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Epidemiological and experimental evidence has supported the notion that solar ultraviolet (UV) radiation is the leading cause of skin cell damage and skin cancer. Non-melanoma skin cancer, one of the malignancies with the most rapidly increasing incidence, is suggested to be directly related to the total exposure to solar UV light. Over the past few years, the mechanisms of cellular responses to UV radiation have received unprecedented attention. Understanding how skin cells respond to UV radiation will undoubtedly help decipher what goes wrong in a variety of clinical skin disorders including skin cancer and will facilitate the development of novel therapeutic strategies. In the past decade, studies have established that UV radiation induces multifarious signal transduction pathways, some of which lead to apoptotic cell death, while others protect against this process. In this review, we summarize some of the most recent progresses regarding the involvement of multiple signal pathways in UV radiation-induced apoptosis in skin cells, especially in keratinocytes. These pathways include pro-apoptosis components such as MAPK, AMPK, and p53 as well as pro-survival components, namely, AKT and mTORC complexes. J. Cell. Physiol. 220: 277–284, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

A polymerase chain reaction-gold magnetic nanoparticles lateral flow assay (PCR-GoldMag LFA) has been developed via integrating multiplex amplification refractory mutation system PCR (multi-ARMS-PCR) with GoldMag-based LFA for the visual detection of single-nucleotide polymorphisms (SNPs). This assay was applied to genotype Apolipoprotein E (ApoE). ApoE genotyping is important due to the predictive value for the development of coronary artery disease and Alzheimer's disease. The method requires two steps: i) Simultaneous amplifications of the two polymorphic codons (ApoE 158 and 112), performed in separated reactions using multi-ARMS-PCR; and ii) detection of the wild-type and mutant PCR products via dual immunoreactions, which can be performed in ~5 min. Within two LFAs, anti-digoxin antibody-conjugated GoldMag probes bind digoxin-labeled wild-type PCR products, and anti-fluorescein isothiocyanate (FITC) antibody-conjugated GoldMag probes bind FITC-labeled mutant PCR products. All PCR products are biotin labeled and are detected by streptavidin-coated regions on the LFA strip, resulting in a red color. The current approach is capable of detecting the SNPs of ApoE in ~1.5 h, with a broad detection range from 10-1,000 ng of genomic DNA. Thus, the present protocol may facilitate simple, fast and cost-effective screening for important SNPs, as demonstrated by the evaluation of the prevalence of ApoE variants in a Han Chinese cohort. [ABSTRACT FROM AUTHOR]

ABO genotyping is commonly used in several situations, including blood transfusion, personal identification and disease detection. The present study developed a novel method for ABO genotyping, using loop-mediated isothermal amplification (LAMP). This method allows the simultaneous determination of six ABO genotypes under 40 min at a constant temperature of 62°C. The genotypes of 101 blood samples were determined to be AA (n=6), AO (n=38), BB (n=12), BO (n=29), AB (n=8) and OO (n=8) by the LAMP assay. The results were compared with the phenotypes determined by serological assay and the genotypes determined by direct sequencing, and no discrepancies were observed. This novel and rapid method, with good accuracy and reasonably cost effective, provides a supplement to routine serological ABO typing and may also be useful in other point-of-care testing. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H2O2-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H2O2 induce AMPK activation (Thr172 phosphorylation) in cultured human skin keratinocytes. UV and H2O2 also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal growth factor receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK serves as a positive regulator for p38 and p53 (Ser15) phosphorylation induced by UV radiation and H2O2 treatment. We also observed that AMPK serves as a negative feedback signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC2-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-apoptotic effect of AMPK on UV- or H2O2-treated cells. Furthermore, activation of AMPK also phosphorylates acetyl-CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK2, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV and H2O2-induced apoptosis via multiple mechanisms in human skin keratinocytes and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer. [ABSTRACT FROM AUTHOR]

Aquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins. Based on the involvement of AQPs in angiogenesis and cell migration as well as our previous studies which show that AQP3 is involved in human skin fibroblasts cell migration, in this study, we investigated whether AQP3 is expressed in cultured human ovarian cancer cell line CaOV3 cells, and whether AQP3 expression in these cells enhances cell migration and metastatic potential. Cultured CaOV3 cells were treated with EGF and/or various reagents and subjected to cell migration assay by phagokinetic track mobility assay or biochemical analysis for expression or activation of proteins by SDS-PAGE/Western blot analysis. In this study, we demonstrate that AQP3 is expressed in CaOV3 cells. EGF induces CaOV3 migration and up-regulates AQP3 expression. EGF-induced cell migration is inhibited by specific AQP3 siRNA knockdown or AQP3 water transport inhibitor CuSO4 and NiCl2. We also find that curcumin, a well known anti-ovarian cancer drug, down-regulates AQP3 expression and reduces cell migration in CaOV3, and the effects of curcumin are mediated, at least in part, by its inhibitory effects on EGFR and downstream AKT/ERK activation. Collectively, our results provide evidence for AQP3-facilitated ovarian cancer cell migration, suggesting a novel function for AQP3 expression in high-grade tumors. The results that curcumin inhibits EGF-induced up-regulation of AQP3 and cell migration, provide a new explanation for the anticancer potential of curcumin. [ABSTRACT FROM AUTHOR]

