The preventive effect of loganin on oxidative stress-induced cellular damage in human keratinocyte HaCaT cells.

Loganin is a type of iridoid glycosides isolated from Corni fructus and is known to have various pharmacological properties, but studies on its antioxidant activity are still lacking. Therefore, in this study, the preventive effect of loganin on oxidative stress-mediated cellular damage in human keratinocyte HaCaT cells was investigated. Our results show that loganin pretreatment in a non-toxic concentration range significantly improved cell survival in hydrogen peroxide (H2O2)-treated HaCaT cells, which was associated with inhibition of cell cycle arrest at the G2/M phase and induction of apoptosis. H2O2-induced DNA damage and reactive oxygen species (ROS) generation were also greatly reduced in the presence of loganin. Moreover, H2O2 treatment enhanced the cytoplasmic release of cytochrome c, upregulation of the Bax/Bcl-2 ratio and degradation of cleavage of poly (ADP-ribose) polymerase, whereas loganin remarkably suppressed these changes. In addition, loganin obviously attenuated H2O2-induced autophagy while inhibiting the increased accumulation of autophagosome proteins, including as microtubule-associated protein 1 light chain 3-II and Beclin-1, and p62, an autophagy substrate protein, in H2O2-treated cells. In conclusion, our current results suggests that loganin could protect HaCaT keratinocytes from H2O2-induced cellular injury by inhibiting mitochondrial dysfunction, autophagy and apoptosis. This finding indicates the applicability of loganin in the prevention and treatment of skin diseases caused by oxidative damage.

Induction of apoptotic cell death in human bladder cancer cells by ethanol extract of Zanthoxylum schinifolium leaf, through ROS-dependent inactivation of the PI3K/Akt signaling pathway.

BACKGROUND/OBJECTIVES: Zanthoxylum schinifolium is traditionally used as a spice for cooking in East Asian countries. This study was undertaken to evaluate the anti-proliferative potential of ethanol extracts of Z. schinifolium leaves (EEZS) against human bladder cancer T24 cells. MATERIALS/METHODS: Subsequent to measuring the cytotoxicity of EEZS, the anti-cancer activity was measured by assessing apoptosis induction, reactive oxygen species (ROS) generation, and mitochondrial membrane potential (MMP). In addition, we determined the underlying mechanism of EEZS-induced apoptosis through various assays, including Western blot analysis. RESULTS: EEZS treatment concentration-dependently inhibited T24 cell survival, which is associated with apoptosis induction. Exposure to EEZS induced the expression of Fas and Fas-ligand, activated caspases, and subsequently resulted to cleavage of poly (ADP-ribose) polymerase. EEZS also enhanced the expression of cytochrome c in the cytoplasm by suppressing MMP, following increase in the ratio of Bax:Bcl-2 expression and truncation of Bid. However, EEZS-mediated growth inhibition and apoptosis were significantly diminished by a pan-caspase inhibitor. Moreover, EEZS inhibited activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway, and the apoptosis-inducing potential of EEZS was promoted in the presence of PI3K/Akt inhibitor. In addition, EEZS enhanced the production of ROS, whereas N-acetyl cysteine (NAC), a ROS scavenger, markedly suppressed growth inhibition and inactivation of the PI3K/Akt signaling pathway induced by EEZS. Furthermore, NAC significantly attenuated the EEZS-induced apoptosis and reduction of cell viability. CONCLUSIONS: Taken together, our results indicate that exposure to EEZS exhibits anti-cancer activity in T24 bladder cancer cells through ROS-dependent induction of apoptosis and inactivation of the PI3K/Akt signaling pathway.

The anti-cancer effect of betulinic acid in u937 human leukemia cells is mediated through ROS-dependent cell cycle arrest and apoptosis.

