Petasin is the main component responsible for the anti-adipogenic effect of Petasites japonicus.

Petasites japonicus is one of the most popular edible wild plants in Japan. Many biological effects of P. japonicus have been reported, including anti-allergy, anti-inflammation, and anticancer effects. Although its anti-obesity effect has been reported in several studies, the most important component responsible for this activity has not been fully elucidated. On screening the components that suppress adipocyte differentiation in 3T3-F442A cells, we found that the extract of the flower buds of P. japonicus has anti-adipogenic effect. Among the known major components of P. japonicus, petasin exhibited a potent anti-adipogenic effect at an IC50 value of 0.95 µM. Quantitative analysis revealed that the active component responsible for most of the anti-adipogenic effects of P. japonicus extract is petasin. Petasin suppressed the expression of markers of mature adipocytes (PPARγ, C/EBPα, and aP2). However, as isopetasin and petasol, analogs of petasin, did not exhibit these effects, it indicates that a double bond at the C11-C12 position and an angeloyl ester moiety were essential for the activity. Petasin affected the late stage of adipocyte differentiation and inhibited the expression of lipid synthesis factors (ACC1, FAS, and SCD1). Additionally, it was revealed that petasin could be efficiently extracted using hexane with minimal amount of pyrrolizidine alkaloids, the toxic components. These findings indicate that P. japonicus extract containing petasin could be a promising food material for the prevention of obesity.

Antiproliferative activity of 3,5-disubstituted tetrahydro-2H-1,3,5-thiadiazine thione (THTT) derivatives and evaluation as potential prodrug.

Four series of tetrahydro-2H-1,3,5-thiadiazine thione derivatives were screened for their in vitro antiproliferative activities against two human cancerous PC3 and HeLa cell lines. The cytotoxicity of all the compounds (series A-D) was also determined on mammalian mouse fibroblast 3T3 cells. Most of the compounds showed significant anticancer potential against both cancer cell lines within the range of IC50 = 6.4-29.9 and 2.4-23.8 M respectively when compared with standard doxorubicin (IC50 = 0.3 M). All compounds demonstrated a notable selectivity for Hela cells and found either non-toxic or relatively less toxic for 3T3 cell lines model. The structure-activity relationship indicated that antiproliferative activity mainly influenced by the nature and position of substituents at thidiazine nucleus. In general, the presence of aryl groups for example 3,4-(OMe) 2.Bzl and CH(Ph)Me at N-3 position resulted in a significant activity. Under enzymatic hydrolysis, complete conversion (100%) of ester derivative of thiadiazine thione (10a) into its acidic counterpart (7c) was achieved during 20 min which indicated that these types of THTT ester derivatives can be a possible lead for future investigations as prodrug anticancer probes.

Resveratrol Attenuates Lipopolysaccharides (LPS)-Induced Inhibition of Osteoblast Differentiation in MC3T3-E1 Cells.

BACKGROUND LPS-inhibited osteoblastic differentiation plays an important role in the pathogenesis of osteomyelitis. Thus, searching for drugs that affect LPS-mediated osteoblastic differentiation may be crucial in developing therapies for osteomyelitis. The purpose of this study was to investigate the role and mechanisms of resveratrol, a natural polyphenol present in red wine, on LPS-inhibited osteoblastic differentiation. MATERIAL AND METHODS Cell viability was measured by MMT assay. Mitochondrial ATP levels, membrane potential, and superoxide production were measured to evaluate the effects of LPS and resveratrol on mitochondrial functions in osteoblast-like MC3T3-E1 cells. Osteoblast-related genes, including ALP, OCN, OPN, and RUNX2, were measured by ELISA analysis and RT-PCR in differentiated osteoblast cells treated with LPS and resveratrol. Cellular Sirt1 and PCG-1α levels were measured by Western blot to probe the impact of resveratrol treatment in LPS-stimulated MC3T3-E1 osteoblasts. RESULTS The results showed that LPS caused significant mitochondrial dysfunctions of MC3T3-E1 cells in a dose-dependent manner, which were attenuated by resveratrol. Furthermore, LPS markedly decreased the expression of ALP, OCN, OPN, and RUNX2 in MC3T3-E1 cells cultivated in osteoblast differentiation medium, suggesting that LPS inhibited the osteoblastic differentiation of MC3T3-E1 cells. However, resveratrol obviously alleviated the suppressive impact of LPS on osteoblast differentiation. In addition, resveratrol increased expression of Sirt1 and PGC-1α in MC3T3-E1 cells treated with LPS. CONCLUSIONS Taken together, these results show that resveratrol alleviated the suppression of LPS on osteoblast differentiation by improving, at least in part, mitochondrial function.

