Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line.     

Design,synthesis and evaluation of novel levoglucosenone derivatives as promising anticancer agents

A series of levoglucosenone-derived 1,2,3-triazoles and isoxazoles featuring a flexible spacer between the heteroaromatic and anhydropyranose cores have been designed and synthesized following an hetero Michael // 1,3-dipolar cycloaddition path. The use of a design of experiments approach allowed the optimization of the oxa-Michael reaction with propargyl alcohol as nucleophile, a key step for the synthesis of the target compounds. All of the compounds were tested for their anticancer activity on MDA-MB-231 cells, featuring mutant p53. The results highlighted the importance of the introduction of the flexible spacer as well as the higher activity of oxa-Michael isoxazole-derivatives. The most prominent compounds also showed anti-proliferative activities against lung and colon cancer cell lines. The compounds showed enhanced cytotoxic effects in the presence of mutant p53, determined both by endogenous mutant p53 knock down (R280K) and by reintroducing p53 R280K in cells lacking p53 expression.     

Synthesis of new chromeno-annulated cis-fused pyrano[4,3-c]isoxazole derivatives via intramolecular nitrone cycloaddition and their cytotoxicity evaluation

New cis-fused chromeno pyrano[4,3-c]isoxazole derivatives have been synthesized by intramolecular [1,3]-cycloaddition of the nitrones generated in situ from hydroxylamine derivatives and 7-O-prenyl derivatives of 8-formyl-2,3-disubstituted chromenones using PEG-400 as a reaction medium under catalyst-free conditions good to excellent yields. The structures were established by spectroscopic data and further confirmed by X-ray diffraction analysis. The results showed that compounds 4b, 4c, 4d, 4e and 4k exhibit very potent antiproliferative activity against MDA-MB-231 breast cancer cells. Compounds 4a, 4c, 4e, 4i and 4k displayed potent inhibitory activity against human MCF-7 breast cancer cell lines. Compounds 4h and 4i exhibited significant anti-proliferative activity against human cervical cancer cell line, HeLa. While 4b, 4d and 4j were active against human lung cancer cell line, A549. In addition, Compound 4j was found to be the most promising against A549 (Lung cancer) with IC₅₀ value of 0.194 μM.     

Inhibition of stearoyl-CoA desaturase activity by the cis-9,trans-11 isomer and the trans-10,cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells

Conjugated linoleic acid (CLA) is a collective term for a group of positional and geometric conjugated dienoic isomers of linoleic acid. CLA has been shown to have strong inhibitory effects on mammary carcinogenesis both in vitro and in vivo. In this study, we investigated the regulation of human stearoyl-CoA desaturase (SCD, EC 1.14.99.5) expression by CLA in human breast cancer cell lines, MDA-MB-231 and MCF-7. Treatment of the cells with the cis-9,trans-11 and trans-10,cis-12 CLA isomers (45 microM) did not repress SCD mRNA in both MDA-MB-231 and MCF-7 cells. However, the cis-9,trans-11 and trans-10,cis-12 CLA isomers significantly decreased SCD protein levels and SCD activity in MDA-MB-231 cells. In MCF-7 cells, both isomers did not affect protein levels, but they inhibited SCD activity. These results suggest that in MDA-MB-231 cells the cis-9,trans-11 and trans-10,cis-12 CLA isomers regulate human SCD by reducing SCD protein levels, while in MCF-7 cells both isomers have a direct inhibitory effect on SCD enzyme activity.     

