Enhanced radiosensitivity of non-small-cell lung cancer (NSCLC) by adenovirus-mediated ING4 gene therapy

Abstract

Radiotherapy is the common treatment of choice for locally advanced lung cancer, but the radioresistance of lung cancer remains a significant therapeutic obstacle. We previously demonstrated that adenovirus-mediated inhibitor of growth 4 (ING4) tumor suppressor gene delivery (AdVING4) can chemosensitize human hepatocarcinoma cells to anticancer drug cisplatin (CDDP). However, its radiosensitizing effects in cancer therapy are largely elusive. In the present study, we investigated the therapeutic efficacy of AdVING4 gene therapy combined with ionizing radiotherapy for SPC-A1 human non-small-cell lung cancer (NSCLC) cells in vitro and in vivo in athymic nude mice, and also elucidated its underlying mechanisms. We found that AdVING4 gene therapy plus radiotherapy induced synergistic tumor suppression and apoptosis in in vitro SPC-A1 human NSCLC cells and in vivo SPC-A1 xenografted tumors s.c. implanted in athymic nude mice. Mechanistically, AdVING4 combined with radiation resulted in a substantial upregulation of Bax, Fas, FasL and Cleaved Caspase-3, and downregulation of Bcl-2 in SPC-A1 human NSCLC xenografted tumors. In addition, AdVING4 plus radiation synergistically reduced the tumor vessel CD34 expression and microvessel density (MVD) in vivo. Most importantly, AdVING4 potentially blocked the radiation-induced enhancement of cyclooxygenase-2 and survivin radioresistant factors, and vascular endothelial growth factor and IL-8 proangiogenic factors. The enhanced antitumor effects elicited by AdVING4 plus radiotherapy were closely associated with the cooperative activation of intrinsic and extrinsic apoptotic pathways, and synergistic inhibition of tumor angiogenesis. Thus, our results suggested that AdVING4 combined with radiotherapy may be a feasible and effective strategy for treatment of radioresistant NSCLC and other cancers.

Introduction

Lung cancer is the leading cause of cancer-related mortality among men and women throughout the world, with non-small-cell lung cancer (NSCLC) accounting for about 80–85% of all cases.1, 2 Approximately 50% of patients have locally advanced (stage III) disease when they are diagnosed with NSCLC. Treatments for lung cancer consist of surgery, chemotherapy, radiotherapy and sequential or concurrent combination.3 Despite advances made in conventional treatments, the therapeutic interventions for lung cancer have achieved only modest benefits, and poor 5-year survival rate of patients with lung cancer has not been significantly improved.4 The resistance to chemotherapeutic drugs and radiation as well as metastatic propensity is one of main reasons for the current treatment failure.4 Therefore, the development of novel treatment strategies to improve the prognosis of lung cancer especially NSCLC are urgently needed. Optimization of combined modality therapy (multimodality management) of standard and novel therapeutic agents may improve the outcome of treatment for lung cancer.3

Inhibitor of growth (ING) represents a type II tumor suppressor family comprising ING1-ING5 five conserved members involved in carcinogenesis, apoptosis, cell cycle control, DNA repair and senescence. Recently, ING4 has attracted more and more attention as a potent tumor suppressor. A number of studies have shown that ING4 is frequently downregulated,5, 6, 7, 8 deleted6, 9 or mutated10, 11 in a large variety of cancers, which is closely correlated with tumorigenesis and development. ING4 can suppress the loss of contact inhibition induced by myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN) and myelocytomatosis viral oncogene homolog (MYC) family oncogenes.12 Forced expression of ING4 can also significantly elicit tumor growth inhibition in a p53-dependent and p53-independent manner via induction of cell cycle alteration and apoptosis.13, 14, 15, 16 Conversely, ING4-dominant mutant can promote MYC-initiated mouse mammary carcinogenesis.10 Furthermore, ING4 can suppress the production of IL-6 and IL-8 proangiogenic factors, and the growth of vascular endothelial cells, and consequently inhibit tumor angiogenesis through repressing the activation of nuclear factor-κB (NF-κB)5, 17 and hypoxia inducible factor-1α (HIF-1α).18, 19 Additionally, ING4 can inhibit tumor cell spreading, migration and invasion15 by interaction with liprin α120 and reduced matrix metalloproteinase-2 and 9.16, 21 Thus, ING4 is a promising candidate tumor suppressor for cancer therapy via targeting multiple pathways.

The combination of gene therapy and some type of conventional anticancer therapies such as chemotherapy and radiotherapy is a promising practice in the treatment of cancers,22, 23, 24 which can improve therapeutic benefits, reduce the side-effects and override the resistance to chemotherapy drugs or radiation. ING4 has been shown to enhance chemosensitivity to DNA-damage agents, such as doxorubicin and etoposide in human hepatocarcinoma cells.14 We also previously reported that adenovirus-mediated ING4 (AdVING4) gene therapy can sensitize human hepatocarcinoma cells to anticancer drug cisplatin (CDDP) and induce enhanced antitumor activity.22 More recently, we have demonstrated that AdVING4 combined with Iodne-125 (125I) intracavitary radiotherapy produced synergistic tumor inhibition in human pancreatic cancer.25 However, its radiosensitizing effect and combined antitumor activity with external beam radiotherapy for human cancers has not been reported. To extend our previous works and improve the therapeutic ratio of radiotherapy for NSCLC, in this study, we further evaluated the therapeutic efficacy of AdVING4 gene therapy combined with ionizing radiation in vitro in radioresistant SPC-A1 human NSCLC cells and in vivo in an athymic nude mouse human NSCLC tumor model, and also elucidated the underlying mechanisms.

