Wen-Hsiung Chan

Curcumin inhibits UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermoid carcinoma A431 cells.

Ultraviolet (UV) light is a strong apoptotic trigger that induces caspase‐dependent biochemical changes in cells. Previously we showed that UV irradiation can activate caspase‐3, and the subsequent cleavage and activation of p21Cdc42/Rac‐activated kinase 2 (PAK2) in human epidermoid carcinoma A431 cells. In this study we demonstrate that curcumin (Cur), the yellow pigment of Curcuma longa with known anti‐oxidant and anti‐inflammatory properties, can prevent UV irradiation‐induced apoptotic changes, including c‐Jun N‐terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP), mitochondrial release of cytochrome C, caspase‐3 activation, and cleavage/activation of PAK2 in A431 cells. Flow cytometric analysis using the cell permeable dye 2′,7′‐dichlorofluorescin diacetate (DCF‐DA) as an indicator of reactive oxygen species (ROS) generation revealed that the increase in intracellular oxidative stress caused by UV irradiation could be abolished by Cur. In addition, we found that SP600125, a JNK‐specific inhibitor, reduced UV irradiation‐induced JNK activation as well as caspase‐3 activation, indicating that JNK activity is required for UV irradiation‐induced caspase activation. Collectively, our results demonstrate that Cur significantly attenuates UV irradiation‐induced ROS formation, and suggest that ROS triggers JNK activation, which in turn causes MMP change, cytochrome C release, caspase activation, and subsequent apoptotic biochemical changes. J. Cell. Biochem. 90: 327–338, 2003.

Apoptotic signaling in methylglyoxal‐treated human osteoblasts involves oxidative stress, c‐Jun N‐terminal kinase, caspase‐3, and p21‐activated kinase 2

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. MG is cytotoxic through induction of cell death, and elevated MG levels in diabetes patients are believed to contribute to diabetic complications. In this report, we show for the first time that MG treatment triggers apoptosis in human osteoblasts. We further show that MG‐induced apoptosis of osteoblasts involves specific apoptotic biochemical changes, including oxidative stress, c‐Jun N‐terminal kinase (JNK) activation, mitochondrial membrane potential changes, cytochrome C release, increased Bax/Bcl‐2 protein ratios, and activation of caspases (caspase‐9, caspase‐3) and p21‐activated protein kinase 2 (PAK2). Treatment of osteoblasts with SP600125, a JNK‐specific inhibitor, led to a reduction in MG‐induced apoptosis and decreased activation of caspase‐3 and PAK2, indicating that JNK activity is upstream of these events. Experiments using anti‐sense oligonucleotides against PAK2 further showed that PAK2 activation is required for MG‐induced apoptosis in osteoblasts. Interestingly, we also found that MG treatment triggered nuclear translocation of NF‐κB, although the precise regulatory role of NF‐κB activation in MG‐induced apoptosis remains unclear. Lastly, we examined the effect of MG on osteoblasts in vivo, and found that exposure of rats to dietary water containing 100–200 µM MG caused bone mineral density (BMD) loss. Collectively, these results reveal for the first time that MG treatment triggers apoptosis in osteoblasts via specific apoptotic signaling, and causes BMD loss in vivo. J. Cell. Biochem. 100: 1056–1069, 2007.

Anti‐apoptotic effects of curcumin on photosensitized human epidermal carcinoma A431 cells

Photodynamic treatment (PDT) can elicit a diverse range of cellular responses, including apoptotic cell death. Previously, we showed that PDT stimulates caspase‐3 activation and subsequent cleavage and activation of p21‐activated kinase 2 (PAK2) in human epidermal carcinoma A431 cells. Curcumin, the yellow pigment of Curcuma longa, is known to have anti‐oxidant and anti‐inflammatory properties. In the present study, using Rose Bengal (RB) as the photosensitizer, we investigated the effect of curcumin on PDT‐induced apoptotic events in human epidermal carcinoma A431 cells. We report that curcumin prevented PDT‐induced JNK activation, mitochondrial release of cytochrome c, caspase‐3 activation, and cleavage of PAK2. Using the cell permeable dye DCF‐DA as an indicator of reactive oxygen species (ROS) generation, we found that both curcumin and ROS scavengers (i.e., l‐histidine, a‐tocopherol, mannitol) abolished PDT‐stimulated intracellular oxidative stress. Moreover, all these PDT‐induced apoptotic changes in cells could be blocked by singlet oxygen scavengers (i.e., l‐histidine, a‐tocopherol), but were not affected by the hydroxyl radical scavenger mannitol. In addition, we found that SP600125, a JNK‐specific inhibitor, reduced PDT‐induced JNK activation as well as caspase‐3 activation, indicating that JNK activity is required for PDT‐induced caspase activation. Collectively, these results demonstrate that singlet oxygen triggers JNK activation, cytochrome c release, caspase activation and subsequent apoptotic biochemical changes during PDT and show that curcumin is a potent inhibitor for this process.

