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XIAP and Survivin As Therapeutic Targets for Radiation Sensitization in Preclinical Models of Lung Cancer

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XIAP and Survivin As Therapeutic Targets for Radiation Sensitization in Preclinical Models of Lung Cancer Oncogene (2004) 23, 7047–7052 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc XIAP and survivin as therapeutic targets for radiation sensitization in preclinical models of lung cancer Carolyn Cao1,YiMu1, Dennis E Hallahan1 and Bo Lu*,1 1Department of Radiation Oncology, Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine, 1301 22nd Avenue South, B-902 The Vanderbilt Clinic, Nashville, TN 37232-5671, USA Survivin and XIAP are members of inhibitors of apoptosis pathway (Salvesen and Duckett, 2002).In addition, (IAPs) family. They are upregulated in various malig- survivin plays an important role in mitosis by interact- nancies. Inactivation of these molecules has resulted in ing with other chromosomal passenger proteins such as chemosensitization. The purpose of this study was to INCEPT and AURORA B (Adams et al., 2001). An determine whether inhibition of survivin, XIAP, or both important feature of these molecules is that there are enhances radiotherapy in a lung cancer model. Transient upregulated in various malignancies (Bao et al., 2002). transfection of H460 cells with antisense oligonucleotides Clinically, high levels of XIAP or survivin have been (ASOs) against either molecule has specifically reduced associated with decreased overall survival, increased their expression, by Western analysis. Results from 3-(4,5- recurrence and resistance to radiotherapy (Holcik et al., methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and 2000; Rodel et al., 2003). Several reports demonstrated clonogenic assays suggest that inhibition of survivin or an association between high levels of survivin and poor XIAP greatly decreased cell survival following irradiation. survival in patients with resected non-small-cell lung A significantly increased number of apoptotic cells were cancer (Monzo et al., 1999; Ikehara et al., 2002). detected when H460 cells were treated with either However, similar association has not been consistently antisurvivin, anti-XIAP or both ASOs (P ¼ 0.03, 0.0003 found for XIAP (Ferreira et al., 2001; Hofmann et al., and 0.01, respectively) plus irradiation. H460 xenografts 2002).Inhibition of survivin or XIAP via antisense that were treated with ASOs plus radiotherapy demon- oligonucleotides (ASOs) has enhanced sensitivity of lung strated growth delay beyond 15 days. Growth delay in the cancer xenografts to chemotherapy.(Olie et al., 2000; groups of combined treatment was greater than that in Hu et al., 2003). Inhibition of survivin via a ribozyme other groups. However, treatment with ASOs alone did not has resulted in radiosensitization of melanoma xeno- affect tumor growth delay in mice, but decreased the grafts (Pennati et al., 2003). XIAP and survivin are also survival of H460 cells in culture. Antisense treatment did involved in tumor angiogenesis (Papapetropoulos et al., not cause any mortality or weight loss during the 32 days 2000; Harfouche et al., 2002). Their levels in endothelial of study. These data suggest that inhibition of survivin or cells are induced by VEGF (Tran et al., 1999, 2002; XIAP radiosensitizes H460 lung cancer cells by upregulat- O’Connor et al., 2000; Mesri et al., 2001). Inhibition of ing apoptosis and downregulating cell survival. Combina- survivin by its T34A dominant-negative mutant sig- tion of radiotherapy and inhibition of survivin and XIAP nificantly reduced tumor-associated angiogenesis through the antisense approach results in improved tumor (Blanc-Brude et al., 2003). Therefore, both XIAP and control by radiotherapy in a mouse model of lung cancer. survivin are logical targets for controlling tumor growth Oncogene (2004) 23, 7047–7052. doi:10.1038/sj.onc.1207929 and angiogenesis (Holick et al., 2001; Altieri, 2003). Published online 19 July 2004 Here, we examined the effects of radiosensitization in the H460 lung cancer model via inhibiting survivin and Keywords: XIAP; survivin; lung cancer; radio therapy XIAP using antisense approach.We found that ASOs against survivin or XIAP reduce survival of H460 cells in culture and increase tumor growth delay of H460 xenografts when combined with radiation. Introduction Inhibitor of apoptosis (IAP) protein is a family of Results proteins that regulate cell death (Salvesen and Duckett, 2002).X-linked IAP and survivin are two important ASOs result in specific attenuation of survivin or XIAP members of the mammalian IAP family.Both were shown to inhibit caspases and block the apoptotic ASOs against survivin or XIAP have been used to inhibit the expression of these molecules in lung cancer *Correspondence: B Lu; E-mail: [email protected] cell lines (Olie et al., 2000; Hu et al., 2003). In