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Purpose: Wogonin, a plant flavonoid, has antitumor activity in various cancers. Dysregulation of GSK-3β has been implicated in tumorigenesis and cancer progression. In this study, we investigated the antitumor activity and the mechanistic action of wogonin in human nasopharyngeal carcinoma (NPC) cells. Methods: The effects of wogonin on the cell survival and apoptosis in NPC cells were investigated by MTS assay, flow cytometry, and PARP cleavage assays. Pharmacological inhibitors (BIO, LiCl, and OA), or small interfering RNA (siRNA) were used to address the expression status of GSK-3β and the anticancer effect of ΔNp63 in NPC cells. Results: Wogonin was shown to induce dose-dependent cell apoptosis due to the induction of sub-G1-phase cells, PARP cleavage, and downregulation of ΔNp63, a survival factor in NPC cells. Strikingly, the apoptotic effect of wogonin involved GSK-3β inactivation via prominent inhibition of phosphorylation at Tyr216 and slightly increment of phosphorylation at Ser9, while there is no change in total GSK-3β proteins. Dysregulation of GSK-3β caused cell apoptosis was confirmed by pharmacological inhibitors (lithium chloroid, LiCl, and 6-bro-moindirubin-3-oxime, BIO). Administration of okadaic acid (OA, a protein phosphatase inhibitor) that significantly inactivated GSK-3β also induced ΔNp63 downregulation and apoptosis. Targeted silencing of ΔNp63 repressed the phosphorylation of GSK-3β at Tyr216 and sensitized NPC cells to wogonin-induced apoptosis. Furthermore, GSK-3β or PP2A inhibitors enhanced wogonin-induced apoptosis via activation of caspase 3/7. Conclusion: These results indicate that GSK-3β, as well as ΔNp63, are novel targets for wogonin action and suggest that wogonin might provide a potential therapeutic option in NPC. Further in vitro and in vivo studies will help to clarify the therapeutic role of wogonin in NPC. [ABSTRACT FROM AUTHOR]

Hatchery produced leptocephalus larvae of the Japanese eel (Anguilla japonica) were reared on a research vessel and fed natural particulate organic matter (POM) collected in the Western North Pacific. A small net (36 cm diameter, 60 µm mesh) was vertically towed from 200 m depth to surface in 2013, and POM-enriched water (POMEW) filtered through 350 µm mesh was fed to the leptocephali for 11 days. Although the swollen gut corroborated the active ingestion of POM by the leptocephali, low survival rate and heavily melanized gut followed by necrosis in the mid-hindgut region of the leptocephali were observed. A large net (1.14 m diameter, 30 µm mesh) was used in 2014 and 2015, which was horizontally drifted subsurface (100–175 m). POMEWs filtered through 53 or 25 µm meshes were fed to the leptocephali for 5–18 days. Neither melanized gut nor necrosis occurred, but considerably low survival rate and little growth comparable with that in a starved condition were observed. No apparent shift in stable carbon and nitrogen isotope ratios was observed in the reared leptocephali. These indicated that the ingested POMs were not assimilated by the leptocephali and suggested that smaller particles may be important for the leptocephali.

BACKGROUND: Given that micronutrient deficiency, neglected intestinal parasitic infections (IPIs) and poor socioeconomic status are closely linked, we conducted a cross-sectional study to assess the relationship between IPIs and nutritional status of children living in remote and rural areas in West Malaysia. METHODS/FINDINGS: A total of 550 children participated, comprising 520 (94.5%) school children aged 7 to 12 years old, 30 (5.5%) young children aged 1 to 6 years old, 254 (46.2%) boys and 296 (53.8%) girls. Of the 550 children, 26.2% were anaemic, 54.9% iron deficient and 16.9% had iron deficiency anaemia (IDA). The overall prevalence of helminths was 76.5% comprising Trichuris trichiura (71.5%), Ascaris lumbricoides (41.6%) and hookworm infection (13.5%). It was observed that iron deficiency was significantly higher in girls (p = 0.032) compared to boys. Univariate analysis demonstrated that low level of mother's education (OR = 2.52; 95% CI = 1.38-4.60; p = 0.002), non working parents (OR = 2.18; 95% CI = 2.06-2.31; p = 0.013), low household income (OR = 2.02; 95% CI = 1.14-3.59; p = 0.015), T. trichiura (OR = 2.15; 95% CI = 1.21-3.81; p = 0.008) and A. lumbricoides infections (OR = 1.63; 95% CI = 1.04-2.55; p = 0.032) were significantly associated with the high prevalence of IDA. Multivariate analysis confirmed that low level of mother's education (OR = 1.48; 95 CI% = 1.33-2.58; p

BACKGROUND: Intestinal parasitic infections (IPIs) have a worldwide distribution and have been identified as one of the most significant causes of illnesses and diseases among the disadvantaged population. In Malaysia, IPIs still persist in some rural areas, and this study was conducted to determine the current epidemiological status and to identify risk factors associated with IPIs among communities residing in rural and remote areas of West Malaysia. METHODS/FINDINGS: A total of 716 participants from 8 villages were involved, comprising those from 1 to 83 years old, 550 (76.8%) participants aged ≤12 years and 166 (23.2%) aged ≥13 years, and 304 (42.5%) male and 412 (57.5%) female. The overall prevalence of IPIs was high (73.2%). Soil-transmitted helminth (STH) infections (73.2%) were significantly more common compared to protozoa infections (21.4%) (p

