| BackgroundGlobal incidence of melanoma has grown dramatically in recent decades.Malignant melanoma is the most lethal cutaneous malignancy.Both immune checkpoint inhibitors and molecular targeted drugs have made great strides despite severe challenges.However,different from the Caucasian,Acral and mucosal types are predominant among Asian patients.Hence fewer benefits have been seen in these studies.Patients witness limited therapeutic effect,severe adverse reactions,high rates of recurrence and metastasis.It remains incredibly resistant to melanoma treatment.With the development of gene therapy,oncolytic viruses have been used more and more widely.Oncolytic viruses are the genetically modified virus which exhibits selective tumor cytotoxicity without affecting normal diploid cells.In previous studies,we constructed a dual cancer-specific oncolytic adenovirus Ad-apoptin-h TERTp-E1a(Ad-VT).Ad-VT based on RAPAD.I system and contained tumor specific promoter(human relomerase reverse transcriptase,h TERTp).Tumor-specific killing gene Apoptin is activated by CMV promoter and the essential for replication of adenovirus gene E1 A is activated by h TERT promoter.Ad-VT can replicate and continuously express apoptin in tumor cells,but not in normal cells.Node mice model is one of important model for studying the development and metastasis of tumors.In order to acquire tumor-related experimental data timely,we need to sacrifice the mice or remove the tumor at different point of time in traditional nude mice model and we cannot supervise the development and metastasis of tumors in real time.Living imaging technology makes it possible to observe the development and metastasis of tumor non-invasively,especially in the xenografted tumor,metastatic tumor and orthotopic tumor models constructed by luciferase-labeled tumor cells.The volume change and metastatic of tumor can be reflected directly through observing the change in position and bioluminescence intensity of tumor using living imaging technology.In this study,we try to use the luc2-type firefly luciferase gene which shows high expression and fluorescence signal intensity to construct stable luciferase-labeled human melanoma cells A375-luc and M14-luc,and to construct melanoma nude mice model for observation by living imaging technology.In order to provide the rational evidence for its clinical application,the suppression effect of Ad-VT luciferaselabeled human melanoma cells will be investigated in vitro and in vivo and the mechanism will be explored meanwhile.ObjectivesTo explore the suppressive effect and mechanism of Ad-VT on luciferase-labeled human melanoma cells in vitro and in vivo,and to provide the rational evidence for its clinical application.Method & Results1.The luciferase-labeled human melanoma A375 cell and melanoma M14 cell were constructed by transfecting with plasmid p GL4.51.These two cells were selected by using G418 selection,luciferase assays and genetic stability analysis.The results showed that the stably expressing luciferase human melanoma cells2.In order to explore whether the insertion of luciferase will affect the biological characteristics of melanoma cells,the growth curve,growth cycle,transwell migration and matrigel invasion were detected in both the constructed luciferase-labeled melanoma cells and normal melanoma cells.The results showed that the biological characteristics of melanoma cells did not be affected by luciferase insertion.3.Expression of luciferase in A375-luc and M14-luc were detected by using living imaging technology in vitro.The results showed that luciferase-labeled cell number and bioluminescence intensity have a little linear relationship.4.A375-luc and M14-luc subcutaneous tumor model in nude mice were constructed and the expression of luciferase in A375-luc and M14-luc were detected by using living imaging technology in vivo.The results showed that melanoma nude mice models for observation by living imaging technology were constructed successfully.As the tumor volume increased,the intensity of bioluminescence gradually increased.Tumor volume and bioluminescence intensity have a little linear relationship.5.Inhibitory effect of Ad-VT on A375-luc and M14-luc was detected by MTS assay.The results showed that the suppression effect of Ad-VT-infected A375-luc and M14-luc was significantly higher than that of the Ad-mock-infected A375-luc and M14-luc.The inhibitory effect was dose-and time-dependent.The supression effect of Ad-VT-infected luciferase-labeled melanoma cells were basically the same as normal