Design,synthesis, and biological evaluation of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-Indole-2-Carbohydrazide derivatives: the methuosis inducer 12A as a Novel and selective anticancer agent

This study describes the synthesis and vacuole-inducing activity of 5-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)-1H-indole-2-carbohydrazide derivatives, including five potent derivatives 12c, 12 g, 12i, 12n, and 12A that exhibit excellent vacuole-inducing activity. Remarkably, 12A effectively induces methuosis in tested cancer cells but not human normal cells. In addition, 12A exhibits high pan-cytotoxicity against different cancer cell lines but is hardly toxic to normal cells. It is found that the 12A-induced vacuoles are derived from macropinosomes but not autophagosomes. The 12A-induced cytoplasmic vacuoles may originate from the endoplasmic reticulum (ER) and be accompanied by ER stress. The MAPK/JNK signalling pathway is involved in the 12A-induced methuotic cell death. Moreover, 12A exhibits significant inhibition of tumour growth in the MDA-MB-231 xenograft mouse model. The excellent potency and selectivity of 12A prompt us to select it as a good lead compound for further development of methuosis inducers and investigation of the molecular and cellular mechanisms underlying methuosis.     

Analysis of a novel human gene, LOC92912, over-expressed in hypopharyngeal tumours

We have identified by differential display a number of novel genes that are expressed in hypopharyngeal head and neck squamous cell carcinoma. We report here the characterisation of one of these novel human genes, LOC92912, that encodes a protein of 375 amino acids. The protein contains a RWD domain, a coiled-coil, and an E2 ubiquitin conjugating enzyme domain. LOC92912 is upregulated in about 85% of tumour samples. It is expressed in tumour masses and in invasive epithelium, and is located in the cytoplasm of cells. To gain insights into its functions, we identified potential interacting partners by immunoaffinity purification of the flag tagged protein followed by MALDI peptide mass fingerprinting mass spectrometry. Actin and six actin-binding proteins were unambiguously identified as potential interacting partners, suggesting that LOC92912's functions may be linked with the cytoskeleton. This novel human gene may represent a new target for cancer therapeutics.     

1H, 13C and 15N resonance assignments for the human E2 conjugating enzyme, UbcH7

UbcH7 is a human E2 conjugating enzyme in the ubiquitin-dependent protein degradation pathway. The resonance assignments of UbcH7 will assist in elucidating the structural basis of interactions that occur within ubiquitination.     

Design,synthesis, characterization and in vitro antimicrobial evaluation of 4,6-diaryl-4,5-dihydro-2-phenyl-2H-indazol-3-ols

New 4,6-diaryl-4,5-dihydro-2-phenyl-2H-indazol-3-ols 25-32 were designed, synthesized and in vitro microbially evaluated using clinically isolated bacterial strains viz Staphylococcus aureus, β-Heamolytic streptococcus, Vibreo cholerae, Salmonella typhii, Shigella felxneri and fungal strains viz Aspergillus flavus, Mucor, Rhizopus and Microsporum gypsuem. Results of this study showed that the nature of the substituents on the phenyl rings viz., methyl, methoxy, chloro, nitro as well as the bromo functions at the meta and para positions of the aryl moieties determined the nature and extent of the activity of the fused indazolonol compounds 25-32.     

Design,synthesis and anticancer activity of novel pyrimidine and pyrimidine-thiadiazole hybrid glycosides

Abstract

New 1,3,4-thiadiazole thioglycosides linked to substituted pyrimidines were synthesized via glycosylation of 1,3,4-thiadiazole thiol compounds. Also, novel 1,2,3-triazole derivatives linked to carbohydrate units were prepared using the standard click chemistry conditions employing the Cu(I)-catalyzed azide-alkyne cycloaddition of substituted-aryl-azides with a selection of alkyne-functionalized sugars. The chemical structures of the new derivatives were verified using various spectroscopic techniques, such as IR, ¹H NMR, ¹³C NMR and elemental analyses. The cytotoxic activities of the prepared compounds were investigated in vitro against human liver cancer (HepG-2) and human breast adenocarcinoma (MCF7) cell lines. In addition, the biological evaluation of the new compounds involved the investigation of their effects on a human normal retinal pigmented epithelial cell line (RPE1) using the MTT assay.     

