Impact of media compositions and culture systems on the immunophenotypes of patient-derived breast cancer cells.

BACKGROUND: Heterogeneous tumor cells are thought to be a significant factor in the failure of endocrine therapy in estrogen receptor-positive (ER+) cancers. Culturing patient-derived breast cancer cells (PDBCCs) provides an invaluable tool in pre-clinical and translational research for the heterogeneity of cancer cells. This study aimed to investigate the effects of different media components and culture methods on the BCSC-associated immunophenotypes and gene expression in ER + PDBCCs. METHODS: Ten patients with ER + breast cancer were employed in this study, six of whom had neoadjuvant chemotherapy and four of whom did not. PDBCCs were isolated by enzymatic methods using collagen I and hyaluronidase. PDBCCs were grown as monolayers in mediums with different compositions and as multicellular spheroid in a suspended condition. Collagen I-coated plate and ultralow attachment plate coated with polymer-X were used for monolayer and spheroid culture. Flow cytometry, immunofluorescent staining, RT-PCR, and RNA-sequencing were employed to examine the immunophenotype and genetic profile of PDBCCs. RESULTS: More than 95% of PDBCCs sustain EpCAM high/+/fibroblast marker- phenotypes in monolayer conditions by subculturing 3-4 times. A83-01 removal induced senescent cells with high ß-galactosidase activity. PDBCCs grown as monolayers were characterized by the majority of cells having an EpCAM+/CD49f + phenotype. Compared to full media in monolayer culture, EGF removal increased EpCAM+/CD49f - phenotype (13.8-fold, p = 0.028), whereas R-spondin removal reduced it (0.8-fold, p = 0.02). A83-01 removal increased EpCAM+/CD24 + phenotype (1.82-fold, p = 0.023) and decreased EpCAM low/-/CD44+/CD24- phenotype (0.45-fold, p = 0.026). Compared to monolayer, spheroid resulted in a significant increase in the population with EpCAM-/CD49+ (14.6-fold, p = 0.006) and EpCAM low/-/CD44+/CD24- phenotypes (4.16-fold, p = 0.022) and ALDH high activity (9.66-fold, p = 0.037). ALDH1A and EMT-related genes were upregulated. In RNA-sequencing analysis between spheroids and monolayers, a total of 561 differentially expressed genes (2-fold change, p < 0.05) were enriched in 27 KEGG pathways including signaling pathways regulating pluripotency of stem cells. In a recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the up-and down-regulated genes identified in spheroids, 15 up-, and 14 down-regulated genes were associated with poor prognosis of breast cancer patients. CONCLUSION: The media composition and spheroid culture method change in the BCSCs and EMT markers of PDBCCs, implying the importance of defining the media composition and culture method for studying PDBCCs in vitro.

The effect of Salix alba L. bark extract on dark circles in vitro and in vivo.

OBJECTIVE: Dark circles in the infraorbital area are a common cosmetic concern among individuals because they exhibit fatigue and are undesirable across all ages. Of the dark circle etiologies, blood stasis by poor-vascular integrity can cause darkening of the lower eyelid skin, which might be alleviated by reduced endothelial permeability. In this study, we investigated the effects of Salix alba bark extract (SABE) on the synthesis of hyaluronic acid (HA) in fibroblasts and vascular integrity protection from inflammatory cytokine. We also performed a clinical trial investigating the effect of SABE on dark circles. METHODS: To confirm the effect of SABE on HA synthesis in human dermal fibroblasts (HDFs), we performed ELISA and real-time PCR. We investigated the interaction HDF-secreted substance with vascular integrity, and human dermal microvascular endothelial cells (HMEC-1) were treated with conditioned medium (CM) from HDF treated with or without SABE. Subsequently, we conducted a clinical study on 29 subjects by having them apply SABE containing cream for 8 weeks. RESULTS: Salix alba bark extract treatment increased HA synthesis and regulated HMW-HA-related gene expressions in HDF. CM from SABE-treated HDF alleviated endothelial permeability and led to improved vascular integrity in HMEC-1 cells. Treatment with the cream containing 2% SABE for 8 weeks improved the parameters measuring dark circles, skin microcirculation and elasticity. CONCLUSION: Our results showed that SABE could protect against dark circles in vitro, and that topical treatment of SABE improved the clinical indexes of dark circles in a clinical study. Therefore, SABE can be used as an active ingredient for improving dark circles.

