Tumor associated macrophages provide the survival resistance of tumor cells to hypoxic microenvironmental condition through IL-6 receptor-mediated signals.

Hypoxia and infiltration of tumor-associated macrophages (TAM) are intrinsic features of the tumor microenvironment. Tumor cells that remain viable in hypoxic conditions often possess an increased survival potential and tend to grow aggressively. TAM also respond to a variety of signals in the hypoxic tumor microenvironment and express a more M2-like phenotype. In this study, the established mouse tumor tissues showed a dense infiltration of CD206+ macrophages at the junctions between the normoxic and hypoxic regions and an increased IL-6 receptor (IL-6R) expression of tumor cells in the areas of CD206+ TAM accumulation, which indicates a role of M2 phenotype TAM in survival adaptation of tumor cells preparing for an impending hypoxic injury before changes in oxygen availability. Cocultured mouse FM3A or human MCF-7 tumor cells with tumor infiltrating macrophages isolated from mouse tumor tissues and M2-polarized macrophages generated from human THP-1 cells, respectively, showed significantly decreased rate of cell death in cultures exposed to hypoxia. The acquisition of survival resistance was attributed to increased IL-6 production by M2 TAM and increased expression of IL-6R in tumor cells in the coculture system. MCF-7 cells cocultured with M2 TAM showed activated JAK1/STAT3 and Raf/MEK/JNK pathways contributing to tyrosine and serine phophorylation of STAT3, respectively. However, only tyrosine phosphorylated STAT3 was detected in the nucleus, which induced upregulation of Bcl-2 and downregulation of Bax and Bak. Finally, knockdown of IL-6R by small interfering RNA significantly counteracted coculture-induced signals and completely abolished the survival resistance to hypoxic injury. Thus, we present evidence for the role of M2 phenotype TAM in IL-6 receptor-mediated signals, particularly tyrosine phosphorylation of STAT3, responsible for the prosurvival adaptation of tumor cells to hypoxia.

Biomarkers of angiogenesis as prognostic factors in myelodysplastic syndrome patients treated with hypomethylating agents.

Angiogenesis occurs in response to tissue ischemia and wound healing, and contributes to the pathogenesis of a variety of diseases, such as benign and malignant neoplasia. Several studies have measured bone marrow microvessel density (MVD) in MDS patients and acute myeloid leukemia (AML) patients transformed from MDS, and MVD was higher in MDS patients than controls, but was lower than in AML patients. Vascular endothelial growth factor (VEGF) is expressed in bone marrow blast cells, and an autocrine VEGF signaling mechanism has been established in MDS. Increased bone marrow angiogenesis and VEGF concentrations are adverse prognostic features in all of these patients. In this study, 69 patients were treated in two groups: hypomethylating agents or supportive care with oxymetholone±pyridoxine. We evaluated the MVD and VEGF expression of paraffin-embedded bone marrow samples from patients. We also investigated the relationship between angiogenesis-related biomarkers including MVD, VEGF expression, and clinical factors. The patient median age was 65 years, and the median follow-up duration was 28 months. MVD assessment among subtypes of WHO MDS classification showed that the MVD of RCUD was significantly lower than in other types (p=0.02). However, there was no significant difference in VEGF expression according to the subtype of MDS. Although MVD and VEGF expression did not differ between risk groups based on the IPSS, the low risk group tended to have lower expression of angiogenesis-related biomarkers. MDS patients receiving hypomethylating agents had significantly lower MVD expression in responders than in non-responders (6.13±3.38 vs. 9.89±2.10, respectively, p=0.039). In a consecutive evaluation at the time of diagnosis and 3 months after the initial treatment, the group with a decrease or no change of MVD had a higher response rate compared to that in the group with an increase of MVD (92.9% vs. 58.8%, respectively, p=0.045). Adverse prognostic factors included older age, MDS type other than RCUD, a higher IPSS risk group, and abnormal cytogenetics. Although angiogenesis-related markers did not demonstrate any significant prognostic association with survival, MVD (≥10n/mm2) and a strong expression of VEGF seemed to be associated with lower survival rate. These data suggested that the MVD value might be helpful in predicting responsiveness to treatment, especially in MDS patients treated with hypomethylating agents. Although angiogenesis-related markers including VEGF did not demonstrate a significant association with survival outcomes, we observed that high MVD and strong VEGF expression seemed to be associated with lower survival rate. Therefore, biologic markers related to angiogenesis might have a potential as prognostic factors for MDS patients.

Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation.

When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼ 200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells.

Amino-terminal arginylation targets endoplasmic reticulum chaperone BiP for autophagy through p62 binding.

We show that ATE1-encoded Arg-transfer RNA transferase (R-transferase) of the N-end rule pathway mediates N-terminal arginylation of multiple endoplasmic reticulum (ER)-residing chaperones, leading to their cytosolic relocalization and turnover. N-terminal arginylation of BiP (also known as GRP78), protein disulphide isomerase and calreticulin is co-induced with autophagy during innate immune responses to cytosolic foreign DNA or proteasomal inhibition, associated with increased ubiquitylation. Arginylated BiP (R-BiP) is induced by and associated with cytosolic misfolded proteins destined for p62 (also known as sequestosome 1, SQSTM1) bodies. R-BiP binds the autophagic adaptor p62 through the interaction of its N-terminal arginine with the p62 ZZ domain. This allosterically induces self-oligomerization and aggregation of p62 and increases p62 interaction with LC3, leading to p62 targeting to autophagosomes and selective lysosomal co-degradation of R-BiP and p62 together with associated cargoes. In this autophagic mechanism, Nt-arginine functions as a delivery determinant, a degron and an activating ligand. Bioinformatics analysis predicts that many ER residents use arginylation to regulate non-ER processes.

Direct cellular delivery of human proteasomes to delay tau aggregation.

The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins.

A neurostimulant para-chloroamphetamine inhibits the arginylation branch of the N-end rule pathway.

In the arginylation branch of the N-end rule pathway, unacetylated N-terminal destabilizing residues function as essential determinants of protein degradation signals (N-degron). Here, we show that a neurostimulant, para-chloroamphetamine (PCA), specifically inhibits the Arg/N-end rule pathway, delaying the degradation of its artificial and physiological substrates, including regulators of G protein signaling 4 (RGS4), in vitro and in cultured cells. In silico computational analysis indicated that PCA strongly interacts with both UBR box and ClpS box, which bind to type 1 and type 2 N-degrons, respectively. Moreover, intraperitoneal injection of PCA significantly stabilized endogenous RGS4 proteins in the whole mouse brain and, particularly, in the frontal cortex and hippocampus. Consistent with the role of RGS4 in G protein signaling, treatment with PCA impaired the activations of GPCR downstream effectors in N2A cells, phenocopying ATE1-null mutants. In addition, levels of pathological C-terminal fragments of TDP43 bearing N-degrons (Arg208-TDP25) were significantly elevated in the presence of PCA. Thus, our study identifies PCA as a potential tool to understand and modulate various pathological processes regulated by the Arg/N-end rule pathway, including neurodegenerative processes in FTLD-U and ALS.

A case of midazolam anaphylaxis.

Midazolam is a type of anesthetic agent frequently used for conscious sedation during a variety of medical procedures. Anaphylactic reactions to midazolam are rarely reported. However, we observed a case of midazolam hypersensitivity in which emergency measures were required to ensure patient recovery after administration of midazolam as a sedative. The occurrence of the anaphylactic reaction to midazolam was confirmed by elevated serum tryptase levels. The current case report presents a discussion of our findings.

Targeting estrogen receptors for the treatment of Alzheimer's disease.

