Bacillus licheniformis-based intensive fermentation of Tibetan tea improved its bioactive compounds and reinforced the intestinal barrier in mice.

Tibetan tea changes during microorganism fermentation. Research on microorganisms in Tibetan tea has focused on their identification, while studies on the influence of specific microorganisms on the components and health functions of Tibetan tea are lacking. Bacillus licheniformis was inoculated into Tibetan tea for intensive fermentation, and the components of B. licheniformis-fermented tea (BLT) were detected by liquid chromatography with tandem mass spectrometry (UHPLC-TOF-MS), and then the effects of BLT on intestinal probiotic functions were investigated by experiments on mice. The results revealed the metabolites of BLT include polyphenols, alkaloids, terpenoids, amino acids, and lipids. Intensified fermentation also improved the antioxidant capacity in vivo and the protective effect on the intestinal barrier of Tibetan tea. In addition, the enhanced fermentation of Tibetan tea exerted intestinal probiotic effects by modulating the relative abundance of short-chain fatty acid-producing bacteria in the intestinal flora. Therefore, intensive fermentation with B. licheniformis can improve the health benefits of Tibetan tea.

Ginseng in white and red processed forms: Ginsenosides and cardiac side effects.

Ginseng (Panax ginseng Meyer) has long been consumed as a medicinal or functional food in East Asia. It is available as dried white ginseng (WG) and steamed red ginseng (RG), which might differ in ginsenoside profiles. We compared ginsenoside types of RG and WG using UPLC-MS/MS and evaluated how they biologically affected heart of healthy rats by recording electrocardiography, measuring biochemical indicators, analyzing cardiac tissue slides, and Ca2+ signaling pathways. About 25 and 29 ginsenosides were detected in WG and RG, respectively, and the total ginsenoside content of RG contained was nearly 1.8 times higher than that of WG. Among them, ginsenoside Rg4, ginsenoside Rg6, ginsenoside Rh4, ginsenoside Rk1, ginsenoside Rg5, and protopanaxadiol were detected only in RG, while 20(R)-ginsenoside Rg2 was detected only in WG. Male SD rats treated by intraperitoneal injection of WG or RG extracts were similar to the control in terms of electrocardiography and heart histology, indicating that both may not significantly affect the rats' myocardial function. However, WG and RG may induce mild cardiac injury resulting in increased cardiac collagen and creatine kinase levels. In addition, upregulated p-CaMKII and PPARδ and downregulated SERCA2a for WG and RG treatments were further associated with increased cardiac contractility. In general, RG had less effect on the heart of healthy rats than WG, which may be due to RG having a high proportion of low-polar ginsenosides.

Effect of Pleurotus eryngii mycelial fermentation on the composition and antioxidant properties of tartary buckwheat.

In this study, we investigated the effect of solid-state fermentation of Pleurotus eryngii on the composition and antioxidant activity of Tartary buckwheat (TB). Firstly, the solid-state fermentation of P. eryngii mycelium with buckwheat was carried out, and the fermentation process was explored. The results of the extraction process and method selection experiments showed that the percolation extraction method was superior to the other two methods. The results of extraction rate, active components and antioxidant activity measurements before and after fermentation of TB extract showed that the extraction rate increased about 1.7 times after fermentation. Total flavonoids, rutin and triterpene contents were increased after fermentation compared to control. Meanwhile, LC-MS results showed an increase in the content of the most important substances in the fermented TB extract and the incorporation of new components, such as oleanolic acid, ursolic acid, amino acids, and D-chiral inositol. The fermented TB extract showed stronger antioxidant activity, while the protein and amino acid contents increased by 1.93-fold and 1.94-fold, respectively. This research was the first to use P. eryngii to ferment TB and prepared a lyophilized powder that could be used directly using vacuum freeze-drying technology. Not only the use of solid-state fermentation technology advantages of edible fungi to achieve value-added buckwheat, but also to broaden the scope of TB applications. This study will provide ideas and directions for the development and application of edible mushroom fermentation technology and TB.

Transcriptome and proteomics conjoint analysis reveal anti-alcoholic liver injury effect of Dianhong Black Tea volatile substances.

