Water-soluble Tetrazolium Salt Research Articles

High expression level of CD44v8-10 in cancer stem-like cells is associated with poor prognosis in esophageal squamous cell carcinoma patients treated with chemoradiotherapy.

BackgroundStrong reactive oxygen species (ROS) suppression in cancer stem-like cell components in various solid tumors is associated with therapeutic resistance. In this study, we investigated the influence of CD44v8-10 expression on the overall survival of esophageal squamous cell carcinoma (E-SCC) patients after definitive chemoradiotherapy (dCRT) and on radio-sensitivities of E-SCC cell lines treated with or without sulfasalazine, a CD44v8-10-xCT-GSH axis inhibitor.MethodsSeventy-three patients with E-SCC who received dCRT were examined retrospectively. CD44v8-10 expression was analyzed immunohistochemically using paraffin-blocked pre-dCRT biopsy specimens obtained by esophagoscopy and was expressed as a histo-score (H-score). The relationship between the H-score and overall survival was analyzed. From human E-SCC cell lines (T.T, T.Tn, or Kyse-3650), we collected CD44v8-10High and CD44v8-10Low subpopulations using a cell sorter. Water-soluble tetrazolium salt-8 (WST), glutathione-SH (GSH) and ROS assays were performed to compare the effect of sulfasalazine on the radio-sensitivities of these subpopulations in T.Tn and Kyse-3650.ResultsHigh CD44v8-10 expression was independently associated with poor prognosis in E-SCC patients treated with dCRT (hazard ratio = 2.906, 95% CI = 1.277–6.611, p = 0.011). In CD44v8-10High cells of each cell line, sulfasalazine decreased cellular GSH levels, resulting in increased radiation-induced ROS and reduced cell viability. In contrast, sulfasalazine had no significant effects in CD44v8-10Low cells.ConclusionHigh CD44v8-10 expression was an independent prognostic factor in E-SCC patients treated with dCRT. CD44v8-10-xCT-GSH axis inhibition sensitized CD44v8-10High E-SCC cells to ROS-inducing treatments such as radiotherapy. Targeting CD44v8-10-xCT-GSH axis may improve the prognosis of post-dCRT E-SCC patients.

A 21.6 kDa tegumental protein of Clonorchis sinensis induces a Th1/Th2 mixed immune response in mice.

Introduction Clonorchis sinensis is a major parasite affecting the Korea population. Despite the high infection rate and pathogenicity, very few studies have been conducted to investigate the immune responses against the proteins of C. sinensis.MethodsIn this study, in vitro immune response induced by a recombinant 21.6 kDa tegumental protein derived from C. sinensis (rCsTegu21.6) was confirmed in murine dendritic cells and T cells. For the in vivo analysis, each mouse was immunized three times. Total serum IgG and T cell cytokine production were determined by ELISA, while T cell proliferation was detected by a WST (Water‐Soluble Tetrazolium salt)‐1 assay.ResultsIn vitro tests indicated that rCsTegu21.6 treatment increased the expression of surface molecules, such as CD40 (77%), CD80 (52%) and CD86 (46%), on murine dendritic cells and the secretion of cytokines (TNF‐α, IL‐6, IL‐1β, IL‐10, and IL‐12p70). Moreover, co‐culturing dendritic cells activated by rCsTegu21.6 with allogenic T cells induced T cell proliferation over time. rCsTegu21.6 also stimulated specific antibody production and cytokine secretion [IL‐2, IL‐4, and interferon (IFN)‐γ)] from T cells following immunization in vivo. Notably, rCsTegu21.6 predominantly induced IgG1 production and secretion of the Th2 cytokine IL‐4, regardless of the type of adjuvant used.ConclusionThese results serve as a foundation for the development of tegumental protein‐based vaccines against C. sinensis.