YES-associated protein (YAP) is a part of the Hippo pathway, with pivotal roles in several developmental processes and dual functionality as both a tumor suppressor and an oncogene. In the present study, we identified YAP activity as a microtubular scaffold protein that maintains the stability of the mitotic spindle and midbody by physically interacting with α-tubulin during mitotic progression. The interaction of YAP and α-tubulin was evident in co-immunoprecipitation assays, as well as observing their co-localization in the microtubular structure of the mitotic spindle and midbody in immunostainings. With YAP depletion, levels of ECT2, MKLP-1, and Aurora B are reduced, which is consistent with YAP functioning in midbody formation during cytokinesis. The concomitant decrease in α-tubulin and increase in acetyl-α-tubulin during YAP depletion occurred at the post-transcriptional level. This suggests that YAP maintains the stability of the mitotic spindle and midbody, which ensures appropriate chromosome segregation during mitotic division. The increase in acetyl-α-tubulin during YAP depletion may provide a lesion-halting mechanism in maintaining the microtubule structure. The depletion of YAP also results in multinuclearity and aneuploidy, which supports its role in stabilizing the mitotic spindle and midbody., (© 2023 Federation of European Biochemical Societies.)

Abnormal expression of p120 catenin is associated with the malignant phenotype in human lung cancer. Numerous studies have focused on the function of p120 catenin in the juxta-membrane compartment. However, the role of nuclear p120 catenin remains unclear. In this study, the dynamic changes in nuclear p120 catenin localization during cell cycle progression were investigated. Immunofluorescent staining, FACS analysis, and western blotting revealed that nuclear p120 catenin is a major architectural constituent of the chromosome periphery during mitosis. During mitosis, granule-like p120 catenin dispersed into a cloudy-like structure and formed cordon-like structures surrounding the condensed chromosomes to create the peri-chromosomal layer. Interestingly, lumican and p120 catenin colocalized at the spindle fiber where the perichromosomal layer connects to the condensed chromosomes during mitosis. Furthermore, downregulation of p120 catenin using a specific siRNA induced cell cycle stalling in the G2/M phase and promoted aneuploidy. This study validates the role of nuclear p120 catenin in the formation of the chromosome periphery and reveals the p120 catenin-lumican interaction may couple orientation of cell division with the segregation of sister chromatids during mitosis. Our data suggest the protective role of p120 catenin in maintaining the integrity of chromosomes, and also warrants further studies to evaluate the contribution of the loss of p120 catenin to the creation of gene rearrangement in cancer evolution and tumor progression., (© 2022. The Author(s).)

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.

Lumican is overexpressed in lung cancer cells and has been implicated in the pathogenesis of tumorigenesis and regulation of cancer cell invasion. Lumican is robustly associated with the binding of p120-catenin protein to modulate cell metastasis. However, its role in cancer cell proliferation is still unclear. This study investigated the effect of lumican on the cell division including mitosis and cytokinesis in non-small lung cancer cells. We found that the downregulation of lumican prolonged the doubling time of cells and retarded the cell growth in H460 and A549 cells. Along with tubulin, lumican localized to the mitotic spindle and centrosome during the metaphase-anaphase stage. The cell cycle was retained in the G2/M phase after the downregulation of lumican. Interestingly, lumican was found to play important roles in central spindle and midbody formation during cytokinesis. Lumican interacted with the midbody-associated proteins such as MKLP1, Aurora B, and ECT2. Notably, the downregulation of lumican decreased the level of MKLP1 accompanied by the retention of midbody-residual that resulted in multi-nucleated cells. Downregulation of lumican promoted the chromosome missegregation and the increment of the bi-/multinucleated cells. The results of this study indicated that lumican associated with tubulin is crucial for spindle fiber formation and midbody assembly in cell division. Downregulation of lumican displayed the defects in mitotic spindle assembly/dynamics and improper kinetochore-microtubules attachment that led to increase aneuploidy. This emerging property of lumican is suggested to tightly control chromosome segregation during cell division in lung cancer cells. Abbreviations: ESCRT: endosomal sorting complex required for transport; PRC1: protein regulator of cytokinesis 1; Nci: negative control siRNA; Lumi: lumican siRNAs; MKLP1: mitotic kinesin-like protein 1; H460LD and A549LD: H460 and A549 cell lines with less expressed lumican.

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.

Caspase-12 (Casp12), an inflammatory caspase, functions as a dominant-negative regulator of inflammatory responses and is associated with the signaling of apoptosis. However, the physiological function of Casp12 presented in cancer cells is still unclear. This study demonstrated that overexpression of Casp12 mediated IκBα degradation and significantly increased NF-κB activity. Exposure of human nasopharyngeal carcinoma (NPC) cells to phorbol-12-myristate-13-acetate (PMA) increased the levels of Casp12 and MMP-9 resulting in NPC cell invasion. Target suppression of Casp12 by small interfering RNA (siRNA) or an inhibitor of Casp12 markedly decreased the level of PMA-induced MMP-9 protein and cell invasion. Moreover, suppression of Casp12 significantly inhibited the basal activity of NF-κB and decreased the PMA-induced NF-κB reporter activity. The effect of Casp12 on NF-κB activation was indicated via the post-translational degradation of IκB. This study revealed that a critical role of Casp12 on the activation of NF-κB via IκBα degradation which provides a link between inflammatory and aggressive invasion in NPC cells.