Materials and methods

Adenoviruses, cell lines, reagents and mice

The recombinant replication-incompetent Ad5E1- and E3-deleted adenovirus with two independent cytomegalovirus (CMV) promoters driving ING4 (1stCMV) and GFP (2ndCMV) expression, AdVING4/GFP (termed AdVING4) and its control blank adenovirus AdVGFP (termed AdV) expressing GFP under the control of 2ndCMV were constructed in our laboratory,16 Cell and Molecular Biology Institute, College of Medicine, Suzhou University (Suzhou, Jiangsu, China). The QBI-293A human embryonic kidney cell line was kindly provided by Professor Jiang Zhong, Fudan University (Shanghai, China). The SPC-A1 human adenocarcinoma cell line, a kind of human NSCLC cell line, was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). The QBI-293A and SPC-A1 cells were cultured in Dulbecco’s modified Eagle medium (Invitrogen, Shanghai, China) supplemented with 10% fetal bovine serum (HyClone, Logan, UT, USA). The Trizol and reverse transcriptase polymerase MuMLV were purchased from Invitrogen. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit were purchased from Sigma (Shanghai, China). The Annexin V-PE/7-AAD apoptosis detection kit was purchased from BD Biosciences (Shanghai, China). The polyclonal anti-ING4 antibody was purchased from Abcam (Shanghai, China). The SuperEnhanced chemiluminescence detection kit was purchased from Applygen Technologies Inc. (Beijing, China). The antibodies specific for Bcl-2, Bax, Fas, FasL and Cleaved Caspase-3, Cyclooxygenase-2 (Cox-2), survivin, CD34, vascular endothelial growth factor (VEGF) and IL-8 were purchased from Cell Signaling Technology. (Boston, MA, USA). The UltraSensitiveTM SP kit was purchased from Maixin (Fuzhou, Fujian, China). The TUNEL apoptosis detection kit was purchased from Beyotime Institute of Biotechnology (Beijing, China). The male athymic BALB/c nude mice were purchased from Shanghai Experimental Animal Center (Shanghai, China) and maintained in the animal facility at Soochow University according to the animal research committee’s guidelines of Soochow University.

Analysis of AdVING4 gene transfer

The AdVING4 and control blank AdV adenoviruses were prepared as previously described.16 The titer of purified adenoviruses was determined using gene transfer unit (GTU) method by calculating the number of the reporter gene GFP-expressing QBI-293A cells within 18 h after adenoviral infection under fluorescence microscopy. To assess the optimal ratio of infectious adenovirus (GTU) to target cells, called multiplicity of infection (MOI), for a maximal infection and transgene expression, the SPC-A1 human lung adenocarcinoma (NSCLC) cells were infected with AdVING4 and AdV at various MOIs (0, 1, 10, 25, 50, 100 and 200) for 24 h, respectively. The adenoviral infection efficiency was then examined according to GFP expression. In addition, the AdVING4 transgene expression in SPC-A1 tumor cells was determined by reverse transcriptase (RT)-PCR and western blot analysis as reported previously.16

Flow cytometric analysis

Before initiating combined treatment experiments, we firstly investigated the radiosensitivity of SPC-A1 human NSCLC cells by flow cytometric analysis of apoptosis. Briefly, the SPC-A1 tumor cells were cultured in T25 flask at 1 × 106 cells per flask. Two days later, the tumor cells were irradiated at various doses of radiation (0, 2, 4, 6, 8 Gy) using 60Co-γ source (1 Gy/min). After another 24 h incubation, the irradiated and unirradiated SPC-A1 tumor cells were harvested, washed with cold phosphate buffer saline (PBS) and then subjected to apoptosis analysis using Annexin V-PE (early apoptotic marker) and 7-AAD (late apoptotic marker) double staining following manufacturer’s instructions by flow cytometry. Briefly, the tumor cells (1 × 105) were incubated in the presence of 5 μl Annexin V-PE and 5 μl 7-AAD in 100 μl of 1X Annexin V binding buffer at room temperature (RT). After 15 min’s incubation, 400 μl of 1x binding buffer was added and the apoptotic cells were then analyzed by flow cytometry. To further assess the combined apoptosis-inducing effects of AdVING4 plus radiotherapy on SPC-A1 tumor cells, the optimal radiation dose of 4 Gy were employed in in vitro combination therapy. In brief, the SPC-A1 tumor cells were cultured in T25 flask at 1 × 106 cells per flask. After 24 h incubation, the SPC-A1 tumor cells were infected with AdVING4 or AdV used as a blank adenovirus control at the MOI of 50. The medium containing PBS without adenovirus was used as a cell control (PBS control). 24 h post infection, the tumor cells were further exposed to radiation at the dose of 4 Gy. There are totally 6 groups: PBS, AdV, AdVING4 and 4 Gy alone, AdV plus 4 Gy and AdVING4 plus 4 Gy. Twenty-four hours after irradiation, tumor cells were then processed to analyze apoptosis by flow cytometry as described above.

