外源性三磷酸腺苷和腺苷对食管癌细胞TE-13和胃癌细胞HGC-27增殖的抑制作用及其作用机制研究
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摘要
胞外嘌呤（包括三磷酸腺苷（ATP），二磷酸腺苷（ADP）和腺苷（ADO））和嘧啶（包括三磷酸尿苷（UTP）和二磷酸尿苷（UDP））是生物体内重要的信号分子，介导了多种细胞的生物学效应。ATP及其相关化合物是许多细胞外信息的传递介质，通过与膜上的特异性嘌呤受体结合，ATP参与调节细胞内的许多生理生化功能。70年代末，Burnstock提出的嘌呤嘧啶受体（P受体）近年来得到了广泛的研究。P受体可分成P1和P2两类，P1受体的内源性配体主要是腺苷，P2受体的内源性配体主要有ATP、ADP、UTP和UDP。P1受体又可分为A1、A2A、A2B和A3四种亚型，属于G蛋白偶联受体，具有独特的组织学分布和药理学特性；P2受体又可分为配体门控离子通道型受体（即P2X受体）和G蛋白偶联型受体（即P2Y受体）。分子生物学研究结果显示，P2X受体包括7种亚型，即P2X1、P2X2、P2X3、P2X4、P2X5、P2X6和P2X7；P2Y受体也有多种亚型，如P2Y1、P2Y2、P2Y4、P2Y6、P2Y11、P2Y12和P2Y13等。
有报道指出，荷瘤动物腹腔注射ATP后具有抗癌活性；ATP抗癌治疗的临床研究国外也已开始报道。但有关ATP体内外抗肿瘤细胞生长的分子机制目前尚不十分清楚，胞外ATP对转化和非转化细胞的作用尚有争议。低浓度的胞外ATP对一些细胞具有促有丝分裂作用，ATP可以和促有丝分裂原一起协同促进细胞生长，此作用可能与P2受体介导的信号级联反应激活细胞早期的生长反应基因有关。但是，在另外一些细胞，特别是肿瘤细胞，高浓度的胞外ATP具有细胞抑制以及细胞毒作用，大多数研究认为此作用可能与诱导细胞凋亡有关。另外，许多文献报道了ATP介导的细胞毒作用的机制。Maaser等报道，中等分化的食管癌细胞Kyse-140表达的P2Y2受体可能介导ATP抑制细胞生长和诱导细胞凋亡的作用。ATP对成纤维细胞的抑制作用则由细胞外膜结合的核苷酸酶水解ATP所生成的ADO介导。众所周知，胞外ATP可通过一系列的胞外NTPDase（胞外核苷三磷酸脱磷酸酶）转化成AMP，AMP又可通过胞外
核苷酸酶的作用，最终生成ADO。越来越多的研究显示，腺苷具有抑制细胞生长的作用，因此，作为ATP逐级脱磷酸的最终产物，ADO可能在抗肿瘤作用中发挥了重要作用。据报道，ADO可通过P1受体引发细胞凋亡的形态学和生物化学改变。近来，Lewis等报道，ADO的细胞毒作用并不总是与细胞表面的P1受体有关，ADO可通过胞膜的转运发挥重要的生物学效应。到目前为止，有关ATP和ADO对人食管低分化鳞癌细胞和人胃未分化腺癌细胞的抑制作用机制以及相关受体的研究尚未见报道。本文以人食管低分化鳞癌细胞TE-13和人胃未分化腺癌细胞HGC-27为研究对象，观察了ATP和ADO对这两种肿瘤细胞生长的抑制作用和诱导凋亡的作用，同时观察了P受体亚型在食管癌和胃癌的分布，以及ATP对TE-13和HGC-27细胞P2Y受体的影响，以探讨ATP和ADO抑制肿瘤细胞生长的分子机制。
第一部分 外源性三磷酸腺苷和腺苷对食管癌细胞TE-13和胃癌细胞HGC-27增殖的影响
本研究采用MTT法观察了ATP和ADO对人胃未分化腺癌细胞株HGC-27和人食管低分化鳞癌细胞株TE-13增殖的影响, 流式细胞术观察ATP和ADO对细胞周期时相分布的影响，应用Wright’s-Giemsa染色观察了细胞形态学的改变。实验结果如下：
1． ATP和ADO对TE-13细胞增殖的抑制作用
与阴性对照组相比，0.1~1mmol·L-1的ATP或0.3~1 mmol·L-1的ADO处理细胞48h及0.01~1 mmol·L-1的ADO处理细胞72h后，明显抑制TE-13细胞的增殖，1 mmol·L-1的ATP和ADO 处理细胞48h的抑制率分别为59.6%和46.5%，72h的抑制率分别为80.5%和74%，ATP和ADO对TE-13细胞的抑制作用呈时间和剂量依赖性。
2． ATP和ADO对HGC-27细胞增殖的抑制作用
与阴性对照组相比，1mmol·L-1的ATP或0.1~1mmol·L-1的ADO处理细胞48h后明显抑制HGC-27细胞增殖，处理细胞72h的抑制浓度二者均为0.3~1mmol·L-1。1 mmol·L-1的ATP和ADO 处理细胞48h的抑制率分别为52.6%和40.3%，72h时分别为58.7%和52%。ATP的作用呈时间和剂量依赖性，而ADO的作用无时间依赖性。此外，低浓度ATP
（0.01~0.1 mmol·L-1）对HGC-27细胞呈促增殖作用，但无统计学意义。
3． ATP和ADO对TE-13细胞周期和增殖指数（PI）的影响
