Fibroin and Spidroin Thin Film to Support the Attachment and Spread of Human Dermal Fibroblast: The Potency of Skin Tissue Engineering

This study aimed to determine the characteristics of scaffolds made of fibroin from Bombyx mori and spidroin from Argiope appensa in supporting the attachment and proliferation of HDF cells on the scaffolds. Thin-film scaffolds were made using the solvent casting technique, where the scaffold is an amalgamation of fibroin, spidroin, PVA, and glycerol. HDF cells were grown on DMEM medium with 10% FBS and 1% antibiotic-antimicotic. Characterization of the scaffolds was performed by using ATR-FTIR, swelling test, contact angle measurement, tensile test, biodegradation, MTT and SEM. The results of the ATR-FTIR analysis showed that the scaffolds contained fibroin, spidroin, PVA, and glycerol. Swelling and contact angle tests showed that all scaffold combinations were hydrophilic. Mechanical properties and in vitro biodegradation tests showed no significant difference among the scaffold combinations. MTT testing showed that all scaffolds could facilitate the attachment of fibroblasts and showed increased viability from day 1, 3, and 5. Scanning electron microscopy showed that the cells in the 70% fibroin and 10% spidroin scaffold had the best cell morphology and the best combination for potential application in skin tissue engineering.

Cynaropicrin Potentiates the Anti-Tumor Effects of Paclitaxel and 5-Fluorouracil on KYSE30 Human Esophageal Carcinoma

Natural products or their use in combination therapy regimens may reduce side effects of chemotherapy and increase the effectiveness of treatments. The cytotoxic effects of cynaropicrin, a sesquiterpene lactone isolated from Centaurea behen were evaluated for the first time against esophageal squamous carcinoma cells (KYSE30). The synergistic effects of cynaropicrin with paclitaxel and 5-fluorouracil (5-Fu), conventional chemotherapeutic drugs used for esophageal cancer treatment, were also investigated. MTT results indicated that the 50% inhibitory concentration (IC50) values of cynaropicrin on KYSE30 cells after 24, 48 and 72 h were 72, 43 and 28 μM, respectively, which were significantly different from those on normal HDF cells. It was also determined that cynaropicrin could induce the cell death via apoptosis. Our results indicated that cynaropicrin had synergism with both drugs. The best effects of cynaropicrin in combination with paclitaxel were observed at 48 h, in which dose reduction of paclitaxel reached 3 times in IC50. Combination with 5-fluorouracil, resulted in 15 times dose reduction of 5-Fu in IC50 at 24 h. In conclusion cynaropicrin has selective cytotoxic effects on KYSE30 cells and we suggest its use in combination therapies with paclitaxel and 5-Fu, which would reduce the side effects of these conventional treatments.

Acetylated Resveratrol and Oxyresveratrol Suppress UVB-Induced MMP-1 Expression in Human Dermal Fibroblasts

Resveratrol (RES) and oxyresveratrol (OXYRES) are considered and utilized as active ingredients of anti-aging skin cosmetics. However, these compounds are susceptible to oxidative discoloration and unpleasant odor in solutions, limiting their use in cosmetics. Accordingly, RES and OXYRES were chemically modified to acetylated derivatives with enhanced stability, and their anti-aging effect on the skin and detailed molecular mechanism of their acetylated derivatives were investigated. Acetylated RES and OXYRES lost their acetyl group and exerted an inhibitory effect on H2O2-induced ROS levels in human dermal fibroblast (HDF) cells. In addition, RES, OXYRES, and their acetylated derivatives suppressed UVB-induced matrix metalloproteinase (MMP)-1 expression via inhibition of mitogen-activated protein kinases (MAPKs) and Akt/mTOR signaling pathways. Furthermore, RES, OXYRES, and their acetylated derivatives suppressed type I collagen in TPA-treated HDF cells. Collectively, these results suggest the beneficial effects and underlying molecular mechanisms of RES, OXYRES, and their acetylated derivatives for anti- skin aging applications.

Metformin-induced ROS upregulation as amplified by apigenin causes profound anticancer activity while sparing normal cells