The significantly higher incidence of Alzheimer's disease (AD) in women than in men has been attributed to loss of estrogen and a variety of related mechanisms at the molecular, cellular, and hormonal levels, which subsequently elucidate neuroprotective roles of estrogen against AD-related pathology. Recent studies have proposed that beneficial effects of estrogen on AD are directly linked to its ability to reduce amyloid-ß peptides and tau aggregates, two hallmark lesions of AD. Despite high expectations, large clinical trials with postmenopausal women indicated that the beneficial effects of estrogen therapies were insignificant and, in fact, elicited adverse effects. Here, we review the current status of AD prevention and treatment using estrogens focusing on recent understandings of their biochemical links to AD pathophysiology. This review also discusses development of selective ligands that specifically target either estrogen receptor α (ERα) or ERß isoforms, which are potentially promising strategies for safe and efficient treatment of AD.

Molecular characteristics of cancer stem-like cells derived from human breast cancer cells.

We characterized the cellular properties of cancer stem-like cells (CSLCs) isolated from immortalized MDA-MB453 human breast cancer cells in culture. We showed that although the expression of Octamer-binding transcription factor-4 (OCT4) correlates to stemness in these CSLCs, OCT4 knockdown does not induce their differentiation. Our results suggest that the differentiation program in MDA-MB453 CSLCs is blocked at a step upstream of the transcription of the OCT4 promoter, allowing CSLCs to maintain their population through asymmetric cell division during many repeated passages. Comparative expression analysis indicates that only a subset of genes and signaling pathways known to be associated with survival and maintenance of CSCs are selectively expressed in CSLCs, as compared with non-CSLCs fractionated from the same parental MDA-MB453 cells. These results suggest that selective expression of a limited number of genes may be sufficient for establishment and maintenance of CSLCs with high tumorigenicity.

UBR2 of the N-end rule pathway is required for chromosome stability via histone ubiquitylation in spermatocytes and somatic cells.

The N-end rule pathway is a proteolytic system in which its recognition components (N-recognins) recognize destabilizing N-terminal residues of short-lived proteins as an essential element of specific degrons, called N-degrons. The RING E3 ligases UBR2 and UBR1 are major N-recognins that share size (200 kDa), conserved domains and substrate specificities to N-degrons. Despite the known function of the N-end rule pathway in degradation of cytosolic proteins, the major phenotype of UBR2-deficient male mice is infertility caused by arrest of spermatocytes at meiotic prophase I. UBR2-deficient spermatocytes are impaired in transcriptional silencing of sex chromosome-linked genes and ubiquitylation of histone H2A. In this study we show that the recruitment of UBR2 to meiotic chromosomes spatiotemporally correlates to the induction of chromatin-associated ubiquitylation, which is significantly impaired in UBR2-deficient spermatocytes. UBR2 functions as a scaffold E3 that promotes HR6B/UbcH2-dependent ubiquitylation of H2A and H2B but not H3 and H4, through a mechanism distinct from typical polyubiquitylation. The E3 activity of UBR2 in histone ubiquitylation is allosterically activated by dipeptides bearing destabilizing N-terminal residues. Insufficient monoubiquitylation and polyubiquitylation on UBR2-deficient meiotic chromosomes correlate to defects in double strand break (DSB) repair and other meiotic processes, resulting in pachytene arrest at stage IV and apoptosis. Some of these functions of UBR2 are observed in somatic cells, in which UBR2 is a chromatin-binding protein involved in chromatin-associated ubiquitylation upon DNA damage. UBR2-deficient somatic cells show an array of chromosomal abnormalities, including hyperproliferation, chromosome instability, and hypersensitivity to DNA damage-inducing reagents. UBR2-deficient mice enriched in C57 background die upon birth with defects in lung expansion and neural development. Thus, UBR2, known as the recognition component of a major cellular proteolytic system, is associated with chromatin and controls chromatin dynamics and gene expression in both germ cells and somatic cells.

A case of partial trisomy 3p syndrome with rare clinical manifestations.

