Tob1 enhances radiosensitivity of breast cancer cells involving the JNK and p38 pathways.

The aim of the present study was to evaluate the effect of Tob1 on the radiosensitivity of breast cancer cells. The results showed that overexpression of Tob1 reduced the clonogenic growth of 231 cells and induced the rate of apoptosis. Tob1 caused an accumulation of cells in the G0 /G1 phase and decreased the percentage of cells in S phase. We also found that overexpression of Tob1 significantly reduced the phosphorylation of JNK and p38. The activator of JNK and p38, anisomycin, attenuated the blockage of Tob1 on the cell cycle and reversed the effect of Tob1 on apoptosis. Taken together, Tob1 enhanced radiosensitivity of breast cancer cells through regulation of the JNK and p38 pathways. The results indicated that Tob1 might be a promising molecular in gene therapy for the treatment of breast cancer.

A comparative study of UV-induced cell signalling pathways in human keratinocyte-derived cell lines.

Ultraviolet (UV) radiation can activate the p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK) and nuclear factor-κB (NFκB) pathways in skin cells. HaCaT cells are widely used as a primary keratinocyte substitute to study these pathways. However, like most squamous cell carcinomas (SCCs), it contains a dysfunctional p53. It is unclear if HaCaT cells activate these signalling pathways similarly to SCC cells (Colo16) or to primary human epidermal keratinocytes (HEK). In this study, the UV activation (UVA, UVB, UVA+B, UVB+A) of p38 MAPK, JNK and NFκB pathways, and TNFα secretion by HEK, HaCaT and Colo16 cells were investigated. The signalling pathway activation was UV-type and dose-dependent with UVB+A radiation inducing a high p38 and JNK activation. HaCaT cells exhibited 2- to 4-fold higher activity of the p38 (771% at 60 min) and JNK (794% at 30 min) pathways following UVB+A radiation than did HEK cells (p38: 367% at 15 min and JNK: 184% at 30 min). While both HaCaT and Colo16 cells did not activate the NFκB pathway, Colo16 cells had a lower p38 and higher JNK activity than HaCaT cells. Irradiated HaCaT cells produced less TNFα (UVB: 3.5 pg/ml), while HEK cells produced the most (UVB: 1,296 pg/ml). When co-exposed to IL1α, irradiated HaCaT had the greatest fold of TNFα release (UVB: 16.2-fold, UVA+B: 8.9-fold and UVB+A: 6.1-fold). The pattern of activation and TNFα secretion of HaCaT cells mirrored that of Colo16 cells. It is likely that the presence of molecular alterations in HaCaT cells may be responsible for its different responses to that seen for HEK cells. The results of this study suggest caution in using HaCaT cells as a substitute for normal keratinocytes in investigating UV-induced cells signalling pathways.

MC1R variant allele effects on UVR-induced phosphorylation of p38, p53, and DDB2 repair protein responses in melanocytic cells in culture.

Variant alleles of the human melanocortin 1 receptor (MC1R) reduce the ability of melanocytes to produce the dark pigment eumelanin, with R alleles being most deficient. Cultured melanocytes of MC1R R/R variant genotype give reduced responses to [Nle(4), D-Phe(7)]α-melanocyte-stimulating hormone (NDP-MSH) ligand stimulation and lower levels of DNA repair than MC1R wild-type strains. p38 controls xeroderma pigmentosum (XP)-C recruitment to DNA damage sites through regulating ubiquitylation of the DNA damage-binding protein 2 (DDB2) protein, and p53 is implicated in the nuclear excision repair process through its regulation of XP-C and DDB2 protein expression. We report the effects of MC1R ligand treatment and UVR exposure on phosphorylation of p38 and p53, and DDB2 protein expression in MC1R variant strains. Wild-type MC1R melanocyte strains grown together with keratinocytes in coculture, when treated with NDP-MSH and exposed to UVR, gave synergistic activation of p38 and p53 phosphorylation, and were not replicated by R/R variant melanocytes, which have lower basal levels of phosphorylated forms of p38. Minor increases in p38 phosphorylation status in R/R variant melanocyte cocultures could be attributed to the keratinocytes alone. We also found that MC1R wild-type strains regulate DDB2 protein levels through p38, but MC1R R/R variant melanocytes do not. This work confirms the important functional role that the MC1R receptor plays in UVR stress-induced DNA repair.