Cell viability assay

The in vitro suppressive effects of AdVING4 plus radiotherapy on SPC-A1 human NSCLC cells were determined by MTT assay. The SPC-A1 tumor cells were dispensed into 96-well culture plates at 1 × 104 cells per well. After 24 h incubation, the SPC-A1 tumor cells were infected with 50 MOI AdVING4 or AdV or without adenovirus (PBS control), followed by irradiation with 4 Gy at day 1 post infection. The experiment also divided six groups: PBS, AdV, AdVING4 and 4 Gy alone, AdV plus 4 Gy, and AdVING4 plus 4 Gy. Before treatment and at different time points after single and/or combined treatment, the viability of SPC-A1 tumor cells was then analyzed using MTT kit according to manufacturer’s protocols.

Animal studies

The male athymic BALB/c nude mice were s.c. inoculated on their armpits of right anterior limbs with 3 × 106 SPC-A1 human NSCLC cells, and then monitored daily for tumor growth. Tumor volume was measured with a caliper and calculated by the formula, tumor size=ab2/2, where a is the larger and b is the smaller of the two dimensions. When the tumors grew up to a mean tumor volume of around 100–200 mm3, SPC-A1 human NSCLC s.c. xenografted tumor-bearing mice were subjected to gene therapy by multi-point intratumoral injection of 1 × 108 GTU of AdVING4 or AdV, or PBS every other day for a total of five times, respectively. On the day 5, the mice bearing SPC-A1 xenografted tumors were further assigned to receive single radiotherapy at a dose of 10 Gy/tumor using 60Co-γ source (1 Gy/min). All the mice for radiation were anesthetized with 10% chloral hydrate (3 μl/g body weight), and positioned in the radiation field so that only the tumor xenografts implanted on the armpits of right anterior limbs were exposed to the irradiation beam and the rest of the mouse’s body was shielded by lead block. Tumor progression and regression was monitored and tumor volume was measured daily. In addition, the tumor-bearing mice were killed 20 days after treatment, and the SPC-A1 human NSCLC s.c. xenografted tumors were removed, weighted, fixed by 10% neutral formalin and embedded in paraffin for hematoxylin and eosin staining and immunohistochemistric analysis.

Immunohistochemistric analysis

The expression of Bcl-2, Bax, Fas, FasL, Cox-2, survivin, cleaved Caspase-3, CD34, VEGF and IL-8 in treated and untreated SPC-A1 human NSCLC s.c. xenografted tumors was examined by immunohistochemistry using UltraSensitiveTM SP kit following manufacturer’s instructions. The presence of buffy or brown diaminobenzidine precipitates is indicative of positive reactivity. The integral optical density (IOD) of immunohistochemical intensity was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). Microvessel density (MVD) was assessed by CD34 immunostaining as described previously by Weidner et al.26 Any endothelial cell cluster immunoreactive for CD34 clearly separated from adjacent microvessels was considered as a single countable vessel. To detect the apoptotic cells in vivo in SPC-A1 human NSCLC xenografted tumors, tumor sections were further analyzed for apoptosis using TUNEL apoptosis detection kit according to company’s protocols. Each value represents IOD, microvessels or apoptotic cells counted at a high-power view ( × 400) by microscopy. The mean value represents for the average number derived from five high-power fields of each case.

Evaluation of combinatorial interaction

The interactive effects of AdVING4 and radiotherapy were evaluated by Q-value calculated by the formula,27 Q=F(A+B)/FA+(1-FA)FB, where F(A+B) represents the fraction affected by treatment with AdVING4 plus radiotherapy compared with the untreated control group, FA represents the fraction affected by AdVING4 alone, and FB represents the fraction affected by radiotherapy alone. A value of Q>1.15 indicates a synergistic effect between AdVING4 and radiotherapy, Q<0.85 indicates an antagonistic effect, and Q between 0.85 and 1.15 indicates an additive effect.

Statistical analysis

All data are presented as the mean±s.d.. The significance of the difference between groups was evaluated by Student’s t-test and one-way or two-way repeated measures analysis of variance and multiple comparisons with SPSS 10.0 software (SPSS, Chicago, IL, USA). A value of P<0.05 was considered statistically significant.

Results

Adenovirus-mediated overexpression of ING4 in SPC-A1 tumor cells

To select the optimal MOI for a maximal transgene expression with minimal adenovirus itself-induced cytotoxicity, the SPC-A1 human lung adenocarcinoma cells were infected with GFP-expressing AdVING4 or AdV at different MOIs and observed under fluorescence microscopy. More than 90% of GFP expression was found in the AdVING4- or AdV-infected SPC-A1 tumor cells at MOI of 50 (Figure 1a) or above (data not shown), whereas the GFP expression was not found in uninfected control SPC-A1 tumor cells (Figure 1a). Furthermore, adenovirus-mediated exogenous ING4 tumor suppressor gene was significantly expressed in 50 MOI AdVING4-infected SPC-A1 tumor cells but not in AdV-infected and uninfected SPC-A1 control cells (Figures 1b and c), indicating that ING4 tumor suppressor gene may be deleted or profoundly downregulated in SPC-A1 human NSCLC cells. In addition, there was almost no adenovirus-elicited cytotoxic effect in 50 MOI blank AdV-infected SPC-A1 tumor cells (Figure 1a). These results suggested that 50 MOI can be used as an optimal dose for the adenovirus-directed ING4 gene transfer and transgene expression in SPC-A1 tumor cells.

