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Objective:To observe the effect of irradiation on the production of IL-8 in lung cancer cell line A549 and explore its possible mechanism.Methods:A549 cells irradiated with different doses of X-rays were used to collect cell supernatant, cellular RNA and protein at different time points after irradiation. The expression level of IL-8 mRNA in A549 cells after irradiation was detected by RT-PCR, which was further validated by real-time quantitative PCR. The expression level of IL-8 in the cell supernatant was quantitatively measured by ELISA. The expression levels of cellular signaling pathway molecules in A549 cells after irradiation were detected by Western Blot. The A549 cells were pretreated with p38 MAPK inhibitor, NF-κB inhibitor and ROS scavenger. The effect of these inhibitors on the expression of IL-8 in A549 cells induced by irradiation was evaluated by ELISA.Results:Irradiation up-regulated the expression of IL-8 in A549 cells in a dose-and time-dependent manner. Irradiation activated the p38 MAPK and NF-κB signaling pathway in A549 cells. p38 MAPK and NF-κB inhibitors blocked the induction of IL-8 of A549 cells by irradiation. Inhibition of ROS failed to inhibit the induction of IL-8 of A549 cells by irradiation.Conclusion:Irradiation can increase the production of IL-8 in lung cancer cells A549, possibly through the activation of p38 MAPK and NF-κB signaling pathways in a ROS-independent pattern.
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Objective To explore the possibility of rapamycin to up-regulate radiosensitivity of nasopharyngeal carcinoma (NPC) and its molecular mechanism.Methods In vitro,with untreated cells as the control,NPC cells were treated with rapamycin,irradiation (IR),or both rapamycin and IR.Phosphorylation of S6 and GSK3β,expression of Cyclin D1,clonogenic survival,number of residual γH2AX foci,and cell cycle status between study groups were compared.In vivo,athymic mice bearing CNE1 tumor were similarly treated.Tumor weight,Cyclin D1 and phosphorylated S6 in the xenograft model were compared between study groups.Results The results showed that rapamycin alone decreased the phosphorylation of S6 and glycogen synthase kinase 3 β (GSK3β),and the expression of Cyclin D1 in NPC cells.Thus,rapamycin-treated NPC cells had lower cell viability,higher DNA damage and more G1 arrest than the control,which was reflected by the in vivo study that rapamycin significantly attenuated tumor growth and decreased the levels of Cyclin D1 and phosphorylated S6.Moreover,the combination of rapamycin and IR caused the highest cell death,DNA damage,G1 arrest and tumor regression compared to those treated either alone.Conclusions Rapamycin up-regulate NPC radiosensitivity by inhibiting signal transduction of Akt/mammalian target of rapamycin (mTOR)/S6 pathway and Akt/GSK3β pathway,and by downregulating Cyclin D1 expression.
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Objective To express and purify the human scFv antibody,SA3,against the hepatoma fused to enhanced green fluorecsent protein,and to observe the targeted capacity of fusion protein EGFP-SA3 in vivo.Methods SA3 and EGFP genes were cloned into plasmid pET-25b( + )to construct the recombinant plasmid EGFP-SA3/pET-25b ( + ),followed by DNA sequencing.Then it was transformed into E.coli BL21 ( DE3 ) and induced for fusion expression of EGFP-SA3with IPTG.The expressed fusion protein EGFP-SA3 was purified and detected with SDS-PAGE.HepG2 cells were incubated with the fusion protein EGFP-SA3 in vitro,and the binding bioactivity was observed under the fluorecsent microscope.Further more,we injected the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observed the whole body fluorescence image of EGFP.Results SA3 and EGFP genes were successfully cloned into pET-25b( + ),which was confirmed by restriction enzyme Nco I-Xho I or Nco I-Eco RI.A band migrated at the position 750 bp,same to EGFP gene,emerged when recombinant plasmid was digested by restriction enzyme Nco I-Eco RI.Similarly,a band,about 1 500 bp,emerged when digested by Nco I-Xho I.The open-reading frame was confirmed by DNA sequencing.Fusion protein EGFP-SA3 was expressed as inclusion body.After purification and refolding,the result of immunofluorecsence detection verified that EGFP-SA3could specifically bind to HepG2 cells and maximum tumor penetration was at 24 h after the injection.Conclusion The purified fusion protein EGFP-SA3 has strong binding capacity to HepG2cells,indicating the scFv SA3 has a potential value as a targeting molecule for diagnosis and targeted therapy for liver cancer.
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OBJECTIVE@#To determine the effect of gamma-aminobutyric acid(GABA) on proliferation and malignant phenotypes of hepatocellular carcinoma cell line HepG-2.@*METHODS@#HepG-2 cells were cultured by routine method, and then treated with different concentrations of GABA. The proliferation of HepG-2 cells was measured through MTT, doubling time and cell cycles by flow cytometry. The malignant phenotypes were investigated by soft agar colony formation assay.@*RESULTS@#Compared with the control group, GABA efficiently stimulated the proliferation of HepG-2 cells in a dose-dependent manner and affected the distribution of cell cycles of HepG-2 cells. The doubling time of the control group and the GABA-treated group were 39.0, 30.6, 30.0, 27.3, 26.6, and 38.2 h, respectively. The colony formation rates were 3.2%, 4.2%, 5.4%, 6.6%,6.5%, and 3.5%, respectively. Tumorigenicity testing showed that the average weights of tumor was 1.382 g, and 0.285 g for the 2 groups. The difference between the control group and the GABA-treated groups was significant (P<0.01).@*CONCLUSION@#GABA can enhance the proliferation and malignant phenotypes of HepG-2 cells.
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Animals , Humans , Mice , Cell Cycle , Cell Proliferation , Dose-Response Relationship, Drug , Hep G2 Cells , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Phenotype , gamma-Aminobutyric Acid , PharmacologyABSTRACT
Objective To screen the anti-nasopharyngeal carcinoma scFv from a human anti-nasopharyngeal carcinoma single-chain phage antibody library, and identify its characteristics. Methods The single-chain phage antibody library was subjected to three rounds of positive and negative cell panning and enrichment, and then it was selected by ELISA. The binding specificity of phage antibodies with naso-pharyngeal carcinoma cells was confirmed by immunohistochemistry. Results After panning, enrichment and testing by ELISA, 3 phage an-tibody clones reacting with CNE2 more strongly than HUVEC and NP69 were picked out from 4212 clones. One clone, HNSAO33, was fur-ther analyzed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. HNSAO33 spe-cifically reacted to nasopharyngeal carcinoma cells in most human nasopharyngeal carcinoma tissue sections except a few human normal naso-pharyngeal tissue sections. The distinction of positive rates was of a great statistical significance. Conclusion ELISA and immunohisto-chemistry results confirmed HNSAO33 specifically bind with nasopharyngeal carcinoma cells. The seFv fragment against nasopharyngeal carci-noma may be further developed and applied in clinical diagnosis and therapy of nasopharyngeal carcinoma.
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Objective To obtain high pure myocardiac Troponin T from myocardiac tissue. Methods After the myocardiac tissues were homogenized and purified with high salt solution, the Sephacryl S-300HR column chromatography was employed to separate the cTnT from cardiac proteins, and identified them with SDS-PAGE and Western blot. Results The crude cTn was purified by column chromatography, and there appeared three peaks. The second peak was single component, which could react with mouse anti-human Troponin T antibody, and its molecular weight was about 34kD. Conclusions In this experiment, high pure cTnT was obtained, which could pave a way for further research.