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Cockroaches live in places where various pathogens exist. Thus, they are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the Periplaneta americana transcriptome and detected periplanetasin-5 using an in silico antimicrobial peptide prediction method. In this study, we investigated whether periplanetasin-5 has anticancer activity against the human leukemia cell line K562. Cell growth and survival of K562 cells treated with periplanetasin-5 were decreased in a dose-dependent manner. By using flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) staining, and DNA fragmentation, we found that periplanetasin-5 induced apoptotic and necrotic cell death in leukemia cells. In addition, these events were associated with increased levels of the proapoptotic proteins Fas and cytochrome c and reduced levels of the anti-apoptotic protein Bcl-2. Periplanetasin-5 induces the cleavage of pro-caspase-9, pro-caspase-8, pro-caspase-3, and poly (ADP-ribose) polymerase (PARP). The above data suggest that periplanetasin-5 induces apoptosis via both the intrinsic and extrinsic pathways. Furthermore, caspase-related apoptosis was further confirmed by using the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK), which reversed the periplanetasin-5-induced reduction in cell viability. In conclusion, periplanetasin-5 caused apoptosis in leukemia cells, suggesting its potential utility as an anticancer therapeutic agent.

Rosuvastatin is a statin used to treat metabolic syndrome conditions, such as hyperlipidemia. It is relatively safe; however, fatal rhabdomyolysis or skeletal myopathy can sometimes occur. Therefore, to investigate the overall effects of rosuvastatin, including lipid lowering and adverse effects, metabolic profiling was performed using metabolomics and lipidomics after rosuvastatin administration. Specifically, the metabolic profiles between healthy subjects and patients with hyperlipidemia were compared and the metabolic changes related to the mechanism of the drug effect were proposed. Healthy volunteers (n = 32) and hyperlipidemic patients (n = 14) were orally administered rosuvastatin (20 mg) once a day for 3–8 weeks, and plasma and urine were collected. Metabolomics and lipidomics were performed using UHPLC-LTQ/Orbitrap/MS/MS for non-targeted analysis and UHPLC-TQ-MS/MS for targeted analysis. Using non-targeted analysis, we successfully profiled and identified 73 and 87 metabolites in healthy subjects and hyperlipidemia subjects, respectively. Through targeted analysis, we have also quantified 188 metabolites, including amino acids, biogenic amines, glycerophospholipids, and sphingolipids. The levels of L-carnitine, diacylglycerol, and acylcarnitines significantly decreased after rosuvastatin administration regardless of the group. The overall levels of fatty acids (FA) and lysophosphatidylcholines (LysoPC) increased, while phosphatidylcholines (PC) decreased only in the patient group. β-Oxidation decreased overall, while the production of polyunsaturated FA increased only in the hyperlipidemic patients. Using metabolic profiling, we have evaluated the alterations in the biochemical pathways, which may aid in a more detailed understanding of the effect of rosuvastatin. Patient-specific metabolomic and lipidomic profiles may serve as valuable markers for the understanding of the adverse effects associated with statin treatment.

The field of gene therapy has evolved over the past two decades after the first introduction of nucleic acid drugs, such as plasmid DNA (pDNA). With the development of in vitro transcription (IVT) methods, synthetic mRNA has become an emerging class of gene therapy. IVT mRNA has several advantages over conventional pDNA for the expression of target proteins. mRNA does not require nuclear localization to mediate protein translation. The intracellular process for protein expression is much simpler and there is no potential risk of insertion mutagenesis. Having these advantages, the level of protein expression is far enhanced as comparable to that of viral expression systems. This makes IVT mRNA a powerful alternative gene expression system for various applications in regenerative medicine. In this review, we highlight the synthesis and preparation of IVT mRNA and its therapeutic applications. The article includes the design and preparation of IVT mRNA, chemical modification of IVT mRNA, and therapeutic applications of IVT mRNA in cellular reprogramming, stem cell engineering, and protein replacement therapy. Finally, future perspectives and challenges of IVT mRNA are discussed.

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

Tetradecanol is a straight-chain saturated fatty alcohol purified from Dendropanax morbifera leaves. We found that tetradecanol (30μM) reduced specifically the growth of T cells such as EL-4 T cell and isolated murine CD4+ T cells. In this study, we investigated the effects of tetradecanol on the regulation of interlukin-2 (IL-2), a potent T cell growth factor. Tetradecanol significantly inhibited IL-2 secretion in EL-4 T cells activated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Io) and also in isolated murine CD4+ T cells activated with anti-CD3 and anti-CD28 antibodies. Next, we examined the effect of tetradecanol on the transcriptional activity related to IL-2 production in T cells. Tetradecanol decreased PMA/Io-induced promoter activity of NF-κB in EL-4 T cells, but did not show any significant effects on the promoters of activator protein 1 (AP-1) and nuclear factor of activated T cells (NF-AT). Tetradecanol inhibited IκBα degradation and nuclear translocation of NF-κB subunit, p65 in PMA/Io-activated EL-4 T cells. These results suggest that tetradecanol might have immunosuppressive effects on T cell mediated disorders. Using a chronic allergic contact dermatitis model induced by repeated application of oxazolone, we showed that tetradecanol reduced ear thickness induced by oxazolone.

To evaluate changes in tryptophan metabolism and discover diagnostic biomarkers for gastric cancer, a quantitative method was developed for tryptophan and its seven metabolites (indole-3-lactic acid, anthranilic acid, serotonin, nicotinic acid, kynurenic acid, kynurenine and 3-indoxyl sulfate) in both human serum and gastric juice using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum and gastric juice were prepared with a simple protein precipitation using aqueous 0.1% formic acid and acetonitrile. As a result, it was found that the kynurenine pathway of tryptophan metabolism was activated in gastric cancer and that the metabolic ratio of kynurenine/tryptophan, which reflects the enzyme activity of indoleamine-2,3-dioxygenase, was associated with the observed metabolic changes. Finally, the investigation of tryptophan metabolites, especially kynurenic acid, in serum and gastric juice might serve as biomarkers for gastric cancer. The findings in this study provide critical information of tryptophan metabolism which can be applied to a serum-based diagnostic test for gastric cancer.

