Synthesis by radical cyclization and cytotoxicity of highly potent bioreductive alicyclic ring fused [1,2-a]benzimidazolequinones

The key step in the synthesis of new five, six and seven-membered alicyclic ring [1,2-a]-fused bioreductive benzimidazolequinones was radical cyclisation. Six and seven-membered tributyltin hydride-mediated homolytic aromatic substitutions of nucleophilic N-alkyl radicals onto the benzimidazole-2-position occurred in high yields (63-70 %) when quaternising the pyridine-like 3-N of imidazole with camphorsulfonic acid and using large excesses of the azo-initiator, 1,1'-azobis(cyclohexanecarbonitrile), to supplement the non-chain reaction. Elaboration of benzimidazoles to the benzimidazolequinones occurred in excellent yields. The IC50 values for the cytotoxicity of benzimidazolequinones towards the human skin fibroblast cell line GM00637 were in the nanomolar range, as determined by using the MTT assay. The benzimidazolequinones were much more cytotoxic than indolequinone analogues. 1,2,3,4-Tetrahydropyrido[1,2-a]benzimidazole-6,9-dione was the most potent compound prepared being more than 300 times more cytotoxic than the clinically used bioreductive drug, mitomycin C. The latter benzimidazolequinone was more potent under hypoxic conditions (associated with solid tumors), being 4.4 times more cytotoxic than under aerobic conditions, while mitomycin C was 1.8 times more selective towards hypoxia. The cyclopropane fused pyrido[1,2-a]benzimidazolequinone, 1a,2,3,9b-tetrahydro-1H-cyclopropa[3,4]pyrido[1,2-a]benzimidazole-5,8-dione was less cytotoxic and selective than the five-membered ring analogue, 1,1a,8,8a-tetrahydrocyclopropa[3,4]pyrrolo[1,2-a]benzimidazole-3,6-dione. Modifying the structure of the most potent pyrido[1,2-a]benzimidazolequinone by attaching methyl substituents onto the quinone moiety increased reductive potentials and decreased cytotoxicity and selectivity towards hypoxia.     

First synthesis of an aziridinyl fused pyrrolo[1,2-a]benzimidazole and toxicity evaluation towards normal and breast cancer cell lines

Anionic aromatic ipso-substitution has allowed an aziridine ring to be fused onto pyrrolo[1,2-a]benzimidazole. This diazole analogue of aziridinomitosene, and N-[(aziridinyl)methyl]-1H-benzimidazole are shown to be significantly more cytotoxic towards the human breast cancer cell lines MCF-7 and HCC1937 than towards a human normal fibroblast cell line (GM00637). The aziridinyl fused pyrrolo[1,2-a]benzimidazole is less cytotoxic than the non-ring fused aziridinyl analogue towards all three cell lines. The BRCA1-deficient HCC1937 cells are more sensitive to mitomycin C (MMC) compared to GM00637 and MCF-7 cells. The evidence provided indicates that different pathways may mediate cellular response to benzimidazole-containing aziridine compounds compared to MMC.     

The Fanconi anemia pathway and ubiquitin

Fanconi anemia (FA) is a rare genetic disorder characterized by aplastic anemia, cancer/leukemia susceptibility and cellular hypersensitivity to DNA crosslinking agents, such as cisplatin. To date, 12 FA gene products have been identified, which cooperate in a common DNA damage-activated signaling pathway regulating DNA repair (the FA pathway). Eight FA proteins form a nuclear complex harboring E3 ubiquitin ligase activity (the FA core complex) that, in response to DNA damage, mediates the monoubiquitylation of the FA protein FANCD2. Monoubiquitylated FANCD2 colocalizes in nuclear foci with proteins involved in DNA repair, including BRCA1, FANCD1/BRCA2, FANCN/PALB2 and RAD51. All these factors are required for cellular resistance to DNA crosslinking agents. The inactivation of the FA pathway has also been observed in a wide variety of human cancers and is implicated in the sensitivity of cancer cells to DNA crosslinking agents. Drugs that inhibit the FA pathway may be useful chemosensitizers in the treatment of cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).     

