A novel recombinant adenovirus expressing apoptin and MEL genes kills hepatocellular carcinoma cells and inhibits the growth and metastasis of ectopic tumors

doi:10.21203/rs.3.rs-4301482/v1

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A novel recombinant adenovirus expressing apoptin and MEL genes kills hepatocellular carcinoma cells and inhibits the growth and metastasis of ectopic tumors

https://doi.org/10.21203/rs.3.rs-4301482/v1

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HCC is the most common fatal malignancy. Although surgical resection is the primary treatment strategy, most patients are not eligible for resection due to tumor heterogeneity, underlying liver disease, or comorbidities. Therefore, this study explores the possibility of multi-molecular targeted drug delivery in treating HCC. In this study, we constructed the recombinant adenovirus co-expressing apoptin and melittin (MEL) genes. The inhibitory effect of recombinant adenovirus on hepatocellular carcinoma cells was detected through experiments on cell apoptosis, migration, invasion, and other factors. The tumor inhibitory effect in vivo was assessed using subcutaneous HCC mice. Results showed that recombinant adenovirus co-expressing anti-tumor genes TAT and apoptin, RGD and MEL can significantly inhibit the proliferation, migration, and invasion of HCC cells by inducing an increase in reactive oxygen species (ROS) levels, upregulation of apoptotic proteins such as Bax, caspase-3, and caspase-9, and downregulation of the anti-apoptotic protein Bcl2. In subcutaneous HCC mice, recombinant adenovirus induced significant apoptosis in tumor cells, inhibited tumor growth. In conclusion, recombinant adenovirus co-expressing apoptin and MEL can inhibit the growth and proliferation of tumor cells both in vivo and in vitro.

recombinant adenovirus

melittin

apoptin

hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the leading cause of primary liver cancer cases [1], with incidence rates increasing annually. More than 70% of the 16 million reported cancer cases each year originate from low- and middle-income countries [2]. Cancer affects the health and well-being of humans and animals. Surgery, radiation therapy, chemotherapy, and immunotherapy are the conventional treatment modalities for HCC. However, there are various limitations, including an increased risk of postoperative complications, incomplete partial tumor resection [3], and the heterogeneity of the patient's immune environment. Nowadays, from systematic chemotherapy to molecular targeted therapy to immunotherapy, as well as various combinations at different stages and in various ways, the treatment of HCC has made significant progress. The treatment of HCC will increasingly emphasize multidisciplinary cooperation and comprehensive care through various approaches. Among them, targeted therapy for tumors has shown promising results in certain types of tumors, indicating potential advancements in the future.

Apoptin, a 13.6 kDa serine-threonine-rich protein, is a 121-amino-acid chicken anemia virus VP3 protein that selectively induces apoptosis in cancer cells [4]. Apoptin has few secondary, tertiary, or quaternary structures and is localized in the cytoplasm in filamentous form. Each apoptin monomer contains two structural domains: the C-terminal and the N-terminal. Different combinations of these domains and their substructural domains can bind DNA and induce apoptosis independently to varying degrees [5]. Apoptin protein has great potential for tumor treatment, and researchers have been continuously studying its mechanism of killing tumor cells. In previous studies, apoptin released Cytochrome C(Cyt-c) and apoptosis-inducing factor(AIF) by regulating Bcl2 and Apaf-1 apoptotic vesicles and activating apoptotic vesicles and Bcl2-regulated death pathways [6]. Apoptin also induces elevated levels of the tumor suppressor lipid ceramides and activates caspase-3 to execute apoptosis prior to Cyt-c release [7]. In cancer cells, anaphase-promoting complex/cyclosome (APC/C) by associating with the APC1 subunit, inducing cell cycle arrest and inhibiting cell proliferation. In normal cells, the protein is located in the cytoplasm and aggregates at the cell edge, where it is degraded by the proteasome without damaging normal cells. In contrast, in tumor cells, apoptin accumulates in the nucleus to form multimers. These multimers prevent dividing cancer cells from repairing their DNA damage and also induce apoptosis through autophagy and mitochondrial apoptosis pathways [8]. Recent studies have identified apoptin as a protein precursor drug that is selectively activated in cancer cells through phosphorylation, thereby disrupting the cytoskeleton and promoting cell death [9]. It has also been found that apoptin induces apoptosis in HepG-2 cells via calcium ions, causing an imbalance and activating their mitochondrial apoptotic pathway [10]. The human immunodeficiency virus (HIV) trans-activated transcription (TAT) protein, the first cell-penetrating peptides discovered in 1998 [11], is derived from the human HIV viral TAT protein. It penetrates the cell membrane and facilitates the entry of macromolecules into the cytoplasm. TAT proteins derived from human immunodeficiency virus (HIV)-1 protein residue 48-60, mainly through the α-helical structural domain of basic amino acids, and play a key role in internalization and nuclear translocation [12]. In this research, TAT will be co-expressed with apoptin protein to enhance the penetration of apoptin into tumor cells.

