Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

Bergamot natural products eradicate cancer stem cells (CSCs) by targeting mevalonate, Rho-GDI-signalling and mitochondrial metabolism.

Here, we show that a 2:1 mixture of Brutieridin and Melitidin, termed "BMF", has a statin-like properties, which blocks the action of the rate-limiting enzyme for mevalonate biosynthesis, namely HMGR (3-hydroxy-3-methylglutaryl-CoA-reductase). Moreover, our results indicate that BMF functionally inhibits several key characteristics of CSCs. More specifically, BMF effectively i) reduced ALDH activity, ii) blocked mammosphere formation and iii) inhibited the activation of CSC-associated signalling pathways (STAT1/3, Notch and Wnt/beta-catenin) targeting Rho-GDI-signalling. In addition, BMF metabolically inhibited mitochondrial respiration (OXPHOS) and fatty acid oxidation (FAO). Importantly, BMF did not show the same toxic side-effects in normal fibroblasts that were observed with statins. Lastly, we show that high expression of the mRNA species encoding HMGR is associated with poor clinical outcome in breast cancer patients, providing a potential companion diagnostic for BMF-directed personalized therapy.

Insulin protects acinar cells during pancreatitis by preserving glycolytic ATP supply to calcium pumps.

Acute pancreatitis (AP) is serious inflammatory disease of the pancreas. Accumulating evidence links diabetes with severity of AP, suggesting that endogenous insulin may be protective. We investigated this putative protective effect of insulin during cellular and in vivo models of AP in diabetic mice (Ins2Akita) and Pancreatic Acinar cell-specific Conditional Insulin Receptor Knock Out mice (PACIRKO). Caerulein and palmitoleic acid (POA)/ethanol-induced pancreatitis was more severe in both Ins2Akita and PACIRKO vs control mice, suggesting that endogenous insulin directly protects acinar cells in vivo. In isolated pancreatic acinar cells, insulin induced Akt-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) which upregulated glycolysis thereby preventing POA-induced ATP depletion, inhibition of the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) and cytotoxic Ca2+ overload. These data provide the first mechanistic link between diabetes and severity of AP and suggest that phosphorylation of PFKFB2 may represent a potential therapeutic strategy for treatment of AP.

Mitochondrial Fission Factor (MFF) Inhibits Mitochondrial Metabolism and Reduces Breast Cancer Stem Cell (CSC) Activity.

Elevated mitochondrial biogenesis and metabolism represent key features of breast cancer stem cells (CSCs), whose propagation is conducive to disease onset and progression. Therefore, interfering with mitochondria biology and function may be regarded as a useful approach to eradicate CSCs. Here, we used the breast cancer cell line MCF7 as a model system to interrogate how mitochondrial fission contributes to the development of mitochondrial dysfunction toward the inhibition of metabolic flux and stemness. We generated an isogenic MCF7 cell line transduced with Mitochondrial Fission Factor (MCF7-MFF), which is primarily involved in mitochondrial fission. We evaluated the biochemical, molecular and functional properties of MCF7-MFF cells, as compared to control MCF7 cells transduced with the empty vector (MCF7-Control). We observed that MFF over-expression reduces both mitochondrial mass and activity, as evaluated using the mitochondrial probes MitroTracker Red and MitoTracker Orange, respectively. The analysis of metabolic flux using the Seahorse XFe96 revealed the inhibition of OXPHOS and glycolysis in MCF7-MFF cells, suggesting that increased mitochondrial fission may impair the biochemical properties of these organelles. Notably, CSCs activity, assessed by 3D-tumorsphere assays, was reduced in MCF7-MFF cells. A similar trend was observed for the activity of ALDH, a well-established marker of stemness. We conclude that enhanced mitochondrial fission may compromise CSCs propagation, through the impairment of mitochondrial function, possibly leading to a quiescent cell phenotype. Unbiased proteomic analysis revealed that proteins involved in mitochondrial dysfunction, oxidative stress-response, fatty acid metabolism and hypoxia signaling are among the most highly up-regulated in MCF7-MFF cells. Of note, integrated analysis of top regulatory networks obtained from unbiased proteomics in MCF7-MFF cells predicts that this cell phenotype activates signaling systems and effectors involved in the inhibition of cell survival and adhesion, together with the activation of specific breast cancer cell death programs. Overall, our study shows that unbalanced and abnormal activation of mitochondrial fission may drive the impairment of mitochondrial metabolic function, leading to inhibition of CSC propagation, and the activation of quiescence programs. Exploiting the potential of mitochondria to control pivotal events in tumor biology may, therefore, represent a useful tool to prevent disease progression.

