[CANCER RESEARCH 60, 2805–2809, June 1, 2000] Advances in Brief A Novel Antisense Oligonucleotide Targeting Survivin Expression Induces Apoptosis and Sensitizes Lung Cancer Cells to Chemotherapy 1 Robert A. Olie, A. Paula Simo ˜es-Wu ¨ st, Bettina Baumann, Sia ˆn H. Leech, Doriano Fabbro, Rolf A. Stahel, and Uwe Zangemeister-Wittke 2 Division of Oncology, Department of Internal Medicine, University Hospital Zu ¨rich, CH-8044 Zu ¨ rich [R. A. O., A. P. S-W., S. H. L., R. A. S., U. Z-W.]; Institute of Biochemistry, Swiss Federal Institute of Technology, CH-8092 Zu ¨rich [B. B.]; and Department of Oncology Research, Novartis Pharma AG, CH-4002 Basel [D. F.], Switzerland Abstract Survivin, an inhibitor of apoptosis protein, deserves attention as a selective target for cancer therapy because it lacks expression in differ- entiated adult tissues but is expressed in a variety of human tumors. We designed 20-mer phosphorothioate antisense oligonucleotides targeting different regions of survivin mRNA and investigated their ability to down-regulate survivin mRNA and induce apoptosis in the lung adeno- carcinoma cell line A549. Oligonucleotide 4003, which targets nucleotides 232–251 of survivin mRNA, was identified as the most potent compound. As measured by real-time PCR, 4003 down-regulated survivin mRNA in a dose-dependent manner with an IC 50 of 200 nM. Its maximum effect was achieved at a concentration of 400 nM, at which mRNA was down- regulated by 70%. As revealed by increased caspase-3-like protease ac- tivity, nuclear condensation and fragmentation, and trypan blue uptake, treatment with 4003 induced apoptosis and sensitized tumor cells to the chemotherapeutic agent etoposide. Oligonucleotide 4003 did not reduce the viability of normal blood leukocytes with marginal levels of survivin mRNA. Introduction Diminished apoptosis plays a critical role in tumor initiation, pro- gression, and drug resistance. Several proteins that inhibit apoptosis have been identified, including bcl-2 family members bcl-2 and bcl-xL and the IAPs. 3 Certain members of the latter family directly inhibit terminal effector caspases engaged in the execution of cell death (1). The gene encoding the IAP survivin was cloned recently, and the protein was characterized (2). Survivin is expressed during embryonal development but lacks expression in terminally differen- tiated adult tissues (2, 3). Interestingly, it becomes reexpressed in transformed cell lines and in a variety of human tumors (1, 2). Survivin is expressed in the G 2 -M phase in a cell cycle-regulated manner, and its interaction with the mitotic spindle apparatus is essential for antiapoptotic function (4). This could imply that the IAP survivin counteracts a default induction of apoptosis in the G 2 -M phase of the cell cycle. Overexpression of survivin has oncogenic potential because it may overcome the G 2 -M-phase checkpoint to enforce progression of cells through mitosis. Because survivin inhibits processing of downstream effector caspase-3 and -7 in cells receiving an apoptotic stimulus (1), its overexpression in tumors is implicated in the resistance to a variety of apoptotic stimuli, including chemo- therapy. Lung and breast cancer are leading causes of cancer death, and their incidence continues to rise. The main reasons for the unfavorable prognosis of these tumors is their propensity to metastasize early and develop resistance to a wide range of functionally unrelated anticancer agents. Interestingly, lung and breast cancer cells express the highest levels of survivin found in human tumors (1, 2), and in agreement with its biological function, survivin expression is correlated with shorter survival in patients with non-small cell lung cancer (5). Al- though survivin has been widely recognized as an attractive target for cancer therapy (2–10), the use of antisense cDNA and oligonucleo- tides to inhibit its expression has only recently been described (11– 13). Whereas these studies were designed to unravel the biological function of survivin, the promise of survivin antisense to facilitate apoptosis of tumor cells and overcome chemoresistance in cancer therapy remains to be determined. In the present study, we developed a series of 20-mer phosphoro- thioate oligonucleotides targeting various regions of survivin mRNA. Using real-time PCR and the survivin-overexpressing lung adenocar- cinoma cell line A549, one antisense oligonucleotide was identified that most efficiently down-regulated survivin mRNA levels and di- rectly induced apoptosis. Moreover, in a combination experiment with the chemotherapeutic agent etoposide, evidence is provided that an- tisense-mediated down-regulation of survivin has the potential to sensitize tumor cells to chemotherapy. Materials and Methods Antisense Oligonucleotides. The secondary structure of the 1619-bp sur- vivin mRNA (GenBank accession number NM001168) was predicted by the RNAdraw program (14) to identify target sites putatively presenting in single- stranded conformation and thus likely to be accessible to antisense oligonu- cleotides (15, 16). Based on the identified sites, a series of six 20-mer antisense sequences targeting different regions of survivin mRNA were designed. An- tisense oligonucleotide sequences are shown in Table 1. A 3-base mismatch control to the most potent antisense oligonucleotide was also used. Oligonu- cleotides were provided by Genset SA (Paris, France) in the form of phos- phorothioate oligonucleotides. A BLASTN search of a database containing all sequences of GenBank, European Molecular Biology Laboratory, DDBJ: DNA Data Base of Japan, and Protein Data Base was performed to exclude homol- ogy of the antisense and control oligonucleotides to other human genes. Tumor Cell Line and PBMCs. The lung adenocarcinoma cell line A549, which is reported to express high levels of survivin (1), was obtained from American Type Culture Collection and maintained in RPMI 1640 supple- mented with 2 mM L-glutamine and 10% FCS at 37°C in a humidified atmosphere containing 5% CO 2 . PBMCs, which are reported to lack survivin expression (2), were isolated from the heparin-treated blood of three healthy donors and used in transfection experiments directly upon isolation using a Ficoll-Hypaque solution (Biochrom KG, Berlin, Germany). Treatment of Cells with Antisense and Etoposide. One day before trans- fection, A549 cells were plated in 6-, 24-, or 96-well tissue culture plates. Oligonucleotides were delivered in the form of complexes with Lipofectin Received 12/10/99; accepted 4/11/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grant 31-40473.94 from the Swiss National Science Foundation, Grant 549-9-1997 from the Krebsforschung Schweiz, and the Stiftung zum Baugarten (Zu ¨rich, Switzerland). 2 To whom requests for reprints should be addressed, at Division of Oncology, Department of Internal Medicine, University Hospital Zu ¨rich, Ha ¨ldeliweg 4, CH-8044 Zu ¨rich, Switzer- land. Phone: 41-1-6342877; Fax: 41-1-6342872; E-mail: uwe.zangemeister@dim.usz.ch. 3 The abbreviations used are: IAP, inhibitor of apoptosis protein; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBMC, peripheral blood mono- nuclear cell; EPR-1, effector cell protease receptor 1. 2805 Research. on December 6, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from