Inhibition of temperature-sensitive TRPV3 channel by two natural isochlorogenic acid isomers for alleviation of dermatitis and chronic pruritus

Genetic gain-of-function mutations of warm temperature-sensitive transient receptor potential vanilloid 3 (TRPV3) channel cause Olmsted syndrome characterized by severe itching and keratoderma, indicating that pharmacological inhibition of TRPV3 may hold promise for therapy of chronic pruritus and skin diseases. However, currently available TRPV3 tool inhibitors are either nonselective or less potent, thus impeding the validation of TRPV3 as therapeutic target. Using whole-cell patch-clamp and single-channel recordings, we report the identification of two natural dicaffeoylquinic acid isomers isochlorogenic acid A (IAA) and isochlorogenic acid B (IAB) that selectively inhibit TRPV3 currents with IC50 values of 2.7 ± 1.3 and 0.9 ± 0.3 μmol/L, respectively, and reduce the channel open probability to 3.7 ± 1.2% and 3.2 ± 1.1% from 26.9 ± 5.5%, respectively. In vivo evaluation confirms that both IAA and IAB significantly reverse the ear swelling of dermatitis and chronic pruritus. Furthermore, the isomer IAB is able to rescue the keratinocyte death induced by TRPV3 agonist carvacrol. Molecular docking combined with site-directed mutations reveals two residues T636 and F666 critical for the binding of the two isomers. Taken together, our identification of isochlorogenic acids A and B that act as specific TRPV3 channel inhibitors and gating modifiers not only provides an essential pharmacological tool for further investigation of the channel pharmacology and pathology, but also holds developmental potential for treatment of dermatitis and chronic pruritus.

Hypocrellin A-based photodynamic action induces apoptosis in A549 cells through ROS-mediated mitochondrial signaling pathway

Over recent decades, many studies have reported that hypocrellin A (HA) can eliminate cancer cells with proper irradiation in several cancer cell lines. However, the precise molecular mechanism underlying its anticancer effect has not been fully defined. HA-mediated cytotoxicity and apoptosis in human lung adenocarcinoma A549 cells were evaluated after photodynamic therapy (PDT). A temporal quantitative proteomics approach by isobaric tag for relative and absolute quantitation (iTRAQ) 2D liquid chromatography with tandem mass spectrometric (LC-MS/MS) was introduced to help clarify molecular cytotoxic mechanisms and identify candidate targets of HA-induced apoptotic cell death. Specific caspase inhibitors were used to further elucidate the molecular pathway underlying apoptosis in PDT-treated A549 cells. Finally, down-stream apoptosis-related protein was evaluated. Apoptosis induced by HA was associated with cell shrinkage, externalization of cell membrane phosphatidylserine, DNA fragmentation, and mitochondrial disruption, which were preceded by increased intracellular reactive oxygen species (ROS) generations. Further studies showed that PDT treatment with 0.08 µmol/L HA resulted in mitochondrial disruption, pronounced release of cytochrome , and activation of caspase-3, -9, and -7. Together, HA may be a possible therapeutic agent directed toward mitochondria and a promising photodynamic anticancer candidate for further evaluation.

Aurone derivatives as Vps34 inhibitors that modulate autophagy

We report in this study the identification of a natural product-like antagonist () of Vps34 as a potent autophagy modulator structure-based virtual screening. Aurone derivative strongly inhibited Vps34 activity in cell-free and cell-based assays. Significantly, prevents autophagy in human cells induced either by starvation or by an mTOR inhibitor. modeling and kinetic data revealed that could function as an ATP-competitive inhibitor of Vps34. Moreover, it suppressed autophagy and without inducing heart or liver damage in mice. could be utilized as a new motif for more selective and efficacious antagonists of Vps34 for the potential treatment of autophagy-related human diseases.

Targeting slug-mediated non-canonical activation of c-Met to overcome chemo-resistance in metastatic ovarian cancer cells

Metastasis-associated drug resistance accounts for high mortality in ovarian cancer and remains to be a major barrier for effective treatment. In this study, SKOV3/T4, a metastatic subpopulation of ovarian cancer SKOV3 cells, was enriched to explore potential interventions against metastatic-associated drug resistance. Quantitative genomic and functional analyses were performed and found that slug was significantly increased in the SKOV3/T4 subpopulation and contributed to the high resistance of SKOV3/T4. Further studies showed that slug activated c-Met in a ligand-independent manner due to elevated levels of fibronectin and provoked integrin V function, which was confirmed by the significant correlation of slug and p-Met levels in 121 ovarian cancer patient samples. Intriguingly, c-Met inhibitor(s) exhibited greatly enhanced anti-cancer effects in slug-positive ovarian cancer models both and . Additionally, IHC analyses revealed that slug levels were highly correlated with reduced survival of ovarian cancer patients. Taken together, this study not only uncovers the critical roles of slug in drug resistance in ovarian cancer but also highlights a promising therapeutic strategy by targeting the noncanonical activation of c-Met in slug-positive ovarian cancer patients with poor prognosis.

