(+)-Catechin prevents ultraviolet B-induced human keratinocyte death via inhibition of JNK phosphorylation

High levels of (+)-catechin are found in the skin and seed of many fruits such as apples and grapes. Dietary supplementation with (+)-catechin has been demonstrated to protect epidermal cells against damage induced by ultraviolet B (UVB) radiation. However, the underlying mechanisms are not well understood yet. To determine whether (+)-catechin protects keratinocytes from UVB-induced damage, the viability of UVB- and H2O2-treated cells was determined by cell viability assay. Intracellular H2O2 level was measured by flow cytometry. UVB- or H2O2-induced signaling pathways were detected by Western blotting. The results indicated that (+)-catechin inhibited UVB- and H2O2-induced keratinocyte death. In parallel, intracellular H2O2 generation in keratinocytes irradiated by UVB was inhibited by (+)-catechin in a concentration-dependent manner. (+)-Catechin also inhibited UVB- and H2O2-induced JNK activation in keratinocytes. However, it had little inhibitory effect on UVB- and H2O2-induced ERK and p38 activation even at a higher concentration, suggesting indirectly that JNK activation is required for the induction of apoptosis in keratinocytes exposed to UVB. Finally, we compared the cytotoxicity of (+)-catechin and (-)-epigallocatechin-3-gallate (EGCG) on keratinocytes. Cell viability assay showed that (+)-catechin was relatively nontoxic at higher doses. Taken together, our results demonstrate that (+)-catechin inhibits UVB- and oxidative stress-induced H2O2 production and JNK activation and enhances human keratinocyte survival. However, although it seems that (+)-catechin and EGCG are equally effective in preventing keratinocyte death, (+)-catechin is relatively nontoxic and thus is suitable for developing as an anti-ageing agent for skin care.     

Detection of poly(ADP-ribose) by immunocytochemistry: a sensitive new method for the early identification of UVB- and H2O2-induced apoptosis in keratinocytes

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Poly (ADP-ribose) (PAR) is formed upon activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), and therefore was suggested as a new marker of apoptosis. Since DNA of epidermal cells represents a well-known chromophore for UVB irradiation, and UVB is known to generate H2O2 in keratinocytes, we hypothesized that PAR is a very sensitive marker of UVB- and H2O2-induced apoptosis in keratinocytes. In order to test this hypothesis, human immortalized keratinocytes (HaCaT) were UVB-irradiated or treated with H2O2, and subsequently apoptosis was identified by comparing conventional parameters such as morphological analysis, DNA laddering, and TUNEL assay, with PAR formation. Both, UVB and H2O2 treatment induced PAR formation in HaCaT cells in a dose-dependent manner, and its formation was detected as early as 4 h after irradiation, and at lower UVB doses (10 mJ/cm2) than observed by DNA laddering and the TUNEL assay. In conclusion, the detection of PAR formation is a very sensitive and early method for the identification of apoptotic cells in UVB-induced apoptosis of human keratinocytes.     

Pre-clinical evaluation of cinobufotalin as a potential anti-lung cancer agent

Lung cancer is a major cause of cancer-related mortality in the United States and around the world. Due to the pre-existing or acquired chemo-resistance, the current standard chemotherapy regimens only show moderate activity against lung cancer. In the current study, we explored the potential anti-lung cancer activity of cinobufotalin in vivo and in vitro, and studied the underlying mechanisms. We demonstrated that cinobufotalin displayed considerable cytotoxicity against lung cancer cells (A549, H460 and HTB-58 lines) without inducing significant cell apoptosis. Our data suggest that mitochondrial protein cyclophilin D (Cyp-D)-dependent mitochondrial permeability transition pore (mPTP) opening mediates cinobufotalin-induced non-apoptotic death of lung cancer cells. The Cyp-D inhibitor cyclosporine A (CsA), the mPTP blocker sanglifehrin A (SfA), and Cyp-D shRNA-silencing significantly inhibited cinobufotalin-induced mitochondrial membrane potential (MMP) reduction and A549 cell death (but not apoptosis). Using a mice xenograft model, we found that cinobufotalin inhibited A549 lung cancer cell growth in vivo. Thus, cinobufotalin mainly induces Cyp-D-dependent non-apoptotic death in cultured lung cancer cells. The results of this study suggest that cinobufotalin might be further investigated as a novel anti-lung cancer agent.     

