Assessment of in vitro anticancer and apoptotic activities of some plant extracts

In this thesis, cytotoxic effects of methanolic extracts of 7 plants, 3 of which are endemic species to Turkey, on Vero [African Green Monkey (Cercopithecus aethiops) kidney fibroblast cell line], HeLa (Human cervical adenocarcinoma cell line), A549 (Human lung carcinoma cell line), Mcf-7 (Human breast adenocarcinoma cell line) and PC-3 (Human prostate adenocarcinoma cell line) cells were determined by Mitochondrial Dehydrogenase Enzyme Activity (MTT) Colorimetric Method. In order to confirm the cytotoxic effect of Centaurea hermannii plant extract having potent anticancer activity and the chemotherapeutic agent Doxorubicin on Vero and HeLa cell lines, cell-index parameters were determined with xCELLigence RTCA DP system. Later, apoptotic activity of the extract with potential anticancer activity was then assessed as biochemically (flow cytometry analysis), fluorometrically (analysis of caspase 3,7 and 9 activation) and molecularly (qRT-PCR analysis). When the results obtained at 48th hour in MTT assay evaluated, the low cytotoxicity of C. hermannii extract in healthy cell line Vero and its selective high cytotoxicity in cervical cancer cell line HeLa showed that C. hermannii extract had potent anticancer activity. IC50 (The extract concentration causing 50% of the cells to die) and SI (Selectivity index; SI= IC50 of Vero cells/ IC50 of HeLa) values of Doxorubicin, the chemotherapeutic agent, and C. hermannii extract on Vero and HeLa cell lines were calculated by using the obtained data from cytotoxic effects of the chemotherapeutic agents Doxorubicin and C. hermanni extract on Vero and HeLa cell lines were calculated. The selectivity index of C. hermannii plant extract and the selectivity index of Doxorubicin were compared. IC50 values were found in the Vero cell line as 599 μg/mL for C. hermannii and 21.2 μM for Doxorubicin, 15.74 μg/mL for C. hermannii and 8.4 μM for Doxorubicin in the HeLa cell line. SI values were found as 38.1 for C. hermannii and 2.5 for Doxorubicin, respectively. As the results of the analysis of cell index parameters with xCELLigence RTCA DP system, IC50 values were found as 557.8 μg/mL for C. hermannii and 19.2 μM for Doxorubicin in the Vero cell line, 18.3 μg/mL for C. hermannii and 7.8 μM for Doxorubicin in the HeLa cell line. SI values were calculated 30.5 for C. hermannii and 2.5 for Doxorubicin. The data obtained from the MTT analysis were consistent with the morphological evaluation as well as the results of the analysis with the xCELLigence RTCA DP system. The determination of the apoptotic activity of C. hermannii extract with potential anticancer activity in HeLa cells was demonstrated by 3 different methods. In the experiments, two concentrations which were nontoxic to Vero cells but toxic to HeLa cells (100 μg/mL) and the concentration causing 50 % of the HeLa cells to die (IC50= 15.74 μg/mL) were applied. The number of apoptotic HeLa cells treated with C. hermannii extract was determined by flow cytometry analysis with FITC-labeled Annexin V. The alive, dead and late apoptotic populations of HeLa cells treated with 100 μg/mL and IC50 concentrations of C. hermannii plant extract were significant in comparison to the control group and this plant extract was found to have apoptotic effect in HeLa cells. The results were found to be consistent with the cytotoxicity results. After determining the apoptotic acvitiy of C. hermannii extract on HeLa cells by flow cytometry analysis, the effect of C. hermannii extract on the activation of caspases 3,7 and 9 in HeLa cells was determined by the caspase activity assay, another important method for determining apoptotic activity. C. hermannii extract enhanced the activation of the lead actors of the apoptotic cascade; caspase-9, the initiator caspase and caspase-3,7 the executer caspases in HeLa cells was determined, spectroflorometrically. Finally, qRT-PCR analysis of expression levels of apoptosis, necrosis and autophage specific genes was determined in HeLa cells treated with C. hermannii extract. According to the results obtained by qRT-PCR analysis, the apoptotic genes FAS, HRK and TNFRSF10B were upregulated in HeLa cells at both concentrations of C. hermannii plant extract. RIPK from necrotic genes, was over-regulated at a highly toxic concentration of 100 μg/mL of C. hermannii plant extract in HeLa cells. RIPK was also upregulated at the IC50 concentration of C. hermannii plant extract. BECN from autophagic genes, was upregulated in HeLa cells at both concentrations of C. hermannii plant extract, ATG3 and ATG5 genes were upregulated only at 100 μg/mL concentration of C. hermannii plant extract. These results suggest that the C. hermannii extract may represent a potentially active new drug for cervical cancer treatment and may support more studies due to promising cytotoxic and apoptotic activity.