Immunoaffinity purification of α-momorcharin from bitter melon seeds (Momordica charantia)

α‐Momorcharin (α‐MMC), a type I ribosome‐inactivating protein (RIP), has shown therapeutic potential such as anti‐tumor and anti‐viral agent. Traditional process of α‐MMC purification from bitter melon seeds was time consuming and low efficient. To take this challenge, we made an affinity matrix by coupling the monoclonal antibody (McAb) with Sepharose 4B. Using this attractive strategy, 196 mg of α‐MMC was obtained from 100 g of bitter melon seeds as the starting material. The yield of the protein was 2.7%. The homogeneity and properties of the protein were assessed by SDS‐PAGE, acidic PAGE, RP‐HPLC and N‐terminal sequence as well as Western blot. Purified α‐MMC showed remarkable inhibition to the melanoma cell line JAR and EMT‐62058. In addition, it also displayed obvious inhibition on hepatitis B virus (HBV). This work provided a simple, rapid and efficient approach for α‐MMC purification from Momordica charantia.     

Industrial case study: Evaluation of a mixed-mode resin for selective capture of a human growth factor recombinantly expressed in E. coli

Mixed-mode chromatography resins are gaining popularity as effective purification tools for challenging feedstocks. This study presents the development of an industrial application to selectively capture recombinant human vascular endothelial growth factor (rhVEGF) on Capto MMC from an alkaline feedstock. Capto MMC resin contains a ligand that has the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. VEGF is a key growth factor involved in angiogenesis and has therapeutic applications for wound healing. In this process, it is expressed in Escherichia coli as inclusion bodies. Solids are harvested from the cell lysate, and the rhVEGF is solubilized and refolded at pH 9.8 in the presence of urea and redox reagents. The unique mixed-mode characteristics of Capto MMC enabled capture of this basic protein with minimal load conditioning and delivered a concentrated pool for downstream processing with >95% yields while reducing host cell protein content to <1.2%. This study explores the impact of loading conditions and residence time on the dynamic binding capacity as well as the development of elution conditions for optimal purification performance. After evaluating various elution buffers, l-arginine HCl was shown to be an effective eluting agent for rhVEGF desorption from the Capto MMC mixed-mode resin since it successfully disrupted the multiple interactions between the resin and rhVEGF. The lab scale effort produced a robust chromatography step that was successfully implemented at commercial manufacturing scale.     

Synthesis of dimethyl substituted benzimidazoles containing cyclopropane fused onto five to eight membered [1,2-a]alicyclic rings and influence of methyl group substituents on cytotoxicity of benzimidazolequinones

Upon thermolysis 5,6-dimethyl-N-[(allyl, but-3-enyl, pent-4-enyl and hex-5-enyl-benzimidazol-2-yl)methylene]-(trans)-2,3-diphenylaziridin-1-amines (Eschenmoser hydrazones) form cyclopropane fused onto pyrrolo-, pyrido-, azepino- and azocino[1,2-a]benzimidazoles in 70, 50, 77 and 11% yield, respectively. The latter reaction also gave carbene insertion products. Dimethyl group substituents were found to significantly reduce the cytotoxicity of benzimidazolequinone towards human skin fibroblast cells.     

Identification of differentially expressed proteins between human hepatoma and normal liver cell lines by two-dimensional electrophoresis and liquid chromatography-ion trap mass spectrometry

In the previous study, the proteomes of the human hepatoma cell line BEL-7404 and the normal human liver cell line L-02 were separated by high resolution two-dimensional electrophoresis (2-DE). Image analysis revealed that 99 protein spots showed quantitative and qualitative variations that were significant (P < 0.01) and reproducible. Here we report the identification results of some of these protein spots. Protein spots excised from 2-D gels were subjected to in-gel digestion with trypsin, and the resulting peptides were measured by microbore high performance liquid chromatography - ion trap - mass spectrometry (LC-IT-MS) to obtain the tandem mass (MS/MS) spectra. Twelve protein spots were identified with high confidence using SEQUEST with uninterpreted MS/MS raw data. Besides inosine-5'-monophosphate dehydrogenase 2, heat shock 27 kDa protein, calreticulin and calmodulin, whose expression was elevated in hepatoma cells, glutathione-S-transferase P was identified from hepatoma cells in which its level was 18-fold higher compared to human liver cells. Two spots were identified as the homologs of reticulocalbin for the first time in hepatoma cells and their expression increased compared to liver cells. However, tubulin beta-1 chain and natural killer cell enhancing factor B were downregulated in hepatoma cells. A tumor suppressing serpin, maspin precursor, was identified from one spot whose quantity was much higher in the normal liver cell line. More interestingly, epidermal fatty acid-binding protein (E-FABP) and fatty acid-binding protein, adipocyte-type (A-FABP), were detected in liver cells but not in hepatoma cells. The functional implication of the identified proteins was discussed.     

Towards a two-dimensional database of common human proteins.

A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.     