0.01、0.1和1 mmol·L-1的ATP和ADO处理细胞48h后，TE-13细胞的细胞周期时相分布和PI均发生了改变。与对照组比较，0.1 mmol·L-1ATP处理组的S期细胞显著增加，G0/G1和G2/M期细胞以及PI值均无显著性差异；而1 mmol·L-1ATP和ADO处理组则表现为G0/G1 期细胞显著增加，S期细胞显著减少，PI值显著降低。与 MTT法测定的细胞增殖实验结果一致，0.01 mmol·L-1ATP和ADO对细胞周期时相和PI值均无显著性影响。不同浓度的ATP对G2/M期细胞均无显著性影响，但1 mmol·L-1的ADO使G2/M期细胞数显著减少。上述结果显示，0.1 mmol·L-1的ATP阻滞TE-13细胞于S期，而1 mmol·L-1的ATP和0.1~1 mmol·L-1ADO则阻滞TE-13细胞于G0/G1期，二者通过改变细胞的周期时相分布，抑制了细胞的增殖。
4． ATP和ADO对HGC-27细胞周期和增殖指数（PI）的影响
0.01，0.1和1 mmol·L-1的ATP和ADO处理HGC-27细胞48h后，亦对肿瘤细胞的细胞周期时相分布和PI产生明显影响。与对?
Extracellular purines (adenosine triphosphate (ATP), adenosine 5’-diphosphate (ADP) and adenosine(ADO)) and pyrimidines (uridine 5’- triphosphate (UTP) and UDP ) are important signaling molecules that activate specific transmembrane receptors in most cell types to mediate diverse biological effects. ATP and related compounds are widespread transmitters for extracellular communication in many cell types. By coupling to specific purinergic receptors ATP is involved in a large variety of cellular function. Receptors for purines and pyrimidines (P receptor), first suggested by Burnstock, are divided into two major classes termed adenosine or P1 receptors, at which adenosine is the principal natural ligand, and P2 receptors, at which ATP, ADP, UTP and UDP is the principal natural ligand. To date four P1 receptors subtypes have been identified, A1, A2A, A2B, A3, all coupled to G proteins, with distinct tissue distribution and pharmacological properties. The P2 receptors are divided into two families: the ligand-gated ion channels (P2X) and the G protein-coupled receptors (P2Y). Cloning studies have revealed seven subtypes of P2X receptors, namely P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7, respectively, and several subtypes of P2Y receptors, including P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12 and P2Y13 et al.
Extracellular ATP has been reported to exhibit anticancer activity in vivo when injected intraperitoneally into tumor bearing animals. Based on those animal model studies, the first clinical trials with cancer patients have been initiated. Moreover, ATP has been reported to inhibit the growth of many tumor cells in vitro. The molecular basis of both in vivo and in vitro tumor growth inhibition by extracellular ATP is still not clearly understood.
