Apoptotic effects of hydrogen peroxide and vitamin C on chicken embryonic fibroblasts: redox state and programmed cell death

The pro-apoptotic effects of hydrogen peroxide and the purported anti-apoptotic effects of Vitamin C on chicken embryonic fibroblasts were investigated. Hydrogen peroxide induced morphological changes in a dose dependent manner, and a myriad of autophagosomes were observed using transmission electron microscopy. Doxorubicin elicited alterations were not inhibited by co-incubation with Vitamin C except that mitochondrial structure was slightly improved. TUNEL assay, cytotoxicity analysis and flow cytometry revealed that the cytotoxicity, DNA fragmentation and apoptotic rates were dose dependent upon treatment with hydrogen peroxide. Calcium homeostasis was disrupted in a dose dependent manner, and cell cycle was blocked at G₂/M checkpoint at low concentration and S/G₂ checkpoint at high concentration respectively upon treatment with hydrogen peroxide. The administration of Vitamin C only has a modest effect against doxorubicin induced apoptosis, calcium homeostasis disruption and cell cycle arrest. This research demonstrated that the elevation of reactive oxygen species is sufficient to induce the apoptosis of chicken embryonic fibroblasts, whereas the administration of Vitamin C does not necessarily have certain anti-apoptotic effects, especially when the stimulus is not directly linked with redox state.     

Potentiation of UVB-induced apoptosis by novel phytosphingosine derivative, tetraacetyl phytosphingosine in HaCaT cell and mouse skin

Inappropriate apoptosis results in the epidermal hyperplasia as in psoriasis and UVB irradiation has been successfully used to treat this kind of skin disorders. Previously, we reported that the novel phytosphingosine derivative, tetraacetyl phytosphingosine (TAPS) induced apoptosis in HaCaT cells. This study examined the effect of UVB irradiation and/or TAPS on the induction of apoptosis in HaCaT. 10 mJ/cm2 of UVB irradiation or 10 microM of TAPS alone exhibited weak cytotoxicity but co-treatment of UVB and TAPS synergistically enhanced the cytotoxicity and apoptosis in HaCaT. The cells treated with UVB and TAPS showed much higher levels of cleaved caspase-3, -8, -9 and Bax than with UVB or TAPS alone, whereas Bcl-2 level was decreased by co-administration of UVB and TAPS. In hairless mice, co-treatment of UVB and TAPS synergistically increased apoptosis, as shown in the HaCaT co-treated with UVB and TAPS. Furthermore, UVB irradiation caused an increase of apoptotic cells in the epidermis and the TAPS-treated mice showed an increase of apoptotic cells in the dermis as well as in the epidermis. These results suggest that the TAPS co-treatment synergistically increases the level of UVB-induced apoptosis via caspase activation by regulating the level of pro-apoptotic Bax and anti-apoptotic Bcl-2.     

Polypeptide from Chlamys farreri protects HaCaT cells from UVB-induced apoptosis

A natural polypeptide from marine Chlamys farreri (a kind of scallop) (PCF), has been recently been found to be an effective photoprotective agent against ultraviolet rays B (UVB)-induced mitochondria damage in normal human fibroblasts. To investigate whether PCF has the antiapoptotic effect on human keratinocytes, in the present study, we established an apoptotic model on HaCaT cell line by means of UVB radiance of 30 mJ/cm(2) and compared the effect of different PCF treatments on UVB-radiated cells. Flow cytometry analyses showed that PCF treatment before UVB-irradiation inhibited UVB-induced apoptosis, the loss of mitochondrial membrane potential (Deltapsim) and the increase of free Ca(2+) level in HaCaT cells. In parallel with these results, UVB-irradiation enhanced activities of caspases-3, 8, 9, while this enhancement was inhibited by PCF treatment prior to irradiation. PCF added after irradiation neither reduced UVB-induced activities of the three caspases nor synergized the effect of pre-added PCF. Cellular ultrastructural features obtained from transmission electron microscopy further confirmed the antiapoptotic effect of PCF pre-treatment. It is concluded that the antiapoptotic effect of PCF is not therapeutic but prophylactic. Caspases-3, 8, 9, Deltapsim and calcium are involved in UVB-induced apoptosis, while prophylactic PCF inhibits apoptosis of UVB-irradiated HaCaT cells by blocking the caspases activities, the Deltapsim lost and the elevation of intracellular free Ca(2+) level.     

