The 'speck'-tacular oversight of the NLRP3-pyroptosis pathway on gastrointestinal inflammatory diseases and tumorigenesis.

The NLRP3 inflammasome is an intracellular sensor and an essential component of the innate immune system involved in danger recognition. An important hallmark of inflammasome activation is the formation of a single supramolecular punctum, known as a speck, per cell, which is the site where the pro-inflammatory cytokines IL-1ß and IL-18 are converted into their bioactive form. Speck also provides the platform for gasdermin D protein activation, whose N-terminus domain perforates the plasma membrane, allowing the release of mature cytokines alongside with a highly inflammatory form of cell death, namely pyroptosis. Although controlled NLRP3 inflammasome-pyroptosis pathway activation preserves mucosal immunity homeostasis and contributes to host defense, a prolonged trigger is deleterious and could lead, in genetically predisposed subjects, to the onset of inflammatory bowel disease, including Crohn's disease and ulcerative colitis, as well as to gastrointestinal cancer. Experimental evidence shows that the NLRP3 inflammasome has both protective and pathogenic abilities. In this review we highlight the impact of the NLRP3-pyroptosis axis on the pathophysiology of the gastrointestinal tract at molecular level, focusing on newly discovered features bearing pro- and anti-inflammatory and neoplastic activity, and on targeted therapies tested in preclinical and clinical trials.

Autotaxin inhibitor IOA-289 reduces gastrointestinal cancer progression in preclinical models.

BACKGROUND: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA stimulates cell proliferation and migration and promotes wound repair following tissue damage. ATX levels are directly correlated with stage and grade in several human cancers. Several small molecule ATX inhibitors have been developed in recent years. IOA-289 is a potent ATX inhibitor, developed to treat cancers containing fibrosis. In this study, we tested IOA-289 treatment on different gastrointestinal tract tumor cell lines, in order to evaluate its effects on viability and motility. METHODS: To determine the effects on cell viability and proliferation of treatment with increasing concentrations of IOA-289, we used the crystal violet assay, a clonogenic assay in matrigel, and we evaluated the inhibitor's effect on formation of 3D spheroids in an in vitro model. The effect of IOA-289 on cell cycle phases was analysed with a redox dye reagent. Cell migration capacity was evaluated by wound healing assay and transwell migration assay. To evaluate the pro-apoptotic effect of the inhibitor, cells were stained with Annexin V and immunofluorescence and flow cytometry analysis were performed. An antibody array was also used, to discriminate, in various samples, the differential expression of 43 proteins involved in the apoptosis pathway. RESULTS: We found that IOA-289 is able to inhibit both growth and migration of gastrointestinal tract tumor cell lines, both in 2D (crystal violet assay) and 3D in vitro models (spheroid formation and clonogenic assay in matrigel). This effect is dose-dependent, and the drug is most effective when administered in FBS-free culture medium. The inhibitory effect on cell growth is due to a pro-apoptotic effect of IOA-289. Staining with FITC-conjugated Annexin V showed that IOA-289 induced a dose-dependent increase in fluorescence following incubation for 24 h, and apoptotic cells were also distinguished in flow cytometry using Annexin/PI staining. The antibody array shows that treatment with IOA-289 causes the increased expression of several pro-apoptotic proteins in all tested cell lines. CONCLUSIONS: These results indicate that IOA-289 may be an effective drug for the treatment of tumors of the gastrointestinal tract, particularly those characterized by a high degree of fibrosis.

Laminin-332 γ2 Monomeric Chain Promotes Adhesion and Migration of Hepatocellular Carcinoma Cells.

Extracellular matrix (ECM) has a well-recognized impact on the progression of solid tumors, including hepatocellular carcinoma (HCC). Laminin 332 (Ln332) is a ECM molecule of epithelial basal lamina, composed of three polypeptide chains (α3, ß3, and γ2), that is usually poorly expressed in the normal liver but is detected at high levels in HCC. This macromolecule was shown to promote the proliferation, epithelial-to-mesenchymal transition (EMT), and drug resistance of HCC cells. The monomeric γ2 chain is up-regulated and localized preferentially at the invasive edge of metastatic intrahepatic HCC nodules, suggesting its potential involvement in the acquisition of invasive properties of HCC cells. HCC cells were tested in in vitro adhesion, scattering, and transwell migration assays in response to fibronectin and the Ln332 and Ln332 γ2 chains, and the activation status of major signaling pathways involved was evaluated. Here, we show that the Ln332 γ2 chain promotes HCC the cell adhesion, migration, and scattering of HCC cells that express the Ln332 receptor α3ß1 integrin, proving to be a causal factor of the EMT program achievement. Moreover, we found that efficient HCC cell adhesion and migration on γ2 require the activation of the small cytosolic GTPase Rac1 and ERKs signaling. These data suggest that the γ2 chain, independently from the full-length Ln332, can contribute to the pro-invasive potential of aggressive HCC cell subpopulations.

Proteoglycans in Cancer: Friends or Enemies? A Special Focus on Hepatocellular Carcinoma.

Proteoglycans are a class of highly glycosylated proteins expressed in virtually all tissues, which are localized within membranes, but more often in the pericellular space and extracellular matrix (ECM), and are involved in tissue homeostasis and remodeling of the stromal microenvironment during physiological and pathological processes, such as tissue regeneration, angiogenesis, and cancer. In general, proteoglycans can perform signaling activities and influence a range of physical, chemical, and biological tissue properties, including the diffusivity of small electrolytes and nutrients and the bioavailability of growth factors. While the dysregulated expression of some proteoglycans is observed in many cancers, whether they act as supporters or limiters of neoplastic progression is still a matter of controversy, as the tumor promoting or suppressive function of some proteoglycans is context dependent. The participation of multiple proteoglycans in organ regeneration (as demonstrated for the liver in hepatectomy mouse models) and in cancer suggests that these molecules actively influence cell growth and motility, thus contributing to key events that characterize neoplastic progression. In this review, we outline the main roles of proteoglycans in the physiology and pathology of cancers, with a special mention to hepatocellular carcinoma (HCC), highlighting the translational potential of proteoglycans as targets or therapeutic agents for the treatment of this disease.

