Chemical mitochondrial uncouplers share common inhibitory effect on NLRP3 inflammasome activation through inhibiting NFκB nuclear translocation.

Activation of NLRP3 inflammasome is implicated in varieties of pathologies, the aim of the present study is to characterize the effect and mechanism of mitochondrial uncouplers on NLRP3 inflammasome activation by using three types of uncouplers, niclosamide, CCCP and BAM15. Niclosamide, CCCP and BAM15 inhibited LPS plus ATP-induced increases of NLRP3 protein and IL-1ß mRNA levels in RAW264.7 macrophages and THP-1 derived macrophages. Niclosamide, CCCP and BAM15 inhibited LPS plus ATP-induced increase of NFκB (P65) phosphorylation, and inhibited NFκB (P65) nuclear translocation in RAW264.7 macrophages. Niclosamide and BAM15 inhibited LPS-induced increase of IκBα phosphorylation in RAW264.7 macrophages, and the inhibitory effect was dependent on increased intracellular [Ca2+]i; however, CCCP showed no significant effect on IκBα phosphorylation in RAW264.7 macrophages stimulated with LPS. In conclusion, chemical mitochondrial uncouplers niclosamide, CCCP and BAM15 share common inhibitory effect on NLRP3 inflammasome activation through inhibiting NFκB nuclear translocation.

A novel gemcitabine derivative-loaded liposome with great pancreas-targeting ability.

Gemcitabine (Gem) is a standard first-line treatment for pancreatic cancer (PC). However, its chemotherapeutic efficacy is hampered by various limitations such as short half-life, metabolic inactivation, and lack of tumor localizing. We previously synthesized a lipophilic Gem derivative (Gem formyl hexadecyl ester, GemC16) that exhibited improved antitumor activity in vitro. In this study, a target ligand N,N-dimethyl-1,3-propanediamine was conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxyl succinimidyl (polyethylene glycol-2000)] (DSPE-PEG-NHS) to form DSPE-PEG-2N. Then, pancreas-targeting liposomes (2N-LPs) were prepared using the film dispersion-ultrasonic method. GemC16-loaded 2N-LPs displayed near-spherical shapes with an average size distribution of 157.2 nm (polydispersity index (PDI) = 0.201). The encapsulation efficiency of GemC16 was up to 97.3% with a loading capacity of 8.9%. In human PC cell line (BxPC-3) and rat pancreatic acinar cell line (AR42J), cellular uptake of 2N-LPs was significantly enhanced compared with that of unmodified PEG-LPs. 2N-LPs exhibited more potent in vitro cytotoxicity against BxPC-3 and AR42J cell lines than PEG-LPs. After systemic administration in mice, 2N-LPs remarkably increased drug distribution in the pancreas. In an orthotopic tumor mouse model of PC, GemC16-bearing liposomes were more effective in preventing tumor growth than free GemC16. Among these treatments, 2N-LPs showed the best curative effect. Together, 2N-LPs represent a promising nanocarrier to achieve pancreas-targeting drug delivery, and this work would provide new ideas for the chemotherapy of PC.

Design of PEI-conjugated bio-reducible polymer for efficient gene delivery.

The poly(cystaminebis(acrylamide)-diaminohexane) (poly(CBA-DAH)) was designed previously as a bio-reducible efficient gene delivery carrier. However, the high weight ratio required to form the polyplexes between poly(CBA-DAH) with pDNA is still a problem that needs to be addressed. To solve this problem and increase the transfection efficiency, poly(ethylenimine) (PEI, 1.8 kDa) was conjugated to poly(CBA-DAH) via disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) can bind with pDNA at a very low weight ratio of 0.5 and above, like PEI 25 kDa, and form the polyplexes with nano-size (102-128 nm) and positive surface charge (27-34 mV). PCDP and PCDP polyplexes had negligible cytotoxicity and indicated similar or better cellular uptake than the comparison groups such as PEI 25 kDa and Lipofectamine® polyplexes. To confirm the transfection efficiency, the plasmid DNA (pDNA) encoded with the luciferase reporter gene (gWiz-Luc) and green fluorescent protein reporter gene (GFP) were used and treated with PCDP into the A549, Huh-7, and Mia PaCa-2 cells. PCDP/pDNA polyplexes showed highest transfection efficiency in all tested cell lines. In the luciferase assay, PCDP polyplexes showed 10.2 times higher gene transfection efficiency than Lipofectamine® polyplexes in mimic in vivo conditions (30% FBS, A549 cells). The VEGF siRNA expressing plasmid (pshVEGF), which is constructed as a therapeutic gene by our previous work, was delivered by PCDP into the cancer cells. The VEGF gene expression of PCDP/pshVEGF polyplexes was dramatically lower than control and the VEGF gene silencing efficiencies of PCDP/pshVEGF (w/w; 10/1) polyplexes were 54% (A549 cells), 77% (Huh-7 cells), and 66% (Mia PaCa-2 cells). In addition, PCDP/pshVEGF had reduced cell viability rates of about 31% (A549 cells), 39% (Huh-7 cells), and 42% (Mia PaCa-2 cells) and showed better results than all comparison groups. In the transfection efficiency and VEGF silencing assay, PCDP polyplexes showed better results than poly(CBA-DAH) at 4-fold lower weight ratio. The data of all experiments demonstrate that the synthesized PCDP could be used for efficient gene delivery and could be widely applied.

