Combination of gemcitabine and erlotinib inhibits recurrent pancreatic cancer growth in mice via the JAK-STAT pathway

Abstract

Compared to single gemcitabine treatment, the combination of gemcitabine and erlotinib has shown effective response in patients with locally advanced or metastatic pancreatic cancer. However, the combination therapy has not proven effective in patients with pancreatic cancer after R0 or R1 resection. In the present study, a nude mice model of orthotopic xenotransplantation after tumor resection was established using pancreatic cancer cell lines, BxPC-3 and PANC‑1. Mice were divided in four groups (each with n=12) and were treated as follows: the control group received a placebo via intraperitoneal injection (i.p.), while the other three groups were treated with gemcitabine (50 mg/kg i.p., twice a week), erlotinib (50 mg/kg oral gavage, once every three days), and combined treatment of gemcitabine and erlotinib, respectively. The treatment lasted for 21 days, after which all mice were sacrificed and tumors were examined ex vivo. We determined that the combination of gemcitabine and erlotinib inhibited recurrent tumor growth and induced apoptosis in vivo by downregulating phosphorylation levels of JAKs and STATs, which in turn downregulated the downstream proteins HIF‑1α and cyclin D1, and upregulated caspase‑9 and caspase‑3 expression. To sum up, the combination of gemcitabine with erlotinib was effective in treating patients with pancreatic cancer after R0 or R1 resection.

Figure 1.

The combination of gemcitabine and erlotinib inhibits tumor growth in vivo. (A and B) The tumor volume data calculated by the standard tumor volume formula [(l × w²)/2]. (C and D) The tumor volume data calculated during the last day of treatment. *P<0.05 indicates the negative controls vs. the treatment groups. ^#P<0.05 indicates the monotherapy (E or G) vs. the combination (E+G) groups. (E-H) The metastatic tumors in the abdominal cavity were observed three weeks after tumor resection in mice bearing BxPC-3 and PANC-1 xenografts. E, erlotinib; G, gemcitabine; N, negative control.

Figure 2.

Tumor apoptosis detection in four experimental groups. (A and B) TUNEL in tumor tissue sections from different treatment groups revealed increased apoptosis in the monotherapy and in the combination treatment groups. Lower panels displayed higher magnification (original magnification, ×100) of the boxed areas in the upper panels. Scale bars, 10 µm. (C and D) The apoptotic index was higher in the E+G group compared with the E or G group, in both BxPC-3 and PANC-1 cells. Quantification of TUNEL-positive cells in pancreatic cancer tumors. A total of ten ×40 fields were examined from three tumors in each of the treatment groups. *P<0.05 denotes the controls vs. the treatment groups. ^#P<0.05 denotes the monotherapy (E or G) vs. the combination (E+G) treatment groups.

Figure 3.

The gemcitabine-erlotinib (E+G) combination group inhibits the activity of the JAK-STAT pathway, as well as the expression of downstream HIF-1α, cyclin D1 and p53. (A and B) The protein levels of phosphorylated STAT3 Tyr⁷⁰⁵ (p-STAT3 Tyr⁷⁰⁵), HIF-1α, p53, cyclin D1 in BxPC-3 and PANC-1 cells were analyzed by western blotting, respectively. (C and D) The protein expression of p-STAT3 Tyr⁷⁰⁵, HIF-1α, p53, cyclin D1 in BxPC-3 and PANC-1 cells was calculated using one-way ANOVA. *P<0.05 denotes the controls vs. the treatment groups. ^#P<0.05 denotes the monotherapy (E or G) vs. the combination (E+G) groups. (E) Phosphorylation levels of JAK1 (Tyr¹⁰²²), JAK2 (Tyr²²¹), JAK3(Tyr⁹⁸¹) and STAT3 (Tyr⁷⁰¹) in BxPC-3 and PANC-1 cells were analyzed by western blotting, respectively. (F and G) The protein intensity of phospho-JAK1 (p-JAK1), p-JAK2 and p-JAK3 in BxPC-3 and PANC-1 cells was calculated using one-way ANOVA. ^#P<0.01; *P<0.05. (H) The protein intensity of p-STAT1 in BxPC-3 and PANC-1 cells was assessed using one-way ANOVA. ^#P<0.01, *P<0.05. E, erlotinib; G, gemcitabine.

Figure 4.

Gemcitabine in combination with erlotinib in recurrent tumors of BxPC-3 cells induces increased activity of caspase-3 and caspase-9. (A and B) Immunohistochemical staining of active caspase-9 and active caspase-3 in tumor tissue sections revealed active caspase-9 activation by combination treatment and active-caspase-3 activation by either monotherapy and by combination treatment in the BxPC-3 cells. Lower panels displayed higher magnification (original magnification, ×400) of the boxed areas in the upper panels. Scale bars, 10 µm. (C and D) Visual fields (–6) in each section of irradiation were photographed. The staining intensity and the percentage of positive cells were calculated and scored in each section. The total scores of each visual field were first analyzed using the homogeneity of variance test, followed by the change of variable test. The scores were presented as the mean ± SD. GraphPad Prism 5 was used to plot cartograms. *P<0.05 denotes the controls vs. the treatment groups. ^#P<0.05 denotes the monotherapy (E or G) vs. the combination (E+G) groups.

Figure 5.

Gemcitabine in combination with erlotinib in recurrent tumors of PANC-1 cells induces increased activity of caspase-3 and caspase-9. (A and B) Immunohistochemical staining of active caspase-9 and active caspase-3 in tumor tissue sections revealed active caspase-9 activation by either the monotherapy and by the combination treatment and active-caspase-3 activation by erlotinib monotherapy and by the combination treatment in PANC-1 cells. Lower panels displayed higher magnification (original magnification, ×400) of the boxed areas in the upper panels. Scale bars, 10 µm. (C and D) Visual fields (–6) in each section of irradiation were photographed. The staining intensity and the percentage of positive cells were calculated and scored in each section. The total scores in each visual field were first analyzed using the homogeneity of variance test, followed by the change of variable test. The scores were presented as the mean ± SD. GraphPad Prism 5 was used to plot cartograms. *P<0.05 indicates the controls vs. the treatment groups. ^#P<0.05 indicates the monotherapy (E or G) vs. the combination (E+G) groups.

Figure 6.

Schematic representation indicates that gemcitabine-erlotinib combination inhibits recurrent pancreatic tumor growth via JAK/STAT signaling. The gemcitabine-erlotinib combination significantly suppressed the phosphorylation levels of JAK1, JAK2, JAK3 as well as downstream STAT3 and STAT1 and eventually inhibited the growth of recurrent pancreatic tumors.