Hypoxia induces proteasome-dependent degradation of estrogen receptor alpha in ZR-75 breast cancer cells

Regulation of estrogen receptor alpha (ERalpha) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O(2)) or hypoxia (1% O(2) or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1alpha protein within 3 h after treatment, whereas ERalpha protein levels were dramatically decreased within 6-12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ERalpha mRNA; however, hypoxic conditions decreased basal and 17beta-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17beta-estradiol and hypoxia induce proteasome-dependent degradation of ERalpha, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.     

Delayed Turnover of Unphosphorylated Ssk1 during Carbon Stress Activates the Yeast Hog1 Map Kinase Pathway

In Saccharomyces cerevisiae, the Hog1 mitogen-activated protein kinase (MAPK) pathway coordinates the adaptation to osmotic stress and was recently reported to respond to acute changes in glucose levels. Similarly as in osmotic stress, glucose starvation leads to a transient accumulation of Hog1 in the nucleus. However, the kinetics and the mechanism of Hog1 activation are different for these stress conditions. During osmotic shock the activation of Hog1 can be transduced by either the Sho1 or the Sln1/Ypd1/Ssk1 branch. During glucose starvation the phosphorylation of Hog1 is slower and is completely dependent on Ssk1, but independent of Sho1. To characterize the mechanism of activation of Hog1 during carbon stress, we examined the turnover of Ssk1 protein levels upon glucose starvation in the presence of cycloheximide and monitored protein levels by western blotting. Our data demonstrate that unphosphorylated Ssk1 was quickly degraded during exponential growth and after osmotic stress but remained remarkably stable during glucose limitation. We conclude that glucose starvation induces a delay in the turnover of unphosphorylated Ssk1, which is sufficient to activate the Hog1 MAPK pathway. Although unphosphorylated Ssk1 is known to be degraded by the proteasome, its stabilization is apparently not due to changes in cellular localization or decrease in ubiquitination levels during glucose limitation.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

Intracellular localization of the proteasome in response to stress conditions

The ubiquitin–proteasome system fulfills an essential role in regulating protein homeostasis by spatially and temporally controlling proteolysis in an ATP- and ubiquitin-dependent manner. However, the localization of proteasomes is highly variable under diverse cellular conditions. In yeast, newly synthesized proteasomes are primarily localized to the nucleus during cell proliferation. Yeast proteasomes are transported into the nucleus through the nuclear pore either as immature subcomplexes or as mature enzymes via adapter proteins Sts1 and Blm10, while in mammalian cells, postmitotic uptake of proteasomes into the nucleus is mediated by AKIRIN2, an adapter protein essentially required for nuclear protein degradation. Stressful growth conditions and the reversible halt of proliferation, that is quiescence, are associated with a decline in ATP and the reorganization of proteasome localization. Cellular stress leads to proteasome accumulation in membraneless granules either in the nucleus or in the cytoplasm. In quiescence, yeast proteasomes are sequestered in an ubiquitin-dependent manner into motile and reversible proteasome storage granules in the cytoplasm. In cancer cells, upon amino acid deprivation, heat shock, osmotic stress, oxidative stress, or the inhibition of either proteasome activity or nuclear export, reversible proteasome foci containing polyubiquitinated substrates are formed by liquid–liquid phase separation in the nucleus. In this review, we summarize recent literature revealing new links between nuclear transport, ubiquitin signaling, and the intracellular organization of proteasomes during cellular stress conditions.     

Reactive oxygen species downregulate glucose transport system in retinal endothelial cells