High-throughput screening for small molecule inhibitors of the type-I interferon signaling pathway

Interferons (IFNs) are cytokines with fundamental roles in resistance to infections, cancer and other diseases. Type-I IFNs, interferon (IFN-) and interferon (IFN-), act through a shared receptor complex (IFNAR) comprised of IFNAR1 and IFNAR2 subunits. Binding of type-I IFN to IFNAR1 will robustly activate Janus activated kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Aberrant activation of the type-I IFN response results in a spectrum of disorders called interferonopathies. The purpose of this research is to develop an assay for high-throughput screening (HTS) of small molecule inhibitors of the type-I IFN signaling pathway. Inhibition of type-I IFN signaling can be beneficial in terms of therapeutic use and understanding the underlying mechanism of action. We report here a HTS campaign with the secreted embryonic alkaline phosphatase (SEAP) reporter gene assay against 32,000 compounds which yielded 25 confirmed hits. These compounds were subsequently characterized for their cytotoxicity, effects on STAT phosphorylation and activities in IFN regulatory factor (IRF) transcription.

Comparação de dois métodos de colheita de medula óssea de equinos e de dois meios de diluição para o isolamento de células mononucleares / Comparison of two methods of equine bone marrow aspiration and two solutions for mononuclear cell isolation

O objetivo do presente estudo foi avaliar a concentração e viabilidade da fração de células mononucleares (FCM) a partir de diferentes técnicas de colheita e processamento de medula óssea (MO) em equinos. Foram avaliados cinco equinos adultos, hígidos e sem raça definida. Obtiveram-se frações de medula óssea (MO) do osso esterno, de acordo com dois protocolos: na colheita A, utilizou-se 10mL de solução de heparina dentro da seringa e em seguida, aspirou-se a MO; na colheita B, 10mL de solução de heparina foi injetada na MO e a aspiração foi realizada após 20 segundos. Todos os animais foram submetidos aos dois protocolos de colheitas, realizadas em sequência, sem intervalo entre os dois procedimentos. Após isolamento da fração de células mononucleares (FCM), das amostras de MO obtidas nas colheitas A e B, cada amostra foi dividida em dois tubos, um contendo solução de DMEM e outro contendo PBS. Assim, alternando-se o tipo de colheita e a solução diluidora, obteve-se quatro tubos de amostras por animal. Os tubos foram centrifugados e os sedimentos foram homogeneizados nos respectivos meios obtendo-se o volume final de 100μL. Realizou-se determinação da concentração e viabilidade celular, obtendo-se as concentrações médias de FCM. Para ambos os meios de diluição, a colheita B apresentou valor numérico maior em comparação à colheita A, porém não foi significativo (p>0,05). Atribui-se tal tendência à menor ocorrência de coagulação da MO no momento da colheita B, sugerindo-se melhor aproveitamento da FCM. Não houve diferença (p>0,05) entre os meios DMEM ou PBS, indicando que os mesmos não alteraram a viabilidade celular. Os protocolos utilizados para colheita de MO e separação da FCM se mostraram eficientes, para o uso em terapia celular em equinos.

The aim of this study was to evaluate mononuclear cells fraction (MCF) concentration and viability from different techniques of bone marrow (BM) aspiration and processing in horses. Five adult horses, healthy and of unknown breed were evaluated. BM was obtained from sternum bone, according two protocols: in aspiration A, 10mL of heparin solution was used inside the syringe and BM was aspirated; in aspiration B, 10mL of heparin solution was injected into the BM, and aspiration was done after 20 seconds. All the animals were submitted by both protocols realized in sequence, without a gap between the procedures. After MCF isolation, of BM samples obtained from A and B aspiration, each sample was divided into two tubes; one contained DMEM solution and the other with PBS solution. Therefore, interchanging the aspiration protocol and the dilution solution, four sample tubes were obtained for each horse. The tubes were centrifuged and the pellet was homogenized with the respectively solution to obtain the final volume of 100μL. Cellular concentration and viability were determined to obtain the FCM medium concentration. For both solutions, the aspiration B had higher numeric values comparing with aspiration A; however, it was not significant (p>0.05). This tendency is attribute for the less BM coagulation observed in the aspiration B, suggesting greater improvement of MCF. No difference (p>0.05) was found between DMEM and PBS solution, indicating that both do not alter the cell viability. The protocols used for BM aspiration and MCF isolation were efficient for application in equine cellular therapy.

