Suppression effect of a dual cancer-specific oncolytic adenovirus on luciferase-labeled human melanoma cells in vitro and in vivo

Abstract

BACKGROUND:

To explore the suppressive effect of Apoptin-loaded oncolytic adenovirus (Ad-VT) on luciferase-labeled human melanoma cells in vitro and in vivo.

METHODS:

The stable luciferase-expressing human melanoma cells A375-luc or M14-luc were obtained by transfecting the plasmid pGL4.51 and selection with G418, followed by luciferase activity, genetic stability and bioluminescence intensity assays. In vitro, the inhibitory effects of Ad-VT on A375-luc or M14-luc were evaluated using the MTS cell proliferation, FITC-Annexin V apoptosis detection, transwell migration, Matrigel invasion and scratch assays. The inhibition pathway in Ad-VT-infected A375-luc and M14-luc cells were analyzed by JC-1 staining and Western-blot detection of mitochondrial apoptosis-related proteins. In vivo, the suppressive effects of Ad-VT on A375-luc or M14-luc were assessed by living imaging technology, tumor volume, bioluminescence intensity, survival curves and immunohistochemical analysis of the tumors from the xenograft tumor model BALB/c nude mice.

RESULTS:

The growth and migration of A375-luc and M14-luc were significantly inhibited by Ad-VT in vitro. The evaluations of A375-luc and M14-luc tumor models in BALB/c nude mice were successfully performed using living imaging technology. Ad-VT had an anti-tumor effect by reducing tumor growth and increasing survival in vivo. Ad-VT significantly changed the mitochondrial membrane potential by triggering the the mitochondrial release of apoptosis-related proteins, AIF (apoptosis inducing factor), ARTS (Apoptosis-Related Proteins), and Cyto-c (cytochrome c) from the mitochondria.

CONCLUSION:

Ad-VT reduced the mitochondrial membrane potential in A375-luc or M14-luc cells and induced the mitochondrial release of AIF, ARTS and Cyto-C. Ad-VT induced apoptosis in A375-luc or M14-luc cells via the mitochondrial apoptotic pathway.

1. Introduction

Melanoma is the most lethal skin cancer. In 2014, the National Central Cancer Registry collected data from local cancer registries and showed that there were approximatively 6,761 new cases and 3,637 yearly deaths in China. With the development of molecular biology techniques and immunology, adenovirus-related biological treatment of melanoma has achieved exciting results [1, 2]. In a report of the melanoma-specific human data from the Advanced Therapy Access Program (ATAP), Ad5/3-D24-GMCSF [3, 4], a serotype 5/3 chimeric oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), showed in vitro and in vivo anti-tumor efficacy in melanoma [5]. Treatment combination of TG1042 [6, 7, 8, 9], a suspension of recombinant type 5 adenoviral particles expressing human interferon- $\gamma$ , and tumor-infiltrating lymphocytes in metastatic melanoma, showed both feasibility and safety in a clinical study [10]. AdCD40L [11, 12, 13, 14, 15], a replication-deficient adenovirus carrying the gene for the human CD40 ligand (CD40L) [16, 17], induced desirable systemic immune effects that correlated with prolonged survival in a phase I/IIa study [18]. Oncolytic adenovirus ICOVIR-5 [19, 20] could reach melanoma metastases upon a single intravenous administration and these studies indicated that disseminated cancer may be treated through the systemic administration of armed oncolytic viruses [21]. However, the pathological types and gene mutations in Chinese melanoma patients may be different from those in other countries. Therefore, more research is needed to provide a reliable basis for the application of biotherapy in Chinese melanoma patients.

