Análise da morfologia de superfície e propriedades mecânicas de células T24 de câncer de bexiga tratadas com quercetina

Abstract

Atomic force microscopy (AFM) analysis has become a powerful tool for studying cells and tissues. The possibility of obtaining information regarding elasticity, roughness, and surface morphology on a nanoscale, in a non-destructive way, has drawn the attention of the scientific community. In cancer cells, mechanical information is an important parameter informing the tumor's essential functions. The physical properties of cancer cells have been linked to adhesion, differentiation, invasiveness, and cellular motility. In the present research, we analyzed quercetin's anticancer activity by assessing the morphological changes in bladder cancer cells (T24). In addition to performing preliminary tests, using T24 cells in their native conditions, to create an adequate protocol for sample preparation to obtain more reliable results. To do so, first, we determine critical periods for cell maintenance in culture, and post-fixation waiting periods, to reduce biases related to the protocol. Analyzes were divided into two parts: 1st - determine the maintenance time in cell culture in which it is possible to observe the normal degradation process of T24 cells by AFM (12h, 24h, and 48h in culture). 2nd - Demonstrate the impact of the elapsed time between sample fixation and analysis in the microscope, in periods of 0h, 12h, 24, and 48h. Our results demonstrate that, after 48hr of maintenance in cell culture, cells begin to undergo morphological changes, decreased elasticity, and increased roughness. Regarding the post-fixation waiting time, we observed slight morphological differences after 12hr from fixation, however, there was a significant decrease in elasticity in addition to increased roughness. In 24h and 48h, the changes related to elasticity and roughness are maintained and are accompanied by drastic changes in surface morphology, such as the collapse of the plasma membrane. Thus, data from the first part of the present study demonstrate the importance of establishing critical periods of culture and postfixation waiting to reduce technical artifacts related to the protocols used. In a second phase, and as the main objective of the study, we analyzed the anticancer effect of quercetin on T24 cells, through AFM, evaluating the mechanical and morphological properties of the cells. Cells were subjected to different quercetin doses (40, 60, and 80 mM) and analyzed by AFM at 24hr and 48hr after treatment. As we increased the quercetin dose, we observed that cells became significantly more rigid and rough as biophysical marks of quercetin anticancer action T24 cells. Our data demonstrate, for the first time, the mechanical and morphological changes of quercetin anticancer effects T24 cells, analyzed by AFM.