Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the Bcl-2 coding sequence.
BACKGROUND
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.


OBJECTIVE
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Ziegler
A
A
University Hospital Zürich, Department of Internal Medicine, Switzerland.
Luedke
G H
GH
Fabbro
D
D
Altmann
K H
KH
Stahel
R A
RA
Zangemeister-Wittke
U
U
D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D015152:Blotting, Northern; D015153:Blotting, Western; D018288:Carcinoma, Small Cell / Q000235:genetics* / Q000628:therapy*; D015342:DNA Probes; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D019254:Genes, bcl-2 / Q000187:drug effects*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000628:therapy*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology* / Q000627:therapeutic use; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis* / Q000187:drug effects* / Q000235:genetics; D012333:RNA, Messenger / Q000187:drug effects; D012334:RNA, Neoplasm / Q000187:drug effects; D014407:Tumor Cells, Cultured; D015854:Up-Regulation / Q000187:drug effects
D000970:Antineoplastic Agents; D015342:DNA Probes; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D012334:RNA, Neoplasm