Anticancer and Apoptotic Activity in Cervical Adenocarcinoma HeLa Using Crude Extract of Ganoderma applanatum.

Cancer is currently one of the foremost health challenges and a leading cause of death worldwide. Cervical cancer is caused by cofactors, including oral contraceptive use, smoking, multiparity, and HIV infection. One of the major and considerable etiologies is the persistent infection of the oncogenic human papilloma virus. G. applanatum is a valuable medicinal mushroom that has been widely used as a folk medicine for the treatment and prevention of various diseases. In this study, we obtained crude extract from G. applanatum mushroom with a subcritical water extraction method; cell viability assay was carried out and the crude extract showed an antiproliferative effect in HeLa cells with IC50 of 1.55 ± 0.01 mg/mL; however, it did not show any sign of toxicity in HaCaT. Protein expression was detected by Western blot, stability of IκBα and downregulation of NFκB, IKKα, IKKß, p-NFκB-65(Ser 536) and p-IKKα/ß(Ser 176/180), suggesting loss of survival in a dose-dependent manner. RT-qPCR revealed RNA/mRNA expression; fold changes of gene expression in Apaf-1, caspase-3, cytochrome-c, caspase-9, Bax and Bak were increased, which implies apoptosis, and NFκB was decreased in a dose-dependent manner. DNA fragmentation was seen in the treatment groups as compared to the control group using gel electrophoresis. Identification and quantification of compounds were carried out by GC-MS and HPLC, respectively; 2(5H)furanone with IC50 of 1.99 ± 0.01 µg/mL could be the responsible anticancer compound. In conclusion, these findings suggest the potential use of the crude extract of G. applanatum as a natural source with anticancer activity against cervical cancer.

In vitro characterization of bioactive compounds extracted from sea urchin (Stomopneustes variolaris) using green and conventional techniques.

This study investigated the in vitro bioactivities of extracts obtained from viscera, spines, shells, and gonads of Stomopneustes variolaris using subcritical water extraction (SWE) at 110 °C, 150 °C, 190 °C, and 230 °C and Soxhlet extraction. The highest amounts of phenolics (22.68 ± 0.05 mg GAE/g), flavonoids (27.11 ± 0.10 mg RE/g), and proteins (40.25 ± 0.84 mg BSA/g) were recorded from gonads at 230 °C, whereas maximum sugar content (23.38 ± 1.30 mg glucose/g) was in viscera at 150 °C. Gonads at 230 °C exhibited the highest DPPH activity (78.68 ± 0.18%), whereas viscera at 150 °C exhibited the highest ABTS+ (98.92 ± 1.27%) and protein denaturation inhibition activity (37.13 ± 9.94%). Viscera at 110 °C claimed the highest amylase inhibition (42.46 ± 0.83%), and spines at 150 °C had the highest anticancer activity (IC50 = 767.47 µg/mL). SWE achieved superior results in bioactive compound recovery and detected higher levels of bioactivities (p < 0.05). Results suggest processing sea urchin extracts via SWE has potential application to the food and pharmaceutical industries.

Green extraction of polyphenolic-polysaccharide conjugates from Pseuderanthemum palatiferum (Nees) Radlk.: Chemical profile and anticoagulant activity.

In this study, pressurized liquid extraction (PLE) of polyphenolic-polysaccharide (PP) from Pseuderanthemum palatiferum (Nees) Radlk. leaves was carried out and compared with a conventional technique using 0.1 M sodium hydroxide. The extracts were purified according to the method reported previously to obtain PP conjugates which were further studied about chemical profiles and anticoagulant activity. Fourier-transform infrared spectroscopy (FTIR), UV-Vis, nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), and spectrophotometry analysis were used to characterize the selected PP conjugates. The results showed that PP conjugates comprised of carbohydrate, phenolic, and protein constituents with the yield ranged from 2.76% to 14.34%. Seven mono sugars containing in all conjugates were determined using high-performance liquid chromatography (HPLC), namely, arabinose, fucose, galactose, glucose, mannose, rhamnose, and xylose. PP conjugates obtained from PLE at 150 °C (PP-PLE5) exhibited better anticoagulant activity than those found at 200 °C and comparable to that of the conventional technique. On gel permeation chromatography, PP-PLE5 showed a broad molecular mass from 6 to 642 kDa. From the obtained results, PLE can be used as a green effective technique for the recovery of PP conjugate from P. palatiferum leaves.