Dianhong Black Tea, a fermented tea containing various bioactive ingredients, has been found to have a significant role in alleviating alcoholic liver injury (ALI). One of its main unique components, Dianhong Black Tea volatile substances (DBTVS), may have potential anti-ALI effects. However, its effects and underlying molecular mechanisms are still unknown. In this study, we aimed to investigate the potential of DBTVS as an anti-ALI agent using alcohol-fed rats. We assessed the effect of DBTVS on ALI by analyzing serum transaminase and lipid levels, as well as conducting hematoxylin-eosin and oil red O staining. Additionally, GC-MS was used to detect the components of DBTVS, while transcriptome, proteomics analysis, Western blot, and molecular docking were employed to uncover the underlying mechanisms. Our results demonstrated that DBTVS significantly reduced serum ALT and AST levels and improved lipid metabolism disorders. Moreover, we identified 14 components in DBTVS, with five of them exhibiting strong binding affinity with key proteins. These findings suggested that DBTVS could be a promising agent for the prevention and treatment of ALI. Its potential therapeutic effects may be attributed to its ability to regulate lipid metabolism through the PPAR signaling pathway.

Plasmon-enhanced fluorescence for ellagic acid detection based on surface structure of gold nanoparticles.

Ellagic acid (EA), as a natural polyphenolic acid, is considered a naturally occurring inhibitor of carcinogenesis. Herein, we developed a plasmon-enhanced fluorescence (PEF) probe for EA detection based on silica-coated gold nanoparticles (Au NPs). A silica shell was designed to control the distance between silica quantum dots (Si QDs) and Au NPs. The experimental results indicated that an 8.8-fold fluorescence enhancement was obtained compared with the original Si QDs. Three-dimensional finite-difference time-domain (3D-FDTD) simulations further demonstrated that the local electric field enhancement around Au NPs led to the fluorescence enhancement. In addition, the fluorescent sensor was applied for the sensitive detection of EA with a detection limit of 0.14 µM. It can be used to detect EA in pomegranate rind with a recovery rate of 100.26-107.93%. It can also be applied to the analysis of other substances by changing the identification substances. These experimental results indicated that the probe provides a good option for clinical analysis and food safety.

Serine/threonine-protein kinase STK24 induces tumorigenesis by regulating the STAT3/VEGFA signaling pathway.

Lung cancer is the most common cause of cancer-related death. Although anti-angiogenesis therapy has been effective in the treatment of nonsmall cell lung cancer (NSCLC), drug-resistance is a common challenge. Therefore, there is a need to develop new therapeutic strategies for NSCLC. Serine/threonine-protein kinase 24 (STK24), also known as MST3, belongs to the germinal center kinase III subfamily, and the biological function of STK24 in NSCLC tumorigenesis and tumor angiogenesis is still unclear. In this study, we demonstrated that STK24 was overexpressed in lung cancer tissues compared with normal lung tissues, and lung cancer patients with higher STK24 expression levels had shorter overall survival time. In addition, our in vitro assays using A549 and H226 cell lines revealed that the STK24 expression level of cancer cells was positively correlated with cancer cells proliferation, migration, invasion, and tumor angiogenesis ability; in vivo assays also demonstrated that silencing of STK24 dramatically inhibited tumor progress and tumor angiogenesis. To investigate a mechanism, we revealed that STK24 positively regulated the signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor A (VEGFA) signaling pathway by inhibiting polyubiquitin-proteasomal-mediated degradation of STAT3. Furthermore, we performed in vivo assays in BALB/c nude mice and in vitro assays to show that STK24-regulated tumor angiogenesis depends on STAT3. These findings deepened our understanding of tumor angiogenesis, and the STK24/STAT3/VEGFA signaling pathway might be a novel therapeutic target for NSCLC treatment.