MEL acts as a non-selective cytolytic peptide that disrupts all prokaryotic and eukaryotic cell membranes. It exhibits biological activities such as antibacterial, antiviral, and anti-inflammatory effects [13]. MEL disrupts the cell membrane by binding to it and forming transmembrane ring pores [14]. This process causes the leakage of substances from the cell and increases the permeability of the cell membrane, ultimately leading to cell lysis. It also allows various other molecular substances with oncogenic activity to enter the tumor cells through the pores, synergistically exerting therapeutic effects [15]. Although many studies have reported the anticancer activity of bee venom peptides in vitro and in vivo, their clinical application has been controversial due to their non-specific cytotoxic and hemolytic activities, which limit their potential in cancer therapy. The Tumor neovascularization targeting peptide RGD peptide is one of the most studied peptides that binds to the integral protein αvβ3 and acts as a targeting ligand [16]. The emergence of targeting peptides that specifically target tumors has brought new opportunities for the application of bee venom peptides. This allows for the exploration of their diverse cell-damaging abilities under the guidance of targeting peptides to develop novel and promising cancer treatment strategies.

Although a large number of target genes have been identified in HCC, the scope of drug therapy solely targeting a single gene is narrow and prone to the emergence of immune escape and drug resistance in tumor cells. Adenovirus, as the most commonly used gene delivery system, offers several advantages and can efficiently express the target protein after inserting exogenous genes. Recombinant viral systems are more efficient at gene delivery than non-viral physicochemical methods of gene delivery. They have larger genomes, higher packaging titers, and are simpler to prepare than other viral vectors, reducing the risk of insertional mutagenesis in the host genome [17]. Therefore, in this experiment, a type 5 recombinant adenovirus capable of expressing TAT, apoptin, RGD and MEL gene sequences will be constructed. The recombinant adenovirus is highly accumulated in tumor tissues by intratumoral injection, leading to the synergistic effect of the dual oncogenes. As a typical tumor with abundant blood perfusion, angiogenesis plays a crucial role in the growth and metastasis of HCC. The tumor-targeting peptide RGD selected for this experiment can specifically target αvβ3 integrin receptors highly expressed in tumor vasculature. MEL can induce apoptosis and lysis of adenovirus-infected tumor cells through non-specific cell membrane lysis, allowing the fusion protein TAT-apoptin to be released into the cytoplasm to exert secondary killing effects. The peptide TAT promotes the internalization of apoptin protein into adjacent cells, triggering tumor cell death through autophagy and mitochondrial apoptosis. Additionally, the RGD targeting peptide guides the specific secondary targeting of MEL to tumor cells, leading to cytolytic effects.

2.1. Cell culture

The human cell lines HEK293A and SMMC-7721 were obtained as a gift from Jilin University, while the Huh7 cell line was maintained in the Prevention Laboratory of Tianjin Agricultural College. The HEK293A cell line was cultured in dulbeccos modified eagle medium (DMEM) medium (Hyclone, China) supplemented with 10% fetal bovine serum (Gibco, China), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco). SMMC-7721 and L-02 cells were cultured in 1640 medium (Hyclone, China) supplemented with 10% fetal bovine serum (Gibco, China), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, China). All cells were maintained in a 37℃, 5% CO₂ incubator.

2.2. Construction of a recombinant adenovirus shuttle vector and recombinant adenovirus packaging

The adenovirus shuttle plasmid GV314-EGFP was linearized by AgeⅠ and NheⅠ for adenovirus packaging and amplification. The target genes were seamlessly cloned, transferred to a linearized adenovirus shuttle vector, and identified through Polymerase Chain Reaction (PCR) and sequencing. The constructed adenovirus shuttle plasmid was co-transfected with the AdGloxde13cre plasmid into HEK 293A cells respectively. The cells were then incubated until a large number of them showed the cytopathic effect (CPE) phenomenon, and 50% of the cells were detached.