OBJECTIF: Les cernes dans la région infra-orbitaire sont un problème cosmétique fréquent chez les patients, car elles témoignent de la fatigue et sont indésirables à tout âge. Parmi les étiologies de cerne, la stase sanguine due à une mauvaise intégrité vasculaire peut entraîner un assombrissement de la peau de la paupière inférieure qui peut être atténué par une réduction de la perméabilité endothéliale. Dans cette étude, nous avons étudié les effets de l'extrait d'écorce de Salix alba sur la synthèse de l'acide hyaluronique (AH) dans les fibroblastes, et la protection de l'intégrité vasculaire contre les cytokines inflammatoires. Nous avons également réalisé une étude clinique portant sur l'effet de l'extrait d'écorce de Salix alba sur les cernes. MÉTHODES: Pour confirmer l'effet de l'extrait d'écorce de Salix alba sur la synthèse de l'AH dans les fibroblastes dermiques humains (Human Dermal Fibroblasts, HDF), nous avons réalisé un test ELISA et un test PCR en temps réel. Nous avons étudié l'interaction entre la substance sécrétée par les HDF et l'intégrité vasculaire, et les cellules endothéliales microvasculaires dermiques humaines (Human Dermal Microvascular Endothelial Cells, HDMEC-1) ont été traitées avec un milieu conditionné pour les HDF traité avec ou sans extrait d'écorce de Salix alba. Par la suite, nous avons mené une étude clinique auprès de 29 sujets en leur demandant d'appliquer une crème à base d'extrait d'écorce de Salix alba pendant 8 semaines. RÉSULTATS: Le traitement par extrait d'écorce de Salix alba a augmenté la synthèse de l'AH et régulé les expressions géniques liées à l'acide hyaluronique à haut poids moléculaire dans les HDF. Les milieux conditionnés pour les HDF traités par extrait d'écorce de Salix alba ont atténué la perméabilité endothéliale et ont permis une amélioration de l'intégrité vasculaire des cellules HMEC-1. Le traitement avec la crème contenant 2% d'extrait d'écorce de Salix alba pendant 8 semaines a amélioré les paramètres de mesure des cernes, la microcirculation cutanée et l'élasticité. CONCLUSION: Nos résultats ont montré que l'extrait d'écorce de Salix alba pouvait protéger contre les cernes in vitro, et que le traitement topique par extrait d'écorce de Salix alba améliorait les indices cliniques des cernes dans une étude clinique. L'extrait d'écorce de Salix alba peut donc être utilisé comme principe actif pour améliorer les cernes.

Effect of Cirsium japonicum Flower Extract on Skin Aging Induced by Glycation.

Advanced glycation end products (AGEs) have recently been increasingly discussed as one factor of skin aging. In this study, we investigated the effects of Cirsium japonicum flower (CFE) extract on glycation in relation to skin aging and skin elasticity. Moreover, we learned the main active constituent of CFE that has effects against glycation. To demonstrate the effects of CFE on glycation, we carried out an in vitro glycation study, 3-dimensional culture, and clinical study. As a result, CFE inhibited formation of AGEs in both bovine serum albumin (BSA)/glucose glycation system and aldehyde-derived glycation system. Moreover, CFE reduced Nε-(carboxymethyl), lysine (CML), and carbonylated proteins that increased by glycation. Furthermore, CFE broke crosslinks of collagen-AGEs and inhibited the increase of matrix metalloproteinase-1 (MMP-1) gene expression by AGEs. In the 3D culture condition, CFE restored the reduction of collagen gel contraction by glycation. Moreover, apigenin was detected as the main active constituent in CFE that has anti-glycation effects. In the clinical study, we confirmed that CFE has effects on skin wrinkles and skin elasticity. Our findings suggest that CFE can be used as a cosmetic or cosmeceutical ingredient for improving skin elasticity and wrinkles. Regulation of AGEs can be an interesting target for anti-aging.

Melanogenesis-promoting effect of Cirsium japonicum flower extract in vitro and ex vivo.

OBJECTIVE: In this study, we examined the effect of C. japonicum flower extract (CFE) on melanogenesis and its mechanism in vitro and ex vivo. METHODS: The effect of CFE on melanogenesis was investigated with lightly (HEMn-LP) and moderately (HEMn-MP) pigmented normal human melanocytes, reconstituted three-dimensional skin (3D skin) model and ex vivo human hair follicles. The melanogenesis-inducing effect of CFE was evaluated using melanin content and intracellular tyrosinase activity assay. The amount and type of eumelanin and pheomelanin were analysed by using HPLC method. The mechanism involved in the effect of CFE on hyperpigmentation was explored by cyclic adenosine monophosphate (cAMP) immunoassay and western blot analysis for tyrosinase, microphthalmia-associated transcription factor (MITF) and phosphorylated CRE-binding protein (pCREB) expression. The degree of pigmentation in 3D skin and L-values were measured using a CR-300 chroma meter. The amount of dissolved melanin was measured using a spectrophotometer. The content of melanin in the hair follicles was evaluated by Fontana Masson staining. RESULTS: C. japonicum flower extract significantly increased the melanin content and cellular tyrosinase activity in both HEMn-LP and HEMn-MP cells. The markers of pheomelanin and eumelanin in HEMn-LP and HEMn-MP were also increased by CFE. We observed that CFE treatment on melanocytes increased intracellular cAMP with inducing pCREB and up-regulating the protein levels of TYR and MITF. Furthermore, CFE considerably increased the melanin content in a 3D skin model and ex vivo human hair follicles. CONCLUSIONS: These results suggest that CFE exerts hyperpigmentation activity through cAMP signalling in human melanocytes that it can improve follicular depigmentation and vitiligo by stimulating the melanin synthesis.