Low doses of ionizing radiation suppress doxorubicin-induced senescence-like phenotypes by activation of ERK1/2 and suppression of p38 kinase in MCF7 human breast cancer cells.

Low-dose radiation has a variety of effects on cellular activities, including the cell division cycle, apoptosis, proliferation and senescence. However, the effects of low doses of radiation remain controversial. In this study, we examined the effects of low-dose radiation on cellular senescence. We treated MCF7 cells with 0.01 microg/ml doxorubicin to induce replicative senescence, 2 h after exposure to low doses of ionizing radiation of 0.05, 0.1, or 0.2 Gy. The status of p53, senescence-associated beta-galactosidase activity, p38 kinase levels, H2AX levels and ERK/MAPK levels were examined. Low doses of ionizing radiation inhibit doxorubicin-induced senescence in human breast cancer MCF7 cells. The phosphorylations of both p38 MAP kinase and p53 induced by doxorubicin were suppressed by low doses of ionizing radiation. The senescence was inhibited without genomic damage, because the level of gamma-H2AX protein was not changed. Moreover, low doses of ionizing radiation inhibited senescence through the activation of ERK1/2. The results thus suggest that low doses of radiation suppress doxorubicin-induced replicative senescence through the inhibition of p38-dependent phosphorylation of p53 and by activation of ERK1/2, without genomic damage. Overall, our results suggest that low doses of ionizing radiation may have a protective role against replicative senescence induced by doxorubicin.

Neodymium-doped yttrium-aluminium-garnet laser irradiation abolishes the increase in interleukin-6 levels caused by peptidoglycan through the p38 mitogen-activated protein kinase pathway in human pulp cells.

The anti-inflammatory effects of low-power laser irradiation have previously been reported. However, how the laser irradiation regulates the expression of inflammatory cytokines remains unknown. In the present study, to elucidate the mechanism behind the anti-inflammatory effect, we examined the effects of low-power neodymium-doped yttrium-aluminium-garnet (Nd:YAG) laser irradiation on interleukin (IL)-6 expression in human pulp (HP) cells stimulated by peptidoglycan (PGN) and focused on intracellular signaling pathways. Low-power Nd:YAG laser irradiation obviated the PGN-induced increase in IL-6 levels in HP cells. A p38 mitogen-activated protein kinase inhibitor, SB203580, also inhibited the increase in IL-6 messenger RNA levels. PGN stimulated the activity of phosphorylated p38 in HP cells. Low-power laser irradiation inhibited the activity. Thus, suppression of the phosphorylated p38 activity by low-power laser irradiation in HP cells culminates in inhibition of the increase in IL-6 induced by PGN, suggesting that low-power laser irradiation regulates intracellular signaling molecule activities to exert its anti-inflammatory effect.

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells.

The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.

MAP kinases: differential activation following in vivo and ex vivo irradiation.

Mitogen activated protein kinases (MAPK) play a critical role in controlling cell survival and repopulation following exposure to ionising radiation. Most investigations on these pathways have been done using cultured cells or by ex vivo treatments. The present study was carried out to determine whether the response of MAPKs in mouse lymphocytes differs following in vivo and ex vivo irradiation with 60Co gamma-rays. We observed that ex vivo treatment resulted in a very significant decrease in the activated p44/42 and p38 MAPK as compared to in vivo. However, stress activated protein kinase (SAPK) response showed no significant difference between in vivo and ex vivo treatments. These observations point towards the differences in response elicited when the treatment is given in vivo as compared to in vitro. Therefore the findings reported from in vitro or ex vivo treatments should be treated with caution especially if it has to be clinically applied.

Ionizing radiation enhances matrix metalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidylinositol 3-kinase/Akt signaling pathways.

