Abstract
Background/Aim: Diffuse-type gastric cancer (GC) frequently exhibits peritoneal metastasis, leading to poor prognosis. However, efforts to develop antitumor strategies for preventing the peritoneal metastasis of GC have been unsuccessful. As diffuse-type GC cells often carry a genetic alteration in the tumor suppressor p53 gene, p53 restoration may be a potent strategy for preventing peritoneal metastasis of GC. In this study, we investigated the therapeutic potential of p53-expressing adenoviral vectors against peritoneal metastasis of diffuse-type GC cells. Materials and Methods: Three diffuse-type human GC cell types with different p53 statuses (p53–wild type NUGC-4, p53–mutant type GCIY, and p53–null type KATOIII) were used to evaluate the therapeutic potential of p53 activation induced by the p53-expressing, replication-deficient adenovirus Ad-p53 and oncolytic adenovirus OBP-702. Viability, apoptosis, and autophagy of virus-treated GC cells were analyzed under normal and sphere-forming culture conditions using the XTT assay and western blot analysis. The in vivo antitumor effects of OBP-702 and Ad-p53 were assessed using xenograft tumor models involving peritoneal metastasis of NUGC-4 and GCIY cells. Results: Under normal and sphere-forming culture conditions, OBP-702 induced a significantly greater antitumor effect in GC cells compared with Ad-p53 by strongly inducing p53-mediated apoptosis and autophagy and receptor tyrosine kinase suppression. In vivo experiments demonstrated that intraperitoneal administration of OBP-702 significantly suppressed the peritoneal metastasis of NUGC-4 and GCIY cells compared with Ad-p53, leading to prolonged survival of mice. Conclusion: Intraperitoneal administration of OBP-702 inhibits the peritoneal metastasis of GC cells by inducing p53-mediated cytopathic activity.
Gastric cancer (GC) is the fifth most common malignancy worldwide, especially in East Asia (1). GC is primarily classified into two histopathological subtypes, intestinal-type and diffuse-type, according to the Lauren classification (2). Intestinal type GC is a well-differentiated type of GC associated with slow-growing tumors, a chemo-sensitive phenotype, inflammatory microenvironment, and relatively better prognosis (3). In contrast, diffuse-type GC involves poorly differentiated or signet-ring cell types of GC with rapidly growing tumors, a chemo-resistant phenotype, stromal microenvironment, and relatively worse prognosis (4). Peritoneal metastasis is the most common form of metastasis in diffuse-type GC (5). Although intraperitoneal chemotherapy has been shown to be more beneficial than systemic chemotherapy in a Phase III clinical trial for treating patients with GC with peritoneal metastasis (6), the clinical outcome of patients with GC with peritoneal metastasis remains unsatisfactory. Therefore, the development of novel therapeutic strategies for treating the peritoneal metastasis of diffuse-type GC is required.
The tumor suppressor p53 gene is the most frequently mutated gene in GC tumors (7-9), suggesting that p53 restoration is a potential antitumor approach for treating GC progression. p53 is a multifunctional protein that induces cell cycle arrest, senescence, apoptosis, and autophagy (10). The replication-deficient adenoviral vector Ad-p53 has been frequently used to induce the expression of exogenous p53 protein in tumor cells in both preclinical and clinical studies (10). Ad-p53 has therapeutic potential for treating human GC involving mutations in p53 (11, 12). However, p53–wild type GC cells are relatively resistant to Ad-p53–induced antitumor effects compared to p53-mutant GC cells (11, 13). Therefore, a novel therapeutic strategy for inducing a stronger antitumor effect against both p53-intact and p53-mutant GC cells than Ad-p53 is necessary.
Oncolytic virotherapy is a novel antitumor therapy that is increasingly being utilized for treating peritoneal metastasis of GC using various types of viruses (14). We developed a telomerase-specific, replication-competent oncolytic adenovirus, OBP-301, to target tumor cells with telomerase activity associated with unlimited cell proliferation (15). Intraperitoneal administration of OBP-301 was shown to enhance the antitumor efficacy of intraperitoneal chemotherapy with cisplatin and paclitaxel in the peritoneal metastasis of human ovarian and GC cells (16, 17). To promote the antitumor effect of OBP-301, we generated a p53-expressing oncolytic adenovirus, OBP-702, in which the wild-type p53 gene cassette was inserted into the E3 region of OBP-301 (18, 19). OBP-702 was shown to induce a greater antitumor effect than OBP-301 and Ad-p53 in epithelial and mesenchymal types of human cancer cells via the induction of apoptosis and autophagy (18, 19). We recently demonstrated that intraperitoneal administration of OBP-702 promotes the antitumor efficacy of intraperitoneal chemotherapy with paclitaxel in the peritoneal metastasis of p53-intact MKN-45 cells (20). Therefore, we hypothesized that OBP-702 would be more effective than Ad-p53 in treating the peritoneal metastasis of both p53-intact and p53-mutant GC cells.
