DESIGNER fraction concept unmasks minor bioactive constituents in red clover (Trifolium pratense L.).

In botanical extracts, highly abundant constituents can mask or dilute the effects of other, and often, more relevant biologically active compounds. To facilitate the rational chemical and biological assessment of these natural products with wide usage in human health, we introduced the DESIGNER approach of Depleting and Enriching Selective Ingredients to Generate Normalized Extract Resources. The present study applied this concept to clinical Red Clover Extract (RCE) and combined phytochemical and biological methodology to help rationalize the utility of RCE supplements for symptom management in postmenopausal women. Previous work has demonstrated that RCE reduces estrogen detoxification pathways in breast cancer cells (MCF-7) and, thus, may serve to negatively affect estrogen metabolism-induced chemical carcinogenesis. Clinical RCE contains ca. 30% of biochanin A and formononetin, which potentially mask activities of less abundant compounds. These two isoflavonoids are aryl hydrocarbon receptor (AhR) agonists that activate P450 1A1, responsible for estrogen detoxification, and P450 1B1, producing genotoxic estrogen metabolites in female breast cells. Clinical RCE also contains the potent phytoestrogen, genistein, that downregulates P450 1A1, thereby reducing estrogen detoxification. To identify less abundant bioactive constituents, countercurrent separation (CCS) of a clinical RCE yielded selective lipophilic to hydrophilic metabolites in six enriched DESIGNER fractions (DFs 01-06). Unlike solid-phase chromatography, CCS prevented any potential loss of minor constituents or residual complexity (RC) and enabled the polarity-based enrichment of certain constituents. Systematic analysis of estrogen detoxification pathways (ERα-degradation, AhR activation, CYP1A1/CYP1B1 induction and activity) of the DFs uncovered masked bioactivity of minor/less abundant constituents including irilone. These data will allow the optimization of RCE with respect to estrogen detoxification properties. The DFs revealed distinct biological activities between less abundant bioactives. The present results can inspire future carefully designed extracts with phytochemical profiles that are optimized to increase in estrogen detoxification pathways and, thereby, promote resilience in women with high-risk for breast cancer. The DESIGNER approach helps to establish links between complex chemical makeup, botanical safety and possible efficacy parameters, yields candidate DFs for (pre)clinical studies, and reveals the contribution of minor phytoconstituents to the overall safety and bioactivity of botanicals, such as resilience promoting activities relevant to women's health.

Breast cancer prevention with liquiritigenin from licorice through the inhibition of aromatase and protein biosynthesis in high-risk women's breast tissue.

Breast cancer risk continues to increase post menopause. Anti-estrogen therapies are available to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer prevention. Natural products such as hops (Humulus lupulus) and three pharmacopeial licorice (Glycyrrhiza) species have demonstrated estrogenic and chemopreventive properties, but little is known regarding their effects on aromatase expression and activity as well as pro-proliferation pathways in human breast tissue. We show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plant extracts. Moreover, phytoestrogens such as liquiritigenin which is common in all licorice species have potent aromatase inhibitory activity, which is further supported by computational docking of their structures in the binding pocket of aromatase. In addition, GI extract and liquiritigenin suppress aromatase expression in the breast tissue of high-risk postmenopausal women. Although liquiritigenin has estrogenic effects in vitro, with preferential activity through estrogen receptor (ER)-ß, it reduces estradiol-induced uterine growth in vivo. It downregulates RNA translation, protein biosynthesis, and metabolism in high-risk women's breast tissue. Finally, it reduces the rate of MCF-7 cell proliferation, with repeated dosing. Collectively, these data suggest that liquiritigenin has breast cancer prevention potential for high-risk postmenopausal women.

Miz1 promotes KRAS-driven lung tumorigenesis by repressing the protocadherin Pcdh10.

