BMX, a specific HDAC8 inhibitor, with TMZ for advanced CRC therapy: a novel synergic effect to elicit p53-, ß-catenin- and MGMT-dependent apoptotic cell death.

BACKGROUND: Despite advances in treatment, patients with refractory colorectal cancer (CRC) still have poor long-term survival, so there is a need for more effective therapeutic options. METHODS: To evaluate the HDAC8 inhibition efficacy as a CRC treatment, we examined the effects of various HDAC8 inhibitors (HDAC8i), including BMX (NBM-T-L-BMX-OS01) in combination with temozolomide (TMZ) or other standard CRC drugs on p53 mutated HT29 cells, as well as wild-type p53 HCT116 and RKO cells. RESULTS: We showed that HDAC8i with TMZ cotreatment resulted in HT29 arrest in the S and G2/M phase, whereas HCT116 and RKO arrest in the G0/G1 phase was accompanied by high sub-G1. Subsequently, this combination approach upregulated p53-mediated MGMT inhibition, leading to apoptosis. Furthermore, we observed the cotreatment also enabled triggering of cell senescence and decreased expression of stem cell biomarkers. Mechanistically, we found down-expression levels of ß-catenin, cyclin D1 and c-Myc via GSK3ß/ß-catenin signaling. Intriguingly, autophagy also contributes to cell death under the opposite status of ß-catenin/p62 axis, suggesting that there exists a negative feedback regulation between Wnt/ß-catenin and autophagy. Consistently, the Gene Set Enrichment Analysis (GSEA) indicated both apoptotic and autophagy biomarkers in HT29 and RKO were upregulated after treating with BMX. CONCLUSIONS: BMX may act as a HDAC8 eraser and in combination with reframed-TMZ generates a remarkable synergic effect, providing a novel therapeutic target for various CRCs. Video Abstract.

NBM-BMX, an HDAC8 Inhibitor, Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the ß-Catenin/c-Myc/SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition.

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of ß-catenin was reversed by proteasome inhibitor via the ß-catenin/ GSK3ß signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the ß-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.

Taiwanese Green Propolis Ethanol Extract Delays the Progression of Type 2 Diabetes Mellitus in Rats Treated with Streptozotocin/High-Fat Diet.

Taiwanese green propolis ethanol extract (TGPE) is produced only in Taiwan and has a different composition from other types of propolis. TGPE is known for its anti-inflammation, anti-oxidation, and anti-microbial properties, but the effects and mechanisms of TGPE in the modulation of diabetes are unclear. In this study, we investigated the effects of TGPE on type 2 diabetes mellitus (T2DM) in a streptozotocin/high-fat-diet (STZ/HFD)-induced T2DM rat model. The results revealed that TGPE delayed the development and progression of T2DM and reduced the severity of β-cell failure. TGPE also attenuated inflammation and reactive oxygen species ROS in the rats. Moreover, there were higher levels of oxidant cytokines, leptin, and adiponectin in the serum of the TGPE-treated group. Unlike Brazilian propolis, TGPE promoted hepatic genes PPAR-α and CYP7A1, which were related to lipid catabolism and removal. TGPE may thus delay the progression of T2DM through anti-inflammation effects, anti-oxidation effects, and balancing lipid metabolism. It is suggested that TGPE can be a potential alternative medicine for T2DM.

Use of urinary metabolomics to evaluate the effect of hyperuricemia on the kidney.

Clinical studies show that hyperuricemia is a risk factor in the progression and development of cardiovascular and metabolic disease. Elevated serum levels of uric acid induce renal injury via an inflammation response, but the detailed mechanism is still under study. To better understand the effect of hyperuricemia on the kidney, we used gas chromatography-mass spectrometry-based metabolomics to investigate the role of uric acid in the mouse kidney. Partial least-squares discriminant analysis revealed significant differences between control and hyperuricemia groups in urine metabolic profiles. We identified 33 metabolites from 76 highly reproducible peaks and found abnormal uric acid levels related to comprehensive kidney injury, including excretive function and energy metabolism. Additionally, inflammation induced by the interleukin 6/signal transducer and activator of transcription 3 signaling pathway participated in hyperuricemia-induced kidney injury. This study helps understand the relationship between hyperuricemia and kidney injury. Metabolomics may be a useful strategy for early diagnosis of kidney damage.