Cytotoxic effect of CdSe quantum dots on mouse embryonic development

AbstractAim:The aim of this study was to examine the cytotoxic effect of quantum dots (QD), a novel luminescent material, on early post-implantation embryonic development.Methods:Mouse blastocysts were incubated in medium with or without CdSe-core QD (250 or 500 nmol/L) for 24 h. Cell apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assay and Annexin V/propidium iodide staining, and proliferation was investigated by dual differential staining. Pre-implantation and post-implantation development was assessed by in vitro and in vivo analyses, respectively.Results:The apoptotic staining analysis showed that CdSe-core QD induced apoptosis in mouse blastocysts in a dose-dependent manner. Pretreatment of blastocysts with CdSe-core QD inhibited cell proliferation, primarily in the inner cell mass. CdSe-core QD also inhibited post-implantation embryonic development; fewer CdSe-core QD-pretreated blastocysts reached the later stages of development compared to the controls. The pre-implantation development of morulas into blastocysts was also inhibited by CdSe-core QD. Furthermore, CdSe-core QD at 500 nmol/L were associated with resorption of post-implantation blastocysts and a decrease in fetal weight. The cytotoxicity of CdSe QD in embryonic development was significantly reduced by the addition of a ZnS coating.Conclusion:Our results show that CdSe-core QD induce apoptosis in mouse blastocysts, inhibit cell proliferation, retard early post-implantation blastocyst development, and increase early-stage blastocyst death in vitro and in vivo.

Cytotoxic effects of CdSe quantum dots on maturation of mouse oocytes, fertilization, and fetal development.

Quantum dots (QDs) are useful novel luminescent markers, but their embryonic toxicity is yet to be fully established, particularly in oocyte maturation and sperm fertilization. Earlier experiments by our group show that CdSe-core QDs have cytotoxic effects on mouse blastocysts and are associated with defects in subsequent development. Here, we further investigate the influence of CdSe-core QDs on oocyte maturation, fertilization, and subsequent pre- and postimplantation development. CdSe-core QDs induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryo development, but not ZnS-coated CdSe QDs. Treatment of oocytes with 500 nM CdSe-core QDs during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. To our knowledge, this is the first study to report the negative impact of CdSe-core QDs on mouse oocyte development. Moreover, surface modification of CdSe-core QDs with ZnS effectively prevented this cytotoxicity.

Methylglyoxal and high glucose co-treatment induces apoptosis or necrosis in human umbilical vein endothelial cells

Hyperglycemia and elevation of methylglyoxal (MG) are symptoms of diabetes mellitus (DM). We previously showed that high glucose (HG; 30 mM) or MG (50–400 µM) could induce apoptosis in mammalian cells, but these doses are higher than the physiological concentrations of glucose and MG in the plasma of DM patients. The physiological concentration of MG and glucose in the normal blood circulation is about 1 µM and 5 mM, respectively. Here, we show that co‐treatment with concentrations of MG and glucose comparable to those seen in the blood circulation of DM patients (5 µM and 15–30 mM, respectively) could cause cell apoptosis or necrosis in human umbilical vein endothelial cells (HUVECs) in vitro. HG/MG co‐treatment directly increased the reactive oxygen species (ROS) content in HUVECs, leading to increases in intracellular ATP levels, which can control cell death through apoptosis or necrosis. Co‐treatment of HUVECs with 5 µM MG and 20 mM glucose significantly increased cytoplasmic free calcium levels, activation of nitric oxide synthase (NOS), caspase‐3 and ‐9, cytochrome c release, and apoptotic cell death. In contrast, these apoptotic biochemical changes were not detected in HUVECs treated with 5 µM MG and 30 mM glucose, which appeared to undergo necrosis. Pretreatment with nitric oxide (NO) scavengers could inhibit 5 µM MG/20 mM glucose‐induced cytochrome c release, decrease activation of caspase‐9 and caspase‐3, and increase the gene expression and protein levels of p53 and p21, which are known to be involved in apoptotic signaling. Inhibition of p53 protein expression using small interfering RNA (siRNA) blocked the activation of p21 and the cell apoptosis induced by 5 µM MG/20 mM glucose. In contrast, inhibition of p21 protein expression by siRNA prevented apoptosis in HUVECs but had no effect on p53 expression. These results collectively suggest that the treatment dosage of MG and glucose could determine the mode of cell death (apoptosis vs. necrosis) in HUVECs, and both ROS and NO played important roles in MG/HG‐induced apoptosis of these cells. J. Cell. Biochem. 103: 1144–1157, 2008.