Nanoparticles in the field of dendritic cell (DC) research are emerging as a promising method of enhancing the efficacy of cancer immunotherapy. We investigated the effect of branched polyethylenimine-superparamagnetic iron oxide nanoparticles (bPEI-SPIONs) on tumor cells loaded onto DCs. The tumor antigens were prepared as follows: (1) apoptotic U266 cells with ultraviolet B (UVB) irradiation followed by a 2 h incubation in the absence (2 h postirradiated cells) or (2) presence of bPEI-SPIONs (bPEI-SPION 2 h postirradiated cells) and (3) apoptotic U266 cells with UVB irradiation followed by an overnight 16 h incubation (16 h postirradiated cells). bPEI-SPIONs render U266 cells sensitive to UVB irradiation through reactive oxygen species production to accelerate apoptotic death. The 2 h postirradiated cells and bPEI-SPION 2 h postirradiated cells released immunogenic proteins, including Hsp70, Hsp90, and HMGB1. The DCs loaded with bPEI-SPION 2 h postirradiated cells showed the highest IL-12p70 production and Th1 polarization compared with other DCs. These results suggest that bPEI-SPIONs are a promising method of enhancing the immunogenicity of tumor cells and promoting Th1 polarization of DCs loaded with these tumor cells.

Na(+)/H(+) exchanger regulatory factor 3 (NHERF3) is a PSD-95/discs large/ZO-1 (PDZ)-based adaptor protein that regulates several membrane-transporting proteins in epithelia. However, the in vivo physiologic role of NHERF3 in transepithelial transport remains poorly understood. Multidrug resistance protein 4 (MRP4) is an ATP binding cassette transporter that mediates the efflux of organic molecules, such as nucleoside analogs, in the gastrointestinal and renal epithelia. Here, we report that Nherf3 knockout (Nherf3(-/-)) mice exhibit profound reductions in Mrp4 expression and Mrp4-mediated drug transport in the kidney. A search for the binding partners of the COOH-terminal PDZ binding motif of MRP4 among several epithelial PDZ proteins indicated that MRP4 associated most strongly with NHERF3. When expressed in HEK293 cells, NHERF3 increased membrane expression of MRP4 by reducing internalization of cell surface MRP4 and consequently, augmented MRP4-mediated efflux of adefovir, a nucleoside-based antiviral agent and well known substrate of MRP4. Examination of wild-type and Nherf3(-/-) mice revealed that Nherf3 is most abundantly expressed in the kidney and has a prominent role in modulating Mrp4 levels. Deletion of Nherf3 in mice caused a profound reduction in Mrp4 expression at the apical membrane of renal proximal tubules and evoked a significant increase in the plasma and kidney concentrations of adefovir, with a corresponding decrease in the systemic clearance of this drug. These results suggest that NHERF3 is a key regulator of organic transport in the kidney, particularly MRP4-mediated clearance of drug molecules.

Aim: The methylation-variable sites around CpG islands are frequently overmethylated in Helicobacter pylori-infected stomachs. Age-related patterns of the overmethylation changes were compared between the fast-growing antrum cells and the slow-growing body cells. Materials & methods: A total of 316 H. pylori-positive tissues and 380 H. pylori-negative tissues were obtained by endoscopic biopsy. The methylation-variable sites of ten housekeeping genes and nine tissue-specific genes were semiquantitatively analyzed, based on the ten-level classification of methylation-specific PCR intensity. The overmethylated genes were scored when their methylation levels were higher than an intermediate level of each gene common in the H. pylori-negative mucosa. Results: The age-dependent methylation level of the inactive APC gene observed similarly in the antrum and the body was used as an age standard of methylation variation in a biopsy tissue. The overmethylation of housekeeping genes and stomach-specific genes rapidly increased to a high plateau frequency in the young-aged APC methylation cases (mean age: 43 years) in the H. pylori-positive antrum. In the H. pylori-positive body, most of the overmethylated housekeeping genes slowly increased to a peak frequency in the middle-aged APC methylation cases (mean age: 53 years). The housekeeping gene pairs showed high correlations (Spearman’s correlation coefficient > 0.4) in both the antrum and the body. Conclusion: The overmethylation of housekeeping genes rapidly and slowly increased to a high frequency in concordance with a rapid and slow growth of epithelial cells in the H. pylori-infected stomach.

Development of a targeted polymeric gene delivery system is essential for reducing the non-specific uptake and toxicity of the gene carriers. In this study, we have tested the specificity of the cell penetrating peptide (DS 4-3), screened by phage display technique, towards metastatic breast cancer cells. This peptide has shown specificity towards metastatic breast cancer cells, which was confirmed through endocytosis inhibition study. Furthermore, the DS 4-3 peptide was conjugated to bPEI, to deliver the therapeutic miR-145. Tumor suppressor miR-145 inhibited tumor cell growth, and significantly suppressed cell invasion. The DS 4-3 peptide conjugated branched PEI (DSbPEI)/pLuc nanoparticles showed increased transfection in malignant murine breast cancer cells at the neutral surface charge (N/P molar ratio of 3), compared to scramble peptide conjugated bPEI/pLuc nanoparticles, without causing any cytotoxicity. This specific increase in transfection with DS-bPEI/pLuc nanoparticles was not found in the cancer cells that originated from different tissue, such as HeLa cervical cancer cells, or in the normal cells, such as NIH-3T3 murine fibroblast cells. Thus, the specific transfection of miR-145 in metastatic breast cancer cells mediated by DS-bPEI resulted in enhanced reduction in proliferation. Open image in new window

Saturated fatty acids (FA) were grafted using tyrosine as a spacer group to the cyclotriphosphazene ring along with equimolar hydrophilic methoxy poly(ethylene glycol) (MPEG) in cis-nongeminal way. Seven new cyclotriphosphazene amphiphiles were prepared from combinations of hydrophilic MPEGs with different molecular weights of 350, 550, 750 and 1000 and four different fatty acids of different hydrophobicity including lauric, myristic, palmitic and stearic acids. These steric amphiphiles bearing fatty acids as a hydrophobic group were found to form more stable micelles with very low critical micelle concentrations (CMC) (2.95-7.80mg/L) compared with oligopeptide analogues, and their highly hydrophobic core environment is unique and potentially useful for various biomedical applications.