Targeting KRAS-mutated non-small-cell lung cancer (NSCLC) remains clinically challenging. Here we show that loss of function of Miz1 inhibits lung tumorigenesis in a mouse model of oncogenic KRAS-driven lung cancer. In vitro, knockout or silencing of Miz1 decreases cell proliferation, clonogenicity, migration, invasion, or anchorage-independent growth in mutant (MT) KRAS murine or human NSCLC cells but has unremarkable impact on non-tumorigenic cells or wild-type (WT) KRAS human NSCLC cells. RNA-sequencing reveals Protocadherin-10 (Pcdh10) as the top upregulated gene by Miz1 knockout in MT KRAS murine lung tumor cells. Chromatin immunoprecipitation shows Miz1 binding on the Pcdh10 promoter in MT KRAS lung tumor cells but not non-tumorigenic cells. Importantly, silencing of Pcdh10 rescues cell proliferation and clonogenicity in Miz1 knockout/knockdown MT KRAS murine or human tumor cells, and rescues allograft tumor growth of Miz1 knockout tumor cells in vivo. Miz1 is upregulated in MT KRAS lung tumor tissues compared with adjacent non-involved tissues in mice. Consistent with this, Miz1 is upregulated while Pcdh10 is downregulated in human lung adenocarcinomas (LUAD) compared with normal tissues, and high Miz1 levels or low Pcdh10 levels are associated with poor survival in lung cancer patients. Furthermore, the Miz1 signature is associated with worse survival in MT but not WT KRAS LUAD, and Pcdh10 is downregulated in MT compared to WT KRAS LUAD. Taken together, our studies implicate the Miz1/Pcdh10 axis in oncogenic KRAS-driven lung tumorigenesis.

miR-221-5p-Mediated Downregulation of JNK2 Aggravates Acute Lung Injury.

Sepsis and acute lung injury (ALI) are linked to mitochondrial dysfunction; however, the underlying mechanism remains elusive. We previously reported that c-Jun N-terminal protein kinase 2 (JNK2) promotes stress-induced mitophagy by targeting small mitochondrial alternative reading frame (smARF) for ubiquitin-mediated proteasomal degradation, thereby preventing mitochondrial dysfunction and restraining inflammasome activation. Here we report that loss of JNK2 exacerbates lung inflammation and injury during sepsis and ALI in mice. JNK2 is downregulated in mice with endotoxic shock or ALI, concomitantly correlated inversely with disease severity. Small RNA sequencing revealed that miR-221-5p, which contains seed sequence matching to JNK2 mRNA 3' untranslated region (3'UTR), is upregulated in response to lipopolysaccharide, with dynamically inverse correlation with JNK2 mRNA levels. miR-221-5p targets the 3'UTR of JNK2 mRNA leading to its downregulation. Accordingly, miR-221-5p exacerbates lung inflammation and injury during sepsis in mice by targeting JNK2. Importantly, in patients with pneumonia in medical intensive care unit, JNK2 mRNA levels in alveolar macrophages flow sorted from non-bronchoscopic broncholaveolar lavage (BAL) fluid were inversely correlated strongly and significantly with the percentage of neutrophils, neutrophil and white blood cell counts in BAL fluid. Our data suggest that miR-221-5p targets JNK2 and thereby aggravates lung inflammation and injury during sepsis.

Auto-hydrolysis of red clover as "green" approach to (iso)flavonoid enriched products.