Cell-targeted platinum nanoparticles and nanoparticle clusters.

Herein, we report the facile preparation of cell-targeted platinum nanoparticles (PtNPs), through the design of peptides that, as a single molecule added in small concentration during the synthesis, control the size of PtNP clusters during their growth, stabilise the PtNPs in aqueous suspension and enable the functionalisation of the PtNPs with a versatile range of cell-targeting ligands. Water-soluble PtNPs targeted respectively at blood group antigens and at integrin receptors are demonstrated.

Cytotoxic activity of chimeric protein PD-L4UWSCI(tr) does not appear be affected by specificity of inhibition mediated by anti-protease WSCI domain.

In a previously study, a type 1 ribosome inactivating protein (PD-L4) and a wheat subtilisin/chymotrypsin inhibitor (WSCI) were engineered into a chimeric protein (PD-L4UWSCI) that presented in addition to the same properties of both domains an intriguing selective cytotoxic action on murine tumor cells. This finding supported the idea that the protection of C-terminal region of PD-L4 could amplify its cytotoxic action by virtue of a greater resistance to proteases. Several authors indeed revealed that the cytotoxicity of RIPs depends not only on the intracellular routing, but also on the intrinsic resistance to proteolysis. In this regard in the present work we have produced a variant of chimeric protein, named PD-L4UWSCI(tr), changing the inhibitory specificity of WSCI domain. The purpose of this approach was to check if the cytotoxicity of the chimeric protein was altered depending on the properties of protease inhibitor domain or by a different fold of whole protein. Data collected supposedly indicate that WSCI domain contributes to cytotoxicity of chimeric protein exclusively from a structural point of view.

In vitro anti-inflammatory and wound-healing potential of a Phyllostachys edulis leaf extract--identification of isoorientin as an active compound.

Extracts prepared from the leaves of Phyllostachys edulis (bamboo) have received attention in pharmacological research due to their potent antitumor, anti-inflammatory, antimicrobial, and anti-ulcerogenic activities. In this study, anti-inflammatory effects of a bamboo leaf extract on tumor necrosis factor alpha-induced overproduction of interleukin 8, vascular endothelial growth factor, and interleukin 6 in immortalized human keratinocytes were investigated for the first time. In addition, wound-healing effects were evaluated in 3T3-swiss albino mouse fibroblasts. Bamboo leaf extract and isoorientin inhibited the tumor necrosis factor alpha-induced release of interleukin 8 and vascular endothelial growth factor. Furthermore, isoorientin dose-dependently reduced levels of interleukin 6 in tumor necrosis factor alpha-α-treated immortalized human keratinocytes cells. Wound healing was evaluated using a modification of the classical scratch assay. For evaluation of the wound gap, a new computerized method based on time-lapse microscopy was developed. It was shown that bamboo leaf extract (10 µg/mL) improved wound closure by 28 % (12 h) and 54 % (24 h), respectively. In concentrations of 50 µg/mL and above, bamboo leaf extract inhibited cell migration without affecting cell viability. Isoorientin (10 µM) improved wound closure by 29 % (12 h) and 56 % (24 h), respectively. Comparable to bamboo leaf extract, higher concentrations of isoorientin prevented cell migration. It is suggested that bamboo leaf extract as well as isoorientin have a dual activity - in higher doses, they show anti-inflammatory effects, and in lower concentrations, they exert anti-angiogenic activities.

Dithiol-PEG-PDLLA micelles: preparation and evaluation as potential topical ocular delivery vehicle.

Thiol-modified nanoparticles have potential applications in mucoadhesive drug delivery and have been examined in this regard for topical ocular delivery. In this paper we provide a simple method for the synthesis of a dithiol terminated amphiphilic diblock copolymer. Bidentate dithiol-poly(ethylene glycol)-poly(d,l-lactide) (SH2-PEG-PDLLA) was synthesized and micelles with dithiol-containing coronas were prepared from this block copolymer via the emulsion method. In vitro release studies indicated that the presence of the thiol groups at the surface did not affect the rate of release of dexamethasone, used as a representative ocular drug. The micelles also showed low cytotoxicity to human corneal epithelial cells (HCEC) and murine fibroblast cells (3T3 cells). A hydrophobic red fluorophore, Nile red, was loaded into the core of micelles and confocal microscopy was used to study HCEC uptake and retention of the micelles. The micelles were rapidly endocytosed by the HCEC, with intracellular micelle levels remaining unchanged with incubation times from 5 to 120 min. Interestingly, Nile red was eliminated significantly more slowly from HCECs treated with the thiolated micelles. These results suggest that these dithiolated micelles may be effective for topical ocular drug delivery.

[Smokeless tobacco extract affects biological properties of the pre-osteoblast cell line MC3T3-E1 ].

OBJECTIVE: To investigate the effects of smokeless tobacco extract (STE) on biological properties of osteoblast, and to identify possible pathological mechanisms of osseointegration. METHODS: MC3T3-E1 Sub-clone 14 cells were cultured in the presence of STE at 0 (control group),0. 01,0. 1,1,5,10 g/L. The cell proliferation was measured by MTT assay 1 d, 3 d, 5 d, and 7 d after exposure. The F-actin cytoskeleton of MC3T3 was stained with Rhodamine and DAPI, and then examined under a confocal laser scanning microscope 24 h after exposure to STE. The mRNA expressions of interleukin-6 (IL-6) and core-binding factor αl(Cbfαl) were quantified by real- time PCR (RT-qPCR) 48 h after exposure to STE. RESULTS: The MTT assay showed that 0. 01-10 g/L STE inhibited MC3T3 proliferation (P<0. 05). Prolonged time enabled 5-10 g/L STE to inhibit MC3T3 proliferation (P<0. 05). Network structure in F-actin cytoskeleton was demonstrated in the controls. In the cells exposed to STE, F-actin cytoskeleton started to change with disruptive structures. As the concentration of STE increased, the changes became more significant. STE increased the mRNA expression of IL-6 at the concentration of 5 g/L and 10 g/L (P<0.05), decreased the mRNA expression of Cbfα1 at the concentration of 0. 1-10 g/L (PO<0. 05). CONCLUSION: Tobacco may inhibit osteoblast proliferation, destroy F-actin cytoskeleton structure, increase the mRNA expression of IL-6 and decrease the mRNA expression of Cbfα1, and inhibit cell differentiation and adhesion accordingly. Smoking is a disadvantage to osseointegration.

In vitro study of the cytotoxicity and antiproliferative effects of surfactants produced by Sphingobacterium detergens.

The application of biosurfactants in the biomedical field is growing due to their antimicrobial activity, low cytotoxicity and ability to induce apoptosis in cancer cells. In the light of this therapeutic potential, as well as possible applications in cosmetics or as drug vehicles in pharmaceutical products, a new biosurfactant produced by Sphingobacterium detergens was investigated for its haemolytic activity and cytotoxic and antiproliferative effects in different cell lines. Fraction A showed 100% haemolysis in rabbit erythrocytes, but in Fraction B the rate was only 83%. When comparing cytotoxicity values (IC50) of the two fractions in model fibroblast and keratinocyte cell cultures, Fraction B was less cytotoxic, showing lower values than the reference compound SDS, indicating low skin irritability. Finally, in non-differentiated intestinal Caco-2 cultures, Fractions A and B reduced cell proliferation and induced apoptosis by 44% and 75%, respectively. According to these results, biosurfactants produced by S. detergens have potential application in cosmetic and pharmaceutical formulations.

PPARγ delivered by Ch-GNPs onto titanium surfaces inhibits implant-induced inflammation and induces bone mineralization of MC-3T3E1 osteoblast-like cells.

OBJECTIVES: To deliver the efficacy and safety of Ch-GNPs (Chitosan gold nanoparticles) conjugated anti-inflammatory molecules peroxisome proliferator activated receptor gamma (PPARγ) on implant surface titanium (Ti) to reduce implant-induced inflammation. MATERIALS AND METHODS: The Ch-GNPs were conjugated with the PPARγ cDNA through a coacervation process. Conjugation was cast over Ti surfaces by dipping, and cells were seeded on different sizes (6 × 6 × 0.1 cm and 1 × 1 × 0.1 cm; n = 3) of Ti surfaces. The size of Ch-GNPs and surface characterization of Ti was performed using UV-vis spectroscopy, TEM (Transmission electron microscopy) and EDX (energy-dispersive X-ray). The DNA conjugation and transfection capacity of Ch-GNPs were simultaneously confirmed by agarose gel electrophoresis, ß-galactosidase staining, and immunoblotting. RESULTS: The Ch-GNPs were well dispersed and spherical in shape, with average size around 10-20 nm. Ti surfaces coated with Ch-GNPs/LacZ, as transfection efficacy molecule, showed strong ß-galactosidase staining in MC-3T3 E1 cells. Cells cultured on Ch-GNPs/PPARγ-coated Ti surfaces were able to inhibit implant-induced inflammation by simultaneously suppressing the expression of tumor necrosis factor- alpha (TNF-α), interleukin-1 beta (IL-1ß), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-2 (MMP-2). The inhibition mechanism of Ch-GNPs/PPARγ was due to inhibition of both reactive oxygen species (ROS) and nitric oxide (NO) secretion (n = 3; P < 0.05). In addition, Ch-GNPs/PPARγ was able to increase expression of bone morphogenetic protein (BMP-7) and runt-related transcription factor-2 (RUNX-2). Furthermore, alkaline phosphatase activity (ALP) was also increased than that in control (n = 3; P < 0.01). Whereas, expression of receptor activator of NF-κB ligand (RANKL) was decreased. CONCLUSIONS: The novel gene delivery materials, like Ch-GNPs, can carry the PPARγ cDNA into the required areas of the implant surfaces, thus aiding to inhibit inflammation and promote osteoblast function. Thus, the PPARγ on implant surfaces may promote its clinical application on peri-implantitis or periodontitis like diseases.

Controlled synergistic delivery of paclitaxel and heat from poly(ß-amino ester)/iron oxide-based hydrogel nanocomposites.

Poly(ß-amino ester) (PBAE) biodegradable hydrogels were investigated for potential combined chemotherapeutic and heat delivery in the synergistic treatment of cancer. Hyperthermia, the heating of cancerous tissue from 41 to 45 °C, increases the efficacy of conventional cancer therapies such as irradiation and chemotherapy. The hydrogel nanocomposites in this work provide a drug delivery vehicle (via the biodegradable PBAE polymer network) and the ability to be heated remotely upon exposure to an alternating magnetic field (via iron oxide nanoparticles incorporated into the hydrogel matrix). PBAE macromers composed of poly(ethylene glycol) (N=400) diacrylate (PEG400DA) or diethylene glycol diacrylate (DEGDA) with isobutylamine (IBA) were synthesized. Hydrogel nanocomposites were fabricated via free-radical polymerization to form a bulk hydrogel matrix entrapping both iron oxide nanoparticles and paclitaxel. The 2EG-IBA hydrogel exhibited complete degradation after approximately 7 weeks whereas the 9EG-IBA hydrogel degraded completely in 11h. The hydrogels heated upon exposure to an alternating magnetic field throughout the degradation process. Additionally, the cytotoxicity of the degradation products was evaluated. Paclitaxel release was controlled via bulk degradation of the hydrogels. The tailorability of these nanocomposites makes them solid candidates for the synergistic treatment of cancer.

Porphyromonas endodontalis lipopolysaccharides induce RANKL by mouse osteoblast in a way different from that of Escherichia coli lipopolysaccharide.

INTRODUCTION: Porphyromonas endodontalis lipopolysaccharide (LPS) has been shown to have a high positive rate in infected root canals and symptomatic apical periodontitis. It may play an integral role as a potent stimulator of inflammatory cytokines involved in apical lesions. The receptor activator of nuclear factor-κB ligand (RANKL) has been proven to be the key regulator of bone remodeling. This study investigated P. endodontalis LPS-induced RANKL production and LPS signaling in mouse osteoblasts. METHODS: LPS-induced RANKL production in mouse osteoblast MC3T3-E1 cells was measured by Western blot and real-time polymerase chain reaction, and the Toll-like receptors (TLRs) were determined by the blocking test using anti-TLRs antibodies. In addition, specific inhibitors were used to analyze the intracellular signaling pathways. Escherichia coli LPS was used as the control. RESULTS: Both of the anti-TLR2 and anti-TLR4 antibodies significantly (P < .05) inhibited the expression of RANKL from osteoblasts stimulated with P. endodontalis LPS; only anti-TLR2 antibody had a significant (P < .05) inhibitory effect on E. coli LPS signaling. SP600125 (c-Jun N-terminal kinase [JNK] inhibitor) prevented the up-regulation of RANKL expression in P. endodontalis LPS-infected osteoblasts (P < .05). The inhibitory effect of wortmannin (phosphatidylinositol 3-kinase inhibitor) and PD98059 (mitogen-activated protein kinase [MAPK]/extracellular signal-regulated kinase [ERK] kinase-1/2 [MEK 1/2] inhibitor) were observed in E. coli LPS-treated mouse osteoblasts (P < .05). CONCLUSIONS: Results from this study showed that P. endodontalis LPS has the ability to promote the expression of RANKL in mouse osteoblasts, and this induction was mainly through the TLR2/4-JNK signaling pathway, a situation quite different from that of typical bacterial endotoxin (E. coli LPS).

PTHrP 1-141 and 1-86 increase in vitro bone formation.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) has anabolic effects in bone, which has led to the clinical use of N-terminal fragments of PTHrP and PTH. Since 10% to 20% of fractures demonstrate healing complications and osteoporosis continues to be a debilitating disease, the development of bone-forming agents is of utmost importance. Due to evidence that regions of PTHrP other than the N-terminus may have bone-forming effects, this study was designed to compare the effects of full-length PTHrP 1-141 to N-terminal PTHrP 1-86 on in vitro bone formation. MATERIALS AND METHODS: MC3T3-E1 pre-osteoblasts were treated once every 6 d for 36 d with 5, 25, and 50 pM of PTHrP 1-141 or 1-86 for 1 or 24 h. Cells were also treated after blocking the N-terminus, the nuclear localization sequence (NLS), and the C-terminus of PTHrP, individually and in combination. Area of mineralization, alkaline phosphatase (ALP), and osteocalcin (OCN) were measured. RESULTS: PTHrP 1-141 and 1-86 increased mineralization after 24-h treatments, but not 1-h. PTHrP 1-141 was more potent than 1-86. Treatment with PTHrP 1-141 for 24-h, but not 1-86, resulted in a concentration-dependent increase in ALP, with no effect after 1-h. Exposure to both peptides for 1- or 24-h induced a concentration-dependent increase in OCN, with 24-h exceeding 1-h. Antibody blocking revealed that the NLS and C-terminus are anabolic. CONCLUSIONS: Both PTHrP 1-141 and 1-86 increased in vitro bone formation; however, PTHrP 1-141 was more effective. The NLS and C-terminus have anabolic effects distinct from the N-terminus. This demonstrates the advantage of PTHrP 1-141 as a skeletal anabolic agent.