Pumpkin (Cucurbita moschata) fruit extract improves physical fatigue and exercise performance in mice.

Pumpkin (Cucurbita moschata) is a popular and nutritious vegetable consumed worldwide. The overall purpose of this study was to evaluate the effects of C. moschata fruit extract (CME) on anti-fatigue and ergogenic functions following physiological challenges. Male ICR mice from four groups designated vehicle, CME-50, CME-100 and CME-250, respectively (n = 8 per group in each test) were orally administered CME for 14 days at 0, 50, 100 and 250 mg/kg/day. The anti-fatigue activity and exercise performance were evaluated using exhaustive swimming time, forelimb grip strength, as well as levels of plasma lactate, ammonia, glucose, and creatine kinase after an acute swimming exercise. The resting muscular and hepatic glycogen was also analyzed after 14-day supplementation with CME. Trend analysis revealed that CME treatments increased grip strength. CME dose-dependently increased 5% body weight loaded swimming time, blood glucose, and muscular and hepatic glycogen levels. CME dose-dependently decreased plasma lactate and ammonia levels and creatine kinase activity after a 15-min swimming test. The mechanism was relevant to the increase in energy storage (as glycogen) and release (as blood glucose), and the decrease of plasma levels of lactate, ammonia, and creatine kinase. Therefore, CME may be potential for the pharmacological effect of anti-fatigue.

Triterpenoid-Rich Extract from Antrodia camphorata Improves Physical Fatigue and Exercise Performance in Mice.

Antrodia camphorata (AC) is an endemic mushroom that grows in Taiwan. We investigated the fatigue-alleviating effects of AC on endurance capacity in swim-exercised and weight-loading mice. Male Institute of Cancer Research (ICR) strain mice from 3 groups (n = 10 per group in each test) were orally administered AC fruiting body extract for 7 days at 0, 50, and 200 mg/kg/day, designated vehicle, AC-50, and AC-200, respectively. Trend analysis revealed that AC treatments increased grip strength. AC dose-dependently increased swim time, blood glucose, and muscular and hepatic glycogen levels and dose-dependently decreased plasma lactate and ammonia levels and creatine kinase activity. The increase in swimming endurance with AC administration was caused by an increase in liver and muscle glycogen deposition. A. camphorata may have potential for use in ergogenic and antifatigue activities.

Metabolomics characterization of energy metabolism reveals glycogen accumulation in gut-microbiota-lacking mice.

Microbiota in the gut are considered an important environmental factor associated with host metabolism and physiology. Although gut microbiota are known to contribute to hepatic lipogenesis and fat storage, little is known about how the condition influences the deposition of glycogen in the liver. To better understand and characterize the host energy metabolism in guts lacking microbiota, we compared the liver metabolome of specific pathogen-free and germ-free mice by gas chromatography-mass spectrometry combined with partial least-squares discriminant analysis. We identified 30 of 52 highly reproducible peaks in chromatograms of liver tissue extracts from the two groups of mice. The two groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids and carbohydrates. The metabolic profile of germ-free mice suggests that they synthesize glycogen and accumulate it in the liver through gluconeogenesis and glycogenesis. Our findings shed light on a new perspective of the role of gut microbiota in energy metabolism and will be useful to help study probiotics, obesity and metabolic diseases.

Echinacea alkamides prevent lipopolysaccharide/D-galactosamine-induced acute hepatic injury through JNK pathway-mediated HO-1 expression.

