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.

Ethanol extract of Kalopanax septemlobus leaf induces caspase-dependent apoptosis associated with activation of AMPK in human hepatocellular carcinoma cells.

The Kalopanax septemlobus leaf (Thunb.) Koidz. has been used as a traditional medicine herb for the treatment of various human diseases for hundreds of years. In this study, we investigated the mechanism underlying the inhibitory effects of an ethanol extract of K. septemlobus leaf (EEKS) on proliferation of HepG2 hepatocellular carcinoma cells. For this study, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, DAPI (4,6-diamidino-2-phenylindole) staining, agarose gel electrophoresis, and flow cytometry. Measurements of the mitochondrial membrane potential (MMP), caspase activity assays and western blots were conducted to determine whether HepG2 cell death occurred by apoptosis. Treatment of HepG2 cells with EEKS concentration-dependently reduced cell survival while significantly increasing the ratio of apoptotic cells. EEKS treatment increased the levels of the death receptors (DRs), DR4 and DR5, and activated caspases, as well as promoting proteolytic degradation of poly(ADP-ribose)-polymerase associated with the downregulation of protein expression of members of the inhibitor of apoptosis protein family. Treatment with EEKS also caused truncation of Bid, translocation of pro-apoptotic Bax to the mitochondria, and loss of mitochondrial membrane permeabilization, thereby inducing the release of cytochrome c into the cytosol. However, treatment of HepG2 cells with a pan-caspase inhibitor reversed EEKS-induced apoptosis and growth suppression, indicating that EEKS appears to induce apoptosis though a caspase-dependent mechanism involving both intrinsic and extrinsic apoptotic pathways. In addition, the phosphorylation level of AMP-activated protein kinase (AMPK) was elevated when cells were exposed to EEKS. A specific inhibitor for AMPK attenuated the EEKS-induced activation of caspases, and consequently prevented the EEKS-induced apoptosis and reduction in cell viability. Overall, our findings suggest that EEKS inhibits the growth of HepG2 cells by inducing AMPK-mediated caspase-dependent apoptosis, suggesting the potential therapeutic application of EEKS in the treatment or prevention of cancers.

An exploration of the antioxidant effects of garlic saponins in mouse-derived C2C12 myoblasts.

In this study, we aimed to confirm the protective effects of garlic saponins against oxidative stress-induced cellular damage and to further elucidate the underlying mechanisms in mouse-derived C2C12 myoblasts. Relative cell viability was determined by 3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide assay. Comet assay was used to measure DNA damage and oxidative stress was determined using 2',7'-dichlorofluorescein diacetate to measure intracellular reactive oxygen species (ROS) generation. Western blot analysis and small interfering RNA (siRNA)-based knockdown were used in order to investigate the possible molecular mechanisms. Our results revealed that garlic saponins prevented hydrogen peroxide (H2O2)-induced growth inhibition and exhibited scavenging activity against intracellular ROS. We also observed that garlic saponins prevented H2O2-induced comet tail formation and decreased the phosphorylation levels of γH2AX expression, suggesting that they can prevent H2O2-induced DNA damage. In addition, garlic saponins increased the levels of heme oxygenase-1 (HO-1), a potent antioxidant enzyme associated with the induction and phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2) and the translocation of Nrf2 from the cytosol into the nucleus. However, the protective effects of garlic saponins on H2O2-induced ROS generation and growth inhibition were significantly reduced by zinc protoporphyrin Ⅸ, an HO-1 competitive inhibitor. In addition, the potential of garlic saponins to mediate HO-1 induction and protect against H2O2­mediated growth inhibition was adversely affected by transient transfection with Nrf2-specific siRNA. Garlic saponins activated extracellular signal­regulated kinase (ERK) signaling, whereas a specific ERK inhibitor was able to inhibit HO-1 upregulation, as well as Nrf2 induction and phosphorylation. Taken together, the findings of our study suggest that garlic saponins activate the Nrf2/HO-1 pathway by enabling ERK to contribute to the induction of phase Ⅱ antioxidant and detoxifying enzymes, including HO-1 in C2C12 cells.

N-benzyl-N-methyldecan-1-amine, a phenylamine derivative isolated from garlic cloves, induces G2/M phase arrest and apoptosis in U937 human leukemia cells.

