Bombesin induces angiogenesis and neuroblastoma growth

Abstract

Gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin (BBS), is a trophic factor for highly vascular neuroblastomas; its mechanisms of action in vivo are unknown. We sought to determine the effects of BBS on the growth of neuroblastoma xenografts and on angiogenesis. BBS significantly increased the growth of SK-N-SH and BE(2)-C human neuroblastomas; tumors demonstrated increased expression of angiogenic markers, PECAM-1 and VEGF, as well as phosphorylated (p)-Akt levels. RC-3095, a BBS/GRP antagonist, attenuated BBS-stimulated tumor growth and angiogenesis in vivo. GRP or GRPR silencing significantly inhibited VEGF as well as p-Akt and p-mTOR expression in vitro. Our findings demonstrate that BBS stimulates neuroblastoma growth and the expression of angiogenic markers. Importantly, these findings suggest that novel therapeutic agents, targeting BBS-mediated angiogenesis, may be useful adjuncts in patients with advanced-stage neuroblastomas.

Figure 1. BBS stimulates cell growth, VEGF and p-Akt expression in SK-N-SH cells

(A) Effects of BBS (10^-7 M) on SK-N-SH cell viability were determined using CCK-8 kit (Data represent mean ± SEM values of eight replicate experiments; * p < 0.05 vs. control). (B) SK-N-SH cells were treated with BBS (10^-7 M) for one day after overnight serum-free conditions. Cell lysates were prepared and analyzed by Western blot for VEGF. (C) SK-N-SH cells were treated with BBS (10^-7 M) for 5 min after overnight serum-free condition. Cell lysates were prepared and analyzed by Western blot for p-Akt and total Akt. Experiments were repeated on two separate occasions.

Figure 2. BBS promotes SK-N-SH growth in vivo

(A, B) Tumor volumes and tumor weights of subscapular SK-N-SH xenografts in mice treated with vehicle or BBS (i.p.) for 45 days as described in “Materials and Methods” (4-6 mice/group). (C, D) Tumor volumes and tumor weights of orthotopic SK-N-SH xenografts at sacrifice, established by injecting cells into left kidney of mice and treated with vehicle or BBS (20 μg/kg/injection; i.p.) for 43 days as described in “Materials and Methods” (5-6 mice/group). Data from all figures represent mean ± SEM; * p < 0.05 vs. control.

Figure 3. BBS induces angiogenesis in SK-N-SH xenografts

(A) Representative sections of SK-N-SH tumors from vehicle- or BBS-treated mice stained with anti-human VEGF antibody (brown); magnification, X400. (B) Representative sections of SK-N-SH tumors from vehicle- or BBS-treated mice stained with anti-human PECAM-1 antibody (brown); magnification X200. (C) Expression of p-Akt in SK-N-SH tumor tissue samples from mice treated with BBS or vehicle; five representative tumors are shown (Data represent mean ± SEM; * p < 0.05 vs. control).

Figure 4. GRP antagonist inhibits BE(2)-C tumor growth and angiogenesis

(A) Tumor volumes in nude mice with BE(2)-C cells treated with vehicle, BBS (20 μg/kg/injection; s.c., t.i.d.), and/or RC-3095 (10 μg/kg/injection; s.c., q 12 h), as described in “Materials and Methods” (5-6 mice/group). (B) Expression of p-Akt and VEGF in BE(2)-C tumor tissue samples (three representative tumor samples from each group are shown). (C) Representative sections of BE(2)-C tumors stained with anti-human VEGF antibody (brown); magnification, X400. (D) VEGF plasma levels from mice detected by ELISA. Data from all figures represent mean ± SEM; * p < 0.05 vs. control.

Figure 5. GRP/GRPR siRNA attenuates angiogenesis in human neuroblastoma cells

(A) Western blot analysis of p-Akt, p-mTOR and VEGF protein expression in SK-N-SH cells at 3 d post-transfection with siNTC or siGRP (left panel). Quantitative RT-PCR for GRP mRNA levels in SK-N-SH cells at 2 d post-transfection with siNTC or siGRP (right panel). (B) Western blot analysis of p-Akt, GRP, p-mTOR and VEGF protein expression in BE(2)-C cells at 3 d post-transfection with siNTC or siGRP. (C) Quantitative RT-PCR analysis for VEGF mRNA level in BE(2)-C cells at 2 d post-transfection with siNTC or siGRP (left panel). VEGF levels in BE(2)-C using human VEGF ELISA kit. Cell culture supernatants were harvested at 3 d post-transfection with siNTC or siGRP (right panel). (D) BE(2)-C cells were transfected with siNTC or siGRPR for various assays. GRPR and VEGF protein expression was determined by Western blot analysis at 3 d post-transfection (left panel). VEGF mRNA levels were assessed by quantitative RT-PCR at 2 d post-transfection (middle panel) and VEGF levels in cell culture supernatants were measured by ELISA at 3 d post-transfection (right panel). Data from all figures represent mean ± SEM; * p < 0.05 vs. control.