Acer okamotoanum Nakai Leaf Extract Inhibits Adipogenesis Via Suppressing Expression of PPAR γ and C/EBP α in 3T3-L1 Cells.

The genus Acer contains several species with various bioactivities including antioxidant, antitumor and anti-inflammatory properties. However, Acer okamotoanum Nakai, one species within this genus has not been fully studied yet. Therefore, in this study, we investigated the anti-adipogenic activities of leaf extract from A. okamotoanum Nakai (LEAO) on 3T3-L1 preadipocytes. Adipogenesis is one of the cell differentiation processes, which converts preadipocytes into mature adipocytes. Nowadays, inhibition of adipogenesis is considered as an effective strategy in the field of anti-obesity research. In this study, we observed that LEAO decreased the accumulation of lipid droplets during adipogenesis and down-regulated the expression of key adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPAR γ) and CCAAT/enhancer binding protein α (C/EBP α). In addition, LEAO inactivated PI3K/Akt signaling and its downstream factors that promote adipogenesis by inducing the expression of PPAR γ. LEAO also activated ß-catenin signaling, which prevents the adipogenic program by suppressing the expression of PPAR γ. Therefore, we found that treatment with LEAO is effective for attenuating adipogenesis in 3T3-L1 cells. Consequently, these findings suggest that LEAO has the potential to be used as a therapeutic agent for preventing obesity.

Induction of apoptosis and G1 phase cell cycle arrest by Aster incisus in AGS gastric adenocarcinoma cells.

In recent decades, various bioactive compounds from plants have been investigated for their potential use in the treatment of diseases in humans. Aster incisus extract (AIE) is the extract of a common plant that is mostly found in Asia. It has traditionally been used for medicinal purposes in South Korea. In this study, we evaluated the potential anticancer effects of a methanolic extract of Aster incisus in a normal human cell line (HaCaT keratinocytes) and in 4 different types of human cancer cell lines (A549, lung cancer; Hep3B, liver cancer; MDA­MB­231, breast cancer; and AGS, gastric cancer). The HaCaT, A549, Hep3B, MDA­MB­231 and AGS cells were treated with various concentrations of AIE and following treatment, cell survival was evaluated. Additional analyses, such as WST-1 assay, western blot analysis, DAPI staining, flow cytometry, immunofluorescence staining and wound healing assay were performed to elucidate the mechanisms and pathways involved in the cell death induced by AIE. Treatment with AIE induced morphological changes and considerably reduced the viability of the both normal and cancer cell lines. Further analysis of the AGS gastric cancer cells revealed that AIE led to the induction of apoptosis and a high accumulation of cells in the G1 cell phase following treatment with AIE in a dose-dependent manner. The results also revealed that AIE successfully suppressed the migration of the AIE-treated AGS cells. The results of western blot analysis indicated that AIE increased the expression of pro-apoptotic proteins, particularly Bid, Bad, Bak, cytochrome c, apoptosis inducing factor (AIF), cleaved caspase­3, -8 and -9 and cleaved poly(ADP-ribose) polymerase (PARP). Additionally, AIE decreased the expression of the anti-apoptotic proteins, Bcl-2 and Bcl-xL. On the whole, the findings of this study demonstrate that AIE induces apoptosis through the activation of the caspase­dependent pathway mediated by the mitochondrial pathway and by arresting the cell cycle in AGS cells.

Antibacterial potential of silver nanoparticles synthesized using Madhuca longifolia flower extract as a green resource.