Retinal endothelial cells are believed to play an important role in the pathogenesis of diabetic retinopathy. In previous studies, we and others demonstrated that glucose transporter 1 (GLUT1) is downregulated in response to hyperglycemia. Increased oxidative stress is likely to be the event whereby hyperglycemia is transduced into endothelial cell damage. However, the effects of sustained oxidative stress on GLUT1 regulation are not clearly established. The objective of this study is to evaluate the effect of increased oxidative stress on glucose transport and on GLUT1 subcellular distribution in a retinal endothelial cell line and to elucidate the signaling pathways associated with such regulation. Conditionally immortalized rat retinal endothelial cells (TR-iBRB) were incubated with glucose oxidase, which increases the intracellular hydrogen peroxide levels, and GLUT1 regulation was investigated. The data showed that oxidative stress did not alter the total levels of GLUT1 protein, although the levels of mRNA were decreased, and there was a subcellular redistribution of GLUT1, decreasing its content at the plasma membrane. Consistently, the half-life of the protein at the plasma membrane markedly decreased under oxidative stress. The proteasome appears to be involved in GLUT1 regulation in response to oxidative stress, as revealed by an increase in stabilization of the protein present at the plasma membrane and normalization of glucose transport following proteasome inhibition. Indeed, levels of ubiquitinated GLUT1 increase as revealed by immunoprecipitation assays. Furthermore, data indicate that protein kinase B activation is involved in the stabilization of GLUT1 at the plasma membrane. Thus subcellular redistribution of GLUT1 under conditions of oxidative stress is likely to contribute to the disruption of glucose homeostasis in diabetes.     

Rox3 and Rts1 Function in the Global Stress Response Pathway in Baker''s Yeast

Yeast respond to a variety of stresses through a global stress response that is mediated by a number of signal transduction pathways and the cis-acting STRE DNA sequence. The CYC7 gene, encoding iso-2-cytochrome c, has been demonstrated to respond to heat shock, glucose starvation, approach-to-stationary phase, and, as we demonstrate here, to osmotic stress. This response was delayed in a the hog1-Δ1 strain implicating the Hog1 mitogen-activated protein kinase cascade, a known component of the global stress response. Deletion analysis of the CYC7 regulatory region suggested that three STRE elements were each capable of inducing the stress response. Mutations in the ROX3 gene prevented CYC7 RNA accumulation during heat shock and osmotic stress. ROX3 RNA levels were shown to be induced by stress through a novel regulatory element. A selection for high-copy suppressors of a ROX3 temperature-sensitive allele resulted in the isolation of RTS1, encoding a protein with homology to the B' regulatory subunit of protein phosphatase 2A(0). Deletion of RTS1 caused temperature and osmotic sensitivity and increased accumulation of CYC7 RNA under all conditions. Over-expression of this gene caused increased CYC7 RNA accumulation in rox3 mutants but not in wild-type cells.     

The p53‐inducible long noncoding RNA TRINGS protects cancer cells from necrosis under glucose starvation

The tumor suppressor p53 is activated in response to cellular stress to prevent malignant transformation. However, several recent studies have shown that p53 can play protective roles in tumor cell survival under adversity. Whether p53‐regulated long noncoding RNAs are involved in this process remains to be fully understood. Here, we show that under glucose starvation condition, p53 directly upregulates a novel lncRNA named TRINGS (Tp53‐regulated inhibitor of necrosis under glucose starvation) in human tumor cells. TRINGS binds to STRAP and inhibits STRAP–GSK3β–NF‐κB necrotic signaling to protect tumor cells from cell death. Interestingly, TRINGS appears to respond to glucose starvation specifically, as it is not activated by serum, serine, or glutamine deprivation. Collectively, our findings reveal that p53‐induced lncRNA TRINGS controls the necrotic pathway and contributes to the survival of cancer cells harboring wild‐type p53 under glucose stress.     

The proapoptotic function of Noxa in human leukemia cells is regulated by the kinase Cdk5 and by glucose

The BH3-only protein, Noxa, is induced in response to apoptotic stimuli, such as DNA damage, hypoxia, and proteasome inhibition in most human cells. Noxa is constitutively expressed in proliferating cells of hematopoietic lineage and required for apoptosis in response to glucose stress. We show that Noxa is phosphorylated on a serine residue (S(13)) in the presence of glucose. Phosphorylation promotes its cytosolic sequestration and suppresses its apoptotic function. We identify Cdk5 as the Noxa kinase and show that Cdk5 knockdown or expression of a Noxa S(13) to A mutant increases sensitivity to glucose starvation, confirming that the phosphorylation is protective. Both glucose deprivation and Cdk5 inhibition promote apoptosis by dephosphorylating Noxa. Paradoxically, Noxa stimulates glucose consumption and may enhance glucose turnover via the pentose phosphate pathway rather than through glycolysis. We propose that Noxa plays both growth-promoting and proapoptotic roles in hematopoietic cancers with phospho-S(13) as the glucose-sensitive toggle switch controlling these opposing functions.     

