Hyperpigmentation can occur in abnormal skin conditions such as melanomas, as well as in conditions including melasma, freckles, age spots, seborrheic keratosis, and café-au-lait spots (flat brown ...spots). Thus, there is an increasing need for the development of depigmenting agents. We aimed to repurpose an anticoagulant drug as an effective ingredient against hyperpigmentation and apply cosmeceutical agents. In the present study, the anti-melanogenic effects of two anticoagulant drugs, acenocoumarol and warfarin, were investigated. The results showed that both acenocoumarol and warfarin did not cause any cytotoxicity and resulted in a significant reduction in intracellular tyrosinase activity and melanin content in B16F10 melanoma cells. Additionally, acenocoumarol inhibits the expression of melanogenic enzymes such as tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2, suppressing melanin synthesis through a cAMP-dependent, protein kinase (PKA)-dependent downregulation of microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. Furthermore, anti-melanogenic effects were exerted by acenocoumarol through downregulation of the p38 and JNK signaling pathway and upregulation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3β (GSK-3β) cascades. In addition, the β-catenin content in the cell cytoplasm and nucleus was increased by acenocoumarol through a reduction in the phosphorylated β-catenin (p-β-catenin content). Finally, we tested the potential of acenocoumarol for topical applications by conducting primary human skin irritation tests. Acenocoumarol did not induce any adverse reactions during these tests. Based on the results, it can be concluded that acenocoumarol regulates melanogenesis through various signaling pathways such as PKA, MAPKs, PI3K/Akt/GSK-3β, and β-catenin. These findings suggest that acenocoumarol has the potential to be repurposed as a drug for treating hyperpigmentation symptoms and could provide new insights into the development of therapeutic approaches for hyperpigmentation disorders.
Syringetin, an active compound present in red grapes, jambolan fruits,
, and
, is a dimethyl myricetin derivative which contains free hydroxyl groups at the C-2' and C-4' positions in ring B. Recent ...studies have revealed that syringetin possesses multiple pharmacological properties, such as antitumor, hepatoprotective, antidiabetic, antioxidative, and cytoprotective activities. To date, there has been no attempt to test the action of syringetin on melanogenesis. In addition, the molecular mechanism for the melanogenic effects of syringetin remains largely unknown. In this study, we investigated the effect of syringetin on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse, B16F10. Our results showed that syringetin markedly stimulated melanin production and tyrosinase activity in a concentration-dependent manner in B16F10 cells. We also found that syringetin increased MITF, tyrosinase, TRP-1, and TRP-2 protein expression. Moreover, syringetin inhibited ERK and PI3K/Akt phosphorylation by stimulating p38, JNK, PKA phosphorylation levels, subsequently stimulating MITF and TRP upregulation, resulting in the activation of melanin synthesis. Furthermore, we observed that syringetin activated phosphorylation of GSK3β and β-catenin and reduced the protein level of β-catenin, suggesting that syringetin stimulates melanogenesis through the GSK3β/β-catenin signal pathway. Finally, a primary skin irritation test was conducted on the upper backs of 31 healthy volunteers to determine the irritation or sensitization potential of syringetin for topical application. The results of the test indicated that syringetin did not cause any adverse effects on the skin. Taken together, our findings indicated that syringetin may be an effective pigmentation stimulator for use in cosmetics and in the medical treatment of hypopigmentation disorders.
The dissemination of circulating tumor cells (CTCs) requires the Epithelial-to-Mesenchymal transition (EMT), in which cells lose their epithelial characteristics and acquire more mesenchymal-like ...phenotypes. Current isolation of CTCs relies on affinity-based approaches reliant on the expression of Epithelial Cell Adhesion Molecule (EpCAM). Here we show EMT-induced breast cancer cells maintained in prolonged mammosphere culture conditions possess increased EMT markers and cancer stem cell markers, as well as reduced cell mass and size by quantitative phase microscopy; however, EpCAM expression is dramatically decreased in these cells. Moreover, CTCs isolated from breast cancer patients using a label-free microfluidic flow fractionation device had differing expression patterns of EpCAM, indicating that affinity approaches reliant on EpCAM expression may underestimate CTC number and potentially miss critical subpopulations. Further characterization of CTCs, including low-EpCAM populations, using this technology may improve detection techniques and cancer diagnosis, ultimately improving cancer treatment.
