Shoei Yn Lin-Shiau

Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade

Tea is a popular beverage. The consumption of green tea is associated with a lower risk of several types of cancer, including stomach, esophagus, and lung. The cancer chemopreventive effect of tea has been attributed to its major phytopolyphenols. The tea polyphenols comprise about one-third of the weight of the dried leaf, and they show profound biochemical and pharmacological activities including antioxidant activities, modulation of carcinogen metabolism, inhibition of cell proliferation, induction of cell apoptosis, and cell cycle arrest. They intervene in the biochemical and molecular processes of multistep carcinogenesis, comprising tumor initiation, promotion, and progression. Several studies demonstrate that most tea polyphenols exert their scavenging effects against reactive oxygen species (ROS); excessive production of ROS has been implicated for the development of cardiovascular diseases, neurodegenerative disorders, and cancer. Recently, we have found that the major tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) suppresses extracellular signals and cell proliferation through epidermal growth factor receptor binding in human A431 epidermoid carcinoma cells; EGCG also blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of the transcription factor NFKB in macrophages. Furthermore, EGCG blocks the cell cycle at the G1 phase in MCF-7 cells. We have demonstrated that EGCG inhibits the activities of cyclin-dependent kinases 2 and 4; meanwhile, EGCG induces the expression of the Cdk inhibitors p21 and p27. These results suggest that tumor promotion can be enhanced by ROS and oxidative mitotic signal transduction, and this enhancement can be suppressed by EGCG or other tea polyphenols.

Suppression of extracellular signals and cell proliferation through EGF receptor binding by (−)-epigallocatechin gallate in human A431 epidermoid carcinoma cells

Tea polyphenols are known to inhibit a wide variety of enzymatic activities associated with cell proliferation and tumor progression. The molecular mechanisms of antiproliferation are remained to be elucidated. In this study, we investigated the effects of the major tea polyphenol (−)‐epigallocatechin gallate (EGCG) on the proliferation of human epidermoid carcinoma cell line, A431. Using a [3H]thymidine incorporation assay, EGCG could significantly inhibit the DNA synthesis of A431 cells. In vitro assay, EGCG strongly inhibited the protein tyrosine kinase (PTK) activities of EGF‐R, PDGF‐R, and FGF‐R, and exhibited an IC50 value of 0.5–1 μg/ml. But EGCG scarcely inhibited the protein kinase activities of pp60v‐src, PKC, and PKA (IC50 > 10 μg/ml). In an in vivo assay, EGCG could reduce the autophosphorylation level of EGF‐R by EGF. Phosphoamino acid analysis of the EGF‐R revealed that EGCG inhibited the EGF‐stimulated increase in phosphotyrosine level in A431 cells. In addition, we showed that EGCG blocked EGF binding to its receptor. The results of further studies suggested that the inhibition of proliferation and suppression of the EGF signaling by EGCG might mainly mediate dose‐dependent blocking of ligand binding to its receptor, and subsequently through inhibition of EGF‐R kinase activity. J. Cell. Biochem. 67:55–65, 1997.

High Glucose–Induced Apoptosis in Human Endothelial Cells Is Mediated by Sequential Activations of c-Jun NH2-Terminal Kinase and Caspase-3

Background—Diabetes mellitus causes multiple cardiovascular complications. High glucose can induce reactive oxygen species and apoptosis in endothelial cells. Little is known about the molecular mechanisms in high glucose–induced endothelial cell apoptosis. Methods and Results—We elucidated the signaling pathway of high glucose–induced apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with media containing 5.5, 19, or 33 mmol/L of glucose in the presence or absence of an antioxidant, ascorbic acid. The level of intracellular H2O2 was measured by flow cytometry. For detection of apoptosis, the cell death detection ELISA assay and the morphological Hoechst staining were used. High glucose was capable of inducing the activity of c-Jun NH2-terminal kinase (JNK) but not extracellular signal–regulated kinase 1/2 or p38 mitogen-activated protein kinase during the treatment periods, as evidenced by immunocomplex kinase assay. Moreover, we found that the interleukin 1β–converting en...

