Phosphorylation of eIF2α suppresses cisplatin-induced p53 activation and apoptosis by attenuating oxidative stress via ATF4-mediated HO-1 expression in human renal proximal tubular cells.

Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of human cancers. However, the nephrotoxicity of cisplatin limits its use as a therapeutic agent. It has been suggested that oxidative stress and p53 activation play important roles in cisplatin-induced nephrotoxicity. It has been demonstrated that the eukaryotic translation initiation factor 2α (eIF2α) may protect HK-2 human renal proximal tubular cells against cisplatin-induced apoptosis through inhibition of reactive oxygen species (ROS)­mediated p53 activation. The aim of the present study was to investigate the effects of siRNA­mediated knockdown of the PKR-like endoplasmic reticulum kinase (PERK) gene, which induces the phosphorylation of eIF2α, or Sal003, a selective inhibitor of eIF2α dephosphorylation, on cisplatin­induced apoptosis in HK-2 cells. Cisplatin induced eIF2α phosphorylation as well as p53 activation. In particular, inhibition of p53 by pifithrin­α, and upregulation of eIF2α phosphorylation by Sal003, reduced cisplatin-induced apoptosis. Of note, Sal003­mediated upregulation of eIF2α phosphorylation suppressed cisplatin­induced p53 activation. Furthermore, reduction of eIF2α phosphorylation by PERK knockdown enhanced cisplatin-induced p53 activation and apoptosis. In addition, the ROS scavenger N-acetyl-L-cysteine inhibited eIF2α phosphorylation as well as p53 activation in HK-2 cells treated with cisplatin, suggesting that oxidative stress induced by cisplatin may lead to apoptosis through p53 activation; furthermore, this stress may confer resistance to apoptosis via eIF2α phosphorylation, which was further supported by the finding that cisplatin­induced ROS generation was attenuated by Sal003, whereas it was enhanced by PERK knockdown. Furthermore, cisplatin induced the expression of activating transcription factor 4 (ATF4) and heme oxygenase-1 (HO-1) that were enhanced by Sal003 and reduced by PERK knockdown. Taken together, these results suggest that phosphorylation of eIF2α suppresses cisplatin­induced p53 activation and apoptosis by attenuating oxidative stress via ATF4-mediated HO-1 expression in HK-2 cells, as ATF4 expression is usually dependent on the phosphorylation of eIF2α and may also transcriptionally induce the expression of HO-1 in response to oxidative stress. Therefore, regulation of eIF2α phosphorylation may play an important role in alleviating cisplatin-induced nephrotoxicity.

Association between betatrophin/ANGPTL8 and non-alcoholic fatty liver disease: animal and human studies.

Betatrophin/angiopoietin-like protein 8 (ANGPTL8) is a liver-secreted protein recently identified as a potent stimulator of beta cell proliferation in mice. However, it is unclear how betatrophin is regulated in humans with non-alcoholic fatty liver disease (NAFLD). We investigated the role of betatrophin in mice and in humans with and without NAFLD. Serum betatrophin levels were examined by ELISA in 164 subjects, including 96 patients with NAFLD. Levels were significantly elevated in subjects with NAFLD compared with controls (1.301 ± 0.617 vs. 0.900 ± 0.574 µg/L, P < 0.001), even after stratification by diabetic or obesity status. Circulating betatrophin positively correlated with obesity or glycemic indices, liver enzyme profiles, and NAFLD status, and was confirmed by multivariate regression analyses (ß = 0.195, P = 0.040). However, when including insulin resistance index in the model, the significant association between betatrophin level and NAFLD was diminished due to a mediation effect of insulin resistance on this relationship. Palmitate or tunicamycin increased betatrophin expression in HepG2 cells, while a chemical chaperone blocked its induction. Hepatic expression of betatrophin was elevated in mice with NAFLD including db/db or ob/ob mice and mice with a high-fat or methionine-choline deficient diet. In conclusion, circulating betatrophin was increased in mice and humans with NAFLD and its expression was induced by endoplasmic reticulum stress in hepatocytes (Clinical trial no. NCT02285218).