Novel quinoline-3-carboxamides (Part 2): Design,optimization and synthesis of quinoline based scaffold as EGFR inhibitors with potent anticancer activity

EGFR has a key role in cell growth. Its mutation and overexpression share in epithelial malignancies and tumor growth. Quinazoline and quinoline derivatives are common anticancer intracellular inhibitors of EGFR kinase, and their optimization is an important issue for development of potent targeted anticancer agents. Based on these facts, different strategies were used for optimizing our reported quinoline-3-carboxamide compound III (EGFR IC₅₀ = 5.283 µM and MCF-7 IC₅₀ = 3.46 µM) through different molecular modeling techniques. The optimized compounds were synthesized and subjected to EGFR binding assay and accordingly some more potent inhibitors were obtained. The most potent quinoline-3-carboxamides were the furan derivative 5o; thiophene derivative 6b; and benzyloxy derivative 10 showing EGFR IC₅₀ values 2.61, 0.49 and 1.73 μM, respectively. Furthermore, the anticancer activity of compounds eliciting potent EGFR inhibition (5o, 5p, 6b, 8a, 8b, and 10) was evaluated against MCF-7 cell line where they exhibited IC₅₀ values 3.355, 3.647, 5.069, 3.617, 0.839 and 10.85 μM, respectively. Compound 6b was selected as lead structure for further optimization hoping to produce more potent EGFR inhibitors.     

Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2

Ube2g2 is an E2 enzyme which functions as part of the endoplasmic reticulum‐associated degradation (ERAD) pathway responsible for identification and degradation of misfolded proteins in the endoplasmic reticulum. In tandem with a cognate E3 ligase, Ube2g2 assembles K48‐linked polyubiquitin chains and then transfers them to substrate, leading ultimately to proteasomal degradation of the polyubiquitin‐tagged substrate. We report here the solution structure and backbone dynamics of Ube2g2 solved by nuclear magnetic resonance spectroscopy. Although the solution structure agrees well with crystallographic structures for the E2 core, catalytically important loops (encompassing residues 95–107 and 130–135) flanking the active site cysteine are poorly defined. ¹⁵N spin relaxation and residual dipolar coupling analysis directly demonstrates that these two loops are highly dynamic in solution. These results suggest that Ube2g2 requires one or more of its protein partners, such as cognate E3, acceptor ubiquitin substrate or thiolester‐linked donor ubiquitin, to assume its catalytically relevant conformation. Within the NMR structural ensemble, interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99, supporting a possible role for His94 as a general base activated by the carboxylate side‐chains of Asp98 or Asp99. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Synthesis,computational studies and antiproliferative activities of coumarin-tagged 1,3,4-oxadiazole conjugates against MDA-MB-231 and MCF-7 human breast cancer cells

A novel library of coumarin tagged 1,3,4 oxadiazole conjugates was synthesized and evaluated for their antiproliferative activities against MDA-MB-231 and MCF-7 breast cancer cell lines. The evaluation studies revealed that compound 9d was the most potent molecule with an IC₅₀ value of <5?µM against the MCF-7 cell line. Interestingly, compounds 10b and 11a showed a similar trend with lower inhibitory concentration (IC₅₀?=?7.07?µM), in Estrogen Negative (ER?) cells than Estrogen Positive (ER+) cells. Structure–activity relationship (SAR) studies revealed that conjugates bearing benzyl moieties (9b, 9c and 9d) had superior activities compared to their alkyl analogues. The most potent compound 9d showed ～1.4?times more potent activity than tamoxifen against MCF-7 cell line; while the introduction of sulfone unit in compounds 11a, 11b and 11c resulted in significant cytotoxicity against both MCF-7 and MDA-MB-231 cell lines. These results were further supported by docking studies, which revealed that the stronger binding affinity of the synthesized conjugates is due to the presence of sulfone unit attached to the substituted benzyl moiety in their pharmacophores.     