Metabolic fate of ultratrace levels of GeCl(4) in the rat and in vitro studies on its basal cytotoxicity and carcinogenic potential in Balb/3T3 and HaCaT cell linesdagger.

The use of germanium (Ge) and the possibility of exposure to trace and ultratrace amounts of this element is increasing. Germanium is widely used in the industrial field as a semiconductor and also as a dietary supplement, an elixir to 'promote health and cure disease' (e.g. cancer and AIDS). More recently, germanium nanoparticles, ranging in size from 60 to 80 nm, have been developed as a potential spleen imaging agent. Like other metal-based nanoparticles used in nanomedicine, Ge nanoparticles may release trace and ultratrace amounts of Ge ions when injected. The metabolic fate and toxicity of these ions still needs to be evaluated. In this study the metabolic fate of a cationic tetravalent Ge species was studied in vivo by injecting rats i.p. with ultratrace amounts of Ge (80 ng kg(-1)) as [(68)Ge]GeCl(4). The cytotoxicity and carcinogenic potential was assessed in vitro using immortalised human skin keratinocytes and mouse fibroblasts (HaCaT and Balb/c 3T3 cell lines, respectively). At 24 h post-exposure Ge was poorly retained in rat tissues (kidney, liver, intestine, femur, spleen and the heart were the organs with the highest Ge concentration). In the blood, Ge was rapidly cleared, being almost equally distributed between plasma and red blood cells. The excretion was mainly via the urine. The hepatic and renal intracellular distribution showed the highest recovery of Ge in the cytosol and the nuclear fractions. Chromatographic separation and ultrafiltration experiments on kidney and liver cytosols showed that the bulk of Ge was associated with low molecular weight components, representing a 'mobile pool' of the element in the body. However, a significant part of the element was able to interact with biological macromolecules which could be responsible for the presence of Ge in the liver and kidney after 7 days. The in vitro experiments confirmed the low degree of cytotoxicity of GeCl(4) both in HaCaT and Balb/3T3. The latter model was more sensitive to the toxic effects induced by Ge as shown by a colony forming efficiency (CFE) greater than 70% at 700 microm of exposure. At the highest exposure concentration tested (700 microm) GeCl(4) failed to induce morphological neoplastic transformation of the cells, suggesting for the first time that a cationic form of Ge ions has no carcinogenic potential. This supports the results of the only study reported in mice, treated orally long-term to an anionic species of Ge such as sodium germanate (Kanisawa and Schroeder, 1967).

A new resin-bonded retrograde filling material.

