Paeoniflorin Potentiates The Inhibitory Effects Of Erlotinib On Pancreatic Cancer Via Reducing ErbB Phosphorylation

Objective: Pancreatic cancer remains one of most prevalent malignancies.60%-80% of pancreatic cancer patients are diagnosed at an advanced stage with only 5% of 5-year survival rate after palliative surgery.In recent years,EGFR receptor tyrosine kinase inhibitor therapy has become new hotspots in pancreatic cancer therapy and erlotinib becomes the only FDA-approved EGFR receptor tyrosine kinase inhibitor for advanced pancreatic cancer.But the inhibition of EGFR tyrosine kinase by erlotinib can be restored through the other member of EGFR receptor family Erb B3,resulting in erlotinib-resistant and leading to limit application of erlotinib.Therefore,to potentiate the efficacy and decrease the resistance of antitumor drugs has become a hot research points at home and abroad in recent years.We studied the common herbs used by patients with pancreatic cancer and found white peony root(bai shao)significantly increased inhibitory activity of erlotinib against pancreatic cancer cells in vitro,so in this study,we further explored the effects of Paeoniflorin on pancreatic cancer cells and the interaction of Paeoniflorin and erlotinib.In this study we explored the effect of Paeoniflorin(PF)on potentiating anti-tumor activity of erlotinib in vivo and in vitro,and the molecular mechanism of the synergy with erlotinib in human pancreatic cancer cell lines.This research provides guidance for enhancing the anti-tumor effect and reversing the resistance of erlotinib clinically.Results 1.The effects of Paeoniflorin on enhancing anti-tumor activity of erlotinib in high Erb B3 expression of pancreatic cancer cell line Bx PC-3 and L3.6pl.Trypan blue assay showed the inhibition rates of Paeoniflorin(50umol)alone in pancreatic cancer cells line Bx PC-3,L3.6pl were 15.2%,25.4% at 48 h.Inhibition rates were 39.7%,43.1% with erlotinib(5nmol)alone.The inhibition rates of combined the two drugs were 55.7%,61.7% respectively.MTS and colony formation assay also showed that the inhibition rate of combined group was greater than that of either erlotinib or Paeoniflorin alone.2.Paeoniflorin potentiated the effect of erlotinib on apoptosis induction and cycle arrest in high Erb B3 expression Bx PC-3 and L3.6pl cell lines.The apoptosis detection showed Paeoniflorin(50umol)potentiated apoptosis induction of erlotinib(5nmol)at 48 h.The apoptosis rate was significantly increased in combined group compared with either drug alone(P < 0.001,P < 0.001).Cellcycle analyzed using flow cytometry showed Paeoniflorin could not enhance cycle arrest in G1 phase of erlotinib in pancreatic cancer cell line Bx PC-3,L3.6pl.(both P values less than 0.05).3.The molecular mechanism of the synergy of Paeoniflorin combination with erlotinib Paeoniflorin reduced Erb B3 protein expression expression and phosphorylation of pancreatic cancer cell lines Bx PC-3 and L3.6pl.After the high expression of phosphorylated EGFR family receptors in pancreatic cancer cell lines S2VP10 were knocked down with micro RNA expression,the down-regulation effects of Paeoniflorin on p-Akt protein disappeared.4.The effect of Paeoniflorin on enhancing anti-tumor activity of erlotinib in vivo.The results showed that Paeoniflorin enhanced tumor growth inhibition of erlotinib in Bx PC-3 xenografts nude mice,and reduced expression of p-Erb B3 protein in vivo(P < 0.001).5.Paeoniflorin and erlotinib did not have obvious inhibition functions in no Erb B3 expressed pancreatic cancer cell line Mia Paca.Mia Paca was treated at same concentrations and hours by Paeoniflorin and erlotinib alone and combined.Trypan blue assay,MTS proliferation assay and colony formation assay showed that neither alone nor combined Paeoniflorin and erlotinib had obvious inhibitory activity on no Erb B3 expressed pancreatic cancer cell line Mia Paca.Flow cytometry analyses of apoptosis and cell cycle showed the same results.Conclusions: 1.Paeoniflorin can dose-dependently inhibited cell viability,induced apoptosis and increased cell cycle arrest in Erb B3 highly expressed pancreatic cancer cell lines.While the no Erb B3 expressed cell lines were not affected.2.Paeoniflorin enhances cell viability inhibition,apoptosis induction and cell cycle arrest activity of erlotinib in Erb B3 highly expressed pancreatic cancer cell lines associated with the inhibition of phosphorylation of Erb B3,inhibition of Erb B3 / PI3 K / Akt signaling pathway activation.