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Objective:To investigate the change of apoptosis-associated genes in human lung adenocarcinoma cell lines A549/DDP cells,which were induced by the recombinant human interleukin-24(rhIL-24) combined with Cisplatin (DDP). Methods: Six genes expression level by GeXP genetic analysis system at the same time,after rhIL-24,DDP and rhIL-24+DDP were used to intervene in A549/DDP cells. Results:rhIL-24 could induce Bax gene,Caspase3 gene and Rb gene transcription up regulation;Bcl-2 gene and survivin gene transcription down regulation. But no regular change in the genes expression level of P53. Bax,Survivin and Rb were more obviously changed after rhIL-24 combined with DDP. Conclusion: RhIL-24 can induce apoptosis of A549/DDP cells through upregulated the genes expression level of Bax,Caspase3,Rb and down regulated of Bcl-2,Survivin.
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Interleukin-24 (IL-24) is a member of IL-10 family, which was cloned from human melanoma cells by the method of subtractive hybridization. IL-24 can inhibit the growth of tumor cells and angiogenesis and enhance radiation sensitivity and immune adjustment with non-toxic effects on normal cells.As a kind of cytokine with multiple anti-tumor functions, IL-24 will become a new weapon of gene therapy against cancer. The application and mechanism of IL-24 in the treatment of cancer will be discussed in this paper.
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Objective To investigate the inhibiting effect of interleukin (IL)‐24 combined with targeted attenuated Salmonella typhimurium vector SL7207/pBud‐VP3 on the growth of gastric cancer cells .Methods The co‐expression eukaryotic expression plasmid pBud‐VP3‐IL‐24 was constructed .The plasmid pBud‐VP3‐IL‐24 was transformed into attenuated Salmonella typhimurium SL7207 by using the high voltage electroporation for constructing the SL 7207/pBud‐VP3‐IL‐24 strain .The mouse gastric cancer transplantation tumor model was established and randomly divided into the normal saline control group ,SL7207/pBud group , SL7207/pBud‐VP3 group and SL7207/pBud‐VP3‐IL‐24 group .The tumor‐bearing mice were fed by oral administration of bacterial strain .The tumor volume was measured and the tumor inhibition rate was calculated .The expression of IL‐24 was detected by Western blotting .The levels of IFN‐γ,IL‐6 and TNF‐αin tumor tissue were detected by using RT‐PCR .The expression of Caspase‐3 and VEGF were detected by using immunohistochemistry .Results The plasmids attenuated Salmonella typhimurium vector carrying the gene IL‐24 was successfully constructed .The IL‐24 protein expression was detected in gastric cancer tissue after 14 d treatment .The tumor volume after 28 d treatment in the SL7207/pBud‐VP3‐IL‐24 group was reduced compared with the other groups ,moreover the tumor growth was significantly inhibited ,and the differences were statistically significant (P<0 .05) .RT‐PCR and immunohistochemistry results showed that IL‐24 combined with SL7207/bBud‐VP3 could significantly increase the expression levels of immune factor IL‐6 ,IFN‐γ and TNF‐αin tumor tissue ,.in addition ,up‐regulated the expression of Caspase‐3 and down‐regulated the VEGF expression(P<0 .05) .Conclu‐sion IL‐24 combined with SL7207/pBud‐VP3 can synergically play the inhibitory effect on the growth of gastric cancer cells ,its mecha‐nism is related with the tumor apoptosis promotion ,tumor vessel inhibition and immune regulation .
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Objective To investigate the effect of adenovirus-mediated IL-24 (Ad-IL-24)gene expression on the apoptosis in a human squamous cell carcinoma cell line COLO 16, and to explore the underlying molecular mechanism. Methods Cultured COLO 16 cells were divided into two groups to be transfected with an adenovirus vector carrying the IL-24 gene (Ad-IL-24 group)or green fluorescent protein (Ad-GFP group), while those receiving no treatment served as the control group. After culture for different durations, qPCR was performed to quantify IL-24 gene expression, methyl thiazolyl tetrazolium (MTT) assay to evaluate the proliferative activity of COLO 16 cells, flow cytometry to detect the apoptosis of COLO 16 cells, laser scanning confocal microscopy (LSCM) to observe the morphological changes of COLO 16 cells, Western blot to determine the levels of Bax and Bcl-2 proteins and to evaluate the activation of caspase-3, qPCR to determine the levels of Bax and Bcl-2 mRNAs, an immunofluorescence assay to observe the expression of Bax and Bcl-2 proteins. Statistical analysis was carried out by a two-sample t-test with the SPSS 19.0 software. Results MTT assay showed that the proliferation of COLO 16 cells in the Ad-IL-24 group was significantly inhibited as early as 4 days after the transfection; thereafter, the inhibitory effect increased in a time-dependent manner, and peaked on day 6(P0.05). Flow cytometry revealed that the apoptosis rate was significantly higher in the Ad-IL-24 group(13.10%± 0.92%)than in the control group(3.69%± 0.36%, P0.05). LSCM demonstrated that the apoptosis of COLO 16 cells was accelerated in the Ad-IL-24 group. The immunofluorescence assay, Western blot and qPCR all showed that the mRNA and protein expressions of Bax were increased, but those of Bcl-2 were decreased in the Ad-IL-24 group compared with the Ad-GFP group and control group. Moreover, Western blot showed a protein band that could specifically bind to the anti-cleaved caspase-3 antibody in the Ad-IL-24 group, but not in the Ad-GFP group or control group. Conclusions Ad-IL-24 can induce apoptosis in human COLO 16 squamous cell carcinoma cells, probably by up-regulating Bax expression, down-regulating Bcl-2 expression, and activating caspase 3.