Fucoidan protects hepatocytes from apoptosis and inhibits invasion of hepatocellular carcinoma by up-regulating p42/44 MAPK-dependent NDRG-1/CAP43

Fucoidan is a traditional Chinese medicine suggested to possess anti-tumor effects. In this study the anti-metastatic effects of fucoidan were investigated in vitro in human hepatocellular carcinoma (HCC) cells (Huh-7 and SNU-761) under normoxic and hypoxic conditions and in vivo using a distant liver metastasis model involving injection of MH134 cells into spleen via the portal vein. Its ability to protect hepatocytes against bile acid (BA)-induced apoptosis was investigated in primary hepatocytes. Fucoidan was found to suppress the invasion of HCC cells through up-regulation of p42/44 MAPK-dependent NDRG-1/CAP43 and partly, under normoxic conditions, through up-regulation of p42/44 MAPK-dependent VMP-1 expression. It also significantly decreased liver metastasis in vivo. As regards its hepatoprotective effect, fucoidan decreased BA-induced hepatocyte apoptosis as shown by the attenuation of caspase-8, and -7 cleavages and suppression of the mobilization of caspase-8 and Fas associated death domain (FADD) into the death-inducing signaling complex. In summary, fucoidan displays inhibitory effects on proliferation of HCC cells and protective effects on hepatocytes. The results suggest fucoidan is a potent suppressor of tumor invasion with hepatoprotective effects.

A simple technique for estimation of phospholipase d and phospholipase c enzymes in smooth muscle and its cells.

This research article describes two novel and simple techniques for the estimation of phospholipase D and phospholipase C enzymes in aortic smooth muscle and cells cultured from the bovine aorta. The techniques encompass the use of ion exchange chromatography and liquid scintillation spectrophotometry.

Proliferation and Functional Activity of Human Adipose Tissue-Derived CD146 Positive Endothelial Cells According to Culture Mediums

Studies on development of serum-free conditioned media using Vero cells and DMEM with controlled concentration of glucose and pyruvate / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to examine in vitro development of early preimplantation mouse embryos in various kind of serum-free conditioned media (SF-VCM) manufactured from DMEM cultured with Vero Cells. METHODS: A total of 846 two-cell mouse embryos were cultured in different kind of SF-VCM. SF-VCMs were divided into SF-VCM-10, -30 and -50 by media volume using DMEM #1 media, and divided into SF-VCM #1, #2 and #3 by controlled concentration of glucose and pyruvate (manufactured by DMEM #1: mixed three volume of DMEM-G (DMEM with glutamine without glucose and pyruvate) and one volume of DMEM-GGP (DMEM with glutamine, glucose, pyruvate), #2: mixed same volume of DMEM-G and DMEM-GGP and #3: mixed one volume of DMEM-G and three volume of DMEM-GGP, respectively). Experimental groups were mainly added 10% SSS, and 20% hFF was added to only Control group co-cultured with Vero cells. Development of embryos was observed every 24 hours. Results between different groups were analyzed using Chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: In vitro developmental rate by each cleavage stages of mouse embryos cultured in SF-VCMs with a various volumes were significantly (P<0.05) higher in SF-VCM-30 (morula< or =: 97.2%, Blastocyst (BL)< or =: 97.2%, Hatching BL< or =: 82.2%) than other groups. In the rate of development on in vitro co-culture vs. a various SF-VCMs manufactured by DMEM controlled concentration of glucose and pyruvate, Group I (SF-VCM #1) was higher than other groups in each cleavage stages (morula< or =: 98.1%, Blastocyst (BL)< or =: 97.1%, hatching BL< or =: 81.7%, respectively). Moreover, specially, in the developmental rate into the hatching blastocyst < or = after 96 hours in vitro culture, Group I (81.7%) was significantly higher than control group (67.6%, P<0.05). CONCLUSION: SF-VCM #1 manufactured by volume of 30 mL DMEM #1 media cultured in vitro for 48 hours in 250 mL flask was the most effective on in vitro developmental rate of mouse preimplantation embryos. Therefore, it is expected that SF-VCM #1 has application to human IVF-ET.