Induction of human aortic myofibroblast-mediated extracellular matrix dysregulation: A potential mechanism of fluoroquinolone-associated aortopathy

ObjectivesFluoroquinolone (FQ) antibiotics are associated with adverse aortic clinical events. We assessed human aortic myofibroblast-mediated extracellular matrix (ECM) dysregulation as a possible cellular mechanism underlying FQ-associated aortopathy. MethodsHuman aortic myofibroblasts were isolated from patients with aortopathy undergoing elective ascending aortic resection (N = 9). The capacity for extracellular matrix degradation in cells exposed to FQ was assessed by multiplex analysis of secreted matrix metalloproteinases relative to tissue inhibitors of matrix metalloproteinases (TIMPs). Direct evaluation of extracellular matrix degradation was investigated in human aortic cells using a 3-dimensional gelatin-fluorescein isothiocyanate fluorescence microgel assay. Aortic cellular collagen-1 expression following FQ exposure was determined by immunoblotting and immunofluorescent staining. Cell apoptosis, necrosis, and metabolic viability was determined by annexin-V, propidium iodide staining, and water-soluble tetrazolium salt (WST1) assay. ResultsFQ exposure significantly decreased aortic cell TIMP-1 (P = .004) and TIMP-2 (P = .0004) protein expression compared with vehicle control. The ratio of matrix metalloproteinase-9/TIMP-2 was increased suggesting an increased capacity for extracellular matrix degradation (P = .01). In collagen gels, we show a trend toward increased aortic myofibroblast-mediated collagen fiber degradation with FQ exposure (P = .09). Similarly, FQ exposure attenuated collagen-1 expression as assessed by immunoblotting (P = .002) and immunofluorescence (P = .02). Cell apoptosis, necrosis, and metabolic viability was not significantly influenced by FQ exposure. ConclusionsFor the first time, we document a putative mechanism underlying FQ-associated aortopathy whereby decreased TIMP expression with impaired compensatory collagen-1 expression results in human aortic myofibroblast-mediated extracellular matrix dysregulation. These novel data may provide a cellular and molecular mechanism to explain the established clinical association between FQ exposure and acute aortic events.

Enhanced biocompatibility of a Ni–Cr alloy prepared by selective laser melting: a preliminary in vitro study

Against a general consensus regarding the high toxicity and low biocompatibility of cast nickel–chromium (Ni–Cr) alloys, the in vitro biocompatibility of selective laser melting (SLM)-fabricated Ni–Cr alloys was investigated and compared with that of cast alloy. Using a single Ni–Cr alloy powder, two SLM alloys with high (SLM_a) and low (SML_b) porosity were prepared by adjusting the laser process parameters in addition to a cast alloy. The alloys were studied in terms of their microstructure, metal ion release, and cell response. All three alloys were composed of only the γ (face-centered cubic) phase. However, both SLM alloys showed more homogeneous dispersion of Ni, Cr, and molybdenum elements and finer grain formation than the cast alloy. Immersion test results indicated that both SLM alloys (in particular, SML_b) exhibited a significantly lower Ni ion release than the cast alloy (P<0.001). A water-soluble tetrazolium salt-8 assay also showed that the viability of L929 mouse fibroblasts was significantly higher for both SLM groups (in particular, SML_b) than for the cast alloy group (P<0.001). Thus, the combined results of this in vitro study suggest that the SLM-processed Ni–Cr alloys exhibited greatly enhanced biocompatibility in comparison with the cast alloy.

The effect of ICRT-3 on Wnt signaling pathway in head and neck cancer.

