Malignant reversion of a human osteosarcoma cell line,Saos-2, by inhibition of NFkappaB

Beyond a pivotal role in neoplastic transformation and malignant progression, NFkappaB is intricately involved in bone biology, pointed up by the osteopetrotic phenotype of NFkappaB (p50-p52) double knock-out mice. Osteopetrosis results from intrinsic defects in osteoclastogenesis, loss of osteoclast bone resorptive activity and, questionably, increased osteoblast activity (bone matrix apposition and mineralization). We here report that inhibition of NFkappaB signaling activity in Saos-2 cells results in a marked decrease in cellular proliferation, assessed by the incorporation of radioactive thymidine into cellular DNA. Decreased cellular proliferation was accompanied by the induction of bone morphogenic proteins (BMP) 4, 7, and the osteoblast specific transciption factor, Cbfa1, heralding osteoblast differentiation, given the induction of alkaline phosphatase, osteopontin, and osteocalcin message levels and the attendant increase in matrix deposition and mineralization in vitro. These results point to the negative regulation of osteoblast differentiation by NFkappaB, with implications in the pathogenesis and progression of osteosarcomas.     

Vitamin C and E supplements to lansoprazole-amoxicillin-metronidazole triple therapy may reduce the eradication rate of metronidazole-susceptible Helicobacter pylori infection

Aim. To test whether vitamin C and E supplements to triple therapy can improve the Helicobacter pylori eradication rate and gastric inflammation. Methods. A total of 104 H. pylori‐infected patients were randomized to receive: either lansoprazole, amoxicillin, and metronidazole twice daily for 1 week (triple‐only group) or lansoprazole, amoxicillin, metronidazole plus vitamin C (250 mg) and vitamin E (200 mg) twice daily for 1 week, followed immediately by vitamin C and E once daily for 6 consecutive weeks (triple‐plus‐vitamin group). Eight weeks after the completion of triple therapy, patients were assessed for the effectiveness of H. pylori eradication. The severity of gastric inflammation in histology was assessed for the acute and chronic inflammation scores. Results. Intention‐to‐treat and per‐protocol eradication rates were 59.1% and 64.4% in the triple‐only group, and 40% and 44% in the triple‐plus‐vitamin group. In the patients infected with metronidazole susceptible isolates, the triple‐only group had a higher intention‐to‐treat eradication rate than those in the triple‐plus‐vitamin group (80% vs. 53.1%, p < .01). However, for the metronidazole resistance isolates, the intention‐to‐treat eradication rates between the two groups were not different (26.3% vs. 21.7%, p = NS). The improvements of both acute and chronic inflammation scores in histology were not different between the two groups. Conclusion. Adding vitamin C and E to triple therapy cannot improve the H. pylori eradication rate and gastric inflammation. For patients with metronidazole susceptible strain infection, adding these vitamins may even reduce the eradication rate of triple therapy.     

Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin

Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.     

Syntaxin-1A binds the nucleotide-binding folds of sulphonylurea receptor 1 to regulate the KATP channel

ATP-sensitive potassium (KATP) channels in neuron and neuroendocrine cells consist of a pore-forming Kir6.2 and regulatory sulfonylurea receptor (SUR1) subunits, which are regulated by ATP and ADP. SNARE protein syntaxin 1A (Syn-1A) is known to mediate exocytic fusion, and more recently, to also bind and modulate membrane-repolarizing voltage-gated K+ channels. Here we show that Syn-1A acts as an endogenous regulator of KATP channels capable of closing these channels when cytosolic ATP concentrations were lowered. Botulinum neurotoxin C1 cleavage of endogenous Syn-1A in insulinoma HIT-T15 cells resulted in the increase in KATP currents, which could be subsequently inhibited by recombinant Syn-1A. Whereas Syn-1A binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1, the functional inhibition of KATP channels in rat islet beta-cells by Syn-1A seems to be mediated primarily by its interactions with NBF-1. These inhibitory actions of Syn-1A can be reversed by physiologic concentrations of ADP and by diazoxide. Syn-1A therefore acts to fine-tune the regulation of KATP channels during dynamic changes in cytosolic ATP and ADP concentrations. These actions of Syn-1A on KATP channels contribute to the role of Syn-1A in coordinating the sequence of ionic and exocytic events leading to secretion.     

Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB protease inhibitors (l-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKalpha/beta and kappaB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85alpha and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.     

Production of xylitol from Candida tropicalis by using an oxidation-reduction potential-stat controlled fermentation

An on-line device, ORP (oxidation-reduction potential)-stat, was used to control glucose-feeding for enhancing xylitol conversion from D-xylose during an oxygen-limited fermentation by Candida tropicalis. The fermentation was carried out in a 5 l jar fermenter. After glucose in the medium was depleted, a switching to a limited aeration and feeding glucose controlled by ORP-stat was performed. The maximum xylitol yield was obtained under a condition at an ORP of - 180 mV and at an aeration rate of 0.2 l min(-1).     