The green and one-step synthesis of silver nanoparticles (AgNPs) has been proposed as simple and ecofriendly. In the present study, a flower extract of Madhuca longifolia was used for the reduction of silver nitrate into AgNPs, with phytochemicals from the flower extract as a reducing and stabilizing agents. The synthesized AgNPs were spherical and oval shaped and about 30-50 nm sizes. The appearance of a brown color in the reaction mixture is a primary indication of AgNPs formation, and it was confirmed by observing UV-visible spectroscopy peak at 436 nm. The Energy Dispersive X-ray spectra and X-ray diffraction analysis results together confirm that the synthesized nanoparticles contain silver and silver chloride nanoparticles. The Zeta potential analysis indicates presence of negative charges on synthesized AgNPs. The FT-IR study represents involvement of functional groups in AgNPs synthesis. Synthesized AgNPs shows potential antibacterial activity against Gram-positive and Gram-negative pathogens. M. longifolia flower is a good source for AgNPs synthesis and synthesized AgNPs are applicable as antibacterial agent in therapeutics.

Morphological changes of bacterial cells upon exposure of silver-silver chloride nanoparticles synthesized using Agrimonia pilosa.

Facile, eco-friendly synthesis of metal nanoparticles has been proposed as a cost effective method. In the present study, we propose the facile synthesis of silver-silver chloride (Ag-AgCl) nanoparticles (NPs) using the medicinally important Agrimonia pilosa plant extract without addition of capping or stabilizing agents. The Ag-AgCl NPs synthesis was observed at 40 °C after 10 min incubation; the synthesis of Ag-AgCl NPs was indicated by color change and confirmed by UV-vis spectroscopic peak at 454 nm. TEM analysis confirmed Ag-AgCl NPs were 10-20 nm in size and spherical, and oval in shape. Elemental composition was determined by energy dispersive X-ray analysis, and crystalline structure was confirmed by X-ray diffraction spectroscopy. Different phytocomponents present in the plant extract were analyzed by Gas Chromatography-Mass spectrometry, and the interaction of biomolecules in reduction process was analyzed by Fourier transform infrared spectroscopy studies. The synthesized Ag-AgCl NPs showed significant antibacterial efficiency, analyzed by well diffusion assay against pathogenic bacteria including Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus saprophyticus, Escherichia coli, Pseudomonas putida. Minimum inhibitory concentration and minimum bactericidal concentration were evaluated by microbroth dilution, and spread plate method, respectively. The possible mechanism of bacterial growth inhibition is due to changes in bacterial cell wall morphology that was studied by FE-SEM analysis.

Anticancer activity of Sasa borealis leaf extract-mediated gold nanoparticles.

Advancements in metal nanoparticle synthesis using plant extracts and their anticancer activity have received significant attention in recent years. The green approach for the synthesis of gold nanoparticles (AuNPs) using leaf extract of Sasa borealis is reported in this study. Synthesis of AuNPs was performed at 50 °C, and nanoparticle formation was observed after 20 min incubation. AuNPs formation was confirmed by the UV-visible spectrum peak at 542 nm. The synthesized AuNPs were oval, spherical with sizes around 10-30 nm observed using the transmission electron microscope. Energy dispersive X-ray analysis was utilized for the detection of elemental compound. The face centered cubic structure was confirmed by X-ray diffraction pattern. The reduction of tetrachloroauric acid into AuNPs by the phytochemical compounds of S. borealis extract was determined by Fourier transform infrared spectroscopy and the presence of biomolecules was studied by GC-MS. The synthesized AuNPs was tested for toxic effect on HEK293 cells and anticancer activity on AGS cells by WST-1® assay. Condensation or fragmentation is a characteristics of apoptosis, which was confirmed by 4,6-diamidino-2-pheynylindole dihydrochoride (DAPI) staining. The S. borealis-mediated AuNPs have good activity as an anticancer agent and it will be beneficial in cancer therapeutics.