melanoma cells and the supression effect of Ad-VT-infected cells did not be affected obviously by luciferase insertion.6.Use scratch test,transwell migration and matrigel invasion assay to detect the migration and matrigel ability of Ad-VT-infected A375-luc and M14-luc.These results showed that the migration and matrigel ability of A375-luc and M14-luc was significantly affected by Ad-VT compared with the control groups.7.In order to explore the inhibitory pathway and mechanism of Ad-VT on A375-luc and M14-luc,the Ad-VT infected cells were detected by Hoechst staining,Annexin V-FITC/PI flow cytometry,JC-1 staining and Western-blot detection of mitochondrial apoptosis pathway related proteins.Use scratch test,transwell migration and matrigel invasion assay to detect the migration and matrigel ability of Ad-VT-infected A375-luc and M14-luc.These results showed that the migration and matrigel ability of A375-luc and M14-luc was significantly affected by Ad-VT compared with the control groups.The results showed that apoptosis was induced in Ad-VT infected A375-luc and M14-luc by a change in the mitochondrial membrane potential and inducing the expression of mitochondrial apoptosis pathway related proteins(AIF,ARTS and Cyto-C).The inhibition pathway and rate of apoptosis were almost the same in Ad-VT infected luciferase-labeled melanoma cells and normal melanoma cells and the inhibition pathway of Ad-VT-infected cells did not be affected obviously by luciferase insertion.8.The subcutaneous tumor model in nude mice was established by using A375-luc and M14-luc.The suppression effect of Ad-VT on A375-luc and M14-luc in vivo was evaluated by measuring tumor growing trend,survival rate and Meanwhile the bioluminescence intensity of tumor region was detected by living imaging technology.The results showed that the average bioluminescence intensity and tumor growth were significantly reduced by Ad-VT compared with the control groups.The average survival rate was prolonged by Ad-VT.9.Immunohistochemical detection of mitochondrial apoptosis pathway related proteins was repeated in vivo for the differential expressed ones,to explore the mechanisms of anti-tumor effect.Collectively,these results showed that the mitochondria apoptosis pathway related proteins can be significantly affected by Ad-VT.Likewise,Ad-VT may significantly enhance the expression of KI67,and induces the expression of Tunel,AIF,ARTS,and Cyto-C.ConclusionsThe experimental data in vitro and in vivo were showed that Ad-VT could suppress the proliferation of A375-luc and M14-luc significantly,and induce human melanoma cells apoptosis through mitochondria apoptosis pathway.The construction of melanoma subcutaneous tumor model in nude mice for observation by living imaging technology provided a research foundation for the development and metastasis of melanoma and the antitumor effect of Ad-VT in vivo.To sum up,recombinant adenovirus Ad-VT can replicate and continuously express apoptin in tumor cells,but not in normal cells.It can inhibit the growth of human melanoma cells and induce their apoptosis.In both constructed two stable luciferase-labeled human melanoma cells(A375-luc and M14-luc)and visual animal model,the antitumor effect of Ad-VT is apparent.That provides a new direction for the comprehensive treatment and mechanism of melanoma,and could be benefit for translating basic science into human therapies.Innovativeness1.Two lines of stable luciferase-labeled human melanoma cells A375-luc and M14-luc were constructed,which share similar biological characteristics with the originals.2.Subcutaneous tumor model in BALB/c nude mice mice were constructed by both A375-luc and M14-luc cells.By detected the expression of luciferase with living imaging technology,the tumorigenesis and tumor progression can be reflected simultaneously,noninvasively and continuously.3.Analyzed the suppressive effect of Ad-VT on luciferase-labeled human melanoma cells in vitro and in vivo.4.Explored the mechanism of suppressive effect of Ad-VT on luciferase-labeled human melanoma cells,and also offer a theoretical basis for clinical applications.LiminationsIn this study,the mechanism of apoptosis induced by oncolytic adenovirus Ad-VT in melanoma cells is relatively weak.In vivo and in vitro experiments have confirmed the involvement of three downstream proteins(AIF,ARTS and Cy TO-C)in mitochondria apoptosis pathway related proteins,but upstream studies on PARP-1/AIF or ROS/Cyto-C/Caspase-3 pathway need to be further explored. |
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