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[Experimental study on A549 cell death mediated by xenoantigen ?-gal ?in human serum].

ABSTRACT: The absence of ?-gal in humans is caused by the inactivity of ?-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing ?-1,3GT gene to synthesize ?-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed ?-gal, and to observe the antitumor mechanisms mediated by human serum.A549 cells were transfected with pEGFP-N1-GT plasmids constructed in a previous study. A stable transgenic cell line, A549-GT, was then selected and cultivated. The biological characteristics of A549-GT cells, including morphology and proliferation, were examined. ?-1,3GT mRNA expression was detected by RT-PCR. Direct immunofluorescence staining and flow cytometry (FCM) were used to analyze the synthesis of ?-gal in A549-GT. The binding of human serum IgM and C3 with A549-GT were also detected.?-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable ?-gal expression rate in A549-GT. Compared with parental A549 cells, the biological characteristics of A549-GT were unaltered. ?-Gal expression was not detected in the human fetal lung fibroblast cell line MRC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between A549-GT treated with human serum and IgM/C3.?-Gal expression in tumor cells by gene transduction can induce complement-dependent cytototic antitumor effects.

SUBMITTER: Zhu S

PROVIDER: S-EPMC6000037 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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[Experimental study on A549 cell death mediated by xenoantigen α-gal  in human serum].

Zhu Shengming S Xie Ling L Zheng Hong H Qin Feng F Liu Mei M Luo Zhiguo Z Wang Yanping Y

Zhongguo fei ai za zhi = Chinese journal of lung cancer 20121101 11

<h4>Background and objective</h4>The absence of α-gal in humans is caused by the inactivity of α-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing α-1,3GT gene to synthesize α-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed α-gal, and to observe the antitumor mechanisms mediated by human ser ...[more]

PMID: 23164348

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Project description:Resveratrol (RSV) has been reported to exhibit cytotoxic activity in multiple types of malignant cells; however, the mechanisms underlying the antitumor effects of RSV in non?small?cell lung cancer (NSCLC) cells remain undetermined. Combining bioinformatics analysis with experimental validation, the present study aimed to examine the effects of RSV on the apoptosis and autophagy of A549 NSCLC cells, and to determine the potential underlying molecular mechanisms. Bioinformatics analysis was used to determine the differentially expressed genes (DEGs) and identify the enriched biological functions and pathways associated with these DEGs following RSV treatment. Cell viability was determined by MTT assay, and flow cytometry and TUNEL assay were used to evaluate cell apoptosis. Monodansylcadaverine staining combined with a transmission electron microscope were used to evaluate the extent of autophagy. The expression levels of apoptosis?, autophagy?, or pathway?associated molecular markers were measured by reverse transcription?quantitative PCR and/or western blot analysis. By bioinformatics analysis, a total of 1,031 DEGs were identified in the RSV?treated A549 cells, which were enriched in apoptosis?, or autophagy?related biological functions and the p53 signaling pathway. In validation experiments, RSV significantly reduced cell viability and initiated apoptosis, with an increase in the number of apoptotic cells; it also upregulated cleaved caspase?3 expression and Bax expression, and downregulated the Bcl?2 expression levels. Additionally, there was an increase in the accumulation of green dot?like structures, indicative of autophagic vesicles, observed under a fluorescence microscope, and an increase in the presence of autophagic vacuoles observed using a transmission electron microscope following RSV treatment. Furthermore, the expression levels of the autophagy?related proteins, LC3?II/LC3?I and Beclin?1, were increased and p62 expression was decreased. 3?methyladenine (3?MA), an inhibitor of autophagy, partially reversed the RSV?induced cytotoxic effects, but did not significantly alter the number of apoptotic cells. RSV elevated the p53 levels and decreased the phosphorylated (p?)Mdm2 and p?Akt levels. Pifithrin??, an inhibitor of p53, partially reduced RSV?induced apoptosis and autophagy. On the whole, the results of the present study demonstrated that RSV initiates the apoptosis and autophagic death of A549 cells via the activation of the p53 signaling pathway, further highlighting the potential of RSV for the treatment of NSCLC.

Dataset Information

[Experimental study on A549 cell death mediated by xenoantigen ?-gal ?in human serum].

Publications

[Experimental study on A549 cell death mediated by xenoantigen α-gal in human serum].

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[Experimental study on A549 cell death mediated by xenoantigen α-gal  in human serum].