TNFSF15 facilitates the differentiation of CD11b+ myeloid cells into vascular pericytes in tumors.

OBJECTIVE: Immature vasculature lacking pericyte coverage substantially contributes to tumor growth, drug resistance, and cancer cell dissemination. We previously demonstrated that tumor necrosis factor superfamily 15 (TNFSF15) is a cytokine with important roles in modulating hematopoiesis and vascular homeostasis. The main purpose of this study was to explore whether TNFSF15 might promote freshly isolated myeloid cells to differentiate into CD11b+ cells and further into pericytes. METHODS: A model of Lewis lung cancer was established in mice with red fluorescent bone marrow. After TNFSF15 treatment, CD11b+ myeloid cells and vascular pericytes in the tumors, and the co-localization of pericytes and vascular endothelial cells, were assessed. Additionally, CD11b+ cells were isolated from wild-type mice and treated with TNFSF15 to determine the effects on the differentiation of these cells. RESULTS: We observed elevated percentages of bone marrow-derived CD11b+ myeloid cells and vascular pericytes in TNFSF15-treated tumors, and the latter cells co-localized with vascular endothelial cells. TNFSF15 protected against CD11b+ cell apoptosis and facilitated the differentiation of these cells into pericytes by down-regulating Wnt3a-VEGFR1 and up-regulating CD49e-FN signaling pathways. CONCLUSIONS: TNFSF15 facilitates the production of CD11b+ cells in the bone marrow and promotes the differentiation of these cells into pericytes, which may stabilize the tumor neovasculature.

TNFSF15 facilitates differentiation and polarization of macrophages toward M1 phenotype to inhibit tumor growth.

Macrophages of the M2 phenotype in malignant tumors significantly aid tumor progression and metastasis, as opposed to the M1 phenotype that exhibits anti-cancer characteristics. Raising the ratio of M1/M2 is thus a promising strategy to ameliorate the tumor immunomicroenvironment toward cancer inhibition. We report here that tumor necrosis factor superfamily-15 (TNFSF15), a cytokine with anti-angiogenic activities, is able to facilitate the differentiation and polarization of macrophages toward M1 phenotype. We found that tumors formed in mice by Lewis lung carcinoma (LLC) cells artificially overexpressing TNFSF15 exhibited retarded growth. The tumors displayed a greater percentage of M1 macrophages than those formed by mock-transfected LLC cells. Treatment of mouse macrophage RAW264.7 cells with recombinant TNFSF15 led to augmentation of the phagocytic and pro-apoptotic capacity of the macrophages against cancer cells. Mechanistically, TNFSF15 activated STAT1/3 in bone marrow cells and MAPK, Akt and STAT1/3 in naive macrophages. Additionally, TNFSF15 activated STAT1/3 but inactivated STAT6 in M2 macrophages. Modulations of these signals gave rise to a reposition of macrophage phenotypes toward M1. The ability of TNFSF15 to promote macrophage differentiation and polarization toward M1 suggests that this unique cytokine may have a utility in the reconstruction of the immunomicroenvironment in favor of tumor suppression.

A new H2S-specific near-infrared fluorescence-enhanced probe that can visualize the H2S level in colorectal cancer cells in mice.

Near-infrared (NIR) fluorescence-based sensors capable of selective detection of H2S in vivo would be useful tools to understand the mechanisms of diseases. A new NIR fluorescence probe 1 was developed for the detection of endogenous H2S in colorectal cancer cells in mice. 1 displayed an 87-fold fluorescence enhancement at 796 nm (with excitation at 730 nm) when reacted with H2S in a buffer (pH 7.4). 1 was water-soluble, cell-membrane-permeable, had low cytotoxicity and high selectivity and sensitivity for H2S. The properties of 1 enable its use in monitoring endogenous H2S in living cells, tissues, and mice. The bioimaging results indicated that (1) d-Cys could induce endogenous H2S production in living cells and stimulate angiogenesis; (2) tail intravenous injection of 1 into mice generated strong fluorescence in the liver while intraperitoneal injection of d-Cys could further enhance fluorescence in the liver in vivo; (3) importantly, endogenous H2S in colorectal cancer cells (HCT116, HT29) in vitro and in murine tumor models could be quickly and selectively detected by intratumoral injection of 1. These results indicated that our new probe could serve as an efficient tool for the detection of cellular H2S in living animals and even for cancer diagnosis.

LMO2 blocks the UBA6-USE1 interaction and downstream FAT10ylation by targeting the ubiquitin fold domain of UBA6.

In eukaryotic cells, the post-translational modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is the most common trigger for protein degradation and is involved in the regulation of a wide range of biological processes. FAT10 (HLA-F-adjacent transcript 10), which belongs to the UBL family, is activated specifically through the UBA6-USE1 cascade and targets substrates covalently for 26S proteasomal degradation. LMO2 is a well-recognized transcriptional regulator in hematopoietic and endothelial systems; however, it is predominantly located in the cytoplasm of epithelium-derived cells. The current study revealed that LMO2 protein interacted with the E1 ubiquitin-activating enzyme UBA6 at the C-terminal ubiquitin fold domain (UFD), which mediates the recognition and recruitment of the E2-conjugating enzyme USE1. Functionally, the LMO2-UBA6 interaction disturbed the interaction between UBA6 and USE1 and led to the decline of the overall cellular FAT10ylation level as well as the FAT10ylation and degradation of a known FAT10 substrate p62. Taken together, this study revealed a novel function of LMO2 involving in the regulatory hierarchy of UBA6-USE1-FAT10ylation pathway by targeting the E1 enzyme UBA6.

In vitro expression and analysis of the 826 human G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are involved in all human physiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826 human GPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.