Although previous studies have shown anti-cancer activity of betulinic acid (BA), a pentacyclic triterpenoid, against various cancer lines, the underlying molecular mechanisms are not well elucidated. In this study, we evaluated the mechanisms involved in the anti-cancer efficacy of BA in U937 human myeloid leukemia cells. BA exerted a significant cytotoxic effect on U937 cells through blocking cell cycle arrest at the G2/M phase and inducing apoptosis, and that the intracellular reactive oxygen species (ROS) levels increased after treatment with BA. The down-regulation of cyclin A and cyclin B1, and up-regulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1 revealed the G2/M phase arrest mechanism of BA. In addition, BA induced the cytosolic release of cytochrome c by reducing the mitochondrial membrane potential with an increasing Bax/Bcl-2 expression ratio. BA also increased the activity of caspase-9 and -3, and subsequent degradation of the poly (ADP-ribose) polymerase. However, quenching of ROS by N-acetyl-cysteine, an ROS scavenger, markedly abolished BA-induced G2/M arrest and apoptosis, indicating that the generation of ROS plays a key role in inhibiting the proliferation of U937 cells by BA treatment. Taken together, our results provide a mechanistic rationale that BA exhibits anti-cancer properties in U937 leukemia cells through ROS-dependent induction of cell cycle arrest at G2/M phase and apoptosis.

ROS-Mediated Anti-Tumor Effect of Coptidis Rhizoma against Human Hepatocellular Carcinoma Hep3B Cells and Xenografts.

Coptidis Rhizoma is the dried rhizome from the Coptis chinensis Franch. that has been shown to have a number of beneficial pharmacological properties including antioxidant, anti-inflammatory, and anti-cancer effects. However, the anti-cancer effects of Coptidis Rhizoma on hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the anti-cancer properties of Coptidis Rhizoma ethanol extract (CR) in HCC Hep3B cells and in a xenograft mouse model. Our results showed that the CR significantly inhibited cell growth and induced apoptosis in Hep3B cells through increased expression of Bcl-2 associated x-protein (Bax) and cleavage of poly-ADP ribose polymerase (PARP), reduced expression of Bcl-2, and activated caspases. CR also increased the generation of intracellular reactive oxygen species (ROS), which caused a loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of the mitochondria-mediated intrinsic apoptosis pathway. Moreover, N-acetylcysteine (NAC), a ROS inhibitor, markedly blocked the effects of CR on apoptotic pathways. CR also induced the expression of light chain 3 (LC3)-I/II, a key autophagy regulator, whereas CR-mediated autophagy was significantly suppressed by NAC. In addition, pre-treatment with NAC perfectly attenuated the inhibition of cell invasion and migration of CR-stimulated Hep3B cells. Furthermore, oral administration of CR suppressed Hep3B tumor growth in xenograft mice without toxicity, alterations to body weight, or changes in hematological and biochemical profiles. Taken together, our findings suggest that CR has anti-tumor effects that result from ROS generation, and may be a potential pharmacological intervention for HCC.

Induction of G2/M Cell Cycle Arrest and Apoptosis by Genistein in Human Bladder Cancer T24 Cells through Inhibition of the ROS-Dependent PI3k/Akt Signal Transduction Pathway.

We examined the anti-cancer effect of genistein, a soy-derived isoflavone, in human bladder transitional cell carcinoma T24 cells. According to our data, genistein induced G2/M phase arrest of the cell cycle and apoptosis. Genistein down-regulated the levels of cyclin A and cyclin B1, but up-regulated the levels of p21WAF1/CIP1, cyclin-dependent kinase (Cdk) inhibitor, that was complexed with Cdc2 and Cdk2. Furthermore, genistein induced the activation of caspases (caspase-3, -8 and -9), and cleavage of poly (ADP-ribose) polymerase cleavage. However, genistein-induced apoptosis was significantly inhibited by a pan-caspase inhibitor, indicating that the induction of apoptosis by genestein was caspase-dependent. In addition, genistein increased the cytosolic release of cytochrome c by increasing the Bax/Bcl-2 ratio and destroying mitochondria integrity. Moreover, genistein inactivated the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, while LY294002, a PI3K/Akt inhibitor, increased the apoptosis-inducing effect of genistein. Genistein further increased the accumulation of reactive oxygen species (ROS), which was significantly suppressed by N-acetyl cysteine (NAC), a ROS scavenger, and in particular, NAC prevented genistein-mediated inactivation of PI3K/Akt signaling, G2/M arrest and apoptosis. Therefore, the present results indicated that genistein promoted apoptosis induction in human bladder cancer T24 cells, which was associated with G2/M phase cell cycle arrest via regulation of ROS-dependent PI3K/Akt signaling pathway.

