Avaliação do fenótipo de célula-tronco tumoral e da dinâmica mitocondrial em células tumorais colorretais refratárias ao tratamento com 5-fluorouracil

Abstract

Colorectal cancer (CRC) is the malignant neoplasm that affects the large intestine, being the second most incident and the third most lethal type of cancer worldwide. Although therapeutic strategies have developed in recent years, almost half of patients suffer from relapses, metastases, and resistance to conventional treatments. 5-fluorouracil (5-FU) is the most widely used chemotherapeutic in CRC therapies, but around half of metastatic patients have resistance to this drug. In this context, cancer stem cells (CSCs) are a tumor subpopulation of cells, with self-renewal and differentiation capabilities, which are closely associated with metastasis and treatment resistance. In its turn, the mitochondria are organelles that play an important role in cancer, presenting a morphological dynamism through the processes of mitochondrial fusion and fission, as well as biogenesis and mitophagy. Changes in the balance of the processes of mitochondrial dynamics are known to have effects on CSCs and therapeutic resistance. Therefore, this work aimed to analyze the CTT phenotype and the mitochondrial dynamics of RCC tumor cells refractory to 5-FU treatment. For this purpose, the colorectal tumor cell line HT-29 was exposed to 5-FU and the refractory cells were analyzed for (1) morphology, by light and fluorescence microscopy, (2) expression of genes related to the “stem phenotype” and mitochondrial dynamics, by RT-qPCR; (3) expression of CD133 (colorectal CTT marker), by flow cytometry and immunocytochemistry; and (4) ultrastructure, by transmission electron microscopy. The results show that refractory cells exhibit greater cell size and complexity, with more vacuolation, multinucleation and structures compatible with entosis. Furthermore, CD133 gene and protein expression decreased in the refractory cell population. However, these cells expressed CD133 much more intensely, also demonstrating greater size and granulosity. As for mitochondrial dynamics, levels of PINK1 mRNA (mitophagy gene) were found to be higher in the treatment-refractory group. Ultrastructure results showed increased vacuoles, lipid inclusions, damaged mitochondria and autophagosome structures in refractory cells. Therefore, this work showed that colorectal tumor cells refractory to treatment with 5-FU have a lower fraction of CD133+ cells, but with high expression per positive cell, as well as increased size and granulosity, in addition to presenting features that suggest an increase in the process of mitophagy.