Circulating tumor cells (CTCs) are highly correlated with the invasive behavior of cancer, so their isolations and quantifications are important for biomedical applications such as cancer prognosis ...and measuring the responses to drug treatments. In this paper, we present the development of a microfluidic device for the separation of CTCs from blood cells based on the physical properties of cells. For use as a CTC model, we successfully separated human breast cancer cells (MCF-7) from a spiked blood cell sample by combining multi-orifice flow fractionation (MOFF) and dielectrophoretic (DEP) cell separation technique. Hydrodynamic separation takes advantage of the massive and high-throughput filtration of blood cells as it can accommodate a very high flow rate. DEP separation plays a role in precise post-processing to enhance the efficiency of the separation. The serial combination of these two different sorting techniques enabled high-speed continuous flow-through separation without labeling. We observed up to a 162-fold increase in MCF-7 cells at a 126 µL min(-1) flow rate. Red and white blood cells were efficiently removed with separation efficiencies of 99.24% and 94.23% respectively. Therefore, we suggest that our system could be used for separation and detection of CTCs from blood cells for biomedical applications.
In this study, the wave attenuation by the inclined porous plates has been evaluated through a numerical approach. As a numerical method, we used the dual boundary element method (DBEM) that solves ...two distinct boundary integral equations (singular and hyper-singular) derived at the coincident source points on each side of the degenerate plate boundary. A quadratic velocity model with the porosity-dependent drag coefficient is adopted to account for energy dissipation through the porous plate. The developed DBEM tool is validated through the comparison with self-conducted experiments at a two-dimensional wave tank. Then it is used to assess the reflection coefficients and wave forces with various design parameters such as porosities, inclined angles, plate arrangements, and wave characteristics. The dual inclined porous plates arranged in either a serial or parallel configuration did not enhance the wave-absorbing efficiency significantly, compared to a single inclined porous plate with optimal values of porosity (P = 0.1) and inclined angle (θ = 10°).
MicroRNAs are reported as promising biomarkers for the diagnosis and treatment of breast cancer. miR‐1260b is identified as a tumor‐associated noncoding microRNA in other cancers, although the role ...of miR‐1260b and its clinical relevance in breast cancer remain unclear. In this study, miR‐1260b as a potential prognostic biomarker was observed by univariate and multivariate Cox regression analyses in 102 breast tumor tissues. The tumorigenic role of miR‐1260b in terms of proliferation, apoptosis, and migration of breast cancer cells was investigated using gain‐ and loss‐of‐function assays in vitro. Additionally, the potential early diagnosis and treatment monitoring marker of miR‐1260b was validated in 129 plasma samples. We found that high miR‐1260b expression was markedly associated with bulky tumor size, advanced stage, and lymph node invasion. Particularly, the high–miR‐1260b‐expression group showed shorter overall survival than the low–miR‐1260b‐expression group. The inhibition of oncogenic miR‐1260b induced apoptosis and decreased migration and invasion of MDA‐MB‐231 cells. CASP8 was revealed as a direct target gene of miR‐1260b, which is closely related to apoptosis. Furthermore, miR‐1260b expression levels in plasma were significantly higher in patients with breast cancer than in healthy controls. The patients who tested positive for miR‐1260b showed 16.3‐ and 18.2‐fold higher risks in the early stage and locally advanced stage, respectively, compared with healthy controls, and the risk was decreased 6.2‐fold after neoadjuvant chemotherapy. Taken together, miR‐1260b may be a potential novel diagnostic, prognostic, and therapeutic target in breast cancer.
High miR‐1260b expression was markedly associated with bulky tumor size, advanced stage, and lymph node invasion. Particularly, the high–miR‐1260b‐expression group showed shorter overall survival than the low–miR‐1260b‐expression group. The inhibition of oncogenic miR‐1260b induced apoptosis and decreased migration and invasion of MDA‐MB‐231 cells. CASP8 was revealed as a direct target gene of miR‐1260b, which is closely related to apoptosis.
Dimethyl sulfoxide (DMSO), an amphipathic molecule composed of one highly polar sulfinyl group and two nonpolar methyl groups, is considered an excellent solvent due to its capability to dissolve ...many polar and nonpolar compounds. Therefore, DMSO is widely used to solubilize drugs for therapeutic applications. DMSO is reported to possess anti-inflammatory, anticancer, and antioxidative capacities, and the anti-inflammatory efficacy of DMSO has been intensively studied in various cell lines and animal models. An in vitro model of mouse macrophage RAW 264.7 cells has been widely used, among several experimental designs, for evaluation during the development of new anti-inflammatory drugs. DMSO, which is used to dissolve samples, is also prone to experimental errors because of its anti-inflammatory properties. Therefore, we systematically confirmed the cytotoxic and anti-inflammatory effects of DMSO and the related signaling pathways in RAW 264.7 cells. The results show that DMSO at 0.25% to 1.5% did not result in cellular toxicity, with results comparable to the control group where DMSO is absent; at concentrations 2.0%, however, it inhibited the viability of RAW264.7 cells (13.25%). The results demonstrate that pretreatment with DMSO profoundly attenuates the lipopolysaccharide (LPS)-stimulated levels of nitric oxide (NO) and prostaglandin (PG)E2, as well as the levels of pro-inflammatory cytokines, cyclooxygenase-2 (COX-2) protein, and inducible nitric oxide synthase (iNOS). Collectively, the DMSO pretreatments appear to notably alleviate LPS-induced damage by reducing phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase proteins (ERKs), nuclear factor-kappa-B (NF-κB) in addition to NF-κB/p65 nuclear translocation. Taken together, the results clearly show that DMSO attenuates the inflammatory response in LPS-induced RAW264.7 cells by regulating the activation of the MAPK and NF-κB signaling pathways. These results contribute to potentially reducing experimental errors or misjudgments when using the LPS-induced RAW 264.7 macrophage cell model for evaluation during the development of new anti-inflammatory drugs.