Increased expression of ATP-binding cassette transporter A1 (ABCA1) as a possible mechanism for the protective effect of cilostazol against hepatic steatosis.

OBJECTIVE: Cilostazol, a phosphodiesterase 3, has been widely used in patients with arterial disease and is known to have additional beneficial effects on dyslipidemia. However, the effect of cilostazol on hepatic steatosis has not been fully elucidated. We investigated the effect of cilostazol on hepatic ABCA1 expression and hepatic steatosis in diet-induced obesity mice model. METHODS: Hepatic ABCA1 expression and lipid accumulation were analyzed in HepG2 cell lines treated with cilostazol. Male C57BL/6 mice were randomly divided into three groups: (1) fed normal chow diet with vehicle; (2) fed high-fat diet (HFD) with vehicle; (3) fed HFD with cilostazol. Cilostazol (30 mg/kg) was orally administered once daily for 9 weeks. RESULTS: Cilostazol significantly enhanced ABCA1 expression and restored ABCA1 expression reduced by palmitate in HepG2 cells. Cilostazol treatment ameliorated lipid accumulation induced by palmitate, and this effect was diminished when ABCA1 or LRP1 was silenced by small interference RNA. After silencing of LRP1, ABCA1 expression was decreased in HepG2 cells. Cilostazol significantly enhanced hepatic ABCA1 expression and decreased hepatic fat in HFD-fed mice. Hepatic expression of cleaved caspase-3 and PARP1 was also decreased in HFD-fed mice treated with cilostazol. CONCLUSIONS: Cilostazol ameliorated hepatic steatosis and increased ABCA1 expression in the hepatocytes. Enhancing ABCA1 expression with cilostazol represents a potential therapeutic avenue for treatment of hepatic steatosis.

The Flavonoid Apigenin Ameliorates Cisplatin-Induced Nephrotoxicity through Reduction of p53 Activation and Promotion of PI3K/Akt Pathway in Human Renal Proximal Tubular Epithelial Cells.

Apigenin is a member of the flavone subclass of flavonoids present in fruits and vegetables. Apigenin has long been considered to have various biological activities, such as antioxidant, anti-inflammatory, and antitumorigenic properties, in various cell types. Cisplatin was known to exhibit cytotoxic effect to renal cells by inducing apoptosis through activation of p53. The present study investigated the antiapoptotic effects of apigenin on the cisplatin-treated human renal proximal tubular epithelial (HK-2) cells. HK-2 cells were pretreated with apigenin (5, 10, 20 µM) for 1 h and then treated with 40 µM cisplatin for various times. Apigenin inhibited the cisplatin-induced apoptosis of HK-2 cells. Interestingly, apigenin itself exerted cytostatic activity because of its ability to induce cell cycle arrest. Apigenin inhibited caspase-3 activity and PARP cleavage in cisplatin-treated cells. Apigenin reduced cisplatin-induced phosphorylation and expression of p53, with no significant influence on production of ROS that is known to induce p53 activation. Furthermore, apigenin promoted cisplatin-induced Akt phosphorylation, suggesting that enhanced Akt activation may be involved in cytoprotection. Taken together, these results suggest that apigenin ameliorates cisplatin-induced apoptosis through reduction of p53 activation and promotion of PI3K/Akt pathway in HK-2 cells.

Nardostachys chinensis induces granulocytic differentiation with the suppression of cell growth through p27(Kip1) protein-related G0/G1 phase arrest in human promyelocytic leukemic cells.