Protective Effects of Fermented Oyster Extract against RANKL-Induced Osteoclastogenesis through Scavenging ROS Generation in RAW 264.7 Cells.

Excessive bone resorption by osteoclasts causes bone loss-related diseases and reactive oxygen species (ROS) act as second messengers in intercellular signaling pathways during osteoclast differentiation. In this study, we explored the protective effects of fermented oyster extract (FO) against receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation in murine monocyte/macrophage RAW 264.7 cells. Our results showed that FO markedly inhibited RANKL-induced activation of tartrate-resistant acid phosphatase and formation of F-actin ring structure. Mechanistically, FO has been shown to down-regulate RANKL-induced expression of osteoclast-specific markers by blocking the nuclear translocation of NF-κB and the transcriptional activation of nuclear factor of activated T cells c1 (NFATc1) and c-Fos. Furthermore, FO markedly diminished ROS production by RANKL stimulation, which was associated with blocking the expression of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) and its regulatory subunit Rac-1. However, a small interfering RNA (siRNA) targeting NOX1 suppressed RANKL-induced expression of osteoclast-specific markers and production of ROS and attenuated osteoclast differentiation as in the FO treatment group. Collectively, our findings suggest that FO has anti-osteoclastogenic potential by inactivating the NF-κB-mediated NFATc1 and c-Fos signaling pathways and inhibiting ROS generation, followed by suppression of osteoclast-specific genes. Although further studies are needed to demonstrate efficacy in in vivo animal models, FO may be used as an effective alternative agent for the prevention and treatment of osteoclastogenic bone diseases.

Protective Effects of Nargenicin A1 against Tacrolimus-Induced Oxidative Stress in Hirame Natural Embryo Cells.

Tacrolimus is widely used as an immunosuppressant to reduce the risk of rejection after organ transplantation, but its cytotoxicity is problematic. Nargenicin A1 is an antibiotic extracted from Nocardia argentinensis and is known to have antioxidant activity, though its mode of action is unknown. The present study was undertaken to evaluate the protective effects of nargenicin A1 on DNA damage and apoptosis induced by tacrolimus in hirame natural embryo (HINAE) cells. We found that reduced HINAE cell survival by tacrolimus was due to the induction of DNA damage and apoptosis, both of which were prevented by co-treating nargenicin A1 or N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, with tacrolimus. In addition, apoptosis induction by tacrolimus was accompanied by increases in ROS generation and decreases in adenosine triphosphate (ATP) levels caused by mitochondrial dysfunction, and these changes were significantly attenuated in the presence of nargenicin A1, which further indicated tacrolimus-induced apoptosis involved an oxidative stress-associated mechanism. Furthermore, nargenicin A1 suppressed tacrolimus-induced B-cell lymphoma-2 (Bcl-2) down-regulation, Bax up-regulation, and caspase-3 activation. Collectively, these results demonstrate that nargenicin A1 protects HINAE cells against tacrolimus-induced DNA damage and apoptosis, at least in part, by scavenging ROS and thus suppressing the mitochondrial-dependent apoptotic pathway.

Sargassum serratifolium Extract Attenuates Interleukin-1ß-Induced Oxidative Stress and Inflammatory Response in Chondrocytes by Suppressing the Activation of NF-κB, p38 MAPK, and PI3K/Akt.