Figure 1
figure1

Adenovirus-mediated ING4 (AdVING4) transgene expression in SPC-A1 tumor cells. The SPC-A1 human non-small-cell lung cancer (NSCLC) cells were infected with AdVING4 or AdV at the MOI of 50 for 24 h, and then observed under GFP fluorescence and differential interference contrast (DIC) images by fluorescence microscopy (a). (b) RT-PCR analysis of ING4 transcriptional expression in SPC-A1 tumor cells. Total cellular RNAs were obtained from AdVING4- or AdV-infected and uninfected SPC-A1 tumor cells. The first-strand cDNAs were synthesized from RNAs using reverse transcriptase; PCRs were conducted using primer sets specific for ING4 and housekeep gene β-actin, respectively. (c) Western blot analysis of ING4 translational expression in SPC-A1 tumor cells. Total cellular lysates derived from AdVING4- or AdV-infected and uninfected SPC-A1 tumor cells were analyzed by immunoblotting with anti-ING4 and anti-β-actin antibody, respectively. Data shown are representative of three independent experiments.

AdVING4 enhances radiosensitivity in SPC-A1 tumor cells in vitro and in vivo

Before performing experiments involving a conjunction of AdVING4 gene therapy plus radiotherapy, we examined the sensitivity of SPC-A1 human lung adenocarcinoma cells to radiation by flow cytometric analysis of apoptosis. As shown in Figures 2a and b, irradiation with various doses resulted in a dose-dependent apoptosis in SPC-A1 tumor cells. The slight induction of apoptosis in SPC-A1 tumor cells was observed at 2 Gy (15.07% apoptosis), moderate apoptotic induction was observed at 4 Gy (26.32%), and significant apoptotic induction was observed starting at 6 Gy (40.82%), with maximum induction observed at 8 Gy (49.96%). The data indicated that SPC-A1 human lung adenocarcinoma cell line is a kind of relatively radioresistant NSCLC cells. Based on the radiosensitivity dose-finding study, we used 4 Gy dose in in vitro combining radiotherapy with AdVING4 gene therapy for SPC-A1 human NSCLC cells. To investigate AdVING4-mediated radiosensitivity and combined antitumor effects in in vitro SPC-A1 tumor cells, the SPC-A1 human NSCLC cells were treated with AdVING4 (50 MOI), AdV (50 MOI) or 4 Gy alone, AdVING4 (50 MOI) plus 4 Gy, or AdV (50 MOI) plus 4 Gy. The tumor cell viability was determined daily for 4 days by MTT assay. As shown in Figure 2c, the combined treatment with AdVING4 gene therapy plus 4 Gy radiotherapy synergistically inhibits in vitro SPC-A1 tumor cell growth in a time-dependent manner, compared with single AdVING4- and 4 Gy-treated group (P<0.05; Q=1.181, 1.185 and 1.164 at day 2, 3 and 4 after treatment, respectively), whereas the phenomenon did not occur in the AdV plus 4 Gy combination treatment, indicating that it is AdVING4 expression that contributes to the radiosensitivity in SPC-A1 tumor cells leading to synergistic antitumor efficacy. To further explore whether the combination of AdVING4 gene therapy plus radiotherapy would exert in vivo enhanced antitumor effects, the athymic nude mice bearing SPC-A1 human NSCLC xenografted tumors were i.t. injected with AdVING4 (1 × 108 GTU), AdV (1 × 108 GTU) or PBS alone, or irradiated with 10 Gy alone or plus intratumoral injection of AdVING4 (1 × 108 GTU) or AdV (1 × 108 GTU). The tumor growth in vivo was monitored daily, and tumor volume and weight were measured. As shown in Figures 2d and e, the tumor growth was more significantly retarded in AdVING4 plus 10 Gy group, compared with single AdVING4- and 10 Gy-treated group (P<0.05; Qvolume=0.896, 1.210 and 1.182 at day 10, 15 and 20 after treatment, and Qweight=1.225, respectively), indicating that AdVING4 plus 10 Gy combination treatment also remarkably suppresses SPC-A1 human NSCLC xenografted tumor growth in vivo in an athymic nude mouse model with synergistic effect.

Figure 2
figure2

Adenovirus-mediated ING4 (AdVING4) combined with radiotherapy induces synergistic suppression in SPC-A1 tumor cells. (a, b) Flow cytometric analysis of radiosensitivity of SPC-A1 tumor cells. The SPC-A1 human non-small-cell lung cancer (NSCLC) cells were irradiated at various doses of radiation (0, 2, 4, 6 and 8 Gy). Twenty-four hours later, the tumor cells were harvested and stained with Annexin V-PE and 7-AAD, and then analyzed by flow cytometry. The Annexin V-positive cells including Annexin V single-positive cells (early apoptosis) and Annexin V/7-AAD double-positive cells (late apoptosis) in the total cell population represented apoptotic cells. *P<0.05 compared with unirradiated (0 Gy) group, Student’s t-test, n=3 replicates per condition. (c) AdVING4 plus radiation induces synergistic tumor inhibition in vitro. The SPC-A1 tumor cells were treated with phosphate buffer saline (PBS), AdV, AdVING4, 4 Gy, AdV plus 4 Gy and AdVING4 plus 4 Gy for the indicated time periods. The survival cells were evaluated at day 0, 1 (that is, 24 h after infection), 2 (that is, 24 h after irradiation), 3 and 4 point after treatment by MTT assay. *P<0.05 compared with PBS and AdV group; **P<0.05 compared with AdVING4 and 4 Gy group, Q=1.181, 1.185 and 1.164, at day 2, 3 and 4 after treatment, respectively, two-way repeated measures analysis of variance (ANOVA) and multiple comparisons, n=4 replicates per condition. (d, e) AdVING4 plus radiation induces synergistic tumor inhibition in vivo. The athymic nude mice bearing SPC-A1 human NSCLC s.c. xenografted tumors were i.t. injected with 1 × 108 GTU of AdVING4 or AdV, or PBS every other day for a total of five times, respectively. The mice were further subjected to single radiotherapy at a dose of 10 Gy/tumor. The SPC-A1 xenografted tumor volume (d) before and after treatment, and tumor weight (e) removed at 20 days after treatment were measured. *P<0.05 compared with PBS and AdV group; **P<0.05 compared with AdVING4 and 10 Gy group, Qvolume=0.896, 1.210 and 1.182 at day 10, 15 and 20, and Qweight=1.225, respectively, one-way and two-way repeated measures ANOVA and multiple comparisons, n=6 mice per condition. Data shown are representative of three independent experiments.