Synthesis,characterization and biological evaluation of some novel sulfonylthiosemicarbazides

Abstract

A series of thiosemicarbazides were synthesized and structurally characterized by spectroscopic techniques (NMR, FT-IR) besides elemental analysis. These compounds were evaluated for their cytotoxicity against human breast cancer cell line MCF7 and prostate cancer cell line PC3 and nonmalignant fibroblast L929 cell line by MTT assay. Among the compounds, N-[2-(4-chlorophenyl)ethyl]-2-[(4-methylphenyl)sulfonyl]hydrazinecarbothioamide (3d) and 2-[(4-methylphenyl)sulfonyl]-N-[4-(trifluoromethoxy)phenyl]hydrazinecarbothioamide (3f) were found to display significant cytotoxicity with IC₅₀ of 13.87?μM (against PC3 cell line) and 1.47?μM (against MCF7 cell line), respectively. These compounds were non-cytotoxic to normal cell line with IC₅₀>100?μM. Western blotting studies demonstrated that compound 3f induced apoptosis and caused cell death in the MCF7 and PC3 cell lines via an increase in Bax protein expression and a slight decrease in Bcl-2 protein expression. The gene expression ratio Bax/Bcl-2 showed the induction of mitochondrial apoptosis in cancer cell lines. All of synthesized compounds have also been tested for antioxidant activity and all compounds achieved strong inhibition of the DPPH radical. These findings showed that compound 3f, displays potential to be further explored in the development of new anticancer agents.     

Proteomic profiling of an undefined microbial consortium cultured in fermented dairy manure: Methods development

The production of polyhydroxyalkanoates (PHA; bioplastics) from waste or surplus feedstocks using mixed microbial consortia (MMC) and aerobic dynamic feeding (ADF) is a growing field within mixed culture biotechnology. This study aimed to optimize a 2DE workflow to investigate the proteome dynamics of an MMC synthesizing PHA from fermented dairy manure. To mitigate the challenges posed to effective 2DE by this complex sample matrix, the bacterial biomass was purified using Accudenz gradient centrifugation (AGC) before protein extraction. The optimized 2DE method yielded high‐quality gels suitable for quantitative comparative analysis and subsequent protein identification by LC‐MS/MS. The optimized 2DE method could be adapted to other proteomic investigations involving MMC in complex organic or environmental matrices.     

Membrane Filtration-Assisted Enzymatic Hydrolysis Affects the Biological Activity of Potato Juice

The results of recently published studies indicate that potato juice is characterized by interesting biological activity that can be particularly useful in the case of gastrointestinal symptoms. Moreover, the studies also described the high nutritional value of its proteins. This article is a report on the impact of the enzymatic hydrolysis of proteins combined with membrane filtration. The obtained potato juice protein hydrolysate (PJPH) and its concentrate (cPJPH) were characterized in terms of their nutritional value and biological activity. The amino acid profile and scoring, the content of mineral compounds, and the antioxidant and in vitro cytotoxic activity were assessed. The study proved that the antioxidant activity of PJPH is higher than that of fresh potato juice, and the cytotoxicity against human gastric carcinoma cell line (Hs 746T), human colon cancer cell line (Caco-2), human colorectal adenocarcinoma cell line (HT-29), and human normal colon mucosa cell line (CCD 841 CoN) showed biological activity specifically targeted against cancer cells. Therefore, it can be concluded that the membrane filtration-assisted enzymatic hydrolysis of potato juice proteins may increase their biological activity and allow for potato juice to be used in the production of medicinal preparations.     

新型单取代两亲性酞菁锌的制备及其光动力活性研究

设计并制备了一种组成和结构单一的新型两亲性酞菁锌光敏剂. 采用固相合成法合成出2-单羧基取代酞菁锌, 并使之与五聚赖氨酸偶联成五聚赖氨酸2-羰基酞菁锌. 综合运用各种分析及光谱方法对所得化合物进行表征和确认. 研究了在光照下五聚赖氨酸2-羰基酞菁锌对3种肿瘤细胞(人源肝癌细胞Bel7402、人源胃癌细胞BGC823和人源白血病细胞K562) 与一种正常细胞(人源胚肺成纤维细胞HELF)的杀灭活性. 该光敏剂不仅克服了酞菁锌在水中溶解度低的问题, 且因所偶联的五聚赖氨酸对肿瘤细胞有靶向作用, 具有较高的杀灭肿瘤细胞活性.     