Cancer cells have been suggested to be more susceptible to oxidative damages and highly dependent on antioxidant capacity in comparison with normal cells, and thus targeting antioxidant enzymes has been a strategy for effective cancer treatment. Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of sulfinylated peroxiredoxins and thereby reactivates them. In this study we developed a Srx inhibitor, K27 (N-[7-chloro-2-(4-fluorophenyl)-4-quinazolinyl]-N-(2-phenylethyl)-β-alanine), and showed that it induces the accumulation of sulfinylated peroxiredoxins and oxidative stress, which leads to mitochondrial damage and apoptotic death of cancer cells. The effects of K27 were significantly reversed by ectopic expression of Srx or antioxidant N-acetyl cysteine. In addition, K27 led to preferential death of tumorigenic cells over non-tumorigenic cells, and suppressed the growth of xenograft tumor without acute toxicity. Our results suggest that targeting Srx might be an effective therapeutic strategy for cancer treatment through redox-mediated cell death.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug. It is mainly metabolized by phase 1 and 2 reactions in the liver, and thus it could be involved in many drug-drug interactions. Therefore, the study of APAP metabolism is important in toxicological and pharmacokinetic studies. The objective of this study was to develop a rapid and sensitive method for the determination of APAP and its six metabolites in rat plasma for the pharmacokinetic studies. APAP and its metabolites were separated through a Capcell Pak MGII C(18) column and quantitated with a 16 min run in a triple-quadruple mass spectrometer. The mobile phases were composed of 0.1% formic acid in either 95% water or 95% acetonitrile and analysis was performed twice in positive and negative modes. Validations such as accuracy, precision, recovery, matrix effect and stability were found to be within acceptance criteria of validation guidelines, indicating that the assay was applicable to the determination of the plasma concentrations of drug and its six metabolites. In conclusion, we developed an LC-MS/MS method for the quantitative analysis of APAP and its six metabolites in rat plasma, and this method appears to be useful for pharmacokinetic/toxicokinetic studies of APAP and its metabolites in rats.

Introduction The lysyl oxidase (LOX) family is an emerging family of amine oxidases responsible for the formation of collagen fibrils in the extracellular matrix. To date, 5 LOX family genes have been identified in humans, encoding LOX and LOX-like proteins (LOXL, LOXL2, LOXL3, and LOXL4). The goal of this study was to evaluate the expression and function of the LOX family genes in odontoblastic differentiation of human dental pulp (HDP) cells. Methods Expression of the LOX family genes was assessed by reverse transcriptase polymerase chain reaction analysis, and the amine oxidase activity of HDP cells was evaluated by peroxidase-coupled fluorometric assays. Mineral nodule formation and expression of odontoblastic marker genes were assessed in the presence and absence of specific small interfering RNAs (siRNAs) of the LOX family genes. Results Among the LOX family genes, only LOX and LOXL showed prominent expression during odontoblastic differentiation of HDP cells. Suppression of LOX and LOXL expression by siRNA-induced interference substantially decreased the amine oxidase activity of the differentiating HDP cells. Furthermore, interference of LOX and LOXL expression inhibited mineral nodule formation and expression of odontoblastic marker genes during odontoblastic differentiation of HDP cells. Conclusions These findings show for the first time that the LOX- and LOXL-mediated organization of collagen fibrils in extracellular matrices of HDP cells might be an important regulator for odontoblastic differentiation of HDP cells.

In this study, we determined the anti-inflammatory activity and mechanism of action of a hexane fraction (hWRF) obtained from white Rosa hybrida flowers by employing various assays such as quantitative real-time PCR, Western blotting, and Electrophoretic-Mobility Shift Assay (EMSA). The results revealed that the hWRF had excellent anti-inflammatory potency by reducing inflammatory repertoires, such as inducible nitric oxide synthase (iNOS), interleukin-, and cyclooxygenase-2 (COX-2) in RAW264.7 cells when stimulated with lipopolysaccharide (LPS), a pro-inflammatory mediator. The reduction of nitric oxide (NO) release from RAW 264.7 cells supported the anti-inflammatory effect of hWRF. Interestingly, hWRF effectively inhibited LPS-mediated nuclear factor- (NF-) p65 subunit translocation into the nucleus and extracellular signal-regulated kinase (ERK)1/2 phosphorylation, suggesting that hWRF anti-inflammatory activity may be based on inhibition of the NF- and MAPK pathways. Based on the findings described in this study, hWRF holds promise for use as a potential anti-inflammatory agent for either therapeutic or functional adjuvant purposes.

Daumone, a pheromone secreted by Caenorhabditis elegans, is an essential regulator of chemosensory processes in development and aging. A quantification method using HPLC/MS-MS was developed for the determination of daumone in mouse plasma. After simple protein precipitation with acetonitrile including methaqualone (an internal standard), the analytes were chromatographed on a reversed-phase column and detected by liquid chromatography/tandem mass spectrometry with electrospray ionization. The accuracy and precision of the assay were in accordance with FDA regulations for validation of bioanalytical methods. This method was applied to measure the plasma daumone concentrations following a 5-week repeated oral administration of daumone in mice.

The transforming growth factor-β1 (TGF-β1) bioassay developed in this study monitors increased luciferase activity in MCF10A cells containing the matrix metalloproteinase-2 (MMP-2) promoter with a luciferase reporter and treated with increasing TGF-β1 concentrations. The response was linear in the concentration range from 75 to 2,500 pg/mL. The abilities of 3 types of TGF-β in inducing MMP-2 were different. The luciferase activity induced by TGF-β1 was about 2 times more than that by TGF-β2 and TGF-β3. The MMP-2 promoter bioassay showed greater reproducibility (coefficient of variation [CV] 10%) than the previously developed anticell proliferation assay of TF-1 cell (CV 16%) and the MMP-2 zymogram assay (CV 40%).

The aim of the present study was to investigate the effects of di- and sesquiterpenoids isolated from the pods of Sindora sumatrana Miq. (Leguminosae) on P-glycoprotein (P-gp) function in an adriamycin-resistant human breast cancer cell line, MCF-7/ADR. Over-expression of P-gp is known to be one of the mechanisms involved in multidrug resistance (MDR), which is a major obstacle in clinical cancer treatment. Among six di- and sesquiterpenoids extracted from S. sumatrana, (+)-7beta-acetoxy-15,16-epoxycleroda-3,13(16),14-trien-18-oic acid (1) showed a strong P-gp inhibitory effect, as great as that of verapamil, a representative P-gp inhibitor. Compound 1 enhanced daunomycin accumulation more than fourfold and significantly decreased daunomycin efflux compared with control, resulting in a decrease in the IC(50) value for daunomycin. These results suggest that compound 1 inhibits the functioning of P-gp and, therefore, can be developed as an MDR-reversing agent.

