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Objective To analyze the type and gene features of 11 unverified HAdVs in China. Methods We downloaded HAdV genome sequences of 67 strains with clear genotypes and the HAdV reference genome sequences of 7 strains from GenBank database, analyzed them together with eleven unverified HAdV gene sequences and classified the species of each sequence based on the genetic structure, sequence similarity and phylogenetic analysis. Results Phylogenetic analysis results predicted the classification of six sequences: two of them belonged to HAdV-B and two belonged to HAdV-D (the gene homology were 97.8% and 92.8% in comparison with reference gene respectively), the rest of two sequences were not conclusive. Multiple sequence alignment was used to predict the species of five HAdVs. All of them belonged to species HAdV-B, the sequence similarity compared with reference gene was 97.9%. Conclusion Genotyping of the unverified HAdV genome from a genetic point of view predicts that seven strains might belong to species HAdV-B, two strains might belong to species HAdV-D and two strains could not be classified. The seven HAdV-B strains might be suitable for the treatment of osteosarcoma as oncolytic adenovirus.
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@#[Abstract] Objective: To explore the anti-tumor activity of oncolytic adenovirus co-expressing lymphocyte activation gene 3 (LAG-3) antibody (aLAG) against glioblastoma. Methods: aLAG sequence was inserted into the skeleton of oncolytic adenovirus Ad3 to obtain recombinant oncolytic adenovirus (Ad3-aLAG). The expression of aLAG in infected gliblastoma GL261 cells was detected by WB. The cytotoxicity of recombinant oncolytic adenovirus against glioblastoma was detected by MTT method. The tumor inhibitory activity of recombinant oncolytic adenovirus against glioblastoma in vivo was evaluated with mice subcutaneous xenograft model. Tumor infiltrating T cells were detected by immunohistochemical staining, and the levels of cytokines TNF-α and IFN-γ secreted by tumor infiltrating T cells were detected by Flow cytometry. Results: The recombinant oncolytic adenovirus was successfully constructed, which could effectively express aLAG and kill GL261 cells in vitro (all P<0.01). Experimental results of mice subcutaneous xenograft model showed that the tumor inhibition ability of recombinant oncolytic adenovirus Ad3-aLAG was stronger than that of Ad3 oncolytic adenovirus (P<0.01), and Ad3-aLAG could effectively enhance the infiltration of CD3+ T cells in tumor tissue (P<0.01) and enhance the IFN-γ secretion ability of infiltrating T cells (P<0.01). Conclusion: Ad3-aLAG recombinant oncolytic adenovirus can significantly inhibit the growth of glioblastoma cells in vivo and in vitro, and enhance the anti-tumor immune response in vivo, which is promising to provide a new scheme for the treatment of glioblastoma.
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Objective: To evaluate the inhibitory role of a novel oncolytic adenovirus (OA), GP73-SphK1sR-Ad5, on the growth of hepatocellular carcinoma (HCC). Methods: GP73-SphK1sR-Ad5 was constructed by integrating Golgi protein 73 (GP73) promoter and sphingosine kinase 1 (SphK1)-short hairpin RNA (shRNA) into adenovirus serotype 5 (Ad5), and transfecting into HCC Huh7 cells and normal human liver HL-7702 cells. The expression of SphK1 and adenovirus early region 1 (E1A) was detected by quantitative real-time PCR (qRT-PCR) and western blot, respectively. Cell viability was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and apoptotic rate was determined by flow cytometry. An Huh7 xenograft model was established in mice injected intratumorally with GP73-SphK1sR-Ad5. Twenty days after injection, the tumor volume and weight, and the survival time of the mice were recorded. The histopathological changes in tumor tissues were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Results: Transfection of GP73-SphK1sR-Ad5 significantly upregulated E1A and downregulated SphK1 in Huh7 cells, but not in HL7702 cells. GP73-SphK1sR-Ad5 transfection significantly decreased the viability and increased the apoptotic rate of Huh7 cells, but had no effect on HL7702 cells. Intratumoral injection of GP73-SphK1sR-Ad5 into the Huh7 xenograft mouse model significantly decreased tumor volume and weight, and prolonged survival time. It also significantly decreased the tumor infiltration area and blood vessel density, and increased the percentages of cells with nucleus deformation and cells with condensed chromatin in tumor tissues. Conclusions: GP73-SphK1sR-Ad5 serves as a novel OA and can inhibit HCC progression with high specificity and efficacy.
