title: The combination of ANT2 shRNA and hNIS radioiodine gene therapy increases CTL cytotoxic activity through the phenotypic modulation of cancer cells: combination treatment with ANT2 shRNA and I-131.
abstract: BACKGROUND
  It is important to simultaneously induce strong cell death and antitumor immunity in cancer patients for successful cancer treatment. Here, we investigated the cytotoxic and phenotypic modulation effects of the combination of ANT2 shRNA and human sodium iodide symporter (hNIS) radioiodine gene therapy in vitro and in vivo and visualized the antitumor effects in an immunocompromised mouse colon cancer model.
  
  
  METHODS
  A mouse colon cancer cell line co-expressing hNIS and the luciferase gene (CT26/hNIS-Fluc, named CT26/NF) was established. CT26/NF cells and tumor-bearing mice were treated with HBSS, scramble, ANT2 shRNA, I-131, and ANT2 shRNA + I-131. The apoptotic rates (%) and MHC class I and Fas gene expression levels were determined in treated CT26/NF cells using flow cytometry. Concurrently, the level of caspase-3 activation was determined in treated cells in vitro. For in vivo therapy, tumor-bearing mice were treated with scramble, ANT2 shRNA, I-131, and the combination therapy, and the anti-tumor effects were monitored using bioluminescence. The killing activity of cytotoxic T cells (CTLs) was measured with a lactate dehydrogenase (LDH) assay.
  
  
  RESULTS
  For the in vitro experiments, the combination of ANT2 shRNA and I-131 resulted in a higher apoptotic cell death rate compared with ANT2 shRNA or I-131 alone, and the levels of MHC class I and Fas-expressing cancer cells were highest in the cells receiving combination treatment, while single treatment modestly increased the level of MHC class I and Fas gene expression. The combination of ANT2 shRNA and I-131 resulted in a higher caspase-3 activation than single treatments. Interestingly, in vivo combination treatment led to increased gene expression of MHC class I and Fas than the respective mono-therapies; furthermore, bioluminescence showed increased antitumor effects after combination treatment than monotherapies. The LDH assay revealed that the CTL killing activity against CT26/NF cells was most effective after combination therapy.
  
  
  CONCLUSIONS
  Increased cell death and phenotypic modulation of cancer cells in vitro and in vivo were achieved simultaneously after combination therapy with ANT2 shRNA and I-131, and this combination therapy induced remarkable antitumor outcomes through improvements in CTL immunity against CT26/NF. Our results suggest that combination therapy can be used as a new therapeutic strategy for cancer patients who show resistance to single therapy such as radiation or immunotherapy.
authors.affiliation: Department of Pathology, Seoul National University College of Medicine, Seoul, Korea.
authors.forename: Yun
authors.initials: Y
authors.lastname: Choi
authors.forename: Ho Won
authors.initials: HW
authors.lastname: Lee
authors.forename: Jaetae
authors.initials: J
authors.lastname: Lee
authors.forename: Yong Hyun
authors.initials: YH
authors.lastname: Jeon
mesh_terms: D033742:Adenine Nucleotide Translocator 2 / Q000235:genetics*; D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D015870:Gene Expression; D005805:Genes, MHC Class I; D015316:Genetic Therapy; D007457:Iodine Radioisotopes / Q000494:pharmacology / Q000627:therapeutic use; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010641:Phenotype; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology / Q000627:therapeutic use*; D019275:Radiopharmaceuticals / Q000627:therapeutic use; D027981:Symporters / Q000235:genetics*; D013602:T-Lymphocytes, Cytotoxic; D019014:fas Receptor / Q000235:genetics; D000099209:Sodium-Iodide Symporters
chemical_list: D033742:Adenine Nucleotide Translocator 2; D053148:Caspase 3; D007457:Iodine Radioisotopes; D007770:L-Lactate Dehydrogenase; D034741:RNA, Small Interfering; D019275:Radiopharmaceuticals; D027981:Symporters; D019014:fas Receptor; D000099209:Sodium-Iodide Symporters; C491899:Fas protein, mouse