A variety of in vitro studies on the effect of extracellular ATP in several
transformed and nontransformed cells revealed controversial results. In some cell systems extracellular ATP at low concentrations was mitogenic. In combination with other known mitogens, ATP synergistically displayed growth stimulation. These stimulatory effects of ATP were probably mediated by P2-purinoceptors in the cell membrane which initiate signaling cascades involving the activation of early growth response genes. However, extracellular ATP at higher concentrations displays cytostatic as well as cytotoxic effects in a variety of cell systems, especially tumor cells. This inhibitory effect is considered to be related to the apoptosis. Several other mechanisms of ATP-mediated cytotoxicity have been discussed. Cells of moderate differentiated esophageal cancer Kyse-140 lineage expressed P2Y2 purinoceptors, which mediate the growth inhibition and apoptosis of ATP. Other cells, like fibroblasts, are inhibited by extracellular ATP through the continuous generation of adenosine from ATP via the action of extracellular as well as membrane-bound nucleotidases. It is well known that extracellular ATP can be transformed in AMP by a variety of enzymes that are called ecto-NTPDases (‘ecto-nucleoside triphosphate diphosphohydrolase’) and finally to adenosine by ecto-nucleotidases. There is growing evidence that adenosine exerts the effects of growth inhibition on many tumor cell types. Therefore, adenosine, the final metabolite in the stepwise dephosphorylation of ATP, may be responsible for the growth inhibition effects of ATP. Adenosine has been reported to induce morphologically and biochemically typical apoptosis in various cells. It was shown to elicit its effects on cell death through adenosine receptor signaling. Recently, Lewis et al reported that adenosine toxicity might not always involve cell-surface receptors, and the nucleoside transport across cell membrane may be involved in its mechanism. However, the effects of ATP and adenosine on human poorly differentiated esophageal and stomach cancer cells, as well as their molecular mechanism have not been reported. To investigate the effects of ATP and adenosine on human poorly differentiated esophageal cancer TE-13 cells, and
有报道指出，荷瘤动物腹腔注射ATP后具有抗癌活性；ATP抗癌治疗的临床研究国外也已开始报道。但有关ATP体内外抗肿瘤细胞生长的分子机制目前尚不十分清楚，胞外ATP对转化和非转化细胞的作用尚有争议。低浓度的胞外ATP对一些细胞具有促有丝分裂作用，ATP可以和促有丝分裂原一起协同促进细胞生长，此作用可能与P2受体介导的信号级联反应激活细胞早期的生长反应基因有关。但是，在另外一些细胞，特别是肿瘤细胞，高浓度的胞外ATP具有细胞抑制以及细胞毒作用，大多数研究认为此作用可能与诱导细胞凋亡有关。另外，许多文献报道了ATP介导的细胞毒作用的机制。Maaser等报道，中等分化的食管癌细胞Kyse-140表达的P2Y2受体可能介导ATP抑制细胞生长和诱导细胞凋亡的作用。ATP对成纤维细胞的抑制作用则由细胞外膜结合的核苷酸酶水解ATP所生成的ADO介导。众所周知，胞外ATP可通过一系列的胞外NTPDase（胞外核苷三磷酸脱磷酸酶）转化成AMP，AMP又可通过胞外