Partial trisomy 3p results from either unbalanced translocation or de novo duplication. Common clinical features consist of dysmorphic facial features, congenital heart defects, psychomotor and mental retardation, abnormal muscle tone, and hypoplastic genitalia. In this paper, we report a case of partial trisomy 3p with rare clinical manifestations. A full-term, female newborn was transferred to our clinic. She had cleft lip-plate, dysgenesis of the corpus callosum, patent ductus arteriosus, pulmonary hypertension, and severe right-sided hydronephrosis, associated with ureteropelvic junction obstruction. Cytogenetic investigation revealed partial trisomy 3p; 46,XX,der(4)t(3;4) (p21.1;p16). The karyotype of her father showed a balanced translocation, t(3;4)(p21.1;p16). Therefore, the size of duplication can be an important factor.

Partners in crime: ubiquitin-mediated degradation and autophagy.

Recent focus on autophagy research has led to new insights on the involvement of ubiquitin (Ub)-mediated signaling as a selectivity factor in autophagy, which is generally considered a nonselective global degradation system. Emerging reports have demonstrated active crosstalk between the Ub-dependent proteolytic system and autophagy. This article highlights recent reports describing Ub-mediated selective autophagy regulated by the Toll-like receptor 4-induced immune response.

Synthesis of copper nanoparticles by solid-state plasma-induced dewetting.

Copper nanoparticles were prepared by the plasma treatment of Cu thin films without extra heating. The Cu nanoparticles were formed through a solid-state dewetting process at temperatures of less than 450 K. The particle sizes, from 10 to 80 nm, were controlled by changing the thickness of the Cu film; the particle size increased linearly with the film thickness. The Cu nanoparticles produced by plasma treatment showed an excellent size uniformity compared to those prepared by heat treatment. In the early stage of the dewetting of the Cu film, uniformly distributed holes nucleated, and the holes grew and coalesced until the Cu nanoparticles were formed. The low operating temperatures used contributed to the production of uniform Cu nanoparticles.

Lysophosphatidic acid induces upregulation of Mcl-1 and protects apoptosis in a PTX-dependent manner in H19-7 cells.

Lysophosphatidic acid (LPA) is a lipid growth factor known to regulate diverse cell functions, including cell proliferation, survival, and apoptosis. Tight regulation of cell survival in neuronal precursor is essential during neurogenesis in both developing and adult brain. Increasing data show that diverse external factors including LPA play roles in controlling cell survival and apoptosis in early developing neurons. However, the underlying control mechanism remains unclear. To explore how LPA regulates cell survival or apoptosis in a developing neuron, mechanisms for cell survival and signaling cascades by LPA were investigated in H19-7 hippocampal progenitor cells. Here, we showed that LPA promotes cell survival by protection from apoptosis. Mcl-1 was demonstrated to be crucial in LPA-induced cell survival by transfection of the siRNA specific for Mcl-1 and overexpression of Mcl-1. LPA-induced cell survival was critically mediated by the upregulation of Mcl-1 which was regulated not only through a post-translational control but a transcriptional control. Mcl-1 stabilization by LPA-induced inhibitory phosphorylation of GSK-3 contributed predominantly to the Mcl-1 upregulation. Both LPA-induced cell survival and the GSK-3 phosphorylation were attenuated by PTX and by siRNA specific for LPA1 or LPA2 receptor. Taken together, these results showed that Mcl-1 stabilization by inhibitory phosphorylation of GSK-3 through Gi/o coupling of the LPA1 and LPA2 receptors following Mcl-1 upregulation plays a critical role in LPA-induced survival of H19-7 cells. In developing neurons, modulation of Mcl-1 levels may constitute a crucial mechanism for controlling their fates.

Difference in growth suppression and apoptosis induction of EGCG and EGC on human promyelocytic leukemia HL-60 cells.

