Briefly, cells were washed with PBS and resuspended in binding buffer followed by the addition of fluorescence conjugated Annexin V and 5 g/ml PI and then a 30 minute incubation in the dark. Cells were observed under a fluorescence microscope at 488 nm. TUNEL assay TUNEL assay was performed using an in situ Cell Death Detection Kit, following the manufacturer,s guidelines. In brief, cells AZD1152-HQPA were fixed with 4% paraformaldehyde and permeabilized after washing. Cells were then rewashed and incubated with the TUNEL assay reaction mixture for 1 hour at 37 and 95% air humidity. Samples were then analysed under a fluorescence microscope at 488 nm. Results A prominent growth inhibitory effect of Syzygium cumini extract on these two cervical cell lines was observed. Different dilutions of the extract exhibited growth inhibition in a dosedependent manner in both cell lines tested with the MTT assay. While the 40% concentration of the extract exhibited 14.
4% and 11.8% growth inhibition, the 80% concentrated extract showed 30.3% and 23.2% growth inhibition, respectively, in HeLa and SiHa cell lines. The 100% concentration did not show any significant effect over the 80% Brivanib alaninate concentration. These results also indicate that the extract is more effective on HeLa than on SiHa cells. Hoechst 33342 staining indicated condensed chromatin, the characteristic pattern of apoptosis, for both the cell lines under experiment. The apoptotic indices for these cell lines were calculated as mentioned earlier. It has been observed that a gradual increase of apoptotic index followed according to the treatment duration. Apoptotic cell counts were carried out at 24 hours, at 36 hours and at 48 hours.
It has also been noticed that the methanolic extract was less effective as compared to the crude extract at the same concentration and the same exposure time. The result for SiHa is shown in. To distinguish the nuclear and membrane changes of the cells during apoptosis and also to investigate the necrotic cell death simultaneously, dual staining of cells with Annexin V and PI was carried out with Annexin V FLUOS/ PI staining. No PI staining was observed after 24 hours treatment, ruling out the possibility of necrosis, but fluorescence due to Annexin V binding to phosphatidylserine on the outer cell membrane of the apoptotic cells was observed. Figure 3B represents the Annexin V binding assay for HeLa cells at 48 hours. The TUNEL assay was performed to detect the DNA strand breaks associated with apoptosis.
The typical DNA strand breaks were detected in apoptotic cells after 24 hours treatment as strong fluorescence, these were absent in controls. Figure 3C represents the TUNEL assay micrograph for SiHa cells at 48 hours. Discussion Cervical cancer is the second cause of death from cancer in women worldwide, and the pathogenesis is well identified. The treatment options generally followed are mainly surgery with or without adjuvant radiotherapy and chemotherapy, which often results in severe side effects. Medicinal plants play a major role in folk medicine in several developing countries.