Modulating Properties of Piroxicam, Meloxicam and Oxicam Analogues against Macrophage-Associated Chemokines in Colorectal Cancer.

The mechanisms underlying the antineoplastic effects of oxicams have not been fully elucidated. We aimed to assess the effect of classic and novel oxicams on the expression/secretion of macrophage-associated chemokines (RTqPCR/Luminex xMAP) in colorectal adenocarcinoma cells, and on the expression of upstream the non-steroidal anti-inflammatory drug (NSAID)-activated genes NAG1, NFKBIA, MYD88, and RELA, as well as at the chemokine profiling in colorectal tumors. Meloxicam downregulated CCL4 9.9-fold, but otherwise the classic oxicams had a negligible/non-significant effect. Novel analogues with a thiazine ring substituted with arylpiperazine and benzoyl moieties significantly modulated chemokine expression to varying degree, upregulated NAG1 and NFKBIA, and downregulated MYD88. They inhibited CCL3 and CCL4, and their effect on CCL2 and CXCL2 depended on the dose and exposure. The propylene linker between thiazine and piperazine nitrogens and one arylpiperazine fluorine substituent characterized the most effective analogue. Only CCL19 and CXCL2 were not upregulated in tumors, nor was CXCL2 in tumor-adjacent tissue compared to normal mucosa. Compared to adjacent tissue, CCL4 and CXCL2 were upregulated, while CCL2, CCL8, and CCL19 were downregulated in tumors. Tumor CCL2 and CCL7 increased along with advancing T and CCL3, and CCL4 along with the N stage. The introduction of arylpiperazine and benzoyl moieties into the oxicam scaffold yields effective modulators of chemokine expression, which act by upregulating NAG1 and interfering with NF-κB signaling.

Transcriptional and Metabolomic Analysis of L-Arginine/Nitric Oxide Pathway in Inflammatory Bowel Disease and Its Association with Local Inflammatory and Angiogenic Response: Preliminary Findings.

L-arginine/nitric oxide pathway in Crohn's disease (CD) and ulcerative colitis (UC) is poorly investigated. The aim of current study is to quantify pathway serum metabolites in 52 CD (40 active), 48 UC (33 active), and 18 irritable bowel syndrome patients and 40 controls using mass spectrometry and at determining mRNA expression of pathway-associated enzymes in 91 bowel samples. Arginine and symmetric dimethylarginine decreased (p < 0.05) in active-CD (129 and 0.437 µM) compared to controls (157 and 0.494 µM) and active-UC (164 and 0.52 µM). Citrulline and dimethylamine increased (p < 0.05) in active-CD (68.7 and 70.9 µM) and active-UC (65.9 and 73.9 µM) compared to controls (42.7 and 50.4 µM). Compared to normal, CD-inflamed small bowel had downregulated (p < 0.05) arginase-2 by 2.4-fold and upregulated dimethylarginine dimethylaminohydrolase (DDAH)-2 (1.5-fold) and arginine N-methyltransferase (PRMT)-2 (1.6-fold). Quiescent-CD small bowel had upregulated (p < 0.05) arginase-2 (1.8-fold), DDAH1 (2.9-fold), DDAH2 (1.5-fold), PRMT1 (1.5-fold), PRMT2 (1.7-fold), and PRMT5 (1.4-fold). Pathway enzymes were upregulated in CD-inflamed/quiescent and UC-inflamed colon as compared to normal. Compared to inflamed, quiescent CD-colon had upregulated DDAH1 (5.7-fold) and ornithine decarboxylase (1.6-fold). Concluding, the pathway is deregulated in CD and UC, also in quiescent bowel, reflecting inflammation severity and angiogenic potential. Functional analysis of PRMTs and DDAHs as potential targets for therapy is warranted.

3-Bromopyruvate as a potential pharmaceutical in the light of experimental data.

