Quercetin decreases the expression of ErbB2 and ErbB3 proteins in HT-29 human colon cancer cells

Quercetin has chemoprotective properties in experimental colon cancer models, and in vitro studies have demonstrated that quercetin inhibits HT-29 colon cancer cell growth. ErbB2 and ErbB3 receptor tyrosine kinases have been associated with the development of human colon cancer, and the expressions of both receptors are high in HT-29 cells. In this study, we assessed quercetin regulation of HT-29 and SW480 cell apoptosis and the influence of quercetin on the protein expression of ErbB2, ErbB3, Akt, Bax and Bcl-2. We cultured HT-29 cells in the presence of various concentrations (0, 25, 50, or 100 micromol/L) of quercetin or rutin. Quercetin inhibited HT-29 cell growth in a dose-dependent manner, whereas rutin had no effect on the cell growth. DNA that was isolated from cells treated with 50 micromol/L of quercetin exhibited an oliogonucleosomal laddering pattern characteristic of apoptotic cell death. Western blot analysis of cell lysates revealed that Bcl-2 levels decreased dose-dependently in cells treated with quercetin, but Bax remained unchanged. Quercetin increased levels of cleaved caspase-3 and the 89-kDa fragment of poly (ADP-ribose) polymerase. In addition, phosphorylated Akt levels were markedly lower in cells treated with 25 micromol/L quercetin, but total Akt levels decreased only at 100 micromol/L quercetin. Furthermore, a dose-dependent decrease in ErbB2 and ErbB3 levels was detected in quercetin-treated cells. The results obtained using SW480 cells were similar to those obtained with HT-29 cells. In conclusion, we have shown that quercetin inhibits cell growth and induces apoptosis in colon cancer cells, and that this may be mediated by its ability to down-regulate ErbB2/ErbB3 signaling and the Akt pathway.     

Synthesis and antifungal activity of noble 5-arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles

5-Arylamino- and 6-arylthio-4,7-dioxobenzoselenazoles 4 and 5 were synthesized and tested for in vitro antifungal activity against Candida and Aspergillus species. 5-Arylamino-4,7-dioxobenzoselenazoles 4 showed, in general, more potent antifungal activity than 6-arylthio-4,7-dioxobenzoselenazoles 5. The results suggest that 5-arylamino-4,7-dioxobenzoselenazoles 4 would be potent antifungal agents.     

Synthesis and preliminary biological evaluation of new anti-tubulin agents containing different benzoheterocycles

A new series of compounds, in which the 2-amino-4-methoxyphenyl ring of phenstatin analogue 5 was replaced with 2- or 3-amino-benzoheterocycles, was synthesized and evaluated for antiproliferative activity and inhibition of colchicine binding. The lack of activity of 3',4'-dimethoxy- and 4'-methoxy-benzoyl derivatives (8 and 9, respectively) indicates that the 3',4',5'-trimethoxybenzoyl moiety is critical for the activity. Two compounds, 7 and 11, displayed potent antiproliferative activity, with IC50 values ranging from 25 to 100 nM against a variety of cancer cell lines. Derivative 11 was more active than CA-4 as an inhibitor of tubulin polymerization. The results demonstrated that the antiproliferative activity was correlated with inhibition of tubulin polymerization.     

2,3-Dimethoxy-5-methyl-1,4-benzoquinones and 2-methyl-1,4-naphthoquinones: glycation inhibitors with lipid peroxidation activity

Anti-glycation activity of our anti-oxidant quinone library was measured and several 2,3-dimethoxy-5-methyl-1,4-benzoquinones and 2-methyl-1,4-naphthoquinones were identified as novel inhibitors of glycation, of which 2,3-dimethoxy-5-methyl-1,4-benzoquinones 13b is the most potent glycation inhibitor with around 50 microM of the IC(50) value.     

Synergistic activation of JNK/SAPK induced by TNF-alpha and IFN-gamma: apoptosis of pancreatic beta-cells via the p53 and ROS pathway