OBJECTIVE: This study determined the physical properties and cytotoxicity of a novel root-end filling material (NRC). STUDY DESIGN: NRC is a powder and liquid system. The liquid is composed of hydroxyethylmethacrylate, benzoyl peroxide, toluidine, and toluenesulfinate. And the powder is made of calcium oxide, calcium silicate, and triphenylbismuth carbonate. The setting time, compressive strength, and pH change of NRC and gray and white mineral trioxide aggregate (MTA) were determined according to ISO standardization. MC3T3-E1 cells were cultured on NRC and white MTA for determining MTT scores. The absorbance of formazan was measured at 570 nm with a spectrophotometer. The MTT assay was performed in triplicate and repeated in 2 cultures. One-way analysis of variance was used to determine statistical differences in physical properties and MTT assay (P < .05). RESULTS: Mean setting time of materials tested were: NRC 12.5 +/- 0.3 minutes, gray MTA 345.5 +/- 96.2 minutes, and white MTA 318.0 +/- 56.0 minutes. After 24 hours, the mean compressive strengths were: NRC, 21.6 +/- 5.5 MPa, gray MTA: 7.7 +/- 3.3 MPa, and white MTA, 18.9 +/- 3.2 MPa. The pH of the test materials were: NRC 12.0, gray MTA 12.2, and white MTA 11.9. There were no statistically significant differences in compressive strength and pH between white MTA and NRC. The compressive strength of gray MTA was significantly lower than white MTA and NRC (P < .05). The setting time of NRC was significantly lower than white and gray MTA. In MTT assay, both NRC and white MTA were not cytotoxic to MC3T3-E1 cells. CONCLUSIONS: It was concluded that the setting time, compressive strength, pH, and initial biocompatibility results of NRC are favorable for a root-end filling material.

Anti-obesity effect of sulfated glucosamine by AMPK signal pathway in 3T3-L1 adipocytes.

In this study, we investigated the effect of sulfated glucosamine (SGlc) on adipogenesis of 3T3-L1 adipocytes during differentiation of preadipocytes into adipocytes by measuring lipid accumulation and adipogenesis related factors. Treatment with SGlc reduced the triglyceride content in Oil-Red O staining and enhanced glycerol secretion in adipocytes in a dose-dependent manner. In addition, SGlc induced the down-regulation of adipogenesis related factors and adipocyte specific gene promoters. Moreover, treatment of 3T3-L1 adipocytes with SGlc activated the phosphorylated adenosine monophosphate-activated protein kinase (AMPK) alpha and beta along with their substrate, acetyl-CoA carboxylase (ACC). These results suggest that inhibitory effect of SGlc on adipocyte differentiation might be mediated through the up-regulation of AMPK pathway.

3,3'-diindolylmethane induction of p75NTR-dependent cell death via the p38 mitogen-activated protein kinase pathway in prostate cancer cells.

The p75(NTR) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with the nonsteroidal anti-inflammatory drug, indomethacin, induced p75(NTR) expression in the T24 cancer cell line leading to p75(NTR)-mediated decreased survival. Utilizing the indole moiety of indomethacin as a pharmacophore, we identified in rank-order with least efficacy, ketorolac, etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, and 3,3'-diindolylmethane (DIM) exhibiting greatest activity for induction of p75(NTR) levels and inhibition of cell survival. Prostate (PC-3, DU-145) and bladder (T24) cancer cells were more sensitive to DIM induction of p75(NTR)-associated loss of survival than breast (MCF7) and fibroblast (3T3) cells. Transfection of the PC-3 prostate cell line with a dominant-negative form of p75(NTR) before DIM treatment significantly rescued cell survival demonstrating a cause and effect relationship between DIM induction of p75(NTR) levels and inhibition of survival. Furthermore, siRNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR) by DIM in the PC-3 prostate cell line. DIM treatment induced phosphorylation of p38 MAPK as early as within 1 minute. Collectively, we identify DIM as an indole capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

Analysis of cellular response to isocyanate using N-succinimidyl N-methylcarbamate exposure in cultured mammalian cells.

Isocyanates (R--N==C==O), one of the highly reactive industrial intermediates, possess the capability to modulate the bio-molecules by forming toxic metabolites and adducts which may cause adverse health effects. Some of their toxic degradations have previously been unknown and overlooked; of which, molecular repercussions underlying their genetic hazards upon occupational/accidental exposures still remain as an intricate issue and are hitherto unknown. To assess the genotoxic potential of methyl isocyanate in cultured mammalian cells after in vitro exposure, we performed a study in three different normal cell lines MM55.K (mouse kidney epithelial), B/CMBA.Ov (mouse ovarian epithelial), and NIH/3T3 (primary mouse embryonic fibroblast). Cellular DNA damage response was studied for qualitative phosphorylation states of ATM, gammaH2AX proteins and quantitative state of p53 phosphorylation; DNA cell cycle analysis and measure of cellular apoptotic index before and after treatment were also investigated. Our results demonstrate that methyl isocyanate by negatively regulating the DNA damage response pathway, might promote cell cycle arrest, and apoptosis in cultured mammalian cells suggestive of causing genetic alterations. We anticipate that these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates. We also predict that increasing knowledge on DNA damage-triggered signaling leading to cell death could provide new strategies for investigating the effects of DNA repair disorders and decreased repair capacity on the toxicity and carcinogenic properties of environmental toxins.

