Blocking glutamate‐mediated signalling inhibits human melanoma growth and migration

Glutamate is an excitatory neurotransmitter that has been shown to regulate the proliferation, migration and survival of neuronal progenitors in the central nervous system through its action on metabotropic and ionotropic glutamate receptors (GluRs). Antagonists of ionotropic GluRs have been shown to cause a rapid and reversible change in melanocyte dendritic morphology, which is associated with the disorganization of actin and tubulin microfilaments in the cytoskeleton. Intracellular expression of microtubule‐associated protein (MAP) 2a affects the assembly, stabilization and bundling of microtubules in melanoma cells; stimulates the development of dendrites; and suppresses melanoma cell migration and invasion. In this study, we investigated the relationship between glutamate‐mediated signalling and microtubules, cell dendritic morphology and melanoma cell motility. We found that metabotropic GluR1 and N‐methyl‐d ‐aspartate receptor antagonists increased dendritic branching and inhibited the motility, migration and proliferation of melanoma cells. We also demonstrated that the invasion and motility of melanoma cells are significantly inhibited by the combination of increased expression of MAP2a and either metabotropic GluR1 or N‐methyl‐d ‐aspartate receptor antagonists. Moreover, the blockade of glutamate receptors inhibited melanoma growth in vivo. Collectively, these results demonstrate the importance of glutamate signalling in human melanoma and suggest that the blockade of glutamate receptors is a promising novel therapy for treating melanoma.     

Proteome analysis of ultraviolet-B-induced protein expression in vitro human dermal fibroblasts

Background: Ultraviolet‐B (UVB) radiation can result in acute photodamage, photoaging and skin cancer through the induction of reactive oxygen species, DNA damage, activation of signaling pathways, and regulation of gene expression. In this study, we investigated UVB‐induced alterations in protein expression in human dermal fibroblasts. Methods: Skin fibroblasts were irradiated with 100 mJ/cm² UVB, and cell viability was monitored by the 3‐(4,5)‐dimethylthiahiazo(‐z‐y1)‐3,5‐diphenytetrazoliumromide assay. Two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy were used to identify differentially expressed proteins. The mRNA and levels of identified proteins were detected using a quantitative real‐time polymerase chain reaction assay and Western blot. Results: UVB decreased the viability of skin fibroblasts. In UVB‐treated cells, eighteen differentially expressed proteins were identified. Among these proteins, the amounts of receptor‐interacting protein (RIP) and vimentin were significantly up‐regulated. However, their mRNA levels decreased and remained relatively stable, respectively. Conclusions: The differential expression of RIP and vimentin was validated in UVB‐irradiated fibroblasts. RIP may promote cell injury, and vimentin may contribute to the resistance of cells to UVB‐induced damage.     

CXC chemokine receptor 4 is essential for Lipo-PGE1-enhanced migration of human dermal fibroblasts

Abstract: Lipo‐PGE1 [EGLANDIN^®; a lipid microsphere‐incorporated prostaglandin E1 (PGE1)] stimulates angiogenesis and promotes the healing of skin ulcers. Because the effects of Lipo‐PGE1 on cutaneous wound healing are not completely understood, we investigated the ability of Lipo‐PGE1 to affect in vivo wound healing and regulate the migration of human dermal fibroblasts (HDFs). In a murine wound model, Lipo‐PGE1 reduced the wound size compared with control mice. Lipo‐PGE1 significantly increased HDF migration in a dose‐ and time‐dependent manner. Lipo‐PGE1 markedly increased the expression of CXC chemokine receptor 4 (CXCR4), which controls the migration of HDFs, at the mRNA and protein levels. Small interfering RNA (siRNA)‐mediated knockdown of CXCR4 inhibited Lipo‐PGE1–enhanced HDF migration. Moreover, Lipo‐PGE1 directly induced the phosphorylation of c‐Jun N‐terminal kinase (JNK), and the JNK‐specific inhibitor Sp6000125 blocked Lipo‐PGE1–enhanced migration and CXCR4 expression of HDFs. Our results demonstrate that Lipo‐PGE1 accelerates wound healing in vivo and increases the CXCR4‐mediated migration of HDFs through the JNK pathway.     

