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Biomedicine & Pharmacotherapy =... Dec 2018Colon cancer is one of the most common digestive malignant tumors that leads to high mortality worldwide, and metastasis is the primary cause of cancer-related death. It...
Colon cancer is one of the most common digestive malignant tumors that leads to high mortality worldwide, and metastasis is the primary cause of cancer-related death. It is well accepted that the epithelial-mesenchymal transition (EMT) plays a key role in the process of metastasis. As a cytokine that macrophage secretes, IL-6 is involved in the progression of tumors, including the invasion and metastasis via kinds of signaling pathways. However, the mechanism of interactions between IL-6, macrophage, EMT and colon cancer is not fully understood. Increased CD68 macrophages and IL-6 level were found in colon tumor as compared to normal colon tissue. Metastatic lymph node showed even more CD68 macrophages and higher IL-6 level than the primary tumor. These results suggested that macrophages and IL-6 play an important role in EMT of colon cancer. In order to investigate the effect of macrophage and IL-6 on EMT of colon cancer, we cultured human colon carcinoma cell line SW48 with conditioned medium (CM) from PMA-stimulated monocyte THP-1 cells and tested for IL-6 dependent EMT pathways. Wound healing assay and Transwell assay were used to analyze cell migration and invasion. Results showed that CM-treated SW48 cells increased IL-6 production and displayed elevated capacity of migration and invasion compared to untreated cells. Increased expressions of EMT markers (N-cadherin, Vimentin and β-catenin) and decreased expression of EMT marker(E-cadherin) were found in CM-treated SW48 cells by Western Blot. The addition of an anti-IL-6 antibody significantly inhibited the increase of EMT markers (Vimentin and β-catenin) as well as cell migration and invasion, suggesting that IL-6 played a critical role in promoting EMT of CM-treated SW48 cells. In addition, we found that the levels of p-STAT3 and p-ERK increased in CM-treated SW48 compared to untreated cells, which can be reversed by AG490, an inhibitor of JAK. In the meantime, the suppression of JAK-associated signaling pathways caused a decrease of β-catenin. In summary, our study suggested that macrophage-induced IL-6 promotes migration and invasion of colon cancer cell via Wnt/β-catenin pathway in STAT3/ERK-dependent way.
Topics: 6-Aminonicotinamide; Antineoplastic Combined Chemotherapy Protocols; Aspartic Acid; Cell Line, Tumor; Cell Movement; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Humans; Interleukin-6; MAP Kinase Signaling System; Macrophages; Methylthioinosine; STAT3 Transcription Factor; THP-1 Cells; Wnt Signaling Pathway; beta Catenin
PubMed: 30243096
DOI: 10.1016/j.biopha.2018.09.067 -
Drug Metabolism and Disposition: the... Aug 2018Mercaptopurine (MP) is a cytotoxic thiopurine important for the treatment of cancer and autoimmune diseases. MP and other thiopurine drugs undergo extensive...
Mercaptopurine (MP) is a cytotoxic thiopurine important for the treatment of cancer and autoimmune diseases. MP and other thiopurine drugs undergo extensive intracellular metabolism, but the mechanisms of action are poorly characterized. In particular, it is unknown how different metabolites contribute to cytotoxicity and incorporation of thiopurine bases into DNA. The aim of this study was to ask whether cytotoxicity results from the incorporation of thioguanosine nucleotides into DNA, an alternative thiopurine metabolite, or a combination of factors. Therefore, we measured the cytotoxicity, metabolism, and incorporation of thioguanosine into DNA in response to MP or MP metabolites. Thiopurine metabolites varied in cytotoxicity, with methyl-thioinosine-mono-phosphate and thioguanosine-tri-phosphate the most toxic, and the methyl-thioguanosine nucleotides the least. We show, using liquid chromatography-tandem mass spectrometry, how different metabolites may perturb biochemical pathways, particularly disrupting guanosine nucleotide homeostasis, that may contribute to the mechanism of action of thiopurines. Although there was no correlation between metabolite cytotoxicity and the levels of 6-methylthioinosine-mono-phosphate or thioguanosine incorporation into DNA as individual factors, a combined analysis suggested that these factors together had a major influence on cytotoxicity. This study emphasizes the importance of enzymes of nucleotide homeostasis, methylation, and demethylation in thiopurine effects. These results will facilitate the development of dynamic biochemical models of thiopurine biochemistry that will improve our understanding of mechanisms of action in relevant target tissues.