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Objective To investigate the synergistic proliferation-inhibiting and apoptosis-inducing effects of recombinant human-derived interleukin-24 (rhIL-24) and recombinant human-derived decorin (rhDCN) on human hepatocellular carcinoma cells HepG2.Methods Cellular growth and morphological changes of HepG2 cells were observed under the inverted microscope at 48 h after being transiently transfected with pcDNA3.1 (+)-IL-24 and pcDNA3.1 (+)-DCN by Lipofectamine.The proliferation-inhibiting effects of IL-24 and DCN on HepG2 cells,respectively and jointly,were observed with MTT assay at 24 h,48 h and 72 h post-transfection.Apoptosis and cell-cycle of HepG2 cells were analyzed by flow cytometry at 48 h post-transfection.Results Compared to control groups,the cells of target gene groups presented typically changes of proliferation inhibition and apoptotic morphology,which occurred obviously in co-transfection group.The results of MTT assay showed that at 48 h and 72 h post-transfection,the profiferation-inhibiting rates in the group of cells co-transfected with IL-24 and DCN were (31.88±6.57) ％ and (36.83±3.76) ％,respectively,displaying significant difference with those of other groups (P ＜ 0.01).The results of flow cytometry showed that IL-24 and DCN can induce HepG2 cells apoptosis to some extent.Compared to the early apoptosis rate of cells of control groups,plasmid (2.98±0.72) ％,blank cell (3.50±0.92) ％,IL-24 (20.01±1.08) ％ and DCN (22.20±0.91) ％,a statistically remarkable apoptosis rate,(32.56±0.90) ％,can be seen in the group of cells treated with IL-24 and DCN jointly (P ＜ 0.01).The result of cell cycle analysis revealed that,compared to control groups,the proportion of cells was higher in the phase of G2/M in the IL-24 group (11.24±0.35) ％ and in the phase of G0/G1 in the DCN group (77.93±0.67) ％.The proportions of cells in the phases of G2/M increased to (71.36±0.60) ％ and that of G0/G1 statistically increased to (10.39±0.67) ％ in the group of cells co-transfected with IL-24 and DCN (P ＜ 0.01).Conclusions Combinatorial treatment of HepG2 cells with IL-24 and DCN can exert stronger synergistic proliferation-inhibiting effect and apoptosisinducing activity-in comparison to single therapies.IL-24 and DCN can induce cell cycle arrest on HepG2 cells,occurred in the phase of G2/M and G0/G1,respectively.Promoting effect of cell cycle arrest in the phase of both G2/M and G0/G1 can be seen on HepG2 cells co-transfected with IL-24 and DCN,which maybe the possible mechanism of the synergistic proliferation-inhibiting and apoptosis-inducing effect.
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Objective:To study the effect of IL-24 on the differentiation and function of osteoclasts.Methods:Mature osteoclasts were isolated from long bones of neonate rats.Optimal multiplicity of infection(MOI)of AdCMV-EGFP was determined.Then osteo-clasts and RAW264.7 cells were transfected with AdCMV-EGFP and AdCMV-IL-24 respectively.The function of osteoblasts was studied by the observation of bone resorption lacunae,the differentiation of RAW264.7 cells was evaluated by the expression of osteo-clast related genes examined with real-time PCR.Results:Osteoclasts were TRAP positive with more than 2 neclei.MOI=400 was suitable for AdCMV-EGFP transfection.The resorption lacunae area in AdCMV-IL24 transfected cells was larger than that in AdCMV-EGFP transfected cells(P<0.05).Real-time PCR showed that under induced conditions,osteoclastic related genes NFATc1,CTSK and TRAP were changed in RAW264.7 cells transfected with AdCMV-IL-24.Conclusion:IL-24 may promote the differentiation and function of osteoclasts.
