三氧化二砷抑制恶性黑色素瘤B_(16)细胞生长及对端粒酶活性的影响

背景与目的:研究三氧化二砷(As2O3)对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的影响,探讨砷及其化合物治疗恶性黑色素瘤的作用机制,为临床治疗恶性黑色素瘤提供新的理论和实验依据。材料与方法:四甲基噻唑氮蓝(Methylthiazolyltetrazolium,MTT)比色法检测As2O3对B16细胞的生长抑制作用:端粒重序列扩增酶联免吸附实验(Telomericrepeatamplificationprotocolenzyme_linkedimmunosorbantassay,TRAR_ELISA)和聚内烯酰胺凝胶电泳银染(Telomericrepeatamplificationprotocol,polyacrylamidegelelectrophoresissilver_staining,TRAP_PAGE)法检测B16细胞的端粒酶活性。结果:As2O3可显著抑制B16细胞的生长并可下调端粒酶活性,其抑制作用有显著的时间和剂量依赖关系。结论:As2O3对恶性黑色素瘤B16细胞生长抑制作用及对端粒酶活性表达的抑制作用随药物浓度的增加和作用时间的延长而增强。     

三氧化二砷对恶性黑色素瘤A375细胞端粒酶活性的抑制作用

背景与目的 :研究三氧化二砷(As2O3)对人恶性黑色素瘤A375 细胞端粒酶活性的抑制作用,探讨砷及其化合物治疗恶性黑色素瘤的作用机制 ,为砷及其化合物治疗恶性黑色素瘤提供新的理论和实验依据。 材料与方法 :用TRAP_ELISA和TRAP_PAGE银染法测定A375 细胞端粒酶活性。结果 :As2O3 对人恶性黑色素瘤A375 细胞端粒酶活性抑制有明显的浓度和时间依赖关系。结论 :As2O3 对恶性黑色素瘤A375 细胞端粒酶活性抑制率随药物浓度的增加或作用时间的延长而提高     

镍化合物诱发恶性转化细胞的端粒酶活性表达

目的：探讨二种镍化合物诱发细胞恶变过程中端粒酶活性的变化。方法：二种镍化合物转化细胞接种BALB／c裸鼠，应用端粒重复序列扩增法(telomeric repeat amplification protocol，TRAP)检测转化细胞和肿瘤细胞的端粒酶活性。结果：结晶型硫化镍和氯化镍诱发的BALB／c-313恶性转化细胞的端粒酶活性均表现为阳性。结论：镍化合物诱发的细胞恶性转化涉及了端粒酶活性改变，提示了端粒酶表达与镍转化作用有关。     

三氧化二砷的抗大肠癌作用及其机制

目的观察三氧化二砷(As2O3)对大肠癌LS-174T细胞生长及端粒酶活性的影响。方法As2O3作用于大肠癌LS-174T细胞和裸鼠移植瘤后,采用噻唑盐(MTT)比色法检测结肠癌细胞生长抑制情况,电镜检测细胞超微结构的改变,荧光染色观察细胞核形态变化,流式细胞术(FCM)检测As2O3对细胞周期的影响,聚合酶链反应-酶联免疫反应(PCR-ELISA)试剂盒检测细胞中端粒酶活性变化。结果MTT法显示,随着As2O3浓度的升高,LS-174T细胞存活率明显下降,IC50= 5.23μmol/L。As2O3作用于LS-174T细胞24 h即出现凋亡峰,凋亡细胞的数量随着作用时间增加而增加。As2O3对LS-174T细胞提取液端粒酶活性有一定的抑制作用,对癌细胞端粒酶活性抑制作用随作用时间的延长而加强。从实验动物瘤体积和瘤重两个指标分析,As2O3组与对照组相比具有明显的抑癌作用(P<0.05)。结论经体外、体内实验证实,As2O3对结肠癌LS-174T细胞生长和裸鼠移植瘤均有抑制作用,其抗癌机制可能与诱导细胞凋亡和抑制端粒酶活性有关。     

三氧化二砷对人大肠癌细胞生长及其端粒酶活性的影响

目的探讨三氧化二砷(As2O3)注射液对人类大肠癌细胞生长及其端粒酶活性的影响。方法采用活细胞观察法、台盘兰拒染法和四甲基偶氮唑蓝比色(MTT)法,严密观察As2O3注射液对人大肠癌细胞株LoVo和CoLo-320生长的影响;应用端粒重复序列扩增法(TRAP)—聚丙烯酰胺凝胶电泳(PAGE)银染法检测As2O3注射液处理前、后大肠癌细胞端粒酶活性的变化。结果As2O3注射液能够显著地抑制LoVo和CoLo-320细胞株的生长,并对癌细胞的端粒酶活性具有明显的抑制作用,该作用呈现出一定的浓度和时间依赖性。结论As2O3注射液的确对于人大肠癌细胞株的生长具有显著的抑制作用,该作用可能与As2O3注射液能够抑制癌细胞的端粒酶活性密切相关。     

