| BackgroundPancreatic cancer is a malignant tumor with very high mortality rates and the5-year survival rate for pancreatic cancer is less than5%. It is one of the most malignant gastrointestinal tumors with poor prognosis. More than80%of pancreatic cancers are advanced cancer at initial diagnosis with invasion of blood vessel or metastasis to distant organs. Pancreatic cancer is resistant to radiotherapy or chemotherapy due to its biological characteristics. Thus, new combinational strategies or methods to improve the sensitivity to radiotherapy and chemotherapy are needed to prolong the survival time and improve the prognosis of patients with pancreatic cancer.Oncolytic virus therapy, a kind of new cancer treatment, is newly arisen. Various types of oncolytic viruses have been evaluated in pre-clinical and clinical settings with promising results. Oncolytic vaccinia virus GLV-1hl51is the latest developed by America Genelux. With inserted into a fluorescent protein marker RUC-GFP gene, removed the TK (thymidine kinase) gene and virulence gene of vaccinia virus, GLV-1hl51is safe and specific replication in tumor cells and has good prospects for clinical application.In this study, oncolytic virus GLV-1h151combined with radiotherapy for intervention on pancreatic cancer in vitro and in vivo was performed to observe the effect of intervention, and to explore its molecular mechanism, providing a theoretical basis for the clinical application in the future.Objectives1) To observe the killing effect of oncolytic virus GLV-1h151combined with radiotherapy on pancreatic cancer cells;2) To explore the molecular mechanism of killing effects of GLV-1h151combined with radiotherapy on pancreatic cancer;3) In vivo expriment further confirmed the killing effect of GLV-1h151combined with radiotherapy on pancreatic cancer.Methods1) Virus with various titers was transfected into human pancreatic cancer cell lines. Expression of green fluorescent protein was confirmed in fluorescence microscopy daily to observe the replication of GLV-1h151in pancreatic cancer cells.2) Cells were treated with media alone (control wells), radiation alone, GLV-1h151alone, or combining radiation and GLV-1h151. Viral cytotoxicity was measured at day0,1,3,5and7. Cytotoxicity data obtained from the experiments were analyzed using CompuSyn software. The combination index (CI) and dose-reduction index (DRI) were calculated to explore the synergistic effect produced by GLV-1h151combined with radiotherapy.3) Viral proliferation assays were performed to assess the impact of radiation on viral replication.4) DNA-binding dye Hoechst33258was performed to image of cell viability and chromatin condensation. Flow cytometry was performed to measure the cells apoptosis in different groups.5) Propidium iodide was performed to measure cell cycle, especially the distribution of G2/M in different groups.6) Two-dimensional gel electrophoresis and mass spectrometry analysis were performed to screen the proteins which were expressed differently in different groups, and further identified the protein that regulated cell apoptosis and cell cycle. Western Blot was performed to confirmed the proteomics results7) Female BALB/c nude mice bearing bilateral tumor xenografts were divided into the following groups:untreated control; radiation alone; GLV-1h151alone and combination treatment with radiation, and to monitor the tumor growth, including tumor growth rate, tumor doubling time and mice body weight. Histological examinations were performed to observe the tissue necrosis in different groups. The virus titer was performed to further verify the results of the experiments in vitro. Immunohistochemistry were performed to explore the expression level of the screening proteins in different groups.Results1) GLV-1h151infected and replicated in all four pancreatic cancer cell lines. All four cell lines showed increasing marker gene expression in parallel with higher virus titers on Day3post-treatment, indicating that oncolytic virus GLV-1h151was transfected into human pancreatic cancer cell lines, and successfully expressed the target gene.2) In single treatment, the four PDAC cell lines showed susceptibility to radiation and GLV-1h151in a dose-dependent fashion. In the combination group, the number of cell survival was significantly less than the single treatment group at the same time. The interactions between the two agents were evaluated by calculating ChouTalalay combination indices (CIs) using the CompuSyn software. For the AsPC-1cell line, the combination treatment yielded a strong synergistic effect at concentrations ranging from IC10to IC97, and synergism was particularly prominent at high effect levels. For MIA PaCa-2and PANC-1cell lines, there was a mild to moderate synergistic effect.3) Viral titers were measured by standard plaque assays on Vero cell monolayers. Radiotherapy has no effect on oncolytic virus replication. There is no significant difference between the radiotherapy group and control group for viral replication, suggesting that the synergistic effect was not caused by enhanced viral replication.4) In contrast to single agent-treated cells, cells stained with Hoechst33342showed a higher