Dados do Resumo

Título

Functional Analysis of MUTYH Expression in Heterozygous and Homozygous Contexts under Chemotherapeutic Exposure and Oxidative Stress

Introdução

MUTYH-associated polyposis (MAP) is an autosomal recessive hereditary condition affecting individuals with biallelic pathogenic variants in the MUTYH gene. This condition is primarily associated with a high risk of developing colorectal cancer with polyposis. The risk for individuals carrying monoallelic pathogenic variants is not fully understood. However, there are reports suggesting that the monoallelic condition may be associated with an increased risk for certain types of cancer, particularly gastric cancer. The protein encoded by the MUTYH gene is involved in DNA repair, correcting single-strand damage caused by oxidative stress.

Objetivo

To assess cell viability and oxidative stress in gastric cancer cell models in wild-type and pathogenic variants, both monoallelic and biallelic, using MTT and immunofluorescence assays.

Métodos

Using previously established cell lines through CRISPR-Cas9 (PE25A7 and PE25D1 – biallelic pathogenic variants; PE25D2 and PE25D3 – monoallelic pathogenic variants; PE25A6 and PE25D4 – wild-type MUTYH), triplicate MTT assays were performed to assess cell viability in the six established lines under exposure to the chemotherapeutic drug fluorouracil at different concentrations (ranging from 10µM to 100µM) over 24, 48, and 72 hours. This assay evaluates the mitochondria’s ability to convert MTT salt into formazan crystals, which can be quantified colorimetrically, allowing assessment of each cell line's viability. Oxidative stress was assessed using triplicate immunofluorescence assays to evaluate nuclear damage by staining for oxidized guanine (8-oxo-G) under oxidative stress induced by potassium bromate (KBrO3) at concentrations of 20mM for 30 minutes, with and without 1 hour of recovery in all six established cell lines.

Resultados

In the cell viability assays, it is expected that cells with intact MUTYH will show higher viability compared to those with partial or complete loss, with a gradual decline over time and increasing concentration. However, in the first 24 hours, no treatment response was observed. After 48 and 72 hours, a decrease in cell viability was noted, though differences between clones and experiments were observed, necessitating further standardization for improved results. In the immunofluorescence assays, it is expected that cells with intact MUTYH will be able to repair the damage induced by KBrO3, resulting in less damage compared to cells with partial or complete loss. Preliminary observations showed that wild-type and heterozygous clones experienced oxidative stress followed by recovery, whereas homozygous clones exhibited stress but unclear recovery, requiring further standardization and reanalysis of the experiment.

Conclusões

Through the functional assays employed, we aim to gain a better understanding of MUTYH function under the established conditions, which may guide future research and therapeutic approaches.

Financiador do resumo

CNPq - National Council for Scientific and Technological Development; INCT – National Institute of Science and Technology

Palavras Chave

MUTYH-associated polyposis; gastric cancer; 8-oxo-G