Hydrogen peroxide-induced cell death in normal human keratinocytes is differentiation dependent

More than other tissues, skin is exposed to numerous external stresses generating ROS that, in addition to endogenous oxygen radicals, cause keratinocyte alterations and contribute in part to photocarcinogenesis and aging. Recent evidence suggests a differentiation-dependent susceptibility of keratinocytes to apoptosis. We explored hydrogen peroxide-induced cell death in normal human keratinocytes according to their differentiation. On H(2)O(2)-exposed skin explants, caspase-3 was strongly activated in basal keratinocytes double stained with beta(1) integrin, whereas DNA fragmentation occurred in suprabasal cells only without caspase-3 activation. In addition, isolated basal keratinocytes, selected by adhesion to type IV collagen, were more sensitive than nonadherent cells to H(2)O(2)-induced apoptosis with regard to mitochondrial transmembrane potential (Deltapsi(mt)) collapse and membrane integrity. Similarly, necrotic/late apoptotic cells were present at low levels only in the adherent epidermal population. Furthermore, in primary cultures of undifferentiated keratinocytes H(2)O(2)-induced cell death appeared via a mitochondrial failure. Deltapsi(mt) collapse was associated with a strong early activation of the initiatory caspase-8, then the executive caspase-3, and, to a lesser extent, the inflammatory caspase-1. Finally, undifferentiated basal cells possess a higher sensitivity than differentiated suprabasal cells to H(2)O(2)-induced cell death, and apoptosis in human keratinocytes occurs via different pathways depending on the cell's differentiation state.     

Oxygen Glucose Deprivation (OGD)/Re-Oxygenation-Induced In Vitro Neuronal Cell Death Involves Mitochondrial Cyclophilin-D/P53 Signaling Axis

Oxidative stress-induced neuronal cell death requires opening of the mitochondrial permeability transition pore. P53 mitochondrial translocation and association with Cyclophilin D (Cyp-D) is required for the pore opening. Here we tested this signaling axis in oxygen glucose deprivation (OGD)/re-oxygenation-induced in vitro neuronal death. Using mitochondrion immunoprecipitation, we found that p53 translocated to mitochondrion and associated with Cyp-D in SH-SY5Y cells exposed to (OGD)/re-oxygenation. Disruption of this complex by Cyp-D inhibitor Cyclosporine A (CsA), or by Cyp-D or p53 deficiency, significantly inhibited OGD/re-oxygenation-induced apoptosis-independent cell death. Conversely, over-expression of Cyp-D in SH-SY5Y cells caused spontaneous cell death, and these cells were more vulnerable to OGD/re-oxygenation. Finally, CsA or Cyp-D RNAi suppressed OGD/re-oxygenation-induced neuronal cell death in primary cultures. Together, our study suggests that OGD/re-oxygenation-induced in vitro cell death involves a mitochondrial Cyp-D/p53 signaling axis.     

Differential apoptotic pathways in human keratinocyte HaCaT cells exposed to UVB and UVC

