[Corrigendum] Hwang-Heuk-San induces apoptosis in HCT116 human colorectal cancer cells through the ROS-mediated activation of caspases and the inactivation of the PI3K/Akt signaling pathway.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that certain of the data panels featured in Figs. 1B, 4A, 6A and 8A, showing DAPI or NAC staining of the cells, appeared to contain overlapping data. The authors have consulted their original data, and realize that errors were made during the compilation of these figures; consequently, they have repeated the affected experiments. The revised versions of Figs. 1, 4, 6 and 8, featuring replacement data for Figs. 1B, 4A, 6A and 8A, are shown on the subsequent pages. The authors regret the errors that were made during the preparation of the published figures, and confirm that these errors did not affect the conclusions reported in the study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum, and all the authors agree to this Corrigendum. Furthermore, they apologize to the readership for any inconvenience caused. [the original article was published in Oncology Reports 36: 205­214, 2016; DOI: 10.3892/or.2016.4812].

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.

A Novel Peptide Oligomer of Bacitracin Induces M1 Macrophage Polarization by Facilitating Ca2+ Influx.

Antimicrobial peptides (AMPs) are components of the innate immune system and form the first defense against pathogens for various organisms. In the present study, we assessed whether CSP32, a novel AMP oligomer of bacitracin isolated from a strain of Bacillus spp., regulates the polarization of murine macrophage-like RAW 264.7 cells. CSP32 stimulated phagocytosis while inducing the appearance of the typical M1 polarized macrophage phenotype; these M1 macrophages play a role in host defense against pathogens. Furthermore, our results showed that CSP32 enhanced the expression and production of pro-inflammatory mediators, such as cytokines and chemokines. In addition, the CSP32-stimulated inflammatory mediators were induced mainly by the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) signaling pathway during M1 macrophage polarization. In particular, CSP32 markedly increased the numbers of Ca2+-positive macrophages while upregulating phospholipase C and activating protein kinase Cε. Furthermore, the inhibition of intracellular Ca2+ by BAPTA-AM, a Ca2+ chelator, significantly suppressed the CSP32-mediated phagocytosis, inflammatory mediator production, and NF-κB activation. In conclusion, our data suggested that CSP32-stimulated M1 macrophage polarization is dependent on the calcium signaling pathway and may result in enhanced immune capacities.

Glutathione Induced Immune-Stimulatory Activity by Promoting M1-Like Macrophages Polarization via Potential ROS Scavenging Capacity.

The present study investigated the immunomodulatory activity of reduced glutathione (GSH) by assessment of the macrophage polarization (MP)-mediated immune response in RAW 264.7 cells. Furthermore, we identified the signal pathway associated with immune regulation by GSH. The expressions of MP-associated cytokines and chemokines were assessed using cytokine array, nCounter Sprit platform, ELISA and immunoblotting. Phagocytosis activity and intracellular reactive oxygen species (ROS) generation were measured using fluorescence-activated cell sorter. As results of the cytokine array and nCounter gene array, GSH not only up-regulated pro-inflammatory cytokines, including interleukins and tumor necrosis factor-α, but also overexpressed neutrophil-attracting chemokines. Furthermore, GSH significantly stimulated the production of immune mediators, including nitric oxide and PGE2, as well as phagocytosis activity through nuclear factor kappa B activation. In addition, GSH significantly decreased LPS-induced ROS generation, which was associated with an activation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2)/ heme oxygenease-1 (HO-1) signaling pathway. Our results suggest that GSH has potential ROS scavenging capacity via the induction of Nrf2-mediated HO-1, and immune-enhancing activity by regulation of M1-like macrophage polarization, indicating that GSH may be a useful strategy to increase the human defense system.

Protective Effect of Glutathione against Oxidative Stress-induced Cytotoxicity in RAW 264.7 Macrophages through Activating the Nuclear Factor Erythroid 2-Related Factor-2/Heme Oxygenase-1 Pathway.