One of the major characteristics of human skin photoaging induced by ultraviolet (UV) radiation is the dehydration of the skin. Water movement across plasma membrane occurs via diffusion through lipid bilayer and via aquaporins (AQPs). We find that UV induces aquaporin-3 (AQP3) down-regulation in human skin keratinocytes. MEK/ERK inhibitors PD98059 and U0126 inhibit UV-induced down-regulation of AQP3. Antioxidant N-acetyl-L-cysteine or NAC blocks UV-induced MEK/ERK activation and down-regulation of AQP3. All-trans retinoic acid or atRA, while alone inducing AQP3 expression, attenuates UV-induced down-regulation of AQP3 and water permeability. Using special inhibitors, we find that activation of EGFR and inhibition on ERK activation are involved in atRA's protective effects against UV-induced AQP3 down-regulation. Using specific AQP3's water transport inhibitors and siRNA knockdown, we observe that AQP3 is involved in cell migration and in vitro wound healing. UV-induced AQP3 down-regulation results in reduced water permeability, decreased cell migration, and delayed wound healing, which are attenuated by atRA pretreatment. We conclude that atRA protects against UV-induced down-regulation AQP3 and decrease in water permeability, reduction in cell migration and delayed in vitro wound healing via trans-activation of EGFR and inhibition on ROS-mediated MEK/ERK pathway. This novel finding provides evidence to support possible involvement of AQP3 in UV induced skin dehydration. J. Cell. Physiol. 215: 506–516, 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Ultraviolet (UV) radiation is related to cataract formation. The dynamics of matrix proteins play crucial roles in cell proliferation, cell migration, and the remodeling of lens capsule and, possibly, cataract formation. However, the change of dynamics of matrix proteins, such as collagens, in lens cells in response to UV radiation has not been investigated. Using cultured human lens epithelial cells, we, for the first time, demonstrate that UV radiation induces a decrease of collagen type I in a time- and dose-dependent manner. Hydrogen peroxide (H2O2) also induces a collagen type I decrease in a similar pattern. We observed that UV and H2O2 induce JNK and its downstream component, c-Jun, activation in both a time- and dose-dependent manner. The pharmacologic inhibitor of JNK or JNKi inhibits UV-induced JNK and c-Jun activation and attenuates a UV-induced decrease of collagen type I. Quercetin, a well known antioxidant, also protects against a UV- and H2O2-induced decrease of collagen type I in a dose-dependent manner. Quercetin inhibits UV- and H2O2-induced JNK and c-Jun activation. Collectively, we conclude that quercetin attenuates both a UV- and H2O2-induced decrease of collagen type I via the inhibiting of JNKc-Jun activity. Understanding the cellular-signaling pathways involved in the UV- and H2O2-induced decrease of collagen type I may reveal potential therapeutic targets for the UV-induced cataract. [ABSTRACT FROM AUTHOR]

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP-sensitive potassium (KATP) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV-induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV-induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of KATP channels are functionally expressed in HaCaT cells and both non-selective KATP channel opener pinacidil and mitoKATP (mitochondrial KATP) channel opener diazoxide attenuated UV-induced keratinocytes cell death. The protective effects were abolished by both non-selective KATP channel blocker glibenclamide and selective mitoKATP channel blocker 5-hydroxydecanoate (5-HD). Also, activation of KATP channel with pinacidil or diazoxide resulted in suppressive effects on UV-induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV-induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5-HD. Taken together, we contend that mitoKATP is likely to contribute the protection against UV-induced keratinocytes cell damage. Our findings suggest that KATP openers such as pinacidil and diazoxide may be utilized to prevent from UV-induced skin aging. J. Cell. Physiol. 212: 252–263, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Cataract is considered as the most common cause of blindness, which is curable only by surgery. Postsurgery, however, many patients gradually develop the complication of posterior capsule opacification (PCO) or secondary cataract, arising from stimulated cell proliferation and cell migration within the lens capsule. The migration of human lens epithelial cells (HLECs) plays crucial roles in the remodeling of lens capsule and cataract formation, but less is known about the cell-signaling mechanism of migration. We observed that epithelial growth factor (EGF) induced cell migration in cultured human lens epithelial cells through the ERK and PI3K/AKT pathways. EGF induced cell migration in a dose-dependent manner; EGF-induced EGFR phosphorylation and downstream activation of c-Jun N-terminal protein kinase (JNK), p38 MAP kinase (p38), extracellular signal-regulated kinase (ERK1/2) and AKT, were inhibited by PD153035 (EGFR inhibitor), JNKi (JNK inhibitor), SB203580 (p38 inhibitor), U0126 (MEK/ERK inhibitor), and LY294002 (PI3K/AKT inhibitor), respectively. Furthermore, we found that EGF induced activity of matrix metalloproteinase-2 (MMP-2) in cultured HLECs. EGF-induced MMP-2 activity was significantly inhibited by treatment of PD153035, U0126, and LY294002, but not SB203580 and JNK inhibitor, suggesting that ERK and the phosphatidylinositol- 3-kinase (PI3K)/AKT pathways selectively mediate EGF-stimulated MMP-2 activity and cell migration in cultured HLECs in vitro. Taken together, our results suggest that the cell-signaling pathways involved in EGF-stimulated cell migration may constitute potential therapeutic targets in the treatment of PCO. [ABSTRACT FROM AUTHOR]