Briefly, HEK 293A cells were seeded at 2 × 10⁵ cells into 6 cm culture plates (Corning, United States) in DMEM medium containing 10% Fetal Bovine Serum (FBS). After HEK293A cells adhered to the dish, 2 µg of adenovirus shuttle plasmid, 4 µg of AdGloxde13cre plasmid, and 8 µl of lipofectine were added to the culture media. The mixture was then incubated for 24 h at 37°C. Following incubation, the culture media were changed every 2–3 d. The infected HEK 293A cells exhibited a significant CPE on day 14. The cells were collected and cracked by repeated freezing and thawing between 37°C and -80°C three times to release the viral particles. The recovered virus was inoculated into HEK 293A cells with a 90% fusion. The CPE was observed and recorded three days post-incubation, and median tissue culture infective dose (TCID50) was calculated using the Karber method. The four recombinant adenoviruses constructed in this study were named Ad-TAT-apoptin, Ad-RGD-MEL, and Ad-TAT-Apoptin-RGD-MEL. The viruses were then collected and stored at -80°C for future use.

2.3. Reverse transcription-polymerase chain reaction

To confirm successful transcription of the target protein, RNA was extracted from SMMC-7721 cells infected with recombinant adenovirus and then reverted to cDNA. Using cDNA as a template, TAT-apoptin and RGD-MEL were amplified by PCR for validation (TAKARA, Japan). The primers were designed using the gene sequences provided in the National Center for Biotechnology Information Gene Bank and synthesized by Beijing Aoko Biological Engineering Co., Ltd. The sequence of primers is shown in Table 1.

2.4. Western Blot

SMMC-7721 cells were seeded in 24-well plates, and various recombinant adenoviruses were added for 36 h. Cellular proteins were extracted from the cell lysate, and the protein concentration was determined. Subsequently, the proteins were separated by electrophoresis using a 20% separating gel. Polyvinylidene fluoride (PVDF) membranes were blocked for 2 h and then incubated overnight at 4°C with anti-rabbit monoclonal antibody against Flag (1:5000, Abcam, USA) and goat anti-mouse monoclonal antibody against MYC (1:5000, Abcam, USA) . After washing with phosphate buffered saline（PBS）, the cells were detected with either an anti-goat IgG secondary antibody or an anti-mouse IgG secondary antibody (1:5000, ZEN BIO, China) and exposed using a gel imaging system.

2.5. Indirect immunofluorescence

SMMC-7721 cells were seeded into 24-well plates until they reached 70% confluence. Subsequently, various recombinant adenoviruses were separately added and incubated for 36 h. After fixation with 4% paraformaldehyde and permeabilization with 0.5% Triton X-100, the cells were treated with 5% Bovine Serum Albumin Solution (BSA) (Solarbio, China) for 60 min at room temperature to block non-specific binding sites. Cells were incubated with anti-rabbit monoclonal antibody Flag (1:200, Abcam, United States) and goat anti-mouse monoclonal antibody MYC (1:200, Abcam, United States) overnight at 4°C in a humidified chamber. After being washed with PBS, cells were incubated with an anti-goat IgG secondary antibody or an anti-mouse IgG secondary antibody (1:250, ZEN BIO, China) at 37°C for 2 h in the dark. Cells were imaged using a fluorescence microscope (Revolve, Echo-Labs, United States).

2.6. Cell proliferation assays

A cell proliferation reagent kit (Beyotime, China) was used to determine the cell proliferation capacity of various cell lines. Cells were seeded in 96-well plates with a density of 5 × 10³ cells/well. 0–1000 multiplicity of infection (MOI) of different recombinant adenoviruses were treated, and cell viability was assessed at 24 h, 48 h, and 72 h according to the manufacturer's instructions and compared between the different groups.

2.7. Wound healing assay

Cells were seeded in a 6-well plate. When cells reached confluency, each well was scratched with a 200 μl pipette tip, creating three lines across each well. The cells were infected with 1000 MOI of recombinant adenovirus, and images were captured using a microscope (Echo Revolve, United States) at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h after treatment.

2.8. Transwell invasion and cell migration test

The Matrixgel thawed slowly on the ice and was diluted with a serum-free medium at a ratio of 1:8. Transwell chambers, which contained a polycarbonate membrane with 8.0 μm pores (Corning, United States), were placed in a 24-well plate. An amount of 200 μl of diluted Matrixgel was added to the transwell chamber and incubated at 37℃ for 2 h. Then, the residual medium was removed. The cell concentration was diluted with RPMI-1640 medium without FBS. Subsequently, 150 μl of cell suspension was added to each transwell chamber with the BD Matrigel Basement Membrance Matrix, and 600 μl of RPMI-1640 medium containing 10% FBS was added under the 24-well plate. The cells were then cultured in the incubator for 48 h. Then, the cells in the upper compartment were wiped with cotton swabs, fixed with methanol for 30 min, and stained with 0.1% crystal violet dye for 10 min. The membrane was removed, fixed on a slide, and randomly photographed under a microscope. In the cell migration experiment, all other conditions were identical to those in the invasion experiment, except for the absence of BD Matrigel Basement Membrance Matrix.