An oncolytic adenovirus is a modified adenovirus that can enter and continuously replicate in tumor cells, where it induces their lysis and infect other tumor cells. Oncolytic adenovirus vectors are often used to express therapeutic genes due to their large load [22, 23]. With the continuous replication of oncolytic adenoviruses in tumor cells, such as Ad3-hTERT-CMV-hCD40L in A549 cells [24], SG635-SF in SK-OV3 and HO9010 cells [25], EnAd in DLD-1 cells [26], ONCOS-102 in AB12 cells [27], and ICO15K-FBiTE in HT1080 cells [28], therapeutic genes are expressed to induce a synergistic anti-tumor effect. The dual cancer-specific oncolytic adenovirus Ad-hTERT-E1a-Apoptin (Ad-VT) and Ad-mock have been constructed in our laboratory [29]. Ad-VT contains the human telomerase reverse transcriptase (hTERT), the adenovirus early region 1a (E1a) gene, the CMV promoter and the chicken anemia virus apoptin gene [30, 31]. Ad-mock that lacks hTERT, the E1a and apoptin genes, was used as a blank control. Ad-VT can replicate and express apoptin in the tumor resulting in accelerated tumor cell apoptosis. Research studies using Ad-VT demonstrated its safety in BALB/c mice, Wistar rats, male Hartley guinea pigs and beagles [32]. We also showed that the growth of many tumor cells [33, 34, 35], such as human gastric cancer (SGC7901) cells, human prostate carcinoma (PC-3) cells, human lung cancer (A549) cells and breast cancer (MCF-7) cells, could be significantly suppressed by Ad-VT in vitro and in vivo. In this study, to investigate the in vitro effects of Ad-VT on the tumorigenesis and progression of human melanoma cells, we generated human melanoma cells A375 Luc or M14 Luc that stably express luciferase. In vivo, these cells were used to establish a BALB/C nude mice models. The in vitro and in vivo anti-tumor effects of Ad-VT were observed and evaluated by using a living imaging technology.

2. Materials and methods

2.1 Virus, cell lines, plasmid, reagents and mice

Ad-VT and Ad-mock were previously constructed in our laboratory. The human melanoma cell lines, A375 and M14, were purchased from the Cell Bank, Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The MTS cell proliferation kit, Plasmid pGL4.51, ONE-Glo ${}^{\text{TM}}$ Luciferase Assay System and VivoGlo ${}^{\text{TM}}$ Luciferin were purchased from Promega (Beijing, China). The X-tremeGENE ${}^{\text{TM}}$ HP DNA Transfection Reagent was purchased from Roche (Beijing, China), Geneticin (G418) from Solarbio (Beijing, China), the Transwells and BioCoat from Corning (Hangzhou, China), and culture inserts from ibidi (Beijing, China), the Annexin V-FITC apoptosis detection kit from BD Biosciences (Beijing, China). Hoechst, JC-1, and the monoclonal anti-ARTS antibody mAb were purchased from Sigma-Aldrich (Shanghai, China), and Sodium pentobarbital from Merck (Beijing, China). The Cytochrome c (D18C7) Rabbit mAb, the Smac/Diablo (D5S3R) Rabbit mAb, and the Endonuclease G Antibody were purchased from Cell Signaling Technology (Shanghai, China). The anti-AIF and anti-HtrA2/Omi antibodies were purchased from Abcam (Shanghai, China).

2.2 Establishment of A375-luc or M14-luc cells

A375 or M14 cells were cultured at a density of 2 $\times$ 10 ${}^5$ cells/well in 6-well plates at 37 ${}^{\circ }$ C with 5% CO ${}_2$ for 24 h. The cells were transfected with 4 $\mu$ g of plasmid pGL4.51 using the X-tremeGENE HP DNA Transfection Reagent and following the manufacturer’s instructions. The transfected cells were cultured at 37 ${}^{\circ }$ C with 5% CO ${}_2$ for 24 h, and then re-cultured at a density of 100 cells/well in 6-well plates after trypsinization and selected using G418 concentrations of 200 $\mu$ g/mL, 400 $\mu$ g/mL and 600 $\mu$ g/mL. The medium containing was changed every 2 days and the optimum G418 concentration of G418 was confirmed by assessing the state of selected cells. Selected monoclonal cells with good growth were used for amplifying and detecting luciferase activity. The optimum concentration of G418 was maintained during the cells’ culture.

