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OBJECTIVE: To investigate apoptosis induction ability of hPP10-Apoptin fusion protein to mouse B16 melanoma cell. METHODS: pET15b-hPP10-Apoptin expression plasmid was constructed, and E. coli BL21 (DE3) was transformed with the plasmid, then the yielded hPP10-Apoptin fusion protein was purified by Ni-NTA His-Bind Resin and confirmed by Western blotting assay. Melanoma cell apoptosis induced by hPP10-Apoptin fusion protein was analyzed by TUNEL assay, and the antitumor effect was examined in melanoma cell-bear mouse model. RESULTS: hPP10-Apoptin fusion protein was highly expressed in BL21 cells, Western blotting analysis result showed that fusion protein was expressed correctly. The fusion protein can induce melanoma cell apoptosis in vivo and in vitro. CONCLUSION: The results confirm that hPP10-Apoptin fusion protein could penetrate into melanoma cell and also has antitumor effect.
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Objective To investigate the effects of PTD4-GFP-Apoptin protein on proliferation inhibition and apoptosis-inducing of different types of leukemia cells. Methods Genetic engineering was used to restructure a carrier containing PTD4-GFP-Apoptin gene, and MTT was applied to detect the expressed PTD4-GFP-Apoptin fusion protein and its effect on the leukemia cell proliferation. Flow cytometry (FCM) was used to detect the effects on cell apoptosis. Results MTT cell proliferation inhibitory experiment showed that PTD4-GFP-Apoptin had different degree of proliferation inhibition on different types of leukemia cells;furthermore, the inhibitory effect presented positive correlation with time and concentration. FCM showed that PTD4-GFP-Apoptin had apoptosis-inducing effect on HL-60 cells, and the apoptotic rate had significant difference compared with the control group (P <0.05). Conclusions PTD4 can carry large proteins to penetrate the cell membrane, and PTD4-GFP-Apoptin may produce the inhibiting proliferation in vitro for a variety of leukemia cells. Apoptin can induce tumor cell apoptosis without affecting normal cells, which might become a new agent for the clinical treatment of leukemia.
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OBJECTIVE:To explore the effects of recombinant adenovirus vector Adxsi-GFP-VP3 carrying apoptin gene VP3 on the apoptosis of human lung squamous carcinoma SK-MES-1 cell lines and human lung adenocarcinoma NCI-H1299 cell lines. METHODS:The exponential phase SK-MES-1 and NCI-H1299 cell lines were respectively divided into a recombinant adenovirus (Adxsi-GFP-VP3) group,a empty virus (Adxsi-GFP) group and a cell control (phosphate buffer) group,which were marked as group A,B and C respectively. Reverse transcription polymerase chain reaction and Western blot method were used to detect the ex-pressions of VP3 mRNA and Apoptin in the cells of groups A and B 48 and 72 h after transfection. The change in the ultrastructure of the cells in group A was observed under transmission electron microscope 72 h thereafter. MTT method was adopted to detect the cell proliferation activities of three groups 24,48,72 and 96 h thereafter and flow cytometry to determine the apoptosis rates and cell cycle changes 24,48 and 72 h thereafter. RESULTS:Compared to group B,group A demonstrated the expression of VP3 mRNA in SK-MES-1 and NCI-H1299 cell lines 48 h after transfection,and Apoptin expression and ultrastructure change for apopto-sis of SK-MES-1 and NCI-H1299 cell lines 72 h thereafter. Compared to groups B and C,group A showed lower proliferation activ-ities and higher apoptosis rates of SK-MES-1 and NCI-H1299 cell lines,which had a positive correlation with transfection time;and in the group A,there was a decrease in the proportion of the SK-MES-1 and NCI-H1299 cell lines in S phase and an increase in the proportion of those in G2/M phase,72 h after transfection. There was statistically difference (P<0.05). CONCLUSIONS:Adxsi-GFP-VP3 can effectively induce the apoptosis of SK-MES-1 and NCI-H1299 cell lines.