Topics: Cell Line, Tumor; DNA; Homeostasis; Humans; Mercaptopurine; Methylation; Methyltransferases; Nucleotides; Thioinosine
PubMed: 29884651
DOI: 10.1124/dmd.118.081844 -
Microbiology (Reading, England) Jul 2018The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic...
The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic reactions. The MSP has been completely elucidated in aerobic organisms, and found to rely on molecular oxygen. Since anaerobic organisms do not use O2, an alternative pathway(s) must be operating. We sought to evaluate whether the functions of two annotated MSP enzymes from Methanocaldococcus jannaschii, a methylthioinosine phosphorylase (MTIP) and a methylthioribose 1-phosphate isomerase (MTRI), are consistent with functioning in a modified anaerobic MSP (AnMSP). We show here that recombinant MTIP is active with six different purine nucleosides, consistent with its function as a general purine nucleoside phosphorylase for both AnMSP and purine salvage. Recombinant MTRI is active with both 5-methylthioribose 1-phosphate and 5-deoxyribose 1-phosphate as substrates, which are generated from phosphororolysis of 5'-methylthioinosine and 5'-deoxyinosine by MTIP, respectively. Together, these data suggest that MTIP and MTRI may function in a novel pathway for recycling the 5'-deoxyadenosine moiety of SAM in M. jannaschii. These enzymes may also enable biosynthesis of 6-deoxy-5-ketofructose 1-phosphate (DKFP), an essential intermediate in aromatic amino acid biosynthesis. Finally, we utilized a homocysteine auxotrophic strain of Methanosarcina acetivorans Δma1821-22Δoahs (HcyAux) to identify potential AnMSP intermediates in vivo. Growth recovery experiments of the M. acetivorans HcyAux were performed with known and proposed intermediates for the AnMSP. Only one metabolite, 2-keto-(4-methylthio)butyric acid, rescued growth of M. acetivorans HcyAux in the absence of homocysteine. This observation may indicate that AnMSP pathways substantially differ among methanogens from phylogenetically divergent genera.
Topics: Bacterial Proteins; Biosynthetic Pathways; Deoxyadenosines; Fructosephosphates; Gene Expression; Genetic Complementation Test; Kinetics; Methanocaldococcus; Methanosarcina; Methionine; Molecular Weight; Recombinant Proteins; S-Adenosylmethionine; Species Specificity; Substrate Specificity
PubMed: 29877790
DOI: 10.1099/mic.0.000670 -
Faraday Discussions Apr 2018S6-Methylthioinosine and O6-methylguanosine are byproducts resulting from the enzymatic reactions of sulfur-substituted prodrugs in cells and from the interaction of...
S6-Methylthioinosine and O6-methylguanosine are byproducts resulting from the enzymatic reactions of sulfur-substituted prodrugs in cells and from the interaction of alkylating agents with cellular DNA, respectively. Their photochemistry has not been investigated, and it is currently unknown whether light absorption by these byproducts may pose any threat to the cell. In this contribution, their photoinduced processes upon absorption of UVB radiation are reported using broadband transient absorption spectroscopy. Plausible electronic relaxation mechanisms are proposed for both biological molecules, which are supported by steady-state absorption and emission measurements, and by singlet and triplet vertical excitation energies performed on a large subset of ground-state optimized conformational isomers in solution. The results are compared to the body of knowledge gathered in the scientific literature about the light-induced processes in the sulfur-substituted and canonical purine monomers. In particular, it is shown that S6-methylation decreases the rate to populate the lowest-energy triplet state and blueshifts the ground-state absorption spectrum compared to those for the sulfur-substituted prodrugs and for the 6-thioguanosine metabolite. Similarly, O6-methylation decreases the rate of internal conversion to the ground state observed in the guanine monomers by more than 10-fold in acetonitrile and 40-fold in aqueous solution, while it redshifts the ground-state absorption spectrum. Collectively, this investigation provides relevant new insights about the relationship between structural modifications of the purine chromophore and the electronic relaxation mechanisms in this important group of biological molecules.