Polycarbonate microspheres containing mitomycin C and magnetic powders as potential hepatic carcinoma therapeutics

The polycarbonate copolymer poly(trimethylene carbonate-co-5,5-dimethyl trimethylene carbonate) (P(TMC-co-DTC)) was synthesized by the polymerization of trimethylene carbonate (TMC) and 5,5-dimethyl trimethylene carbonate (DTC) using tin (II) 2-ethylhexanoate [Sn(Oct)(2)] as a catalyst. In vitro degradation tests indicated this polycarbonate copolymer degraded slowly in phosphate buffer saline solution (PBS, 0.1 mol/L, at 37°C). Magnetic polymer microspheres (MMC-PC-M) generated from the P(TMC-co-DTC) copolymer and containing Fe(3)O(4) magnetic ultrafine powders and an anticancer drug, mitomycin C (MMC) were prepared by a solvent evaporation technique. These anticancer magnetic polycarbonate microspheres showed strong magnetic responsiveness and high MMC loading capacity. In vitro drug release studies indicated that these microspheres sustained steady release rates of MMC in PBS. In vitro cytotoxicity assays demonstrated the microspheres were strongly inhibitory to human hepatic carcinoma (Bel-7204) cells. In vivo site-specific therapy in nude mice with human hepatic carcinoma indicated that the microspheres possessed markedly high antitumor activity against human hepatic carcinoma (Bel-7204).     

Greener synthesis using hydrogen peroxide in ethyl acetate of alicyclic ring-fused benzimidazoles and anti-tumour benzimidazolequinones

Environmentally-friendly and cost effective hydrogen peroxide in ethyl acetate was used to prepare in high yields pyrrolo[1,2-a]benzimidazoles from commercial o-(pyrrolidin-1-yl)anilines without the requirement for organic-aqueous extraction and chromatography. Six, seven and eight membered ring-fused analogues were similarly obtained in high yields with methanesulfonic acid required for the pyrido[1,2-a]benzimidazole. Anti-tumour benzimidazolequinone derivatives were obtained in high yield via the cyclization of 3,6-dimethoxy-2-(cycloamino)anilines.     

Potentiation of photodynamic therapy with hypericin by mitomycin C in the radiation-induced fibrosarcoma-1 mouse tumor model

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been shown to be a promising photosensitizer. We investigated the combination of hypericin-photodynamic therapy (PDT) and a bioreductive drug mitomycin C (MMC) in the present study. The radiation-induced fibrosarcoma-1 tumors were exposed to laser light (120 J/cm2 at 595 nm) 24 h after an intravenous injection of hypericin (1 mg/kg). Hypericin-PDT alone significantly decreased tumor perfusion and oxygen tension as demonstrated by India ink staining technique and OxyLite pO2 measurement, respectively. The in vivo-in vitro cell-survival assay revealed about 60% direct tumor cell killing immediately after PDT. No significant delayed tumor cell death was observed after PDT, which suggests that vascular damage does not contribute significantly to the overall tumor cell death. Injection of a 2.5 mg/kg dose of MMC 20 min before light application significantly decreased tumor cell survival and delayed tumor growth compared with PDT or MMC alone. No greater skin reaction was observed after the combination of MMC and PDT than after PDT alone. Our study demonstrates that combining hypericin-PDT with MMC can be effective in enhancing tumor response with little side effect.     

Global proteome analysis of a human gastric carcinoma

An approach that combines analysis of global protein digests (GPDs) of various subcellular fractions with a novel chromatographic-based method to map protein expression profiles is described. The KATO III gastric carcinoma cell line was fractionated into membrane and cytosol fractions. Each subcellular fraction was digested with trypsin to yield complex mixtures of global protein tags (GPTs). These mixtures were fractionated by two dimensions of chromatography, and GPTs were sequenced by microcapillary liquid chromatography-tandem mass spectrometry (LC-MS/MS), using two further complementary dimensions of chromatography. Additionally, a novel method of protein expression profiling was used to map the KATO III human gastric carcinoma cell line. This method uses the cells' natural proteolytic processes to derive in vivo peptide tags that represent proteins of every functional class and from all subcellular compartments. In one example, expressed protein tags (EPTs) are naturally displayed on the surface of cells by multiligand receptors. Isolation and sequence identification of EPTs is an efficient approach for protein profiling that is complementary to GPT analysis. The EPT approach also provides a further unique subcellular fraction of the biological starting material. Isolation of the multiligand receptors was by immunoaffinity chromatography (IAC). In the current study, five individual peptide maps (two EPTs and three GPTs) of the KATO III cell line were fractionated by multimodal chromatography, and sequenced by on-line multimodal microcapillary LC-MS/MS. This analysis led to the identification of 4291 individual peptide sequences, which defined 1966 unique proteins expressed by this human carcinoma cell line.     