Enhanced apoptosis induction by AdVING4 plus radiation

To explore the mechanism by which combination therapy with AdVING4 and radiation synergistically inhibits tumor cell growth, the in vitro apoptosis of SPC-A1 human NSCLC cells treated with AdVING4, AdV or 4 Gy alone, AdVING4 plus 4 Gy, or AdV plus 4 Gy for 48 h were analyzed using Annexin V-PE and 7-AAD double staining by flow cytometry. As shown in Figures 3a and 3b, AdVING4 plus 4 Gy radiation combined treatment induced 52.54% apoptosis in SPC-A1 tumor cells, whereas there was only 2.76, 4.71, 17.97, 26.32 and 29.83% apoptotic SPC-A1 tumor cells in the PBS, AdV, AdVING4, 4 Gy and AdV plus 4 Gy group. Compared with single AdVING4- and 4 Gy-treated group, AdVING4 plus 4 Gy more potentially induced SPC-A1 tumor cell apoptosis with synergistic effect (P<0.05; Q=1.328). However, the enhanced apoptosis-inducing effect did not occur in the AdV plus 4 Gy group. To further assess the induction of apoptosis in vivo, we perform TUNEL assay in the treated and untreated SPC-A1 human NSCLC s.c. xenografted tumors by immunohistochemistric analysis (Figure 3c). Consistent with the flow cytometric results in vitro, AdVING4 plus radiation (10 Gy) also has a synergistic effect on in vivo apoptosis induction of SPC-A1 human NSCLC cells s.c. implanted in athymic nude mice (P<0.05; Q=1.187).

Figure 3
figure3

Adenovirus-mediated ING4 (AdVING4) combined with radiotherapy elicits synergistic apoptosis in SPC-A1 tumor cells. (a, b) In vitro apoptosis analyzed by flow cytometry. The SPC-A1 human NSCLC cells were treated with phosphate buffer saline (PBS), AdV, AdVING4, 4 Gy, AdV plus 4 Gy and AdVING4 plus 4 Gy. Forty-eight hours after infection (that is, 24 h after irradiation), the tumor cells were harvested and stained with Annexin V-PE and 7-AAD, and then analyzed by flow cytometry. The Annexin V-positive cells including Annexin V single-positive cells (early apoptosis) and Annexin V/7-AAD double-positive cells (late apoptosis) in the total cell population represented apoptotic cells. *P<0.05 compared with PBS and AdV group; **P<0.05 compared with AdVING4 and 4 Gy group, Q=1.328, respectively, one-way repeated measures analysis of variance (ANOVA) and multiple comparisons, n=3 replicates per condition. (c) In vivo apoptosis in SPC-A1 human non-small-cell lung cancer (NSCLC) xenografted tumors analyzed by TUNEL assay. *P<0.05 compared with PBS and AdV group; **P<0.05 compared with AdVING4 and 10 Gy group, Q=1.187, respectively, one-way repeated measures ANOVA and multiple comparisons, n=6 mice per condition, n=5 observations per representative section. Data shown are representative of three independent experiments.

AdVING4 plus radiation cooperatively regulates intrinsic and extrinsic apoptotic pathways

To further address the underlying molecular mechanism responsible for AdVING4 plus radiation combined therapy-mediated synergistic antitumor effects, the expression of apoptosis-related proteins such as Bcl-2, Bax, Fas, FasL and Cleaved Caspase-3 in SPC-A1 human NSCLC s.c. xenografted tumors was assessed by immunohistochemistric analysis (Figure 4). Compared with PBS and AdV group, the expression of Bax, Fas, FasL and Cleaved Caspase-3 in AdVING4, 10 Gy, AdV plus 10 Gy or AdVING4 plus 10 Gy was significantly increased, whereas the expression of Bcl-2 was decreased (P<0.05). Moreover, AdVING4 plus 10 Gy combined treatment elicited an enhanced effect on the altered expression of Bcl-2, Bax, Fas, FasL and Cleaved Caspase-3 involved in the cooperative activation of intrinsic and extrinsic apoptotic pathways (P<0.05; QBcl-2=0.993, QBax=1.192, QFas=1.338, QFasL=1.762 and QCleaved Caspase-3=1.062). These results indicated that AdVING4 plus radiation synergistically suppresses in vitro and in vivo SPC-A1 human NSCLC cell growth and induces apoptosis closely associated with the coordinate regulation of intrinsic and extrinsic apoptotic pathways.

