Emodin sensitizes human pancreatic cancer cells to EGFR inhibitor through suppressing Stat3 signaling pathway

Abstract

Background: Excessive expression of EGFR is closely related to tumor formation, transfer and deterioration, which has attracted much attention. EGFR overexpression may be detected in up to 90% of pancreatic tumors. However, drug resistance of EGFR inhibitors targeting treatment severely limits its clinical application.

Methods: In this study, Western blotting was used to detect the expression of p-Stat3, EGFR, Bcl-2, cleaved-caspase3 and Bax. Cell apoptosis was evaluated via flow cytometry. The colon assay and MTT assay were applied for detecting the cell proliferation in vitro. The xenograft mouse model was used to examine the cell proliferation in vivo.

Results: Emodin remarkably enhanced the anti-cancer effect of EGFR inhibitor on pancreatic cancer cells. In addition, emodin promoted afatinib-induced apoptosis by inhibiting the Stat3 signaling pathway. Meanwhile, siRNAs against Stat3 significantly increased the apoptosis of pancreatic cancer cells. EGFR inhibitor promoted phosphorylation of Stat3 in pancreatic cancer cells. Interestingly, emodin combined with EGFR inhibitor inhibited the proliferation of pancreatic cancer cells in vitro. The tumor xenograft mice model was further confirmed that emodin possessed a synergy anticancer effect with afatinib on pancreatic cancer cells by regulating the Stat3 expression.

Conclusion: These results indicate that the combination of emodin with EGFR inhibitor is an effective therapeutic strategy to sensitize human pancreatic cancer.

Keywords: EGFR inhibitors; Stat3; afatinib; pancreatic cancer.

Figure 1

Emodin inhibits phosphorylation of Stat3 in pancreatic cancer cells. The PANC-1 and BxPC-3 cells were incubated with emodin at the concentration of 30, 60 or 90 μM. p-Stat3 expression in the cells was detected by Western blotting.

Figure 2

Emodin promotes apoptosis of pancreatic cancer cells. The PANC-1 and BxPC-3 cells were incubated with emodin at the concentration of 30, 60 or 90 μM. (A) Flow cytometry was used to detect the apoptosis of cells, and the right is the quantitative analysis of flow cytometry data. (B) Bcl-2, cleaved-caspase3, and Bax expressions in the cells were detected with Western blotting. *P<0.05; **P<0.01.

Figure 3

The overexpression of Stat3 promotes proliferation of pancreatic cancer cells. (A) The mRNA and (B) protein expression of Stat3 were detected with qRT-PCR and Western blotting, respectively. (C) The proliferation of cells was detected with MTT assay.

Figure 4

EGFR inhibitor promotes phosphorylation of Stat3 in pancreatic cancer cells. The EGFR inhibitor (erlotinib, gefitinib, afatinib and cetuximab) was used to treat the PANC-1 and BxPC-3 cells at the concentration of 20 nM. (A) The mRNA and (B) protein expressions of EGFR were detected with qRT-PCR and Western blotting, respectively. (C) And the protein expression of p-Stat3 was detected with Western blotting.

Figure 5

Emodin combined with EGFR inhibitor inhibits proliferation of pancreatic cancer cells in vitro. (A) The colon and (B) MTT assays were used to detect the proliferation in PANC-1 and BxPC-3 cells.

Figure 6

Emodin combined with EGFR inhibitor inhibits proliferation of pancreatic cancer cells in vivo. Mice were administrated with vehicle (saline), emodin at 50 mg/kg/day, afatinib at 50 mg/kg/day, or the combination of the 2 drugs for 4 weeks. Tumor size and tumor weight (A), body weight (B) and tumor volume (C) of mice were measured. The protein of EGFR (D) and p-Stat3 (E) were detected with Western blotting. TUNEL assay detected the apoptosis in the tumor tissues (F). *P<0.05; **P<0.01.