title: X-shaped DNA potentiates therapeutic efficacy in colitis-associated colon cancer through dual activation of TLR9 and inflammasomes.
abstract: BACKGROUND
  Immunotherapy has been extensively pursed as a promising strategy for the treatment of cancer. Pattern-recognition receptors (PRRs) play important roles in triggering activation of innate and adaptive immunity. Therefore, agents that stimulate PRRs could be useful for cancer immunotherapy. We developed two kinds of X-shaped double-stranded oligodeoxynucleotides (X-DNA), a single unit of X-DNA (XS-DNA) composed of four strands of DNA and a ligated X-DNA complex (XL-DNA) formed by crosslinking each XS-DNA to the other, and investigated if they had immunostimulatory activity and could be applied to anti-cancer immunotherapy.
  
  
  METHODS
  Activation of MAPKs and NF-κB was determined by immunoblotting in bone marrow-derived primary dendritic cells (BMDCs). Immune cytokines and co-stimulatory molecules were measured by ELISA and flow cytometry analysis. Anti-cancer efficacy was examined in an azoxymethane/dextran sulfate sodium-induced colitis-associated colon cancer mouse model. Association of X-DNA and TLR9 was determined by co-immunoprecipitation followed by immunoblotting. The involvement of TLR9 and inflammasomes was determined using TLR9- or caspase-1-deficient BMDCs. Inflammasome activation was examined by degradation of pro-caspase-1 to caspase-1 and cleavage of pro-IL-1β to IL-1β in BMDCs.
  
  
  RESULTS
  XL-DNA and XS-DNA induced activation of MAPKs and NF-κB and production of immune cytokines and co-stimulatory molecules in BMDCs. BMDCs stimulated by XL-DNA induced differentiation of naïve CD4(+) T cells to TH1 cells. Intravenous injection of XL-DNA into mice resulted in increased serum IFN-γ and IL-12 levels, showing in vivo efficacy of XL-DNA to activate TH1 cells and dendritic cells. XL-DNA greatly enhanced the therapeutic efficacy of doxorubicin, an anti-cancer drug, in colitis-associated colon cancer. XL-DNA directly associated with TLR9. In addition, immunostimulatory activities of X-DNA were abolished in TLR9-deficient dendritic cells. Furthermore, X-DNA induced caspase-1 degradation and IL-1β secretion in BMDCs, which were abolished in caspase-1-deficient cells.
  
  
  CONCLUSIONS
  X-DNA induced the activation of dendritic cells as shown by the expression of immune-cytokines and co-stimulatory molecules, resulting in the differentiation of TH1 cells, mediated through dual activation of TLR9 and inflammasomes. X-DNA represents a promising immune adjuvant that can enhance the therapeutic efficacy of anti-cancer drugs by activating PRRs.
authors.affiliation: Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, 420-743, Bucheon, Republic of Korea. hohoje@nate.com.
authors.forename: Jung Eun
authors.initials: JE
authors.lastname: Koo
authors.affiliation: School of Chemical Engineering and SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, 440-746, Suwon, Republic of Korea. seung3885@skku.edu.
authors.forename: Seung Won
authors.initials: SW
authors.lastname: Shin
authors.affiliation: School of Chemical Engineering and SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, 440-746, Suwon, Republic of Korea. sh.um@skku.edu.
authors.forename: Soong Ho
authors.initials: SH
authors.lastname: Um
authors.affiliation: Integrated Research Institute of Pharmaceutical Sciences, College of Pharmacy, The Catholic University of Korea, 420-743, Bucheon, Republic of Korea. joolee@catholic.ac.kr.
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Lee
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D003092:Colitis / Q000150:complications / Q000188:drug therapy* / Q000473:pathology; D003110:Colonic Neoplasms / Q000150:complications / Q000188:drug therapy* / Q000473:pathology; D016207:Cytokines / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use*; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D004195:Disease Models, Animal; D004317:Doxorubicin / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D058847:Inflammasomes / Q000378:metabolism*; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D009690:Nucleic Acid Conformation; D018417:Th1 Cells / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000378:metabolism*; D016896:Treatment Outcome
chemical_list: D000970:Antineoplastic Agents; D016207:Cytokines; D007155:Immunologic Factors; D058847:Inflammasomes; D051217:Toll-Like Receptor 9; D004317:Doxorubicin; D004247:DNA