Nuclear proteasome activation and degradation of carboxymethylated histones in human keratinocytes following glyoxal treatment

Nuclear DNA damage has been studied in detail, but much less is known concerning the occurrence and fate of nuclear protein damage. Glycoxidation, protein damage that results from a combination of protein glycation and oxidation, leads to the formation of protein-advanced glycation end products (AGE) of which N(epsilon)-carboxymethyllysine (CML) is a major AGE. We have used glyoxal, a product of environmental exposures that readily leads to the formation of CML, to study nuclear protein glycoxidation in HaCaT human keratinocytes. Glyoxal treatment that did not affect cell viability but inhibited cell proliferation in a dose-dependent manner that led to accumulation of CML-modified histones. Modified histones were slowly degraded but persisted for more than 3 days following treatment. Preincubation of cells with a proteasome inhibitor following glyoxal treatment led to an increase in CML-modified histones. While glyoxal treatment resulted in a slight decrease in total cellular proteasome activity, a dose dependent increase of up to 4-fold in nuclear proteasome activity was observed. The increase in nuclear proteasome activity was due to both increased nuclear proteasome protein content and increased activity, neither of which were affected by cyclohexamide. The increase also was unaffected by inhibitors of poly(ADP-ribose) polymerases, which have been previously implicated in nuclear proteasome activation by oxidizing agents. Accumulation of CML-modified histones over time may lead to epigenetic changes that contribute to various pathologies including aging and cancer, and upregulation of nuclear proteasome activity under conditions of glyoxidative stress may function to limit such damage.     

ALIS are stress-induced protein storage compartments for substrates of the proteasome and autophagy

Misfolded proteins can be directed into cytoplasmic aggregates such as aggresomes and dendritic cell aggresome-like induced structures (DALIS). DALIS were originally identified in lipopolysaccharide-stimulated dendritic cells and act as storage compartments for polyubiquitinated Defective Ribosomal Products (DRiPs) prior to their clearance by the proteasome. Here we demonstrate that ubiquitinated protein aggregates that are similar to DALIS, and not related to aggresomes, can be observed in several cell types in response to stress, including oxidative stress, transfection, and starvation. Significantly, both immune and nonimmune cells could form these aggresome-like induced structures (ALIS). Protein synthesis was essential for ALIS formation in response to oxidative stress, indicating that DRiP formation was required. Furthermore, puromycin, which increases DRiP formation, was sufficient to induce ALIS formation. Inhibition of either proteasomes or of autophagy interfered with ALIS clearance in puromycin treated cells. Autophagy inhibition enhanced ALIS formation under a variety of stress conditions. During starvation, ALIS formation in autophagy-deficient cells was only partially inhibited by protein synthesis inhibitors, indicating that both long-lived proteins and DRiPs can be targeted to ALIS. Together, these findings demonstrate that ALIS act as generalized stress-induced protein storage compartments for substrates of the proteasome and autophagy.     

Epithelial-Mesenchymal Transition Induces Endoplasmic-Reticulum-Stress Response in Human Colorectal Tumor Cells

Tumor cells are stressed by unfavorable environmental conditions like hypoxia or starvation. Driven by the resulting cellular stress tumor cells undergo epithelial-mesenchymal transition. Additionally, cellular stress is accompanied by endoplasmic reticulum-stress which induces an unfolded protein response. It is unknown if epithelial-mesenchymal transition and endoplasmic reticulum-stress are occurring as independent parallel events or if an interrelationship exists between both of them. Here, we show that in colorectal cancer cells endoplasmic reticulum-stress depends on the induction of ZEB-1, which is a main factor of epithelial-mesenchymal transition. In the absence of ZEB-1 colorectal cancer cells cannot mount endoplasmic reticulum-stress as a reaction on cellular stress situations like hypoxia or starvation. Thus, our data suggest that there is a hierarchy in the development of cellular stress which starts with the presence of environmental stress that induces epithelial-mesenchymal transition which allows finally endoplasmic reticulum-stress. This finding highlights the central role of epithelial-mesenchymal transition during the process of tumorigenesis as epithelial-mesenchymal transition is also associated with chemoresistance and cancer stemness. Consequently, endoplasmic reticulum-stress might be a well suited target for chemotherapy of colorectal cancers.     