In the present study, we investigated the therapeutic potential of Ad-p53 and OBP-702 against human diffuse-type GC cells with different p53 statuses (NUGC-4, GCIY, and KATOIII cells). Virus-induced cytopathic activity was analyzed under normal and spheroid culture conditions using the XTT assay. Virus-mediated p53 activation, apoptosis, autophagy, and modulation of receptor tyrosine kinases (RTKs) were analyzed using western blotting. In addition, xenograft tumor models of peritoneal dissemination of diffuse-type GC cells were used to assess the antitumor effects of Ad-p53 and OBP-702.
Materials and Methods
Cell lines. The human diffuse-type GC cell line NUGC-4 (p53–wild type) was purchased from the Japanese Collection Research Bioresources (Osaka, Japan). NUGC-4 cells expressing red fluorescent protein (RFP) (NUGC-4-RFP) were obtained from AntiCancer, Inc. (San Diego, CA, USA). The human diffuse-type GC cell lines GCIY (p53–mutant type) and KATOIII (p53–null type) were purchased from RIKEN Cell Bank (Tsukuba, Japan) and the Human Science Research Resources Bank (Osaka, Japan), respectively. Cells were cultured for no longer than 5 months following resuscitation. Authentication was not performed by the authors. NUGC-4 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). NUGC-4-RFP cells were maintained in Dulbecco’s Modified Essential Medium supplemented with 10% FBS. GCIY cells were maintained in Eagle’s Minimum Essential Medium supplemented with 15% FBS. GCIY cells transfected with the firefly luciferase (Luc) plasmid vector (GCIY-Luc) were maintained in medium containing 0.2 mg/ml geneticin (G418; Invitrogen, Carlsbad, CA, USA), as previously reported (17). KATOIII cells were maintained in a 1:1 mixture of Eagle’s Minimum Essential Medium and RPMI-1640 medium supplemented with 10% FBS. All media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were routinely maintained at 37°C in a humidified atmosphere with 5% CO2.
Recombinant adenoviruses. The telomerase-specific replication-competent adenovirus OBP-301 (suratadenoturev), in which the promoter element of the human telomerase reverse transcriptase (hTERT) gene drives expression of the E1A and E1B genes, was previously constructed and characterized (15, 21) (Figure 1A). OBP-702 was generated by modifying OBP-301 via insertion of a human wild-type p53 gene expression cassette into the E3 region of OBP-301 (18, 19) (Figure 1B). Replication-deficient Ad-p53 was used to induce p53 expression in target cells (Figure 1C).
Schematic diagrams of structures of the replication-deficient and conditionally replicating adenoviral vectors. A) OBP-301 is a telomerase-specific, replication-competent oncolytic adenovirus in which the hTERT promoter drives expression of the E1A and E1B genes, which are linked by an IRES. B) OBP-702 is a p53-armed OBP-301 variant in which the Egr1 promoter drives expression of the human wild-type p53 gene. C) Ad-p53 is non-replicative E1/E3-deleted adenovirus expressing the human wild-type p53 gene under the control of CMV promoter. Ad5: Adenovirus serotype 5; CMV: cytomegalovirus; hTERT: human telomerase reverse transcriptase; IRES: internal ribosome entry site; ITR: inverted terminal repeat.
Flow cytometric analysis. To analyze the expression of CAR protein, cells were incubated with anti-CAR antibody (Upstate Biotechnology, Lake Placid, NY, USA) or isotype control IgG for 60 min on ice. The cells were then labeled with fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA, USA) for 30 min and analyzed using FACS array (BD Biosciences). The mean fluorescence intensity (MFI) for each cell line was determined by calculating the difference between the MFI of the antibody-treated and isotype control IgG-treated cells.