level of cell morphological changes in the combination treatment group, such as chromatin condensation, nuclear fragmentation and widespread cell death.5) Flow cytometry was performed to test the quantification of the degree of apoptosis at24h and48h. For AsPC-1, the apoptosis rate was significantly more in combined treatment group than that in single treatment group (0.47±0.07vs.0.16±0.10vs.0.31±0.04, P<0.05;0.54±0.04vs.0.19±0.02vs.0.40±0.04, P<0.05). Similar results were observed in MIAPaCa-2and PANC-1.6) The effect of GLV-1h151infections and/or irradiation on cell cycle distribution was investigated by means of FACS analysis with PI staining. G2/M population in control, single radiation, single virus and combination group was0.10±0.02vs.0.77±0.01vs.0.08±0.02vs.0.24±0.01;0.21±0.04vs.0.78±0.04vs.0.09±0.02vs.0.06±0.02respectively. Number of phase G2/M was significantly lower in combination group than single radiation group. Similar results were observed in MIAPaCa-2and PANC-1.7) Two-dimensional gel electrophoresis and mass spectrometry identify the protein that expressed differently between radiation group and combination group. Total20proteins were selected. In combination group,3proteins were up-regulated and the rest of17were down-regulated than in single radiation group. After the literature review, we found that the14-3-3σ played an important role in regulation of cell cycle and apoptosis. Western Blot results confirmed the proteomic results. The expression of14-3-3σ in combination group was significantly lower than the single radiation group, and with the extension of time, the protein expression was lower in24hours than12hours. In addition we found that the cyclinBl and cdc25(CDK1), which interact closely with14-3-3σ, changed accordingly. CyclinB1and CDK1expression in the combination group decreased than in single radiation group.8) Female BALB/c nude mice bearing bilateral tumor xenografts were divided into the following groups:untreated control; radiation alone; GLV-1h151alone; combination treatment with radiation plus GLV-1h151. Tumor volume was measured at baseline and then at weekly intervals. The tumor volumes in the control, GLV-1h151alone, radiation alone, and combination group differed significantly at4and5weeks after treatment. Five weeks after treatment, the mean tumor volume changed to649.0±195.8mm3from117.0±14.7mm3in mice with combination therapy compared with mice treated with GLV-1h151alone (1574.6±96.4mm3), radiation alone (1795.7±202.3mm3) or the control group (2478.6±242.9mm3). Consistent with this, tumor doubling time in the combination group (33.0±6.47days) was significantly increased compared with GLV-1h151alone (28.6±5.81days), radiation alone (15.2±4.23days), or the control group (13.5±2.31days). The therapeutic effect of using the combined protocol was statistically significant compared with either modality alone. No evidence of excessive local or systemic toxicity was observed in the mice.9) Histological examination showed that tumors in the control or radiation alone group showed little or no signs of necrosis. Less necrotic tissue was seen in the G1V-1h151alone treatment group. The tumors in the combination treatment group contained the highest level of necrotic tissue.10) Consistent with the results from in vitro experiments, replication of GLV-1h151was attenuated in the presence of radiation. Moreover, replication of viruses in4Gy dose group was significantly lower than that in2Gy dose group, indicating that increased replication of viruses can be excluded as a mechanism for the sensitization of pancreatic cancer to radiation.11) Immunohistochemistry was performed on sample harvested from mice post7day intervention to observe the expression level of14-3-3a, CyclinBl and CDK1in different groups. Results showed that expression of14-3-3σ, CyclinBl and CDK1were strong positive in radiation alone while decreased expression or no expression of14-3-3σ, CyclinB1and CDK1were observed in combination group. The results were further confirmed that expression of14-3-3σ in combination group was decreased compared with radiation alone. Consistently, the expression of its downstream regulatory protein CyclinB1/CDK1complex was also reduced in combination group.Conclusions1) Oncolytic viruses GLV-1h151can be replicated in pancreatic cancer. GLV-1h151combined with radiation have a synergistic effect on pancreatic cancer.2) Oncolytic virus GLV-1h151reduce the expression of14-3-3σ, CyclinBl and CDK1protein, which decrease pancreatic cancer cell G2/M checkpoint number induced by radiation damage. Decreased the G2/M checkpoint number lead DNA failed to be repaired before entered mitosis, and apoptosis occurs via mitotic catastrophe.3) Mice subcutaneous pancreatic cancer xenografts model further confirmed that the response of pancreatic cancer to GLV-1h151combining with radiation treatment was stronger than single treatment. The in vivo experiments also confirm that GLV-1hl51increases the sensitivity of pancreatic cancer to radiation by reducing14-3-3σ, CyclinB1/CDK1complex expression. The study provides a theoretical basis for the clinical application of GLV-1h151in future. |
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