This study aimed to shed light on the anti-inflammatory and hepatoprotective effect of the major alkamides dodeca-2E,4E,8Z,10Z(E)-tetraenoic acid isobutylamides (Alk-8/9), isolated from Echinacea purpurea roots, against acute fulminant hepatitis induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in mice. The results show that Alk-8/9 dose-dependently induced heme oxygenase (HO)-1 protein expression in LPS-stimulated murine macrophages that was likely regulated by the JNK-mediated pathway through increasing SAPK/JNK phosphorylation, c-jun protein expression, and phosphorylation, and transcription factor AP-1 binding consensus DNA activity. The HO-1 inhibitor or CO scavenger significantly reversed the inhibitory effect of Alk-8/9 on TNF-α expression, whereas N-acetyl-L-cysteine was observed to reduce Alk-8/9-induced HO-1 expression in LPS-treated macrophages. Furthermore, Alk-8/9 markedly induced c-jun and HO-1 protein expression and suppressed serum aminotransferase activities, TNF-α expression, and hepatocyte damage in liver tissues of LPS/d-GalN-treated mice. This paper suggests a new application of Echinacea, a top-selling herbal supplement, as a hepatoprotective agent.

Anti-Hyperglycemic Properties of Crude Extract and Triterpenes from Poria cocos.

Poria cocos, Bai Fu Ling in Chinese, is used in traditional Chinese medicine to treat diabetes. However, its claimed benefits and mechanism are not fully understood. This study aimed to investigate the effect and action of P. cocos on type 2 diabetes. We first performed phytochemical analysis on the crude extract and factions of P. cocos. P. cocos crude extract at 50 mg/kg body weight or more significantly decreased blood glucose levels in db/db mice. Based on a bioactivity-directed fractionation and isolation (BDFI) strategy, chloroform fraction and subfractions 4 and 6 of the P. cocos crude extract possessed a blood glucose-lowering effect. Dehydrotumulosic acid, dehydrotrametenolic acid, and pachymic acid were identified from the chloroform sub-fractions 4, 3, and 2, respectively. Dehydrotumulosic acid had anti-hyperglycemic effect to a greater extent than dehydrotrametenolic acid and pachymic acid. Mechanistic study on streptozocin- (STZ-) treated mice showed that the crude extract, dehydrotumulosic acid, dehydrotrametenolic acid, and pachymic acid of P. cocos exhibited different levels of insulin sensitizer activity. However, the P. cocos crude extract and triterpenes appeared not to activate PPAR-γ pathway. Overall, the data suggest that the P. cocos extract and its triterpenes reduce postprandial blood glucose levels in db/db mice via enhanced insulin sensitivity irrespective of PPAR-γ.

Anti-inflammatory properties of phenolic compounds and crude extract from Porphyra dentata.

ETHNOPHARMACOLOGICAL RELEVANCE: Porphyra dentata, a red edible seaweed, has long been used worldwide in folk medicine for the treatment of inflammatory diseases such as hypersensitivity, lymphadenitis, bronchitis. AIMS OF STUDY: To clarify the anti-inflammatory role of Porphyra dentata crude extract and its identified phenolic compounds by investigating their effect on the nitric oxide (NO)/inducible nitric oxide synthase (iNOS) transcription pathway in macrophage RAW 264.7 cells. MATERIALS AND METHODS: Porphyra dentata crude extract was prepared with methanol. High performance liquid chromatography (HPLC) hyphenated to electrospray ionization mass spectrometry (ESI-MS) and UV detection were utilized to analyze the extract fingerprints. Nitrite measurement, iNOS promoter activity and nuclear factor-kappaB (NF-kappaB) enhancer activity were used to assess the anti-inflammatory effect in lipopolysaccharide (LPS) challenged mouse RAW 264.7 cell line. RESULTS: Phenolic compounds (catechol, rutin and hesperidin) were identified in the crude extract of Porphyra dentata. The crude extract and the phenolic compounds inhibited the production of NO in LPS-stimulated RAW 264.7 cells. Catechol was a more potent suppressor of the up-regulation of iNOS promoter and NF-kappaB enhancer than rutin and yet, hesperidin alone failed to inhibit either activity. CONCLUSION: Our results indicate that catechol and rutin, but not hesperidin, are primary bioactive phenolic compounds in the crude extract to suppress NO production in LPS-stimulated macrophages via NF-kappaB-dependent iNOS gene transcription. The data also explain the anti-inflammatory use and possible mechanism of Porphyra dentata in iNOS implicated diseases.