Cell viability assay. To analyze the antitumor effect under normal culture conditions, the cells were seeded in 96-well plates at a density of 103 cells/well. To analyze the antitumor effect under spheroid culture conditions, cells were seeded in Corning™ 96-well, round-bottom, ultra-low attachment microplates at a density of 5×103 cells/well. After 24 h, cells were infected with OBP-702 or Ad-p53 at a multiplicity of infection (MOI) of 0, 1, 5, 10, 50, or 100 plaque-forming units (PFU)/cell. Cell viability was determined on day 3 or 6 after virus infection under normal or spheroid culture conditions, respectively, using a Cell Proliferation Kit II (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s protocol.
Western blot analysis. Cells were seeded in a 100-mm dish at a density of 105 cells/dish 24 h before virus infection. The cells were then infected for 72 h with Ad-p53 or OBP-702 at the indicated MOI. Whole-cell lysates were prepared in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100] containing a protease inhibitor cocktail (Complete Mini; Roche Applied Science, Mannheim, Germany). Proteins were electrophoresed on 10% SDS polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (Hybond-P; GE Health Care, Buckinghamshire, UK). Blots were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) at room temperature for 30 min. The primary antibodies used were as follows: mouse anti-p53 monoclonal antibody (mAb) (18032; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-poly (ADP-ribose)polymerase (PARP) polyclonal antibody (pAb) (9542; Cell Signaling Technology), rabbit anti-EGFR pAb (2232; Cell Signaling Technology), rabbit anti–hepatocyte growth factor receptor (c-Met) mAb (8198; Cell Signaling Technology), rabbit anti–fibroblast growth factor receptor 2 (FGFR2) mAb (11835; Cell Signaling Technology), and mouse anti–β-actin mAb (A5441; Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were horseradish peroxidase–conjugated antibodies against mouse IgG (NA931; GE Healthcare) or rabbit IgG (NA934; GE Healthcare). Immunoreactive bands on the blots were visualized using enhanced chemiluminescence (ECL Prime; GE Healthcare).
Transfection of cells with siRNA and cell viability assay. Cells were seeded in 96-well plates at a density of 103 cells/well 24 h before transfection. The cells were transfected with small interfering RNAs (siRNAs) against EGFR (s563; Thermo Fisher Scientific, Waltham, MA, USA), c-Met (s8700; Thermo Fisher Scientific), and FGFR2 (s5174; Thermo Fisher Scientific) or with negative control siRNA (4390943; Thermo Fisher Scientific) at a concentration of 30 nM using Lipofectamine™ RNAi MAX transfection reagent (Thermo Fisher Scientific). A mixture of three siRNAs (10 nM each siRNA) was used as si-MIX. Cell viability was determined on day 3 after transfection using a Cell Proliferation Kit II (Roche Molecular Biochemicals), according to the manufacturer’s protocol.
Live and dead cell assay. Cells were seeded in Corning™ 96-well, round-bottom, ultra-low attachment microplates at a density of 5×103 cells/well. After 24 h, the cells were infected with OBP-702 or Ad-p53 at a MOI of 0, 1, 10, or 100 PFU/cell. Cell viability was determined on day 6 after virus infection using a solution of calcein (4 μM) and EthD-1 (4 μM) dissolved in phosphate-buffered saline (PBS). Cells that formed spheroids were imaged using a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan).
Fluorescence assay of apoptotic cells using cleaved caspase-3. Cells were seeded in a Corning™ 96-well, round-bottom, ultra-low attachment microplate at a density of 5×103 cells/well. Twenty-four hours later, the cells were treated with Ad-p53 or OBP-702 at a multiplicity of infection (MOI) of 0, 1, 10, or 100 plaque-forming units (PFU)/cell. Cells with activated caspase-3 were evaluated on day 6 after virus infection using CellEvent™ Caspase-3/7 Green reagent according to the manufacturer’s protocol. Cells that formed spheroids were imaged using a BZ-X700 fluorescence microscope system (Keyence, Osaka, Japan).