The effect of Wnt pathway in head and neck cancer could not be elucidated, even though the aberrant Wnt signaling plays a key role in the development of many types of cancer. The inhibitor of β-catenin responsive transcription (ICRT-3) blocks the Wnt signaling pathway by binding toβ-catenin, which is a coactivator of the Wnt signaling pathway anda promising agent for inhibiting aberrant signaling. In our study, we aimed to evaluate the effect of ICRT-3 on the cytotoxicity, apoptosis, cell cycle progression, migration, and gene expressions in head and neck cancer stem cell (HNCSC) and hypopharynx cancer. The effect of this compound on cytotoxicity and cell viability in FaDu and HNCSC line was assessed by using the water-soluble tetrazolium salt-1 method. The effect of ICRT-3 on apoptosis was detected by using Annexin V and caspase-3, caspase-9 kit, on cell cycle progression by cycle test plus DNA reagent kit, on gene expression by dual luciferase reporter assay, and on migration activity by wound healing assay in bothcell lines. ICRT-3 was determined to have cytotoxic and apoptotic effect in bothcell lines. In addition, it was also found that the administration of ICRT-3 caused cell cycle arrest and significant decrease in gene expression level and migration ability of the cells.

Triazine- and Heptazine-Based Carbon Nitrides: Toxicity

Graphitic carbon nitride has attracted extensive interests recently because of its potential in biosensing, photocatalytic, and biomedical applications. Similar to graphene, it is a two-dimensional carbon and nitrogen-based nanomaterial with weak van der Waals forces between each layer. Carbon nitrides can have various structural moieties such as triazine and heptazine. Unlike graphene-substituted nanosheets, the toxicity of graphitic carbon nitrite is largely unknown. In respond to that, toxicological study was carried out to determine its toxicity toward human lung carcinoma epithelial cells (A549). Two formazan-based cell viability assays (water-soluble tetrazolium salt (WST-8) assay and methylthiazolyldiphenyltetrazolium bromide (MTT) assay) were utilized on the A549 cell line to derive the cytotoxicity profile. Both materials demonstrated a dose-dependent toxicological effect with triazine-based carbon nitrides being more cytotoxic than heptazine-based carbon nitrides. These findings are of great imp...

High glucose-induced oxidative stress impairs proliferation and migration of human gingival fibroblasts.

Delayed gingival wound healing is widely observed in periodontal patients with diabetes. However, the molecular mechanisms of the impaired function of gingival fibroblasts in diabetes remain unclear. The purpose of this study was to investigate changes in the properties of human gingival fibroblasts (HGFs) under high-glucose conditions. Primary HGFs were isolated from healthy gingiva and cultured with 5.5, 25, 50, and 75 mM glucose for 72 h. In vitro wound healing, 5-ethynyl-2′-deoxyuridine (EdU), and water-soluble tetrazolium salt (WST-8) assays were performed to examine cell migration and proliferation. Lactase dehydrogenase (LDH) levels were measured to determine cytotoxicity. The mRNA expression levels of oxidative stress markers were quantified by real-time PCR. Intracellular reactive oxygen species (ROS) were also measured in live cells. The antioxidant N-acetyl-l-cysteine (NAC, 1 mM) was added to evaluate the involvement of ROS in the glucose effect on HGFs. As a result, the in vitro wound healing assay showed that high glucose levels significantly reduced fibroblast migration and proliferation at 6, 12, 24, 36, and 48 h. The numbers of cells positive for EdU staining were decreased, as was cell viability, at 50 and 75 mM glucose. A significant increase in LDH was proportional to the glucose concentration. The mRNA levels of heme oxygenase-1 and superoxide dismutase-1 and ROS levels were significantly increased in HGFs after 72 h of exposure to 50 mM glucose concentration. The addition of NAC diminished the inhibitory effect of high glucose in the in vitro wound healing assay. The results of the present study show that high glucose impairs the proliferation and migration of HGFs. Fibroblast dysfunction may therefore be caused by high glucose-induced oxidative stress and may explain the delayed gingival wound healing in diabetic patients.

In Vitro and In Vivo Characterization of N-Acetyl-L-Cysteine Loaded Beta-Tricalcium Phosphate Scaffolds.