Fluorofenidone inhibits UV‐A induced senescence in human dermal fibroblasts via the mammalian target of rapamycin‐dependent SIRT1 pathway

The aim of this study was to investigate the protective effect of fluorofenidone (5‐methyl‐1‐[3‐fluorophenyl]‐2‐[1H]‐pyridone, AKF‐PD) on ultraviolet (UV)‐A‐induced senescence in human dermal fibroblasts (HDF) and examine the mechanisms involved. HDF were treated with AKF‐PD. Senescence‐associated (SA)‐β‐galactosidase level, cell viability and expression of p16 were evaluated. In addition, UV‐A‐irradiated HDF were treated with AKF‐PD, rapamycin and MHY1485; SA‐β‐galactosidase staining, 3‐(4 5‐dimethylthiazol‐2‐yl)‐2 5‐diphenyltetrazolium bromide assay and western blot for SIRT1 were performed; and phosphorylated mammalian target of rapamycin (p‐mTOR) expression and reactive oxygen species (ROS) levels were measured. Intracellular ROS was detected by the 2′,7′‐dichlorofluroescein diacetate probe. Our results showed that AKF‐PD substantially attenuated the changes of p16 expression, SA‐β‐galactosidase staining and cellular proliferation induced by UV‐A irradiation in HDF. AKF‐PD rescued the increased mTOR phosphorylation and reduced SIRT1 expression induced by UV‐A irradiation in HDF. AKF‐PD and rapamycin together had a synergistic effect on p‐mTOR reduction and SIRT1 increase. mTOR activator MHY1485 partly blocked the above effects. Moreover, intracellular ROS level induced by UV‐A irradiation could partly decrease by AKF‐PD, and MHY1485 could reduce this effect. Our results indicated that AKF‐PD could alleviate HDF senescence induced by UV‐A‐irradiation by inhibiting the p‐mTOR and increasing SIRT1. Moreover, AKF‐PD may be a potential treatment material for skin.     

Blue light inhibits transforming growth factor‐β1‐induced myofibroblast differentiation of human dermal fibroblasts

Transforming growth factor‐β1 (TGF‐β1) is the major promoter of phenotypic shift between fibroblasts and myofibroblasts accompanied by the expression and incorporation of α‐smooth muscle actin (α‐SMA). This differentiation is crucial during normal wound healing and wound closure; however, myofibroblasts are considered as the main effecter cell type in fibrosis, for example in scleroderma and hypertrophic scarring. As blue light has exerted antiprolific and toxic effects in several cell types, we investigated whether blue light irradiations with a light‐emitting diode array (420 nm) were able to affect proliferation and differentiation of human dermal fibroblasts (HDF). We found that repeated irradiation with non‐toxic doses significantly inhibits TGF‐β1‐induced differentiation of HDF into myofibroblasts shown by α‐SMA immunocytochemistry and Western blotting. Additionally, used doses reduced proliferation and myofibroblast contractibility measured by resazurin and collagen gel contraction assays. It could be demonstrated that blue light mediates cell toxicity by oxidative stress due to the generation of singlet oxygen. We postulate that irradiations at non‐toxic doses induce low‐level oxidative stress and energy‐consuming cellular responses, which both may effect proliferation stop and interfere with myofibroblast differentiation. Thus, targeting differentiation, proliferation and activity of myofibroblasts by blue light may represent a useful strategy to prevent or reduce pathological fibrotic conditions.     

人真皮网状层成纤维细胞在瘢痕疙瘩皮损组织中的表达与分布

目的 探讨人真皮乳头层成纤维细胞(Fp)、网状层成纤维细胞(Fr)和肌成纤维细胞(MFB)在瘢痕疙瘩皮损组织中的表达与分布.方法 2019年5-12月在武汉大学人民医院皮肤科门诊确诊的15例瘢痕疙瘩患者,男8例,女7例,年龄20 ～ 50岁,取皮损组织,以15例年龄匹配的女性乳房整形术正常皮肤组织为对照.采用双重免疫荧...     

Curcumin inhibits UVB‐induced matrix metalloproteinase‐1/3 expression by suppressing the MAPK‐p38/JNK pathways in human dermal fibroblasts

Curcumin (diferuloylmethane) is a polyphenol derived from turmeric (Curcuma longa), which is commonly used as a spice. Recent studies have shown that curcumin has a wide range of pharmacological activities, including anticarcinogenic, antioxidant, anti‐inflammatory and antiangiogenic activities. However, the antiphotoageing effects of curcumin have yet to be characterized. In this study, we investigated the inhibitory effects of curcumin on matrix metalloproteinase (MMP)‐1 and MMP‐3 expression in human dermal fibroblast cells. Western blot analysis revealed that curcumin inhibited ultraviolet (UV) B‐induced MMP‐1 and MMP‐3 expression. Furthermore, curcumin significantly blocked UVB‐induced reactive oxygen species generation in fibroblasts. Curcumin treatment significantly blocked the UVB‐induced activation of nuclear factor (NF)‐κB and activator protein (AP)‐1. Additionally, curcumin strongly repressed the UVB‐induced phosphorylation of p38 and c‐Jun N‐terminal kinase. Curcumin prevented UVB‐induced MMP expression through mitogen‐activated protein kinase/NF‐κB inhibition and AP‐1 activation. In conclusion, curcumin may be useful for preventing and treating skin photoageing.     

