Concomitant reduction of matrix metalloproteinase-2 secretion and intracellular reactive oxygen species following anti-sense inhibition of telomerase activity in PC-3 prostate carcinoma cells
Concomitant reduction of matrix metalloproteinase-2 secretion and intracellular reactive oxygen species following anti-sense inhibition of telomerase activity in PC-3 prostate carcinoma cells
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DOI：
10.1007/s11010-005-8158-7
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发表时间：
2005-05-01
影响因子：
4.3
通讯作者：
Ebrahimkhani, M
中科院分区：
文献类型：
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作者：
Shariftabrizi, A;Khorramizadeh, M;Ebrahimkhani, M
背景：端粒酶的活性水平已被证明与几种肿瘤类型的侵袭程度相关。此外，细胞氧化还原状态被认为调节基质金属蛋白酶-2的分泌。目的：研究反义端粒酶处理对前列腺癌细胞基质金属蛋白酶-2活性的影响，以及细胞氧化还原状态的两个效应物活性氧和氮物种的变化。方法：用Fugene-6基因转染剂将针对人端粒酶RNA组分的反义寡核苷酸导入细胞内。端粒重复序列扩增法(TRAP法)检测端粒酶活性。酶谱法检测基质金属蛋白酶-2(MMP2)活性。用二氯荧光素-二乙酸酯(DCFH-DA)染色和Griess试剂分别检测细胞内活性氧(ROS)和一氧化氮代谢产物的水平。结果：TRAP法检测细胞端粒酶活性，72h后端粒酶活性抑制率达90%以上。原-基质金属蛋白酶-2活性下降到对照水平的50%。细胞内的活性氧也明显减少。结论：端粒酶抑制PC-3细胞后，伴随着原-基质金属蛋白酶-2分泌和ROS的减少，提示端粒酶的过度激活可能通过增强细胞内氧化应激，增加基质金属蛋白酶-2的水平，直接参与PC-3细胞的侵袭过程。
Background: The level of activity of the telomerase has been shown to correlate with the degree of invasiveness in several tumor types. In addition, cellular redox state is believed to regulate the secretion of matrix metalloproteinase-2 (MMP-2).Aims: To determine the effect of anti-sense telomerase treatment of prostate cancer cells on MMP-2 activity, and the reactive oxygen and nitrogen species (two effectors of cellular redox state).Methods: Anti-sense oligonucleotide against RNA component of human telomerase (hTR) was introduced into the cells using Fugene-6 transfection reagent. The activity of telomerase was assessed using Telomere Repeat Amplification Protocol (TRAP assay). Activity of matrix metalloproteinase-2 (MMP-2) was determined by zymography. Levels of intracellular reactive oxygen species (ROS) and nitric oxide metabolites were measured by dichlorofluorescein diacetate (DCFH-DA) staining and Griess reagent, respectively. The level of apoptosis was determined using TUNEL assay.Results: TRAP assay showed more than 90% inhibition of telomerase activity after 72 h of transfection. Pro-MMP-2 activity was decreased down to 50% of the control levels. Intracellular reactive oxygen species were also significantly decreased. Neither apoptosis rate nor the level of nitric oxide metabolites was significantly different between anti-sense treated and control cells.Conclusions: Concomitant reduction of the pro-MMP-2 secretion and ROS in PC-3 cells following hTR inhibition suggests that over-activity of telomerase in cancer cells might increase the level of matrix metalloproteinase-2 and thus, be directly involved in the invasion process through enhancement of intracellular oxidative stress.