Wing-Keung Chu

The protective effect of resveratrols on ischaemia‐reperfusion injuries of rat hearts is correlated with antioxidant efficacy

Dietary antioxidants are thought to be beneficial in reducing the incidence of coronary heart disease. In this study, we compared resveratrol and analogues on their antioxidation and free radical scavenging activities to their protective effects on ischaemia‐reperfusion induced injuries of rat hearts. Astringinin (3,3′,4′,5‐tetrahydroxystilbene) was shown to be a more potent inhibitor than other analogues against Cu2+‐induced LDL (low‐density lipoprotein) oxidation, as measured by the formation of conjugated diene and TBARS (thiobarbituric acid‐reactive substance) and by the electrophoretic mobility of the oxidized LDL. Resveratrol (trans‐3,4′,5‐trihydroxystilbene) and astringinin scavenged the stable free radical DPPH (1,1‐diphenyl‐2‐picryl‐hydrazyl) with an IC0.200 of 7.1 and 4.3 μM, respectively. Astringinin has a superoxide anion scavenging activity about 160 fold more potent than resveratrol. After a 30 min global ischemia followed by 2 h reperfusion, astringinin (10 μM) significantly reduced infarct size, superoxide anion production and increased functional recovery of the coronary flow in Langendorff‐perfused rat hearts. The result showed there is a positive correlation between the anti‐oxidation and cardioprotective activities among these phenolic compounds. Our finding together with the fact that astringinin is more water‐soluble than resveratrol suggest that astringinin could potentially be used as an anti‐oxidant and cardioprotective agent in biological systems.

Transcriptional Activity of the ΔNp63 Promoter Is Regulated by STAT3

The N terminus-truncated splicing variant of TAp63 is known as ΔNp63. ΔNp63 lacks transactivation function and is thought to antagonize the transcriptional regulation of the p53 and TAp63 target genes. Overexpression of ΔNp63 has been observed in a number of human cancers, suggesting a role in carcinogenesis. In the present study we present data showing that the ΔNp63 gene promoter activity is positively regulated by ΔNp63α, and such positive autoregulation is mediated via activation of STAT3 activity. We show that expression of ΔNp63α in Hep3B cells induces Stat3 phosphorylation on Tyr-705 and Ser-727. A putative STAT3-responsive element (STAT3-RE) is identified in the ΔNp63 promoter region. Electrophoretic mobility shift and avidin biotin-Conjugated DNA assays show direct binding of STAT3 to STAT3-RE of the ΔNp63 promoter, and such binding is stimulated by ΔNp63α. Binding of the endogenous STAT3 to the ΔNp63 promoter in Hep3B cells was demonstrated by a chromatin immunoprecipitation assay. The stimulation of the ΔNp63 transcriptional activity by ΔNp63α is abolished by Janus kinase 2 (JAK2)/STAT3 inhibitor AG490, dominant-negative STAT3, STAT3 small interfering RNA, and deletion of the STAT3-RE sequence from ΔNp63 promoter. Taken together these observations clearly indicated that autoregulation of ΔNp63 gene transcription is mediated through activation of STAT3 and its subsequent binding to the STAT3RE. Because the activation of STAT3 by interleukin-6 also leads to ΔNp63 up-regulation and the blockade of ΔNp63 or STAT3 expression by siRNA leads to repression of the cell growth, the identified regulatory pathway is presumably of cell physiological significance.

Overexpression of ΔNp63 in a human nasopharyngeal carcinoma cell line downregulates CKIs and enhances cell proliferation

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The ΔNp63 isoform lacks the N‐terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. ΔNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of ΔNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a ΔNp63 overexpressing human NPC cell line (NPC‐076) to explore the possible roles of ΔNp63 in cell proliferation and cell‐cycle regulation. We found that the proliferation of NPC‐076 cell is greatly suppressed when the overexpressed ΔNp63 is silenced by specific ΔNp63 siRNA. Further studies show that ΔNp63 silencing results in the upregulation of CKIs, including p27kip1 and p57kip2 in both mRNA and protein levels. Cell‐cycle analysis shows that ΔNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that ΔNp63 plays important roles in the regulation of NPC‐076 cell‐cycle progression, and may play a role in the maintenance of NPC‐076 tumor cell phenotype. J. Cell. Physiol. 219: 117–122, 2009.

