## 4 Discussion
Although several plant species belonging to the Lamiaceae family have shown in vivo antitumor activity [36; 37; 38], thus far, the _C. vulgare_ plant extracts have not been studied in animal cancer models. The results obtained in our study demonstrating selective anticancer effects on a panel of cell lines and the observed inhibition of cancer cell migration could serve as a good basis for conducting future in vivo experiments with laboratory animals to confirm the safety and efficacy of _C. vulgare_ extracts and validate their antitumor effects in more complex cancer model systems. Despite the increasing amount of experimental evidence revealing the potential of _C. vulgare_ extracts to decrease tumor cell survival and proliferation, the mechanisms underlying their anticancer effects remain unclear. A recently published study investigated the involvement of the apoptosis pathway as a possible mechanism of the anticancer effects of a methanolic extract of the plant on the A549 human lung carcinoma cell line [25]. The results of the performed analysis showed that the treatment of the tumor cells with _C. vulgare_ methanolic extracts did not lead to discernible alteration in the expression of the apoptosis-related genes Bax and Bcl-2. Moreover, the flow cytometric analysis of treated tumor cells stained with Annexin V/propidium iodide indicated that the observed anticancer effects were due to the induction of necrosis rather than apoptosis [25]. In contrast, applying three different fluorescent staining methods in combination with cell cycle analysis, the present study clearly demonstrates the ability of the tested aqueous extract obtained from in vitro cultured _C. vulgare_ plants to induce cancer cell apoptosis. The discrepancies between our findings and the lack of detectable apoptotic alterations in the previous study [25] could be explained with the use of different extraction procedures and different in vitro cancer models. Apoptotic cells are characterized by the occurrence of specific alterations in the cellular and nuclear morphology, allowing their identification by cytomorphological methods [39]. The AO/EB and DAPI staining used in our study showed chromatin condensation, nuclear fragmentation, membrane blabbing and the formation of apoptotic bodies in the HeLa cells treated with the extract from in vitro cultured _C. vulgare_ plants, demonstrating the ability of the tested extract to induce apoptosis. The proapoptotic activity of the tested _C. vulgare_ extract was also confirmed by fluorescence microscopy after staining with Annexin V/propidium iodide. This method allows the detection of apoptosis-specific alterations in the cell membrane expressed in the translocation of phosphatidylinositolserine from the inner to the outer surface of the plasma membrane. The results of our analysis reveal the presence of early and late apoptotic cells in the HeLa cell cultures treated with the _C. vulgare_ extract. Cell cycle analysis was performed to further elaborate on the molecular mechanisms of the detected cytotoxicity, inhibition of cell migration and proapoptotic activity. The flow cytometry results additionally suggest the induction of apoptosis by the _C. vulgar_ extract as evidenced by the increase in the sub-G1 population. Moreover, statistically significant and concentration-dependent alterations in the distribution of cells in the different phases of the cell cycle were identified in the extract-treated cell cultures compared to the untreated control. The accumulation of cells in the S and G2/M phases and the decrease in the G1 cell population could be explained by the activation of checkpoint and repair mechanisms in response to cellular stress or damage induced by the _C. vulgar_ extract. Further detailed molecular studies of the potential of _C.