2.9. Scanning electron microscopy (SEM)

Cells grown on slides were washed with a PBS buffer and fixed with 2.5% glutaraldehyde at 4°C overnight. The sample was dehydrated using a gradient of ethanol concentrations (50%, 70%, 80%, 95%, and 100%). The sample was then sputter-coated with gold-palladium and imaged using a scanning electron microscope (Gemini 300, ZEISS, Germany).

2.10. Flow cytometry analysis

SMMC-7721 cells were infected with different recombinant adenoviruses. After 36 h of infection, each group of cells was digested with 0.25% trypsin. The digestion process was terminated by adding a culture medium, and the cells were washed twice with PBS. Cells were collected by centrifugation and suspended in 500 μl of binding buffer. An amount of 5 μl of Annexin APC and 5 μl of 7AAD (Annexin V-APC/7-AAD double-stained apoptosis detection kit, Key GEN Bio Tech, China) were added to the mixture, and cells from each group were incubated in the dark for 15 min. Uninfected SMMC-7721 cells were used as a blank control. The apoptosis rate was determined by flow cytometry (Verse, BD, United States).

2.11. Reactive oxygen species staining

The reactive oxygen species (ROS) was detected using a ROS assay kit (Key GEN Bio Tech, China) according to the manufacturer's protocol. The cells were collected and suspended in DCFH-DA. After incubation at 37℃ for 20 min, the cells were observed under a fluorescence microscope (Revolve, Echo-Labs, United States).

2.12. TdT-mediated dUTP nick end labeling

TdT-mediated dUTP nick end labeling (TUNEL) staining of SMMC-7721 cells began by seeding the cells on a 6-well plate and treating them with 1000 MOI recombinant adenovirus for 48 h. After the cells were fixed in 4% paraformaldehyde for 30 min, a 1% Triton X-100 permeable solution was promoted at room temperature for 3–5 min. The endogenous peroxidase-blocking solution was incubated at room temperature for 10 min. Terminal nucleotidyl transferase (TdT )enzyme reaction solution was added and incubated in the dark at 37℃ for 1 h, followed by the addition of Streptavidin-Horseradish Peroxidase working solution, which was also incubated in the dark at 37℃ for 30 min. The Diaminobenzidine (DAB) working solution was color-developed at room temperature for 0.5–5 min. After washing with PBS, the sample was observed under the microscope and photographed.

TUNEL staining of tumor tissues was performed by dewaxing tumor sections in water, washing with PBS, adding Proteinase K working solution, and incubating at 37℃ for 30 min. Equilibration buffer was added to the 1 × equilibration buffer and incubated at room temperature for 10–30 min. An amount of 50 μL of TdT solution was added to the slices, incubated at 37℃ for 1 h, and washed with PBS three times for 5 min each time. After DAPI staining, the film was sealed and observed under a fluorescence microscope.

2.13. Animal xenograft model

SMMC-7721 cells (1 × 10⁷/100 μl) were injected subcutaneously into the right axilla of BALB/c nude mice (Charles River, Beijing, China). When the tumors grew to an average diameter of 5 mm, the animals were randomized into five groups and received intratumoral injections of either PBS (100μl), Ad_TA(2 × 10⁸pfu/100μl), Ad_RD(2 × 10⁸pfu/100μl), Ad_TARD(2 × 10⁸pfu/100μl), or Cisplatin (DDP) (60μg/kg), respectively, every two days. The tumor size was measured with a Vernier caliper every two days. Tumor volume was calculated using the formula: V = length × width² ÷ 2. All experiments were approved by the Ethics Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences. All manipulations were carried out in accordance with the requirements of the Regulations of the Experimental Animal Administration of China.

2.14. Hematoxylin-eosin staining

The samples were fixed with 10% formalin, dehydrated through a graded series of alcohol (50%, 70%, 80%, 95%, and 3 × 100%), embedded in paraffin, and cut at a thickness of 5 µm using a microtome. The slides were stained with hematoxylin and eosin (H&E). The HE-stained slides were visualized under a light microscope (Echo Revolve, United States) at 400× magnification. Cell nuclei in the HE stain appeared to be blue.

2.15. Statistical analyses

GraphPad Prism 6 (GraphPad, San Diego, United States) was used to generate figures. All quantitative experiments were performed in triplicate and/or repeated three times. Data were expressed as mean ± standard deviation (SD). The statistical significance between groups was determined by a t-test of variance. A value of P < 0.05 was considered statistically significant, while a value of P < 0.01 was considered highly statistically significant.