Beta-tricalcium phosphate bioceramics are widely used as bone replacement scaffolds in bone tissue engineering. The purpose of this study is to develop beta-tricalcium phosphate scaffold with the optimum mechanical properties and porosity and to identify the effect of N-acetyl-L-cysteine loaded to beta-tricalcium phosphate scaffold on the enhancement of biocompatibility. The various interconnected porous scaffolds were fabricated using slurries containing various concentrations of beta-tricalcium phosphate and different coating times by replica method using polyurethane foam as a passing material. It was confirmed that the scaffold of 40 w/v% beta-tricalcium phosphate with three coating times had optimum microstructure and mechanical properties for bone tissue engineering application. The various concentration of N-acetyl-L-cysteine was loaded on 40 w/v% beta-tricalcium phosphate scaffold. Scaffold group loaded 5 mM N-acetyl-L-cysteine showed the best viability of MC3T3-E1 preosteoblastic cells in the water-soluble tetrazolium salt assay test.

Anti-inflammatory and anti-aging effects of hydroxytyrosol on human dermal fibroblasts (HDFs)

BackgroundHydroxytyrosol is discovered in a form of elenolic acid ester oleuropein in olive leaf and oil. The anti-carcinogenic, antioxidant, and anti-inflammatory effects of hydroxytyrosol have been reported in the food, medical, pharmaceutical, and life science fields. But it has not been studied on skin biology field. This study is demonstrated using UVA-induced cellular aging model of human dermal fibroblast.MethodsThe cell survival rate was measured using the principle of water-soluble tetrazolium salt-1 assay, which is a measurement method for cell survival rate. The quantitative real-time PCR (qRT-PCR) was used to quantitatively analyze the gene expression changes in human dermal fibroblasts (HDFs) by hydroxytyrosol.Senescence-associated β-galactosidase (SA-β-gal) assay was implemented to dye β-galactosidases (used as a cell aging biomarker) to measure HDF cell aging by UVA.ResultsHydroxytyrosol decreased the SA-β-galactosidase activity in a dose-dependent manner in UVA-exposed HDFs. Also, the elevated expression of MMP-1 and MMP-3 by UVA were decreased by hydroxytyrosol in a dose-dependent manner. To examine the anti-inflammatory effect of hydroxytyrosol in UV exposed HDFs, the expression of inflammatory interleukins IL-1β, IL-6, and IL-8 were analyzed. Quantitative RT-PCR showed that hydroxytyrosol decreased the expression of IL-1β, IL-6, and IL-8 gene.ConclusionThrough this research, we demonstrate that hydroxytyrosol has effects on anti-inflammatory and anti-aging in HDFs damaged by UVA. We suggest that hydroxytyrosol is fully worthy of using as a cosmetic material effective to anti-inflammatory and to delay cellular senescence on HDFs.

IL-1α and IL-1β as alternative biomarkers for risk assessment and the prediction of skin sensitization potency

ABSTRACTPotential biomarkers of skin sensitization in RAW264.7 mouse macrophages were investigated as alternatives to animal experiments and risk assessment. The concentrations that resulted in a cell viability of 90% (CV90) and 75% (CV75) were calculated by using a water-soluble tetrazolium salt (WST)-1 assay and used to analyze the skin sensitization potency of 23 experimental materials under equivalent treatment conditions. In addition, the expression of interleukin (IL)-1α, IL-1β, IL-31, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2) was analyzed utilizing Western blotting. In the cell viability analysis, skin sensitizers were generally more cytotoxic and exhibited increased skin sensitization potency. However, nonsensitizers did not show any marked cytotoxic tendency. Biomarker analysis demonstrated that IL-1α, IL-1β, and the combination of IL-1α and IL-1β (IL-1α + IL-1β) predicted reliably skin sensitization potential (1) sensitivities of 94.4%, 83.3%, and 83.3%, specificities of 100%, 100%, and 100%, and (2) accuracies of 95.7%, 87%, and 87%, respectively. These observations correlated most reliably as indicators for skin sensitization potency. Data suggest that IL-1α and IL-1β may serve as potential biomarkers for skin sensitization and provide an alternative method to animal experiments for prediction of skin sensitization potency and risk assessment.

Role of the tumour protein P53 gene in human cervical squamous carcinoma cells: Discussing haematopoietic cell-specific protein 1-associated protein X-1-induced survival, migration and proliferation.