Production of the chemokine eotaxin-1 in osteoarthritis and its role in cartilage degradation

The expression of the chemokine, eotaxin-1, and its receptors in normal and osteoarthritic human chondrocytes was examined, and its role in cartilage degradation was elucidated in this study. Results indicated that plasma concentrations of eotaxin-1 as well as the chemokines, RANTES, and MCP-1alpha, were higher in patients with osteoarthritis (OA) than those in normal humans. Stimulation of chondrocytes with IL-1beta or TNF-alpha significantly induced eotaxin-1 expression. The production of eotaxin-1 induced expression of its own receptor of CCR3 and CCR5 on the cell surface of chondrosarcomas, suggesting that an autocrine/paracrine pathway is involved in eotaxin-1's action. In addition, eotaxin-1 markedly increased the expressions of MMP-3 and MMP-13 mRNA, but had no effect on TIMP-1 expression in chondrocytes. However, pretreatment of anti-eotaxin-1 antibody significantly decreased the MMP-3 expression induced by IL-1beta. These results first demonstrate that human chondrocytes express the chemokine, eotaxin-1, and that its expression is induced by treatment with IL-1beta and TNF-alpha. The cytokine-triggered induction of eotaxin-1 further results in enhanced expressions of its own receptor of CCR3, CCR5, and MMPs, suggesting that eotaxin-1 plays an important role in cartilage degradation in OA.     

Mechanisms involved in the antiplatelet activity of ketamine in human platelets

The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100–350 µM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca²⁺ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 µM) significantly inhibited thromboxane (Tx) A₂ formation stimulated by collagen. Moreover, ketamine (200 and 350 µM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein ofMr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 µM). These results indicate that the antiplatelet activity of ketamine may be involved in the following pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca²⁺ mobilization and TxA₂ formation, ultimately resulting in inhibition of platelet aggregation.     

Expression of estrogen receptor-α and Ki67 in relation to pathological and molecular features in early-onset infiltrating ductal carcinoma

Estrogen causes breast cancer by triggering proliferation via an estrogen receptor (ER)-mediated mechanism. However, paradoxically, ER, one of the two known ER subtypes, and the proliferation marker, Ki67, are not usually expressed in the same breast tumor. To explore whether ER-positive tumors and proliferating (Ki67-positive) tumors have different tumorigenic characteristics, we performed an immunohistochemical study on 74 early-onset infiltrating ductal carcinomas of the breast. To test this hypothesis, we examined whether ER-positive and Ki67-positive tumors showed differences in (i) pathological grade, (ii) three indices of tumor grade (tubule formation, nuclear pleomorphism, and mitotic number), and (iii) expression of important proteins implicated in breast tumorigenesis (cyclin D1, ErbB2, ATM, BRCA1, Rb, p53, and p21). The results of the multigenic analysis showed that ER and Ki67 were the only two important markers significantly and independently associated with tumor grade, consistent with the above hypothesis. ER-positive, Ki67-negative tumors frequently displayed a low tumor grade (i.e. being well differentiated), whereas Ki67-positive, ER-negative tumors were more likely to exhibit a high tumor grade. In addition, positive ER expression (46 of 74 cases, 62%) correlated well with positive cyclin D1 expression (p<0.005), less nuclear pleomorphism (p<0.001), and a low mitotic count (p < 0.005), whereas positive Ki67 expression (36 of 74 cases, 49%) correlated with reduced BRCA1 expression (p < 0.01) and high mitotic activity (p<0.01). These findings suggest that the expressions of ER and Ki67 might be involved in distinct pathological and molecular features during breast cancer development.     

Predominant Th2/Tc2 polarity of tumor-infiltrating lymphocytes in human cervical cancer

Cytotoxic T lymphocytes (Tc) play a central role in cellular immunity against cancers. The cytotoxic potential of freshly isolated tumor-infiltrating lymphocytes (TILs) is usually not expressed. This suggests the possible existence of as yet unspecified and perhaps complex immunosuppressive factors or cytokines that affect the anti-tumor capacity of these TILs in the tumor milieu. In the present study, we demonstrated for the first time that TILs derived from human cervical cancer tissue consist mainly of Th2/Tc2 phenotypes. In vitro kinetic assays further revealed that cancer cells could direct the tumor-encountered T cells toward the Th2/Tc2 polarity. Cancer cells promote the production of IL-4 and down-regulate the production of IFN-gamma in cancer-encountered T cells. The regulatory effects of cervical cancer cells are mediated mainly by IL-10, and TGF-beta plays only a synergistic role. The cancer-derived effects can be reversed by neutralizing anti-IL-10 and anti-TGF-beta Abs. IL-10 and TGF-beta are present in cancer tissue and weakly expressed in precancerous tissue, but not in normal cervical epithelial cells. Our study strongly suggests important regulatory roles of IL-10 and TGF-beta in cancer-mediated immunosuppression.