IFN-γ and TNF-α are major proinflammatory cytokines implicated in islet β-cell destruction, which results in type-1 diabetes; however, the underlying mechanism is not clear. Using pancreatic β-cell line MIN6N8 cells, co-treatment with TNF-α and IFN-γ, but neither cytokine alone, synergistically induced apoptosis, correlated with the activation of the JNK/SAPK, which resulted in the production of reactive oxidative species (ROS) and loss of mitochondrial transmembrane potential (ΔΨm). Additionally, cells transfected with wild-type JNK1 became more susceptible to apoptosis induced by TNF-α/IFN-γ through ROS production and loss of Δψm, while cascading apoptotic events were prevented in dominant-negative JNK1-transfected or JNK inhibitor SP600125-treated cells. As the antioxidant, N-acetyl-cysteine, failed to completely suppress apoptosis induced by TNF-α/IFN-γ, an additional pathway was considered to be involved. The level of p53 was significantly increased through synergistic activation of JNK by TNF-α/IFN-γ. Furthermore, the synergistic effect of TNF-α/IFN-γ on apoptosis and ROS production was further potentiated by the overexpression of wild-type p53, but not with mutant p53. This synergistic activation of JNK/SAPK by TNF-α/IFN-γ was also induced in insulin-expressing pancreatic islet cells, and increased ROS production and p53 level, which was significantly inhibited by SP600125. Collectively, these data demonstrate that TNF-α/IFN-γ synergistically activates JNK/SAPK, playing an important role in promoting apoptosis of pancreatic β-cell via activation of p53 pathway together with ROS.     

Stimulation of various functions in murine peritoneal macrophages by glucans produced by glucosyltransferases from Streptococcus mutans

The glucan that was produced by glucosyltransferases (GTFs) from Streptococcus mutans was examined for its stimulating functions toward murine peritoneal macrophages. Soluble glucan was obtained by the reaction with cell-free crude GTFs and sucrose, followed by ethanol precipitation, dispersion in water and re-precipitation by ethanol. Soluble glucan, those average molecular weight was about 3 x 10(5), was composed of mixture of alpha-1,6 and alpha-1,3 linkages in a 3:1 ratio. When 30 and 60 microg/ml of the glucan was incubated with peritoneal macrophages, the lysosomal phosphatase activity was increased in a dose-dependent manner, indicating that soluble glucan may activate macrophages. To examine its effects on the various functions of macrophages, soluble glucan was orally administered daily at a level of 100 mg/kg of body weight to C57BL/6 mice. Significant stimulation of the production of H2O2 by the macrophages was observed without any increase in NO production. The production of tumor necrosis factor-alpha (TNF-alpha) by the macrophages was also stimulated from 538.73-555.06 pg/ml to 585.73-596.40 pg/ml during 15 days of oral administration of soluble glucan. The cytotoxicity of peritoneal macrophages against B16 tumor cells was significantly enhanced by 25-38% during 15 days of oral administration. These results may indicate that soluble glucan stimulates the immune functions of macrophages.     

Sphingosylphosphorylcholine generates reactive oxygen species through calcium-, protein kinase Cdelta- and phospholipase D-dependent pathways

Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in numerous biological processes. Treatment of MS1 pancreatic islet endothelial cells with SPC increased phospholipase D (PLD) activity in a time- and dose-dependent manner. In addition, treatment of the MS1 cells with 10 microM SPC induced stimulation of phospholipase C (PLC) activity and transient elevation of intracellular Ca2+. The SPC-induced PLD activation was prevented by pretreatment of the MS1 cells with a PLC inhibitor, U73122, and an intracellular Ca2+-chelating agent, BAPTA-AM. This suggests that PLC-dependent elevation of intracellular Ca2+ is involved in the SPC-induced activation of PLD. The SPC-dependent PLD activity was also almost completely prevented by pretreatment with pan-specific PKC inhibitors, GF109203X and RO-31-8220, and with a PKCdelta-specific inhibitor, rottlerin, but not by pretreatment with GO6976, a conventional PKC isozymes-specific inhibitor. Adenoviral overexpression of a kinase-deficient mutant of PKCdelta attenuated the SPC-induced PLD activity. These results suggest that PKCdelta plays a crucial role for the SPC-induced PLD activation. The SPC-induced PLD activation was preferentially potentiated in COS-7 cells transfected with PLD2 but not with PLD1, suggesting a specific implication of PLD2 in the SPC-induced PLD activation. SPC treatment induced phosphorylation of PLD2 in COS-7 cells, and overexpression of the kinase-deficient mutant of PKCdelta prevented the SPC-induced phosphorylation of PLD2. Furthermore, SPC treatment generated reactive oxygen species (ROS) in MS1 cells and the SPC induced production of ROS was inhibited by pretreatment with U73122, BAPTA-AM, and rottlerin. In addition, pretreatment with a PLD inhibitor 1-butanol and overexpression of a lipase-inactive mutant of PLD2 but not PLD1 attenuated the SPC-induced generation of ROS. These results suggest that PLC-, Ca2+-, PKCdelta-, and PLD2-dependent pathways are essentially required for the SPC induced ROS generation.     