Metformin increased cellular ROS levels in AsPC-1 pancreatic cancer cells, with minimal effect in HDF, human primary dermal fibroblasts. Metformin reduced cellular ATP levels in HDF, but not in AsPC-1 cells. Metformin increased AMPK, p-AMPK (Thr172), FOXO3a, p-FOXO3a (Ser413), and MnSOD levels in HDF, but not in AsPC-1 cells. p-AMPK and p-FOXO3a also translocated from the cytosol to the nucleus by metformin in HDF, but not in AsPC-1 cells. Transfection of si-FOXO3a in HDF increased ROS levels, while wt-FOXO3a-transfected AsPC-1 cells decreased ROS levels. Metformin combined with apigenin increased ROS levels dramatically and decreased cell viability in various cancer cells including AsPC-1 cells, with each drug used singly having a minimal effect. Metformin/apigenin combination synergistically decreased mitochondrial membrane potential in AsPC-1 cells but to a lesser extent in HDF cells. Metformin/apigenin combination in AsPC-1 cells increased DNA damage-, apoptosis-, autophagy- and necroptosis-related factors, but not in HDF cells. Oral administration with metformin/apigenin caused dramatic blocks tumor size in AsPC-1-xenografted nude mice. Our results suggest that metformin in cancer cells differentially regulates cellular ROS levels via AMPK-FOXO3a-MnSOD pathway and combination of metformin/apigenin exerts anticancer activity through DNA damage-induced apoptosis, autophagy and necroptosis by cancer cell-specific ROS amplification.

Insulin Smart Drug Delivery Nanoparticles of Aminophenylboronic acid–POSS Molecule at Neutral pH

Self-regulated “smart” insulin administration system that mimic pancreatic endocrine function would be highly desirable for diabetes management. Here, a glucose-responsive continuous insulin delivery system is developed, where novel polyhedral oligosilsesquioxane (POSS) modified with 3-aminophenylboronic acid (APBA) were used to encapsulate insulin (insulin entrapment efficiency : 73.2%; loading capacity : 50.5%) to prepare a fast response, high stability, good distribution, and excellent biocompatible system. Micelles self‐assembled from these molecules possess glucose‐responsiveness at varying glucose concentrations. The interaction of the PBA and diol containing insulin via boronate ester bond and its interchange with glucose was investigated by FT-IR, 1H NMR and XPS. Furthermore, the successful glucose-triggered release of insulin from the POSS-APBA micelles was investigated at neutral pH. A linear graph was plotted with the measured released insulin vs glucose concentrations, with a linear correlation coefficient (R2) value close to 1. When confirming intracellular apoptosis signaling, cleaved caspase 3 and caspase 9 were not increased by 640 µg/ml POSS-APBA and POSS-APBA@Insulin in HeLa cells and HDF cells. Application in the biomedical field for controlled delivery of insulin appear to be promising.

Dieckol, an Algae-Derived Phenolic Compound, Suppresses UVB-Induced Skin Damage in Human Dermal Fibroblasts and Its Underlying Mechanisms

Ultraviolet (UV) irradiation is considered to be the primary environmental factor that causes skin damage. In the present study, we investigated the protective effect of dieckol (DK), a compound isolated from the brown seaweed Ecklonia cava, against UVB-induced skin damage in human dermal fibroblasts (HDF cells). The results indicated that DK effectively inhibited the activity of collagenase. DK remarkably reduced the intracellular reactive oxygen species level and improved the viability of UVB-irradiated HDF cells. Besides, DK significantly and dose-dependently improved collagen synthesis and inhibited intracellular collagenase activity in UVB-irradiated HDF cells. In addition, DK markedly reduced the expression of proinflammatory cytokines and matrix metalloproteinases. Further analyses revealed that these processes were mediated through the regulation of nuclear factor kappa B, activator protein 1, and mitogen-activated protein kinase signaling pathways in the UVB-irradiated HDF cells. In conclusion, these results indicate that DK possesses strong in vitro photoprotective effects and therefore has the potential to be used as an ingredient in the cosmeceutical industry.

Encapsulation and Characterization of Meropenem in Niosome Nanoparticles and Inhibitory Effects of Antibiofilm and Antibacterial on Methicillin and Vancomycin Resistant Strains of Staphylococcus Aureus

Background: We aim to assess the antibacterial and anti-biofilm properties of niosome-encapsulated meropenem. Methods: After isolating S. aureus isolates and determining their microbial sensitivity, their ability to form biofilms was examined using plate microtiter assay. Various formulations of niosome-encapsulated meropenem were prepared using the thin-film hydration method, Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Inhibitory Concentration (MIC) were determined, and biofilm genes expression was examined. Drug formulations’ toxicity effect on HDF cells were determined using MTT assay.Results: Out of the 162 separated Staphylococcus aureus, 106 were resistant to methicillin. 87 MRSA isolates were vancomycin-resistant, all of which could form biofilms. The F1 formulation of neoplastic meropenem with a size of 51.3 ± 5.84 and an encapsulation index of 84.86 ± 3.14 was detected, which prevented biofilm growth with a BDI index of 69% and reduced icaD, FnbA, Ebps biofilms’ expression with p ≤0.05 in addition to reducing MBIC and MIC by 4-6 times. Interestingly, F1 formulation of neoplastic meropenem indicated cell viability over 90% at all tested concentrations. Conclusions: Results of the present study indicate that niosome-encapsulated meropenem reduces the resistance of Staphylococcus aureus MRSA to antibiotics in addition to increasing its anti-biofilm and antibiotic activity, and could prove useful as a new strategy for drug delivery.