Mucosal-associated invariant T cells reduce and display tissue-resident phenotype with elevated IL-17 producing capacity in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the important causes of cancer-related mortality worldwide. Previous studies have demonstrated the crucial roles of mucosal-associated invariant T (MAIT) cells in regulating tumor immunity, while their roles in NSCLC remain largely unknown. The aim of this study is to evaluate clinical relevance of MAIT cells in blood and tumor tissues of patients with NSCLC. Here, we find that there is no significant difference in the frequency of circulating MAIT cells between NSCLC patients and healthy donors. However, the MAIT-frequency is significantly declined in lung tumor tissues compared to their peri-tumor counterparts, which relates to Tumor-Node-Metastasis (TNM) stage. The MAIT-frequency in lung tumor tissues is higher in node-negative patients compared to node-positive patients. Furthermore, tumor-infiltrating MAIT cells display a tissue-resident effector-memory phenotype and exhibit upregulated levels of exhaustion markers. The percentage of tissue-resident cells in MAIT tends to be higher in tumor tissues than in peri-tumor tissues. In addition, the percentage of IL-17A+ MAIT cells is significantly higher in lung tumor tissues than that in peri-tumor tissues. In summary, our results indicate the possible detrimental role of MAIT cells in the development and progression of NSCLC.

Chinese Baijiu: The Perfect Works of Microorganisms.

Chinese Baijiu is one of the famous distilled liquor series with unique flavors in the world. Under the open environment, Chinese Baijiu was produced by two solid-state fermentation processes: jiuqu making and baijiu making. Chinese Baijiu can be divided into different types according to the production area, production process, starter type, and product flavor. Chinese Baijiu contains rich flavor components, such as esters and organic acids. The formation of these flavor substances is inseparable from the metabolism and interaction of different microorganisms, and thus, microorganisms play a leading role in the fermentation process of Chinese Baijiu. Bacteria, yeasts, and molds are the microorganisms involved in the brewing process of Chinese Baijiu, and they originate from various sources, such as the production environment, production workers, and jiuqu. This article reviews the typical flavor substances of different types of Chinese Baijiu, the types of microorganisms involved in the brewing process, and their functions. Methods that use microbial technology to enhance the flavor of baijiu, and for detecting flavor substances in baijiu were also introduced. This review systematically summarizes the role and application of Chinese Baijiu flavor components and microorganisms in baijiu brewing and provides data support for understanding Chinese Baijiu and further improving its quality.

Recent Developments in Surface-Enhanced Raman Spectroscopy and Its Application in Food Analysis: Alcoholic Beverages as an Example.

Surface-enhanced Raman spectroscopy (SERS) is an emerging technology that combines Raman spectroscopy and nanotechnology with great potential. This technology can accurately characterize molecular adsorption behavior and molecular structure. Moreover, it can provide rapid and sensitive detection of molecules and trace substances. In practical application, SERS has the advantages of portability, no need for sample pretreatment, rapid analysis, high sensitivity, and 'fingerprint' recognition. Thus, it has great potential in food safety detection. Alcoholic beverages have a long history of production in the world. Currently, a variety of popular products have been developed. With the continuous development of the alcoholic beverage industry, simple, on-site, and sensitive detection methods are necessary. In this paper, the basic principle, development history, and research progress of SERS are summarized. In view of the chemical composition, the beneficial and toxic components of alcoholic beverages and the practical application of SERS in alcoholic beverage analysis are reviewed. The feasibility and future development of SERS are also summarized and prospected. This review provides data and reference for the future development of SERS technology and its application in food analysis.

G protein-coupled estrogen receptor promotes acrosome reaction via regulation of Ca2+ signaling in mouse sperm†.

G protein-coupled estrogen receptor (GPER), a seven-transmembrane G protein-coupled receptor, mediates the rapid pre-genomic signaling actions of estrogen and derivatives thereof. The expression of GPER is extensive in mammal male reproductive system. However, the functional role of GPER in mouse sperm has not yet been well recognized. This study revealed that GPER was expressed at the acrosome and the mid-flagellum of the mouse sperm. The endogenous GPER ligand 17ß-estradiol and the selective GPER agonist G1 increased intracellular Ca2+ concentration ([Ca2+]i) in mouse sperm, which could be abolished by G15, an antagonist of GPER. In addition, the G1-stimulated Ca2+ response was attenuated by interference with the phospholipase C (PLC) signaling pathways or by blocking the cation channel of sperm (CatSper). Chlortetracycline staining assay showed that the activation of GPER increased the incidence of acrosome-reacted sperm. Conclusively, GPER was located at the acrosome and mid-flagellum of the mouse sperm. Activation of GPER triggered the elevation of [Ca2+]i through PLC-dependent Ca2+ mobilization and CatSper-mediated Ca2+ influx, which promoted the acrosome reaction of mouse sperm.