3-Bromopyruvate (3-BrPA) is an halogenated analogue of pyruvic acid known for over four decades as an alkylating agent reacting with thiol groups of many proteins. It enters animal cells like a lactate: via monocarboxylic acid transporters. Increasing interest in this compound, in recent times, is mainly due to hopes associated with its anticancer action. It is based on the impairment of energy metabolism of tumor cells by inhibiting enzymes in the glycolysis pathway (hexokinase II, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase) and the oxidative phosphorylation (succinate dehydrogenase). Two cases of clinical application of this compound in the treatment of advanced cancers were reported. By using 3-BrPA, rheumatoid arthritis in SKG mice has been reduced. This compound has also antiparasitic activity: lowers cell viability of Trypanosoma brucei, decreases intracellular proliferation of Toxoplasma gondii and reduces the metabolic activity of Schistosoma mansoni. It also has antifungal properties; particularly it acts strongly on Cryptococcus neoformans, as well as Saccharomyces cerevisiae. An inhibitory effect on bacterial enzymes was also described on: isocitrate lyase from Escherichia coli, Mycobacterium tuberculosis, Pseudomonas indigofera and 2-methylisocitrate lyase, succinate dehydrogenase and acetohydroxylic acid synthase from Escherichia coli. Wherever undesirable (cancer, parasitic) cells differ from normal by more intense glycolysis and higher energy needs, there is a good chance of successful 3-BrPA use. However, this compound acts on all cells and it, therefore, seems that its future as a pharmaceutical is dependent upon the development of appropriate methods for its effective and safe application.

Can Shockwave Treatment Elicit a Molecular Response to Enhance Clinical Outcomes in Pressure Ulcers? The SHOck Waves in wouNds Project.

Wound healing requires the coordinated interaction of dermis cells, the proper deposition of extracellular matrix, re-epithelialization, and angiogenesis. Extracorporeal shock wave (ESW) is a promising therapeutic modality for chronic wounds. This study determined the biological mechanisms activated under ESW, facilitating the healing of pressure ulcers (PUs). A group of 10 patients with PUs received two sessions of radial ESW (300 + 100 pulses, 2.5 bars, 0.15 mJ/mm2, 5 Hz). Histomorphological and immunocytochemical assessments were performed on tissue sections obtained from the wound edges before the ESW (M0) and after the first (M1) and second (M2) ESW. The proliferation index of keratinocytes and fibroblasts (Ki-67), the micro-vessels' density (CD31), and the number of myofibroblasts (α-SMA) were evaluated. The involvement of the yes-associated protein (YAP1) in sensing mechanical strain, and whether the nuclear localization of YAP1, was shown. The increased proliferative activity of epidermal cells and skin fibroblasts and the increased number of myofibroblasts, often visible as integrated cell bands, were also demonstrated as an effect of wound exposure to an ESW. The results indicate that the major skin cells, keratinocytes, and fibroblasts are mechanosensitive. They intensify proliferation and extracellular matrix remodeling in response to mechanical stress. A significant improvement in clinical wound parameters was also observed.

The Effect of Oleanolic Acid and Its Four New Semisynthetic Derivatives on Human MeWo and A375 Melanoma Cell Lines.

This study aimed to synthesize four new semisynthetic derivatives of natural oleanolic acid (OA) and, based on an analysis of their cytotoxic and anti-proliferative effects against human MeWo and A375 melanoma cell lines, select those with anti-cancer potential. We also screened the treatment time with the concentration of all four derivatives. We synthesized oxime 2 and performed its acylation with carboxylic acids into new derivatives 3a, 3b, 3c and 3d according to the methods previously described. Colorimetric MTT and SRB assays were used to measure the anti-proliferative and cytotoxic activity of OA and its derivatives 3a, 3b, 3c and 3d against melanoma cells. Selected concentrations of OA, the derivatives, and different time periods of incubation were used in the study. The data were analyzed statistically. The present results revealed the possible anti-proliferative and cytotoxic potential of two selected OA derivatives 3a and 3b, on A375 and MeWo melanoma cells, especially at concentrations of 50 µM and 100 µM at 48 h of incubation (p < 0.05). Further studies will be necessary to analyze the proapoptotic and anti-cancer activities of 3a and 3b against skin and other cancer cells. The bromoacetoxyimine derivative (3b) of OA morpholide turned out to be the most effective against the tested cancer cells.

The effect of biologically active compounds in the mucus of slugs Limax maximus and Arion rufus on human skin cells.

Molluscs are one of the sources of biologically active substances, which are now intensively studied, especially for their anti-cancer properties. Malignant melanoma originates from melanocytes, develops very quickly and is associated with poor prognosis. Therefore, the aim of the study was to assess the properties of biologically active compounds in sterile mucus isolated from slugs Limax maximus and Arion rufus. Tested mucus were isolated using the new self-developed method which is safe for the environment and the animal donors. The impact of the mucus on human keratinocytes CCD 1106 KERTr and malignant melanoma cells A-375 was examined using MTT assay and SRB assay, which allowed us to determine the cell metabolic activity and cell number after treating them with slug mucus isolated from Limax maximus and Arion rufus decreased human keratinocytes and melanoma cells metabolic activity as well as manifested properties of reducing the number of cells in both tested cell lines, and therefore can be a source of biologically active substances with anticancer potential.