Figure 4
figure4

Adenovirus-mediated ING4 (AdVING4) combined with radiotherapy coordinately activates intrinsic and extrinsic apoptotic pathways and blocks radiation-induced upregulation of cyclooxygenase-2 (Cox-2) and survivin. (a) Representative pictures of immunohistochemical detection of Bcl-2, Bax, Fas, FasL, Cox-2, survivin and cleaved Caspase-3 in SPC-A1 human non-small-cell lung cancer (NSCLC) xenografted tumors. (b) The integral optical density (IOD) of immunostaining intensity quantified by Image-Pro Plus 6.0 software. *P<0.05 compared with phosphate buffer saline (PBS) and AdV group; Bcl-2, Bax, Fas, FasL and cleaved Caspase-3, **P<0.05 compared with AdVING4 and 10 Gy group, QBcl-2=0.993, QBax=1.192, QFas=1.338, QFasL=1.762 and QCleaved Caspase-3=1.062; Cox-2 and survivin, **P<0.05 compared with 10 Gy group, respectively, one-way repeated measures analysis of variance (ANOVA) and multiple comparisons, n=6 replicates per condition, n=5 observations per representative section. Data shown are representative of three independent experiments.

AdVING4 blunts radiation-induced enhancement of Cox-2 and survivin

Cox-2 and survivin have been shown to be overexpressed in a variety of tumor cells, which is closely related to carcinogenesis and involved in radioresistance of tumor cells.28, 29 Therefore, we were interested in analyzing the effects of AdVING4, radiation, or AdVING4 plus radiation on the expression of Cox-2 and survivin in in vivo SPC-A1 human NSCLC s.c. xenografted tumors by immunohistochemistry. As shown in Figure 4, radiation alone abundantly induce the expression of Cox-2 and survivin compared with PBS and AdV group (P<0.05), whereas AdVING4 alone downregulated their expression (P<0.05). Interestingly, AdVING4 gene therapy can efficiently block radiation-induced upregulation of Cox-2 and survivin when combined with radiotherapy, suggesting that AdVING4 can sensitize SPC-A1 human NSCLC cells to radiation very possibly through targeting radioresistant factors including Cox-2 and survivin.

Synergistic reduction of MVD by AdVING4 plus radiation

The positive expression of CD34 was mainly presented as brownish yellow or brownish granules in vascular endothelial cells of SPC-A1 human NSCLC s.c. xenografted tumors (Figure 5a). Compared with PBS and AdV group, the CD34 expression of tumor vascular endothelial cells in AdVING4, 10 Gy, AdV plus 10 Gy and AdVING4 plus 10 Gy group was weaker or less (Figures 5a and b) (P<0.05). In addition, the MVD (Figure 5c) counted in AdVING4, 10 Gy, AdV plus 10 Gy and AdVING4 plus 10 Gy group was significantly less than that in PBS and AdV group (P<0.05). Moreover, AdVING4 plus 10 Gy but not AdV plus 10 Gy has a synergistic effect on downregulation of CD34 and reduction of MVD in SPC-A1 human NSCLC xenografted tumors (P<0.05; QCD34=1.161 and QMVD=1.238), which may be involved in AdVING4 and 10 Gy combined therapy-induced in vivo synergistic growth suppression of SPC-A1 human NSCLC xenografted tumors in athymic nude mice.

Figure 5
figure5

Adenovirus-mediated ING4 (AdVING4) combined with radiotherapy synergistically reduces tumor angiogenesis. (a) Representative pictures of immunohistochemical detection of CD34 in SPC-A1 human non-small-cell lung cancer (NSCLC) xenografted tumors. (b) The integral optical density (IOD) of CD34 immunostaining intensity quantified by Image-Pro Plus 6.0 software. *P<0.05 compared with phosphate buffer saline (PBS) and AdV group; **P<0.05 compared with AdVING4 and 10 Gy group, Q=1.161, respectively, one-way repeated measures analysis of variance (ANOVA) and multiple comparisons, n=6 replicates per condition, n=5 observations per representative section. (c) The MVD in SPC-A1 human NSCLC xenografted tumors. *P<0.05 compared with PBS and AdV group; **P<0.05 compared with AdVING4 and 10 Gy group, Q=1.238, respectively, one-way repeated measures ANOVA and multiple comparisons, n=6 replicates per condition, n=5 observations per representative section. Data shown are representative of three independent experiments.

AdVING4 suppresses radiation-elicited upregulation of VEGF and IL-8

It has been shown that tumors express proangiogenic factors, such as VEGF and IL-8, that promote the formation of tumor blood vessels.30 To examine whether AdVING4, radiation or their combination affected the expression of those factors, we assessed VEGF and IL-8 expression in in vivo SPC-A1 human NSCLC s.c. xenografted tumors with different treatments by immunohistochemistric analysis. As shown in Figure 6, some constitutive expression of VEGF and IL-8 was apparent in PBS- or AdV-treated group. AdVING4 alone significantly downregulated expression levels of IL-8 and modestly inhibited the expression of VEGF (P<0.05). However, radiation alone substantially enhanced their expression (P<0.05), especially IL-8, in SPC-A1 human NSCLC xenografted tumors. Most importantly, AdVING4 potentially impaired radiation-induced enhancement of VEGF and IL-8 expression, indicating that AdVING4 gene transfer is capable of attenuating the radiation-elicited proangiogenic activity via overriding the production of proangiogenic factors such as VEGF and IL-8.

