Cell surface expression of TRAIL R1 and TRAIL R2 The susceptibility/resistance of cells to TRAIL/zVAD/ CHX or TNF/zVAD/CHX induced programmed selleck AZD9291 necro sis is initially dependent on the expression of the corre sponding receptors on the cell surface. We limited our analyses to cell surface expression of TRAIL R1 and TRAIL R2 on the tumor cell lines used in Inhibitors,Modulators,Libraries this study since TNF R1 is known to be expressed on the surface of every cell type in the human body except red blood cells. Whereas TRAIL R1 differentially showed pro nounced or low to no cell surface expression, TRAIL R2 was highly expressed on the sur face of all analyzed tumor cell lines. Since Guo and coworkers have shown that TRAIL R2 can mediate programmed necrosis by itself, this in dicates that susceptibility or resistance of the investi gated tumor cell lines to programmed necrosis is not determined at the level of receptor expression.
Expression of RIPK3 is a primary determinant Inhibitors,Modulators,Libraries for the susceptibility or resistance of tumor cell lines to programmed necrosis Programmed necrosis elicited through death receptors critically depends on the protein kinase RIPK1 and the related downstream kinase RIPK3. We therefore next determined the role of RIPK1 in the investigated tumor cell lines. Inhibitors,Modulators,Libraries In Inhibitors,Modulators,Libraries line with its essential role in pro grammed necrosis, pharmacological inhibition of RIPK1 by necrostatin 1 protected the same subset of sensitive tumor cell lines that we had used for analysis in Figure 1d from both TRAIL/zVAD/CHX and TNF/zVAD/CHX in duced programmed necrosis.
However, Western blots revealed ubiquitous expression of RIPK1 in all 14 can cer cell lines demonstrating that their sensitivity Inhibitors,Modulators,Libraries or resistance against programmed necrosis is not determined by presence or ab sence of RIPK1. In contrast to RIPK1, we found a differen tial expression of RIPK3 that largely correlated with the sensitivity of the tumor cell lines to TRAIL/zVAD/CHX or TNF/zVAD/CHX, Figure 1a and b. Specifically, RIPK3 was clearly expressed in the highly sensitive cell lines U 937, Mz ChA 1, BxPC 3 and HT 29 but barely detectable in the fully resistant cell lines SK OV 3, KNS 62, Pt45P1 and SK Mel28, whereas the less sensitive cell lines Colo357, Panc89 and PancTu I showed correspondingly reduced levels of RIPK3.
Pointing to factors independent from RIPK3 that additionally regu late the resistance of tumor cells against programmed ne crosis, MKN 28 cells expressed similar levels of RIPK3 as Colo357 or Panc89 cells but were resistant kinase inhibitor Perifosine to both TRAIL/ zVAD/CHX and TNF/zVAD/CHX. Likewise, despite strong expression of RIPK3, A818 4 cells responded only poorly to both TRAIL/zVAD/CHX or TNF/zVAD/CHX induced programmed necrosis, and CCRF CEM cells were select ively resistant to TRAIL/zVAD/CHX induced programmed necrosis, Figure 1a and b.