Chelidonine Induces Apoptosis via GADD45a-p53 Regulation in Human Pancreatic Cancer Cells.

Chelidonium majus has been used as a traditional medicine in China and western countries for various diseases, including inflammation and cancer. However, the anti-cancer effect of chelidonine, a major compound of C. majus extracts, on pancreatic cancer remains poorly understood. In this study, we found that treatment with chelidonine inhibited proliferation of BxPC-3 and MIA PaCa-2 human pancreatic cancer cells. Annexin-V/propidium iodide staining assay showed that this growth inhibitory effect of chelidonine was induced through apoptosis. We found that chelidonine treatment upregulated mRNA levels and transcription factor activity in both cell lines. Increases in protein expression levels of p53, GADD45A, p21 and cleaved caspase-3 were also observed, with more distinct changes in MIA PaCa-2 cells compared to the BxPC-3 cells. These results suggest that chelidonine induces pancreatic cancer apoptosis through the p53 and GADD45A pathways. Our findings provide new insights into the use of chelidonine for the treatment of pancreatic cancer.

Nectandrin B-mediated activation of the AMPK pathway prevents cellular senescence in human diploid fibroblasts by reducing intracellular ROS levels.

Nectandrin B (NecB) is a bioactive lignan compound isolated from Myristica fragrans (nutmeg), which functions as an activator of AMP-activated protein kinase (AMPK). Because we recently found that treatment with NecB increased the cell viability of old human diploid fibroblasts (HDFs), the underlying molecular mechanism was investigated. NecB treatment in old HDFs reduced the activity staining of senescence-associated ß-galactosidase and the levels of senescence markers, such as the Ser15 phosphorylated p53, caveolin-1, p21waf1, p16ink4a, p27kip1, and cyclin D1. NecB treatment increased that in S phase, indicating a enhancement of cell cycle entry. Interestingly, NecB treatment ameliorated age-dependent activation of AMPK in old HDFs. Moreover, NecB reversed the age-dependent expression and/or activity changes of certain sirtuins (SIRT1-5), and cell survival/death-related proteins. The transcriptional activity of Yin-Yang 1 and the expression of downstream proteins were elevated in NecB-treated old HDFs. In addition, NecB treatment exerted a radical scavenging effect in vitro, reduced cellular ROS levels, and increased antioxidant enzymes in old HDFs. Moreover, NecB-mediated activation of the AMPK pathway reduced intracellular ROS levels. These results suggest that NecB-induced protection against cellular senescence is mediated by ROS-scavenging through activation of AMPK. NecB might be useful in ameliorating age-related diseases and extending human lifespan.

Adsorptive removal of harmful algal species Microcystis aeruginosa directly from aqueous solution using polyethylenimine coated polysulfone-biomass composite fiber.

In recent times, the treatment of harmful algal blooms (HABs) became an important environmental issue to preserve and remediate water resources globally. In the present study, the adsorptive removal of harmful algal species Microcystis aeruginosa directly from an aqueous medium was attempted. Waste biomass (Escherichia coli) was immobilized using polysulfone and coated using the cationic polymer polyethylenimine (PEI) to generate PEI-coated polysulfone-biomass composite fiber (PEI-PSBF). The density of M. aeruginosa in an aqueous medium (BG11) was significantly decreased by treatment with PEI-PSBF. additionally, analysis using FE-SEM, confirmed that the removal of M. aeruginosa algal cells by PEI-PSBF was caused by the adsorption mechanism. According to the profiles of phosphorus for the algal cell growth in M. aeruginosa cultivating samples, we found that the adsorbed M. aeruginosa onto the PEI-PSBF lost their biological activity compared to the non-treated M. aeruginosa cells.

Restoring Effects of Natural Anti-Oxidant Quercetin on Cellular Senescent Human Dermal Fibroblasts.

