14K prolactin derived 14-mer antiangiogenic peptide targets bradykinin-/nitric oxide-cGMP-dependent angiogenesis.

Over the past few decades, VEGF-targeted antiangiogenic therapy for cancers has gained increasing attention. Nevertheless, there are still several limitations such as the potential resistance mechanisms arising in cancer cells against these therapies and their potential adverse effects. These limitations highlight the need for novel anti-angiogenesis molecules and better understanding of the mechanisms of tumor angiogenesis. In the present study, we investigated the antiangiogenic properties of a novel 14-mer antiangiogenic peptide (14-MAP) derived from N-terminal 14 kDa buffalo prolactin and characterized its mode of action. 14-MAP at the picomolar concentration inhibited VEGF- and bradykinin (an autacoid peptide expressed in vascular tissues in pathophysiology, BK)-stimulated endothelial nitric oxide (eNO) production, cell migration, and proliferation in endothelial cells and vessel development in the chick embryo. Although this peptide inhibited both VEGF- and BK-dependent angiogenic processes, its action was more pronounced in the latter. Moreover, the interference of 14-MAP with the eNO synthase (eNOS)-cyclic GMP pathway was also identified. A combination of a low dose of Avastin, a widely used drug targeting VEGF-dependent angiogenesis, and 14-MAP significantly reduced tumor size in an in vivo model of human colon cancer. Taken together, our results suggest that 14-MAP, a BK- and eNOS-dependent antiangiogenic peptide, might be useful for overcoming the limitation of VEGF-targeted antiangiogenic therapy in cancer patients. However, further studies will be required to further characterize its mode of action and therapeutic potential.

Anti­adipogenic effect and underlying mechanism of lignan­enriched nutmeg extract on 3T3­L1 preadipocytes.

Nutmeg is the seed derived from Myristica fragrans. Nutmeg seeds contain alkylbenzene derivatives such as myristicin, which are toxic to the human organism, and lignan compounds such as nectandrin B, which possess anti-aging and anti-diabetic properties. However, the anti-adipogenic, prolipolytic and anti-inflammatory effects of lignan-enriched nutmeg extract (LNX) on preadipocytes remain unclear. In the present study, the effects of LNX on lipid accumulation, glycerol release and inflammatory cyclooxygenase-2 (COX-2) expression in differentiated 3T3-L1 preadipocytes were investigated. Oil red O staining demonstrated that treatment with LNX resulted in a concentration-dependent reduction in lipid accumulation in differentiating 3T3-L1 preadipocytes without affecting cell growth. Mechanistically, LNX treatment at 6 µg/ml led to a reduction in phosphorylation levels of signal transducer and activator of transcription 3 (STAT3), whereas it did not influence the peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT enhancer binding protein alpha (C/EBP-α) expression levels during 3T3-L1 preadipocyte differentiation. In addition, LNX treatment at 6 µg/ml led to a decrease in fatty acid synthase (FAS) expression levels on day (D) 2, but not D5 and D8, during preadipocyte differentiation. Treatment with LNX at 6 µg/ml did not affect the expression levels of perilipin A during preadipocyte differentiation. In differentiated 3T3-L1 adipocytes, LNX treatment at 6 µg/ml did not stimulate glycerol release and hormone-sensitive lipase phosphorylation, which are known lipolysis hallmarks. Furthermore, LNX treatment at the doses tested had no effect on tumor necrosis factor alpha-induced COX-2 expression in 3T3-L1 preadipocytes. Collectively, these results demonstrated that LNX has an anti-adipogenic effect on differentiating 3T3-L1 preadipocytes, which is mediated by the downregulation of STAT3 phosphorylation and FAS expression.

Nectandrin B significantly increases the lifespan of Drosophila - Nectandrin B for longevity.

Phytochemicals are increasingly recognized in the field of healthy aging as potential therapeutics against various aging-related diseases. Nutmeg, derived from the Myristica fragrans tree, is an example. Nutmeg has been extensively studied and proven to possess antioxidant properties that protect against aging and alleviate serious diseases such as cancer, heart disease, and liver disease. However, the specific active ingredient in nutmeg responsible for these health benefits has not been identified thus far. In this study, we present evidence that Nectandrin B (NecB), a bioactive lignan compound isolated from nutmeg, significantly extended the lifespan of the fruit fly Drosophila melanogaster by as much as 42.6% compared to the control group. NecB also improved age-related symptoms including locomotive deterioration, body weight gain, eye degeneration, and neurodegeneration in aging D. melanogaster. This result represents the most substantial improvement in lifespan observed in animal experiments to date, suggesting that NecB may hold promise as a potential therapeutic agent for promoting longevity and addressing age-related degeneration.