In vivo xenograft tumor model with peritoneal dissemination. Animal experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of the Okayama University School of Medicine (no. OKU-2018334). NUGC-4-RFP cells (5×106 cells/mouse) or GCIY-Luc cells (2×106 cells/mouse) were inoculated into the peritoneal cavity of 7-week-old female BALB/c-nu/nu mice (CLEA Japan, Tokyo, Japan). NUGC-4–RFP tumor-bearing mice were treated with PBS (mock control) (n=9), Ad-p53 (1×108 PFU) (n=7), or OBP-702 (1×108 PFU) (n=9) on days 14, 16, and 18 after inoculation. Red fluorescence photographs of NUGC-4–RFP tumor-bearing mice were taken in the prone position after laparotomy under a fluorescence microscope on day 28 after inoculation. Tumor volume was calculated based on red fluorescence intensity using ImageJ software. In contrast, GCIY-Luc tumor-bearing mice were treated with PBS (mock control) (n=8), Ad-p53 (1×108 PFU) (n=8), or OBP-702 (1×108 PFU) (n=8) on days 14, 21, and 28 after inoculation. Images of GCIY-Luc tumor-bearing mice were collected in the prone position after luciferin injection using a Xenogen In Vivo Imaging System (IVIS) Lumina (Caliper Life Sciences, Cheshire, UK) on days 13, 20, 27, 34, 41, and 48 after inoculation. Photons emitted from the abdominal region were quantified using Xenogen Living Image Software (Caliper Life Science). The survival of the mice was monitored for 160 days after inoculation.
Statistical analysis. Data are expressed as the mean±SD. The significance of differences between two groups was assessed using the Student’s t-test. One-way analysis of variance followed by Tukey’s multiple-group comparison test was used to compare differences between groups in animal experiments. The Kaplan–Meier method and log-rank test were used to compare the differences in survival rates between groups. Statistical significance was defined as p<0.05.
Results
OBP-702 induced a greater cytopathic effect in association with apoptosis and autophagy than Ad-p53 in diffuse-type GC cells. The therapeutic potential of the p53-expressing adenoviral vectors Ad-p53 and OBP-702 against diffuse-type GC cells was investigated using three human diffuse-type GC cell lines with different p53 status: NUGC-4 (p53–wild type), GCIY (p53–mutant type), and KATOIII (p53–null type). All GC cells exhibited high expression of the coxsackie and adenovirus receptor (CAR), a primary receptor for adenovirus serotype 5 (Ad5) (Figure 2A). The viability of GC cells was analyzed using the XTT assay 72 h after infection with Ad-p53 or OBP-702. Ad-p53 significantly decreased the viability of p53-inactivated GCIY and KATOIII cells in a dose-dependent manner, whereas p53-intact NUGC-4 cells were resistant to Ad-p53 (Figure 2B). By contrast, OBP-702 significantly decreased the viability of all GC cell lines in a dose-dependent manner to a greater extent than Ad-p53 did (Figure 2B). These results indicate that OBP-702 is superior to Ad-p53 in terms of inducing cytopathic activity in diffuse-type GC cells.
Cytopathic effect of Ad-p53 and OBP-702 against human diffuse-type GC cells. A) Expression of CAR protein on the surface of human GC cells. Expression level of CAR on NUGC-4, GCIY, and KATOIII cells as assessed using flow cytometry. The mean fluorescence intensity (MFI) of each cell line was determined by calculating the difference between the MFI of anti-CAR antibody-treated and isotype control IgG-treated cells. B) Cells were seeded in 96-well plates at a density of 103 cells/well 24 h before virus infection. Cell viability was determined using the XTT assay on day 3 after infection with Ad-p53 or OBP-702 at the indicated MOI. Cell viability was calculated relative to that of mock infected cells, the viability of which was set at 1.0. Data are expressed as mean±SD (n=5). *p<0.05; **p<0.01 (vs. 0 MOI). C) Expression of p53, PARP, cleaved PARP (C-PARP), and p62 proteins in diffuse-type GC cells infected with Ad-p53 for 72 h at the indicated MOI. D) Expression of p53, PARP, cleaved PARP (C-PARP), and p62 proteins in diffuse-type GC cells infected with OBP-702 for 72 h at the indicated MOI. β-Actin was assayed as a loading control.
To investigate whether the virus-mediated cytopathic activity in diffuse-type GC cells is associated with the induction of p53, apoptosis, or autophagy, the expression of p53, PARP, and cleaved PARP (apoptosis markers), and p62 (autophagy marker) was analyzed using western blotting in GC cells treated with Ad-p53 or OBP-702 for 72 h. Ad-p53 induced an increase in the expression of p53 and cleaved PARP and a decrease in the expression of p62 in p53-inactivated GCIY and KATOIII cells, but this effect was not observed in p53-intact NUGC-4 cells (Figure 2C). In contrast, OBP-702 up-regulated the expression of p53 and cleaved PARP and down-regulated the expression of p62 in all GC cells more strongly than Ad-p53 (Figure 2D). These results suggest that the virus-induced cytopathic effect is associated with the induction of p53, apoptosis, and autophagy in diffuse-type GC cells.