The oxidative damage initiated by reactive oxygen species (ROS) is a major contributor to the functional decline and disability that characterizes aging. The anti-oxidant flavonoid, quercetin, is a plant polyphenol that may be beneficial for retarding the aging process. We examined the restoring properties of quercetin on human dermal fibroblasts (HDFs). Quercetin directly reduced either intracellular or extracellular ROS levels in aged HDFs. To find the aging-related target genes by quercetin, microarray analysis was performed and two up-regulated genes LPL and KCNE2 were identified. Silencing LPL increased the expression levels of senescence proteins such as p16INK4A and p53 and silencing KCNE2 reversed gene expressions of EGR1 and p-ERK in quercetin-treated aged HDFs. Silencing of LPL and KCNE2 decreased the expression levels of anti-oxidant enzymes such as superoxide dismutase and catalase. Also, the mitochondrial dysfunction in aged HDFs was ameliorated by quercetin treatment. Taken together, these results suggest that quercetin has restoring effect on the cellular senescence by down-regulation of senescence activities and up-regulation of the gene expressions of anti-oxidant enzymes in aged HDFs.

Allergen-Removed Rhus verniciflua Extract Induces Ovarian Cancer Cell Death via JNK Activation.

Nuclear factor-[Formula: see text]B (NF-[Formula: see text]B)/Rel transcription factors are best known for their central roles in promoting cell survival in cancer. NF-[Formula: see text]B antagonizes tumor necrosis factor (TNF)-[Formula: see text]-induced apoptosis through a process involving attenuation of the c-Jun-N-terminal kinase (JNK). However, the role of JNK activation in apoptosis induced by negative regulation of NF-[Formula: see text]B is not completely understood. We found that allergen-removed Rhus verniciflua Stokes (aRVS) extract-mediated NF-[Formula: see text]B inhibition induces apoptosis in SKOV-3 ovarian cancer cells via the serial activation of caspases and SKOV-3 cells are most specifically suppressed by aRVS. Here, we show that in addition to activating caspases, aRVS extract negatively modulates the TNF-[Formula: see text]-mediated I[Formula: see text]B/NF-[Formula: see text]B pathway to promote JNK activation, which results in apoptosis. When the cytokine TNF-[Formula: see text] binds to the TNF receptor, I[Formula: see text]B dissociates from NF-[Formula: see text]B. As a result, the active NF-[Formula: see text]B translocates to the nucleus. aRVS extract (0.5[Formula: see text]mg/ml) clearly prevented NF-[Formula: see text]B from mobilizing to the nucleus, resulting in the upregulation of JNK phosphorylation. This subsequently increased Bax activation, leading to marked aRVS-induced apoptosis, whereas the JNK inhibitor SP600125 in aRVS extract treated SKOV-3 cells strongly inhibited Bax. Bax subfamily proteins induced apoptosis through caspase-3. Thus, these results indicate that aRVS extract contains components that inhibit NF-[Formula: see text]B signaling to upregulate JNK activation in ovarian cancer cells and support the potential of aRVS as a therapeutic agent for ovarian cancer.

Growth inhibitory and apoptosis-inducing effects of allergen-free Rhus verniciflua Stokes extract on A549 human lung cancer cells.

Evidence suggests that Rhus verniciflua Stokes (RVS) or its extract has the potential to be used for the treatment of inflammatory and neoplastic diseases. However, direct use of RVS or its extract as a herbal medicine has been limited due to the presence of urushiol, an allergenic toxin. In the present study, we prepared an extract of the allergen­removed RVS (aRVS) based on a traditional method and investigated its inhibitory effect on the growth of various types of human cancer cells, including lung (A549), breast (MCF-7) and prostate (DU-145) cancer cell lines. Notably, among the cell lines tested, treatment with the aRVS extract strongly inhibited proliferation of the A549 cells at a 0.5 mg/ml concentration for 24 h that was not cytotoxic to normal human dermal fibroblasts. Furthermore, aRVS extract treatment largely reduced the survival and induced apoptosis of the A549 cells. At the mechanistic levels, treatment with the aRVS extract led to the downregulation of Bcl-2 and Mcl-1 proteins, the activation of caspase-9/-3 proteins, an increase in cytosolic cytochrome c levels, the upregulation of Bax protein, an increase in phosphorylated p53 protein but a decrease in phosphorylated S6 protein in the A549 cells. Importantly, treatment with z-VAD­fmk, a pan-caspase inhibitor attenuated aRVS extract-induced apoptosis in the A549 cells. These results demonstrate firstly that aRVS extract has growth inhibitory and apoptosis-inducing effects on A549 human lung cancer cells through modulation of the expression levels and/or activities of caspases, Bcl-2, Mcl-1, Bax, p53 and S6.

