Human manganese superoxide dismutase (hMnSOD) is a new type of cancer suppressor. Nonamer of arginine (R9) is an efficient protein transduction domain (PTD). The aim of the study was to improve the transduction efficiency of hMnSOD and investigate its activity in vitro. In this study, we designed, constructed, expressed, and purified a novel fusion protein containing the hMnSOD domain and R9 PTD (hMnSOD–R9). The DNA damaged by Fenton’s reagent was found to be significantly reduced when treated with hMnSOD–R9. hMnSOD–R9 fusion protein was successfully delivered into HeLa cells. The MTT assay showed that proliferation of various cancer cell lines were inhibited by hMnSOD–R9 in a dose-dependent manner. In addition, the cell cycle of HeLa cells was arrested at the sub-G0 phase by hMnSOD–R9. hMnSOD–R9 induced apoptosis of HeLa cells in a dose-dependent manner. With hMnSOD–R9 treatment, Bax, JNK, TBK1 gene expression was increased and STAT3 gene expression was gradually down-regulated in HeLa cells. We also found that apoptosis was induced by hMnSOD–R9 in HeLa cells via up-regulation of cleaved caspase-3 and down-regulation phospho-STAT3 pathway. These results indicated that hMnSOD–R9 may provide benefits to cervical cancer treatment.
Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1) cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1). SaCSD1 comprised a complete open reading frame (ORF) of 459 bp which encoded a protein of 152 amino acids. Its mature protein is predicted to be 15.32 kDa and the deduced isoelectric point is 5.78. SaCSD1 has high sequence similarity (85%–90%) with the superoxide dismutase (CSD) of some other plant species. SaCSD1 was expressed with 30.6% yield regarding total protein content after being introduced into the pET-15b (Sma I) vector for expression in Rosetta-gami and being induced with IPTG. After affinity chromatography on Ni-NTA, recombinant SaCSD1 was obtained with 3.2-fold purification and a specific activity of 2200 U/mg. SaCSD1 showed good activity as well as stability in the ranges of pH between 3 and 7 and temperature between 25 and 55 °C. The activity of recombinant SaCSD1 was stable in 0.25 M NaCl, Dimethyl Sulphoxide (DMSO), glycerol, and chloroform, and was reduced to a great extent in β-mercaptoethanol, sodium dodecyl sulfate (SDS), H2O2, and phenol. Moreover, the SaCSD1 protein was very susceptive to pepsin digestion. Real-time Quantitative Polymerase Chain Reaction (PCR) assay demonstrated that SaCSD1 was expressed in leaf, stem, flower, and fruit organs, with the highest expression in fruits. Under 0.25 M and 0.5 M salt stress, the expression of SaCSD1 was down-regulated in roots, but up-regulated in leaves.
Growing evidences have demonstrated alternative splicing makes great contribution to tumor metastasis since multiple protein isoforms from a single gene that often display different functions in the cell. Human manganese superoxide dismutase (hMnSOD) was revealed dysregulation in progress of tumor metastasis, while the effect of hMnSOD isoforms on metastasis remained unclear. In this study, we showed a novel truncated hMnSOD isoform hMnSOD183, which lacked 39 amino acids compared with hMnSOD222. We expressed two hMnSOD protein isoforms in Escherichia coli, respectively, and found that the MnSOD activity of truncated hMnSOD isoform was especially weaker. In 95-D cells, mRNA levels of hMnSOD variants and MnSOD activity were significantly increased than that in A549 cells. Furthermore, the hMnSODc exhibited lower mRNA level than hMnSODa/b in A549 and 95-D cells. Additionally, the effects of two isoforms were assessed about cell invasion, overexpression of hMnSOD222 but not hMnSOD183 promoted 95-D cells metastasis, and hMnSOD knockdown significantly reduced cells invasive behavior. Overexpression of hMnSOD isoforms also caused changes of metastasis associated proteins, such as up-regulation of MMPs, VEGF and Vimentin and down-regulation of E-cadherin. Moreover, overexpression of hMnSOD183 had weaker effect on metastasis related signaling proteins, such as Akt, JNK and IKKβ, compared to hMnSOD222. In conclusion, our study identified that hMnSOD isoforms induced lung cancer cells invasion through Akt-JNK-IKKβ signaling pathways and the hMnSOD183 isoform played a weaker role than hMnSOD222. Characterization of hMnSOD isoforms is useful for future investigation on metastasis of lung cancer cells.
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