Fig. 4

GLIPR1-ΔΤΜ synergized with docetaxel in activating JNK/c-Jun through JNK phosphorylation, whereas addition of GLIPR1-ΔΤΜ to docetaxel reduced ERK1/2 signaling. a,b. VCaP and PC-3 cells were treated with 1nM docetaxel, 10 μg/ml GLIPR1-ΔTM, or both and then, we added JNK inhibitor (SP600125) 1 μΜ to single agents or to their combination. Total treatment administration lasted for 24 h (VCaP cells) or 48 h (PC-3 cells), and the effects on JNK and ERK1/2 signaling were evaluated via western blot. JNK phosphorylation was increased synergistically with the combination of docetaxel and GLIPR1-ΔΤΜ, a pathway that leads to apoptosis; whereas ERK1/2 phosphorylation, which results in drug resistance and migration through c-Myc-CXCR4, was reduced by administration of this combination. These activities were reversed by the JNK inhibitor, SP600125. Western blot experiments were conducted three times and the quantitative data are presented as supplementary data. c,d. Under the same conditions and treatments in both cell lines, we performed MTS and DNA fragmentation assay to evaluate the percentage of survived cells and cell apoptosis, respectively. We found that, in both cell lines, the combination treatment of docetaxel and GLIPR1-ΔΤΜ resulted in statistically significant decrease in the percentage of survived cells and increase of apoptotic cells (VCaP: p < 0.001 compared to both single agents in MTS assay, p = 0.02 compared to GLIPR1-ΔΤΜ and p < 0.001 compared to docetaxel according to DNA fragmentation assay, PC-3: p = 0.001 compared to both single agents in MTS assay, p < 0.001 compared to both single agents according to DNA fragmentation assay) but this observation was reversed significantly when we added the JNK inhibitor (SP600125) to the combination docetaxel and GLIPR1-ΔΤΜ (p < 0.001 for both cells lines according to both techniques). The results are presented as the mean ± standard error from at least three independent experiments. e. Signaling effects of the combination treatment of docetaxel and GLIPR1-ΔΤΜ. Docetaxel induces JNK phosphorylation (apoptosis pathway); whereas it concomitantly induces ERK1/2 phoshorylation (drug resistance and migration pathway). JNK and ERK1/2 pathways can demonstrate reciprocal inhibition. GLIPR-ΔΤΜ induces JNK signaling but inhibits the ERK1/2-c-Myc-CXCR4 resistance loop. Thus, docetaxel and GLIPR1-ΔΤΜ combination treatment leads to JNK pathway dominance over the ERK1/2 pathway, and apoptosis dominates over drug resistance and invasion/migration