## 3 Results and Discussion
### SG4 Increased DNA Fragmentation and Annexin V Binding to Membranes in HT-29 Cells  
The activation of caspase-3 seems to play a critical role in inducing apoptosis and DNA fragmentation (an indicator of late-stage apoptosis) [37]. Here, the detection of chromatin condensation and DNA fragmentation in cells was performed by DAPI staining and flow cytometric-based TUNEL staining, respectively. The results of DAPI staining in Figure 5a suggested that treatment of HT-29 cells with 200 ug/mL SG4 for 48 h resulted in nuclear condensation (bright blue), indicating that they were undergoing apoptosis (white arrows in Figure 5a). In addition, as shown in Figure 5b,c, when HT-29 cells were treated with 200 ug/mL SG4 for 48 h, the high fluorescent cell population significantly increased from 3.17% \(\pm\) 0.25% (control) to 21.4% \(\pm\) 0.5% (\(p<0.001\)), indicating SG4-mediated DNA fragmentation occurs. Previous investigations showed that, when inducing apoptosis, cells lose plasma membrane asymmetry, and the phosphatidylinositol(PS) residues become exposed at the outer plasma membrane [38]. Annexin V specifically binds to PS, and this can be utilized to detect apoptosis by monitoring the loss of plasma membrane integrity [38]. Here, we used an annexin V-FITC/PI staining analysis for determining the percentage of dead cells (cells in early-stage or late-stage apoptosis, as well as in necrosis) by flow cytometry. As shown in Figure 6a,b, exposure of HT-29 cells to 200 ug/mL SG4 for 48 h resulted in an increase from 0.03% \(\pm\) 0.02% (control) to 9.42% \(\pm\) 0.02% (\(p<0.001\)) in the percentages of early apoptotic cells, an increase from 0.08% \(\pm\) 0.02% (control) to 25.3% \(\pm\) 0.5% (\(p<0.001\)) in the percentages of late apoptotic cells, an increase from 0.73% \(\pm\) 0.13% (control) to 9.90% \(\pm\) 0.23% (\(p<0.001\)) in the percentages of necrotic cells, and a reduction from 99.1% \(\pm\) 0.2% (control) to 55.4% \(\pm\) 0.3% (\(p<0.001\)) in the percentages of live cells. These findings clearly suggest that SG4 causes  
Figure 4: The expression of active caspase-9 and -3 in HT-29 cells treated with 200 ug/mL SG4 for 48 h: (**a**) In the histograms, \(x\) axis and \(y\) axis denote caspase-9 content and cell counts, respectively. The percentage of cells with mean channel fluorescence values above the marker border is indicated in each histogram; (**b**) the percentage of cells with high fluorescence of caspase-9 is presented as determined for (**a**). (**c**) In the histograms, \(x\) axis and \(y\) axis denote caspase-3 content and cell counts, respectively. The percentage of cells with mean channel fluorescence values above the marker border is indicated in each histogram; (**d**) the percentage of cells with high fluorescence of caspase-3 is presented as determined for (**c**). The experiments were performed in triplicate. *** \(p<0.001\).  
HT-29 cell death (consisting of apoptosis and necrosis) and apoptosis is predominantly involved. Taken together, SG4 induced colon cancer cell apoptosis as revealed by DNA fragmentation and annexin V-FITC/PI staining, and therefore, SG4 may have potential for use as a natural agent in a preventive therapy for colon cancer.  
Figure 5: The induction of chromatin condensation and DNA fragmentation in HT-29 cells treated with 200 \(\upmu\)g/mL SG4 for 48 h. (**a**) Morphological and DAPI-staining assessments of HT-29 cells treated with 200 \(\upmu\)g/mL SG4 for 48 h. Arrows in the graph indicate nuclear condensation (bright blue) (scale bar, 50 \(\upmu\)m). (**b**) In the histograms, \(x\) axis and \(y\) axis denote BrdU content and cell counts, respectively. The percentage of cells with mean channel fluorescence values above the marker border is indicated in each histogram; (**c**) the percentage of cells with high fluorescence of BrdU is presented as determined for (**b**). The experiments were performed in triplicate. ***\(p<0.001\).