2.3 Detection of A375-luc and M14-luc cells

Stable luciferase-expressing human melanoma cell lines (A375-luc or M14-luc) were detected via their luciferase activity, analysis of their genetic stability and intensity of bioluminescence. To detect luciferase activity, all monoclones that had a good cell growth, were cultured at a density of 5 $\times$ 10 ${}^4$ /100 $\mu$ L in 96-well plates for 48 h. The luciferase activity of the monoclones was detected using the ONE-Glo ${}^{\text{TM}}$ Luciferase Assay System. After addition of 100 $\mu$ L detection reagent, the monoclones were cultured at room temperature away from light for 3 min, and the luciferase activity was measured using a microplate reader.

To analyze the genetic stability of A375 Luc or M14 Luc cell lines, the cells with the highest luciferase activity were subcultured 40 times and their genetic stability determined every 10 generations. The monoclone with the highest luciferase activity and the best genetic stability was named A375-luc or M14-luc.

To evaluate the intensity of bioluminescence in vitro, A375-luc or M14-luc cells were seeded in 96-well plates and cultured in 5% CO2 at 37 ${}^{\circ }$ C for 24 h. The cell density was set at two-fold serial dilutions starting from 5 $\times$ 10 ${}^4$ /100 $\mu$ L. After adding 100 $\mu$ L luciferin, the medium was discarded, and the cells cultured at room temperature and away from light for 10 min. The intensity of bioluminescence was evaluated using a living imaging technology. A375-luc or M14-luc cells without luciferin addition were used as negative controls.

2.4 In vitro evaluation of the suppressive effect of Ad-VT on A375-luc or M14-luc cells

The suppressive effects of Ad-VT on A375-luc or M14-luc were evaluated using the MTS cell proliferation assay, FITC-Annexin V apoptosis detection, transwell migration, Matrigel invasion, and the scratch test. To analyze the inhibitory effects of Ad-VT on infected cells, A375-luc or M14-luc cells were seeded in 96-well cell culture plates at 1 $\times$ 10 ${}^4$ per well, cultured at 37 ${}^{\circ }$ C with 5% CO ${}_2$ for 24 h and infected with Ad-VT at a multiplicity of infection (MOI) of 10, 50 and 100. Growth suppression of Ad-VT-infected cells was measured using the MTS cell proliferation kit at 24, 48 and 72 h, and following the manufacturer’s instructions. Ad-mock-infected A375 or M14 cells were used as negative controls.

For the transwell migration and Matrigel invasion assays, Ad-VT-infected A375-luc or M14-luc cells were seeded in 24 well plates with at a concentration of 5 $\times$ 10 ${}^4$ cells/well, cultured in 5% CO ${}_2$ at 37 ${}^{\circ }$ C for 24 h and infected with Ad-VT at a MOI of 100 for 72 h. After trypsinization, the infected cells were seeded in the upper chamber of the cell culture inserts and cultured for 24 h. Cells that had migrated through the membrane were fixed with carbinol, stained with crystal violet and counted under a microscope. The experimental procedure for the Matrigel invasion assay was the same as for the transwell migration assay, except for the incubation of the upper chamber with Matrigel for 1 h prior the cells’ seeding step. Uninfected and Ad-mock-infected A375 or M14 cells were used as negative controls.

For the scratch test, Ad-VT-infected A375-luc or M14-luc cells were seeded in 6-well plates at a concentration of 2 $\times$ 10 ${}^5$ cells/well. The cells were cultured in 5% CO ${}_2$ at 37 ${}^{\circ }$ C for 24 h and then the cell layer was scratched from one side to the other in each well of the 6-well plate. The scratched cells were washed with PBS three times and infected with Ad-VT at a MOI of 100 for 72 h. The scratch widths were measured by ImageJ (version 1.51j8; National Institutes of Health, USA). Uninfected and Ad-mock-infected A375 or M14 cells were used as negative controls.

