酒石酸锑钾诱导人胃癌细胞凋亡
郭光云,徐少勇,郧阳医学院附属人民医院消化内科湖北省十堰市442000
邓长生,武汉大学中南医院消化内科湖北省武汉市430071
郭光云,女, 1971-08-16生,湖北省十堰市人,汉族, 武汉大学在读硕士.
湖北省教育厅资助项目,No. 2000A43003
项目负责人:徐少勇,442000, 湖北省十堰市,郧阳医学院附属人民医院消化内科.xushaoyong195571@sina.com.cn
, 百拇医药
电话:0719-8652119-6208
收稿日期:2003-10-09接受日期:2003-11-06
Potassiumantimonyl tartrate induces apoptosis in human gastric carcinoma cells
Guang-YunGuo, Shao-Yong Xu, Chang-Sheng Deng
Guang-YunGuo, Shao-Yong Xu, Department of Gastroenterology, People,sHospital of Yunyang Medical College, Shiyan442000, Hubei Province, China
, 百拇医药
Chang-ShengDeng, Department of Gastroenterology, Zhongnan Hospital of WuhanUniversity, Wuhan 430071, Hubei Province, China
Supported by the Education Department Foundation of Hubei Province,No. 2000A43003
Correspondence to: Shao-Yong Xu, Department of Gastroenterology,People,sHospital of Yunyang Medical College, Shiyan442000, Hubei Province, China.xushaoyong195571@sina.com.cn
, http://www.100md.com
Received:2003-10-09Accepted: 2003-11-06
AbstractAIM:Potassium antimonyl tartrate (PAT), an antiparasitic agent, has rencentlybeen shown to induce apoptosis of leukaemia cells. Its effects on humangastric cancer SGC-7901 cells and possible mechanism were investigated inthis study.
METHODS:The growth inhibition of cells induced by various concentrations of PAT indifferent time course was analyzed by using MTT assay. The nuclei werestained by Hoechst 33 258 and the morphologic changes were observed byfluorescence microscope. TUNEL staining, double-staining, and flowcytomery were used to detect apoptosis of cells.
, 百拇医药
RESULTS:Growth inhibitory rates of SGC-7901 cells by PAT were significantlydifferent in dose-and time-dependent manners (analysis of variance,P <0.01).Chromatin condensation and nuclear fragmentation were seen underfluorescence microscope in the cells treated with Hoechst 33 258, andapoptotic cells were also detected by flow cytometry and TUNEL staining.
CONCLUSION:PAT can induce the apoptosis of SGC-7901 cells, and PAT may be a promisingapoptosis-inducer in gastric cancer therapy.
, 百拇医药
Guo GY, XuSY, Deng CS. Potassium antimonyl tartrate induces apoptosis in humangastric carcinoma cells. Shijie Huaren Xiaohua Zazhi2004;12(3):520-522
摘要
目的:用于治疗寄生虫病的酒石酸锑钾能抑制白血病细胞及淋巴瘤细胞生长，并诱导其凋亡.本文探讨其对人胃癌SGC-7901细胞株的作用及可能的机制.
方法:采用MTT法检测不同浓度酒石酸锑钾对胃癌SGC-7901细胞的生长抑制作用;经 Hoechst 33258染色后用荧光显微镜观察细胞核的形态变化;DNA末端原位标记染色法、双染法(annexin-ⅴ/PI)和流式细胞术检测细胞凋亡.
, http://www.100md.com
结果:酒石酸锑钾能明显抑制胃癌细胞生长，抑制作用呈时间和剂量依赖性(方差分析，P<0.01); Hoechst 33 258染色后荧光显微镜观察细胞核固缩，呈致密的强荧光;DNA末端原位标记染色法、流式细胞仪检测均检测到凋亡细胞.
结论:酒石酸锑钾能诱导胃癌SGC-7901细胞凋亡.有望作为一种新的凋亡诱导剂用于胃癌的治疗.
郭光云,徐少勇, 邓长生.酒石酸锑钾诱导人胃癌细胞凋亡.世界华人消化杂志2004;12(3):520-522
: 对照组; B: 酒石酸锑钾.
图2(PDF)Annexinⅴ-FITC和PI双染检测SGC-7901细胞早期凋亡.A: 对照组; B:酒石酸锑钾.
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图3 SGC-7901细胞凋亡TUNEL×400. A: 对照组;B:20 mmol/L酒石酸锑钾.
