Xanthohumol inhibits angiogenesis by suppressing nuclear factor-κB activation in pancreatic cancer

Abstract

Xantohumol, a prenylated chalcone from hops (Humulus lupulus L.), has been shown to inhibit proliferation in some cancers. However, little is known regarding the effects of xanthohumol in pancreatic cancer. We have previously reported that activation of the transcription factor nuclear factor-κB (NF-κB) plays a key role in angiogenesis in pancreatic cancer. In this study, we investigated whether xanthohumol inhibited angiogenesis by blocking NF-κB activation in pancreatic cancer in vitro and in vivo. We initially confirmed that xanthohumol significantly inhibited proliferation and NF-κB activation in pancreatic cancer cell lines. Next, we demonstrated that xanthohumol significantly suppressed the expression of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) at both the mRNA and protein levels in pancreatic cancer cell lines. We also found that coculture with BxPC-3 cells significantly enhanced tube formation in human umbilical vein endothelial cells, and treatment with xanthohumol significantly blocked this effect. In vivo, the volume of BxPC-3 subcutaneous xenograft tumors was significantly reduced in mice treated with weekly intraperitoneal injections of xanthohumol. Immunohistochemistry revealed that xanthohumol inhibited Ki-67 expression, CD31-positive microvessel density, NF-κB p65 expression, and VEGF and IL-8 levels. Taken together, these results showed, for the first time, that xanthohumol inhibited angiogenesis by suppressing NF-κB activity in pancreatic cancer. Accordingly, xanthohumol may represent a novel therapeutic agent for the management of pancreatic cancer.

Keywords: NF-κB; angiogenesis; interleukin-8; pancreatic cancer; xanthohumol.

Figure 1

A, Effects of xanthohumol on the proliferation of pancreatic cancer cell lines. Pancreatic cancer cell lines (BxPC‐3, AsPC‐1 and MIA PaCa‐2) were treated with xanthohumol at the indicated concentrations for 72 h, and cytotoxicity was measured using WST‐1 assays. Values are expressed as means ± SD (n = 6; *P < .05; **P < .01 compared with the control in all cell lines). XN, xanthohumol; C, control. B, C, Xanthohumol inhibited NF‐κB activity in pancreatic cancer cell lines. Pancreatic cancer cell lines (BxPC‐3, AsPC‐1 and MIA PaCa‐2) were treated with xanthohumol at the indicated concentrations for 24 h (B) and then stimulated with TNF‐α (10 ng/mL) for 30 min (C). The activity of NF‐κB in nuclear extracts was measured using Trans AM NF‐κB p65/p50 Transcription Factor Assays. Values are expressed as means ± SD (n = 3; *P < .05; **P < .01 compared with the control in all cell lines). XN, xanthohumol

Figure 2

Xanthohumol suppressed VEGF and IL‐8 mRNA expression in pancreatic cancer cells. Pancreatic cancer cell lines (BxPC‐3 and AsPC‐1) were treated with xanthohumol at the indicated concentrations for 48 h. VEGF and IL‐8 mRNA levels were measured using real‐time qPCR (normalized to β‐actin expression). Values are expressed as means ± SDs (n = 3; *P < .05; **P < .01 compared with the control in all cell lines). C, control

Figure 3

Xantohumol suppressed the secretion of VEGF and IL‐8 in pancreatic cancer cell lines. Pancreatic cancer cell lines (BxPC‐3 and AsPC‐1) were treated with xanthohumol at the indicated concentrations for 72 h, and the supernatants were then collected. The concentrations of secreted VEGF and IL‐8 were measured using ELISA. Values are expressed as means ± SD (n = 3; *P < .05; **P < .01 compared with the control in all cell lines). C, control

Figure 4

Xanthohumol inhibited BxPC‐3‐induced tube formation by HUVEC in vitro. A, Effects of anti‐VEGF (10 μg/mL) and anti‐IL‐8 (10 μg/mL) antibody on tube formation by HUVEC. B, Effects of xanthohumol on tube formation by HUVEC. Tube formation assays were performed using an angiogenesis kit. Values are expressed as means ± SD (n = 3; *P < .05; **P < .01 compared with the untreated group). C, (i) control; (ii) coculture with BxPC‐3 cells; (iii) coculture with BxPC‐3 cells treated with xanthohumol (0.5 μmol/L); and (iv) coculture with BxPC‐3 cells treated with xanthohumol (1 μmol/L). Representative images are shown (40×)

Figure 5

Xanthohumol inhibited tumor growth in a subcutaneous xenograft model of pancreatic cancer. BxPC‐3 cells (5 × 10⁶ cells in 100 μL Hank's balanced salt solution) were injected subcutaneously, and tumors were developed. Mice were divided into 2 groups: untreated (group I) and treated with xanthohumol (10 mg/kg, ip, injection, weekly; group II). A, Images of mice and solid tumors removed from each group: (i) control and (ii) xanthohumol. B, Measurement of tumor volumes in each group once per week. C, Measurement of tumor weights in nude mice after sacrifice. D, Measurement of body weights in each group once per week. Values in (B), (C) and (D) represent the means ± SD of each group (n = 5; *P < .05, **P < .01 compared with the untreated group). NS, not significant

Figure 6

Xanthohumol inhibited proliferation and angiogenesis and reduced the activation of NF‐κB subunit p65 in pancreatic cancer tumors. A, Representative immunohistochemical analysis. Representative images are shown (200×: Ki‐67 and CD31, 400×: p65). B, Quantification of Ki‐67‐positive cells. C, Quantification of microvessel density. D, Quantification of the activation of NF‐κB p65. Values in (B)‐(D) represent the means ± SD of each group. *P < .05, **P < .01 compared with the untreated group

Figure 7

Xanthohumol reduced the expression of VEGF and IL‐8 in pancreatic cancer tumors. A, Representative immunohistochemical analysis. Representative images are shown (200×). B, Quantification of VEGF. C, Quantification of IL‐8. D, E, Xanthohumol suppressed VEGF and IL‐8 mRNA expression in pancreatic cancer tumors. Values in (B)‐(E) represent the means ± SD of each group. *P < .05, **P < .01 compared with the untreated group