Clogging the Ubiquitin-Proteasome Machinery with Marine Natural Products: Last Decade Update.

The ubiquitin-proteasome pathway (UPP) is the central protein degradation system in eukaryotic cells, playing a key role in homeostasis maintenance, through proteolysis of regulatory and misfolded (potentially harmful) proteins. As cancer cells produce proteins inducing cell proliferation and inhibiting cell death pathways, UPP inhibition has been exploited as an anticancer strategy to shift the balance between protein synthesis and degradation towards cell death. Over the last few years, marine invertebrates and microorganisms have shown to be an unexhaustive factory of secondary metabolites targeting the UPP. These chemically intriguing compounds can inspire clinical development of novel antitumor drugs to cope with the incessant outbreak of side effects and resistance mechanisms induced by currently approved proteasome inhibitors (e.g., bortezomib). In this review, we report about (a) the role of the UPP in anticancer therapy, (b) chemical and biological properties of UPP inhibitors from marine sources discovered in the last decade, (c) high-throughput screening techniques for mining natural UPP inhibitors in organic extracts. Moreover, we will tell about the fascinating story of salinosporamide A, the first marine natural product to access clinical trials as a proteasome inhibitor for cancer treatment

Correlation between hormonal homeostasis and morphogenic responses in Arabidopsis thaliana seedlings growing in a Cd/Cu/Zn multi-pollution context

To date, almost no information is available in roots and shoots of the model plant Arabidopsis thaliana about the hierarchic relationship between metal accumulation, phytohormone levels, and glutathione/phytochelatin content, and how this relation affects root development. For this purpose, specific concentrations of cadmium, copper and zinc, alone or in triple combination, were supplied for 12 days to in vitro growing seedlings. The accumulation of these metals was measured in roots and shoots, and a significant competition in metal uptake was observed. Microscopic analyses revealed that root morphology was affected by metal exposure, and that the levels of trans-zeatin riboside, dihydrozeatin riboside, indole-3-acetic acid, and the auxin/cytokinin ratio varied accordingly. By contrast, under metal treatments, minor modifications in gibberellic acid and abscisic acid levels occurred. RT-PCR analysis of some genes involved in auxin and cytokinin synthesis (e.g., AtNIT in roots and AtIPT in shoots) showed on average a metal up-regulated transcription. The production of thiol-peptides was induced by all the metals, alone or in combination, and the expression of the genes involved in thiol-peptide synthesis (AtGSH1, AtGSH2, AtPCS1 and AtPCS2) was not stimulated by the metals, suggesting a full post-transcriptional control. Results show that the Cd/Cu/Zn-induced changes in root morphology are caused by a hormonal unbalance, mainly governed by the auxin/cytokinin ratio

Deferasirox drives ROS-mediated differentiation and induces interferon-stimulated gene expression in human healthy haematopoietic stem/progenitor cells and in leukemia cells