三氧化二砷联合氟尿嘧啶体外对人类大肠癌细胞生长及端粒酶活性的影响

目的:探讨三氧化二砷(As2O3)注射液与氟尿嘧啶(5-FU)联合应用对人类大肠癌细胞的生长及其端粒酶活性的影响。方法:采用四甲基偶氮唑蓝比色(MTT)法观察As2O3注射液和/或5-FU对人类大肠癌细胞株LoVo和CoLo-320生长的影响;应用端粒重复序列扩增法(TRAP)—聚丙烯酰胺凝胶电泳(PAGE)银染法检测As2O3注射液和/或5-FU处理前后大肠癌细胞端粒酶活性的变化。结果:与As2O3或5-FU单药作用相比,As2O3与5-FU联合应用可显著增强抑制人结肠癌细胞增殖作用,并对细胞端粒酶活性具有增强抑制作用。结论:与各单药相比较,As2O3注射液联合5-FU能够明显增强抗大肠癌作用,其机制可能与增强抑制癌细胞的端粒酶活性有关。     

三氧化二砷和5-氟脲嘧啶对人结肠癌裸鼠移植瘤端粒酶及其催化亚单位的影响

目的：研究三氧化二砷(As2O3)注射液、5-氟脲嘧啶(5-Fu)注射液单用和两药联合使用埘人结肠癌裸鼠移植瘤端粒酶活性及其催化亚单位表达的影响：方法：建立人类结肠癌裸鼠移植瘤模型,随机分为4组：牛理盐水组;As2O3组(2．5mg／kg);As2O3(2．5mg／kg) 5-Fu组(20mg／kg);5-Fu组(20mg／kg)。采用腹腔注射给药。利用银染-端粒重复扩增(TRAP)法测定人结肠癌裸鼠移植瘤端粒酶活性、RT—PCR法检测端粒酶催化亚基(human telomerase reverse transeriptases,hTERT)的表达：结果：生理盐水组和5-Fu组的端粒酶活性及其催化亚单位表达不受影响．As2O3单药组和联合用药组均明显抑制端粒酶活性及其催化亚单位表达。结论：As2O3单用以及联合5-Fu对于人结肠癌裸鼠移植瘤细胞的端粒酶活性及其催化亚单位的表达均具有明显的抑制作用。     

三氧化二砷诱导HeLa细胞凋亡与其抑制HPV18 E6表达和端粒酶活性有关

目的 探讨三氧化二砷(As2O3)诱导人宫颈癌HeLa细胞凋亡时,对14PV18 E6致瘤基因和端粒酶活性的影响。方法 用不同浓度的As2O3作用于HeLa细胞不同时间段后,光镜和电镜观察细胞形态改变,流式细胞仪检测细胞周期改变,MTT法测定细胞生长抑制情况。RT-PCR方法检测HPV18 E6的表达,TRAP-ELISA方法测定细胞内端粒酶活性变化。结果 2μmol／L As2O3处理HeLa细胞48h后,可降低细胞存活率,诱导细胞凋亡,抑制HPV18 E6的表达,细胞内端粒酶活性明显下降。As2O3对HeLd细胞的这种作用呈时间-剂量依赖关系。结论 As2O3诱导HeLa细胞凋亡可能与其抑制HPV18 E6致瘤基因的表达有关,细胞内端粒酶活性的抑制可能与这种E6致瘤基因的抑制有关。     

三氧化二砷对肝癌细胞survivin基因和端粒酶活性的影响

目的:探讨三氧化二砷(As2O3)诱导肝癌细胞凋亡时survivin基因表达和端粒酶活性的变化。方法:采用倒置相差显微镜观察细胞形态,流式细胞仪分析细胞亚二倍体百分率,逆转录-多聚酶链式反应检测细胞survivin mRNA表达,免疫组织化学染色检测细胞survivin蛋白的表达,端粒酶检测试剂盒检测端粒酶活性。结果:0.25~2.00μmol/L As2O3明显抑制肝癌Bel-7402细胞生长,诱导细胞凋亡,survivin mRNA和蛋白表达上调,端粒酶活性下降,呈时间、剂量依赖关系。结论:As2O3抑制肝癌Bel-7402细胞增殖,诱导细胞凋亡,可能与其降低端粒酶活性有关;而survivin基因表达上调,可能是抵抗As2O3诱导凋亡的途径之一。     

