Necroptosis inhibits autophagy by regulating the formation of RIP3/p62/Keap1 complex in shikonin-induced ROS dependent cell death of human bladder cancer.

BACKGROUND: Shikonin, a natural naphthoquinone compound, has a wide range of pharmacological effects, but its anti-tumor effect and underlying mechanisms in bladder cancer remain unclear. PURPOSE: We aimed to investigate the role of shikonin in bladder cancer in vitro and in vivo in order to broaden the scope of shikonin's clinical application. STUDY DESIGN AND METHODS: We performed MTT and colony formation to detect the inhibiting effect of shikonin on bladder cancer cells. ROS staining and flow cytometry assays were performed to detect the accumulation of ROS. Western blotting, siRNA and immunoprecipitation were used to evaluate the effect of necroptosis in bladder cancer cells. Transmission electron microscopy and immunofluorescence were used to examine the effect of autophagy. Nucleoplasmic separation and other pharmacological experimental methods described were used to explore the Nrf2 signal pathway and the crosstalk with necroptosis and autophagy. We established a subcutaneously implanted tumor model and performed immunohistochemistry assays to study the effects and the underlying mechanisms of shikonin on bladder cancer cells in vivo. RESULTS: The results showed that shikonin has a selective inhibitory effect on bladder cancer cells and has no toxicity on normal bladder epithelial cells. Mechanically, shikonin induced necroptosis and impaired autophagic flux via ROS generation. The accumulation of autophagic biomarker p62 elevated p62/Keap1 complex and activated the Nrf2 signaling pathway to fight against ROS. Furthermore, crosstalk between necroptosis and autophagy was present, we found that RIP3 may be involved in autophagosomes and be degraded by autolysosomes. We found for the first time that shikonin-induced activation of RIP3 may disturb the autophagic flux, and inhibiting RIP3 and necroptosis could accelerate the conversion of autophagosome to autolysosome and further activate autophagy. Therefore, on the basis of RIP3/p62/Keap1 complex regulatory system, we further combined shikonin with late autophagy inhibitor(chloroquine) to treat bladder cancer and achieved a better inhibitory effect. CONCLUSION: In conclusion, shikonin could induce necroptosis and impaired autophagic flux through RIP3/p62/Keap1 complex regulatory system, necroptosis could inhibit the process of autophagy via RIP3. Combining shikonin with late autophagy inhibitor could further activate necroptosis via disturbing RIP3 degradation in bladder cancer in vitro and in vivo.

Anthocyanin-enriched polyphenols from Hibiscus syriacus L. (Malvaceae) exert anti-osteoporosis effects by inhibiting GSK-3ß and subsequently activating ß-catenin.

BACKGROUND: The bark and petal of Hibiscus syriacus L. (Malvaceae) have been used to relieve pain in traditional Korean medicine. Recently, we identified anthocyanin-enriched polyphenols from the petal of H. syriacus L. (AHs) and determined its anti-melanogenic, anti-inflammatory, and anti-oxidative properties. Nevertheless, the osteogenic potential of AHs remains unknown. PURPOSE: This study was aimed to investigating the effect of AHs on osteoblast differentiation and osteogenesis in osteoblastic cell lines and zebrafish larvae. Furthermore, we investigated whether AHs ameliorates prednisolone (PDS)-induced osteoporosis. STUDY DESIGN AND METHODS: Cell viability was assessed by cellular morphology, MTT assay, and flow cytometry analysis, and osteoblast differentiation was measured alizarin red staining, alkaline phosphatase (ALP) activity, and osteoblast-specific marker expression. Osteogenic and anti-osteoporotic effects of AHs were determined in zebrafish larvae. RESULTS: AHs enhanced calcification and ALP activity concomitant with the increased expression of osterix (OSX), runt-related transcription factor 2 (RUNX2), and ALP in MC3T3-E1 preosteoblast and MG-63 osteosarcoma cells. Additionally, AHs accelerated vertebral formation and mineralization in zebrafish larvae, concurrent with the increased expression of OSX, RUNX2a, and ALP. Furthermore, PDS-induced loss of osteogenic activity and vertebral formation were restored by treatment with AHs, accompanied by a significant recovery of calcification, ALP activity, and osteogenic marker expression. Molecular docking studies showed that 16 components in AHs fit to glucagon synthase kinase-3ß (GSK-3ß); particularly, isovitexin-4'-O-glucoside most strongly binds to the peptide backbone of GSK-3ß at GLY47(O), GLY47(N), and ASN361(O), with a binding score of -7.3. Subsequently, AHs phosphorylated GSK-3ß at SER9 (an inactive form) and released ß-catenin into the nucleus. Pretreatment with FH535, a Wnt/ß-catenin inhibitor, significantly inhibited AH-induced vertebral formation in zebrafish larvae. CONCLUSION: AHs stimulate osteogenic activities through the inhibition of GSK-3ß and subsequent activation of ß-catenin, leading to anti-osteoporosis effects.