Synthesis, spectral, structural characterization and biological investigation of m-Xylylenediaminium-bis (p-toluenesulfonate) monohydrate.

Novel organic charge transfer complex, m-xylylenediaminium-bis (p-toluenesulfonate) monohydrate (XDPTS) have been synthesized and crystallized to the triclinic system with space group P-1 and the lattice parameters obtained are a=9.9265(7) Å, b=9.9676(6) Å, c=13.4948(10) Å, α=71.95(6)°, ß=77.02(6)°, γ=76.851(5)°. The synthesized complex structure was confirmed by IR, (1)H NMR and (13)C NMR spectral analysis. Pharmacology activities of charge transfer complex were evaluated through antimicrobial, DNA binding/cleavage, antioxidant and cytotoxicity studies. The results reveal that the compound shows good antimicrobial activity against various antibacterial and antifungal species. The DNA interaction indicated that the compound could interact with DNA through intercalation, which is further confirmed by viscosity measurements. The compound should have weak to moderate capacity of scavenging with DPPH, Hydroxyl and ABTS radicals. The cytotoxicity has been evaluated by MTT assay method against MCF-7 cancer cell line.

The in vitro impact of toothpaste extracts on cell viability.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Covalent IR820-PEG-diamine nanoconjugates for theranostic applications in cancer.

Near-infrared dyes can be used as theranostic agents in cancer management, based on their optical imaging and localized hyperthermia capabilities. However, their clinical translatability is limited by issues such as photobleaching, short circulation times, and nonspecific biodistribution. Nanoconjugate formulations of cyanine dyes, such as IR820, may be able to overcome some of these limitations. We covalently conjugated IR820 with 6 kDa polyethylene glycol (PEG)-diamine to create a nanoconjugate (IRPDcov) with potential for in vivo applications. The conjugation process resulted in nearly spherical, uniformly distributed nanoparticles of approximately 150 nm diameter and zeta potential -0.4±0.3 mV. The IRPDcov formulation retained the ability to fluoresce and to cause hyperthermia-mediated cell-growth inhibition, with enhanced internalization and significantly enhanced cytotoxic hyperthermia effects in cancer cells compared with free dye. Additionally, IRPDcov demonstrated a significantly longer (P<0.05) plasma half-life, elimination half-life, and area under the curve (AUC) value compared with IR820, indicating larger overall exposure to the theranostic agent in mice. The IRPDcov conjugate had different organ localization than did free IR820, with potential reduced accumulation in the kidneys and significantly lower (P<0.05) accumulation in the lungs. Some potential advantages of IR820-PEG-diamine nanoconjugates may include passive targeting of tumor tissue through the enhanced permeability and retention effect, prolonged circulation times resulting in increased windows for combined diagnosis and therapy, and further opportunities for functionalization, targeting, and customization. The conjugation of PEG-diamine with a near-infrared dye provides a multifunctional delivery vector whose localization can be monitored with noninvasive techniques and that may also serve for guided hyperthermia cancer treatments.

A novel selenadiazole derivative induces apoptosis in human glioma cells by dephosphorylation of AKT.