The induction of apoptosis in keratinocytes by ultraviolet (UV)-irradiation is considered to be a protective function against skin cancer. UV-induced DNA damage is a crucial event in UVB- and UVC-mediated apoptosis. However, the differences between the UVB- and UVC-induced apoptotic pathways remain unclear. Here we examine the differential mechanisms by which UVB and UVC irradiations induce keratinocyte apoptosis using human keratinocyte HaCaT cells. Differences in the production of (6-4)photoproducts ((6-4)PPs) and cyclobutane pyrimidine dimers (CPDs) were measured following irradiation with UVB and UVC at doses causing the same extent of apoptotic cell death. In addition, main apoptotic features, such as caspase activation and its regulation, were compared between UVB- and UVC-induced apoptosis. Exposures of 500 J/m² UVB and 100 J/m² UVC resulted in apoptosis to almost the same extent. At these apoptotic doses, the amounts of both (6-4)PPs and CPDs were significantly larger in the case of UVC irradiation than UVB irradiation; in parallel, the release of cytochrome c and Smac/DIABLO and the activation of caspases-9 following UVC irradiation were greater than after UVB irradiation. Importantly, caspase-8 activation occurred only in UVB-irradiated cells. Furthermore, the activation of caspase-8 was not inhibited by caspases-9 and -3 specific tetrapeptide inhibitors, indicating that the caspase-8 cleavage is not due to feedback from activation of caspases-9 and -3. Thus, these results clearly suggest that the reason apoptosis is induced to the same extent by UVB irradiation as by UVC irradiation, despite the lower production of photoproducts in DNA by UVB irradiation, is attributable to the additional activation of the caspase-8 pathway. Thus, UVB irradiation induces apoptosis through both mitochondrial (intrinsic) and caspase-8 activation (extrinsic) pathways, while UVC induces apoptosis only via the intrinsic pathway.     

Normal function of HERG K+ channels expressed in HEK293 cells requires basal protein kinase B activity

In this study, we show that ultraviolet B radiation (UVB)-induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl-2 inhibited UVB-induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)-induced cell death. It also prevented the subsequent activation of procaspase-3 and -8 as well as Bid cleavage in UVB-treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB-induced activation of procaspase-8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL-induced cell death. Our results suggest that Bcl-2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase-8 in UVB-irradiated keratinocytes.     

Cisplatin-induced non-apoptotic death of pancreatic cancer cells requires mitochondrial cyclophilin-D-p53 signaling

The pancreatic cancer remains a fatal disease for the majority of patients. Cisplatin has displayed significant cytotoxic effects against the pancreatic cancer cells, however the underlying mechanisms remain inconclusive. Here, we found that cisplatin mainly induced non-apoptotic death of the pancreatic cancer cells (AsPC-1 and Capan-2), which was associated with a significant p53 activation (phosphorylation and accumulation). Further, activated p53 was found to translocate to mitochondria where it formed a complex with cyclophilin D (Cyp-D). We provided evidences to support that mitochondrial Cyp-D/p53 complexation might be critical for cisplatin-induced non-apoptotic death of pancreatic cancer cells. Inhibition of Cyp-D by its inhibitor cyclosporine A (CsA), or by shRNA-mediated knockdown suppressed cisplatin-induced pancreatic cancer cell death. Both CsA and Cyp-D knockdown also disrupted the Cyp-D/p53 complex formation in mitochondria. Meanwhile, the pancreatic cancer cells with p53 knockdown were resistant to cisplatin. On the other hand, HEK-293 over-expressing Cyp-D were hyper-sensitive to cisplatin. Interestingly, camptothecin (CMT)-induced pancreatic cancer cell apoptotic death was not affected CsA or Cyp-D knockdown. Together, these data suggested that cisplatin-induced non-apoptotic death requires mitochondria Cyp-D-p53 signaling in pancreatic cancer cells.     

Release of cytokines/chemokines and cell death in UVB-irradiated human keratinocytes, HaCaT

Ultraviolet (UV) B can lead to inflammatory responses such as sunburn, which involves the production of various inflammatory cytokines and chemokines, and the induction of cell death. Keratinocytes in the skin has one of the highest risks of exposure to UV. However, the detailed mechanisms underlying UVB irradiation-induced inflammation and cell death are not well known. Thus, we investigated the effect of UVB irradiation on the production of various cytokines/chemokines and the induction of cell death in UVB-irradiated human keratinocytes (HaCaT cells). We evaluated 11 cytokines/chemokines in cell culture supernatants from HaCaT cells exposed to 0-400 mJ/cm(2) UVB irradiation. UVB at a dose 400 mJ/cm(2) induced the release of various cytokines; interleukin (IL)-1beta, IL-6, IL-8, interferon (IFN)-gamma, granulocyte-colony stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, and tumor necrosis factor (TNF)-alpha. These results suggest that UVB irradiation-induced the release of several cytokines/chemokines and led to cell death in human keratinocytes. UV exposure may be associated with multiple physiological events in the human skin.     

Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore

The mitochondrial permeability transition pore (mPTP) opening plays a critical role in mediating cell death during ischemia/reperfusion (I/R) injury. Our previous studies have shown that cysteine 203 of cyclophilin D (CypD), a critical mPTP mediator, undergoes protein S-nitrosylation (SNO). To investigate the role of cysteine 203 in mPTP activation, we mutated cysteine 203 of CypD to a serine residue (C203S) and determined its effect on mPTP opening. Treatment of WT mouse embryonic fibroblasts (MEFs) with H(2)O(2) resulted in an 50% loss of the mitochondrial calcein fluorescence, suggesting substantial activation of the mPTP. Consistent with the reported role of CypD in mPTP activation, CypD null (CypD(-/-)) MEFs exhibited significantly less mPTP opening. Addition of a nitric oxide donor, GSNO, to WT but not CypD(-/-) MEFs prior to H(2)O(2) attenuated mPTP opening. To test whether Cys-203 is required for this protection, we infected CypD(-/-) MEFs with a C203S-CypD vector. Surprisingly, C203S-CypD reconstituted MEFs were resistant to mPTP opening in the presence or absence of GSNO, suggesting a crucial role for Cys-203 in mPTP activation. To determine whether mutation of C203S-CypD would alter mPTP in vivo, we injected a recombinant adenovirus encoding C203S-CypD or WT CypD into CypD(-/-) mice via tail vein. Mitochondria isolated from livers of CypD(-/-) mice or mice expressing C203S-CypD were resistant to Ca(2+)-induced swelling as compared with WT CypD-reconstituted mice. Our results indicate that the Cys-203 residue of CypD is necessary for redox stress-induced activation of mPTP.     

H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation

Ultraviolet radiation (UVR)-induced receptor phosphorylation is increasingly recognized as a widely occurring phenomenon. However, the mechanisms, mediators, and sequence of events involved in this process remain ill-defined. We have recently shown that exposure of human keratinocytes to physiologic doses of ultraviolet B radiation (UVB) activates epidermal growth factor receptor (EGFR)/extracellular-regulated kinase 1 and 2 (ERK1/2), and p38 signaling pathways via reactive oxygen species. Here we demonstrate that UVB exposure increased intra- and extracellular H2O2 production rapidly in a time-dependent manner. An EGFR-specific monoclonal antibody abrogated EGFR autophosphorylation and markedly decreased the phosphorylation of ERK1/2 whereas p38 activation was unaffected. Overexpression of catalase strongly inhibited UVB-induced EGFR/ERK1/2 pathway activation. These findings establish the sequence of events after UVB irradiation: (i) H2O2 generation, (ii) EGFR phosphorylation, and (iii) ERK activation. Our results identify UVB-induced H2O2 as a second messenger that is required for EGFR and dependent downstream signaling pathways activation.     

Sestrin2 Protein Positively Regulates AKT Enzyme Signaling and Survival in Human Squamous Cell Carcinoma and Melanoma Cells

Skin cancer is the most common cancer in the United States and is mainly caused by environmental UV radiation. Reducing skin cancer incidence is becoming an urgent issue. The stress-inducible protein Sestrin2 (Sesn2) plays an important role in maintaining redox and metabolic homeostasis and their related pathologies. However, the role of Sesn2 in cancer remains unclear. Here we show that UVB radiation induces Sesn2 expression in normal human keratinocytes, mouse skin, normal human melanocytes, and melanoma cells. In addition, Sesn2 promotes AKT activation through a PTEN-dependent mechanism. Sesn2 deletion or knockdown sensitizes squamous cell carcinoma (SCC) cells to 5-fluorouracil-induced apoptosis and melanoma cells to UVB- and vemurafenib-induced apoptosis. In mice Sesn2 knockdown suppresses tumor growth from injected human SCC and melanoma cells. Last, as compared with normal skin, Sesn2 is up-regulated in both human skin SCC and melanoma. Our findings demonstrate that Sesn2 promotes AKT activation and survival in response to UVB stress and chemotherapeutics and suggest that Sesn2 is oncogenic in skin SCC and melanoma.     

