Adenovirus-mediated transfer of siRNA against survivin enhances the radiosensitivity of human non-small cell lung cancer cells

Cancer Gene Therapy (2010) 17, 120–130 r 2010 Nature Publishing Group All rights reserved 0929-1903/10 $32.00 www.nature.com/cgt ORIGINAL ARTICLE Adenovirus-mediated transfer of siRNA against survivin enhances the radiosensitivity of human non-small cell lung cancer cells C-T Yang1,2, J-M Li1,3, H-H Weng4, Y-C Li1, H-C Chen5 and M-F Chen6 1 Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, Chiayi, Taiwan; Department of Respiratory Care, College of Medicine, Chang Gung University, Taoyuan, Taiwan; 3Graduate Institute of Animal Science, College of Agriculture, National Chiayi University, Chiayi, Taiwan; 4Department of Diagnostic Radiology, Chang Gung Memorial Hospital, Chiayi, Taiwan; 5Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan and 6Department of Radiation Oncology, Chang Gung Memorial Hospital, Chiayi, Taiwan 2 Expression of survivin has been reported to be correlated with shorter survival in patients with non-small cell lung cancer (NSCLC), and overexpression of survivin may lead to radioresistance in various human cancers. In this study, we inhibited survivin expression by using an adenoviral vector (AdsiSurvivin)-mediated RNA interference to elucidate the combined effect of survivin-targeting gene therapy and radiotherapy on the NSCLC cells. Our data showed that AdsiSurvivin exerted survivin gene silencing, induced apoptosis, and significantly attenuated the growth potential in NSCLC cells within 72 h after infection. The combined treatment modalities with AdsiSurvivin infection and radiation were significantly more potent on cell-growth inhibition than monotherapy. In H1650, H460, A549, and H1975 human NSCLC cells, the survival ratios of AdsiSurvivin-treated groups at multiplicity of infection of 25 and 50 were significantly lower than those of control groups at varying radiation dose (0–8 Gy; three-way analysis of variance, Po0.05). The cytotoxicity of combined AdsiSurvivin infection and irradiation increased in a dose-dependent manner in both the virus and the irradiation treatment. Knockdown of the survivin gene expression seems to be a promising treatment strategy for NSCLC. Our data warrant the need for further effort to develop survivin-targeted radiosensitizer for lung cancer treatment. Cancer Gene Therapy (2010) 17, 120–130; doi:10.1038/cgt.2009.55; published online 4 September 2009 Keywords: adenovirus; RNA interference; survivin; radiotherapy; non-small cell lung cancer Introduction Survivin is a member of the inhibitor of apoptosis family that acts as a suppressor of apoptosis and has a critical role in tumor initiation, progression, and drug resistance.1 High levels of survivin have been found in most human cancers, including cancers of the lung, colon, pancreas, prostate, breast, and stomach.2–5 Expression of survivin has been reported to be correlated with shorter survival in patients with non-small cell lung cancer (NSCLC).3 Survivin is expressed in the G2/M phase in a cell-cycleregulated manner, and its interaction with the microtubules of mitotic spindle at the beginning of mitosis is essential for its anti-apoptotic function. Disruption of the survivin–microtubules interaction results in the loss of Correspondence: Dr C-T Yang, Division of Pulmonary and Critical Care Medicine, Chang Gung Memorial Hospital, 6 West Chiapu Road, Putzu, 613 Chiayi, Taiwan. E-mail: Received 10 December 2008; revised 12 April 2009; accepted 21 June 2009; published online 4 September 2009 survivin’s anti-apoptotic function and increases the caspase-3 activity.6,7 Overexpression of survivin has oncogenic potential because it may overcome the G2/M-phase checkpoint to compel the cell cycle to progress through mitosis.6 In addition, survivin overexpression has been shown to result in accelerated S-phase shift, resist G1 arrest, activate cyclin-dependent kinase 2/cyclin E complex and thereby result in Rb phosphorylation.8 Moreover, the expression of survivin could be significantly upregulated by a sublethal dose of irradiation, which might cause radioresistance.9 Lu et al.10 reported that overexpression of survivin in human embryonic kidney cells (293 cells) prevented irradiation-induced apoptosis and increased cell viability after irradiation. Increasing evidences indicated that the expression of survivin correlated with radioresistance in various cancers, thereby implying that survivin may be a potential target for radiosensitization during cancer treatment.11–14 Radiotherapy is commonly used in NSCLC treatment for either curative or palliative purposes. However, majority of patients are most likely to suffer regional failure as a part of their disease recurrence process.15,16 Survivin inhibition and radiation in NSCLC cells C-T Yang et al Recent researches to improve the clinical outcome in such patients include altered irradiation fraction schedule and the introduction of chemotherapy, biotherapy, immunotherapy, virotherapy, or gene therapy on a concurrent or adjuvant basis.17–22 Survivin gene expression has been identified in a majority of NSCLC.3 Additionally, Tamm et al.23 reported that among all the human tumor cells screened, lung cancer cells expressed the highest levels of survivin. As survivin overexpression may lead to resistance to radiotherapy by inhibiting apoptosis and enhancing cell viability, knockdown of the survivin gene expression by RNA interference should be a promising approach to ameliorate the efficacy of radiotherapy during NSCLC treatment. In this study, we inhibited survivin expression by using RNA interference to elucidate the combined effect of survivin-targeting gene therapy and radiotherapy on NSCLC cells. To achieve sufficiently high level of gene suppression in adequately large numbers of target cells, we constructed an adenoviral vector expressing short hairpin RNA (shRNA).24 We show here the impact of adenovirus-mediated transfer of small interfering RNA (siRNA) targeting survivin on human NSCLC cells and the additive efficacy with irradiation in cell killing. Materials and methods Cell lines The NSCLC cell lines A549 (ATCC CCL-185), H460 (ATCC HTB-177), H1650 (ATCC CRL-5883), and H1975 (ATCC CRL-5908) were purchased from American Type Culture Collection (ATCC; Manassas, VA, US). All were cultured in Dulbecco’s modified eagle medium (DMEM) complete media containing 10% fetal bovine serum. Development of stable 293 cell lines with high survivin expression PcDNA3-survivin plasmid was constructed by subcloning the fragment of the survivin-encoding cDNA from plasmid pORF5-hSurvivin (Invivogene, San Diego, CA) into the pcDNA3-flag at BamHI site. The vector of pcDNA3-flag was the same as described earlier.25 Human embryonic kidney cells (E1-transformed; 293 cells) were subsequently transfected with pcDNA3-survivin; transfection was conducted by using a liposome-mediated transfection technique with lipofectamine 2000 reagent (Invitrogen, Grand Island, NY) and in accord (...truncated)