Tryptophan 2,3-dioxygenase (TDO) from Xanthomonas campestris is a highly specific heme-containing enzyme from a small family of homologous enzymes, which includes indoleamine 2,3-dioxygenase (IDO). The structure of wild type (WT TDO) in the catalytically active, ferrous (Fe (2+)) form and in complex with its substrate l-tryptophan ( l-Trp) was recently reported [Forouhar et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 473-478] and revealed that histidine 55 hydrogen bonds to l-Trp, precisely positioning it in the active site and implicating it as a possible active site base. In this study the substitution of the active site residue histidine 55 by alanine and serine (H55A and H55S) provides insight into the molecular mechanism used by the enzyme to control substrate binding. We report the crystal structure of the H55A and H55S mutant forms at 2.15 and 1.90 A resolution, respectively, in binary complexes with l-Trp. These structural data, in conjunction with potentiometric and kinetic studies on both mutants, reveal that histidine 55 is not essential for turnover but greatly disfavors the mechanistically unproductive binding of l-Trp to the oxidized enzyme allowing control of catalysis. This is demonstrated by the difference in the K d values for l-Trp binding to the two oxidation states of wild-type TDO (3.8 mM oxidized, 4.1 microM reduced), H55A TDO (11.8 microM oxidized, 3.7 microM reduced), and H55S TDO (18.4 microM oxidized, 5.3 microM reduced).

Substance P (SP) regulates various physiologic and pathophysiologic responses predominantly by acting through its primary receptor, the neurokinin-1 receptor (NK1R). There are two naturally occurring forms of NK1R: full-length NK1R-FL and truncated NK1R-Tr. SP-coupled NK1R can directly or indirectly regulate the proliferation and metastatic progression of many types of human cancer cells. However, the exact roles played by the two isoforms of NK1R in breast carcinogenesis still remain largely unclear. In the present study, we first examined the expression profile of total NK1Rs, NK1R-FL and NK1R-Tr in multiple breast cancer cell lines as well as in breast tumor samples. We found that total NK1Rs are present in normal, benign and breast tumor tissues; while, NK1R-FL expression are significantly decreased in tumor specimens, particularly in metastatic carcinomas. More interestingly, NK1R-FL is highly expressed in nontumorigenic HBL-100 breast cells, whereas MDA-MB-231, MCF-7 and T47D breast cancer cells express only NK1R-Tr. To further investigate potential implications of NK1R-FL and NK1R-Tr in the malignant phenotypes of breast cancer, we studied the impacts of ectopically overexpressed NK1R-FL and NK1R-Tr in MDA-MB-231 and HBL-100 cells, respectively. Our in vitro and in vivo data showed that NK1R-FL expression was inversely associated with proliferation, invasiveness and metastasis of MDA-MB-231 cells, but overexpression of NK1R-Tr was able to promote malignant transformation of HBL-100 cells and NK1R-Tr may contribute to tumor progression and promote distant metastasis in human breast cancer. A long-term treatment of NK1R antagonist ASN-1377642 exerted antitumor action in breast cancer cells with NK1R-Tr high expression.

MicroRNAs are short endogenous non-coding RNAs, which can frequently emerge as regulators in many cancer types. It wasdemonstrated in the research that miR-185 expression levels were remarkably down-regulated in NSCLC tissues than in adjacent normal tissues. Lower miR-185 expression levels were definitely associated with lymph node metastasis. Functional assays showed that up-regulated miR-185 expression in NSCLC cells could inhibit the cell propagation, cell colony formation and incursion capacities in vitro. Furthermore, it was researched that miR-185 could suppress the epithelial-mesenchymal transition (EMT) process with the way of down-regulating E-cadherin expression and upregulating N-cadherin expression. Bioinformatics analysis and luciferase reporter gene assays revealed that Kruppel-like factor 7 (KLF7) acted as straightforward target of miR-185. Overexpression of miR-185 reduced the expression of KLF7 in NSCLC cells. Up-regulated KLF7 partly rescued the inhibiting effects induced by miR-185 on cell proliferation and invasion of NSCLC. Additionally, it was confirmed that miR-185 expression suppressed tumor growth using a tumor xenograft model . Thus, it was concluded that miR-185 may serve as a potential target of NSCLC treatment.

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Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer. MicroRNAs have been increasingly implicated in NSCLC and may serve as novel therapeutic targets to combat cancer. Here we investigated the functional implication of miR-188 in NSCLC. We first analyzed miR-188 expression in both NSCLC clinical samples and cancer cell lines. Next we investigated its role in A549 and H2126 cells with cell proliferation, migration, and apoptosis assays. To extend the in vitro study, we employed both xenograft model and LSL- K-ras G12D lung cancer model to examine the role of miR-188 in tumorigenesis. Last we tested MAP3K3 as miR-188 target in NSCLC model. MiR-188 expression was significantly downregulated at the NSCLC tumor sites and lung cancer cells. In vitro transfection of miR-188 reduced cell proliferation and migration potential and promoted cell apoptosis. In xenograft model, miR-188 inhibited tumor growth derived from cancer cells. Intranasal miR-188 administration reduced tumor formation in NSCLC animal model. MAP3K3 was validated as direct target of miR-188. Knocking down MAP3K3 in mice also inhibited tumorigenesis in LSL- K-ras G12D model. Our results demonstrate that miR-188 and its downstream target MAP3K3 could be a potential therapeutic target for NSCLC.