三氧化二砷与顺铂合用抗人肝癌细胞株QGY-7701的实验研究

目的　探讨三氧化二砷 (As2 O3)与顺铂 (PDD)联合抗人肝癌细胞株QGY 770 1作用性质及其作用机制。方法　应用MTT法、流式细胞术分析和端粒酶活性检测等实验方法。结果　As2 O3与PDD联合应用在抑制肝癌细胞的生长繁殖、诱导肝癌细胞凋亡和抑制其端粒酶活性等方面均较各自单药的作用明显增强。结论　As2 O3与PDD联合应用具有明显的协同抗肝癌作用 ,其主要机制可能在于增强了抑制端粒酶活性。     

小鼠黑色素瘤某些生物学特性与其侵袭潜能的相关性研究

目的 研究具有相同起源而转移能力不同的小鼠黑色素瘤细胞系（B16、B16F10、B16BL6）的某些生物学特性与其侵袭潜能的相关性。方法 用重组基质膜实验考察细胞的侵袭能力;用分光光度法测定黑色素瘤细胞的黑色素含量;利用细胞运动记录分析系统研究细胞在三维胶原基质中的运动状态;用明胶底物酶谱法分析细胞分泌Ⅳ型胶原酶的能力;用端粒重复扩增（TRAP）－PCR法测定细胞的端粒酶活性。结果 B16B6力B16F10具有较高的侵袭能力,其运动能力及分泌Ⅳ型胶原酶的能力亦较强,但B16F10的黑色素含量却较低;端粒酶活性在三者之间无明显差异。结论在所研究的小鼠黑色素瘤不同亚系的声东击西 些生物学特性中,侵袭能力与细胞运动能力、分泌Ⅳ型胶原酶的能力之间有较好的相关性,而与黑色素含量及端粒酶活性无直接相关性。     

Migration and metalloproteinases determine the invasive potential of mouse melanoma cells,but not melanin and telomerase

The biological characteristic cell locomotion and invasion, melanin content, metalloproteinases and telomerase activity were studied in a parental mouse melanoma cell line B16 and two descendents B16BL6 and B16F10. The invasive potential of melanoma cells was assayed in a transwell cell culture chamber. Melanin content was determined by the absorbance value at 470 nm per 10⁶ cells. Tumor cells migration within the 3-D collagen matrix was microscopically recorded with a time-lapse video recorder and analyzed by computer-assisted cell tracking. Gelatin zymography was adopted to assay the metalloproteinases secretion. A polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) was used for measuring telomerase activity. The results demonstrated that B16BL6 and B16F10 cells were highly invasive compared to B16 cells, but the melanin content of B16F10 was very low. B16F10 and B16BL6 were hypermotile and secreted much more metalloproteinases than B16. No differences were observed in telomerase activity among the three melanoma cell lines. Invasion of mouse melanoma was closely correlated to tumor cell migration and secretion of metalloproteinases. Melanin content and telomerase activity were phenotypically not related to invasiveness in these three mouse melanoma cell lines.     

二氟甲基鸟氨酸对K562细胞生长特性的影响及其与端粒酶活性的相关性

目的研究二氟甲基鸟氨酸(DFMO)对K562细胞生长特性、凋亡及端粒酶活性的影响。方法采用细胞计数、形态观察、FCM分析及DNA电泳分别检测DFMO处理的细胞生长速率、周期分布及细胞凋亡。按TRAP法对细胞端粒酶活性进行检测。结果DFMO处理的细胞生长速率明显减慢;G1期细胞增多而S期细胞减少;细胞表现凋亡诱导,且其端粒酶活性受到抑制。结论DFMO引起的K562细胞生长抑制及凋亡诱导与端粒酶活性抑制有关。端粒酶的失活是抑制多胺生物合成的抗癌分子机制重要事件之一。     

As2 O3对人胃癌BGC-823 细胞端粒酶的影响

 目的 观察三氧化二砷（As₂ O₃）对人胃癌BGC-823细胞端粒酶活性及端粒酶逆转录酶基因转录的影响。方法 用不同浓度的三氧化二砷作用于人胃癌BGC-823细胞，通过MTT法测定细胞活力；流式细胞术检测细胞凋亡变化；TRAP PCR ELIsA方法检测细胞端粒酶活性的变化；RT-PCR半定量方法检测细胞端粒酶hTEI汀基因mRNA的转录水平。结果 三氧化二砷可明显抑制人胃癌BGC-823细胞的生长，诱导细胞发生凋亡，抑制作用随三氧化二砷浓度的升高及作用时间的延长而增强。细胞的端粒酶活性明显下降，细胞hTERT基因的转录表达显著抑制。BGC-823细胞端粒酶活性的下调与As₂ O₃作用时间和浓度有关，呈现明显的时间-剂量效应关系。结论 As₂ O₃能够抑制人胃癌BGC-823细胞的增殖、促使细胞凋亡、抑制细胞的端粒酶活性。As₂ O₃是一种良好的端粒酶抑制剂，在肿瘤的治疗中具有一定应用价值。     