2.3细胞凋亡检测结果 酒石酸锑钾作用于SGC7901细胞，用Annexinⅴ和PI染色流式细胞仪检测双参数图上可见对照组细胞主要分布于第三象限(Annexinⅴ-PI-)，酒石酸锑钾处理后细胞分布发生明显改变，第四象限细胞(Annexinⅴ+PI-)增加，代表早期凋亡细胞，第二象限细胞(Annexinⅴ+PI+)分布也增加，代表坏死细胞(图2).酒石酸锑钾处理SGC7901细胞24h后，早期细胞的凋亡率3.4%，8.2%和12.9%，呈一定的剂量效应关系.TUNEL检查细胞核中有棕红色颗粒为阳性细胞(图3).细胞的凋亡指数为7.5%，16.7%，38.1%，阴性对照组为4.6%，(dP<0.01，x2=47.454).可见在一定时间内，随着作用浓度增大，凋亡细胞的百分率也随着增加.
, 百拇医药
3 讨论凋亡异常在肿瘤发生发展中具有重要作用，逃避凋亡机制的调控在肿瘤细胞对常规化疗药物耐药中起关键作用.随着凋亡研究的深入，人们致力于开发新的药物以选择性诱导肿瘤细胞凋亡[20].酒石酸锑钾能抑制急性早幼粒细胞白血病细胞NB4和NB4R4生长，通过降解PML/PML-RARa混合蛋白，而诱导NB4和NB4R4凋亡.其微摩尔浓度可诱导NB4，NB4R4细胞凋亡，机体能很好耐受而无明显毒副作用，锑剂与RA之间无交叉耐药，副作用比三氧化二砷小.Lecureur et al [18]研究表明，酒石酸锑钾能诱导其他白血病细胞(K562，HL60，U937和KGLa)凋亡.他可能作用于谷光甘肽过氧化物酶或巯基，导致氧化应激，线粒体跨膜电位下降，触发细胞凋亡的级联反应;因诱导的凋亡不能被广谱caspase酶的抑制剂所阻滞，推测凋亡过程可能不需caspase酶的激活，但凋亡诱导因子是否参于凋亡过程尚需研究.Lecureur et al [19]研究亦发现酒石酸锑钾能抑制多种淋巴细胞株的生长，包括来源于急性淋巴细胞白血病的Jurkat，Molt-4，Nalm-6细胞和来源于淋巴瘤Dudi，Raji，Recl细胞.并能诱导Dudi，Jurkat细胞凋亡.凋亡依赖于caspase酶的活性，并与线粒体跨膜电位下降和细胞内活性氧类物质增多有关.酒石酸锑钾能显著抑制恶性淋巴细胞生长，而对正常的淋巴细胞无影响.提示酒石酸锑钾诱导凋亡对正常组织的影响小，使其在治疗血液系统恶性肿瘤中具有一定的应用前景[18-19].
, http://www.100md.com
我们发现微摩尔浓度酒石酸锑钾能明显抑制体外培养的胃癌SGC-7901细胞的生长，且其抑制细胞增生在一定范围内，随酒石酸锑钾的浓度而增加，作用时间延长，细胞增生抑制率相应增加，具有时效及量效关系.通过荧光显微镜观察到酒石酸锑钾作用后，细胞的染色质浓缩、边聚，等凋亡的形态改变;用异硫氢酸荧光素(fluorescienisothiocyanate，FITC)标记的连接素(Annexinⅴ-FITC)和PI双染法检测细胞凋亡，因Annexinⅴ能与暴露在凋亡早期细胞膜外表面的磷脂酰丝氨酸特异性结合与其连接的FITC发出绿色荧光，而此时细胞维持膜的完整性，变性染色质着色的荧光染料PI不能进入细胞;凋亡晚期及继发性坏死细胞膜失去完整性，故FITC和PI双染;坏死、机械性损伤的细胞只能PI与结合.故通过双染可区分早期的凋亡细胞与凋亡晚期坏死、继发性坏死细胞.酒石酸锑钾作用后SGC-7901细胞呈典型的凋亡细胞分布图.TUNEL法亦检测到凋亡细胞.TUNEL法和Annexinⅴ/PI法检测细胞凋亡率有一差别，TUNEL法检测凋亡率稍高，可能因为TUNEL无法区分死亡细胞与凋亡细胞，少数坏死细胞也呈阳性染色有关[3].