CONTEXT: Nardostachys chinensis Batalin (Valerianaceae) has been used in Korean traditional medicine to elicit stomachic and sedative effects. However, the anti-leukemic activities of N. chinensis have not been well examined. OBJECTIVE: To investigate the effect of N. chinensis on differentiation and proliferation in the human promyelocytic leukemic HL-60 cells. MATERIALS AND METHODS: The dried roots and stems of N. chiensis are extracted using hot water and then freeze-dried. The yield of extract was 12.82% (w/w). The HL-60 cells were treated with 25-200 µg/ml of N. chinensis for 72 h or 100 µg/ml of N. chinensis for 24-72 h. RESULTS: Nardostachys chinensis significantly inhibited cell viability dose dependently with an IC50 of 100 µg/ml in HL-60 cells. Nardostachys chinensis induced differentiation of the cells as measured by reduction activity of NBT and expression of CD11b but not of CD14 as analyzed by flow cytometry, which indicates a differentiation toward the granulocytic lineage. Nardostachys chinensis also induced growth inhibition through G0/G1 phase arrest in the cell cycle of HL-60 cells. Among the G0/G1 phase in the cell cycle-related protein, the expression of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) was increased in N. chinensis-treated HL-60 cells, whereas the expression levels of CDK2, CDK4, CDK6, cyclin D1, cyclin D3, cyclin E, and cyclin A were decreased. Interestingly, N. chinensis markedly enhanced the binding of p27(Kip1) with CDK2 and CDK6. DISCUSSION AND CONCLUSION: This study demonstrated that N. chinensis is capable of inducing cellular differentiation and growth inhibition through p27(Kip1) protein-related G0/G1 phase arrest in HL-60 cells.

The hypolipidemic effect of cilostazol can be mediated by regulation of hepatic low-density lipoprotein receptor-related protein 1 (LRP1) expression.

OBJECTIVES: Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is a vasodilator and an anti-thrombotic agent. The mechanism whereby cilostazol reduces plasma triglyceride is not completely understood. Here we investigated the effect of cilostazol on a remnant lipoprotein receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which has been reported to play an essential role in clearance of circulating triglyceride in the liver. MATERIALS/METHODS: Total cellular expression, and functional and transcriptional regulation of LRP1 were analyzed in human hepatocarcinoma cell lines incubated with cilostazol. Also, C57BL/6 mice were subjected to high-fat diet (60% kcal) and cilostazol (30 mg/kg) treatment for 10 weeks. RESULTS: Cilostazol increased both mRNA and protein expression of LRP1 in HepG2 and Hep3B cells. In addition, enhanced transcriptional activity of the LRP1 promoter containing a peroxisome proliferator response element (PPRE) was observed after cilostazol exposure. Cilostazol treatment enhanced the uptake of lipidated apoE3, and this effect was abolished when LRP1 was silenced by siRNA knockdown. High-fat diet induced hyperglycemia with high level of plasma triglycerides, and reduced hepatic LRP1 expression in mice. Treatment with cilostazol for the same period of time, however, successfully prevented this down-regulation of LRP1 expression and reduced plasma triglycerides. CONCLUSION: Taken together, our results demonstrated that cilostazol enhances LRP1 expression in liver by activating PPARγ through the PPRE in the LRP1 promoter. Increased hepatic LRP1 may be essential for the reduction of circulating triglycerides brought about by cilostazol.

Nardostachys chinensis induces the differentiation of human promyelocytic leukemic cells through the activation of the protein kinase C-dependent extracellular signal-regulated kinase signaling pathway.