Optimal parameters for the auto-hydrolysis of (iso)flavone glycosides to aglycones in ground Trifolium pratense L. plant material were established as a "green" method for the production of a reproducible red clover extract (RCE). The process utilized 72-h fermentation in DI water at 25 and 37 °C. The aglycones obtained at 25 °C, as determined by UHPLC-UV and quantitative 1H NMR (qHNMR), increased significantly in the auto-hydrolyzed (ARCE) (6.2-6.7% w/w biochanin A 1, 6.1-9.9% formononetin 2) vs a control ethanol (ERCE) extract (0.24% 1, 0.26% 2). After macerating ARCE with 1:1 (v/v) diethyl ether/hexanes (ARCE-d/h), 1 and 2 increased to 13.1-16.7% and 14.9-18.4% w, respectively, through depletion of fatty components. The final extracts showed chemical profiles similar to that of a previous clinical RCE. Biological standardization revealed that the enriched ARCE-d/h extracts produced the strongest estrogenic activity in ERα positive endometrial cells (Ishikawa cells), followed by the precursor ARCE. The glycoside-rich ERCE showed no estrogenic activity. The estrogenicity of ARCE-d/h was similar to that of the clinical RCE. The lower potency of the ARCE compared to the prior clinical RCE indicated that substantial amounts of fatty acids/matter likely reduce the estrogenicity of crude hydrolyzed preparations. The in vitro dynamic residual complexity of the conversion of biochanin A to genistein was evaluated by LC-MS-MS. The outcomes help advance translational research with red clover and other (iso)flavone-rich botanicals by inspiring the preparation of (iso)flavone aglycone-enriched extracts for the exploration of new in vitro and ex vivo bioactivities that are unachievable with genuine, glycoside-containing extracts.

SAR Study on Estrogen Receptor α/ß Activity of (Iso)flavonoids: Importance of Prenylation, C-Ring (Un)Saturation, and Hydroxyl Substituents.

Many botanicals used for women's health contain estrogenic (iso)flavonoids. The literature suggests that estrogen receptor beta (ERß) activity can counterbalance estrogen receptor alpha (ERα)-mediated proliferation, thus providing a better safety profile. A structure-activity relationship study of (iso)flavonoids was conducted to identify ERß-preferential structures, overall estrogenic activity, and ER subtype estrogenic activity of botanicals containing these (iso)flavonoids. Results showed that flavonoids with prenylation on C8 position increased estrogenic activity. C8-prenylated flavonoids with C2-C3 unsaturation resulted in increased ERß potency and selectivity [e.g., 8-prenylapigenin (8-PA), EC50 (ERß): 0.0035 ± 0.00040 µM], whereas 4'-methoxy or C3 hydroxy groups reduced activity [e.g., icaritin, EC50 (ERß): 1.7 ± 0.70 µM]. However, nonprenylated and C2-C3 unsaturated isoflavonoids showed increased ERß estrogenic activity [e.g., genistein, EC50 (ERß): 0.0022 ± 0.0004 µM]. Licorice (Glycyrrhiza inflata, [EC50 (ERα): 1.1 ± 0.20; (ERß): 0.60 ± 0.20 µg/mL], containing 8-PA, and red clover [EC50 (ERα): 1.8 ± 0.20; (ERß): 0.45 ± 0.10 µg/mL], with genistein, showed ERß-preferential activity as opposed to hops [EC50 (ERα): 0.030 ± 0.010; (ERß): 0.50 ± 0.050 µg/mL] and Epimedium sagittatum [EC50 (ERα): 3.2 ± 0.20; (ERß): 2.5 ± 0.090 µg/mL], containing 8-prenylnaringenin and icaritin, respectively. Botanicals with ERß-preferential flavonoids could plausibly contribute to ERß-protective benefits in menopausal women.

Is rifampin resistance a reliable predictive marker of multidrug-resistant tuberculosis in China: A meta-analysis of findings.

OBJECTIVES: Systematic review of multidrug-resistant tuberculosis (MDR-TB) prevalence among rifampicin (RIF)-resistant tuberculosis (RR-TB) patients in 34 provinces of China was conducted to correlate RIF resistance with concurrent isoniazid (INH) resistance. METHODS: Database searches (PubMed, Embase, China National Knowledge Infrastructure, Chinese Scientific Journal, Wanfang), identified drug resistance surveillance studies conducted between January 1, 2000 and June 30, 2018. Of 1554 records, random-effects meta-analysis of 34 studies of adequate methodological quality yielded 108,366 TB cases for MDR-TB prevalence analysis of RR-TB cases. RESULTS: MDR-TB prevalence among RR-TB cases varied from 57% (Xinjiang; 95% CI 47%, 67%) to 95% (Taiwan; 95% CI 92%, 98%), for a pooled national rate of 77% (95% CI 75%, 80%). Subgroup and meta-regression analyses revealed greater MDR-TB prevalence in previously treated versus new RR-TB cases (P < 0.001), with no significant differences of regional initial drug resistance rates or sampling methods. Regional MDR-TB prevalence among RR-TB cases was lowest (69%) in the Northeast Region (95% CI 65%, 73%) and highest (90%) in Hong Kong, Macao and Taiwan (95% CI 81%, 98%). CONCLUSIONS: In China, ∼77% of RR-TB cases are MDR-TB. Thus, RIF resistance cannot effectively predict MDR-TB. Highly variable RR-TB prevalence across China warrants improved TB management.

