Molecular and functional characterization of PACAP/VIP receptors in Lewis lung carcinoma cells

Recent evidence suggests that the pleiotropic neuropeptide PACAP plays a role in cancer initiation, progression and metastasis. Here we explore expression patterns and functions of PACAP receptors in LLC1 cells. RT-PCR analysis revealed the expression of the PAC1 receptor splice variants PAC1null, PAC1 hip, PAC1 hop, PAC1 3a and PAC1s. No VPAC1 and only traces of VPAC2 mRNA were detectable. Measurement of cAMP by FRET live cell imaging with EPAC-camps1 revealed that the PAC1 receptors in LLC1 cells were functional and that PACAP binding was nearly irreversible with an EC50 of about 60 pM indicating high affinity binding. Stimulation of LLC1 cells with PACAP also induced robust calcium signaling, measured as Fluo4 fluorescence. Treating LLC1 cells with PACAP or maxadilan prior to exposure to H2O2-induced oxidative stress enhanced LLC1 viability. In contrast, LLC1 cells treated with PACAP or maxadilan concomitantly with H2O2 stress exhibited reduced cell viability. LLC1 tumor transplants showed robust PAC1 expression revealed by RT-PCR and in situ hybridization. LLC1 tumor transplants are proposed as new models to explore the role of PACAP and PACAP receptors in tumor progression and metastasis and to test whether they are targets for tumor therapy and tumor imaging.

Supported by the LOEWE-Schwerpunkt “Tumor and Inflammation” of the state of Hesse.