The underground parts of Nardostachys chinensis (N. chinensis), which belongs the genus Valerianaceae, have been used as sedative and analgesic agents in traditional Korean medicine for centuries. The mitogen-activated protein kinases (MAPKs) are serine/threonine kinases involved in the regulation of various cellular responses, such as cell proliferation, differentiation and apoptosis. Protein kinase C (PKC) plays a key role in the regulation of proliferation and differentiation. In this study, we investigated the signaling pathways involved in the differentiation of the HL-60 human leukemic cells induced by N. chinensis extract. Treatment with N. chinensis extract resulted in the activation of the extracellular signal-regulated kinase (ERK) pathway and induced the differentiation of HL-60 cells into granulocytes. The activation of p38 MAPK was also observed 24 h after treatment; however, the activation of c-Jun N-terminal kinase (JNK) was unaffected. Treatment with an inhibitor of ERK (PD98059) blocked the nitrotetrazolium blue chloride (NBT) reducing activity and CD11b expression in the N. chinensis-treated HL-60 cells, whereas treatment with an inhibitor of p38 MAPK (SB203580) had no significant effect on NBT reducing activity and CD11b expression. In addition, N. chinensis extract increased PKC activity and the protein levels of PKCα, PKCßI and PKCßII isoforms, without a significant change in the protein levels of the PKCγ isoform. PKC inhibitors (GF 109203X, chelerythrine and H-7) inhibited the differentiation of HL-60 cells into granulocytes, as well as ERK activation in the N. chinensis-treated HL-60 cells. These results indicate that the PKC and ERK signaling pathways may be involved in the induction, by N. chinensis extract, of the differentiation of HL-60 cells into granulocytes.

Anti-cancer activity of highly purified sulfur in immortalized and malignant human oral keratinocytes.

Sulfur is commonly used in Asia as an herbal medicine to treat inflammation and cancer, and potent chemopreventive effects have been demonstrated in various in vivo and in vitro models for sulfur-containing compounds found in naturally occurring products. Here, we report the growth inhibitory and apoptosis-related effects of a newly developed highly purified sulfur (HPS) on immortalized human oral keratinocytes (IHOKs) and on oral cancer cells representing two stages of oral cancer (HN4, HN12) based on a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Western blotting, cell cycle analysis, and nuclear staining. The purity of the sulfur preparation was verified by high-performance liquid chromatography. HPS inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. FITC-annexin V staining, DNA fragmentation testing, and Hoechst 33258 staining revealed that HPS inhibited cell growth via apoptosis. HPS increased the sub-G1 cell cycle fraction, with decreased expression of cyclins D1, D2, and E and their activating partners cdk2, cdk4, and cdk6, and a concomitant induction of p53 and p21/WAF1. Furthermore, HPS treatment increased the cytosolic level of cytochrome c and resulted in caspase-3 activation; this effect was correlated with Bax up-regulation and Bcl-2 down-regulation. Thus, these data suggest that HPS is a potential candidate for anti-cancer therapy in oral cancer.

Verticinone induces cell cycle arrest and apoptosis in immortalized and malignant human oral keratinocytes.

Although verticinone, a major alkaloid isolated from the bulbus of Fritillaria ussuriensis, has been shown to induce differentiation in human leukemia cells, the exact mechanism of this action is not completely understood in cancer cells. Verticinone was used to conduct growth and apoptosis-related experiments for two stages of oral cancer on immortalized human oral keratinocytes (IHOKs) and primary oral cancer cells (HN4). The procedures included MTT assay, three-dimensional (3-D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression related to the apoptosis signaling pathway. Verticinone inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. In 3-D organotypic culture, verticinone-treated cells were less mature than the control cells, displaying low surface keratinization and decreased epithelial thickness. The major mechanism by which verticinone inhibits growth appears to be induced apoptosis and G(0)G(1) cell cycle arrest. This finding is supported by the results of the cell cycle analysis, FITC-Annexin V staining, DNA fragmentation assay and Hoechst 33258 staining. Furthermore, the cytosolic level of cytochrome c was increased, while the expression of Bcl-2 protein was gradually down-regulated and Bax was up-regulated, accompanied by caspase-3 activation. The data suggests that verticinone may induce apoptosis through a caspase pathway mediated by mitochondrial damage in immortalized keratinocytes and oral cancer cells.

Antiplatelet and antithrombotic activity of indole-3-carbinol in vitro and in vivo.