Metabolomics investigation of exercise-modulated changes in metabolism in rat liver after exhaustive and endurance exercises.

Exhaustive exercise and endurance exercise training modify the physiological status of the body differently. The present study aimed to evaluate the alteration in biochemical composition with exhaustive and endurance exercises in rats using metabolomics strategy. The metabolite profile of liver tissue was investigated on gas chromatography-mass spectrometry (GC-MS). Data further underwent partial least-squares-discriminant analysis (PLS-DA) to compare the effects on metabolites in sedentary control, exhaustively exercised and endurance trained rats. GC-MS detected 115 highly reproducible peaks in chromatograms from individual liver tissue extracts, and we identified 55 of them. The three groups showed significant differences in metabolic profile. Changes in liver metabolism involved metabolites such as amino acids, fatty acids, organic acids, and carbohydrates. Endurance training elevated the greater rate of tricarboxylic acid cycle and antioxidant activity, and exhaustive exercise led to accumulated urea markers and an inflammation response in liver. In addition, GC-MS-based metabolomic analysis is a promising tool to investigate a pathological status with different exercise programs.

Comparative metabolomics approach coupled with cell- and gene-based assays for species classification and anti-inflammatory bioactivity validation of Echinacea plants.

Echinacea preparations were the top-selling herbal supplements or medicines in the past decade; however, there is still frequent misidentification or substitution of the Echinacea plant species in the commercial Echinacea products with not well chemically defined compositions in a specific preparation. In this report, a comparative metabolomics study, integrating supercritical fluid extraction, gas chromatography/mass spectrometry and data mining, demonstrates that the three most used medicinal Echinacea species, Echinacea purpurea, E. pallida, and E. angustifolia, can be easily classified by the distribution and relative content of metabolites. A mitogen-induced murine skin inflammation study suggested that alkamides were the active anti-inflammatory components present in Echinacea plants. Mixed alkamides and the major component, dodeca-2E,4E,8Z,10Z(E)-tetraenoic acid isobutylamides, were then isolated from E. purpurea root extracts for further bioactivity elucidation. In macrophages, the alkamides significantly inhibited cyclooxygenase 2 (COX-2) activity and the lipopolysaccharide-induced expression of COX-2, inducible nitric oxide synthase and specific cytokines or chemokines [i.e., TNF-α, interleukin (IL)-1α, IL-6, MCP-1, MIP-1ß] but elevated heme oxygenase-1 protein expression. Cichoric acid, however, exhibited little or no effect. The results of high-performance liquid chromatography/electron spray ionization/mass spectrometry metabolite profiling of alkamides and phenolic compounds in E. purpurea roots showed that specific phytocompound (i.e., alkamides, cichoric acid and rutin) contents were subject to change under certain post-harvest or abiotic treatment. This study provides new insight in using the emerging metabolomics approach coupled with bioactivity assays for medicinal/nutritional plant species classification, quality control and the identification of novel botanical agents for inflammatory disorders.

Genomics and proteomics of immune modulatory effects of a butanol fraction of echinacea purpurea in human dendritic cells.