核苷酸酶的作用，最终生成ADO。越来越多的研究显示，腺苷具有抑制细胞生长的作用，因此，作为ATP逐级脱磷酸的最终产物，ADO可能在抗肿瘤作用中发挥了重要作用。据报道，ADO可通过P1受体引发细胞凋亡的形态学和生物化学改变。近来，Lewis等报道，ADO的细胞毒作用并不总是与细胞表面的P1受体有关，ADO可通过胞膜的转运发挥重要的生物学效应。到目前为止，有关ATP和ADO对人食管低分化鳞癌细胞和人胃未分化腺癌细胞的抑制作用机制以及相关受体的研究尚未见报道。本文以人食管低分化鳞癌细胞TE-13和人胃未分化腺癌细胞HGC-27为研究对象，观察了ATP和ADO对这两种肿瘤细胞生长的抑制作用和诱导凋亡的作用，同时观察了P受体亚型在食管癌和胃癌的分布，以及ATP对TE-13和HGC-27细胞P2Y受体的影响，以探讨ATP和ADO抑制肿瘤细胞生长的分子机制。
第一部分 外源性三磷酸腺苷和腺苷对食管癌细胞TE-13和胃癌细胞HGC-27增殖的影响
本研究采用MTT法观察了ATP和ADO对人胃未分化腺癌细胞株HGC-27和人食管低分化鳞癌细胞株TE-13增殖的影响, 流式细胞术观察ATP和ADO对细胞周期时相分布的影响，应用Wright’s-Giemsa染色观察了细胞形态学的改变。实验结果如下：
1． ATP和ADO对TE-13细胞增殖的抑制作用
与阴性对照组相比，0.1~1mmol·L-1的ATP或0.3~1 mmol·L-1的ADO处理细胞48h及0.01~1 mmol·L-1的ADO处理细胞72h后，明显抑制TE-13细胞的增殖，1 mmol·L-1的ATP和ADO 处理细胞48h的抑制率分别为59.6%和46.5%，72h的抑制率分别为80.5%和74%，ATP和ADO对TE-13细胞的抑制作用呈时间和剂量依赖性。
2． ATP和ADO对HGC-27细胞增殖的抑制作用
与阴性对照组相比，1mmol·L-1的ATP或0.1~1mmol·L-1的ADO处理细胞48h后明显抑制HGC-27细胞增殖，处理细胞72h的抑制浓度二者均为0.3~1mmol·L-1。1 mmol·L-1的ATP和ADO 处理细胞48h的抑制率分别为52.6%和40.3%，72h时分别为58.7%和52%。ATP的作用呈时间和剂量依赖性，而ADO的作用无时间依赖性。此外，低浓度ATP
（0.01~0.1 mmol·L-1）对HGC-27细胞呈促增殖作用，但无统计学意义。
3． ATP和ADO对TE-13细胞周期和增殖指数（PI）的影响
0.01、0.1和1 mmol·L-1的ATP和ADO处理细胞48h后，TE-13细胞的细胞周期时相分布和PI均发生了改变。与对照组比较，0.1 mmol·L-1ATP处理组的S期细胞显著增加，G0/G1和G2/M期细胞以及PI值均无显著性差异；而1 mmol·L-1ATP和ADO处理组则表现为G0/G1 期细胞显著增加，S期细胞显著减少，PI值显著降低。与 MTT法测定的细胞增殖实验结果一致，0.01 mmol·L-1ATP和ADO对细胞周期时相和PI值均无显著性影响。不同浓度的ATP对G2/M期细胞均无显著性影响，但1 mmol·L-1的ADO使G2/M期细胞数显著减少。上述结果显示，0.1 mmol·L-1的ATP阻滞TE-13细胞于S期，而1 mmol·L-1的ATP和0.1~1 mmol·L-1ADO则阻滞TE-13细胞于G0/G1期，二者通过改变细胞的周期时相分布，抑制了细胞的增殖。
4． ATP和ADO对HGC-27细胞周期和增殖指数（PI）的影响
0.01，0.1和1 mmol·L-1的ATP和ADO处理HGC-27细胞48h后，亦对肿瘤细胞的细胞周期时相分布和PI产生明显影响。与对?
Extracellular purines (adenosine triphosphate (ATP), adenosine 5’-diphosphate (ADP) and adenosine(ADO)) and pyrimidines (uridine 5’- triphosphate (UTP) and UDP ) are important signaling molecules that activate specific transmembrane receptors in most cell types to mediate diverse biological effects. ATP and related compounds are widespread transmitters for extracellular communication in many cell types. By coupling to specific purinergic receptors ATP is involved in a large variety of cellular function. Receptors for purines and pyrimidines (P receptor), first suggested by Burnstock, are divided into two major classes termed adenosine or P1 receptors, at which adenosine is the principal natural ligand, and P2 receptors, at which ATP, ADP, UTP and UDP is the principal natural ligand. To date four P1 receptors subtypes have been identified, A1, A2A, A2B, A3, all coupled to G proteins, with distinct tissue distribution and pharmacological properties. The P2 receptors are divided into two families: the ligand-gated ion channels (P2X) and the G protein-coupled receptors (P2Y). Cloning studies have revealed seven subtypes of P2X receptors, namely P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7, respectively, and several subtypes of P2Y receptors, including P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12 and P2Y13 et al.
