We then performed xenograft experiments in which HCT116 p53+/+ or p53�C/�C cell lines expressing shMyD88 were implanted subcutaneously into nude mice. Only in HCT116 p53+/+ cells, a marked reduction in tumor growth (fold tumor increase: 4.9��0.1 vs 23.1��4.7; P = .01) (Figure 2A), accompanied by TUNEL staining (Figure 2B), was observed upon doxycycline administration, Cabozantinib manufacturer indicating that p53 is required for MyD88 knockdown-mediated apoptosis of cancer cells in vivo. To rule out the possibility that the different outcomes of MyD88 knockdown are due to the differences in growth kinetics (Figure 2A), this parameter was measured in five cell lines used in this study. We found that HCT116 p53�C/�C and LS513 cells had similarly slow rates of proliferation (Supplementary Figure 2B, available online).

However, the susceptibility of LS513 cells is similar to that of HTC116 p53+/+, and not that of p53�C/�C, cells (Figure 1, ,AA and andC).C). This makes it highly unlikely that the difference in the proapoptotic effect of MyD88 inhibition between p53+/+ and p53�C/�C cells is due to differences in growth kinetics. Figure 2. Effect of MyD88 silencing on p53-dependent tumor inhibition in vivo. A) Growth of HCT116 p53+/+ or p53�C/�C cells expressing doxycycline-inducible MyD88 short hairpin RNA (shMyD88) implanted subcutaneously in nude mice, treated or not treated … Mechanisms of Apoptosis Following MyD88 Silencing We then silenced MyD88 using siRNA in HCT116 p53+/+ and p53�C/�C cell lines in the presence or absence of Z-VAD-FMK, a general caspase inhibitor.

We show that Z-VAD treatment statistically significantly inhibits the apoptosis (Figure 3A) induced by MyD88 silencing (controls in Supplementary Figure 3A) in the HCT116 p53+/+ cell line (29.9%��3.8% vs 66.4%��4.9%; P = .001), indicating that this apoptosis is caspase-dependent. This is consistent with the observation that cleavage Carfilzomib of caspase 3 and 9, as well as of PARP, is only detected upon MyD88 silencing in the HCT116 p53+/+, but not in p53�C/�C cells (Figure 3B), further underscoring a role for p53 in apoptosis induction. Figure 3. MyD88 extinction, Erk-MAPK signaling, and caspase-dependent apoptosis. A) Apoptosis of HCT116 p53+/+ or p53�C/�C cell lines upon MyD88 silencing by small interfering RNA (siRNA) and treatment with either caspase inhibitor ZVAD or its control … We had previously shown that MyD88 is implicated in two major pathways: the TLR/IL-1R pathway known to predominantly activate NF-��B, and the canonical Ras pathway, which activates the Erk/MAP kinase (5).