Glioblastoma is a severe type of primary brain tumor, and its highly invasive character is considered to be a major therapeutic obstacle. Several recent studies have reported that ionizing radiation (IR) enhances the invasion of tumor cells, but the mechanisms for this effect are not well understood. In this study, we investigated the possible signaling mechanisms involved in IR-induced invasion of glioma cells. IR increased the matrix metalloproteinase (MMP)-2 promoter activity, mRNA transcription, and protein secretion along with the invasiveness of glioma cells lacking functional PTEN (U87, U251, U373, and C6) but not those harboring wild-type (WT)-PTEN (LN18 and LN428). IR activated phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin, and blockade of these kinases by specific inhibitors (LY294002, Akt inhibitor IV, and rapamycin, respectively) and transfection of dominant-negative (DN) mutants (DN-p85 and DN-Akt) or WT-PTEN suppressed the IR-induced MMP-2 secretion in U251 and U373 cells. In addition, inhibitors of epidermal growth factor receptor (EGFR; AG490 and AG1478), Src (PP2), and p38 (SB203580), EGFR neutralizing antibody, and transfection of DN-Src and DN-p38 significantly blocked IR-induced Akt phosphorylation and MMP-2 secretion. IR-induced activation of EGFR was suppressed by PP2, whereas LY294002 and SB203580 did not affect the activations of p38 and PI3K, respectively. Finally, these kinase inhibitors significantly reduced the IR-induced invasiveness of these cells on Matrigel. Taken together, our findings suggest that IR induces Src-dependent EGFR activation, which triggers the p38/Akt and PI3K/Akt signaling pathways, leading to increased MMP-2 expression and heightened invasiveness of PTEN mutant glioma cells.

Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo.

Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-beta expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent.

Regulation of Drosophila p38 activation by specific MAP2 kinase and MAP3 kinase in response to different stimuli.

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in cellular responses to inflammatory stimuli and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. However, the mechanism of how different upstream MAP2Ks and MAP3Ks specifically contribute to p38 activation in response to different stimuli is still not clearly understood. By using double-stranded RNA-mediated interference (RNAi) in Drosophila cells, we demonstrate that D-MKK3 is a major MAP2K responsible for D-p38 activation by UV, heat shock, NaCl or peptiodglycan (PGN). Stimulation of UV and PGN activates D-p38 through D-MEKK1, heat shock-induced activation of D-p38 signals through both D-MEKK1 and D-ASK1. On the other hand, maximal activation of D-p38 by NaCl requires the expression of four MAP3Ks.

Ultraviolet irradiation suppresses T cell activation via blocking TCR-mediated ERK and NF-kappa B signaling pathways.

UV irradiation is carcinogenic and immunosuppressive. Previous studies indicate that UV-mediated alteration of APCs and induction of suppressor T cells play a critical role in UV-induced immune suppression. In this study, we show that UV irradiation can directly (independently of APCs and suppressor T cells) inhibit T cell activation by blocking TCR-mediated phosphorylation of ERK and IkappaB via overactivation of the p38 and JNK pathways. These events lead to the down-modulation of c-Jun, c-Fos, Egr-1, and NF-kappaB transcription factors and thereby inhibit production of cytokines, e.g., IL-2, IL-4, IFN-gamma, and TNF-alpha, upon TCR stimulation. We also show that UV irradiation can suppress preactivated T cells, indicating that UV irradiation does not only impair T cell function in response to T cell activation, but can also have systemic effects that influence ongoing immune responses. Thus, our data provide an additional mechanism by which UV irradiation directly suppresses immune responses.

Activation of ERK and p38 kinase mediated keloid fibroblast apoptosis after flashlamp pulsed-dye laser treatment.