Glycogen synthase kinase GSK3α promotes tumorigenesis by activating HIF1/VEGFA signaling pathway in NSCLC tumor.

BACKGROUND: Lung cancer is one of the most common cancers and the leading cause of cancer-related death. Glycogen synthase kinase-3 (GSK-3) α, a member of the glycogen synthase kinase-3 family, reportedly plays a role in tumorigenesis. However, its biological function in tumorigenesis requires deeper exploration. Hypoxia is a major feature of solid tumor, along with decreasing availability of oxygen, inducing treatment resistance, and tumor progress. METHODS: Levels of GSK3α expression in clinical samples were detected using western blot and IHC assays, while its biological function and underlying mechanism of action in tumor progression were investigated using western blot, CCK8, cell cycle, colony formation, Transwell, ELISA and tube formation assays. Furthermore, we investigated the relationship between GSK3α expression and the HIF1α/VEGFA signaling pathway in vivo using a mouse xenograft model. RESULTS: GSK3α was significantly upregulated in NSCLC patients with cases that exhibited high GSK3α levels recording shorter survival times. Moreover, GSK3α overexpression promoted proliferation, migration, invasion and clone formation ability of NSCLC cells, while its silencing resulted in an opposite phenomenon. Moreover, GSK3α not only activated the HIF1α/VEGFA signaling pathway, but also regulated HIF1α stabilization independently via the PHDs-pVHL signaling pathway. Moreover, GSK3α-mediated tumor angiogenesis depended on HIF1α expression both in vitro and in vivo. CONCLUSION: GSK3α functioned as an oncogene in NSCLC tumorigenesis by regulating the HIF1/VEGFA signaling pathway in an independent manner through the PHDs-pVHL signaling pathway. These findings were expected to provide novel sights to guide future development of therapies for effective treatment of NSCLC. Video abstract.

Salvage surgery following downstaging of advanced non-small cell lung cancer by targeted therapy.

BACKGROUND: Advanced non-small cell lung cancer (NSCLC) accounts for a high proportion of lung cancer cases. Targeted therapy improve the survival in these patients, but acquired drug resistance will inevitably occur. If tumor downstaging is achieved after targeted therapy, could surgical resection before drug resistance improve clinical benefits for patients with advanced NSCLC? Here, we conducted a clinical trial showing that for patients with advanced driver gene mutant NSCLC who did not progress after targeted therapy, salvage surgery (SS) could improve progression-free survival (PFS). Herein, we retrospectively reviewed our former clinical trial and thoracic cancer database in our medical institutions. METHODS: We identified patients with advanced driver gene mutant NSCLC treated with targeted therapy plus SS or targeted therapy alone in our former clinical trial and our thoracic cancer database from July 2016 to July 2019. PFS was compared between the targeted therapy plus SS group and the targeted therapy only group using the log-rank test. RESULTS: We identified 73 patients with driver gene mutant NSCLC who were treated with targeted therapy and 18 treated with targeted therapy plus SS.Among the 18 patients treated with targeted therapy plus SS, there were no obvious perioperative complications and deaths. Targeted therapy followed by SS resulted in a significantly longer PFS compared with targeted therapy alone (23.4 months VS 12.9 months, p = 0.0004). CONCLUSIONS: Salvage surgery after tumor downstaging is a promising therapeutic strategy for some patients with advanced (stage IIIB-IV) NSCLC and may offer a new therapeutic option for multidisciplinary comprehensive treatment of lung cancer.

Overexpression of KDM4D promotes acute myeloid leukemia cell development by activating MCL-1.