Evidence for Chemopreventive and Resilience Activity of Licorice: Glycyrrhiza Glabra and G. Inflata Extracts Modulate Estrogen Metabolism in ACI Rats.

Women are increasingly using botanical dietary supplements (BDS) to reduce menopausal hot flashes. Although licorice (Glycyrrhiza sp.) is one of the frequently used ingredients in BDS, the exact plant species is often not identified. We previously showed that in breast epithelial cells (MCF-10A), Glycyrrhiza glabra (GG) and G. inflata (GI), and their compounds differentially modulated P450 1A1 and P450 1B1 gene expression, which are responsible for estrogen detoxification and genotoxicity, respectively. GG and isoliquiritigenin (LigC) increased CYP1A1, whereas GI and its marker compound, licochalcone A (LicA), decreased CYP1A1 and CYP1B1 The objective of this study was to determine the distribution of the bioactive licorice compounds, the metabolism of LicA, and whether GG, GI, and/or pure LicA modulate NAD(P)H quinone oxidoreductase (NQO1) in an ACI rat model. In addition, the effect of licorice extracts and compounds on biomarkers of estrogen chemoprevention (CYP1A1) as well as carcinogenesis (CYP1B1) was studied. LicA was extensively glucuronidated and formed GSH adducts; however, free LicA as well as LigC were bioavailable in target tissues after oral intake of licorice extracts. GG, GI, and LicA caused induction of NQO1 activity in the liver. In mammary tissue, GI increased CYP1A1 and decreased CYP1B1, whereas GG only increased CYP1A1 LigC may have contributed to the upregulation of CYP1A1 after GG and GI administration. In contrast, LicA was responsible for GI-mediated downregulation of CYP1B1 These studies highlight the polypharmacologic nature of botanicals and the importance of standardization of licorice BDS to specific Glycyrrhiza species and to multiple constituents.

Estrogen Receptor (ER) Subtype Selectivity Identifies 8-Prenylapigenin as an ERß Agonist from Glycyrrhiza inflata and Highlights the Importance of Chemical and Biological Authentication.

Postmenopausal women are increasingly using botanicals for menopausal symptom relief due to the increased breast cancer risk associated with traditional estrogen therapy. The deleterious effects of estrogens are associated with estrogen receptor (ER)α-dependent proliferation, while ERß activation could enhance safety by opposing ERα effects. Three medicinal licorice species, Glycyrrhiza glabra ( G. glabra), G. uralensis, and G. inflata, were studied for their differential estrogenic efficacy. The data showed higher estrogenic potency for G. inflata in an alkaline phosphatase induction assay in Ishikawa cells (ERα) and an estrogen responsive element (ERE)-luciferase assay in MDA-MB-231/ß41 breast cancer cells (ERß). Bioassay-guided fractionation of G. inflata led to the isolation of 8-prenylapigenin (3). Surprisingly, a commercial batch of 3 was devoid of estrogenic activity. Quality control by MS and qNMR revealed an incorrect compound, 4'- O-methylbroussochalcone B (10), illustrating the importance of both structural and purity verification prior to any biological investigations. Authentic and pure 3 displayed 14-fold preferential ERß agonist activity. Quantitative analyses revealed that 3 was 33 times more concentrated in G. inflata compared to the other medicinal licorice extracts. These data suggest that standardization of G. inflata to 3 might enhance the safety and efficacy of G. inflata supplements used for postmenopausal women's health.