Osteoarthritis (OA) is a degenerative joint disease that is characterized by irreversible articular cartilage destruction by inflammatory reaction. Among inflammatory stimuli, interleukin-1ß (IL-1ß) is known to play a crucial role in OA pathogenesis by stimulating several mediators that contribute to cartilage degradation. Recently, the marine brown alga Sargassum serratifolium has been reported to exhibit antioxidant and anti-inflammatory effects in microglial and human umbilical vein endothelial cell models using lipopolysaccharide and tumor necrosis factor-α, but its beneficial effects on OA have not been investigated. This study aimed to evaluate the anti-osteoarthritic effects of ethanol extract of S. serratifolium (EESS) in SW1353 human chondrocytes and, in parallel, primary rat articular chondrocytes. Our results showed that EESS effectively blocked the generation of reactive oxygen species in IL-1ß-treated SW1353 and rat primary chondrocytes, indicating that EESS has a potent antioxidant activity. EESS also attenuated IL-1ß-induced production of nitric oxide (NO) and prostaglandin E2, major inflammatory mediators in these cells, which was associated with the inhibition of inducible NO synthase and cyclooxygenase-2 expression. Moreover, EESS downregulated the level of gene expression of matrix metalloproteinase (MMP)-1, -3 and -13 in SW1353 chondrocytes treated with IL-1ß, resulting in their extracellular secretion reduction. In addition, the IL-1ß-induced activation of nuclear factor-kappa B (NF-κB) was restored by EESS. Furthermore, EESS reduced the activation of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways upon IL-1ß stimulation. These results indicate that EESS has the potential to exhibit antioxidant and anti-inflammatory effects through inactivation of the NF-κB, p38 MAPK, and PI3K/Akt signaling pathways. Collectively, these findings demonstrate that EESS may have the potential for chondroprotection, and extracts of S. serratifolium could potentially be used in the prevention and treatment of OA.

Inhibition of Adipocyte Differentiation by Anthocyanins Isolated from the Fruit of Vitis coignetiae Pulliat is Associated with the Activation of AMPK Signaling Pathway.

Anthocyanins are naturally occurring water-soluble polyphenolic pigments in plants that have been shown to protect against cardiovascular diseases, and certain cancers, as well as other chronic human disorders. However, the anti-obesity effects of anthocyanins are not fully understood. In this study, we investigated the effects of anthocyanins isolated from the fruit of Vitis coignetiae Pulliat on the adipogenesis of 3T3-L1 preadipocytes. Our data indicated that anthocyanins attenuated the terminal differentiation of 3T3-L1 preadipocytes, as confirmed by a decrease in the number of lipid droplets, lipid content, and triglyceride production. During this process, anthocyanins effectively enhanced the activation of the AMP-activated protein kinase (AMPK); however, this phenomenon was inhibited by the co-treatment of compound C, an inhibitor of AMPK. Anthocyanins also inhibited the expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein a and b, and sterol regulatory element-binding protein-1c. In addition, anthocyanins were found to potently inhibit the expression of adipocyte-specific genes, including adipocyte fatty acid-binding protein, leptin, and fatty acid synthase. These results indicate that anthocyanins have potent anti-obesity effects due to the inhibition of adipocyte differentiation and adipogenesis, and thus may have applications as a potential source for an anti-obesity functional food agent.

Spermidine Protects against Oxidative Stress in Inflammation Models Using Macrophages and Zebrafish.

Spermidine is a naturally occurring polyamine compound that has recently emerged with anti-aging properties and suppresses inflammation and oxidation. However, its mechanisms of action on anti-inflammatory and antioxidant effects have not been fully elucidated. In this study, the potential of spermidine for reducing pro-inflammatory and oxidative effects in lipopolysaccharide (LPS)-stimulated macrophages and zebrafish was explored. Our data indicate that spermidine significantly inhibited the production of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines including tumor necrosis factor-α and interleukin-1ß in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects of spermidine accompanied by a marked suppression in their regulatory gene expression at the transcription levels. Spermidine also attenuated the nuclear translocation of NF-κB p65 subunit and reduced LPS-induced intracellular accumulation of reactive oxygen species (ROS) in RAW 264.7 macrophages. Moreover, spermidine prevented the LPS-induced NO production and ROS accumulation in zebrafish larvae and was found to be associated with a diminished recruitment of neutrophils and macrophages. Although more work is needed to fully understand the critical role of spermidine on the inhibition of inflammation-associated migration of immune cells, our findings clearly demonstrate that spermidine may be a potential therapeutic intervention for the treatment of inflammatory and oxidative disorders.