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Objective To investigate the inhibition effects of an hTERT-promoter-dependent oncolytic adenovirus Ad-VT that expresses apoptin on human prostatic carcinoma cell PC-3. Methods MTT assay was used to measure viability of PC-3 cell which was infected by recombinant adenovirus.The viability was measured at time points of 12,24,36,48,60,72,84 and 96 h after infection.AO/EB staining,DAPI staining,Annexin V assay were used to investigate the lethal effect and style of Ad-VT on PC-3 cell in vitro.The Caspases were measured by whole cell extraction of PC-3 cells 48hrs after infection. Results Ad-VT,Ad-VP3 and Ad-GT inhibited the proliferation of PC-3 cell in vitro.Ad-VT and Ad-GT were more effective than Ad-VP3 on cell growth,P ＜ 0.05.At 48,72,96 h time points,the inhibition effect of Ad-VT on PC-3 cell exhibited a dose related manner.When infection at MOI 100,the inhibition effect of Ad-VT on PC-3 cells exhibited time related manner.The AO/EB staining,DAPI staining,Annexin V assay,Annexin V assays and Caspase assays showed that Ad-VT inhibited the proliferation of PC-3 cells by inducing apoptosis of prostate cancer cells,Loss of cytoplasmic membrane integrity. Conclusions The hTERT-promoterdependent oncolytic adenovirus Ad-VT could effectively suppress prostate cancer cells PC-3 growth.
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AIM: To investigate the variation and significance of mRNA and protein concentration of myeloid cell leukemin-1 (Mcl-1) in apoptotic HepG2 cells induced by apoptin. METHODS: The apoptin expression vector pCDNA3.0-VP3 was transfected into HepG2 cells via liposome. Mcl-1 mRNA was analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction. The protein of apoptin, Mcl-1 and cytochrome C were detected by Western blotting. RESULTS: The VP3 gene was transfected into HepG2 cells successfully and expressed steadily. Compared to blank control, Mcl-1 mRNA and protein levels of VP3 positive cells were decreased (mRNA: 0.09%±0.00% vs 0.41%±0.14%, P<0.05; protein: 0.43%±0.01% vs 0.90%±0.04%, P<0.01). Released cytochrome C from mitochondrion was increased (0.98%±0.02% vs 0.62%±0.03%, P<0.01). CONCLUSION: In the course of the apoptosis of HepG2 cells induced by apoptin, the amount of Mcl-1 mRNA and protein is decreased, and released cytochrome C from mitochondrion is increased. The apoptosis induced by apoptin may be correlated with the down-regulation of Mcl-1 mRNA and protein.
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Objective Present study aimed to observe the effects of Apoptin gene on killing retinoblastoma HXO-RB_(44) cells and illustrates its mechanisms. Methods Human retinoblastoma cells strain, HXO-RB_(44), was cultured and passaged in RPMI 1640 medium containing bovine serum. Apoptin gene was transfected into HXO-RB_(44) cells by liposome into HXO-RB_(44)/Apoptin, and pcDNA_3 was transfected in HXO-RB_(44)/peDNA_3 group. The expression of Apoptin mRNA was detected using Reverse Transcription Polymerase Chain Reaction (RT-PCR). The expression of protein of Apoptin and p53 were detected by SABC immunohistochemistry. The growth rate of HXO-RB_(44) cells was studied by constructing the growth curve and calculated as the formula: inhibitory rate = 1-cell number in experiment group/cell number in control group x 100%. Cellular apoptosis was determined by flow cytometry. Results The RT-PCR result showed the 450 kb specific band in UXO-RB_(44)/Apoptin group and absent amplification result in HXO-RB_(44) group and HXO-RB_(44)/pcDNA_3 group. The difference in SABC-positive cell number between HXO-RB_(44)/Apoptin group and control group was statistically significant (P ＜ 0. 05). The growth of HXO-RB_(44) cells was significantly inhibited in HXO-RB_(44)/Apoptin group compared with control group (P ＜ 0.05). Apoptosis cells increased significantly. The apoptosis rate was 38. 5% . Conclusion Apoptin gene could inhibit the growth of HXO-RB_(44) cells effectively. Up-regulation of expression of p53 gene might not be one of cell apoptosis mechanisms.
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Apoptin,a chicken anemia virus-derived protein.selectively induces apoptosis in trans-formed or tumor cells but not in normal cells.The tumor specificity of apoptin is thought to be related to its cel-lular localization.The apoptin-induced apoptosis is p53-independent and can not be blocked by overexpressionof Bcl-2.But activation of Caspase-3 is essential for apoptin-induced apoptosis.These features make apoptin a potential candidate as a novel therapeutic and diagnostic tool in cancer treatment.
