MUTANT P53 AGGREGATES IN HEPATOCELLULAR CARCINOMA CELL LINES AND THEIR IMPORTANCE IN PATHOGENESIS: CHARACTERIZATION OF A NEW PHARMACOLOGICAL TARGET

Vol 1, 2020 - 131588
MS - Master's student
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Abstract

Introduction and objective: Hepatocellular carcinoma (HCC) is the sixth most recurrent cancer and the fourth responsible for mortality among all cancers in the world. Depending on the tumor’s stage, treatments may vary from chemotherapy, ablation therapy and transplant. Nonetheless, even with all these options, the treatment is arduous and frequently unsuccessful due to the advanced stage of identification of tumors and high mortality rate. Before this scenario, it is necessary to search for new targets, such as p53. The tumor suppressor protein, p53, is also described as "the guardian of the genome" owing to its capacity to protect the genome and to avoid the appearance of cancer. However, mutations in this protein are present in more than 50% of cancers, contributing to tumor progression, reduced patient survival and therapeutic resistance. In most cases, these mutations result in amyloid aggregation, supporting oncogenic gains-of-function and negative dominance, which promotes wild-type p53, loss of function and aggregation. Therefore, the aim of this study is to evaluate the role of mutant p53 aggregation in HCC, regarding the formation, transmission, and functions of these aggregates in these cells, in order to contribute to the characterization of this new pharmacological target.
Material and methods: The total cell extracts of Hep3B (p53 null), HepG2 (wild-type p53), Huh-7 (p53 Y220C) and PLC/PRF/5 (p53 R249S) cell lines were analyzed and incubated with anti-amyloid antibody (A11) and anti-p53 antibody (DO-1) for immunoprecipitation (IP) assay, western blotting (WB), and dot-blot (DB) analyses. The cells were fixed and labeled with antibodies for confocal fluorescence microscopy. The viability of HepG2, in spheroids and monolayer, treated with the conditioned medium (CM) was evaluated by acid phosphatase (APH) and MTT assay, respectively. Wound healing assay was performed to observe the migration of cells and analyzed with the ImageJ software. Clonogenic assay was staines with crystal violet and colonies were counted manually.
Results and conclusion: HepG2, Huh-7 and PLC/PRF/5 cell lines express p53 in distinct levels, whereas Hep3B does not express, confirmed by WB. DB results revealed that all cell lines have amyloid aggregates, although the IP assays showed the coimmunoprecipitation of p53 in its amyloid state only in mutant cell lines. It was possible to observe the presence of p53 amyloid aggregates in Huh-7 and PLC/PRF/5 cell lines by confocal fluorescence microscopy. In MTT and APH assays, changes in viability in cells expressing wild-type p53 treated with CM, both in monolayer and spheroid were analyzed. In the WH experiment, HepG2 migration increased after treatment and, in the clonogenic assay, there was a reduction of the formation of MC-treated HepG2 colonies. With these results, we expect to increasingly understand this phenomenon in order to elucidate a new pharmacological target for a new more effective HCC treatment.

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Author

Mariana Muniz da Paz

Boa tarde, Daniela.

Obrigada pelas perguntas!

Ainda não existem muitos trabalhos abordando os efeitos desses agregados sobre o microambiente tumoral. Inclusive, no nosso trabalho, pretendemos avaliar o papel desses agregados no microambiente tumoral.  Com o intuito de elucidar se há transmissão e alguma alteração no funcionamento de células que compõem o microambiente.

A p53 mutante consegue alterar a conformação da p53 selvagem, por um fenômeno chamado de dominância negativa, levando à perda de função e, por outro lado, a ganhos de função oncogênica (através da indução da transcrição de novos genes e da interação com proteínas diferentes).

Author

Nathalia Oliveira da Silva

Olá! Boa tarde!

Nosso objetivo é fazer esses mesmos experimentos utilizando outras linhagens celulares, inclusive de carcinoma hepatocelular de humanos! 

Temos outros projetos no laboratório que utilizam essas linhagens inclusive. 

Author

Mariana Muniz da Paz

Boa tarde, Antonio

  • Acha que seria valido, avaliar o impacto do tratamento quimioterápico concomitante com o tratamento com MC?  

Com certeza! Estamos realizando os experimentos com a doxorrubicina e a cisplatina.

  • Em sua opinião, o que foi observado no trabalho, pode representar um mecanismo amplo de carcinogênese, ou seria algo restrito apenas ao hepatocarcinoma?  

Já foi descrito na literatura sobre os efeitos prion-like da p53 mutante em outros modelos de linhagem celular. Em relação a capacidade de transmissão dos agregados, até o momento, parece representar um mecanismo amplo. Mas, em relação ao comportamento funcional das células receptoras, os resultados de cada experimento podem variar conforme o ganho de função daquela mutação específica.

  • Não seria interessante utilizar outras linhagens com as mesmas mutações, uma vez que a utilização de uma única linha poderia ter um viés biológico “linhagem específico”?

Ótima sugestão. Podemos avaliar futuramente, uma vez que a mutação p53 Y220C, por exemplo, pode ocorrer em carcinoma de células escamosas da orofaringe, carcinoma ductal invasivo da mama, adenocarcinoma seroso ovariano de alto grau, entre outros.

 

Obrigada pelas perguntas!

Institutions
  • 1 Universidade Federal do Rio de Janeiro
Track
  • Cellular Biology
Keywords
hepatocellular carcinoma
p53 amyloid aggregation