Enhanced effect of recombinant adenoviruses co‐expression of ING4 and OSM on anti‐tumour activity of laryngeal cancer

Abstract The inhibitor of growth family member 4 (ING4) is one of the ING family genes, serves as a repressor of angiogenesis or tumour growth and suppresses loss of contact inhibition. Oncostatin M (OSM) is a multifunctional cytokine that belongs to the interleukin (IL)‐6 subfamily with several biological activities. However, the role of recombinant adenoviruses co‐expressing ING4 and OSM (Ad‐ING4‐OSM) in anti‐tumour activity of laryngeal cancer has not yet been identified. Recombinant Ad‐ING4‐OSM was used to evaluate their combined effect on enhanced anti‐tumour activity in Hep‐2 cells of laryngeal cancer in vivo. Moreover, in vitro function assays of co‐expression of Ad‐ING4‐OSM were performed to explore impact of co‐expression of Ad‐ING4‐OSM on biological phenotype of laryngeal cancer cell line, that is Hep‐2 cells. In vitro, Ad‐ING4‐OSM significantly inhibited the growth, enhanced apoptosis, altered cell cycle with G1 and G2/M phase arrest, and upregulated the expression of P21, P27, P53 and downregulated survivin in laryngeal cancer Hep‐2 cells. Furthermore, in vivo functional experiments of co‐expressing of Ad‐ING4‐OSM demonstrated that solid tumours in the nude mouse model were significantly suppressed, and the co‐expressing Ad‐ING4‐OSM showed a significant upregulation expression of P21, P53, Bax and Caspase‐3 and a downregulation of Cox‐2, Bcl‐2 and CD34. This study for the first time demonstrated the clinical value and the role of co‐expressing Ad‐ING4‐OSM in biological function of laryngeal cancer. This work suggested that co‐expressing Ad‐ING4‐OSM might serve as a potential therapeutic target for laryngeal cancer patients.

growth, 7 loss of contact inhibition 2 and angiogenesis. 8 Oncostatin M (OSM) is a multifunctional cytokine that belongs to the interleukin (IL)-6 subfamily. Human OSM (hOSM) was initially recognized by its activity to inhibit the proliferation of A375 melanoma cells and numerous other tumour cells. 9 hOSM is a secreted glycoprotein of 28 kDa that was originally isolated from phorbol 12-myristate 13-acetate (PMA)-stimulated human histiocytic lymphoma U937 cells. Furthermore, OSM is a unique cytokine that functions in various biological systems, such as inflammatory response, haematopoiesis, tissue remodelling and development. 10 It also inhibits tumour cell growth and induces cell cycle alteration and apoptosis in different tumour types, such as melanomas, 11,12 glioblastomas, 13 lung carcinomas, 14 ovarian carcinomas 15 and breast tumours. 16,17 In addition, human OSM induces differentiation of several tumour cell types. 13,18 Laryngeal cancer is the most common type of head and neck cancer in most countries; tobacco smoking and alcohol consumption are the major risk factors. The common treatments include surgery, chemotherapy, radiation and combinations of two or three of the above methods. The effects of ING4 and OSM emphasize their potential application as gene therapeutic agents. In this study, we ex-

| In vitro treatment
The experiment was designed with following five groups: (1) Phosphate-buffered saline (PBS): treated with PBS as a cell control; (2) Ad-GFP: treated with Ad-GFP at the optimal multiplicity of infections as a blank adenovirus (Ad) control; (3) Ad-ING4: treated with Ad-ING4 at the optimal multiplicity of infections; (4) Ad-OSM: treated with Ad-OSM at the optimal multiplicity of infections; and (5) Ad-ING4-OSM: treated with Ad-ING4-OSM at the optimal multiplicity of infections. For each group, the cells were cultured in RPMI1640 supplemented with 10% FBS incubated overnight at 37°C in humidified 5% CO 2 atmosphere. After treatment with gene recombinant adenoviruses, 2% FBS was used instead.

| Adenoviral infection efficiency
In order to assess the optimal multiplicity of infections for a maximal

| Ad-ING4-OSM transgene expression
The Ad-directed ING4 and OSM transgene expression in Hep-2 cells was analysed by RT-PCR and Western blot. For the RT-PCR, the total cellular RNA was extracted using TRIzol, and the first-strand cDNA was reverse transcribed with RNA as a template and Oligo d(T) 18 as a primer. The PCR amplification was carried out using cDNA as the template and primers specific for ING4 and OSM, respectively. For the Western blot, proteins were isolated from infected and uninfected The membrane was developed using a SuperEnhanced chemiluminescence detection kit. The immunoreactive bands were visualized after exposure of the membranes to Kodak X-ray film.

| MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay
The cytotoxic activity of each group of Hep-2 cells was determined by MTT assay. Briefly, the Hep-2 cells were dispensed in a 96-well culture plate at a density of 0.5 × 10 4 cells/well and incubated at 37°C in a humidified 5% CO 2 atmosphere. After incubation for overnight, the cells were treated for 0-4 days. After treatments with different periods, the cells were incubated with 10 μl MTT (5 mg/ml) at 37°C for 4 h. The formazan crystals in the cells were solubilized with 10% SDS-HCl (100 μl/well). The plate was read at 570 nm using a Microplate Reader Model 550 (Bio-Rad). The cell inhibition rate was calculated as (A control group−A treatment group)/A control group × 100%.

| Hochest 33258 fluorescence staining
The Hep-2 cells from exponentially growing cultures were seeded

| Flow cytometry of cell cycle by PI (propidium iodide)
After the Hep-2 cells were treated, respectively, for 48 h, each group of cells (1 × 10 5 ) were harvested, washed in cold PBS, fixed in 70% cold alcohol for >24 h at 4°C, washed in cold PBS and stained with PI solution containing 0.1 mg/ml RNase A at 4°C in dark for 30 min.
The DNA content and cell cycle were analysed by flow cytometry.