OBJECTIF: Dans cette étude, nous avons examiné l'effet de l'extrait de fleur de C. japonicum (EFC) sur la mélanogenèse et son mécanisme in vitro et ex vivo. MÉTHODES: L'effet du EFC sur la mélanogenèse a été étudié avec des mélanocytes humains normaux légèrement (HEMn-LP) et modérément (HEMn-MP) pigmentés, un modèle de peau reconstituée en 3 dimensions (peau 3D) et des follicules pileux ex vivo. L'effet inducteur de la mélanogénèse de la EFC a été évalué en utilisant la teneur en mélanine et le dosage de l'activité de la tyrosinase intracellulaire. La quantité et le type d'eumélanine et de phéomélanine ont été analysés en utilisant la méthode HPLC. Le mécanisme impliqué dans l'effet de la EFC sur l'hyperpigmentation a été exploré par immunoessai à l'adénosine monophosphate cyclique (AMPc) et Western blot pour l'expression de la tyrosinase, du facteur de transcription associé à la microphtalmie (MITF) et l'expression de la protéine CREB phosphorylée. Le degré de pigmentation de la peau 3D, les valeurs L ont été mesurées à l'aide d'un chromamètre CR-300. La quantité de mélanine dissoute a été mesurée à l'aide d'un spectrophotomètre. La teneur en mélanine des follicules pileux a été évaluée par coloration Fontana Masson. RÉSULTATS: EFC a augmenté de manière significative la teneur en mélanine et l'activité de la tyrosinase cellulaire dans les cellules HEMn-LP et HEMn-MP. Les marqueurs de phéomélanine et d'eumélanine dans HEMn-LP et HEMn-MP ont également été augmentés par EFC. Nous avons observé que le traitement EFC sur les mélanocytes augmentait l'AMPc intracellulaire en induisant pCREB et en régulant à la hausse les niveaux de protéines de TYR et MITF. De plus, le EFC a considérablement augmenté la teneur en mélanine dans un modèle de peau 3D et dans les follicules pileux humains ex vivo. CONCLUSIONS: Ces résultats suggèrent que la EFC exerce une activité d'hyperpigmentation via la signalisation de l'AMPc dans les mélanocytes humains qu'elle peut améliorer la dépigmentation folliculaire et le vitiligo en stimulant la synthèse de mélanine.

NAD(P)-dependent steroid dehydrogenase-like is involved in breast cancer cell growth and metastasis.

BACKGROUND: The cholesterol biosynthesis pathway is typically upregulated in breast cancer. The role of NAD(P)-dependent steroid dehydrogenase-like (NSDHL) gene, which is involved in cholesterol biosynthesis, in breast cancer remains unknown. This study aimed to uncover the role of NSDHL in the growth and metastasis of breast cancer. METHODS: After NSDHL knockdown by transfection of short interfering RNA into human breast cancer cell lines (MCF-7, MDA-MB-231 and BT-20) and human breast epithelial cell line (MCF10A), cell proliferation assay, cell cycle analysis, three-dimensional cell culture, clonogenic assay, transwell migration and invasion assays, and wound healing assay were performed. Erlotinib was used as the target drug for epidermal growth factor receptor. Immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1wjl /SzJ) were used as orthotropic breast tumor models by injecting them with NSDHL-knockdown MDA-MB-231 cells using lentivirus-carrying NSDHL short hairpin RNA. Clinical data from 3951 breast cancer patients in Gene Expression Omnibus databases were used to investigate the potential prognostic role of NSDHL by survival analysis. RESULTS: NSDHL knockdown in BT-20, and MDA-MB-231 resulted in a significant decrease in their viability, colony formation, migration, and invasion abilities (p < 0.05). Total cholesterol levels were observed to be significantly decreased in NSDHL-knockdown BT-20 and MDA-MB-231 (p < 0.0001). NSDHL knockdown significantly increased the rate of erlotinib-induced cell death, especially in MDA-MB-231 (p = 0.01). NSDHL knockdown led to significantly decreased tumor growth and lung metastasis in the MDA-MB-231 xenograft model (p < 0.01). Clinically, high NSDHL expression in tumors of patients with breast cancer was associated with significantly reduced recurrence-free survival (p < 0.0001). CONCLUSIONS: NSDHL might have a role in promoting breast cancer progression. The usage of NSDHL as a therapeutic target in breast cancer needs to be clarified in further studies.