3.1. Construction of adenovirus vectors

The DNA fragments of the TAT, apoptin gene, and RGD, MEL genes were separately inserted into the vector pMD-19T. In addition, the MYC tag was fused with the TAT-apoptin gene, and the Flag tag was fused with the RGD-MEL gene for the detection of target gene expression. Subsequently, the desired DNA fragments were transferred to the adenovirus shuttle vector GV314-CMV-EGFP, yielding the vectors GV314-TAT-apoptin, GV314-RGD-MEL, and GV314-TAT-apoptin-RGD-MEL (Figure 1a). The positive clones were identified through PCR and sequencing. Three bands for the adenovirus shuttle vector were obtained by electrophoresis, with molecular weights of 492 bp, 1424 bp, and 1875 bp, respectively, which were consistent with the theoretical molecular weight of the shuttle vector. This indicates the successful construction of the adenovirus shuttle vectors GV314-TAT-apoptin, GV314-RGD-MEL, and GV314-TAT-apoptin-RGD-MEL (Figure 1b).

3.2. Packaging and titer determination of recombinant adenovirus

The successfully constructed recombinant adenovirus plasmids, identified by PCR and DNA sequencing, were selected and transfected into HEK293A cells for packaging with GloxdelE13cre plasmids, respectively. Following conventional culture for 14 d, infected HEK293A cells show a significant CPE and clear fluorescence (Supplementary Figure 1). Following four rounds of amplification, the titers of the successfully packaged Ad_TA, Ad_RM, Ad_TARM, and Ad were 10^10.75 pfu/mL, 10^10.35 pfu/mL, 10^10.25 pfu/mL, and 10¹¹ pfu/mL, respectively.

3.3. Successful transcription and expression of recombinant adenovirus in SMMC-7721 cells

Reverse transcription-polymerase chain reaction (RT-PC), Western Blot (WB), and Immunofluorescence (IF) were used to determine the successful transcription and expression of the recombinant adenovirus target gene in SMMC-7721 cells. Four recombinant adenovirus-infected SMMC7721 cells were collected for RNA extraction and reverse transcription. The TAT-apoptin gene (492 bp), RGD-MEL (1424 bp), and TAT-apoptin-RGD-MEL (1875 bp) were amplified by PCR using specific primers (Figure 1c). The expression of TAT-apoptin and RGD-MEL genes in SMMC-7721 cells infected with recombinant adenovirus was detected using the MYC label expressed through fusion with TAT-apoptin and the Flag label expressed through fusion with RGD-MEL. WB showed that Ad_TA, Ad_RM, and Ad_TARM targeted bands at 7 kDa and 16 kDa, respectively (Figure 1e). IF showed that specific blue and red fluorescence was observed for TAT-apoptin and RGD-MEL, respectively, which was not detected in the negative control Ad. These results demonstrate that the recombinant genes could be expressed in vitro following recombinant adenovirus infection in SMMC-7721 cells and that the protein could retain its antigenic reactivity (Figure 1d).

3.4. Inhibition of hepatocellular carcinoma cell proliferation by recombinant adenovirus in vitro

To investigate the effect of recombinant adenovirus on SMMC-7721, Huh 7, and L-02 cells, the cells were individually treated with Ad_TA, Ad_RM, Ad_TARM, and Ad. After treating Huh7 and SMMC-7721 cells with the recombinant adenoviruses Ad_TA, Ad_RM, and Ad_TARM for 48 h, the cell survival rate decreased as the virus dose increased. The Ad_TARM group significantly decreased cell survival in Huh7 cells at 200 MOI (P < 0.01) and in SMMC-7721 cells at 500 MOI (P < 0.01), while the same dose had no significant inhibitory effect on L-02 cells (Figure 2a).

As shown in Supplementary Figures 2A and 2B, recombinant adenoviruses Ad_TA, Ad_RM, and Ad_TARM showed a time-dependent dependence on Huh7 and SMMC-7721 cells. Overall, Ad_TARM exhibited the highest inhibitory effects, followed by Ad_TA and Ad_RM (P < 0.01). There was no significant difference between the Ad group and the control group. Overall, the inhibitory effects of Ad_TA, Ad_RM, and Ad_TARM on SMMC-7721 and Huh7 cells increased in a dose-dependent and time-dependent manner throughout the experiment, demonstrating significant differences from the Ad control group.