2.5 Analysis of the inhibitiory pathway in Ad-VT-infected A375-luc or M14-luc cells

The inhibitiory pathway that relied on detecting mitochondrial apoptosis-associated proteins was analyzed using JC-1 staining and Western-blot. To detect changes in the mitochondrial membrane potential in Ad-VT-infected A375-luc or M14-luc, the two cell types were seeded at 2 $\times$ 10 ${}^5$ cells/well into 6-well plates and at 5 $\times$ 10 ${}^4$ /100 $\mu$ L into 96-well plates and cultured at 37 ${}^{\circ }$ C with 5% CO ${}_2$ for 24 h. The cells were infected with Ad-VT at a MOI of 100 for 24, 48 and 72 h. The Ad-VT-infected cells were stained with 1000-fold dilutions of JC-1 at 37 ${}^{\circ }$ C, away from light for 15 min, and washed twice with PBS. The cells that were cultured in 6-well plates were observed via a fluorescence microscope for qualitative analysis. The cells that were cultured in 96-well plates were used for quantitative analysis using the TECAN Infinite ${}^{\mathrm{\circledR }}$ 200 microplate reader. Uninfected and Ad-mock-infected A375-luc or M14-luc cells were used as negative controls.

To detect the levels of mitochondrial apoptosis-related proteins in Ad-VT-infected A375-luc or M14-luc, the two cell types were seeded at 2 $\times$ 10 ${}^5$ cells/well into 6-well plates, cultured at 37 ${}^{\circ }$ C with 5% CO ${}_2$ for 24 h, and infected with Ad-VT at a MOI of 100 for 72 h. Total cellular lysates were prepared with RIPA lysis buffer (Bryotime, Shanghai, China), electrophoresed through a 10% SDS-polyacrylamide gel, transferred onto nitrocellulose membranes (Bio-Rad, California, US), incubated with corresponding antibodies and revealed for examination of protein expression by Western blotting.

2.6 Xenograft tumor model in BALB/c nude mice and treatment strategy.

Six-week-old BALB/c nude mice were purchased from the Experimental Animal Center, Academy of Military Medical Sciences of the PLA (Beijing, China) and were housed under standard pathogen-free conditions. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Military Medical Science, Changchun, China (10ZDGG007). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. The xenograft models were established via subcutaneous injection of A375-luc or M14-luc cells (3 $\times$ 10 ${}^5$ /100 $\mu$ L) into the legs of the mice. When the tumors were clearly observable using a living imaging technology (usually at day 8), the mice were randomly divided into three groups ( $n=$ 39). Group 1 was injected with 1 $\times$ 10 ${}^8$ plaque forming units (PFU) of Ad-VT in 100 $\mu$ L of Normal Saline. Group 2 was injected with 1 $\times$ 10 ${}^8$ PFU of Ad-Mock in 100 $\mu$ L of Normal Saline. Group 3 was injected with 100 $\mu$ L of Normal Saline. All groups were injected on day 8 and the injections were given every 4 days for 16 days. The length and width of the xenograft tumors were measured and observed every 4 days for 36 days using Vernier calipers and a living imaging technology. Before observation, all mice were intraperitoneally injected with 15 mg/kg luciferin and 40 mg/kg 1% sodium pentobarbital. After 5 minutes, each mouse was anesthetized by intraperitoneal injection of 40 mg/kg 1% sodium pentobarbital before observing the intensity of the tumors’ bioluminescence using a living imaging technology. During the experiment, the tumors from mice in each group ( $n=$ 3 mice) were harvested. Immunohistochemical staining was used to investigate a range of mitochondrial apoptosis-related proteins, including AIF, ARTS and Cyto-C. All immunohistochemical detections were performed by Servicebio.

2.7 Statistical analysis

The statistical analysis was performed using data from at least three independent experiments using the Statistical Package for the Social Sciences (SPSS) statistical software package (version 15.0; SPSS Inc., Chicago, IL, USA), and the results were obtained using GraphPad Prism version 7.0 (GraphPad Software Inc., La Jolla, CA, USA). Student’s $t$ -test or one-way analysis of variance followed by Tukey’s post hoc test were used. Differences with a $p<$ 0.05 or $p<$ 0.01 were considered statistically significant. Data were presented as mean $\pm$ standard deviation (SD) values.

