Basic Study On Application Of Attenuated Mutants Of Shiga-like Toxin Ⅰ In Cancer Therapy

Objects:This study aimed to evaluate the potential anticancer and antiangiogenesis effect ofStx1^W203F and Stx1^R170H, two attenuated mutants of Shiga-like toxinâ… (Stx1), incancer gene therapy.Methods:The genes encoding attenuated Shiga-like toxinâ… mutants were amplified byoverlap PCR and then cloned into pMD18-T vectors. Thereafter, the genes were cutfrom pMD18-T vectors and cloned into eukaryotic expression plasmid pcDNA3.1.The N-terminal part of Stx1 A-or B-subunit protein sequence was submitted to theinternet server SignalP 3.0 to study whether the original signal peptides of Stx1 canbe recognized by eukaryotic cell. Human ovarian carcinoma cell line SKOV3 cellsand Chinese hamster ovary cell line CHO cells were transfected with theserecombinant pcDNA3.1 plasmids by liposomal mediated gene transfer. The cell deathafter the transfection was monitored under microscope and by FACS. Three CHO celllines which can express native or attenuated mutants of Stx1, respectively, wereestablished by G418 selection. The integrated mutant Stx1 genes in these CHO celllines were determined by PCR. The transcription and secretory expression of Stx1mutants in these CHO cell lines were screened by RT-PCR and by Western blotanalysis. Antiproliferative effects of these Stx1 mutants were tested in SKOV3 andStx1-susceptible human umbilical vein endothelial cells (HUVECs) in vitro. Effect ofthese Stx1 mutants on inducing cell death and cell cycle arrest was analyzed inmutant Stx1 gene-transfected SKOV3 cells. Wound migration assay and tubeformation assay were performed to test the effect of these Stx1 mutants on HUVECsfunction. In vivo therapeutic effect of these Stx1 mutants on transplanted SKOV3tumors was explored using xenograft models in nude mice by intratumoral injection of the recombinant pcDNA3.1 plasmids. Base on the results of these studies, we alsoconstruct two replication defective adenoviral vectors harboring the coding sequenceof attenuated Stx1 mutant Stx1^R170H or Stx1^R170L, respectively, for further study.Results:The genes encoding Stx1^W203F and Stx1^R170H were successfully cloned intopcDNA3.1. Their mRNA was verified by RT-PCR in transfected CHO cells andtransfected SKOV3 cells. Bioinformatics analysis suggests that the original signalpeptide of Stx1 A- or B-subunit can be recognized by eukaryotic cell. Thetranscription and secretory expression of Stx1 mutants were verified by RT-PCR andby Western blot analysis in mutant Stx1 gene-integrated CHO cells. The supernatantsof mutant Stx1 gene-integrated CHO cells have antiproliferative effect on SKOV3cells and HUVECs, and this effect can be abrogated by treatment with monoclonalantibody to Stx1 B-subunit. These recombinant pcDNA3.1 plasmids causedconsiderable cell death of SKOV3 cells in 24 hour, neither caspase activity nor DNAfragmentation was observed and necrosis is the major mode of cell death. These Stx1mutants can induce cell cycle arrest of SKOV3 cells in G₂-M or S phase dependingon the dosage of gene transfer. Furthermore, these attenuated Stx1 mutantsignificantly decreased migration and capillary tube formation of HUVECs at lowdose. In vivo study showed that Stx1^W203F but not Stx1^R170H significantly suppressedtransplanted SKOV3 tumor growth in nude mice model. Interestingly, the microvasseldensities of tumors treated with these two recombinant pcDNA3.1 plasmids wereboth significantly reduced. In addition, the genes encoding Stx1^R170H and Stx1^R170Lwere cloned into replication defective adenoviral vectors.Conclusion:1. The genes encoding Stx1^W203F and Stx1^R170H have been successfully expressed ineukaryotic expression system. 2. Stx1^W203F or Stx1^R170H gene-integrated CHO cell lines have been establishedsuccessfully. The findings suggest that Stx1 mutants synthesized within eukaryoticcells can be secreted and may act in a similar way to native Stx1.3. These Stx1 mutants can induce necrosis and cell cycle arrest of SKOV3 cells.Taken together, we can conclude that these attenuated mutants of Stx1 inhibit tumorgrowth, at least partly, by causing tumor cell necrosis and inducing tumor cell cyclearrest.4. These Stx1 mutants have showed antiangiogenic activity in vitro and in vivo,suggesting that the tumors were inhibited by a combination of antineoplastic andantiangiogenic activity.5. We successful constructed the replication defective adenoviral vectors harboringthe coding sequence of attenuated Stx1 mutant Stx1^R170H or Stx1^R170L, respectively,which may be more helpful to further researches.