Effect of resveratrol on high glucose-induced stress in human leukemia K562 cells.

Hyperglycemia, a symptom of diabetes mellitus, induces hyperosmotic responses, including apoptosis, in vascular endothelial cells and leukocytes. Hyperosmotic shock elicits a stress response in mammalian cells, often leading to apoptotic cell death. In a previous report, we showed that hyperosmotic shock induced apoptosis in various mammalian cells. Importantly, apoptotic biochemical changes (i.e., caspase‐3 activation and DNA fragmentation) were blocked by antioxidant pretreatment during hyperosmotic shock‐induced cell death. In the present study, we report that resveratrol, a phytoalexin present in grapes with known antioxidant and anti‐inflammatory properties, attenuates high glucose‐induced apoptotic changes, including c‐Jun N‐terminal kinase (JNK) activation and caspase‐3 activation in human leukemia K562 cells. Experiments with the cell permeable dye, 2′,7′‐dichlorofluorescein diacetate (DCF‐DA), an indicator of reactive oxygen species (ROS) generation, revealed that high glucose treatment directly increased intracellular oxidative stress, which was attenuated by resveratrol. In addition, high glucose‐treated K562 cells displayed a lower degree of attachment to collagen, the major component of vessel wall subendothelium. In contrast, cells pretreated with resveratrol followed by high glucose exhibited higher affinity for collagen. The results of this report collectively imply the involvement of oxidative stress in high glucose‐induced apoptosis and alterations in attachment ability. Moreover, resveratrol blocks these events by virtue of its antioxidant property.

Embryonic toxicity of sanguinarine through apoptotic processes in mouse blastocysts.

In this study, we examined the cytotoxic effects of sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation via embryo transfer. Blastocysts treated with 0.5-2 μM sanguinarine exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with sanguinarine were lower than that of their control counterparts. Moreover, in vitro treatment with 0.5-2 μM sanguinarine was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that sanguinarine induces apoptosis and retards early post-implantation development in vitro and in vivo. In addition, sanguinarine induces apoptotic injury effects on mouse blastocysts through intrinsic and extrinsic apoptotic signaling processes to impair sequent embryonic development. However, the extent to which sanguinarine exerts teratogenic effects on early human development is not known at present, and further studies are required to establish effective protection strategies against its cytotoxic effects.

Methylglyoxal has injurious effects on maturation of mouse oocytes, fertilization, and fetal development, via apoptosis.

Methylglyoxal (MG) is a metabolite of glucose. The serum MG level is increased in diabetic patients, and MG is implicated in diabetic complications related to embryonic development injury. We previously reported cytotoxic effects of MG on mouse embryonic stem cells and blastocysts, and a further association with defects in subsequent development. Here, we further investigate the effects of MG on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, MG induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with MG during in vitro maturation (IVM) led to increased resorption of post-implantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 10-20 microM MG led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented MG-triggered injury effects, suggesting that embryo impairment by MG occurs via a caspase-dependent apoptotic process.

Effect of genistein on mouse blastocyst development in vitro

AbstractAim:To examine the cytotoxic effects of genistein, an isoflavone compound, on early postimplantation embryonic development in vitro.Methods:Mouse blastocysts were incubated in medium with or without genistein (25 or 50 μmol/L) or daidzein (50 μmol/L) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, and apoptotic or necrotic cells were visualized by Annexin-V and propidium iodide (PI) staining. Implantation and postimplantation development of embryos were measured by in vitro development analysis.Results:TUNEL staining and Annexin-V/PI staining showed that genistein dose-dependently increased apoptosis in mouse blastocysts, while daidzein, another soy isoflavone, had no such effect. The pretreatment of the blastocysts with genistein caused fewer cells than the control group and this effect was primary in the inner cell mass. The genistein-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer genistein-pretreated embryos reached the later stages of embryonic development versus the controls, with many of the former embryos dying at relatively early stages of development. In addition, genistein treatment decreased the development of morulas into blastocysts, and dietary genistein was found to induce cell apoptosis and decrease cell proliferation in an animal assay model of embryogenesis.Conclusions:Our results collectively indicate that genistein treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death in vitro, while dietary genistein appears to negatively affect mouse embryonic development in vivo by inducing cell apoptosis and inhibiting cell proliferation. These novel findings provide important new insights into the effect of genistein on mouse blastocysts.