Lonicera japonica Thunb. Induces caspase-dependent apoptosis through death receptors and suppression of AKT in U937 human leukemic cells.

Decoctions obtained from the dried flowers of Lonicera japonica Thunb. (Indongcho) have been utilized in folk remedies against inflammatory diseases. Recently, many agents that have used for inflammatory diseases are showing anticancer effects. Here, we have isolated polyphenols extracted from lyophilized Lonicera japonica Thunb (PELJ) and investigated the anticancer effects of PELJ on U937 cells. Here, we demonstrated that PELJ induced apoptosis by upregulation of DR4 and Fas, and further it is augmented by suppression of XIAP. In addition, The PELJ-induced apoptosis is at least in part by blocking PI3K/Akt pathway. These findings suggest that PELJ may provide evidence of anticancer activities on U937 cells. Further study for detailed mechanism and the effects on animal models is warranted to determine whether PELJ provide more conclusive evidence that PELJ which may provide a beneficial effect for treating cancer.

Anti-inflammatory effects of Daehwangmokdantang, a traditional herbal formulation, in lipopolysaccharide-stimulated RAW 264.7 macrophages.

Daehwangmokdantang (DHMDT) is a traditional polyherbal formulation that has known antidiarrheal and anti-inflammatory activities. However, the underlying mechanisms of these activities are poorly understood. In the present study, the inhibitory effects of DHMDT on the production of proinflammatory mediators and cytokines in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were investigated. The inhibitory effects of DHMDT on LPS-induced nitric oxide (NO), prostaglandin (PG)E2, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß production were examined using Griess reagent and ELISA detection kits. The effects of DHMDT on the expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2, IL-1ß and TNF-α, and their upstream signal proteins, including nuclear factor (NF)-κB, mitogen-activated protein kinases (MAPKs) and RAC-α serine/threonine-protein kinase (Akt), a phosphatidylinositol 3-kinase (PI3K) downstream effector, were investigated using western blotting and immunofluorescence staining. The results revealed the pretreatment with DHMDT significantly inhibited the LPS-induced production of NO, PGE2, TNF-α, and IL-1ß, and expression of iNOS, COX-2 TNF-α, and IL-1ß, without any significant cytotoxicity. DHMDT also efficiently prevented the translocation of the NF-κB subunit p65 into the nucleus by interrupting the activation of the upstream mediator inhibitor of NF-κB kinase α/ß. Furthermore, the anti-inflammatory effects of DHMDT were associated with the suppression of LPS-induced phosphorylation of Akt and MAPKs in RAW 264.7 macrophages. Therefore, the results of the present study indicate that DHMDT exhibited anti-inflammatory activity via the inhibition of proinflammatory mediators and cytokines, in which the inactivation of NF-κB, PI3K/Akt, and MAPKs may be involved. These results suggest that DHMDT may be a potential anti-inflammatory drug candidate.