Anti-Inflammatory Effects of Aster incisus through the Inhibition of NF-κB, MAPK, and Akt Pathways in LPS-Stimulated RAW 264.7 Macrophages.

Aster incisus is a common flower found in almost all regions of South Korea. In the current study, we investigated the potential antioxidant and anti-inflammatory properties of the Aster incisus methanol extract in LPS-stimulated RAW 264.7 cells. We analyzed the phytochemicals contained in the extract by GC-MS. GC-MS results showed that the Aster incisus extract contains 9 known compounds. Later on, DPPH assay, WST-1 assay, nitric oxide (NO) assay, Western blot, and RT-PCR were conducted to investigate the anti-inflammatory effects of the extract. Our WST-1 assay results revealed that Aster incisus did not affect the viability of all tested cell lines up to a concentration of 200 µg/ml; therefore, lower concentrations (50 µg/ml and 150 µg/ml) were used for further assays. Aster incisus scavenged DPPH and inhibited the production of NO. Aster incisus also reduced significantly the production of inflammation-related enzymes (iNOS, Cox-2) and cytokines (TNFα, IL-1ß, and IL-6) and the gene expression of the proinflammatory cytokines. Additionally, further Western blot results indicated that Aster incisus inhibited the expression of p-PI3K, p-IκBα, p-p65 NF-κB, p-ERK1/2, p-SAPK/JNK, and p-Akt. Our results demonstrated that Aster incisus suppressed the expression of the inflammation mediators through the regulation of NF-κB, MAPK, and Akt pathways.

Cyperus amuricus induces G1 arrest and apoptosis through endoplasmic reticulum stress and mitochondrial signaling in human hepatocellular carcinoma Hep3B cells.

ETHNOPHARMACOLOGY RELEVANCE: Cyperus amuricus (C. amuricus), belongs to the family Cyperaceae, was used to exert wound healing, diuretic, astringent and other intestinal problems in oriental medicine. However, only a few studies have reported its anticancer activities. AIM OF THE STUDY: In this study, we determined the activity of C. amuricus on ER stress-induced cell death and G1 cell cycle arrest in human hepatocellular carcinoma (HCC) Hep3B cells. MATERIALS AND METHODS: The in vitro cell proliferation assay of C. amuricus was tested on Hep3B and human embryonic kidney HEK293 cells. Subsequently, the cell cycle distribution in the indicated stages using flow cytometric analysis, the expression of cell cycle-related proteins by western blot analysis and immunofluorescence detection of p21CIP1/WAF1 were determined for the comprehensive identification of cell cycle arrest in Hep3B cells. The effect of C. amuricus on the expression of apoptosis-related proteins in Hep3B cells was also performed by western blot analysis. Furthermore, induction of the ER stress mediators in C. amuricus-treated Hep3B cells were observed by western blot analysis, intracellular Ca2+ mobilization assay and immunofluorescence detection of caspase-12. RESULTS: C. amuricus strongly exhibited cytotoxic activity on Hep3B cells, but not on HEK293 cells. C. amuricus affected the phosphorylation levels of endoplasmic reticulum sensors and increased the expression of GRP78/BiP and CHOP, resulting in the accumulation of unfolded proteins in the ER and triggering the unfolded protein response. These changes occurred by the elevation of intracellular Ca2+ concentrations, which contributed to ER stress-induced apoptosis in C. amuricus-treated Hep3B cells. C. amuricus also coordinated the stimulation of ER chaperones, which initiated G1 cell cycle arrest through the induction of CDKIs and the inhibition of cyclins and CDKs. Furthermore, C. amuricus triggered apoptosis through the activation of mitochondrial-dependent pathway in Hep3B cells. CONCLUSIONS: Our results suggest that C. amuricus is an effective apoptosis inducing agent for Hep3B cells via the G1 arrest, ER stress and mitochondrial mediated apoptotic pathways.