Suppression of receptor tyrosine kinases by OBP-702 and Ad-p53 in diffuse-type GC cells. Recent studies on diffuse-type GC have identified activated RTKs, EGFR, c-Met, and FGFR2 as potential molecular targets (22). To determine whether Ad-p53 or OBP-702 suppressed the expression of RTKs in diffuse-type GC cells, the expression of EGFR, c-Met, and FGFR2 was analyzed in GC cells treated with Ad-p53 or OBP-702 for 72 h. Ad-p53 infection at high doses (MOI of 50 or 100) decreased the expression of EGFR and c-Met in p53-inactivated GC cells (Figure 3A). However, the expression of FGFR2 in KATOIII cells was increased by Ad-p53 infection (Figure 3A). By contrast, OBP-702 infection at low and high doses (MOI >5) decreased the expression of EGFR, c-Met, and FGFR2 in p53-intact and p53-inactivated GC cells (Figure 3B). The expression of FGFR2 in KATOIII cells was also suppressed by non-armed OBP-301 without p53-inducing activity (Figure 3C).
Virus-mediated down-regulation of receptor tyrosine kinases is involved in the decreased viability of human diffuse-type GC cells. A) Expression of epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-Met), and fibroblast growth factor receptor 2 (FGFR2) proteins in diffuse-type GC cells infected for 72 h with Ad-p53 at the indicated MOI. B) Expression of EGFR, c-Met, ERK1/2, and FGFR2 proteins in diffuse-type GC cells infected for 72 h with OBP-702 at the indicated MOI. C) Expression of FGFR2 in KATOIII cells infected for 72 h with OBP-301 at the indicated MOI. β-Actin was assayed as a loading control. D) Effect of siRNA against EGFR (si-EGFR), c-Met (si-Met), and FGFR2 (si-FGFR2) on the viability of diffuse-type GC cells was determined using the XTT assay on day 3 after transfection. The viability of siRNA-transfected cells was calculated relative to that of negative control siRNA (si-NC)–transfected cells, which was set at 1.0. *p<0.05; **p<0.01 (vs. si-NC).
To further evaluate the effect of RTKs on diffuse-type GC cells, the viability of GC cells was analyzed using the XTT assay 72 h after transfection with EGFR siRNA, c-Met siRNA, FGFR2 siRNA, or a mixture of each siRNA. si-EGFR significantly suppressed the viability of all GC cells (Figure 3D). The viability of NUGC-4 and KATOIII cells was significantly decreased by treatment with si-Met and si-FGFR2, respectively (Figure 3D). A mixture of each siRNA significantly decreased the viability of NUGC-4 and KATOIII cells more strongly than treatment with the single siRNAs (Figure 3D). These results suggest that down-regulation of RTKs plays a role in the virus-mediated decrease in diffuse-type GC cell viability.
Cytopathic effect of Ad-p53 and OBP-702 in sphere formation by diffuse-type GC cells. Three-dimensional spheroid cultures are frequently used to mimic the physical tumor microenvironment of cancer cells, including GC cells (23). To examine the therapeutic potential of Ad-p53 and OBP-702 against sphere-forming diffuse-type GC cells, GC cells were seeded in low-attachment 96-well plates to form tumor spheroids and then treated with Ad-p53 or OBP-702. The viability of sphere-forming GC cells was analyzed using the XTT assay on day six after virus infection. Ad-p53 decreased the viability of p53-inactivated GCIY and KATOIII cells, whereas p53-intact NUGC-4 cells were resistant to Ad-p53 (Figure 4A). By contrast, OBP-702 significantly suppressed the viability of all sphere-forming GC cells (Figure 4A). The distribution of virus-affected dead cells in GC tumor spheres was analyzed using live and dead assays on day 6 after virus infection. Treatment with Ad-p53 resulted in the appearance of red-colored dead cells within the central area of GCIY and KATOIII spheres but not NUGC-4 spheres (Figure 4B). Moreover, treatment with OBP-702 resulted in the appearance of red-colored dead cells within the central area of all GC spheres (Figure 4B).