BACKGROUND: Echinacea spp. extracts and the derived phytocompounds have been shown to induce specific immune cell activities and are popularly used as food supplements or nutraceuticals for immuno-modulatory functions. Dendritic cells (DCs), the most potent antigen presenting cells, play an important role in both innate and adaptive immunities. In this study, we investigated the specific and differential gene expression in human immature DCs (iDCs) in response to treatment with a butanol fraction containing defined bioactive phytocompounds extracted from stems and leaves of Echinacea purpurea, that we denoted [BF/S+L/Ep]. RESULTS: Affymetrix DNA microarray results showed significant up regulation of specific genes for cytokines (IL-8, IL-1beta, and IL-18) and chemokines (CXCL 2, CCL 5, and CCL 2) within 4 h after [BF/S+L/Ep] treatment of iDCs. Bioinformatics analysis of genes expressed in [BF/S+L/Ep]-treated DCs revealed a key-signaling network involving a number of immune-modulatory molecules leading to the activation of a downstream molecule, adenylate cyclase 8. Proteomic analysis showed increased expression of antioxidant and cytoskeletal proteins after treatment with [BF/S+L/Ep] and cichoric acid. CONCLUSION: This study provides information on candidate target molecules and molecular signaling mechanisms for future systematic research into the immune-modulatory activities of an important traditional medicinal herb and its derived phytocompounds.

Specific plant terpenoids and lignoids possess potent antiviral activities against severe acute respiratory syndrome coronavirus.

In this study, 221 phytocompounds were evaluated for activity against anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) activities using a cell-based assay measuring SARS-CoV-induced cytopathogenic effect on Vero E6 cells. Ten diterpenoids (1-10), two sesquiterpenoids (11 and 12), two triterpenoids (13 and 14), five lignoids (15-19), curcumin (20), and reference controls niclosamide (21) and valinomycin (22) were potent inhibitors at concentrations between 3.3 and 10 microM. The concentrations of the 22 compounds to inhibit 50% of Vero E6 cell proliferation (CC50) and viral replication (EC50) were measured. The selective index values (SI = CC50/EC50) of the most potent compounds 1, 5, 6, 8, 14, and 16 were 58, >510, 111, 193, 180, and >667, respectively. Betulinic acid (13) and savinin (16) were competitive inhibitors of SARS-CoV 3CL protease with Ki values = 8.2 +/- 0.7 and 9.1 +/- 2.4 microM, respectively. Our findings suggest that specific abietane-type diterpenoids and lignoids exhibit strong anti-SARS-CoV effects.

A galactolipid possesses novel cancer chemopreventive effects by suppressing inflammatory mediators and mouse B16 melanoma.

Crassocephalum rabens (Asteraceae) is a popular anti-inflammatory folk medicine and food supplement. We investigated the cancer chemopreventive bioactivity of C. rabens phytocompounds in vitro and in vivo using cell- and gene-based bioassays and a mouse B16 melanoma model. The bioactive glyceroglycolipid 1,2-di-O-alpha-linolenoyl-3-O-beta-galactopyranosyl-sn-glycerol (dLGG) that was identified from C. rabens was found in vitro and in vivo to be a potent nitric oxide (NO) scavenger. dLGG treatment inhibited both mRNA and protein expression of inducible NO synthase and cyclooxygenase-2 (COX-2) in murine macrophages and inhibited COX-2 gene transcription in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B16 cells. In immunohistochemical studies, dLGG inhibited TPA-induced expression of COX-2 and nitration of proteins in mouse skin. dLGG could also significantly inhibit lipopolysaccharide-induced prostaglandin E(2) production in murine macrophages. Furthermore, dLGG prevented nuclear translocation of cytoplasmic nuclear factor-kappaB (NF-kappaB) by suppressing IkappaBalpha phosphorylation and degradation. Structure-activity relationship study by electrophoretic mobility shift assay indicated that the dilinolenoylglycerol moiety in dLGG is the essential structural feature preventing NF-kappaB.DNA complex formation. A dLGG-enriched extract from C. rabens (10 mg/kg) markedly suppressed B16 melanoma growth in C57BL/6J mice following i.p. administration, an effect comparable with that of cisplatin, a cancer chemotherapeutic drug. This study shows the detailed molecular mechanism(s) underlying the anti-inflammatory and tumor-suppressive effects of a natural galactolipid.