亚砷酸钠对人肺癌Spc-A1细胞端粒酶活性的影响

目的:研究亚砷酸钠( NaAsO2)对人肺癌Spc-A1细胞株端粒酶活性及其催化亚单位hTERT表达的影响,探讨其抗癌机制.方法:四甲基偶氮唑蓝法(MTT)检测亚砷酸钠对Spc-A1细胞增殖的抑制作用;端粒酶活性采用端粒末端重复序列扩增-酶联免疫吸附法(TRAP-ELISA)测定;端粒酶催化亚单位hTERT mRNA表达采用反转录聚合酶链式反应法(RT-PCR)测定.结果:亚砷酸钠对人肺癌细胞株Spc-A1的增殖具有一定程度的抑制作用.在12h、24h和48h三个时间段均可见Spc-A1细胞端粒酶活性随着药物浓度的增加逐渐下降.并且,hTERT mRNA表达下调与端粒酶活性下降一致.结论:亚砷酸钠对Spc-A1细胞的增殖具有一定的抑制作用,下调癌细胞hTERT mRNA的表达来抑制端粒酶活性可能是其中一种机制.     

Spontaneous regression of cutaneous melanoma in sinclair swine is associated with defective telomerase activity and extensive telomere erosion

Recently we proposed the hypothesis that extensive telomeric association of chromosomes is an early manifestation of cell death and asked whether there are extensive telomeric associations present in metaphases of the spontaneously regressing Sinclair swine cutaneous melanoma (SSCM). Our results indicate that early passage SSCMs, in the accelerated growth phase, do not show telomeric associations but do have numerical and other specific structural abnormalities. However, the same melanoma cell lines at late passages or melanomas obtained from middle- and old-aged Sinclair swine show extensive telomeric associations in the form of dicentric, multicentric, and ring configurations. Such abnormal structures are present mostly in metaphases that are hyperploids. Increasing frequencies of apoptotic bodies were also observed in higher passage tumor cell lines obtained from younger animals or in melanomas obtained from older animals. The polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay shows no detectable telomerase activity in any of these regressing swine melanoma cell lines, neither in normal swine skin fibroblasts nor in nevi. However, the fetal swine (i.e., non-regressing) melanoma cells show telomerase activity. Fluorescence in situ hybridization (FISH) results using the commercially available human telomeric repeat DNA probe indicate a reduction of telomeric signals in metaphase and interphase cells of regressing melanomas. From these observations we conclude that spontaneous regression of SSCM is associated with the loss of telomerase activity and a reduction of telomeric repeats that results in the formation of multicentric and ring configurations. Such abnormal chromosome configurations are lost, following the breakage-fusion-bridge-cycles, and result in extensive DNA fragmentation, as shown by laddering experiments, and, finally, cell death.     

Antitumor activity of systemically delivered ribozymes targeting murine telomerase RNA.

PURPOSE: To test ribozymes targeting mouse telomerase RNA (mTER) for suppression of the progression of B16-F10 murine melanoma metastases in vivo. EXPERIMENTAL DESIGN: Hammerhead ribozymes were designed to target mTER. The ribozyme sequences were cloned into a plasmid expression vector containing EBV genomic elements that substantially prolong expression of genes delivered in vivo. The activity of various antitelomerase ribozymes or control constructs was examined after i.v. injection of cationic liposome:DNA complexes containing control or ribozyme constructs. Expression of ribozymes and mTER at various time points were evaluated by quantitative real-time PCR. Telomerase activity was examined using the telomeric repeat amplification protocol. RESULTS: Systemic administration of cationic liposome:DNA complexes containing a plasmid-expressed ribozyme specifically targeting a cleavage site at mTER nucleotide 180 significantly reduced the metastatic progression of B16-F10 murine melanoma. The antitumor activity of the anti-TER 180 ribozyme in mice was abolished by a single inactivating base mutation in the ribozyme catalytic core. The EBV-based expression plasmid produced sustained levels of ribozyme expression for the full duration of the antitumor studies. In addition to antitumor activity, cationic liposome:DNA complex-based ribozyme treatment also produced reductions in both TER levels and telomerase enzymatic activity in tumor-bearing mice. CONCLUSIONS: Systemic, plasmid-based ribozymes specifically targeting TER can reduce both telomerase activity and metastatic progression in tumor-bearing hosts. The work reported here demonstrates the potential utility of plasmid-based anti-TER ribozymes in the therapy of melanoma metastasis.