Yan-Der Hsuuw

Curcumin prevents methylglyoxal-induced oxidative stress and apoptosis in mouse embryonic stem cells and blastocysts

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG is cytotoxic through induction of apoptosis. Curcumin, the yellow pigment of Curcuma longa, is known to have antioxidant and anti‐inflammatory properties. In the present study, we examined the effect of curcumin on apoptotic biochemical events caused by incubation of ESC‐B5 cells with MG. Curcumin inhibited the MG‐induced DNA fragmentation, caspase‐3 activation, cleavage of PARP, mitochondrial cytochrome c release, and JNK activation. Importantly, curcumin also inhibited the MG‐stimulated increase of reactive oxygen species (ROS) in these cells. In addition, we demonstrated that curcumin prevented the MG‐induced apoptosis of mouse blastocysts isolated from pregnant mice. Moreover, curcumin significantly reduced the MG‐mediated impairment of blastocyst development from mouse morulas. The results support the hypothesis that curcumin inhibits MG‐induced apoptosis in mouse ESC‐B5 cells and blastocysts by blocking ROS formation and subsequent apoptotic biochemical events.

Epigallocatechin Gallate Dose-Dependently Induces Apoptosis or Necrosis in Human MCF-7 Cells

Abstract:  The catechins, a family of polyphenols found in tea, can evoke various responses, including cell death. However, the precise molecular mechanisms of these effects are unknown. Here, we demonstrate that treatment of human MCF‐7 cells with 50 μM (‐)‐Epigallocatechin‐3‐gallate (EGCG), a catechin that is highly abundant in green tea, can induce apoptotic changes, including mitochondrial membrane potential changes and activation of c‐Jun N‐terminal kinase (JNK), caspase‐9, and caspase‐3. In contrast, higher concentrations of EGCG (100–400 μM) do not induce apoptosis, but rather trigger necrotic cell death in MCF‐7 cells. Investigations of the possible mechanisms underlying these differences revealed that treatment with lower concentrations of EGCG (10–50 μM) directly increased intracellular oxidative stress, while higher concentrations (100–400 μM) did not. Immunoblotting revealed that treatment of MCF‐7 cells with 10–50 μM EGCG caused increases in Bax protein levels and decreases in Bcl‐2 protein levels, shifting the Bax–Bcl‐2 ratio to favor apoptosis, while treatment with 100–400 μM EGCG had no such effect. Moreover, we observed a dose‐dependent decrease in intracellular ATP levels in cells treated with high‐dose EGCG. Blockade of reactive oxygen species (ROS) generation and ATP synthesis using antioxidants and ATP synthesis inhibitors revealed that ROS and ATP play important roles to switch cell death types with apoptosis or necrosis. Collectively, these results indicate for the first time that EGCG treatment has a dose‐dependent effect on ROS generation and intracellular ATP levels in MCF‐7 cells, leading to either apoptosis or necrosis, and that the apoptotic cascade involves JNK activation, Bax expression, mitochondrial membrane potential changes, and activation of caspase‐9 and caspase‐3.

Higher LDL Oxidation at Rest and after a Rugby Game in Weekend Warriors

Background/Aim: Weekend warriors (WW), people who only exercise vigorously once a week, are a fast-growing population. The present study investigates LDL oxidation and activities of antioxidant enzymes in WW (n = 6) at rest and after a rugby game. The results are compared with those of sedentary controls (CON, n = 10) and trained rugby players (TR, n = 15). Methods: LDL + VLDL conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) as well as erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were analyzed in WW and TR groups before and after a rugby game. Results: The WW showed higher LDL+VLDL CD and TBARS levels than TR and CON groups at rest (p < 0.05). LDL+VLDL CD and TBARS were also significantly higher in the WW group than in the TR group after exercise (p < 0.05). Both WW and TR groups showed significant exercise-induced elevations in LDL+VLDL TBARS (p < 0.05), with the WW having higher degrees of increase (15.8 ± 7.9 vs. 6.3 ± 9.0%, WW group vs. TR group; p < 0.05). Erythrocyte SOD and GPx activities at rest were similar in all three groups. Conclusion: These results suggest that WW may have a higher LDL oxidation than TR and CON persons at rest and higher exercise-induced LDL oxidation levels than TR athletes.

Ochratoxin A Inhibits Mouse Embryonic Development by Activating a Mitochondrion-Dependent Apoptotic Signaling Pathway

Ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 μM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish) or after in vivo embryo transfer. However, in vitro treatment with 10 μM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.

Hazardous Effects of Curcumin on Mouse Embryonic Development through a Mitochondria-Dependent Apoptotic Signaling Pathway

In this study, we examined the cytotoxic effects of curcumin, the yellow pigment of Curcuma longa, on the blastocyst stage of mouse embryos, subsequent embryonic attachment, and outgrowth in vitro and in vivo implantation by embryo transfer. Mouse blastocysts were incubated in medium with or without curcumin (6, 12 or 24 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining, apoptosis was analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and implantation and post-implantation development of embryos were measured by in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 24 μM curcumin displayed significantly increased apoptosis and decreased total cell number. Interestingly, we observed no marked differences in the implantation success rates between curcumin-pretreated and control blastocysts during in vitro embryonic development through implantation with a fibronectin-coated culture dish. However, in vitro treatment with 24 μM curcumin was associated with decreased implantation rate and increased resorption of postimplantation embryos in mouse uterus, as well as decreased fetal weight in the embryo transfer assay. Our results collectively indicate that in vitro exposure to curcumin triggers apoptosis and retards early postimplantation development after transfer to host mice. In addition, curcumin induces apoptotic injury effects on mouse blastocysts through ROS generation, and further promotes mitochondria-dependent apoptotic signaling processes to impair sequent embryonic development.