Bufalin sensitizes human bladder carcinoma cells to TRAIL-mediated apoptosis.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, has garnered interest as it is relatively non-toxic to normal cells, but selectively induces apoptotic cell death in multiple types of transformed or malignant cells. Bufalin is the major digoxin-like immunoreactive component of Sum Su, which is obtained from the skin and parotid venom gland of the toad. Bufalin is known to inhibit cell proliferation and induce apoptosis in a variety of cancer cells. The present study investigated whether bufalin promoted TRAIL-induced apoptotic cell death. In the present study, a combined treatment using bufalin and TRAIL significantly increased TRAIL-mediated inhibition of cell viability and increased apoptosis in T24 human bladder cancer cells. The apoptotic effects were associated with the upregulation of death receptor proteins and the downregulation of cellular Fas-associated death domain-like interleukin-1ß-converting enzyme inhibitory protein and X-linked inhibitor of apoptosis protein. Furthermore, the data revealed that bufalin and TRAIL activated caspase-3, -8 and -9 and subsequently increased the degradation of poly (ADP-ribose) polymerase. Taken altogether, the nontoxic doses of bufalin and TRAIL sensitized T24 cells to TRAIL-mediated apoptosis. Therefore, bufalin may provide an effective therapeutic strategy for the safe treatment of human bladder cancers that are resistant to TRAIL.

Induction of apoptosis by Moutan Cortex Radicis in human gastric cancer cells through the activation of caspases and the AMPK signaling pathway

Abstract Moutan Cortex Radicis, the root bark of Paeonia × suffruticosa Andrews, Paeoniaceae, has been widely used in traditional medicine therapy. Although it has been shown to possess many pharmacological activities, the molecular mechanisms of its anti-cancer activity have not been clearly elucidated. In the present study, we investigated the pro-apoptotic effects of the ethanol extract of Moutan Cortex Radicis in human gastric cancer AGS cells. Moutan Cortex Radicis treatment inhibited the cell viability of AGS cells in a concentration-dependent manner, which was associated with apoptotic cell death. Moutan Cortex Radicis's induction of apoptosis was connected with the upregulation of death receptor 4, death receptor 5, tumor necrosis factor-related apoptosis-inducing ligand, Fas ligand, and Bax, and the downregulation of Bcl-2 and Bid. Moutan Cortex Radicis treatment also induced the loss of mitochondrial membrane potential (Δψm), the proteolytic activation of caspases (-3, -8, and -9), and the degradation of poly(ADP-ribose) polymerase, an activated caspase-3 substrate protein. However, the pre-treatment of a caspase-3 inhibitor significantly attenuated Moutan Cortex Radicis-induced apoptosis and cell viability reduction. In addition, Moutan Cortex Radicis treatment effectively activated the adenosine monophosphate-activated protein kinase signaling pathway; however, a specific inhibitor of AMPK significantly reduced Moutan Cortex Radicis-induced apoptosis. Overall, the results suggest that the apoptotic activity of Moutan Cortex Radicis may be associated with a caspase-dependent cascade through the activation of both extrinsic and intrinsic signaling pathways connected with adenosine monophosphate-activated protein kinase activation, and Moutan Cortex Radicis as an activator of adenosine monophosphate-activated protein kinase could be a prospective application to treat human cancers.

Induction of p53-Independent Apoptosis and G1 Cell Cycle Arrest by Fucoidan in HCT116 Human Colorectal Carcinoma Cells.

It is well known that fucoidan, a natural sulfated polysaccharide present in various brown algae, mediates anticancer effects through the induction of cell cycle arrest and apoptosis. Nevertheless, the role of tumor suppressor p53 in the mechanism action of fucoidan remains unclear. Here, we investigated the anticancer effect of fucoidan on two p53 isogenic HCT116 (p53+/+ and p53-/-) cell lines. Our results showed that inhibition of cell viability, induction of apoptosis and DNA damage by treatment with fucoidan were similar in two cell lines. Flow cytometric analysis revealed that fucoidan resulted in G1 arrest in the cell cycle progression, which correlated with the inhibition of phosphorylation of retinoblastoma protein (pRB) and concomitant association of pRB with the transcription factor E2Fs. Furthermore, treatment with fucoidan obviously upregulated the expression of cyclin-dependent kinase (CDK) inhibitors, such as p21WAF1/CIP1 and p27KIP1, which was paralleled by an enhanced binding with CDK2 and CDK4. These events also commonly occurred in both cell lines, suggesting that fucoidan triggered G1 arrest and apoptosis in HCT116 cells by a p53-independent mechanism. Thus, given that most tumors exhibit functional p53 inactivation, fucoidan could be a possible therapeutic option for cancer treatment regardless of the p53 status.