小鼠泛素结合酶UBE2W的抗体制备及组织表达谱分析

泛素结合酶(E2)是蛋白泛素化修饰所需的第二个连接酶, 在泛素转移和底物特异性识别过程中发挥着重要作用。UBE2W是一种新发现的E2酶, 其果蝇属同源蛋白可能在光转导或视网膜变性过程中发挥作用, 鼠和人同源蛋白功能未见报道。生物信息学分析UBE2W鼠源氨基酸序列, 发现UBE2W具有典型的UBC结构域并在多种物种高度保守。通过构建UBE2W原核表达质粒, 在大肠杆菌中表达并纯化了GST-UBE2W融合蛋白。以此纯化蛋白作为抗原免疫新西兰白兔制备抗UBE2W多抗血清, 并利用制备的UBE2W抗原柱亲和纯化UBE2W多抗。为了检测纯化抗体特异性, 在真核细胞中瞬时表达了myc-UBE2W融合蛋白, 分别用myc单抗和UBE2W多抗进行Western blotting分析, 结果表明获得了特异性的UBE2W抗体。利用此特异性抗体在小鼠脑、心脏、肾脏、肝脏、肺、肌肉、脾脏和睾丸等组织中均检测到了UBE2W的表达, 且在小鼠睾丸中成年期表达最高。     

小鼠泛素结合酶UBE2W的抗体制备及组织表达谱分析

泛素结合酶(E2)是蛋白泛素化修饰所需的第二个连接酶, 在泛素转移和底物特异性识别过程中发挥着重要作用。UBE2W是一种新发现的E2酶, 其果蝇属同源蛋白可能在光转导或视网膜变性过程中发挥作用, 鼠和人同源蛋白功能未见报道。生物信息学分析UBE2W鼠源氨基酸序列, 发现UBE2W具有典型的UBC结构域并在多种物种高度保守。通过构建UBE2W原核表达质粒, 在大肠杆菌中表达并纯化了GST-UBE2W融合蛋白。以此纯化蛋白作为抗原免疫新西兰白兔制备抗UBE2W多抗血清, 并利用制备的UBE2W抗原柱亲和纯化UBE2W多抗。为了检测纯化抗体特异性, 在真核细胞中瞬时表达了myc-UBE2W融合蛋白, 分别用myc单抗和UBE2W多抗进行Western blotting分析, 结果表明获得了特异性的UBE2W抗体。利用此特异性抗体在小鼠脑、心脏、肾脏、肝脏、肺、肌肉、脾脏和睾丸等组织中均检测到了UBE2W的表达, 且在小鼠睾丸中成年期表达最高。     

Green synthesis of selenium-N-heterocyclic carbene compounds: Evaluation of antimicrobial and anticancer potential

Three benzimidazolium salts (III-V) and respective selenium adducts (VI-VIII) were designed, synthesized and characterized by various analytical techniques (FT-IR and NMR ¹H, ¹³C). Selected salts and respective selenium N-Heterocyclic carbenes (selenium-NHC) adducts were tested in vitro against Cervical Cancer Cell line (Hela), Breast Adenocarcinoma cell line (MCF-7), Retinal Ganglion Cell line (RGC-5) and Mouse Melanoma Cell line (B16F10) using MTT assay and the results were compared with standard drug 5-Fluorouracil. Se-NHC compounds and azolium salts showed significant anticancer potential. Molecular docking studies of compounds (VI, VII and VIII) showed strong binding energies and ligand affinity toward following angiogenic factors: VEGF-A (vascular endothelial growth factor A), EGF (human epidermal growth factor), HIF (Hypoxia-inducible factor) and COX-1 (Cyclooxygenase-1) suggesting that the anticancer activity of adducts (VI, VII and VIII) may be due to their strong anti-angiogenic effect. In addition, compounds III-VIII were screened for their antibacterial and antifungal potential. Adduct VI was found to be potent anti-fungal agent against A. Niger with zone of inhibition (ZI) value 27.01 ± 0.251 mm which is better than standard drug Clotrimazole tested in parallel.     