Acute myeloid leukemia (AML) is regarded as a fatal cancer in the world. The overall survival in adult patients with AML is still poor. As lysine demethylases, the KDM4 family is found highly expressed in many kinds of tumors. In this study, we demonstrate that KDM4D is overexpressed in AML and knockdown of KDM4D not only inhibits the proliferation of AML cells, but also induces cell cycle arrest and apoptosis. Furthermore, our research shows that KDM4D can regulate the expression of MCL-1 by demethylating H3K9me3 at the promoter region in AML cells. Besides, we find that high expression of KDM4D is correlated with poor overall survival in AML patients. Taken together, our study demonstrated that KDM4D can promote MCL-1 expression in AML and may serve as a novel target for the treatment of AML.

NSD2 promotes tumor angiogenesis through methylating and activating STAT3 protein.

Tumor angiogenesis plays vital roles in tumorigenesis and development; regulatory mechanism of angiogenesis is still not been fully elucidated. NSD2, a histone methyltransferase catalyzing di-methylation of histone H3 at lysine 36, has been proved a critical molecule in proliferation, metastasis, and tumorigenesis. But its role in tumor angiogenesis remains unknown. Here we demonstrated that NSD2 promoted tumor angiogenesis in vitro and in vivo. Furthermore, we confirmed that the angiogenic function of NSD2 was mediated by STAT3. Momentously, we found that NSD2 promoted the methylation and activation of STAT3. In addition, mass spectrometry and site-directed mutagenesis assays revealed that NSD2 methylated STAT3 at lysine 163 (K163). Meanwhile, K to R mutant at K163 of STAT3 attenuated the activation and angiogenic function of STAT3. Taken together, we conclude that methylation of STAT3 catalyzed by NSD2 promotes the activation of STAT3 pathway and enhances the ability of tumor angiogenesis. Our findings investigate a NSD2-dependent methylation-phosphorylation regulation pattern of STAT3 and reveal that NSD2/STAT3/VEGFA axis might be a potential target for tumor therapy.

Estrogen promotes the metastasis of non­small cell lung cancer via estrogen receptor ß by upregulation of Toll­like receptor 4 and activation of the myd88/NF­κB/MMP2 pathway.

Estrogen promotes non­small cell lung cancer (NSCLC) metastasis via estrogen receptor ß (ERß)­mediated invasiveness­associated matrix metalloprotease 2 (MMP2) upregulation. However, how ERß increases the aggressiveness of NSCLC cells remains unclear. Recently, MMP2 was found to be upregulated by Toll­like receptor 4 (TLR4) signaling activation and to promote NSCLC metastasis. Our present study aimed to examine the role of ERß in the activation of TLR4 signaling and in tumor progression and metastasis, and to explore the synergistic metastatic effect of a combination of ERß and TLR4 activation on human NSCLC cells in vitro and in vivo. Here, we found that ERß is associated with TLR4 in metastatic lymph nodes. Western blot analysis and immunofluorescence revealed that ERß overexpression upregulated TLR4 protein expression and activated downstream targets, myeloid differentiation primary response 88 (myd88)/nuclear factor (NF)­κB/MMP2, enhancing NSCLC cell migration and invasion in vitro. A novel ERß­TLR4 interaction in cell plasma was identified by co­immunoprecipitation and confocal immunofluorescence. The combination of estradiol and specific TLR4 agonist lipopolysaccharide (LPS) synergistically promoted metastatic behaviors in NSCLC cells. In cell culture and murine lung metastasis models, exposure to estradiol and LPS induced increased matrix degradation and accelerated invadopodia and metastasis formation in NSCLC cells compared with that in cells treated with estradiol or LPS alone. Together, we showed that estrogen promoted NSCLC metastasis via ERß by upregulating TLR4 and activating its downstream signaling axis myd88/NF­κB/MMP2. The combined targeting of ERß and TLR4 may be a novel therapeutic strategy against advanced metastatic lung cancer.

