Silibinin up-regulates DNA-protein kinase-dependent p53 activation to enhance UVB-induced apoptosis in mouse epithelial JB6 cells.
In the present study, we employed a well established JB6 mouse epithelial cell model to define the molecular mechanism of efficacy of a naturally occurring flavonoid silibinin against ultraviolet B (UVB)-induced skin tumorigenesis. UVB exposure of cells caused a moderate phosphorylation of ERK1/2 and Akt and a stronger phosphorylation of p53 at Ser(15), which was enhanced markedly by silibinin pretreatment. Kinase activity of ERK1/2 for Elk-1 and Akt for glycogen synthase kinase-3beta was also potently enhanced by silibinin pretreatment. Furthermore, silibinin increased the UVB-induced level of cleaved caspase 3 as well as apoptotic cells. Based on these observations, next we investigated the role of upstream kinases, ATM/ATR and DNA-PK, which act as sensors for UVB-induced DNA damage and transduce signals leading to DNA repair or apoptosis. Whereas UVB strongly activated ATM as observed by Ser(1981) phosphorylation, it was not affected by silibinin pretreatment. However, pretreatment of cells with the DNA-protein kinase (PK) inhibitor LY294002 strongly reversed silibinin-enhanced Akt-Ser(473) and p53-Ser(15) as well as ERK1/2 phosphorylation together with a dose-dependent decrease in cleaved caspase 3 and apoptosis (p < 0.05). In addition, silibinin pretreatment strongly enhanced H2A.X-Ser(139) phosphorylation and DNA-PK-associated kinase activity as well as the physical interaction of p53 with DNA-PK; pretreatment of cells with LY294002 but not caffeine abolished the silibinin-caused increase in both DNA-PK activation and p53-Ser(15) phosphorylations. Together, these findings suggest that silibinin preferentially activates the DNA-PK-p53 pathway for apoptosis in response to UVB-induced DNA damage, and that this could be a predominant mechanism of silibinin efficacy against UVB-induced skin cancer.
Dhanalakshmi
Sivanandhan
S
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Agarwal
Chapla
C
Singh
Rana P
RP
Agarwal
Rajesh
R
D000818:Animals; D017209:Apoptosis / Q000528:radiation effects*; D064007:Ataxia Telangiectasia Mutated Proteins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004847:Epithelial Cells; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D000077385:Silybin; D012838:Silymarin / Q000494:pharmacology*; D012878:Skin Neoplasms / Q000209:etiology; D014157:Transcription Factors / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D014466:Ultraviolet Rays; D050807:ets-Domain Protein Elk-1
D018797:Cell Cycle Proteins; D002867:Chromones; D004268:DNA-Binding Proteins; C494113:Elk1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D011518:Proto-Oncogene Proteins; D012838:Silymarin; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D050807:ets-Domain Protein Elk-1; C015482:protein kinase modulator; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000077385:Silybin; D004247:DNA; D011494:Protein Kinases; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D038362:Glycogen Synthase Kinase 3; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases