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Human insulin-like growth factor 1 (IGF-I) is the primary mediator of the effects of the growth hormone (GH). Therefore, it has been used as a biomarker to detect the abuse of GH in sports. The measurement of IGF-I relies on mass-based and immunological approaches to analysis. Among the mass-based analysis methods, liquid chromatography-mass spectrometry (LC-MS) has a number of functional advantages. LC-MS measurements based on the quantification of IGF-I, according to trypsin digestion, are used in the most common method of analyzing doping. However, this method is time-consuming and subject to experimental variability. In this study, we optimized a rapid method for detecting IGF-I without the trypsin digestion step. This method of analysis uses an ultra-centrifugal filter and an LC-HRMS through narrow-range mass scan method. To verify the validity of this method, eight categories of validation testing were applied with the following results: linearity, R2 > 0.99; limit of detection, 15 ng/ml; limit of quantification, 20 ng/ml; accuracy, >99%; recovery rate, >95%; carryover

Summary Background Total breast reconstruction involves a long process consisting of mastectomy, breast reconstruction, and adjuvant therapy. For various reasons, some patients refuse the final step of nipple-areola reconstruction. Some patients have potential risk factors for poor outcome after undergoing conventional techniques. We have performed tattoo-only nipple-areola complex (NAC) reconstruction in these situations and accomplished satisfactory results for both patients and surgeons. Methods Ninety-five patients who underwent NAC reconstruction between October 2017 and June 2018 were included. We retrospectively evaluated the breast reconstruction timing and type, history of a secondary breast procedure or other operations, history of adjuvant therapy, reasons for performing a tattoo-only procedure, and average operative time. Overall patient satisfaction was assessed and compared. Results Twenty patients (21%) underwent tattoo-only NAC reconstruction. The main reasons for performing the tattoo-only technique were patient reluctance to undergo another operation, thin and/or tight breast skin in patients with implant-based reconstruction, radiation therapy after implant-based reconstruction, adverse chemotherapy effects, scar across the central breast mound, and smoking habit. The average time for tattooing was 29 min (range, 15–45 min). The average overall satisfaction score was 8.1 on a 10-point scale, which was significantly the highest compared with that of other techniques. Conclusion The tattoo-only NAC reconstruction technique is an essential option to consider and utilize in selected patients who refuse another nipple reconstruction operation for various reasons. Moreover, tattoo-only NAC reconstruction has distinct advantages for patients with potential risk factors and poor outcome after conventional NAC reconstruction.

A series of 2,4-disubstituted-5-(6-alkylpyridin-2-yl)-1H-imidazoles, 7a–c, 11a–h, and 16a–h has been synthesised and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. Incorporation of a quinoxalin-6-yl moiety and a methylene linker at the 4- and 2-position of the imidazole ring, respectively, and a m-CONH2 substituent in the phenyl ring generated a highly potent and selective ALK5 inhibitor 11e. Docking model of ALK5 in complex with 11e showed that it fitted well in the ATP-binding pocket with favourable interactions.

Isocudraxanthone K (IK) is a novel, natural compound from a methanol extract of the root bark of Cudrania 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-α in 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-1α activator, 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-1α inhibitor and suggest it may be a drug candidate for chemotherapy against oral cancer.

Cockroaches live in places where various pathogens exist and thus are more likely to use antimicrobial compounds to defend against pathogen intrusions. We previously performed an in silico analysis of the

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.

Objectives The inhibitors of P-glycoprotein (P-gp) which limits an access of exogenous compounds in the luminal membrane of the intestine have been studied to enhance the intestinal P-gp-mediated absorption of anticancer drugs. Methods Inhibition of the efflux pump by synthesized benzoxanthone derivatives was investigated in vitro and in vivo. MCF-7/ADR cell line was used for cytotoxicity assay and [3H]-daunomycin (DNM) accumulation/efflux study. Eight benzoxanthone analogues were tested for their effects on DNM cytotoxicity. Among them, three analogues were selected for the accumulation/efflux and P-gp ATPase studies. Paclitaxel (PTX), a P-gp substrate anticancer drug, was orally administered to rats with/without compound 1 (8,10-bis(thiiran-2-ylmethoxy)-7H-benzo[c]xanthen-7-one). The pharmacokinetic parameters of PTX in the presence/absence of compound 1 were evaluated from the plasma concentration-time profiles. Key-findings Compound 1 increased the DNA accumulation to 6.5-fold and decreased the DNM efflux to approximately 1/2 in the overexpressing P-gp cell line. Relative bioavailability (RB) of PTX in rats was significantly increased up to 3.2-fold by compound 1 (0.5 or 2 mg/kg). Conclusions Benzoxanthone analogue, compound 1 is strongly suggested to be a promising inhibitor of P-gp to improve an oral absorption of compounds for cancer therapy.

Yong Joo Jun,1,* Jung Hyun Park,2,* Prakash G Avaji,1 Kyung Su Park,3 Kyung Eun Lee,3 Hwa Jeong Lee,2 Youn Soo Sohn1 1C & Pharm, Ewha Womans University, Seodaemun-gu, Seoul, Republic of Korea; 2Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seodaemun-gu, Seoul, Republic of Korea; 3Advanced Analysis Center, Korea Institute of Science and Technology, Seongbuk-gu, Seoul, Republic of Korea *These authors contributed equally to this work Abstract: To prepare an efficient theranostic polyphosphazene–docetaxel (DTX) conjugate, a new drug delivery system was designed by grafting a multifunctional lysine ethylester (LysOEt) as a spacer group along with methoxy poly(ethylene glycol) (MPEG) to the polyphosphazene backbone ([NP]n), and then DTX was conjugated to the carrier polymer using acid-cleavable cis-aconitic acid (AA) as a linker. The resultant polyphosphazene–DTX conjugate, formulated as [NP(MPEG550)3(Lys-OEt)(AA)(DTX)]n and named “Polytaxel”, exhibited high water solubility and stability by forming stable polymeric micelles as shown in its transmission electron microscopy image and dynamic light scattering measurements. Another important aspect of Polytaxel is that it can easily be labeled with various imaging agents using the lysine amino group, enabling studies on various aspects, such as its organ distribution, tumor-targeting properties, pharmacokinetics, toxicity, and excretion. The pharmacokinetics of Polytaxel was remarkably improved, with prolonged elimination half-life and enhanced area under the curve. Ex vivo imaging study of cyanine dye-labeled Polytaxel showed that intravenously injected Polytaxel is long circulating in the blood stream and selectively accumulates in tumor tissues. Polytaxel distributed in other organs was cleared from all major organs at ~6weeks after injection. The invitro study of DTX release from the carrier polymer showed that >95% of conjugated DTX was released at pH 5.4 over a period of 7days. Xenograft trials of Polytaxel using nude mice against the human gastric tumor cell line MKN-28 showed complete tumor regression, with low systemic toxicity. Polytaxel is currently in preclinical study. Keywords: docetaxel, polyphosphazene, anticancer drug, nanomedicine, theranostics

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.

Basavaraj R Patil,1 Su Yeon Kang,2 Da Hee Jung,2 Prakash G Avaji,1 Yong Joo Jun,1 Hwa Jeong Lee,2 Youn Soo Sohn1 1C & Pharm, Ewha Womans University, Seoul 03760, Republic of Korea; 2Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 03760, Republic of KoreaCorrespondence: Youn Soo SohnC & Pharm, Ewha Womans University, Room 304, University-Industry Cooperation Building, 52 Ewhayeodae-Gil, Seodaemun-Gu, Seoul 03760, Republic of KoreaTel +82 70 7008 2120Fax +82 2 362 8813Email yssohn@ewha.ac.krHwa Jeong LeeGraduate School of Pharmaceutical Sciences, Ewha Womans University, 52 Ewhayeodae-Gil, Seodaemun-Gu, Seoul 03760, Republic of KoreaTel +82 2 3277 3409Fax +82 2 3277 2851Email hwalee@ewha.ac.krPurpose: A new theranostic nanomedicine involving anticancer-active cisplatin moiety was designed to study its tumor-targeting properties as well as its drug efficacy and toxicity.Methods: A cisplatin carrier polymer was prepared by grafting equimolar polyethylene glycol of a molecular weight of 550 (PEG550) and aminoethanol to the poly(dichlorophosphazene) backbone. Cisplatin was conjugated to the carrier polymer using cis-aconitic acid as a linker.Results: The cisplatin-loaded polyphosphazene, named “Polycisplatin” was found to be amphiphilic in aqueous solution and self-assembled into nanoparticles with an average particle size of 18.6 nm in diameter. The time-dependent organ distribution study of Cy5.5-labeled Polycisplatin in the A549-tumor-bearing mice exhibited a high tumor selectivity of Polycisplatin by EPR effect despite the relatively small particle size. In order to compare the in vivo efficacy of Polycisplatin and cisplatin, their xenograft trials were performed using nude mice against the human gastric cell line MKN-28. Polycisplatin exhibited slightly less tumor suppression effect compared with cisplatin at the same dose of 1.95 mg Pt/kg, which is the maximum tolerate dose of cisplatin, but at the higher double dose of 3.9 mg Pt/kg, Polycisplatin exhibited a little better efficacy than cisplatin. Furthermore, mice treated with cisplatin at the dose of 1.95 mg Pt/kg exhibited severe body weight decrease by about 25%, while mice treated with Polycisplatin did not show serious body weight decrease even at its double dose of 3.9 mg Pt/kg. Furthermore, kidney indicators including kidney index, BUN, and creatinine values measured displayed that Polycisplatin is much less nephrotoxic than cisplatin.Conclusion: Nanoparticular Polycisplatin was successfully prepared by conjugating cisplatin to a hydrophilic polyphosphazene carrier polymer using the acid-cleavable cis-aconitic acid. Polycisplatin nanoparticles exhibit excellent tumor-targeting properties by EPR effect. The xenograft trials exhibited excellent antitumor efficacy and reduced systemic toxicity of Polycisplatin.Keywords: cisplatin, polyphosphazene, platinum drug, drug delivery, nanomedicine

Recent studies have shown that many types of cancer cells have increased levels of reactive oxygen species (ROS) and enhance antioxidant capacity as an adaptation to intrinsic oxidative stress, suggesting that cancer cells are more vulnerable to oxidative insults and are more dependent on antioxidant systems compared with normal cells. Thus, disruption of redox homeostasis caused by a decline in antioxidant capacity may provide a method for the selective death of cancer cells. Here we show that ROS-mediated selective death of tumor cells can be caused by inhibiting sulfiredoxin (Srx), which reduces hyperoxidized peroxiredoxins, leading to their reactivation. Srx inhibitor increased the accumulation of sulfinic peroxiredoxins and ROS, which led to oxidative mitochondrial damage and caspase activation, resulting in the death of A549 human lung adenocarcinoma cells. Srx depletion also inhibited the growth of A549 cells like Srx inhibition, and the cytotoxic effects of Srx inhibitor were considerably reversed by Srx overexpression or antioxidants such as N-acetyl cysteine and butylated hydroxyanisol. Moreover, Srx inhibitor rendered tumorigenic ovarian cells more susceptible to ROS-mediated death compared with nontumorigenic cells and significantly suppressed the growth of A549 xenografts without acute toxicity. Our results suggest that Srx might serve as a novel therapeutic target for cancer treatment based on ROS-mediated cell death.

This meta-analysis investigated the association between the risk of breast cancer and hormone replacement therapy (HRT). Various stratified analyses were performed according to race (Asian/Westerner), HRT type [all hormone therapies, estrogen-only therapy (ET), or combined estrogen–progestin therapy (EPT)], histological breast cancer type (ductal/lobular/mixed ductal–lobular), and estrogen receptor status (ER-positive/ER-negative). A literature search was performed using Pubmed, Embase, and KoreaMed. Twenty-five epidemiological studies including 23 cohort studies and two randomized controlled trials were included in this meta-analysis. Using a random-effects model, HRT use was found to be positively associated with the risk of breast cancer with a pooled hazard ratio (HR) of 1.33 [95% confidence interval (CI) 1.24, 1.44]. Compared with ET, EPT was more strongly associated with breast cancer risk. EPT was associated with both ductal and lobular breast cancer risks [for ductal breast cancer, HR = 1.51 (95% CI 1.28, 1.78); for lobular breast cancer, HR = 1.38 (95% CI 1.20, 1.60)]. According to ER status, all HRTs were associated with the risk of ER-positive breast cancer, but not with that of ER-negative breast cancer. Asian HRT users had a higher risk of breast cancer than western HRT users. Both ET and EPT were significantly associated with the risk of all breast cancer histological types and ER-positive breast cancer.

Cudraxanthone H (CH) is a natural compound isolated from a methanol extract of the root bark of Cudrania tricuspidata, a herbal plant also known as Moraceae. However, the effect of CH on human cancer cells has not been reported previously. The aim of this study was to investigate the anticancer effects and mechanism of action of CH on oral squamous cell carcinoma (OSCC) cells. CH exerted significant antiproliferative effects on OSCC cells in dose- and time-dependent manners. CH also induced apoptosis in OSCC cells, as evidenced by an increased percentage of cells in the sub-G1 phase of the cell cycle, annexin V-positive/propidium iodide-negative cells, and nuclear morphology. This antiproliferative effect of CH was associated with a marked reduction in the expression of cyclin D1 and cyclin E, with a concomitant induction of cyclin-dependent kinase inhibitor (CDKI) expression (p21 and p27). CH inhibited the phosphorylation and degradation of I[Formula: see text]B-[Formula: see text]and the nuclear translocation of NF-[Formula: see text]B p65. Furthermore, CH treatment down-regulated PIN1 mRNA and protein expression in a dose-dependent manner. PIN1 overexpression by infection with adenovirus-PIN1 (Ad-PIN1) attenuated the CH-induced growth-inhibiting and apoptosis-inducing effects, blocked CH-enhanced CDKI expression and restored cyclin levels. In contrast, inhibiting PIN1 expression via juglone exerted the opposite effects. The present study is the first to demonstrate antiproliferative and apoptosis-inducing effects of CH, which exerts its effects by inhibiting NF-[Formula: see text]B and PIN1. These data suggest that it might be a novel alternative chemotherapeutic agent for use in the treatment of oral 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.

A series of 2-substituted-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)imidazoles was synthesized and evaluated to optimize a prototype inhibitor of TGF-β type I receptor kinase (ALK5), 6. Combination of replacement of a quinoxalin-6-yl moiety of 6 with a [1,2,4]triazolo[1,5-a]pyridin-6-yl moiety, insertion of a methyleneamino linker, and a o-F substituent in the phenyl ring markedly increased ALK5 inhibitory activity, kinase selectivity, and oral bioavailability. The 12b (EW-7197) inhibited ALK5 with IC50 value of 0.013 μM in a kinase assay and with IC50 values of 0.0165 and 0.0121 μM in HaCaT (3TP-luc) stable cells and 4T1 (3TP-luc) stable cells, respectively, in a luciferase assay. Selectivity profiling of 12b using a panel of 320 protein kinases revealed that it is a highly selective ALK5/ALK4 inhibitor. Pharmacokinetic study with 12b·HCl in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Rational optimization of 6 has led to the identification of a highly potent, selective, and orally bioavailable ALK5 inhibitor 12b.

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.

Since P-glycoprotein (P-gp) acts as a barrier to intestinal absorption of various drugs, P-gp inhibitors have been studied as oral absorption enhancers of P-gp substrate drugs. Here, we investigated the in vitro and in vivo effects of a novel coumarin derivative (LL-348) for its P-gp inhibitory activity. With LL-348, accumulation of daunomycin (DNM) increased 270% and efflux of DNM decreased 63% compared to that of DNM alone. Paclitaxel (PTX, 25mg/kg) after oral administration with LL-348 (5mg/kg), the optimal dose of LL-348 as an oral absorption enhancer of PTX, improved the relative bioavailability (RB) of PTX to 961%. In a xenograft animal model, PTX (40mg/kg) treated with LL-348 (10mg/kg) significantly increased the efficacy of PTX. The results collectively demonstrate that LL-348 can provide a therapeutic benefit in the oral absorption of P-gp substrate anticancer drugs.

The expression of P-glycoprotein (P-gp), an ATP-dependent efflux transporter, is closely associated with the failure of chemotherapy and drug absorption. Two synthesized optically active phenylbutenoid dimers, 3S-(3,4-dimethoxyphenyl)-4R-{(E)-3,4-dimethoxystyryl}cyclohex-1-ene (1) and 3R-(3,4-dimethoxyphenyl)-4S-{(E)-3,4-dimethoxystyryl}cyclohex-1-ene (2), were tested for their P-gp inhibitory effects by measuring cellular accumulation and efflux of daunomycin in P-gp-overexpressed human breast cancer cells (MCF-7/ADR). Compound 2 significantly increased the accumulation of daunomycin (539%) and decreased the efflux of this compound (55.4%), and similar results were observed for 1. ATPase assays and Western blot analysis were performed to identify the mechanisms by which compounds 1 and 2 inhibit P-gp. In addition, changes in the pharmacokinetic profile of paclitaxel coadministered with 2 in rats were evaluated. Paclitaxel (25 mg/kg) when orally administered with 2 (5 mg/kg) improved its relative bioavailability by 185%. Compound 2 effectively improved cellular accumulation by reducing the efflux of daunomycin and significantly enhanced oral exposure to paclitaxel. Therefore, compound 2 may be useful for improving oral exposure and cellular availability of drugs that are also substrates of P-gp.

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.

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.

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.

In this study, we present anti-cancer drug containing nanofiber-mediated gene delivery to treat liver cancer. Electro-spun nanofibers have big potential for local delivery and sustained release of therapeutic gene and drugs. We reported a temperature-responsive nanofibers mainly compounded by branched poly(ε-caprolactone) (PCL) macro-monomers and anti-cancer drug paclitaxel. The nanofiber could be administrated into liver tumors to dramatically hinder their growth and prevent their metastasis. As a result, paclitaxel encapsulated PCL (PTX/PCL) nanofibers with diameters of around several tens nanometers to 10 nm were successfully obtained by electro-spinning andobserved in scanning electron microscopy (SEM). Nanoparticles composed of disulfide cross-linked branched PEI (ssPEI) and anti-cancer therapeutic gene miRNA-145 were complexed based on the electrostatic interaction and coated over the paclitaxel-loaded nanofiber. MicroRNA 145/ssPEI nanoparticles (MSNs) immobilized on the PTX/PCL nanofiber showed time-dependent sustained release of the microRNA for enhanced uptake in neighboring liver cancer cells without any noticeable cytotoxicity. From this study we are expecting a synergistic effect on the cancer cell suppression since we have combined the drug and gene delivery. This approach uses the nanofibers and nanoparticles together for the treatment of cancer and the detailed investigation in vitro and in vivo must be conducted for the practicality of this study. The polymer is biodegradable and the toxicity issues must be cleared by our approach.

Cancer gene therapy involves the replacement of missing or altered genes with healthy ones. In this paper, we have proposed tumor suppressor gene-carrying superparamagnetic iron oxide nanoparticles (SPIONs) for anti-cancer gene therapy. Thermally crosslinked SPIONs (TCL-SPIONs) were conjugated with branched polyethylenimine (PEI 1800 Da) by EDC-NHS chemistry for p53 plasmid DNA delivery. The morphology of the bPEI conjugated TCL-SPIONs (bPEI-TCL-SPION) and pDNA-loaded bPEI-TCL-SPION nanoparticles was measured using transmission electron microscopy (TEM). The particle sizes of the pDNA-loaded bPEI-TCL-SPION nanoparticles were also confirmed by dynamic light scattering, and ranged from 100 to 130 nm, depending on the molar charge ratio. The fluorescently labeled pDNA was complexed with bPEI-TCL-SPION and its intracellular internalization was investigated using confocal microscopy. The p53 plasmid-loaded bPEI-TCL-SPION nanoparticles achieved significantly higher p53 tumor suppressor gene expression and cellular viability compared to positive controls. The expressed wild-type p53 protein suppressed tumor cell proliferation as compared to the mutant control. When transgene expression of the p53 tumor suppressor gene was evaluated at the mRNA level and quantified using real-time PCR, the results were highly dependent on the molar charge ratio (N/P) as well as the cancer cell type. SPIONs internalized within cancer cells were tracked by magnetic resonance (MR) imaging. It was concluded that bPEI-TCL-SPION could be used as efficient gene delivery carriers that can be tracked by MR imaging.

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.

The aims of the present study were to investigate the effects of silymarin, an inhibitor of the P-glycoprotein efflux pump, on oral bioavailability of paclitaxel in rats, and to compare pharmacokinetic parameters of paclitaxel between a commercial formulation of paclitaxel (Taxol®) and a paclitaxel microemulsion. Oral bioavailability of paclitaxel in a Taxol® formulation was enhanced in the combination with silymarin (10 and 20mg/kg). In particular, the mean maximum plasma concentration (C(max)) and the mean area under the plasma concentration-time curve (AUC(0-)(t)) of paclitaxel in the Taxol® formulation were significantly increased 3-fold and 5-fold compared with control, respectively, following oral co-administration with 10mg/kg of silymarin (p

The total synthesis of optically active phenylbutenoid dimers 1, 3, and ent-3 is described. The key step to access optically active cyclohexene rings was achieved by Diels-Alder reaction of chiral acryloyloxazolinone 9 and phenylbetadiene 10.

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.

Summary Background and objective: CYP2C19 is clinically important in Korea because of the relatively high incidence of poor metabolizers in the population. To fully understand the genetic mechanism of the CYP2C19 defect in poor metabolizers, all variants need to be studied simultaneously. The aim of this study was to investigate the usefulness of CYP2C19 haplotypes as a marker of CYP2C19 enzyme activity in Koreans. Methods: We analysed the single nucleotide polymorphisms and haplotypes of the CYP2C19 gene in 150 healthy Koreans and found three major (frequency > 0·1) haplotypes (H1, H2 and H3). One oral dose of 40 mg omeprazole (Losec®) was administered to 30 subjects grouped as H1/H1, H2/H2, H1/H2, H1/H3 and H2/H3. The pharmacokinetics of omeprazole and its metabolites, 5-hydroxyomeprazole and omeprazole sulphone, in those groups was analysed. Results and discussion: The area under the plasma concentration–time curve (AUC0∞) and elimination half-life (T1/2) of omeprazole were significantly greater in the H2/H2 and H2/H3 groups than in the H1/H1 group (P

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 AND OBJECTIVE Genetic polymorphism of CYP2D6 leads to differences in pharmacokinetics of CYP2D6 substrates. The CYP2D6*10 allele is clinically important in Koreans because of its high frequency in Asians. We investigated whether the pharmacokinetics of metoprolol was altered by the presence of the CYP2D6*10 allele in Korean subjects. METHODS One hundred and seven volunteers were recruited and grouped as CYP2D6*1/*1, CYP2D6*1/*10 and CYP2D6*10/*10 according to their genotypes. Metoprolol tartrate 100 mg (Betaloc) was administered orally once to each subject in these three groups (n = 6, 7 and 5, respectively). The pharmacokinetic parameters of metoprolol and its metabolite, alpha-hydroxymetoprolol, and the metabolic ratio for the three groups were estimated and compared. RESULTS AND DISCUSSION The area under the plasma concentration-time curve (AUC(0-->infinity)), the maximum plasma concentration (C(max)) and the elimination half-life (T(1/2)) of metoprolol and alpha-hydroxymetoprolol for the CYP2D6*10/*10 group were all significantly different from those of the CYP2D6*1/*1 group (P infinity)s of metoprolol were 443.7 +/- 168.1, 995.6 +/- 321.4 and 2545.3 +/- 632.0 ng.h/mL, and the AUC(0-->infinity)s of alpha-hydroxymetoprolol were 1232.0 +/- 311.2, 1344.0 +/- 288.1 and 877.4 +/- 103.4 ng.h/mL for groups CYP2D6*1/*1, *1/*10 and *10/*10, respectively. The corresponding T(1/2) values of metoprolol were 2.7 +/- 0.5, 3.2 +/- 1.3 and 5.0 +/- 1.1 h, while those of alpha-hydroxymetoprolol were 5.4+/-1.5, 6.0 +/- 1.4 and 10.5 +/- 4.2 h, respectively. The metabolic ratios of the three groups were significantly different (P < 0.05). CONCLUSION The CYP2D6*10 allele altered the pharmacokinetics of metoprolol in Korean subjects and is likely to affect other drugs metabolized by the CYP2D6 enzyme, similarly.

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.

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.

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.

A specific and rapid high performance liquid chromatographic (HPLC) method with UV detection (254 nm) was developed for the determination of cefroxadine in human plasma. The sample extraction was performed by a simple procedure, vortexing and centrifugation of sample following addition of 60% trichloroacetic acid. Cephalexin was used as an internal standard (I.S.). The HPLC analysis was carried out on a Capcell Pak C18 analytical column with a mobile phase of 50 mM ammonium formate buffer/pH 3.5 and acetonitrile (90:10, v/v). No interference was observed near the peaks of cefroxadine and I.S. The calibration curve was linear over the range of 0.5-40 microg/mL and the lower limit of quantification (LLOQ) was 0.5 microg/mL. The method was validated with excellent sensitivity, accuracy, precision and stability. This assay was successfully applied to determine the pharmacokinetic parameters of cefroxadine in Korean healthy volunteers after an oral administration of two 250 mg cefroxadine capsules. As a result, the plasma half-life was 1.00+/-0.26 h and the mean AUC(0-6 h) was 46.25+/-6.41microgh/mL. The maximum plasma concentration (C(max)) of 17.62+/-4.87 microg/mL reached 1.44+/-0.39 h after administration.

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.

The roles of IL-1alpha and TNF-alpha in early pulp inflammation were investigated by determining the alkaline phosphatase (ALP) activity, the osteonectin (ON), osteocalcin (OC), bone sialoprotein (BSP), and heme oxygenase-1 (HO-1) expression using an immunoblot method. Primary cultured dental pulp cells were treated with IL-1alpha, TNF-alpha, or both for 3, 7, and 14 days. The pulp cells treated with IL-1alpha for 3 days showed elevated ALP activity and increased ON, OC, and HO-1 expression, whereas TNF-alpha treatment did not increase the ALP activity and no BSP was expressed until day 14. The pulp cells treated with both IL-1alpha and TNF-alpha for 3 days showed increased HO-1 expression compared with that of the control. These data suggest that IL-1alpha and TNF-alpha produced in the early inflammatory reaction have different functions in human pulp cells. IL-1alpha induces ALP, ON, and OC in tooth mineralization and it may play a role in the cytoprotection of pulp cells via HO-1 expression, while long-term treatment of TNF-alpha may inhibit the tooth mineralization.

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.

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 .

Gender differences have been well described in pharmacokinetics and contribute to the interindividual variation in drug disposition, therapeutic response, and drug toxicity. Sex-related differences in the membrane transport of endogenous substrates and xenobiotics have been reported in various organs of the body including kidney, liver, intestine, and brain. These gender-related differences in transport systems could also contribute to interindividual variability in pharmacokinetics and pharmacodynamics. This review will focus on current knowledge of gender-associated differences in the transport of endogenous and exogenous compounds in a variety of body organs and will discuss the implications and the clinical significance of these observations.

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).