Investigation of anticancer and antioxidant properties of bioactive compounds from different macroalgae species

The discovery of new anticancer agents plays a significant role in cancer treatment. Seaweeds (macroalgae) are among the natural sources of chemically active and cytotoxic metabolites. Therefore, the identification of stronger and more selective anticancer compounds isolated from brown, green, and red seaweeds, as well as studies on their mechanisms of action, have recently attracted attention and represent an important advancement for pharmacological applications. The aim of this thesis study is to investigate the anti-cancer effects, antioxidant and anti-inflammatory activities of some commercially available, purified and characterized secondary metabolites, hordenine, tyramine and 2-phenylethylamine, obtained from some macroalgae species, on cancer cell lines. The results of the in silico ADME/T analyses of these natural compounds showed that they can pass through both the gastrointestinal tract and the blood-brain barrier (BBB) and have pharmacologically active groups and properties with relatively high drug scores. In the initial phase of the study, the cytotoxic activies of the metabolites hordenine, tyramine, and 2-phenylethylamine, along with the positive control paclitaxel, were tested on 10 human cancer cell lines (HCT-116, CaCo-2, HT-29, HepG2, PANC-1, Ishikawa, HeLa, A172, A549, and TT) and 2 control cell lines (HEK293 and CCD-18Co). No cytotoxic activity of hordenine was detected in any of the tested cancer cell lines. On the other hand, tyramine and 2-phenylethylamine showed the highest cytotoxic activity on colon cancer cell lines (HCT-116, CaCo-2 and HT-29) and endometrial cancer cell line (Ishikawa). The EC50 concentrations for tyramine and 2-phenylethylamine were calculated as 64.63 μM and 84.50 μM, respectively, in the HCT-116 cell line, 126.90 μM and 156.23 μM in the CaCo-2 cell line, 204.90 μM and 208.71 μM in the HT-29 cell line, and 169.40 μM and 212.30 μM in the Ishikawa cell line. Tyramine treatment inhibited colony formation in HCT-116, CaCo-2 and HT-29 cell lines by 92%, 95% and 62%, and 2-phenylethylamine inhibited cell migration by 60%, 30% and 17%, respectively. Wound healing test results showed that tyramine and 2-phenylethylamine treatment inhibited HCT-116 cell migration by 20% and 18%, respectively, and CaCo-2 cell migration by 49% and 43%. Computational analyses suggest that this effect may have occurred through the inhibition of the MMP-9 enzyme. The determination of the apoptotic activities of the compounds tyramine and 2-phenylethylamine were carried out using two different methods. Flow cytometry results showed that EC50 concentrations of these compounds caused a significant difference in the number of live, dead, early and apoptotic cell populations in HCT-116, CaCo-2 and HT-29 cells compared to the negative control group. qRT-PCR analyses revealed that the expressions of the CASP 3, CASP 8, CASP 9, BAX, and P53 genes were induced, while the expression of the BCL-2 gene was suppressed, in a manner that indicates anticancer potential. The Griess test demonstrated that the increased NO levels in LPS-induced RAW264.7 cells showed a significant decrease with the application of hordenine, tyramine, and 2-phenylethylamine compared to the positive control group. Additionally, qRT-PCR analyses indicated that the expressions of the iNOS, COX2, IL-6, and TNF-α genes were significantly suppressed, while the expression of the IL-10 gene was increased, in a manner that suggests an anti-inflammatory effect. Antioxidant activities of hordinin, tyramine and 2-phenylethylamine were tested by five different methods, namely DPPH, ABTS, Galvinoxyl, FRAP and Phosphomolybdenum. The tested compounds were determined to have antioxidant activity only by the ABTS method. Using this method, the EC50 values for the standard antioxidants trolox and ascorbic acid were calculated to be 5.23 μM and 6.52 μM, respectively, while for hordinin, tyramine and 2-phenylethylamine, they were calculated to be 15.78 μM, 9.09 μM and 137.64 μM, respectively. The total GST, GSTT1 and GSTM1 isoenzymes, GPX and SOD enzyme activities and total thiol levels in HCT-116, CaCo-2 and HepG2 cells treated with EC50 doses of tyramine and 2-phenylethylamine were evaluated comparatively compared to the control group. Treatment with these alkaloids caused an increase in total GST, GPX and SOD enzyme activities and a decrease in total thiol levels in the HCT-116 cell line. The results of this thesis indicate that the metabolites tyramine and 2-phenylethylamine, obtained from macroalgae, exhibit significant cytotoxic, anti-metastatic, and apoptotic effects, demonstrating their high anticancer potential. Supporting this fact, hordenine, tyramine, and 2-phenylethylamine also showed significant anti-inflammatory and antioxidant properties.