A proteomic approach to paclitaxel chemoresistance in ovarian cancer cell lines

Ovarian cancer is the leading cause of gynaecological cancer mortality. Paclitaxel is used in the first line treatment of ovarian cancer, but acquired resistance represents the most important clinical problem and a major obstacle to a successful therapy. Several mechanisms have been implicated in paclitaxel resistance, however this process has not yet been fully explained. To better understand molecular resistance mechanisms, a comparative proteomic approach was undertaken on the human epithelial ovarian cancer cell lines A2780 (paclitaxel sensitive), A2780TC1 and OVCAR3 (acquired and inherently resistant). Proteins associated with chemoresistance process were identified by DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS). Out of the 172 differentially expressed proteins in pairwise comparisons among the three cell lines, 151 were identified and grouped into ten main functional classes. Most of the proteins were related to the category of stress response (24%), metabolism (22%), protein biosynthesis (15%) and cell cycle and apoptosis (11%), suggesting that alterations of those processes might be involved in paclitaxel resistance mechanisms. This is the first direct proteomic comparison of paclitaxel sensitive and resistant ovarian cancer cells and may be useful for further studies of resistance mechanisms and screening of resistance biomarkers for the development of tailored therapeutic strategies.     

Olive embryo in vitro germination potential: role of explant configuration and embryo structure among cultivars

The in vitro germination of excised embryos can break dormancy rapidly and shorten the time required to produce seedlings, speeding up olive breeding programmes as well as rootstock production. In this study, the in vitro germination potential of four Sicilian olive cultivars was evaluated during two years of experiments, using explants with three different morphological configurations that represent three different degrees of embryo exposure: (1) intact stoneless seeds containing the embryo, the endosperm and the seed coat (Emb+En+SC), (2) seeds without the seed coat (Emb+En) and (3) naked, isolated embryos (seed coat and endosperm both removed: Emb). Differences were found in the germination percentages and timing due to both genotype and explant type. The root and shoot meristems, the radicle-hypocotyl axis, the provascular tissues and embryo storage reserves were identified as embryo anatomical structures which could influence germination capacity. Observation of these structures, however, indicated similar germination potential among cultivars, suggesting possible differences in other dormancy factors. In spite of variation in cultivar performance, after 60 days of in vitro culture all cultivars demonstrated the highest germination of naked embryos (explant type 3) and lowest for stoneless seeds (explant type 1); stoneless seeds without the seedcoat (explant type 2) showed intermediate germination percentages.     

Cytotoxic alpha-bromoacrylic derivatives of low molecular weight

In vitro and in vivo activities of a small series of alpha-bromoacrylic derivatives of low molecular weight (MW) are described and compared with those of alpha-bromoacrylic derivatives of distamycin-like frames. Low MW compounds, when lacking of a strong basic moiety, are potent cytotoxics, while analogues bearing a strong basic moiety are not. This suggests the existence of an active transport mechanism for distamycin-derived cytotoxics characterized by strong basic amidino or guanidino moieties. Low MW compounds are inactive in vivo, possibly because of the metabolic lability of alpha-bromoacrylic moiety. The same moiety is however present in a series of potent anticancer distamycin-like minor groove binders, for example, PNU-166196 (brostallicin), a fact that underlines the features of the latter.     

ELF-MF attenuates quercetin-induced apoptosis in K562 cells through modulating the expression of Bcl-2 family proteins

This study investigated the effects of sinusoidal ELF-MF (1 mT; 50 Hz) on the apoptosis induced by four different compounds, namely vinblastine, etoposide, quercetin, and resveratrol, in human K562 chronic myeloid leukemia cells. The exposure to ELF-MF did not affect growth and viability of untreated K562 cells and did not influence the anti-proliferative effects of resveratrol, vinblastine, and etoposide. On the contrary, in quercetin-treated cells, exposure to ELF-MF significantly reduced the percentage of apoptotic cells and the caspase-3 activity and modified the cell cycle profile especially after 48 h of exposure. In addition, the simultaneous treatments for 24 h with quercetin plus ELF-MF increased Bcl-2 protein expression and prevented quercetin-induced downregulation of Mcl-1 and Bcl-xL. Finally, an increase of HSP70 expression was also observed after prolonged ELF-MF treatment. The ELF-MF-dependent modulation of the expression of anti-apoptotic Bcl-2 family and Hsp70 proteins could act as a pro-survival mechanism in K562 cells.     