Effects of Vero Cells Co-culture System on The In Vitro Development of Mouse Preimplantation Embryos in Media with Different Composition of Glucose and Pyruvate / 대한산부인과학회지

OBJECTIVE: The purpose of this study was to examine the effects of vero cells co-culture system on the in vitro development of mouse preimplantation embryos in culture media with different composition of glucose and pyruvate. METHODS: Two-cell embryos were collected from 4-5 weeks old ICR mice. Seven hundreds twenty two embryos were cultured with or without vero cells monolayer in four media with different compositions that was manufactured by two DMEM media with (DMEM-GGP) or without (DMEM-G) glucose and pyruvate. In control, DMEM-G medium which is currently using for human embryo culture in our infertility clinic was used. Group I (DMEM-G(1/4)GP) was cultured in medium which was mixed three volume of DMEM-G and one volume of DMEM-GGP, and group II (DMEM-G(1/2)GP) was cultured in medium which was mixed same volume of DMEM-G and DMEM-GGP, and group III was cultured in DMEM-GGP. All media were added to 20% hFF. Results between different groups were analyzed using a Chi-square test, and considered statistically significant when p value was less than 0.05. RESULTS: The developmental rate into 3-cell

The Influences of Different Composition of Glucose and Pyruvate on In Vitro Development of Mouse Preimplantation Embryos in Medium with Glutamine / 대한산부인과학회잡지

OBJECTIVE: The purpose of this study was to examine the effects on in vitro development of early preimplantation mouse embryos in DMEM medium with glutamine which was controlled by different composition of glucose and pyruvate. METHODS: Four hundred and nineteen mouse 2-cell embryos were cultured in four different media with different composition of glucose and pyruvate for 96 hours. The DMEM-G contained L-glutamine for energy sources was used for control group. Group I embryos were cultured in the medium that mixed one volume of DMEM-GGP contained L-glutamine, D-glucose and sodium pyruvate for energy sources with three volume of DMEM-G, and group II embryos were cultured in the medium that mixed with same volume of DMEM-G and DMEM-GGP, and group III embryos were cultured in DMEM-GGP. RESULTS: At 24 hours, the development into >or=3-cell was significantly higher (por=8-cell was significantly higher in group I (73.1%) than control (44.2%), group II (59.6%) and III (45.8%), and also group II was significantly higher than control and group III. At 48 hours, the development into >or=morula was significantly higher in group I (90.4%) and II (86.5%) than control (73.0%). However, the development into blastocyst, in group III (15.0%) was significantly lower than control, group I and II. At 72 hours, the development into >or=expanded blastocyst was significantly higher in group I (69.2%) than group III (47.7%), and total blastocyst was significantly higher in group I (80.8%) than control (66.3%) and group III (67.3%). At 96 hours, the development into >or=hatching blastocyst was significantly higher in group I (78.8%) than control (61.5%) and group III (57.9%), and also, total blastocyst was significantly higher in group I (85.6%) than control (69.2%) and group III (72.0%). CONCLUSION: The development of early preimplantation mouse embryos cultured in group I medium that mixed one volume of DMEM-GGP with three volume of DMEM-G was better than other groups during the culture period.

Study on Development of Mouse Preimplantation Embryos in Culture Media with Different Composition of Energy Sources / 대한산부인과학회잡지

OBJECTIVE: The objective of this study was to examine the effect on development of mouse preimplantation embryos in culture media with different composition of energy sources in vitro culture. METHODS: Two hundred and seventy one two-cell embryos were cultured in four different culture system for 96 hours. Group I (n=61) was cultured in DMEM-G (DMEM with glutamine) only, groupII (n=64) was cultured in DMEM-GGP (DMEM with glutamine, glucose and pyruvate) only, group III (n=72) was cultured for 48 hours in DMEM-G and then transferred to DMEM-GGP and group IV (n=74) was cultured for 48 hours in DMEM-GGP and then transferred to DMEM-G. Development of embryos in each group was observed every 24 hours. RESULTS: After 24 hours, the rate of development > or = 3-cell was significantly higher in groupII (87.5%) and IV (86.5%) compared with group I (59.0%) and III (62.5%). After 48 hours, the rate of development into > or = morula stage was significantly higher in GroupII (79.7%) and IV (86.5%) compared with group I (34.4%) and III (37.5%). After 72 hours, the rate of development into blastocyst was significantly higher in group IV (74.3%) compared with group I (49.2%) and III (45.8%). After 96 hours, the rate of development into > or = expanded blastocyst was significantly higher in group IV (70.3%) compared with group I (32.8%),II (53.1%), and group III (40.3%). CONCLUSION: Mouse preimplantation embryos development was the most effective in culture system with DMEM-GGP for 48 hours and then transferred to DMEM-G.