HPV-16 E6/7 immortalization sensitizes human keratinocytes to ultraviolet B by altering the pathway from caspase-8 to caspase-9-dependent apoptosis

UVB from solar radiation is both an initiating and promoting agent for skin cancer. We have found that primary human keratinocytes undergo an apoptotic response to UVB. To determine whether these responses are altered during the course of immortalization, we examined markers of apoptosis in primary human foreskin keratinocytes (HFK) transduced with either a retroviral vector expressing the E6 and E7 genes of HPV-16 or with empty vector alone (LXSN-HFK). Whereas LXSN-HFK as well as early passage keratinocytes expressing HPV-16 E6 and E7 (p7 E6/7-HFK) were both moderately responsive to UVB irradiation, late passage-immortalized keratinocytes (p27 E6/7-HFK) were exquisitely sensitive to UVB-induced apoptosis. After exposure to UVB, enhanced annexin V-positivity and internucleosomal DNA fragmentation were observed in p27 E6/7-HFK compared with either LXSN- or p7 E6/7-HFK. Caspase-3 fluorometric activity assays as well as immunoblot analysis with antibodies to caspase-3 and poly(ADP-ribose) polymerase revealed elevated caspase-3 activity and processing at lower UVB doses in p27 E6/7-HFK compared with LXSN- or p7 E6/7-HFK. In addition, the caspase inhibitor DEVD-CHO reduced the apoptotic response and increased survival of all three HFK types. Immunoblot analysis revealed that caspase-8 was activated in all three cell types, but caspase-9 was only activated in p27 E6/7-HFK. Cell cycle analysis further showed that only p27 E6/7-HFK exhibit G(2)/M accumulation that is enhanced by UVB treatment. This accumulation was associated with a rapid down-regulation of Bcl-2 in these cells. The immortalization process subsequent to the expression of HPV E6 and E7 may therefore determine UVB sensitivity by switching the mode of apoptosis from a caspase-8 to a Bcl-2-caspase-9-mediated pathway of apoptosis.     

Defining the DNA mismatch repair-dependent apoptotic pathway in primary cells: Evidence for p53-independence and involvement of centrosomal caspase 2

Many studies have shown that DNA mismatch repair (MMR) has a role beyond that of repair in response to several types of DNA damage, including ultraviolet radiation (UV). We have demonstrated previously that the MMR-dependent component of UVB-induced apoptosis is integral to the suppression of UVB-induced tumorigenesis. Here we demonstrate that Msh6-dependent UVB-induced apoptotic pathway is both activated via the mitochondria and p53-independent. In addition, we have shown for the first time that caspase 2, an initiator caspase, localizes to the centrosomes in mitotic primary mouse embryonic fibroblasts, irrespective of MMR status and UVB treatment.     

p38 mitogen-activated protein kinase regulates a novel, caspase-independent pathway for the mitochondrial cytochrome c release in ultraviolet B radiation-induced apoptosis

The mechanisms of UVB-induced apoptosis and the role of p38 mitogen-activated protein kinase (MAPK) were investigated in HaCaT cells. UVB doses that induced apoptosis also produced a sustained activation of p38 MAPK and mitochondrial cytochrome c release, leading to pro-caspase-3 activation. Late into the apoptotic process, UVB also induced a caspase-mediated cleavage of Bid. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone substantially blocked the UVB-induced apoptosis without preventing the release of mitochondrial cytochrome c and the p38 MAPK activation. The inhibition of p38 MAPK counteracted both apoptosis and cytochrome c release as well as the DEVD-amino-4-methylcoumarin cleavage activity without affecting the processing of pro-caspase-8. These results indicate that UVB induces multiple and independent apoptotic pathways, which culminate in pro-caspase-3 activation, and that the initial cytochrome c release is independent of caspase activity. Importantly, we show that a sustained p38 MAPK activation contributes to the UVB-induced apoptosis by mediating the release of mitochondrial cytochrome c into the cytosol.     

