Discussion MPM is typically refractory to current treatment

7 dichlorohydrofluorescein diacetate, a specific ROS detecting fluorescent dye. As a result, staining intensity of carboxy H2DCFDA increased in both cell lines after poly transfection, and this increase was inhibited by adding the ROS scavenger N acetyl L cysteine. The NAC treatment apparently decreased the percentages of Annexin V cells in poly transfected SKRC 1 and SKRC 44 cells, indicating KU-55933 臨床試験 that ROS comprise a key mediator of apoptosis after poly transfection. We next examined the level of Ψm in these cells be cause its level is reversely associated with the intrinsic apoptotic pathway. We examined Ψm levels using the cyanine dye DiOC2 and found that the percent ages of cells with a high Ψm decreased by poly transfection. these decreased percentages were partially restored by NAC treatment.<br><br> We further in vestigated the relationship between Ψm level and Annexin V staining. As buy Linifanib shown in Figure 2d, poly transfection increased Annexin V RCC cells with a low Ψm, and the NAC treatment conversely decreased such cells but increased Annexin V− RCC cells with a high Ψm. These results indicate that poly transfec tion induced ROS generation and thereby induced apop tosis in RCC cells in association with decreased Ψm. ROS mediated DNA damage in poly transfected RCC cells ROS induce DNA damage, and phosphorylation of his tone H2A. X is an indicator of DNA double strand breaks. Therefore, we next determined whether DNA damage was induced in poly trans fected RCC cells in a ROS dependent manner. Figure 3a shows that poly transfection increased phosphoryl ation ofH2A.<br><br> X in both cell lines, and that adding of NAC alleviated its expression. Similar results were observed in immunoblotting. These results indicate that ROS are responsible for DNA dam age in poly transfected RCC cells. LY3009104 1187594-09-7 DNA damage induces p53 activation, which can ultim ately lead to apoptosis. Therefore, we examined the levels of total and phosphorylated p53, as well as NOXA and Puma in poly transfected RCC cells. Although p53 protein levels de creased 12 or 24 h after poly transfection, phos phorylated p53 increased transiently in both cell lines from 2 to 12 h after poly transfection, but decreased thereafter, likely due to degradation of total p53. Although NOXA expression increased shortly after p53 activation, Puma expression began to decrease 12 h after poly transfection, exhibiting similar kinetics to those of total p53.<br><br> Additionally, tBid expression began to increase 4 or 12 h after poly transfection. These re sults indicate that poly transfection initially trig gered p53 activation in conjunction with NOXA, but degraded p53 thereafter. Furthermore, poly trans fection caused the conversion of Bid into tBid, leading to caspase 8 activation in poly transfected RCC cells. RNA interference was then performed to examine the role of p53 in poly transfection induced apop tosis. Transfection of p53 siRNA decreased p53 protein expression in both cell lines. Additionally, p53 knockdown decreased apoptosis in SKRC 44 cells, but not in SKRC 1 cells. Moreover, the ef fects of p53 knockdown on p53 target molecules were also examined.