Figure 6
figure6

Adenovirus-mediated ING4 (AdVING4) combined with radiotherapy impairs radiation-induced expression of proangiogenic factors. (a) Representative pictures of immunohistochemical detection of vascular endothelial growth factor (VEGF) and IL-8 in SPC-A1 human non-small-cell lung cancer (NSCLC) xenografted tumors. (b) The integral optical density (IOD) of VEGF and IL-8 immunostaining intensity quantified by Image-Pro Plus 6.0 software. *P<0.05 compared with phosphate buffer saline (PBS) and AdV group; **P<0.05 compared with 10 Gy group, respectively, one-way repeated measures of analysis of variance (ANOVA) and multiple comparisons, n=6 replicates per condition, n=5 observations per representative section. Data shown are representative of three independent experiments.

Discussion

NSCLC is the deadliest and most common type of human lung cancer. External ionizing radiotherapy remains an important local treatment modality in NSCLC. Unfortunately, its therapeutic effectiveness is modest and disappointing as evidenced by the radioresistance of the tumor and high-dose radiation-induced toxicity to normal tissues, including pneumonitis, esophagitis and chronic conditions such as lung fibrosis, esophageal stricture and fistula. Although the extensive efforts have been made to understand the reasons for tumor radioresistance, the molecular mechanisms underlying radioresistance are still incompletely clear. Some studies have suggested the potential involvements of p53 mutation,31 aberrant overexpression of survival genes such as Cox-2,28, 32 XIAP and survivin33 or activation of PI3K/Akt,34 NK-κB35 and HIF-1α36 signaling pathways in the radioresistance of lung cancer. It has also been shown that radiation-induced secretion of proangiogenic factors can cause radioprotection of tumor vasculature,37, 38 which is a crucial determinant of overall radioresponses.

Gene therapy is an alternative radiosensitizing approach for treating radioresistant cancer including lung cancer. The identification of genes responsible for carrying or override radioresistance would be of paramount importance to discover novel molecular targets whose regulation can enhance radioresponses. Previous studies have shown that tumor suppressor ING4 can suppress the activity of NF-κB and HIF-1α transcriptional factors that are critical for cancer progression and radioresistance via directly associating with NF-κB p65 subunit5 and HIF prolyl hydroxylase,18 resulting in the inhibition of production of radioresistance-associated prosurvival factors. Hence, in this report, we followed-up our previous studies and were interested in assessing the combined antitumor effects of AdVING4 gene therapy and ionizing radiotherapy in radioresistant SPC-A1 human NSCLC cells in vitro and in vivo in athymic nude mice, and investigating whether AdVING4 would sensitize SPC-A1 tumor cells to radiation. We demonstrated that AdVING4 gene therapy combined with ionizing radiation-induced in vitro synergistic tumor suppression and apoptosis in SPC-A1 human NSCLC cells. Furthermore, AdVING4 plus radiation also synergistically inhibited the growth of in vivo SPC-A1 human NSCLC s.c. xenografted tumors and induced apoptosis in athymic nude mice. To delineate the molecular mechanisms responsible for the AdVING4-mediated radiosensitizing effects and improved therapeutic efficacy of their combination, the expression of apoptosis-related proteins, such as Bcl-2, Bax, Fas, FasL and cleaved Caspase-3, radioresistance-associated factors, including Cox-2 and survivin, vascular endothelial marker CD34 and proangiogenic factors, such as VEGF and IL-8, in SPC-A1 human NSCLC s.c. xenografted tumors were determined by immunohistochemistric analysis. Our study shown that AdVING4 combined with radiotherapy (i) additively increased Bax, Fas, FasL and Cleaved Caspase-3 as well as decreased Bcl-2; (ii) synergistically reduced CD34 expression and MVD; and (iii) efficiently blocked the radiation-induced enhancement of Cox-2 and survivin radioresistant factors, and VEGF and IL-8 proangiogenic factors.

Initiation of apoptosis induced by irreparable cellular damage is a key mechanism by which ionizing radiation kills cancer cells. There are at least two common types of apoptosis: one elicited by intrinsic apoptotic pathway and the other by extrinsic apoptotic pathways. Bcl-2 protein family is known to be pivotal regulator of apoptosis and important determinant of cell fate.39 The ratio of anti- to pro-apoptotic Bcl-2 family molecules such as Bcl-2/Bax constitutes a rheostat that sets the threshold of susceptibility to apoptosis for the intrinsic pathway, which promotes pore formation in the mitochondrial outer membrane, loss of mitochondrial integrity and the release into the cytosol of cytochrome c followed by the cleavage of Caspase-9, leading to the activation of intrinsic apoptotic pathway. In addition, Fas and FasL as important apoptotic markers have been shown to regulate FasL-Fas extrinsic apoptotic pathway.39 Upregulation of Fas and/or FasL triggers the cleavage of downstream targets including Caspase-8 and Bid, resulting in the activation of extrinsic apoptotic pathway. Furthermore, Cleaved Bid can translocate to the mitochondria and then mediate cytochrome c release, thereby further causing the activation of intrinsic apoptotic pathway. Thus, the additive effect of AdVING4 combined with radiotherapy on altered expression of Bcl-2, Bax, Fas and FasL involved in the cooperative activation of extrinsic and intrinsic apoptotic pathways may be closely accountable for the enhanced apoptosis induction and synergistic antitumor efficacy of AdVING4 plus radiation in SPC-A1 human NSCLC cells.