Growth suppression and apoptosis inducing effect of (-)-epigallocatechin 3-gallate (EGCG) and (-)-epigallocatechin (EGC) were studied against human promyeolcytic leukemia, HL-60 cells. EGCG showed higher growth suppression against HL-60 cells than EGC. IC(50) values for EGCG were 60.0 microM and EGC was 107.7 microM, respectively. Both EGCG and EGC induced apoptosis evidenced by nuclei fragmentation. Nuclear fragmentation was observed as a time-dependent manner and the extent of nuclear fragmentation was slightly higher in EGCG-treated cells than EGC-treated cells. The expression level of Bcl-2 was decreased and caspase-3 was activated by EGCG or EGC treatment. The extent in decrease of Bcl-2 and activation caspase-3 were more extensively occurred in EGCG-treated cells than in EGC-treated cells. These data corresponded to the growth suppression data. EGC showed no cytotoxicity to a normal V79-4 cell line and EGCG showed slight cytotoxicity at higher concentrations.

Anti-proliferative and apoptosis induction activity of green tea polyphenols on human promyelocytic leukemia HL-60 cells.

BACKGROUND: Anti-proliferation and apoptosis inducing effects of green tea polyphenols (GTPs) were studied against human promyeolcytic leukemia HL-60 cells. MATERIALS AND METHODS: Anti-proliferation activity of GTPs against HL-60 cells and cytotoxicity against normal cells, V79-4 were measured with the MTT assay. Nuclear fragmentation of GTP-treated cells was observed with a fluorescence microscope and flow-cytometric analysis was performed to observe the appearance of apoptotic bodies. Changes in apoptosis-related protein levels were investigated by Western blot analysis. RESULTS: GTPs significantly reduced the HL-60 proliferation, however, they did not show significant inhibition in proliferation of normal V79-4 cells. GTP treatment induced apoptosis in HL-60 cells, evidenced by nuclei fragmentation and apoptotic body appearance. The expression level of Bcl-2 was decreased and caspase-3 was activated by GTP treatment. CONCLUSION: GTPs revealed anti-proliferative and apoptosis-inducing activity against HL-60 cells through the down-regulation of Bcl-2 and activation of caspase-3.

Antioxidant and apoptosis-inducing activities of ellagic acid.

BACKGROUND: Antioxidant, antiproliferative and apoptosis inducing activities of a natural polyphenolic compound, ellagic acid, were studied. MATERIALS AND METHODS: DPPH radical scavenging and lipid peroxidation inhibitory activities were observed. Activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) were measured in ellagic acid-treated V79-4 cells. For apoptotic inducing activity, human osteogenic sarcoma (HOS) cell proliferation, chromosomal DNA degradation and changes in apoptosis-related protein levels were measured. RESULTS: Ellagic acid showed high DPPH radical scavenging and lipid peroxidation inhibition activities. SOD, CAT and GPX activities were significantly increased in ellagic acid-treated V79-4 cells. Ellagic acid significantly reduced HOS cell proliferation, and induced apoptosis evidenced by chromosomal DNA degradation and apoptotic body appearance. Bax expression was induced and caspase-3 was activated by ellagic acid treatment. CONCLUSION: Ellagic acid exhibited both antioxidant activity in V79-4 cells and apoptosis-inducing activity in HOS cells through the up-regulation of Bax and activation of caspase-3.

Inhibition of proliferation and induction of apoptosis by EGCG in human osteogenic sarcoma (HOS) cells.

EGCG [(-)-epigallocatechin-3-gallate], a major component of green tea has been considered as a major antioxidant constituent. In addition to having been considered for cancer treatment as a chemopreventive and chemotherapeutic agent, EGCG has recently been attributed an anti-proliferative effect. We re-examined the latter finding in this study and added specific focus on the ability of EGCG to induce apoptosis in human osteogenic sarcoma (HOS) cells. Antiproliferative action of EGCG (IC50 = 35.3 +/- 6.0 microg/mL) appeared to be linked to apoptotic cell death based on morphological changes, chromosomal DNA degradation, and an increase in the sub-G1 apoptotic cell population. Treatment of HOS cells with EGCG gradually activated caspase-3, an established inducer of apoptotic cell death.