A novel metformin derivative,HL010183, inhibits proliferation and invasion of triple-negative breast cancer cells

Mounting evidence suggests that metformin (N,N-dimethylbiguanide), a widely prescribed drug for the treatment of type II diabetes, exerts an anti-tumor effect on several cancers including breast cancer. Breast cancer has been estimated as one of the most commonly diagnosed types of cancer among women. In particular, triple-negative breast cancers are associated with poor prognosis and metastatic growth. In the present study, we synthesized a novel metformin derivative 5 (HL010183) and metformin salts, 9a, 9b, and 9c (metformin gamma-aminobutyric acid (GABA) salt, metformin pregabalin salt and metformin gabapentin salt), which exerted more potent inhibitory effects on the proliferation and invasiveness of Hs578T triple-negative breast carcinoma cells than metformin. Importantly, 5 showed approximately 100-fold more potent effects compared to metformin. In a triple-negative breast cancer xenograft model, 5 showed a comparable degree of inhibitory effect on in vivo tumor growth at the 100 mg/kg dose to that of metformin at 500 mg/kg. Our results clearly demonstrate that 5 exerts a potent anti-tumor effect both in vitro and in vivo, paving the way for a strategy for treatment of triple-negative breast cancer.     

Crystal structure of hexakis(2,6-di-O-methyl)-alpha-cyclodextrin-acetonitrile dihydrate: a channel formed by methyl groups harbors a chain of five partially occupied water sites

Hexakis(2,6-di-O-methyl)-alpha-cyclodextrin (DIMEA) crystallizes from 1:1 water-acetonitrile as DIMEA-acetonetril-dihydrate in the orthorhombic space group P2(1)2(1)2(1), unit cell dimensions a = 14.2775(5), b = 15.7312(5), c = 31.1494(11) A. Refinement of the structure against 5540 X-ray diffraction data converged at an R factor of 0.083. The macrocycle exhibits a 'round' conformation and is stabilized by intramolecular, interglucose O-3-H(n)...O-2(n + 1) and C-6-H(n)...O-5(n + 1) hydrogen bonds. Acetonitrile is included in the central cavity of DIMEA and held in position by C-5-H...N interactions. The two water molecules in the asymmetric units are distributed over six sites. One is fully occupied due to hydrogen bonding to O-3 groups of two symmetry-related DIMEA molecules, whereas the five remaining sites show occupancies between 0.15 and 0.25. These sites are in hydrogen bonding contact with O...O distances between 2.59 and 3.50 A and are located in infinite, hydrophobic channels parallel to the alpha-axis, which are coated with methyl groups of symmetry-related DIMEA.     

Oral mucosal lesions in denture wearers

doi:10.1111/j.1741‐2358.2009.00289.x
Oral mucosal lesions in denture wearers Objective: To determine the prevalence of oral mucosal lesions (OMLs) and denture‐related mucosal lesions (DMLs) in denture wearers and to co‐relate the prevalence with age, gender, type of denture and any systemic conditions. Materials and methods: Dental records of 380 denture wearers were retrospectively reviewed for OMLs and DMLs. Results: We found 45% of the denture wearers had DMLs and 60.8% had OMLs not related to denture wearing. Although the prevalence of DMLs was higher in complete denture wearers than in partial denture wearers (49% vs. 42.2%), this difference was not significant. The most common DMLs were traumatic ulcer (19.5%) and denture‐induced stomatitis (18.1%). When analysed by type, traumatic ulcer, denture hyperplasia, frictional keratosis and candidiasis were more common in complete denture wearers, whereas denture‐induced stomatitis was more common in partial denture wearers. Frictional keratosis was more common in men than in women. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers, and the most common OML was fissured tongue (27.6%). No association between DMLs and systemic conditions or xerostomic drugs was noted. Conclusion: No differences in the prevalence of DMLs in association with denture type were found. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers. This difference was affected by age, and the data were similar to the findings observed in the elderly.     

