PAC1 and PACAP expression, signaling, and effect on the growth of HCT8, human colonic tumor cells

Abstract

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.

Cryptosporidium parvumat Different Developmental Stages Modulates Host Cell Apoptosis In Vitro

“…5); in the sorted noninfected cells, FasL mRNA was not detected at any time (data not shown). Fas expression did not differ when comparing the sorted infected cells with the sorted noninfected cells (data not shown), although Fas is known to be expressed at a low level in HCT-8 cells (16). This finding, together with the observation that FasL was expressed only in infected cells and only early in the culture period, suggests that the parasite regulates apoptosis by interfering with effector enzymes downstream of the receptor.…”

Changes in the Expression of Pituitary Adenylate Cyclase-Activating Polypeptide in the Human Placenta during Pregnancy and Its Effects on the Survival of JAR Choriocarcinoma Cells

“…For example, in human leukemic myeloid cells and multiple myeloma-derived cells, PACAP decreased cell proliferation (Hayez et al 2004;Li et al 2006). In contrast, PACAP treatment increased the number of viable cells in colon and prostate cancer cell lines (Gutierrez-Canas et al 2003;Le et al 2002) and stimulated proliferation in a carcinoid tumor cell line (Hofsli et al 2005). PACAP also stimulated colony formation of non small cell lung cancer cells (Zia et al 1995).…”

Effects of PACAP on Oxidative Stress-Induced Cell Death in Rat Kidney and Human Hepatocyte Cells

“…Studies have also shown that the protective effects of PACAP are not limited to neuronal cells, but can be observed in numerous other cell lines and tissues. Among others, PACAP could prevent or attenuate cell death in cardiomyocytes (Racz et al 2008(Racz et al , 2010, endothelial cells (Racz et al 2007), ovarian follicles (Vaccari et al 2006), T cells (Delgado and Ganea 2000), and prostate as well as colon cancer cells (Gutierrez-Canas et al 2003;Le et al 2002).…”