Figure 3

Poly(A:U) and RMT5265 have cooperative cytotoxic effects on NPC cells. (A) MTT cell-growth assay for NPC cells and non-tumorigenic nasopharyngeal epithelial cells treated with poly(I:C) or poly(A:U) (0.5 μg/mL for C666-1 and HONE1, 1 μg/mL for CNE1 and NP69) used either alone or in combination with a Smac-mimetic RMT5265 (5 nM for C666-1, 50 nM for CNE1, HONE1 and NP69). Poly(A:U) is known to have greater specificity for TLR3 than poly(I:C) which also stimulates the MDA5 and RIG-I receptors[19]. Despite this more restricted action, poly(A:U) combined with RMT5265 had cytotoxic effects on NPC cells almost equivalent to poly(I:C) and no effect on NP69 cells. Excess over Bliss additivism was calculated as indicated in the Materials and Methods section. The results are given as mean +/− SD. (B) Detection of PARP cleavage by Western blot analysis. Proteins were extracted after 24 h of culture from NPC cells treated with poly(I:C) or poly(A:U) used either alone or in combination with RMT5265. The drugs were used at the same concentrations as for the WST/MTT assays. 40 μg of protein extracts were loaded on PAGE gels. The blotted membranes stained with anti-PARP antibodies were also stained with anti-tubulin for protein loading control. No massive PARP cleavage was observed in any experimental condition. However the most abundant cleaved products were seen when poly(A:U) or poly(I:C) were combined with RMT5265