order to Received 17 May 2004; revised 25 May 2004; accepted 25 May 2004; determine whether there is reduction of survivin published online 19 July 2004 or XIAP following transfection of these ASO (ASOs), XIAP and survivin as therapeutic targets C Cao et al 7048 a a MS Anti-Survivin 35.00 30.00 25.00 Survivin 20.00 15.00 10.00 5.00 percentage of apoptotic cells β-actin 0.00 l T M R 0n +RT in+RT X+ v 300nM AP 150nM) Contro n30 I nti- vi AP X -X( A MS MS-RT 2Gy vi survi ti- ti r -XI S+ ti An b nti- n +An A A ) nti- nti-su M A MS Anti-XIAP A n 150 S( ti- n XIAP A b 1.00 0.90 β-actin 0.80 0.70 Figure 1 Inhibition of XIAP and survivin expression in H460 lung cancer cells: H460 lung cancer cells were transfected with anti- 0.60 survivin (a) or anti-XIAP (b) ASOs.MS is the control oligo.After 48 h, the transfected cells were collected and analysed by Western 0.50 Blotting. b-Actin was probed to show equal loading of protein MTT activity extracts 0.40 0.30 anti-survivin, anti-XIAP or a missense (MS) control 0.20 oligo were transiently transfected into H460 cells using Lipofectin.Protein levels of survivin or XIAP were 0.10 determined by Western immunoblots 48 h following 0.00 S vin T P T X transfection.As shown in Figure 1, the ASO against M RT A RT -XI + R nti- + S + R i X X i- i- +A MS+X ti-survi Ant survivin attenuated survivin expression and the ASO nt -S nti- An A Ant ti A + An against XIAP reduced the level of XIAP, as compared -S ti to b-actin, which was not affected by either ASO. An Figure 2 Inhibition of XIAP or survivin sensitizes H460 cells to radiation by increasing apoptosis and decreasing cell viability. Inhibition of survivin or XIAP sensitizes H460 cells to H460 cells were transfected with ASOs as indicated in the bar radiation by increasing apoptosis and decreasing cell graphs.The transfected cells were irradiated with either 0 or 3 Gy. viability After 48 h, the percentage of apoptotic cells was determined by flow cytometry of 7AAD-stained cells (a) and the viable cells were Inhibition of survivin or XIAP sensitizes lung cancer determined by the MTT assay (b).Each bar represents the cells to chemotherapeutic agents (Olie et al., 2000; Hu mean7s.d., from three repeated experiments et al., 2003). To determine whether inhibition of survivin or XIAP sensitizes H460 cells to radiotherapy, the ASOs against survivin, XIAP or both were transfected into To determine the cell viability following the various H460 cells.This was followed by either 0 or 2 Gy.After treatments described, 3-(4,5-methylthiazol-2-yl)-2,5-di- 48 h, the percentage of apoptotic cells was determined phenyl-tetrazolium bromide (MTT) assay was per- by flow-cytometry analysis of 7-aminoactinomycin D formed at 48 h after irradiation.As shown in (7AAD)-stained cells.As shown in Figure 2a, radiation Figure 2b, radiation alone decreased the MTT prolif- alone increased apoptotic cells from 5 to 10%, while erative activity from 95 to 85%, while either anti- either anti-survivin or anti-XIAP oligo alone increased survivin or anti-XIAP oligo alone decreased the MTT apoptotic population to 15%.In comparison, irradia- activity to 45–50% (P ¼ 0.00015 and 0.00078, respec- tion of cells that were transfected with either anti- tively).However, irradiation of cells transfected with survivin or anti-XIAP oligo significantly increased the either anti-survivin or anti-XIAP oligo decreased the apoptotic population to almost 30% (P ¼ 0.035 and viable cells to 30–35%.Interestingly, only 20% viable 0.0003, respectively). To test the potential synergism cells were present following simultaneous inhibition of between the two ASOs, they were equally mixed survivin and XIAP by both ASOs.Irradiation of cells (150 nM þ 150 nM) to have the same final concentration transfected with both ASOs resulted in the maximal of 300 nM as that of single ASO and transfected into reduction of MTT activity, that is, only less than 10% of H460 cells.As shown in Figure 2b, no further increase in viable activity was detected. apoptosis was detected when the anti-survivin and anti- To confirm the results from the MTT assay, XIAP oligos were combined. clonogenic assays were performed.H460 cells were Oncogene XIAP and survivin as therapeutic targets C Cao et al 7049 1 a 2.600 2.400 2.200 2.000 ) 1.800 3 1.600 1.400 Control 1.200 MS Control 1.000 Anti-XIAP Tumor Volume (cm 0.800 AS-Survivin 0.1 Anti-X+Anti-S 0.600 Anti-X + XRT 0.400 XRT alone AS-S+ XRT 0.200 Anti-X+Anti-S+XRT Control 0.000 MS control 0 2 4 6 8 10121416182022242628303234 Days Anti-Survivin 300nM Anti-XIAP 300nM b BW change should be less than 10% Anti-S150nM+Anti-X 150nM 1.20 0.01 012345 1.10 Figure 3 Inhibition of XIAP and survivin decreased clonogenic survival of H460 cells following irradiation: H460 cells were 1.00 Control transfected by ASOs as indicated.The transfected cells or MS Anti-XIAP nontransfected control were treated with 0–5 Gy.After 2 weeks, B.
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