Activation of P2X(7)-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice.
Activation of P2X(7)-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice.
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DOI：
10.1186/1471-2407-9-114
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发表时间：
2009-04-20
期刊：
影响因子：
3.8
通讯作者：
Gorodeski GI
中科院分区：
文献类型：
--
作者：
Fu W;McCormick T;Qi X;Luo L;Zhou L;Li X;Wang BC;Gibbons HE;Abdul-Karim FW;Gorodeski GI
这项研究验证了细胞凋亡可以预防和控制肿瘤细胞生长的假设。先前的体外研究表明，促凋亡的P2X7受体调节上皮细胞的生长。本研究的具体目的是了解P2X7系统在体内在多大程度上控制皮肤癌的发生和生长，以及P2X7的作用涉及哪些细胞和分子机制。局部应用DMBA/TPA诱导小鼠皮肤肿瘤(乳头状瘤，随后是鳞状梭形细胞癌)。体外实验利用来自野生型或来自P2X7缺失小鼠的培养的表皮角质形成细胞。检测方法包括蛋白免疫染色和免疫印迹、实时定量定量聚合酶链式反应和细胞凋亡率(用原位末端标记法或酶联免疫吸附试验对培养的角质形成细胞进行DNA定量)。激光共聚焦显微镜观察细胞内钙离子或溴化乙锭内流(P2X7孔形成)的变化。(A)在皮肤上联合应用P2X7特异性激动剂BzATP可抑制DMBA/TPA诱导的皮肤乳头状瘤和癌症的形成。在研究结束时(第28周)，DMBA/TPA组患癌症的活动物比例为100%，而DMBA/TPA+BzATP组为43%。(B)在正常皮肤中，BzATP主要影响表达P2X7受体的增殖性角质形成细胞，在此过程中它促进了细胞的凋亡，而不引起炎症变化。(C)在BzATP治疗的小鼠中，癌细胞的凋亡程度低于正常或乳头状瘤角质形成细胞。(4)癌组织中P2X7受体、蛋白和mRNA的表达水平比正常小鼠组织低4-5倍。(5)在培养的小鼠角质形成细胞中，BzATP诱导细胞凋亡，在质膜上形成孔洞，并促进长时间的钙内流。(F)BzATP诱导的细胞凋亡、成孔和钙内流增加具有相似的剂量依赖关系。(G)成孔和钙内流的增加依赖于P2X7受体的表达，而BzATP诱导的细胞凋亡依赖于钙内流。(H)caspase-9和caspase-3抑制剂联合作用可阻断BzATP诱导的细胞凋亡，但不能阻断caspase-8的作用。(A)依赖于P2X7的细胞凋亡是控制小鼠表皮肿瘤发生发展的重要机制。(B)依赖于P2X7的细胞凋亡是由钙离子通过P2X7毛孔内流介导的，并涉及caspase-9(线粒体)途径。(C)BzATP对小鼠肿瘤角质形成细胞促凋亡作用的减弱可能是由于P2X7受体低表达所致。(D)激活依赖于P2X7的细胞凋亡，例如用BzATP，可能是体内乳头状瘤和上皮性癌的一种新的化疗生长预防方式。
The study tested the hypothesis that apoptosis can prevent and control growth of neoplastic cells. Previous studies in-vitro have shown that the pro-apoptotic P2X7 receptor regulates growth of epithelial cells. The specific objective of the present study was to understand to what degree the P2X7 system controls development and growth of skin cancer in vivo, and what cellular and molecular mechanisms are involved in the P2X7 action. Skin neoplasias in mice (papillomas, followed by squamous spindle-cell carcinomas) were induced by local application of DMBA/TPA. Experiments in-vitro utilized cultured epidermal keratinocytes generated from wild-type or from P2X7-null mice. Assays involved protein immunostaining and Western blots; mRNA real-time qPCR; and apoptosis (evaluated in situ by TUNEL and quantified in cultured keratinocytes as solubilized DNA or by ELISA). Changes in cytosolic calcium or in ethidium bromide influx (P2X7 pore formation) were determined by confocal laser microscopy. (a) Co-application on the skin of the P2X7 specific agonist BzATP inhibited formation of DMBA/TPA-induced skin papillomas and carcinomas. At the completion of study (week 28) the proportion of living animals with cancers in the DMBA/TPA group was 100% compared to 43% in the DMBA/TPA+BzATP group. (b) In the normal skin BzATP affected mainly P2X7-receptor – expressing proliferating keratinocytes, where it augmented apoptosis without evoking inflammatory changes. (c) In BzATP-treated mice the degree of apoptosis was lesser in cancer than in normal or papilloma keratinocytes. (d) Levels of P2X7 receptor, protein and mRNA were 4–5 fold lower in cancer tissues than in normal mouse tissues. (e) In cultured mouse keratinocytes BzATP induced apoptosis, formation of pores in the plasma membrane, and facilitated prolonged calcium influx. (f) The BzATP-induced apoptosis, pore-formation and augmented calcium influx had similar dose-dependence for BzATP. (g) Pore formation and the augmented calcium influx were depended on the expression of the P2X7 receptor, while the BzATP-induced apoptosis depended on calcium influx. (h) The BzATP-induced apoptosis could be blocked by co-treatment with inhibitors of caspase-9 and caspase-3, but not of caspase-8. (a) P2X7-dependent apoptosis is an important mechanism that controls the development and progression of epidermal neoplasia in the mouse. (b) The P2X7-dependent apoptosis is mediated by calcium influx via P2X7 pores, and involves the caspase-9 (mitochondrial) pathway. (c) The diminished pro-apoptotic effect of BzATP in mouse cancer keratinocytes is possibly the result of low expression of the P2X7 receptor. (d) Activation of P2X7-dependent apoptosis, e.g. with BzATP could be a novel chemotherapeutic growth-preventive modality for papillomas and epithelial cancers in vivo.
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影响因子：
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作者：
Fu, W;Cockerell, CJ
通讯作者：
Cockerell, CJ
影响因子：
4.8
作者：
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通讯作者：
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影响因子：
6.5
作者：
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通讯作者：
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影响因子：
3.5
作者：
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通讯作者：
Dahl, Gerhard
影响因子：
6.5
作者：
Greig, AVH;Linge, C;Burnstock, G
通讯作者：
Burnstock, G