Selenadiazole derivatives are synthetic organoselenium compounds with improved anticancer activity and greater selectivity than inorganic selenium. In this study, 4-(benzo[c][1,2,5]selenadiazol-6-yl)-benzene-1,2-diamine (BSBD) was shown to induce time- and dose-dependent apoptosis in SWO-38 human glioma cells by accumulation of a sub-G1 cell population, DNA fragmentation, nuclear condensation, caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage. Further mechanistic investigation showed that BSBD treatment induced dephosphorylation of AKT and DNA damage-mediated activation of p53, leading to extensive apoptosis through the mitochondrial pathway. Our findings suggest that BSBD represents a potential human glioma therapeutic.

N-Polybenzylated alicyclic 1,2-diamines: cytotoxicity and G1 phase arrest in cancer cell line.

Cytotoxicity in the µM range was observed in cancer cell lines treated with N,N,N',N'-tetrabenzyl-4,5-diamino-2-cyclopentenone. Cell cycle analysis on HeLa cells showed a clear G1 phase arrest. A preliminary SAR on structural analogs was performed in order to identify the pharmacophores.

Labeling of human hepatocellular carcinoma cells by hexamethylene diamine modified fluorescent carbon dots.

Fluorescent carbon dots (CDs) were synthesized by a solvothermal method with glucose as carbon source and surface-modified with 1,6-hexamethylene diamine. In this hybrid CDs, the modification played important role for improving the fluorescent performance by introducing nitrogenous compound to passivate CD's surface, making the CDs emit strong fluorescence. The as-prepared CDs were linked with mouse anti-human Alpha fetoprotein (AFP) antibody and goat anti-mouse immunoglobulin (IgG) to directly and indirectly label fixed human hepatocellular carcinoma cells, respectively. The cytotoxicity of these CDs were also tested using the human hepatocellular carcinoma cells. No apparent cytotoxicity was observed, which suggested the potential application of the as-prepared CDs in bioimaging.

Synthesis of N(4)-(substituted phenyl)-N(4)-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines and identification of new microtubule disrupting compounds that are effective against multidrug resistant cells.

A series of fourteen N(4)-(substituted phenyl)-N(4)-alkyl/desalkyl-9H-pyrimido[4,5-b]indole-2,4-diamines was synthesized as potential microtubule targeting agents. The synthesis involved a Fisher indole cyclization of 2-amino-6-hydrazinylpyrimidin-4(3H)-one with cyclohexanone, followed by oxidation, chlorination and displacement with appropriate anilines. Compounds 6, 14 and 15 had low nanomolar potency against MDA-MB-435 tumor cells and depolymerized microtubules. Compound 6 additionally had nanomolar GI(50) values against 57 of the NCI 60-tumor panel cell lines. Mechanistic studies showed that 6 inhibited tubulin polymerization and [(3)H]colchicine binding to tubulin. The most potent compounds were all effective in cells expressing P-glycoprotein or the ßIII isotype of tubulin, which have been associated with clinical drug resistance. Modeling studies provided the potential interactions of 6, 14 and 15 within the colchicine site.

Novel lipoidal amine-based nanocarrier formulations for siRNA delivery.

BACKGROUND: Lack of safe and efficient delivery of siRNA remains the greatest hurdle for the therapeutic application of siRNA. This article reports synthesis and evaluation of novel lipoidal amine-based nanocarrier (LANC) formulations for siRNA delivery. METHOD: Physicochemical properties were analyzed for LANC formulations. siRNA delivery efficiency of LANC-siRNA complexes was determined using a luciferase reporter gene assay. Cytotoxicity of the LANC-siRNA complexes was measured by the MTS assay. Finally, cellular uptake and cytoplasmic release of siRNA were analyzed using flow cytometry. CONCLUSION: The LANC formulation facilitated siRNA uptake and release into the cytoplasm, mediating significant luciferase knockdown (70% inhibition).

Synthesis, cytotoxicity, and in vitro antileishmanial activity of mono-t-butyloxycarbonyl-protected diamines.