3.5. Recombinant adenovirus inhibits migration and invasion of SMMC-7721 cells in vitro

The effects of recombinant adenoviruses Ad, Ad_TA, Ad_RM, Ad_TARM, and PBS on SMMC-7721 cell migration were analyzed using transwell. SMMC-7721 cells infected with recombinant adenovirus were added to the upper chamber of the transwell chamber, and the chamber was removed 24 h later and stained with crystal violet. After statistical analysis, the results showed that the number of cell migrations in the recombinant adenovirus treatment group was significantly reduced compared to that in the Ad and control groups (P < 0.01), Ad_TARM had the most significant effect on FBS-mediated cell chemotactic, and the number of cell migrations was significantly different compared to the recombinant adenovirus Ad_TA and Ad_RM treatment groups (P < 0.01). In the cell migration experiment, we pre-coated Matrigel in a transwell chamber and incubated it at 37℃ for 2 h. After discarding any excess unsolidified Matrigel glue, we added SMMC-7721 cells infected with recombinant adenovirus to the upper chamber of the transwell chamber. The chamber was removed 48 h later, followed by crystal violet staining. The results of the statistical analysis revealed a significant reduction in the number of cell migrations in the recombinant adenovirus treatment group compared to the Ad and control groups (P < 0.01). Moreover, the cell migration number in the co-expression group was notably lower than that in the recombinant adenovirus Ad_TA and Ad_RM treatment groups (P < 0.01). The co-expression group exhibited the most effective inhibition of invasion in SMMC-7721 cells (Figure 2b).

In addition, we evaluated the effect of recombinant adenovirus on SMMC-7721 cell migration. In the cell scratch experiment, SMMC-7721 cells were infected with recombinant adenoviruses Ad, Ad_TA, Ad_RM, and Ad_TARM. The changes in the scratch area at different time points were compared and analyzed. The results showed that, compared with the control group, recombinant adenoviruses Ad_TA, Ad_RM, and Ad_TARM significantly inhibited cell migration in SMMC-7721 cells after 12 h (P < 0.01). The recombinant adenovirus Ad_TARM treatment group exhibited a lower degree of wound healing and significantly inhibited the migration of SMMC-7721 cells compared to all other treatment groups (Figure 2c, P < 0.01).

The Annexin V flow cytometry method was used to further analyze the apoptosis of SMMC-7721 cells induced by recombinant adenovirus. We found that Ad_TA, Ad_RM, and Ad_TARM all induced apoptosis of SMMC-7721 cells at 36 h (Figure 2d, P < 0.01). In conclusion, compared with the control group, Ad_TA, Ad_RM, and Ad_TARM can induce apoptosis in SMMC-7721 cells.

3.6. Recombinant adenovirus induces apoptosis in SMMC-7721 cells by increasing ROS content

Subsequently, to study the changes in oxidative stress in SMMC-7721 cells, those infected with 1000 MOI of Ad_TA, Ad_RM, Ad_TARM, and Ad, respectively, were stained with a DHE probe staining solution at 36 h. Compared with the Ad and control groups, ROS content was significantly increased in all recombinant adenovirus treatment groups (P > 0.05), especially in the Ad_TARM treatment group (Figure 3a). In summary, the recombinant adenoviruses tested showed a significant increase in DHE-specific ROS in SMMC-7721 cells.

An apoptosis-activated endonuclease cleaved nuclear DNA. The TUNEL reagent was used to detect nuclear DNA breakage in SMMC 7721 cells treated with recombinant adenovirus and the PBS control group. The nuclei of the PBS and Ad control groups were almost unstained, while the recombinant adenovirus treatment group showed different degrees of nuclear staining post-administration. The recombinant adenovirus treatment group induced the production of DNA fragments in SMMC-7721 cells (Figure 3b).

Scanning electron microscopy (SEM) results showed that after recombinant adenovirus infected SMMC-7721 cells for 48 h, the cell folds became rounded, and the pseudopodia microvilli disappeared in the Ad_TA treatment group. The cells in the Ad_RM-treated group were slightly atrophic, while those in the Ad_TARM-treated group exhibited the two deformations mentioned above (Figure 3c).

WB was used to detect the expression of apoptotic proteins in SMMC-7721 cells treated with the recombinant adenoviruses Ad_TA, Ad_RM, and Ad_TARM. Results showed that, compared with PBS and Ad controls, the Ad_TA, Ad_RM, and Ad_TARM groups all induced activation of cleaved caspase-3 protein, increased Bax expression, and decreased Bcl-2 protein (Figure 3d).

3.7. Recombinant adenovirus inhibits ectopic tumor growth in vivo

Ad_TA, Ad_RM, Ad_TARM, and Ad were administered via intra-tumor injection at a dose of 2 × 10⁸ pfu/100 μl/mouse once every two days. As shown in Figures 4A and 4B, after five doses, the tumor volume and weight of the recombinant adenovirus-treated group were smaller and lighter than those of the PBS group, indicating that the recombinant adenovirus significantly inhibited the growth of xenografted tumors in mice (P < 0.01). However, there was no difference between the recombinant adenovirus groups, possibly because of the effect of low-dose administration. The tumor growth rate, final tumor volume, and tumor weight in the DDP group were significantly lower than those in other groups. However, the weight of nude mice was significantly lower than that in other groups, indicating serious side effects (Figure 4a-c).

