title: Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention.
abstract: Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anticancer effects are still being elucidated. We hypothesized that aspirin's chemopreventive actions may involve cell-cycle regulation through modulation of the levels or activity of cyclin A2/cyclin-dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The downregulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. First, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Second, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, preincubation of CDK2 with salicylic acid dose-dependently quenched the fluorescence due to ANS. Third, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid downregulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention.
  
  
  CONCLUSIONS
  Biochemical and structural studies indicate that the antiproliferative actions of aspirin are mediated through cyclin A2/CDK2.
authors.affiliation: Department of Pharmaceutical Sciences and Translational Cancer Research Center, South Dakota State University College of Pharmacy, Brookings, South Dakota.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Dachineni
authors.affiliation: Department of Pharmaceutical Sciences and Translational Cancer Research Center, South Dakota State University College of Pharmacy, Brookings, South Dakota.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Ai
authors.affiliation: Department of Pharmaceutical Sciences and Translational Cancer Research Center, South Dakota State University College of Pharmacy, Brookings, South Dakota.
authors.forename: D Ramesh
authors.initials: DR
authors.lastname: Kumar
authors.affiliation: Department of Pharmaceutical Sciences and Translational Cancer Research Center, South Dakota State University College of Pharmacy, Brookings, South Dakota.
authors.forename: Satya S
authors.initials: SS
authors.lastname: Sadhu
authors.affiliation: Department of Pharmaceutical Sciences and Translational Cancer Research Center, South Dakota State University College of Pharmacy, Brookings, South Dakota.
authors.forename: Hemachand
authors.initials: H
authors.lastname: Tummala
authors.affiliation: Department of Pharmaceutical Sciences and Translational Cancer Research Center, South Dakota State University College of Pharmacy, Brookings, South Dakota. Jayarama.gunaje@sdstate.edu.
authors.forename: G Jayarama
authors.initials: GJ
authors.lastname: Bhat
mesh_terms: D001241:Aspirin / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D056751:Cyclin A2 / Q000235:genetics / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D061986:MCF-7 Cells; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control*; D011485:Protein Binding; D020156:Salicylic Acid / Q000494:pharmacology*
chemical_list: C503721:CCNA2 protein, human; D056751:Cyclin A2; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D020156:Salicylic Acid; D001241:Aspirin