The haematopoietic cell-specific protein 1-associated protein X-1 (HAX-1), as a mitochondrial membrane protein, induces cancer progression and metastasis. The present study aimed to investigate the role of HAX-1-induced survival, migration and proliferation of human cervical squamous carcinoma cells and to elucidate its potential molecular mechanisms. The level of HAX-1 was examined by quantitative polymerase chain reaction and western blot analyses. The survival, migration and proliferation of the human cervical squamous carcinoma SiHa cell line were measured by the water-soluble tetrazolium salt (WST-1) assay, Transwell assay and 3H-thymidine incorporation into DNA (3H-TdR) assay, respectively. The intracellular reactive oxygen species (ROS) was estimated by the fluorescence of H2DCFDA, and the mitochondrial membrane potential was tested using a JC-1 probe. The expression of the HAX-1 gene was significantly increased in human cervical carcinoma tissues relative to non-cancerous cervix tissues. Overexpression of HAX-1 increased the survival, migration and proliferation ability of SiHa cells, decreased the production of ROS, and maintained the integrity of the mitochondrial membrane and morphology. The effect brought on these cells could be abrogated by the addition of wild-type tumour protein P53 (p53) or carbonyl cyanide-p-trifluoro methoxyphenylhydrazone-induced mitochondrial dysfunction. In summary, these data support the notion that HAX-1 induced the survival, migration and proliferation of human cervical squamous carcinoma cells by inhibiting its downstream regulatory factor p53 in SiHa cells.

The intraocular staining potential of anthocyanins and their retinal biocompatibility: a preclinical study

Purpose: To perform preclinical studies to determine the efficacy and safety of anthocyanins as stains for the internal limiting membrane (ILM) of the eye.Materials and methods: Cyanidin (Cya), delphinidin (Del), luteolinidin (Lut), peonidin (Peo) and pelargonidin (Pel) were evaluated. These natural dyes were used to stain the lens capsule and ILM of pig eyes. The effects of these dyes on retinal cell viability was determined using a water-soluble tetrazolium salt assay, and oxidative stress was measured in vitro. Histopathology, in situ TUNEL labelling, transmission electronic microscopy (TEM), and electroretinography (ERG) were performed on rats following the intravitreal and subretinal injection of the neuroprotective dyes.Results: All anthocyanins stained the lens capsule and ILM of the pigs at a concentration of 1 mg/ml. Del, Lut and Peo were non-toxic and produced survival rates in the ARPE19 and RGC5 cells that were similar to those in control cells. We treated eyes with H2O2 and three dyes (Del, Lut, and Peo) to explore the possible neuroprotective effects and observed significantly higher survival rates in the ARPE19 cells treated with Del, Lut or Peo and the RGC5 cells treated with Lut or Peo than those in the control cells. Three dyes were intravitreally and subretinally injected into rats in vivo, and the histology showed mildly disorganized retinal cell layers. TUNEL staining and TEM examinations did not reveal additional toxic effects. Rat ERGs were not altered after intravitreal injections.Conclusions: This preclinical study, Del, Lut, and Peo show potential as staining agents and warrant further investigation as vital dyes.

