Expression Of Manganese Superoxide Dismutase Influences The Radioresistance Of Nasopharyngeal Carcinoma

Background Carcinoma of the nasopharynx is prevalent in the South China region. The poorly-differentiated subtype is most common histologic type of na-sopharyngeal carcinoma, which is radiosensitive. The most effective means of treatment is generally radiation therapy. The five-year survival rate of nasopha-ryngeal carcinomas is about 50% overall and failure to control the cancer is mainly due to a portion of radioresistant phenotype of nasopharyngeal carcinoma. To in-crease the dose of radiation that can be delivered to a tumor is limited by the damage caused to surrounding normal tissues and the consequent risk of com-plications. Hence, therapeutic efficacy can be improved either by increasing the effective radiation dose delivered to the tumor relative to that given to surround-ing normal tissues or by devising an approach that will increase the response of the tumor relative to that of the surrounding normal tissues. The former ap-proach implies improvement in the physical aspects of radiation therapy. The second approach involves exploiting biological factors that result in differences in the response of tumors and normal tissues to radiation therapy. Thanks to the advancements in genomics, proteomics and bioinformatics in recent decades, more understanding of the disease carcinogenesis and progression has been gained. In the era of molecular medicine, specific treatment to the potential target using tech-nologies such as immunotherapy and RNAi becomes formulating from bench to bedside application and thus makes a potential genetic marker of radiosensitivity discovery more meaningful for NPC management. Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species that are associated with radiation-induced cytotoxicity. The antioxidant enzyme manganese super-oxide dismutase (SOD2) catalyzes the dismutation of the superoxide anions into hydrogen peroxide, to enhance the radioresistance of a mammalian cell.Methods Two human nasopharyngeal carcinoma cell lines CNE1 and CNE2 were characterized using the clonogenic survival assay after irradiation. The multitar-get, single-hit cell survival equation and the linear-quadratic cell survival equation were fit to the radiation dose response data using the programme SigmaPlot9. Ra-diosensitivity parameters of the nasopharyngeal carcinoma cell lines (CNE1 and CNE2) obtained from the fitted data were D0(the slope of in the straight-line region), Dq(a measure of the size of the shoulder), a(a measure of the initial slope),β(a measure of the final slope), and SF2(surviving fraction at 2 Gy).Activities of SOD2 gene on nasopharyngeal carcinoma cells were investigated in comparison with the relative radiosensitivity of NPC cell line and the relative radioresistance of NPC cell line. SOD enzymatic activity was assessed by the reduction of WST-1 using water-soluble tetrazolium salt microplate assay.Expression of SOD2 gene on nasopharyngeal carcinoma cells was investigated in comparison with the relative radiosensitivity of NPC cell line and'the relative radioresistance of NPC cell line. Western blots were carried out essentially to assess SOD2 protein expression and the optic density (OD) value of each band was estimated by Quantity One(?) 1-D Analysis Software.We have investigated the potential low expression of SOD2, through expres-sion of miRNA for RNAi using Gateway(?)-adapted expression vector in mam-malian cells, to attenuate the radioresistance of a nasopharyngeal carcinoma cell line. Using these as in vitro models, we have investigated whether SOD2 gene therapy may be suitable for the improvement of the effects of radiotherapy on nasopharyngeal carcinoma.·Designed and synthesized three pairs of complementary DNA oligos for SOD2 messenger RNA, with each containing 4 nucleotide overhangs nec-essary for directional cloning.·Annealed DNA oligos to generate three double-strand oligos.·Clone the ds oligos into pcDNATM6.2-GW/EmGFP-imR expression vectors using T4 DNA Ligase.·Transformed E.coli and analyzed colonies for the desired expression clone.·Transfected the expression clone for transient and stable RNAi analysis.