Co-Transplantation of Endothelial Progenitor Cells and Pancreatic Islets to Induce Long-Lasting Normoglycemia in Streptozotocin-Treated Diabetic Rats

Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3–5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals'' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards.     

Urinary CD133+ Extracellular Vesicles Are Decreased in Kidney Transplanted Patients with Slow Graft Function and Vascular Damage

Extracellular vesicles (EVs) present in the urine are mainly released from cells of the nephron and can therefore provide information on kidney function. We here evaluated the presence of vesicles expressing the progenitor marker CD133 in the urine of normal subjects and of patients undergoing renal transplant. We found that EV expressing CD133 were present in the urine of normal subjects, but not of patients with end stage renal disease. The first day after transplant, urinary CD133⁺ EVs were present at low levels, to increase thereafter (at day 7). Urinary CD133⁺ EVs significantly increased in patients with slow graft function in respect to those with early graft function. In patients with a severe pre-transplant vascular damage of the graft, CD133⁺ EVs did not increase at day 7. At variance, the levels of EVs expressing the renal exosomal marker CD24 did not vary in the urine of patients with end stage renal disease or in transplanted patients in respect to controls. Sorted CD133⁺ EVs were found to express glomerular and proximal tubular markers. These data indicate that urinary CD133⁺ EVs are continuously released during the homeostatic turnover of the nephron and may provide information on its function or regenerative potential.     

Evidence of Uncoupling between Renal Dysfunction and Injury in Cardiorenal Syndrome: Insights from the BIONICS Study

Objective

The objective of the study was to assess urinary biomarkers of renal injury for their individual or collective ability to predict Worsening renal function (WRF) in patients with acutely decompensated heart failure (ADHF).

Methods

In a prospective, blinded international study, 87 emergency department (ED) patients with ADHF were evaluated with biomarkers of cardiac stretch (B type natriuretic peptide [BNP] and its amino terminal equivalent [NT-proBNP], ST2), biomarkers of renal function (creatinine, estimated glomerular filtration rate [eGFR]) and biomarkers of renal injury (plasma neutrophil gelatinase associated lipocalin [pNGAL], urine kidney injury molecule-1 [KIM-1], urine N-acetyl-beta-D-glucosaminidase [NAG], urine Cystatin C, urine fibrinogen). The primary endpoint was WRF.

Results

26% developed WRF; baseline characteristics of subjects who developed WRF were generally comparable to those who did not. Biomarkers of renal function and urine biomarkers of renal injury were not correlated, while urine biomarkers of renal injury correlated between each other. Biomarker concentrations were similar between patients with and without WRF except for baseline BNP. Although plasma NGAL was associated with the combined endpoint, none of the biomarker showed predictive accuracy for WRF.

Conclusions

In ED patients with ADHF, urine biomarkers of renal injury did not predict WRF. Our data suggest that a weak association exists between renal dysfunction and renal injury in this setting (Clinicaltrials.gov NCT#0150153).     

A pro-survival effect of polyamine depletion on norepinephrine-mediated apoptosis in cardiac cells: role of signaling enzymes

Recent studies report that the primary transmitter of sympathetic nervous system norepinephrine (NE), which is actively produced in failing human heart, is able to induce apoptosis of rat cardiomyocytes. Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy and heart failure, therefore representing a potential therapeutic target. The natural occurring polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. Thus, we have studied the involvement of polyamines in the apoptosis of cardiac cells induced by the treatment with NE. The results indicate that NE caused an early induction of the activity of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis, followed by a later increase of apoptotic cell death. This effect was prevented in the presence of α-difluoromethylornithine, an irreversible inhibitor of ODC. Moreover, the study of some key signal transduction pathways revealed an involvement of AMP-activated protein kinase, AKT and p38 mitogen-activated protein kinases, in the modulation by polyamines of the response of cardiomyocytes to NE. In fact, polyamine-depleted cells showed an altered activation pattern of these kinases that may contrast apoptosis and appeared to result from a differential effect on the specific phosphatases that dephosphorylate and switch off these signaling proteins. In conclusion, these results indicate that in cardiac cells polyamines are involved in the execution of the death program activated by NE, and suggest that their apoptosis facilitating action is mediated by a network of specific phosphatases and kinases.