, http://www.100md.com
4 参考文献1 Zhou YN, Xu CP, Han B, Li M, Qiao L, Fang DC, Yang JM. Expressionof E-cadherin and beta-catenin in gastric carcinoma
and its correlation with theclinicopathological features and patient survival. World J Gastroenterol 2002;8:987-993
2 Song ZJ, Gong P, Wu YE. Relationshipbetween the expression of iNOS, VEGF, tumor angiogenesis and gastriccancer.
, http://www.100md.com World J Gastroenterol 2002;8:591-595
3 林万隆,李定国, 陈强,陆汉民, 马小明,孙培龙. 奥沙利铂综合治疗胃癌的疗效及机制.世界华人消化杂志
2003;11:1535-1539
4 孙利平,李岩, 张宁,姜乃佳, 付伟,薛一雪. MDM2基因扩增和蛋白表达与胃癌相关性的研究.世界华人消化杂志
2003;11:1800-1801
5 任群, 王振宁,罗阳, 敖杨,鲁冲, 姜莉,徐惠绵, 张学.胃癌中18号染色体的杂合性丢失研究.世界华人消化杂志
2003;11:310-313
, 百拇医药
6 潘传敬,刘宽宇. 胃癌增生凋亡与调节基因的表达.世界华人消化杂志 2003;11:526-530
7 Qiu GB, Gong LG, Hao DM, Zhen ZH, Sun KL. Expression of MTLC genein gastric carcinoma. World J Gastroenterol
2003;9:2160-2163
8 Ding YB, Chen GY, Xia JG, Zang XW, Yang HY, Yang L. Association ofVCAM-1 overexpression with oncogenesis, tumor
angiogenesis and metastasis of gastriccarcinoma. World J Gastroenterol 2003;9:1409-1414
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9 张晓兵,刘泽军. 胃癌DNA甲基化谱研究进展.世界华人消化杂志 2002;10:1454-1457
10 Shi H, Xu JM, Hu NZ, Xie HJ. Prognostic significance of expressionof cyclooxygenase-2 and vascular endothelial growth
factor in human gastric carcinoma. World JGastroenterol 2003;9:1421-1426
11 Zheng HC, Sun JM, Wei ZL, Yang XF, Zhang YC, Xin Y. Expression ofFas ligand and caspase-3 contributes to formation
, 百拇医药
of immune escape in gastric cancer. World JGastroenterol 2003;9:1415-1420
12 Yang L, Kuang LG, Zheng HC, Li JY, Wu DY, Zhang SM, Xin Y. PTENencoding product: a marker for tumorigenesis and
progression of gastric carcinoma. World JGastroenterol 2003;9:35-39
13 Sun L, Wang X. Effects of allicin on both telomerase activity andapoptosis in gastric cancer SGC-7901 cells. World J
, 百拇医药
Gastroenterol 2003;9:1930-1934
14 刘文超,穆怀兴, 任军,张学庸, 潘伯荣.防御素对胃癌细胞系体外的杀伤作用.世界华人消化杂志 2001;9:622-626
15 Li JY, Wang XZ, Chen FL, Yu JP, Luo HS. Nimesulide inhibitsproliferation via induction of apoptosis and cell cycle arrest
in human gastric adenocarcinoma cell line.World J Gastroenterol 2003;9:915-920
16 Wang ZY. Arsenic compounds as anticancer agents.Cancer ChemotherPharmacol 2001;48(Suppl1):S72-76
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17 Ratnaike RN. Acute and chronic arsenic toxicity. Postgrad Med J 2003;79:391-396
18 Lecureur V, Lagadic-Gossmann D, Fardel O. Potassium antimonyltartrate induces reactive oxygen species 杛elated
apoptosis in human myeloid leukemic HL60cells. Int J Oncol 2002;20:1071-1076
19 Lecureur V, Le Thiec A, Le Meur A, Amiot L, Drenou B, Bernard M,Lamy T, Fauchet R, Fardel O. Potassium antimonyl
tartrate induces caspase- and reactiveoxygen species-dependent apoptosis in lymphoid tumoral cells. Br JHaematol
2002;119:608-615
20 Kasibhatla S, Tseng B. Why target apoptosis in cancer treatment?Mol Cancer Ther 2003;2:573-580, http://www.100md.com( 郭光云, 徐少勇, 邓长生)
邓长生,武汉大学中南医院消化内科湖北省武汉市430071
郭光云,女, 1971-08-16生,湖北省十堰市人,汉族, 武汉大学在读硕士.