UVB-based mate-choice cues used by females of the jumping spider Phintella vittata

Although there are numerous examples of animals having photoreceptors sensitive to UVA (315-400 nm) [1] and relying on UVA-based mate-choice cues [2-5], here we provide the first evidence of an animal using UVB (280-315 nm) for intraspecific communication. An earlier study showed that Phintella vittata, a jumping spider (Salticidae) from China, reflects UVB [6]. By performing six series of binary mate-choice experiments in which we varied lighting conditions with filters (UVB+ [no filter] versus UVB-, UVB+ versus ND1, UVB+ versus ND2, UVB- versus ND1, UVB- versus ND2, and UVB- versus UVA-), we show that significantly more UVB + males than UVB- males are chosen by females as preferred mates. Female preference for UVB-reflective males is not affected by differences in brightness or by UVA.     

Vitamin E analog modulates UVB-induced signaling pathway activation and enhances cell survival

We have recently shown that exposure of human keratinocytes to physiologic doses of ultraviolet B (UVB) activates epidermal growth factor receptor (EGFR)/extracellular-regulated kinases 1 and 2 (ERK1/2) and p38 signaling pathways via reactive oxygen species, an effect that can be modulated by antioxidants. Trolox, a water-soluble vitamin E analog, is among the antioxidants that are currently being investigated for their preventive and protective potential against harmful effects of UV radiation to the skin. We found that Trolox inhibits both basal and UVB-induced intracellular H(2)O(2) generation in primary keratinocytes in a concentration-dependent manner. Trolox did not significantly affect UVB-induced phosphorylation of EGFR. Stronger inhibition was observed for ERK1/2 activation at lower, and for p38 activation at higher, concentrations of Trolox added to cells before exposure to UVB. Similarly different effects were found with regard to length of pretreatment with Trolox before UVB exposure-increasing inhibition for ERK1/2 activation at shorter, and for p38 activation at longer, pretreatment intervals. UVB-induced c-jun-N-terminal kinase activation was potently suppressed by Trolox. Also, increasing the pretreatment time of Trolox decreased the rate of cell death following UVB. In conclusion, UVB-induced signaling pathway activation is differentially modulated by Trolox. Further investigation into the time-dependent biologic activation of Trolox and its metabolic products, and modulation of signal transduction with cell outcome should facilitate development of rational strategies for pharmacologic applications.     

Novel anti-inflammatory peptides as cosmeceutical peptides

Ultraviolet (UV) radiation induced inflammation plays an important role in the aging of human skin. Prostaglandin (PG) E₂ is the primary mediator of UVB induced photoinflammation. We screened an internal library for dipeptides that inhibited UVB induced PGE₂ synthesis but showed no cytotoxicity toward human keratinocytes. We identified three highly active inhibitory sequences, LE (Leu + Glu), MW (Met + Trp) and MY (Met + Tyr). To evaluate their efficacy in human skin, 24 sites of abdomen skin were irradiated with a 308 nm excimer laser (300 mJ/cm²), after which 2% LE, MW, MY or a control were applied to the irradiated sites for 24 h. The erythema index (EI) was measured before and 24 h after treatment. The results showed that LE and MW significantly decreased UVB induced erythema (p = 0.041 and p = 0.036, respectively), but ME did not. Overall, LE and MW are candidate cosmeceutical peptides that can protect skin from UVB induced photoinflammation.     

Protective effects of silkworm hemolymph extract and its fractions on UV-induced photoaging

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.     

Oxidative stress mediates cell surface expression of SS-A/Ro antigen on keratinocytes

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.