Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells

Abstract

We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 14,000/cell) when NCI-H838 cells were used. Specific 125I-labeled PACAP binding was inhibited with high affinity by PACAP-27 and PACAP-38, with moderate affinity by PACAP(6-38), and with low affinity by vasoactive intestinal polypeptide, PACAP(28-38), and PACAP(16-38). PACAP-27 elevated cAMP in a dose-dependent manner, and the increase in cAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasoactive intestinal polypeptide, elevated cytosolic Ca2+ in individual NCI-H838 cells. PACAP-27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(6-38) reduced colony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. These data suggest that biologically active type 1 PACAP receptors are present on human non-small cell lung cancer cells, which exhibit dual signal transduction pathways and regulate cell proliferation.

Fig. 1.

The amount of specifically bound ¹²⁵I-PACAP-27 is indicated as a function of PACAP-27 concentration. The mean of four deteminations is indicated. The line is point to point.

Fig. 2.

A Scatchard plot of the specific binding data is shown. The line represents the best fit.

Fig. 3.

The percent of specific ²⁵I-PACAP bound was determined as a function of PACAP-27 (Δ), PACAP-38 (●), PACAP(6–38) (○), and VIP (■) concentration. The mean of three experiments each repeated in quadruplicate is indicated. Bars, SE.

Fig. 4.

Northern blot analysis. A main band of radioactivity at 7.5 kb was detected using guanidine isothiocyanate extracts of NCl-H838 cells.

Fig. 5.

Left, cAMP was determined in NCI-H838 cells as a function of PACAP-27 (○) and PACAP-38 (●) concentration. Right, cAMP was determined as a function of PACAP(6–38) concentration in the absence (Δ) and presence (▲) of 10 nM PACAP. The mean of four determinations is indicated. Bars, SE.

Fig. 6.

NCI-H838 cells were loaded with Indo-1AM, and the basal cytosolic Ca²⁺ was determined in a field of 13 cells. PACAP-27 (100 nM) was added, and cytosolic Ca²⁺ determined after 0 min (A), 0.5 min (B), 1 min (C), 1.5 min (D), 2 min (E), and 2.5 min (F). This experiment is representative of two others.

Fig. 7.

Nude mice received injections of NCI-H838 cells, and, after 2 weeks, a xenograft formed. PBS and PACAP(6–38) (10 µg/day s.c.) were injected daily during weeks 2–6, and the xenografts were measured weekly.