Background: Administration of the iron chelator deferasirox (DFX) in transfusion-dependent patients occasionally results in haematopoiesis recovery by a mechanism remaining elusive. This study aimed to investigate at a molecular level a general mechanism underlying DFX beneficial effects on haematopoiesis, both in healthy and pathological conditions. Methods: Human healthy haematopoietic stem/progenitor cells (HS/PCs) and three leukemia cell lines were treated with DFX. N-Acetyl cysteine (NAC) and fludarabine were added as antioxidant and STAT1 inhibitor, respectively. In vitro colony-forming assays were assessed both in healthy and in leukemia cells. Intracellular and mitochondrial reactive oxygen species (ROS) as well as mitochondrial content were assessed by cytofluorimetric and confocal microscopy analysis; mtDNA was assessed by qRT-PCR. Differentiation markers were monitored by cytofluorimetric analysis. Gene expression analysis (GEA) was performed on healthy HS/PCs, and differently expressed genes were validated in healthy and leukemia cells by qRT-PCR. STAT1 expression and phosphorylation were assessed by Western blotting. Data were compared by an unpaired Student t test or one-way ANOVA. Results: DFX, at clinically relevant concentrations, increased the clonogenic capacity of healthy human CD34+ HS/PCs to form erythroid colonies. Extension of this analysis to human-derived leukemia cell lines Kasumi-1, K562 and HL60 confirmed DFX capacity to upregulate the expression of specific markers of haematopoietic commitment. Notably, the abovementioned DFX-induced effects are all prevented by the antioxidant NAC and accompanied with overproduction of mitochondria-generated reactive oxygen species (ROS) and increase of mitochondrial content and mtDNA copy number. GEA unveiled upregulation of genes linked to interferon (IFN) signalling and tracked back to hyper-phosphorylation of STAT1. Treatment of leukemic cell lines with NAC prevented the DFX-mediated phosphorylation of STAT1 as well as the expression of the IFN-stimulated genes. However, STAT1 inhibition by fludarabine was not sufficient to affect differentiation processes in leukemic cell lines. Conclusions: These findings suggest a significant involvement of redox signalling as a major regulator of multiple DFX-orchestrated events promoting differentiation in healthy and tumour cells. The understanding of molecular mechanisms underlying the haematological response by DFX would enable to predict patient's ability to respond to the drug, to extend treatment to other patients or to anticipate the treatment, regardless of the iron overload

po 031 naa induces antitumoral effects in bxpc3 pancreatic cancer cell line

Introduction Pancreatic adenocarcinoma is a tumour with poor prognosis. Usually diagnosed at a late stage, the high mortality is linked to resistance to conventional chemotherapy. Combination therapy and targeted therapies proved to be not very effective. Thus, a better understanding of the molecular mechanisms underlying drug resistance in pancreatic cancer could lead to the development of more effective therapeutic strategies. Material and methods BX-PC3 pancreatic tumour cells were treated with increasing doses of NAA (2,4,8 and 16 mM) for 72 hour and cell viability was assessed by xCELLigence system technology. The gene expression profile induced by NAA treatment in BX-PC3 cells was examined using Real-Time qPCR. Anti-proliferative and differentiating effects of NAA in BX-PC3 treated cells were evaluated by flow cytometric analysis. Acetyl CoA levels after 72 hour NAA treatment was mesured by HPLC/HRMS. The expression of proteins involved in acetylation mechanism were measured by Western Blotting. The metabolic analysis were performed by the Seahorse Bioanalyzer. The effects of NAA in 3D cultures were studied morphologically by inverted microscope. Results and discussions NAA treatment in BX-PC3 pancreatic tumour cells elicited anti-proliferative and differentiating effects evident with the arrest of proliferation and decreased expression of specific stemness markers such as cMyc, Klf4, Lin28 and Oct4 . Exposure of cells to NAA induced down-regulation of CD133 and CD184 surface markers, arrest of cell cyle at G0/G1 phase, associated to increased levels of p53 , p21 and p27 genes. Moreover, NAA-treated BX-PC3 cells showed decreased levels of the central metabolite Coenzyme A, which correlates with alterations in protein acetylation. In addition, an overall impairment of mitochondrial function was observed following NAA treatment, resulting in a revised feeding of metabolic substrates. Finally, NAA showed a strong effect on tumour spheroid growth, with reduction in colony size. Conclusion To our knowledge, this is the first study that demonstrates the differentiating effects of NAA treatment in pancreatic tumour cells and its ability to reduce the size of 3D pancreatic carcinoma spheroids

po 268 adipose derived mesenchymal stem cells microenvironment promotes the tumorigenic phenotype in triple negative breast cancer cell line

