Abstract
In response to ultraviolet B damage, keratinocytes undergo apoptosis to eliminate damaged cells, thereby preventing tumorigenic transformation. Caffeine, the most widely consumed psychoactive substance, produces complex pharmacological actions; it has been shown to be chemopreventive in non-melamona skin cancer in mice through increasing apoptosis. Here we have investigated the molecular and cellular mechanisms in the pro-apoptotic effect of caffeine on UVB-irradiated human HaCaT keratinocytes. Pretreatment with caffeine increased UVB-induced apoptosis in HaCaT cells. Caffeine blocked UVB-induced Chk1 phosphorylation. In addition, similar to the effect of the PI3K inhibitor LY294002, caffeine also inhibited phosphorylation of AKT and up-regulation of COX-2, two critical oncogenic pathways in skin tumorigenesis. However, phosphorylation of EGFR or ERK was unaffected. Inhibiting ATR pathways by siRNA targeting ATR had little effect on UVB-induced apoptosis or AKT activation, indicating that the inhibitory effect of caffeine on apoptosis and the AKT pathway does not require the ATR pathway. Inhibiting AKT by caffeine blocked UVB-induced COX-2 up-regulation. Expression of constitutively active AKT that was not inhibited by caffeine was found to protect cells from caffeine-promoted apoptosis post-UVB irradiation, indicating that AKT is an essential inhibitory target for caffeine to promote apoptosis. Caffeine specifically sensitized cells with unrepaired DNA damage to UVB-induced apoptosis. These findings indicate that in HaCaT keratinocytes, inhibiting the AKT/COX-2 pathways through an ATR-independent pathway is a critical molecular mechanism by which caffeine promotes UVB-induced apoptosis of unrepaired keratinocytes for elimination.
Highlights
The major environmental factor in skin cancer is ultraviolet radiation B (UVB)2 in sunlight, which causes the formation of DNA damage products, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6 – 4) pyrimidone photoproducts (6 – 4PPs) [3, 4]
To determine whether the decrease in cell viability is caused by increased apoptosis, we used the annexin V/propidium iodide assay followed by flow cytometric analysis to compare apoptosis and necrosis between vehicle and caffeine-treated cells post-UVB
To determine whether inhibiting the ATR/checkpoint kinase-1 (Chk1) pathway is critical for the pro-apoptotic effect of caffeine, we investigated the consequence of ATR inhibition by siRNA targeting ATR on UVB-induced apoptosis of HaCaT, Normal human epidermal keratinocytes (NHEK), and HeLa cells
Summary
The major environmental factor in skin cancer is ultraviolet radiation B (UVB) in sunlight, which causes the formation of DNA damage products, i.e. cyclobutane pyrimidine dimers (CPD) and pyrimidine (6 – 4) pyrimidone photoproducts (6 – 4PPs) [3, 4]. Failure to repair these major DNA damage products is the principal cause of skin cancer. The major regulators of the DNA damage response are the phosphoinositide 3-kinase (PI3K)-related protein kinases (PIKKs), including ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR). ATM activates another checkpoint protein, checkpoint kinase-2 (Chk2) [12,13,14,15] These pathways coordinate the DNA damage checkpoint function. The serine/threonine kinase AKT, known as protein kinase B (PKB), is a downstream effector of phosphatidylinositol 3-kinase (PI3K) that has recently been a focus of intense research It appears that AKT lies at the cross-roads of multiple cellular signaling pathways and acts as a transducer of many functions initiated by growth factors and other receptors that activate PI3K [22,23,24]. Extensive studies have identified multiple mechanisms mediating UVB-induced COX-2 up-regulation [1, 26]
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