WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma.

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12-Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.

TIAF1 self-aggregation in peritumor capsule formation, spontaneous activation of SMAD-responsive promoter in p53-deficient environment, and cell death.

Self-aggregation of transforming growth factor ß (TGF-ß)1-induced antiapoptotic factor (TIAF1) is known in the nondemented human hippocampus, and the aggregating process may lead to generation of amyloid ß (Aß) for causing neurodegeneration. Here, we determined that overexpressed TIAF1 exhibits as aggregates together with Smad4 and Aß in the cancer stroma and peritumor capsules of solid tumors. Also, TIAF1/Aß aggregates are shown on the interface between brain neural cells and the metastatic cancer cell mass. TIAF1 is upregulated in developing tumors, but may disappear in established metastatic cancer cells. Growing neuroblastoma cells on the extracellular matrices from other cancer cell types induced production of aggregated TIAF1 and Aß. In vitro induction of TIAF1 self-association upregulated the expression of tumor suppressors Smad4 and WW domain-containing oxidoreductase (WOX1 or WWOX), and WOX1 in turn increased the TIAF1 expression. TIAF1/Smad4 interaction further enhanced Aß formation. TIAF1 is known to suppress SMAD-regulated promoter activation. Intriguingly, without p53, self-aggregating TIAF1 spontaneously activated the SMAD-regulated promoter. TIAF1 was essential for p53-, WOX1- and dominant-negative JNK1-induced cell death. TIAF1, p53 and WOX1 acted synergistically in suppressing anchorage-independent growth, blocking cell migration and causing apoptosis. Together, TIAF1 shows an aggregation-dependent control of tumor progression and metastasis, and regulation of cell death.

Effects of sebum on drug transport across the human stratum corneum in vivo.

The objective of this study was to investigate the effect of sebum on drug transport across the human stratum corneum (SC) in vivo for two model compounds, 4-cyanophenol (CP) and cimetidine (CM), of different lipophilicity and molecular size by utilizing noninvasive tape-stripping techniques, in conjunction with an unsteady-state diffusion model for data analysis. The results demonstrated that the SC permeability of the relatively hydrophilic CM on the forehead may be as much as four times the permeability on the forearm. The administration of sebum supplementation to the forearm increased the SC permeability of CM more than threefold, but did not have the same effect with regard to CP. Removal of sebum from the forehead demonstrated a small but significant effect (-22%) on the SC permeability of CM. The presence of sebum on the forehead or forearm increased the diffusion of both molecules, but the effect on partition varied between sites and drugs. The change in the SC permeability of the relatively hydrophilic drug using sebum treatment may be attributable to the altered barrier function of the SC due to the disordering structures of the intercellular lipid molecules.

Analysis of Taiwanese ichthyosis vulgaris families further demonstrates differences in FLG mutations between European and Asian populations.

BACKGROUND: Mutations in the gene encoding filaggrin (FLG) were identified to underlie ichthyosis vulgaris (IV) and also shown to predispose to atopic eczema. Until now, no FLG mutations have been described in the Taiwanese population. OBJECTIVES: To elucidate filaggrin mutations in the Taiwanese population and further to clarify the population genetics of filaggrin gene mutations in the Asian populations. METHODS: In the present study, 12 individuals from four unrelated Taiwanese IV families were examined for FLG mutations. We carried out comprehensive sequencing of the entire FLG coding region using an overlapping polymerase chain reaction strategy. RESULTS: We identified three FLG mutations in the Taiwanese IV families. One mutation E1795X was a previously unidentified FLG mutation, which might be specific to the Taiwanese. Interestingly, another FLG mutation 3321delA is prevalent in the Japanese population and the other mutation Q2417X was found in the Singaporean Chinese population. No FLG mutation identified in the white European population was found in the Taiwanese population. CONCLUSIONS: The present findings suggest that the Taiwanese population, as an East Asian group, share FLG mutations with both the Japanese and the Singaporean Chinese population. In addition, these results exemplify differences in the population genetics of filaggrin between Europe and Asia.

Downregulation of HER2/neu receptor by solamargine enhances anticancer drug-mediated cytotoxicity in breast cancer cells with high-expressing HER2/neu.

