A combination of BR101801 and venetoclax enhances antitumor effect in DLBCL cells via c-Myc/Bcl-2/Mcl-1 triple targeting.

Double hit diffuse large B-cell lymphoma (DLBCL) with rearrangement and overexpression of both c-Myc and Bcl-2 responds poorly to standard R-CHOP therapy. In a recent phase I study, Venetoclax (ABT-199) targeting Bcl-2 also exhibited disappointing response rates in patients with relapsed/refractory DLBCL, suggesting that targeting only Bcl-2 is not sufficient for achieving successful efficacy due to the concurrent oncogenic function of c-Myc expression and drug resistance following an increase in Mcl-1. Therefore, co-targeting c-Myc and Mcl-1 could be a key combinatorial strategy to enhance the efficacy of Venetoclax. In this study, BR101801 a novel drug for DLBCL, effectively inhibited DLBCL cell growth/proliferation, induced cell cycle arrest, and markedly inhibited G0/G1 arrest. The apoptotic effect of BR101801 was also observed by increased Cytochrome C, cleaved PARP, and Annexin V-positive cell populations. This anti-cancer effect of BR101801 was confirmed in animal models, where it effectively inhibited tumor growth by reducing the expression of both c-Myc and Mcl-1. Furthermore, BR101801 exhibited a significant synergistic antitumor effect even in late xenograft models when combined with Venetoclax. Our data strongly suggest that c-Myc/Bcl-2/Mcl-1 triple targeting through a combination of BR101801 and Venetoclax could be a potential clinical option for double-hit DLBCL.

Synergistic radiosensitizing effect of BR101801, a specific DNA-dependent protein kinase inhibitor, in various human solid cancer cells and xenografts.

DNA-dependent protein kinase (DNA-PK), an essential component of the non-homologous end-joining (NHEJ) repair pathway, plays an important role in DNA damage repair (DDR). Therefore, DNA-PK inhibition is a promising approach for overcoming radiotherapy or chemotherapy resistance in cancers. In this study, we demonstrated that BR101801, a potent DNA-PK inhibitor, acted as an effective radiosensitizer in various human solid cancer cells and an in vivo xenograft model. Overall, BR101801 strongly elevated ionizing radiation (IR)-induced genomic instability via induction of cell cycle G2/M arrest, autophagic cell death, and impairment of DDR pathway in human solid cancer cells. Interestingly, BR101801 inhibited not only phosphorylation of DNA-PK catalytic subunit in NHEJ factors but also BRCA2 protein level in homologous recombination (HR) factors. In addition, combination BR101801 and IR suppressed tumor growth compared with IR alone by reducing phosphorylation of DNA-PK in human solid cancer xenografts. Our findings suggested that BR101801 is a selective DNA-PK inhibitor with a synergistic radiosensitizing effect in human solid cancers, providing evidence for clinical applications.

Anti-inflammatory activity of compounds isolated from Astragalus sinicus L. in cytokine-induced keratinocytes and skin.

Inflammation is a part of the complex biological responses of a tissue to injury that protect the organ by removing injurious stimuli and initiating the healing process, and is considered as a mechanism of innate immunity. To identify biologically active compounds against pathogenic inflammatory and immune responses, we fractionated water, aqueous methanol and n-hexane layers from nine kinds of leguminosae and examined anti-inflammatory activity of the fractions in human keratinocytes and mouse skin. Among the fractions, rf3 and rf4, isolated from the aqueous methanol layer of Astragalus sinicus L., exhibited the strongest reactive oxygen species (ROS)-scavenging and anti-inflammatory activities as measured by inhibition of the intracellular ROS production, nuclear factor-kappaB (NF-κB), janus kinase (JAK)/signal transducer and activator of transcription (STAT), and phosphatidylinositol 3-kinase/Akt signaling in cytokine-stimulated human keratinocytes, as well as by effects on T-cell differentiation in mouse CD4(+) T cells. In addition, topical application of rf3 and rf4 suppressed the progression of psoriasis-like dermatitis and expression of pro-inflammatory mediators in interleukin (IL)-23-injected mouse ears. Our results suggest that Astragalus sinicus L. may ameliorate chronic inflammatory skin diseases due to its antioxidant and anti-inflammatory activities via regulation of the intracellular ROS production, NF-κB, JAK/STAT and PI3/Akt signaling cascades as well as immune responses, and these results are the first report that Astragalus sinicus L. exhibits pharmacological activity.

IFNγ-mediated inhibition of cell proliferation through increased PKCδ-induced overexpression of EC-SOD.

Extracellular superoxide dismutase (EC-SOD) overexpression modulates cellular responses such as tumor cell suppression and is induced by IFNγ. Therefore, we examined the role of EC-SOD in IFNγ-mediated tumor cell suppression. We observed that the dominant-negative protein kinase C delta (PKCδ) suppresses IFNγ-induced EC-SOD expression in both keratinocytes and melanoma cells. Our results also showed that PKCδ-induced ECSOD expression was reduced by pretreatment with a PKCspecific inhibitor or a siRNA against PKCδ. PKCδ-induced ECSOD expression suppressed cell proliferations by the up-regulation of p21 and Rb, and the downregulation of cyclin A and D. Finally, we demonstrated that increased expression of EC-SOD drastically suppressed lung melanoma proliferation in an EC-SOD transgenic mouse via p21 expression. In summary, our findings suggest that IFNγ-induced EC-SOD expression occurs via activation of PKCδ. Therefore, the upregulation of EC-SOD may be effective for prevention of various cancers, including melanoma, via cell cycle arrest.

EC-SOD induces apoptosis through COX-2 and galectin-7 in the epidermis.

BACKGROUND: Extracellular superoxide dismutase (EC-SOD) is an anti-oxidant enzyme found in the extracellular matrix of tissues, and plays an important role in the prevention of many diseases caused by oxidative stress. However, other functions of EC-SOD in epidermis are not well known. OBJECTIVE: We investigated the functions of EC-SOD in epidermis using keratinocyte cell line and EC-SOD transgenic mice. METHODS: Expression of galectin-7 in pEC-SOD transfected cells or skin of EC-SOD transgenic mice was detected by western blot analysis. The percentage of apoptotic cells was determined by propidium iodide staining and subsequent FACS analysis. COX-2 siRNA or scrambled siRNA was transfected into HaCaT cells and western blot analysis was performed to detect pro-apoptotic protein levels. RESULTS: The epidermis of EC-SOD transgenic mice was thinner than wild type mice. In addition, we showed that the thin epidermis of EC-SOD transgenic mice results from the apoptosis of epidermal cells. To elucidate which molecules are involved in EC-SOD-induced apoptosis, we utilized two-dimensional electrophoresis; the results showed that the epidermis of EC-SOD transgenic mice produces more galectin-7, a pro-apoptotic factor, than the wild type. Furthermore, we showed that the transfection of EC-SOD-expressing plasmids induces the production of galectin-7, and pro-apoptotic proteins in keratinocytes. This suggests that EC-SOD induces apoptosis through increased galectin-7 expression. Finally, we demonstrated that EC-SOD-induced galectin-7 results from the production of COX-2. CONCLUSION: Our results imply that EC-SOD plays a role not only as a reactive oxygen species scavenger, but also as a pro-apoptotic factor via COX-2/galectin-7 pathways in the epidermis.

Regulation of skin inflammation and angiogenesis by EC-SOD via HIF-1α and NF-κB pathways.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that breaks down superoxide anion into oxygen and hydrogen peroxide in extracellular spaces and plays key roles in controlling pulmonary and vascular diseases in response to oxidative stresses. We aimed to investigate the role of EC-SOD in angiogenesis and inflammation in chronic inflammatory skin disorders such as psoriasis. Overexpressed EC-SOD reduced expression of angiogenic factors and proinflammatory mediators in hypoxia-induced keratinocytes and in ultraviolet B-irradiated mice, whereas the expression of the antiangiogenic factor tissue inhibitor of metalloproteinase-1 and anti-inflammatory cytokine interleukin-10 were increased. EC-SOD decreased new vessel formation, epidermal edema, and inflammatory cell infiltration in UVB-irradiated transgenic mice. Moreover, cells treated with recombinant human EC-SOD showed inhibited endothelial tube formation and cell proliferation. Overall, the antiangiogenic and anti-inflammatory effects of EC-SOD might be due to suppression of hypoxia-inducible factor-1α, protein kinase C, and nuclear factor-κB expression. Furthermore, EC-SOD expression in tissue from psoriasis patients was markedly decreased in psoriatic lesional and nonlesional skins from psoriasis patients in comparison to normal skin from healthy volunteers. Together, these results suggest that EC-SOD may provide a novel therapeutic approach to treating angiogenic and inflammatory skin diseases such as psoriasis.

Inactive extracellular superoxide dismutase disrupts secretion and function of active extracellular superoxide dismutase.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that protects cells and tissues from extracellular damage by eliminating superoxide anion radicals produced during metabolism. Two different forms of EC-SOD exist, and their different enzyme activities are a result of different disulfide bond patterns. Although only two folding variants have been discovered so far, five folding variants are theoretically possible. Therefore, we constructed five different mutant EC-SOD expression vectors by substituting cysteine residues with serine residues and evaluated their expression levels and enzyme activities. The mutant EC-SODs were expressed at lower levels than that of wild-type EC-SOD, and all of the mutants exhibited inhibited extracellular secretion, except for C195S ECSOD. Finally, we demonstrated that co-expression of wild-type EC-SOD and any one of the mutant EC-SODs resulted in reduced secretion of wild-type EC-SOD. We speculate that mutant EC-SOD causes malfunctions in systems such as antioxidant systems and sensitizes tissues to ROS-mediated diseases.

Increased yield of high-purity and active tetrameric recombinant human EC-SOD by solid phase refolding.

Superoxide dismutase (SOD) removes damaging reactive oxygen species from the cellular environment by catalyzing the dismutation of two superoxide radicals to hydrogen peroxide and oxygen. Extracellular superoxide dismutase (EC-SOD) is a tetramer and is present in the extracellular space and to a lesser extent in the extracellular fluids. Increasing therapeutic applications for recombinant human extracellular superoxide dismutase (rEC-SOD) has broadened interest in optimizing methods for its purification, with a native conformation of tetramer. We describe a solid phase refolding procedure that combines immobilized metal affinity chromatography (IMAC) and gel filtration chromatography in the purification of rEC-SOD from Escherichia coli. The purified rEC-SOD tetramer from the Ni(2+)-column chromatography is refolded in Tris buffer. This method yields greater than 90% of the tetramer form. Greater than 99% purity is achieved with further purification over a Superose 12PC 3.2/30 column to obtain the tetramer and specific activities as determined via DCFHDA assay. The improved yield of rEC-SOD in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2.

Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.

Reciprocal regulation of 12- and 15-lipoxygenases by UV-irradiation in human keratinocytes.

The 12/15-lipoxygenase (12/15-LOX) pathways of arachidonate metabolism have been implicated in the pathogenesis of psoriasis. Since UV photo-therapy is a commonly used technique for inhibiting cell proliferation and inflammation in skin diseases, we hypothesized that UV-irradiation may affect 12/15-LOX expression which might regulate cell proliferation. In this study, we showed that UV-irradiation suppressed 12-LOX expression, whereas up-regulated 15-LOX expression. Treatment with the 15-LOX metabolites sufficiently suppressed insulin-like growth factor II-induced 12-LOX expression and blocked cell cycle progression. On the basis of our findings, we think that the 15-LOX metabolites may inhibit epidermal hyperplasia in psoriasis by regulating 12-LOX expression.