Although antiviral assays have been the most widely available biological assays for interferons (IFNs), they are less sensitive and provide considerable interassay variation. In this study, we demonstrate a new reporter cell line, which is based on HeLa cells transfected with a plasmid containing a human Mx2 promoter driving a luciferase (Luc) cDNA. To characterize the specific gene expression profiles induced by interferon alpha, we analyzed the microarray results of interferon response gene expression induced by IFN-α2a or IFN-α2b treatment with HeLa cells. We found that the Mx2 gene increased the most by treatment with both IFN-α2a and IFN-α2b. Based on this result, we designed a reporter cell line, HeLa-Mx2, suitable for determination of IFN-α. HeLa cells were stably transfected with the luciferase gene under the control of Mx2 promoter. The expression of luciferase can be easily measured for luminescence using a 96-well luminometer and has been correlated with the concentration of added IFN and cell density. In the validation results, our reporter cell line had specificity for type I IFN, but the significant effects of a number of other cytokines such as tumor necrosis factor-α, interleukin (IL)-1β, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-γ) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa-Mx2 cell culture’s age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-α2a in the range of 1–10,000 IU/ml. The 95% confidential limit and total coefficient of variation estimates ranged between 96 and 116 and 10.51% in the reducible range mentioned above, respectively. In conclusion, we established a stable IFN-responsible HeLa-Mx2 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-α activity.

A novel water soluble and biodegradable cyclotriphosphazene-paclitaxel conjugate was prepared by reacting 2'-succinyl paclitaxel with cyclotriphosphazenes bearing equimolar glycyl-L-lysine and methoxy poly(ethylene glycol) as side groups. The macromolecular conjugate was found to self-assemble in aqueous solution to form stable micelles with a mean hydrodynamic diameter of 24.7 nm and a low critical micelle concentration of 10 mg/L. The present conjugate exhibited lower than free paclitaxel but reasonably high in vitro cytotoxicity against selected human tumor cells due to their hydrolytic degradation in PBS solution.

Background Although hydrogen peroxide may play an important role in the development of cancer, it can be an efficient inducer of apoptosis in cancer cells; the exact mechanism by which this action occurs is not completely understood in oral cancer cells. Method In this study, the mechanisms by which H(2)O(2) inhibited growth and induced apoptosis were differentially investigated using HPV-immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). Results H(2)O(2) treatment sensitively and dose-dependently induced growth inhibition and typical apoptosis in IHOK and HN4 cells, as demonstrated by a decreased level of cell viability, an increased population of cells in the sub-G(0)/G(1) phase, ladder formation of the genomic DNA, chromatin condensation and accumulation of Annexin V(+)/PI(+) cells. Furthermore, the expression of Bax, p53 and p21(WAF1/CIP1) increased, whereas the expression of Bcl-2 decreased in immortalized and malignant keratinocytes that were treated with H(2)O(2). In addition, cytochrome-c from the mitochondria was observed in H(2)O(2)-treated IHOK and oral cancer cells, and this was accompanied by the activation of caspase-3 and -9. Additionally, H(2)O(2) treatment induced upregulation of CHOP, GRP78 and several representative endoplasmic reticulum (ER) stress-responsive proteins, including heme oxygenase-1. Conclusion Overall, these results suggest that H(2)O(2) triggers apoptosis via the mitochondrial and ER stress pathway in IHOK and HN4 cells, and that increasing the cellular levels of H(2)O(2) sufficiently may lead to selective killing of oral cancer cells and therefore be therapeutically useful.

Citral is a major compound of lemongrass (Cymbopogon citratus L.) that has many pharmacological activities such as anti-fungal and anti-bacterial effects. In this study, we investigated the anti-inflammatory effect of citral and defined its mechanism of action in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Citral (3-12 microg/mL) significantly inhibited LPS-induced nitric oxide (NO) production in a concentration-dependent manner (IC50: 6.5 microg/mL). Furthermore, it was found that citral effectively inhibited the transcriptional activity and expression of iNOS, and potently suppressed the DNA binding activity and nuclear translocation of NF-kappa B as well as I kappa B phosphorylation in a concentration dependent manner. These results suggest that citral is anti-inflammatory, and its effects may be due to the inhibition of NO production through the suppression of NF-kappa B activation.

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.

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.

In order to develop a new gene delivery vector, a novel cationic poly(organophosphazene) was synthesized by stepwise nucleophilic substitutions of poly(dichlorophosphazene) with a hydrophilic methoxy-poly(ethylene glycol) (MPEG) as a shielding group and a branched tetra( l -lysine), LysLys(LysEt) 2 , as a cationic moiety. The cationic polymer has shown to form a polyplex by DNA condensation and very low in vitro cytotoxicity probably due to the shielding effect of MPEG, which provides a basis for improving the low gene transfection yield of cationic polyphosphazenes.

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.

The substituted chloroisoquinolinediones and pyrido[3,4-b]phenazinediones were synthesized, and the cytotoxic activity and topoisomerase II inhibitory activity of the prepared compounds were evaluated. Chloroisoquinolinediones have been prepared by the reported method employing 6,7-dichloroisoquinoline-5,8-dione. The cyclization to pyrido[3,4-b]phenazinediones was achieved by adding the aqueous sodium azide solution to the dimethylformamide solution of corresponding chloroisoquinoline-5,8-dione. The cytotoxicity of the synthesized compounds was evaluated by a SRB (Sulforhodamine B) assay against various cancer cell lines such as A549 (human lung cancer cell line), SNU-638 (human stomach cancer cell), Col2 (human colon cancer cell line), HT1080 (human fibrosarcoma cell line), and HL-60 (human leukemia cell line). Almost all the synthesized pyrido[3,4-b]phenazinediones showed greater cytotoxic potential than ellipticine (IC(50)=1.82-5.97 microM). In general, the cytotoxicity of the pyrido[3,4-b]phenazinediones was higher than that of the corresponding chloroisoquinolinediones. The caco-2 cell permeability of selected compounds was 0.62 x 10(-6)-35.3 x 10(-6)cm/s. The difference in cytotoxic activity among tested compounds was correlated with the difference in permeability to some degree. To further investigate the cytotoxic mechanism, the topoisomerase II inhibitory activity of the synthesized compounds was estimated by a plasmid cleavage assay. Most of compounds showed the topoisomerase II inhibitory activity (28-100%) at 200 microM. IC(50) values for the most active compound 6a were 0.082 microM. However, the compounds were inactive for DNA relaxation by topoisomerase I at 200 microM.