Tumor angiogenesis is a prerequisite for successful tumor growth and formation of metastasis, which is regulated through the balance of tumor-derived proangiogenic (VEGF, bFGF, TGFβ and IL-8) and antiangiogenic (endostatin and angiostatin) factors.30 Thus, tumor angiogenesis is a potential therapeutic target in anticancer therapy. Accumulating evidences have suggested that ionizing radiation can upregulate the expression of proangiogenic factors such as VEGF,23, 38, 40 bFGF23, 38 and IL-8,23 and subsequently facilitate the formation of tumor vessels, leading to tumor regrowth and accelerated metastasis.37 It has been reported that combined therapy with radiotherapy and angiogenesis inhibitor can display enhanced antitumor activity for cancers. In our study, we demonstrated that AdVING4 gene therapy plus ionizing radiotherapy synergistically inhibited CD34 expression and reduced MVD in SPC-A1 human NSCLC xenografted tumors, which may be another important mechanism involved in AdVING4 plus radiation-mediated in vivo synergistic growth inhibition of SPC-A1 human NSCLC xenografted tumors in athymic nude mice. Consistent with the previous reports,23, 38, 40 ionizing radiation alone abundantly elevated the levels of proangiogenic factors such as VEGF and IL-8. Interestingly, AdVING4 efficiently blocked the production of VEGF and IL-8 induced by radiation, likely contributing to the enhanced antiangiogenic effects. Previous studies have shown that radiation can induce HIF-1α expression and increase the prosurvival and proangiogenic activity.38, 41 Irradiation also can activate NF-κB survival transcription factor via degradation of IκBα.42, 43 The radiation-induced activation of HIF-1α and NF-κB has been proposed as dominant governor triggering radioprotection of tumor vessels and critical determinant of radioresistance. ING4 can potentially inhibit the transcriptional activity of NF-κB and HIF-1α leading to the inhibition of production of proangiogenic factors.5, 18, 19 Therefore, AdVING4 impaired the radiation-induced proangiogenic activity and radioprotection of tumor vessels through targeting NF-κB and HIF-1α, and thereby inhibiting the production of proangiogenic factors including VEGF and IL-8, which may be involved in the combined antitumor effects and AdVING4 gene therapy-mediated enhancement of tumor radioresponses.

Cox-2, a mitogen-inducible isoform of cyclooxygenase, and Cox-2-derived products particularly prostaglandin E2 have been found to be implicated in cell proliferation, apoptosis inhibition, tumor angiogenesis, immune suppression and tumor invasion.28, 44, 45 Survivin as the smallest member of inhibitor of apoptosis protein family can also promote cell division, suppress apoptosis and facilitate cell survival.29 Extensive studies have shown that Cox-2 and survivin are highly expressed in a large variety of cancers including NSCLC, and appear to be closely associated with carcinogenesis, tumor progression and malignancy, poor progronsis and increased tumor recurrence, chemotherapy and radiotherapy resistance, and reduced overall survival rates, suggesting that Cox-2 and survivin are essential tumor survival factors and attractive therapeutic targets in anticancer therapy.28, 46 To further address the underlying mechanism responsible for AdVING4 gene therapy-induced enhanced radioresponses, therefore, we analyzed the expression of two important radioresistant factors Cox-2 and survivin in human SPC-A1 NSCLC xenografted tumors. We found that radiation profoundly elevated the Cox-2 and survivin expression, indicating that they may function as inducible radioresistant factors. Notably, AdVING4 almost completely eliminated the increased Cox-2 and survivin induced by ionizing radiation. It has been shown that ionizing radiation can enhance the transcriptional activity of NF-κB42, 43 and HIF-1α38 and positively modulate the expression of downstream genes Cox-247, 48 and survivin.49, 50 It has also been reported that ING4 can efficiently suppress the activity of NF-κB and HIF-1α.5, 18, 19 Thus, AdVING4 gene therapy potentially radiosensitize SPC-A1 human NSCLC cells very possibly by blunting the radiation-induced upregulation of radioresistant factors Cox-2 and survivin, at least in part, via blocking NF-κB and HIF-1α signaling pathways.

In summary, AdVING4 gene therapy plus ionizing radiotherapy-induced synergistic tumor suppression and apoptosis in in vitro SPC-A1 human NSCLC cells and in vivo SPC-A1 xenografted tumors s.c. implanted in athymic nude mice. Mechanistically, AdVING4 combined with radiation resulted in a substantial upregulation of Bax, Fas, FasL and Cleaved Caspase-3, and downregulation of Bcl-2 in SPC-A1 human NSCLC xenografted tumors. In addition, AdVING4 plus radiation synergistically reduced the tumor vessel CD34 expression and MVD in vivo. Most importantly, AdVING4 potentially blocked the radiation-induced enhancement of Cox-2 and survivin radioresistant factors, and VEGF and IL-8 proangiogenic factors. The enhanced antitumor effects elicited by AdVING4 plus radiotherapy were closely associated with the cooperative activation of intrinsic and extrinsic apoptotic pathways, and synergistic inhibition of tumor angiogenesis.

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Acknowledgements

This research work was supported by grants from the National Natural Science Foundation of China (no. 81001016) and Medicine Research Foundation of Department of Public Health of Jiangsu Province (no. H200914).

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Correspondence to J Yang.

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The authors declare no conflict of interest.

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Keywords

  • gene therapy
  • inhibitor of growth 4 (ING4)
  • non-small-cell lung cancer (NSCLC)
  • radiosensitivity
  • radiotherapy

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