Neuropoietin attenuates adipogenesis and induces insulin resistance in adipocytes.

Recent findings have implicated gp130 receptor ligands, particularly ciliary neurotrophic factor (CNTF), as potential anti-obesity therapeutics. Neuropoietin (NP) is a recently discovered cytokine in the gp130 family that shares functional and structural features with CNTF and signals via the CNTF receptor tripartite complex comprised of CNTFRalpha, LIF receptor, and gp130. NP plays a role in the development of the nervous system, but the effects of NP on adipocytes have not been previously examined. Because CNTF exerts anti-obesogenic effects in adipocytes and NP shares the same receptor complex, we investigated the effects of NP on adipocyte development and insulin action. Using cultured 3T3-L1 adipocytes, we observed that NP has the ability to block adipogenesis in a dose- and time-dependent manner. We also observed that cultured adipocytes, as well as murine adipose tissue, are highly responsive to acute NP treatment. Rodents injected with NP had a substantial increase in STAT3 tyrosine phosphorylation and ERK 1 and 2 activation. We also observed the induction of SOCS-3 mRNA in 3T3-L1 adipocytes following NP treatment. Unlike CNTF, our studies have revealed that NP also substantially attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes. In addition, NP blocks insulin action in adipose tissue in vivo. These observations are supported by data demonstrating that NP impairs insulin signaling via decreased activation of both IRS-1 and Akt. In summary, we have observed that both adipocytes in vitro and in vivo are highly responsive to NP, and this cytokine has the ability to affect insulin signaling in fat cells. These novel observations suggest that NP, unlike CNTF, may not be a viable obesity therapeutic.

Different S/M checkpoint responses of tumor and non tumor cell lines to DNA replication inhibition.

Cell cycle checkpoint abrogation, especially the inhibition of Chk1 in combination with DNA-damaging treatments, has been proposed as a promising way of sensitizing cancer cells. However, less is known about the possibility to selectively affect tumor cells when they are treated with agents that block DNA synthesis in combination with replication checkpoint inhibitors. Here, we present clear insights in the different responses of tumor and non-transformed cells to the inhibition of DNA replication with hydroxyurea in combination with checkpoint abrogation via inhibition of Ataxia telangiectasia-mutated- (ATM) and Rad3-related/ATM (ATR/ATM) and Chk1 kinases. Interestingly, we find that non-transformed cell lines activate ATR/ATM- and Chk1-independent pathways in response to replication inhibition to prevent mitotic entry with unreplicated DNA. In contrast, tumor cell lines such as HCT116 and HeLa cells rely entirely on Chk1 activity for a proper response to replication inhibitors. Our results show that p38 is activated in response to hydroxyurea treatment and collaborates with Chk1 to prevent mitotic entry in non-transformed cell lines by maintaining cyclin B1/Cdk1 complexes inactive. Furthermore, DNA replication arrest down-regulates cyclin B1 promoter activity in non-transformed cells, but not in tumor cells in a Chk1- and p38-independent way. Thus, our data show that non-transformed cells present a more robust DNA replication checkpoint response compared with tumor cells that involves activation of the p38 pathway. We show that some of these responses to replication block can be lost in tumor cells, causing a defective checkpoint and providing a rationale for tumor-selective effects of combined therapies.