Long-term effects of alendronate on fracture healing and bone remodeling of femoral shaft in ovariectomized rats
Abstract
Ling-jie FU1, 2, Ting-ting TANG1, Yong-qiang HAO1, Ke-rong DAI1, 2, *
1Department of Orthopedic Surgery, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China; 2Orthopedic Cellular and Molecular Biology Laboratory, Institute of Health Sciences, Chinese Academy of Sciences, and Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
Aim: Benzothiophene compounds are selective estrogen receptor modulators (SERMs), which are recently found to activate antioxidant signaling. In this study the molecular mechanisms of antioxidant signaling activation by benzothiophene compound BC-1 were investigated.
Methods: HepG2 cells were stably transfected with antioxidant response element (ARE)-luciferase reporter (HepG2-ARE cells). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in HepG2-ARE cells was suppressed using siRNA. The metabolites of BC-1 in rat liver microsome incubation were analyzed using LC-UV and LC-MS.
Results: Addition of BC-1 (5 μmol/L) in HepG2-ARE cells resulted in a 17-fold increase of ARE-luciferase activity. Pretreatment with the estrogen receptor agonist E2 (5 μmol/L) or antagonist ICI 182,780 (5 μmol/L) did not affect BC-1-induced ARE-luciferase activity. However, transfection of the cells with anti-Nrf2 siRNA suppressed this effect by 79%. Addition of BC-1 in rat microsome incubation resulted in formation of di-quinone methides and o-quinones, followed by formation of GSH conjugates. BC-1 analogues with hydrogen (BC-2) or fluorine (BC-3) at the 4' position did not form the di-quinone methides. Both BC-2 and BC-3 showed comparable estrogenic activity with BC-1, but did not induce ARE-luciferase activity in HepG2-ARE cells.
Conclusion: Benzothiophene compound BC-1 activates ARE signaling via reactive metabolite formation that is independent of estrogen receptors.
Keywords: selective estrogen receptor modulator; benzothiophene compound; E2; ICI 182,780; antioxidant response element (ARE); reactive metabolites; quinoid formation; nuclear factor erythroid 2-related factor 2 (Nrf2); siRNA
This work was supported by the Key Project of the Science and Technology Commission of Shanghai Municipality (Grant No 05JC14034), the Selecting and Training Outstanding Young Teachers in Shanghai Universities (Grant No jdy08068), the National Natural Science Foundation of China (Grant No 81201424), and the Program for Shanghai Key Laboratory of Orthopaedic Implant (Grant No 08DZ2230330).
* To whom correspondence should be addressed.
E-mail krdai@163.com
Received 2012-07-29 Accepted 2012-11-15
Keywords:
1Department of Orthopedic Surgery, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China; 2Orthopedic Cellular and Molecular Biology Laboratory, Institute of Health Sciences, Chinese Academy of Sciences, and Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
Aim: Benzothiophene compounds are selective estrogen receptor modulators (SERMs), which are recently found to activate antioxidant signaling. In this study the molecular mechanisms of antioxidant signaling activation by benzothiophene compound BC-1 were investigated.
Methods: HepG2 cells were stably transfected with antioxidant response element (ARE)-luciferase reporter (HepG2-ARE cells). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in HepG2-ARE cells was suppressed using siRNA. The metabolites of BC-1 in rat liver microsome incubation were analyzed using LC-UV and LC-MS.
Results: Addition of BC-1 (5 μmol/L) in HepG2-ARE cells resulted in a 17-fold increase of ARE-luciferase activity. Pretreatment with the estrogen receptor agonist E2 (5 μmol/L) or antagonist ICI 182,780 (5 μmol/L) did not affect BC-1-induced ARE-luciferase activity. However, transfection of the cells with anti-Nrf2 siRNA suppressed this effect by 79%. Addition of BC-1 in rat microsome incubation resulted in formation of di-quinone methides and o-quinones, followed by formation of GSH conjugates. BC-1 analogues with hydrogen (BC-2) or fluorine (BC-3) at the 4' position did not form the di-quinone methides. Both BC-2 and BC-3 showed comparable estrogenic activity with BC-1, but did not induce ARE-luciferase activity in HepG2-ARE cells.
Conclusion: Benzothiophene compound BC-1 activates ARE signaling via reactive metabolite formation that is independent of estrogen receptors.
Keywords: selective estrogen receptor modulator; benzothiophene compound; E2; ICI 182,780; antioxidant response element (ARE); reactive metabolites; quinoid formation; nuclear factor erythroid 2-related factor 2 (Nrf2); siRNA
This work was supported by the Key Project of the Science and Technology Commission of Shanghai Municipality (Grant No 05JC14034), the Selecting and Training Outstanding Young Teachers in Shanghai Universities (Grant No jdy08068), the National Natural Science Foundation of China (Grant No 81201424), and the Program for Shanghai Key Laboratory of Orthopaedic Implant (Grant No 08DZ2230330).
* To whom correspondence should be addressed.
E-mail krdai@163.com
Received 2012-07-29 Accepted 2012-11-15