Connexin43 is involved in the effect of endothelin-1 on astrocyte proliferation and glucose uptake.

In previous studies, we showed that endothelin-1 increased astrocyte proliferation and glucose uptake. These effects were similar to those observed with other gap junction inhibitors, such as carbenoxolone (CBX). Because 24-h treatment with endothelin-1 or CBX downregulates the expression of connexin43, the main protein forming astrocytic gap junctions, which can also be involved in proliferation, in this study, we addressed the possible role of connexin43 in the effects of endothelin-1. To do so, connexin43 was silenced in astrocytes by siRNA. The knock down of connexin43 increased the rate of glucose uptake, characterized by the upregulation of GLUT-1 and type I hexokinase. Neither endothelin-1 nor CBX were able to further increase the rate of glucose uptake in connexin43-silenced astrocytes. In agreement, no effects of endothelin-1 and CBX on GLUT-1 and type I hexokinase were observed in connexin-43 silenced astrocytes or in astrocytes from connexin43 knock-out (KO) mice. Our previous studies suggested a close relationship between glucose uptake and astrocyte proliferation. Consistent with this, connexin43-silenced astrocytes exhibited an increase in Ki-67, a marker of proliferation. The effects of ET-1 on retinoblastoma phosphorylation on Ser780 and on the upregulation of cyclins D1 and D3 were affected by the levels of connexin43. In conclusion, our results indicate that connexin43 participates in the effects of endothelin-1 on glucose uptake and proliferation in astrocytes. Interestingly, although the rate of growth in connexin43 KO astrocytes has been reported to be reduced, we observed that an acute reduction in connexin43 by siRNA increased proliferation and glucose uptake.

The ER-alpha mutation Y537S confers Tamoxifen-resistance via enhanced mitochondrial metabolism, glycolysis and Rho-GDI/PTEN signaling: Implicating TIGAR in somatic resistance to endocrine therapy.

Naturally-occurring somatic mutations in the estrogen receptor gene (ESR1) have been previously implicated in the clinical development of resistance to hormonal therapies, such as Tamoxifen. For example, the somatic mutation Y537S has been specifically associated with acquired endocrine resistance. Briefly, we recombinantly-transduced MCF7 cells with a lentiviral vector encoding ESR1 (Y537S). As a first step, we confirmed that MCF7-Y537S cells are indeed functionally resistant to Tamoxifen, as compared with vector alone controls. Importantly, further phenotypic characterization of Y537S cells revealed that they show increased resistance to Tamoxifen-induced apoptosis, allowing them to form mammospheres with higher efficiency, in the presence of Tamoxifen. Similarly, Y537S cells had elevated basal levels of ALDH activity, a marker of "stemness", which was also Tamoxifen-resistant. Metabolic flux analysis of Y537S cells revealed a hyper-metabolic phenotype, with significantly increased mitochondrial respiration and high ATP production, as well as enhanced aerobic glycolysis. Finally, to understand which molecular signaling pathways that may be hyper-activated in Y537S cells, we performed unbiased label-free proteomics analysis. Our results indicate that TIGAR over-expression and the Rho-GDI/PTEN signaling pathway appear to be selectively activated by the Y537S mutation. Remarkably, this profile is nearly identical in MCF7-TAMR cells; these cells were independently-generated in vitro, suggesting a highly conserved mechanism underlying Tamoxifen-resistance. Importantly, we show that the Y537S mutation is specifically associated with the over-expression of a number of protein markers of poor clinical outcome (COL6A3, ERBB2, STAT3, AFP, TFF1, CDK4 and CD44). In summary, we have uncovered a novel metabolic mechanism leading to endocrine resistance, which may have important clinical implications for improving patient outcomes.