Design,synthesis, spectral analysis and in vitro microbiological evaluation of 2-phenyl-3-(4,6-diarylpyrimidin-2-yl)thiazolidin-4-ones

A series of novel 2-phenyl-3-(4,6-diarylpyrimidin-2-yl)thiazolidin-4-ones 23-33 were synthesized, and studied for their in vitro antibacterial and antifungal activities against clinically isolated strains. Generally compounds possessing electron donating groups showed good antibacterial activity. Compound 31, which contain both electron withdrawing chloro and electron donating methyl groups showed potent activity against all the tested Gram positive and Gram negative bacterial strains whereas compounds 32 and 33 which contain electron donating methoxy functional group at the para position of the phenyl ring attached to pyrimidine ring showed promising activity against S.aureus, S.typhii and E.coli. Compounds 32 and 33, both containing electron withdrawing groups (-Cl, -F) showed excellent activities against all the tested A. flavus, Mucor, Rhizopus and M.gypsuem fungal strains. while against Mucor, compound 27 which contains an electron donating methyl group at the para position of the phenyl ring attached to pyrimidine ring showed promising activity. Also compound 31, which contains both electron withdrawing chloro and electron donating methyl groups showed potent activity against A. flavus and Rhizopus.     

Copper modulates activities of genistein, nitric oxide, and curcumin in breast tumor cells

Several papers have reported that low level of genistein (<8 microM), the major bioactive component of isoflavones, stimulates the growth of MCF-7 cells. In the present study, we found that genistein-induced growth stimulation of MCF-7 cells is inhibited in the presence of Cu(2+) (5 microM). Genistein induces the release of nitric oxide in MCF-7 cells in a concentration-dependent manner. The release of nitric oxide was inhibited by N(G)-nitro-L-arginine methyl ester, suggesting the possibility of the activation of nitric oxide synthase. The growth of MCF-7 cells also increases in the presence of low levels of sodium nitriprusside (<10 microM), a nitric oxide donor compound, while high levels (>25 microM) are toxic. The sodium nitroprusside-induced growth of MCF-7 cells is drastically suppressed in the presence of Cu(2+) (5 microM). This parallel behavior between Cu(2+)-genistein and Cu(2+)-sodium nitroprusside mixtures suggests that Cu(2+) and/or copper-protein complexes, that may be formed in the media, may be reacting with nitric oxide or nitric oxide-derived reactive species. The products of these reactions may be responsible for the toxic effects of these mixtures. In contrast, the effect of curcumin that inhibits the growth of both estrogen receptor-positive and -negative breast tumor cells appreciably decreased in the presence of Cu(2+). Since copper is known to overwhelmingly bind with proteins, present data suggest that an increase in copper-protein moieties or complexes formed in the serum containing media and their reactions with nitric oxide may be responsible for their toxic effects. Further studies are needed to characterize these reactions.     

Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application

The Eph family of receptors, with 14 members in humans, makes up the largest group of receptor tyrosine kinases. These Eph receptors, along with their ligands, the 8 members of the ephrin family of ligands are involved in diverse developmental functions, including hindbrain development in vertebrates, tissue patterning, and angiogenesis. These Eph receptors and ephrin ligands have also been identified as important regulators in the development and progression of cancer. We have presented here a systematic and comprehensive investigation of the Eph/ephrin expression profiles of MCF-10A, MCF-7, and MDA-MB-231 cells representing normal breast, non-invasive breast tumor, and invasive tumor, respectively, based on their characteristic phenotypes in Matrigel matrix. The data have allowed us to correlate the gene expression profile with the cell phenotype that has potential application in tumor diagnostics. We demonstrate here that upregulation of EphA2, A7, A10, and ephrinA2 and B3 is likely involved in tumorigenesis and/or invasiveness, while downregulation of EphA1, A3, A4, A8, B3, B4, B6, and ephrinA1 and B1 may be particularly important in invasiveness. Based on these results we discuss the role of EphA2 and ephrinA1 combination in malignancy. The data have provided clues as to the importance of these molecules in the progression of breast cancer and specifically identified EphB6, a kinase-deficient receptor, which is downregulated in the most aggressive cell line, as reported for several other cancer types including neuroblastoma and melanoma suggesting its potential as a prognostic indicator in breast cancer as well.