Shengxiang Xiao

Apoptosis induced by synthetic retinoic acid CD437 on human melanoma A375 cells involves RIG-I pathway

Human malignant melanoma is notoriously resistant to currently available pharmacological modulation. Our aim was to evaluate the anti-tumor effect of a novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carbo-xylic acid (CD437) on melanoma cell line A375. Analysis of cell morphology showed that CD437 promoted marked apoptosis in A375 cells. To explore the mechanisms of CD437-induced apoptosis, an NF-κB-luciferase reporter assay was performed, demonstrating that apoptosis induction by CD437 required activation of transcription factor NF-κB. Importantly, based on the findings that RIG-I (retinoic acid inducible gene I) can be induced by retinotic acid and can activate NF-κB through a CARD-containing adaptor protein VISA, we proposed a hypothesis that RIG-I was involved in the signal pathway of NF-κB activation induced by CD437 through the adaptor protein VISA. By specially cleaving VISA with hepatitis C virus (HCV) non-structural (NS)3/4A, the RIG-I pathway was blocked, with subsequent simultaneous inhibition of CD437-induced NF-κB activation and cell apoptosis in A375 cells. These results support our hypothesis and suggest that RIG-I may be a useful intermediate biologic marker for retinoid chemoprevention and treatment studies.

Activation of Erk and p53 regulates copper oxide nanoparticle-induced cytotoxicity in keratinocytes and fibroblasts

Copper oxide nanoparticles (CuONP) have attracted increasing attention due to their unique properties and have been extensively utilized in industrial and commercial applications. For example, their antimicrobial capability endows CuONP with applications in dressings and textiles against bacterial infections. Along with the wide applications, concerns about the possible effects of CuONP on humans are also increasing. It is crucial to evaluate the safety and impact of CuONP on humans, and especially the skin, prior to their practical application. The potential toxicity of CuONP to skin keratinocytes has been reported recently. However, the underlying mechanism of toxicity in skin cells has remained unclear. In the present work, we explored the possible mechanism of the cytotoxicity of CuONP in HaCaT human keratinocytes and mouse embryonic fibroblasts (MEF). CuONP exposure induced viability loss, migration inhibition, and G2/M phase cycle arrest in both cell types. CuONP significantly induced mitogen-activated protein kinase (extracellular signal-regulated kinase [Erk], p38, and c-Jun N-terminal kinase [JNK]) activation in dose- and time-dependent manners. U0126 (an inhibitor of Erk), but not SB 239063 (an inhibitor of p38) or SP600125 (an inhibitor of JNK), enhanced CuONP-induced viability loss. CuONP also induced decreases in p53 and p-p53 levels in both cell types. Cyclic pifithrin-α, an inhibitor of p53 transcriptional activity, enhanced CuONP-induced viability loss. Nutlin-3α, a p53 stabilizer, prevented CuONP-induced viability loss in HaCaT cells, but not in MEF cells, due to the inherent toxicity of nutlin-3α to MEF. Moreover, the experiments on primary keratinocytes are in accordance with the conclusions acquired from HaCaT and MEF cells. These data demonstrate that the activation of Erk and p53 plays an important role in CuONP-induced cytotoxicity, and agents that preserve Erk or p53 activation may prevent CuONP-induced cytotoxicity.

Isolation and antitumor activities of acidic polysaccharide from Gynostemma pentaphyllum Makino

Two acidic polysaccharides (GP-B1 and GP-C1) were obtained from Gynostemma pentaphyllum. The molecular weights (Mw) of the two fractions were 79 kDa for GP-B1 and 126 kDa for GP-C1. GP-B1 was composed of Gal, Ara, Man, Rha, Xyl, Glc, GalA and GlcA in a molar ration of 3.5:3.2:0.6:0.9:0.3:0.5:0.6:0.4. GP-C1 consisted of Gal, Ara, Man, Rha, Glc, and GlcA in the proportions of 2.1:1.0:0.3:0.5:0.4:0.9. Among them, GP-B1 treatment had a significant inhibitory effect on the growth of melanoma B16 in vivo and in vitro. Meanwhile GP-B1 could increase the relative spleen weight and stimulate the splenocyte proliferation alone or combined with ConA. Moreover, GP-B1 treatment induced an evident increase in the level of serum TNF-α, IFN-γ, and IL-12 and a reduction for IL-10 production. These results indicate that the antitumor effects of GP-B1 are associated with immunostimulation.

