control; #p 0

control; #p 0.05 vs. stained using Hoechst 33342, and apoptotic body was counted under a fluorescence microscope. The number of apoptotic cells was expressed as a percentage of the total number of cells counted. *p 0.05 vs. control; #p 0.05 vs. MDL-12330A treated and CTL RNAi cells. MDL-12330A upregulates DR5 expression Since the extrinsic apoptotic pathway seems to play an important role in MDL-12330A-induced apoptosis, we then explored whether membrane death receptors are involved in the apoptotic mechanism. When we measured the levels of mRNAs encoding the apoptosis-inducing death receptors, DR4 and DR5, in SNU601 cells, a concentration-dependent increase in the mRNA expression of DR5, but not DR4, was observed. In agreement with this result, the protein level of DR5 was also strongly induced in all three GC cell lines upon exposure to MDL-12330A. In order to confirm the role of DR5 in MDL-12330A-induced apoptotic cell death, we knocked down the expression of DR5 using a small interference RNA specifically targeting DR5 and examined the ability of MDL-12330A to induce apoptosis. Upon exposure of SNU601 cells to 20 M MDL-12330A, the apoptotic rate was approximately 27%; silencing of DR5 partially inhibited apoptosis, Fendiline hydrochloride whereas, as expected, DR4 silencing had no effect on Fendiline hydrochloride apoptosis. It should be noted that DR5 silencing only partially prevented apoptosis induced by 20 M MDL-12330A (about 34.4%), whereas at a lower concentration of MDL-12330A (10 M) the effect on apoptosis was more apparent (about 78.1%), although the apoptotic rate induced by 10 M MDL-12330A was lower than that induced by 20 M MDL-12330A. These results suggest that at low concentrations of MDL-12330A apoptosis is primarily mediated via a DR5-mediated pathway whereas at higher concentrations of MDL-12330A additional DR5-independent apoptotic pathways also operate. CHOP mediates MDL-12330A-induced DR5 induction in gastric cancer cells To search for the factors responsible for mediating MDL-12330A-induced DR5 expression, we selected several candidate proteins based on published data and evaluated their potential role in MDL-12330A-induced DR5 expression using RNA interference. Previously published reports have shown that several transcription factors regulate the expression of DR5 including nuclear factor-kB, p53, and C/EBP homologous protein (CHOP) [17,18,19,20]. In Fendiline hydrochloride this study, we excluded p53 as a possible regulator of DR5 expression since SNU601 and SNU638 cells express mutant p53 proteins. As shown in Fig. 4A, we observed that silencing of CHOP suppressed the MDL-12330A-induced DR5 induction in SNU601 and SNU638 cells. Furthermore, MDL-12330A increased CHOP expression level in both of these cells (Fig. 4B). Therefore, MDL-12330A appears to increase DR5 expression through a CHOP-activated pathway. Since the activation of CHOP is suggested to be regulated by ER stress, we then examined whether MDL-12330A induces ER stress by detecting ER stress markers such as glucose regulate protein (GRP) 78/BiP and PERK. As detected in Fig. 4C, MDL-12330A induced Fendiline hydrochloride BiP and p-PERK levels in SNU601 and SNU638 cells, indicating that MDL-12330A can activate ER stress response. Open in a separate window Fig. 4 MDL-12330A-mediated DR5 expression is regulated by CHOP.(A) SNU601 or SNU638 cells were transfected with a scrambled small interfering RNA (CTL RNAi), RNAi, CHOP RNAi, and em C-EBP /em RNAi, and then treated with 20 M MDL-12330A (MDL) for 24 h. Cell lysates were prepared and analyzed by immunoblotting to assess DR5 expression. Silencing effect of each siRNA was confirmed by immunoblotting in vehicle treated control samples. Rabbit Polyclonal to EDG4 (B, C) SNU601 or SNU638 cells were exposed to the indicated concentrations of MDL-12330A for 16 h and cell lysates were Fendiline hydrochloride analyzed by immunoblotting with an antibody to CHOP (B), and to BiP and p-PERK (C). Alpha-tubulin was used as a loading control. Antitumor effect of MDL-12330A is independent from inhibition of AC activity Since MDL-12330A is developed to be an AC inhibitor, we explored whether other AC inhibitors can induce similar effects on GC cells. However, cell viability and DR5 expression were not affected in the presence of AC inhibitors NB001 or NKY80 in SNU601 and SNU638 cells (Fig. 5). Furthermore, NB001 or NKY80 did not induce BiP expression in these cells (Fig. 5B). Thus, these results suggest that the anticancer effects and ER stress response induced by MDL-12330A may not result from inhibition of AC activity. Open in a separate.