Fucosterol, isolated from Ecklonia stolonifera, inhibits adipogenesis through modulation of FoxO1 pathway in 3T3-L1 adipocytes.

OBJECTIVES: The purpose of this study was to investigate the effects of fucosterol on adipogenesis of 3T3-L1 preadipocytes and its underlying mechanisms. METHODS: Fucosterol, isolated from brown algae, Ecklonia stolonifera. We investigated the levels of lipid accumulation using Oil Red O staining. We conducted Western blot analysis to investigate regulatory effects of fucosterol on expression of phosphoinositide 3-kinase (PI3K), Akt, extracellular signal-regulated kinase (ERK), forkhead box protein O 1 (FoxO1) in 3T3-L1 adipocytes. KEY FINDINGS: Fucosterol significantly reduced intracellular lipid accumulation of 3T3-L1 adipocytes at concentrations of 25 and 50 µm. Fucosterol downregulated insulin-triggered PI3K/Akt, and ERK pathways. It subsequently decreased expression of adipogenic transcription factors, including PPARγ, C/EBPα and SREBP-1. In addition, fucosterol enhanced SirT1 expression while decreased phospho-FoxO1 expression which resulted in the activation of FoxO1. CONCLUSIONS: We revealed that fucosterol inhibited adipogenesis of 3T3-L1 preadipocytes through modulation of FoxO signalling pathway. Therefore, our results suggest that fucosterol may be used for novel agents for the treatment of obesity.

Eco-friendly approach for nanoparticles synthesis and mechanism behind antibacterial activity of silver and anticancer activity of gold nanoparticles.

This review covers general information about the eco-friendly process for the synthesis of silver nanoparticles (AgNP) and gold nanoparticles (AuNP) and focuses on mechanism of the antibacterial activity of AgNPs and the anticancer activity of AuNPs. Biomolecules in the plant extract are involved in reduction of metal ions to nanoparticle in a one-step and eco-friendly synthesis process. Natural plant extracts contain wide range of metabolites including carbohydrates, alkaloids, terpenoids, phenolic compounds, and enzymes. A variety of plant species and plant parts have been successfully extracted and utilized for AgNP and AuNP syntheses. Green-synthesized nanoparticles eliminate the need for a stabilizing and capping agent and show shape and size-dependent biological activities. Here, we describe some of the plant extracts involved in nanoparticle synthesis, characterization methods, and biological applications. Nanoparticles are important in the field of pharmaceuticals for their strong antibacterial and anticancer activity. Considering the importance and uniqueness of this concept, the synthesis, characterization, and application of AgNPs and AuNPs are discussed in this review.

The herbal medicine Cyperus amuricus inhibits proliferation of human hepatocellular carcinoma Hep3B cells by inducing apoptosis and arrest at the G0/G1 cell cycle phase.

Cyperus amuricus (C. amuricus) is one of the most common herbs in Oriental folk medicine for exerting astringent, diuretic, wound healing and other intestinal problems. However, little is known about the molecular mechanism of C. amuricus on anticancer activity. In the present study, the underlying mechanism of the anticancer effect of C. amuricus were elucidated. The methyl alcohol extract from the whole plant of C. amuricus exhibited cytotoxicity against Hep3B cells, but not against A549 and HaCaT cells. Consistent with an acceleration of the sub-G1 phase, downregulation of cdc25A, cyclin D1 and cyclin E, CDK4 and 2 as well as E2F-1, phospho-Rb, with concomitant of upregulation of p21CIP1/WAF1, p27KIPI and p16INK4a proteins, as evidenced by the appearance of cell cycle arrest, were detected in C. amuricus-treated Hep3B cells. Additionally, the sequential activation of various caspases (cleaved caspase-8, -9, -3, -7 and -6, and cleaved PARP) and the changed expression of other proteins related to the apoptosis pathway were observed after C. amuricus exposure. An increment in the pro-apoptotic proteins (Bim, tBid, Bax and Bak) and a reduction of anti-apoptotic protein (Bcl-2) regulate Hep3B cell death by controlling the permeability of mitochondrial membranes and the release of cytochrome c from mitochondria into the cytosol with Apaf-1 after C. amuricus treatment. This is the first study indicating the potential of C. amuricus as a complementary agent for prevention and treatment of human liver cancer.