Background: N-cadherin is a trans-membrane adhesion molecule associated with advanced carcinoma progression and poor prognosis. The effect of N-cadherin on matrix metalloproteinase 9 (MMP-9) regulation is implicated in human nasopharyngeal carcinoma (NPC) cell invasion., Methods and Results: Exposure of NPC cells to phorbol-12-myristate-13-acetate (PMA) or macrophage conditioned media (CM) upregulated MMP-9 and N-cadherin cleavage, which resulted in NPC cell invasion. MMP-9 cleaved the extracellular domain of N-cadherin, which was further cleaved by γ-secretase with PMA or macrophage-CM treatment. The extracellular cleavage of N-cadherin was inhibited with treatment with an MMP inhibitor and MMP-9 siRNA, whereas the intracellular cleavage of N-cadherin was inhibited by treatment with a γ-secretase inhibitor (γI), which resulted in enhanced accumulation of N-cadherin C-terminal fragment (CTF1, ~40 kDa). CTF2/N-cad (CTF2), a product of the γ-secretase cleavage of N-cadherin, was released and translocated into the nuclear compartment in PMA-treated cells. Moreover, CTF2 enhanced the effect of PMA-mediated MMP-9 gene expression as assessed by treatment with γI or overexpression with exogenous CTF2. Additionally, siRNA silencing of N-cadherin decreased PMA-mediated MMP-9 expression and cell invasion. The outside-in signaling effect of MMP-9 in macrophage CM- or PMA-treated cell cultures significantly enhanced NPC cell invasion via N-cadherin cleavage., Conclusion: Extracellular and intracellular cleavage of N-cadherin might be involved in elevated MMP-9 expression enhancing tumor cell invasion. Furthermore, N-cadherin-affected tumor progression might be via enhanced MMP-9 signaling in a cross-talk regulatory mechanism. N-cadherin might contribute to the invasive characteristics of carcinoma cells by upregulating MMP-9, thereby leading to increased aggressive metastasis.

A negative-pressure of 125mmHg (NP) has been widely used to treat chronic wounds in modern medicine. Keratinocytes under NP treatment have shown accelerated cell movement and decreased E-cadherin expression. However, the molecular mechanism of E-cadherin regulation under NP remains incompletely understood. Therefore, we investigated the E-cadherin regulation in keratinocytes (HaCaT cells) under NP. HaCaT cells were treated at ambient pressure (AP) and NP for 12h. Cell movement was measured by traditional and electric wound healing assays at the 2 different pressures. Mutants with overexpression of p120-catenin (p120(ctn)) were used to observe the effect of NP on p120(ctn) and E-cadherin expression during wound healing. Cell fractionation and immunoblotting data showed that NP increased Y228-phosphorylated p120(ctn) level and resulted in the translocation of p120(ctn) from the plasma membrane to cytoplasm. Immunofluorescence images revealed that NP decreased the co-localization of p120(ctn) and E-cadherin on the plasma membrane. Knockdown of p120(ctn) reduced E-cadherin expression and accelerated cell movement under AP. Overexpression of the Y228-phosphorylation-mimic p120(ctn) decreased E-cadherin membrane expression under both AP and NP. Phosphorylation-deficient mutants conferred restored adherens junctions (AJs) under NP. The Src inhibitor blocked the phosphorylation of p120(ctn) and impeded cell migration under NP. In conclusion, Src-dependent phosphorylation of p120(ctn) can respond rapidly to NP and contribute to E-cadherin downregulation. The NP-induced disassembly of the AJ further accelerates wound healing., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Negative-pressure of 125 mmHg (NP) has been shown to accelerate wound healing. Effects of NP on human keratinocyte behaviors during wound healing process were highlighted in this study., Methods: An NP incubator incorporating the electric cell-substrate impedance sensing (ECIS) technique has been built to quantify monolayer keratinocytes movement in serum-free media at the ambient pressure (AP) and NP for 12 h. Monolayer cell motions were continuously recorded by ECIS in the frequency range of 22.5-64 kHz. Membrane capacitance (Cm), cell-substratum resistance (α), and cell-cell junction resistance (Rb) were evaluated in cells at the different pressures., Results: A greater monolayer cell migration distance was found in cells at NP. Decreased cell-substratum adhesion reflected in the significantly low α (AP:NP = ∼5 Ω(0.5):∼3 Ω(0.5)⋅cm), decreased integrin expression, and increased cell-substratum distance were seen in cells at NP. A significantly increased Cm (AP:NP = ∼4:∼8 μF/cm(2)) in association with increased membrane ruffling and microtubule filaments were observed early in the monolayer cell movement at NP. A progressive drop in the Rb from 1.2 Ω·cm(2) to 0.8 Ω·cm(2) corresponding to the gradually decreased E-cadherin expressions were observed 6 h after wound closure after NP treatment., Conclusion: A quick membrane ruffling formation, an early cell-substratum separation, and an ensuing decrease in the cellular interaction occur in cells at NP. These specific monolayer cell behaviors at NP have been quantified and possibly accelerate wound healing., (Copyright © 2016 Chang Gung University. Published by Elsevier B.V. All rights reserved.)

Autophagy and endoplasmic reticulum (ER) stress response is important for cancer cells to maintain malignancy and resistance to therapy. trans-Resveratrol (RSV), a non-flavonoid agent, has been shown to induce apoptosis in human nasopharyngeal carcinoma (NPC) cells. In this study, the involvements of tumor-specific ER stress and autophagy in the RSV-mediated apoptosis were investigated. In addition to traditional autophagosomes, the images of transmission electron microscopy (TEM) indicated that RSV markedly induced larger, crescent-shaped vacuoles with single-layered membranes whose the expanded cisternae contains multi-lamellar membrane structures. Prolonged exposure to RSV induced a massive accumulation of ER expansion. Using an EGFP-LC3B transfection and confocal laser microscopy approach, we found RSV-induced EGFP-LC3 puncta co-localized with ER-tracker red dye, implicating the involvement of LC3II in ER expansion. The proapoptotic effect of RSV was enhanced after suppression of autophagy by ATG7 siRNA or blocking the autophagic flux by bafilomycin A1, but that was not changed after targeted silence of IRE1 or CHOP by siRNA. Using caspase inhibitors, we demonstrated the upregulation of caspase-12 (casp12) and the activation of casp4 were associated with the proapoptotic induction of RSV through the caspase-9/caspase-3 pathway. Intriguingly, siRNA knockdown of casp12, but not caspase-4, decreased the susceptibility of the NPC cells to RSV-mediated apoptosis. Further, we showed that RSV dose-dependently increased the ceramide accumulation as assessed by LC-MS/MS system. Using serine palmitoyltransferase (SPT, a key enzyme of de novo ceramide biosynthesis) inhibitors (L-cycloserine and myriocin), we found the increased ceramide accumulation was strongly correlated with the proapoptotic potential of RSV. This study revealed the ER expansion and upregulation of ER casp12 together may indicate profound biological effects of RSV and contributed to NPC cell death. Targeting the different status of ER stress may provide a possible strategy for cancer treatments.

