Le Sang V, Yamaguchi Dean J, McArdle Craig A, Tachiki Ken, Pisegna Joseph R, Germano Patrizia
CURE: Digestive Diseases Research Center, VA Greater Los Angeles Healthcare System, University of California, Los Angeles 90073, USA.
Regul Pept. 2002 Nov 15;109(1-3):115-25. doi: 10.1016/s0167-0115(02)00194-5.
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+), 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+), 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.
垂体腺苷酸环化酶激活多肽（PACAP）1型受体（PAC1）是一种七螺旋的G蛋白偶联受体，已证实在非鳞状肺癌和乳腺癌细胞系中表达，并与这些肿瘤的生长相关。我们之前已表明PACAP及其受体PAC1在大鼠结肠组织中表达。在本研究中，我们使用针对PAC1羧基末端的多克隆抗体以及荧光标记的PACAP（Fluor-PACAP），通过流式细胞术分析和共聚焦激光扫描显微镜来证明PAC1在HCT8人结肠肿瘤细胞上的表达。同样，抗PACAP多克隆抗体用于确认该细胞系中PACAP激素的表达。然后我们研究了PAC1在这些肿瘤细胞中的信号转导特性。PACAP-38以剂量依赖方式提高细胞内cAMP水平，半最大（EC(50)）刺激浓度约为3 nM。此外，PACAP-38刺激导致胞质Ca(2+)浓度Ca(2+)增加，这被PACAP拮抗剂PACAP-(6 - 38)部分抑制。最后，我们研究了PACAP对这些肿瘤细胞生长的潜在作用。我们发现，通过MTT活性测定，PACAP-38而非VIP增加了存活的HCT8细胞数量。我们还证明HCT8细胞表达Fas受体（Fas-R/CD95），在用PACAP-38激活后其表达随后下调，进一步表明PACAP在这些肿瘤细胞的生长和存活中可能发挥作用。这些数据表明HCT8人结肠肿瘤细胞表达PAC1并产生PACAP激素。此外，PAC1激活与腺苷酸环化酶偶联，增加胞质Ca(2+)并促进细胞增殖。因此，PACAP能够增加人结肠癌细胞系中存活细胞的数量并调节Fas-R表达，表明PACAP可能通过Fas-R/Fas-L凋亡途径在结肠癌生长调节和T淋巴细胞抗肿瘤反应调节中发挥作用。