Inhibition of Lipopolysaccharide-Induced Inflammatory and Oxidative Responses by Trans-cinnamaldehyde in C2C12 Myoblasts.

Background: Trans-cinnamaldehyde (tCA), a bioactive component found in Cinnamomum cassia, has been reported to exhibit anti-inflammatory and antioxidant effects, but its efficacy in muscle cells has yet to be found. In this study, we investigated the inhibitory effect of tCA on inflammatory and oxidative stress induced by lipopolysaccharide (LPS) in C2C12 mouse skeletal myoblasts. Methods: To investigate the anti-inflammatory and antioxidant effects of tCA in LPS-treated C2C12 cells, we measured the levels of pro-inflammatory mediator, cytokines, and reactive oxygen species (ROS). To elucidate the mechanism underlying the effect of tCA, the expression of genes involved in the expression of inflammatory and oxidative regulators was also investigated. We further evaluated the anti-inflammatory and antioxidant efficacy of tCA against LPS in the zebrafish model. Results: tCA significantly inhibited the LPS-induced release of pro-inflammatory mediators and cytokines, which was associated with decreased expression of their regulatory genes. tCA also suppressed the expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor, and attenuated the nuclear translocation of nuclear factor-kappa B (NF-κB) and the binding of LPS to TLR4 on the cell surface in LPS-treated C2C12 cells. Furthermore, tCA abolished LPS-induced generation of ROS and expression levels of ROS producing enzymes, NADPH oxidase 1 (NOX1) and NOX2. However, tCA enhanced the activation of nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) in LPS-stimulated C2C12 myoblasts. In addition, tCA showed strong protective effects against NO and ROS production in LPS-injected zebrafish larvae. Conclusions: Our findings suggest that tCA exerts its inhibitory ability against LPS-induced inflammatory and antioxidant stress in C2C12 myoblasts by targeting the TLR4/NF-κB, which might be mediated by the NOXs and Nrf2/HO-1 pathways.

Yeast Synthetic Biology for the Production of Lycium barbarum Polysaccharides.

The fruit of Lycium barbarum L. (goji berry) is used as traditional Chinese medicine, and has the functions of immune regulation, anti-tumor, neuroprotection, anti-diabetes, and anti-fatigue. One of the main bioactive components is L. barbarum polysaccharide (LBP). Nowadays, LBP is widely used in the health market, and it is extracted from the fruit of L. barbarum. The planting of L. barbarum needs large amounts of fields, and it takes one year to harvest the goji berry. The efficiency of natural LBP production is low, and the LBP quality is not the same at different places. Goji berry-derived LBP cannot satisfy the growing market demands. Engineered Saccharomyces cerevisiae has been used for the biosynthesis of some plant natural products. Recovery of LBP biosynthetic pathway in L. barbarum and expression of them in engineered S. cerevisiae might lead to the yeast LBP production. However, information on LBP biosynthetic pathways and the related key enzymes of L. barbarum is still limited. In this review, we summarized current studies about LBP biosynthetic pathway and proposed the strategies to recover key enzymes for LBP biosynthesis. Moreover, the potential application of synthetic biology strategies to produce LBP using engineered S. cerevisiae was discussed.

Metabolic Engineering for Glycyrrhetinic Acid Production in Saccharomyces cerevisiae.

Glycyrrhetinic acid (GA) is one of the main bioactive components of licorice, and it is widely used in traditional Chinese medicine due to its hepatoprotective, immunomodulatory, anti-inflammatory and anti-viral functions. Currently, GA is mainly extracted from the roots of cultivated licorice. However, licorice only contains low amounts of GA, and the amount of licorice that can be planted is limited. GA supplies are therefore limited and cannot meet the demands of growing markets. GA has a complex chemical structure, and its chemical synthesis is difficult, therefore, new strategies to produce large amounts of GA are needed. The development of metabolic engineering and emerging synthetic biology provide the opportunity to produce GA using microbial cell factories. In this review, current advances in the metabolic engineering of Saccharomyces cerevisiae for GA biosynthesis and various metabolic engineering strategies that can improve GA production are summarized. Furthermore, the advances and challenges of yeast GA production are also discussed. In summary, GA biosynthesis using metabolically engineered S. cerevisiae serves as one possible strategy for sustainable GA supply and reasonable use of traditional Chinese medical plants.