Reactive oxygen species (ROS), products of oxidative stress, contribute to the initiation and progression of the pathogenesis of various diseases. Glutathione is a major antioxidant that can help prevent the process through the removal of ROS. The aim of this study was to evaluate the protective effect of glutathione on ROS-mediated DNA damage and apoptosis caused by hydrogen peroxide, H2O2, in RAW 264.7 macrophages and to investigate the role of the nuclear factor erythroid 2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. The results showed that the decrease in the survival rate of RAW 264.7 cells treated with H2O2 was due to the induction of DNA damage and apoptosis accompanied by the increased production of ROS. However, H2O2-induced cytotoxicity and ROS generation were significantly reversed by glutathione. In addition, the H2O2-induced loss of mitochondrial membrane potential was related to a decrease in adenosine triphosphate (ATP) levels, and these changes were also significantly attenuated in the presence of glutathione. These protective actions were accompanied by a increase in the expression rate of B-cell lymphoma-2 (Bcl-2)/Bcl-2-associated X protein (Bax) and poly(ADP-ribose) polymerase cleavage by the inactivation of caspase-3. Moreover, glutathione-mediated cytoprotective properties were associated with an increased activation of Nrf2 and expression of HO-1; however, the inhibition of the HO-1 function using an HO-1 specific inhibitor, zinc protoporphyrin IX, significantly weakened the cytoprotective effects of glutathione. Collectively, the results demonstrate that the exogenous administration of glutathione is able to protect RAW 264.7 cells against oxidative stress-induced mitochondria-mediated apoptosis along with the activity of the Nrf2/HO-1 signaling pathway.

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.

Corni Fructus attenuates testosterone-induced benign prostatic hyperplasia by suppressing 5α-reductase and androgen receptor expression in rats.

BACKGROUND/OBJECTIVES: Benign prostatic hypertrophy (BPH) is a major cause of abnormal overgrowth of the prostate mainly in the elderly. Corni Fructus has been reported to be effective in the prevention and treatment of various diseases because of its strong antioxidant effect, but its efficacy against BPH is not yet known. This study was designed to evaluate the therapeutic efficacy of Corni Fructus water extract (CF) in testosterone-induced BPH rats. MATERIALS/METHODS: To induce BPH, rats were intraperitoneal injected with testosterone propionate (TP). Rats in the treatment group were orally administered with CF with TP injection, and finasteride, which is a selective inhibitor of 5α-reductase type 2, was used as a positive control. RESULTS: Our results showed that the increased prostate weight and histopathological changes in BPH model rats were suppressed by CF treatment. CF, similar to the finasteride-treated group, decreased the levels of testosterone and dihydrotestosterone by TP treatment in the serum, and it also reduced 5α-reductase expression and concentration in prostate tissue and serum, respectively. In addition, CF significantly blocked the expression of the androgen receptor (AR), AR co-activators, and proliferating cell nuclear antigen in BPH rats, and this blocking was associated with a decrease in prostate-specific antigen levels in serum and prostate tissue. CONCLUSIONS: These results suggest that CF may weaken the BPH status through the inactivation of at least 5α-reductase and AR activity and may be useful for the clinical treatment of BPH.

Induction of Apoptosis by Citrus unshiu Peel in Human Breast Cancer MCF-7 Cells: Involvement of ROS-Dependent Activation of AMPK.

The fruit of Citrus unshiu MARKOVICH used for various purposes in traditional medicine has various pharmacological properties including antioxidant, anti-inflammatory, and antibacterial effects. Recently, the possibility of anti-cancer activity of the extracts or components of this fruit has been reported; however, the exact mechanism has not yet been fully understood. In this study, we evaluated the anti-proliferative effect of water extract of C. unshiu peel (WECU) on human breast cancer MCF-7 cells and investigated the underlying mechanism. Our results showed that reduction of MCF-7 cell survival by WECU was associated with the induction of apoptosis. WECU-induced apoptotic cell death was related to the activation of caspase-8 and -9, representative initiate caspases of extrinsic and intrinsic apoptosis pathways, respectively, and increase in the Bax : Bcl-2 ratio accompanied by cleavage of poly(ADP-ribose) polymerase (PARP). WECU also increased the mitochondrial dysfunction and cytosolic release of cytochrome c. In addition, AMP-activated protein kinase (AMPK) and its downstream target molecule, acetyl-CoA carboxylase, were activated in a concentration-dependent manner in WECU-treated cells. In contrast, compound C, an AMPK inhibitor, significantly inhibited WECU-induced apoptosis, while inhibiting increased expression of Bax and decreased expression of Bcl-2 by WECU and inhibition of WECU-induced PARP degradation. Furthermore, WECU provoked the production of reactive oxygen species (ROS); however, the activation of AMKP and apoptosis by WECU were prevented, when the ROS production was blocked by antioxidant N-acetyl cysteine. Therefore, our data indicate that WECU suppresses MCF-7 cell proliferation by activating the intrinsic and extrinsic apoptosis pathways through ROS-dependent AMPK pathway activation.