Inhibition of cyclin‐dependent kinases 2 and 4 activities as well as induction of cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (−)‐epigallocatechin‐3‐gallate

(−)‐Epigallocatechin‐3‐gallate (EGCG) potently inhibits cell proliferation and suppresses tumor growth both in vitro and vivo, but little is known regarding the cell cycle regulatory proteins mediating these effects. This study investigated the effects of EGCG and other catechins on the cell cycle progression. DNA flow cytometric analysis indicated that 30 μM of EGCG blocked cell cycle progression at G1 phase in asynchronous MCF‐7 cells. In addition, cells exposed to 30 μM of EGCG remained in the G1 phase after release from aphidicolin block. Over a 24‐h exposure to EGCG, the Rb protein changed from hyper‐ to hypophosphorylated form and G1 arrest developed. The protein expression of cyclin D1, and E reduced slightly under the same conditions. Immunocomplex kinase experiments showed that EGCG inhibited the activities of cyclin‐dependent kinase 2 (Cdk2) and 4 (Cdk4) in a dose‐dependent manner in the cell‐free system. As the cells were exposed to EGCG (30 μM) over 24 h a gradual loss of both Cdk2 and Cdk4 kinase activities occurred. EGCG also induced the expression of the Cdk inhibitor p21 protein and this effect correlated with the increase in p53 levels. The level of p21 mRNA also increased under the same conditions. In addition, EGCG also increased the expression of the Cdk inhibitor p27 protein within 6 h after EGCG treatment. These results suggest that EGCG either exerts its growth‐inhibitory effects through modulation of the activities of several key G1 regulatory proteins such as Cdk2 and Cdk4 or mediates the induction of Cdk inhibitor p21 and p27. J. Cell. Biochem. 75:1–12, 1999.

High Glucose Induces Human Endothelial Cell Apoptosis Through a Phosphoinositide 3-Kinase–Regulated Cyclooxygenase-2 Pathway

Objectives—Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenase-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)–induced apoptosis in human umbilical vein endothelial cells (HUVECs). Methods and Results—For detection of apoptosis, the morphological Hoechst staining and Annexin V/propidium iodide staining were used. Glucose upregulated COX-2 protein expression, which was associated with the induction of prostaglandin (PG) E2 (PGE2), caspase-3 activity, and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE2 production, caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the downstream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and nuclear factor &kgr;B (NF-&kgr;B) activation. Blocking the PI3K and Akt activities with the dominant-negative vectors greatly diminished the HG-triggered NF-&kgr;B activation and COX-2 expression and apoptosis. Conclusions—These results suggest that HG, via PI3K/Akt signaling, induces NF-&kgr;B–related upregulation of COX-2, which in turn triggers the caspase-3 activity that facilitates HUVEC apoptosis. Also, HG may cause ROS generation in HUVECs through a PI3K/Akt–dependent pathway.

Suppression of protein kinase C and nuclear oncogene expression as possible molecular mechanisms of cancer chemoprevention by apigenin and curcumin

Apigenin, a less‐toxic and non‐mutagenic flavonoid, suppressed 12‐0‐tetradecanoyl‐phorbol‐13‐acetate‐(TPA)‐mediated tumor promotion of mouse skin. TPA had the ability to activate protein kinase C (PKC) and induced nuclear proto‐oncogene expression. Our study indicates that apigenin inhibited PKC by competing with adenosine triphosphate (ATP). Apigenin also reduced the level of TPA‐stimulated phosphorylation of cellular proteins and inhibited TPA‐induced c‐jun and c‐fos expression. Curcumin, a dietary pigment phytopolyphenol, is also a potent inhibitor of tumor promotion induced by TPA in mouse skin. When mouse fibroblast cells were treated with TPA alone, PKC translocated from the cytosolic fraction to the particulate fraction. Treatment with 15 or 20 μM curcumin for 15 min inhibited TPA‐induced PKC activity in the particulate fraction by 26–60%. Curcumin also inhibited PKC activity in vitro by competing with phosphatidylserine. Curcumin (10 μM) suppressed the expression of c‐jun in TPA‐treated cells. Fifteen flavonoids were examined for their effects on morphological changes in soft agar and cellular growth in v‐H‐ras transformed NIH3T3 cells. The results demonstrated that only apigenin, kaempferol, and genistein exhibited the reverting effect on the transformed morphology of these cells. Based on these findings, it is suggested that the suppression of PKC activity and nuclear oncogene expression might contribute to the molecular mechanisms of inhibition of TPA‐induced tumor promotion by apigenin and curcumin. J. Cell. Biochem. Suppls. 28/29:39–48.

Cytogenetic toxicity of uranyl nitrate in Chinese hamster ovary cells.

The effects of uranyl nitrate (UO2+2) on viability, cell cycle kinetics (CCK), micronuclei (MN), chromosome aberrations (CA), and sister-chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells were investigated. Uranyl nitrate decreased the viability of CHO cells in a dose-related fashion. The concentration for 50% inhibition (IC50) of uranyl nitrate on viability was 0.049 mM. Uranyl nitrate at concentrations ranging from 0.01 to 0.3 mM decreased CCK and increased frequencies of MN and SCE. CA were also significantly augmented by uranyl nitrate. This finding indicates that uranyl nitrate has the property of causing genotoxicity and cytotoxicity in CHO cells. It appears that this cytogenetic toxicity of uranyl nitrate provides a biological basis for the potential teratogenic effect of uranium on developing fetal mice.