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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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PURPOSE: The melanoma differentiation-associated gene-7 (MDA-7) protein, also known as interleukin 24 (IL-24), is a novel candidate of tumor suppressor that has been found to experimentally induce apoptosis and growth inhibition in a variety of human malignant cells. However, there have been few studies about its role in lung adenocarcinoma. Even at the same stage and with similar pathologic characteristics, lung adenocarcinomas with a diameter of 3 cm or less can have a variable prognosis depending on their biologic characteristics. The purpose of this study is to define the relationship between MDA-7/IL-24 expression and the progression of small-sized lung adenocarcinomas. MATERIALS AND METHODS: We performed immunohistochemical detection of MDA-7/IL-24 in forty-seven tissue samples from primary lung adenocarcinomas of 2 cm or < or =3 cm in diameter. A statistically significant association was found between MDA-7/IL-24 expression and tumor size (p=0.03). Although this difference did not reach statistical significance, tumors with a negative MDA-7/IL-24 expression tended to more frequently show lymph node metastasis (p=0.07). There were no significant associations for other clinicopathologic characteristics. CONCLUSION: These results suggest the possible involvement of MDA-7/IL-24 in the growth and progression of small-sized lung adenocarcinoma. MDA-7/IL-24 immunoreactivity could be used to identify a subset of adenocarcinomas of the lung of 3 cm or less in diameter that have different biologic behavior.
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Humans , Adenocarcinoma , Apoptosis , Immunohistochemistry , Interleukins , Lung , Lung Neoplasms , Lymph Nodes , Melanoma , Neoplasm Metastasis , Population Characteristics , PrognosisABSTRACT
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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Studies have shown that IL-24 is the only cytokine capable of inhibiting tumor growth and neovascularization as well as stimulating immune system. IL-24 exerts its inhibition effects through pathways independent of p53, Rb, p16 and other tumor suppressor genes and has no effect on normal cells. IL-24 has emerged as a hot topic in cancer therapy research.
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Objective: To construct an E1 A-deleted 24-bp triple regulated replicative adenovirus vector SG600/interleukin24 (IL24), which was driven by both hTERT promoter and HRE promoter. The level of IL24 in liver cancer cells was determined and the replication capacity of SG600/ IL24 and its killing effects on liver cancer cells were observed. Methods: SG600-IL24 vector was constructed using DNA cloning and recombination techniques. The IL24 gene expression in liver cancer cell lines SMMC-7721 and BEL-7404 and normal cell line BJ was detected by ELISA assay. The replications of SG600/IL-24 in different cell lines were determined by evaluating TCID50 (50% tissue culture infectious dose) at 49 and 96 h. In vitro cell-killing effects of SG600/IL24 on the three liver cancer cell lines were analyzed by MTT assay and CPE (cytopathic effect) staining method at different MOI values. Results: IL24 was over-expressed in both SMMC-7721 and BEL-7404 cells but was weakly expressed in BJ cells. At 48 and 96 h post infection the replication of SG600/IL-24 were 794 and 7940 folds in SMMC-7721 cells; 622 and 7 810 folds in BEL-7404 cells; 20 and 200 folds in BJ cells. MTT assay showed that the MOI values of SG600/IL24 for killing 50% and 90% cells were 0.3 and 5 for SMMC-7721 cells; 3 and 20 for BEL-7404 cells; 50 and 150 for BJ cells. CPE staining demonstrated that SG600/IL24 had significant killing effects on both liver cancer cells SMMC-7721 and BEL-7404 but had no significant influence on BJ cells. The cell-killing capability of SG600/IL24 was superior than that of replicative adenovirus ZD55/IL24 and non replicative adenovirus Ad-IL24. Conclusion: After SMMC-7721 and BEL-7404 liver cancer cells are infected with SG600/1124 at high efficiency, the virus replication is active and the expression of IL24 increases greatly. SG600/IL24 has specific cell-killing effects on the two liver cancer cell lines but has no significant influence on normal cells. This study provides a basis for further investigating the effect of SG600/IL24 on liver cancer in vivo.
