The treatment of MGC803 and
HGC27 cells with SPARC siRNA increased early apoptotic cells as well as late apoptotic cells, compared with negative control siRNA treatment (Figure 4A) as measured by the Annexin V assay. As expected, the decreased survival #PI3K activator randurls[1|1|,|CHEM1|]# of the cells transfected with SPARC siRNA was associated with increased rates of apoptosis by 91% in MGC803 and 92% in HGC27 cells (Figure 4B). These findings suggest that SPARC is involved in apoptosis to maintain cellular survival in some gastric cancer cells. Figure 4 SPARC knockdown results in induction of apoptosis in gastric cancer cell lines. For flow cytometric analysis, cells were harvested 96 h after transfection with SPARC siRNA or negative control siRNA, then stained with annexin V-FITC and propidium iodide (PI). the left half data represent data obtained from MGC 803 cells and the right ones represent data obtained from HGC 27 cells. The percentages SAHA HDAC in vivo of annexin V/PI(early apoptotic) and annexin V/PI(late apoptotic) cells is shown
in each panel. Values in bold indicate decreasing SPARC expression increased apoptosis by 65% in MGC803 and 92% in HGC27 compared with negative control siRNA. Apoptotic effect of SPARC siRNA transfected treatment in MGC 803 and HGC27 cells In an effort to elucidate the mechanism of SPARC siRNA induced apoptosis in MGC 803 cells and HGC27 cells, expression levels of apoptotic-regulation proteins such as Bcl-2, Bax and caspase-3 and PARP were evaluated. MGC 803 cells and HGC27 cells were transfected with SPARC siRNA. As shown in Figure 5, There were significant differences in the expressions of Bax
and Bcl-2 in MGC 803 cells and HGC27 cells in comparison with the negative control group (P < 0.05 and P < 0.01). In response to apoptotic stimuli, procaspase-3 is cleaved into a 20 kDa fragment, and the subsequent autocatalytic reaction leads to the formation of the active 17 kDa fragment. When Olopatadine the caspase-3 is activated, PARP is cleaved. Thus cleavage of PARP is used as an indicator of apoptosis. In order to obtain direct evidence showing the relationship of caspase activation and apoptosis, procaspase-3 cleavage and PARP were examined in MGC 803 cells and HGC27 cells after SPARC siRNA transfected. As shown in Figure 5, SPARC SiRNA induced the cleavage of 32 kDa procaspase-3 into its active 17 kDa form and cleavage of PARP appeared in MGC 803 cells and HGC27 cells. Figure 5 The expression of apoptosis proteins in MGC 803 and HGC27 cells after transfection with either control or SPARC siRNA. The cell lysates were separated on 10% SDS-PAGE gel, transferred to nitrocellulose membrane and probed with anti-PARP, anti-caspase-3, anti-Bcl-2, and anti-Bax. Protein contents were normalized by probing the same membrane with anti-β-actin.