PAC1 and PACAP expression, signaling, and effect on the growth of HCT8, human colonic tumor cells.
The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) is a heptahelical, G protein-coupled receptor that has been shown to be expressed by non-squamous lung cancer and breast cancer cell lines, and to be coupled to the growth of these tumors. We have previously shown that PACAP and its receptor, PAC1, are expressed in rat colonic tissue. In this study, we used polyclonal antibodies directed against the COOH terminal of PAC1, as well as fluorescently labeled PACAP, Fluor-PACAP, to demonstrate the expression of PAC1 on HCT8 human colonic tumor cells, using FACS analysis and confocal laser scanning microscopy. Similarly, anti-PACAP polyclonal antibodies were used to confirm the expression of PACAP hormone by this cell line. We then investigated the signal transduction properties of PAC1 in these tumor cells. PACAP-38 elevated intracellular cAMP levels in a dose-dependent manner, with a half-maximal (EC(50)) stimulation of approximately 3 nM. In addition, PACAP-38 stimulation caused an increase in cytosolic Ca(2+) concentration [Ca(2+)](i), which was partially inhibited by the PACAP antagonist, PACAP-(6-38). Finally, we studied the potential role of PACAP upon the growth of these tumor cells. We found that PACAP-38, but not VIP, increased the number of viable HCT8 cells, as measured by MTT activity. We also demonstrated that HCT8 cells expressed the Fas receptor (Fas-R/CD95), which was subsequently down-regulated upon activation with PACAP-38, further suggesting a possible role for PACAP in the growth and survival of these tumor cells. These data indicate that HCT8 human colon tumor cells express PAC1 and produce PACAP hormone. Furthermore, PAC1 activation is coupled to adenylate cyclase, increase cytosolic [Ca(2+)](i), and cellular proliferation. Therefore, PACAP is capable of increasing the number of viable cells and regulating Fas-R expression in a human colonic cancer cell line, suggesting that PACAP might play a role in the regulation of colon cancer growth and modulation of T lymphocyte anti-tumoral response via the Fas-R/Fas-L apoptotic pathway.
Le
Sang V
SV
CURE: Digestive Diseases Research Center, VA Greater Los Angeles Healthcare System, University of California, Los Angeles 90073, USA.
Yamaguchi
Dean J
DJ
McArdle
Craig A
CA
Tachiki
Ken
K
Pisegna
Joseph R
JR
Germano
Patrizia
P
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D015536:Down-Regulation; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
C495401:ADCYAP1 protein, human; C495503:ADCYAP1R1 protein, human; C495402:Adcyap1 protein, mouse; C495504:Adcyap1r1 protein, mouse; D001426:Bacterial Proteins; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C414536:FasR protein, Rhodococcus fascians; C505658:Fasl protein, mouse; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D009479:Neuropeptides; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D014157:Transcription Factors; C075647:pituitary adenylate-cyclase-activating-peptide (6-38); D002118:Calcium