3. Results

3.1 Construction and identification of A375-luc or M14-luc cells

The optimum concentration of G418 was determined as 200 $\mu$ g/mL. The luciferase activity of every monoclone of A375 cells is shown in Fig. 1A. The highest luciferase activity was observed in clone 11 that was continuously passaged 40 times, and its luciferase activity assessed every tenth passage. This result demonstrates that clones 11, that was named A375-luc, has a good genetic stability (Fig. 1B). The in vitro bioluminescence intensity of A375-luc increased with the increase in the clone cell number (Fig. 1C and D), suggesting a linear relationship between bioluminescence intensity and the number of cells (R2 $=$ 0.9982). Regarding M14 cells, the luciferase activity of each clone is shown in Fig. 1E. Clone 5, which was named M14-luc had the highest luciferase activity and showed a similar genetic stability as A375-luc (Fig. 1F). The bioluminescence intensity also showed a linear relationship with the number of cells (R2 $=$ 0.996). These results demonstrate that the bioluminescence of A375-luc and M14-luc could be detected by living imaging technology.

Figure 1.

Identification of A375-luc or M14-luc cells. (A) Luciferase activity of each A375-luc monoclone of A375-luc. Clone 11 that had the highest luciferase activity, was screened with 200 $\mu$ g/mL of G418. (B) Genetic stability analysis of clones 11. Clone 11 stably expressed luciferase and was named A375-Luc. (C–D) The bioluminescence of A375-luc cells was detected by living imaging technology in vitro. The bioluminescence intensity of cells increased with the increase of the number of cells. (E) The luciferase activity of each M14-luc monoclone. Clone 5 that had the highest luciferase activity, was screened with 200 $\mu$ g/mL of G418. (F) Genetic stability analysis of clones 5. Clone 5 stably expressed luciferase and was named M14-Luc. (G–H) The bioluminescence of M14-luc was detected by living imaging technology in vitro. The bioluminescence intensity of cells increased with the increase of the number of cells.

Figure 2.

The inhibitory effects of Ad-VT on A375-luc or M14-luc cells. (A–B) MTS proliferation assay showing the suppression of the proliferation of A375-luc and M14-luc cells following infection with Ad-VT or Ad-mock at a MOI of 10, 50 and 100. (C–D) Detection of apoptosis rates in Ad-VT-infected A375-luc or M14-luc cells. FITC annexin V flow cytometry was used to detect the apoptosis rate of Ad-VT infected, Ad-mock infected, and control cells at 72 h. (E–F) Transwell migration assay showing the inhibition of the migration of A375-luc or M14-luc cells infected with Ad-VT at MOI of 100 for 72 hours ( $\times$ 100). (G–H) Matrigel invasion assay showing the inhibition of the invasion of A375-luc and M14-luc cells that were infected with Ad-VT. The infection time of A375-luc and M14-luc cells with Ad-VT was 72 hours, and the MOI was 100 hours ( $\times$ 100). (I–J) Migration scratch test that the inhibition of A375-luc or M14-luc cells that were infected with Ad-VT or Ad-mock at MOI of 100 at 0 and 72 hours ( $\times$ 100). All values represent the mean $\pm$ SD. ${}^{*}p<$ 0.05, ${}^{**}p<$ 0.01.

3.2 Effects of Ad-VT on A375-luc or M14-luc cells in vitro

Ad-VT infection of A375 Luc and M14-luc cells inhibited their proliferation (Fig. 2A and B). There was no significant difference in growth suppression between Ad-VT-infected and Ad-Mock-infected cells at a MOI of 10 ( $p>$ 0.05), while growth suppression in Ad-VT-infected cells was significantly higher than that in Ad-mock-infected cells following infection at a MOI of 50 or 100 for 48 h and 72 h ( $P<$ 0.05), but the difference was not significant at 24 h. The proliferation of A375-luc was significantly suppressed by Ad-VT infection at a MOI of 100.