RNAi-mediated inhibition of COL1A1 and COL3A1 in human skin fibroblasts.

Abstract:  Types I and III collagens are the major collagens comprising skin connective tissue. Defects in these collagens lead to diseases of dermal connective tissue and fibre hyperplasia. RNA interference (RNAi) provides a powerful tool to inhibit specific gene expression. In this study, we generated small interfering RNAs (siRNA) expression cassettes (SECs) by polymerase chain reaction (PCR) as a method to quickly screen the efficacy of siRNAs. We then cloned the most efficient SECs into vectors, using a rapid and novel method intrinsic to the design of the SEC, and transfected human skin fibroblasts (HSF) to generate stable lines. We show that the transfection of SECs into HSFs resulted in specific and effective repression of COL1A1 and COL3A1 expression (5.00% and 6.48% of control levels) provided a rapid method for testing candidate siRNA sequences. We report the use of vector‐based RNAi to establish stable HSF cell lines with persistent knockdown over at least 30 days (25.21% and 22.12% of control levels). These stably modified HSF cell lines may be used for the study of other types of collagen or proteins of the extracellular matrix (ECM).

Increased βTrCP are associated with imiquimod-induced psoriasis-like skin inflammation in mice via NF-κB signaling pathway

Psoriasis is a common inflammatory skin disease characterized by T cell-mediated hyperproliferation of keratinocytes, increased angiogenesis and inflammation. Accumulating evidence suggests that some keratinocyte differentiation events are controlled by the ubiquitin/proteasome system. β-transducin repeat-containing protein (βTrCP) serve as substrate recognition component of E3 ubiquitin ligases that control stability of important regulators of signal transduction including the nuclear factor (NF)-κB signaling, a key regulatory element in inflammatory pathways related to psoriasis, suggesting a potential role of βTrCP in psoriasis pathogenesis. However, no published study has investigated the role of βTrCP in the etiology of psoriasis. Here, we combined an in vitro cell model of tumor necrosis factor (TNF)-α-induced keratinocyte inflammation and an animal model of imiquimod (IMQ)-induced psoriasis-like inflammation to investigate the pathogenic mechanisms in psoriasis-like dermatitis and assess its βTrCP/NF-κB dependency. Daily application of IMQ on mouse back skin induced inflamed scaly skin lesions resembling plaque type psoriasis. These lesions were associated with elevated βTrCP levels, reduced inhibitor κB (IκB), and enhanced NF-κB activation in epidermal tissues. Furthermore, βTrCP knockdown via siRNA in in TNF-α-stimulated HaCaT and normal human epidermal keratinocytes (NHEK) cells significantly inhibited the over-activation of NF-κB and expression of intercellular adhesion molecule 1 (ICAM-1), demonstrating a pivotal role of βTrCP in regulation the TNF-α-activated NF-κB inflammatory pathways. Moreover, downregulation of βTrCP through lentiviral shRNA ameliorates IMQ-induced psoriasis-like skin lesions in vivo. In conclusion, βTrCP is involved in the NF-κB signaling mediated-, psoriasis-related inflammation and represent a novel target for developing agents to treat psoriasis.

microRNA-216b inhibits cell proliferation and migration in human melanoma by targeting FOXM1 in vitro and in vivo†