[Bushen Tiaojing Recipe Regulated Expressions of BMPR I[/ALK6-Smads in Mouse Oocytes Cul- ture in vitro].

Objective To observe the effects of Bushen Tiaojing Recipe (BTR) on the counts of survival preantral follicles and the bone morphogenetic protein receptor II (BMPR II )/activin receptor- like kinase 6-drosophila mothers against decapentaplegic proteins (ALK6-Smads) signal pathway in oocytes cultured in vitro, and to study its mechanism for improving the quality of oocytes. Methods Prean- tral follicles were mechanically isolated from 65 female 12-day old healthy Kunming mice, which were inoculated by normal rats' serum (as the control group) , high, medium, low dose BTR containing serums (as Shen-supplementing groups) , high dose BTR containing serum + K02288 (as the inhibitor group) , respectively. All were cultured by common method in vitro. On the 6th day the counts of survival preantral follicles were compared between each Shen-supplementing group and the control group respectively. mR- NA expressions of BMPR II, ALK6, Smad1 , Smad5, and Smad8 were detected by Real-time fluorescence quantitative PCR. The protein expressions of indices mentioned above and phospho-Smadl/5/8 (p- Smadl/5/8) were detected by cellular immunofluorescence test. Results Compared with the control group, the quantity of survival preantral follicles increased in the high dose BTR containing serum group; mRNA expressions of BMPR II, ALK6, Smad5, and Smad8 were elevated, protein expressions of indi- ces mentioned above and p-Smadl/5/8 were increased in the 3 Shen-supplementing groups (P <0. 05) ; mRNA and protein expressions of Smad1 were increased in high and medium dose BTR containing serum groups (P<0.05). Compared with the high dose BTR containing serum group, protein expressions of Smad1/5/8 were reduced in the inhibitor group (P <0.05). Conclusion BTR could elevate the quantity of survival preantral follicles cultured in vitroand improve the quality of oocytes, which might be possibly as- sociated to regulating the BMPR II/ALK6-Smads signal pathway in oocytes.

Differentiation of bel-7402 human hepatocarcinoma cells induced by aqueous extracts of fresh gecko (AG) and its anti-tumor activity in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Gecko, a kind of reptile, has been widely used as a traditional Chinese medicine to treat various diseases including cancer in China for thousands of years. The aim of this study was to investigate the anti-tumor effect of AG (aqueous extracts of fresh gecko) on human hepatocellular carcinoma cell Bel-7402 in vitro and mouse H22 hepatocellular in vivo. Further to underlie the molecular mechanism of AG inducing the differentiation of Bel-7402 cells. MATERIALS AND METHODS: AG was obtained by water extracting method and qualitatively analyzed through High Performance Liquid Chromatography. The total protein concentration of AG was measured by BCA (bicinchoninic acid disodium) assay. The anti-tumor activities in vivo were analyzed through H22 (mouse hepatocellular carcinoma cell line H22) tumor xenografts mice. The cytotoxic activity of AG on Bel-7402 cells was evaluated by MTT assays. AFP (alpha fetoprotein) was detected by radioimmunoassay. ALB (albumin), ALP (alkaline phosphatase) and γ-GT (γ-glutamyl transpeptidase) were detected by biochemical methods with commercial kits. While morphological changes were observed through an inverted microscope. Moreover, the expression level of the proteins involved in MAPK (mitogen-activated protein kinase) signal pathway which was closely related to cellular differentiation was assessed by Western blot. RESULTS: AG showed obviously anti-tumor activity in vivo and anti-proliferative activity on Bel-7402 cells in vitro both dose-dependently. The number of clones of Bel-7402 cells treated with AG reduced and the cells were displaying differentiation state such as relatively bigger size and dispersed growth. The biochemical function markers of the cells were significantly changed after being treated with AG. The data showed that AFP secretion of the cells decreased 42.5%, ALB secretion increased 58.9%, the activity of ALP and γ-GT markedly decreased 67.0% and 48.5% separately when the concentration of AG was 10µl/ml, and those effects were all in a dose-dependent manner. The major original and phosphorylated signal proteins (ERK1/2 (extracellular sigal-regualted kinase 1/2), P38 (p38 MAPK) and JNK1/2 (c-Jun N-terminal kinase 1/2)) involved in MAPK signal pathway were measured and the results showed that AG activated the ERK1/2 of Bel-7402 cells. CONCLUSIONS: AG has anti-tumor activity in vivo and inhibits Bel-7402 cell proliferation in vitro through inducing cell differentiation, and the mechanism involves the activation of ERK1/2.

[Antitumor effects of fresh gecko hydrolysate on H22 transplanted tumor in mice].

OBJECTIVE: To compare the inhibitory effects of fresh gecko crude extract and its hydrolysate on H22 transplanted tumor in mice. METHODS: The content of soluble nitrogen (SN-TCA index) was used to determine the degree of enzymolysis. The hydrolysate of gecko was obtained from fresh gecko crude extract by pepsin and papain hydrolyzing. H22 transplanted tumor mouse models were established and divided into negative group, positive group,crude extract group and hydrolysate group. RESULTS: The inhibition rate of the H22 tumor-bearing mice was 29.17%, 48.99% respectively for the crude extract group and the hydrolysate group. The inhibition rate of hydrolysate group and the negative group were significantly different (P < 0.05). The spleen and thymus index for the crude extract group and the hydrolysate group didn't show different compared with the negative group. CONCLUSION: The crude extract of the fresh gecko and the hydrolysate can inhibit the growth of the H22 transplanted tumor. The enzymolysis by pepsin and papain can increase the antitumor activity of the crude extract of fresh gecko.