The apoptosis of Ad-VT-infected A375-luc and M14-luc cells at 72 h was evaluated by flow cytometry (Fig. 2C and D). The rate of apoptosis in Ad-VT-infected cells was significantly higher than that in Ad-mock-infected cells and controls at 72 h ( $p<$ 0.01) demonstrating that Ad-VT induces apoptosis in A375-luc or M14-luc cells.

Using the transwell migration assay, we observed that the migration rates of A375-luc and M14-luc cells that were infected with Ad-VT at MOI of 100 for 72 h, were significantly lower than those of other groups (Fig. 2E and F) ( $p<$ 0.01), demonstrating an inhibitory effect of Ad-VT on the migration of A375-luc and M14-luc cells The Matrigel invasion assay showed similar results to those observed with migration assay (Fig. 2G and H). In addition, the effect of Ad-VT on the migration of A375-luc or M14-luc cells was confirmed using the scratch assay following an infection (Fig. 2I and J) ( $p<$ 0.01).

Taken together, the results of the MTS cell proliferation assay, transwell migration, Matrigel invasion and scratch test demonstrate that the proliferation, migration, and invasion of A375-luc or M14-luc cells are inhibited by Ad-VT in a dose- and time-dependent manner.

Figure 3.

The inhibition pathway in Ad-VT-infected A375-luc or M14-luc cells. (A–B) JC-1 staining of Ad-VT-infected A375-luc and M14-luc cells at 24, 48 and 72 h ( $\times$ 100). Relative fluorescence (Red/Green) of JC-1 stained cells at 24, 48 and 72 h. JC-1 staining of A375-luc or M14-luc cells infected with Ad-VT, Ad-Mock and control at 24, 48 and 72 h ( $\times$ 100). The relative fluorescence (Red/Green) of Ad-VT-infected cells was significantly higher than that of Ad-mock-infected cells and controls. (C–D) Western-blot analysis of the expression of proteins related to the mitochondrial apoptotic pathwaya. All values represent the mean $\pm$ SD. ${}^{*}p<$ 0.05, ${}^{**}p<$ 0.01.

Figure 4.

Effects of Ad-VT on BALB/C nude mice A375-luc model. (A) Living imaging of the xenograft tumor BALB/c nude mice model on day 8 day and day 36. (B) Survival rates of xenograft tumor BALB/c nude mice. (C–D) The tumor growth inhibition effect in transplanted nude mice. The tumor volume in the Ad-VT group was significantly smaller than that in Ad-mock and Normal Saline groups ( $p<$ 0.01). All values represent the mean $\pm$ SD. ${}^{**}p<$ 0.01.

Figure 5.

Effects of Ad-VT on BALB/C nude mice M14-luc model. (A) Living imaging of the xenograft tumor BALB/c nude mice model on day 8 and day 36. (B) Survival rates of xenograft tumor BALB/c nude mice. (C–D) Inhibition of tumor in transplanted nude mice. The tumor volume in the Ad-VT group was significantly smaller than that in Ad-mock and Normal Saline groups ( $p<$ 0.01). All values represent the mean $\pm$ SD. ${}^{**}p<$ 0.01.

Figure 6.

The immunohistochemical detection of proteins related to the mitochondrial apoptotic pathway. (A–B) Expression levels of proteins related to the mitochondrial apoptotic pathway (AIF, ARTS Cyto-C) were detected by immunocytochemistry (800 $\times$ ). Data are presented as mean $\pm$ SD, ${}^{**}p<$ 0.01.

3.3 Inhibition pathway in Ad-VT-infected A375-luc or M14-luc cells

The results of the inhibition pathway analysis in A375-luc or M14-luc cells are shown in Fig. 3A–D. The fluorescence of A375-luc and M14-luc cells that were stained with JC-1 changed from red to green within 24 and 72 hours after infection with Ad-VT at a MOI of 100 (Fig. 3A and B). The relative fluorescence (Red/Green) of Ad-VT-infected cells was significantly higher than that of Ad-mock-infected cells and controls ( $p<$ 0.01). These results indicated that a decline in mitochondrial membrane potential of A375-luc or M14-luc cells fowllowing Ad-VT infection.