湖北省教育厅资助项目,No. 2000A43003
项目负责人:徐少勇,442000, 湖北省十堰市,郧阳医学院附属人民医院消化内科.xushaoyong195571@sina.com.cn
, 百拇医药
电话:0719-8652119-6208
收稿日期:2003-10-09接受日期:2003-11-06
Potassiumantimonyl tartrate induces apoptosis in human gastric carcinoma cells
Guang-YunGuo, Shao-Yong Xu, Chang-Sheng Deng
Guang-YunGuo, Shao-Yong Xu, Department of Gastroenterology, People,sHospital of Yunyang Medical College, Shiyan442000, Hubei Province, China
, 百拇医药
Chang-ShengDeng, Department of Gastroenterology, Zhongnan Hospital of WuhanUniversity, Wuhan 430071, Hubei Province, China
Supported by the Education Department Foundation of Hubei Province,No. 2000A43003
Correspondence to: Shao-Yong Xu, Department of Gastroenterology,People,sHospital of Yunyang Medical College, Shiyan442000, Hubei Province, China.xushaoyong195571@sina.com.cn
, http://www.100md.com
Received:2003-10-09Accepted: 2003-11-06
AbstractAIM:Potassium antimonyl tartrate (PAT), an antiparasitic agent, has rencentlybeen shown to induce apoptosis of leukaemia cells. Its effects on humangastric cancer SGC-7901 cells and possible mechanism were investigated inthis study.
METHODS:The growth inhibition of cells induced by various concentrations of PAT indifferent time course was analyzed by using MTT assay. The nuclei werestained by Hoechst 33 258 and the morphologic changes were observed byfluorescence microscope. TUNEL staining, double-staining, and flowcytomery were used to detect apoptosis of cells.
, 百拇医药
RESULTS:Growth inhibitory rates of SGC-7901 cells by PAT were significantlydifferent in dose-and time-dependent manners (analysis of variance,P <0.01).Chromatin condensation and nuclear fragmentation were seen underfluorescence microscope in the cells treated with Hoechst 33 258, andapoptotic cells were also detected by flow cytometry and TUNEL staining.
CONCLUSION:PAT can induce the apoptosis of SGC-7901 cells, and PAT may be a promisingapoptosis-inducer in gastric cancer therapy.
, 百拇医药
Guo GY, XuSY, Deng CS. Potassium antimonyl tartrate induces apoptosis in humangastric carcinoma cells. Shijie Huaren Xiaohua Zazhi2004;12(3):520-522
摘要
目的:用于治疗寄生虫病的酒石酸锑钾能抑制白血病细胞及淋巴瘤细胞生长，并诱导其凋亡.本文探讨其对人胃癌SGC-7901细胞株的作用及可能的机制.
方法:采用MTT法检测不同浓度酒石酸锑钾对胃癌SGC-7901细胞的生长抑制作用;经 Hoechst 33258染色后用荧光显微镜观察细胞核的形态变化;DNA末端原位标记染色法、双染法(annexin-ⅴ/PI)和流式细胞术检测细胞凋亡.
, http://www.100md.com
结果:酒石酸锑钾能明显抑制胃癌细胞生长，抑制作用呈时间和剂量依赖性(方差分析，P<0.01); Hoechst 33 258染色后荧光显微镜观察细胞核固缩，呈致密的强荧光;DNA末端原位标记染色法、流式细胞仪检测均检测到凋亡细胞.
结论:酒石酸锑钾能诱导胃癌SGC-7901细胞凋亡.有望作为一种新的凋亡诱导剂用于胃癌的治疗.
郭光云,徐少勇, 邓长生.酒石酸锑钾诱导人胃癌细胞凋亡.世界华人消化杂志2004;12(3):520-522
: 对照组; B: 酒石酸锑钾.
图2(PDF)Annexinⅴ-FITC和PI双染检测SGC-7901细胞早期凋亡.A: 对照组; B:酒石酸锑钾.
, 百拇医药
图3 SGC-7901细胞凋亡TUNEL×400. A: 对照组;B:20 mmol/L酒石酸锑钾.