Anti-inflammatory effects of prunin on UVB-irradiated human keratinocytes

BackgroundThe flavonoid prunin is a flavanone glycoside found in cherry trees, including the flowering cherry Prunus yedoensis Matsumura (Rosacea). Although this compound has been studied for its antioxidant, anti-bacterial, and blood-sugar-lowering effects, no studies address its use in cosmetics. This study investigates whether prunin exhibits anti-inflammatory effects in cells exposed to ultraviolet B (UVB) radiation.MethodsThe effects of prunin were assessed by measuring cell viability using the water-soluble tetrazolium salt-1 assay and free radical damage using the dichlorofluorescein diacetate assay as well as by quantitative real-time PCR.ResultsUVB-induced decrease in cell viability diminished by pretreatment with prunin in a concentration-dependent manner. Intracellular reactive oxygen species (ROS) quantitative analysis revealed that the expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is associated with the inflammatory response, and mRNA expression of interleukin-6 (IL-6), interleukin-8 (IL-8), cyclooxygenase-2 (COX2), tumor necrosis factor-alpha (TNF-α), and protease-activated receptor (PAR2) decreased with prunin pretreatment in a concentration-dependent manner.ConclusionsPrunin increased the survival rate of UVB-treated human keratinocyte HaCaT cells. Prunin protected the HaCaT cells, eliminated ROS, and demonstrated anti-inflammatory effects. Thus, prunin is worthy of investigation for use as a cosmetic ingredient that protects the skin and has anti-inflammatory effects.

Arsenic trioxide induces apoptosis and inhibits the growth of human liver cancer cells

AimsAs a fifth most common cancer type, Hepatocellularcarcinoma (HCC) ranked third leading cause of cancer deaths worldwide. Arsenic trioxide (As2O3) is known as chemotherapeutic agent against few cancer including Acute promyelocyticleukemia and solid tumors. But its effect and possible associated mechanism in HCC is meager. Present study aimed to assess As2O3 modulatory effect on liver cancer by assessing cell growth and viability. MethodsLiver normal (Chang liver) and cancerous cells (Hep3B) were exposed to different concentration's (0, 1, 5, 10 & 15 μM) of As2O3 at different intervals (24, 48 & 72 h). Cell growth was assessed microscopically, and Cytotoxicity assays were done through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Water-soluble tetrazolium salt (WST) growth inhibition assays. Cell viability was studied by trypan blue staining. Apoptosis was analyzed by Annexin V/PI assay, and expression of genes (Notch and anti-apoptotic) were determined through western blotting and Q-PCR method. Key findingsA significant reduction in cell growth and viability was reported in liver cancerous cells as compare to normal cells at 5 μM As2O3. Consistently, As2O3 induced apoptosis along with down-regulation of anti-apoptotic protein Bcl-xL, and up regulates expression of Notch that leads towards apoptosis. SignificanceResults clearly suggest that As2O3 restricted growth and induces apoptosis more in liver cancer cells as compared to normal cells. This finding suggests that it could be a promising potential therapeutic agent against liver cancer which need further testing by in-vivo investigations.

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

Antioxidant and skin protection effect of morin upon UVA exposure

BackgroundMorin is a family of phenolic compounds and is a bioflavonoid ingredient in fruits and vegetables. Morin exhibits various biological activities, including antioxidant cell protection and antimutagenic and anti-inflammatory effects; these activities safely minimize free radical-mediated damage biologically. However, the photoaging mitigation effect of morin on skin cells remains unknown. To investigate the effect of the morin on cell senescence mitigated against photoaging, cell viability, antioxidation, and anti-inflammation experiments were conducted. As a natural result of oxygen consumption, reactive oxygen species (ROS) in the form of harmful superoxides and hydroxyl radicals are generated through an oxidation reaction involving heavy metal cations such as iron. This adversely affects DNA, lipids, and proteins; therefore, organisms have a self-protective mechanism against oxidative stress via enzymes such as catalase (CAT) and superoxide dismutase (SOD), molecules such as glutathione, and proteins such as thioredoxin. Therefore, in this study, the antioxidative and skin protection functions of morin were examined to investigate the possibility of cosmetics.MethodsTo examine morin-mediated anti-photoaging mechanisms, human dermal fibroblasts (HDFs) were selected as the model cell line and UVA was selected as the stimulus source. The water-soluble tetrazolium salt-1 assay was performed to assess cell viability and cytoxicity in UV-exposed HDFs. To examine the molecular mechanism underlying the antioxidation capacity of morin, genes were analyzed using qRT-PCR, the expressions of several antioxidant enzymes were monitored, and the effect of morin on GPx1, CAT, HO-1, and NRF2 expressions in UV-exposed HDFs was assessed.ResultsThe results of the morin toxicity showed the cell viability was above 100% when the concentration of morin was set at 20 and 50 μM. The cytotoxicity test for oxidative stress through UVA showed that the appropriate intensity of UVA 10 J/cm2 was set as the cell viability reduced by 10 J/cm2. And the cell survival over 100% rate after the morin treatment was 20 and 50 μM cell. A result of mRNA experiments verified that the expression of the antioxidant enzyme genes GPx1, CAT, HO-1, and NRF2 increased with morin, in a concentration-dependent manner.ConclusionMorin increases the expression of antioxidant enzymes, which facilitates the antioxidant mechanism to respond to oxidative stress associated with exposure to UV and heat, which are considered to be the most harmful factors damaging the skin cells. This results in ROS removal, a byproduct of the natural metabolism of oxygen and the protection of neurons and proteins from toxicity. In conclusion, this study verified the applicability of morin as a cosmetic ingredient for the protection of cells against oxidization and UV.

