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Estudo do efeito do Palbociclib e do Tipifarnib em linhas celulares de carcinomas anaplásicos da tiróide
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O cancro da tiróide é a neoplasia mais comum do sistema endócrino. Os carcinomas da tiróide que derivam das células foliculares englobam os carcinomas bem-diferenciados (WDTC), os carcinomas pouco diferenciados (PDTC) e os carcinomas indiferenciados ou anaplásicos (ATC). Os WDTC subdividem-se em carcinomas papilares (PTC) e foliculares (FTC). Os ATC representam 2-3 % dos carcinomas da tiróide, sendo considerados os tumores mais agressivos da tiróide, para os quais não existem actualmente terapêuticas eficazes. O nosso grupo identificou, em estudos anteriores, os genes TP53, RAS, CDKN2A (p14ARF/p16INK4A) e CDKN2B e (p15INK4B) e vias relacionadas, como desempenhando papéis essenciais no desenvolvimento de ATC.
As proteínas p15INK4B e p16 INK4A funcionam como supressores tumorais inibindo as CDK4/6 e levando à paragem do ciclo celular. O Palbociclib inibe as CDK4/6, sendo esperado que a sua acção compense a perda funcional dos genes CDKN2A e CDKN2B. Por outro lado, mutações no gene HRAS conduzem à activação constitutiva da via MAPK-ERK e, consequentemente, ao desenvolvimento tumoral. A isoforma HRAS tem de ser farnesilada para adquirir actividade oncogénica, representando um alvo molecular do composto Tipifarnib, um inibidor da farnesil transferase (FTI).
O objectivo principal deste projecto foi identificar novas terapêuticas direccionadas para alvos moleculares específicos envolvidos no desenvolvimento de ATC, tendo sido estudados dois novos compostos, o Palbociclib e o Tipifarnib.
Para estudo do seu efeito nas diferentes linhas celulares foram realizados ensaios de viabilidade metabólica celular (pelo método MTS), ensaios de ciclo e morte celular (por citometria de fluxo) e viabilidade celular (pelo método de exclusão com azul de tripano). Foram usadas três linhas celulares de ATC: C643 (mutações nos genes TP53, HRAS e delecção dos genes CDKN2A e CDKN2B), T238 (mutações nos genes TP53, CDKN2A, BRAF e PIK3CA) e T235 (mutação no gene BRAF); uma linha celular de PTC designada BCPAP (mutações nos genes BRAF e TP53); e células normais de tiróide PCCL3.
Nos ensaios de ciclo celular e de viabilidade celular realizados com o Palbociclib observou-se, respectivamente, um aumento significativo de células na fase G0/G1 do ciclo celular e uma diminuição do número de células viáveis ao longo do tempo, nas linhas celulares de ATC e PTC, mas não nas células normais de tiróide.
Nos ensaios realizados com o Tipifarnib observou-se um efeito do composto apenas na linha celular de ATC, com mutação no gene HRAS, que consistiu no aumento significativo de células na fase G2/M e na diminuição do número de células viáveis ao longo do tempo. Os resultados preliminares do Tipifarnib quanto à expressão do mRNA de genes envolvidos na regulação do ciclo celular sugeriram, em concordância com os resultados dos ensaios de ciclo celular, que este composto levou à paragem do ciclo na transição G2/M, pelo aumento observado na expressão dos genes CCNA2, CDK2 e CDC2.
Os resultados deste estudo sugerem que o Palbociclib e o Tipifarnib poderão ter um papel importante na inibição da progressão tumoral. A continuação deste trabalho poderá contribuir para clarificar quais os mecanismos de acção e a relevância destes compostos como terapêuticas para os ATC.
