## 3 Results
### Fluorescence Microscopy  
To investigate the mechanisms underlying the detected suppressive effects on cancer cell growth, HeLa cells treated for 24 h with the extract of in vitro cultured plants and stained with AO/EtBr were analyzed by fluorescence microscopy. The cellular morphology of the cancer cells treated with _C. vulgare_ extract markedly differed compared to the control (Figure 4). Untreated HeLa cells were homogenously green-stained and displayed normal monolayer growth and typical morphological characteristics (Figure 4a). Fluorescent staining with the DNA-binding dye AO allowed the detection of numerous cells in a phase of mitosis. In contrast, in the cell cultures treated with _C. vulgare_ extract, a large number of red-stained dead and late apoptotic cells with condensed chromatin and fragmented nuclei were observed, and the monolayer confluency was greatly reduced (Figure 4b). The observed morphological alterations demonstrate the ability of the tested extract to inhibit proliferation and induce apoptotic cell death.  
For a more detailed study of the nuclear morphology of tumor cells treated with the extract from in vitro cultured _C. vulgare_, fluorescence microscopic analysis was performed after staining with the DNA-binding dye DAPI. The nuclei of untreated cells were oval-shaped, with smooth edges and homogenous blue staining (Figure 4d). After treatment with the _C. vulgare_ extract, cells with shrunk nuclei, condensed chromatin, and intense fluorescent staining (early apoptotic cells), as well as nuclear fragmentation and the formation of apoptotic bodies (late apoptosis), occurred (Figure 4e).  
Figure 4: Cytomorphological alterations induced by the extract of in vitro cultured _C. vulgare_ plants in HeLa cervical carcinoma cells. (**a**,**d**) Negative control (untreated cells); (**b**,**e**) cells treated with _C. vulgare_ extract (600 ug/mL); (**c**,**f**) positive control (cells treated with 2.25 ug/mL doxorubicin); (**a**–**c**) AO/EB staining; and (**c**–**f**) DAPI staining. Fluorescence microscopy; objective 40\(\times\).  
The cytomorphological alterations induced by the tested extract were similar to the changes observed in HeLa cells treated with the standard cytostatic drug doxorubicin (Figure 4c,f). Doxorubicin was also applied at a concentration equal to the IC\({}_{50}\) value determined by the MTT assay.  
To confirm the ability of the studied extract to induce apoptotic cell death, annexin V/propidium iodide fluorescent staining was applied (Figure 5). In control untreated tumor cells, no fluorescence was observed (Figure 5a), as the cell membrane was impermeable to propidium iodide and FITC-conjugated annexin V. After treatment with the extract from in vitro cultured _C. vulgare_, the tumor cell cultures showed the presence of early apoptotic cells with green, fluorescent staining (Figure 5b) due to the translocation of phosphatidylserine from the inner to the outer surface of the cell membrane, which makes possible the binding of fluorescently labeled annexin V.  
Late apoptotic cells were also identified, with green-labeled membranes and red-stained nuclei (Figure 5c), as in the late stages of apoptosis, the loss of membrane integrity allows propidium iodide to enter the cell and bind to nuclear DNA. The results of the AV/PI fluorescence microscopic analysis of HeLa cervical carcinoma cells are consistent with the results obtained by the other cytomorphological methods used and are proof of the ability of the studied extract of in vitro cultured _C. vulgare_ plants to induce tumor cell death via apoptosis.