Auranofin, an inhibitor of thioredoxin reductase, induces apoptosis in hepatocellular carcinoma Hep3B cells by generation of reactive oxygen species.

Mammalian thioredoxin reductase (TrxR) plays a vital role in restoring cellular redox balance disrupted by reactive oxygen species (ROS) generation and oxidative damage. Here, we evaluated whether auranofin, a selective inhibitor of TrxR, could serve as a potential anti-cancer agent through its selective targeting of TrxR activity in Hep3B hepatocellular carcinoma cells. Auranofin treatment reduced the TrxR activity of these cells and induced apoptosis, which were accompanied by up-regulation of death receptors (DRs) and activation of caspases, as well as promotion of proteolytic degradation of poly(ADP-ribose)-polymerase. Treatment with a pan-caspase inhibitor reversed the auranofin-induced apoptosis and growth suppression, indicating that auranofin may induce apoptosis through a caspase-dependent mechanism involving both the intrinsic and extrinsic apoptotic pathways. Auranofin also significantly altered mitochondrial function, promoting mitochondrial membrane permeabilization and cytochrome c release by regulating Bcl-2 family proteins; these events were accompanied by an accumulation of ROS. Inhibition of ROS generation with the ROS quencher significantly attenuated the inactivation of TrxR in auranofin-treated cells and almost completely suppressed the auranofin-induced up-regulation of DRs and activation of caspases, thereby preventing auranofin-induced apoptosis and loss of cell viability. Taken together, these findings indicate that auranofin inhibition of TrxR activity in Hep3B cells activates ROS- and caspase-dependent apoptotic signaling pathways and triggers cancer cell death.

Fucoidan Induces ROS-Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway.

Preclinical Research Fucoidan, a sulfated polysaccharide, is a compound found in various species of seaweed that has anti-viral, anti-bacterial, anti-oxidant, anti-inflammatory, and immunomodulatory activities; however, the underlying relationship between apoptosis and anti-telomerase activity has not been investigated. Here, we report that fucoidan-induced apoptosis in 5637 human bladder cancer cells was associated with an increase in the Bax/Bcl-2 ratio, the dissipation of the mitochondrial membrane potential (MMP, Δψm), and cytosolic release of cytochrome c from the mitochondria. Under the same experimental conditions, fucoidan-treatment decreased hTERT (human telomerase reverse transcriptase) expression and the transcription factors, c-myc and Sp1. This was accompanied by decreased telomerase activity. Fucoidan-treatment also suppressed activation of the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling enhanced fucoidan-induced apoptosis and anti-telomerase activity. Meanwhile, fucoidan treatment increased the generation of intracellular ROS, whereas the over-elimination of ROS by N-acetylcysteine, an anti-oxidant, attenuated fucoidan-induced apoptosis, inhibition of hTERT, c-myc, and Sp1 expression, and reversed fucoidan-induced inactivation of the PI3K/Akt signaling pathway. Collectively, these data indicate that the induction of apoptosis and the inhibition of telomerase activity by fucoidan are mediated via ROS-dependent inactivation of the PI3K/Akt pathway. Drug Dev Res 78 : 37-48, 2017. © 2016 Wiley Periodicals, Inc.

Flavonoids Isolated from Flowers of Lonicera japonica Thunb. Inhibit Inflammatory Responses in BV2 Microglial Cells by Suppressing TNF-α and IL-ß Through PI3K/Akt/NF-kb Signaling Pathways.