Nanofibers are one-dimensional nanomaterial in fiber form with diameter less than 1 µm and an aspect ratio (length/diameter) larger than 100:1. Among the different types of nanoparticle-loaded nanofiber systems, nanofibers loaded with magnetic nanoparticles have gained much attention from biomedical scientists due to a synergistic effect obtained from the unique properties of both the nanofibers and magnetic nanoparticles. These magnetic nanoparticle-encapsulated or -embedded nanofiber systems can be used not only for imaging purposes but also for therapy. In this review, we focused on recent advances in nanofibers loaded with magnetic nanoparticles, their biomedical applications, and future trends in the application of these nanofibers.

Nitric oxide (NO) inactivates iron-sulfur enzymes in bacterial amino acid biosynthetic pathways, causing amino acid auxotrophy. We demonstrate that exogenous supplementation with branched-chain amino acids (BCAA) can restore the NO resistance of hmp mutant Salmonella Typhimurium lacking principal NO-metabolizing enzyme flavohemoglobin, and of mutants further lacking iron-sulfur enzymes dihydroxy-acid dehydratase (IlvD) and isopropylmalate isomerase (LeuCD) that are essential for BCAA biosynthesis, in an oxygen-dependent manner. BCAA supplementation did not affect the NO consumption rate of S. Typhimurium, suggesting the BCAA-promoted NO resistance independent of NO metabolism. BCAA supplementation also induced intracellular survival of ilvD and leuCD mutants at wild-type levels inside RAW 264.7 macrophages that produce constant amounts of NO regardless of varied supplemental BCAA concentrations. Our results suggest that the NO-induced BCAA auxotrophy of Salmonella, due to inactivation of iron-sulfur enzymes for BCAA biosynthesis, could be rescued by bacterial taking up exogenous BCAA available in oxic environments.

Orthodontic tooth movement is recognized as a pro-inflammatory stressor of human periodontal ligament (hPDL) cells. However, the cell-signaling pathways linking interleukin-8 (IL-8), intercellular adhesion molecule-1 (ICAM-1), pro-inflammatory cytokines, and dexamethasone in hPDL cells have not been well elucidated. In this study, we investigated the role of mitogen-activated protein (MAP) kinases in dexamethasone- and TNF-alpha-induced IL-8 and ICAM-1 expression in hPDL cells. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. MAP kinase activation and IkappaB degradation were determined by Western blot analysis, and ICAM-1 expression was determined by RT-PCR and FACS analysis. TNF-alpha increased IL-8 mRNA expression and protein secretion in a dose- and time-dependent manner. Dexamethasone suppressed TNF-alpha-induced IL-8 production in a dose-dependent manner. In addition, dexamethasone inhibited TNF-alpha-induced phosphorylation of p38 MAP kinase and extracellular-regulated kinases (ERKs), IkappaB degradation, and NF-kappaB activation. Selective inhibitors for ERKs and p38 attenuated TNF-alpha-induced IL-8 and ICAM-1 expression in the presence and absence of dexamethasone, indicating that MAP kinases play a role in the response of hDPL cells to TNF-alpha. Furthermore, these results suggest that inflammatory cytokine- and dexamethasone-induced IL-8 and ICAM-1, produced via a MAP kinase pathway, may serve as an important mediator of PDL immunoregulation involved in bone remodeling during orthodontic tooth movement.

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-G1 phase arrest and Annexin V-FITC (fluorescein isothiocyanate) staining. Furthermore, Bax expression increased together with p53 and p21WAF1/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.

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.

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 .

The 528 murine monoclonal antibody (MAb) to the human epidermal growth factor receptor (EGFR) was sequentially cationized with hexamethylenediamine and conjugated with diethylenetriaminepentaacetic acid (DTPA) as a potential antibody radiopharmaceutical for imaging EGFR-expressing cancer. The cationized 528 MAb was characterized with isoelectric focusing and electrophoresis, and an immunoradiometric assay, which showed the affinity of the 528 MAb for the human EGFR was retained following cationization. The native or cationized 528 MAb, labeled with (111)In, was injected intravenously in scid mice bearing human U87 flank tumors, which express the EGFR, and tumor imaging was performed with both external detection in live animals and with whole body autoradiography. However, the tumor signal was not increased with the cationized MAb, relative to the native MAb, and this was due to a serum inhibition phenomenon that was confirmed by a pharmacokinetics analysis in control mice. In an attempt to block the serum inhibition, the cationized 528 MAb was pegylated with 2000 Da poly(ethylene glycol), and the cationized/pegylated MAb was conjugated with DTPA and labeled with (111)In. However, a pharmacokinetics analysis showed the pegylation did not reverse the serum inhibition of the cationic charge on the MAb. These studies describe methods for reformulating monoclonal antibodies to develop improved radiopharmaceuticals, but show that radiolabeling a cationized MAb with DTPA produces a serum neutralization of the initial cationization modification.

MicroRNAs (miRNA) are short oligonucleotides of endogenous origin involved in post-transcriptional regulation and are altered in disease, making them potential therapeutic targets. miRNA replacement is necessary in cells with downregulated miRNAs levels in response to disease. miRNA 145 is a novel tumor suppressor gene involved in cell suppression, invasion and migration of cancer cells; it is downregulated in most cancers. Delivery of therapeutic miRNA using nanoparticles enhances the chances of successful delivery and expression of genes at the target site. We evaluated polysorbitol-mediated transporter (PSMT) in the cellular delivery of miRNA 145. The polysorbitol backbone possesses osmotic properties and leads to enhanced cellular uptake. PSMT delivers genes into cells by a caveolae-mediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Physicochemical characterization, and the transfection efficiency and transgene expression capability of PSMT/reporter plasmid DNA nanoparticles, were determined. GFP-tagged miRNA 145 delivery with PSMT was confirmed by confocal microscopy and Western blotting. The functional effects of miRNA 145 delivered with PSMT were analyzed by confocal microscopy, as well as in apoptosis, proliferation and wound healing assays. Finally, the expression of an miRNA 145 target protein, c-myc, was determined by Western blotting after intracellular delivery of PSMT/miRNA 145 nanoparticle (NP).