Overexpression of HER2/neu is associated with drug resistance and poor outcome in breast cancer. Solamargine (SM), a glycoalkaloid purified from the herb Solanum incanum, exhibits HER2/neu gene modulation of HER2/neu high-expressing human breast cancer cell line ZR-75-1. SM downregulation of HER2/neu gene expression was determined by RT-PCR and Southern hybridization. Additionally, the membrane-bound HER2/neu receptor in highly HER2/neu-expressing breast cancer cells was determined by radioimmunoassay, immunocytochemistry, fluorescent immunocytochemistry, and flow cytometry. SM significantly decreased the number of HER2/neu receptors on the cell membrane. Methotrexate (MTX), 5-florouracil (5-Fu), and cisplatin (CDDP) are commonly used for breast carcinoma treatment in clinics; however, patients with HER2/neu overexpression exhibit resistance to these anticancer drugs. Notably, combination of MTX, 5-Fu, and CDDP with SM individually increased the susceptibility of breast cancer cells to these chemotherapeutic agents. Experimental results indicated that downregulation of HER2/neu by SM might be an effective strategy for enhancing drug susceptibility of breast cancer cells expressing high levels of HER2/neu.

Environmental factors, parental atopy and atopic eczema in primary-school children: a cross-sectional study in Taiwan.

BACKGROUND: Parental atopy and environmental exposure are recognized risk factors for atopic eczema (AE) in childhood. However, the relative contributions of specific risk factors and the overall contributions of hereditary and environmental exposure remain unexplored. OBJECTIVES: To identify risk factors, estimate the population attributable risk (PAR) of environmental exposure, and compare the AE data for boys vs. girls in primary-school children. METHODS: During a February to June 2001 cross-sectional, Taiwan-based questionnaire survey, we investigated 23 980 children from 22 primary schools, all located within 1 km of an air-monitoring station. RESULTS: The 12-month prevalence of AE was reported as 6.1% in boys and 4.9% in girls. In both sexes, the risk of AE was strongly associated with parental atopy and perceived ambient air pollution. The presence of cockroaches [odds ratio (OR) 1.18, 95% confidence interval (CI) 1.00-1.40] and visible mould on walls at home (OR 1.46, 95% CI 1.22-1.70) were also significantly related to AE for girls; however, only visible mould on walls (and not the presence of cockroaches) at home was related to AE for boys (OR 1.40, 95% CI 1.18-1.66). While mutually adjusted models were applied, we found adjusted ORs and PARs were similar in boys and girls in hereditary and outdoor environmental factors. The PAR of indoor environmental factors was higher in girls (8.4%) than in boys (5.5%). There was no interaction between parental atopy and environmental factors. CONCLUSIONS: Parental atopy contributed more to AE than indoor or outdoor environmental factors. Girls may be more susceptible to indoor environmental factors than boys.

Solamargine induces apoptosis and sensitizes breast cancer cells to cisplatin.

Solamargine (SM), a major steroidal alkaloid glycoside, was purified from Solanum incanum plant. SM exhibited the most cytotoxic effect comparing with that of cisplatin (cDDP), methotrexate (MTX), 5-fluorouracil (5-FU), epirubicin (EPI) and cyclophosphamide (CP) against human breast cancer cells. In this study, SM induces apoptosis of the breast cancer cells and the mechanism was characterized. SM up-regulated the expressions of external death receptors, such as tumor necrosis factor receptor I (TNFR-I), Fas receptor (Fas), TNFR-I-associated death domain (TRADD), and Fas-associated death domain (FADD). SM also enhanced the intrinsic ratio of Bax to Bcl-2 by up-regulating Bax and down-regulating Bcl-2 and Bcl-xL expressions. These effects resulted in the release of mitochondrial cytochrome c and activation of caspase-8, -9 and -3 in the cells, indicating that SM triggered extrinsic and intrinsic apoptotic pathways of breast cancer cells. Similar to function way of SM, cDDP causes cancer cell apoptosis though caspase-8/caspase-3 and Bax/cytochrome c pathways, but the resistance to cDDP is correlated with Bcl-2 and Bcl-xL overexpression. However, the overexpression of Bcl-2 and Bcl-xL can be broken through by SM. The combined treatment of SM and cDDP significantly reduced Bcl-2 and Bcl-xL expressions, and enhanced Bax, cytochrome c, caspase-9 and -3 expressions in breast cancer cells. Thus, the combined use of SM and cDDP may be effective in cDDP-resistant breast cancer.