Proteoglycan-4 is correlated with longer survival in HCC patients and enhances sorafenib and regorafenib effectiveness via CD44 in vitro.

Sorafenib and regorafenib administration is among the preferential approaches to treat hepatocellular carcinoma (HCC), but does not provide satisfactory benefits. Intensive crosstalk occurring between cancer cells and other multiple non-cancerous cell subsets present in the surrounding microenvironment is assumed to affect tumor progression. This interplay is mediated by a number of soluble and structural extracellular matrix (ECM) proteins enriching the stromal milieu. Here we assess the HCC tumor expression of the ECM protein proteoglycan 4 (PRG4) and its potential pharmacologic activity either alone, or in combination with sorafenib and regorafenib. PRG4 mRNA levels resulted strongly correlated with increased survival rate of HCC patients (p = 0.000) in a prospective study involving 78 HCC subjects. We next showed that transforming growth factor beta stimulates PRG4 expression and secretion by primary human HCC cancer-associated fibroblasts, non-invasive HCC cell lines, and ex vivo specimens. By functional tests we found that recombinant human PRG4 (rhPRG4) impairs HCC cell migration. More importantly, the treatment of HCC cells expressing CD44 (the main PRG4 receptor) with rhPRG4 dramatically enhances the growth-limiting capacity of sorafenib and regorafenib, whereas not significantly affecting cell proliferation per se. Conversely, rhPRG4 only poorly potentiates drug effectiveness on low CD44-expressing or stably CD44-silenced HCC cells. Overall, these data suggest that the physiologically-produced compound PRG4 may function as a novel tumor-suppressive agent by strengthening sorafenib and regorafenib effects in the treatment of HCC.

Radical reduction of aromatic azides to amines with triethylsilane.

Aromatic azides are inert toward triethylsilane under thermal conditions in the presence of a radical initiator, but in the presence of additional catalytic amounts of tert-dodecanethiol, they afford anilinosilanes and thence the corresponding anilines in virtually quantitative yields.

Reaction of azides with dichloroindium hydride: very mild production of amines and pyrrolidin-2-imines through possible indium-aminyl radicals.

Organic azides are easily reduced to the corresponding amines by reaction with dichloroindium hydride under very mild conditions and in a highly chemoselective fashion. Gamma-azidonitriles give rise to outstanding five-membered cyclizations affording pyrrolidin-2-imines. A rationalization of the overall experimental data cannot exclude the occurrence of competitive radical and nonradical pathways, but certain results are, however, soundly consistent with the intermediacy of indium-bound nitrogen-centered radicals. [reaction: see text]

Generation and cyclization of unsaturated carbamoyl radicals derived from S-4-pentynyl carbamothioates under tin-free conditions.

The radical reaction of benzenethiol with S-4-pentynyl carbamothioates provides a valuable protocol for the tin-free generation of carbamoyl radicals, which arise from intramolecular substitution at sulfur by the initial sulfanylvinyl radicals. This procedure can be usefully employed to achieve N-benzylcarbamoyl radical 5-exo and 4-exo cyclizations leading, respectively, to pyrrolidinones and azetidinones, although, for the latter, it seems of lesser utility. Novel evidence is presented that N-tosyl-substituted carbamoyl radicals display a peculiar tendency to yield the corresponding isocyanate by beta-elimination of the tosyl radical.

Radical reduction of aromatic azides to amines with tributylgermanium hydride.

[reaction: see text] Aromatic azides are inert toward tributylgermanium hydride under thermal conditions in the absence and in the presence of a radical initiator but in the presence of catalytic amounts of benzenethiol undergo fast reaction, yielding reduced anilines and 2-germylated derivatives in high overall yields.

Thermal reactions of tributyltin hydride with alpha-azido esters: unexpected intervention of tin triazene adducts under both nonradical and radical conditions.

Thermal reaction of various alpha-azido esters with Bu(3)SnH in refluxing benzene results in smooth production of 3-(tributylstannyl)-1-triazene adducts affording cyclized 1,2,3-triazol-4-ones in preference to reduced amines and thence provides a new useful method for the preparation of these triazole derivatives. In the presence of AIBN the occurrence of triazene products still remains important or even exclusive and, consequently, generation of the expected stannylaminyl radicals is seriously limited. With 2-azidomalonates and alpha-azido-beta-keto esters stannyltriazenes can similarly occur in the absence of the radical initiator, but in the latter cases the ensuing triazenes undergo preferential cyclization onto the ketone moiety to give reactive hydroxytriazolines. Contrary to alpha-azido esters, in the presence of AIBN alpha-azido-beta-keto esters as well as azidomalonates give rise only to the usual stannylaminyl radicals. A possible explanation for the different behavior of the mono- and dicarbonyl azides in the presence of AIBN is put forward.

Cyclizations of N-stannylaminyl radicals onto nitriles.

[reaction: see text] Stannylaminyl radicals derived from radical reactions of Bu(3)SnH with azidoalkylmalononitriles exhibit highly efficient 5- and 6-exo cyclization onto either nitrile group to give aminoiminyl radicals that in turn are reduced to amidines or undergo successive 5-exo cyclization onto an internal alkene.