Reactive oxygen species-dependent apoptosis induction by water extract of Citrus unshiu peel in MDA-MB-231 human breast carcinoma cells.

BACKGROUND/OBJECTIVES: Although several recent studies have reported the anti-cancer effects of extracts or components of Citrus unshiu peel, which has been used for various purposes in traditional medicine, the molecular mechanisms for their effects remain unclear. In the present study, the anti-cancer activity of a water-soluble extract of C. unshiu peel (WECU) in MDA-MB-231 human breast carcinoma cells at the level of apoptosis induction was investigated. MATERIALS/METHODS: Cytotoxicity was evaluated using the MTT assay. Apoptosis was detected using DAPI staining and flow cytometry analyses. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, caspase activity and Western blotting were used to confirm the basis of apoptosis. RESULTS: The results indicated that WECU-induced apoptosis was related to the activation of caspase-8, and -9, representative initiator caspases of extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3 accompanied by proteolytic degradation of poly(ADP-ribose) polymerase and down-regulation of the inhibitors of apoptosis protein family members. WECU also increased the pro-apoptotic BAX to anti-apoptotic BCL-2 ratio, loss of mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm. Furthermore, WECU provoked the generation of ROS, but the reduction of cell viability and induction of apoptosis by WECU were prevented when ROS production was blocked by antioxidant N-acetyl cysteine. CONCLUSIONS: These results suggest that WECU suppressed proliferation of MDA-MB-231 cells by activating extrinsic and intrinsic apoptosis pathways in a ROS-dependent manner.

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.

Induction of apoptosis by ethanol extract of Citrus unshiu Markovich peel in human bladder cancer T24 cells through ROS-mediated inactivation of the PI3K/Akt pathway.

Citrus unshiu peel has been used to prevent and treat various diseases in traditional East-Asian medicine including in Korea. Extracts of C. unshiu peel are known to have various pharmacological effects including antioxidant, anti-inflammatory, and antibacterial properties. Although the possibility of their anti-cancer activity has recently been reported, the exact mechanisms in human cancer cells have not been sufficiently studied. In this study, the inhibitory effect of ethanol extract of C. unshiu peel (EECU) on the growth of human bladder cancer T24 cells was evaluated and the underlying mechanism was investigated. The present study demonstrated that the suppression of T24 cell viability by EECU is associated with apoptosis induction. EECU-induced apoptosis was found to correlate with an activation of caspase-8, -9, and -3 in concomitance with a decrease in the expression of the inhibitor of apoptosis family of proteins and an increase in the Bax:Bcl-2 ratio accompanied by the proteolytic degradation of poly(ADP-ribose) polymerase. EECU also increased the generation of reactive oxygen species (ROS), collapse of mitochondrial membrane potential, and cytochrome c release to the cytosol, along with a truncation of Bid. In addition, EECU inactivated phosphatidylinositol 3-kinase (PI3K) as well as Akt, a downstream molecular target of PI3K, and LY294002, a specific PI3K inhibitor significantly enhanced EECU-induced apoptosis and cell viability reduction. However, N-acetyl cysteine, a general ROS scavenger, completely reversed the EECU-induced dephosphorylation of PI3K and Akt, as well as cell apoptosis. Taken together, these findings suggest that EECU inhibits T24 cell proliferation by activating intrinsic and extrinsic apoptosis pathways through a ROS-mediated inactivation of the PI3K/Akt pathway.

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.

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.

An ethanol extract of Aster yomena (Kitam.) Honda inhibits lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages.