The tumors from the recombinant adenoviruses Ad_TA, Ad_RM, Ad_TARM, PBS, and cisplatin groups were selected for immunohistochemical staining and TUNEL assay. The TUNEL experiment showed that the apoptosis rate of tumor tissue cells in the Ad_TARM group was 77.92 ± 2.86%, which was higher than that in the Ad_TAgroup (74.42 ± 3.14%) and the Ad_RM group (59.38 ± 9.03%). The apoptotic cells were distributed in a flaky pattern, indicating that DNA damage was induced in the tumor tissue after treatment (Figure 4d). Immunohistochemical staining results showed that the expressions of caspase-3, caspase-9, and P53 were increased in tumor tissues treated with recombinant adenovirus. Compared with the Ad_TA and Ad_RM treatment groups, the expression of caspase-9 protein was the highest in the Ad_TARM treatment group (Figure 4f). H&E results showed that there were no obvious heart lesions in the administration groups. Pulmonary congestion was worse than that in the PBS control group, but the symptoms of alveolar wall thickening were alleviated. Mild renal edema was observed in the cisplatin group, but no obvious abnormalities were observed in the other groups. A large number of neutrophils infiltrated the liver in the cisplatin group, while the liver lobular structure was destroyed in the PBS group. No significant abnormalities were observed in the other groups. These results indicate that the recombinant adenovirus can inhibit liver metastasis caused by subcutaneous HCC mice. The boundary of the white medulla of the spleen in the recombinant adenovirus group was clearer and healthier than that in the PBS and cisplatin groups. Tumor tissues in all groups were grade II heterozygous. The tumor cells exhibited moderate aberrations, were susceptible to nucleolar division, and were arranged in ring, or block formations (Supplementary Figure 2d).

In this study, recombinant adenovirus was used to co-express the apoptin and MEL genes. The transmembrane peptide TAT was fused with apoptin to promote the second internalization of apoptin protein into cancer cells in HCC cells. The targeting peptide RGD was used to target the integrin receptor in HCC vessels and fused with MEL to instruct MEL to specifically kill tumor cells. The non-specific damage to normal tissue was reduced. In conclusion, apoptin was used to specifically induce apoptosis and kill tumor cells through mitochondrial pathways and autophagy. At the same time, apoptin was combined with MEL to lyse the cell membrane of cancer cells and disrupt the complete structural characteristics of the cells, aiming to achieve tumor inhibition through dual-gene coordination.

Apoptin protein has been widely studied in colorectal cancer [18], HCC [19], lung cancer [20], and other types of cancer. Its potential applications are vast. The process of purifying the obtained protein is complex, expensive, unstable, and not easily absorbed by cells, which limits the direct use of the apoptin protein. Delivery of adenovirus vectors can be highly expressed in tumor cells without the risk of gene integration, and it is simpler than exosomes or self-assembled nanomaterials. This study evaluated the inhibitory effect of recombinant adenovirus on SMMC-7721 cells in vitro. In vitro experiments involved inflecting SMMC-7721 cells with 1000 MOI for 72 h, resulting in a 40% inhibitory effect. In the cell migration and invasion experiment, migration and invasion were significantly inhibited by 50% compared to the control group. In the ectopic mouse models of HCC, tumor tissue growth was inhibited, DNA damage occurred in cells, and the levels of apoptotic proteins caspase-9, caspase-3, and P53 increased. Similar to our results, Yiquan Li et al. constructed apoptin into a recombinant type 5 adenovirus vector to achieve stable and effective protein expression in cells. The results showed a significant increase in the apoptosis and autophagy of HCC cells. Additionally, there was a notable elevation in the level of cell ROS, which mediated autophagy and apoptosis in HCC cells. The apoptosis induced by apoptosis at 24 h was about 30% [21]. Xiaoyang Yu et al. found that after incubating HepG-2 cells with apoptin for 72 h, 25% apoptosis was observed. The expression levels of cleaved-PARP and cleaved-caspase-3 were significantly higher, and the ROS levels in HepG-2 cells were significantly increased [22]. The above studies confirm the great potential and application value of apoptin in the treatment of HCC.

