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Role of microRNAs and Epigenetics Modifications in Paclitaxel Resistance in a Breast Cancer Cell Line Model
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Resumo(s)
RESUMO: O cancro da mama (CM) teve a maior incidência de novos casos de cancro
mundialmente em 2020, representando 11,7% do total de novos casos de cancro. Para
além disso, o CM é a maior causa de mortalidade relacionada com cancro em
mulheres. Apesar do progresso no desenvolvimento de novas terapias, vários doentes
recidivam após algum tempo devido à aquisição de resistência a fármacos em cancro
(RFC), sendo um dos maiores obstáculos para o sucesso do tratamento. Atualmente
é estimado que mais de 90% dos doentes com cancro metastático é devido à RFC. Os
mecanismos associados a RFC incluem o aumento do efluxo de fármacos,
principalmente por transportadores ATP-binding cassete (ABC), e modificações
epigenéticas, como a metilação aberrante do ADN e alterações na expressão de
microRNAs (miRNAs).
Neste projeto, desenvolvemos uma linha celular (MDA-MB-231) resistente ao
paclitaxel (PAX) a diferentes concentrações, nomeadamente, 400 nM, 800 nM e 1000
nM. A resistência foi monitorizada pelo ensaio MTT, que permite determinar a
viabilidade celular, e por alterações na morfologia das células, nomeadamente nos
núcleos, lisossomas e citoplasma, tendo sido detetadas através de microscopia
confocal. As células resistentes apresentam um aumento no número e tamanho dos
núcleos e na dimensão do citoplasma. Na concentração 1000 nM, foi detetado um
aumento acentuado no número de lisossomas. De seguida, estudou-se o efeito do
PAX no ciclo celular das células resistentes através de citometria de fluxo, onde foi
observada uma paragem do ciclo celular das células MDA-MB-231/ 1000 nM na fase
G2. Adicionalmente, a relação entre a metilação do ADN e PAX foi estudada através
de um kit de 5-mC ELISA, onde foi observado um comportamento oscilatório. Por
último, obtivemos um perfil de expressão de 118 miRNAs, diferencialmente expressos
em MDA-MB-231/ 1000 nM e MDA-MB-231, juntamente com os potenciais genes alvo
(1922 genes).
O conjunto de miRNAs, assim como os seus potenciais genes alvo,
identificados nos nossos resultados poderão vir a ser usados para uma deteção
precoce da resistência ao PAX em CM.
ABSTRACT: Breast cancer (BC) had the highest incidence of new cases of cancer worldwide in 2020, representing 11.7% of total new cases of cancer. Moreover, BC is the largest cause of cancer-related mortality in women. Despite the improvements in patients’ outcomes with chemotherapy, the development of cancer drug resistance (CDR) is an obstacle to a successful treatment. In fact, CDR is responsible for treatment failure in over 90% of patients with metastatic cancer. The mechanisms associated with CDR include the increase of drug efflux, mainly by ATP-binding cassette (ABC) transporters, and epigenetic modifications, such as aberrant DNA methylation and alterations in microRNAs (miRNAs) expression. In this project, we developed resistance to 400 nM, 800 nM, and 1000 nM of paclitaxel (PAX) in the MDA-MB-231 cell line. The resistance was monitored by the measurement of cell viability, through MTT, and changes in cell morphology, namely in the nucleus, lysosomes, and cytoplasm, which were detected by confocal microscopy. Resistant cells showed an increase in the size and number of nuclei and enlarged cytoplasm. At 1000 nM, an increase in the number of lysosomes was also detected. The effect of PAX in the cell cycle of resistant cells was evaluated by flow cytometry, where an arrest of the cell cycle of MDA-MB-231/ 1000 nM cells in the G2 phase was clear. Moreover, the relationship between DNA methylation and PAX was studied using a specific 5-mC ELISA kit, where an oscillatory behavior was observed. Finally, a profile of 118 miRNAs differentially expressed in MDA-MB-231/ 1000 nM and MDA-MB-231 was obtained, as well as their 1922 potential genes targets. The signature of miRNAs and their putative targets identified in our analysis might be used as biomarkers for an early-detection of PAX resistance in BC.
ABSTRACT: Breast cancer (BC) had the highest incidence of new cases of cancer worldwide in 2020, representing 11.7% of total new cases of cancer. Moreover, BC is the largest cause of cancer-related mortality in women. Despite the improvements in patients’ outcomes with chemotherapy, the development of cancer drug resistance (CDR) is an obstacle to a successful treatment. In fact, CDR is responsible for treatment failure in over 90% of patients with metastatic cancer. The mechanisms associated with CDR include the increase of drug efflux, mainly by ATP-binding cassette (ABC) transporters, and epigenetic modifications, such as aberrant DNA methylation and alterations in microRNAs (miRNAs) expression. In this project, we developed resistance to 400 nM, 800 nM, and 1000 nM of paclitaxel (PAX) in the MDA-MB-231 cell line. The resistance was monitored by the measurement of cell viability, through MTT, and changes in cell morphology, namely in the nucleus, lysosomes, and cytoplasm, which were detected by confocal microscopy. Resistant cells showed an increase in the size and number of nuclei and enlarged cytoplasm. At 1000 nM, an increase in the number of lysosomes was also detected. The effect of PAX in the cell cycle of resistant cells was evaluated by flow cytometry, where an arrest of the cell cycle of MDA-MB-231/ 1000 nM cells in the G2 phase was clear. Moreover, the relationship between DNA methylation and PAX was studied using a specific 5-mC ELISA kit, where an oscillatory behavior was observed. Finally, a profile of 118 miRNAs differentially expressed in MDA-MB-231/ 1000 nM and MDA-MB-231 was obtained, as well as their 1922 potential genes targets. The signature of miRNAs and their putative targets identified in our analysis might be used as biomarkers for an early-detection of PAX resistance in BC.
Descrição
Palavras-chave
Cancro da mama (CM) Resistência a fármacos em cancro (RFC) Paclitaxel (PAX) Metilação do ADN microRNA (miRNA) Epigenética Breast cancer (BC) Cancer drug resistance (CDR) Paclitaxel (PAX) DNA methylatio microRNA (miRNA) Epigenetics