Expression of sex-determining genes in human sebaceous glands and their possible role in the pathogenesis of acne.

BACKGROUND: The human skin, especially the sebaceous gland, is a steroidogenic organ similar to the gonads and adrenal cortex, possessing all the enzymes required for steroid sex-hormone synthesis and metabolism. Factors regulating cutaneous steroidogenesis associated with disease status remain largely unknown. OBJECTIVE: We hypothesized that transcription factors involved in sex formation and regulation of steroidogenesis in the classical steroidogenic organs are also expressed in the sebaceous glands. Their possible role in the pathogenesis of acne were investigated. METHODS: We used reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization and Western blotting to analyse the expression of SF-1, WT-1, SRY, SOX-9 and DAX-1 mRNAs and their proteins in cultured human sebocytes and the facial skin of acne patients. RESULTS: The in situ hybridization study showed SOX-9 mRNA mainly localized in basal keratinocytes, the basal layer of the sebaceous glands and eccrine glands. Immortalized human sebaceous gland cells (SZ95) expressed mRNA for SOX-9, WT-1 and DAX-1 but not for SF-1 or SRY. The expression of DAX-1 protein was slightly inhibited by 10(-6) m oestradiol (E2) at 6 h but enhanced by 10(-6) m dihydrotestosterone (DHT) at 48 h. The facial expression of SOX-9 seemed to be higher in the acne-prone male patients, while DAX-1 was stronger in subjects without acne, although both were statistically insignificant. CONCLUSION: Our findings confirm the expression of some sex-determining genes in human sebaceous glands. Further studies on a larger patient population including the normal controls are needed to elucidate the functional significance of these transcription factors in the pathogenesis of acne.

Tape stripping and sodium dodecyl sulfate treatment increase the molecular weight cutoff of polyethylene glycol penetration across murine skin.

Prior studies in hairless mice have demonstrated that acute barrier disruption by acetone treatment increases the molecular weight (MW) cutoff of polyethylene glycol (PEG) penetration through the skin. The objective of the present study was to further investigate the dependence of permeability on MW with different forms of barrier disruption. A series of PEGs ranging in MW from near 300 to over 1000 Da were used to study the effects of tape stripping and sodium dodecyl sulfate (SDS) treatment on the MW permeability profiles of mouse skin in vitro. The 12-h percutaneous penetration of all the PEG 300, 600, and 1000 oligomers generally increased as a function of transepidermal water loss (TEWL) of the skin, either tape-stripped or SDS-treated. In addition, the total penetration of PEG oligomers across control skin, and skin tape-stripped and SDS-treated to different degrees of barrier disruption progressively decreased with increasing MW. There were no significant differences in the percutaneous penetration of the PEG oligomers between skin tape-stripped and SDS-treated to the same degree of barrier disruption. The penetration enhancement relative to control skin was more prominent with larger molecules. The MW cutoff for skin penetration increased with the degree of barrier disruption irrespective of the treatment applied, and was 986 Da (tape stripping) and 766 Da (SDS treatment) at TEWL levels in the range 10-20 g/m(2) per h in comparison with 414 Da for control skin. In accordance with previous findings in acetone-treated mouse skin, the results strongly suggest that, irrespective of the form of barrier disruption applied, not only higher amounts but also more varieties of chemicals (larger molecules) may penetrate skin with a compromised barrier than normal skin.

Effect of barrier disruption by acetone treatment on the permeability of compounds with various lipophilicities: implications for the permeability of compromised skin.