·assessed protein expression and mRNA expression of SOD2 in the transfected cells using Western blot analysis and RT-PCR.·assessed the effect of clonogenic cell kill of established stable cell line ex-pressing miRNA of SOD2 following exposure to ionizing radiation.·determined if radiation efficiency increased with reduced expression SOD2 through miRNA expression construct transfer. Results The radiobiological parameter of nasopharyngeal carcinoma cell lines Surviving fraction of CNE1 was higher than those of CNE2 at each dose point on radiation cell survival curves. The value of Do, Dq, a,β, SF2 of cell line CNE1 and CNE2 were 1.398 Gy,0.881 Gy,0.228 Gy-1,0.104 Gy-2,0.403 and 0.926 Gy, 0.266 Gy,0.763 Gy-1,0.066 Gy-2,0.151, respectively.SOD enzyme activity and SOD2 expression of nasopharyngeal carcinoma cell lines The levels of total SOD activity and SOD2 activity in cell lines CNEl and CNE2 were 234.04 and 146.10 U/108cell,143.20 and 97.82U/108cell, respectively. Compared to CNE2 cells, levels of SOD2 protein expression were increased 1.94-fold in CNE1 cells.Construction of the plasmid vectors and stable cell lines The plasmid vectors, pcDNATM6.2-GW/EmGFP-imR-SOD2(411,424 and 700), express miRNA for use in RNAi analysis of SOD2 gene in nasopharyngeal carcinoma cells. EmGFP en-codes Green Fluorescent Protein. Following plasmid/LipofectamineTM2000 trans-duction, transduced CNE1, uninduced cell and cell transfected with pcDNATM6.2-GW/EmGFP-imR-neg down-regulated the expression of SOD2 protein to 86.57%, 48.24%,58.64%,100% and 90% respectively; down-regulated the level of SOD2 mRNA to 63%,47.81%,54.88%,100% and 92% respectively. Transfected pcDNATM6.2-GW/Em GFP-imR-SOD2424 inhibited tumor cell proliferation. All transduced plasmids did not influence cell cycles.Selected for stable cell line transfected with pcDNATM6.2-GW/EmGFP-imR-SOD2700 using Blasticidin. Assay for SOD2 gene knockdown, compare to unin-duced CNE1 cell and cell stably transfected with pcDNATM6.2-GW/EmGFP-imR-neg showed the level of SOD2 mRNA were down-regulated to 27.82%,100% and 98.28%; the protein expression of SOD2 gene were down-regulated to 30.05%, 100% and 94.88%.The radiobiological parameter of SOD2 gene knockdown NPC cell lines Sur-viving fraction of SOD2 gene knockdown CNE1 was lower than those of uninduced CNE1 at each dose point on radiation cell survival curves. The value of Do, Dq, a, 0, SF2 of both were 1.446 Gy,0.856 Gy,0.256 Gy-1,0.085 Gy-2,0.407 and 0.927 Gy,0.276 Gy,0.790 Gy-1,0.043 Gy-2,0.153, respectively. The radiation cell sur-vival curves of cell stably transfected with pcDNATM6.2-GW/EmGFP-imR-neg was similar to that of uninduced CNE1 cell.The effect of miRNA interference silencing SOD2 in CNEl cells on efficiency of radiation CNE1 cells transduced with miR-SOD2 or miR-neg control or parental CNE1 cells were irradiated at either 2 or 4 Gy. Cells transduced with miR-SOD2 demonstrated an increased efficiency of treatment of carcinoma cell with ionizing radiation as seen by a significant increase in colony number at both dosesConclusions CNE1 is more radioresistance than CNE2. The levels of total SOD activity and SOD2 activity in CNEl cell were significantly(1.5-fold) higher than CNE2 cell. There is marked correlation observed between the activity of SOD, SOD2 and radiosensitivity of the nasopharyngeal carcinoma cell lines.The protein expression of SOD2 in CNE1 cell is significantly (2-fold) higher than that of CNE2 cell. There is marked correlation observed between the quantity of SOD2 protein and radiosensitivity of the nasopharyngeal carcinoma cell lines. x-irradiation can result in increasing in total quantity of SOD2 protein.We construct successfully the recombination plasmid vectors which target SOD2 gene, the plasmid vectors which express miRNA can down-regulate the expression of SOD2 gene efficiently, the miRNA of SOD2 gene loci from 700 to 721 is effective to down-regulate SOD2 gene.In vitro, miRNA expression of SOD2 led to a decrease in mRNA and protein of SOD2 in transduced cell in comparison to contols, the Corresponding cell is very lower radioresistance than uninduced CNE1. The results presented suggest that miRNA interference silencing SOD2 gene therapy may be applicable to the radioresistant nasopharyngeal carcinoma, enabling radiosensitivity escalation in cancer radiotherapy.