DESIGNER Extracts as Tools to Balance Estrogenic and Chemopreventive Activities of Botanicals for Women's Health.

Botanical dietary supplements contain multiple bioactive compounds that target numerous biological pathways. The lack of uniform standardization requirements is one reason that inconsistent clinical effects are reported frequently. The multifaceted biological interactions of active principles can be disentangled by a coupled pharmacological/phytochemical approach using specialized ("knock-out") extracts. This is demonstrated for hops, a botanical for menopausal symptom management. Employing targeted, adsorbent-free countercurrent separation, Humulus lupulus extracts were designed for pre- and postmenopausal women by containing various amounts of the phytoestrogen 8-prenylnaringenin (8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis of their estrogenic (alkaline phosphatase), chemopreventive (NAD(P)H-quinone oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that the estrogenicity of hops is a function of 8-PN, whereas their NQO1 induction and cytotoxic properties depend on XH levels. Antagonization of the estrogenicity of 8-PN by elevated XH concentrations provided evidence for the interdependence of the biological effects. A designed postmenopausal hop extract was prepared to balance 8-PN and XH levels for both estrogenic and chemopreventive properties. An extract designed for premenopausal women contains reduced 8-PN levels and high XH concentrations to minimize estrogenic while retaining chemopreventive properties. This study demonstrates the feasibility of modulating the concentrations of bioactive compounds in botanical extracts for potentially improved efficacy and safety.

Clinical significance of the correlation between PLCE 1 and PRKCA in esophageal inflammation and esophageal carcinoma.

Esophagitis and Barrett's esophagus are linked to esophageal squamous cell carcinoma and adenocarcinoma, respectively. However, the underlying mechanisms are still unclear. This study analyzed the expression levels of and correlation between PLCE1 and PRKCA in human esophagitis, carcinogen NMBA-induced rat esophagus, PLCE1 genetic deficient mouse esophageal epithelial tissues and human esophageal cancer cell line, integrated with Online oncology data sets. We found that the expression levels of both PLCE1 and PRKCA were significantly elevated in human esophagitis, esophageal squamous cell carcinoma, Barrett's esophagus, esophageal adenocarcinoma and in NMBA-treated rat esophageal epithelia. However, PRKCA and cytokines were significantly downregulated in PLCE1-deficient mouse esophageal epithelia, and knockdown of PLCE1 in human esophageal cancer cells led to reduction of PRKCA and cytokines. Finally, high expression of both PLCE1 and PRKCA is significantly associated with poor outcomes of the patients with esophageal cancers. In conclusion, this study defined the initiation and progression of esophageal inflammation and malignant transformation, in which the positive correlation of PLCE1 and PRKCA exhibits critical clinical significance.

[TIE's flying acupuncture for acute cerebral infarction hemiplegia: a randomized controlled trial].

OBJECTIVE: To compare the efficacy difference between TIE 's flying acupuncture combined with conventional treatment and conventional treatment alone on acute cerebral infarction hemiplegia. METHODS: A total of 120 patients were randomly divided into an observation group and a control group, 60 cases in each one. The control group was treated with conventional treatment, including anti-platelet aggregation, lipid-lowering, formula of traditional Chinese medicine which could promote circulation and remove stasis, neurotrophic medication and symptomatic treatment; mannitol was used for cerebral infarction with large area or increased intracranial pressure. Based on the conventional treatment applied in the control group, the observation group was treated with flying acupuncture at the affected Jianyu (LI 15), Quchi (LI 11), Shousanli (LI 10), Waiguan (TE 5), Hegu (LI 4), Huantiao (GB 30), Biguan (ST 31), Futu (ST 32), Zusanli (ST 36), etc. The treatment was given once a day, six days per week, for totally 2 weeks. The simplified Fugl-Meyer score, National Institute of Health Stroke Scale (NIHSS) and ADL-Bathel index (BI) score were evaluated before and after treatment in the two groups. RESULTS: After the treatment, the simplified Fugl-Meyer and BI were significantly increased in both groups (all P<0.05), which was significantly higher in the observation group (both P<0.05); after the treatment, the NIHSS was significantly lowered in both groups (both P<0.05), which was significantly lower in the observation group (P<0.05). CONCLUSION: TIE 's flying acupuncture combined with conventional treatment were effective for acute cerebral infarction hemiplegia, which have better efficacy than conventional treatment on improving motor function, neurological deficit and daily living ability, and the pain is mild.