Indole-3-carbinol, a natural compound found in cruciferous vegetables, is known to have anticancer activity. In the present study, the antiplatelet and antithrombotic activities of indole-3-carbinol were investigated in vitro and in vivo. Indole-3-carbinol significantly inhibited collagen-induced platelet aggregation in human platelet rich plasma (PRP) in a concentration-dependent manner. Indole-3-carbinol significantly inhibited fibrinogen binding to the platelet surface glycoprotein IIb/IIIa (GP IIb/IIIa) receptor by flow cytometric analysis. In addition, the levels of thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) in collagen stimulated PRP were significantly inhibited in a concentration-dependent manner by indole-3-carbinol. Furthermore, indole-3-carbinol dose-dependently suppressed the death of mice with pulmonary thrombosis induced by intravenous injection of collagen and epinephrine. These results suggest that indole-3-carbinol can be a potent antithrombotic agent with antiplatelet activity through the inhibition of GP IIb/IIIa receptor and thromboxane B2 formation.

Functional interaction between nitric oxide-induced iron homeostasis and heme oxygenase-1 in immortalized and malignant oral keratinocytes.

Heme oxygenase-1 (HO-1) is involved in a variety of regulatory and protective cellular mechanisms as a stress-responsive protein. Whether HO-1 plays a protective role against NO-induced cytotoxicity in oral cancer cells has not yet been established. We used sodium nitroprusside (SNP) as a source of exogenous NO in studies of NO-induced cytotoxicity in immortalized (IHOK) and malignant oral keratinocytes (HN12). The roles of the caspase pathway, of regulatory proteins of iron metabolism (iron regulatory protein (IRP)1, IRP2, transferrin receptor (TfR), and ferritin), and of HO-1 in protection against NO-induced cytotoxicity were assessed. The SNP-induced growth inhibition and apoptosis of IHOK and HN12 cells was reduced by addition of ferric citrate (FC). At low concentrations (< 1 mM), SNP up-regulated cellular iron metabolism by increasing expression of IRP1, IRP2, and TfR, whereas at high concentrations (> 2 mM), SNP down-regulated expression of these proteins. A consistent correlation between decreased levels of IRP1, IRP2, and TfR and increased NO-induced cytotoxicity and apoptosis was observed. Addition of FC inhibited the NO-induced decrease in IRP1, IRP2, and TfR levels. Moreover, SNP increased the expression of HO-1 and ferritin in IHOK and HN12 cells in a concentration-dependent manner. NO-induced cytotoxicity was also inhibited by hemin (an HO-1 agonist) and was enhanced by zinc protoporphyrin IX (an HO-1 inhibitor). Based on these results, we conclude that HO-1 plays a major role in mediating cytoprotection and iron homeostasis against NO toxicity in immortalized and malignant oral keratinocytes.

Extract of Coptidis rhizoma induces cytochrome-c dependent apoptosis in immortalized and malignant human oral keratinocytes.

Coptidis rhizoma (C. rhizoma) had been demonstrated as an antioxidant and anticancer agent, however, its antioral cancer mechanism still remains unclear. Using water extracts of C. rhizoma, growth and apoptosis-related experiments for the treatment of multi-stage of oral cancer were carried out on immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12) and human skin keratinocytes (HaCaT) by MTT assay, three-dimensional (3-D) raft cultures, western blotting, cell cycle analysis, nuclear staining and cytochrome c expression related to the apoptosis signaling pathway. C. rhizoma inhibited the proliferation of immortalized and malignant oral keratinocytes in a dose- and time-dependent manner. In 3-D organotypic culture, C. rhizoma-treated cells showed less maturation than the control cells, displaying low surface keratinization and decreased epithelial thickness. The major mechanism of growth inhibition by C. rhizoma appears to be the induction of apoptosis, which is supported by the results of the cell cycle analysis, FITC-annexin V staining, DNA fragmentation assay and DAPI staining. The induction of apoptosis by C. rhizoma was more prominent in immortalized keratinocytes than in malignant oral keratinocytes. Cytochrome-c release from mitochondria, accompanied by the activation of caspase-3, was observed in C. rhizoma-treated IHOK and oral cancer cells. These results suggest that C. rhizoma has apoptotic effects in immortalized and malignant oral keratinocytes via the mitochondrial signaling pathway.

p38 and ERK MAP kinase mediates iron chelator-induced apoptosis and -suppressed differentiation of immortalized and malignant human oral keratinocytes.

