A novel cisplatin mediated apoptosis pathway is associated with acid sphingomyelinase and FAS proapoptotic protein activation in ovarian cancer.

Platinum-based anticancer drugs, including cisplatin and carboplatin, have been cornerstones in the treatment of solid tumors. We report here that these DNA-damaging agents, particularly cisplatin, induce apoptosis through plasma membrane disruption, triggering FAS death receptor via mitochondrial (intrinsic) pathways. Our objectives were to: quantify the composition of membrane metabolites; and determine the potential involvement of acid sphingomyelinase (ASMase) in the FAS-mediated apoptosis in ovarian cancer after cisplatin treatment. The resulting analysis revealed enhanced apoptosis as measured by: increased phosphocholine, and glycerophosphocholine; elevated cellular energetics; and phosphocreatine and nucleoside triphosphate concentrations. The plasma membrane alterations were accompanied by increased ASMase activity, leading to the upregulation of FAS, FASL and related pro-apoptotic BAX and PUMA genes. Moreover FAS, FASL, BAX, PUMA, CASPASE-3 and -9 proteins were upregulated. Our findings implicate ASMase activity and the intrinsic pathways in cisplatin-mediated membrane demise, and contribute to our understanding of the mechanisms by which ovarian tumors may become resistant to cisplatin.

Reduced apoptosis in term placentas from gestational diabetic pregnancies.

Gestational diabetic mellitus (GDM) pregnancies have an increased risk of macrosomic infants and large placental mass, though the mechanisms explaining each of these is uncertain. We sought to evaluate the contribution of apoptosis to placental size and the expression of glucose transporters (SLC2A) in GDM pregnancies. Maternal age and pre-pregnancy body weight were documented. Newborn weights were recorded after delivery. Placentas 37-40-week gestation from control patients (no pregnancy complication) (n = 5), or with GDM (n = 5) were weighed immediately after delivery. Villous samples (4 mm diameter) were collected and divided into specimens; one was fixed in 4% paraformaldehyde for immunostaining using terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) and activated caspase-3. The other specimen was snap frozen in liquid nitrogen and stored at -80°C for active caspase-3, poly(ADP-ribose) polymerase (PARP), SLC2A1 and SLC2A3 gene expression analysis. Our results showed that maternal age and pre-pregnancy body weight were significantly higher in the GDM group when compared with those from the controls (P < 0.05). The mean neonatal birth weight and placenta weight were significantly higher in the GDM group compared with that from the controls (P < 0.05). The apoptotic index of placentas (0.05 ± 0.01 v. 0.17 ± 0.04, P < 0.04), active caspase-3 polypeptide fragments and PARP protein were significantly decreased in GDM placentas as compared with controls. Further, the level of placental SLC2A1 protein expression was ∼3-fold higher in GDM placentas. Our results suggest that reduced apoptosis in GDM placentas may contribute to increased placental tissue, which together with enhanced SLC2A1 expression, could play a role in fetal macrosomia.

Increased placental apoptosis in maternal food restricted gestations: role of the Fas pathway.

The placenta has a pivotal role, shuttling nutrients to the developing fetus and producing hormones essential to pregnancy. Maternal food restriction (MFR) during pregnancy results in growth restricted newborns, a consequence attributed primarily to maternal nutrient supply. We hypothesized that MFR further compromises fetal growth by decreased placental growth or increased placental apoptosis. We determined the potential pathway (Fas-Fas-ligand; Fas-FasL) of placental apoptosis in MFR pregnancies. We assessed the two morphologically and functionally distinct zones (basal and labyrinth) at embryonic age 20 (E20) in ad libitum fed controls (AdLib) and 50% MFR placentas. We studied fetuses and placentas from both proximal and mid-horn positions to evaluate any differential impact by MFR. Placenta apoptosis was quantified using terminal dUTP nick-end labeling (TUNEL) assay and the data were compared to immunodetection of cleaved caspase-3, Fas and FasL followed by Western blot quantification of Fas, FasL, caspase-8 and -3, tBID, and poly-ADP-ribose polymerase (PARP). MFR reduced maternal, fetal and placental basal and labyrinth weights. The results suggest that the increased apoptosis may be mediated, in part, via Fas pathway and the defective placental development in the MFR may be a major contributor to the decreased fetal growth.

Differential expression of membrane and soluble adenylyl cyclase isoforms in cytotrophoblast cells and syncytiotrophoblasts of human placenta.

Adenylyl cyclase (AC) activity is ubiquitous in mammalian cells, and various forms of this enzyme exist that widely differ with regard to tissue distribution, abundance, and modes of regulation. Human placenta is made, among others, of cytotrophoblast cells and syncytiotrophoblasts. This latter is a polynucleate structure that originates from the differentiation of proliferative mononucleated cytotrophoblast cells, the placental stem cell, into syncytiotrophoblasts. In vitro, this differentiation process is associated with a concomitant increase in cellular levels of cAMP and enhanced expression of genes representative of syncytiotrophoblasts endocrine activity. Thus, in this study we evaluated the differential distribution of AC isoforms in cytotrophoblast cells and syncytiotrophoblasts by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA or purified mRNA. Our results demonstrate that all membrane and soluble AC mRNA isoforms are present in both cell types. Interestingly in syncytiotrophoblasts, AC4 and AC8 mRNA are highly expressed, while AC1, AC2 mRNA are less abundant when compared to cytotrophoblast cells. Additionally, the soluble AC is expressed in both trophoblast cells, but its expression is greatly reduced in differentiated cells, syncytiotrophoblasts. The presence of these AC proteins in both cells was confirmed by Western blotting. Taken together, these data help us to characterize the different AC isoforms in human cytotrophoblast cells and syncytiotrophoblasts, and demonstrate that the AC isoforms expression seems to be mainly modulated in groups 1 and 2. Moreover, the important decrease of the soluble AC isoform in syncytiotrophoblasts as compared to cytotrophoblast cells could suggest an important role of this AC in the extravillous trophoblast formation.

Determination of optimum growth conditions for gliotoxin production by Aspergillus fumigatus and development of a novel method for gliotoxin detection.

Gliotoxin is a toxic metabolite of Aspergillus fumigatus Fresenius and other fungi. It has been suggested that this toxin may play an important role in the pathogenesis of aspergillosis as gliotoxin has immunosuppressive activity both in vitro and in vivo. We have determined the optimum growth conditions for the production of gliotoxin by selected isolates of A. fumigatus using a number of defined media. Gliotoxin was detected by thin layer chromatography and high performance liquid chromatography. The carbohydrate source, concentration of carbohydrate in the growth medium and incubation temperature were all found to influence gliotoxin production. Optimum growth conditions for gliotoxin production in our study were Czapek-Dox broth containing 30% glucose and incubation at 37 degrees C. Most of the gliotoxin was produced after 29 h incubation, during the exponential phase of growth. A novel method for screening large numbers of A. fumigatus isolates for gliotoxin production, which is both quick and easy, has also been developed, based on the ability of gliotoxin to inhibit the adherence of lung fibroblast (L929) cells to plastic microtitre plates.

Cryopreservation of Aspergillus fumigatus stock cultures with a commercial bead system.

A method using beads and storage at -80 degrees C was used to maintain isolates of the pathogenic mould Aspergillus fumigatus. Viability was assessed after 6 and 15 months and all isolates were recovered in culture. This inexpensive technique was found to be an effective and easy method of preserving A. fumigatus and other pathogenic moulds.