Cytopathic effect of Ad-p53 and OBP-702 against sphere-forming gastric cancer (GC) cells. A) cells were seeded in ultra-low attachment, round-bottom 96-well plates at a density of 5×103 cells/well 24 h before virus infection. Cell viability was determined using the XTT assay on day 6 after infection with Ad-p53 or OBP-702 at the indicated multiplicity of infection (MOI). Cell viability was calculated relative to that of mock infected cells, the viability of which was set at 1.0. Data are expressed as mean±SD (n=5). *p<0.05; **p<0.01 (vs. 0 MOI). B) Distribution of green-colored live and red-colored dead cells within GC spheres was determined using the live and dead assay on day 6 after infection with Ad-p53 or OBP-702 at the indicated MOI. C) Apoptosis induction was determined using CellEvent™ Caspase-3/7 Green reagent on day 6 after infection with Ad-p53 or OBP-702 at the indicated MOI. Scale bars: 200 μm.
The distribution of virus-induced apoptotic cells in GC tumor spheres was analyzed using the activated caspase-3 assay on day 6 after virus infection. Treatment with Ad-p53 resulted in the appearance of green-colored apoptotic cells within the central area of GCIY and KATOIII spheres but not NUGC-4 spheres (Figure 4C). Furthermore, treatment with OBP-702 resulted in the appearance of green-colored apoptotic cells within the central area of all GC spheres (Figure 4C). These results suggest that OBP-702 exhibits more potent cytopathic activity against diffuse-type GC spheres than Ad-p53.
Antitumor effect of OBP-702 against peritoneal metastasis of p53-intact NUGC-4 cells. A xenograft peritoneal metastasis model involving p53-intact NUGC-4 cells was used to investigate the therapeutic potential of Ad-p53 and OBP-702 in peritoneal metastasis of diffuse-type GC cells. RFP-expressing NUGC-4–RFP cells were inoculated intraperitoneally into mice, and after 14 days, the mice were injected intraperitoneally with Ad-p53 or OBP-702 every other day for three times (Figure 5A). Intraperitoneal administration of OBP-702, but not Ad-p53, significantly suppressed the intensity of RFP in the peritoneal metastasis of NUGC-4 cells (Figure 5B and C). The incidence of hemorrhage ascites was decreased by administration of OBP-702 but not Ad-p53 (Figure 5D). These results suggest that intraperitoneal administration of OBP-702 has therapeutic potential to suppress the peritoneal metastasis of p53-intact diffuse-type GC cells.
Antitumor effect of Ad-p53 and OBP-702 against peritoneal metastasis of p53-intact NUGC-4 cells. A) NUGC4-RFP cells (5×106 cells) were inoculated into the peritoneal cavity of 7-week-old female athymic nude mice. Fourteen days after tumor inoculation, mice were treated with mock control (PBS) (n=9), Ad-p53 (108 PFU/mouse) (n=7), or OBP-702 (108 PFU/mouse) (n=9) every other day for three times. Twenty-eight days after inoculation, red fluorescence photographs were taken under a fluorescent microscope from 7-9 mice in each group after laparotomy. B) Macroscopic appearance and fluorescence images of the abdominal cavity at 28 days after inoculation. Scale bars: 10 mm. C) Relative fluorescence intensity of peritoneally disseminated tumors in mice treated with mock control (PBS), Ad-p53, or OBP-702. Data are expressed as mean±SD (n=7-9). *p<0.05. D) Frequency of hemorrhagic ascites in mice with peritoneally metastatic NUGC-4-RFP tumors after treatment with Ad-p53 or OBP-702.
Antitumor effect of OBP-702 against peritoneal metastasis of p53-mutant GCIY cells. Finally, a xenograft peritoneal metastasis model with luciferase-expressing GCIY-Luc cells was used to evaluate the therapeutic potential of Ad-p53 and OBP-702 in terms of the survival of mice with peritoneal metastasis of diffuse-type GC cells. Mice were injected intraperitoneally with GCIY-Luc cells, and after 14 days, the mice were injected intraperitoneally with Ad-p53 or OBP-702 every week for 3 weeks (Figure 6A). IVIS assay demonstrated that OBP-702 significantly suppressed the peritoneal metastasis of GCIY-Luc cells on day 48 after initial treatment compared to administration of PBS or Ad-p53 (Figure 6B and C). The survival time of mice with intraperitoneal metastasis of GCIY-Luc cells was significantly increased by treatment with OBP-702 compared with Ad-p53 treatment (Figure 6D). These results suggest that intraperitoneal administration of OBP-702 has therapeutic potential to suppress the peritoneal metastasis of p53-inactivated diffuse-type GC cells in mice, resulting in prolonged survival.