Effect of rutin from tartary buckwheat sprout on serum glucose-lowering in animal model of type 2 diabetes.

This study investigates the anti-diabetic effects of rutin from tartary buckwheat sprout in type 2 diabetes mouse model. The rutin content in tartary buckwheat sprout (TBS) is five times higher than that found in common buckwheat sprout (CBS) as evident from high-performance liquid chromatography analysis. Administration of either rutin or TBS ethanolic extract to diabetes mice decreased the serum glucose level significantly. Rutin down-regulated the expression levels of protein-tyrosine phosphatase 1B; it is negative regulator of insulin pathway, both transcriptionally and translationally in myocyte C2C12 in a dose-dependent manner. In conclusion, rutin can play a critical role in down-regulation of serum glucose level in type 2 diabetes.

Anti-proliferative effects of ginsenosides extracted from mountain ginseng on lung cancer.

OBJECTIVE: To investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer. METHODS: The inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis. RESULTS: The abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed. CONCLUSION: Major ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.

Identification of regulators of the early stage of viral hemorrhagic septicemia virus infection during curcumin treatment.

The effect of curcumin pretreatment (15-240 µM) in fathead minnow cells infected with viral hemorrhagic septicemia virus (VHSV) was evaluated. Cell viability, apoptosis and viral copy number were analyzed using Cell Counting Kit-8 assay, Annexin V staining, and reverse transcription-PCR, respectively. Pretreatment with 120 µM curcumin showed an increase in viability (>90% of mock) of VHSV-infected cells and reduction in the copy number (0.2-log reduction in VHSV N gene expression), reactive oxygen species and apoptosis in the cells without cytotoxic effects. To understand the mechanisms underlaying the antiviral effects of curcumin pretreatment, a comparative proteomic analysis was performed in four samples (M, mock; C, curcumin-treated; V, VHSV-infected; and CV, curcumin-treated VHSV-infected) in triplicate. In total, 185 proteins were detected. The analysis showed that three proteins, including heat shock cognate 71 (HSC71), actin, alpha cardiac muscle (ACTC1) and elongation factor 1 (EEF1) were differentially expressed between V and CV samples. Network analysis performed by Ingenuity Pathways Analysis (IPA) showed that HSC71 was the primary protein interacting with fibronectin (FN) 1, actins (ACTB, ACTG, F-actin) and gelsolin (GSN) in both V and CV samples and thus is a strong target candidate for the protection from VHSV infection at the viral entry stage. Our proteomics data suggest that curcumin pretreatment inhibits entry of VHSV in cells by downregulating FN1 or upregulating F-actin. For both proteins, HSC71 acts as a binding protein that modulates their functions. Furthermore, consistent with the effect of a heat shock protein inhibitor (KNK437), curcumin downregulated HSC71 expression with increasing viability of VHSV-infected cells and inhibited VHSV replication, suggesting that the downregulation of HSC71 could be responsible for the antiviral activity of curcumin. In conclusion, this study indicates that the suppression of viral entry by rearrangement of the F-actin/G-actin ratio via downregulating HSC71 is a plausible mechanism by which curcumin pretreatment controls the early stages of VHSV infection.

An enzymatically fortified ginseng extract inhibits proliferation and induces apoptosis of KATO3 human gastric cancer cells via modulation of Bax, mTOR, PKB and IκBα.