Cancer is a genomic functional disease with features of oncogene activation and tumor suppressor inactivation. These genomic features have resulted in the limited effectiveness of conventional therapies and therefore forced considerable efforts to explore new types of anticancer agents. It has been clear that chemically synthesized or in vivo-expressed short interfering RNA (siRNA) can specifically and effectively direct homology-dependent post-transcriptional gene silencing. In the present study, we intended to investigate whether siRNA could suppress the proliferation of human cancer cells through interfering oncogene activities and recovering the functions of tumor-suppressor gene. Single siRNA or combinatorial siRNAs were successfully transfected into HeLa cells, lung adenocarcinoma cells, hepatoma cells, ovarian carcinoma cells, and melanoma cells with cationic lipid complexes. These siRNA molecules not only specifically knocked down their cognate targets such as bcl-2, cdk-2, mdm-2, pkc-alpha, tgf-beta1, H-ras, vegf, and GFP mRNAs, but also effectively suppressed the proliferation of cancer cells to different extents. These data suggest that (1) all these human cancer cells preserve RNAi machinery; (2) chemically synthesized and vector-driven siRNAs can be incorporated into intrinsic RNAi system for silencing target mRNA molecules; and (3) the combination of different siRNAs inhibits the growth and proliferation of cancer cells. [ABSTRACT FROM AUTHOR]

Determines whether differentially expressed in chondrocytes (DEC)1 and DEC2 displayed similar expression patterns among paired tumor-normal tissues from the colon, lung, and kidney. Tissue collection and processing; Site-directed mutagenesis; Evidence that forced expression of DEC1 proportionally decreases the expression of DEC2.

Aquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins. Based on the involvement of AQPs in angiogenesis and cell migration as well as our previous works that AQP3 is involved human skin fibroblast cell migration, we tested whether AQP3 is expressed in ovarian cancer line Caov3 and SKOV3 cells, and whether AQP3 expression in these cells enhances cell migration and metastatic potential. Our studies demonstrated that AQP3 is over-expressed in human ovarian cancer cell line Caov3 and SKOV3 cells. Our data, using specific AQP3 water transport inhibitors as well as siRNA knockdown, indicated that AQP3, or AQP3-mediated water transport, is involved in ovarian cell migration. EGF induced ovarian cancer cell migration, up-regulated AQP3 expression. EGF-induced cell migration was inhibited by AQP3 siRNA knockdown or by treatment with CuSO4 and NiCl2, both known as AQP3 water transport inhibitors. Mechanistic analysis suggests that EGFR activation is involved in EGF-induced AQP3 up-regulation and cell migration. Curcumin down-regulated AQP3 expression, and reduced cell migration in ovarian cancer cells. And the effects of curcumin were mediated, at least in part, by its inhibitory roles on EGFR activation. Collectively, our results provide evidence for AQP3-facilitated ovarian cancer cell migration, suggesting a novel function for AQP3 expression in high-grade tumors. [ABSTRACT FROM AUTHOR]

Overexpression of EGFR is associated with advanced-stage disease and poor prognosis of ovarian cancer, and activation of EGFR signaling pathway is involved in increased cell proliferation, angiogenesis, metastasis, and decreased apoptosis. Tyrosine kinase activity is essential for signal transduction and for receptor down-regulation. However, we found in this, study that tyrosine kinase activity is not necessary in ligand-induced EGFR down-regulation in Caov3 cells. EGFR tyrosine kinase inhibitors, such as PD153035, AG1478, as well as non-specific tyrosine kinase inhibitor PP2 did not reverse EGF-induced down-regulation of EGFR. These findings permit us to develop the following exciting but unconventional strategy, namely, priming ovarian cancer cell with EGF together with EGFR inhibitor PD153035. This priming down-regulates EGFR without induction of mitogenic signals. EGF plus EGFR inhibitor-primed ovarian cancer cells displayed more sensitive to Taxol-induced cell death and resistant to EGF-induced cell migration, cell proliferation as well as ERK, PI3K activation. Mechanistic analysis indicated that PD153035, which did not reverse ligand-induced EGFR down-regulation, blocked EGF-induced EGFR activation as well as EGFR binding with c-cb1 and Grb2. Taken together, we contend that combination of EGF priming with EGFR inhibitors would ultimately result in better chemotherapeutical outcome. [ABSTRACT FROM AUTHOR]