Extracellular ATP has been reported to exhibit anticancer activity in vivo when injected intraperitoneally into tumor bearing animals. Based on those animal model studies, the first clinical trials with cancer patients have been initiated. Moreover, ATP has been reported to inhibit the growth of many tumor cells in vitro. The molecular basis of both in vivo and in vitro tumor growth inhibition by extracellular ATP is still not clearly understood.
A variety of in vitro studies on the effect of extracellular ATP in several
transformed and nontransformed cells revealed controversial results. In some cell systems extracellular ATP at low concentrations was mitogenic. In combination with other known mitogens, ATP synergistically displayed growth stimulation. These stimulatory effects of ATP were probably mediated by P2-purinoceptors in the cell membrane which initiate signaling cascades involving the activation of early growth response genes. However, extracellular ATP at higher concentrations displays cytostatic as well as cytotoxic effects in a variety of cell systems, especially tumor cells. This inhibitory effect is considered to be related to the apoptosis. Several other mechanisms of ATP-mediated cytotoxicity have been discussed. Cells of moderate differentiated esophageal cancer Kyse-140 lineage expressed P2Y2 purinoceptors, which mediate the growth inhibition and apoptosis of ATP. Other cells, like fibroblasts, are inhibited by extracellular ATP through the continuous generation of adenosine from ATP via the action of extracellular as well as membrane-bound nucleotidases. It is well known that extracellular ATP can be transformed in AMP by a variety of enzymes that are called ecto-NTPDases (‘ecto-nucleoside triphosphate diphosphohydrolase’) and finally to adenosine by ecto-nucleotidases. There is growing evidence that adenosine exerts the effects of growth inhibition on many tumor cell types. Therefore, adenosine, the final metabolite in the stepwise dephosphorylation of ATP, may be responsible for the growth inhibition effects of ATP. Adenosine has been reported to induce morphologically and biochemically typical apoptosis in various cells. It was shown to elicit its effects on cell death through adenosine receptor signaling. Recently, Lewis et al reported that adenosine toxicity might not always involve cell-surface receptors, and the nucleoside transport across cell membrane may be involved in its mechanism. However, the effects of ATP and adenosine on human poorly differentiated esophageal and stomach cancer cells, as well as their molecular mechanism have not been reported. To investigate the effects of ATP and adenosine on human poorly differentiated esophageal cancer TE-13 cells, and
引文
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20 吕桂芝，林仲翔，孟松娘et al. 三磷酸腺苷对胃癌细胞Na+-K+-三磷酸
腺苷酶活性和c-myc基因表达的影响. 中华医学杂志，1995, 75（9）：563~564
21 Twentyman PR, Luscombe M. A study of some variables in a tetrazolirm dye (MTT) based assay for cell growth and chemosensitivity . Br J Cancer, 1987, 56(3)：279~285
22 司徒镇强，吴军正. 四唑盐（MTT）比色实验. 见：司徒镇强，吴军正主编. 细胞培养，西安：世界图书出版公司，1996：186~187
23 Fredholm B, Abbracchio M, Burnstock G et al. Nomenclature and classification of purinoceptors. Pharmacol Rev, 1994, 46：143~156
24任雷鸣. 嘌呤和嘧啶受体的心血管药理学. 见：苏定冯主编. 心血管药理学，北京：科学出版社, 2001：130~149
25 Vandewalle B，Hornez L，Revillin F，Lefebvre J. Effect of extracellular ATP on breast tumour cell growth: Implication of intracellular calcium. Cancer Lett，1994, 85：47~54
26 Batra S，Fadeel I. Release of intracellular calcium and stimulation of cell growth by ATP and histamine in human ovarian cancer cells (SK-OV3) . Cancer Lett，1994, 78(1)：57~63
27 吕桂芝，林仲翔，张志谦，et al. ATP抑制人胃腺癌（MGC-803）细胞增殖的机理研究. 实验生物学报，1994, 27(1)：137~139