| Flow cytometry of cell apoptosis by Annexin V-PE/7-AAD
Apoptosis was assessed using Annexin V-PE/7-AAD double staining following manufacturer's instructions. For each group, the Hep-2 cells were cultured 24 h and treated, respectively. Subsequently, 1 × 10 5 cells were harvested, washed in cold PBS for twice, incubated for 15 min at room temperature in the presence of 5 μl Annexin V-PE and 5 μl 7-AAD in 100 μl of 1× binding buffer in dark. After incubation, 400 μl of 1 × binding buffer was added, and the apoptotic cells were analysed by flow cytometry.

| Real-time reverse transcription (RT)-PCR
To further determine the expression levels of P21, P27, P53 and Survivin in Hep-2 cells for each group, total RNA was prepared for the two-step real-time RT-PCR analysis based on SYBR Green I detection. The RT-PCR assay was performed using the MJ Research OpticonTM2 system (MJ Research). Briefly, total RNA was isolated using TRIzol reagent (Invitrogen). The first-strand cDNA synthesis was performed as described above in larger volumes, such that each sample could be tested in different subsequent PCR reactions. The PCR reaction was performed using the following program: 95°C for 5 min, then 72℃ for 5 min, followed by 50 cycles of 95°C for 15 s, 58°C for 20 s and 72°C for 30 s. The cDNA quantities were normalized to that of the internal control gene Gapdh measured in the same samples. The relative gene expression of the target gene was calculated using the 2 −△△CT method with pooled cDNA from all samples as a reference. 19 The authenticity of the PCR products was verified by melting curve analysis and agarose gel electrophoresis. Each sample was analysed in duplicate in independent reactions, and experiments were repeated at least three times.

| Animal experiments
Male athymic nude mice were subcutaneously inoculated on the armpits of the right anterior limbs with 1-2 ×
Then, the integral optical density (IOD) of immunohistochemistry was calculated by Image-Pro Plus 6.0 software. Each value represents IOD at a power view (200×) by microscopy. The mean value represents the average number derived from five high-power fields in each group.

| Statistical analysis
All data were presented as the mean ± SD. The significance of differences between groups was evaluated by one-way and two-way

| Ad-ING4, Ad-OSM and Ad-ING4-OSM had inhibitory effect of Hep-2 cells in vitro
For each group, the Hep-2 cells were treated at the indicated periods (0-4 days), respectively. The cell viability was evaluated at days 0, 1, 2, 3 and 4 after MTT assay, respectively (Figure 2A

| Ad-ING4-OSM arrested Hep-2 cells in both G2/M and G0/G1 phase while decrease the proportion of the S phase by PI
To further explore the mechanisms of Hep-2 cell growth inhibition, we analysed the cell cycle distribution, as assessed by PI staining shown in Figure 3A,B, the Ad-ING4 arrested Hep-2 cells in the G2/M phase, and the proportion of the G2/M phase was 12.3% ± 2.2% higher than the PBS and Ad-GFP groups (4.6% ± 1.0% and 5.5% ± 1.4%, respectively; p < 0.05). Ad-OSM arrested Hep-2 cells in G0/G1 phase, and the proportion of the G0/G1 phase was 67.1% ± 2.5%, which was higher than those of PBS and Ad-GFP (55.1% ± 1.7% and 56.8%

| Flow cytometry of cell apoptosis by Hoechst 33258 fluorescence staining and Annexin V-PE/7-AAD
The Hep-2 cells in each group were stained with fluorescent dye Hoechst 33528 and visualized under a fluorescence microscope. To explore the apoptosis of Hep-2 cells, we used Annexin V-PE/7-AAD double staining by flow cytometry. As shown in Figure 3D,E, the apoptotic ratio was 2.3% and 3.5% in the PBS and Ad-GFP groups, respectively, with no significant difference (p > 0.05).  In addition, it enhances chemosensitivity in HepG2 hepatocarcinoma cells. 26 A recent study reported that ING4 exerts a marked inhibitory effect on tumour cell spread, migration and invasion. 7,30 Oncostatin M plays a critical role in various biological systems, such as inflammatory response, haematopoiesis, tissue remodelling and development. 10  and STAT5) and the STAT3-SMAD3. 10,34 Furthermore, OSM induces STAT3 and ERK signalling and promotes the proliferation and migration of malignant keratinocyte PDVC57 cells. 35 These phenomena suggested that growth inhibition in these cells by cytokines could F I G U R E 6 Expression of P21, P53, Bax, Bcl-2, CD34, Cox-2 and Caspase-3, respectively, in Hep-2 cells xenografted tumours tested by immunohistochemistry analysis be attributed to the suppressed activity of cyclin-dependent kinase (CDK) by p27kip1. Recent studies implicated OSM in macrophage M2 polarization, which might promote tumour progression. [36][37][38] OSM reportedly increases the level of metastasis-related proteins, including matrix metalloproteinase-1 (MMP-1) in osteogenic differentiation of mouse MC3T3 osteoblasts, 39 MMP-2 in human trophoblast cell line 40 and cathepsin L in osteosarcoma cells. 41 Moreover, OSM is cytostatic for high-grade chondrosarcomas, independent of p53 and presumably through the JAK3/STAT1 pathway. 42 Both ING4 and OSM exert a negative effect on cancer cells rather than normal cells. Han et al. 43  not been fully clarified in this study. Therefore, we will validate these findings and expand our investigation by including additional laryngeal cancer cell lines in future.

| CON CLUS ION
Overall, our study suggests that recombinant adenoviruses co-