Dosage Effects of Ginkgolide B on Ethanol-Induced Cell Death in Human Hepatoma G2 Cells

Abstract:  Ginkgolide B is a major active component of Ginkgo biloba extracts, which has been shown to confer anticancer effects by inducing apoptosis or inhibiting oxidative stress generation. Ethanol induces a wide range of cellular toxicities, many of which have been linked to free radical generation. To further elucidate the cellular effects of ginkgolide B, we examined the dose–response effect of ginkgolide B on ethanol‐induced toxicity in human Hep G2 cells. TUNEL and MTT assays revealed that ethanol (50–400 mM) induced apoptotic cell death in human Hep G2 cells, and that this effect was inhibited by low (5–25 μM) doses of ginkgolide B, but enhanced by high (50–100 μM) doses of ginkgolide B. Additional experiments revealed that ethanol treatment directly increased intracellular oxidative stress; this effect was enhanced by high doses of ginkgolide B but decreased following treatment with low concentrations of ginkgolide B. The dose–response effects of ginkgolide B on reactive oxygen species (ROS) generation were directly correlated with cell apoptotic biochemical changes including c‐Jun N‐terminal kinase (JNK) activation, caspase‐3 activation, and DNA fragmentation. These results indicate that treatment dosage may determine the effect of ginkgolide B on ethanol‐induced ROS generation and cell apoptosis, and support the notion that an appropriate dosage of ginkgolide B may aid in decreasing the toxic effects of ethanol.

Characterization of Apoptosis Induced by Emodin and Related Regulatory Mechanisms in Human Neuroblastoma Cells

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10–20 μM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 μM. Treatment with emodin at concentrations of 10–20 μM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 μM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10–20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca2+, NO, p53, caspase-9 and caspase-3.

Ginkgolide B Induces Apoptosis via Activation of JNK and p21‐Activated Protein Kinase 2 in Mouse Embryonic Stem Cells

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signaling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards the proliferation and development of mouse embryonic stem cells and blastocysts in vitro, and causes developmental injury in vivo. However, the precise molecular mechanisms underlying its actions are currently unknown. Here, our study further revealed that GKB induced apoptotic biochemical changes, including activation of JNK, caspase‐3, and p21‐activated protein kinase 2 (PAK2), in ESC‐B5 mouse embryonic stem cells. Treatment of ESC‐B5 cells with a JNK‐specific inhibitor (SP600125) reduced GKB‐induced activation of both JNK and caspase‐3, indicating that JNK activity is required for GKB‐induced caspase activation. Experiments using caspase‐3 inhibitors and antisense oligonucleotides against PAK2 showed that caspase‐3 activation is required for PAK2 activation and both of these activations are required for GKB‐induced apoptosis in ESC‐B5 cells.

Effect of curcumin on in vitro early post-implantation stages of mouse embryo development §

OBJECTIVES This study was designed to examine the embryotoxic potential of the curcumin at the blastocyst stage and during early post-implantation development of mouse embryos in vitro. STUDY DESIGN Curcumin was administered to ICR mice embryos at a dose of 0, 6, 12, 24 μM throughout in vitro culture. A total of 1015 embryos were randomly assigned to the different dosage groups. The embryotoxic effects were studied by the exposure of curcumin at the blastocyst, implanted blastocyst and early egg cylinder stages, respectively. For assessment of implantation in vitro and further embryonic differentiation, blastocysts were cultured for 8 days. The cell proliferation of outgrowth blastocysts was analysed by Giemsa staining. RESULTS Exposure to 24 μM of curcumin at the implanted blastocyst stage or early egg stage cause adverse effects on development. The percentage of embryos in the later stages of development was changed depending upon the dose of curcumin used. Furthermore, exposure to 24 μM of curcumin at the blastocyst stage was lethal to all embryos. The number of nuclei per outgrowth of the blastocyst decreased significantly after curcumin pre-treatment. The percentage of trophoblastic giant cells per outgrowth increased significantly after curcumin pre-treatment. CONCLUSIONS These findings demonstrate that curcumin exerts an adverse effect on mouse embryos during the early post-implantation stages of development, equivalent to day 3-day 8 of gestation in vivo. Curcumin treatment or administration should be used carefully at the early post-implantation stage of gestation.

Apoptotic effects of dillapiole on maturation of mouse oocytes, fertilization and fetal development

Abstract Previously, we reported that dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal and acaricidal activities, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to impaired embryonic development and cell viability. In the current study, we investigated the deleterious effects of dillapiole on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, dillapiole induced significant impairment of mouse oocyte maturation, decrease in the IVF rate and inhibition of subsequent embryonic development in vitro. Pre-incubation of oocytes with dillapiole during in vitro maturation led to an increase in post-implantation embryo resorption and decrease in mouse fetal weight. In an in vivo animal model, 2.5, 5 or 10 μM dillapiole provided in drinking water caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked dillapiole-triggered deleterious effects, clearly implying that embryonic injury induced by dillapiole is mediated via a caspase-dependent apoptotic mechanism. To the best of our knowledge, this is the first study to establish the impact of dillapiole on maturation of mouse oocytes, fertilization and sequential embryonic development.