Targeted Induction of Endogenous VDUP1 by Small Activating RNA Inhibits the Growth of Lung Cancer Cells.

Recent studies have reported that small double-strand RNAs (dsRNAs) can activate endogenous genes via an RNA-based promoter targeting mechanism termed RNA activation (RNAa). In the present study, we showed that dsVDUP1-834, a novel small activating RNA (saRNA) targeting promoter of vitamin D3 up-regulated protein 1 (VDUP1) gene, up-regulated expression of VDUP1 at both mRNA and protein levels in A549 lung cancer cells. We also demonstrated that dsVDUP1-834 inhibited cell proliferation in A549 lung cancer cells. Further studies showed that dsVDUP1-834 induced cell-cycle arrest by increasing p27 and p53 and decreasing cyclin A and cyclin B1. In addition, knockdown of VDUP1 abrogated dsVDUP1-834-induced up-regulation of VDUP1 gene expression and related effects. The activation of VDUP1 by dsVDUP1-834 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 3 (H3ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) at the target site of VDUP1 promoter. Moreover, the enrichment of Ago2 was detected at the dsVDUP1-834 target site, and Ago2 knockdown significantly suppressed dsVDUP1-834-mediated inhibition of cell proliferation and modulation of cell-cycle regulators. Taken together, the results presented in this report demonstrate that dsVDUP1-834 induces VDUP1 gene expression by epigenetic changes, resulting in cell growth inhibition and cell-cycle arrest. Our results suggest that targeted induction of VDUP1 by dsVDUP1-834 might be a promising therapeutic strategy for the treatment of lung cancer.

Inhibition of Skin Inflammation by Scytonemin, an Ultraviolet Sunscreen Pigment.

Scytonemin is a yellow-green ultraviolet sunscreen pigment present in different genera of aquatic and terrestrial blue-green algae, including marine cyanobacteria. In the present study, the anti-inflammatory activities of scytonemin were evaluated in vitro and in vivo. Topical application of scytonemin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear swelling in BALB/c mice. The expression of tumor necrosis factor-a (TNF-a) and inducible nitric oxide synthase (iNOS) was also suppressed by scytonemin treatment in the TPA-treated ear of BALB/c mice. In addition, scytonemin inhibited lipopolysaccharide (LPS)-induced production of TNF-a and nitric oxide (NO) in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expressions of TNF-a and iNOS were also suppressed by scytonemin in LPS-stimulated RAW 264.7 cells. Further study demonstrated that LPS-induced NF-kB activity was significantly suppressed by scytonemin treatment in RAW 264.7 cells. Our results also showed that the degradation of IkBa and nuclear translocation of the p65 subunit were blocked by scytonemin in LPS-stimulated RAW 264.7 cells. Collectively, these results suggest that scytonemin inhibits skin inflammation by blocking the expression of inflammatory mediators, and the anti-inflammatory effect of scytonemin is mediated, at least in part, by down-regulation of NF-kB activity. Our results also suggest that scytonemin might be used as a multi-function skin care ingredient for UV protection and anti-inflammation.

Design and synthesis of novel benzoxazole analogs as Aurora B kinase inhibitors.

A novel series of benzoxazole analogs was designed and synthesized, and their inhibitory activities against Aurora kinases were evaluated. Some of the tested compounds exhibited a promising activity with respect to the inhibition of Aurora B kinase. A structure-activity relationship study indicated that linker length, regiochemistry, and halogen substitution play important roles in kinase inhibitory potency. The binding modes between representative compounds and Aurora kinases were interpreted through a molecular docking study to explain the inhibitory activity and selectivity for Aurora A and B kinases. Compounds 13l and 13q also show an antiproliferative effect on the human tumor cell lines in a dose-dependent manner. The most potent 13q demonstrated good efficacy in the prostate cancer PC-3 tumor xenograft model.

Protection against Lipopolysaccharide-Induced Endotoxemia by Terrein Is Mediated by Blocking Interleukin-1ß and Interleukin-6 Production.