Accumulative evidence suggests ginseng extract and/or its major components, ginsenosides and compound K, a metabolized ginseng saponin, have anti-cancer effects. In the present study, the effects of a ginseng butanolic extract (GBX) and an enzymatically fortified ginseng extract (FGX), with enriched ginsenosides and compound K, on the growth of KATO3 human gastric cancer cells were investigated using a cell viability assay. While treatment with GBX at 31.25-125 mg/ml for 24 h did not affect the proliferation of KATO3 cells, FGX under the same conditions inhibited cell proliferation in a concentration-dependent manner. Furthermore, Annexin V/PI-staining and flow cytometric analysis demonstrated that the population of apoptotic KATO3 cells was increased following treatment with FGX, which was greater than in the GBX-treated cells, suggesting that FGX had a stronger apoptotic effect than GBX. To investigate the underlying mechanism of the cytostatic and cytotoxic effects of the ginseng extracts, apoptosis-associated proteins were assessed using western blot analysis. The data revealed higher expression levels of B-cell lymphoma 2-associated X protein (Bax), lower expression of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) and reduced phosphorylation of mammalian target of rapamycin (mTOR) and protein kinase B (PKB) in the FGX-treated KATO3 cells than in the GBX-treated cells. Collectively, these results demonstrated for the first time, to the best of our knowledge, that FGX had stronger anti-proliferative and pro-apoptotic effects on KATO3 cells than GBX. The anti-proliferative and/or pro-apoptotic effects of FGX appeared to be mediated via the upregulation of Bax, IκBα proteolysis (activation of nuclear factor-κB) and the blocking of mTOR and PKB signals.

Identification of KMU-3, a novel derivative of gallic acid, as an inhibitor of adipogenesis.

Differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. Excessive adipogenesis, however, is largely linked to the development of obesity. Herein we investigated a library of 53 novel chemicals, generated from a number of polyphenolic natural compounds, on adipogenesis. Strikingly, among the chemicals tested, KMU-3, a derivative of gallic acid, strongly suppressed lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. On mechanistic levels, KMU-3 inhibited expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) during adipocyte differentiation. Moreover, KMU-3 reduced expressions of adipokines, including retinol binding protein-4 (RBP-4), leptin, and regulated on activation, normal T cell expressed and secreted (RANTES) during adipocyte differentiation. Of further note, KMU-3 rapidly blocked the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) during the early stage of adipogenesis. Importantly, pharmacological inhibition studies revealed that AG490, a JAK-2/STAT-3 inhibitor suppressed adipogenesis and STAT-3 phosphorylation, implying that early blockage of STAT-3 activity is crucial for the KMU-3-mediated anti-adipogenesis. These findings demonstrate firstly that KMU-3 inhibits adipogenesis by down-regulating STAT-3, PPAR-γ, C/EBP-α, and FAS. This work shows that KMU-3 is an inhibitor of adipogenesis and thus may have therapeutic potential against obesity.

Lactobacillus casei extract induces apoptosis in gastric cancer by inhibiting NF-κB and mTOR-mediated signaling.

Lactobacillus casei extract (LBX) has been reported to prevent gastric cancer, but the underlying mechanism remains unclear. The proliferation and cell death of gastric cancer KATO3 cells were examined after treatment with LBX for various times and at various doses. LBX inhibited the growth of gastric cancer cells and induced apoptosis by inactivating NF-κB promoter activity. Apoptosis induced by LBX, however, is not directly associated with the intrinsic mitochondrial pathway. Immunoblot analysis revealed that LBX decreased the expressions of NF-κB and IκB. The reduced NF-κB levels led to the decreased phosphorylation of mTOR signaling components, such as PI3K, Akt, and (p70)S6 kinase. These results showed for the first time that LBX induced apoptosis in gastric cancer cells by inhibiting NF-κB and mTOR-mediated signaling.

Dynamic proteomic profile of potato tuber during its in vitro development.

