A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells

Background

Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays.

Principal Findings

LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis.

Conclusion

LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.     

Ultrastructural immuno-localization of tropoelastin in the chick eye

Summary Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.     

Involvement of state transitions in the switch between linear and cyclic electron flow in Chlamydomonas reinhardtii

The energetic metabolism of photosynthetic organisms is profoundly influenced by state transitions and cyclic electron flow around photosystem I. The former involve a reversible redistribution of the light-harvesting antenna between photosystem I and photosystem II and optimize light energy utilization in photosynthesis whereas the latter process modulates the photosynthetic yield. We have used the wild-type and three mutant strains of the green alga Chlamydomonas reinhardtii—locked in state I (stt7), lacking the photosystem II outer antennae (bf4) or accumulating low amounts of cytochrome b₆f complex (A-AUU)—and measured electron flow though the cytochrome b₆f complex, oxygen evolution rates and fluorescence emission during state transitions. The results demonstrate that the transition from state 1 to state 2 induces a switch from linear to cyclic electron flow in this alga and reveal a strict cause–effect relationship between the redistribution of antenna complexes during state transitions and the onset of cyclic electron flow.     

Experimental hybridization within the genus Triturus (Urodela: Salamandridae)

The spermatogenesis of 9 F₁ hybrids of Triturus cristatus carnifex × T. vulgaris meridionalis was studied in squash preparations of testicular fragments, treated by the C-staining method. The chromosome number of these hybrids was examined in spermatogonial metaphases and found to be diploid. The two parental sets were always recognized, which means that a regular, although heterospecific, amphimixis occurred (2n=n+n). Meiotic prophase I is greatly altered owing to a failure of typical chromosome pairing and chiasma formation. At metaphase I and/or meta-anaphase I, the effects of the hybrid combination of the 2 specific parental sets are clearly visible. Most primary spermatocytes contain only univalents. A few show chromosome associations (bivalents, trivalents and, more rarely, quadrivalent chains) besides univalents. Such associations are of 2 types: (a) intragenomal associations = associations of 2 chromosomes by a terminal (a¹) or subterminal chiasma (a²); (b) intergenomal associations = associations of 2 chromosomes by a terminal (b¹) or subterminal chiasma (b²). Univalents segregate at random while the associations often lag on the equatorial plane or migrate entire to a spindle pole. Primary spermatocytes with chromosome multivalents can encounter greater difficulties in accomplishing the first cytokinesis. Secondary spermatocytes are numerically and qualitatively unbalanced; however, some of them undergo spermiogenesis and can give rise to a small number of sperms, generally abnormal and never united in bundles. — Problems related to the occurrence of anomalous chiasmata and of intra- and inter-genomal homologies are discussed.     

Enhanced activity of the plasma membrane oxidoreductase in circulating lymphocytes from insulin-dependent diabetes mellitus patients

Circulating human lymphocytes contain a transmembrane oxidoreductase (PMOR) capable of reducing dichlorophenol indophenol (DCIP) by endogenous reductants, presumably NADH. Membranes from lymphocytes obtained from buffy coats contain a NADH DCIP reductase having a K(m) of about 1 microM and almost insensible to dicoumarol. The PMOR of lymphocytes from insulin-dependent diabetic patients is higher than that from age-matched controls and, in addition, has a dicoumarol-sensitive component, lacking in most controls, presumably due to membrane association of DT-diaphorase. The increase of PMOR in diabetes is likely due to overexpression of the enzyme, in view of the very low K(m) for NADH indicating that, in intact cells, the enzyme is practically saturated with the reductant substrate.     

Video tracking and analysis of sleep in Drosophila melanogaster

In the past decade, Drosophila has emerged as an ideal model organism for studying the genetic components of sleep as well as its regulation and functions. In fruit flies, sleep can be conveniently estimated by measuring the locomotor activity of the flies using techniques and instruments adapted from the field of circadian behavior. However, proper analysis of sleep requires degrees of spatial and temporal resolution higher than is needed by circadian scientists, as well as different algorithms and software for data analysis. Here I describe how to perform sleep experiments in flies using techniques and software (pySolo and pySolo-Video) previously developed in my laboratory. I focus on computer-assisted video tracking to monitor fly activity. I explain how to plan a sleep analysis experiment that covers the basic aspects of sleep, how to prepare the necessary equipment and how to analyze the data. By using this protocol, a typical sleep analysis experiment can be completed in 5-7 d.