Background
Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within breast cancer tumors is used to determine the prognosis and select ...therapies, additional markers need to be identified. Circulating tumor cells (CTCs) are constituent cells that have detached from a primary tumor to circulate in the bloodstream. CTCs are considered the main source of breast cancer metastases; therefore, detection of CTCs could be a promising diagnostic method for metastatic breast cancer.
Methods
In this study, the CircleGen CTC RT-qDx assay was used to analyze the mRNA expression levels of six CTC-specific markers including EpCAM, CK19, HER2, Ki67, hTERT, and vimentin with a total of 692 peripheral whole blood samples from 221 breast cancer patients and 376 healthy individuals.
Results
This assay showed high specificity with multiple markers; none of the healthy controls were detected positive, whereas 21.7 and 14 % of breast cancer patients were positive for EpCAM and CK19, respectively. Of the 221 breast cancer patients, 84 (38 %), 46 (20.8 %), 83 (37.6 %), and 39 (17.6 %) were positively for HER2, Ki67, hTERT, and vimentin mRNA, respectively. Of the 84 patients who were HER2 positive, nine (4 %) were also positive for EpCAM, CK19, Ki67, hTERT, and vimentin. Of the 139 breast cancer patients who were HER2 negative, 65 (29.1 %) were negative for EpCAM, CK19, Ki67, hTERT, and vimentin. Furthermore, the EpCAM-positive population decreased from 21.5 to 8.3 % after completion of anti-tumor treatment (TP4). Similarly, the CK19, HER2, hTERT, and vimentin positives also decreased from 13.9 to 9.5 %, from 37.7 to 21.4 %, from 37.2 to 33.3 %, and from 17.5 to 14.3 %, respectively, after completion of anti-tumor treatment. In contrast, the Ki67 positives increased from 20.6 to 41.7 % after completion of anti-tumor treatment.
Conclusions
mRNA overexpression of six CTC-specific markers was detected by the CircleGen CTC RT-qDx assay with high specificity, and the obtained mRNA expression levels of CTC-specific markers might provide useful criteria to select appropriate anti-tumor treatment for breast cancer patients.
In this study, the wave attenuation by the inclined porous plates has been evaluated through a numerical approach. As a numerical method, we used the dual boundary element method (DBEM) that solves ...two distinct boundary integral equations (singular and hyper-singular) derived at the coincident source points on each side of the degenerate plate boundary. A quadratic velocity model with the porosity-dependent drag coefficient is adopted to account for energy dissipation through the porous plate. The developed DBEM tool is validated through the comparison with self-conducted experiments at a two-dimensional wave tank. Then it is used to assess the reflection coefficients and wave forces with various design parameters such as porosities, inclined angles, plate arrangements, and wave characteristics. The dual inclined porous plates arranged in either a serial or parallel configuration did not enhance the wave-absorbing efficiency significantly, compared to a single inclined porous plate with optimal values of porosity (P = 0.1) and inclined angle (θ = 10°).
Circulating tumor cells (CTCs) are dissociated from primary tumor and circulate in peripheral blood. They are regarded as the genesis of metastasis. Isolation and enumeration of CTCs serve as ...valuable tools for cancer prognosis and diagnosis. However, the rarity and heterogeneity of CTCs in blood makes it difficult to separate intact CTCs without loss. In this paper, we introduce a parallel multi-orifice flow fractionation (p-MOFF) device in which a series of contraction/expansion microchannels are placed parallel on a chip forming four identical channels. CTCs were continuously isolated from the whole blood of breast cancer patients by hydrodynamic forces and cell size differences. Blood samples from 24 breast cancer patients were analyzed (half were from metastatic breast cancer patients and the rest were from adjuvant breast cancer patients). The number of isolated CTCs varied from 0 to 21 in 7.5ml of blood. Because our devices do not require any labeling processes (e.g., EpCAM antibody), heterogeneous CTCs can be isolated regardless of EpCAM expression.
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