Background: Negative-pressure wound therapy (NPWT) is developed to facilitate wound healing at controlled subatmospheric pressures in modern medicine. Molecular mechanism for this therapy is still undefined., Objective: This study highlights the localization and time-course of the cell division control protein 42 (Cdc42) in the cell membrane at ambient pressure (AP) and negative pressures of 75mmHg (NP75), 125mmHg (NP125) and 175mmHg (NP175)., Methods: The prepared cells were cultured in a negative pressure incubator with the same O2 and CO2 tensions at the four different pressures. The effective time, complete wound closure time, cell volume, cell viability, and the fluorescence of proliferating cell nuclear antigens (PCNA) and actins were evaluated in cells at different pressures. Wound-healing process and Cdc42 fluorescence were examined in cells with the knockdown of Cdc42. Cdc42 pathway proteins in cell membranes were analyzed after incubation at different pressures for 6 and 12h., Results: The cells at NP125 had less wound closure time and obvious cell podia. Similar PCNA fluorescent intensity was observed in cells at different pressures. The Cdc42, neural Wiskott-Aldrich syndrome protein, and actin expression increased significantly (p<0.05) in plasma membranes of cells at NP125 for 12h. The knockdown of active Cdc42 resulted in the absence of Cdc42 expression at the cell leading edge., Conclusions: The activation and localization of Cdc42 pathway proteins in the cell membrane are involved in the cell podia formation in keratinocytes at NP125. NPWT may facilitate cell migration to accelerate wound healing., (Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Autophagy as well as apoptosis is an emerging target for cancer therapy. Wogonin, a flavonoid compound derived from the traditional Chinese medicine of Huang-Qin, has anticancer activity in many cancer cells including human nasopharyngeal carcinoma (NPC). However, the involvement of autophagy in the wogonin-induced apoptosis of NPC cells was still uninvestigated. In this study, we found wogonin-induced autophagy had interference on the process of apoptosis. Wogonin-induced autophagy formation evidenced by LC3 I/II cleavage, acridine orange (AO)-stained vacuoles and the autophagosome/autolysosome images of TEM analysis. Activation of autophagy with rapamycin resulted in increased wogonin-mediated autophagy via inhibition of mTOR/P70S6K pathway. The functional relevance of autophagy in the antitumor activity was investigated by annexin V-positive stained cells and PARP cleavage. Induction of autophagy by rapamycin ameliorated the wogonin-mediated apoptosis, whereas inhibition of autophagy by 3-methyladenine (3-MA) or bafilomycin A1 increased the apoptotic effect. Interestingly, this study also found, in addition the mTOR/P70S6K pathway, wogonin also inhibited Raf/ERK pathway, a variety of Akt pathways. Inactivation of PI(3) K/Akt by their inhibitors significantly induced apoptosis and markedly sensitized the NPC cells to wogonin-induced apoptosis. This anticancer effect of Akt was further confirmed by SH6, a specific inhibitor of Akt. Importantly, inactivation of its downstream molecule ERK by PD98059, a MEK inhibitor, also induced apoptosis. This study indicated wogonin-induced both autophagy and apoptosis through a variety of Akt pathways and suggested modulation of autophagy might provide profoundly the potential therapeutic effect., (Copyright © 2012 Wiley Periodicals, Inc.)

The members of Rho family are well known for their regulation of actin cytoskeleton to control cell migration. The Cip/kip members of cyclin-dependent (CDK) inhibitors have shown to implicate in cell migration and cytoskeletal dynamics. p57(kip2) , a CDK inhibitor, is frequently down-regulated in several malignancy tumors. However, its biological roles in human nasopharyngeal carcinoma (NPC) cells remained to be investigated. Here, we found p57(kip2) has nuclear and cytoplasm distributions and depletion of endogenous p57(kip2) did not change the cell-cycle progression. Inhibition of cell proliferation by mitomycin C promoted FBS-mediated cell migration and accompanied with the downregulation of ΔNp63α and p57(kip2), but did not change the level of p27(kip1) , another CDK inhibitor. By using siRNA transfection and cell migration/invasion assays, we found that knockdown of p57(kip2) , but not ΔNp63α, involved in promotion of NPC cell migration and invasion via decrease of phospho-cofilin (p-cofilin). Treatment with Y-27632, a specific ROCK inhibitor, we found that dysregulation of ROCK/cofilin pathway decreased p-cofilin expression and induced cell migration. This change of p-cofilin induced actin remodeling and pronounced increase of membrane protrusions. Further, silence of p57(kip2) not only decreased the interaction between p57(kip2) and LIMK-1 assayed by immunoprecipitation but also reduced the level of phospho-LIMK1/2. Therefore, this study indicated that dysregulation of p57(kip2) promoted cell migration and invasion through modulation of LIMK/cofilin signaling and suggested this induction of inappropriate cell motility might contribute to promoting tumor cell for metastasis., (Copyright © 2011 Wiley Periodicals, Inc.)