Topics: Guanosine; Methylthioinosine; Photochemical Processes; Solutions; Spectrophotometry, Ultraviolet
PubMed: 29372193
DOI: 10.1039/c7fd00193b -
International Journal of Antimicrobial... Dec 2017
Topics: Humans; Methylthioinosine; Nucleosides; Virus Replication; Zika Virus; Zika Virus Infection
PubMed: 29032113
DOI: 10.1016/j.ijantimicag.2017.10.002 -
International Journal of Antimicrobial... Nov 2017
Topics: Animals; Antiviral Agents; Female; Fetal Development; Humans; Methylthioinosine; Pregnancy
PubMed: 28890394
DOI: 10.1016/j.ijantimicag.2017.09.003 -
Chemico-biological Interactions Sep 2017To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity.
AIM
To apply an innovative LC-MS/MS method to quantify thiopurine metabolites in human hepatocytes and to associate them to cytotoxicity.
METHODS
Immortalized human hepatocytes (IHH cells) were treated for 48 and 96 h, with 1.4 × 10 M azathioprine and 1.1 × 10 M mercaptopurine, concentrations corresponding to the IC values calculated after 96 h exposure in previous cytotoxicity analysis. After treatments, cells were collected for LC-MS/MS analysis to quantify 11 thiopurine metabolites with different level of phosphorylation and viable cells were counted by trypan blue exclusion assay to determine thiopurines in vitro effect on cell growth and survival. Statistical significance was determined by analysis of variance (ANOVA).
RESULTS
Azathioprine and mercaptopurine had a significant time-dependent cytotoxic effect (p-value ANOVA = 0.012), with a viable cell count compared to controls of 55.5% and 67.5% respectively after 48 h and 23.7% and 36.1% after 96 h; no significant difference could be observed between the two drugs. Quantification of thiopurine metabolites evidenced that the most abundant metabolite was TIMP, representing 57.1% and 40.3% of total metabolites after 48 and 96 h. Total thiopurine metabolites absolute concentrations decreased over time: total mean content decreased from 469.9 pmol/million cells to 83.6 pmol/million cells (p-value ANOVA = 0.0070). However, considering the relative amount of thiopurine metabolites, TGMP content significantly increased from 11.4% cells to 26.4% (p-value ANOVA = 0.017). A significant association between thiopurine effects and viable cell counts could be detected only for MeTIMP: lower MeTIMP concentrations were associated with lower cell survival (p-value ANOVA = 0.011). Moreover, the ratio between MeTIMP and TGMP metabolites directly correlated with cell survival (p-value ANOVA = 0.037).
CONCLUSION
Detailed quantification of thiopurine metabolites in a human hepatocytes model provided useful insights on the association between thioguanine and methyl-thioinosine nucleotides with cell viability.
Topics: Azathioprine; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Hepatocytes; Humans; Mercaptopurine; Purines; Tandem Mass Spectrometry
PubMed: 28811125
DOI: 10.1016/j.cbi.2017.08.009 -
International Journal of Antimicrobial... Dec 2017Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the...
Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the virus. ZIKV-associated neurological manifestations and congenital defects make the development of safe and effective antivirals against ZIKV of utmost importance. Here we evaluated the antiviral activity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analogue derived from the prodrug azathioprine, against the epidemic ZIKV strain circulating in Brazil. In all of the assays, an epithelial (Vero) and a human neuronal (SH-SY5Y) cell line were used to evaluate the cytotoxicity and effective concentrations of 6MMPr against ZIKV. Levels of ZIKV-RNA, viral infectious titre and the percentage of infected cells in the presence or absence of 6MMPr were used to determine antiviral efficacy. 6MMPr decreased ZIKV production by >99% in both cell lines in a dose- and time-dependent manner. Interestingly, 6MMPr was 1.6 times less toxic to SH-SY5Y cells compared with Vero cells, presenting a 50% cytotoxic concentrations (CC) of 460.3 µM and 291 µM, respectively. The selectivity index of 6MMPr for Vero and SH-SY5Y cells was 11.9 and 22.7, respectively, highlighting the safety profile of the drug to neuronal cells. Taken together, these results identify, for the first time, the thiopurine nucleoside analogue 6MMPr as a promising antiviral candidate against ZIKV that warrants further in vivo evaluation.
Topics: Animals; Antiviral Agents; Brazil; Cell Line; Cell Survival; Epithelial Cells; Humans; Methylthioinosine; Neurons; Virus Replication; Zika Virus; Zika Virus Infection
PubMed: 28803932
DOI: 10.1016/j.ijantimicag.2017.08.016 -
Virology Journal Jun 2017Canine distemper (CD) is a widespread infectious disease that can severely impact a variety of species in the order Carnivora, as well as non-carnivore species such as...
BACKGROUND
Canine distemper (CD) is a widespread infectious disease that can severely impact a variety of species in the order Carnivora, as well as non-carnivore species such as non-human primates. Despite large-scale vaccination campaigns, several fatal outbreaks have been reported in wild and domestic carnivore populations. This, in association with expansion of the disease host range and the development of vaccine-escape strains, has contributed to an increased demand for therapeutic strategies synergizing with vaccine programs for effectively controlling canine distemper. 6-methylmercaptopurine riboside (6MMPr) is a modified thiopurine nucleoside with known antiviral properties against certain RNA viruses.
METHODS
We tested the inhibitory effects of 6MMPr against a wild-type CDV strain infection in cell culture. We measured infectious particle production and viral RNA levels in treated and untreated CDV-infected cells. Ribavirin (RIB) was used as a positive control.
RESULTS
Here, we report for the first time the antiviral effects of 6MMPr against canine distemper virus (CDV) in vitro. 6MMPr was able to reduce viral RNA levels and to inhibit the production of infectious CDV particles. The therapeutic selectivity of 6MMPr was approximately six times higher than that of ribavirin.
CONCLUSION
Our results indicate that 6MMPr has high anti-CDV potential and warrants further testing against other paramyxoviruses, as well as clinical testing of the compound against CDV.
Topics: Animals; Antiviral Agents; Cell Line; Distemper Virus, Canine; Dogs; Methylthioinosine; Microbial Viability
PubMed: 28651549
DOI: 10.1186/s12985-017-0785-6 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... Apr 2015Eleven compounds were isolated from the culture of Streptomyces sp. CPCC 202950 by a combination of various chromatographic techniques including column chromatography...
Eleven compounds were isolated from the culture of Streptomyces sp. CPCC 202950 by a combination of various chromatographic techniques including column chromatography over macroporous resin HP-20, MCI, and reversed-phase HPLC. Their structures were identified as 1H-pyrrole-2-carboxamide(1),5'-deoxy-5'-methylthioinosine(2), vanillamide(3), trans-3-methylthioacrylamide(4), 1,2,3,4-Tetraydro-1H-pyrido[3,4-b]indole-3-carboxylic acid(5), cyclo(L-pro-L-tyr) (6), N-[2-(4-hydroxyphenyl)]ethylacetamide(7), benzamide (8), cyclo ('L-leucyl-trans-4-hydroxy-L-proline)(9), cyclo-(Phe-Gly) (10), and tryptophan (11). Among them, compounds 1 and 2 were new natural products. In the preliminary assays, none of the compounds exhibited obvious inhibition of HIV-1 protease activity (IC50 > 10 micromol x L(-1)).
Topics: Culture Media; HIV Protease; HIV Protease Inhibitors; Molecular Structure; Spectrometry, Mass, Electrospray Ionization; Streptomyces
PubMed: 26281555
DOI: No ID Found