2.3细胞凋亡检测结果 酒石酸锑钾作用于SGC7901细胞，用Annexinⅴ和PI染色流式细胞仪检测双参数图上可见对照组细胞主要分布于第三象限(Annexinⅴ-PI-)，酒石酸锑钾处理后细胞分布发生明显改变，第四象限细胞(Annexinⅴ+PI-)增加，代表早期凋亡细胞，第二象限细胞(Annexinⅴ+PI+)分布也增加，代表坏死细胞(图2).酒石酸锑钾处理SGC7901细胞24h后，早期细胞的凋亡率3.4%，8.2%和12.9%，呈一定的剂量效应关系.TUNEL检查细胞核中有棕红色颗粒为阳性细胞(图3).细胞的凋亡指数为7.5%，16.7%，38.1%，阴性对照组为4.6%，(dP<0.01，x2=47.454).可见在一定时间内，随着作用浓度增大，凋亡细胞的百分率也随着增加.
, 百拇医药
3 讨论凋亡异常在肿瘤发生发展中具有重要作用，逃避凋亡机制的调控在肿瘤细胞对常规化疗药物耐药中起关键作用.随着凋亡研究的深入，人们致力于开发新的药物以选择性诱导肿瘤细胞凋亡[20].酒石酸锑钾能抑制急性早幼粒细胞白血病细胞NB4和NB4R4生长，通过降解PML/PML-RARa混合蛋白，而诱导NB4和NB4R4凋亡.其微摩尔浓度可诱导NB4，NB4R4细胞凋亡，机体能很好耐受而无明显毒副作用，锑剂与RA之间无交叉耐药，副作用比三氧化二砷小.Lecureur et al [18]研究表明，酒石酸锑钾能诱导其他白血病细胞(K562，HL60，U937和KGLa)凋亡.他可能作用于谷光甘肽过氧化物酶或巯基，导致氧化应激，线粒体跨膜电位下降，触发细胞凋亡的级联反应;因诱导的凋亡不能被广谱caspase酶的抑制剂所阻滞，推测凋亡过程可能不需caspase酶的激活，但凋亡诱导因子是否参于凋亡过程尚需研究.Lecureur et al [19]研究亦发现酒石酸锑钾能抑制多种淋巴细胞株的生长，包括来源于急性淋巴细胞白血病的Jurkat，Molt-4，Nalm-6细胞和来源于淋巴瘤Dudi，Raji，Recl细胞.并能诱导Dudi，Jurkat细胞凋亡.凋亡依赖于caspase酶的活性，并与线粒体跨膜电位下降和细胞内活性氧类物质增多有关.酒石酸锑钾能显著抑制恶性淋巴细胞生长，而对正常的淋巴细胞无影响.提示酒石酸锑钾诱导凋亡对正常组织的影响小，使其在治疗血液系统恶性肿瘤中具有一定的应用前景[18-19].
, http://www.100md.com
我们发现微摩尔浓度酒石酸锑钾能明显抑制体外培养的胃癌SGC-7901细胞的生长，且其抑制细胞增生在一定范围内，随酒石酸锑钾的浓度而增加，作用时间延长，细胞增生抑制率相应增加，具有时效及量效关系.通过荧光显微镜观察到酒石酸锑钾作用后，细胞的染色质浓缩、边聚，等凋亡的形态改变;用异硫氢酸荧光素(fluorescienisothiocyanate，FITC)标记的连接素(Annexinⅴ-FITC)和PI双染法检测细胞凋亡，因Annexinⅴ能与暴露在凋亡早期细胞膜外表面的磷脂酰丝氨酸特异性结合与其连接的FITC发出绿色荧光，而此时细胞维持膜的完整性，变性染色质着色的荧光染料PI不能进入细胞;凋亡晚期及继发性坏死细胞膜失去完整性，故FITC和PI双染;坏死、机械性损伤的细胞只能PI与结合.故通过双染可区分早期的凋亡细胞与凋亡晚期坏死、继发性坏死细胞.酒石酸锑钾作用后SGC-7901细胞呈典型的凋亡细胞分布图.TUNEL法亦检测到凋亡细胞.TUNEL法和Annexinⅴ/PI法检测细胞凋亡率有一差别，TUNEL法检测凋亡率稍高，可能因为TUNEL无法区分死亡细胞与凋亡细胞，少数坏死细胞也呈阳性染色有关[3].
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