Apigenin enhances the cytotoxic effects of tumor necrosis factor-related apoptosis-inducing ligand in human rheumatoid arthritis fibroblast-like synoviocytes.

Activated rheumatoid arthritis (RA) fibroblast-like synoviocytes (RAFLSs) play a central role in both initiating and driving RA. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been documented to induce apoptosis only in a small proportion of RAFLSs, which is followed by an induction of proliferation in surviving cells. Apigenin, a chemopreventive bioflavonoid, exhibits proapoptotic activity in many types of cells. In the present study, we sought to determine whether apigenin could enhance the cytotoxic effect of TRAIL on activated RAFLSs. Human RAFLSs isolated from patients with RA were treated with TRAIL (1 nM), apigenin (20 µM), or their combination, and subjected to apoptosis analysis after a 24-h incubation and proliferation analysis after a 72-h incubation. Apoptosis assay revealed that TRAIL or apigenin alone induced a marked apoptosis in RAFLS and their combination yielded a synergistic increase in RAFLS apoptosis. Immunoblotting analysis of apoptosis regulators demonstrated that combined treatment with apigenin increased caspase-3 expression and activity and decreased the Bcl-2/Bax ratio relative to treatment with TRAIL alone. The presence of apigenin significantly restrained TRAIL-induced RAFLS proliferation, coupled with restoration of the expression of two cell-cycle inhibitors p21 and p27. Moreover, the combination with apigenin blunted TRAIL-induced activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Our data collectively demonstrate that apigenin sensitizes RAFLS to TRAIL-induced apoptosis and counteracts TRAIL-dependent RAFLS proliferation, which is likely mediated through inactivation of PI3-K/Akt signaling pathway.

Effects of berberine on human rheumatoid arthritis fibroblast-like synoviocytes.

Activated rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) play an important role in the initiation and progression of rheumatoid arthritis (RA). Rapid proliferation and defective apoptosis of RAFLSs are two main mechanisms contributing to synovial hyperplasia. Berberine, the major constituent of Coptidis Rhizoma, has been widely used as an antitumor and anti-inflammation agent. Here we show that berberine significantly inhibited cell proliferation of serum-starved human RAFLSs in a dose-dependent manner. Cell cycle analysis of berberine-treated RAFLSs indicated a cell cycle arrest at the G0/G1 phase. The inhibitory effects of berberine correlated with an induction of cyclin-dependent kinase (CDK) inhibitors Cip1/p21 and Kip1/p27 and a reduction of CDK2, CDK4 and CDK6, and cyclins D1, D2 and E. Furthermore, an apoptosis assay showed that berberine treatment increased apoptotic death of RAFLSs, which was associated with an increased expression of proapoptotic protein Bax and decreased expression of antiapoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspase-3, caspase-9 and poly (ADP-ribose) polymerase. Taken together, these results demonstrate that berberine exerts antiproliferative effects against RAFLSs, likely through deregulation of numerous cell cycle and apoptosis regulators, thus having potential therapeutic implications in the treatment of RA.

[Research progresses on septin family].

The septins are a family of proteins that are broadly distributed in almost all of eukaryotes except plants. septin was first identified in yeast as a protein that played a role in cytokinesis. With the recent advances in the field, the functions of these proteins become diverse in many organisms. In particular, the number of known mammalian septin family members has increased dramatically. They are now known to have many cellular roles such as cytokinesis, polarity determination, vesicle trafficking and membrane dynamics. Recently, more and more data suggest that some septin family members participate in the pathogenesis of different diseases including neoplasia, neurodegeneration and infections. These make the research of septins a hallmark in cell biology and pathology. In this review, we will summarize the major research progresses about septins in their classification, structure, biological function and the relationship with human diseases.

Studies on the beta(l-4)Galactosyltransferase of Cell Surface in Induced HL60 Cells.

A fluorescene assay method for beta(1-4) galactosyltransferase (beta1-4GT) of cell surface has been developed using a pyridylaminated sugar as an acceptor substrate. A fluorescent sugar chain, whose reducing end of the Gnbeta1-2Malpha1-6(Gnbeta1-2Malpha1-3) Mbeta1-4Gnbeta1-4(Fucalpha1-6) Gn has been aminated with 2-aminopyridine. beta1-4GT activity of cell surface varied in different stages of the cell cycle with the highest activity at interphase. The enzyme activity of cell surface took a change when HL60 cell line was induced to differentiate by PMA or RA. The cell surface enzyme activity was 1.30 times as much as tile control group at 24 h in the case of PMA induction, and a maximum increase of 70% over the control on the third day with RA induction.

Studies on the Method for the Determination of beta1-4 Galactosyltransferase and Its Kinetics.

A fluorescence assay method for beta1-4galactosyltransferase (beta1-4GT) has been developed involving a pyridylaminated sugar as an acceptor substrate, a fluorescent sugar chain, with the reducing end of the Gnbeta1 - 2Malpha1 - 6(Gnbeta1-2Malpha1-3)Mbeta1 - 4Gnbeta1 - 4Gn - PA aminated with 2-aminopyridine. Microsome was prepared from the liver of normal male rats as an enzyme sample. Then the fluorescent reaction product was separated by reverse-phase HPLC. The kinetic experiments were carried out using crude enzyme extracts of the Golgi complex from the rat liver. The enzyme has a pH optimum of 6.5,and optimal concentration of Triton X-100 of 0.5%. The K(m) and V(max) values for the sugar acceptor substrates were found to be 2.31x10(-3)M(-1) and 5.75x10(-2) &mgr;mol/(min.mg) respectively. Furthermore, our research revealed that beta1-4GT had branch specificity. The Gn of alpha1-3 mannose branch of the acceptor substrate was more susceptible to galactosylation than that of the alpha1-6 branch by 2.10 times.