Acute Toxicity and Gastroprotection Studies of a New Schiff Base Derived Copper (II) Complex against Ethanol-Induced Acute Gastric Lesions in Rats

Background

Copper is an essential element in various metabolisms. The investigation was carried out to evaluate acute gastroprotective effects of the Copper (II) complex against ethanol-induced superficial hemorrhagic mucosal lesions in rats.

Methodology/Principal Findings

Rats were divided into 7 groups. Groups 1 and 2 were orally administered with Tween 20 (10% v/v). Group 3 was orally administered with 20 mg/kg omeprazole (10% Tween 20). Groups 4–7 received 10, 20, 40, and 80 mg/kg of the complex (10% Tween 20), respectively. Tween 20 (10% v/v) was given orally to group 1 and absolute ethanol was given orally to groups 2–7, respectively. Rats were sacrificed after 1 h. Group 2 exhibited severe superficial hemorrhagic mucosal lesions. Gastric wall mucus was significantly preserved by the pre-treatment complex. The results showed a significant increase in glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), and Prostaglandin E2 (PGE₂) activities and a decrease in malondialdehyde (MDA) level. Histology showed marked reduction of hemorrhagic mucosal lesions in groups 4–7. Immunohistochemical staining showed up-regulation of Hsp70 and down-regulation of Bax proteins. PAS staining of groups 4–7 showed intense stain uptake of gastric mucosa. The acute toxicity revealed the non-toxic nature of the compound.

Conclusions/Significance

The gastroprotective effect of the Copper (II) complex may possibly be due to preservation of gastric wall mucus; increase in PGE₂ synthesis; GSH, SOD, and NO up-regulation of Hsp70 protein; decrease in MDA level; and down-regulation of Bax protein.     

Establishment and characterization of two human breast carcinoma cell lines by spontaneous immortalization: Discordance between Estrogen, Progesterone and HER2/neu receptors of breast carcinoma tissues with derived cell lines

Background

Breast cancer is one of the most common cancers among women throughout the world. Therefore, established cell lines are widely used as in vitro experimental models in cancer research.

Methods

Two continuous human breast cell lines, designated MBC1 and MBC2, were successfully established and characterized from invasive ductal breast carcinoma tissues of Malaysian patients. MBC1 and MBC2 have been characterized in terms of morphology analysis, population doubling time, clonogenic formation, wound healing assay, invasion assay, cell cycle, DNA profiling, fluorescence immunocytochemistry, Western blotting and karyotyping.

Results

MBC1 and MBC2 exhibited adherent monolayer epithelial morphology at a passage number of 150. Receptor status of MBC1 and MBC2 show (ER⁺, PR⁺, HER2⁺) and (ER⁺, PR^-, HER2⁺), respectively. These results are in discordance with histopathological studies of the tumoral tissues, which were triple negative and (ER^-, PR^-, HER2⁺) for MBC1 and MBC2, respectively. Both cell lines were capable of growing in soft agar culture, which suggests their metastatic potential. The MBC1 and MBC2 metaphase spreads showed an abnormal karyotype, including hyperdiploidy and complex rearrangements with modes of 52–58 chromosomes per cell.

Conclusions

Loss or gain in secondary properties, deregulation and specific genetic changes possibly conferred receptor changes during the culturing of tumoral cells. Thus, we hypothesize that, among heterogenous tumoral cells, only a small minority of ER⁺/PR⁺/HER2⁺ and ER⁺/PR^-/HER2⁺ cells with lower energy metabolism might survive and adjust easily to in vitro conditions. These cell lines will pave the way for new perspectives in genetic and biological investigations, drug resistance and chemotherapy studies, and would serve as prototype models in Malaysian breast carcinogenesis investigations.     

Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity

Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.     