Decoctions of the dried flowers of Lonicera japonica Thunb. (Indongcho) have been utilized in folk remedies against various inflammatory diseases, and it is reported neuroprotective effects. The cytokines release from microglia is closely linked to various chronic neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. It is still unknown whether the neuroprotective effects are associated with the antiinflammatory effects. Here, we determined whether polyphenols extracted from lyophilized Lonicera japonica Thunb. (PELJ) would inhibit inflammatory cytokines and mediators. We stimulated microglia with lipopolysaccharide (LPS) to produce inflammatory cytokines, and then assessed the effects of PELJ on these cytokines. PELJ significantly inhibited LPS-induced interleukin-1ß and tumor necrosis factor-α expressions and LPS-induced nitric oxide (NO) and prostaglandin E2 expressions by down-regulating inducible enzyme NO synthase and cyclooxygenase-2 at the protein and mRNA levels. All the suppression of these mediators did not cause any significant cytotoxicity. PELJ also inhibited the nuclear translocation of nuclear factor-kappa B and phosphorylated Akt. These findings suggest that PELJ may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases by inhibiting pro-inflammatory cytokines through inhibiting phosphoinositol 3-kinase /Akt/nuclear factor-kappa B signaling pathway. Copyright © 2016 John Wiley & Sons, Ltd.

Polyphenols from Korean prostrate spurge Euphorbia supina induce apoptosis through the Fas-associated extrinsic pathway and activation of ERK in human leukemic U937 cells.

The Korean prostrate spurge Euphorbia supina (Euphorbiaceae family) has been used as a folk medicine in Korea against a variety of ailments such as bronchitis, hemorrhage, jaundice and multiple gastrointestinal diseases. Polyphenols from Korean E. supina (PES) which include quercetin and kaempferol derivatives have anticancer properties. Hence, we investigated the anticancer effects of PES on U937 human leukemic cells. Firstly, PES significantly inhibited the proliferation of U937 cells in a dose-dependent manner. PES induced accumulation of the sub-G1 DNA content (apoptotic cell population), apoptotic bodies and chromatin condensation and DNA fragmentation in the U937 cells. PES also induced activation of caspase-3, -8 and -9, subsequent cleavage of PARP, and significantly suppressed XIAP, cIAP-1 and cIAP-2 in a dose-dependent manner. Furthermore, PES activated Bid, and induced the loss of mitochondrial membrane potential (MMP, ΔΨm) along with upregulation of pro-apoptotic proteins (Bax and Bad), and downregulation of anti-apoptotic proteins (Bcl-2 and Bcl-xL) and cytochrome c release. The Fas receptor was upregulated by PES in a dose-dependent manner, suggesting that the extrinsic pathway was also involved in the PES-induced apoptosis. Moreover, the PES-induced apoptosis was at least in part associated with extracellular signal-regulated kinase (ERK) activation in the U937 human leukemic cells. This study provides evidence that PES may be useful in the treatment of leukemia.

Baicalein Induces Caspase-dependent Apoptosis Associated with the Generation of ROS and the Activation of AMPK in Human Lung Carcinoma A549 Cells.

Baicalein is one of the main bioactive flavonoids found in the roots of Scutellaria baicalensis Georgi. Here, we report that baicalein-induced growth inhibition was associated with the induction of apoptosis in human lung carcinoma A549 cells. Baicalein stimulated the expression of DR5, FasL, and FADD, and activated caspase-8 by reducing the levels of FLIPs (FLICE-inhibitory proteins). The apoptotic cell death was also connected with an activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase; however, a blockage of caspase activation abolished baicalein-induced apoptotic potentials. Additionally, baicalein caused a mitochondrial membrane potential (MMP), the truncation of Bid, and the translocation of pro-apoptotic Bax to the mitochondria, thereby inducing the release of cytochrome c into the cytosol. In turn, baicalein increased the generation of reactive oxygen species (ROS); however, an ROS scavenger, N-acetylcysteine, notably attenuated baicalein-mediated loss of MMP and activation of caspases. Furthermore, baicalein activated the AMP-activated protein kinase (AMPK) signaling pathway. Consequently, baicalein-triggered cell death was attenuated by an AMPK inhibitor, but increased by an AMPK activator, compound C. Overall, the results suggest that the apoptotic activity of baicalein may be associated with caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways connected with ROS generation and AMPK activation.