The expression levels of AIF, ARTS, and Cyto-C, in Ad-VT-infected A375-luc and M14-luc cells were significantly higher than those in Ad-Mock-infected cells at 72 h (Fig. 3C and D) ( $p<$ 0.05). There were no significant differences in the expression levels of HtrA, Endo G, and Smac/Diablo between A375-luc or M14-luc cells infected with Ad-VT and Ad-mock ( $p>$ 0.05). Overall, these investigations of the inhibition pathway in Ad-VT-infected A375-luc or M14-luc cells indicated that the apoptosis of Ad-VT infected cells was induced by changes in the mitochondrial membrane potential and increases in the expression levels of AIF, ARTS and Cyto-C. Ad-VT may induce apoptosis in A375-luc or M14-luc cells via the mitochondrial apoptotic pathway.

3.4 Tumor growth suppression and survival rate in BALB/c nude mice model

The proliferation of A375-luc or M14-luc cells can be significantly suppressed by Ad-VT in tumor bearing nude mice. The results of the tumor growth suppression experiment are shown in Figs 4A–D and 5A–D. Tumor growth in the Ad-VT group was significantly lower than that in the Ad-mock and Normal Saline groups ( $p<$ 0.01). Living imaging of the xenograft tumor BALB/c nude mice model for each group was performed on day 8 and 36 after injection of A375-luc (Fig. 4A) or M14-luc cells (Fig. 5A). The survival rate in the Ad-VT group was significantly higher than that in the Ad-Mock and Normal Saline groups when analyzed 36 days after tumor xenografting (Figs 4B and 5B) ( $p<$ 0.01). The tumor volume (Figs 4C and 5C) and intensity of bioluminescence (Figs 4C and 5D) in the Ad-VT group were significantly lower than those in the Ad-Mock and Normal Saline groups ( $p<$ 0.01).

These results indicate that we have successfully established a tumor model of A375 Luc or M14 Luc in BALB/c nude mice and demonstrated that Ad-VT inhibits tumor growth and improve survival rate in vivo.

Immunohistochemical studies of the tumors showed that Ad-VT significantly inhibits the proliferation of A375-luc and M14-luc cells and causes significant changes in proteins related to mitochondria (Fig. 6A and B). The positive rates for Ki67 staining in tumors grown by the Ad-VT group were significantly lower than those in the Ad-Mock and Normal Saline groups ( $p<$ 0.01). The positive rates of TUNEL staining in Ad-VT group were significantly higher than those in the Ad-Mock and Normal Saline groups ( $p<$ 0.01). The areal densities of AIF, ARTS, and Cyto-C in Ad-VT group were significantly higher than those in the Ad-Mock and Normal Saline groups ( $p<$ 0.01). Collectively, these in vivo results showed that the mitochondria apoptosis-related proteins can be significantly affected by Ad-VT, which also Ad-VT may significantly induces the expression of AIF, ARTS, and Cyto-C.

4. Discussion

Melanoma is a type of skin cancer that originates from the abnormal proliferation of melanocytes in the neural crest located in the epidermal layer of the skin [36]. It is highly malignant and invasive, and prone to blood and lymph node metastasis. Once metastasis occurs, the mortality is extremely high and approximatively 80% of skin cancer patients die of melanoma [37]. The treatment of melanoma patients involves surgery, radiation, chemical, and molecular targeted therapies; however, after chemotherapy, there are often adverse reactions. Although melanoma diagnosis technology is well advanced, late detection of the disease in some patients with recurrence and metastasis results in poor treatment. In recent years, with the development of biomedical and genetic engineering technologies, many new therapeutic programs have been developed, including gene-targeted therapy and oncolytic virotherapy [38].