ΔNp63, the N-terminal truncated isoform of p63, has been found to be overexpressed in several human epithelial cancers, including nasopharyngeal carcinomas (NPCs), suggesting a function in carcinogenesis. Trans-resveratrol (RSV) has been shown to exert proapoptotic activities through a p53-dependent or p53-independent pathway in various cancer cells. However, the effects of RSV on NPC are still unexplored. In this study, we investigated the apoptotic effects of RSV on ΔNp63-overexpressing NPC cell lines. We showed that RSV (12-100 μ) induced dose-dependent growth suppression, cell-cycle arrest in the S phase and caspase-dependent apoptosis in NPC-TW076 and NPC-TW039 cells. The RSV effect was accompanied by the downregulation of ΔNp63 and the upregulation of p53 protein in a dose-dependent manner. By using small-interfering RNA (siRNA) technology, we found that the targeted silencing of ΔNp63 induced apoptosis and sensitized the NPC cells to RSV-induced apoptosis through caspase-3 activation, whereas suppression of p53 by siRNA did not inhibit RSV-induced apoptosis. Furthermore, transfection with p53 siRNA or pretreatment with caspase inhibitors (Z-VAD-fmk or Z-DEVD-fmk) had no influence on the RSV downregulation of ΔNp63. Interestingly, ecoptic expression of ΔNp63 did not significantly block RSV-induced cell death and was also downregulated after RSV treatment. Downregulation of ΔNp63 by RSV was shown to occur at the mRNA transcript and post-translational levels. Importantly, RSV enhanced chemotheraptic drug-induced apoptosis in NPC and two human carcinoma cell lines, HT1376 and Hep3B cells. These results suggested that ΔNp63, but not p53, is a molecular target of RSV-induced apoptosis and the regulation of ΔNp63 expression by RSV may provide a therapeutic effect of RSV in human NPC.

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 DeltaNp63 isoform lacks the N-terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. DeltaNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of DeltaNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a DeltaNp63 overexpressing human NPC cell line (NPC-076) to explore the possible roles of DeltaNp63 in cell proliferation and cell-cycle regulation. We found that the proliferation of NPC-076 cell is greatly suppressed when the overexpressed DeltaNp63 is silenced by specific DeltaNp63 siRNA. Further studies show that DeltaNp63 silencing results in the upregulation of CKIs, including p27(kip1) and p57(kip2) in both mRNA and protein levels. Cell-cycle analysis shows that DeltaNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that DeltaNp63 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., ((c) 2008 Wiley-Liss, Inc.)

Purpose: Natural killer cells (NK) induce the death of tumor cells by perforin/granzyme-mediated cytotoxicity, whereas platelets reduce the capacity of NK to destroy tumor cells. Physical exercise affects both immune function and platelet activity because responses depend on type, intensity, and duration of exercise. This investigation explores how various exercise regimens influence platelet-impeded cytotoxicity of NK to nasopharyngeal carcinoma cells (NPC)., Methods: Thirty sedentary men performed on three occasions moderate exercise (60% .VO2max for 40 min), severe exercise (up to .VO2max), and severe exercise after warm-up exercise (60% .VO2max for 20 min). NK count and perforin/granzyme B contents, NK-NPC binding, and NK-induced NPC apoptosis were measured using a flow cytometer, whereas NK-induced NPC detachment from collagen-coated surface was determined using an electrical cell-substrate impedance sensing., Results: Severe exercise enhanced NK-induced NPC caspase-3 activation, phosphatidylserine (PS) exposure, DNA fragmentation, and detachment and was accompanied by increased NK count and perforin/granzyme B contents (P < 0.05). Moreover, severe exercise simultaneously promoted the suppression of platelet to NK-NPC binding and NK-induced NPC caspase-3 and -8 activations, PS exposure, DNA fragmentation, and detachment (P < 0.05). However, warm-up exercise pretreatment diminished the effects of severe exercise in platelet-impeded NK-NPC binding and NK-induced NPC apoptosis. Although moderate exercise also suppressed platelet-impeded NK-NPC interaction (P < 0.05), no significant changes occurred in NK count and perforin/granzyme B contents after this exercise., Conclusions: Severe exercise enhances the cytotoxicity of NK to NPC and simultaneously promotes the platelet-impeded apoptosis of NPC induced by NK. However, warm-up exercise reduces the resistance of platelet to NK-NPC interaction, increasing the efficiency of anti-NPC cytotoxicity by NK after severe exercise.

Interaction between platelet and carcinoma cell contributes to pathogenesis of cancer-related thrombosis and metastasis. This study investigated whether physical exercise affects platelet-nasopharyngeal carcinoma cell (NPC) interaction and platelet-promoted tissue factor (TF) and matrix metalloproteinase (MMP) activities of NPC. Thirty sedentary men performed on three occasions moderate-intensity exercise [MIE, 60% maximal oxygen consumption (V(.)o(2max)) for 40 min] and high-intensity exercise (HIE, up to V(.)o(2max)), with and without warm-up exercise (WUE, 60% V(.)o(2max) for 20 min) on a bicycle ergometer. Before and immediately after exercise, platelet-NPC aggregation, the TF, MMP-2 and MMP-9 expressions and activities, and TF pathway inhibitor (TFPI) and tissue inhibitor of MMP-1 levels of NPC and platelet were measured. The results of this study demonstrated that HIE enhanced platelet-NPC aggregation in the presence of fibrinogen and was accompanied by increased platelet-promoted TF activity, expression of NPC, decreased platelet-promoted MMP-2 and MMP-9 activities, and TFPI release of NPC, whereas these alterations to HIE on platelet-NPC interactions were ameliorated by WUE pretreatment. Conversely, MIE reduced the formation of platelet-NPC aggregates, but did not change the TF, TFPI, MMP-2, MMP-9, tissue inhibitor of MMP activities, and/or levels of NPC mediated by platelet. It is concluded that HIE may enhance aggregation and coagulation and reduce MMP bioactivity related to platelet-NPC interactions, by raising the binding affinity to fibrinogen and TF activity and expression and lowering TFPI release and MMP-2 and -9 activities. These effects on HIE diminish after WUE. However, MIE minimizes the risk of thrombosis induced by platelet-NPC interactions.

