Investıgatıon of the potentıal effects of Erıodıctyol and Brusatol wıth 5-Florouracıl on cell vıtalıty and apoptosis ın HT-29 human colon cancer cell

Cancer is one of the major causes of death. Due to the side effects of traditional methods used for the treatment of cancer, studies on developing new strategies to prevent or treat cancer have gained momentum. In recent studies, the importance of a combined approach in cancer treatment has been emphasized. Nature has a great wealth of active and safe anti-cancer agents, which mankind has applied to since ancient times. Colorectal cancer is among the most frequently diagnosed cancers. Combination or multicomponent therapy is traditionally indicated for the treatment of this disease alongside surgery and chemotherapy as a potential way to improve treatment and delay side effects. In this study, it was aimed to determine the effects of commercial Eriodictyol and Brusatol together with 5-FU anticancer drug in human colon cancer/colorectal cancer cell line (HT-29 cell line) on cell viability for the first time as far as we know, as well as to evaluate the potential effects on apoptosis. For this purpose, first of all, the effects of separate and combined applications of active substances on cell viability in the HT-29 cell line were investigated depending on dose and time with the MTT experiment, and the IC50 value of each active substance was determined. Considering the IC50 values, the effects of the individual and combined applications of the active substances on cell viability in the HT-29 cell line were evaluated for 24, 48 and 72 hours, and the combination index values were calculated. The effects of combination treatments with IC50 and IC50/2 concentrations of Brusatol or Eriodictyol and IC50 concentrations of 5-FU on apoptosis were investigated for 48 hours by Annexin V/PI staining and Real-Time PCR analysis of apoptotic genes. In addition, the effect of the combined applications of the active substances on the cell cycle profile was also investigated for 48 hours. The lowest IC50 values were calculated for Brusatol. The IC50 values of Brusatol calculated for 24, 48 and 72 hours are 109.5, 1.15 and 0.96 µM, respectively. CI values calculated in combination applications differ. Among the combined applications of Brusatol and 5-Fluorouracil, it was determined that the IC50 and IC50/2 value of Brusatol and the combined applications of 5-FU with the IC50 and IC50/2 value of the combined applications showed a synergistic effect in 24 and 48 hours. It has been observed that the effect of combined applications with the IC50/2 concentration of Eriodictyol and the IC50 and IC50/2 concentrations of 5-FU in 48 hours, as well as the IC50 and IC50/2 value of Eriodictyol concentrations, is synergistic. Unlike Brusatol, a synergistic effect was determined in the combination of 5-FU with Eriodictyol for 72 hours. The triple combinations of Brusatol, Eriodictyol and 5-FU tested were found to be antagonists for each incubation time. It was determined that the percentage of total apoptotic cells in combined applications of Brusatol with 5-FU was higher than the percentage of total apoptotic cells caused by the active substances alone. According to the results of real time PCR analysis, a significant increase in the level of bax and caspase-3 apoptotic genes among the tested genes was detected in combination with the IC50 concentrations of Brusatol and 5-FU. However, it was determined that Brusatol and Eriodictyol alone caused an increase in the percentage of cells in HT-29 cells compared to the control, especially in the S phase. In the combination applications of Brusatol with 5-FU, it was observed that the percentage of cells, especially in the S phase, was higher than the control. Data from experiments for 48 hours showed that Brusatol, alone or in combination with 5-FU, can cause an increase in the percentage of apoptotic cells in HT-29 cells compared to Eriodictyol. The data obtained from this study can provide basic data for combination or other alternative approaches, especially against colon cancer. In vivo studies will contribute to studies on the use of these active substances.