Oxidative stress impairs HIF1α activation: a novel mechanism for increased vulnerability of steatotic hepatocytes to hypoxic stress

Steatosis increases the sensitivity of hepatocytes to hypoxic injury. Thus, this study was designed to elucidate the role of hypoxia-inducible factor-1α (HIF1α) in steatotic hepatocytes during hypoxia. AML12 hepatocytes and isolated rat hepatocytes were treated with a free fatty acid mixture of oleate and palmitate (2:1, 1 mM) for 18 h, which generated intrahepatocyte fat accumulation. The cells were then exposed to hypoxia (1% oxygen, 6-24 h). After hypoxia, a further increase in cellular fat accumulation was seen. In steatotic hepatocytes, a decreased HIF1α activation by hypoxia was observed. The capacity of these cells to express HIF1α-dependent genes responsible for the utilization of nutrients for energy was also impaired. This resulted in significantly lower intracellular ATP levels and greater cell death in steatotic hepatocytes compared with control hepatocytes. In contrast, overexpression of constitutively active HIF1α significantly increased cell viability as well as ATP and GLUT1 mRNA levels in steatotic hepatocytes under hypoxia. Hypoxia significantly enhanced HIF1α mRNA levels in control but not in steatotic hepatocytes. Concomitantly, an increase in oxidative stress was found in steatotic hepatocytes under hypoxic conditions compared with control cells. This included higher reactive oxygen species generation, lower cellular and nuclear GSH levels, and higher accumulation of 4-hydroxynonenal protein adducts. Hypoxia-mediated oxidative stress was accompanied by inactivation of basal nuclear factor-κB (NF-κB) DNA binding. Treatment with N-acetyl-l-cysteine, a reducing agent, improved NF-κB DNA-binding capacity and restored HIF1α induction. Conversely, overexpression of an NF-κB super-suppressor in control hepatocytes (IκBαΔN-transfected cells) resulted in complete inhibition of HIF1α expression, confirming that indeed NF-κB regulates HIF1α expression in hypoxic hepatocytes. In conclusion, hypoxia in combination with hepatic steatosis was shown to promote augmented oxidative stress, leading to NF-κB inactivation and impaired HIF1α induction and thereby increased susceptibility to hypoxic injury.     

Biochemical changes during sucrose deprivation in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

An experimental arrangement was described that enables nuclear magnetic resonance spectra of compressed plant cells to be recorded while circulating a medium through the sample. The system provided a convenient arrangement for monitoring by 31P NMR the behavior of plant cells over a long period of time under different conditions such as sucrose starvation. Perfusion of compressed sycamore cells with sucrose-free culture medium triggered a progressive decrease in the glucose 6-P and uridine-5'-diphosphate-alpha-D-glucose resonances over 30 h. When almost all the intracellular carbohydrate pool had disappeared the nucleotide triphosphate resonances decline progressively. These changes were accompanied by a Pi accumulation in the vacuole and a phosphorylcholine (P-choline) accumulation in the cytoplasm. The very long lag phase observed for ATP and P-choline evolution was comparable with that observed for the progressive intracellular digestion of cytoplasmic constituents (Journet, E., Bligny, R. and Douce, R. (1986) J. Biol. Chem. 261, 3193-3199). Addition of sucrose in the circulating system after a long period of sucrose starvation led to a disappearance of the cytoplasmic Pi resonance and a marked increase in that of glucose 6-P. Under these conditions the vacuolar Pi pool did not fluctuate to buffer the Pi in the cytoplasm. The results suggest that Pi which has been sequestered in the vacuole during the course of sucrose starvation is not restored to the cytoplasm for rapid metabolic processes. Furthermore, the presence of P-choline in plant cells in large excess should be considered as a good marker of membrane utilization after a long period of sucrose starvation and is very likely related to stress.     