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Objective To construct a recombinant expression vector of human interleukin-24(hIL-24) gene and express it in E.coli M15,and to evaluate the bioactivity of IL-24 fusion protein.Methods The human IL-24 cDNA fragment was amplified from plasmid by polymerase chain reaction(PCR),and sequenced.PQE/hIL-24 was constructed by gene rearrangement,then it was transformed into E.coli M15.The expression of the target protein was induced with IPTG and purified by Ni2+-NTA agarose column.The expressed recombinant hIL-24(rhIL-24) was identified by SDS-PAGE and Western blotting.Normal peripheral blood mononuclear cells(PBMCs) were cultivated with the expression protein for 48 and 72 h,the levels of IL-6,IFN-? and TNF-? of PBMCs stimulated with rhIL-24 were detected by ELISA.Results The recombinant prokaryotic expression vector PQE/IL-24 was constructed successfully and expressed stably in E.coli M15.At about 18 400 of molecular weight,there was an induced protein band.The levels of IFN-?,IL-6 and TNF-? in the group of cultivated with the expression protein were obviously higher than those in the groups without the expresson protein(P
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0.05).The apoptotic percentage was increased significantly after X-rays irradiation with the dose of 6 Gy(P
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Aim To obtain recombinant human IL-24 secretorily expressed in Pichia pastoris,and study the activity of inducing tumor cells apoptosis of this N-glycosylation protein. Methods By the recombinant plasmid ?/pUC18,the confirmed IL-24 gene was inserted between the sites of BamH Ⅰ and EcoR Ⅰ of expression plasmid pPIC9K. The recombinant plasmid IL-24/pPIC9K was transformed into P. pastoris strain GS115. Yeast transformants were induced for expression of recombinant human IL-24 with methanol. The desired protein was identified with Tricine-SDS-PAGE and Western blot.Amount of IL-24 was assayed with ELISA and the glycosylation was analyzed by PNGase F.The activity of inducing tumor cells apoptosis was confirmed by MTT assay and Hoechst assay in vitro.Results Recombinant expression plasmid IL-24/pPIC9K was successfully constructed. 5 transformants were screened with G418 and PCR. Induced with methanol,the expression level of IL-24 was (81.31?14.46) mg?L-1 at flask fermentation,and 70 % IL-24 generated N-glycosylation.Recombinant IL-24 induced apoptosis in MCF-7 cells,but not in NHLF.Conclusion The secretorily expression of the N-glycosylation IL-24 protein in P. pastoris and the study of inducing tumor cells apoptosis lay the foundation for the further study of molecular mechanism of IL-24 on anti-tumors and the potential application.
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Objective To prepare recombinant mda-7/IL-24 adenovirus to study its function on the proliferation of small cell lung cancer NCI-H446 cells. Methods According to the manufacturer's instructions of AdEasy vector system, mda-7/IL-24 cDNA was subcloned into the adenoviral shuttle vector pAdTrack-CMV. The efficient recombination of adenoviral backbone vector pAdEasy-1 and pAdTrack-CMV-mda-7/IL-24 was achieved in bacteria E. coli BJ 5183. The recombinant adenoviral vector pAd-mda-7/IL-24 linearized by Pac Ⅰ was transfected into HEK293 cells with lipofectamine 2000. To generate higher titer viral stocks, the amplification of recombinant adenovirus was accomplished in packing cells. Viral titers were measured by tissue culture infectious dose 50 (TCID_ 50 ) method. Ad.mda-7/IL-24 was identified by PCR. The expression of pAd-mda-7/IL-24 in NCI-H446 cells was detected by Western-blot analysis. The function of Ad.mda-7/IL-24 on the proliferation of NCI-H446 cells was assayed by MTT after cells were infected by 50 pfu/ml adenovirus. Results The recombinant adenoviral shutter vector pAdTrack-CMV-mda-7/IL-24 and recombinant adenoviral vector pAd-mda-7/IL-24 were constructed successfully as identified by sequence analysis. PCR assay showed that adenovirus Ad.mda-7/IL-24 contained mda-7/IL-24 cDNA. After amplification in packing cell HEK293, the titer of virus was 2?10~ 10 pfu/ml measured by TCID_ 50 assay. Western-blot results identified that MDA-7/IL-24 could be expressed in NCI-H446 cells. After infected by 50 pfu/ml adenovirus, the proliferation of NCI-H446 cells was inhibited by 21.37% with MTT method. Conclusion Ad.mda-7/IL-24 can inhibit the growth of NCI-H446 cell obviously. This result lays foundation to study its function mechanism and to apply it in gene therapy of the small cell lung cancer.
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Interleukin 24(IL 24),also called the melanoma differentiation associated gene 7(MDA 7),is stably expressed in human tissues associated with the immune system such as the spleen,thymus,peripheral blood leukocytes and normal melanocytes. IL 24 binds to IL 20 and IL 22 receptor complexes(IL 22R1/IL 20R2 and IL 20R1/IL 20R2),and induces secretion of high levels of IL 6,TNF alpha,and IFN gamma and low levels of IL 1beta,IL 12,and GM CSF from human PBMC. Adenoviral IL 24(Ad IL 24) induces growth suppression and apoptosis selectively in diverse human cancers and tumor cell lines without producing any apparent harmful effect in normal cells. The effects of Ad IL 24 are associated to the ratio of pro apoptotic(BAX,BAK) to anti apoptotic(Bcl 2) proteins,and the up regulation of p38 MAPK and a family of growth arrest and DNA damage(GADD) inducible genes. These demonstrate the potential therapeutic effects of Ad IL 24 in human cancer.