Apoptotic Effects of Bilirubin on Skin Cancer Cell Lines SK-MEL-3 (Melanoma) and A431 (Non-Melanoma)

Background:: Bilirubin has long been exclusively considered as a potentially dangerous sign of liver diseases, but it is currently regarded as a reliable signaling molecule as well. Objective:: This study investigated the effects of unconjugated bilirubin on survival, proliferation, apoptotic and cell arrest capacities of melanoma SKMEL-3 and non-melanoma A431 skin cancer cells in comparison with normal human dermal fibroblast (HDF) cells. Methods:: The MTT assay test was used to identify survival and the IC50 at various concentrations of bilirubin on SKMEL-3, A431, and HDF cells for 24h and 48h. The comet assay technique was used to investigate genotoxicity effects and flow cytometry was run to investigate apoptotic and cell arresting effects of bilirubin on the cells. The gene expression of cyclin D1, cyclin E1, survivin, Bcl-2, and p53 was investigated by qRT-PCR. The molecular docking of bilirubin on CDKs (Cyclin-dependent kinases 2, 4, and 6) and pro-apoptotic factors Bad, Bak, Bax, Bid, Bik, and Bim were done by Autodock software version 2. Results:: The IC50 of bilirubin on HDF, A431, and SKMEL-3 cells were 125, 115, and 95 μM at 24h and 115, 100, and 75 μM at 48h, respectively. Although cell arrest in the G1 phase occurred in all cells, bilirubin induced the genotoxicity and apoptosis in SKMEL-3 and A431 cancer cells more pronouncedly than those in normal HDF cells. Conclusion:: Bilirubin led to cell arrest in the G1 phase in SKMEL-3, A431, and HDF cells. Additionally, bilirubin induced apoptotic pathways in SKMEL-3 and A431 cancer cells.

Analysis of the Epigenetic Signature of Cell Reprogramming by Computational DNA Methylation Profiles

Background: DNA methylation plays an important role in the reprogramming process. Understanding the underlying molecular mechanism of reprogramming is crucial for answering fundamental questions regarding the transition of cell identity. Methods: In this study, based on the genome-wide DNA methylation data from different cell lines, comparative methylation profiles were proposed to identify the epigenetic signature of cell reprogramming. Results: The density profile of CpG methylation showed that pluripotent cells are more polarized than Human Dermal Fibroblasts (HDF) cells. The heterogeneity of iPS has a greater deviation in the DNA hypermethylation pattern. The result of regional distribution showed that the differential CpG sites between pluripotent cells and HDFs tend to accumulate in the gene body and CpG shelf regions, whereas the internal differential methylation CpG sites (DMCs) of three types of pluripotent cells tend to accumulate in the TSS1500 region. Furthermore, a series of endogenous markers of cell reprogramming were identified based on the integrative analysis, including focal adhesion, pluripotency maintenance and transcription regulation. The calcium signaling pathway was detected as one of the signatures between NT cells and iPS cells. Finally, the regional bias of DNA methylation for key pluripotency factors was discussed. Our studies provide new insight into the barrier identification of cell reprogramming. Conclusion: Our studies analyzed some epigenetic markers and barriers of nuclear reprogramming, hoping to provide new insight into understanding the underlying molecular mechanism of reprogramming.

The Evaluation of the Effects of Nanoemulsion Formulations Containing Boron and/or Zinc on the Wound Healing in Diabetic Rats

Wound healing remains a challenging clinical problem, especially in the presence of diabetes. Diabetic patients have the impaired ability to fight infection and insufficient inflammatory response. The aim of this study was to evaluate the effects of boronophenylalanine (BFA) and/or Zn-containing nanoemulsion (NE) formulations on wound healing in diabetic rats. MTT and scratch assays were performed to evaluate the proliferative effects of BFA and/or Zn on human dermal fibroblast (HDF) cells and the migration of these cells, respectively. The BFA and/or Zn-NE were prepared, and the effects of NEs on wound healing in diabetic rats were evaluated by applying once a day for 14 days. MTT assay showed that 10 to 25 µM BFA and/or 50 µM Zn had very significant positive effects on cell proliferation. In the scratch assay, 10 µM BFA significantly increased the migration of HDF cell compared with control. The droplet sizes of all the NEs were <115 nm and their zeta potential values were in range of (−) 23.9 ± 2.356 to (−) 33.1 ± 1.438 mV. There was a significant reduction in the wound contraction values (%) of the groups treated with the BFA and/or Zn-NE on the 14th day compared with the untreated diabetic rats group. According to histopathological findings, wound healing was nearly complete in BFA and/or Zn-NE compared with untreated diabetic rats. Especially, the group treated with the NE containing the low concentration of BFA showed highly promising results in wound healing of diabetic rats within 14 days with complete epithelialization and the completely closed wound area.