Oleanolic Acid Glycosides from Scabiosa caucasica and Scabiosa ochroleuca: Structural Analysis and Cytotoxicity.

In the field of research on medicinal plants from the Armenian flora, the phytochemical study of two Scabiosa L. species, S. caucasica M. Bieb. and S. ochroleuca L. (Caprifoliaceae), has led to the isolation of five previously undescribed oleanolic acid glycosides from an aqueous-ethanolic extract of the roots: 3-O-α-L-rhamnopyranosyl-(1→3)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid, 3-O-ß-D-xylopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-ß-D-xylopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester, 3-O-α-L-rhamnopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1→4)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyloleanolic acid 28-O-ß-D-glucopyranosyl-(1→6)-ß-D-glucopyranosyl ester. Their full structural elucidation required extensive 1D and 2D NMR experiments, as well as mass spectrometry analysis. For the biological activity of the bidesmosidic saponins and the monodesmosidic saponin, their cytotoxicity on a mouse colon cancer cell line (MC-38) was evaluated.

IL-1RAP, a Key Therapeutic Target in Cancer.

Cancer is a major cause of death worldwide and especially in high- and upper-middle-income countries. Despite recent progress in cancer therapies, such as chimeric antigen receptor T (CAR-T) cells or antibody-drug conjugate (ADC), new targets expressed by the tumor cells need to be identified in order to selectively drive these innovative therapies to tumors. In this context, IL-1RAP recently showed great potential to become one of these new targets for cancer therapy. IL-1RAP is highly involved in the inflammation process through the interleukins 1, 33, and 36 (IL-1, IL-33, IL-36) signaling pathways. Inflammation is now recognized as a hallmark of carcinogenesis, suggesting that IL-1RAP could play a role in cancer development and progression. Furthermore, IL-1RAP was found overexpressed on tumor cells from several hematological and solid cancers, thus confirming its potential involvement in carcinogenesis. This review will first describe the structure and genetics of IL-1RAP as well as its role in tumor development. Finally, a focus will be made on the therapies based on IL-1RAP targeting, which are now under preclinical or clinical development.

Nanofitins targeting heat shock protein 110: An innovative immunotherapeutic modality in cancer.

The presence of an inactivating heat shock protein 110 (HSP110) mutation in colorectal cancers has been correlated with an excellent prognosis and with the ability of HSP110 to favor the formation of tolerogenic (M2-like) macrophages. These clinical and experimental results suggest a potentially powerful new strategy against colorectal cancer: the inhibition of HSP110. In this work, as an alternative to neutralizing antibodies, Nanofitins (scaffold ~7 kDa proteins) targeting HSP110 were isolated from the screening of a synthetic Nanofitin library, and their capacity to bind (immunoprecipitation, biolayer interferometry) and to inhibit HSP110 was analyzed in vitro and in vivo. Three Nanofitins were found to inhibit HSP110 chaperone activity. Interestingly, they share a high degree of homology in their variable domain and target the peptide-binding domain of HSP110. In vitro, they inhibited the ability of HSP110 to favor M2-like macrophages. The Nanofitin with the highest affinity, A-C2, was studied in the CT26 colorectal cancer mice model. Our PET/scan experiments demonstrate that A-C2 may be localized within the tumor area, in accordance with the reported HSP110 abundance in the tumor microenvironment. A-C2 treatment reduced tumor growth and was associated with an increase in immune cells infiltrating the tumor and particularly cytotoxic macrophages. These results were confirmed in a chicken chorioallantoic membrane tumor model. Finally, we showed the complementarity between A-C2 and an anti-PD-L1 strategy in the in vivo and in ovo tumor models. Overall, Nanofitins appear to be promising new immunotherapeutic lead compounds.

Skin surface temperature of the mammary gland, measured by infrared thermography, in primiparous Girolando cows fed diets containing different lipid sources.

This research communication describes the influence of diet, mammary quarter position and milking process on the temperature of teats and udder of cows fed diets containing different lipid sources. Five primiparous cows were fed diets containing cottonseed, sunflower seed, soybeans or soybean oil as a source of lipids and a reference diet without the inclusion of lipid sources in a 5 × 5 Latin Square design. Milk yield was determined in the last five days of each period. Milk samples were collected for SCC analysis on the last two days of each experimental period. The images of the mammary gland were obtained using an infrared camera and were analyzed with appropriate computer software. Milk yield was 14.8% higher for cows fed soybeans as a source of lipids. Diets and somatic cell counts did not influence the temperature of teats and udder. The milking process reduced the temperature of teats and udder by 0.79°C. Rear teats and rear quarters had higher surface temperatures than front teats and fore quarters. Changes in temperature of teats and mammary quarters occurred as a function of the milking process and quarter position. However, the diet and the SCC did not influence the temperature of teats and mammary quarters in this experiment.