Anthocyanins isolated from Hibiscus syriacus L. attenuate lipopolysaccharide-induced inflammation and endotoxic shock by inhibiting the TLR4/MD2-mediated NF-κB signaling pathway.

BACKGROUND: Hibiscus syriacus L. has been used as a medicinal plant in many Asian countries. However, anti-inflammatory activity of H. syriacus L. remains unknown. PURPOSE: This study was aimed to investigating the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. variety Pulsae (PS) on the lipopolysaccharide (LPS)-induced inflammation and endotoxic shock. STUDY DESIGN AND METHODS: MTT assay and flow cytometry analysis were performed to determine cytotoxicity of PS. RT-PCR, western blotting, and ELISA were conducted to evaluate the expression of proinflammatory mediators and cytokines. Molecular docking study predicted the binding scores and sites of PS to TLR4/MD2 complex. Immunohistochemical assay was conducted to evaluate the binding capability of PS to TLR4/MD2 and nuclear translocation of NF-κB p65. A zebrafish endotoxic shock model was used to evaluate anti-inflammatory activity of PS in vivo. RESULTS: PS suppressed LPS-induced nitric oxide and prostaglandin E2 secretion concomitant with the downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in LPS-stimulated RAW 264.7 macrophages. Additionally, molecular docking data showed that PS mostly fit into the hydrophobic pocket of MD2 and bound to TLR4. In particular, apigenin-7-O-glucoside powerfully bound to MD2 and TLR4 via hydrogen bonding. Additionally, immunohistochemistry assay revealed that PS inhibited LPS-induced TLR4 dimerization or expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation, resulting in the inhibition of NF-κB activity. PS also attenuated LPS-mediated mortality and abnormality in zebrafish larvae and diminished the recruitment of neutrophils and macrophages at the inflammatory site accompanied by the low levels of proinflammatory mediators and cytokines. CONCLUSION: PS might be a novel immunomodulator for the effective treatment of LPS-mediated inflammatory diseases.

Cordycepin induces apoptosis in human bladder cancer T24 cells through ROS-dependent inhibition of the PI3K/Akt signaling pathway.

Cordycepin, a derivative of nucleoside adenosine, is one of the active ingredients extracted from the fungi of genus Cordyceps, which have been used for traditional herbal remedies. In this study, we examined the effect of cordycepin on the proliferation and apoptosis of human bladder cancer T24 cells and its mechanism of action. Cordycepin treatment significantly reduced the cell survival rate of T24 cells in a concentration-dependent manner, which was associated with the induction of apoptosis. Cordycepin activated caspase-8 and -9, which are involved in the initiation of extrinsic and intrinsic apoptosis pathways, respectively, and also increased caspase-3 activity, a typical effect caspase, subsequently leading to poly (ADP-ribose) polymerase cleavage. Additionally, cordycepin increased the Bax/Bcl-2 ratio and truncation of Bid, and destroyed the integrity of mitochondria, which contributed to the cytosolic release of cytochrome c. Moreover, cordycepin effectively inactivated the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, while LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of cordycepin. Cordycepin further enhanced the intracellular levels of reactive oxygen species (ROS), while the addition of N-acetyl cysteine (NAC), a ROS inhibitor, significantly diminished cordycepin-induced mitochondrial dysfunction and growth inhibition, and also blocked the inactivation of PI3K/Akt signaling pathway. Furthermore, the presence of NAC significantly attenuated the enhanced apoptotic cell death and reduction of cell viability by treatment with cordycepin and LY294002. Collectively, the data indicate that cordycepin induces apoptosis through the activation of extrinsic and intrinsic apoptosis pathways and the ROS-dependent inactivation of PI3K/Akt signaling in human bladder cancer T24 cells.

Oleifolioside B-mediated autophagy promotes apoptosis in A549 human non-small cell lung cancer cells.

The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.

Oleifolioside A mediates caspase-independent human cervical carcinoma HeLa cell apoptosis involving nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.