Epidemiological studies indicate that components of garlic (Allium sativum) have anti-proliferative effects against various types of cancer. In the present study, we investigated the effect of newly isolated phenylamine derivative N-benzyl-N-methyldecan-1-amine (NBNMA) from garlic cloves on the inhibition of the growth and apoptosis of human leukemia U937 cells and its potential anticancer mechanism. NBNMA exhibited an antiproliferative effect in U937 cells by inducing cell cycle arrest at the G2/M phase and apoptotic cell death. Western blot analyses revealed that NBNMA decreased the expression of the regulator genes of G2/M phase progression, cyclin dependent kinase (Cdk) 2 and Cdc2 and elevated the expression of the Cdk inhibitor p21WAF1/CIP1 in a p53-independent manner. In addition, NBNMA activated caspase-8 and caspase-9, initiator caspases of the extrinsic and intrinsic pathways of apoptosis, respectively, which led to activation of executioner caspase-3 along with degradation of poly(ADP-ribose) polymerase. NBNMA-induced apoptosis was observed in parallel with an increased ratio of pro-apoptotic Bax and Bad/anti-apoptotic Bcl-2 and Bcl-xL, and inhibition of inhibitor of apoptosis protein (IAP) family members XIAP and cIAP-1. Furthermore, NBNMA-treated cells displayed enhanced release of cytochrome c from the mitochondria into the cytosol concomitant with a loss of mitochondrial membrane potential and downregulation of Bid, suggesting that NBNMA-induced apoptosis occurred via the extrinsic and intrinsic apoptotic pathways with a possible link to Bid protein activity between the two pathways. These results indicate that NBNMA has promising potential to become a novel anticancer agent for the treatment of leukemia. We provide new insight into the mechanisms underlying the anticancer effect of NBNMA.

Resveratrol analogue HS-1793 induces the modulation of tumor-derived T cells.

Recent advances in the understanding of the mechanisms responsible for tumor progression suggest the possibility to control cancer growth, not only through chemotherapy-induced cancer cell destruction, but also by stimulating anticancer immunity. However, immune tolerance against tumor antigens disturbs diverse forms of immunotherapy. One of the most potent and well-studied tumor-induced immunosuppressive phenotypes found in the tumor microenvironment is the regulatory subpopulation cells (CD4(+)CD25(+)FoxP3(+) Treg cells). Among the great number of natural agents derived from plants and potentially useful for application in the complementary therapy of cancer, resveratrol is gaining attention for its immunomodulating properties in breast cancer, since the ineffectiveness of numerous immunotherapy strategies may be related, in part, to their negative effects on Treg cells. The present study was undertaken to examine whether HS-1793, a synthetic resveratrol analogue free from the restriction of the metabolic instability and high dose requirement of resveratrol, shows a direct effect on immune responses by enhancing lymphocyte proliferation or an immunomodulatory effect by inducing changes in the Treg cell population in FM3A breast tumor-bearing mice. Although HS-1793 had no direct immunostimulatory effect, it dose-dependently decreased IL-2 secretion and increased IL-4 secretion of concanavalin A-stimulated lymphocytes from tumor-bearing mice, which suggest that HS-1793 may induce changes in the subpopulations of tumor-derived T lymphocytes. The CD4(+)CD25(+) cell population from tumor-bearing mice decreased after HS-1793 treatment in a dose-dependent manner, while the CD4(+) T cell population remained unchanged. FoxP3(+)-expressing cells among the CD4(+)CD25(+) population showed a similar pattern. In contrast, the CD8(+) T cell population as well as the interferon (IFN)-γ-expressing CD8(+) T cell population and IFN-γ secretion of splenocytes from tumor-bearing mice were significantly upregulated by HS-1793 treatment. These results suggest that HS-1793 induces the modulation of tumor-derived T lymphocytes, particulary having a suppressive effect on the Treg cell population, likely contributing to enhanced tumor-specific cytotoxic T lymphocyte responses and CD4(+) T cells involving antitumor immunity. Therefore, HS-1793 may serve as a promising adjuvant therapeutic reagent in breast cancer immunotherapy.

Resveratrol analog, HS-1793 enhance anti-tumor immunity by reducing the CD4+CD25+ regulatory T cells in FM3A tumor bearing mice.

Natural agents with the immunomodulating property have been gaining traction to be employed in the complementary therapy of cancer because the ineffectiveness of numerous therapeutic strategies may be related in part to the tumor-induced immunosuppressive phenotypes, especially regulatory T (Treg) cells found in the tumor microenvironment. The present study was undertaken to examine whether HS-1793, synthetic resvertrol analog free from the restriction of metabolic instability and high dose requirement of resveratrol, induces an in vivo anti-tumor effect in FM3A tumor bearing mice through the suppression of Treg cells, which contribute to an increase in tumor specific cytotoxic T cell responses. Intraperitoneal injections of HS-1793 showed not only therapeutic benefits on established tumors, but also preventive anti-tumor effects. Treg cells (CD4+CD25+Foxp3+ cells) were significantly reduced in the total splenocytes as well as tumor tissues from HS-1793-administered mice, and the production of TGF-ß inducing Treg showed a similar pattern. On the contrary, the administration of HS-1793 increased IFN-γ-expressing CD8+ T cells, upregulated IFN-γ production, and enhanced the cytotoxicity of splenocytes against FM3A tumor cells both in therapeutic and preventive experimental animals. These results demonstrated the suppressive role of HS-1793 on the function of Treg cells contributing to tumor specific cytotoxic T lymphocyte responses in tumor-bearing mice, which explained the underlying mechanism of the anti-tumor immunity of HS-1793.

Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI-H460 human lung carcinoma cells.

The root of Mori cortex has traditionally been used in Korea for the treatment of cutaneous inflammation, pulmonary asthma, and congestion for thousands of years. The present study was designed to validate the anticancer effects of methylene chloride extracts of the M. cortex root (MEMC) in NCI-H460 human lung carcinoma cells. Exposure to MEMC was found to result in growth inhibition by the induction of caspase­dependent apoptosis in NCI-H460 cells, which correlated with upregulated expression of death receptor (DR)4, DR5 and FasL, downregulation of anti-apoptotic Bcl-2 and Bcl-xL expression, cleavage of Bid, and loss of mitochondrial membrane potential. In addition, autophagosomes, a characteristic finding of autophagy, and markers of autophagy, conversion of microtubule-associated protein light chain-3 (LC3)-I to LC3-II and increased beclin-1 accumulation, were observed in MEMC-treated NCI-H460 cells. Inhibition of autophagy by 3-methyladenine or LC3B small interfering (siRNA) resulted in enhanced apoptotic cell death, suggesting that MEMC-induced autophagy functions as a suppressor of apoptosis. MEMC-induced autophagy was also blocked by N-acetyl-cysteine (NAC) and catalase, indicating that H2O2 can regulate autophagy. Our data demonstrate that MEMC triggers both ROS-mediated autophagy and caspase-dependent apoptosis, and that autophagy plays a protective role against apoptotic cell death.

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.

Efficacy on anaplastic thyroid carcinoma of valproic acid alone or in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative, or lactacystin.

The present study investigated the mechanism underlying the antitumor activity of the histone deacetylases inhibitor valproic acid (VPA), alone and in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative (HS-1200), or the proteasome inhibitor lactacystin on cultured anaplastic thyroid carcinoma KAT-18 cells. Cell viability was evaluated by trypan-blue exclusion. Western blotting determined caspase and histone deacetylase activities and expression of poly(ADP)-ribose polymerase. Induction of apoptosis was identified by Hoechst staining, DNA electrophoresis, DNA hypoploidy and cell cycle phase analysis, and measurement of mitochondrial membrane potential. Subcellular translocation of apoptosis inducing factor and caspase-activated DNase after treatment was determined by confocal microscopy following immunofluorescent staining. VPA treatment increased apoptotic death of KAT-18 cells. VPA treatment was also associated with degradation of procaspase-3, procaspase-7, and poly(ADP)-ribose polymerase; induction of histone hyperacetylation; condensation of peripheral chromatin; decreased mitochondrial membrane potential and DNA content; and decreased translocation of apoptosis inducing factor and caspase-activated DNase. VPA in combination with doxorubicin, HS-1200, or lactacystin, applied at the highest concentrations that did not induce KAT-18 cell death, efficiently induced apoptosis in KAT-18 cells. The results suggest VPA combination therapy may represent an alternative therapeutic strategy for anaplastic thyroid carcinoma.

A purified extract from Clematis mandshurica prevents staurosporin-induced downregulation of 14-3-3 and subsequent apoptosis on rat chondrocytes.

OBJECTIVE: To dissect the mechanism of the protection of staurosporin-induced apoptosis on rat chondrocytes by a purified extract from Clematis mandshurica. DESIGN: Primary cultured rat articular chondrocytes as well as RCJ3.1C.18 cells were incubated with 1 microM staurosporin and 300 microg/ml purified extract from Clematis mandshurica. Western blot assay, silencing 14-3-3 gene and immunoprecipitation were conducted. RESULTS: Clematis mandshurica prevented staurosporin-induced downregulation of several antiapoptotic bcl-2 family proteins Bcl-xL and Bcl-2, and staurosporin-induced upregulation of an apoptotic bcl-2 family protein Bax. Clematis mandshurica also prevented staurosporin-induced downregulation of a premitochondrial antiapoptotic protein 14-3-3. It is noticeable that siRNA to 14-3-3 abolished the prevention of caspase-3 activation by Clematis mandshurica. Furthermore viability assay corroborated that silencing of 14-3-3 gene abolished this apoptosis protection efficacy by Clematis mandshurica. Immunoprecipitation assay elucidated that Clematis mandshurica prevented the staurosporin-induced reduction of the interactions between 14-3-3 with phospho-ser112-Bad and Bcl-xL to phospho-ser155-Bad. CONCLUSIONS: Clematis mandshurica prevents staurosporin-induced apoptosis of rat chondrocytes via 14-3-3.

