p38 MAP kinase plays a functional role in UVB-induced mouse skin carcinogenesis.

UVB irradiation of epidermal keratinocytes results in the activation of the p38 mitogen-activated protein kinase (MAPK) pathway and subsequently activator protein-1 (AP-1) transcription factor activation and cyclooxygenase-2 (COX-2) expression. AP-1 and COX-2 have been shown to play functional roles in UVB-induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH-1 hairless mice and assess UVB-induced AP-1 activation, COX-2 expression, and the skin carcinogenesis response in these mice compared to wild-type littermates. We observed a significant inhibition of UVB-induced AP-1 activation and COX-2 expression in p38DN transgenic mice, leading to a significant reduction of UVB-induced tumor number and growth compared to wild-type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and growth rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki-67 staining in p38DN mice compared to wild-type. Although we detected no difference in chronic apoptotic rates between transgenic and nontransgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB-induced apoptosis of keratinocytes. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV-induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX-2 expression and proliferation of UVB-irradiated cells.

Quercetin potentiates UVB-Induced c-Fos expression: implications for its use as a chemopreventive agent.

Quercetin (Qu) is currently being investigated as a chemopreventive agent for several cancers, including nonmelanoma skin cancer induced by UV light. We previously reported that Qu degradation has important consequences on signaling and cell biology. In the current study, we report that Qu induces c-Fos mRNA and protein expression through activation of p38 and cAMP-responsive element binding protein (CREB), and Qu potentiates UVB-induced c-Fos expression. Inclusion of ascorbic acid (AA) in cell culture medium stabilizes Qu and completely prevents both Qu- and UVB-induced p38 and CREB activation, leading to a blockade of c-fos gene expression through reduced CREB/cAMP-responsive element binding. AA stabilizes c-Fos mRNA, increasing steady-state levels even when c-fos gene expression is suppressed, but this has no effect on c-Fos protein levels in either mock- or UVB-irradiated cells. We report that Qu blocks mammalian target of rapamycin signaling and inhibits c-Fos protein expression directly through this mechanism because cotreatment with Qu and AA resulted in the complete suppression of UVB-induced c-Fos protein expression even in the presence of significantly increased mRNA levels. We further confirmed that this was not due to increased protein turnover because inhibition of proteasome activity with MG-132 did not raise c-Fos protein levels in Qu+AA-treated cells. Together, these data indicate that although Qu has been reported to have some beneficial properties as a chemopreventive agent, it is also capable of inducing c-fos expression, a cellular event important for the promotion phase of tumor development, if it is not stabilized.

Inhibition of activator protein-1 by sulforaphane involves interaction with cysteine in the cFos DNA-binding domain: implications for chemoprevention of UVB-induced skin cancer.

Sulforaphane is an isothiocyanate derived from cruciferous vegetables that has been linked to decreased risk of certain cancers. Although the role of sulforaphane in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor activator protein-1 (AP-1) may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that sulforaphane is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinoma in a mouse model using cotreatment with the compound and the carcinogen. We also show that sulforaphane pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by sulforaphane. Electrophoretic mobility shift assay analysis of nuclear proteins also shows that sulforaphane and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun, we show that mutation of critical cysteines in the DNA-binding domain of these proteins (Cys(154) in cFos and Cys(272) in cJun) results in loss of sensitivity to both sulforaphane and diamide in electrophoretic mobility shift assay analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of squamous cell carcinoma by sulforaphane.

Stabilization of quercetin paradoxically reduces its proapoptotic effect on UVB-irradiated human keratinocytes.

UVB light promotes survival of initiated keratinocytes, in part, by the direct activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway. Novel chemopreventative agents targeting UVB-induced signaling pathways are needed to reduce the incidence of nonmelanoma skin cancer. Quercetin (Qu) is a dietary flavonoid and a known inhibitor of PI3K. We determined that Qu degrades rapidly when diluted in DMEM and incubated under normal cell culture conditions. Degradation was delayed by supplementing the medium with 1 mmol/L ascorbic acid (AA), and as expected, stabilization actually increased the effectiveness of Qu as a PI3K inhibitor because basal and UVB-induced Akt phosphorylation were reduced compared with Qu treatment in the absence of AA. Although AA stabilization increased Qu-induced apoptosis in mock-irradiated HaCaT cells, consistent with it acting as a PI3K inhibitor (13.4% Annexin V-positive cells for AA-stabilized Qu versus 6.3% for Qu), AA stabilization of Qu actually reduced the ability of the compound to induce apoptosis of UVB-irradiated HaCaTs (29.7% of Qu-treated cells versus 15.5% of AA + Qu-treated cells). Similar trends were seen in the analysis of caspase-3 and poly(ADP-ribose) polymerase cleavage. Qu is known to oxidize to form reactive products, and we found that dihydroethidium is oxidized by Qu regardless of whether or not it was stabilized. Although redox cycling occurs even in the presence of AA, stabilization reduces the accumulation of reactive Qu products that contribute to the proapoptotic effect of the compound, and thus reduces the ability of the compound to induce apoptosis of UVB-irradiated HaCaT cells.