Presence of the Panton-Valentine Leukocidin Genes in Methicillin-Resistant Staphylococcus aureus Is Associated with Severity and Clinical Outcome of Hospital-Acquired Pneumonia in a Single Center Study in China.

The Panton-Valentine leukocidin (PVL) genes of methicillin-resistant Staphylococcus aureus (MRSA) have previously been associated with severe infections. Here, the impact of the PVL genes on severity of disease and clinical outcome of patients with hospital-acquired pneumonia (HAP) or ventilator-associated pneumonia (VAP) due to MRSA was investigated in a single center observational study in a hospital in China. HAP due to MRSA was diagnosed in 100 patients and 13 of the patients were PVL positive, while VAP was diagnosed in 5 patients and 2 were PVL positive. The PVL positive patient group showed a significantly higher Acute Physiology and Chronic Health Evaluation (APACHE) II score (14.3 ±7.8 vs. 10.1 ±4.7, P = 0.005) and significantly more patients with CRP levels >80 mg/L (8/15 vs. 12/90, P = 0.006) or WBC counts >15x109/L (7/15 vs. 12/90, P = 0.006), indicating that the severity of disease is affected by the presence of the PVL genes. The outcome of the study was defined by 30-day mortality. Four (27%) of the PVL positive patients and four (4%) of the PVL negative patients died within 30 days (P = 0.01, Fisher exact test). Kaplan-Meier survival curves were generated for the PVL positive and PVL negative patient groups, which differed significantly (P = 0.003). Among the patients that died, the mean interval between diagnosis and death was shorter for the PVL positive patients (9.3 ±5.6 vs. 40.8 ±6.6 days, P = 0.013). Further analysis within the HAP and VAP patient groups showed that the presence of PVL in MRSA impacted the severity of disease and clinical outcome of HAP, but for VAP the number of patients included in the study was too low. In conclusion, in this single center study in a Chinese hospital the presence of the PVL genes in MRSA impacted the severity of disease and clinical outcome in patients with HAP due to MRSA.

c-Jun NH2-teminal kinase 1 interacts with vitamin D receptor and affects vitamin D-mediated inhibition of cancer cell proliferation.

BACKGROUND: Vitamin D is a chemopreventive agent that acts against colorectal carcinogenesis in vivo and in vitro through vitamin D receptor (VDR). Previous studies showed that stress-activated protein kinase JNKs (c-Jun NH2-terminal kinases) and p38 cooperated to activate VDR and increase vitamin D3-dependent growth inhibition in breast cancer cells. This study is to determine whether vitamin D-mediated inhibition of cell proliferation is associated with JNK1 in colorectal cancer cells. METHODS AND RESULTS: Human colon cancer cells were treated with calcitriol, an active vitamin D3. The results showed that calcitriol significantly inhibited cell proliferation and caused cell cycle arrest in HT29 cells, which was associated with induction of phosphorylated JNK1 (p-JNK). The induction of VDR and p-JNK by calcitriol was also observed in Caco-2 cells. Furthermore, VDR expression was significantly downregulated in JNK1-/- mouse intestinal epithelial cells, and VDR reporter activity was reduced in JNK1-/- mouse embryonic fibroblasts (MEFs). However, increasing activated JNK1 upregulated VDR expression and transcriptional activity in vitro. Moreover, JNK1 co-localized with VDR in nuclei and cytoplasm and physically bound together. Reduced expression of JNK1 and VDR in HT29 and Caco-2 cells and JNK1 absence in JNK1-/- MEFs attenuated calcitriol-mediated inhibition of cell proliferation. CONCLUSION: JNK1 physically and functionally interacted with VDR and positively regulated VDR expression at transcriptional and translational levels, which influenced calcitriol-mediated inhibition of cancer cell proliferation.