Antitumor effect of Ad-p53 and OBP-702 against peritoneal metastasis of p53-mutant GCIY cells. A) GCIY-Luc cells (2×106 cells) were inoculated into the peritoneal cavity of 7-week-old female athymic nude mice. Fourteen days after tumor inoculation, mice were treated with mock control (PBS), Ad-p53 (108 PFU/mouse), or OBP-702 (108 PFU/mouse) every week for 3 times. Luminescence in peritoneal tumors was analyzed using the IVIS system on days 13, 20, 27, 34, 41, and 48 after tumor inoculation. B) Photographs of mice on days 13, 20, 27, 34, 41, and 48 after tumor inoculation. C) Luminescence in peritoneal tumors was analyzed using the IVIS system on days 13, 20, 27, 34, 41, and 48 after tumor inoculation. D) Survival rate in each group of mice bearing peritoneal GCIY-Luc tumors (n=8 in each group) determined using the Kaplan–Meier method. Statistical significance was determined using the log-rank test.
Discussion
Overcoming peritoneal metastasis is the most critical obstacle in improving the poor prognosis of patients with GC (5). However, the therapeutic efficacy of systemic chemotherapy and intraperitoneal chemotherapy for GC in patients with peritoneal metastasis is unsatisfactory (6). Therefore, novel therapeutic strategies for inhibiting the peritoneal metastasis of diffuse-type GC cells are needed. In this study, we investigated the in vitro and in vivo therapeutic potential of p53-expressing adenoviral vectors, replication-deficient Ad-p53 and telomerase-specific, replication-competent OBP-702, against diffuse-type GC cells. Ad-p53 significantly suppressed the viability of p53-inactivated GCIY and KATOIII cells but not p53-intact NUGC-4 cells. By contrast, OBP-702 significantly suppressed the viability of all GC cells more strongly than Ad-p53. Up-regulation of p53, induction of apoptosis and autophagy, and down-regulation of RTK were involved in the virus-mediated cytopathic activity. OBP-702 significantly suppressed the viability of sphere-forming GC cells more strongly than Ad-p53. Intraperitoneal administration of OBP-702 (but not Ad-p53) significantly suppressed the peritoneal metastasis of p53-intact NUGC-4 and p53-inactivated GCIY cells. Thus, OBP-702–mediated p53 gene therapy appears to be a promising antitumor strategy for the treatment of peritoneal metastasis of diffuse-type GC cells with different p53 status.
Diffuse-type GC tumors frequently exhibit p53 inactivation due to genetic alterations or chromosomal abnormalities (7, 8). As molecular classification of GC tumors demonstrated worse prognosis for p53-inactivated GC compared with p53-intact GC (8), targeting p53-mutant GC cells represents a potentially beneficial therapeutic approach for suppressing GC progression. In this study, p53-mutant type GCIY cells were sensitive to the cytopathic activity of Ad-p53 and OBP-702 (Figure 2). However, the peritoneal metastasis of GCIY cells was significantly suppressed by intraperitoneal administration of OBP-702, but not by administration of Ad-p53 (Figure 6). We previously reported that OBP-702 activates p53 and induces apoptosis and autophagy more potently than Ad-p53 in human cancer cells via E1-mediated suppression of p21, which normally suppresses apoptosis and autophagy (18, 19). Thus, OBP-702–mediated p53 gene therapy is a promising antitumor approach for suppressing the peritoneal metastasis of p53-mutant GC cells via strong induction of apoptosis and autophagy.
A recent report showed that metastatic GC cells established from malignant ascites exhibit activation of RTKs; thus, suppression of RTKs could be a beneficial therapeutic approach (24). OBP-702 and Ad-p53 suppressed the expression of EGFR and c-Met in GC cells (Figure 3), suggesting the involvement of p53 in the down-regulation of EGFR and c-Met. Bhede et al. demonstrated that p53 inhibits EGFR promoter activity in normal human keratinocytes (25). Hwang et al. showed that p53 negatively regulates c-Met expression in human ovarian epithelial cells by activating c-MET–targeting microRNA (miRNA)-34 and inhibiting c-Met promoter activity (26). We also revealed that OBP-301 inhibits EGFR expression via adenoviral E1-mediated activation of EGFR-targeting miRNA-7 in human lung cancer cells (27). In this study, OBP-702 and OBP-301 (but not E1-deleted Ad-p53) suppressed FGFR2 expression in KATOIII cells (Figure 3). Suppression of RTKs in GC cells may be associated with OBP-702–mediated activation of p53 and adenoviral E1.
