibidores de tirosina quinase têm demonstrado excelentes resultados no tratamento
da maioria dos doentes com leucemia mieloide crónica (LMC). Contudo, cerca de 20 a 30%
dos doentes acabam por desenvolver resistência ao tratamento ao longo do tempo. A sobre-
expressão da glicoproteína-P (P-gp), um transportador capaz de expulsar uma variedade fár-
macos, incluindo o imatinib (IM), através da membrana celular, é considerado um dos meca-
nismos predominantes no fenótipo de quimiorresistência em diversas neoplasias como é o
caso da LMC.
Nesse sentido, este projeto teve como principal objetivo aumentar a sensibilidade de
células LMC resistentes através da combinação de um inibidor de tirosina quinase tradicional
com a nanovetorização de oligonucleotídeos antisense (ASOs, do inglês antisense oligonucleoti-
des) que visam o silenciamento génico de MDR1. Para isso, desenhou-se uma sequência de
ssDNA complementar ao mRNA de MDR1 e procedeu-se à funcionalização e caracterização
dos nanoconjugados para silenciamento génico. Posteriormente, avaliou-se a expressão de
MDR1 e de P-gp em células LMC resistentes a IM (K562-IM) incubadas na presença dos na-
noconjugados por RT-qPCR e Western Blot respetivamente. Por fim, a viabilidade celular após
exposição aos nanoconjugados em combinação com IM foi avaliada através do ensaio padrão
3-(4,5- dimetiltiazol-2-il) -5- (3-carboximetoxifenil) -2- (4-sulfofenil) -2H-tetrazólio (MTS).
Utilizando nanopartículas de ouro funcionalizadas com polietilenoglicol como vetores
de entrega de oligonucleotídeos complementares ao mRNA de MDR1 foi possível levar a uma
redução em cerca de 45% da expressão de MDR1 após 48 horas de incubação em células K562-
IM. Adicionalmente, a redução do mRNA de MDR1 também se repercute ao nível da dimi-
nuição da expressão de P-gp em cerca de 60%. No entanto, apesar dos efeitos demonstrados,
não foi possível observar uma reversão do fenótipo de resistência das células K562-IM quando
incubadas na presença dos nanoconjugados e do IM em simultâneo durante 48 horas ou des-
fasadamente, expondo as células primeiramente aos nanoconjugados durante 24 horas e de
seguida ao IM durante mais 24 horas, perfazendo um período total de 48 horas.
Em suma, apesar da nanovetorização de ASOs ter levado ao silenciamento de MDR1
não se conseguiu aumentar a sensibilidade das células resistentes ao IM, impossibilitando a
reversão do fenótipo resistente.
Tyrosine kinase inhibitors have shown excellent results in the treatment of most patients with chronic myeloid leukemia (CML). However, about 20 to 30% of patients end up devel- oping resistance over time. Overexpression of P-glycoprotein (P-gp), a transporter capable of expelling a variety of drugs, including imatinib (IM), across the cell membrane, is considered one of the main mechanisms in the chemoresistance phenotype in several cancers, such as CML. In this sense, this project aimed to increase the sensitivity of CML cells resistant by com- bining traditional chemotherapy with nanovectorization of antisense oligonucleotides (ASOs) targeting MDR1 silencing. To this end, a complementary ssDNA sequence to MDR1 mRNA was designed and the nanoconjugates were functionalized and characterized for gene silenc- ing. Subsequently, it was evaluated the expression of MDR1 and P-gp in CML cells resistant to IM (K562-IM) incubated in the presence of nanoconjugates by RT-qPCR and Western Blot respectively. Finally, the cell viability after exposure to the nanoconjugates in combination with IM was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- fophenyl)-2H-tetrazolium (MTS) assay. Using gold nanoparticles functionalized with polyethylene glycol as delivery vectors of oligonucleotides complementary to MDR1 mRNA, it was possible to lead to a 45% reduction of MDR1 mRNA expression after 48 hours of exposure. Additionally, exposure to the nano- conjugates also seemed to have an effect at the protein level, decreasing its expression (≈ 60%). However, it was not possible to observe a reversal of the resistance phenotype of the cells when incubated in the presence of the nanoconjugates and IM simultaneously for 48 hours or staggered by exposing the cells first to the nanoconjugates for 24 hours and then to IM for additional 24 hours. In conclusion, although the nanovectorization of ASOs led to MDR1 silencing, it was not possible to increase the sensitivity of IM resistant cells.
Tyrosine kinase inhibitors have shown excellent results in the treatment of most patients with chronic myeloid leukemia (CML). However, about 20 to 30% of patients end up devel- oping resistance over time. Overexpression of P-glycoprotein (P-gp), a transporter capable of expelling a variety of drugs, including imatinib (IM), across the cell membrane, is considered one of the main mechanisms in the chemoresistance phenotype in several cancers, such as CML. In this sense, this project aimed to increase the sensitivity of CML cells resistant by com- bining traditional chemotherapy with nanovectorization of antisense oligonucleotides (ASOs) targeting MDR1 silencing. To this end, a complementary ssDNA sequence to MDR1 mRNA was designed and the nanoconjugates were functionalized and characterized for gene silenc- ing. Subsequently, it was evaluated the expression of MDR1 and P-gp in CML cells resistant to IM (K562-IM) incubated in the presence of nanoconjugates by RT-qPCR and Western Blot respectively. Finally, the cell viability after exposure to the nanoconjugates in combination with IM was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- fophenyl)-2H-tetrazolium (MTS) assay. Using gold nanoparticles functionalized with polyethylene glycol as delivery vectors of oligonucleotides complementary to MDR1 mRNA, it was possible to lead to a 45% reduction of MDR1 mRNA expression after 48 hours of exposure. Additionally, exposure to the nano- conjugates also seemed to have an effect at the protein level, decreasing its expression (≈ 60%). However, it was not possible to observe a reversal of the resistance phenotype of the cells when incubated in the presence of the nanoconjugates and IM simultaneously for 48 hours or staggered by exposing the cells first to the nanoconjugates for 24 hours and then to IM for additional 24 hours. In conclusion, although the nanovectorization of ASOs led to MDR1 silencing, it was not possible to increase the sensitivity of IM resistant cells.
Descrição
Palavras-chave
Leucemia Mieloide Crónica Resistência a múltiplos fármacos Glicoproteína- P Silenciamento génico oligonucleotídeos ssDNA