Min-Fu Tsan

Resveratrol induces Fas signalling-independent apoptosis in THP-1 human monocytic leukaemia cells

Resveratrol, a natural product present in wine, has recently been shown to inhibit the growth of a number of cancer cell lines in vitro. In the current study, we have demonstrated that resveratrol inhibits the growth of THP‐1 human monocytic leukaemia cells in a dose‐dependent manner with a median effective dose of 12 μm. It did not induce differentiation of THP‐1 cells and had no toxic effect on THP‐1 cells that had been induced to differentiate into monocytes/macrophages by phorbol myristate acetate. A significant fraction of resveratrol‐treated cells underwent apoptosis as judged by flow cytometric analysis of DNA content, DNA fragmentation and caspase‐specific cleavage of poly(ADP‐ribosyl) polymerase. Resveratrol treatment had no effect on the expression of Fas receptor or Fas ligand (FasL) in THP‐1 cells, nor did it induce clustering of Fas receptors. In addition, THP‐1 cells were resistant to activating anti‐Fas antibody, and neutralizing anti‐Fas and/or anti‐FasL antibodies had no protective effect against resveratrol‐induced inhibition of THP‐1 cell growth. The effect of resveratrol on THP‐1 cells was reversible after its removal from the culture medium. These results suggest that (1) resveratrol inhibits the growth of THP‐1 cells, at least in part, by inducing apoptosis, (2) resveratrol‐induced apoptosis of THP‐1 cells is independent of the Fas/FasL signalling pathway and (3) resveratrol does not induce differentation of THP‐1 cells and has no toxic effect on differentiated THP‐1 cells. Thus, resveratrol may be a potential chemotherapeutic agent for the control of acute monocytic leukaemia.

Anti-leukemia Effect of Resveratrol

Resveratrol is a phytoalexin naturally present in fruits, medicinal plants and wines. It has a diversity of biological activities. While its role in the protection against coronary heart disease (CHD) in people with moderate wine consumption, remains unclear, resveratrol preferentially inhibits the growth of leukemia cells in culture. Potential mechanisms for its anti-leukemia effect include induction of leukemia cell differentiation, apoptosis, and cell cycle arrest at S-phase; and inhibition of DNA synthesis by inhibiting ribonucleotide reductase or DNA polymerase. Preliminary results suggest that resveratrol also inhibits the viability of freshly isolated leukemia cells, especially promyelocytic leukemia cells. Because of its low in vivo toxicity, resveratrol deserves further investigation as an anti-leukemia agent.

L-2-Oxothiazolidine-4-carboxylate protects cultured endothelial cells against hyperoxia-induced injury

When bovine pulmonary artery endothelial monolayers were exposed to hyperoxia (95% O2 and 5% Co2), they responded by selectively elevating the intracellular concentration of glutathione without affecting the activities of glutathione peroxidase or glutathione reductase.l-2-Oxothiazolidine-4-carboxylate, an intracellular cysteine-delivering agent, further enhanced the intracellular concentration of glutathione in oxygen-exposed endothelial cells and protected them from the lethal effect of hyperoxia. In contrast, buthionine sulfoximine, a potent inhibitor ofγ-glutamylcysteine synthetase, reduced the glutathione concentration and rendered the cells more sensitive to the toxic effect of oxygen. Bothl-2-oxothiazolidine-4-carboxylate and buthionine sulfoximine had no effect on the activities of glutathione peroxidase or glutathione reductase. Our results suggest thatl-2-oxothiazolidine-4-carboxylate may have the potential of preventing oxygen toxicity.

Direct staining and visualization of endothelial monolayers cultured on synthetic polycarbonate filters.

Endothelial and epithelial cells cultured on synthetic filter supports have been used to study permeability and transport under various experimental conditions. However, because of the non-transparent nature of these filters, morphological studies using light microscopy are not possible. Presently, investigators circumvent this problem by using cells cultured on glass coverslips, extrapolating morphological data from a system clearly different from that used for functional studies. We describe here a useful technique for direct staining and visualization of cells grown on polycarbonate filter supports, using fluorochrome probes and fluorescence microscopy. We have utilized acridine orange, rhodamine phalloidin, and an anti-vimentin monoclonal antibody to provide information about cell shape, monolayer configuration, and cytoskeletal protein distribution in cultured calf pulmonary artery endothelial cell monolayers. Comparison staining of coverslip and filter preparations revealed a number of clear differences in these parameters. This technique should enable investigators to perform the necessary studies to obtain direct correlations between functional and morphological data.

Membrane endopeptidases of human neutrophil

Recent studies using inhibitors or synthetic substrates of serine protease suggest that membrane protease activity may be essential for neutrophil chemotaxis, phagocytosis, degranulation, and superoxide production. However, little is known about the nature and localization of the proteases. In this study, we demonstrated that intact human neutrophils hydrolyzed [125I] glucagon. The degradation of glucagon was temperature dependent and was not dependent on the release of lysosomal enzymes. Two endopeptidases were demonstrated: a metalloendopeptidase which accounted for two thirds of the intact cell activity, and a serine endopeptidase, accounting for the rest of the activity. Both enzymes had a neutral to alkaline pH optimum (pH 7–9). The metalloendopeptidase had aKm of 15.3μM andVmax of 5.9 nmol/5 × 106 cells/45 min. The corresponding values for the serine endopeptidase were 33.3 μM and 5.0 nmol/5 x 106 cells/45 min, respectively. Inhibition of the membrane metalloendopeptidase or serine endopeptidase by 1,10-phenanthroline or diisopropylfluorophosphate, respectively, did not inhibit the production of superoxide by phorbol myristate acetate-stimulated neutrophils.

The substrate-associated protein p5 of porcine endothelial cells: Multiple isoforms, cytoskeletal-like properties and induction by hyperoxic stress

1. Cultured mesenchymal cells respond to hyperoxic (hyper-O2) stress with increased cell flattening/substrate adhesion and overall 47-69% reductions in total matrix-associated (i.e. saponin-resistant [SAP fraction]) protein. 2. Electrophoretic analysis revealed a selective hyper-O2-related 2.7- to 4-fold increase in SAP and cytoskeletal fraction deposition of the protein p45 beginning early (within 12 hr) after initial exposure of porcine endothelial cells to hyper-O2 and increasing over a 48 hr period. 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-O2-augmented matrix deposition of 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-02-augmented matrix deposition of p45 involved all 8 isoforms with the more basic subtypes exhibiting slightly greater net increases. 4. Both the specificity and time course of p45 induction, relative to the onset of hyper-O2 cytoarchitectural remodeling, indicate that p45 up-regulation constitutes an early aspect of the hyper-O2 adaptive response.