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@#Objective: To explore the difference in the proliferation inhibition of doxorubicin and dual specific oncolytic adenoviruses (Ad-VT, Ad-T, Ad-VP3 and d-Mock) on breast cancer cells and normal mammary cells. Methods: The proliferation inhibition rates of doxorubicin and recombinant adenovirus(Ad-VT, Ad-T, Ad-VP3and Mock) on breast cancer cells were detected through WST-1 experiment, and the effects of two drugs on the inhibitory rates of normal mammary epithelial cells were also detected. Moreover, the apoptosis rates of doxorubicin and oncolytic adenoviruses on breast cancer cells and normal mammary epithelial cells were evaluated by Annexin V flow cytometry, Hoechst and JC-1 staining, and the difference in the apoptosis rates were also compared. Results: All the recombinant adenovirus could effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), the inhibition effects followed the order ofAd-VT>Ad-T>Ad-VP3>Ad-MOCK, and the inhibition effect was positively correlated with time. Doxorubicin could also effectively suppress the proliferation of breast cancer cells (P<0.05 or P<0.01), and the inhibition effect was markedly enhanced with the increases in does and time. However, doxorubicin also showed strong inhibition effect on the normal mammary epithelial cells, and the inhibition rate achieved 80% under 72 h and 5 ug/ml doxorubicin, while that of oncolytic adenovirus Ad-VT on MCF-10A was 20% at 72 h. The apoptosis effects of oncolytic adenoviruses-induced breast cancer cellwere increased with time, and the apoptosis rate efficiency followed the order of Ad-VT>Ad-T>Ad-VP3>Ad-MOCK, but they displayed low ability to induce normal mammary cell apoptosis. The apoptosis effects of doxorubicin-induced breast cancer cell were similar to that of the normal mammary epithelial cell (P <0.05 or P<0.01), which followed the dose of 0.05<0.5<5 μg/ml. Conclusion: Dual specific oncolytic adenoviruses can effectively suppress the proliferation of breast cancer cells, but they have low inhibition on normal mammary cells, which have displayed superior safety and provide a new method for the biotherapy of tumor.
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@# Objective: To investigate the inhibitory effect of recombinant oncolytic adenovirusAd-Apoptin-hTERTp-E1A(ATV) on luciferase-labeled human lung cancer cells (A549-luc) and human lung cancerA549 cells, and to compare the differences in the inhibitory effect on two cell lines. Methods:ATV was used to infectA549-luc cells andA549 cells respectively. WST-1 and crystal violet staining were used to determine the difference in the inhibitory effect of ATV. Hoechst and Annexin V-FITC/PI staining were used to verify the inhibition mode ofATV. Results: WST-1 and crystal violet staining showed thatATV had significant inhibitory effect on bothA549-luc and A549 cells ( P <0.05). ATV showed significant inhibitory effect on both cells at 24, 48 and 72 h ( P <0.05 or P <0.01), and reached the peak at 72 h; ATV at concentrations of 1, 10 and 100 MOI all showed inhibitory effect on both cells, and reached the peak at 100 MOI. Hoechst staining showed that A549-luc cells and A549 cells infected with ATV showed typical nuclear fragmentation and marginal set. The results of Annexin V-FITC/PI Flow cytometry showed that ATV infection resulted in apoptosis of A549-luc and A549 cells, which was in a time-dependent manner and reached the peak at 72 h( P <0.05 or P <0.01). Conclusion: Insertion of luciferase didn’t significantly change the inhibitory effect and inhibitory mode ofATV onA549-luc cells.ATV exerted its in vitro inhibitory effect onA549-luc and A549 cells by inducing cell apoptosis.
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Objective To investigate whether IL-24 can inhibit the growth of pancreatic cancer in immune-competent mice,which could be mediated by tumor-targeting oncolytic adenovirus (ZD55-IL-24).Methods Crystal violet staining assay was used to evaluate the anti-proliferative influence of ZD55-IL-24 on Panc02.The tumor growth curve,the tumor formation curve,TUNEL assay,cytotoxic T lymphocyte (CTL) killing assay and serum cytokines detection were used to assess anti-tumor efficacy of ZD55-IL-24 for pancreatic cancer in immune-competent mice.Results ZD55-IL-24 significantly inhibited the growth of pancreatic cancer cells in vitro.Lower tumor formation and slower tumor growth were identified in ZD55-IL-24 group,compared with ZD55-EGFP group or PBS group.ZD55-IL-24 induced pancreatic cancer cells'apoptosis,and mediated anti-cancer immunity by increasing killing responses of CTL to cancer cells and up-regulating serum γ-IFN and IL-6 levels.Conclusion ZD55-IL-24 can induce anti-tumor immunity and suppress pancreatic cancer growth in immune-competent mice.