The permeability of compromised skin barrier was investigated in vitro using acetone-disrupted hairless mouse skin as a model membrane. The effect of compound lipophilicity was studied using sucrose, caffeine, hydrocortisone, estradiol, and progesterone as model compounds. The results demonstrated that permeability barrier disruption by acetone treatment significantly enhanced the permeability of the skin to both hydrophilic and amphipathic compounds, including sucrose, caffeine and hydrocortisone. This effect was more prominent with caffeine and hydrocortisone at different transepidermal water loss (TEWL) levels. Acetone treatment, however, didn't appear to alter the percutaneous penetration of highly lipophilic compounds, such as estradiol and progesterone. The characteristics of skin permeability were described by parabolic relationships between log P(WS) (permeability coefficient of whole skin) and log K(O/W) (octanol/water partition coefficient) at different degrees of permeability barrier disruption. The optimal log K(O/W) of compounds for skin penetration appeared to decrease with an increase in TEWL levels. The maximal permeability achieved was similar through skin displaying different TEWL levels. In an attempt to explore the underlying mechanisms for the changes in skin permeability, the stratum corneum/normal saline partition coefficients of water, caffeine, and hydrocortisone either decreased or remained unaffected with an increase in TEWL. Electron microscopic examinations have revealed reductions in stratum corneum lipid content and alterations in intercellular membrane structures as a result of acetone treatment, whereas negligible changes in the number of horny layers were observed by safranin staining of the stratum corneum. We have concluded that the enhancement in skin permeability to both hydrophilic and amphipathic compounds by acetone treatment arose mainly because of the increase in stratum corneum diffusivity at higher TEWL levels. The results imply the possibility of using both TEWL and drug lipophilicity to predict alterations in skin permeability and hence the dose adjustment of topically applied medication for patients with impaired skin barrier function.

Molecular weight dependence of polyethylene glycol penetration across acetone-disrupted permeability barrier.

Previous studies have demonstrated that permeability barrier disruption by acetone treatment significantly enhances skin permeability to both hydrophilic and amphipathic compounds, but not to highly lipophilic compounds. The purpose of the present study was to investigate the dependence of permeability on molecular weight (MW) in acetone-disrupted hairless mouse skin in contrast to normal skin. Penetration of polyethylene glycol (PEG) 300, 600, and 1,000 over 12 h was measured using diffusion cells. High-performance liquid chromatographic methods with refractive index detection were used to separate and quantitate the individual oligomeric species in the PEG samples. Percutaneous penetration of PEGs exhibited slightly steeper MW dependency at a transepidermal water loss (TEWL) of 30-41 g/m2 per h in comparison with TEWLs of 0-10 (control skin), 10-20, and 20-30 g/m2 per h, with a higher percentage of smaller oligomer PEGs penetrating than larger ones. Increasing the TEWL of the skin increased the penetration of all the PEG oligomers, and the degree of the enhancement relative to penetration through control skin increased with MW and was maximal for oligomers with a MW ranging from 326 to 414 Da. Within the limit of quantitation of the assay, the MW cut-off for PEG penetration across mouse skin with TEWLs of 0-10, 10-20, and 20-30 g/m2 per h was 414, 590, and 942 Da, respectively, while all the measurable oligomers up to MW 1,074 Da were able to penetrate skin with TEWLs in the range 30-41 g/m2 per h. The results suggest that not only higher amounts but also more varieties of chemicals may penetrate skin with a compromised barrier than normal skin, implying a higher risk of intoxication and hypersensitization by environmental agents through diseased skin with impaired barrier function.

Morphology of glass fibers in electronics workers with fiberglass dermatitis--a scanning electron microscopy study.

BACKGROUND: Fiberglass is used as a reinforcement filler material in printed circuit boards (PCBs) which are widely used in the electronics industry. In a recent survey, we demonstrated that fiberglass dermatitis is the most common occupational dermatosis among electronics industry workers in Taiwan. Little is known, however, about the morphologic structures of the glass fibers which induce dermatitis. The purpose of this study was to assess the morphology of fiber spicules and to determine the relationship of this structure to fiberglass dermatitis. METHODS: Fourteen female patients with a diagnosis of fiberglass dermatitis were selected for study. The diagnosis was confirmed in all patients by positive skin stripping for glass fibers and matching with glass fibers from dust collected in work areas and from samples collected by scraping the edge of PCBs. Samples of collected glass fibers were analyzed by scanning electron microscopy (SEM). RESULTS: SEM of the fiberglass samples revealed that fibers were approximately 10 microm in diameter. In samples from both the edge of PCBs and from dust collected in work areas, SEM revealed that most of the fibers were in bundles of various sizes and lengths. All fibers collected from patients' skin by tape stripping showed a singular spicule, most had a sharp free end, and the lengths were in the range 50-150 microm . CONCLUSIONS: Singular glass fibers with a sharp free end and a length of 50-150 microm are most likely to induce fiberglass dermatitis.

Photodynamic therapy for Bowen's disease (squamous cell carcinoma in situ) of the digit.