Potato tuberization is a complicated biochemical process, which is dependent on external environmental factors. Tuber development in potato consists of a series of biochemical and morphological processes at the stolon tip. Signal transduction proteins are involved in the source-sink transition during potato tuberization. In the present study, we examined protein profiles under in vitro tuber-inducing conditions using a shotgun proteomic approach involving denaturing gel electrophoresis and liquid chromatography-mass spectrometry. A total of 251 proteins were identified and classified into 9 groups according to distinctive expression patterns during the tuberization stage. Stolon stage-specific proteins were primarily involved in the photosynthetic machinery. Proteins specific to the initial tuber stage included patatin. Proteins specific to the developing tuber stage included 6-fructokinase, phytoalexin-deficient 4-1, metallothionein II-like protein, and malate dehydrogenase. Novel stage-specific proteins identified during in vitro tuberization were ferredoxin-NADP reductase, 34 kDa porin, aquaporin, calmodulin, ripening-regulated protein, and starch synthase. Superoxide dismutase, dehydroascorbate reductase, and catalase I were most abundantly expressed in the stolon; however, the enzyme activities of these proteins were most activated at the initial tuber. The present shotgun proteomic study provides insights into the proteins that show altered expression during in vitro potato tuberization.

Mountain ginseng extract exhibits anti-lung cancer activity by inhibiting the nuclear translocation of NF-κB.

Administration of mountain ginseng (MG) extract can restore advanced cancer to a normal state. To elucidate the mechanism by which MG extract prevents the progression of lung cancer, the processes of proliferation and death of lung cancer cells (A549) were examined after treatment with MG extract. Butanol-extracted MG (BX-MG) showed a high inhibitory effect (IC(50) = 2 mg/ml) by attenuating proliferation and inducing apoptosis in lung cancer cells. By HPLC-UV analysis of BX-MG, ginsenosides, Rb1 was identified as the most abundant ginsenoside, followed by Rg1, Re, Rc and Rb2. BX-MG induced caspase-3 dependent apoptosis by inhibiting NF-κB. In addition, BX-MG activated p53 and p21, resulting in the attenuated proliferation of A549 cells. Reduced activity of the NF-κB promoter and increased activity of the p53 promoter indicate that BX-MG regulates apoptosis at the level of transcription in lung cancer cells. Furthermore, BX-MG blocked the nuclear translocation of RelA and the associated reduction in surviving. These results suggest that BX-MG inhibits lung cancer cell growth by activating tumor suppressors and inhibiting nuclear translocation of NF-κB.

Panax ginseng improves survival and sperm quality in guinea pigs exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin.

OBJECTIVES: To further assess the effect of Panax ginseng on survival and sperm quality of guinea pigs exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). MATERIALS AND METHODS: Eighty male guinea pigs were divided into eight equal groups. The normal control (NC) group received vehicle and saline; one dose of 1 micro g/kg body weight TCDD was injected intraperitoneally into the single TCDD-treated (TT) and test groups (P100, P200, C100, C200); G and NC groups received vehicle instead of TCDD. P. ginseng water extract (PG-WE) was injected intraperitoneally at daily doses of 100 (G100, P100, C100) or 200 mg/kg body weight (G200, P200, C200). The PG-WE was administered to the P and G groups for 12 weeks from 1 week before TCDD exposure, and to the C groups for 10 weeks from 1 week after TCDD exposure. After a 4-week discontinuation of PG-WE treatment after the 13th week the surviving males were then tested for fertility by mating them with females. The litter size, death rate, male/female birth ratio and physical abnormalities of the progeny were investigated. After confirming delivery of the offspring, the parent males were killed at 40 weeks, their testes weighed and sperm quality assessed. RESULTS: All TT animals died within 18 days after TCDD exposure, but 40-70% of the PG-WE-treated groups, depending on the group, survived until death at 40 weeks. All the surviving males were fertile regardless of TCDD exposure; there was no difference in litter size between the NC and test groups. Notably the death rate of progeny born to PG-WE-treated groups was lower than that of progeny born to the NC group. The progeny born to TCDD-exposed groups (P200 and C groups) had a preponderance of females. G Group animals had higher sperm quality than that of NCs even long after discontinuing PG-WE. CONCLUSION: P. ginseng improves the survival rate and sperm quality in guinea pigs exposed to TCDD.