Targeting flavin-containing enzymes eliminates cancer stem cells (CSCs), by inhibiting mitochondrial respiration: Vitamin B2 (Riboflavin) in cancer therapy.

Here, we performed high-throughput drug-screening to identify new non-toxic mitochondrial inhibitors. This screening platform was specifically designed to detect compounds that selectively deplete cellular ATP levels, but have little or no toxic side effects on cell viability. Using this approach, we identified DPI (Diphenyleneiodonium chloride) as a new potential therapeutic agent. Mechanistically, DPI potently blocks mitochondrial respiration by inhibiting flavin-containing enzymes (FMN and FAD-dependent), which form part of Complex I and II. Interestingly, DPI induced a chemo-quiescence phenotype that potently inhibited the propagation of CSCs, with an IC-50 of 3.2 nano-molar. Virtually identical results were obtained using CSC markers, such as CD44 and CD24. We further validated the effects of DPI on cellular metabolism. At 10 nM, DPI inhibited oxidative mitochondrial metabolism (OXPHOS), reducing mitochondrial driven ATP production by >90%. This resulted in a purely glycolytic phenotype, with elevated L-lactate production. We show that this metabolic inflexibility could be rapidly-induced, after only 1 hour of DPI treatment. Remarkably, the mitochondrial inhibitory effects of DPI were reversible, and DPI did not induce ROS production. Cells maintained in DPI for 1 month showed little or no mitochondrial activity, but remained viable. Thus, it appears that DPI behaves as a new type of mitochondrial inhibitor, which maintains cells in a state of metabolic-quiescence or "suspended animation".In conclusion, DPI treatment can be used to acutely confer a mitochondrial-deficient phenotype, which we show effectively depletes CSCs from the heterogeneous cancer cell population. These findings have significant therapeutic implications for potently targeting CSCs, while minimizing toxic side effects. We also discuss the possible implications of DPI for the aging process. Interestingly, previous studies in C. elegans have shown that DPI prevents the accumulation of lipofuscin (an aging-associated hallmark), during the response to oxidative stress. Our current results are consistent with data showing that flavins (FAD, FMN and/or Riboflavin) are auto-fluorescent markers of i) increased mitochondrial "power" (OXPHOS) and ii) elevated CSC activity.Finally, we believe that DPI is one of the most potent and highly selective CSC inhibitors discovered to date. Therefore, our current findings suggest a new impetus to create novel analogues of i) DPI (Diphenyleneiodonium chloride) and ii) DPI-related compounds (Diphenyliodonium chloride), using medicinal chemistry, to optimize this very promising and potent anti-CSC activity. We propose to call these new molecules "Mitoflavoscins".For example, DPI is ~30 times more potent than Palbociclib (IC-50 = 100 nM), which is an FDA-approved CDK4/6 inhibitor, that broadly targets proliferation in any cell type, including CSCs.

The increase in gap junctional communication decreases the rate of glucose uptake in C6 glioma cells by releasing hexokinase from mitochondria.

