Bidirectional effect of MnSOD overexpression on the proliferation of esophageal cancer cells in vitro

Abstract

To construct a recombinant lentiviral vector for manganese superoxide dismutase (MnSOD) gene expression, and observe its effect on the proliferation of esophageal cancer cells in vitro. Chemical methods were employed for synthesis of the MnSOD cDNA sequence sections, along with the attB sites. Target gene fragment was constructed on the pMD-18T vector, and the recombinant plasmid pDONR221 was obtained after BP recombination reaction. Sequencing was followed by LR recombination reaction between the plasmid and DEST to obtain the lentiviral vector, which worked with helper plasmid for co-transfection of human embryonic kidney epithelial cells (293T cells). Amplification was done to determine its titer, and both transfection and selection procedures were made to get two stable transfected esophageal cancer TE-1 cell lines with medium MnSOD expression (TE-1Mm cells) and high MnSOD expression (TE-1Mh cell), and empty vector cell (TE-1Mn cells). Reverse transcription polymerase chine reaction (RT-PCR), immunofluorescence, immunocytochemistry and Western blot were used to detect the target gene with respect to its expression in the TE-1 cells. Additionally, colorimetric 3-[4,5-dimethy thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, agar colony formation assay, annexin V-FITC/PI staining and flow cytometry experiments were also conducted as to observe the influence of the medium and high MnSOD overexpressions on the proliferation of esophageal cancer cells. RT-PCR indicated that the transfected TE-1 cells showed positive MnSOD expression at different levels. Immunofluorescence, immunocytochemistry and Western blot suggested that TE-1Mm cells and TE-1Mh cells had MnSOD protein expression at different levels. MTT assay indicated that TE-1Mm cells had a significantly decreased survival rate compared with that of the two control cells (TE-1 cells and TE-1Mn cells), and TE-1 Mh cells had an significantly increased survival rate (P<0.05). The colony formation ability of TE-1Mm cells was (23.0 +/- 2.7)%, and that of TE-1Mh cells was (45.3 +/- 4.5)%, significantly different form the (34.7 +/- 4.2)% in TE-1 cells and (33.7 +/- 4.7)% in TE-1Mn cells (P<0.05). Annexin V-FITC/PI double staining experiment of the stably transfected cells cultured for 48 h showed that the early apoptosis rate in TE-1Mm cells was (10.6 +/- 1.0)%, significantly higher than (2.6 +/- 0.2)% in the TE-1 cells, (2.5 +/- 0.6)% in the empty vector cells and (1.0 +/- 0.1)% in the TE-1Mh cels (P<0.05). The fluorescence index (FI) of mitochondrial apoptosis of TE-1Mm cells was 0.948 +/- 0.019, significantly lower than that of TE-1 cell (1.000 +/- 0.022) and empty vector The fluorescence index of TE-1Mn cells (0.997 +/- 0.023) and TE-1 cells (1.000 +/- 0.022) were significant different from that of 0.948 +/- 0.019 in TE-1Mm cells and 1.076 +/- 0.022 in TE-1Mh cells, indicating a significant difference of mitochondrial apoptosis between the cell groups. FCM results indicated that the ROS fluorescence index of TE-1Mm cells was 0.859 +/- 0.040, that of TE-1Mh cells was 0.763 +/- 0.039, significantly lower than that of TE-1 cells (1.000 +/- 0. 042) and empty vector cells (1.002 +/- 0.047) (P<0.05). Stably transfected cell lines with MnSOD expression have been successfully established. MnSOD overexpression shows bidirectional effect on the proliferation of esophageal cancer cells.