Silk sericin protein of tropical tasar silkworm inhibits UVB-induced apoptosis in human skin keratinocytes

The silk protein sericin has been identified as a potent antioxidant and photoprotective agent against ultraviolet B (UVB) irradiation in mouse skin model. In this study, we have investigated the anti-apoptotic effect of sericin in UVB (30 mJ/cm²)-irradiated human keratinocytes. Flow cytometry analysis has shown that pre-treatment with sericin inhibits UVB-induced apoptosis. The pre-treatment with sericin suppresses bax expression, up-regulates the expression of bcl-2, prevents both the activation of caspase-3 and cleavage of Poly (ADP-ribose) polymerase. Generation of intracellular hydrogen peroxide in UVB-treated keratinocytes is inhibited through pre-treatment with sericin suggesting that sericin probably prevents mitochondrial damage. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Insulin-like growth factor-1-mediated AKT activation postpones the onset of ultraviolet B-induced apoptosis,providing more time for cyclobutane thymine dimer removal in primary human keratinocytes

Insulin-like growth factor-1 (IGF-1) acts as a potent survival factor in numerous cell lines, primarily through activation of the AKT signaling pathway. Although some targets of this pathway have known anti-apoptotic functions, its relationship with the improved survival of cells after exposure to environmental stresses, including UVB, remains largely unclear. We report that in growth factor-deprived keratinocytes, IGF-1 significantly and consistently delayed the onset of UVB-induced apoptosis by >7 h. This delay allowed IGF-1-supplemented keratinocytes to repair significantly more cyclobutane thymine dimers than their growth factor-deprived counterparts. This increase in cyclobutane thymine removal resulted in enhanced survival if the amount of DNA damage was not too high. To increase cell survival after UVB irradiation, IGF-1 supplementation was required only during this initial time period in which extra repair was executed. Finally, we show that IGF-1 mediated this delay in the onset of UVB-induced apoptosis through activation of the AKT signaling pathway. We therefore believe that the AKT signaling pathway increases cell survival after a genotoxic insult such as UVB irradiation not by inhibiting the apoptotic stimulus, but only by postponing the induction of apoptosis, giving the DNA repair mechanism more time to work.     

Hepatocyte growth factor/scatter factor inhibits UVB-induced apoptosis of human keratinocytes but not of keratinocyte-derived cell lines via the phosphatidylinositol 3-kinase/AKT pathway

Acute irreparable UV-induced DNA damage leads to apoptosis of epidermal keratinocytes (KC) and the formation of sunburn cells, whereas less severely damaged cells survive but harbor the potential of tumor formation. Here we report that hepatocyte growth factor/scatter factor (HGF/SF) prevents UVB-induced apoptosis in primary KC cultured in vitro. When we analyzed the signaling pathways initiated by the HGF/SF receptor c-met, we found that the phosphatidylinositol (PI) 3-kinase and its downstream-element AKT and the mitogen-activated protein (MAP) kinase were activated. Inhibition of PI 3-kinase led to a complete abrogation of the anti-apoptotic effect of HGF/SF, whereas blockade of the MAP kinase pathway had no effect. In contrast to the observation with primary KC, HGF/SF could not enhance survival after UVB irradiation of HaCaT and A431 cell lines, despite the fact that in these cells the PI 3-kinase and MAP kinase pathways were also activated by HGF/SF. Cell cycle analysis of KC revealed a G(2)/M arrest after UVB irradiation and a complete loss of proliferating cells. Because HGF/SF in the skin is produced by dermal fibroblasts, our findings suggest that the HGF/SF-mediated rescue of KC from apoptosis represents an important paracrine loop by which UVB-damaged KC can be kept alive to maintain the epidermal barrier function but cannot further proliferate, thereby preventing the induction of epithelial skin tumors.     

Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice

UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.     

UVB induced oxidative stress in human keratinocytes and protective effect of antioxidant agents

This study aims at exploring the oxidative stress in keratinocytes induced by UVB irradiation and the protective effect of nutritional antioxidants. Cultured Colo-16 cells were exposed to UVB in vitro followed by measurement of reactive oxygen species (ROS), endogenous antioxidant enzyme activity, as well as cell death in the presence or absence of supplementation with vitamin C, vitamin E, or Ginsenoside Panoxatriol. Intracellular ROS content was found significantly reduced 1 h after exposure, but increased at later time points. After exposure to 150–600 J m⁻² UVB, reduction of ROS content was accompanied by increased activity of catalase and CuZn-superoxide dismutase at early time points. Vitamins C and E, and Ginsenoside Panoxatriol counteracted the increase of ROS in the Colo-16 cells induced by acute UVB irradiation. At the same time, Ginsenoside Panoxatriol protected the activity of CuZn-superoxide dismutase, while vitamin E showed only a moderate protective role. Vitamins C and E, and Ginsenoside Panoxatriol in combination protected the Colo-16 cells from UVB-induced apoptosis, but not necrosis. These findings suggest that vitamins C and E as well as Ginsenoside Panoxatriol are promising protective agents against UVB-induced damage in skin cells.     

Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes

The p38 MAPK pathway controls critical premitochondrial events culminating in apoptosis of UVB-irradiated human keratinocytes, but the upstream mediators of this stress signal are not completely defined. This study shows that in human keratinocytes exposed to UVB the generation of reactive oxygen species (ROS) acts as a mediator of apoptosis signal regulating kinase-1 (Ask-1), a redox-sensitive mitogen-activated protein kinase kinase kinase (MAP3K) regulating p38 MAPK and JNK cascades. The NADPH oxidase antagonist diphenylene iodonium chloride and the EGFR inhibitor AG1487 prevent UVB-mediated ROS generation, the activation of the Ask-1-p38 MAPK stress response pathway, and apoptosis, evidencing the link existing between the early plasma membrane-generated ROS and the activation of a lethal cascade initiated by Ask-1. Consistent with this, Ask-1 overexpression considerably sensitizes keratinocytes to UVB-induced mitochondrial apoptosis. Although the JNK pathway is also stimulated after UVB, the killing effect of Ask-1 overexpression is reverted by p38 MAPK inhibition, suggesting that Ask-1 exerts its lethal effects mainly through the p38 MAPK pathway. Moreover, p38alpha(-/-) murine embryonic fibroblasts are protected from UVB-induced apoptosis even if JNK activation is fully preserved. These results argue for an important role of the UVB-generated ROS as mediators of the Ask-1-p38 MAPK pathway that, by culminating in apoptosis, restrains the propagation of potentially mutagenic keratinocytes.     

Elevated miR-34c-5p Mediates Dermal Fibroblast Senescence by Ultraviolet Irradiation

Previous studies showed that several miRNAs can regulate pathways involved in UVB-induced premature senescence and response to ultraviolet irradiation. It has also been reported that miR-34c-5p may be involved in senescence-related mechanisms. We propose that miR-34c-5p may play a crucial role in senescence of normal human primary dermal fibroblasts. Here, we explored the roles of miR-34c-5p in UVB-induced premature senescence on dermal fibroblasts. MiR-34c-5p expression was increased in dermal fibroblasts after repeated subcytotoxic UVB treatments. Underexpression of miR-34c-5p in dermal fibroblasts led to a marked delay of many senescent phenotypes induced by repeated UVB treatments. Furthermore, underexpression of miR-34c-5p in dermal fibroblasts can antagonize the alteration of G1-arrested fibroblasts. Moreover, E2F3, which can inactivate p53 pathway and play a role in cell cycle progression, is a down-stream target of miR-34c-5p. Forced down-expression of miR-34c-5p decreased the expression of UVB-SIPS induced P21 and P53 at both mRNA and protein levels. Our data demonstrated that down-regulation of miR-34c-5p can protect human primary dermal fibroblasts from UVB-induced premature senescence via regulations of some senescence-related molecules.     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

