Ziegler A, Luedke G H, Fabbro D, Altmann K H, Stahel R A, Zangemeister-Wittke U
University Hospital Zürich, Department of Internal Medicine, Switzerland.
J Natl Cancer Inst. 1997 Jul 16;89(14):1027-36. doi: 10.1093/jnci/89.14.1027.
The emergence of resistance to chemotherapy remains a major problem in the treatment of patients with small-cell lung cancer. Elevated expression of Bcl-2, a protein that inhibits programmed cell death or apoptosis, has been associated with radiation and drug resistance and has been observed in the majority of small-cell lung cancer specimens and cell lines.
To test the hypothesis that Bcl-2 expression levels are critical for inhibiting apoptosis in small-cell lung cancer cells, we used an antisense strategy to reduce Bcl-2 expression in these cells in an attempt to restore the natural occurrence of apoptosis.
Thirteen antisense oligodeoxynucleotides (ODNs) targeting various regions of the bcl-2 messenger RNA and a control scrambled-sequence ODN were tested to identify the most effective sequence(s) for reducing Bcl-2 protein levels. Northern and western blot analyses were used to examine basal bcl-2 messenger RNA and protein levels, respectively, in four human small-cell lung cancer cell lines (SW2, NCI-H69, NCI-H82, and NCI-N417). SW2 cells were treated with the antisense ODNs in the presence of cationic lipids (to facilitate uptake), and cytotoxic effects were measured by use of a cell viability assay. Flow cytometric analysis of DNA fragmentation and cell morphology was also performed. The cytotoxic effect of the most potent antisense ODN was also tested on the three other cell lines.
The viability of SW2 cells was effectively reduced by ODNs that targeted the translation initiation and termination sites of the bcl-2 messenger RNA, but ODN 2009 that targeted the coding region was the most cytotoxic. Treatment of SW2 cells with 0.15 microM ODN 2009 for 96 hours reduced their viability by 91% (95% confidence interval [CI] = 88%-94%) and caused a dose-dependent reduction in Bcl-2 levels that became detectable 24 hours after treatment and persisted up to 96 hours; analysis of cellular morphology demonstrated that viability was reduced through apoptosis. Moreover, ODN 2009 at 0.15 microM was cytotoxic to NCI-H69, NCI-H82, and NCI-N417 cells, resulting in decreases in cell viability of 82% (95% CI = 78%-86%), 100%, and 100%, respectively, after 96 hours of treatment. The cytotoxic effects were inversely correlated with the basal Bcl-2 levels in the cell lines (r = -9964). A control scrambled-sequence oligodeoxynucleotide had no statistically significant effect on the cell lines (P values ranging from .38 to .89).
We have identified a novel antisense ODN sequence (ODN 2009) that effectively reduces the viability of small-cell lung cancer cells by reducing Bcl-2 levels and facilitating apoptosis.
化疗耐药的出现仍是小细胞肺癌患者治疗中的一个主要问题。Bcl-2是一种抑制程序性细胞死亡或凋亡的蛋白质，其表达升高与放疗和耐药相关，并且在大多数小细胞肺癌标本和细胞系中均有观察到。
为了验证Bcl-2表达水平对抑制小细胞肺癌细胞凋亡至关重要这一假说，我们采用反义策略降低这些细胞中的Bcl-2表达，试图恢复凋亡的自然发生。
测试了13种靶向bcl-2信使核糖核酸不同区域的反义寡脱氧核苷酸（ODN）和一种对照乱序序列ODN，以确定降低Bcl-2蛋白水平最有效的序列。分别使用Northern印迹和Western印迹分析检测4种人小细胞肺癌细胞系（SW2、NCI-H69、NCI-H82和NCI-N417）中基础bcl-2信使核糖核酸和蛋白水平。在阳离子脂质存在的情况下（以促进摄取）用反义ODN处理SW2细胞，并通过细胞活力测定法测量细胞毒性作用。还进行了DNA片段化的流式细胞术分析和细胞形态分析。最有效的反义ODN对其他三种细胞系的细胞毒性作用也进行了测试。
靶向bcl-2信使核糖核酸翻译起始和终止位点的ODN有效降低了SW2细胞的活力，但靶向编码区的ODN 2009细胞毒性最强。用0.15微摩尔/升ODN 2009处理SW2细胞96小时后，其活力降低了91%（95%置信区间[CI]=88%-94%），并导致Bcl-2水平呈剂量依赖性降低，处理后24小时可检测到，持续至96小时；细胞形态分析表明活力通过凋亡降低。此外，0.15微摩尔/升的ODN 2009对NCI-H69、NCI-H82和NCI-N417细胞具有细胞毒性，处理96小时后，细胞活力分别降低了82%（95%CI=78%-86%）、100%和100%。细胞毒性作用与细胞系中的基础Bcl-2水平呈负相关（r=-0.9964）。对照乱序序列寡脱氧核苷酸对细胞系无统计学显著影响（P值范围为0.38至0.89）。
我们确定了一种新的反义ODN序列（ODN 2009），其通过降低Bcl-2水平和促进凋亡有效降低小细胞肺癌细胞的活力。