Thyroid cancer is the most common malignancy of the endocrine system. Thyroid carcinomas, which derivate from follicular cells include well-differentiated carcinomas (WDTC), poorly differentiated carcinomas (PDTC) and undifferentiated or anaplastic carcinomas (ATC). The WDTC are subdivided into papillary carcinomas (PTC) and follicular carcinomas (FTC). ATC represent 2-3% of thyroid carcinomas, being considered the most aggressive thyroid tumours, for which there are currently no effective therapies. In previous studies, our group identified the genes TP53, RAS, CDKN2A (p14ARF/p16INK4A), CDKN2B, (p15INK4B) and related pathways as playing essential roles in the development of ATC. The p15INK4B and p16 INK4A proteins act as tumour suppressors by inhibiting CDK4/6 and leading to cell cycle arrest. Palbociclib inhibits CDK4/6, so it is expected that its action compensates the functional loss of CDKN2A and CDKN2B genes. On the other hand, HRAS gene mutations lead to constitutive activation of the MAPK-ERK pathway, and thus to tumour development. The HRAS isoform have to be farnesylated to acquire oncogenic activity, representing a molecular target for Tipifarnib compound, a farnesyl transferase inhibitor (FTI). The main objective of this project was to identify novel therapies directed at specific molecular targets involved in the development of ATC. In particular, we aimed to study the effect of two compounds, Palbociclib and Tipifarnib, in ATC cell lines. Metabolic cell viability (MTS assay), cell cycle and death (flow cytometry) and cell viability (trypan blue exclusion method) were assessed to study the effect of these compounds on the different cell lines. Three ATC cell lines were used: C643 (with TP53 and HRAS gene mutations and deletion of CDKN2A and CDKN2B genes), T238 (with TP53, CDKN2A, BRAF and PIK3CA gene mutations) and T235 (with BRAF gene mutation); one PTC cell line designated BCPAP (with BRAF and TP53 gene mutations); and normal thyroid cells (PCCL3 cells). In the cell cycle and viability assays performed with Palbociclib, a significant cell arrest in the G0/G1 phase and a decreased number of viable cells through time, were observed in ATC and PTC cell lines. These effects were not observed in normal thyroid cells. In the study of Tipifarnib, significant effects were only observed in the ATC cell line with an HRAS mutation, which consisted in an arrest in the G2/M phase of cell cycle, and in a decrease in the number of viable cells through time. Preliminary results of the study of Tipifarnib effect in mRNA expression of genes involved in cell cycle regulation (CCNA2, CDK2 and CDC2 genes), were in agreement with the results obtained in the cell cycle assays, suggesting that this compound led to cycle arrest in the G2/M transition. The results of this study suggest that Palbociclib and Tipifarnib may play a major role in inhibiting tumour progression. The continuation of this work may help to clarify the mechanisms of action and the relevance of these compounds as therapies for ATC.
Thyroid cancer is the most common malignancy of the endocrine system. Thyroid carcinomas, which derivate from follicular cells include well-differentiated carcinomas (WDTC), poorly differentiated carcinomas (PDTC) and undifferentiated or anaplastic carcinomas (ATC). The WDTC are subdivided into papillary carcinomas (PTC) and follicular carcinomas (FTC). ATC represent 2-3% of thyroid carcinomas, being considered the most aggressive thyroid tumours, for which there are currently no effective therapies. In previous studies, our group identified the genes TP53, RAS, CDKN2A (p14ARF/p16INK4A), CDKN2B, (p15INK4B) and related pathways as playing essential roles in the development of ATC. The p15INK4B and p16 INK4A proteins act as tumour suppressors by inhibiting CDK4/6 and leading to cell cycle arrest. Palbociclib inhibits CDK4/6, so it is expected that its action compensates the functional loss of CDKN2A and CDKN2B genes. On the other hand, HRAS gene mutations lead to constitutive activation of the MAPK-ERK pathway, and thus to tumour development. The HRAS isoform have to be farnesylated to acquire oncogenic activity, representing a molecular target for Tipifarnib compound, a farnesyl transferase inhibitor (FTI). The main objective of this project was to identify novel therapies directed at specific molecular targets involved in the development of ATC. In particular, we aimed to study the effect of two compounds, Palbociclib and Tipifarnib, in ATC cell lines. Metabolic cell viability (MTS assay), cell cycle and death (flow cytometry) and cell viability (trypan blue exclusion method) were assessed to study the effect of these compounds on the different cell lines. Three ATC cell lines were used: C643 (with TP53 and HRAS gene mutations and deletion of CDKN2A and CDKN2B genes), T238 (with TP53, CDKN2A, BRAF and PIK3CA gene mutations) and T235 (with BRAF gene mutation); one PTC cell line designated BCPAP (with BRAF and TP53 gene mutations); and normal thyroid cells (PCCL3 cells). In the cell cycle and viability assays performed with Palbociclib, a significant cell arrest in the G0/G1 phase and a decreased number of viable cells through time, were observed in ATC and PTC cell lines. These effects were not observed in normal thyroid cells. In the study of Tipifarnib, significant effects were only observed in the ATC cell line with an HRAS mutation, which consisted in an arrest in the G2/M phase of cell cycle, and in a decrease in the number of viable cells through time. Preliminary results of the study of Tipifarnib effect in mRNA expression of genes involved in cell cycle regulation (CCNA2, CDK2 and CDC2 genes), were in agreement with the results obtained in the cell cycle assays, suggesting that this compound led to cycle arrest in the G2/M transition. The results of this study suggest that Palbociclib and Tipifarnib may play a major role in inhibiting tumour progression. The continuation of this work may help to clarify the mechanisms of action and the relevance of these compounds as therapies for ATC.
Descrição
Palavras-chave
Biologia molecular Tumores Cancro da tiróid Neoplasia Alvos moleculares Palbococlib Tipifarnib Linhas celulares
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical