Characterization of missense mutations in the NADPH oxidase partner p22phox in the A22° subtype of chronic granulomatous disease.

Defective superoxide production by NADPH oxidase 2 (Nox2) in phagocyte cells results in the development of chronic granulomatous disease (CGD), a hereditary disease characterized by recurrent and life-threatening infections. The partner protein p22phox is a membrane-spanning protein which forms a stable heterodimer with Nox2 in the endoplasmic reticulum. This interaction ensures the stability of each protein and their accurate trafficking to the cell membrane. The present paper describes the characterization of p22phox missense mutations that were identified in a patient with CGD who presented with undetectable levels of p22phox . Using a reconstitution system, it was found that p22phox expression decreased when R90Q, A117E, S118R, A124S, A124V, A125T, or E129K mutations were introduced, suggesting that these mutations destabilize the protein. In contrast, introducing an L105R mutation did not affect protein expression, but did inhibit p22phox binding to Nox2. Thus, the missense mutations discussed here contribute to the development of CGD by either disrupting protein stability or by impairing the interaction between p22phox and Nox2.

Coculture in vitro with endothelial cells induces cytarabine resistance of acute myeloid leukemia cells in a VEGF-A/VEGFR-2 signaling-independent manner.

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway-dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling-dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR-Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling-dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche.

The NADPH oxidase NOX4 promotes the directed migration of endothelial cells by stabilizing vascular endothelial growth factor receptor 2 protein.

Directed migration of endothelial cells (ECs) is an important process during both physiological and pathological angiogenesis. The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the EC surface is necessary for directed migration of these cells. Here, we used TAXIScan, an optically accessible real-time horizontal cell dynamics assay approach, and demonstrate that reactive oxygen species (ROS)-producing NADPH oxidase 4 (NOX4), which is abundantly expressed in ECs, mediates VEGF/VEGFR-2-dependent directed migration. We noted that a continuous supply of endoplasmic reticulum (ER)-retained VEGFR-2 to the plasma membrane is required to maintain VEGFR-2 at the cell surface. siRNA-mediated NOX4 silencing decreased the ER-retained form of VEGFR-2, resulting in decreased cell surface expression levels of the receptor. We also found that ER-localized NOX4 interacts with ER-retained VEGFR-2 and thereby stabilizes this ER-retained form at the protein level in the ER. We conclude that NOX4 contributes to the directed migration of ECs by maintaining VEGFR-2 levels at their surface.

The rRNA synthesis inhibitor CX-5461 may induce autophagy that inhibits anticancer drug-induced cell damage to leukemia cells.

Autophagy induced in cancer cells during chemotherapy is classified into two types, which differ depending on the kind of cells or anticancer drugs. The first type of autophagy contributes to the death of cells treated with drugs. In contrast, the second type plays a crucial role in preventing anticancer drug-induced cell damages; the use of an autophagy inhibitor is considered effective in improving the efficacy of chemotherapy. Thus, it is important to determine which type of autophagy is induced during chemotherapy. Here, we showed that a novel inhibitor of RNA polymerase I, suppresses growth, induces cell cycle arrest and promotes apoptosis in leukemia cell lines. The number of apoptotic cells induced by co-treatment with CX-5461 and chloroquine, an autophagy inhibitor, increased compared with CX-5461 alone. Thus, the autophagy which may be induced by CX-5461 was the second type.

Withaferin A suppresses the growth of myelodysplasia and leukemia cell lines by inhibiting cell cycle progression.

Treatment outcomes for acute myeloid leukemia and myelodysplastic syndromes (MDS) remain unsatisfactory despite progress in various types of chemotherapy and hematopoietic stem cell transplantation. Therefore, there is a need for the development of new treatment options. We investigated the growth-suppressive effects of withaferin A (WA), a natural plant steroidal lactone, on myelodysplasia and leukemia cell lines. WA exhibited growth-suppressive effects on the cell lines, MDS-L, HL-60, THP-1, Jurkat and Ramos, and induction of cell cycle arrest at G2/M phase at relatively low doses. Evaluation by annexin V/PI also confirmed the induction of partial apoptosis. Gene expression profiling and subsequent gene set enrichment analysis revealed increased expression of heme oxygenase-1 (HMOX1). HMOX1 is known to induce autophagy during anticancer chemotherapy and is considered to be involved in the treatment resistance. Our study indicated increased HMOX1 protein levels and simultaneous increases in the autophagy-related protein LC3A/B in MDS-L cells treated with WA, suggesting increased autophagy. Combined use of WA with chloroquine, an autophagy inhibitor, enhanced early apoptosis and growth suppression. Together with the knowledge that WA had no apparent suppressive effect on the growth of human normal bone marrow CD34-positive cells in the short-term culture, this drug may have a potential for a novel therapeutic approach to the treatment of leukemia or MDS.

Rigosertib induces cell death of a myelodysplastic syndrome-derived cell line by DNA damage-induced G2/M arrest.

A multi-kinase inhibitor, rigosertib (ON 01910.Na) has recently been highlighted as a novel type of anti-cancer agent for the treatment of the myelodysplastic syndromes (MDS), but its action mechanisms remain to be clarified. We investigated the in vitro effects of rigosertib on an MDS-derived cell line MDS-L and a myeloid leukemia cell line HL-60. Rigosertib suppressed the proliferation of both HL-60 and MDS-L cells and induced apoptosis by inhibition of the PI3 kinase/Akt pathway. As the effects on cell cycle, rigosertib treatment promoted the phosphorylation of histone H2AX and led to the DNA damage-induced G2/M arrest. In addition, an immunofluorescence staining study demonstrated the abnormal localization of aurora A kinase, suggesting that rigosertib causes perturbation of spindle assembly and deregulated mitotic patterns towards cell cycle arrest and apoptosis. We also found that rigosertib exerted growth inhibitory effects on two lymphoid cell lines, Jurkat and Ramos. We further examined the molecular pathways influenced by rigosertib from the gene expression profiling data of MDS-L cells and found a possible involvement of rigosertib treatment in the upregulation of the genes related to microtubule kinetics and the downregulation of the mRNA degradation system. The gene set enrichment analysis showed the suppression of "nonsense-mediated mRNA decay (NMD)" as the most significantly affected gene set. These data provide a new aspect and a potential utility of rigosertib for the treatment of refractory hematopoietic malignancies.

Anti-fatigue activity of methyl dihydrojasmonate and linalool in a rat model evaluated by a novel index for neuro-immune and oxidative stress interactions.

Avoiding fatigue is a long-standing challenge in both healthy and diseased individuals. Establishing objective standard markers of fatigue is essential to evaluate conditions in spatiotemporally different locations and individuals and identify agents to fight against fatigue. Herein, we introduced a novel method for evaluating fatigue using nervous system markers (including dopamine, adrenaline, and noradrenaline), various cytokine levels (such as interleukin [IL]-1ß, tumor necrosis factor [TNF]-α, IL-10, IL-2, IL-5 and IL-17A), and oxidative stress markers (such as diacron-reactive oxygen metabolites [d-ROMs] and biological antioxidant potential [BAP]) in a rat fatigue model. Using this method, the anti-fatigue effects of methyl dihydrojasmonate (MDJ) and linalool, the fragrance/flavor compounds used in various products, were assessed. Our method evaluated the anti-fatigue effects of the aforementioned compounds based on the changes in levels of the nerves system markers, cytokines, and oxidative stress markers. MDJ exerted more potent anti-fatigue effects than linalool. In conclusion, the reported method could serve as a useful tool for fatigue studies and these compounds may act as effective therapeutic agents for abrogating fatigue symptoms.

Fine definition of the epitopes on the human gp91phox/NOX2 for the monoclonal antibodies CL-5 and 48.

Superoxide-producing NADPH oxidase, gp91phox/NOX2, in phagocytes plays a critical role in the host defenses against pathogens. Moreover, gp91phox/NOX2 contributes to the oxidative stress in endothelial cells. Therefore, investigating the level of gp91phox/NOX2 with immunoblotting is important for estimating the amount of superoxide produced. Here, we showed that the epitopes in human gp91phox/NOX2 recognized by monoclonal antibodies (mAbs) CL-5 and 48 were in amino acids 132-147 and 136-144, respectively. Although the epitopes were close to the N-glycosylation sites, N-glycan maturation did not affect mAbs recognition. When Pro-136 was substituted with Arg, the corresponding mouse residue, human gp91phox/NOX2 was not recognized by mAbs CL-5 and 48; however, the substitution did not affect gp91phox/NOX2-based oxidase activity. This finding explains why these mAbs specifically recognize the human but not mouse gp91phox/NOX2. Hence, these mAbs are useful for investigating the level of gp91phox/NOX2 without amino acid substitutions in the epitopes.

Regulation of Derlin-1-mediated degradation of NADPH oxidase partner p22phox by thiol modification.

The transmembrane protein p22phox heterodimerizes with NADPH oxidase (Nox) 1-4 and is essential for the reactive oxygen species-producing capacity of oxidases. Missense mutations in the p22phox gene prevent the formation of phagocytic Nox2-based oxidase, which contributes to host defense. This results in chronic granulomatous disease (CGD), a severe primary immunodeficiency syndrome. In this study, we characterized missense mutations in p22phox (L51Q, L52P, E53V, and P55R) in the A22° type (wherein the p22phox protein is undetectable) of CGD. We demonstrated that these substitutions enhanced the degradation of the p22phox protein in the endoplasmic reticulum (ER) and the binding of p22phox to Derlin-1, a key component of ER-associated degradation (ERAD). Therefore, the L51-L52-E53-P55 sequence is responsible for protein stability in the ER. We observed that the oxidation of the thiol group of Cys-50, which is adjacent to the L51-L52-E53-P55 sequence, suppressed p22phox degradation. However, the suppression effect was markedly attenuated by the serine substitution of Cys-50. Blocking the free thiol of Cys-50 by alkylation or C50S substitution promoted the association of p22phox with Derlin-1. Derlin-1 depletion partially suppressed the degradation of p22phox mutant proteins. Furthermore, heterodimerization with p22phox (C50S) induced rapid degradation of not only Nox2 but also nonphagocytic Nox4 protein, which is responsible for redox signaling. Thus, the redox-sensitive Cys-50 appears to determine whether p22phox becomes a target for degradation by the ERAD system through its interaction with Derlin-1.

Identification of anti-SARS-CoV-2 agents based on flavor/fragrance compositions that inhibit the interaction between the virus receptor binding domain and human angiotensin converting enzyme 2.

Coronavirus disease 2019 (COVID-19) pandemic poses a threat to human beings and numerous cases of infection as well as millions of victims have been reported. The binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (RBD) to human angiotensin converting enzyme 2 (hACE2) is known to promote the engulfment of the virus by host cells. Employment of flavor/fragrance compositions to prevent SARS-CoV-2 infection by inhibiting the binding of viral RBD (vRBD) to hACE2 might serve as a favorable, simple, and easy method for inexpensively preventing COVID-19, as flavor/fragrance compositions are known to directly interact with the mucosa in the respiratory and digestive systems and have a long history of use and safety assessment. Herein we report the results of screening of flavor/fragrance compositions that inhibit the binding of vRBD to hACE2. We found that the inhibitory effect was observed with not only the conventional vRBD, but also variant vRBDs, such as L452R, E484K, and N501Y single-residue variants, and the K417N+E484K+N501Y triple-residue variant. Most of the examined flavor/fragrance compositions are not known to have anti-viral effects. Cinnamyl alcohol and Helional inhibited the binding of vRBD to VeroE6 cells, a monkey kidney cell line expressing ACE2. We termed the composition with inhibitory effect on vRBD-hACE2 binding as "the molecularly targeted flavor/fragrance compositions". COVID-19 development could be prevented by using these compositions with reasonable administration methods such as inhalation, oral administration, and epidermal application.

Inhibition of pancreatic cancer-cell growth and metastasis in vivo by a pyrazole compound characterized as a cell-migration inhibitor by an in vitro chemotaxis assay.

Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.

The downregulation of NADPH oxidase Nox4 during hypoxia in hemangioendothelioma cells: a possible role of p22phox on Nox4 protein stability.

NADPH oxidase (Nox) 4 produces H2O2 by forming a heterodimer with p22phox and is involved in hemangioendothelioma development through monocyte chemoattractant protein-1 (MCP-1) upregulation. Here, we show that Nox4 protein levels were maintained by p22phox in hemangioendothelioma cells and Nox4 protein stability was dependent on p22phox coexpression. Conversely, the degradation of Nox4 monomer was enhanced by p22phox knockdown. Under hypoxic conditions in hemangioendothelioma cells, p22phox was downregulated at the mRNA and protein levels. Downregulation of p22phox protein resulted in the enhanced degradation of Nox4 protein in hypoxia-treated hemangioendothelioma cells. In contrast, Nox2, a Nox isoform, was not altered at the protein level under hypoxic conditions. Nox2 exhibited a higher affinity for p22phox compared with Nox4, suggesting that when coexpressed with Nox4 in the same cells, Nox2 acts as a competitor. Nox2 knockdown restored Nox4 protein levels partially reduced by hypoxic treatment. Thus, Nox4 protein levels were attenuated in hypoxia-treated cells resulting from p22phox depletion. MCP-1 secretion was decreased concurrently with hypoxia-induced Nox4 downregulation compared with that under normoxia.

Constitutive activity of NADPH oxidase 1 (Nox1) that promotes its own activity suppresses the colon epithelial cell migration.

Superoxide producing NADPH oxidase 1 (Nox1), abundantly expressed in the colon epithelium, plays a crucial role in mucosal host defenses. In this study, we found that pre-treatment of cells with edaravone, a free radical scavenger, inhibited Nox1 constitutive activity even after washout without affecting Nox1 trafficking to the plasma membrane and membrane recruitment of the cytosolic regulators Noxo1 and Noxa1. These results suggest that a Nox1-derived product is involved in the step that initiates the electron transfer reaction after the formation of the Nox1-Noxo1-Noxa1 complex. Furthermore, we show that the mean migration directionality and velocity of epithelial cells were significantly enhanced by the inhibition of constitutive Nox1 activity. Thus, the constitutive Nox1 activity limits undesired cell migration in resting cells while participating in a positive feedback loop toward its own oxidase activity.

Five-aza-2'-deoxycytidine-induced hypomethylation of cholesterol 25-hydroxylase gene is responsible for cell death of myelodysplasia/leukemia cells.

DNA methyltransferase inhibitors (DNMT inhibitors) are administered for high-risk MDS, but their action mechanisms are not fully understood. Hence, we performed a genome-wide DNA methylation assay and focused on cholesterol 25-hydroxylase (CH25H) among the genes whose expression was up-regulated and whose promoter region was hypomethylated after decitabine (DAC) treatment in vitro. CH25H catalyzes hydroxylation of cholesterol and produces 25-hydroxycholesterol (25-OHC). Although CH25H mRNA expression level was originally low in MDS/leukemia cell lines, exposure to DNMT inhibitors enhanced CH25H mRNA expression. The promoter region of CH25H was originally hypermethylated in HL-60 and MDS-L cells, but DAC treatment induced their hypomethylation together with increased CH25H mRNA expression, activation of CH25H-oxysterol pathway, 25-OHC production and apoptotic cell death. We further confirmed that normal CD34-positive cells revealed hypomethylated status of the promoter region of CH25H gene. CH25H-knockdown by transfection of shRNA lentiviral vector into the cell lines partially protected the cells from DAC-induced cell death. Exogenous addition of 25-OHC suppressed leukemic cell growth. The present study raises a possibility that DNMT inhibitors activate CH25H-oxysterol pathway by their hypomethylating mechanism and induce leukemic cell death. Further investigations of the promoter analysis of CH25H gene and therapeutic effects of DNMT inhibitors on MDS/leukemia will be warranted.

Derivative (5;19)(p10;q10): a rare but recurrent whole-arm translocation in acute myeloid leukemia.

A previous study of cases of myelodysplastic syndrome harboring der(5;19)(p10;q10) found that they displayed common characteristics including predominance in elderly men, dysplasia involving three hematopoietic lineages and CD7 expression in blasts. However, the whole-arm translocation der(5;19)(p10;q10) has not been fully analyzed because of its rarity. In this study we used flow cytometry to evaluate the immunophenotype of two patients' bone marrow mononuclear cells. Both patients had involved der(5;19)(p10;q10) in their karyotype analyzed by standard G-banding technique. Both patients had the CD7+ and CD41+ phenotype, and the CD41 positivity suggested that the myeloid neoplasms involving der(5;19)(p10;q10) were of megakaryoblastic origin. The der(5;19)(p10;q10) abnormality is associated with unique characteristics of the immunophenotype. We address the clinical, immunophenotypic and morphological aspects of hematological malignancy involving der(5;19)(p10;q10), along with a review of the literature.

A rare der(Y)t(Y;1)(q12;q12) in a patient with post-polycythemic myelofibrosis: a case report.

We describe a case of post-polycythemic myelofibrosis harboring der(Y)t(Y;1)(q12;q12). The patient was a 69-year-old man and was initially diagnosed with polycythemia vera. During the clinical course of his condition, the polycythemia developed into myelofibrosis. Chromosome analysis detected der(Y)t(Y;1)(q12;q12). We discuss the association between der(Y)t(Y;1)(q11~12;q12~21) and tumorigenesis along with a review of literature.

Efficacy and safety of intravenous itraconazole as empirical antifungal therapy for persistent fever in neutropenic patients with hematological malignancies in Japan.

Recently, empirical antifungal therapy with intravenous itraconazole (ITCZ) for neutropenic patients with antibiotics-resistant fever has been approved by Japanese Ministry of Health, Labour and Welfare on the bases of previous multicenter trials of foreign countries. In this study, we conducted a single-arm, multicenter, prospective study in order to evaluate the efficacy of empirical ITCZ injection on Japanese patients. Sixty-eight patients with hematological diseases who underwent anticancer chemotherapy or stem cell transplantation were enrolled. In this study, we found that the overall clinical response rate to ITCZ injection was 67.6% and success rate of achieving composite endpoints including survival, defervescence during neutropenia, no breakthrough fungal infections, and no premature discontinuation of drug was 50.0%. Mild adverse reactions were observed in 6 patients (8.8%). Further analysis revealed that possible/probable deep fungal infection according to the 2002 and 2008 criteria defined by EORTC/MSG were found in 19.1 and 7.5% of the patients, respectively. Interestingly, response rate to ITCZ injection of possible/probable cases according to the 2002 and 2008 criteria was 61.5% (8/13) and 100% (5/5), respectively. These results not only proved the good efficacy and safety of empirical ITCZ injection for Japanese patients, but also indicated a utility of the drug on future "presumptive" approach.