Kee-Min Woo

Nickel(II)-induced apoptosis and G2/M enrichment

Treatment with certain DNA-damaging agents induce a complex cellular response comprising pertubation of cell cycle progression and/or apoptosis on proliferating mammalian cells. Our studies were focused on the cellular effects of nickel (II) acetate, DNA-damaging agent, on Chinese hamster ovary (CHO) cells. Fragmented DNAs were examined by agarose gel electrophoresis and cell cycle was determined by DNA flow cytometry using propidium iodide fluorescence. Apparent DNA laddering was observed in cells treated with 240 µM nickel (II) and increased with a concentration-dependent manner. Treatment of nickel (II) acetate resulted in apoptosis which was accompanied by G2/M cell accumulation. Proportion of CHO cells in G2/M phase was also significantly increased in cells exposed to at least 480 µM nickel (II) from 57.7% of cells in the G0/G1 phase, 34.7% in the S phase, and 7.6% in the G2/M1 phase for 0 microM nickel (II), to 58.6%, 14.5%, and 26.9% for 640 µM nickel (II). These findings suggest that nickel (II) can modulate cellular response through some common effectors involving in both apoptotic and cell cycle regulatory pathways.

Relationship between pulmonary surfactant protein and lipid peroxidation in lung injury due to paraquat intoxication in rats.

Background Pulmonary damage resulting from lipid peroxidation is a principal effect of paraquat intoxication. The host-defense functions of surfactant are known to be mediated by the surfactant proteins A and D (SP-A and SP-D, respectively). The primary objective of this study was to evaluate the variations over time in levels of surfactant protein and lipid peroxidation (LPO) in lung tissue following free-radical-induced injury. Methods 42 adult, male, Sprague-Dawley rats were administered intraperitoneal injections of paraquat (35 mg/kg body weight). SP-A and SP-D levels were determined via Western blot. LPO in the left lung homogenate was measured via analyses of the levels of thiobarbituric acid-reactive substances. Results LPO levels peaked at 6 hours, with no associated histological changes. SP-D levels increased until hour 12 and declined until hour 48; SP-D levels subsequently began to increase again, peaking at hour 72. SP-A levels peaked at hour 6, declining thereafter. Conclusions We suggest that in the early phase of paraquat injury, SP-D levels reflect alveolar damage and that de novo synthesis of SP-D takes 72 hours. Levels of SP-A, on the other hand, reflect abnormalities in the surfactant system in the late stage of paraquat intoxication. Surfactant proteins may play a role in protecting the lungs from reactive oxygen injury. A time-dependent variation has been observed in the levels of surfactant proteins A and D following paraquat injury, and it has been suggested that these proteins play a role in the protection of lung tissue against ROS-induced injuries.

Nickel(II)-induced nasal epithelial toxicity and oxidative mitochondrial damage

In probing the underlying mechanisms of nickel(II)-induced cytotoxicity on nasal epithelium, we investigated the effects of nickel(II) acetate on nasal epithelial RPMI-2650 cells. Nickel(II) elicited apoptosis, as signified by pyknotic and fragmented nuclei, increased caspase-3/7 activity, and an increase in annexin V binding, hypodiploid DNA, and Bax/Bcl-2 protein ratio. Nickel(II)-induced G2/M arrest was associated with up-regulation of p21(WAF1/CIP1) expression, decrease in phosphorylation at Thr(161) of Cdc2, and down-regulation of cyclin B1. Associated with these responses, ROS generation and mitochondrial depolarization increased in a nickel(II) concentration-dependent fashion. Pretreatment with N-acetylcysteine (NAC) attenuated these changes. p53 reporter gene assay and analyses of p53, Puma, Bax, and Bcl-2 protein levels indicated that NAC inhibited nickel(II)-induced activation of p53-mediated mitochondrial apoptotic pathway. Collectively, our study provides evidences that nickel(II) may induce oxidative damage on nasal epithelium in which antioxidant NAC protects cells against nickel(II)-induced apoptosis through the prevention of oxidative stress-mediated mitochondrial damage.