We have previously shown that the enhancement of glucose uptake caused by the inhibition of gap junctional communication is a consequence of the increase in astrocyte proliferation. Since C6 glioma cells are highly proliferative and are poorly coupled through gap junctions, we used these cells to investigate the effect of increasing gap junctional communication on the rate of glucose uptake. Previous work by us had shown that tolbutamide increases gap junctional communication in C6 glioma cells, as does dbcAMP, a classical activator of gap junctional communication. In this work, our results show that both tolbutamide and dbcAMP reduce the rate of glucose uptake in C6 glioma cells and that their effects are additive. The main glucose transporters expressed in C6 glioma cells are GLUT-1 and GLUT-3. Neither the expression nor the cellular localization of either GLUT-1 or GLUT-3 were modified by increasing gap junctional communication. The estimation of glucose uptake with 2-deoxyglucose includes not only glucose transport but also glucose phosphorylation, which in C6 glioma cells is mainly catalyzed by type I and type II hexokinase. Our results reveal that the increase in gap junctional communication caused by tolbutamide and dbcAMP is associated with a decrease in the activity of hexokinase. In agreement with this, tolbutamide and dbcAMP caused a rapid change in the localization of both type I and type II hexokinase, which were detached from the mitochondria to the cytosol.

Mitochondrial biogenesis is required for the anchorage-independent survival and propagation of stem-like cancer cells.

Here, we show that new mitochondrial biogenesis is required for the anchorage independent survival and propagation of cancer stem-like cells (CSCs). More specifically, we used the drug XCT790 as an investigational tool, as it functions as a specific inhibitor of the ERRα-PGC1 signaling pathway, which governs mitochondrial biogenesis. Interestingly, our results directly demonstrate that XCT790 efficiently blocks both the survival and propagation of tumor initiating stem-like cells (TICs), using the MCF7 cell line as a model system. Mechanistically, we show that XCT790 suppresses the activity of several independent signaling pathways that are normally required for the survival of CSCs, such as Sonic hedgehog, TGFß-SMAD, STAT3, and Wnt signaling. We also show that XCT790 markedly reduces oxidative mitochondrial metabolism (OXPHOS) and that XCT790-mediated inhibition of CSC propagation can be prevented or reversed by Acetyl-L-Carnitine (ALCAR), a mitochondrial fuel. Consistent with our findings, over-expression of ERRα significantly enhances the efficiency of mammosphere formation, which can be blocked by treatment with mitochondrial inhibitors. Similarly, mammosphere formation augmented by FOXM1, a downstream target of Wnt/ß-catenin signaling, can also be blocked by treatment with three different classes of mitochondrial inhibitors (XCT790, oligomycin A, or doxycycline). In this context, our unbiased proteomics analysis reveals that FOXM1 drives the expression of >90 protein targets associated with mitochondrial biogenesis, glycolysis, the EMT and protein synthesis in MCF7 cells, processes which are characteristic of an anabolic CSC phenotype. Finally, doxycycline is an FDA-approved antibiotic, which is very well-tolerated in patients. As such, doxycycline could be re-purposed clinically as a 'safe' mitochondrial inhibitor, to target FOXM1 and mitochondrial biogenesis in CSCs, to prevent tumor recurrence and distant metastasis, thereby avoiding patient relapse.

JNK1 stress signaling is hyper-activated in high breast density and the tumor stroma: connecting fibrosis, inflammation, and stemness for cancer prevention.

