Effects and mechanisms of combined suppression of epidermal growth factor receptor and hedgehog signaling on proliferation in pancreatic cancer cells

Abstract

To explore the synergistic effects on proliferation and apoptosis by targeted suppression of epidermal growth factor receptor (EGFR) in combination with blockade of Hedgehog signaling pathways in pancreatic cancer cells and examine the synergistic mechanism of Hedgehog and EGFR signaling pathways. The sequences of RNA interference targeting EGFR gene were designed, synthesized and cloned into the pFU-GW-RNAi vector. And a stable transfection cell line was obtained by transfecting the human Panc-1 cells with lentivirus. The expressions of Shh and Gli1 were tested by real-time polymerase chain reaction (PCR). The antiproliferative effect was examined by the assays of colony formation and methyl thiazolyl tetrazolium (MTT). Fluorescence activated cell sorter (FACS) was applied to assay the apoptotic rate in all experimental groups. Western blot was applied to detect the phosphorylation levels of ERK and AKT.In vivo nude mice tumorigenicity model was used to test the effect of growth inhibition. The RNAi technology with lentivirus could restrain the expression of EGFR gene. After the blocking of EGFR and Hedgehog signaling pathways by RNAi silencing, the chemosensitivity to cyclopamine significantly increased in human pancreatic cancer cells. The half-inhibitory concentration (IC 50) of cyclopamine declined from (2.978 ± 0.336) to (1.698 ± 0.057) µmol/L (P < 0.05). The prophase apoptotic rate of co-treated group was as high as 38.75% and it was significantly higher than the RNAi silencing EGFR (17.65%) and control groups (3.02%) (P < 0.05). The results of tumor xenografts assay showed that the tumor volume of co-treated group (394.8 ± 87.5 mm(3)) was significantly lower than that of simple EGFR RNAi (594.7 ± 86.1 mm(3)) and single cyclopamine treated group (771.3 ± 82.9 mm(3)); the combination treatment could also produce obviously synergistic antiproliferative effect in colony formation assays. After RNAi silencing EGFR, the phosphorylation levels of ERK and AKT decreased significantly versus the control group. Further reduction was obtained with the combined use of cyclopamine in the co-treated group. The blocking of EGFR and Hedgehog signaling pathways by RNAi silencing may further inhibit cell proliferation and increase apoptosis in vivo and in vitro in human pancreatic cancer cells. The synergism of Hh and EGFR signaling pathways may be correlated with the phosphorylation levels of ERK and AKT.