Phosphoinositol 3-kinase, a novel target molecule for the inhibitory effects of juglone on TPA-induced cell transformation

Juglone (5-hydroxy-1,4-naphthalenedione) from black walnut trees induces apoptosis and inhibits proliferation of various malignant cells. Here, we investigated whether juglone affects 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell transformation through the phosphoinositol 3-kinase (PI3K) pathway. The results showed that TPA- and endothelial growth factor (EGF)-induced anchorage-independent colony formation were suppressed in a dose-dependent manner by treatment of JB6 CI41 mouse skin epidermal cells with juglone (2.5 and 5 µM). We demonstrated that juglone suppressed PI3K activity via direct binding to PI3K by sepharose 4B pull-down assay and western blot analysis. Juglone significantly suppressed TPA-induced protein kinase B (AKT) and c-Jun phosphorylation and c-fos activation, but not mitogen-activated protein-kinase kinase (MEK), extracellular signaling-regulated kinase (ERK) or 90 kDa ribosomal protein S6 kinase (RSK) phosphorylation. Juglone significantly blocked activator protein-1 (AP-1) and cyclooxygenase-2 (COX-2) activation more than the PI3K inhibitors LY294002 and wortmannin. Overall, these results showed the anticancer efficacy of juglone targeting PI3K to prevent TPA-induced tumorigenesis.

Quercetin exerts preferential cytotoxic effects on malignant mesothelioma cells by inducing p53 expression, caspase-3 activation, and apoptosis

Quercetin, a naturally occurring flavonoid, has been heralded as a promising chemopreventive agent. This study was undertaken to probe the molecular mechanisms underlying the anti-cancer activity of quercetin in malignant mesothelioma (MM) cells. Quercetin at low doses elicited apoptotic cell death on MM MSTO-211H cells, as signified by pyknotic and fragmented nuclei, increased annexin V binding, and increased proportion of cells with hypodiploid DNA. Preceding these changes, quercetin induced up-regulation of p53 at both mRNA and protein levels without altering its ubiquitination, and increased caspase-3/7 activity with the resultant cleavages of procaspase-3 and PARP. Analyses of nuclear p53 level, p53 reporter gene, and RT-PCR toward p53-regulated genes demonstrated that induced p53 is transcriptionally active. The proportion of cells at sub-G0/G1 peak and G2/M phase increased in a quercetin concentration-dependent fashion, which were blocked by the pan-caspase inhibitor Z-VAD. Additionally, quercetin and gemcitabine produced a significant synergistic effect on inhibiting MS-TO-211H cell growth. Given that quercetin induced preferential p53-upregulating, growth-inhibiting, and apoptosis-activating effects on MM cells, the use of quercetin may be a potential therapeutic strategy for enhancing anti-cancer efficacy of existing chemotherapy in MM.

Cariporide Enhances the DNA Damage and Apoptosis in Acid-tolerable Malignant Mesothelioma H-2452 Cells

The Na+/H+ exchanger is responsible for maintaining the acidic tumor microenvironment through its promotion of the reabsorption of extracellular Na+ and the extrusion of intracellular H+. The resultant increase in the extracellular acidity contributes to the chemoresistance of malignant tumors. In this study, the chemosensitizing effects of cariporide, a potent Na+/H+-exchange inhibitor, were evaluated in human malignant mesothelioma H-2452 cells preadapted with lactic acid. A higher basal level of phosphorylated (p)-AKT protein was found in the acid-tolerable H-2452AcT cells compared with their parental acid-sensitive H-2452 cells. When introduced in H-2452AcT cells with a concentration that shows only a slight toxicity in H-2452 cells, cariporide exhibited growth-suppressive and apoptosis-promoting activities, as demonstrated by an increase in the cells with pyknotic and fragmented nuclei, annexin V-PE(+) staining, a sub-G0/G1 peak, and a G2/M phase-transition delay in the cell cycle. Preceding these changes, a cariporide-induced p-AKT down-regulation, a p53 up-regulation, an ROS accumulation, and the depolarization of the mitochondrial-membrane potential were observed. A pretreatment with the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 markedly augmented the DNA damage caused by the cariporide, as indicated by a much greater extent of comet tails and a tail moment with increased levels of the p-histone H2A.X, p-ATMSer1981, p-ATRSer428, p-CHK1Ser345, and p-CHK2Thr68, as well as a series of pro-apoptotic events. The data suggest that an inhibition of the PI3K/AKT signaling is necessary to enhance the cytotoxicity toward the acid-tolerable H-2452AcT cells, and it underlines the significance of proton-pump targeting as a potential therapeutic strategy to overcome the acidic-microenvironment-associated chemotherapeutic resistance.