Ultrasonic Treatment Enhanced Astaxanthin Production of Haematococcus pluvialis.

In this study, effects of ultrasonic treatment on Haematococcus pluvialis (H. pluvialis) were investigated. It has been confirmed that the ultrasonic stimulation acted as stress resources in the red cyst stage H. pluvialis cells containing astaxanthin, resulting in additional astaxanthin production. With the increase in production of astaxanthin, the average diameter of H. pluvialis cells increased accordingly. In addition, to determine how ultrasonic stimulation had an effect on the further biosynthesis of astaxanthin, genes related to astaxanthin synthesis and cellular ROS level were measured. As a result, it was confirmed that astaxanthin biosynthesis related genes and cellular ROS levels were increased, and thus ultrasonic stimulation acts as an oxidative stimulus. These results support the notion on the effect of the ultrasonic treatment, and we believe our novel approach based on the ultrasonic treatment would help to enhance the astaxanthin production from H. pluvialis.

Ginsenoside Rb2 suppresses cellular senescence of human dermal fibroblasts by inducing autophagy.

Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated ß-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased ß-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-Ⅰ to LC3-Ⅱ and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

Novel electrochemical PMI marker biosensor based on quantum dot dissolution using a double-label strategy.

A novel and facile post-mortem interval (PMI) biosensor was fabricated using a double-label strategy to detect the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) biomarker. A monoclonal anti-GAPDH antibody was immobilized on a surface label containing cadmium selenide quantum dots (CdSe QDs) on a cysteamine graphene oxide (Cys-GO) self-assembled monolayer. Glucose oxidase (GOx) was used as a signal label to conjugate with GAPDH. GAPDH recognition was achieved through the dissolution of the surface-attached CdSe QDs by hydrogen peroxide generated through GAPDH-conjugated GOx-catalyzed ß-glucose oxidation. To enhance sensitivity, a competitive interaction was introduced between free and conjugated GAPDH to the active site of the anti-GAPDH antibody. The electrochemical response due to CdSe dissolution decreased proportionally with the concentration of free GAPDH. Differential pulsed voltammetry was conducted to determine the analytical characteristics of the immunosensor, including the limit of detection, linear dynamic range, target selectivity, system stability, and applicability toward the analysis of real samples.

FBXW7-mediated ERK3 degradation regulates the proliferation of lung cancer cells.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family, members of which play essential roles in diverse cellular processes during carcinogenesis, including cell proliferation, differentiation, migration, and invasion. Unlike other MAPKs, ERK3 is an unstable protein with a short half-life. Although deubiquitination of ERK3 has been suggested to regulate the activity, its ubiquitination has not been described in the literature. Here, we report that FBXW7 (F-box and WD repeat domain-containing 7) acts as a ubiquitination E3 ligase for ERK3. Mammalian two-hybrid assay and immunoprecipitation results demonstrated that ERK3 is a novel binding partner of FBXW7. Furthermore, complex formation between ERK3 and the S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) E3 ligase resulted in the destabilization of ERK3 via a ubiquitination-mediated proteasomal degradation pathway, and FBXW7 depletion restored ERK3 protein levels by inhibiting this ubiquitination. The interaction between ERK3 and FBXW7 was driven by binding between the C34D of ERK3, especially at Thr417 and Thr421, and the WD40 domain of FBXW7. A double mutant of ERK3 (Thr417 and Thr421 to alanine) abrogated FBXW7-mediated ubiquitination. Importantly, ERK3 knockdown inhibited the proliferation of lung cancer cells by regulating the G1/S-phase transition of the cell cycle. These results show that FBXW7-mediated ERK3 destabilization suppresses lung cancer cell proliferation in vitro.

Kaempferol sensitizes cell proliferation inhibition in oxaliplatin-resistant colon cancer cells.