Regulation of proteasome-mediated protein degradation during oxidative stress and aging

Protein degradation is a physiological process required to maintain cellular functions. There are distinct proteolytic systems for different physiological tasks under changing environmental and pathophysiological conditions. The proteasome is responsible for the removal of oxidatively damaged proteins in the cytosol and nucleus. It has been demonstrated that proteasomal degradation increases due to mild oxidation, whereas at higher oxidant levels proteasomal degradation decreases. Moreover, the proteasome itself is affected by oxidative stress to varying degrees. The ATP-stimulated 26S proteasome is sensitive to oxidative stress, whereas the 20S form seems to be resistant. Non-degradable protein aggregates and cross-linked proteins are able to bind to the proteasome, which makes the degradation of other misfolded and damaged proteins less efficient. Consequently, inhibition of the proteasome has dramatic effects on cellular aging processes and cell viability. It seems likely that during oxidative stress cells are able to keep the nuclear protein pool free of damage, while cytosolic proteins may accumulate. This is because of the high proteasome content in the nucleus, which protects the nucleus from the formation and accumulation of non-degradable proteins. In this review we highlight the regulation of the proteasome during oxidative stress and aging.     

Adrenomedullin in Ovarian Cancer: Foe In Vitro and Friend In Vivo?

Stromal elements within a tumor interact with cancer cells to create a microenvironment that supports tumor growth and survival. Adrenomedullin (ADM) is an autocrine/paracrine factor produced by both stromal cells and cancer cells to create such a microenvironment. During differentiation of macrophages, ADM is produced in response to pro-inflammatory stimuli and hypoxia. In this study we investigated the role of ADM as a growth factor for ovarian cancer cells and as a modulator of macrophages. We also analyzed ADM expression levels in a retrospective clinical study using nanofluidic technology and assessment of ADM at the gene level in 220 ovarian cancer patients. To study the effects of ADM, ovarian cancer cell lines A2780, OVCAR-3, and HEY and their drug-resistant counterparts were used for proliferation assays, while monocytes from healthy donors were differentiated in vitro. ADM was a weak growth factor, as revealed by proliferation assays and cell cycle analysis. After culturing cancer cells under stressing conditions, such as serum starvation and/or hypoxia, ADM was found to be a survival factor in HEY but not in other cell lines. In macrophages, ADM showed activity on proliferation/differentiation, primarily in type 2 macrophages (M2). Unexpectedly, the clinical study revealed that high expression of ADM was linked to positive outcome and to cancer with low Ca125. In conclusion, although in vitro ADM was a potential factor in biological aggressiveness, this possibility was not confirmed in patients. Therefore, use of an ADM antagonist would be inappropriate in managing ovarian cancer patients.     

Investigation of glucose catabolism in hypoxic Mcf 7 breast cancer culture

Hypoxia plays an important role in tumor phenotype and progression and alters glycolysis, with changes in signaling pathways that develop in response to hypoxia. In this study, the effects of oxygen (normoxia/hypoxia) and of glucose levels on the glucose metabolism was investigated in MCF-7 cancer cells. Under either normoxia or hypoxia conditions, the cells were exposed to glucose at different concentrations (0, 5.5, 15 or 55 mM) for either 3, 6, 12, 24 or 48 h. In all groups, cell viability, levels of key enzymes reflecting glycolytic metabolism in cell lysates, glucose consumed in the medium and extracellular lactate levels and wound closure percentages were determined. In hypoxic cells, intracellular consumption of glucose, and extracellular lactate levels due to increased glucose concentration were observed to be higher (compared to normoxia) and as a result of prolonged exposure to hypoxia, cells were observed to develop resistance to the prolonged exposure to hypoxia. The number of glycolytic enzymes obtained at different levels proved that cells had different potential capacities and changing mechanisms for the metabolic needs of the cell depending on the glucose amount in the medium and time in adapting to the oxygen tension. This study showed that there was an important interaction between hypoxia and glucose metabolism in general, and it was concluded that metabolic processes activated by hypoxia could offer new therapeutic targets.