BACKGROUND: Surgical excision is the preferred method of eradicating Bowen's disease (BD). However, when BD occurs on the digit, surgical intervention can sometimes lead to scar contracture and loss of function of the digit. OBJECTIVE: To evaluate the effectiveness of photodynamic therapy (PDT) in eradicating BD of the digit while preserving the full function of the digit. METHODS: Four patients of chronic arsenism with biopsy-proven BD on the digit were treated with PDT by using a newly designed light-emitting diode (LED) array with a peak wavelength of 630 nm (630 +/- 40 nm; 40 mW/cm2 at skin surface). After partial removal of the thickened horny layer and 16 hours of occlusion with a 2% aminolevulinic acid (ALA) solution, each lesion was irradiated with 240 J/cm2 in two fractions with a 90-minute interval. RESULTS: All patients experienced a significant burning, tingling sensation that was tolerable during the procedure except one who needed local anesthesia. All treated digits healed without scarring in 2 weeks. Posttreatment biopsy in one patient showed normal epidermis and a slight fibrosis in the papillary dermis. Three patients remained free of recurrence (75%) at 15-17 months (average 16 months) after one treatment. One patient's BD recurred at 8 months, but was successfully treated without recurrence after 20 months. CONCLUSION: Our preliminary study suggests that PDT using 2% 5-ALA solution and an LED array is an effective, noninvasive method to treat digital BD with the benefit of scar-free contracture and loss of digital function. Among the various factors that would affect the results of PDT, we feel that partial removal of the thickened horny layer is the most important step to achieve sufficient therapeutic effect in digital BDs.

Anticancer activity evaluation of the solanum glycoalkaloid solamargine. Triggering apoptosis in human hepatoma cells.

Solamargine, an herbal and molluscicidal medicine derived from Solanum incanum, is a steroidal alkaloid glycoside. To characterize the anticancer mechanism of solamargine on human hepatoma cells (Hep3B), changes of cell morphology, DNA content, and gene expression of cells after solamargine treatment were studied. The appearance in solamargine-treated cells of chromatin condensation, DNA fragmentation, and a sub-G(1) peak in a DNA histogram suggests that solamargine induces cell death by apoptosis. The maximum number of dead Hep3B cells was detected within 2 hr of incubation with constant concentrations of solamargine, and no further cell death was observed after an extended incubation with solamargine, indicating that the action of solamargine was irreversible. To determine the susceptibility of cell phases to solamargine-mediated apoptosis, Hep3B cells were synchronized at defined cell cycles by cyclosporin A, colchicine, and genistein, followed by solamargine treatment. The IC(50) values of solamargine for control, G(0)/G(1)-, M-, and G(2)/M-synchronized Hep3B cells were 5.0, > 10, 3.7, and 3.1 microg/mL, implying that cells in the G(2)/M phases are relatively susceptible to solamargine-mediated apoptosis. In addition, a parallel up-regulation of tumor necrosis factor receptor (TNFR)-I and -II on Hep3B cells was detected after solamargine treatment, and the solamargine-mediated cytotoxicity could be neutralized with either TNFR-I or -II specific antibody. Therefore, these results reveal that the actions of TNFR-I and -II on Hep3B cells may be independent, and both are involved in the mechanism of solamargine-mediated apoptosis.

Quantitative determination of the expression of xeroderma pigmentosum F gene in human nonmelanoma skin cancers.

Nonmelanoma skin cancers (NMSC) has been evidenced with an impaired function in nucleotide excision repair (NER). However, malfunction of NER elements in NMSC has not been identified. Xeroderma pigmentosum F (XPF) is an essential subunit in NER and functions as a 5'-incision enzyme when repairing damaged DNA. So far, neither XPF's protein nor antibody is commercially available. To explore the expression of XPF in NMSC, the gene was determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). All the designed primers specifically amplified XPF cDNA as demonstrated by nested PCR, and one set of the primers was mimic constructed to form a controlled cDNA for the semiquantification of XPF gene in NMSC. The results indicated that the quantities of XPF expression of BCC and SCC specimens were approximately 57.0 and 76.4% less than that of normal skins, respectively. This paper indicates that the decrease expression of XPF gene may be one of mechanisms for impaired NER in NMSC, and the feasible and quantitative primers used in the experiments may explore the study of XPF in etiology of carcinogenesis.