Heat Shock Proteins HSPA1 and HSP90AA1 Are Upregulated in Colorectal Polyps and Can Be Targeted in Cancer Cells by Anti-Inflammatory Oxicams with Arylpiperazine Pharmacophore and Benzoyl Moiety Substitutions at Thiazine Ring.

Heat shock proteins HSPA1/Hsp70α and HSP90AA1/Hsp90α are crucial for cancer growth but their expression pattern in colorectal polyps or whether they can be modulated by oxicams is unknown. We quantified (RTqPCR) HSPA1 and HSP90AA1 expression in 50 polyp-normal pairs in relation to polyp malignancy potential and examined the effect of piroxicam, meloxicam and five novel analogues on HSPA1 and HSP90AA1 expression (mRNA/protein) in colorectal adenocarcinoma lines. HSPA1 and HSP90AA1 were upregulated in polyps by 3- and 2.9-fold. Expression ratios were higher in polyps with higher dysplasia grade and dominant villous growth pattern, mostly a result of diminished gene expression in normal tissue. Classic oxicams had negligible/non-significant effect on HSP expression. Their most effective analogue inhibited HSPA1 protein and gene by 2.5-fold and 5.7-fold in Caco-2 and by 11.5-fold and 6.8-fold in HCT116 and HSPA1 protein in HT-29 by 1.9-fold. It downregulated HSP90AA1 protein and gene by 1.9-fold and 3.7-fold in Caco-2 and by 2-fold and 5.0-fold in HCT116. HSPA1 and HSP90AA1 are upregulated in colorectal polyps reflecting their potential for malignancy. HSPA1 in cancer cells and, to lesser degree, HSP90AA1 can be reduced by oxicam analogues with thiazine ring substituted via propylene linker by arylpiperazine pharmacophore with fluorine substituents and by benzoyl moiety.

The effect of 3-bromopyruvate on the properties of cathepsin B in the aspect of metastatic potential of colon cancer cells.

BACKGROUND: Cathepsin B (CTSB, EC 3.4.22.1) is a protease that physiologically resides in lysosomes and whose biosynthesis, cell surface location, intracellular distribution, and enzymatic activity undergo changes during the pathogenesis of cancer; it plays an important role in metastasis. Due to its active center structure, it is theoretically susceptible to the action of 3-bromopyruvate - an analogue of pyruvic acid and an alkylator that has been studied in depth in recent years for its anti-cancer activity, mainly through the inhibition of glycolytic enzymes. OBJECTIVES: To investigate the effects of 3-bromopyruvate on the tumor cell properties in selected colorectal carcinoma cell lines that are widely attributed to the dysregulation of CTSB. Moreover, the effect of direct action of 3-bromopyruvate on the CTSB molecule was investigated in vitro. MATERIAL AND METHODS: The research on the effect of 3-bromopyruvate on Caco-2/HCT 116 cells and purified human CTSB included a scratch/wound healing assay, a cell invasion assay, spectrofluorimetric measurements of enzymatic activity of cathepsin B, indirect immunofluorescence and flow cytometry, zymography, and liquid chromatography/mass spectrometry methods. RESULTS: 3-bromopyruvate reduced the activity and secretion of active CTSB and lowered the motility and invasiveness of Caco-2/HCT 116 human colorectal cancer cells. It decreased the exposure of CTSB on the outer surface of the cell membrane in both cell lines. 3-bromopyruvate inhibited the activity of CTSB reversibly and did not alkylate the molecule of the enzyme. CONCLUSIONS: This is the first report on the effect of 3-bromopyruvate directly on CTSB and indirectly on the mechanisms leading to its distinct pathophysiological properties, resulting in increased metastatic potential of cancer cells, among others. Although detailed mechanisms of the interaction between 3-bromopyruvate and the active site of CTSB require further research, the results provide a new perspective from which to study the antitumor effect of 3-bromopyruvate.

Interleukins 4 and 13 and Their Receptors Are Differently Expressed in Gastrointestinal Tract Cancers, Depending on the Anatomical Site and Disease Advancement, and Improve Colon Cancer Cell Viability and Motility.