trans-Resveratrol (RSV) has been shown to have cardioprotective effect during ischemia-reperfusion through reactive oxygen species (ROS)-scavenging activity. Elevated ROS has been implicated in the initiation and progression of atherosclerosis. The nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of vascular ROS formation. In the present study, we show that exposure of vascular endothelial cells (EC) to oxidized low-density lipoproteins (oxLDL) results in elevations of NOX activity and cellular ROS levels. The oxLDL effects are effectively suppressed by RSV or astringinin (AST), either before or after oxLDL exposure. In this study, we show that RSV or AST treatment appears to suppress NOX activity by reducing the membrane association of gp91(phox) and Rac1, two protein species required for the assembly of active NOX complex. Exposure to RSV or AST protects EC from oxidative functional damages, including antiplatelet activity and mononucleocyte adhesion. In addition, ANG II-induced NOX activation is also attenuated. These results suggest that RSV or AST protects EC from oxLDL-induced oxidative stress by both direct ROS scavenging and inhibition of NOX activity.

Dietary antioxidants are thought to be beneficial in reducing the incidence of coronary heart disease. In this study, the antithrombogenic endothelial cells (EC) defense was investigated in an experiment model in which cultured endothelial cells were incubated with aggregating platelets in the aggregometer. We examined the possible protective effect of trans-resveratrol (RSV) on oxidized low density lipoprotein (ox-LDL)-induced insults on the antithrombogenic activity of the vascular EC. EC were treated with ox-LDL (25-100 microg/ml) for 1 h with or without a 30 min-preexposure to RSV. The antiplatelet property of the endothelial cells was then shown by measuring platelet aggregation, [Ca2+]i and cGMP contents in the platelets and EC. Exposure of EC to ox-LDL reduced the antiplatelet aggregating property of EC, and this effect was attenuated by pretreatment with RSV. Further studies revealed that exposure of EC to ox-LDL reduced the protein contents of endothelial nitric oxide synthase (NOS). The effect of ox-LDL on the NOS protein content was abrogated by pretreating EC with RSV. The results suggest that ox-LDL acts via reducing the endothelial NOS activity to suppress the antithrombogenic activity of the EC.

Exercise significantly influences the progression of atherosclerosis. Oxidized LDL (ox-LDL), as a stimulator of oxidative stress, facilitates monocyte-related atherogenesis. This study investigates how exercise intensity impacts ox-LDL-mediated redox status of monocytes. Twenty-five sedentary healthy men exercised mildly, moderately, and heavily (i.e., 40, 60, and 80% maximal oxygen consumption, respectively) on a bicycle ergometer. Reactive oxygen species (ROS) production, cytosolic and mitochondrial superoxide dismutase (c-SOD and m-SOD, respectively) activities, and total and reduced-form gamma-glutamylcysteinyl glycine (t-GSH and r-GSH, respectively) contents in monocytes mediated by ox-LDL were measured. This experiment obtained the following findings: 1) ox-LDL increased monocyte ROS production and was accompanied by decreased c-SOD and m-SOD activities, as well as t-GSH and r-GSH contents, whereas treating monocytes with diphenyleneiodonium (DPI) (a NADPH oxidase inhibitor) or rotenone/2-thenoyltrifluoroacetone (TTFA) (mitochondrial complex I/II inhibitors) hindered ox-LDL-induced monocyte ROS production; 2) production of ROS and reduction of m-SOD activity and r-GSH content in monocyte by ox-LDL were enhanced by heavy exercise and depressed by mild and moderate exercise; and 3) heavy exercise augmented the inhibition of ox-LDL-induced monocyte ROS production by DPI and rotenone/TTFA, whereas these DPI- and rotenone/TTFA-mediated monocyte ROS productions were unchanged in response to mild and moderate exercise. We conclude that heavy exercise increases ox-LDL-induced monocyte ROS production, possibly by decreasing m-SOD activity and r-GSH content in monocytes. However, mild and moderate exercise likely protects individuals against suppression of anti-oxidative capacity of monocyte by ox-LDL.

p63 splicing variants lacking NH(2)-terminal transactivating domain, known as DeltaNp63, are thought to antagonize p53 and p63 functions and are suggested to play roles in keratinocyte differentiation. Here, we show that DeltaNp63 has a dual-regulatory effect on the activity of its own promoter in NPC-076 cell. Down-regulation of the transcriptional activity is observed when DeltaNp63 is present in low levels. In contrast, up-regulation of DeltaNp63 transcriptional activity is observed when DeltaNp63 is expressed at higher levels. The down-regulation effect is abolished when the p53-binding site of the DeltaNp63 promoter is mutated. In sharp contrast, similar mutation does not affect the up-regulation of the DeltaNp63 transcriptional activity under the same experimental conditions. Further study shows that the up-regulation is correlated with the activation of the STAT3, as the blockade of STAT3 nuclear translocation abolishes the up-regulation by DeltaNp63. Thus, DeltaNp63 exerts a bidirectional regulation of the DeltaNp63 transcriptional activity in NPC-076 cell.