Isocudraxanthone K (IK) is a novel, natural compound from a methanol extract of the root bark ofCudrania tricuspidata. It has not been shown previously that IK possessed antitumor activity. We investigated the antitumor effects and molecular mechanism of IK and related signal transduction pathway(s) in oral squamous cell carcinoma cells (OSCCCs). The MTT assay revealed that IK had an antiproliferative effect on OSCCCs, in a dose- and time-dependent manner. IK induced apoptosis in OSCCCs, as identified by a cell-cycle analysis, annexin V-FITC and propidium iodide staining, and the nuclear morphology in cell death. IK caused time-dependent phosphorylation of Akt, p38, and ERK (extracellular signal-regulated kinase). In addition, IK increased the cytosolic to nuclear translocation of nuclear factor-κB (NF-κB) p65 and the degradation and phosphorylation of IκB-αin HN4 and HN12 cells. Furthermore, IK treatment downregulated hypoxia-inducible factor 1α(HIF-1α) and its target gene, vascular endothelial growth factor (VEGF). Cobalt chloride (CoCl2), a HIF-1αactivator, attenuated the IK-induced growth-inhibiting and apoptosis-inducing effects, and blocked IK-induced expression of apoptosis regulatory proteins, such as Bax, Bcl-2, caspase-3, caspase-8, and caspase-9, and cytochrome c. Collectively, these data provide the first evidence of antiproliferative and apoptosis-inducing effects of IK as a HIF-1αinhibitor and suggest it may be a drug candidate for chemotherapy against oral cancer.

We recently reported that overexpression of thymosin beta-4 (Tβ4) in transgenic mice promotes abnormal hair growth and tooth development, but the role of Tβ4 in dental pulp regeneration was not completely understood. The aim of this study was to investigate the role of Tβ4 on odontoblastic differentiation and the underlying mechanism regulating pulp regeneration in human dental pulp cells (HDPCs). Our results demonstrate that mRNA and protein expression of Tβ4 is upregulated during odontogenic differentiation in HDPCs. Transfection with Tβ4 siRNA decreases OM-induced odontoblastic differentiation by decreasing alkaline phosphatase (ALP) activity, mRNA expression of differentiation markers, and calcium nodule formation. In contrast, Tβ4 activation with a Tβ4 peptide promotes these processes by enhancing the phosphorylation of p38, JNK, and ERK mitogen-activated protein kinases (MAPKs), bone morphogenetic protein (BMP) 2, BMP4, phosphorylation of Smad1/5/8 and Smad2/3, and expression of transcriptional factors such as Runx2 and Osterix, which were blocked by the BMP inhibitor noggin. The expression of integrin receptors α1, α2, α3, and β1 and downstream signaling molecules including phosphorylated focal adhesion kinase (p-FAK), p-paxillin, and integrin-linked kinase (ILK) were increased by Tβ4 peptide in HDPCs. ILK siRNA blocked Tβ4-induced odontoblastic differentiation and activation of the BMP and MAPK transcription factor pathways in HDPCs. In conclusion, this study demonstrates for the first time that Tβ4 plays a key role in odontoblastic differentiation of HDPCs and activation of Tβ4 could provide a novel mechanism for regenerative endodontics.

Superparamagnetic iron oxide nanoparticle (SPION)-based diagnostic properties with accompanying therapeutics such as drugs or genes have been explored for improvement of their therapeutic efficacy. Positively charged SPION-loaded polymersomes was prepared to deliver genes to the target sites; this process was concomitantly monitored by magnetic resonance imaging (MRI). The surface characteristics and morphology were respectively measured by dynamic light scattering and transmission electron microscopy. The complex between the polymer and the pDNA was confirmed by a gel retardation assay. The transfection efficiency and cytotoxicity in vitro were tested by treating of the CT-26 colon cancer cell line with luciferase-expressing plasmids/SPION complex. MRI was also used to check the detectability of SPION in vitro and in vivo. A SPION-loaded polymersome carrying genetic materials was delivered and then accumulated in the tumor site of the murine colon cancer xenograft model after intravenous injection, possibly through a passive targeting mechanism. The accumulation was monitored using clinical MRI. This result indicates that the SPION-loaded polymersome can be applied to MR imageguided gene therapy.

Metastatic lymph nodes (LN) originate from primary cancer cells that metastasize to the lymphatic system. It is difficult to non-invasively discriminate between metastatic LN and normal LN because of their similarities in size and shape. Magnetic resonance (MR) contrast agents are widely utilized to enhance the image contrast among different tissues. Currently available dextran-based contrast agents are non-specifically internalized by macrophages. Therefore, the aim of this study was to develop mannan-coated superparamagnetic iron oxide nanoparticles (mannan-SPION) for specific delivery to immune cells in LN by receptor-mediated endocytosis for facilitated uptake in the target cells and faster acquisition of MR images. Mannan is a water soluble polysaccharide with a high content of D-mannose residues that can be recognized by mannose receptors on activated macrophages and dendritic cells. Mannan-SPION are proven to be suitable for MRI due to their small size, excellent aqueous stability, and lower cytotoxicity. Mannan-SPION are taken up by antigen-presenting cells such as macrophages and dendritic cells, which could be confirmed by Prussian blue and fluorescent staining. In addition, mannan-SPION exhibit enhanced delivery efficiency in targeting macrophages in LN in vivo compared with polyvinyl alcohol (PVA)-SPION. More specifically, the enhancement of MRI of LN by mannan-SPION increased dramatically during the earlier stages after intravenous injection, compared with PVA-SPION as a control, which indicates the potential for successful and early detection of metastastatic LN.

Background: A specific and effective strategy is in demand to treat ovarian cancer successfully. Epidermal growth factor receptor (EGFR) is highly expressed in ovarian cancer, and thus EGFR antisense gene therapy can be a potential therapeutic strategy. Method: L-Arginine-grafted-polyamidoamine dendrimer (PAMAMArg) has been reported to be a novel nonviral gene delivery carrier. Therefore, the ability of PAMAM-Arg in transferring a luciferase gene to ovarian carcinoma SK-OV3 cells has been examined, and the cytotoxicity of the cationic polymer has been investigated. In addition, the suppression of cell proliferation has been evaluated by transferring an EGFR antisense gene to SK-OV3 cells using PAMAM-Arg. Polyethyleneimine (PEI) 25K was used as a positive control. Results: As a result, in vitro gene transfection efficiency of PAMAMArg was enhanced with increasing transfection time and N/P ratios. PAMAM-Arg transferred the luciferase gene into cells more efficiently than PEI. In addition, PAMAM-Arg was minimally toxic to the cells whereas PEI 25K was highly toxic. The polyplexes formed by the EGFR antisense gene and PAMAM-Arg significantly reduced thymidine incorporation into the cells suggesting the suppression of cancer cell proliferation. Conclusion: These results suggest that a PAMAM-Arg/EGFR antisense gene complex can be used as a safe and efficient therapeutic agent for cancer gene therapy.