Resolution to chemoresistance is a major challenge in patients with advanced-stage malignancies. Thus, identification of action points and elucidation of molecular mechanisms for chemoresist human cancer are necessary to overcome this challenge. In this study, we provide important evidence that kaempferol targeting RSKs might be a strategy to reduce the oxaliplatin-resistant colon cancer cells. We found that MAPK and PI3K-AKT signaling were increased in oxaliplatin (Ox)-resistant HCT116 (HCT116-OxR) cells compared to Ox-sensitive HCT116 (HCT116-OxS) cells. Comparison of cell sensitivities using SP600125 (JNK inhibitor), SB206580 (p38 kinase inhibitor), or MK-2206 (AKT inhibitor) revealed that cell proliferation inhibition was strongly observed in HT29 cells compared to that in HCT116 cells in both OxS and OxR cells. Interestingly, SP600125, SB206580, and MK-2206 treatment showed higher cell proliferation inhibition in OxS cells than that in OxR cells in both HCT116 and HT29 cells, except following treatments with 10 µM of SP600125, and 30 µM of SB206580. In comparison to magnolin and aschantin, kaempferol showed the strongest inhibitory effect on cell proliferation in both HCT116 and HT29 cells. Importantly, HCT116- and HT29-OxR cells showed higher sensitivities to cell proliferation inhibition than those of HCT116- and HT29-OxS cells, resulting in the accumulation of cells at the G2/M-phases of the cell cycle. Finally, we showed that AP-1 transactivation activity was markedly decreased by kaempferol in HCT116- and HT29-OxR cells compared to the activity levels in HCT116- and HT29-OxS cells. Taken together, the results demonstrate that kaempferol-mediated AP-1 inhibition might be an important signaling mechanism to resolve the chemoresistance of Ox-resistant colon cancer cells.

Anti-growth and pro-apoptotic effects of dasatinib on human oral cancer cells through multi-targeted mechanisms.

Dasatinib is an inhibitor of Src that has anti-tumour effects on many haematological and solid cancers. However, the anti-tumour effects of dasatinib on human oral cancers remain unclear. In this study, we investigated the effects of dasatinib on different types of human oral cancer cells: the non-tumorigenic YD-8 and YD-38 and the tumorigenic YD-10B and HSC-3 cells. Strikingly, dasatinib at 10 µM strongly suppressed the growth and induced apoptosis of YD-38 cells and inhibited the phosphorylation of Src, EGFR, STAT-3, STAT-5, PKB and ERK-1/2. In contrast, knockdown of Src blocked the phosphorylation of EGFR, STAT-5, PKB and ERK-1/2, but not STAT-3, in YD-38 cells. Dasatinib induced activation of the intrinsic caspase pathway, which was inhibited by z-VAD-fmk, a pan-caspase inhibitor. Dasatinib also decreased Mcl-1 expression and S6 phosphorylation while increased GRP78 expression and eIF-2α phosphorylation in YD-38 cells. In addition, to its direct effects on YD-38 cells, dasatinib also exhibited anti-angiogenic properties. Dasatinib-treated YD-38 or HUVEC showed reduced HIF-1α expression and stability. Dasatinib alone or conditioned media from dasatinib-treated YD-38 cells inhibited HUVEC tube formation on Matrigel without affecting HUVEC viability. Importantly, dasatinib's anti-growth, anti-angiogenic and pro-apoptotic effects were additionally seen in tumorigenic HSC-3 cells. Together, these results demonstrate that dasatinib has strong anti-growth, anti-angiogenic and pro-apoptotic effects on human oral cancer cells, which are mediated through the regulation of multiple targets, including Src, EGFR, STAT-3, STAT-5, PKB, ERK-1/2, S6, eIF-2α, GRP78, caspase-9/3, Mcl-1 and HIF-1α.

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.

Stereoisomer-specific ginsenoside 20(S)-Rg3 reverses replicative senescence of human diploid fibroblasts via Akt-mTOR-Sirtuin signaling.

BACKGROUND: The replicative senescence of human dermal fibroblasts (HDFs) is accompanied by growth arrest. In our previous study, the treatment of senescent HDFs with Rg3(S) lowered the intrinsic reactive oxygen species (ROS) levels and reversed cellular senescence by inducing peroxiredoxin-3, an antioxidant enzyme. However, the signaling pathways involved in Rg3(S)-induced senescence reversal in HDFs and the relatedness of the stereoisomer Rg3(R) in corresponding signaling pathways are not known yet. METHODS: We performed senescence-associated ß-galactosidase and cell cycle assays in Rg3(S)-treated senescent HDFs. The levels of ROS, adenosine triphosphate (ATP), and cyclic adenosine monophosphate (cAMP) as well as the mitochondrial DNA copy number, nicotinamide adenine dinucleotide (NAD)+/1,4-dihydronicotinamide adenine dinucleotide (NADH) ratio, and NAD-dependent sirtuins expression were measured and compared among young, old, and Rg3(S)-pretreated old HDFs. Major signaling pathways of phosphatidylinositol 3-kinase/Akt, 5' adenosine monophosphate-activated protein kinase (AMPK), and sirtuin 1/3, including cell cycle regulatory proteins, were examined by immunoblot analysis. RESULTS: Ginsenoside Rg3(S) reversed the replicative senescence of HDFs by restoring the ATP level and NAD+/NADH ratio in downregulated senescent HDFs. Rg3(S) recovered directly the cellular levels of ROS and the NAD+/NADH ratio in young HDFs inactivated by rotenone. Rg3(S) mainly downregulated phosphatidylinositol 3-kinase/Akt through the inhibition of mTOR by cell cycle regulators like p53/p21 in senescent HDFs, whereas Rg3(R) did not alter the corresponding signaling pathways. Rg3(S)-activated sirtuin 3/PGC1α to stimulate mitochondrial biogenesis. CONCLUSION: Cellular molecular analysis suggests that Rg3(S) specifically reverses the replicative senescence of HDFs by modulating Akt-mTOR-sirtuin signaling to promote the biogenesis of mitochondria.