MicroRNAs (miRNAs) play an increasingly important role in cancer growth by coordinately suppressing genes that control cell migration, proliferation, and invasion. The above results can be achieved through the regulation of gene expression by miRNAs by suppressing translation or the direct sequence‐specific degradation of the targeted mRNA. In the present study, we indicate that the expression of miR‐216b could be effectively repressed both in human melanoma tissues through a comparison with primary melanoma and in human melanoma cell lines through a comparison with a normal human keratinocyte line. Moreover, miR‐216b induced a clear decrease in melanoma cell proliferation and migration in vitro. Forkhead box M1 (FOXM1) was confirmed as a target gene of miR‐216b, and the overexpression of miR‐216b markedly repressed the luciferase activity of reporter plasmids containing the FOXM1 3′‐UTR (untranslated region). Furthermore, miR‐216b suppressed melanoma cell growth in nude mice in vivo, with the effects of miR‐216b overexpression on melanoma cell growth and proliferation reversed by FOXM1 overexpression. The results demonstrated that miR‐216b is a tumor suppressor in melanoma, identified the FOXM1 signaling pathway as a target of miR‐216b action, and suggested a potential therapeutic role for miR‐216b in melanoma.

Two novel mutations of the ATP2C1 gene in Chinese patients with Hailey–Hailey disease

Hailey–Hailey disease (HHD; OMIM 169600) is an autosomal dominant blistering disease. Pathogenic mutations in ATP2C1 encoding the human secretory pathway Ca2+/Mn2+-ATPase protein 1 (hSPCA1) have been identified since 2000. The aim of this study was to report a Chinese pedigree and a sporadic case of HHD and to explore the genetic mutations. The Chinese pedigree and the sporadic case of typical HHD were subjected to mutation detection of ATP2C1. The 27 coding exons and their flanking sequences were amplified and sequenced. The heterozygous C to T transition at nucleotide 2753 in exon 26 and G to T transition at nucleotide 2090 in exon 21 of the ATP2C1 gene were identified in a pedigree and a sporadic case of HHD, respectively. The C2753T transition resulted in a novel nonsense mutation of glutamine codon (CAG) to a stop codon (TAG) at amino acid residue 865 (Q865X) and the G2090T transition resulted in a novel missense mutation of glycine condon (GGA) to Valine (GUA) at amino acid residue 645 (G645V) in hSPCA1. This study should be useful for genetic counseling and prenatal diagnosis for affected families and in expanding the repertoire of ATP2C1 mutations underlying HHD.

Wnt/β-Catenin and Wnt5a/Ca Pathways Regulate Proliferation and Apoptosis of Keratinocytes in Psoriasis Lesions.

Background/Aims: Wnt5a is overexpressed in psoriasis lesions, however the mechanism by which Wnt5a is involved in the pathogenesis of psoriasis is not clear. To address this, the expression of Wnt5a in psoriatic lesions and its effect on keratinocyte cell proliferation and apoptosis was examined in vitro. Methods: The expression levels of WNT5A, and genes encoding its receptors frizzled2 (FZD2) and frizzled5 (FZD5) were examined in samples obtained from individuals with psoriasis and healthy controls. Knockdown of Wnt5a with short interfering (si)RNAs was performed in cultured HaCaT keratinocytes and normal human keratinocytes (NHK), and the expression of Wnt5a, protein kinase C (PKC), and β-catenin were determined, and cell cycle activity, proliferation and apoptosis were assessed. Results: The expression of WNT5A, FZD2 and FZD5 mRNA and protein were increased in psoriatic lesions. Wnt5a knockdown suppressed proliferation and induced apoptosis in HaCaT and NHK cells. Additionally, expression of PCNA, MKI67, CCND1, BCL2, CTNNB1, and genes encoding PKC and survivin were downregulated, whereas CASP3 was upregulated. The mRNA levels of the Wnt pathway inhibitors DKK1 and SFRP1 were upregulated, Western blotting analyses demonstrated reduction in β-catenin and PKC protein levels. Conclusion: Knockdown of Wnt5a suppresses the proliferation of keratinocytes and induces apoptosis by inhibiting the Wnt/β-catenin or Wnt5a/Ca2+ pathways.

Overexpression of S100A7 protects LPS-induced mitochondrial dysfunction and stimulates IL-6 and IL-8 in HaCaT cells.