Aster yomena (Kitam.) Honda has been widely used as a traditional herbal medicine for centuries to treat cough, asthma, insect bites, etc. Recent reports indicate that A. yomena possesses a wide spectrum of pharmacological activities; however, few experiments have described its anti-inflammatory properties. The present study examined the anti-inflammatory effects of an ethanol extract of A. yomena leaves (EEAY) on lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages. Treatment with EEAY significantly reduced the secretion of pro-inflammatory molecules, such as nitric oxide and interleukin-1ß, in LPS-stimulated RAW 264.7 cells, without incurring any significant cytotoxicity. These protective effects were accompanied by a marked reduction in the expression of regulatory genes at the transcription level. Treatment with EEAY also inhibited the DNA-binding activity of nuclear factor-κB (NF-κB) by suppression of nuclear translocation of NF-κB and by degradation of the inhibitor of NF-κB; these effects were associated with suppression of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase signaling pathways. The EEAY treatment also potently suppressed LPS-induced toll like receptor (TLR) 4 expression and attenuated the binding of LPS to the macrophage cell surface. In addition, EEAY treatment markedly inhibited LPS-induced accumulation of intracellular reactive oxygen species in RAW 264.7 macrophages. Therefore, the inhibitory effects of EEAY on LPS-stimulated inflammatory responses in RAW 264.7 macrophages were apparently associated with suppression of the TLR-mediated NF-κB signaling pathway. More work is needed to fully understand the critical role and clinical usefulness of EEAY treatment, but the findings of the present study provide some insights into the potential of EEAY as a therapeutic agent for treatment of inflammatory disorders.

Protective effects of Fructus sophorae extract on collagen-induced arthritis in BALB/c mice.

Styphnolobium japonicum (L.) is utilized in Korean medicine for the treatment of various inflammatory diseases. The aim of the present study was to explore the effects of Fructus sophorae extract (FSE) isolated from the dried ripe fruit of S. japonicum (L.) on the development of type II collagen-induced arthritis (CIA) in BALB/c mice. The CIA mice were orally administered FSE or saline daily for 2 weeks. The incidence and severity of disease and the inflammatory response in the serum and the joint tissues were assessed. Macroscopic and histological investigation indicated that FSE protected against CIA development. FSE was associated with a significant reduction in the levels of total immunoglobulin G2a and proinflammatory cytokines and mediators in the serum. In addition, FSE suppressed the gene expression levels of proinflammatory cytokines and mediators, the mediator of osteoclastic bone remodeling, the receptor activator of nuclear factor κ-B ligand and matrix metalloproteinases in the joint tissues. The present results suggest that FSE may protect against inflammation and bone damage, and would be a valuable candidate for further investigation as a novel anti-arthritic agent.

Cytoprotective effects of esculetin against oxidative stress are associated with the upregulation of Nrf2-mediated NQO1 expression via the activation of the ERK pathway.

Esculetin, a coumarin derivative isolated from a variety of medicinal herbs, has been reported to possess multiple therapeutic and pharmacological actions. Although several studies have demonstrated the antioxidant activity of esculetin, its mechanisms of action have not been clearly established. The aim of this study was to evaluate the effects of esculetin against hydrogen peroxide (H2O2)­induced oxidative stress in C2C12 myoblasts and to investigate the mechanisms involved in this process. Our data indicated that esculetin preconditioning significantly attenuated H2O2­induced growth inhibition and DNA damage and the apoptosis of C2C12 cells by suppressing intracellular reactive oxygen species (ROS) accumulation. Treatment with esculetin effectively increased the phosphorylation of nuclear factor erythroid 2­related factor 2 (Nrf2) and the expression of NAD(P)H:quinone oxidoreductase 1 (NQO1). Esculetin treatment also activated extracellular signal­regulated kinase (ERK), and pre­treatment with PD98059, an ERK­specific inhibitor, blocked esculetin-mediated phosphorylation of Nrf2 and the induction of NQO1 expression. In addition, the protective effects of esculetin against H2O2­induced ROS accumulation, apoptosis and growth inhibition were abrogated in the C2C12 cells pre­treated with PD98059. Thus, the present study demonstrates that esculetin protects C2C12 cells against oxidative stress-induced injury, possibly through the activation of the Nrf2/NQO1 pathway.

Morin exerts cytoprotective effects against oxidative stress in C2C12 myoblasts via the upregulation of Nrf2-dependent HO-1 expression and the activation of the ERK pathway.