The Anti-Adipogenic Activity of a New Cultivar, Pleurotus eryngii var. ferulae 'Beesan No. 2', through Down-Regulation of PPAR γ and C/EBP α in 3T3-L1 Cells.

Adipogenesis is one of the cellular processes and a highly controlled program. Nowadays, inhibition of adipogenesis has received attention as an effective way to regulate obesity. In the current study, we investigated the inhibition effect of a chloroform extract of Pleurotus eryngii var. ferulae 'Beesan No. 2' (CEBT) on adipogenesis in 3T3-L1 murine preadipocytes. Pleurotus eryngii var. ferulae is one of many varieties of King oyster mushroom and has been reported to have various biological activities, including antitumor and anti-inflammation effects. Biological activities of 'Beesan No. 2', a new cultivar of Pleurotus eryngii var. ferulae, have not yet been reported. In this study, we found that CEBT suppressed adipogenesis in 3T3-L1 cells through inhibition of key adipogenic transcription factors, such as peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. Additionally, CEBT reduced the expression of the IRS/PI3K/Akt signaling pathway and its downstream factors, including mammalian target of rapamycin and p70S6 kinase, which stimulate adipogenesis. Furthermore, ß-catenin, a suppressor of adipogenesis, was increased in CEBT-treated cells. These results indicate that Pleurotus eryngii var. ferulae 'Beesan No. 2' effectively inhibited adipogenesis, so this mushroom has potential as an anti-obesity food and drug.

The Anti-inflammatory Effect of Gnaphalium affine Through Inhibition of NF-κB and MAPK in Lipopolysaccharide-Stimulated RAW264.7 Cells and Analysis of Its Phytochemical Components.

Gnaphalium affine is an annual herbaceous plant that is used as a traditional medicine in some Latin American and Asian countries. However, systematic studies on its anti-inflammatory activity and signaling pathways have not yet been reported. In this study, we investigated the anti-inflammatory effect of G. affine methanol extract in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cells and fractioned the methanol extract into hexane, chloroform, ethyl acetate (EtOAc), butyl alcohol (BuOH), and distilled water (DW) by measuring the generation of nitric oxide (NO). G. affine inhibited the generation of NO and prostaglandin E2. The chloroform-soluble fraction most effectively inhibited LPS-stimulated NO production. We also examined the cytotoxicity of G. affine in three normal cell lines: RAW264.7, HEK293, and HaCaT. Cell viability assays showed that the methanol extract and chloroform-soluble fraction of G. affine had no cytotoxic effect on normal cell lines. The expression of pro-inflammatory mediators was also investigated. Western blotting and immunofluorescence showed that G. affine reduces the expression of iNOS, COX-2, and MAPKs, as well as activation of NF-κB in LPS-stimulated RAW264.7 cells. RT-PCR showed that G. affine also negatively regulates inflammatory cytokines at the gene expression level. Taken together, G. affine exerts its anti-inflammatory activity through inhibition of NO generation as a result of inhibiting NF-κB and MAPKs-related inflammatory signaling pathways. In addition, the result of GC-MS analysis revealed the presence of nineteen different types of constituents including guaiacol in the chloroform-soluble fraction of G. affine.

Anti-adipogenic effect of epiberberine is mediated by regulation of the Raf/MEK1/2/ERK1/2 and AMPKα/Akt pathways.