Extruded urea could reduce true protein source in beef cattle diets.

Rumen micro-organisms are capable of producing microbial protein from ammonia and carbon skeleton, and non-protein nitrogen (NPN) may be one of the sources of ammonia. Alternative source of NPN is the slow release of ammonia sources in which the product is the extrusion of starch with urea. This work aimed to determine the effects on nutrient intake, ingestive behaviour, digestibility, nitrogen balance, ruminal pH, rumen ammonia nitrogen, volatile fatty acids (VFA) and blood parameters with increased levels of extruded urea (50, 60, 70 and 80 g/100 kg of body weight [BW]) in beef cattle diet. Four rumen cannulated crossbred steers with initial mean weight of 336 ± 47 kg in a 4 × 4 Latin square design were distributed. Diets were formulated with 400:600 g/kg roughage:concentrate ratio on dry matter based and provided once per day, being used whole corn silage as roughage. There were no effects on nutrient intake (kg/day), ingestive behaviour, apparent digestibility, nitrogen balance, volatile fatty acid (VFA) and blood parameters in extruded urea treatment groups. Similar results were observed on time spent on feeding, rumination and idleness. There were positive linear effects (p = 0.022) on rumen pH in the time of 8 hr after feeding and also on plasma concentration of the extruded urea levels (p = 0.039); whereas a linear negative effect (p = 0.030) was observed on ammonia nitrogen for the collection time of 2 hr after feeding. Increased levels of extruded urea could maintain nutrient intake, digestibility, ingestive behaviour, rumen pH and blood parameters in normal conditions. In conclusion, we recommend the extruded urea use with values up to 80 g/100 kg BW in confined beef cattle that receive balanced diets with 140 g/kg of crude protein.

Beta3 adrenergic receptor stimulation in human macrophages inhibits NADPHoxidase activity and induces catalase expression via PPARγ activation.

The beta3 adrenergic receptor (ß3-AR) stimulation plays a protective role against preterm labor by blocking myometrial contraction, cytokine production, remodeling and apoptosis. We previously demonstrated that macrophage-induced ROS production in the myometrium was a key element leading to the induction of all these labor-associated features. We thus aimed to investigate if the ß3-AR could be expressed in human macrophages and could trigger its protective role in the myometrium by directly inhibiting ROS production. Using lipopolysaccharide (LPS)-stimulated myometrial samples and cell co-culture experiments, we demonstrated that ß3-AR stimulation inhibits the activation of the NADPH oxidase, leading to the subsequent inhibition of ROS production by macrophages. This antioxidant effect was associated with a potent anti-inflammatory response in macrophages. Furthermore, we observed that ß3-AR leads to the expression of catalase not only in macrophages but also in myometrial cells, thereby preventing the transactivation of myometrial cells by hydrogen peroxide. Pharmacological experiments allowed us to demonstrate that these effects were driven by an Erk1/2-mediated activation of the antioxidant transcription factor PPARγ. These results suggest that ß3-AR protective effects in the myometrium could be due to its dual antioxidant properties. Further, the effects observed in a macrophage could highlight new applications in chronic inflammatory diseases.

Peroxisomal ATP-binding cassette transporters form mainly tetramers.

ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.

Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers.

ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.

LXR antagonists induce ABCD2 expression.

X-linked adrenoleukodystrophy (X-ALD) is a rare neurodegenerative disorder characterized by the accumulation of very-long-chain fatty acids resulting from a beta-oxidation defect. Oxidative stress and inflammation are also key components of the pathogenesis. X-ALD is caused by mutations in the ABCDI gene, which encodes for a peroxisomal half ABC transporter predicted to participate in the entry of VLCFA-CoA into the peroxisome, the unique site of their beta-oxidation. Two homologous peroxisomal ABC transporters, ABCD2 and ABCD3 have been proven to compensate for ABCD1 deficiency when overexpressed. Pharmacological induction of these target genes could therefore represent an alternative therapy for X-ALD patients. Since LXR activation was shown to repress ABCD2 expression, we investigated the effects of LXR antagonists in different cell lines. Cells were treated with GSK(17) (a LXR antagonist recently discovered from the GlaxoSmithKline compound collection), 22(S)-hydroxycholesterol (22S-HC, another LXR antagonist) and 22R-HC (an endogenous LXR agonist). We observed up-regulation of ABCD2,ABCD3 and CTNNB1 (the gene encoding for beta-catenin, which was recently demonstrated to induce ABCD2 expression) in human HepG2 hepatoma cells and in X-ALD skin fibroblasts treated with LXR antagonists. Interestingly, induction in X-ALD fibroblasts was concomitant with a decrease in oxidative stress. Rats treated with 22S-HC showed hepatic induction of the 3 genes of interest. In human, we show by multiple tissue expression array that expression of ABCD2 appears to be inversely correlated with NR1H3 (LXRalpha) expression. Altogether, antagonists of LXR that are currently developed in the context of dyslipidemia may find another indication with X-ALD.

Superparamagnetic Iron Oxide Nanoparticles for Immunotherapy of Cancers through Macrophages and Magnetic Hyperthermia.

Cancer immunotherapy has tremendous promise, but it has yet to be clinically applied in a wider variety of tumor situations. Many therapeutic combinations are envisaged to improve their effectiveness. In this way, strategies capable of inducing immunogenic cell death (e.g., doxorubicin, radiotherapy, hyperthermia) and the reprogramming of the immunosuppressive tumor microenvironment (TME) (e.g., M2-to-M1-like macrophages repolarization of tumor-associated macrophages (TAMs)) are particularly appealing to enhance the efficacy of approved immunotherapies (e.g., immune checkpoint inhibitors, ICIs). Due to their modular construction and versatility, iron oxide-based nanomedicines such as superparamagnetic iron oxide nanoparticles (SPIONs) can combine these different approaches in a single agent. SPIONs have already shown their safety and biocompatibility and possess both drug-delivery (e.g., chemotherapy, ICIs) and magnetic capabilities (e.g., magnetic hyperthermia (MHT), magnetic resonance imaging). In this review, we will discuss the multiple applications of SPIONs in cancer immunotherapy, focusing on their theranostic properties to target TAMs and to generate MHT. The first section of this review will briefly describe immune targets for NPs. The following sections will deal with the overall properties of SPIONs (including MHT). The last section is dedicated to the SPION-induced immune response through its effects on TAMs and MHT.

Lactobacillus stress protein GroEL prevents colonic inflammation.

BACKGROUND: We previously showed that supernatants of Lactobacillus biofilms induced an anti-inflammatory response by affecting the secretion of macrophage-derived cytokines, which was abrogated upon immunodepletion of the stress protein GroEL. METHODS: We purified GroEL from L. reuteri and analysed its anti-inflammatory properties in vitro in human macrophages isolated from buffy coats, ex vivo in explants from human biopsies and in vivo in a mouse model of DSS induced intestinal inflammation. As a control, we used GroEL purified (LPS-free) from E. coli. RESULTS: We found that L. reuteri GroEL (but not E. coli GroEL) inhibited pro-inflammatory M1-like macrophages markers, and favored M2-like markers. Consequently, L. reuteri GroEL inhibited pro-inflammatory cytokines (TNFα, IL-1ß, IFNγ) while favouring an anti-inflammatory secretome. In colon tissues from human biopsies, L. reuteri GroEL was also able to decrease markers of inflammation and apoptosis (caspase 3) induced by LPS. In mice, we found that rectal administration of L. reuteri GroEL (but not E. coli GroEL) inhibited all signs of haemorrhagic colitis induced by DSS including intestinal mucosa degradation, rectal bleeding and weight loss. It also decreased intestinal production of inflammatory cytokines (such as IFNγ) while increasing anti-inflammatory IL-10 and IL-13. These effects were suppressed when animals were immunodepleted in macrophages. From a mechanistic point of view, the effect of L. reuteri GroEL seemed to involve TLR4, since it was lost in TRL4-/- mice, and the activation of a non-canonical TLR4 pathway. CONCLUSIONS: L. reuteri GroEL, by affecting macrophage inflammatory features, deserves to be explored as an alternative to probiotics.

Selecting the first chemical molecule inhibitor of HSP110 for colorectal cancer therapy.

Pro-survival stress-inducible chaperone HSP110 is the only HSP for which a mutation has been found in a cancer. Multicenter clinical studies demonstrated a direct association between HSP110 inactivating mutation presence and excellent prognosis in colorectal cancer patients. Here, we have combined crystallographic studies on human HSP110 and in silico modeling to identify HSP110 inhibitors that could be used in colorectal cancer therapy. Two molecules (foldamers 33 and 52), binding to the same cleft of HSP110 nucleotide-binding domain, were selected from a chemical library (by co-immunoprecipitation, AlphaScreening, Interference-Biolayer, Duo-link). These molecules block HSP110 chaperone anti-aggregation activity and HSP110 association to its client protein STAT3, thereby inhibiting STAT3 phosphorylation and colorectal cancer cell growth. These effects were strongly decreased in HSP110 knockdown cells. Foldamer's 33 ability to inhibit tumor growth was confirmed in two colorectal cancer animal models. Although tumor cell death (apoptosis) was noted after treatment of the animals with foldamer 33, no apparent toxicity was observed, notably in epithelial cells from intestinal crypts. Taken together, we identified the first HSP110 inhibitor, a possible drug-candidate for colorectal cancer patients whose unfavorable outcome is associated to HSP110.

HSP110 translocates to the nucleus upon genotoxic chemotherapy and promotes DNA repair in colorectal cancer cells.

A multicenter clinical study demonstrated the presence of a loss-of-function HSP110 mutation in about 15% of colorectal cancers, which resulted from an alternative splicing and was produced at the detriment of wild-type HSP110. Patients expressing low levels of wild-type HSP110 had excellent outcomes (i.e. response to an oxaliplatin-based chemotherapy). Here, we show in vitro, in vivo, and in patients' biopsies that HSP110 co-localizes with DNA damage (γ-H2AX). In colorectal cancer cells, HSP110 translocates into the nucleus upon treatment with genotoxic chemotherapy such as oxaliplatin. Furthermore, we show that HSP110 interacts with the Ku70/Ku80 heterodimer, an essential element of the non-homologous end joining (NHEJ) repair machinery. We also demonstrate by evaluating the resolved 53BP1 foci that depletion in HSP110 impairs repair steps of the NHEJ pathway, which is associated with an increase in DNA double-strand breaks and in the cells' sensitivity to oxaliplatin. HSP110-depleted cells sensitization to oxaliplatin-induced DNA damage is abolished upon re-expression of HSP110. Confirming a role for HSP110 in DNA non-homologous repair, SCR7 and NU7026, two inhibitors of the NHEJ pathway, circumvents HSP110-induced resistance to chemotherapy. In conclusion, HSP110 through its interaction with the Ku70/80 heterodimer may participate in DNA repair, thereby inducing a protection against genotoxic therapy.

Restoring Anticancer Immune Response by Targeting Tumor-Derived Exosomes With a HSP70 Peptide Aptamer.

BACKGROUND: Exosomes, via heat shock protein 70 (HSP70) expressed in their membrane, are able to interact with the toll-like receptor 2 (TLR2) on myeloid-derived suppressive cells (MDSCs), thereby activating them. METHODS: We analyzed exosomes from mouse (C57Bl/6) and breast, lung, and ovarian cancer patient samples and cultured cancer cells with different approaches, including nanoparticle tracking analysis, biolayer interferometry, FACS, and electron microscopy. Data were analyzed with the Student's t and Mann-Whitney tests. All statistical tests were two-sided. RESULTS: We showed that the A8 peptide aptamer binds to the extracellular domain of membrane HSP70 and used the aptamer to capture HSP70 exosomes from cancer patient samples. The number of HSP70 exosomes was higher in cancer patients than in healthy donors (mean, ng/mL ± SD = 3.5 ± 1.7 vs 0.17 ± 0.11, respectively, P = .004). Accordingly, all cancer cell lines examined abundantly released HSP70 exosomes, whereas "normal" cells did not. HSP70 had higher affinity for A8 than for TLR2; thus, A8 blocked HSP70/TLR2 association and the ability of tumor-derived exosomes to activate MDSCs. Treatment of tumor-bearing C57Bl/6 mice with A8 induced a decrease in the number of MDSCs in the spleen and inhibited tumor progression (n = 6 mice per group). Chemotherapeutic agents such as cisplatin or 5FU increase the amount of HSP70 exosomes, favoring the activation of MDSCs and hampering the development of an antitumor immune response. In contrast, this MDSC activation was not observed if cisplatin or 5FU was combined with A8. As a result, the antitumor effect of the drugs was strongly potentiated. CONCLUSIONS: A8 might be useful for quantifying tumor-derived exosomes and for cancer therapy through MDSC inhibition.