Oxidative stress induced neuronal cell death by accumulation of β-amyloid (Aβ) is a critical pathological mechanism of Alzheimer's disease (AD). Intracerebroventrical infusion of Aβ(1-42) (300 pmol/day per mouse) for 14 days induced neuronal cell death and memory impairment, but pre-treatment of 4-O-methylhonokiol (4-O-MH), a novel compound extracted from Magnolia officinalis for 3 weeks (0.2, 0.5 and 1.0 mg/kg) prior to the infusion of Aβ(1-42) and during the infusion dose dependently improved Aβ(1-42)-induced memory impairment and prevented neuronal cell death. Additionally, 4-O-MH reduced Aβ(1-42) infusion-induced oxidative damages of protein and lipid but reduced glutathione levels in the cortex and hippocampus. Aβ(1-42) infusion-induced activation of astrocytes and p38 mitogenic activated protein (MAP) kinase was also prevented by 4-O-MH in mice brains. In further study using culture cortical neurons, p38 MAP kinase inhibitor abolished the inhibitory effect of 4-O-MH (10 μM) on the Aβ(1-42) (5 μM)-induced reactive oxidative species generation and neuronal cell death. These results suggest that 4-O-MH might prevent the development and progression of AD through the reduction of oxidative stress and neuronal cell death via inactivation of p38 MAP kinase pathway.

The objective of this study was to compare the pharmacokinetic parameters of zaltoprofen and those of its sodium salt in rats. Zaltoprofen, a potent non-steroidal anti-inflammatory agent, was virtually insoluble in water, but its sodium salt had excellent water solubility. To investigate the effect of aqueous solubility differences upon their pharmacokinetic parameters, minicapsules containing the drug powders were administrated orally to rats, and blood samples were taken via the common carotid artery. A column-switching high-performance liquid chromatographic analytical procedure was developed and validated for the quantitation of zaltoprofen in rat plasma samples. Our study demonstrated that the time required to reach maximum plasma concentration (Tmax) of zaltoprofen sodium was significantly reduced and its maximum plasma concentration (Cmax) was increased 1.5-fold, relative to the values for zaltoprofen. It is anticipated that the sodium salt of zaltoprofen will allow the rapid onset of the drug's action in the treatment of inflammatory diseases. Copyright © 2008 John Wiley & Sons, Ltd.

A simple high-performance liquid chromatographic (HPLC) method was developed and validated for the quantification of mizoribine in human serum. After the addition of 70% perchloric acid and 3-methylxanthine (50 microg/mL, internal standard) to human serum, the samples were mixed and centrifuged at 12,000 rpm (1432 g) for 10 min. The supernatant was injected onto a C(18) column eluted with a mobile phase of 20 mm Na2HPO4 and methanol (93:7, v/v, pH 3) containing 0.04% octanesulfonic acid and detected utilizing an ultraviolet detector at 275 nm. The linear calibration curve was obtained in the concentration range of 0.1-4.0 microg/mL and the lower limit of quantification was 0.1 microg/mL. This method was validated with selectivity, linearity, precision and accuracy. In addition, the method was successfully applied to estimate the pharmacokinetic parameters of mizoribine in Korean subjects following an oral administration of 100 mg mizoribine (two Bredinine 50 mg tablets). The maximum serum concentration (C(max)) of 2.30 +/- 0.83 microg/mL was reached 2.27 +/- 0.66 h after an oral dose. The mean AUC(0-12 h) and the elimination half-life (t(1/2)) were 13.2 +/- 4.79 microg h/mL and 3.10 +/- 0.74 h, respectively.

Although substance P (SP) is associated with osteoclast differentiation and bone resorption, little is known about the osteogenic differentiation-inducing effects of SP in periodontal ligament (PDL) cells. This study investigated whether PDL cells could differentiate into osteoblastic-like cells by SP. The expression of osteoblastic differentiation markers such as osteopontin (OPN), osteonectin (ON), osteocalcin (OCN) and bone sialoprotein (BSP) were evaulated by Western blotting. Additionally, SP-mediated heme oxygenase-1 (HO-1) pathways were further clarified. SP increased HO-1 and osteogenic differentiation in concentration- and time-dependent manners, as determined by OPN, ON, OCN and BSP expression. Furthermore, treatment with inhibitors of p38, ERK MAPK, and NF-κB abolished SP-induced osteogenic differentiation and HO-1 expression. SP-induced translocation of Nrf-2 was also observed. The combined results suggest that SP activates the stress-response enzymes HO-1 and Nrf-2, subsequently leading to upregulation of osteogenic differentiation in human PDL cells.

Although the induction of heme oxygenase-1 (HO-1) by hydrogen peroxide (H2O2) has been reported, the HO-1 and odontoblastic differentiation-inducing effects against H2O2 have not been clarified in human pulp cells. In this study, we investigated whether HO-1 is involved in the protective mechanisms against the cytotoxic effects of H2O2 by using a cell viability assay, and we examined the production of dentin sialophosphoprotein (DSPP) and other mineralization markers by using reverse transcriptase-polymerase chain reaction in human pulp cells. H2O2 decreased cell viability but increased HO-1 and DSPP expression in a concentration- and time-dependent manner. Antioxidants and inhibitors of HO-1, phosphatidylinositol-3'-kinase, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase blocked H2O2-induced cytotoxicity and the expression of HO-1 and DSPP mRNA in pulp cells. These data suggest that the induction of HO-1 by H2O2 in pulp cells plays a protective role against the cytotoxic effects of H2O2 and stimulates DSPP expression, resulting in premature odontoblast differentiation through pathways that involve phosphatidylinositol-3'-kinase, p38, and extracellular signal-regulated kinase.