Apoptosis, the main type of programmed cell death, plays an essential role in a variety of biological events. Whereas "classical" apoptosis is dependent on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. To develop new anticancer agents, oleifolioside A was isolated from Dendropanax morbifera Leveille and the biochemical mechanisms of oleifolioside A-induced apoptosis in HeLa cells were investigated. Exposure to oleifolioside A resulted in caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Oleifolioside A treatment induced up-regulation of Bad, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, apoptosis-inducing factor (AIF), endonuclease G (EndoG), and apoptosis induction. This is the first report of anticancer activity of oleifolioside A, and nuclear translocation of AIF and EndoG in oleifolioside A-treated HeLa cells might represent an alternative death signaling pathway in the absence of caspase activity.

Induction of apoptosis by ethanol extract of Prunus mume in U937 human leukemia cells through activation of caspases.

Prunus mume (P. mume), a traditional drug and health food in Korea, Japan and China, possesses various pharmacological activities that include a potential source of free radical scavenging, anti-viral, anti-microbial, anti-inflammatory and anti-cancer activities. However, the cellular and molecular mechanisms of apoptosis induction by P. mume in human cancer cells are poorly understood. In the present study, we conducted an investigation of the pro-apoptotic effects of an ethanol extract of P. mume (EEPM) in U937 human leukemia cells. Exposure to EEPM was found to result in a concentration-dependent growth inhibition by induction of apoptosis. Induction of apoptotic cell death of U937 cells by EEPM showed a correlation with the down-regulation of members of the inhibitor of apoptosis protein (IAP) family, including X-linked inhibitor of apoptosis protein (XIAP) and survivin, and anti-apoptotic Bcl-2, up-regulation of FasL, and cleavage of Bic. EEPM treatment induced proteolytic activation of caspase-3, -8 and -9, and degradation of caspase-3 substrate proteins, including poly(ADP-ribose) polymerase (PARP) and ß-catenin. In addition, apoptotic cell death induced by EEPM was significantly inhibited by z-DEVD-fmk, a caspase-3-specific inhibitor, which demonstrated the important role played by caspase-3 in the process. Taken together, these findings suggest that EEPM may be a potential chemotherapeutic agent for use in the control of human leukemia U937 cells and that further studies are needed for the identification of the active compounds.

Anti-inflammatory effects of fucoidan through inhibition of NF-κB, MAPK and Akt activation in lipopolysaccharide-induced BV2 microglia cells.

Fucoidan, a sulfated polysaccharide extracted from brown seaweed, displays a wide variety of internal biological activities; however, the cellular and molecular mechanisms underlying fucoidan's anti-inflammatory activity remain poorly understood. In this study, we investigated the inhibitory effects of fucoidan on production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators in BV2 microglia. Our data indicated that fucoidan treatment significantly inhibited excessive production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated BV2 microglia. It also attenuated expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, monocyte chemoattractant protein-1 (MCP-1), and pro-inflammatory cytokines, including interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF)-α. Moreover, fucoidan exhibited anti-inflammatory properties by suppression of nuclear factor-kappa B (NF-κB) activation and down-regulation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and AKT pathways. These finding suggest that fucoidan may offer substantial therapeutic potential for treatment of neurodegenerative diseases that are accompanied by microglial activation.

Induction of apoptosis by cordycepin via reactive oxygen species generation in human leukemia cells.

Cordycepin (3'-deoxyadenosin), a specific polyadenylation inhibitor, is the main functional component in Cordyceps militaris, one of the top three renowned traditional Chinese medicines. Cordycepin has been shown to possess many pharmacological activities including immunological stimulation, and anti-bacterial, anti-viral, and anti-tumor effects. However, the mechanisms underlying its anti-cancer mechanisms are not yet understood. In this study, the apoptotic effects of cordycepin were investigated in human leukemia cells. Treatment with cordycepin significantly inhibited cell growth in a concentration-dependent manner by inducing apoptosis but not necrosis. This induction was associated with generation of reactive oxygen species (ROS), mitochondrial dysfunction, activation of caspases, and cleavage of poly(ADP-ribose) polymerase protein. However, apoptosis induced by cordycepin was attenuated by caspase inhibitors, indicating an important role for caspases in cordycepin responses. Administration of N-acetyl-l-cysteine, a scavenger of ROS, also significantly inhibited cordycepin-induced apoptosis and activation of caspases. These results support a mechanism whereby cordycepin induces apoptosis of human leukemia cells through a signaling cascade involving a ROS-mediated caspase pathway.