It has been reported that alkaloids derived from Coptis chinensis exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating peroxisome proliferation-activity receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α). However, the signaling-based mechanism of the inhibitory role of epiberberine in the early stages of 3T3-L1 adipocyte differentiation is uncharacterized. Here, we show that epiberberine had inhibitory effects on adipocyte differentiation and significantly decreased lipid accumulation by downregulating an adipocyte-specific transcription factor, sterol regulatory element-binding protein-1 (SREBP-1). Furthermore, we observed that epiberberine markedly suppressed the differentiation-mediated phosphorylation of components of both the Raf/mitogen-activated protein kinase 1 (MEK1)/extracellular signal-regulated protein kinase 1/2 (ERK1/2) and AMP-activated protein kinase-α1 (AMPKα)/Akt pathways. In addition, gene expression of fatty acid synthase (FAS) was significantly inhibited by treatment with epiberberine during adipogenesis. These results indicate that the anti-adipogenic mechanism of epiberberine is associated with inhibition of phosphorylation of Raf/MEK1/ERK1/2 and AMPKα/Akt, followed by downregulation of the major transcription factors of adipogenesis, such as PPAR-γ, C/EBP-α, and SREBP-1, and FAS. Taken together, this study suggests that the anti-adipogenic effect of epiberberine is mediated by downregulation of the Raf/MEK1/ERK1/2 and AMPKα/Akt pathways during 3T3-L1 adipocyte differentiation. Moreover, the anti-adipogenic effects of epiberberine were not accompanied by modulation of ß-catenin.

Anti-inflammatory potential of peat moss extracts in lipopolysaccharide-stimulated RAW 264.7 macrophages.

The aim of the present study was to identify the anti-inflammatory and anti-oxidative effects of peat moss aqueous extract (PME) on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. To demonstrate the anti-inflammatory and antioxidant effects of PME, the levels of nitric oxide (NO) and cytokines were measured using Griess reagent and cytokine ELISA kits, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis were conducted to evaluate the expression of genes and proteins. Immunofluorescence was used to measure the expression and translocation of transcription factors. Pre-treatment with PME inhibited the production of prostaglandin E(2) and NO by suppressing the gene expression of cyclooxygenase-2 and inducible NO synthase, respectively. The LPS-stimulated gene expression and the production of tumor necrosis factor-α and interleukin-1ß were significantly reduced by PME. In the LPS-stimulated RAW 264.7 cells, nuclear factor­κB (NF-κB) translocated from the cytosol to the nucleus, while pre-treatment with PME induced the sequestration of NF-κB in the cytosol through the inhibition of IκBα degradation. In the same manner, PME contributed to the inhibition of the activation of mitogen-activated protein kinases. In addition, the PME-treated RAW 264.7 cells facilitated the activation of nuclear factor-like 2 (Nrf2) , and in turn, enhanced heme oxygenase-1 (HO-1) expression. These results indicate that PME exerts anti-inflammatory and antioxidant effects, and suggest that PME may neutralize inflammation and prevent cellular damage by oxidative stress.

Coptis chinensis alkaloids exert anti-adipogenic activity on 3T3-L1 adipocytes by downregulating C/EBP-α and PPAR-γ.

Obesity is a complex, multifactorial, and chronic disease that increases the risk for type 2 diabetes, coronary heart disease and hypertension, and has become a major worldwide health problem. Developing novel anti-obesity drugs from natural products is a promising solution to the global health problem of obesity. While screening anti-obesity potentials of natural products, the methanol extract of the rhizome of Coptis chinensis (Coptidis Rhizoma) was found to significantly inhibit adipocyte differentiation and lipid contents in 3T3-L1 cells, as assessed by Oil-Red O staining. Five known alkaloids, berberine, epiberberine, coptisine, palmatine, and magnoflorine, were isolated from the n-BuOH fraction of the methanol extract of Coptidis Rhizoma. We determined the chemical structure of these alkaloids through comparisons of published nuclear magnetic resonance (NMR) spectral data. Furthermore, we screened these alkaloids for their ability to inhibit adipogenesis over a range of concentrations (12.5-50 µM). All five Coptidis Rhizoma alkaloids significantly inhibited lipid accumulation in 3T3-L1 cells without affecting cell viability in a concentration dependent manner. In addition, the five alkaloids significantly reduced the expression levels of several adipocyte marker genes including proliferator activated receptor-γ (PPAR-γ) and CCAAT/enhancer-binding protein-α (C/EBP-α). In the present study, we found that the isolated alkaloids inhibited adipogenesis in a dose-dependent manner in 3T3-L1 cells; this inhibition was attributed to their abilities to downregulate the protein levels of the adipocyte marker proteins PPAR-γ and C/EBP-α. Thus, these results suggest that Coptidis Rhizoma extract and its isolated alkaloids may be of therapeutic interest with respect to the treatment of obesity.