Glycogen synthase kinase‐3β regulates ΔNp63 gene transcription through the β‐catenin signaling pathway

Overexpression of ΔNp63 has been observed in a number of human cancers, suggesting a role for ΔNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase‐3β (GSK‐3β) by lithium chloride (LiCl) elicited a stimulatory effect on ΔNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced ΔNp63 promoter activation as well as ΔNp63 protein expression in the cells. The effect of GSK‐3β on ΔNp63 expression was further confirmed by the use of two highly specific GSK‐3β inhibitors, SB216763 and SB415286. Further study showed the presence of a putative β‐catenin responsive element (β‐catenin‐RE) in the ΔNp63 promoter region, and the stimulation of ΔNp63 promoter activity by GSK‐3β inhibitor is markedly abolished by mutation or deletion of the putative β‐catenin‐RE. Data are also presented to show that β‐catenin acts together with Lef‐1 to influence ΔNp63 promoter activity and protein expression. J. Cell. Biochem. 105: 447–453, 2008.

STAT3 Regulates the Proliferation and Differentiation of Rabbit Limbal Epithelial Cells via a ΔNp63-Dependent Mechanism

PURPOSE To explore the roles of STAT3 in the regulation of ΔNp63-dependent proliferation and differentiation of rabbit limbal keratinocytes. METHODS siRNAs were designed to specifically suppress the expression of STAT3 and ΔNp63, and their effects on limbal epithelial cell proliferation and differentiation were examined. Ectopically expressed ΔNp63 was used to compensate for the decreased endogenous ΔNp63. Immunoblot was used to examine the expressions of STAT3, ΔNp63, K3, integrin β1, and involucrin. RESULTS Limbal tissue expresses higher level of phosphorylated and nuclear translocated STAT3 compared with that of the cornea. Knockdown of STAT3 expression reduces the expression of ΔNp63, inhibits the expansion of limbal epithelial outgrowth, suppresses the expression of integrin β1, and promotes the expression of involucrin. CONCLUSIONS STAT3 enhances the proliferation of limbal keratinocytes through a ΔNp63-dependent mechanism. Suppression of this pathway inhibits cell proliferation with a concomitant increase of cell differentiation.

Ischemic preconditioning ameliorates microcirculatory disturbance through downregulation of TNF-alpha production in a rat cremaster muscle model

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IĸB-α-NF-ĸB-TNF-α (tumor necrosis factor-α) pathway in IPC’s ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n = 8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF-α expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF-α protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IĸB-α phosphorylation and NF-ĸB (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-ĸB activation and subsequently reduced TNF-α expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles.

Preservation of human limbal epithelial progenitor cells on carbodiimide cross-linked amniotic membrane via integrin-linked kinase-mediated Wnt activation.

UNLABELLED The Wnt pathway is a major signaling pathway that regulates corneal epithelial stem cells. However, little is known about how the ultrastructure of the limbal epithelial basement membrane (EBM) affects Wnt activity. Due to its enhanced matrix stability, the cross-linked amniotic membrane (AM) has gained increasing interest in the field of regenerative medicine. For the first time, we used EDC/NHS cross-linked denuded AM (CLDAM) as a simulated EBM substrate to investigate this mechanism. Human limbal epithelial (HLE) cells were cultured on dishes (HLE/dish), denuded AM (HLE/DAM) or CLDAM (HLE/CLDAM). Compared with HLE/dish or HLE/DAM cultures, HLE/CLDAM cultures showed greater BrdU retention and colony formation efficiency and expressed higher levels of p63, ABCG2, integrin β1, and integrin-linked kinase (ILK). Nuclear β-catenin and TCF-4 levels were higher in HLE/CLDAM cultures compared with HLE cells cultured on collagen IV, laminin, Matrigel, or DAM. Silencing of ILK in HLE/CLDAM cultures resulted in decreased levels of nuclear β-catenin, TCF-4 and deltaNp63α, whereas cytokeratin 12 expression increased. Over-expression of ILK in HLE/dish cultures had the opposite effects. Accordingly, we proposed that the CLDAM matrix, with its higher rigidity and rougher ultrastructure, better preserved HLE progenitor cells in vitro, possibly by activating integrin β1/ILK, which indirectly activated Wnt/β-catenin and subsequently deltaNp63α. Crosstalk between the integrin β1/ILK and Wnt/β-catenin pathways appears to play a crucial role in limbal progenitor cell survival on EBM. STATEMENT OF SIGNIFICANCE We demonstrated the superior capability of carbodiimide cross-linked denuded amniotic membrane (CLDAM) than natural DAM to preserve limbo-corneal epithelial progenitor cells in vitro, then we used CLDAM as a simulated epithelial basement membrane (EBM) to study how EBM maintains limbal epithelial stem cells (LESCs). We found that integrin-linked kinase (ILK) is an important mediator that transfers survival signals detected by integrin β1 to the Wnt/β-catenin pathway, which in turn up-regulates deltaNp63α, a master gene that regulates LESC function. The rougher surface of the limbal EBM suggests that the surface complexity of the LESC niche may be important in regulating LESC function, which is triggered by the recognition of topographic cues by integrin β1, followed by activation of the ILK/Wnt/β-catenin/p63 cascade.