Introduction Breast cancer is one of the common cause of cancer-related deaths in women, and the vast majority of breast cancer-related deaths involve metastatic disease. Thus, an understanding of the molecular and cellular mechanism supporting tumour progression is essential for developing targeted treatments. Recently, several studies have recognised the critical role of the metabolic setting and of microenvironmental cues in tumour development and therapeutic responses. In the present study we investigated the tumor-stroma metabolic interplay in MDA-MB-231 (high metastatic) cultured with conditioned medium (CM) from mammary adipose tissue-derived mesenchymal stem cells (MSC) either under high (HG) and low glucose (LG) regimen. Material and methods Metabolic fluxes analyses were performed with Seahorse Bioanalyzer providing both mitochondrial respiratory activity, as oxygen consumption rate (OCR), and glycolysis-related extracellular acidification rate (ECAR). Live cell imaging for mitochondrial membrane potential Δψ m , was performed by confocal microscopy by using TMRE as selective probe. Cell migration was evaluated by scratch assay monitoring the wound's closure after 24 hour. Results and discussions Metabolic fluxes analysis showed that HG growth condition caused a significant reduction of OCR and ECAR in MDA cell line as well as in MSC. MDA grown in MSC-derived CM revealed a dampening of OCR and ECAR and this effect was attained irrespectively of the glucose concentration. Analysis of Δψ m showed a significant decrease only in MDA cultured with CM from HG-MSC. Intriguingly, in a specular setup MSC cultured with CM from theMDA, a significant reduction of ECAR, both in HG and LG was observed whereas OCR resulted to be strongly reduced only in HG. Our results would indicate a reciprocal interplay between the one of the most aggressive breast cancer cell line and MSC in rewiring the mitochondrial oxidative metabolism and likely the tumour phenotype. Consistently, in vitro scratch assay showed an increasing migration of MDA cultured with CM from MSC HG, thus suggesting a key role of MSC in facilitating cancer progression/invasiveness. Conclusion Our results strongly support the involvement of stromal MSC in tumour metabolism of malignant phenotypes. Further experiments are needed to define the factors released from MDA acting on MSC metabolism. Understanding these interactions is fundamental to develop therapeutic interventions addressing the MSC-tumour interaction

po 238 dichloroacetate dca treatment affects mitochondrial activity and stemness in pancreatic cancer pc cell lines

Introduction Targeting metabolism represents a new approach to treat cancer, expecially when conventional chemoterapy fails. In this study, we tested a metabolic approach to treat PC, investigating, in vitro and in vivo, its response to DCA treatment. Material and methods Two PC cell lines, BXPC3 and PANC1, were treated with DCA 4 and 10 mM for 24 hour. Cell viability and proliferation were assessed by MTS assay and xCELLigence, apoptosis and ROS by flow-cytometry; pPDH Ser293 /tot PDH, LC3B, DRP1, MFN1, MNF2, OPA1 and TOMM20 protein expression was evaluated by western blotting, lin28 gene expression by qPCR. The oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) were measured by Seahorse Technology. Ultra-low attachment plates were used to form spheroids. In vivo, DCA was administered to BXPC3-luc tumor-bearing nude mice. After measuring bioluminescence signalling, the tumour masses were harvested, photographed and weighed. Results and discussions DCA treatment reduced cell proliferation, decreasing cell survival with an increase in ROS production and apoptosis in both cell lines. Despite PDH activation by dephosphorylation, DCA did not restore bioenergetic profile but decreased OCR, a measure of oxidative phosphorylation efficiency. ECAR was not affected, suggesting that the glicolytic capacity was not modified by DCA treatment. These observations led us to explore mitophagy, whose activation was confirmed by LC3B protein overexpression and TOMM20 downregulation, and mitochondrial dynamics also altered following DCA treatment as shown by the downregulation of MFN1, MFN2, OPA1, key proteins of mitochondrial fusion. Interestingly, DCA was able to negatively affect the cancer stem cell (CSCs) fraction in both cell lines, reducing the expression of stemness genes such as Lin28 and inhibiting spheroid formation. When added to 3D cultures already formed, it was able to downregulate stemness genes expression, leading to a significant size reduction and affecting spheroid viability. Finally, DCA efficacy was confirmed in a xenograft pancreatic cancer mouse model in which DCA treatment displayed a significant retarded progression of PC, reducing diameter of the tumour mass. Conclusion Our data suggest that DCA is able to strongly affect PC cells metabolism counteracting mitochondrial activity. This effect is not related to PDH activity stimulation. In addition, the ability of DCA to hit CSCs offers a further rationale to candidate this drug for PC treatment, trying to reach a complete tumour eradication

po 266 metabolic profiling of osteosarcoma cancer stem cells as tool to identify potential target for cancer therapy