Extract of Saccharina japonica induces apoptosis companied by cell cycle arrest and endoplasmic reticulum stress in SK-Hep1 human hepatocellular carcinoma cells.

Saccharina japonica is a family member of Phaeophyceae (brown macro-alga) and extensively cultivated in China, Japan and Korea. Here, the potential anti-cancer effect of n-hexane fraction of S. japonica was evaluated in SK-Hep1 human hepatocellular carcinoma cells. The N-hexane fraction reduced cell viability and increased the numbers of apoptotic cells in a both dose- and time-dependent manner. Apoptosis was activated by both caspase-dependent and independent pathways. The caspase-dependent cell death pathway is mediated by cell surface death receptors and activated caspase-8 amplified the apoptotic signal either through direct activation of downstream caspase-3 or pro-apoptotic proteins (Bad, Bax and Bak) subsequently leading to the release of cytochrome c. On the other hand, caspase-independent apoptosis appeared mediated by disruption of mitochondrial membrane potential and translocation of AIF to the nucleus where they induced chromatin condensation and/or large-scale DNA fragmentation. In addition, the n-hexane fraction induced endoplasmic reticulum (ER)-stress and cell cycle arrest. The results suggested that potential anti-cancer effects of n-hexane extract from S. japonica on SK-Hep1 cells.

Actionable gene expression-based patient stratification for molecular targeted therapy in hepatocellular carcinoma.

BACKGROUND: The effectiveness of molecular targeted agents is modest in hepatocellular carcinoma (HCC). Efficacy of molecular targeted therapies has been better in cancer patients with high expression of actionable molecules defined as cognate target molecules. However, patient stratification based on the actionable molecules dictating the effectiveness of targeted drugs has remained understudied in HCC. EXPERIMENTAL DESIGN & RESULTS: Paired tumor and non-tumoral tissues derived from a total of 130 HCC patients were studied. Real-time RT-PCR was used to analyze the mRNA expression of actionable molecules in the tissues. mRNA levels of EGFR, VEGFR2, PDGFRß, FGFR1, and mTOR were up-regulated in tumors compared to non-tumors in 35.4, 42.3, 61.5, 24.6, and 50.0% of patients, respectively. Up-regulation of EGFR was observed at early stage and tended to gradually decrease toward late stages (BCLC stage A: 41.9%; B: 30.8%; C: 17.6%). Frequency of VEGFR2 expression in tumors at stage C was lower than that in the other stages (BCLC stage A: 45.9%; B: 41.0%; C: 29.4%). PDGFRß and mTOR were observed to be up-regulated in more than 50% of tumors in all the stages whereas FGFR1 was up-regulated in only about 20% of HCC irrespective of stages. A cluster analysis of actionable gene expression revealed that HCC can be categorized into different subtypes that predict the effectiveness of molecular targeted agents and combination therapies in clinical trials. Analysis of in vitro sensitivity to sorafenib demonstrated that HCC cells with up-regulation of PDGFRß and c-Raf mRNA are more susceptible to sorafenib treatment in a dose and time-dependent manner than cells with low expression of the genes. CONCLUSIONS: mRNA expression analysis of actionable molecules could provide the rationale for new companion diagnostics-based therapeutic strategies in the treatment of HCC.