Protective effects of Helicobacter pylori against gastroesophageal reflux disease may be due to a neuroimmunological anti-inflammatory mechanism

There is some evidence that Helicobacter pylori infection has a protective effect against gastroesophageal reflux disease (GORD) and its complications such as Barrett's oesophagus and oesophageal adenocarcinoma. In this paper, we propose that a neuroimmunological mechanism is responsible for the protective effect of H. pylori on GORD. H. pylori infection of the gastric mucosa induces a T helper1-like immune response and production of pro-inflammatory cytokines. These cytokines can inhibit local sympathetic tone, whereas they increase systemic sympathetic tone. Increased sympathetic tone can induce an anti-inflammatory milieu, which in turn can inhibit inflammation in the oesophagus and lower oesophageal sphincter (LOS). Furthermore, H. pylori infection may stimulate the cholinergic anti-inflammatory pathway. It has been suggested that reflux-induced oesophageal inflammation plays an important role in the pathogenesis of reflux oesophagitis. Reduction of oesophageal inflammation by increased systemic sympathetic tone and vagal activity may lead to a decrease in reflux-induced oesophageal injury and LOS dysfunction in GORD.     

Tissue invasion by Entamoeba histolytica: evidence of genetic selection and/or DNA reorganization events in organ tropism

Entamoeba histolytica infection may have various clinical manifestations. Nine out of ten E. histolytica infections remain asymptomatic, while the remainder become invasive and cause disease. The most common form of invasive infection is amebic diarrhea and colitis, whereas the most common extra-intestinal disease is amebic liver abscess. The underlying reasons for the different outcomes are unclear, but a recent study has shown that the parasite genotype is a contributor. To investigate this link further we have examined the genotypes of E. histolytica in stool- and liver abscess-derived samples from the same patients. Analysis of all 18 paired samples (16 from Bangladesh, one from the United States of America, and one from Italy) revealed that the intestinal and liver abscess amebae are genetically distinct. The results suggest either that E. histolytica subpopulations in the same infection show varying organ tropism, or that a DNA reorganization event takes place prior to or during metastasis from intestine to liver.     

Bio-stretch, a computerized cell strain apparatus for three-dimensional organotypic cultures

Summary In the present study, a unique mechanical strain apparatus for three-dimensional organotypic cultures was developed into a computerized system. It consists of a personal computer running Windows-based software, the Bio-Stretch Manager, a Bio-Stretch Controller, and three sets of magnet boards. Cells are cultured on a Gelfoam? sponge that is placed in a 35-mm petri dish with one end glued to the dish, and the other end attached to a coated steel bar. The petri dish is placed in front of a magnet, and the movement of the steel bar is controlled by dynamically changing the magnetic field. Up to five stretch patterns of variable frequency, duty cycle, and magnitude can be designed for each stretch regimen. Three different stretch regimens can be tested simultaneously. The operational characteristics of sponges were examined. Attachment of cells to the sponges was observed on several cell types. These features provide wide options for using this system to study the effects of mechanical stretch on cells.     

PKB mediates c-erbB2-induced epithelial beta1 integrin conformational inactivation through Rho-independent F-actin rearrangements

Signalling from the growth factor receptor subunit and proto-oncogene c-erbB2 has been shown to inhibit the adhesive function of the collagen receptor integrin alpha(2)beta(1) in human mammary epithelial cells. This anti-adhesive effect is mediated by the MAP ERK kinase 1/2 (MEK1/2) and protein kinase B (PKB) pathways. Here, we show that both pathways mediate suppression of matrix adhesion by causing the extracellular domain of the beta(1) integrin subunit to adopt an inactive conformation. The conformational switch was also dependent on rapid and extensive actin depolymerisation. While neither activation nor inhibition of the Rho GTPase affected this rearrangement, Rho was found to be activated by c-erbB2 and to be necessary for conformation-dependent integrin inactivation and, apparently by a different mechanism, a delayed re-formation of stress fibers which did not restore integrin function. Interestingly, the initial actin depolymerisation as well as its effects on integrin function was shown to be mediated by PKB. These results demonstrate how oncogenic growth factor signalling inhibits matrix adhesion by multiple pathways converging on integrin conformation and how Rho signalling can profoundly influence integrin activation in a cytoskeleton-independent manner.     

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.