Oncolytic adenoviruses are a type of selective tumor-killing viruses that have many advantages compared with other types of tumor therapies, including a multi-path killing mechanism and a broad anti-tumor spectrum, safe and lower toxic side effects, and a low cost compared with cell immunotherapy such as CAR-T Because of the outstanding performance, adenoviruses have attracted a significant attention in the field of oncolytic virotherapy and tumors’ targeting strategies using adenoviruses as vectorshave been shown to have great applications’prospect. In previous studies, we used the CMV promoter, apoptin, the hTERT promoter and E1a, to generate a tumor-specific replicative recombinant adenovirus [39, 40]. Apoptin has a broad range of antitumor properties that can specifically induce apoptosis of tumor cells without affecting normal cells [41]. Most normal human cells lack telomerase activity; however, hTERT expression and telomerase activation have been observed in most human malignances [42]. Therefore, Ad-VT can replicate and continuously express apoptin in tumor cells, but not in normal cells.

In this study, we investigated the effects of Ad-VT on the tumorigenesis and tumor progression of human melanoma cells tumorigenesis and progression, and we plan to establish a visualized human melanoma bearing nude mouse model using living imaging technology, one of the most common techniques. At present, luciferase that has the advantages of luminescence without excitation, strong penetrability, high sensitivity, and specificificity of interfence excitation, is one of the most commonly used marker gene in living imaging technology that is widely used in anti-tumor research. Tumorigenesis, tumor progression and metastasis in luciferase-labeled tumor bearing nude mouse model can be detected using living imaging technology. The bioluminescence intensity in the tumor area is detected using living imaging technology in orthotopic, metastatic and xenograft luciferase-labeled tumor models, where it can directly reflect the change in tumors’ volume. Tumor metastasis can be traced according to the location of bioluminescent regions [43, 44]. In our model, with the increase of tumor volume, the intensity of bioluminescence increased gradually and according to the bioluminescence localization, no metastasis occurred during the observation period.

In the present study, we used the plasmid pGL4.51 (Promega), which expresses the luciferase type 2 gene at a high level, to establish A375-luc and M14-luc stable luciferase-expressing human melanoma cell lines, that were used to visualize tumor growth in our in vivo model. Throughout the experiment, we found that the average tumor luminescence intensity of the control group significantly increased and was significantly higher than that in the Ad-VT group. At 24-36 days, the the tumor volume and average bioluminescence intensity of the Ad-VT group were lower than those of the other groups ( $p<$ 0.01). From the results of mice survival rates, we found that the survival of the mice was significantly prolonged. The average survival times of A375-luc or M14-luc xenograft tumor model BALB/c nude mice that were treated with Ad-VT, were 34.8 and 37 days, respectively. At 38 days, the survival rates in these two groups were 80% and 70%, respectively. These results show that Ad-VT significantly suppresses tumor growth in vivo and increases the survival rate of mice. Whereas, we did not establish A375-luc or M14-luc metastatic tumor model BALB/c nude mice, which may be a limitation to this study.

The inhibition pathway in Ad-VT-infected A375-luc and M14 cells was analyzed using FITC-Annexin V apoptosis flow assays, JC-1 staining and Western-blot. These results showed that after infection with Ad-VT, the apoptosis levels of A375-luc and M14-luc cells were significantly increased, reaching 40.54% and 23.7%, respectively. The Western-blot results demonstrated that the mitochondrial membrane potential and apoptotic-related protein expression levels in Ad-VT-infected A375-luc or M14-luc cells were significantly higher than those of the control group ( $p<$ 0.05), suggesting that Ad-VT treatment combination with mitochondrial specific drugs could improve efficacy and reduce toxicity for melanoma treatment. These results indicate that Ad-VT may induce apoptosis of human melanoma cells by activating the mitochondrial apoptotic pathway.

In conclusion, the oncolytic adenovirus Ad-VT has a significant inhibitory effect on A375-luc and M14-luc stably expressing luciferase cells on vitro. In vivo, the effects of Ad-VT on A375-luc or M14-luc tumorigenesis and tuomr progression can be reflected using living imaging technology. The findings not only provide a foundation for further research on human melanoma tumorigenesis and progression, but also offer a theoretical basis for the treatment of human melanoma using oncolytic adenoviruses.