Anticarcinogenic and antimitotic effects of Turkish propolis and mitomycin-C on tissue cultures of bladder cancer

The in vitro anticarcinogenic and antimitotic effects of propolis and mitomycin-C (MMC) on transitional carcinoma cell cultures were investigated. Tissue samples were obtained from 22 patients with bladder carcinomas, which were exposed to propolis (0.1 mL) and MMC (1.6 microL). The mean MI rates for control, propolis and MMC were 8.327 +/- 0.624, 6.990 +/- 0.519 and 5.423 +/- 0.479, respectively. The differences between the control and exposed cells were significant (p < 0.05). We conclude that exposure to propolis can decrease cell division and it may be used as an antimitotic and anticarcinogenic agent.     

Effect of dextran layer on protein uptake to dextran-grafted adsorbents for ion-exchange and mixed-mode chromatography

In the current research, a series of dextran-grafted adsorbents were prepared using sulfopropyl and 4-(1H-imidazol-1-yl) aniline as chromatographic ligands for ion-exchange (IEC) and mixed-mode chromatography (MMC) to respectively investigate the influence of dextran layer on adsorption of γ-globulin. Experimental evidences of static adsorption on dextran-grafted IEC adsorbents showed that adsorption capacity of γ-globulin increased with dextran content. It could be attributed to the multilayer adsorption of charged protein in dextran layer and thus further induced a significant electrical potential gradient at the boundary of adsorbed area and its proximity, improving mass transfer in combination with concentration gradient. In contrast to IEC adsorbents, adsorption capacity and effective diffusivity of dextran-grafted MMC adsorbents did not change obviously with dextran grafting. It was considered that hydrophobic ligands immobilized onto dextran-grafted MMC adsorbents were stuck together at pH 8.0, resulting in the collapse of dextran layer. In concert with measured effective porosity for γ-globulin at pH 4.0, it was confirmed that dextran layer in MMC adsorbent was more complicated and influenced significantly by buffer pH. It was also manifested by protein adsorption at different pHs. Thus, it revealed the complexity in intraparticle mass transfer of the protein in dextran-grafted MMC adsorbent.     

Evaluation of Antiproliferative Palladium(II) Complexes of Synthetic Bisdemethoxycurcumin towards In Vitro Cytotoxicity and Molecular Docking on DNA Sequence

Metallodrugs form a large family of therapeutic agents against cancer, among which is cisplatin, a paradigmatic member. Therapeutic resistance and undesired side effects to Pt(II) related drugs, prompts research on different metal–ligand combinations with potentially enhanced biological activity. We present the synthesis and biological tests of novel palladium(II) complexes containing bisdemethoxycurcumin (BDMC) 1 and 2. Complexes were fully characterized and their structures were determined by X-ray diffraction. Their biological activity was assessed for several selected human tumor cell lines: Jurkat (human leukaemic T-cell lymphoma), HCT-116 (human colorectal carcinoma), HeLa (human cervix epitheloid carcinoma), MCF-7 (human breast adenocarcinoma), MDA-MB-231 (human mammary gland adenocarcinoma), A549 (human alveolar adenocarcinoma), Caco-2 (human colorectal carcinoma), and for non-cancerous 3T3 cells (murine fibroblasts). The cytotoxicity of 1 is comparable to that of cisplatin, and superior to that of 2 in all cell lines. It is a correlation between IC₅₀ values of 1 and 2 in the eight studied cell types, promising a potential use as anti-proliferative drugs. Moreover, for Jurkat cell line, complexes 1 and 2, show an enhanced activity. DFT and docking calculations on the NF-κB protein, Human Serum Albumin (HSA), and DNA were performed for 1 and 2 to correlate with their biological activities.     

Antitumor,cytotoxic, and antioxidant evaluation of six heterocyclic compounds containing different heterocycle moieties

Heterocyclic compounds with different heterocycle moieties, namely benzoxazinone, benzimidazole, quinazolinone, and benzofuranone heterocyclic rings, were synthesized, characterized, and evaluated for their anticancer activity against human hepatocellular carcinoma cell line (HepG2) using sulforhodamine B (SRB) and dimethylthiazol-diphenyltetrazolium bromide (MTT) assays. Also, their cytotoxic activities were tested against human epithelioid carcinoma (Hela) cell line in comparison with normal cell, amniotic epithelial (WISH) cell line, as an in vitro toxicity estimation model. The results showed clearly that 2-(2-benzyl-4-oxoquinazolin-3(4H)-yl)acetohydrazide 4 is the most potent antioxidant and anticancer agents. Although, 3-amino-2-benzylquinazolin-4(3H)-one 5 is less potent anticancer agent against Hela but it is more safe against normal cell (WISH).