title: In vitro enhancement of tumor cell radiosensitivity by a selective inhibitor of cyclooxygenase-2 enzyme: mechanistic considerations.
abstract: OBJECTIVE
  Selective cyclooxygenase-2 inhibitors have been reported to enhance the tumor response to radiation in vivo, but the cellular mechanisms underlying the radiosensitizing effect are not understood. In the present study, we investigated several possible mechanisms using a murine sarcoma cell culture system.
  
  
  METHODS
  Cells derived from a murine sarcoma, designated NFSA, were cultured in vitro and exposed to different (either single or split) doses of radiation with and without a pretreatment of SC-236 (4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-l-yl] benzene sulfonamide), a selective cyclooxygenase-2 (COX-2) inhibitor. The cells were assayed for clonogenic survival to determine the radiosensitizing effect of SC-236. In addition, MTT assay and TUNEL assay were performed to determine the effects of SC-236 and radiation on the cell survival and cell cycle distribution. RNase protection assay was performed on the total RNA extract using probes that encoded for selected cell cycle regulatory proteins, such as cyclins and cyclin-dependent kinases. To monitor the extent of COX-2 activity and its role in radiosensitization, the cellular content of prostaglandin E2, a major metabolite of COX-2 activity on arachidonic acid, was also determined.
  
  
  RESULTS
  The cell clonogenic survival assay showed that SC-236 significantly enhanced tumor cell radiosensitivity: 50 microM SC-236 increased it by a factor of 1.51 at the 0.1 cell survival level. Treatment with SC-236 (50 microM, 3 days) removed the "shoulder" region on the radiation survival curve, suggesting that the drug inhibited repair of sublethal radiation damage. The inhibition was confirmed by split-dose experiments where two doses (3 Gy each) of radiation were given 4 h apart. The cells exposed to radiation only repaired the damage by a factor of 1.44, whereas those treated with SC-236 plus radiation repaired it by a factor of 1.1 only. Whereas SC-236 induced apoptosis in these NFSA cells, radiation did not. No further increase in apoptosis was observed when the cells were exposed to both SC-236 and radiation, suggesting that SC-236 did not render tumor cells more susceptible to radiation-induced apoptosis. The RNase protection assay showed that SC-236 (50 microM, 3 days) inhibited the expression of cyclins A and B, as well as cyclin-dependent kinase-1. Inhibition of these cell cycle regulatory elements by SC-236 was associated with the arrest of cells in the radiosensitive G2-M phase (67%), determined by flow cytometry.
  
  
  CONCLUSIONS
  SC-236 significantly enhanced radiosensitivity of tumor cells; the magnitude of sensitivity was dependent on the drug's concentration. The likely mechanisms involve accumulation of cells in the radiosensitive G2-M phase of the cell cycle and inhibition of repair from sublethal radiation damage.
authors.affiliation: Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA. uraju@mdanderson.org
authors.forename: Uma
authors.initials: U
authors.lastname: Raju
authors.forename: Eiko
authors.initials: E
authors.lastname: Nakata
authors.forename: Peiying
authors.initials: P
authors.lastname: Yang
authors.forename: Robert A
authors.initials: RA
authors.lastname: Newman
authors.forename: Kian K
authors.initials: KK
authors.lastname: Ang
authors.forename: Luka
authors.initials: L
authors.lastname: Milas
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004396:Coloring Agents; D019925:Cyclin A / Q000032:analysis; D056750:Cyclin A1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D004249:DNA Damage; D015232:Dinoprostone / Q000032:analysis; D004354:Drug Screening Assays, Antitumor; D020287:In Situ Nick-End Labeling; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D011829:Radiation Dosage; D011836:Radiation Tolerance / Q000187:drug effects*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000528:radiation effects*; D000096926:Benzenesulfonamides
chemical_list: D004396:Coloring Agents; D019925:Cyclin A; D056750:Cyclin A1; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D015232:Dinoprostone; D007527:Isoenzymes; D011451:Prostaglandin-Endoperoxide Synthases; D011720:Pyrazoles; D012333:RNA, Messenger; D011838:Radiation-Sensitizing Agents; D013449:Sulfonamides; D013778:Tetrazolium Salts; D013844:Thiazoles; C119130:4-(5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide; D000096926:Benzenesulfonamides; C503747:Ccna1 protein, mouse; C022616:thiazolyl blue