MS-5, a Naphthalene Derivative, Induces the Apoptosis of an Ovarian Cancer Cell CAOV-3 by Interfering with the Reactive Oxygen Species Generation.

Reactive oxygen species (ROS) are widely generated in biological processes such as normal metabolism and response to xenobiotic exposure. While ROS can be beneficial or harmful to cells and tissues, generation of ROS by diverse anti-cancer drugs or phytochemicals plays an important role in the induction of apoptosis. We recently identified a derivative of naphthalene, MS-5, that induces apoptosis of an ovarian cell, CAOV-3. Interestingly, MS-5 induced apoptosis by down-regulating the ROS. Cell viability was evaluated by water-soluble tetrazolium salt (WST-1) assay. Apoptosis was evaluated by flow cytometry analysis. Intracellular ROS (H2O2), mitochondrial superoxide, mitochondrial membrane potential (MMP) and effect on cycle were determined by flow cytometry. Protein expression was assessed by western blotting. The level of ATP was measured using ATP Colorimetric/Fluorometric Assay kit. MS-5 inhibited growth of ovarian cancer cell lines, CAOV-3, in a concentration- and time-dependent manner. MS-5 also induced G1 cell cycle arrest in CAOV-3 cells, while MS-5 decreased intracellular ROS generation. In addition, cells treated with MS-5 showed the decrease in MMP and ATP production. In this study, we found that treatment with MS-5 in CAOV-3 cells induced apoptosis but decreased ROS level. We suspect that MS-5 might interfere with the minimum requirements of ROS for survival. These perturbations appear to be concentration-dependent, suggesting that MS-5 may induce apoptosis by interfering with ROS generation. We propose that MS-5 may be a potent therapeutic agent for inducing apoptosis in ovarian cancer cell through regulation of ROS.

Cytoprotective effects of sinapic acid on human keratinocytes (HaCaT) against ultraviolet B

BackgroundSinapic acid is a phenolic compound in various plants, including spices, berries, citrus fruits, vegetables, grains, and oil seeds. It has been studied mainly for the various pathological symptoms it induces related to its anticancer, antioxidant, and neuroprotective effects. However, little has been studied about sinapic acid in human keratinocytes (HaCaT). Therefore, this study examined its reactive oxygen species (ROS) scavenging capacity, protective effect against DNA damage, and DNA repair mechanism.MethodsTo confirm the protective effects of sinapic acid on HaCaT irradiated by ultraviolet (UV) B, this study used the water-soluble tetrazolium salts (WST)-1 assay to determine the cytotoxicity of sinapic acid for verifying the cell survival rate and dichlorofluorescein diacetate (DCF-DA) to measure the changes in the concentration of ROS generated by UVB. This study also used the comet assay, cyclobutane pyrimidine dimer (CPD) formation, and malondialdehyde (MDA) assay to measure the degree of protection against cell damage. In addition, this study used quantitative real-time polymerase chain reaction (qRT-PCR) to measure the expression of repaired genes from cell damage.ResultsSinapic acid protects cells from UVB-induced cytotoxicity and reduces ROS generation by UVB. This study showed that sinapic acid reduced the generation of tailed DNA, CPD, and lipid peroxidation, which are the indicators of DNA damage. Depending on the increase in the concentration of sinapic acid, the gene expressions of XPC and ERCC1 (repair factors for damaged DNA) increased.ConclusionsThe findings of this study confirmed that sinapic acid is effective in removing ROS generated by UVB, reducing intracellular damage, and repairing damaged DNA in HaCaT. These results also showed that sinapic acid is effective in DNA damage repair, indicating its protective effect on HaCaT against UVB damage.