Physical exercise can affect the risk of cardiovascular disease. Oxidized-low density lipoprotein (ox-LDL) promotes transendothelial migration (TEM) of monocyte, thereby accelerating the pathogenesis of atherosclerosis. This study investigated how exercise intensity affects monocyte/EC interactions under ox-LDL-mediated condition. Light- (LIE), moderate- (MIE) and high- (HIE) intensity exercise (i.e., 40%, 60%, and 80% VO2max, respectively) on a bicycle ergometer in 18 sedentary healthy men were performed on three separate occasions. Before and immediately after exercise, ox-LDL-promoted expressions of monocyte adhesion molecules and TEM of monocyte, as well as oxidation of LDL and amounts of soluble adhesion molecules in plasma were measured. Analytical results showed that (1) ox-LDL furthered monocyte L-selectin shedding and Mac-1 expression, and an attendant increase in TEM of monocyte, while treating the monocyte with Mac-1 antibody inhibited the ox-LDL-promoted TEM of monocyte; (2) under ox-LDL-treated condition, MIE increased monocyte Mac-1 and LFA-1 expressions, enhancing the TEM of monocyte, whereas HIE downregulated monocyte Mac-1 expression, suppressing the TEM of monocyte; (3) LIE decreased basal LFA-1 expression as well as basal and ox-LDL-promoted TEM of monocyte; and (4) MIE and HIE, but not LIE, elevated plasma ox-LDL level, while there were no significant changes in sL-selectin, sE-selectin, sICAM-1, and sVCAM-1 following these exercises. Therefore, we conclude that monocyte activation and subsequent TEM promoted by ox-LDL are changed by short-term exercise in an intensity-dependent manner. These findings provide a new insight into the may aid the development of suitable exercise intensity enable people to prevent early atherogenesis.

Animal studies have demonstrated that restoration of blood flow to severely ischemic myocardium is a prerequisite for myocardial salvage. However, it has been shown that the restoration of blood flow to ischemic myocardium may be associated with deleterious changes of the myocardium, including arrhythmias, enzyme release, and contractile dysfunction. These changes were considered to be additional injuries to the myocardium manifested at the time of reperfusion. The reperfusion was accompanied by a burst of oxygen free radical generation and their role as main mediators of the reperfusion injury have been well accepted. Reactive oxygen species (ROS) and cellular redox status regulate many important cellular activities. The role of antioxidant as a therapy for reperfusion injury has thus been tried with mixed and mostly negative results. Further studies are needed if the antioxidant therapies for ischemia reperfusion injury were to be effective.

Objective: To investigate how exercise training and detraining affect oxidized low-density lipoprotein (Ox-LDL)-potentiated platelet function in men., Design: Cohort study., Setting: Department of physical medicine and rehabilitation., Participants: Ten sedentary men (mean age +/- standard error of the mean, 21.6+/-0.2 y) who did not engage in any regular physical activity for at least 1 year before the study., Interventions: Subjects cycled on an ergometer at about 50% of maximal oxygen consumption for 30 minutes daily, 5 days a week, for 8 weeks, then detrained for 12 weeks., Main Outcome Measures: During the experimental period, blood samples from the subjects were collected before and immediately after a progressive exercise test (ie, strenuous, acute exercise) every 4 weeks. The following measurements were taken when the subjects were at rest and immediately after exercise: plasma lipid profile, plasma Ox-LDL level, and platelet aggregation and intracellular calcium concentration ([Ca2+]i) elevation induced by adenosine disphosphate (ADP) alone or simultaneous ADP and Ox-LDL addition., Results: Analytical results indicated that: (1) plasma total cholesterol and LDL levels were reduced after exercise training from 151+/-7 mg/dL and 58+/-2 mg/dL to 133+/-6 mg/dL and 46+/-2 mg/dL (P<.05), respectively, whereas the plasma Ox-LDL level remained unchanged; (2) platelet aggregation and [Ca2+]i elevation promoted by 100 microg/mL of Ox-LDL were significantly increased from 70%+/-5% and 91%+/-7% of resting level to 108%+/-4% and 125%+/-3% after strenuous, acute exercise (P<.05); (3) exercise training decreased resting and postexercise 100 microg/mL Ox-LDL-potentiated platelet aggregation (ie, 31%+/-4% and 82%+/-4%, respectively; P<.05) and [Ca2+]i elevation (ie, 35%+/-6% and 71%+/-4%, respectively; P<.05); (4) detraining reversed the training effects on lipid profile and platelet function; and (5) treating the platelet with L-arginine-inhibited Ox-LDL-potentiated platelet activation during the experimental period., Conclusions: Our results suggest that 8 weeks of exercise training decreased the plasma LDL level, but failed to influence production of plasma Ox-LDL. Importantly, resting and exercise-induced Ox-LDL-potentiated platelet activation was decreased by exercise training. However, this was reversed by detraining to the pretraining level.

Vigorous exercise transiently increases the risk of primary cardiac arrest. The reciprocal modulation of platelet and polymorphonuclear leukocyte (PMN) activities is important in the pathogenesis of thrombosis. This study investigates how strenuous, acute exercise affects platelet-PMN reciprocal modulation by closely examining 18 sedentary men who exercised strenuously on a bicycle ergometer. Shear-induced platelet activation, PMN interaction with surface-adherent platelets under shear flow, and PMN-dependent inhibition of platelet activation were measured both before and immediately after exercise. Analytical results can be summarized as follows: (i) shear-induced platelet adhesion on fibronectin-coated surface as well as ADP-induced release of platelet soluble P-selectin release and elevation of [Ca2+]i significantly increases after strenuous exercise; (ii) strenuous exercise is associated with higher velocity and percentage of rolling PMNs and lower numbers of PMNs remaining bound to surface-adherent platelets under shear flow than at rest; (iii) PMN-dependent inhibition of platelet [Ca2+]i elevation and soluble P-selectin release after strenuous exercise is much greater than that at rest; and (iv) strenuous exercise increases PMN-derived nitric oxide metabolite level and reduces oxidized low-density lipoprotein-promoted interaction between platelets and PMNs. Therefore, we conclude that platelet activity may be sensitized by strenuous exercise. However, strenuous exercise can also simultaneously enhance the antiplatelet effect of PMNs. The finding provides a new insight into the negative feedback of PMNs against exercise-evoked platelet-related thrombotic risk.

