On the contrary, the strategy of targeted apoptosis to neoplastic cells using dietary phytochemicals having immunostimulative, antioxidant, anti neoplastic, apoptotic and other physiological benefits are showing promising results in in vitro studies for several cancers. Apoptotic and growth inhibitory effects of curcumin, aloe emodin, resveratrol, Cyclopamine retinoic acid, lycopene and the tea polyphenol EGCG in HeLa and other cervical cancer cell lines is well documented. In this study, we evaluated the potential growth inhibitory and apoptotic effects of Syzygium cumini extract on two cervical cancer cell lines. Materials and methods Syzygium cumini extract Crude extracts were isolated from wild type partially ripe fruit skin along with the outermost layer of the berry, and then serial dilutions of the extract from 100% to 10% were made using PBS for MTT assays. Methanol extracts were prepared for other experiments by blending 5 g of skin with 50 ml of methanol at 4. After incubation at 37 for 15 minutes, the extract was centrifuged at 3000 rpm for 10 minutes at 4.
The supernatant was filtered, and the filtrate was vacuum dried and stored at 4. For use the dried extract was dissolved BMS-582664 in DMSO and diluted with culture medium to a final concentration of 80%. Cell culture Human cervical carcinoma cell lines HeLa and SiHa were cultured in DMEM medium in 96 well plates at 37 with 5% CO2 and air humidity 95%. Exponentially, growing cells were used for experiments. A haemocytometer was used for cell counting using the formula: Average count/4 ? 104 ? 10 1.05 ? 106 cells/ml Total number of cells 1.05 ? 106 cells/ml ? 5ml 5.25 ? 107 cells MTT assay The cytotoxic effect of Syzygium cumini crude extract on HeLa and SiHa cells was initially determined by MTT assay. In a 96 well plate, 0.25 ml cultures were added to 0.1 ml medium and incubated for two hours.
Fifty l of 100% or 10% extract were added to specific test wells, 50 l PBS was used as a control. After 24 hours of culture 100 l of MTT solution was added to each well and left for 90 minutes. Culture were then washed with 0.1 ml of 10% SDS in a low speed shaker for 3 hours and then centrifuged at 100 rpm for 5 minutes. The absorbance of 0.9 ml of cell supernatant was read at 570 nm against a blank. The growth inhibition was measured using the following formula: Percentage inhibition ? 100. The average growth inhibition percentages of triplicates are represented in Figure 1. Morphological features of apoptosis were evaluated with Hoechst 33342 staining, TUNEL catalysed dUTP nick end labelling, and Annexin V FLUOS/PI binding assays using phase contrast and fluorescence microscopy.
Hoechst 33342 staining HeLa and SiHa cells seeded in chamber slides overnight were treated with 80% Syzygium cumini extract. After 24, 36 and 48 hours of treatment, the medium was removed and the cells were washed twice with PBS. Cells were then fixed in 3% paraformaldehyde for 30 minutes, washed and stained with Hoechst 33342 at 37 for 30 minutes in the dark. After three washes with PBS, mounted slides were viewedunder a fluorescence microscope at 450 490 nm. The percentage of apoptotic cells was determined by cell counting, taking the mean count of five microscopic fields. Annexin V FLUOS/PI assay Annexin V FLUOS/PI Staining Kit was used to determine membrane morphology of treated cells according to the manufacturer,s protocol.