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Oncolytic adenoviruses have been used in cancer gene therapy because of their ability to selectively transfect and repli-cate in tumor cells. However, systemically administering oncolytic adenoviruses is often inefficient because the host's immunity also at-tacks the viruses. Delivering oncolytic adenoviruses by loading them into mesenchymal stem cells effectively solves this problem. Mes-enchymal stem cells have been described to possess a tumor-homing ability, as well as the capacity to deliver oncolytic adenoviruses. This review discusses the current challenges faced by oncolytic adenovirus delivery via mesenchymal stem cells.
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Objective To evaluate the ability to kill human urothelial carcinoma T24 cells selectively in vitro by celecoxib combined with ZD55-IL-24 and explore the effectiveness for this combination use.Methods The EGFP expression of cells was observed by fluorescence microscope.The human umbilical vein endothelial cells (HUVEC) were used as crotrols.The expression of IL-24 mRNA was detected by RT-PCRwhen the cells were transfected by ZD55-EGFP or ZD55-IL-24.After transfected by ZD55-IL-24 and treated by celecoxib,the inhibition effect on cells was measured by MTT assay,and the apoptosis rate was examined by flow cytometry.Results The fluorescence in T24 and HUVEC can be observed 24h after ZD55-EGFP transfection and the fluorescence intensity was increased corresponding with the times.Fluorescence intensity in T24 cells showed higher than that in HUVEC group at the same times.The result of RT-PCR showed that the T24 cells expressed higher level of IL-24 mRNA than HUVEC group at the same time when the cells were transfect by ZD55-IL-24 (P ＜ 0.01).The inhibition rate of ZD55-IL-24 combined with celecoxib group was significantly higher than other groups (P ＜ 0.001).The inhibition rate of T24 cells in each group was significantly higher than HUVEC group (P ＜ 0.01).The flow cytometry results indicated that celecoxib combined with ZD55-IL-24 had the highest apoptosis rate on T24 cells than other single use group.Apoptosis rate of T24 cells showed a higher than HUVEC cells (P ＜ 0.01).Conclusion Celecoxib combined with ZD55-IL-24 can inhibit T24 cells proliferation at a greatest degree and this effect may be contributed to apoptosis.
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Objective To construct an oncolytic adenovirus CNHK600-IL24, and to observe the in vivo effects of CNHK600-IL24 in treating breast cancer. Methods The IL-24 gene was cloned into adenovirus shuttle vector SG502-△CR2, and CNHK600-IL24 was obtained by cotransfection of SG502-INSIL24 and pPE3 plasmids and subsequent recombination in 293 cells. Based on the establishment of the athymic mice model of breast cancer in situ and imitated metastatic breast cancer by injection in the vena caudalis and the left artrium, we administered the virus by the tail vein. We used the optical imaging in vivo system to monitor the effects. Results The oncolytic adenovirus CNHK600-IL24 was correctly constructed and confirmed by restriction DNA sequence analysis and PCR. The titer of CNHK600-IL24 reached 1.9 ×1010pfu/ml. Establishing athymic mice model of breast cancer in situ, the volume and photon number of the tumors in the control group was significantly larger than those of the CNHK600-IL24 group( P ＜0. 05). The tumor had conspicuous necrosis after the treatment of CNHK600-IL24. There was noticeable apoptosis of the tumor cells. Immunohistochemistry showed the expression of IL-24 and the Hexon protein in the tumor cells.In athymic mice model of imitated metastatic breast cancer by infusion into the vena caudalis, most of the mice in the control group died before 38 days, the mice of the CNHK600-IL24 group survived significantly longer(P ＜0. 05 ). Using athymic mice model of imitated metastatic breast cancer by infusion in the left artrium, the optical imaging in vivo system showed obvious difference between the control group and the CNHK600-IL24 group. Conclusions The high-titer oncolytic adenovirus CNHK600-IL24 was successfully constructed and purified. The oncolytic adenovirus had obvious antitumor effect on breast cancer.