A vasoactive intestinal peptide antagonist inhibits the growth of glioblastoma cells

Abstract

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (125I-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific 125I-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptase-polymerase chain reaction (RT-PCR), a major 305 bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration-dependent manner. Using a clonogenic assay in vitro, 10 microM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 microg/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.

Fig. 1.

Specificity of ¹²⁵I-PACAP-27 binding. The ability of varying doses of PACAP (○), VIP (●) and VIPhyb (▲) to inhibited specific ¹²⁵I-PACAP-27 to U87 cells was determined. The mean value ±S.D. of 3 determinations each repeated in quadruplicate is indicated.

Fig. 2.

RT-PCR. A major 305 base pair PCR product was observed for U87, U118, and U373 cell lines.

Fig. 3.

cAMP. The ability of PACAP to elevate the cAMP was determined as a function of concentration in the absence (g) or presence (1) of 10 mM VIPhyb. The mean value + S.D. of 4 determinations is shown.

Fig. 4.

Cytosolic Ca²⁺. PACAP-27 (30 nM) caused cytosolic Ca²⁺ elevation using Fura-2 AM loaded U87 cells. The ability of 10 and 30 mM VIPhyb to inhibit the cytosolic Ca²⁺ increase caused by PACAP-27 was determined. This experiment is representative of two others.

Fig. 5.

MTT assay. U373 cells were incubated with varying concentrations of VIPhyb. The mean value ± S.D. of 8 determinations is indicated; p < 0.05,* using Student’s t-test.

Fig. 6.

U87 xenografts in nude mice. A palpable mass formed after 1 wk and animals were subsequently injected with 100 mL of PBS daily s.c. (●), 10 μg of VIPhyb daily s.c. (○), and 0.1 μg VIP hyb (▲); p < 0.05, *. The mean value ± S.E. of 5 determinations is indicated.