Ethyl alcohol extracts of Hizikia fusiforme sensitize AGS human gastric adenocarcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been reported in some cancer cells, including AGS human gastric adenocarcinoma cells. Hizikia fusiforme is a commonly used brown seaweed species in Korea that possesses potent antibacterial, antifungal, and anti-inflammatory activities. In this study, we demonstrated that treatment with TRAIL in combination with subtoxic concentrations of ethyl alcohol extract of H. fusiforme (EAHF) sensitized TRAIL-resistant AGS cells to TRAIL-mediated apoptosis. Combined treatment with EAHF and TRAIL increased chromatin condensation, DNA fragmentation, and sub-G1-phase DNA content. The restored sensitivity to TRAIL-induced apoptosis appeared to be correlated with the modulation of Bcl-2 family proteins and activation of caspases, which resulted in the cleavage of poly(ADP-ribose)polymerase. Taken together, the use of EAHF in combination with TRAIL may be an effective and selective anticancer strategy via suppressing the resistance to TRAIL-induced apoptosis in some tumor cell lines, including AGS cells.

Induction of apoptosis and inhibition of telomerase activity in human lung carcinoma cells by the water extract of Cordyceps militaris.

Cordyceps militaris is well known as a traditional medicinal mushroom and is a potentially interesting candidate for use in cancer treatment. In this study, the potential of the water extract of C. militaris (WECM) to induce apoptosis in human lung carcinoma A549 cells and its effects on telomerase activity were investigated. The growth inhibition and apoptosis induction by WECM treatment in A549 cells was associated with the induction of Fas, catalytic activation of caspase-8, and Bid cleavage. Activation of caspases, downregulation of anti-apoptotic Bcl-2 expression, and upregulation of pro-apoptotic Bax protein were also observed in WECM-treated cells. However, the cytotoxic effects and apoptotic characteristics induced by WECM were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. In addition, WECM exerted a dose-dependent inhibition of telomerase activity via downregulation of human telomerase reverse transcriptase (hTERT), c-myc and Sp1 expression. Taken together, the data from this study indicate that WECM induces the apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic and mitochondria-mediated intrinsic caspase pathways. It was also conclude that apoptotic events due to WECM were mediated with diminished telomerase activity through the inhibition of hTERT transcriptional activity.

Induction of apoptosis by aqueous extract of Cordyceps militaris through activation of caspases and inactivation of Akt in human breast cancer MDA-MB-231 Cells.

Cordyceps militaris is well known as a traditional medicinal mushroom and has been shown to exhibit immunostimulatory and anticancer activities. In this study, we investigated the apoptosis induced by an aqueous extract of C. militaris (AECM) via the activation of caspases and altered mitochondrial membrane permeability in human breast cancer MDA-MB-231 cells. Exposure to AECM induced apoptosis, as demonstrated by a quantitative analysis of nuclear morphological change and a flow cytometric analysis. AECM increased hyperpolarization of mitochondrial membrane potential and promoted the activation of caspases. Both the cytotoxic effect and apoptotic characteristics induced by AECM treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role of caspase-3 in the observed cytotoxic effect. AECM-induced apoptosis was associated with the inhibition of Akt activation in a time-dependent manner, and pretreatment with LY294002, a PI3K/Akt inhibitor, significantly increased AECM-induced apoptosis. The results indicated that AECM-induced apoptosis may relate to the activation of caspase-3 and mitochondria dysfunctions that correlate with the inactivation of Akt.

Development of Species-specific Primers for Rapid Detection of Phellinus linteus and P. baumii.

Genus Phellinus taxonomically belongs to Aphyllophorales and some species of this genus have been used as a medicinal ingredients and Indian folk medicines. Especially, P. linteus and morphological-related species are well-known medicinal fungi that have various biological activities such as humoral and cell-mediated, anti-mutagenic, and anti-cancer activities. However, little is known about the rapid detection for complex Phellinus species. Therefore, this study was carried out to develop specific primers for the rapid detection of P. linteus and other related species. Designing the species-specific primers was done based on internal transcribed spacer sequence data. Each primer set detected specifically P. linteus (PL2/PL5R) and P. baumii (PB1/PB4R). These primer sets could be useful for the rapid detection of specific-species among unidentified Phellinus species. Moreover, restriction fragment length polymorphism analysis of the ITS region with HaeIII was also useful for clarifying the relationship between each 5 Phellinus species.