In the present study, we investigated the cytoprotective efficacy of morin, a natural flavonoid, against oxidative stress and elucidated the underlying mechanisms in C2C12 myoblasts. Our results indicated that morin treatment prior to hydrogen peroxide (H2O2) exposure significantly increased cell viability and prevented the generation of reactive oxygen species. H2O2-induced comet-like DNA formation and γH2AX phosphorylation were also markedly suppressed by morin with a parallel inhibition of apoptosis in C2C12 myoblasts, suggesting that morin prevented H2O2-induced cellular DNA damage. Furthermore, morin markedly enhanced the expression of heme oxygenase-1 (HO-1) associated with the induction and phosphorylation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and the inhibition of Kelch-like ECH-associated protein 1 (Keap1) expression. Notably, these events were eliminated by transient transfection with Nrf2­specific small interfering RNA. Additional experiments demonstrated that the activation of the Nrf2/HO-1 pathway by morin was mediated by the extracellular signal­regulated kinase (ERK) signaling cascade. This phenomenon was confirmed with suppressed Nrf2 phosphorylation and consequently diminished HO-1 expression in cells treated with a pharmacological inhibitor of ERK. Collectively, these results demonstrated that morin augments the cellular antioxidant defense capacity through the activation of Nrf2/HO­1 signaling, which involves the activation of the ERK pathway, thereby protecting C2C12 myoblasts from H2O2-induced oxidative cytotoxicity.

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.

Induction of apoptosis by ethanol extract of Evodia rutaecarpa in HeLa human cervical cancer cells via activation of AMP-activated protein kinase.

The fruit of Evodia rutaecarpa (Juss.) Benth has been used widely 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 an ethanol extract isolated from immature fruits of E. rutaecarpa (EEER) in HeLa human cervical cancer cells. EEER treatment decreased the cell viability of HeLa cells in a concentration-dependent manner, which was related to apoptotic cell death resulting from apoptotic body formation, DNA fragmentation, and an increased population of annexin V+-positive cells. EEER treatment significantly suppressed anti-apoptotic Bcl-2 expression, leading to subsequent loss of mitochondrial membrane potential (MMP), while it did not change expression levels of death receptor (DR)-related proteins. EEER treatment increased activity of caspase-3 and -9 but not caspase-8, and pretreatment of a caspase-3 inhibitor markedly attenuated EEER-induced apoptosis. Furthermore, EEER activated the AMP-activated protein kinase (AMPK) signaling pathway; however, inhibition of AMPK markedly abrogated EEER-induced apoptosis. Overall, the results suggest that the apoptotic activity of EEER may be associated with a caspase-dependent cascade through activation of the intrinsic signaling pathway connected with AMPK activation. E. rutaecarpa could be a prospective clinical application to treat human cervical cancer.

Baicalein Inhibits the Migration and Invasion of B16F10 Mouse Melanoma Cells through Inactivation of the PI3K/Akt Signaling Pathway.

Baicalein, a natural flavonoid obtained from the rhizome of Scutellaria baicalensis Georgi, has been reported to have anticancer activities in several human cancer cell lines. However, its antimetastatic effects and associated mechanisms in melanoma cells have not been extensively studied. The current study examined the effects of baicalein on cell motility and anti-invasive activity using mouse melanoma B16F10 cells. Within the noncytotoxic concentration range, baicalein significantly inhibited the cell motility and invasiveness of B16F10 cells in a concentration-dependent manner. Baicalein also reduced the activity and expression of matrix metalloproteinase (MMP)-2 and -9; however, the levels of tissue inhibitor of metalloproteinase-1 and -2 were concomitantly increased. The inhibitory effects of baicalein on cell motility and invasiveness were found to be associated with its tightening of tight junction (TJ), which was demonstrated by an increase in transepithelial electrical resistance and downregulation of the claudin family of proteins. Additionally, treatment with baicalein markedly reduced the expression levels of lipopolysaccharide-induced phosphorylated Akt and the invasive activity in B16F10 cells. Taken together, these results suggest that baicalein inhibits B16F10 melanoma cell migration and invasion by reducing the expression of MMPs and tightening TJ through the suppression of claudin expression, possibly in association with a suppression of the phosphoinositide 3-kinase/Akt signaling pathway.