Pectobacterium carotovorum elicits plant cell death with DspE/F but the P. carotovorum DspE does not suppress callose or induce expression of plant genes early in plant-microbe interactions

The broad-host-range bacterial soft rot pathogen Pectobacterium carotovorum causes a DspE/F-dependent plant cell death on Nicotiana benthamiana within 24 h postinoculation (hpi) followed by leaf maceration within 48 hpi. P. carotovorum strains with mutations in type III secretion system (T3SS) regulatory and structural genes, including the dspE/F operon, did not cause hypersensitive response (HR)-like cell death and or leaf maceration. A strain with a mutation in the type II secretion system caused HR-like plant cell death but no maceration. P. carotovorum was unable to impede callose deposition in N. benthamiana leaves, suggesting that P. carotovorum does not suppress this basal immunity function. Within 24 hpi, there was callose deposition along leaf veins and examination showed that the pathogen cells were localized along the veins. To further examine HR-like plant cell death induced by P. carotovorum, gene expression profiles in N. benthamiana leaves inoculated with wild-type and mutant P. carotovorum and Pseudomonas syringae strains were compared. The N. benthamiana gene expression profile of leaves infiltrated with Pectobacterium carotovorum was similar to leaves infiltrated with a Pseudomonas syringae T3SS mutant. These data support a model where Pectobacterium carotovorum uses the T3SS to induce plant cell death in order to promote leaf maceration rather than to suppress plant immunity.     

H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells

Human tumors frequently exhibit constitutively activated Ras signaling, which contributes to the malignant phenotype. Mounting evidence suggests unique roles of the Ras family members, H-Ras, N-Ras and K-Ras, in normal and pathological conditions. In an effort to dissect distinct Ras isoform-specific functions in malignant phenotypic changes, we previously established H-Ras- and N-Ras-activated MCF10A human breast epithelial cell lines. Using these, we showed that p38 kinase is a key signaling molecule differentially regulated between H-Ras and N-Ras, leading to H-Ras-specific induction of invasive and migrative phenotypes. The present study is to further investigate H-Ras- and N-Ras-mediated signaling pathways and to unveil how these pathways are integrated for regulation of invasive/migrative phenotypic conversion of human breast epithelial cells. Here we report that the Rac-MAPK kinase (MKK)3/6-p38 pathway is a unique signaling pathway activated by H-Ras, leading to the invasive/migrative phenotype. In contrast, Raf-MEK-ERK and phosphatidylinositol 3-kinase-Akt pathways, which are fundamental to proliferation and differentiation, are activated by both H-Ras and N-Ras. A significant role for p38 in cell invasion is further supported by the observation that p38 activation by MKK6 transfection is sufficient to induce invasive and migrative phenotypes in MCF10A cells. Activation of the MKK6-p38 pathway results in a marked induction of matrix metalloproteinase (MMP)-2, whereas it had little effect on MMP-9, suggesting MMP-2 up-regulation by MKK6-p38 pathway as a key step for H-Ras-induced invasion and migration. We also provide evidence for cross-talk among the Rac, Raf, and phosphatidylinositol 3-kinase pathways critical for regulation of MMP-2 and MMP-9 expression and invasive phenotype. Taken together, the present study elucidated the role of the Rac-MKK3/6-p38 pathway leading to H-Ras-specific induction of malignant progression in breast epithelial cells, providing implications for developing therapeutic strategies for mammary carcinoma to target Ras downstream signaling molecules required for malignant cancer cell behavior but less critical for normal cell functions.     

Biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source

Biosurfactant production by Pseudomonas aeruginosa A41, a strain isolated from seawater in the gulf of Thailand, was examined when grown in defined medium containing 2% vegetable oil or fatty acid as a carbon source in the presence of vitamins, trace elements and 0.4% NH(4)NO(3), at pH 7 and 30 degrees C with 200 rpm-shaking for 7 days. The yield of biosurfactant steadily increased even after a stationary phase. Under such conditions the surface tension of the medium was lowered from 55-70 mN/m to 27.8-30 mN/m with every carbon source tested. However, types of carbon sources were found to affect biosurfactant yield. The yields of rhamnolipid biosurfactant were 6.58 g/L, 2.91 g/L and 2.93 g/L determined as rhamnose content when olive oil, palm oil and coconut oil, respectively, were used as a carbon source. Among them, biosurfactant obtained from palm oil was the best in lowering surface tension of the medium. Increase in biosurfactant activities in terms of oil displacement test and rhamnose content were observed to be higher with shorter chain fatty acids than that of the longer chains (C12>C14>C16). In addition, we found that C18:2, highly unsaturated fatty acid, showed higher oil displacement activity and rhamnose content than that of C18:1. The optimal oil displacement activity was found at pH 7-9 and in the presence of 0.5-3% NaCl. The oil displacement activity was stable to temperatures up to 100 degrees C for 15 h. Surface tension reduction activity was relatively stable at pH 2-12 and 0-5% of NaCl. Emusification activity tested with various types of hydrocarbons and vegetable oils showed similarity of up to 60% stability. The partially purified biosurfactant via TLC and silica gel column chromatography gave three main peaks on HPLC with mass spectra of 527, 272, and 661 m/z respectively, corresponding to sodium-monorhamnodecanoate, hydroxyhexadecanoic acid and an unknown compound, respectively.     