Antioxidant and inhibition of senescence effects of embelin

BackgroundEmbelin is a major active ingredient of Embelia ribes Burm. belonging to Myrsinaceae, an important traditional medicinal plant of Indian origin. Embelin has a benzoquinone derivative structure and has been studied for anticancer, anti-inflammatory, and antioxidative effects. However, there have been no studies on the use of cosmetic raw materials for embelin. In this study, antioxidant and inhibition of senescence effects of embelin were examined using hydrogen peroxide (H2O2)-induced cellular aging model of human dermal fibroblast.MethodsCell viability was measured using the principle of water-soluble tetrazolium salt (WST-1) assay. qRT-PCR was used to quantitatively analyze gene expression patterns in human dermal fibroblasts (HDFs) by embelin. Dichlorofluorescein diacetate (DCF-DA) was used to measure changes in intracellular reactive oxygen species (ROS) concentration. Cell senescence was measured using the senescence-associated β-galactosidase (SA-β-galactosidase) assay, a method of staining β-galactosidase. A wound healing assay was used to observe cell mobility.ResultsThe WST-1 assay demonstrated that decreased cell viability by H2O2 was restored by embelin in a dose-dependent manner. In addition, the expression of SOD1, GPx1, and CAT genes were upregulated by embelin suggesting that embelin-induced antioxidant capability may be enhanced through the upregulation of intracellular antioxidant-related genes. To determine whether embelin inhibits cell senescence in H2O2-induced senescence model of HDFs, the SA-β-galactosidase assay was performed. Embelin decreased the SA-β-galactosidase activity in a dose-dependent manner in H2O2-treated HDFs. Moreover, the expression of p21 and MMP1 genes were reduced by embelin in H2O2-treated HDFs in a dose-dependent manner. However, COL1A1 genes were increased by embelin in H2O2-treated HDFs in a dose-dependent manner.ConclusionsThese results suggest that embelin has a potential as an antiaging cosmetic ingredient with antioxidant and anti-senescence properties.

In vitro cytotoxicity of covalently protected layered molybdenum disulfide

Layered molybdenum disulfide (MoS2) is in forefront of materials research. MoS2 has been reported to display various unique electronic, catalytic and sensing properties. Functionalization of MoS2 is common as it can improve biocompatibility, electrochemical behaviour, catalytic performance, and sensing abilities. In this study, a cytotoxicity study of MoS2 functionalized with an organic molecule, thiobarbituric acid (TBA) was conducted. The functionalization of MoS2 with TBA (MoS2-TBA) is unique as it was reported to retain metallic 1T-type character which is important for electrochemical applications. In addition, MoS2-TBA could possibly be utilized to a wider range of applications as it can be further modified due to the presence of alcohol groups. Thus, investigating the toxicity of MoS2-TBA is essential since it has promising properties for several applications. Functionalized MoS2-TBA is expected to have different toxicity profile than non-functionalized MoS2 as several physicochemical properties may have changed after functionalization. The cytotoxicity study has been conducted via two common cell viability assays which are methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and water-soluble tetrazolium salt (WST-8) assay. Human lung carcinoma epithelial cell line (A549) was chosen for this study it is highly probable for nanomaterials to enter the lungs and interact with the body via inhalation through the respiratory tract from the environment. This study has found that MoS2-TBA has become less toxic as compared to MoS2.