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.

Meridianin C inhibits the growth of YD-10B human tongue cancer cells through macropinocytosis and the down-regulation of Dickkopf-related protein-3.

Meridianin C is a marine natural product known for its anti-cancer activity. At present, the anti-tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD-8, YD-10B, YD-38 and HSC-3. Among the cells tested, meridianin C most strongly reduced the growth of YD-10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD-10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC-dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD-10B cells treated with meridianin C, pointing out that meridianin C-induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf-related protein-3 (DKK-3), a known negative regulator of macropinocytosis. A role for DKK-3 in regulating macropinocytosis in the YD-10B cells was confirmed by siRNA knockdown of endogenous DKK-3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK-3 overexpression, which resulted in a considerable inhibition of the meridianin C-induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti-proliferative effects via macropinocytosis in the highly tumorigenic YD-10B cell line and the effects are mediated in part through down-regulation of DKK-3.

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.

AZD1208, a pan-Pim kinase inhibitor, inhibits adipogenesis and induces lipolysis in 3T3-L1 adipocytes.

The proviral integration moloney murine leukaemia virus (Pim) kinases, consisting of Pim-1, Pim-2 and Pim-3, are involved in the control of cell growth, metabolism and differentiation. Pim kinases are emerging as important mediators of adipocyte differentiation. AZD1208 is a pan-Pim kinase inhibitor and is known for its anti-cancer activity. In this study, we investigated the effect of AZD1208 on adipogenesis and lipolysis in 3T3-L1 cells, a murine preadipocyte cell line. AZD1208 markedly suppressed lipid accumulation and reduced triglyceride contents in differentiating 3T3-L1 cells, suggesting the drug's anti-adipogenic effect. On mechanistic levels, AZD1208 reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and perilipin A but also the phosphorylation of signal transducer and activator of transcription-3 (STAT-3) in differentiating 3T3-L1 cells. Remarkably, AZD1208 increased cAMP-activated protein kinase (AMPK) and LKB-1 phosphorylation while decreased intracellular ATP contents in differentiating 3T3-L1 cells. Furthermore, in differentiated 3T3-L1 adipocytes, AZD1208 also partially promoted lipolysis and enhanced the phosphorylation of hormone-sensitive lipase (HSL), a key lipolytic enzyme, indicating the drug's HSL-dependent lipolysis. In summary, the findings show that AZD1208 has anti-adipogenic and lipolytic effects on 3T3-L1 adipocytes. These effects are mediated by the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, ACC, perilipin A, STAT-3, AMPK and HSL.

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.

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.

Degradation of Kidney and Psoas Muscle Proteins as Indicators of Post-Mortem Interval in a Rat Model, with Use of Lateral Flow Technology.

We investigated potential protein markers of post-mortem interval (PMI) using rat kidney and psoas muscle. Tissue samples were taken at 12 h intervals for up to 96 h after death by suffocation. Expression levels of eight soluble proteins were analyzed by Western blotting. Degradation patterns of selected proteins were clearly divided into three groups: short-term, mid-term, and long-term PMI markers based on the half maximum intensity of intact protein expression. In kidney, glycogen synthase (GS) and glycogen synthase kinase-3ß were degraded completely within 48 h making them short-term PMI markers. AMP-activated protein kinase α, caspase 3 and GS were short-term PMI markers in psoas muscle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a mid-term PMI marker in both tissues. Expression levels of the typical long-term PMI markers, p53 and ß-catenin, were constant for at least 96 h post-mortem in both tissues. The degradation patterns of GS and caspase-3 were verified by immunohistochemistry in both tissues. GAPDH was chosen as a test PMI protein to perform a lateral flow assay (LFA). The presence of recombinant GAPDH was clearly detected in LFA and quantified in a concentration-dependent manner. These results suggest that LFA might be used to estimate PMI at a crime scene.