Mammography is an important screening modality for the early detection of DCIS and breast cancer lesions. More specifically, high mammographic density is associated with an increased risk of breast cancer. However, the biological processes underlying this phenomenon remain largely unknown. Here, we re-interrogated genome-wide transcriptional profiling data obtained from low-density (LD) mammary fibroblasts (n = 6 patients) and high-density (HD) mammary fibroblasts (n = 7 patients) derived from a series of 13 female patients. We used these raw data to generate a "breast density" gene signature consisting of>1250 transcripts that were significantly increased in HD fibroblasts, relative to LD fibroblasts. We then focused on the genes that were increased by ≥ 1.5-fold (P<0.05) and performed gene set enrichment analysis (GSEA), using the molecular signatures database (MSigDB). Our results indicate that HD fibroblasts show the upregulation and/or hyper-activation of several key cellular processes, including the stress response, inflammation, stemness, and signal transduction. The transcriptional profiles of HD fibroblasts also showed striking similarities to human tumors, including head and neck, liver, thyroid, lung, and breast cancers. This may reflect functional similarities between cancer-associated fibroblasts (CAFs) and HD fibroblasts. This is consistent with the idea that the presence of HD fibroblasts may be a hallmark of a pre-cancerous phenotype. In these biological processes, GSEA predicts that several key signaling pathways may be involved, including JNK1, iNOS, Rho GTPase(s), FGF-R, EGF-R, and PDGF-R-mediated signal transduction, thereby creating a pro-inflammatory, pro-proliferative, cytokine, and chemokine-rich microenvironment. HD fibroblasts also showed significant overlap with gene profiles derived from smooth muscle cells under stress (JNK1) and activated/infected macrophages (iNOS). Thus, HD fibroblasts may behave like activated myofibroblasts and macrophages, to create and maintain a fibrotic and inflammatory microenvironment. Finally, comparisons between the HD fibroblast gene signature and breast cancer tumor stroma revealed that JNK1 stress signaling is the single most significant biological process that is shared between these 2 data sets (with P values between 5.40E-09 and 1.02E-14), and is specifically associated with tumor recurrence. These results implicate "stromal JNK1 signaling" in the pathogenesis of human breast cancers and the transition to malignancy. Augmented TGF-ß signaling also emerged as a common feature linking high breast density with tumor stroma and breast cancer recurrence (P = 5.23E-05). Similarities between the HD fibroblast gene signature, wound healing, and the cancer-associated fibroblast phenotype were also noted. Thus, this unbiased informatics analysis of high breast density provides a novel framework for additional experimental exploration and new hypothesis-driven breast cancer research, with a focus on cancer prevention and personalized medicine.

Ethanol diverts early neuronal differentiation trajectory of embryonic stem cells by disrupting the balance of lineage specifiers.

BACKGROUND: Ethanol is a toxin responsible for the neurodevelopmental deficits of Fetal Alcohol Spectrum Disorders (FASD). Recent evidence suggests that ethanol modulates the protein expression of lineage specifier transcription factors Oct4 (Pou5f1) and Sox2 in early stages of mouse embryonic stem (ES) cell differentiation. We hypothesized that ethanol induced an imbalance in the expression of Oct4 and Sox2 in early differentiation, that dysregulated the expression of associated and target genes and signaling molecules and diverted cells from neuroectodermal (NE) formation. METHODOLOGY/PRINCIPAL FINDINGS: We showed modulation by ethanol of 33 genes during ES cell differentiation, using high throughput microfluidic dynamic array chips measuring 2,304 real time quantitative PCR assays. Based on the overall gene expression dynamics, ethanol drove cells along a differentiation trajectory away from NE fate. These ethanol-induced gene expression changes were observed as early as within 2 days of differentiation, and were independent of cell proliferation or apoptosis. Gene expression changes were correlated with fewer ßIII-tubulin positive cells of an immature neural progenitor phenotype, as well as a disrupted actin cytoskeleton were observed. Moreover, Tuba1a and Gapdh housekeeping genes were modulated by ethanol during differentiation and were replaced by a set of ribosomal genes with stable expression. CONCLUSIONS/SIGNIFICANCE: These findings provided an ethanol-response gene signature and pointed to the transcriptional dynamics underlying lineage imbalance that may be relevant to FASD phenotype.

Ethanol exposure induces the cancer-associated fibroblast phenotype and lethal tumor metabolism: implications for breast cancer prevention.