Cell-Penetrating Peptide-Patchy Deformable Polymeric Nanovehicles with Enhanced Cellular Uptake and Transdermal Delivery.

We herein propose a polymeric nanovehicle system that has the ability to remarkably improve cellular uptake and transdermal delivery. Cell-penetrating peptide-patchy deformable polymeric nanovehicles were fabricated by tailored coassembly of amphiphilic poly(ethylene oxide)- block-poly(ε-caprolactone) (PEO- b-PCL), mannosylerythritol lipid (MEL), and YGRKKRRQRRR-cysteamine (TAT)-linked MEL. Using X-ray diffraction, differential scanning calorimetry, and nuclear magnetic resonance analyses, we revealed that the incorporation of MEL having an asymmetric alkyl chain configuration was responsible for the deformable phase property of the vehicles. We also discovered that the nanovehicles were mutually attracted, exhibiting a gel-like fluid characteristic due to the dipole-dipole interaction between the hydroxyl group of MEL and the methoxy group of PEO- b-PCL. Coassembly of TAT-linked MEL with the deformable nanovehicles significantly enhanced cellular uptake due to macropinocytosis and caveolae-/lipid raft-mediated endocytosis. Furthermore, the in vivo skin penetration test revealed that our TAT-patchy deformable nanovehicles remarkably improved transdermal delivery efficiency.

Application of immunotherapy based on dendritic cells stimulated by tumor cell-derived exosomes in a syngeneic breast tumor mouse model.

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-ß, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8+ cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4+ or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy.

Experience with a Hybrid Procedure Involving Laparoscopic Fundoplication with Percutaneous Endoscopic Gastrostomy in Chronically Ill Children.

Gastrostomy with concurrent laparoscopic Nissen fundoplication (LNF) is often performed as a laparoscopic gastrostomy (LG) by surgeons. Since 2014, we started performing percutaneous endoscopic gastrostomy (PEG) as gastrostomy with LNF. This study aims to compare the outcomes of LG and PEG with LNF. Patients were recruited into two groups: LNF with LG (historical control) or PEG. Demographic data, operation time, time to start feeding, time to full feeding, length of hospital stay (LOS), and complications were compared between the groups. Fourteen patients underwent LNF with LG and 49 underwent LNF with PEG. The median age and body weight of patients were 4.25 years and 14.15 kg in the LG group and 2.58 years and 10.60 kg in the PEG group, respectively. Operation times were significantly shorter in the PEG group (1.81 vs. 2.61 h). The times to start feeding and full feeding as well as LOS were shorter in the PEG group. Nevertheless, complications were similar in both groups. In conclusion, PEG with LNF was associated with significantly shorter operation times, times to start feeding and reach full feeding, and LOS. PEG is a suitable method for LNF in chronically ill children.

Skin Barrier Recovery by Protease-Activated Receptor-2 Antagonist Lobaric Acid.

Atopic dermatitis (AD) results from gene and environment interactions that lead to a range of immunological abnormalities and breakdown of the skin barrier. Protease-activated receptor 2 (PAR2) belongs to a family of G-protein coupled receptors and is expressed in suprabasal layers of the epidermis. PAR2 is activated by both trypsin and a specific agonist peptide, SLIGKV-NH2 and is involved in both epidermal permeability barrier homeostasis and epithelial inflammation. In this study, we investigated the effect of lobaric acid on inflammation, keratinocyte differentiation, and recovery of the skin barrier in hairless mice. Lobaric acid blocked trypsin-induced and SLIGKV-NH2-induced PAR2 activation resulting in decreased mobilization of intracellular Ca²âº in HaCaT keratinocytes. Lobaric acid reduced expression of interleukin-8 induced by SLIGKV-NH2 and thymus and activation regulated chemokine (TARC) induced by tumor necrosis factor-a (TNF-α) and IFN-γ in HaCaT keratinocytes. Lobaric acid also blocked SLIGKV-NH2-induced activation of ERK, which is a downstream signal of PAR2 in normal human keratinocytes (NHEKs). Treatment with SLIGKV-NH2 downregulated expression of involucrin, a differentiation marker protein in HaCaT keratinocytes, and upregulated expression of involucrin, transglutamase1 and filaggrin in NHEKs. However, lobaric acid antagonized the effect of SLIGKV-NH2 in HaCaT keratinocytes and NHEKs. Topical application of lobaric acid accelerated barrier recovery kinetics in a SKH-1 hairless mouse model. These results suggested that lobaric acid is a PAR2 antagonist and could be a possible therapeutic agent for atopic dermatitis.