Iron is essential for neoplastic cell growth, and iron chelators have been tested for potential anti-proliferative and anti-cancer effects, but the effects of iron chelators on oral cancer have not been clearly elucidated. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during iron chelator-induced apoptosis and differentiation of immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). The iron chelator deferoxamine (DFO) exerted potent time- and dose-dependent inhibitory effects on the growth and apoptosis of IHOK and HN4 cells. DFO strongly activates p38 MAP kinase and extracellular signal-regulated kinase (ERK), but does not activate c-Jun N-terminal kinase/stress-activated protein kinase. Of the three MAP kinase blockers used, the selective p38 MAP kinase inhibitor SB203580 and ERK inhibitor PD98059 protected IHOK and HN4 cells against iron chelator-induced cell death, which indicates that the p38 and ERK MAP kinase is a major mediator of apoptosis induced by this iron chelator. Interestingly, treatment of IHOK and HN4 cells with SB203580 and PD98059 abolished cytochrome c release, as well as the activation of caspase-3 and caspase-8. DFO suppressed the expression of epithelial differentiation markers such as involucrin, CK6, and CK19, and this suppression was blocked by p38 and ERK MAP kinase inhibitors. Collectively, these data suggested that p38 and ERK MAP kinase plays an important role in iron chelator-mediated cell death and in the suppression of differentiation of oral immortalized and malignant keratinocytes, by activating a downstream apoptotic cascade that executes the cell death pathway.

Iron chelator-induced growth arrest and cytochrome c-dependent apoptosis in immortalized and malignant oral keratinocytes.

BACKGROUND: Many studies have shown the anti-proliferative effects of iron deprivation on cancer cells, but the effects of iron-chelators on oral cancer have not been clearly elucidated. METHODS: To investigate the effects of an iron chelator, desferrioxamine (DFO), on the growth of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12) and human skin keratinocytes (HaCaT) in the MTT assay, three-dimensional (3D) raft cultures, Western blotting, cell cycle analysis, nuclear staining and cytochrome c expression for apoptosis signaling pathway were used. RESULTS: Desferrioxamine inhibited the growth of immortalized IHOK and HaCaT and malignant HN4 and HN12 keratinocytes in a time- and dose-dependent manner according to the MTT assay. The 3D organotypic culture also revealed that DFO-treated cells showed less epithelial maturation, less surface keratinization and decreased epithelial thickness. The major mechanism of growth inhibition with the micromolar DFO treatment was by the induction of apoptosis, which was supported by nuclear DAPI staining, DNA fragmentation analysis and flow cytometric analysis for sub-G(1) phase arrest and Annexin V-FITC (fluorescein isothiocyanate) staining. Furthermore, Bax expression increased together with p53 and p21(WAF1/CIP1), while the Bcl-2 expression decreased in the immortalized and malignant keratinocytes treated with DFO. Time-dependent cytochrome c from mitochondria was observed in DFO-treated IHOK and oral cancer cells and was accompanied by the activation of caspase-3 in IHOK cells. CONCLUSION: These results demonstrate that DFO has growth inhibitory effects on immortalized and malignant oral keratinocytes through the induction of apoptosis and suggest that further evaluation of DFO as a potential therapeutic agent for human oral precancerous lesions is warranted.

Caesalpinia sappan induces cell death by increasing the expression of p53 and p21WAF1/CIP1 in head and neck cancer cells.