Little is known about how alcohol consumption promotes the onset of human breast cancer(s). One hypothesis is that ethanol induces metabolic changes in the tumor microenvironment, which then enhances epithelial tumor growth. To experimentally test this hypothesis, we used a co-culture system consisting of human breast cancer cells (MCF7) and hTERT-immortalized fibroblasts. Here, we show that ethanol treatment (100 mM) promotes ROS production and oxidative stress in cancer-associated fibroblasts, which is sufficient to induce myofibroblastic differentiation. Oxidative stress in stromal fibroblasts also results in the onset of autophagy/mitophagy, driving the induction of ketone body production in the tumor microenvironment. Interestingly, ethanol has just the opposite effect in epithelial cancer cells, where it confers autophagy resistance, elevates mitochondrial biogenesis and induces key enzymes associated with ketone re-utilization (ACAT1/OXCT1). During co-culture, ethanol treatment also converts MCF7 cells from an ER(+) to an ER(-) status, which is thought to be associated with "stemness," more aggressive behavior and a worse prognosis. Thus, ethanol treatment induces ketone production in cancer-associated fibroblasts and ketone re-utilization in epithelial cancer cells, fueling tumor cell growth via oxidative mitochondrial metabolism (OXPHOS). This "two-compartment" metabolic model is consistent with previous historical observations that ethanol is first converted to acetaldehyde (which induces oxidative stress) and then ultimately to acetyl-CoA (a high-energy mitochondrial fuel), or can be used to synthesize ketone bodies. As such, our results provide a novel mechanism by which alcohol consumption could metabolically convert "low-risk" breast cancer patients to "high-risk" status, explaining tumor recurrence or disease progression. Hence, our findings have clear implications for both breast cancer prevention and therapy. Remarkably, our results also show that antioxidants [such as N-acetyl cysteine (NAC)] can effectively reverse or prevent ethanol-induced oxidative stress in cancer-associated fibroblasts, suggesting a novel strategy for cancer prevention. We also show that caveolin-1 and MCT4 protein expression can be effectively used as new biomarkers to monitor oxidative stress induced by ethanol.

Mitochondrial dysfunction in breast cancer cells prevents tumor growth: understanding chemoprevention with metformin.

Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak "mitochondrial poison," as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of "mitochondrial health" in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases ß-oxidation, ketone body production and the release of ATP-rich vesicles, which "fuels" tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.

Increased levels of cyclins D1 and D3 after inhibition of gap junctional communication in astrocytes.

We showed previously that the inhibition of gap junctional communication in astrocytes increased bromodeoxyuridine (BrdU) incorporation and promoted changes in the metabolic phenotype destined to fulfil the requirements of cell proliferation. In the present study we investigated the changes in the cell cycle of astrocytes promoted by the inhibition of intercellular communication through gap junctions. Thus, the presence of endothelin-1 and carbenoxolone, two gap junction uncouplers, promoted an increase in the percentage of astrocytes found in the S, G2 and M phases of the cell cycle, with a concomitant decrease in G0 and G1 phases. In addition, the levels of Ki-67, a protein present during all active phases of the cell cycle but absent from resting cells, increased after the inhibition of gap junctional communication. These effects were not observed when the inhibition of gap junctions was prevented with tolbutamide, indicating that the inhibition of gap junctional communication promotes the entry of astrocytes into the cell cycle. The passage of the cells from a quiescent state to the cell cycle is ultimately regulated by the degree of retinoblastoma phosphorylation. Inhibition of gap junctions increased the phosphorylation of retinoblastoma at Ser 780 but not at Ser 795 or Ser 807/811. In addition, the levels of cyclins D1 and D3 increased, whereas those of p21 and p27 were not significantly modified. Because D-type cyclins are key regulators of retinoblastoma protein phosphorylation, it is suggested that the phosphorylation of retinoblastoma protein at Ser 780, observed under our experimental conditions, is a consequence of the increase in the levels of cyclins D1 and D3. Our work provides evidence for the involvement of cyclins D1 and D3 as sensors of the inhibition of gap junctional communication in astrocytes.

Tolbutamide reduces glioma cell proliferation by increasing connexin43, which promotes the up-regulation of p21 and p27 and subsequent changes in retinoblastoma phosphorylation.