Leishmania amazonensis is the etiologic agent of the cutaneous and diffuse leishmaniasis. This species is often associated with drug resistance, and the conventional treatments exhibit high toxicity for patients. Therefore, the search for new antileishmanial compounds is urgently needed since there is no vaccine available. In this study, using the in vitro traditional drug screening test, we have analyzed the effects of a series of diaminoalkanes monoprotected with t-butyloxycarbonyl (BOC) against L. amazonensis. Among the 18 tested compounds, 6 exhibited antileishmanial activity (2, 7-9, 17, and 18). Best IC(50) values (10.39 ± 0.27 and 3.8 ± 0.42 µg/mL) were observed for compounds 17 and 18 (H(2)N(CH(2))nNHBoc, n = 10 and 12), respectively. Although those compounds had higher lipophilicity as indicated by their cLog P values, compound 17 was very toxic. Determination of the selective indexes indicated that 50% of the active compounds were very toxic for HepG2 cells. However, compounds 2, 8, and 18 had good lipophilicity and were less toxic among all polyamine derivatives tested. The chemical properties of antileishmanial diamine derivatives, such as lipophilicity and cytotoxicity, are relevant factors for the design of new drugs. A higher lipophilicity is likely to improve the chances of reaching this intracellular parasite.

Synthesis and cytotoxic activity of 5,6-heteroaromatically annulated pyridine-2,4-diamines.

A series of 5,6-heteroaromatically annulated pyridine-2,4-diamines have been synthesized and their in vitro cytotoxic activities evaluated against six human cancer cell lines. Benzo[g] annulated pyrido[2,3-b]indolediamines 7a-b and 8 showed relatively high cytotoxic activity as well as most of the diamines with pyrrolo[2,3-b]pyridine 17, thieno[2,3-b]pyridine and furo[2,3-b]pyridine 26-28, 1,8-naphthyridine 32 and 34 and benzo[h]quinoline 37 skeletons. Surprisingly, pyrido[2,3-b]indolediamines 13 and 14 without benzo[g] annulation were inactive. None of the new compounds were as potent as ellipticine, the reference compound.

Synthesis and evaluation of some lipidic aminoalcohols and diamines as immunomodulators.

Lymphoproliferation inhibition and cytotoxicity of a number of lipidic aminoacids, aminoalcohols and diamines were evaluated as a preliminary screening to select potential immunomodulators. The four most potent/less toxic compounds were submitted to delayed hypersensibility (DTH) assays to define the best to be evaluated further Graft-vs-Host, NO production and other immunoevaluation (CD4(+), CD45, CD8, CD11b, I-Ek, and NK cells) assays, to establish their immunomodulation potential for being further considered as auxiliary agents for vaccination against some parasitic infections. Compounds 5d, 6d, 6f, 7a, and 9a, fairly inhibited the lymphoproliferation (71.6-79.5%, at 3.2-2.4 nM), while the aminoalcohol derivative 6f and the diamine 7a gave the most promising results in the DTH assays. Diamine derivative 8b induced nitrite production on normal macrophages, whereas compounds 6f and 7a induced nitrite production on LPS pre-stimulated macrophages. These two last compounds have been selected to follow in vivo vaccination assays.

Toxicity of hexamethylenediamine.

Hexamethylendiamine (HMDA; CAS No. 124-09-4; 6055-52-3 for the dihydrochloride salt) is moderately toxic following acute doses/exposures with oral lethal doses in rats ranging from 750 to 1500 mg/kg. HMDA is extremely irritating to the skin and eyes and is not a sensitizer in guinea pigs. Repeated exposure inhalation studies have defined the upper respiratory tract to be the first target of HMDA. The irritation seen is proportional to the exposure concentration. Systemic damage is limited. Genetic testing is not extensive, but there is no indication of activity. HMDA is neither a developmental nor a reproductive toxin, but in one developmental study, the fetal No-observed-adverse-effect-level (NOAEL) was lower than that of the maternal animal. No carcinogenicity studies have been conducted. Documented human experience is limited, but indications of HMDA's irritative properties are found in the literature. HMDA does not persist or bioaccumulate in the environment. The chemical is not particularly toxic to fish and aquatic invertebrates but is quite toxic to algae. HMDA is rapidly absorbed and metabolized by the rat with little tissue storage.

Synthesis and in vitro cytotoxicity of long chain 2-amino alcohols and 1,2-diamines.

The synthesis of enantiomerically pure unsaturated long chain 1,2-diamines and amino alcohols was carried out starting from the corresponding non-natural alpha-amino acids. The in vitro cytotoxicity of the compounds prepared was evaluated against six solid tumor cell lines (A2780, H322, LL, WiDr, C26-10 and UMSCC-22B). Free 1, 2-diamines proved to be the most active compounds exhibiting IC50 values between 2.0 mM and 3.3 mM.

