Silibinin Up-regulates DNA-Protein Kinase-dependent p53 Activation to Enhance UVB-induced Apoptosis in Mouse Epithelial JB6 Cells

Abstract

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-3␤ was also potently enhanced by silibinin pretreatment. Furthermore, silibinin increased the UVBinduced 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-PKassociated 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. Ultraviolet B (UVB)1 radiation plays a major role in the development of non-melanoma skin cancer, which is the most common human malignancy in the United States (1). In recent years, chemoprevention is considered to be a promising strategy for the control of this malignancy, where several studies have shown that various phytochemicals from both nutritive and non-nutritive sources protect against UVB-induced skin cancer (2-6). In this regard, silymarin (a polyphenolic flavonoid antioxidant from milk thistle) and silibinin (the bioactive component in silymarin) have also shown strong potential in preventing UV-induced skin damages and photocarcinogenesis (2, 5-7). We have demonstrated the chemopreventive efficacy of silymarin/silibinin in the SKH-1 mouse skin model, where these agents showed strong protection against UVBinduced tumor initiation, tumor promotion, and complete carcinogenesis (2). We also found that silymarin protects against chemical carcinogenesis in SENCAR mouse skin by modulating mitogen-activated protein kinases (MAPKs) and inducing apoptosis (3). In a more recent study, we observed a strong protective effect of silibinin against UVB-induced skin carcinogenesis in SKH-1 mice where topical applications before o...

Silibinin attenuates methotrexate-induced pulmonary injury by targeting oxidative stress

“…Previous studies have reported that silibinin targets multiple signaling pathways, including those associated with oxidative stress and inflammation, to subsequently prevent tissue injuries and cancer by genotoxicity and other agents, which are similar to the pathways triggered following vesicant exposure (22,24,25). Therefore, silibinin was hypothesized to exhibit notable efficacy in attenuating MTX-induced lung injury, since MTX is known to trigger oxidative stress.…”

Silymarin exacerbates p53-mediated tubular apoptosis in glycerol-induced acute kidney injury in rats

“…Several studies have shown their capacity to decrease tumor cell proliferation and induce apoptosis in cancer cell lineages, endothelial cells, or normal cells exposed to carcinogenic stimuli. [5][6][7][8][9] The cornerstone of the proapoptotic action of silymarin/silibinin is the activation of the tumor suppressor gene p53. [6][7][8][9] Several downstream effectors of p53 activation have been implicated in silymarin-induced apoptosis, such as increased expression of proapoptotic Bax protein as well as reduction of the anti-apoptotic proteins Bcl-2, Bcl-xl, and survivin.…”

“…[5][6][7][8][9] The cornerstone of the proapoptotic action of silymarin/silibinin is the activation of the tumor suppressor gene p53. [6][7][8][9] Several downstream effectors of p53 activation have been implicated in silymarin-induced apoptosis, such as increased expression of proapoptotic Bax protein as well as reduction of the anti-apoptotic proteins Bcl-2, Bcl-xl, and survivin. [6][7][8][9] In this study, we evaluated the effects of silymarin treatment on renal function and histopathology in glycerol-induced acute kidney injury (Gly-AKI) in rats.…”

Silibinin synergizes with mitoxantrone to inhibit cell growth and induce apoptosis in human prostate cancer cells

“…Recent data using mouse epidermal keratinocyte JB6 cells and ultraviolet light B have given insight into silibinin's activity with DNA-damaging agents. 41 In these experiments, the addition of silibinin increased the percentage of apoptotic cells when compared to the ultraviolet light B treatments alone. Silibinin's enhancement of ultraviolet light B- induced apoptosis was seen with an upregulation of DNA protein kinase, an important element in sensing genotoxic stress, with this effect abrogated by the addition of a semi-selective inhibitor of DNA protein kinase.…”

“…41,42 Of the 3 cell types examined here, LNCaP cells are considered to have wild type p53, PC-3 cells have a deletion in codon 138 with negative immunohistochemical staining for p53, and DU145 cells have 2 mutations in codons 223 and 274 of p53, but stain positive for p53 expression. 43 Notably, the PC-3 cells showed little apoptosis in response to either low-dose silibinin or mitoxantrone; however, the combination demonstrated a significant increase.…”