Caesalpinia sappan L. (C. sappan) has been used in Oriental medicine as an antitumor agent. The present study shows the effects of the chloroform extract of C. sappan on cell death in head and neck cancer cell lines. The viability of HNSCC4 and HNSCC31 cells (head and neck cancer cell lines) was noticeably decreased compared to that of HaCaT cells (control group) in the presence of chloroform extract. No significant difference was observed in the viability of HNSCC4 and HNSCC31 cells when compared with HaCaT cells in the presence of n-butanol, methanol, and water extracts. Exposure to the chloroform extract of C. sappan resulted in an increase in the Sub-G1 phase of the cell cycle and condensation and shrinkage of nuclei in the HNSCC4 and HNSCC31 cells. The levels of p53 and p21WAF1/CIP1 were also increased in the HNSCC4 and HNSCC31 cells. The results suggest that the chloroform extract of C. sappan may increase cell death in the HNSCC4 and HNSCC31 cells, which is linked to increased cellular levels of p53 and p21WAF1/CIP1.

Effects of nicotine on proliferation, cell cycle, and differentiation in immortalized and malignant oral keratinocytes.

BACKGROUND: Numerous epidemiological studies have reported that tobacco smoking is a major risk factor for oral cancer, but relatively little is known about the effect of nicotine, a major product of cigarette smoking, on immortalized oral keratinocytes and cancer cells. METHODS: We investigated the effects of nicotine on the growth and differentiation of immortalized human oral keratinocytes (IHOK), primary oral cancer cells (HN4), metastatic oral cancer cells (HN12), and human skin keratinocytes (HaCaT), in the monolayer and in the three-dimensional (3D) raft cultures using the MTT assay, Western blotting, and cell cycle analysis. RESULTS: Nicotine inhibited the proliferation of immortalized and malignant keratinocytes in dose- and time-dependent manners as determined by MTT assay. The 3D organotypic culture showed that nicotine at high concentration (300 microM) inhibits epithelial maturation, surface keratinization, and decreased epithelial thickness. Flow cytometry showed that nicotine inhibited cell cycle progression by inducing G(0)/G(1) arrest of HaCaT, IHOK, HN4, and HN12 cells without causing apoptosis. Nicotine treatment increased p21 expression in immortalized cells (HaCaT, IHOK) and oral cancer cells (HN4, HN12), but decreased pRb and p53 expression in oral cancer cells. Moreover, after high-dose nicotine treatment, the involucrin expression increased markedly in immortalized cells, but not in oral cancer cells. CONCLUSIONS: We demonstrated that nicotine inhibits growth through cell cycle arrest at G(0)/G(1) phase probably by increasing the expression of p21(WAF1/CIP1). Nicotine also affects epithelial differentiation in immortalized and malignant oral keratinocytes. Malignant oral keratinocytes appear to be more resistant to the effects of nicotine on epithelial growth and differentiation as compared to the immortalized cells.

Inhibitory effect of GBH on platelet aggregation through inhibition of intracellular Ca2+ mobilization in activated human platelets.

Geiji-Bokryung-Hwan (GBH) was studied on antiplatelet activity in human platelet suspensions. GBH consists of the 5 herbs Cinnamomi Ramulus, Poria Cocos, Mountan Cortex Radicis, Paeoniae Radix, and Persicae Semen, which have been used in herbal medicine for thousands of years for atherosclerosis. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH inhibited platelet aggregation and Ca2+ mobilization in a concentration-dependent manner without increasing intracellular cyclic AMP and cyclic GMP. GBH had no inhibitory effect on thromboxane B2 (TXB2) production in cell-free systems. Collagen-related peptide (CRP)-induced Ca2+ mobilization is regulated by phospholipase C-2 (PLC-gamma2) activation. We evaluated the effect of GBH on tyrosine phosphorylation of PLC-gamma2 and the production of inositol-1,4,5-trisphosphate (IP3). GBH at concentrations that inhibited platelet aggregation and Ca2+ mobilization had no effects on tyrosine phosphorylation of PLC-gamma2 or on the formation of IP3 induced by CRP. Similar results were obtained with thrombin-induced platelet activation. GBH inhibited platelet aggregation and Ca2+ mobilization induced by thrombin without affecting the production of IP3. We then evaluated the effect of GBH on the binding of IP3 to its receptor. GBH at high concentrations partially blocked the binding of IP3 to its receptor. Therefore, the results suggested that GBH suppresses Ca2+ mobilization at a step distal to IP3 formation. GBH may provide a good tool for investigating Ca2+ mobilization.