Co-administration of 20(S)-protopanaxatriol (g-PPT) and EGFR-TKI overcomes EGFR-TKI resistance by decreasing SCD1 induced lipid accumulation in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with sensitive epidermal growth factor receptor (EGFR) mutations are successfully treated with EGFR tyrosine kinase inhibitors (EGFR-TKIs); however, resistance to treatment inevitably occurs. Given lipid metabolic reprogramming is widely known as a hallmark of cancer and intimately linked with EGFR-stimulated cancer growth. Activation of EGFR signal pathway increased monounsaturated fatty acids (MUFA) and lipid metabolism key enzyme Stearoyl-CoA Desaturase 1 (SCD1) expression. However the correlation between EGFR-TKI resistance and lipid metabolism remains to be determined. METHODS: In this study the differences in lipid synthesis between paired TKI-sensitive and TKI-resistant patient tissues and NSCLC cell lines were explored. Oleic acid (OA, a kind of MUFA, the SCD1 enzymatic product) was used to simulate a high lipid metabolic environment and detected the affection on the cytotoxic effect of TKIs (Gefitinib and osimertinib) in cell lines with EGFR-activating mutations. (20S)-Protopanaxatriol (g-PPT), an aglycone of ginsenosides, has been reported to be an effective lipid metabolism inhibitor, was used to inhibit lipid metabolism. Additionally, synergism in cytotoxic effects and signal pathway activation were evaluated using CCK-8 assays, Western blotting, flow cytometry, Edu assays, plate clone formation assays and immunofluorescence. Furthermore, two xenograft mouse models were used to verify the in vitro results. RESULTS: Gefitinib-resistant cells have higher lipid droplet content and SCD1 expression than Gefitinib-sensitive cells in both NSCLC cell lines and patient tissues. Additionally oleic acid (OA, a kind of MUFA, the SCD1 enzymatic product) abrogates the cytotoxic effect of both Gefitinib and osimertinib in cell lines with EGFR-activating mutations. As a reported effective lipid metabolism inhibitor, g-PPT significantly inhibited the expression of SCD1 in lung adenocarcinoma cells, and then down-regulated the content of intracellular lipid droplets. Combined treatment with Gefitinib and g-PPT reverses the resistance to Gefitinib and inhibits the activation of p-EGFR and the downstream signaling pathways. CONCLUSIONS: Our findings uncover a link between lipid metabolic reprogramming and EGFR-TKI resistance, confirmed that combination target both EGFR and abnormal lipid metabolism maybe a promising therapy for EGFR-TKI resistance and highlighting the possibility of monitoring lipid accumulation in tumors for predicting drug resistance.

Cellular Mechanism Underlying Hydrogen Sulfide Mediated Epithelial K+ Secretion in Rat Epididymis.

As a novel gasotransmitter, hydrogen sulfide (H2S) elicits various physiological actions including smooth muscle relaxation and promotion of transepithelial ion transport. However, the pro-secretory function of H2S in the male reproductive system remains largely unclear. The aim of this study is to elucidate the possible roles of H2S in modulating rat epididymal intraluminal ionic microenvironment essential for sperm storage. The results revealed that endogenous H2S-generating enzymes cystathionine ß-synthetase (CBS) and cystathionine γ-lyase (CSE) were both expressed in rat epididymis. CBS located predominantly in epithelial cells whilst CSE expressed primarily in smooth muscle cells. The relative expression level of CBS and CSE escalated from caput to cauda regions of epididymis, which was paralleled to the progressively increasing production of endogenous H2S. The effect of H2S on epididymal epithelial ion transportation was investigated using short-circuit current (I SC), measurement of intracellular ion concentration and in vivo rat epididymal microperfusion. Our data showed that H2S induced transepithelial K+ secretion via adenosine triphosphate-sensitive K+ (KATP) channel and large conductance Ca2+-activated K+ (BKCa) channel. Transient receptor potential vanilloid 4 (TRPV4) channel-mediated Ca2+ influx was implicated in the activation of BKCa channel. In vivo studies further demonstrated that H2S promoted K+ secretion in rat epididymal epithelium. Inhibition of endogenous H2S synthesis caused a significant decrease in K+ concentration of cauda epididymal intraluminal fluid. Moreover, our data demonstrated that high extracellular K+ concentration actively depressed the motility of cauda epididymal sperm in a pH-independent manner. Collectively, the present study demonstrated that H2S was vital to the formation of high K+ concentration in epididymal intraluminal fluid by promoting the transepithelial K+ secretion, which might contribute to the maintenance of the cauda epididymal sperm in quiescent dormant state before ejaculation.

miR-19a contributes to gefitinib resistance and epithelial mesenchymal transition in non-small cell lung cancer cells by targeting c-Met.

Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is used as a first-line treatment for advanced non-small cell lung cancer (NSCLC). However, most NSCLC patients inevitably develop gefitinib resistance, and the mechanisms underlying this resistance are not fully understood. In this study, we show that miR-19a is significantly down-regulated in gefitinib-resistant NSCLC cell lines compared with gefitinib-sensitive cell lines. In addition, the down-regulation of miR-19a suppressed the expression of epithelial markers but induced the expression levels of mesenchymal markers. A mechanistic analysis revealed that miR-19a regulated c-Met expression by directly targeting the c-Met 3'UTR. Overexpression of miR-19a decreased c-Met expression and re-sensitized gefitinib-resistant NSCLC cells in vitro and in vivo. Consistent with the in vitro findings, the miR-19a serum level was significantly decreased in NSCLC patients with acquired gefitinib resistance compared with the level observed prior to the acquisition of resistance in each patient, indicating that miR-19a expression may be a valuable biomarker for the prediction of acquired gefitinib resistance in a clinical setting. Our data demonstrate that the miR-19a/c-Met pathway plays a critical role in acquired resistance to gefitinib and that the manipulation of miR-19a might provide a therapeutic strategy for overcoming acquired gefitinib resistance.

Role of GPR30 in estrogen-induced prostate epithelial apoptosis and benign prostatic hyperplasia.

Several studies have implicated estrogen and the estrogen receptor (ER) in the pathogenesis of benign prostatic hyperplasia (BPH); however, the mechanism underlying this effect remains elusive. In the present study, we demonstrated that estrogen (17ß-estradiol, or E2)-induced activation of the G protein-coupled receptor 30 (GPR30) triggered Ca2+ release from the endoplasmic reticulum, increased the mitochondrial Ca2+ concentration, and thus induced prostate epithelial cell (PEC) apoptosis. Both E2 and the GPR30-specific agonist G1 induced a transient intracellular Ca2+ release in PECs via the phospholipase C (PLC)-inositol 1, 4, 5-triphosphate (IP3) pathway, and this was abolished by treatment with the GPR30 antagonist G15. The release of cytochrome c and activation of caspase-3 in response to GPR30 activation were observed. Data generated from the analysis of animal models and human clinical samples indicate that treatment with the GPR30 agonist relieves testosterone propionate (TP)-induced prostatic epithelial hyperplasia, and that the abundance of GPR30 is negatively associated with prostate volume. On the basis of these results, we propose a novel regulatory mechanism whereby estrogen induces the apoptosis of PECs via GPR30 activation. Inhibition of this activation is predicted to lead to abnormal PEC accumulation, and to thereby contribute to BPH pathogenesis.

Functional expression of G protein-coupled receptor 30 in immature rat epididymal epithelium.

The aim of this study is to investigate the functional role of G protein-coupled receptor 30 (GPR30) in the epididymis. We found that GPR30 is expressed in the epithelium of the immature rat epididymis and is involved in chloride secretion into the caudal epididymis lumen. The short-circuit current (Isc) experiments showed that in primary cultured caudal epididymis epithelium, activation of GPR30 by its specific agonist G1 induced a mono-phasic current increase, and G15, the specific antagonist of GPR30, could completely inhibit the current induced by G1. The G1-induced Isc was largely blocked by application of the non-specific chloride channel inhibitor diphenylamine-dicarboxylic acid (DPC), or by the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 , suggesting that the current was mainly mediated through CFTR. In addition, after stimulating GPR30 by G1, the intracellular concentration of cAMP in the epithelium was significantly increased, indicating that the cAMP signal pathway is involved and could be responsible for the CFTR activation. Finally, to further investigate the function of GPR30 in vivo, G15 was administrated into rats subcutaneously. The osmotic pressure of the micro perfusion solution from epididymis was measured and the sperms were collected. Results showed that there was an osmotic pressure increase of the perfusion solution from G15 treated rats. When the GPR30 was inhibited by G15 endogenously, the motility of sperms decreased. Our data demonstrated that GPR30 is involved in the formation of caudal epididymis fluid micro-environment thus affecting sperm motility.