The Role of Phosphoinositide 3-Kinase/Akt Signaling in Low-Dose Mercury–Induced Mouse Pancreatic β-Cell Dysfunction In Vitro and In Vivo

The relationship between oxidation stress and phosphoinositide 3-kinase (PI3K) signaling in pancreatic β-cell dysfunction remains unclear. Mercury is a well-known toxic metal that induces oxidative stress. Submicromolar-concentration HgCl2 or methylmercury triggered reactive oxygen species (ROS) production and decreased insulin secretion in β-cell–derived HIT-T15 cells and isolated mouse islets. Mercury increased PI3K activity and its downstream effector Akt phosphorylation. Antioxidant N-acetyl-l-cysteine (NAC) prevented mercury-induced insulin secretion inhibition and Akt phosphorylation but not increased PI3K activity. Inhibition of PI3K/Akt activity with PI3K inhibitor or by expressing the dominant-negative p85 or Akt prevented mercury-induced insulin secretion inhibition but not ROS production. These results indicate that both PI3K and ROS independently regulated Akt signaling–related, mercury-induced insulin secretion inhibition. We next observed that 2- or 4-week oral exposure to low-dose mercury to mice significantly caused the decrease in plasma insulin and displayed the elevation of blood glucose and plasma lipid peroxidation and glucose intolerance. Akt phosphorylation was shown in islets isolated from mercury-exposed mice. NAC effectively antagonized mercury-induced responses. Mercury-induced in vivo effects and increased blood mercury were reversed after mercury exposure was terminated. These results demonstrate that low-dose mercury–induced oxidative stress and PI3K activation cause Akt signaling–related pancreatic β-cell dysfunction.

Suppression of TNFα-mediated NFκb activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells

Flavonoids are a group of naturally‐occurring phenolic compounds in the plant kingdom, and many flavonoids are found with vascular protective properties. Nevertheless how the protective response is exerted by flavonoids is not well characterized. In view of the nuclear factor‐κB (NFκB) may play a central role in the initiation of atherosclerosis, prevention of the activation of NFκB represents an important role in protecting vascular injury. In this study, the effects of flavonoids on NFκB/inhibitor‐κB (IκB) system in ECV304 cells activated with tumor necrosis factor‐α (TNFα) were examined. We investigated the inhibitory action of six flavonoids on IκB kinase (IKK) activity, an enzyme recently found to phosphorylate critical serine residues of IκB for degradation. Of six flavonoids tested, myricetin was found to strongly inhibit IKK kinase activity, and prevent the degradation of IκBα and IκBβ in activated endothelial cells. Furthermore, myricetin was also found to inhibit NFκB activity correlated with suppression of monocyte adhesion to ECV304 cells. Therefore we conclude that flavonoids may be of therapeutic value for vascular disease through down regulation of NFκB/IκB system. J. Cell. Biochem. 74:606–615, 1999.

Nitric oxide prevents apoptosis of human endothelial cells from high glucose exposure during early stage

Hyperglycemia is a major cause of diabetic vascular disease. High glucose can induce reactive oxygen species (ROS) and nitric oxide (NO) generation, which can subsequently induce endothelial dysfunction. High glucose is also capable of triggering endothelial cell apoptosis. Little is known about the molecular mechanisms and the role of ROS and NO in high glucose‐induced endothelial cell apoptosis. This study was designed to determine the involvement of ROS and NO in high glucose‐induced endothelial cell apoptosis. Expression of endothelial nitric oxide synthase (eNOS) protein and apoptosis were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to control‐level (5.5 mM) and high‐level (33 mM) glucose at various periods (e.g., 2, 12, 24, 48 h). We also examined the effect of high glucose on H2O2 production using flow cytometry. The results showed that eNOS protein expression was up‐regulated by high glucose exposure for 2–6 h and gradually reduced after longer exposure in HUVECs. H2O2 production and apoptosis, which can be reversed by vitamin C and NO donor (sodium nitroprusside), but enhanced by NOS inhibitor (NG‐nitro‐L‐arginine methyl ether), were collated to a different time course (24–48 h) to HUVECs. These results provide the molecular basis for understanding that NO plays a protective role from apoptosis of HUVECs during the early stage (<24 h) of high glucose exposure, but in the late stage (>24 h), high glucose exposure leads to the imbalance of NO and ROS, resulting to the observed apoptosis. This may explain, at least in part, the impaired endothelial function and vascular complication of diabetic mellitus that would occur at late stages. J. Cell. Biochem. 75:258–263, 1999.