Terrein is a fungal metabolite and has been known to exert anti-melanogenesis, anti-cancer, and anti-bacterial activities. However, its role in endotoxemia has never been investigated until now. In the present study, we examined the effect of terrein on lipopolysaccharide (LPS)-induced endotoxemia in mice and characterized the potential mechanisms of action. Treatment with terrein increased the survival of mice and decreased the production of inflammatory cytokines, including interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in an LPS-induced endotoxemia model. In addition, terrein suppressed the LPS-induced production of IL-1ß and IL-6 in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expression of IL-1ß and IL-6 was also inhibited by terrein in LPS-stimulated RAW 264.7 cells. Further study demonstrated that terrein blocked LPS-induced phosphorylation of p65 subunit of nuclear factor (NF)/κB and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) was also suppressed by terrein treatment. Collectively, these results suggest that terrein exerts a protective effect again LPS-induced endotoxemia in mice by blocking the production of inflammatory cytokines. Our results also suggest that the anti-inflammatory effect of terrein might be mediated, at least in part, by blocking the activation of NF-κB, JNK, and p38 MAPK signaling pathways.

Combination Treatment with GSK126 and Pomalidomide Induces B-Cell Differentiation in EZH2 Gain-of-Function Mutant Diffuse Large B-Cell Lymphoma.

Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates genes involved in cell lineage and differentiation through methylating lysine 27 on histone H3 (H3K27me3). Recurrent gain-of-function mutations of EZH2 have been identified in various cancer types, in particular, diffuse large B-cell lymphoma (DLBCL), through large-scale genome-wide association studies and EZH2 depletion or pharmacological inhibition has been shown to exert an antiproliferative effect on cancer cells, both in vitro and in vivo. In the current study, a combination of pomalidomide and GSK126 synergistically inhibited the growth of EZH2 gain-of-function mutant Diffuse large B-cell lymphoma (DLBCL) cells. Furthermore, this synergistic effect appeared to be dependent on cereblon (CRBN), a cellular receptor of pomalidomide, but not degradation of IKAROS family zinc finger 1 (IKZF1) or IKAROS family zinc finger 3 (IKZF3). RNA sequencing analyses revealed that co-treatment with GSK126 and pomalidomide induced specific gene sets involved in B-cell differentiation and apoptosis. Synergistic growth inhibition and B-cell differentiation were further validated in xenograft mouse models. Our collective results provide a molecular basis for the mechanisms underlying the combined therapeutic effects of PRC2 inhibitors and pomalidomide on EZH2-mutated DLBCL.

A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells.

Glutamine is an essential nutrient that regulates energy production, redox homeostasis, and signaling in cancer cells. Despite the importance of glutamine in mitochondrial metabolism, the mitochondrial glutamine transporter has long been unknown. Here, we show that the SLC1A5 variant plays a critical role in cancer metabolic reprogramming by transporting glutamine into mitochondria. The SLC1A5 variant has an N-terminal targeting signal for mitochondrial localization. Hypoxia-induced gene expression of the SLC1A5 variant is mediated by HIF-2α. Overexpression of the SLC1A5 variant mediates glutamine-induced ATP production and glutathione synthesis and confers gemcitabine resistance to pancreatic cancer cells. SLC1A5 variant knockdown and overexpression alter cancer cell and tumor growth, supporting an oncogenic role. This work demonstrates that the SLC1A5 variant is a mitochondrial glutamine transporter for cancer metabolic reprogramming.

Testing Cell-Based Immunotherapy for Colorectal Cancer.

Cell-based immunotherapy for cancer is emerging as an attractive alternative to conventional small-molecule or antibody-based treatment. Due to the characteristics of cell-based therapy, validation of test materials before in vivo administration is required. Here we describe general validation steps for preclinical evaluation of cell-based immunotherapy. We also describe a xenograft model of human colorectal cancer. This model can be used for applied to preclinical evaluation of various cell-based therapy regimens for colorectal cancer treatment.

Small activating RNA induced expression of VHL gene in renal cell carcinoma.

Recent studies have reported that chemically synthesized double-stranded RNAs (dsRNAs), also known as small activating RNA (saRNAs), can specifically induce gene expression by targeting promoter sequences by a mechanism termed RNA activation (RNAa). In the present study, we designed 4 candidate saRNAs targeting the Von Hippel-Lindau (VHL) gene promoter. Among these saRNAs, dsVHL-821 significantly inhibited cell growth by up-regulating VHL at both the mRNA and protein levels in renal cell carcinoma 769-P cells. Functional analysis showed that dsVHL-821 induced apoptosis by increasing p53, decreasing Bcl-xL, activating caspase 3/7 and poly-ADP-ribose polymerase in a dose-dependent manner. Chromatin immunoprecipitation analysis revealed that dsVHL-821 increased the enrichment of Ago2 and RNA polymerase II at the dsVHL-821 target site. In addition, Ago2 depletion significantly suppressed dsVHL-821-induced up-regulation of VHL gene expression and related effects. Single transfection of dsVHL-821 caused long-lasting (14 days) VHL up-regulation. Furthermore, the activation of VHL by dsVHL-821 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 4 (H4ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) in the dsVHL-821 target region. Taken together, these results demonstrate that dsVHL-821, a novel saRNA for VHL, induces the expression of the VHL gene by epigenetic changes, leading to inhibition of cell growth and induction of apoptosis, and suggest that targeted activation of VHL by dsVHL-821 may be explored as a novel treatment of renal cell carcinoma.