Insufficient expression of the melanocortin‐1 receptor by human dermal fibroblasts contributes to excess collagen synthesis in keloid scars

Activation of the α‐melanocyte‐stimulating hormone (αMSH)/melanocortin‐1 receptor (MC1R) signalling pathway exerts antagonistic actions on cutaneous inflammatory and fibrogenic responses in addition to promoting pigment production. Herein, the expression of MC1R by keloid‐derived fibroblasts and keloid scar tissue was investigated using a range of techniques. MC1R mRNA expression levels in five different keloid fibroblast cell lines were significantly reduced to less than half compared with five normal fibroblast cell lines (P < 0.05). Immunohistological analysis of tissue samples indicated that MCR1 immunoreactivity in both epidermal and dermal compartments of five keloid tissue samples was dramatically decreased compared with normal skin (P < 0.05). Insufficient expression of MC1R on human dermal fibroblasts might abolish the αMSH‐mediated suppression of collagen production and myofibroblast transformation elicited by the profibrotic cytokine‐transforming growth factor‐β1. Restoration of reduced MC1R by dermal fibroblasts may lead to novel scar‐reducing therapeutic approaches for treating this refractory fibrotic disease.     

Melan‐A positive dermal cells in malignant melanoma in situ

The presence of Melan‐A positive dermal cells in excisions for melanoma in situ represents a frequent conundrum for pathologists. These cells may represent superficially invasive melanoma, benign, incidental, dermal nevi or non‐specific staining of dermal melanophages. Occasionally, rare, Melan‐A positive dermal cells are present which do not clearly correspond to the above three categories. Our objective was to further characterize these Melan‐A positive dermal cells. To do this, immunoperoxidase staining for Melan‐A and SOX‐10 was performed on 188‐cutaneous excisions, including examples of melanoma in situ, atypical junctional melanocytic hyperplasia and non‐melanocytic tumors. These were evaluated for the presence of Melan‐A and SOX‐10 positive dermal cells. Dermal cells, positive for both markers, were identified in 17% of the excisions. The cells were present in 10% of cases from the melanocytic group and 31% of the cases from the non‐melanocytic group. These cells did not exhibit cytologic atypia and resembled neither the co‐existing neoplasm nor melanophages. We conclude that positivity of these rare Melan‐A positive cells for SOX‐10 argues that they represent true melanocytes and not non‐specific staining. The absence of cytologic atypia in these cells and their presence in excisions of non‐melanocytic neoplasms argues that they are benign, reactive, dermal melanocytes.     

UVA致人皮肤成纤维细胞急性和慢性光损伤模型的建立

目的:探讨人皮肤成纤维细胞(human dermal fibroblasts,HDFs)急性和慢性光损伤模型建立的方法。方法:体外培养原代人皮肤成纤维细胞,选取第4~8代的细胞进行实验。用长波紫外线(UVA)单次照射建立HDFs急性光损伤模型,荧光倒置显微镜观察不同剂量UVA照射后第1、2、3天HDFs的形态变化;CCK-8法检测照射后HDFs的增殖活性。慢性光损伤HDFs模型采用8-甲氧沙林(8-MOP)避光孵育细胞24h,随后以含8-MOP的磷酸盐缓冲液(PBS)置换培养基,行9J/cm~2 UVA照射,照射完成后换Dulbecco改良Eagle培养基(DMEM)(含10%胎牛血清)避光传代培养,21d后显微镜下观察HDFs形态;衰老相关-β-半乳糖苷酶(SA-β-Gal)染色法计算衰老细胞率。结果:单次UVA照射导致HDFs增殖率下降,与UVA剂量呈正相关。UVA在剂量为≤10J/cm~2时,细胞存活率保持在85%;而UVA剂量≥15J/cm~2时细胞存活率明显降低;≥20 J/cm~2时存活率为50%左右,至25 J/cm~2时仅为约25%。慢性光损伤HDFs诱导组(即UVA+MOP组)几乎所有细胞均出现体积变大、细胞颗粒增加等细胞老化的特征性改变;SA-β-Gal染色细胞的阳性率95%。结论:UVA单次照射可成功建立HDFs急性光损伤模型,UVA联合8-MOP构建HDFs慢性光损伤模型。