Nanog expression is negatively regulated by protein kinase C activities in human cancer cell lines

Nanog is a transcription factor that is essential for the maintenance of pluripotency of the embryonic stem cells. Nanog has been shown to be expressed in various kinds of human tumors, suggesting a role in tumorigenesis. In this study, we found that Nanog expression was upregulated by inhibition of protein kinase C (PKC) activity in six human cancer cell lines examined. In a Nanog non-expressing human nasopharyngeal carcinoma cell line, NPC-076, Nanog mRNA level and protein level were both induced and dose-dependently promoted by exposure to PKC inhibitors. Knockdown experiments showed that PKCα and PKCδ were two subtypes exerted most of the effect. The reporter assay showed that Nanog promoter activity was promoted by exposure of the cells to PKC inhibitors and the effect was dependent on the presence of the Octamer-Sox composite element. The involvement of Octamer-Sox composite element was further supported by the observation that silencing of Oct4 and Sox2 in NPC-076 cells attenuated the effects of PKC inhibitors. In Nanog-expressing human embryonal carcinoma cell lines, NT2/D1 and NCCIT, Nanog expression was suppressed by exposure to PKC activator Phorbol-12-myristate-13-acetate (PMA). Further study showed that overexpression of PKCα elicited a repressive effect on Nanog expression in NT2/D1 cells. Consistently, mutation of the Octamer-Sox composite element abolished the suppressive effect by PKC activator. Nanog expression was of cellular significance in that ectopic expression in NPC-076 stimulated cell proliferation and knockdown of the endogenous Nanog expression in NT2/D1-suppressed cell proliferation.

Exposure to oxidized low-density lipoprotein reduces activable Ras protein in vascular endothelial cells.

SummaryOxidized low-density lipoprotein (ox-LDL) has been shown to alter the migratory and proliferative activities of the vascular endothelial cells (EC) in response to serum and growth factors. The mechanism underlying the antiproliferative effect of ox-LDL on vascular EC has not been fully elucidated. In this report, we show that exposure of vascular EC to ox-LDL results in a marked reduction of the membrane-associated Ras protein. Further study shows that in ox-LDL-treated EC, reduction of the membrane-associated Ras protein is correlated with a reduced amount of active Ras (Ras-guanosine triphosphate), indicating that the Ras signaling pathway is attenuated. The attenuation of the Ras signaling pathway in ox-LDL-treated EC may thus be responsible for the retarded response to the mitogenic stimulation of serum and growth factors.

Downregulation of lumican accelerates lung cancer cell invasion through p120 catenin

The overexpression of lumican has been found in lung cancer cells; however, the functional role of lumican in lung cancer cells remains unclear. In this study, we found lumican functioned as a tubulin-binding protein and the depletion of lumican by transfection with its specific shRNA increased lung cancer cell invasion. Such alterations led to morphological changes and actin cytoskeleton remodeling, including the induction of membrane ruffling or protrusion and stress fiber formation, correlated with the increased activities of Rac and Rho. The downregulation of lumican was also implicated in macrophage-conditioned media (maCM)-induced cell invasion. Immunofluorescence images and immunoprecipitation assays revealed the co-localization of p120-catenin (p120ctn) and lumican. Reduction in the levels of p120ctn induced membrane ruffling and the activation of the Rho family, which accelerated cell invasion. Our data indicated that lumican is associated with microtubule-modulated p120ctn signaling, providing important insights into lung cancer progression.