Our previous work has shown that tolbutamide increases gap junctional permeability in poorly coupled C6 glioma cells and that this effect is similar and additive to that found with dbcAMP, a well-known activator of gap junctional communication. Furthermore, the increase in gap junctional communication promoted by tolbutamide or dbcAMP is concurrent with the inhibition of proliferation of C6 glioma cells. In the present work, we show that tolbutamide and dbcAMP increase the synthesis of the tumor suppressor protein Cx43 and that they decrease the level of Ki-67, a protein expressed when cells are proliferating. These effects were accompanied by a reduction in the phosphorylation of pRb, mainly on Ser-795, a residue critical for the control of cell proliferation. The decrease in the phosphorylation of pRb is not likely to be mediated by a reduction in the levels of D-type cyclins, since instead of decreasing the expression of cyclins, D1 and D3 increased slightly after treatment with tolbutamide or dbcAMP. However, the Cdk inhibitors p21 and p27 were up-regulated after treatment with tolbutamide and dbcAMP, suggesting that they would be involved in the decrease in pRb phosphorylation. When Cx43 was silenced by siRNA, neither tolbutamide nor dbcAMP were able to up-regulate p21 and consequently to reduce glioma cell proliferation, as judged by Ki-67 expression. In conclusion, tolbutamide and dbcAMP inhibit C6-glioma cell proliferation by increasing Cx43, which correlates with a reduction in pRb phosphorylation due to the up-regulation of the Cdk inhibitors p21 and p27.

Endothelin-1 stimulates the translocation and upregulation of both glucose transporter and hexokinase in astrocytes: relationship with gap junctional communication.

We have previously shown that endothelin-1 increases glucose uptake in astrocytes. In the present work we investigate the mechanism through which endothelin-1 (ET-1) increases glucose uptake. Our results show that ET-1 activates a short-term and a long-term mechanism. Thus, ET-1 induced a rapid change in the localization of both GLUT-1 and type I hexokinase. These changes are probably aimed at rapidly increasing the entry and phosphorylation of glucose. In addition, ET-1 upregulated GLUT-1 and type I hexokinase and induced the expression of isoforms not normally expressed in astrocytes, such as GLUT-3 and type II hexokinase. These changes provide astrocytes with the machinery required to sustain a high rate of glucose uptake for a longer period of time. Our previous work had suggested that the effect of ET-1 on glucose uptake was associated with the inhibition of gap junctions. In this work, we compare the effect of ET-1 with that of carbenoxolone, a classical inhibitor of gap junction communication. Carbenoxolone increased glucose uptake to the same extent as ET-1 following the same mechanisms. Thus, carbenoxolone induced a rapid change in the localization of both GLUT-1 and type I hexokinase, upregulated GLUT-1 and type I hexokinase and induced the expression of GLUT-3 and type II hexokinase. When the inhibition of gap junction was prevented by tolbutamide, neither ET-1 nor carbenoxolone were able to increase the levels of GLUT-1, GLUT-3, type I hexokinase or type II hexokinase, indicating that these events are closely related to gap junctions.

Oxidative stress in preterm rat brain is due to mitochondrial dysfunction.

Prematurity-mediated cerebral damage has been associated with oxidative stress. The aim of the present work was to study the possible role played by free oxygen radicals generated by mitochondrial respiratory function in cerebral injury in preterm neonates. Our results show that whereas total glutathione concentrations are similar in term and preterm neonates, the GSH/GSSG ratio decreases sharply in preterm neonates immediately after birth. This effect is not due to a lack of enzymes involved in GSH regeneration, such as glutathione reductase and glucose-6-phosphate dehydrogenase, but to a significant increase in free-radical generation in preterm rat brain as shown by the increase in lipoperoxidation. Because the mitochondrion is the main source of free radicals in the cell, mitochondrial respiratory function was studied in the brain of preterm neonates. Our results show that prematurity prevented the postnatal increases in complex II-III activity and ATP concentrations that occur in term neonates at 5 min after delivery. All these effects were counteracted by the oxygen supply, suggesting that the inhibition of mitochondrial function is caused by restricted oxygen availability. Consequently, cerebral damage associated with prematurity may be mediated by mitochondrial free-radical generation as a consequence of hypoxia undergone by preterm neonates at birth.