Suppression of arthritis by the inhibitors of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (DP IV, CD26) is a serine exoprotease which selectively cleaves the penultimate proline residue of polypeptides. This enzyme is also expressed as a surface marker on activated T cells. In order to assess the relevance of DP IV in immunological disorders, we evaluated the in vivo effects of specific DP IV inhibitors using two arthritis models, one which was induced by collagen one by alkyldiamine. These animal models share several pathological features associated with rheumatoid arthritis. The transition state substrate analog of DP IV, (S)-Alanylpyrrolidine-boronic Acid (Ala-boroPro), suppressed hind paw swelling, which was associated with collagen-induced and alkyldiamine-induced arthritis. A competitive inhibitor of DP IV, Lys(Z(NO2))-thiazolidide and an irreversible inhibitor, Ala-Pro-nitrobenzoylhydroxylamine also suppressed alkyldiamine-induced arthritis dose-dependently. We also analyzed the pharmacological effects of Lys(Z(NO2))-thiazolidide on several immune responses in vitro, in order to determine its mode of action. This inhibitor suppressed mitogen-induced and antigen-induced proliferation of T cells. However, studies using splenic cells from DP IV deficient rats showed that the inhibition of lymphocyte proliferation was not exerted through the inhibition of DP IV.

Ornithine decarboxylase inhibitor attenuates NaCl enhancement of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.

The effects of combined administration of NaCl and the ornithine decarboxylase (ODC) inhibitor 1,3-diaminopropane (DAP) on the incidence and number of gastric cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and on the ODC activity of the gastric wall and the labeling index of the gastric mucosa were investigated in inbred Wistar rats. Rats were given drinking water with or without 2.5 g/l DAP and chow pellets with and without 10% NaCl ad libitum after 25 weeks of oral administration of MNNG. At week 52 feeding 10% NaCl resulted in significant increases in the incidence of gastric cancers, in the ODC activity of the antral portion of the gastric wall and in the labeling index of antral epithelial cells. Administration of both NaCl and DAP significantly reduced the enhancements by NaCl of gastric carcinogenesis, ODC activity of the antral wall and the labeling index of antral epithelial cells. These results suggest that inhibition of ODC attenuates NaCl enhancement of gastric carcinogenesis and that enhancement by NaCl of gastric carcinogenesis is mediated by polyamine biosynthesis.

Anti-mitochondrial effects of bisethyl polyamines in mammalian cells.

The effects of three bisethyl polyamine analogs on mitochondrial structure and function were examined in human HeLa and L1210 murine leukemia cells. N, N' Bis-[3(ethylamino)-propyl]1-7- heptane diamine (BEPH), and its octane (BEPO), and butane (BESPM) derivative, were shown by electron microscopy and/or Rhodamine 123 uptake studies to alter the structural integrity of mitochondria when both cell lines were treated at the approximate IC50 dose of each drug. At this dose, BEPH had no marked effects on levels of the naturally occurring polyamines, putrescine, spermidine or spermine, in either cell line whereas BEPO and BESPM treatment did result in pool depletion. Southern blot analysis demonstrated a time and dose-dependent loss of mitochondrial DNA from BEPH-treated L1210 cultures suggesting that loss of mitochondrial integrity extended to the DNA level. Treatment of L1210 cells with all three analogs revealed marked reductions in the activity of two mitochondrial enzymes citrate synthase and cytochrome C oxidase. HeLa cells treated with all three analogs exhibited markedly reduced levels of ATP, complete loss of cytidine triphosphate (CTP) and near total depletion of uridine triphosphate (CTP) and near total depletion of uridine triphosphate (UTP). There was also a loss of colony forming ability in HeLa cells which could be nearly completely reversed by the addition of either uridine or cytidine suggesting that NTP reduction may be the primary antiproliferative determinant in these cells. Growth inhibition by BEPH in L1210 cells was markedly potentiated by the glycolysis inhibitor, 2-deoxyglucose, which had no such effect in otherwise untreated cells. This suggests that BEPH treatment of L1210 cells results in impairment of mitochondrial ATP synthesis and activation of the glycolytic pathway for energy production. 2-deoxyglucose treatment also completely prevented the increase of ATP by BEPH treatment of L1210 cells. It is concluded that all three bisethyl polyamines alter HeLa and L1210 mitochondria both structurally and functionally and that these alterations may play a primary role in the antiproliferative activity of these agents in HeLa cells. In L1210, the different spectra of cellular biochemical changes following bisethyl polyamine treatment suggests that additional mechanisms may be in effect.