Multi-paratopic VEGF decoy receptor have superior anti-tumor effects through anti-EGFRs and targeted anti-angiogenic activities.

Limitation of current anti-Vascular Endothelial Growth Factor (VEGF) cancer therapy is transitory responses, inevitable relapses and its insufficient tumor-targeting. Thus, multifaceted approaches, including the development of bispecific antibodies and combination strategies targeting different pathways have been proposed as an alternative. Here, we developed a novel multi-paratopic VEGF decoy receptor, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab, by genetically fusing VEGF decoy receptor (VEGF-Grab) to a single chain Fv of anti-Epidermal Growth Factor Receptor (EGFR) antibody (Cetuximab and Trastuzumab). These multi-paratopic VEGF decoy receptor, which recognize VEGF and EGFR family (EGFR or HER2), effectively suppressed both VEGF and EGFR pathways in vitro, to levels similar to those of the parental VEGF-Grab and anti-EGFR antibodies. In addition, the concurrent binding of multi-paratopic VEGF decoy receptor to VEGF and EGFR family enabled their specific localization to EGFR + tumor in vitro and in vivo. Furthermore, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab exhibited the enhanced anti-tumor activities compared to VEGF-Grab in EGFR + tumor xenograft mouse model via anti-EGFR and the targeted anti-angiogenic activities. These results indicate that multi-paratopic VEGF decoy receptor can be a promising agent, combining tumor-targeted anti-angiogenic therapy with efficient blockade of proliferative signals mediated by EGFR family.

Control of leucine-dependent mTORC1 pathway through chemical intervention of leucyl-tRNA synthetase and RagD interaction.

Leucyl-tRNA synthetase (LRS) is known to function as leucine sensor in the mammalian target of rapamycin complex 1 (mTORC1) pathway. However, the pathophysiological significance of its activity is not well understood. Here, we demonstrate that the leucine sensor function for mTORC1 activation of LRS can be decoupled from its catalytic activity. We identified compounds that inhibit the leucine-dependent mTORC1 pathway by specifically inhibiting the GTPase activating function of LRS, while not affecting the catalytic activity. For further analysis, we selected one compound, BC-LI-0186, which binds to the RagD interacting site of LRS, thereby inhibiting lysosomal localization of LRS and mTORC1 activity. It also effectively suppressed the activity of cancer-associated MTOR mutants and the growth of rapamycin-resistant cancer cells. These findings suggest new strategies for controlling tumor growth that avoid the resistance to existing mTOR inhibitors resulting from cancer-associated MTOR mutations.Leucyl-tRNA synthetase (LRS) is a leucine sensor of the mTORC1 pathway. Here, the authors identify inhibitors of the GTPase activating function of LRS, not affecting its catalytic activity, and demonstrate that the leucine sensor function of LRS can be a new target for mTORC1 inhibition.

miR-6734 Up-Regulates p21 Gene Expression and Induces Cell Cycle Arrest and Apoptosis in Colon Cancer Cells.

Recently, microRNAs have been implicated in the regulation of gene expression in terms of both gene silencing and gene activation. Here, we investigated the effects of miR-6734, which has a sequence homology with a specific region of p21WAF1/CIP1 (p21) promoter, on cancer cell growth and the mechanisms involved in this effect. miR-6734 up-regulated p21 expression at both mRNA and protein levels and chromatin immunoprecipitation analysis using biotin-labeled miR-6734 confirmed the association of miR-6734 with p21 promoter. Moreover, miR-6734 inhibited cancer cell growth and induced cell cycle arrest and apoptosis in HCT-116 cells, which was abolished by knockdown of p21. The phosphorylation of Rb and the cleavage of caspase 3 and PARP were suppressed by miR-6734 transfection in HCT-116 cells and these effects were also reversed by p21 knockdown. In addition, miR-6734 transfection caused prolonged induction of p21 gene and modification of histones in p21 promoter, which are typical aspects of a phenomenon referred to as RNA activation (RNAa). Collectively, our results demonstrated that miR-6734 inhibits the growth of colon cancer cells by up-regulating p21 gene expression and subsequent induction of cell cycle arrest and apoptosis, suggesting its role as an important endogenous regulator of cancer cell proliferation and survival.