Taninos de diferentes especies vegetales en la prevención del fotoenvejecimiento / Tannins from different vegetal species in the prevention of photoaging

La utilización de algunas sustancias naturales, en específico estructuras polifenólicas como las catequinas oligoméricas y flavonoides, ha demostrado ser una fuente de protección para el organismo. Son reconocidas sus propiedades antimicrobianas, antioxidantes, fotoprotectoras, así como inhibidores de proteasas como la elastasa. Dada la similitud estructural, fueron estudiados los taninos vegetales condensados de diferentes especies forestales a saber: pino, casuarina, mimosa, conos de pinos, eucalipto y soplillo. En los últimos años se ha prestado especial atención al estudio de nuevas sustancias con características fisicoquímicas capaces de prevenir trastornos que conducen a mutaciones cutáneas provocadas por las radiaciones ultravioletas; por causa fundamentalmente del incremento de la contaminación ambiental y el desgaste de la capa de ozono. El objetivo de este trabajo fue determinar la capacidad fotoprotectora de los taninos y dilucidar su posible mecanismo de acción. Para ello se realizaron 3 técnicas: fotoprotección en bacterias (E. coli); capacidad antioxidante espontánea e inducida en homogenato de cerebro de rata y actividad antielastasa. En los resultados se observó que los taninos de todas las especies vegetales eran capaces de proteger a las bacterias contra el daño de las radiaciones ultravioletas, lo que coincide con una buena actividad antioxidante y antielastasa; resultaron menores estas 2 últimas para el eucalipto

Caracterización de indicadores bioquímicos de estrés oxidativo en pacientes quemados muy graves / Characterization of biochemical indicators of oxidative stressin very critical burned patients

Se caracterizó el comportamiento de algunos indicadores de estrés oxidativo sistémico en 33 pacientes quemados muy graves, y se tomaron muestras de sangre venosa durante 4 puntos experimentales a partir de su admisión. Se cuantificaron las concentraciones de malonildialdehído, productos de oxidación de las proteínas, vitamina A y b-carotenos así como la actividad superóxido dismutasa, catalasa, fosfolipasa A2 (FLA2), elastasa y el porcentaje de inhibición de tripsina. En los pacientes quemados las concentraciones de malonildialdehído fueron mayores respecto al control en todos los tiempos. De igual manera se evidenciaron diferencias estadísticamente significativas entre los pacientes fallecidos y los sobrevivientes, y resultaron siempre mayores los valores de este indicador en los primeros. Las proteínas, también dianas del daño oxidativo, en general se encontraron elevadas en los pacientes quemados con respecto al control. La actividad de las enzimas FLA2 y elastasa fue mayor en los pacientes quemados que en los sujetos supuestamente sanos. Contrario a esto, la superóxido dismutasa exhibió una mayor actividad en estos últimos, sin mostrarse diferencias significativas con respecto a los pacientes quemados no fallecidos en los días 3 y 14. La actividad de la catalasa fue mayor en todos los tiempos de estudio en los pacientes que fallecieron respecto al control y a los que sobrevivieron a la agresión térmica. Estos últimos no fueron significativamente diferentes del control. Los valores de vitamina A fueron superiores en los pacientes quemados mientras que las concentraciones de b-carotenos fueron menores. El porcentaje de inhibición de tripsina fue mayor en los pacientes que no fallecieron respecto a los fallecidos y a los individuos que integraron el grupo control, este aumento fue significativo en los días 3 y 7. Los resultados permiten concluir que los indicadores de daño oxidativo aumentan en el paciente quemado muy grave y que los indicadores bioquímicos de defensa antioxidante se modifican con el tiempo de evolución