Introduction Recently, several studies have highlighted the key role of cancer stem cells (CSC) in tumour initiation, metastasis, and relapses. The CSC pool generally exhibits higher resistance to conventional chemo-radiotherapy and a different cell metabolism. Aim of our study is to investigate the main metabolic differences in the human osteosarcoma stem-like cells (3ABOS) and differentiated osteosarcoma cells (MG63) to unveil new metabolic therapeutic targets. Material and methods Metabolic analyses were performed with Seahorse Bioanalyzer. Live cell imaging for ROS content, mitochondrial membrane potential and morphology, were performed by confocal microscopy by using DCF-DA, Mito-Tracker Red and NAO as selective probes, respectively. Protein expression was revealed by qPCR and western blot. Results and discussions Our results showed a significant reduction of the mitochondrial oxygen consumption rate in 3ABOS compared with MG63 cells. Next, we assessed the specific contributions of glucose, fatty acid and glutamine to the respiratory phenotype, unveiling larger reliance on oxidation of these three main fuels with a significant reduction in mitochondrial flexibility in 3ABOS. The lower OXPHOS is compensated by a shift in glucose metabolism demonstrated by increased extracellular acidification rate.These results were further supported by a significant reduction of 3ABOS proliferation in glucose shortage. According to this scenario, confocal microscopy highlighted reduced mitochondrial membrane potential, and increased ROS content in 3ABOS compared to MG63. Additionally, 3ABOS displayed a lower mitochondrial DNA amount associated with more elongated mitochondrial network confirmed by both live cell imaging and Mitofusin expression analysis. Moreover, members of the NADPH oxidases family resulted to be differently expressed in the two cell lines, thus suggesting a potential role of ROS mediated signalling in cancer cell phenotype. Conclusion Overall our results demonstrated that the oxidative metabolic phenotype hallmarks cancer biology. Further investigations are ongoing to define specific drugs acting on metabolic target and their effectiveness as a therapeutic approach

po 213 high glucose affects er breast cancer cell metabolism

Introduction In vitro high glucose (HG) level, mimicking hyperglycemia condition in vivo , has been reported to influence breast cancer cells growth, proliferation and survival suggesting glucose as being crucial for breast cancer progression and response to therapy in diabetic patients. Diabetes, in turn, might have a direct effect on breast cancer prognosis. This study investigated the impact of HG on MCF-7 breast cancer cell metabolism and phenotype. Material and methods MCF-7 breast cancer cell line were cultured in DMEM with high glucose (HG 25 mM) and low glucose (LG 5 mM). Metabolic fluxes analyses were performed with Seahorse Bioanalyzer. Live cell imaging for reactive oxygen species (ROS) content was performed by confocal microscopy by using DCF-DA as selective probe. Mitochondrial DNA (mtDNA) and protein expression were evaluated by qPCR and western blotting respectively. Results and discussions Using a metabolic fluxes analyses, we showed a significant reduction of the mitochondrial oxygen consumption rate (OCR) and glycolysis-related extracellular acidification rate (ECAR) in MCF-7 cultured in HG- as compared in LG-medium. According with these results, MCF-7 in HG displayed lower mtDNA amount and increased ROS level. Furthermore, the analysis of stemness markers revealed a significant upregulation of Nanog, Lin28 and Myc thus suggesting an increased stem-like phenotype due to growth in HG. Conclusion Overall our results indicate that glucose may foster breast cancer progression promoting stem cell-like phenotype strongly affecting the metabolic profile in MCF-7 cell line. Further investigations are ongoing to define the mechanism underlying the switch towards an undifferentiated state to be exploited as therapeutic target