Chang Mike, Xiao Lin, Shulkes Arthur, Baldwin Graham S, Patel Oneel
Department of Surgery, University of Melbourne, Austin Health, Heidelberg 3084, Victoria, Australia.
Endocrinology. 2016 Dec;157(12):4706-4719. doi: 10.1210/en.2016-1270. Epub 2016 Oct 31.
Gastrin, acting via the cholecystokinin-2 receptor (CCK2R), activates its own promoter in a positive-feed-forward loop that may result in hypergastrinemia. Activity of the gastrin promoter is also stimulated by exogenous Zn ions. Here, the role of intracellular zinc and calcium signaling in the gastrin positive-feed-forward loop was investigated. Gastrin promoter activity was measured in the human gastric carcinoma cell line AGS-CCK2R and in Jurkat cells transfected with various gastrin promoter-luciferase constructs after treatment with gastrin in the presence and absence of zinc- and calcium-chelating agents. The free intracellular zinc ion concentrations were measured in the same cells with the fluorescent indicator FluoZin-3. Cell proliferation and migration/invasion were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell proliferation assay and in Boyden chamber assays, respectively. The zinc chelator N,N,N,N-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) abolished gastrin-stimulated gastrin promoter activity, and the inhibition was completely reversed by exogenous Zn ions. In contrast, the calcium chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) potentiated gastrin-stimulated gastrin promoter activity. Treatment with gastrin increased the intracellular concentration of free Zn ions, and the increase was blocked by TPEN, but not by BAPTA-AM. TPEN also inhibited the stimulation of cell proliferation and migration/invasion by gastrin, but BAPTA-AM had no effect. These results, which are the first report of the existence of Zn signaling downstream of CCK2R activation, suggest that zinc chelation therapies may be effective in counteracting gastrin-dependent tumor growth.
胃泌素通过胆囊收缩素-2受体（CCK2R）发挥作用，在一个可能导致高胃泌素血症的正反馈前馈环中激活其自身启动子。胃泌素启动子的活性也受到外源性锌离子的刺激。在此，研究了细胞内锌和钙信号在胃泌素正反馈前馈环中的作用。在用胃泌素处理且存在或不存在锌和钙螯合剂的情况下，在人胃癌细胞系AGS-CCK2R以及转染了各种胃泌素启动子-荧光素酶构建体的Jurkat细胞中测量胃泌素启动子活性。使用荧光指示剂FluoZin-3在相同细胞中测量游离细胞内锌离子浓度。分别通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐细胞增殖试验和博伊登小室试验测量细胞增殖以及迁移/侵袭。锌螯合剂N,N,N,N-四-(2-吡啶甲基)-乙二胺（TPEN）消除了胃泌素刺激的胃泌素启动子活性，并且这种抑制作用被外源性锌离子完全逆转。相反，钙螯合剂1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸四钾盐（BAPTA-AM）增强了胃泌素刺激的胃泌素启动子活性。用胃泌素处理可增加游离锌离子的细胞内浓度，并且这种增加被TPEN阻断，但未被BAPTA-AM阻断。TPEN还抑制胃泌素对细胞增殖和迁移/侵袭的刺激作用，但BAPTA-AM没有影响。这些结果是CCK2R激活下游存在锌信号的首次报道，表明锌螯合疗法可能对对抗胃泌素依赖性肿瘤生长有效。