Polymorphism in beta-cyclodextrin-benzoic acid inclusion complex: a kinetically controlled crystal growth according to the Ostwald's rule

Crystal form II of the beta-cyclodextrin-benzoic acid (beta-CD-BA) inclusion complex was obtained from the 1.5-year stored aqueous EtOH solution of beta-CD and BA as 2beta-CD.1.5BA.0.7EtOH.21H(2)O in the monoclinic space group C2 with unit cell constants: a=19.413(3), b=24.306(4), c=32.975(1)A, beta=104.41(1) degrees . By contrast, the desired crystal form I in the triclinic space group P1 that ever grew up from the fresh solution as 2beta-CD.2BA.0.7EtOH.20.65H(2)O was not reproducible any more [Aree, T.; Chaichit, N. Carbohydr. Res.2003, 338, 439-446]. In the two crystal forms, beta-CDs are isostructural with a 'round' conformation stabilized by intramolecular O-3(n)cdots, three dots, centeredO-2(n+1) hydrogen bonds. The BA inclusion geometries are similar with a preferred orientation, that is, BAs are situated above the O-4 plane, point their COOH groups to the beta-CD O-6 side, incline 52 degrees with respect to the O-4 plane and are mainly maintained in positions by hydrogen bonding with the surrounding water molecules. beta-CDs form dimers as structural motif of different packing modes: the screw-channel type in form II and the average of intermediate and tetrad types in form I. Polymorphism in the beta-CD-BA inclusion complex is a kinetically controlled crystal growth following the Ostwald's rule: the less stable crystal form I grew up first within one week from the fresh solution, whereas the more stable crystal form II appeared after 1.5-year storage.     

Plasma and synovial fluid inflammatory cytokine profiles in primary knee osteoarthritis

Objective: The objective of this study is to compare inflammatory cytokine levels in primary knee osteoarthritis (OA) patients and healthy controls.

Methods: A total of 32 knee OA patients and 14 healthy controls were enrolled. A multiplex immunoassay was utilized for 10 cytokines in plasma and synovial fluid.

Results: Plasma IL-2, IL-4, and IL-6 concentrations were significantly greater in knee OA patients than controls. Moreover, both plasma IL-4 and IL-6 were positively correlated with the radiographic severity of knee OA.

Conclusions: Plasma IL-4 and IL-6 may serve as biomarkers reflecting the severity of OA.     

Global gene expression profiling unveils S100A8/A9 as candidate markers in H-ras-mediated human breast epithelial cell invasion

The goal of the present study is to unveil the gene expression profile specific to the biological processes of human breast epithelial cell invasion and migration using an MCF10A model genetically engineered to constitutively activate the H-ras or N-ras signaling pathway. We previously showed that H-Ras, but not N-Ras, induces MCF10A cell invasion/migration, whereas both H-Ras and N-Ras induce cell proliferation and phenotypic transformation. Thus, these cell lines provide an experimental system to separate the gene expression profile associated with cell invasion apart from cell proliferation/transformation. Analysis of whole human genome microarray revealed that 412 genes were differentially expressed among MCF10A, N-Ras MCF10A, and H-Ras MCF10A cells and hierarchical clustering separated 412 genes into four clusters. We then tested whether S100A8 and S100A9, two of the genes which are most highly up-regulated in an H-Ras-specific manner, play a causative role for H-Ras-mediated MCF10A cell invasion and migration. Importantly, small interfering RNA-mediated knockdown of S100A8/A9 expression significantly reduced H-Ras-induced invasion/migration. Conversely, the induction of S100A8/A9 expression conferred the invasive/migratory phenotype to parental MCF10A cells. Furthermore, we provided evidence of signaling cross-talk between S100A8/A9 and the mitogen-activated protein kinase signaling pathways essential for H-Ras-mediated cell invasion and migration. Taken together, this study revealed S100A8/A9 genes as candidate markers for metastatic potential of breast epithelial cells. Our gene profile data provide useful information which may lead to the identification of additional potential targets for the prognosis and/or therapy of metastatic breast cancer.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.