Tumor suppressor PRSS8 targets Sphk1/S1P/Stat3/Akt signaling in colorectal cancer.

PRSS8 is a membrane-anchored serine protease prostasin and has been shown an association with carcinogenesis. Herein we found that PRSS8 expression was significantly reduced in colorectal adenomas and adenocarcinomas. The decreased PRSS8 was well correlated with clinical stages, poor differentiation and shorter survival time of colorectal cancer. Furthermore, increase of PRSS8 led to the inhibition of colorectal cancer cell proliferation, knockdown of PRSS8 accelerated cell proliferation in vitro, and overexpressing PRSS8 retarded cancer cell growth in nude mice. Mechanistic studies revealed that PRSS8 inhibited Sphk1/S1P/Stat3/Akt signaling pathway, in terms of inverse association between PRSS8 and Sphk1 in human colorectal cancers and in Sphk1-/- mice. In conclusion, PRSS8 acts as a tumor suppressor by inhibiting Sphk1/S1P/Stat3/Akt signaling pathway, and could be used as a biomarker to monitor colorectal carcinogenesis and predict outcomes.

PRSS8 methylation and its significance in esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common cancers worldwide, and the incidence and mortality is increasing rapidly in recent years in China, but the underlying mechanisms are largely unclear. Herein we found that the expression of PRSS8, a serine protease prostasin, is significantly decreased in esophageal squamous cell carcinomas (ESCC) at mRNA and protein levels. The reduction of PRSS8 was well correlated with poor differentiation and shorter survival time. Interestingly, ESCC stromal expression of PRSS8 was significantly correlated with stromal lymphocyte infiltration and cancer progression. Methylation specific PCR showed that PRSS8 was hypermethylated in ESCC tissues and ESCC cell lines, which was linked to the downregulation of PRSS8 expression and decreased activities of PRSS8 promoter. De-methylation agent decitabine was able to restore PRSS8 expression, leading to the inhibition of cancer cell proliferation, motility, migration and cell cycle arrest. However, the restored PRSS8 and its tumor inhibition could be reversed by small interfering RNA targeting PRSS8. Mechanistic study showed that tumor inhibition of PRSS8 may be associated with proliferation- and epithelial mesenchymal transition - related proteins in ESCC cells. In conclusion, our finding showed that PRSS8 methylation and its stromal expression had important clinical significance in ESCC.

Induction of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) by Glycyrrhiza Species Used for Women's Health: Differential Effects of the Michael Acceptors Isoliquiritigenin and Licochalcone A.