BACKGROUND AND OBJECTIVES: Flashlamp pulsed-dye lasers (PDLs) revealed effective regression or arrest in patients with keloids in our clinical studies [Kuo YR et al., Laser Surg Med 2004;34:104-108]. In this study, we further investigated whether the induction of keloid regression seen with PDL treatment through activation in mitogen-activated protein (MAP) kinase and caspase promotes cell apoptosis and reduces fibroblast proliferation. STUDY DESIGN/MATERIALS AND METHODS: Keloid tissues were obtained from 10 patients with intralesional or punch biopsies prior to and 7 days after PDL treatments [fluence per pulse was 10-18 J/cm2 (mean 14 J/cm2)]. Prior to and after PDL treatments, the proliferating fibroblasts in keloid tissue were immunohistochemically detected by proliferating cell nuclear antigen (PCNA) expression. The apoptotic cell was detected by terminal deoxynucleotidyl transferase dUTP-nick end labeling (TUNEL) staining and fragmented caspase-3 expression. MAP kinase activation as represented by extracellular signal-regulated kinase (ERK), p38 kinase (p38), and c-Jun N-terminal kinase (JNK) expression of keloid tissues was investigated by immunohistochemical (IHC) staining, respectively. RESULTS: IHC staining indicated that PCNA expression of fibroblasts was significantly reduced in keloid tissue after PDL irradiation. TUNEL assay revealed lower apoptotic cells expression in the keloid tissue prior to laser treatment. Following laser treatment, apoptotic cells with relatively strong DNA damage and fragmentation were seen in all keloid biopsy samples, especially in the keloid fibroblast population. The activation of ERK and p38 MAP kinase increased significantly in keloid tissue after PDL treatment. JNK was shown to be unchanged. CONCLUSIONS: The PDL treatment is shown to induce keloid regression through suppression of keloid fibroblast proliferation, induction of apoptosis, and upregulation of ERK and p38 MAP kinase activity.

Xanthophylls and alpha-tocopherol decrease UVB-induced lipid peroxidation and stress signaling in human lens epithelial cells.

Epidemiological studies suggest that consumption of vegetables rich in the xanthophylls lutein (LUT) and zeaxanthin (ZEA) reduces the risk for developing age-related cataract, a leading cause of vision loss. Although LUT and ZEA are the only dietary carotenoids present in the lens, direct evidence for their photoprotective effect in this organ is not available. The present study examined the effects of xanthophylls and alpha-tocopherol (alpha-TC) on lipid peroxidation and the mitogen-activated stress signaling pathways in human lens epithelial (HLE) cells following ultraviolet B light (UVB) irradiation. When presented with LUT, ZEA, astaxanthin (AST), and alpha-TC as methyl-beta-cyclodextrin complexes, HLE cells accumulated the lipophiles in a concentration- and time-dependent manner with uptake of LUT exceeding that of ZEA and AST. Pretreatment of cultures with either 2 micromol/L xanthophyll or 10 micromol/L alpha-TC for 4 h before exposure to 300 J/m(2) UVB radiation decreased lipid peroxidation by 47-57% compared with UVB-treated control HLE cells. Pretreatment with the xanthophylls and alpha-TC also inhibited UVB-induced activation of c-JUN NH(2)-terminal kinase (JNK) and p38 by 50-60 and 25-32%, respectively. There was substantial inhibition of UVB-induced JNK and p38 activation for cells containing <0.20 and approximately 0.30 nmol xanthophylls/mg, respectively, whereas >2.3 nmol alpha-TC/mg protein was required to significantly decrease UVB-induced stress signaling. These data suggest that xanthophylls are more potent than alpha-TC for protecting human lens epithelial cells against UVB insult.

[Exposure to power-frequency magnetic fields can induce activation of P38 mitogen-activated protein kinase].

OBJECTIVE: To study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of P38 mitogen-activated protein kinase (P38 MAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields. METHODS: Chinese hamster lung (CHL) cell line was exposed to power-frequency magnetic fields with two intensities(0.1 and 0.4 mT) for different exposure durations. The cytoplasmic protein was extracted. The phosphorylated(activated) and non-phosphorylated P38 MAPK and MKK3/MKK6 were measured by Western blotting analysis with their specific corresponding antibodies. RESULTS: Power-frequency magnetic fields at 0.4 mT for 10 min could transitorily induce the activation of P38 MAPK and after 15 min the phosphorylation of P38 MAPK restored to control level, while 0.1 mT power-frequency magnetic fields could not induce the activation of P38 MAPK within 24 h. However, both 0.1 mT and 0.4 mT power-frequency magnetic fields could not phosphorylate(activate) the MKK3/MKK6, which is a general upstream kinase of P38 MAPK. CONCLUSION: Power-frequency magnetic fields could transitorily activate the P38 MAPK, but not MKK3/MKK6. The activation mechanism of P38 MAPK needs to be further identified.