Inflexinol, anent-kaurane diterpenoid, was isolated from the leaves ofIsodon excisus. Many diterpenoids isolated from the genusIsodon(Labiatae) have antitumor and antiinflammatory activities. We investigated the antiinflammatory effect of inflexinol in RAW 264.7 cells and astrocytes. As a result, we found that inflexinol (1, 5, 10μM) suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) as well as the production of nitric oxide (NO) in LPS-stimulated RAW 264.7 cells and astrocytes. Consistent with the inhibitory effect on iNOS and COX-2 expression, inflexinol also inhibited transcriptional and DNA binding activity of NF-κBvia inhibition ofIκBdegradation as well as p50 and p65 translocation into nucleus. These results suggest that inflexinol inhibits iNOS and COX-2 expression through inhibition of NF-κBactivation, thereby inhibits generation of inflammatory mediators in RAW 264.7 cells and astrocytes, and may be useful for treatment of inflammatory diseases.

(-)-Syringaresinol and tricin, isolated from the AcOEt-soluble extract of the whole plants of Sasa borealis (Gramineae), showed inhibitory effects on the P-glycoprotein in adriamycin-resistant human breast cancer cells, MCF-7/ADR.

Introduction: The application of nanoparticles in dendritic cell (DC)-based cancer immunotherapy represents a promising strategy to enhance antigen-specific T cell immune responses. This study was to investigate the effect of bPEI-SPIONs on antigen-specific T cell responses elicited by DCs loaded with apoptotic U266 myeloma tumor antigen in the presence or absence of bPEI-SPIONs. Materials and Methods: The myeloma tumor antigens were prepared as following: 1) apoptotic U266 cells by UBV-irradiation and overnight incubation (16 h irradiated cells) and 2) apoptotic U266 cells by UVB-irradiation and 2 h incubation in the absence (2 h irradiated cells) or 3) presence of bPEI-SPIONs (bPEI-SPION 2 h irradiated cells). Monocyte-derived immature DCs were activated with TNF-α, loaded with several kinds of myeloma tumor antigens 2 h after TNF-α stimulation, and cultured for 2 days. Results: Optimal concentration of bPEI-SPIONs was 16 µg/mL to uptake into tumor cells and the bPEI-SPIONs render U266 cells sensitive to UVB-irradiation through reactive oxygen species (ROS) induction pathway hence accelerated the apoptotic cell death. 2 h irradiated cells and bPEI-SPION 2 h irradiated cells released immunogenic proteins, including HSP70, HSP90, and HMGB1. bPEI-SPION 2 h irradiated cells were easily up-taken by DCs without alteration of surface phenotypes and migration capacities. DCs loaded with bPEI-SPION 2 h irradiated cells showed highest IL-12p70 production level and Th1 polarization compared to other DCs. Conclusion: These results suggest that bPEI-SPIONs are a promising tool to improve immunogenicity of myeloma tumor cells and to enhance Th1 polarization of DCs loaded with these tumor cells. Disclosures No relevant conflicts of interest to declare.

Background The goal of this work was the development of a gene targeting technology that will enable the delivery of therapeutic genes to brain cancer cells in vivo following intravenous administration. High-grade brain gliomas overexpress the epidermal growth factor receptor (EGFR) and EGFR antisense gene therapy could reduce the growth of EGFR-dependent gliomas. Methods A human EGFR antisense gene driven by the SV40 promoter in a non-viral plasmid carrying elements that facilitate extra-chromosomal replication was packaged in the interior of 85 nm pegylated immunoliposomes (PILs). The PILs were targeted to U87 human glioma cells with the 83-14 murine monoclonal antibody (MAb) to the human insulin receptor (HIR). Results Confocal fluorescent microscopy demonstrated that the unconjugated HIR MAb is rapidly internalized by the glioma cells. Endocytosis followed by entry into the nucleus was also demonstrated for the HIR MAb conjugated PILs carrying fluorescein-labeled plasmid DNA. The PILs delivered exogenous genes to virtually all cells in culture, based on β-galactosidase histochemistry. The targeting of a luciferase gene to the U87 cells with the PILs resulted in luciferase levels in excess of 150 pg/mg protein after 72 h of incubation. The level of luciferase gene expression in the U87 cells achieved with the PIL gene targeting system was comparable to that with lipofectamine. Targeting the EGFR antisense gene to U87 glioma cells with the PILs resulted in more than 70% reduction in [3H]thymidine incorporation into the cells; this was paralleled by a 79% reduction in the level of immunoreactive EGFR. Conclusion The present work describes the targeting of an EGFR antisense gene to human brain cancer cells, which results in a 70–80% inhibition in cancer cell growth. PILs provide a new approach to gene targeting that is effective in vivo following intravenous administration without viral vectors. Copyright © 2002 John Wiley & Sons, Ltd.

The membrane permeation in vivo of therapeutic proteins may be enhanced by conjugation of the protein to cationic import peptides, such as the tat protein of the human immune deficiency virus. The organ uptake, expressed as a percent of injected dose (ID) per gram of tissue, is a function of both membrane permeability and the area under the plasma concentration curve (AUC), which is a function of the plasma pharmacokinetics. The purpose of the present studies was to examine the effect of the tat peptide on the plasma AUC of a model exogenous protein, streptavidin, and to examine the extent to which changes in the plasma AUC influence organ uptake (%ID/g) of the protein. The cationic portion of the tat protein is comprised of a lysine/arginine-rich sequence, designated tat48-58. A biotin analogue of this cationic peptide, tat-biotin, was radioiodinated and injected intravenously into rats with or without conjugation to streptavidin. The unconjugated tat-biotin peptide was nearly instantaneously cleared from plasma by all tissues with a very high systemic clearance of 29 +/- 4 mL/min/kg and a high systemic volume of distribution of 4160 m+/- 450 mL/kg. The plasma clearance of the tat-biotin/streptavidin conjugate, 1.37 +/- 0.01 mL/min/kg, was reduced relative to the clearance of unconjugated tat peptide, but was higher than the plasma clearance of the unconjugated streptavidin, 0.058 +/- 0.005 mL/min/kg. Conjugation of cationic import peptides such as tat48-58 to higher molecular weight proteins results in a marked increase in the rate of removal of the protein from the circulation, which is reflected in the reduced plasma AUC. In summary, tat conjugation of a protein has opposing effects on membrane permeation and the plasma AUC. Therefore, the organ %ID/g is not increased in proportion to the increase in membrane permeation caused by tat conjugation of proteins.