Oxidized 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.

This study investigates how strenuous arm exercise affects oxidized-low density lipoprotein (O(X)-LDL) mediated-platelet activation in patients with SCI. Ten patients with SCI and ten age- and sex-matched healthy subjects exercised strenuously using an arm crank ergometer. The following measurements were taken both when the subjects were at rest, and immediately after exercise: plasma lipid profile, O(X)-LDL mediated platelet aggregability and [Ca(2+)]i, urinary 11-dehydro-thromboxane B2 (11-dehydro-TXB2) and 8-iso-prostaglandin F(2alpha), (8-iso-PG F(2alpha)) contents, and plasma NO metabolite (nitrite plus nitrate) level. Based on these measurements, the major findings of this study can be summarized as follows: 1) the SCI group had higher urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB2 contents, but a lower plasma nitrite plus nitrate level than the control group; 2) at rest, the SCI group had a higher platelet aggregability and [Ca(2+)]i, and O(X)-LDL-potentiated platelet activation than the control group; 3) O(X)-LDL-potentiated platelet aggregation was enhanced by strenuous arm exercise in both groups, but the effect of exercise was more pronounced in the SCI group than in the control group; 4) treating the platelet with L-arginine inhibited O(X)-LDL-potentiated platelet activation in both groups. The study concludes that individuals with SCI had more extensive resting and exercise-enhanced O(X)-LDL-potentiated platelet activation and greater amounts of preformed lipid peroxides than those without SCI. Therefore, supplementation therapy with antioxidants may be needed for patients with SCI, especially in a strenuous arm exercise period.

This study investigates how exercise training affects Oxidized LDL (Ox-LDL) mediated-platelet activation. Five-week-old male Wistar rats were assigned to either control or trained groups. Trained rats were treadmill-trained for 10 weeks after familiarization. The following measurements were taken in both control and trained groups: Ox-LDL-mediated platelet aggregability and [Ca2+]i, plasma and platelet-derived nitric oxide (NO) metabolite (nitrite plus nitrate) levels, and antiaggregating activity of NO derived from endothelial cells. Based on those measurements, major findings in this study can be summarized as follows: 1) the trained group had a higher plasma -NO metabolite level than the control group; 2) the trained group had a lower platelet aggregability and [Ca2+]i elevation and a higher platelet derived-NO metabolite level than the control group; 3) the trained group had lower Ox-LDL-potentiated platelet aggregability and [Ca2+]i elevation and Ox-LDL-attenuated NO metabolite in platelet than the control group; 4) treating the platelet with L-arginine inhibited Ox-LDL-potentiated platelet activation in both control and trained groups; 5) Ox-LDL enhances platelet aggregation directly although impairing NO bioactivity but not guanylate cyclase activity in both control and trained groups. Results in this study demonstrate that exercise training decreases Ox-LDL-potentiated platelet activation most likely by enhancing platelet-derived NO release.

Human low density lipoprotein (LDL) is prepared in the presence of antioxidants and is oxidized to different levels (measured by thiobarbituric acid reactive substance) with copper ion. The effects of unoxidized LDL and oxidized LDL (ox-LDL) on stress fiber formation, cell membrane ruffling, and pinocytosis (measured by [14C]sucrose uptake) in cultured human umbilical cord vein endothelial cells (EC) are compared. We show that at a concentration range of 100 to 200 microg cholesterol/ml, both unoxidized LDL and ox-LDL promote EC elongation and stress fiber formation, but the effect by the latter is more prominent when compared at the same dose range. In addition, ox-LDL also induces EC membrane ruffling and promotes pinocytosis. These effects are positively correlated with the extent of LDL oxidation and depend on the dose of ox-LDL. Ox-LDL-promoted membrane ruffling and pinocytosis are effectively blocked by brief preexposure of the cells to antioxidants. In contrast, stress fiber formation is not affected by antioxidant pretreatment. Although unoxidized LDL also promotes [14C]sucrose uptake, it is less potent than ox-LDL and significantly higher concentrations are required to produce a detectable effect. Unlike ox-LDL, unoxidized LDL-enhanced pinocytosis is not accompanied by the appearance of membrane ruffling; therefore, they may act via different mechanisms. Elevated pinocytosis may increase transcytotic activity of the endothelium, leading to an increased influx of plasma components such as LDL into the subendothelial space.

Accumulating evidence indicates that telomerase activity is stringently repressed in normal human somatic cells but reactivated in cancers and immortal cells, suggesting that activation of telomerase activity may play a role in carcinogenesis and immortalization. Recently, down-regulation of telomerase activity by induction of differentiation has been reported for cells of pre-myelocytic and myelocytic leukemia as well as embryonic carcinoma. To gain further insight about the regulation of telomerase activity following induction of differentiation, telomerase activity was examined in a human hematopoietic progenitor cell line (D2), a melanoma cell line (CM73-36) and a glioma cell line (Ast812) before and after addition of differentiation inducing agents. The state of differentiation was assessed by growth inhibition and cell morphological maturation. Telomerase activity was assayed by a PCR-based telomeric repeat amplification protocol (TRAP). Our data show that telomerase activity was inhibited only in differentiation-induced D2 cells but not in differentiation-induced melanoma and glioma cells. A model for the differential inhibition of telomerase activity following induction of differentiation in different cancer cells will be presented.