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Objective To explore the induction of apoptin on apoptosis in C6 glioma cells.Methods After having successfully cultivated C6 glioma cells and had an inoculation in Wistar rats,the animal models of Wistar rats bearing C6 glioma were set up.The rats were randomly divided into three groups:tumor control,chemotherapy and pvVP3 groups.Different treatment methods were performed in different groups.The ultrastructural changes and the apoptotic rates were detected with TUNEL.Results Typical changes of apoptosis in pvVP3 group were found through ultrastructural observation,while they could not be found in tumor control group.The apoptotic rate in pvVP3 group(51.11%) was higher than that in control group(28.96%)(P
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Objective To construct an Apoptin prokaryotic vector,aiming to produce antigenic fusion protein Apoptin. Methods The Apoptin gene was amplified from the template of plasmid pSSCHG/NT4-Apoptin-HA2-TAT by PCR.The Apoptin was sub-cloned into the multiple clone sites of plasmid pET-28a(+) to get the prokaryotic vector of pET-28a(+)-Apoptin,which was transformed into E.coli BL21(DE3).Expression of E.coli BL21(DE3) was induced by IPTG.The specific protein expression was detected by SDS-PAGE. Results The fusion protein was expressed with high efficiency in E.coli BL21(DE3) transformed by pET-28a(+)-Apoptin after induction with IPTG.The specific fusion protein had an apparent related molecular weight of about 17 000 ku as indicated by SDA-PAGE analysis. Conclusion The Apoptin prokaryotic expression vector with pET-28a(+)-Apoptin can effectively express Apoptin fusion protein,laying a foundation for further study of Apoptin and preparation of antibodies against Apoptin.
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Objective: To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy. Methods: Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3. 1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI. Results: Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G 2-M phase increased dramatically after Ad/apoptin(+) infection. Conclusion: Apoptin can induce cell cycle G 2-M arrest and chromatin condensation in cancer cells.
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Objective To investigate the apoptosis of human bile duct carcinoma cell induced by Apoptin gene transfection in vitro. Methods Apoptin gene was amplified by polymerase chain reaction (PCR) and then inserted into pAdtrack-CMV containing green fluorescent protein gene through T4 DNA ligase. The reconstructed plasmids were identified by enzyme digestion and sequencing. Apoptin expression vector was transfected into human bile duct carcinoma cell line QBC_(939). The apoptosis of human bile duct carcinoma cell induced by Apoptin gene was detected by TUNEL. Results Reconstructed pAdtrack-CMV containing Apoptin gene was obtained successfully. TUNEL test showed that there was significant difference in apoptosis index between Apoptin gene transfection and the control group (P
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Objective To study the therapy for xenograft human bile duct cancer in mice mediated by adenovirus containing Apoptin gene.Methods Subcutaneous human bile duct cancer was established in nude mice.Variations of tumor volume and histomorphology,side effects were observed after intratumoral injection of adenovirus containing Apoptin gene.Finally the mice were sacrificed for calculating the ratio of antitumor.Results Twelve days after treatment,the mean volume of the xenograft human bile duct cancer in the group of intratumoral injection of adenovirus containing Apoptin gene was(92.31?28.31)mm,which was reduced significantly compared with that of adenovirus infection without apoptin gene(288.86?113.13)mm and control group(344.86?113.87)mm.The ratio of antitumor was 72.10%,which was significantly higher than that in control group(11.9%).During the whole experimental course,no side effect was observed.The histological results demonstrated that the reduction of tumor growth was the result of apoptosis in bile duct cells,which was reduced by transfection of Apoptin gene.Conclusion The adenovirus vectors containing Apoptin gene may constitute a safe tool for the treatment of cholangiocarcinoma.
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Objective: To investigate the curative effects of apoptin on the multidrug-resistant model, of human osteosarcoma cell line (R-OS-732). Methods: 363 bpVP 3 gene was cloned into the vector pIRES1 and the recombinant eukaryon expression vector pIRVP3 was transfected into R-OS-732 cells with liposome. The expression of apoptin gene at transcription level was proved by RT-PCR. The pathological changes of the cells was observed by light microscope and electronmicroscope. The transfected R-OS-732 cells were collected after 48 hours. The genomic DNA extracted from the cells was observed by agarose gel electrophoresis. The apoptosis rate of the cells was analysed by flow cytometry. Results: The expression of apoptin gene at transcription level had been proved by RT-PCR. The construction changes of the two groups were obviously different under light microscope: most of R-OS-732 cells in the transfected group existed in form of being away from the bottom of the culture dish after 24 hours, in form of apoptosis after 48 hours and in form of necrosis after 72 hours; but those cells in the controlled group grow luxuriantly. And under electronmicroscope there was much change of cell nucleus between the two groups. DNA ladder of the transfected cells was observed through agarose gel electrophoresis only one band was observed in the controlled group; but bands of fragmented DNA observed in ositive control group. Flow cytometry analysis showed that the apoptosis rate of the cells in the transfected group was relatively higher than that of the controlls( P