Kolon kanseri hücreleri üzerine timokinon ve 5-fluorouracilin terapotik etkisinin in vitro ve in vivo araştırılması / In vitro and in vivo investigation of the therapeutic effect of thymoquinone and 5-fluorouracil on colon cancer cells

Abstract

Cancer is a major public health issue worldwide. In developed countries, colon cancer is the 2nd most common observed type of cancer in women and the 3rd most common in men. The 30% success of 5-FU, which is a routine treatment for colon cancer, has led the researchers to seek different treatment modalities. Therefore, plant-derived natural substances have become the target of a significant amount of attention. In recent years, increasing efforts have been made to discover the effects of combined plant-derived substances with conventional therapy methods on cancer. This study aimed to investigate the cytotoxic, genotoxic, apoptotic, and anti-cancer effects of thymoquinone – the active ingredient of Nigella sativa – and 5-Fluorouracil in combination against in vitro and in vivo colon cancer while illuminating possible action mechanisms. Initially, Luciferase transfection was performed in LoVo colon cancer cells to which TQ, 5-FU, and combinations of different concentrations were given, and they were incubated for 24 hours. Luminometric cytotoxicity by ATP method, DNA damage by comet assay, luminometric intracellular glutathione level, fluorometric mitochondrial membrane potential, apoptosis by fluorescence microscopy with acridine orange/ethidium bromide dye, apoptosis in flow cytometry by annexin V-FITC dye, expression of pro-apoptotic and anti-apoptotic proteins with western blot method have been investigated. Transfected LoVo cells have been injected subcutaneously to five groups of nude mice for the following: control, TQ, 5-FU, and combined. 3 weeks of treatment with TQ, 5-FU, and combined therapy have been initiated 3 weeks after the injection. At the end of this period, tumor size was measured with the IVIS device and caliper. Also, oxidative stress and inflammation markers were examined in blood and growth factors, and vascularization markers were examined in tissue samples at the end of the treatment. Compared to the monotherapy with TQ and 5-FU on colon cancer, combined treatment has been found in low doses to increase cytotoxicity, DNA damage, apoptosis and intracellular reactive oxygen species in the cell culture studies, while decreasing mitochondrial membrane potential and glutathione levels. Also, the expression of apoptotic proteins Bax, Caspase-3, Caspase-9, p53, and p21 increased while the anti- apoptotic protein Bcl-2 expression decreased. Combination therapy was found to be more effective than mono therapies in vivo colon cancer, which was formed by the xenographic method. While tumor size decreased, TGFβ1 levels and VEGF levels – a vascularization indicator – were decreased in the tissue. In the plasma taken after treatment, oxidative stress levels decreased compared to the positive control group. According to the data obtained through this study, in colon cancer TQ has been found to increase the anti-tumor properties of the routine therapy of 5-FU in vitro and in vivo while diminishing the side effects profile.