A new class of molecular targeted radioprotectors: GSK-3beta inhibitors.
OBJECTIVE
Development of new treatments is critical to effective protection against radiation-induced injury. We investigate the potential of developing small-molecule inhibitors of glycogen synthase kinase 3beta (GSK-3beta)-SB216763 or SB415286-as radioprotective agents to attenuate intestinal injury.


METHODS
A survival study was done by use of C57BL/6J mice to evaluate the radioprotective effect of GSK-3beta inhibitors. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemical staining for Bax and Bcl-2 were used to assess apoptosis in the small intestines of the treated mice. A clonogenic survival study, apoptosis assays (staining with annexin V or 4',6-diamidino-2-phenylindole), and immunoblot analysis of beta-catenin, Bcl-2, Bax, and caspase 3 were done by use of Rat intestinal epithelial cell line IEC-6 cells.


RESULTS
Pretreatment with SB415286 significantly improved survival of mice irradiated with 8 and 12 Gy. Mice pretreated with SB216763 or SB415286 showed a significant reduction in TUNEL- and Bax-positive cells and an increase in Bcl-2-positive cells in intestinal crypts at 4 and/or 12 h after radiation with 4 and/or 8 Gy compared with radiation alone. Pretreatment of irradiated IEC-6 cells with GSK-3beta inhibitors significantly increased clonogenic survival compared with cells treated with radiation alone. This increase was due to the attenuation of radiation-induced apoptosis, as shown by annexin V and 4',6-diamidino-2-phenylindole assays, as well as immunoblot analysis of Bcl-2, Bax, and caspase 3.


CONCLUSIONS
Glycogen synthase kinase 3beta small-molecule inhibitors protect mouse intestine from radiation-induced damage in cell culture and in vivo and improve survival of mice. Molecular mechanisms of this protection involve attenuated radiation-induced apoptosis regulated by Bcl-2, Bax, and caspase 3. Therefore GSK-3beta inhibitors reduce deleterious consequences of intestinal irradiation and thereby improve quality of life during radiation therapy.
Thotala
Dinesh K
DK
Department of Radiation Oncology, Vanderbilt University School of Medicine, Nashville, TN, USA.
Geng
Ling
L
Dickey
Amy K
AK
Hallahan
Dennis E
DE
Yazlovitskaya
Eugenia M
EM
D000627:Aminophenols / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000032:analysis; D002460:Cell Line; D002470:Cell Survival; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D000071679:Glycogen Synthase Kinase 3 beta; D020287:In Situ Nick-End Labeling / Q000379:methods; D007211:Indoles / Q000494:pharmacology*; D007422:Intestines / Q000528:radiation effects*; D008301:Maleimides / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D011833:Radiation Injuries, Experimental / Q000378:metabolism / Q000517:prevention & control*; D011837:Radiation-Protective Agents / Q000494:pharmacology*; D051381:Rats; D051028:bcl-2-Associated X Protein / Q000032:analysis; D051176:beta Catenin / Q000032:analysis
C417520:3-(3-chloro-4-hydroxyphenylamino)-4-(4-nitrophenyl)-1H-pyrrole-2,5-dione; D000627:Aminophenols; C495267:CTNNB1 protein, mouse; D007211:Indoles; D008301:Maleimides; D011837:Radiation-Protective Agents; C417521:SB 216763; D051028:bcl-2-Associated X Protein; D051176:beta Catenin; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3; D053148:Caspase 3