Immunosuppressive interleukins (IL)-4 and 13 may directly promote cancer but neither their status nor role in gastrointestinal tract is clarified. We aim at quantifying ILs and their receptors in paired normal-tumor samples (n = 49/51) and sera (n = 263), using immunoassays and RTqPCR, and screening for their effect on colonic cancer cells. Both ILs were elevated locally at protein level in all cancers but only IL13 transcripts in colon were upregulated. Interleukin and their receptor expression reflected cancer pathology to varying degrees, with the association frequently inverse and manifested in non-cancerous tissue. Positive correlation with cancer-promoting genes BCL2, BCLxL, HIF1A, VEGFA, ACTA2, CCL2, PTGS2, and CDKN1A, but not Ki67, was demonstrated, particularly for ILs' receptors. Circulating IL-4 was elevated in all, while IL-13 only in colorectal or esophageal cancers, reflecting their advancement. IL4Ra and IL13Ra1 transcripts were downregulated by hypoxia and, in Caco-2, also by IL-4. Interleukin stimulation slightly improved colonic cancer cell viability, weakly upregulating BCL2 and Ki67 in HCT116 and HT-29. It affected cell motility more markedly and was consistently accompanied by upregulation of claudin-2. Gastrointestinal tract cancers are associated with IL-4 and IL-13 upregulation, which may facilitate cancer growth. Targeting both interleukins as an antineoplastic strategy warrants further investigation.

L-Arginine/Nitric Oxide Pathway Is Altered in Colorectal Cancer and Can Be Modulated by Novel Derivatives from Oxicam Class of Non-Steroidal Anti-Inflammatory Drugs.

L-arginine/nitric oxide pathway metabolites are altered in colorectal cancer (CRC). We evaluated underlying changes in pathway enzymes in 55 paired tumor/tumor-adjacent samples and 20 normal mucosa using quantitative-PCR and assessed the impact of classic and novel oxicam analogues on enzyme expression and intracellular metabolite concentration (LC-MS/MS) in Caco-2, HCT116, and HT-29 cells. Compared to normal mucosa, ARG1, PRMT1, and PRMT5 were overexpressed in both tumor and tumor-adjacent tissue and DDAH2 solely in tumor-adjacent tissue. Tumor-adjacent tissue had higher expression of ARG1, DDAH1, and DDAH2 and lower NOS2 than patients-matched tumors. The ARG1 expression in tumors increased along with tumor grade and reflected lymph node involvement. Novel oxicam analogues with arylpiperazine moiety at the thiazine ring were more effective in downregulating DDAHs and PRMTs and upregulating ARG2 than piroxicam and meloxicam. An analogue distinguished by propylene linker between thiazine's and piperazine's nitrogen atoms and containing two fluorine substituents was the strongest inhibitor of DDAHs and PRMTs expression, while an analogue containing propylene linker but no fluorine substituents was the strongest inhibitor of ARG2 expression. Metabolic reprogramming in CRC includes overexpression of DDAHs and PRMTs in addition to ARG1 and NOS2 and is not restricted to tumor tissue but can be modulated by novel oxicam analogues.

Early induction of stress-associated Src activator/Homo sapiens chromosome 9 open reading frame 10 protein following photodynamic therapy.

BACKGROUND: There are proteins, responsible for many basic cell functions (transmission of extracellular signals to cytoplasm or nucleus, cell growth, proliferation, migration, survival), which are activated and overexpressed in response to acute oxidative stress, especially tyrosine kinases. The oxidative stress-associated Src activator/Homo sapiens chromosome 9 open reading frame 10 protein (Ossa/C9orf10) protects cancer cells from oxidative stress-induced apoptosis by Src family kinases activation. METHODS: In this study precursor of protoporphyrin IX, 5-aminolevulinic acid and its encapsulated form were used in treating MCF-7 human breast cancer cells. After light illumination, cells were collected at different time points and used for evaluation (immunocytochemistry, Western blot analysis) of expression of above proteins, c-Src and Ossa. RESULTS: Our results showed that 5-aminolevulinic acid-mediated photodynamic therapy caused decrease of c-Src expression at 7h after irradiation. The strongest expression was observed at 24h after treatment. Encapsulated form of 5-aminolevulinic acid in terms of PDT caused similar changes of expression of c-Src protein. Furthermore, we observed strong Ossa expression at 7h after treatment in comparison to very low expression at time points 0, 18 and 24h. CONCLUSION: We would like to emphasize that our results showed high expression of Ossa at early time interval after PDT, which was accompanied by a low expression of c-Src kinase, what could protect cancer cells from PDT through activation of c-Src in response to oxidative stress.