Monitoring of petroleum hydrocarbon degradative potential of indigenous microorganisms in ozonated soil

This study was performed to investigate the petroleum hydrocarbon (PH) degradative potential of indigenous microorganisms in ozonated soil to better develop combined pre-ozonation/bioremediation technology. Diesel-contaminated soils were ozonated for 0–900min. PH and microbial concentrations in the soils decreased with increased ozonation time. The greatest reduction of total PH (TPH, 47.6%) and aromatics (11.3%) was observed in 900-min ozonated soil. The number of total viable heterotrophic bacteria decreased by three orders of magnitude in the soil. Ozonated soils were incubated for 9weeks for bioremediation. The number of microorganisms in the soils increased during the incubation period, as monitored by culture- and nonculture-based methods. The soils showed additional PH-removal during incubation, supporting the presence of PH-degraders in the soils. The highest removal (25.4%) of TPH was observed during the incubation of 180-min ozonated soil during the incubation while a negligible removal was shown in 900-min ozonated soil. This negligible removal could be explained by the existence of relatively few or undetected PH-degraders in 900-min ozonated soil. After a 9-week incubation of the ozonated soils, 180-min ozonated soil showed the lowest TPH concentration, suggesting that appropriate ozonation and indigenous microorganisms survived ozonation could enhance remediation of PH-contaminated soil. Microbial community composition in 9-week incubated soils revealed a slight difference between 900-min ozonated and unozonated soils, as analyzed by whole cell hybridization. Taken together, this study provided insight into indigenous microbial potential to degrade PH in ozonated soils.     

Molecular characterization of spontaneous and growth-factor-augmented chondrogenesis in periosteum–bone tissue transferred into a joint

Multilineage potential of progenitor cells from periosteum is well established, but conditions for differentiation within their native niche are unclear. We evaluated at cellular and molecular levels whether chondrogenesis of periosteal progenitor cells is promoted spontaneously or by growth-factor mixture (GFM) application when transferring periosteum–bone cylinders into cartilage defects. Osteochondral defects in the patellar groove of minipigs were filled with periosteum–bone cylinders and randomly supplemented with GFM. Neochondrogenesis was characterized by histology, immunohistology, and quantitative gene expression analysis. According to morphology and glycosaminoglycan accumulation, spontaneous neocartilage formation occurred in the cambium layer already at 6 weeks, increased after 12 weeks, but declined until 52 weeks, independent of GFM. Multiple cartilage differentiation markers were induced after transfer. Expression of aggrecan, COMP, decorin, and Col10a1 increased significantly within 52 weeks. Sox 9 and Col2a1 mRNA levels were elevated at 6 versus 52 weeks in the GFM group and resulted in higher collagen type II protein accumulation. Neochondrogenesis was promoted in lower periosteum layers by transfer of periosteum–bone plugs into a joint, and collagen type II protein deposition was enhanced by GFM. The final tissue subsumed typical features of periosteum and fibrocartilage but lacked an intact tide mark and features of hyaline cartilage desired for cartilage repair.     

Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells

Estrogen stimulates cell proliferation in breast cancer. The biological effects of estrogen are mediated through two intracellular receptors, estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta). However, the role of ERs in the proliferative action of estrogen is not well established. Recently, it has been known that ER activates phosphatidylinositol-3-OH kinase (PI3K) through binding with the p85 regulatory subunit of PI3K. Therefore, possible mechanisms may include ER-mediated phosphoinositide metabolism with subsequent formation of phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), which is generated from phosphatidylinositol 4,5-bisphosphate via PI3K activation. The present study demonstrates that 17beta-estradiol (E2) up-regulates PI3K in an ERalpha-dependent manner, but not ERbeta, and stimulates cell growth in breast cancer cells. In order to study this phenomenon, we have treated ERalpha-positive MCF-7 cells and ERalpha-negative MDA-MB-231 cells with 10nM E2. Treatment of MCF-7 cells with E2 resulted in a marked increase in PI3K (p85) expression, which paralleled an increase in phospho-Akt (Ser-473) and PIP(3) level. These observations also correlated with an increased activity to E2-induced cell proliferation. However, these effects of E2 on breast cancer cells were not observed in the MDA-MB-231 cell line, indicating that the E2-mediated up-regulation of PI3K/Akt pathway is ERalpha-dependent. These results suggest that estrogen activates PI3K/Akt signaling through ERalpha-dependent mechanism in MCF-7 cells.