Fas Antigen Modulates Ultraviolet B-Induced Apoptosis of SVHK Cells: Sequential Activation of Caspases 8, 3, and 1 in the Apoptotic Process

Interferon-gamma (IFN-gamma) induces various apoptosis-related proteins, including Fas antigen (Fas) in keratinocytes. Ultraviolet B (UVB) irradiation produces "sunburn cells," a specific type of apoptosis. Previously, we reported that IFN-gamma augments Fas-dependent apoptosis of SV40-transformed human keratinocytes (SVHK cells). Caspases are a new class of cysteine proteinases that play an important role in apoptosis. We investigated the mechanism of UVB-induced apoptosis by examining activation of the caspase cascade. UVB irradiation of SVHK cells increased the activities of caspases 1, 3, and 8, which were detected at 3 h, and peak activities occurred at 6 h. Pretreatment of SVHK cells with IFN-gamma significantly increased the activity of caspases 1, 3, and 8. UVB-induced caspase 8 stimulation was significantly suppressed only by caspase 8 inhibitor, while inhibitors of caspases 1, 3, and 8 significantly suppressed UVB-induced caspase 1 stimulation. Caspase 3 and 8 inhibitors, but not caspase 1 inhibitor, significantly suppressed UVB-induced caspase 3 activity, suggesting sequential activation of caspases 8, 3, and 1 in UVB-irradiated SVHK cells. Cross-linking and immunoprecipitation analyses showed multimerization of Fas antigen following UVB irradiation of SVHK cells. Pretreatment of SVHK cells with IFN-gamma significantly augmented UVB-induced apoptosis that was accompanied by increased Fas expression. The susceptibility to UVB-induced apoptosis was also increased in Fas-transfected SVHK cells (F2 cells). Neutralizing anti-Fas antibody significantly suppressed caspase activation and Fas-dependent apoptosis of SVHK cells and F2 cells. In contrast, UVB-induced caspase activation and apoptosis were not inhibited by neutralizing anti-Fas antibody in both cell lines. Our results suggest that UVB directly activates Fas and subsequent caspase cascade resulting in apoptosis of SVHK cells. Furthermore, the expression level of Fas antigen in keratinocytes influenced their susceptibility to UVB-induced apoptosis.     

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.     

Up-Regulation and redistribution of Bax in ultraviolet B-irradiated melanocytes

Ultraviolet B (UVB) radiation may activate or deteriorate cultured human epidermal melanocytes, depending on the doses and culture conditions. It is also reported that cultured human epidermal melanocytes derived from different pigmentary phenotypes showed different responses to UVB radiation. In this study, we examined whether apoptosis of melanocytes can be induced by physiologic doses of UVB irradiation using cultured human epidermal melanocytes derived from oriental males of skin types III and IV. Propidium iodide staining for DNA condensation and flow cytometric analyses demonstrated the apoptotic cell death of melanocytes following UVB irradiation (0-30 mJ/cm2). The levels of p53, Bax, and Bcl-2, determined by immunoblotting, revealed a dose-dependent increase in p53 and Bax, but the level of Bcl-2 remained unchanged. Confocal microscopic examination showed that Bax moved from a diffuse to a punctate distribution after UVB irradiation. However, there were no changes in the pattern of distribution of Bcl-2. These data suggest that the high constitutional level of Bcl-2 may protect melanocytes from UVB-induced injury, and that apoptotic death of melanocytes may be induced by the elevation and redistribution of Bax.