Three-dimensional cell culture techniques can be used to mimic the tumor microenvironment as a preclinical model of oncolytic virotherapy (28). OBP-702 significantly suppressed the viability of sphere-forming GC cells in three-dimensional culture (Figure 4A). Peritoneal metastasis of GC can be promoted by various tumor microenvironment factors, including cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) (29). Zhao et al. showed that the proliferation and peritoneal metastasis of NUGC-4 and GCIY cells were enhanced by exposure to hepatocyte growth factor secreted from CAFs (30). Yasumoto et al. demonstrated that the EGFR ligands secreted by TAMs promote the peritoneal metastasis of NUGC-4 cells (31). Multicellular spheroids containing CAFs and TAMs may be a more suitable model to evaluate the therapeutic potential of Ad-p53 and OBP-702 against sphere-forming GC cells.
Previous reports have shown that p53–wild type MKN-45 cells are resistant to Ad-p53 in vitro and in vivo (11, 13). Consistent with previous reports, p53–wild type NUGC-4 cells were resistant to Ad-p53 due to the lack of p53 activation (Figure 2). Regarding the underlying mechanism of impaired p53 activation in p53–wild type GC cells, high expression of murine double minute (MDM)2 and MDM4 is expected to negatively regulate p53 expression induced by chemotherapy via the proteasomal degradation of p53 (32). Another study showed that p53–wild type human cancer cells, including NUGC-4 cells, frequently express MDM2 and MDM4 (33). Zhang et al. demonstrated that over-expression of MDM4 in NUGC-4 cells enhances their resistance to the chemotherapeutic drugs 5-fluouracil (5-FU) and oxaliplatin (34). In contrast, Imanishi et al. showed that down-regulation of MDM2 and MDM4 via siRNAs promotes 5-FU–induced p53 activation in NUGC-4 cells (35). Hirose et al. also demonstrated that knockdown of MDM2 and MDM4 synergistically reactivates p53 expression in NUGC-4 cells (33). We previously demonstrated that OBP-702 suppresses MDM2 expression and subsequently induces marked p53 expression in tumor cells (18). Thus, suppression of MDM2 and MDM4 may be involved in the antitumor efficacy of OBP-702 against NUGC-4 cells via activation of p53 expression.
In conclusion, we demonstrated that a telomerase-specific, replication-competent oncolytic adenovirus, OBP-702, inhibits the peritoneal metastasis of diffuse-type GC cells more strongly than Ad-p53. This inhibition involves p53-mediated induction of apoptosis and autophagy and suppression of RTKs. Thus, p53 gene therapy mediated by intraperitoneally administered OBP-702 represents a novel antitumor strategy to treat the peritoneal metastasis of diffuse-type GC cells.
Acknowledgements
The Authors thank Tomoko Sueishi, Yuko Hoshijima, and Tae Yamanishi for their excellent technical support.
Footnotes
Authors’ Contributions
Conception and design: H.T., S.Ka., T.F.; development of methodology: N.H., Y.L., T.Ok.; acquisition of data: N.H., Y.L., T.Ok.; analysis and interpretation: N.H., Y.L., T.Ok.; writing, review, and/or revision of the manuscript: N.H., H.T., T.F.; administrative, technical, or material support: Y.U.; study supervision: H.T., S.Ki., S.Ku, T.Oh., K.N., M.N., S.Ka., T.F.
Conflicts of Interest
Y.U. is the President & CEO of Oncolys BioPharma Inc. H.T. and T.F. are consultants of Oncolys BioPharma Inc. The other Authors have no potential conflicts of interest to disclose in relation to this study.
Funding
This study was supported in part by grants from the Japan Agency for Medical Research and Development (17ck0106285h0001 and 20ck0106569h0001 to T.F.) and JSPS KAKENHI grants JP16K10596 to H.T., JP16K21185 and JP18K16362 to S.Ki., and JP26461978 to M.N.
- Received September 15, 2023.
- Revision received October 8, 2023.
- Accepted October 9, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
