Native LDL‐induced oxidative stress in human proximal tubular cells: multiple players involved

Dyslipidemia is a well‐established condition proved to accelerate the progression of chronic kidney disease leading to tubulo‐interstitial injury. However, the molecular aspects of the dyslipidemia‐induced renal damage have not been fully clarified and in particular the role played by low‐density lipoproteins (LDLs). This study aimed to examine the effects of native non‐oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS‐induced ROS release mechanism. The LDL‐dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca²⁺ uptake and loss of cytochrome c. All the above LDL‐induced effects were completely abrogated by chelating extracellular Ca²⁺ as well as by inhibition of the Ca²⁺‐activated cytoplas‐mic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL‐induced intracellular redox unbalance is triggered by a Ca²⁺ inward flux‐dependent commencement of cPLA2 followed by activation of a lipid‐ and ROS‐based cross‐talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox‐ and Ca²⁺‐dependent dysfunctions leading to cell‐harming conditions. These findings may help to clarify the mechanism of dyslipidemia‐induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.     

Acylpeptide hydrolase inhibition as targeted strategy to induce proteasomal down-regulation

Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy.Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed.Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression.     

Enhancing chemotherapy response with Bmi-1 silencing in ovarian cancer

Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and therapeutic options are limited. Although the polycomb group gene, Bmi-1 that regulates the self-renewal of normal stem and progenitor cells has been implicated in the pathogenesis of many human malignancies, yet a role for Bmi-1 in influencing chemotherapy response has not been addressed before. Here we demonstrate that silencing Bmi-1 reduces intracellular GSH levels and thereby sensitizes chemoresistant ovarian cancer cells to chemotherapeutics such as cisplatin. By exacerbating ROS production in response to cisplatin, Bmi-1 silencing activates the DNA damage response pathway, caspases and cleaves PARP resulting in the induction apoptosis in ovarian cancer cells. In an in vivo orthotopic mouse model of chemoresistant ovarian cancer, knockdown of Bmi-1 by nanoliposomal delivery significantly inhibits tumor growth. While cisplatin monotherapy was inactive, combination of Bmi-1 silencing along with cisplatin almost completely abrogated ovarian tumor growth. Collectively these findings establish Bmi-1 as an important new target for therapy in chemoresistant ovarian cancer.     

Targeting glutathione-S transferase enzymes in musculoskeletal sarcomas: a promising therapeutic strategy

Recent studies have indicated that targeting glutathione-S-transferase (GST) isoenzymes may be a promising novel strategy to improve the efficacy of conventional chemotherapy in the three most common musculoskeletal tumours: osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma. By using a panel of 15 drug-sensitive and drug-resistant human osteosarcoma, Ewing's sarcoma, and rhabdomyosarcoma cell lines, the efficay of the GST-targeting agent 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) has been assessed and related to GST isoenzymes expression (namely GSTP1, GSTA1, GSTM1, and MGST). NBDHEX showed a relevant in vitro activity on all cell lines, including the drug-resistant ones and those with higher GSTs levels. The in vitro activity of NBDHEX was mostly related to cytostatic effects, with a less evident apoptotic induction. NBDHEX positively interacted with doxorubicin, vincristine, cisplatin but showed antagonistic effects with methotrexate. In vivo studies confirmed the cytostatic efficay of NBDHEX and its positive interaction with vincristine in Ewing's sarcoma cells, and also indicated a positive effect against the metastatisation of osteosarcoma cells. The whole body of evidence found in this study indicated that targeting GSTs in osteosarcoma, Ewing's sarcoma and rhabdomyosarcoma may be an interesting new therapeutic option, which can be considered for patients who are scarcely responsive to conventional regimens.     

A global assessment of freshwater fish introductions in mediterranean-climate regions

Mediterranean-climate regions (med-regions) are global hotspots of endemism facing mounting environmental threats associated with human-related activities, including the ecological impacts associated with non-native species introductions. We review freshwater fish introductions across med-regions to evaluate the influences of non-native fishes on the biogeography of taxonomic and functional diversity. Our synthesis revealed that 136 freshwater fish species (26 families, 13 orders) have been introduced into med-regions globally. These introductions, and local extirpations, have increased taxonomic and functional faunal similarity among regions by an average of 7.5% (4.6–11.4%; Jaccard) and 7.2% (1.4–14.0%; Bray–Curtis), respectively. Faunal homogenisation was highest in Chile and the western Med Basin, whereas sw Cape and the Aegean Sea drainages showed slight differentiation (decrease in faunal similarity) over time. At present, fish faunas of different med-regions have widespread species in common (e.g. Gambusia holbrooki, Cyprinus carpio, Oncorhynchus mykiss, Carassius auratus, and Micropterus salmoides) which are typically large-bodied, non-migratory, have higher physiological tolerance, and display fast population growth rates. Our findings suggest that intentional and accidental introductions of freshwater fish have dissolved dispersal barriers and significantly changed the present-day biogeography of med-regions across the globe. Conservation challenges in med-regions include understanding the ecosystem consequences of non-native species introductions at macro-ecological scales.     