Modulatory effects of Echinacea purpurea extracts on human dendritic cells: a cell- and gene-based study.

Echinacea spp. are popularly used as an herbal medicine or food supplement for enhancing the immune system. This study shows that plant extracts from root [R] and stem plus leaf [S+L] tissues of E. purpurea exhibit opposite (enhancing vs inhibitory) modulatory effects on the expression of the CD83 marker in human dendritic cells (DCs), which are known as professional antigen-presenting cells. We developed a function-targeted DNA microarray system to characterize the effects of phytocompounds on human DCs. Down-regulation of mRNA expression of specific chemokines (e.g., CCL3 and CCL8) and their receptors (e.g., CCR1 and CCR9) was observed in [S+L]-treated DCs. Other chemokines and regulatory molecules (e.g., CCL4 and CCL2) involved in the c-Jun pathway were found to be up-regulated in [R]-treated DCs. This study, for the first time, demonstrates that E. purpurea extracts can modulate DC differentiation and expression of specific immune-related genes in DCs.

Antidiabetic dimeric guianolides and a lignan glycoside from Lactuca indica.

Three novel sesquiterpene lactones, lactucain A (1), B (2), and C (3), and a new furofuran lignan, lactucaside (4), were isolated from Lactuca indica along with nine known compounds, 11beta,13-dihydrolactucin, cichoriosides B, quercetin, quercetin 3-O-glucoside, rutin, apigenin, luteolin, luteolin 7-O-glucuronide, and chlorogenic acid. Among these compounds, latucain C (3) and lactucaside (4) showed significant antidiabetic activity.

Bacopaside III, bacopasaponin G, and bacopasides A, B, and C from Bacopa monniera.

Two new saponins, 3-O-[6-O-sulfonyl-beta-d-glucopyranosyl-(1-->3)]-alpha-l-arabinopyranosyl pseudojujubogenin (1) and 3-O-[alpha-l-arabinofuranosyl-(1-->2)]-alpha-l-arabinopyranosyl jujubogenin (2), a new matsutaka alcohol derivative, (3R)-1-octan-3-yl-(6-O-sulfonyl)-beta-d-glucopyranoside (3), a new phenylethanoid glycoside, 3,4-dihydroxyphenylethyl alcohol (2-O-feruloyl)-beta-d-glucopyranoside (4), and a new glycoside, phenylethyl alcohol [5-O-p-hydroxybenzoyl-beta-d-apiofuranosyl-(1-->2)]-beta-d-glucopyranoside (5), were isolated from Bacopa monniera. Their structures were established by NMR, MS, and chemical methods.

Microbial transformations of isosteviol.

Microbial transformations of the tetracyclic diterpenoid isosteviol (ent-16-ketobeyeran-19-oic acid) (2) have revealed that isosteviol is metabolized by Cunninghamella bainieri, Actinoplanes sp., Mucor recurvatus, and Cunninghamella blakesleeana to yield five new metabolites, ent-11alpha,12alpha-dihydroxy-16-ketobeyeran-19-oic acid (5), ent-11alpha,12alpha,17-trihydroxy-16-ketobeyeran-19-oic acid (6), ent-12alpha,15alpha-dihydroxy-16-ketobeyeran-19-oic acid (7), ent-7alpha,15alpha-dihydroxy-16- ketobeyeran-19-oic acid (8), and ent-9alpha-hydroxy-16-ketobeyeran-19-oic acid (9), together with three known metabolites, ent-7alpha-hydroxy-16-ketobeyeran-19-oic acid (3), ent-7beta-hydroxy-16-ketobeyeran-19-oic acid (4), and ent-12alpha-hydroxy-16-ketobeyeran-19-oic acid (10). The structures of these metabolites were established on the basis of HRFABMS and 1D and 2D NMR spectral data. In addition, metabolites 3-10 were tested for antihypertensive activity and were found to be less active than the parent compound 2.