Stylopine from Chelidonium majus inhibits LPS-induced inflammatory mediators in RAW 264.7 cells.

Stylopine is a major component of the leaf of Chelidonium majus L. (Papaveraceae), which has been used for the removal of warts, papillomas and condylomas, as well as the treatment of liver disease, in oriental countries. Stylopine per se had no cytotoxic effect in unstimulated RAW 264.7 cells, but concentration-dependently reduced nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and the IL-6 production and cyclooxygenase-2 (COX-2) activity caused by the LPS stimulation. The levels of inducible nitric oxide synthase (iNOS) and COX-2 protein expressions were markedly suppressed by stylopine in a concentration dependent manner. These results suggest that stylopine suppress the NO and PGE2 production in macrophages by inhibiting the iNOS and COX-2 expressions. These biological activities of stylopine may contribute to the anti-inflammatory activity of Chelidonium majus.

Methanol extract of Dioscoreae Rhizoma inhibits pro-inflammatory cytokines and mediators in the synoviocytes of rheumatoid arthritis.

Dioscoreae Rhizoma (MDR), the root of Dioscorea tokoro MAKINO, has been used for the treatment of arthritis, muscular pain and urinary diseases in oriental medicine. The present work evaluates a methanol extract of Dioscoreae Rhizoma (MDR). MDR did not show any cytotoxic effect on mouse lung fibroblast cells (mLFCs) or human fibroblast-like synovial cells (hFLSCs). However, it significantly reduced the proliferation of hFLSCs stimulated by interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). MDR significantly inhibited the production of TNF-alpha and IL-1beta as well as down-regulating the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in IL-1beta- and TNF-alpha-stimulated hFLSCs. MDR also effectively reduced the level of reactive oxygen species (ROS) in these cells. Taken together, these findings provide evidence that MDR may be a candidate for the treatment of rheumatoid arthritis (RA).

Cloning and characterization of a new cysteine proteinase secreted by Paragonimus westermani adult worms.

The cysteine proteinases of Paragonimus westermani are known to play important roles in invasion and pathogenesis to hosts and in immune modulation and nutrient uptake. In this study, we have cloned a new cysteine proteinase of P. westermani, PwCP2, from adult worms and tested its diagnostic usefulness. The PwCP2 gene had an open reading frame of 816 base pairs and a conserved catalytic triad of cysteine, histidine, and asparagine residues. The mature form of recombinant PwCP2 (rPwCP2) lacking a proregion was overexpressed in Escherichia coli and used to produce antiserum. Western blot and immunohistochemical analyses using this antiserum showed that PwCP2 was expressed as a mature form, 24-kD product in a crude extract and in the excretory-secretory product of P. westermani, and was localized mainly in the intestinal epithelium of the adult worm. Western blot analysis using the rPwCP2 showed not only high sensitivity (90%) and specificity (100%) to sera from patients with paragonimiasis westermani, but also no cross-reactivity with sera from patients with clonorchiasis, sparganosis, or cysticercosis. Furthermore, an enzyme-linked immunosorbent assay using rPwCP2 exhibited a sensitivity of 93% and a specificity of 93% with sera of rats infected with P. westermani metacercariae. These results suggest that the excretory-secretory PwCP2 can be used for the diagnosis of paragonimiasis.