Caspase-dependent and caspase-independent apoptosis induced by evodiamine in human leukemic U937 cells.

Evodiamine is one of the major bioactive compounds that have been isolated and purified from the fruit of Evodiae fructus. Evodiamine exhibits antitumor activities against the human tumor cells, including multidrug-resistant tumor cells. However, the molecular mechanism involved in cell death induced by evodiamine treatment remains poorly understood. In the present study, we showed that evodiamine activated the caspase-dependent apoptotic pathway. This apoptosis was only partially inhibited by a pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, which suggested that evodiamine-induced apoptosis in leukemic U937 cells is partially caspase independent. We observed the nuclear translocation of apoptosis-inducing factor in evodiamine-induced apoptosis of U937 cells, which may be responsible for the caspase-independent apoptotic execution. We next showed that evodiamine induced the substantial amount of apoptosis both in Bcl-2- and Akt-overexpressing U937 cells but not in human peripheral blood mononuclear cells. Although benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited caspase activity in Bcl-2-overexpressing U937 cells, it completely prevented neither the induction of apoptosis or the nuclear translocation of apoptosis-inducing factor, which suggests that evodiamine is, at least in part, able to bypass the resistance of leukemia cells via caspase-independent apoptotic pathways. Thus, therapeutic strategy using evodiamine may warrant further evaluation.

Poncirus trifoliata fruit induces apoptosis in human promyelocytic leukemia cells.

BACKGROUND: Substances inducing apoptosis have shown efficacy in the treatment of cancers. Poncirus trifoliata (L.) Raf. (Rutaceae) fruits (PTF) has been used for the treatment of various cancers among Korean Oriental Medical doctors. METHODS: PTF-induced cytotoxicity of human leukemia HL-60 cells was monitored by the MTT assay. The apoptosis was determined by (a) apoptotic morphology in microscopy; (b) DNA fragmentation in electrophoresis and FACS analysis; and (c) activation of caspase-3 and poly-ADP-ribose polymerase (PARP) cleavage assay. RESULTS: The cytotoxic activity of PTF in HL-60 cells was increased in a concentration- and time-dependent manner. PTF caused the cell shrinkage, cell membrane blebbing, apoptotic body and DNA fragmentation. PTF-induced apoptosis is accompanied by the activation of caspase-3 and the specific proteolytic cleavage of PARP. However, PTF did not show cytotoxicity in normal peripheral blood mononuclear cells. CONCLUSIONS: Our novel finding provides evidence that PTF could be a candidate as an anti-leukemic agent through apoptosis of cancer cells.

Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

Magnoliae flos induces apoptosis of RBL-2H3 cells via mitochondria and caspase.

BACKGROUND: Magnoliae flores (MF), the buds of Magnolia denudata Desrousseaux, have been successfully used for the management of allergic diseases in Korea. The purpose of the present study was to determine their causal role in inducing apoptosis of mast cells and to verify the underlying mechanism. METHODS: The viability of mast cells was assessed by the trypan blue exclusion test. Induction of apoptosis was confirmed by DNA fragmentation, nuclear staining and DNA hypoploidy. Western blotting and immunofluorescent staining were performed to study the alterations in expression level and translocation of apoptosis-related proteins. Mitochondrial membrane potential (MMP) change and cytochrome C release were assayed. RESULTS: We present several lines of evidence indicating that MF induce apoptosis. Changes in cell morphology, generation of DNA fragmentation, cell cycle arrest, activation of caspase-3, and PARP and DFF degradations were demonstrated. The reduction of MMP and the release of cytochrome C to cytosol were also shown. Either PTP blockers, bongkrekic acid and cyclosporin A, or pancaspase inhibitors, Boc.D-fmk and zVAD-fmk, did not prevent the release of cytochrome C. Bax protein content was increased, and Bax was translocated from cytosol into mitochondria at early time points after MF treatment. CONCLUSIONS: We have demonstrated that MF induce mitochondria- and caspase-dependent mast cell apoptosis. Our observations contribute new insights to the role of MF and support the view that the clinical effect of MF may depend on their pharmacological efficacy in regulating mast cell apoptosis.