For the alleviation of menopausal symptoms, women frequently turn to botanical dietary supplements, such as licorice and hops. In addition to estrogenic properties, these botanicals could also have chemopreventive effects. We have previously shown that hops and its Michael acceptor xanthohumol (XH) induced the chemoprevention enzyme, NAD(P)H: quinone oxidoreductase 1 (NQO1), in vitro and in vivo. Licorice species could also induce NQO1, as they contain the Michael acceptors isoliquiritigenin (LigC) found in Glycyrrhiza glabra (GG), G. uralensis (GU), G. inflata (GI), and licochalcone A (LicA) which is only found in GI. These licorice species and hops induced NQO1 activity in murine hepatoma (Hepa1c1c7) cells; hops ≫ GI > GG ≅ GU. Similar to the known chemopreventive compounds curcumin (turmeric), sulforaphane (broccoli), and XH, LigC and LicA were active dose-dependently; sulforaphane ≫ XH > LigC > LicA ≅ curcumin ≫ liquiritigenin (LigF). Induction of the antioxidant response element luciferase in human hepatoma (HepG2-ARE-C8) cells suggested involvement of the Keap1-Nrf2 pathway. GG, GU, and LigC also induced NQO1 in nontumorigenic breast epithelial MCF-10A cells. In female Sprague-Dawley rats treated with GG and GU, LigC and LigF were detected in the liver and mammary gland. GG weakly enhanced NQO1 activity in the mammary tissue but not in the liver. Treatment with LigC alone did not induce NQO1 in vivo most likely due to its conversion to LigF, extensive metabolism, and its low bioavailability in vivo. These data show the chemopreventive potential of licorice species in vitro could be due to LigC and LicA and emphasize the importance of chemical and biological standardization of botanicals used as dietary supplements. Although the in vivo effects in the rat model after four-day treatment are minimal, it must be emphasized that menopausal women take these supplements for extended periods of time and long-term beneficial effects are quite possible.

Biological and chemical standardization of a hop (Humulus lupulus) botanical dietary supplement.

Concerned about the safety of conventional estrogen replacement therapy, women are using botanical dietary supplements as alternatives for the management of menopausal symptoms such as hot flashes. Before botanical dietary supplements can be evaluated clinically for safety and efficacy, botanically authenticated and standardized forms are required. To address the demand for a standardized, estrogenic botanical dietary supplement, an extract of hops (Humulus lupulus L.) was developed. Although valued in the brewing of beer, hop extracts are used as anxiolytics and hypnotics and have well-established estrogenic constituents. Starting with a hop cultivar used in the brewing industry, spent hops (the residue remaining after extraction of bitter acids) were formulated into a botanical dietary supplement that was then chemically and biologically standardized. Biological standardization utilized the estrogen-dependent induction of alkaline phosphatase in the Ishikawa cell line. Chemical standardization was based on the prenylated phenols in hops that included estrogenic 8-prenylnaringenin, its isomer 6-prenylnaringenin, and pro-estrogenic isoxanthohumol and its isomeric chalcone xanthohumol, all of which were measured using high-performance liquid chromatography-tandem mass spectrometry. The product of this process was a reproducible botanical extract suitable for subsequent investigations of safety and efficacy.

A sequence variant in the phospholipase C epsilon C2 domain is associated with esophageal carcinoma and esophagitis.

A single-nucleotide polymorphism (rs2274223: A5780G:His1927Arg) in the phospholipase C epsilon gene (PLCϵ) was recently identified as a susceptibility locus for esophageal cancer in Chinese subjects. To determine the underlying mechanisms of PLCϵ and this SNP in esophageal carcinogenesis, we analyzed PLCϵ genotypes, expression, and their correlation in esophageal cancer cell lines, non-transformed esophageal cells, 58 esophageal squamous cell carcinomas and 10,614 non-cancer subjects from China. We found that the G allele (AG or GG) was associated with increased PLCϵ mRNA and protein expression in esophageal cancer tissues and in esophageal cancer cell lines. G allele was also associated with higher enzyme activity, which might be associated with increased protein expression. Quantitative analysis of the C2 domain sequences revealed that A:G allelic imbalance was strongly linked to esophageal malignancy. Moreover, the analysis of 10,614 non-cancer subjects demonstrated that the G allele was strongly associated with moderate to severe esophagitis in the subjects from the high-incidence areas of China (OR 6.03, 95% CI 1.59-22.9 in high-incidence area vs. OR 0.74, 95% CI 0.33-1.64 in low-incidence area; P = 0.008). In conclusion, the PLCϵ gene, particularly the 5780G allele, might play a pivotal role in esophageal carcinogenesis via upregulating PLCϵ mRNA, protein, and enzyme activity, and augmenting inflammatory process in esophageal epithelium. Thus, 5780G allele may constitute a promising biomarker for esophageal squamous cell carcinoma risk stratification, early detection, and progression prediction.