Investigation of synergistic anticancer effects of vulpinic acid and different chemotherapeutic drugs on breast cancer cell lines at gene and protein levels

This thesis was conducted to analyze the synergistic anticancer effect of different chemotherapeutic drugs (cisplatin, docetaxel, doxorubicin, and mitoxantrone) on human breast cancer cell lines (MCF-7, MDA-MB-231, and MDA-MB-453) at the gene and protein levels. In the first step, the cytotoxic effects of vulpinic acid and each of the chemotherapeutic drugs on the above-mentioned cell lines were investigated in a dose- and time-dependent manner. The absorbance values obtained here were then processed in the CompSyn program and the combination index (CI) value of vulpinic acid with each chemotherapeutic drug was determined. The CI values showed that vulpinic acid had a synergistic effect with doxorubicin and mitoxantrone on MDA-MB-231 and MDA-MB-453 cells, and an antagonistic effect on MCF-7 cells. In the second phase, changes in ROS levels, mitochondrial membrane potential, multi-caspase activity, cell cycle, apoptosis and PI3K/AKT signalling pathway were examined by flow cytometric analysis in cell lines in which the compounds showed synergistic effects. The results showed that there was an increase in the amount of ROS, mitochondrial membrane depolarization, multicaspase activity and apoptotic cell rate in both cell lines, as well as G0/G1 cell cycle arrest and PI3K/AKT pathway blockade. In the third step, to confirm the results of the flow cytometry analysis at the molecular level, quantitative changes in marker proteins of the apoptotic pathway (BAX, BCL2, P53, Caspase-3, Cleaved caspase-3, Cytochrome-c) and the PI3K/AKT signaling pathway (PI3K, mTOR and AKT) were examined at the protein and gene levels by Western blot and real-time PCR (qPCR). The data obtained showed that apoptotic effects in cells occurred at both the gene and protein levels, while the suppression of the PI3K/AKT signaling pathway occurred at the protein levels. Finally, as a result of migration tests using the wound healing method, it was found that the combinations of compounds applied to both cell lines had significant anti-migratory effects. As a result, these data have opened the door to in vivo studies of combinations of vulpinic acid with doxorubicin and mitoxantrone, which show synergistic effects in vitro on breast cancer cell lines.