MEL has been regarded as a promising broad-spectrum antitumor drug, but it still shows obvious toxic side effects in the treatment process, such as non-specific cytolytic activity, hemolytic toxicity, coagulation dysfunction, and allergic reactions, which seriously hinder its wide clinical application [13]. In recent years, the anti-tumor mechanism and targeted delivery strategy of MEL have made great progress, providing the possibility for clinical application. There have been attempts to alter the sequence of MEL or fine-tune the conformation of MEL [23]. Studies have also been conducted on the delivery of MEL using smart nanocarrier strategies to achieve passive or active targeting for the treatment of recurrent and refractory malignancies [24]. The tumor-homing peptide RGD recognizes global proteins present on the surface of cancer cells and specifically targets the vascular region of tumors. In this study, RGD and MEL were fused to reduce the toxic side effects of MEL by leveraging the targeting effect of RGD. Studies have shown that melittin nanoparticles induce apoptosis and necrosis of B16F10 cells in vitro, inhibit liver metastasis of B16F10, 4T-1, and CT26, prevent metastasis to other organs, and extend the survival of multiple tumor models [25]. In addition, similar to our results, Mao Jie et al. found that melittin nanoliposomes can significantly induce apoptosis, with IC50 ranges of 1.44 to 2.1 μM for five HCC cell lines (Bel-7402, SMMC-7721, HepG2, LM-3, and Hepa 1-6 cells). Melittin nano-liposomes (2μM) increased the expression levels of pro-apoptotic proteins (such as Bax and cleaved caspase-3) and decreased the expression levels of anti-apoptotic proteins (including Bcl-2 and PARP) in HepG2 cells. Significant inhibition of HCC growth was also shown in the HepG2 cell ectopic mouse models and the LM-3 cell orthotopic model in nude mouse models [26].

This project investigated the inhibitory effect of recombinant adenovirus on SMMC-7721 cells and an HCC ectopic tumor model in vitro, elaborated on the expression pattern of the recombinant adenovirus in tumor animal models, and confirmed the targeted therapeutic effect of the recombinant adenovirus on HCC. The results provide ideas and methods for comparative medicine and tumor-targeted gene therapy. In the future, we will increase the dose of subcutaneous ectopic tumors to explore the optimal therapeutic effect of the co-expression treatment group. In addition, we will also perform experiments on orthotopic HCC mouse models and other tumor models.

In summary, we designed and packaged a recombinant adenovirus that co-expresses apoptin and MEL therapeutic genes. We verified its ability to induce HCC cell death through in vitro and in vivo experiments. Through multi-level studies, we found that the recombinant adenovirus co-expressing apoptin and MEL therapeutic genes could inhibit the invasion and migration of tumor cells, increase the production of ROS in tumor cells, up-regulate the tumor cell apoptosis-related proteins Bax, caspase-3, and caspase-9, induce tumor cell apoptosis, and inhibit the growth of ectopic hepatocellular carcinoma. Our design utilized two different mechanisms of cytotoxic proteins to synergistically treat tumors, allowing for the possibility of treating multiple tumors regardless of tumor type and heterogeneity.

Author Contributions

T.M.J., J.Q.W., and D.C.Z. conceived and designed the study. Z.Y.L., X.L., and H.J.L. analyzed the data. J.Q.W. drafted the manuscript. T.M.J. and D.C.Z. revised the manuscript. Funding were provided by D.C.Z. and J.Q.W. All authors reviewed and approved the submitted version of the manuscript.

Funding

This work was supported by the Youth Program of the National Natural Science Foundation of China [grant number 32202760]; the Key Program of Postgraduate Research Innovation of Tianjin [grant number 2019YJSS093]; the Open Fund of Key Laboratory of Smart Breeding (Co-construction by Ministry and Province), Ministry of Agriculture and Rural Affairs [grant number 2023-TJAUKLSBF-2103]; the Research Project of Tianjin Education Commission [grant number 2019KJ032].

Institutional Review Board Statement

The study protocol has been reviewed and approved by the Animal Ethical and Welfare Committee of Tianjin Agricultural University (Approval number 2022LLSC25) on 16 March 2022.

Declarations Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Table1 Primer sequence of target gene

Name	Sequence
TAT-Apoptin F	GGAGGTGGAGGATCAATG
TAT-Apoptin R	CCTCTTCTGAGATGAGTTT
RGD-MEL F	GACTGCTTCTGCGGTATT
RGD-MEL R	TTGTCGTCATCATCCTTATAGT

No competing interests reported.

SupplementaryFigure1.tif
Supplementary Figure 1: Packaging of recombinant adenovirus vectors containing the TAT-apoptin gene, UBI-RGD-MEL, and TAT-apoptin gene + UBI-RGD-MEL.
SupplementaryFigure2.tif
Supplementary Figure 2 A, B, and C. Cell survival rate of L-02 cells, SMMC-7721 and Huh7, after treatment with recombinant adenovirus for 24 h and 48 h. D. HE staining of tissue sections of a mouse model of a subcutaneous tumor of HCC.

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A novel recombinant adenovirus expressing apoptin and MEL genes kills hepatocellular carcinoma cells and inhibits the growth and metastasis of ectopic tumors

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