Cardiovascular protective effect of glabridin: Implications in LDL oxidation and inflammation.

Atherosclerosis is one of the most common causes of death in Western countries and now considered as a chronic inflammatory disease in broad outline. Glaridin, a flavonoid isolated from licorice root, has been shown to exert a variety of biological activities, including antimicrobial, antioxidant, anti-inflammatory and cardiovascular protective effects. Among these, the most extensive research area in the past two decades was a cardiovascular protection-related activity of glabridin. The protective effect of glabridin on LDL oxidation, which is one of the important processes involved in the development of atherosclerosis, was demonstrated in vitro and in vivo and the mechanisms involved in this process were established well. Structure-activity relationship of glabridin derivatives on LDL oxidation was also reported. In addition, the inhibitory effects of glabridin on early inflammatory processes, including the expression of adhesion molecules on endothelial cells and the activation of macrophages and dendritic cells, were also demonstrated previously. In this review, we summarized the cardiovascular protection-related activities of glabridin and the mechanism of action involved in these activities. Collectively, it is hoped that glabridin or glabridin derivatives might be used as a therapeutic agent for the treatment of cardiovascular diseases in the future.

Hypothemycin inhibits tumor necrosis factor-α production by tristetraprolin-dependent down-regulation of mRNA stability in lipopolysaccharide-stimulated macrophages.

Hypothemycin, a resorcylic acid lactone polyketide, has been shown to inhibit oncogenic ras-transformation and T cell activation. In the present study, we investigated the effect of hypothemycin on tumor necrosis factor-α (TNF-α) production in macrophages and the molecular mechanisms involved in this effect. Hypothemycin potently suppressed the TNF-α production without affecting nitric oxide production in lipopolysaccharide (LPS)-stimulated macrophages. However, hypothemycin had no effect on the activity of TNF-α-converting enzyme, a key enzyme for converting membrane-bound pro-TNF-α into soluble TNF-α. Further study demonstrated that the stability of TNF-α mRNA was decreased by hypothemycin treatment. In addition, hypothemycin suppressed LPS-induced phosphorylation of p38 MAPK and ERK. Moreover, knockdown of tristetraprolin (TTP), which is an important trans-acting regulator of TNF-α mRNA stability and downstream target of p38 MAPK and ERK, reversed hypothemycin-mediated inhibition of TNF-α mRNA expression. Collectively, our results suggest that hypothemycin suppresses TNF-α production by TTP-dependent destabilization of TNF-α mRNA and this is mediated, at least in part, by blocking the activation of p38 MAPK and ERK.

Differential anti-inflammatory and analgesic effects by enantiomers of zaltoprofen in rodents.

In the present study, we investigated the effect of zaltoprofen enantiomers on inflammation and pain and compared their effect with racemic zaltoprofen. S(+)-zaltoprofen potently inhibited the inflammatory response in carrageenan-induced paw edema model, whereas R(-)-zaltoprofen did not. Moreover, the anti-inflammatory effect of S(+)-zaltoprofen was stronger than that of racemic zaltoprofen, suggesting that S(+)-zaltoprofen is an active component of racemic zaltoprofen in terms of anti-inflammatory activity. In contrast, the results of acetic acid-induced writhing model demonstrated that no significant analgesic effect was observed by racemic zaltoprofen and zaltoprofen enantiomers at doses used in carrageenan-induced paw edema model. However, racemic zaltoprofen and zaltoprofen enantiomers all exerted an analgesic effect at higher doses, which is inconsistent with the result of carrageenan-induced paw edema model. Gastric ulcers induced by racemic zaltoprofen and zaltoprofen enantiomers were minimal. Taken together, these results suggest that S(+)-zaltoprofen is a potent and active anti-inflammatory component of racemic zaltoprofen, but both S(+)-zaltoprofen and R(-)-zaltoprofen might seem to contribute to the analgesic effect of racemic zaltoprofen.