MicroRNAs (miRNAs) are some short RNAs that regulate multiple biological functions at post-transcriptional levels, such as tumorigenic processes, inflammatory lesions and cell apoptosis. Zinc finger E-box binding homeobox factor 1 (ZEB1) is a crucial mediator of epithelial-mesenchymal transition (EMT). It induces malignant progression of various cancers including human esophageal squamous-cell carcinoma (ESCC). In this study, we found that miR-128-3p was downregulated in ESCC tissues and cells by using PCR. Moreover, down-regulated expression of miR-128-3p was testified to be associated with poor prognosis of ESCC patients and might be regarded as an independent prognostic factor. Then, we examined the role of miR-128-3p in ESCC cells, and found that miR-128-3p could suppress the cell migration and invasion in vitro. Furthermore, ZEB1 was confirmed to be a direct target of miR-128-3p by luciferase reporter assay. Rescue experiments proved that EMT was regulated by miR-128-3p via suppression of ZEB1. Taken all together, we conclude that miR-128-3p suppresses EMT and metastasis via ZEB1, and miR-128-3p may be a critical mediator in ESCC.

This study aimed to investigate whether lidocaine, alone or in combination with other chemotherapeutic agents, inhibits the growth of human bladder cancer cells in vitro and orthotopically transplanted bladder tumors in vivo. The effects of lidocaine (1.25, 2.5 or 5 mg/mL), mitomycin C (MMC, 0.66 mg/mL), pirarubicin (0.75 mg/mL) and Su Fu’ning lotion (SFN, 0.0625 mg/mL) on the proliferation of human bladder cancer (BIU-87) cells were studied using the MTT assay. A Balb/c nude mouse model of bladder cancer was developed by orthotopic transplantation of BIU-87 cells, and the effects of intravesical instillation of lidocaine and MMC on bladder wet weight (a measure of tumor size) and survival (over 60 days) were studied. Lidocaine inhibited proliferation of BIU-87 cells in a concentration-dependent manner and (when given in combination) enhanced the actions of each of the other antiproliferative agents. In tumor-bearing mice, MMC alone had no effect on mean survival or bladder wet weight. However, the combination of 0.66 mg/mL MMC and 5 mg/mL lidocaine prolonged survival (from 34.62 ± 6.49 to 49.30 ± 6.72 days; n = 8, P < 0.05) and reduced bladder wet weight (from 68.94 ± 53.61 to 20.26 ± 6.07; n = 8, P < 0.05). Intravesical instillation of lidocaine combined with other chemotherapeutic agents potentially could be an effective therapy for bladder cancer.

The microRNAs (miRNAs) have been shown to play important roles in the development of the immune system and in regulation of host inflammation responses. Probiotics can effectively alleviate the inflammation caused by Salmonella in chickens. However, whether and how miRNAs are involved in modulation of the inflammation response in the gut of chickens have not been reported. In this study, the impact of a probiotics, Lactobacillus plantarum Z01 (LPZ01), was investigated on the cecal miRNAs and cytokine secretions in Salmonella Typhimurium (S. Typhimurium)-infected chickens at the age of 3 days. Newly hatched chicks were assigned to four groups (1): NC (basal diet) (2): S (basal diet + S. Typhimurium challenged) (3): SP (basal diet + S. Typhimurium challenged + LPZ01) (4): P (basal diet + LPZ01). In comparison with the S group, chicks in the SP group reduced the number of S. Typhimurium and had lower levels of interferon-γ and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in ceca post challenge. Expression of 14 miRNAs was significantly affected by the presence of S. Typhimurium and/or lactobacillus. Five differential expression miRNAs (gga-miR-215-5p, gga-miR-3525, gga-miR-193a-5p, gga-miR-122-5p, and gga-miR-375) were randomly selected for confirmation by the RT-PCR. Predicted target genes of differentially expressed miRNAs were enriched in regulation of cAMP-dependent protein kinase activity, stress-activated MAPK cascade, immune system development and regulation of immune system process as well as in immune related pathways such as MAPK and Wnt signaling pathways. The relationship between changes of miRNAs and changes of cytokines was explored. Finally, 119 novel miRNAs were identified in 36 libraries totally. Identification of novel miRNAs significantly expanded the repertoire of chicken miRNAs and provided the basis for understanding the function of miRNAs in the host. Our results suggest that the probiotics reduce the inflammation of the S. Typhimurium infection in neonatal broiler chicks, at least partially, through regulation of miRNAs expression.