Background S100A7 (or psoriasin) is distributed in the cytoplasm of keratinocytes of normal human epidermis, and it is overexpressed in many epidermal inflammatory diseases. Lipopolysaccharide (LPS) induces mitochondrial function changes, which play important roles in multiple cellular mechanisms including inflammation. Although S100A7 expression is regulated by various factors in the human epidermis during inflammation, whether S100A7 interacts with mitochondria in keratinocytes is not clear. Objectives Our study was designed to investigate whether S100A7 could prohibit mitochondrial dysfunction and stimulate cytokines in cultured normal HaCaT cells treated with LPS. Results We generated HaCaT cells that constitutively express enhanced green fluorescence protein (EGFP)-S100A7 (S100A7-EGFP) or EGFP alone, as a control. Here, we show that S100A7-EGFP HaCaT cells exhibit an increase in mitochondrial DNA (mtDNA) copy number and mitochondrial membrane potential (MMP). qRT-PCR revealed that expression of three main mitochondrial biogenesis-associated genes was significantly increased: PPAR-coactivator-1alpha (PGC-1α), the mitochondrial transcription factor A (Tfam) and nuclear respiratory factor-1 (NRF1). S100A7 overexpression increased mtDNA content and effectively increased intracellular adenosine 5′-triphosphate (ATP) production, while decreasing reactive oxygen species (ROS) generation. S100A7 overexpression also significantly decreased the expression of Mfn2 and increased DRP1 expression compared with control EGFP cells. S100A7 down-regulated the expression of the autophagy-related proteins Beclin-1 and LC3B. S100A7 also increased expression of IL-6 and IL-8 cytokines. Knockdown of S100A7 decreased MMP and disrupted mitochondrial homeostasis. Conclusions These findings demonstrate that S100A7 stimulates mitochondrial biogenesis and increases mitochondrial function in HaCaT cells treated with LPS; and S100A7 also promotes secretion of IL-6 and IL-8.

Predictive factors for adverse dermatological events during pegylated/interferon alpha and ribavirin treatment for hepatitis C

BACKGROUND Treatment of chronic hepatitis C (CHC) with pegylated interferon-alpha/ribavirin is associated with well-characterized dermatological adverse events (AEs), which can lead to premature discontinuation of treatment. OBJECTIVE To investigate the incidence and spectrum of dermatological AEs during CHC treatment with interferon-alpha plus ribavirin and analyzed factors predisposing patients to such reactions. STUDY DESIGN Between January 2008 and December 2012, 152 CHC patients who had received interferon/pegylated interferon plus ribavirin therapy were enrolled in this retrospective study. To determine which factors were associated with dermatological AE development, a Cox proportional-hazards regression analysis was performed. RESULTS Thirty dermatological AEs were recorded in 28 (18.4%) patients. These reactions included 14 (9.2%) patients with eczematous reactions, four (2.6%) patients with xerosis, three (2.0%) patients with new-onset or exacerbation of psoriasis, two (1.3%) patients with lichenoid eruption, two (1.3%) patients with diffuse folliculitis and one patient with lichen planus, alopecia areata, hypermelanosis, and necrosis of the skin and toenails. Application of the Cox proportional-hazards model revealed that age older than 60 years (HR=1.070; 95% CI: 1.043-1.096), pre-existing anaphylaxis/skin disease (HR=2.612; 95% CI: 1.593-3.324), cirrhosis (HR=1.863; 95% CI: 1.047-3.013), and treatment with pegylated interferon formulations (HR=1.930; 95% CI: 1.052-3.687) were associated with occurrence of dermatologic AEs. Twenty-seven (90%) skin conditions were classified as mild to moderate, while one case (3.3%) warranted premature discontinuation of treatment. CONCLUSION Dermatological AEs resulting from interferon-alpha/ribavirin treatment of CHC contribute to a wide spectrum involve the skin, mucous membrane, hair, and nails. These dermatological AEs correlated with older age, previous skin condition, cirrhosis, and use of pegylated interferon formulations.