Resveratrol Couples Apoptosis with Autophagy in UVB-Irradiated HaCaT Cells

UVB radiation causes about 90% of non-melanoma skin cancers by damaging DNA either directly or indirectly by increasing levels of reactive oxygen species (ROS). Skin, chronically exposed to both endogenous and environmental pro-oxidant agents, contains a well-organised system of chemical and enzymatic antioxidants. However, increased or prolonged free radical action can overwhelm ROS defence mechanisms, contributing to the development of cutaneous diseases. Thus, new strategies for skin protection comprise the use of food antioxidants to counteract oxidative stress. Resveratrol, a phytoalexin from grape, has gained a great interest for its ability to influence several biological mechanisms like redox balance, cell proliferation, signal transduction pathways, immune and inflammatory response. Therefore, the potential of resveratrol to modify skin cell response to UVB exposure could turn out to be a useful option to protect skin from sunlight-induced degenerative diseases. To investigate into this matter, HaCaT cells, a largely used model for human skin keratinocytes, were treated with 25 or 100 µM resveratrol for 2 and 24 hours prior to UVB irradiation (10 to 100 mJ/cm²). Cell viability and molecular markers of proliferation, oxidative stress, apoptosis, and autophagy were analyzed. In HaCaT cells resveratrol pretreatment: reduces UVB-induced ROS formation, enhances the detrimental effect of UVB on HaCaT cell vitality, increases UVB-induced caspase 8, PARP cleavage, and induces autophagy. These findings suggest that resveratrol could exert photochemopreventive effects by enhancing UVB-induced apoptosis and by inducing autophagy, thus reducing the odds that damaged cells could escape programmed cell death and initiate malignant transformation.     

Inhibiting the Growth of Pancreatic Adenocarcinoma In Vitro and In Vivo through Targeted Treatment with Designer Gold Nanotherapeutics

Background

Pancreatic cancer is one of the deadliest of all human malignancies with limited options for therapy. Here, we report the development of an optimized targeted drug delivery system to inhibit advanced stage pancreatic tumor growth in an orthotopic mouse model.

Method/Principal Findings

Targeting specificity in vitro was confirmed by preincubation of the pancreatic cancer cells with C225 as well as Nitrobenzylthioinosine (NBMPR - nucleoside transporter (NT) inhibitor). Upon nanoconjugation functional activity of gemcitabine was retained as tested using a thymidine incorporation assay. Significant stability of the nanoconjugates was maintained, with only 12% release of gemcitabine over a 24-hour period in mouse plasma. Finally, an in vivo study demonstrated the inhibition of tumor growth through targeted delivery of a low dose of gemcitabine in an orthotopic model of pancreatic cancer, mimicking an advanced stage of the disease.

Conclusion

We demonstrated in this study that the gold nanoparticle-based therapeutic containing gemcitabine inhibited tumor growth in an advanced stage of the disease in an orthotopic model of pancreatic cancer. Future work would focus on understanding the pharmacokinetics and combining active targeting with passive targeting to further improve the therapeutic efficacy and increase survival.     

Identification and Functional Characterization of Three NoLS (Nucleolar Localisation Signals) Mutations of the CDC73 Gene

Hyperparathyroidism Jaw-Tumour Syndrome (HPT-JT) is characterized by primary hyperparathyroidism (PHPT), maxillary/mandible ossifying fibromas and by parathyroid carcinoma in 15% of cases. Inactivating mutations of the tumour suppressor CDC73/HRPT2 gene have been found in HPT-JT patients and also as genetic determinants of sporadic parathyroid carcinoma/atypical adenomas and, rarely, typical adenomas, in familial PHPT. Here we report the genetic and molecular analysis of the CDC73/HRPT2 gene in three patients affected by PHPT due to atypical and typical parathyroid adenomas, in one case belonging to familial PHPT. Flag-tagged WT and mutant CDC73/HRPT2 proteins were transiently transfected in HEK293 cells and functional assays were performed in order to investigate the effect of the variants on the whole protein expression, nuclear localization and cell overgrowth induction. We identified four CDC73/HRPT2 gene mutations, three germline (c.679_680delAG, p.Val85_Val86del and p.Glu81_Pro84del), one somatic (p.Arg77Pro). In three cases the mutation was located within the Nucleolar Localisation Signals (NoLS). The three NoLS variants led to instability either of the corresponding mutated protein or mRNA or both. When transfected in HEK293 cells, NoLS mutated proteins mislocalized with a predeliction for cytoplasmic or nucleo-cytoplasmic localization and, finally, they resulted in overgrowth, consistent with a dominant negative interfering effect in the presence of the endogenous protein.