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Abstract Background Cancer-associated fibroblasts (CAFs), mainly responsible for the desmoplastic reaction hallmark of intrahepatic Cholangiocarcinoma (iCCA), likely have a role in tumor aggressiveness and resistance to therapy, although the molecular mechanisms involved are unknown. Aim of the study is to investigate how targeting hCAF/iCCA cross-talk with a Notch1 inhibitor, namely Crenigacestat, may affect cancer progression. Methods We used different in vitro models in 2D and established new 3D hetero-spheroids with iCCA cells and human (h)CAFs. The results were confirmed in a xenograft model, and explanted tumoral tissues underwent transcriptomic and bioinformatic analysis. Results hCAFs/iCCA cross-talk sustains increased migration of both KKU-M213 and KKU-M156 cells, while Crenigacestat significantly inhibits only the cross-talk stimulated migration. Hetero-spheroids grew larger than homo-spheroids, formed by only iCCA cells. Crenigacestat significantly reduced the invasion and growth of hetero- but not of homo-spheroids. In xenograft models, hCAFs/KKU-M213 tumors grew significantly larger than KKU-M213 tumors, but were significantly reduced in volume by Crenigacestat treatment, which also significantly decreased the fibrotic reaction. Ingenuity pathway analysis revealed that genes of hCAFs/KKU-M213 but not of KKU-M213 tumors increased tumor lesions, and that Crenigacestat treatment inhibited the modulated canonical pathways. Cell cycle checkpoints were the most notably modulated pathway and Crenigacestat reduced CCNE2 gene expression, consequently inducing cell cycle arrest. In hetero-spheroids, the number of cells increased in the G2/M cell cycle phase, while Crenigacestat significantly decreased cell numbers in the G2/M phase in hetero but not in homo-spheroids. Conclusions The hCAFs/iCCA cross-talk is a new target for reducing cancer progression with drugs such as Crenigacestat. Graphical abstract

Abstract Background Intrahepatic cholangiocarcinoma (iCCA) is a lethal primary liver tumor characterized by clinical aggressiveness, poor prognosis, and scarce therapeutic possibilities. Therefore, new treatments are urgently needed to render this disease curable. Since cumulating evidence supports the oncogenic properties of the Heat Shock Factor 1 (HSF1) transcription factor in various cancer types, we investigated its pathogenetic and therapeutic relevance in iCCA. Methods Levels of HSF1 were evaluated in a vast collection of iCCA specimens. The effects of HSF1 inactivation on iCCA development in vivo were investigated using three established oncogene-driven iCCA mouse models. In addition, the impact of HSF1 suppression on tumor cells and tumor stroma was assessed in iCCA cell lines, human iCCA cancer-associated fibroblasts (hCAFs), and patient-derived organoids. Results Human preinvasive, invasive, and metastatic iCCAs displayed widespread HSF1 upregulation, which was associated with a dismal prognosis of the patients. In addition, hydrodynamic injection of a dominant-negative form of HSF1 (HSF1dn), which suppresses HSF1 activity, significantly delayed cholangiocarcinogenesis in AKT/NICD, AKT/YAP, and AKT/TAZ mice. In iCCA cell lines, iCCA hCAFs, and patient-derived organoids, administration of the HSF1 inhibitor KRIBB-11 significantly reduced proliferation and induced apoptosis. Cell death was profoundly augmented by concomitant administration of the Bcl-xL/Bcl2/Bcl-w inhibitor ABT-263. Furthermore, KRIBB-11 reduced mitochondrial bioenergetics and glycolysis of iCCA cells. Conclusions The present data underscore the critical pathogenetic, prognostic, and therapeutic role of HSF1 in cholangiocarcinogenesis.

Abstract Neurons are highly dependent on mitochondria to meet their bioenergetic needs and understanding the metabolic changes during the differentiation process is crucial in the neurodegeneration context. Several in vitro approaches have been developed to study neuronal differentiation and bioenergetic changes. The human SH-SY5Y cell line is a widely used cellular model and several differentiation protocols have been developed to induce a neuron-like phenotype including retinoic acid (RA) treatment. In this work we obtained a homogeneous functional population of neuron-like cells by a two-step differentiation protocol in which SH-SY5Y cells were treated with RA plus the mitotic inhibitor 2-deoxy-5-fluorouridine (FUdr). RA-FUdr treatment induced a neuronal phenotype characterized by increased expression of neuronal markers and electrical properties specific to excitable cells. In addition, the RA-FUdr differentiated cells showed an enrichment of long chain and unsaturated fatty acids (FA) in the acyl chain composition of cardiolipin (CL) and the bioenergetic analysis evidences a high coupled and maximal respiration associated with high mitochondrial ATP levels. Our results suggest that the observed high oxidative phosphorylation (OXPHOS) capacity may be related to the activation of the cyclic adenosine monophosphate (cAMP) pathway and the assembly of respiratory supercomplexes (SCs), highlighting the change in mitochondrial phenotype during neuronal differentiation.

Abstract 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.

Abstract Background Intrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-β1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA. Methods We investigated Crenigacestat’s effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA. Results In silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted “liver fibrosis” as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p

Aggressive hepatocellular carcinoma (HCC) overexpressing Angiopoietin-2 (ANG-2) (a protein linked with angiogenesis, proliferation, and epithelial–mesenchymal transition (EMT)), shares 95% of up-regulated genes and a similar poor prognosis with the proliferative subgroup of intrahepatic cholangiocarcinoma (iCCA). We analyzed the pro-invasive effect of ANG-2 and its regulator vascular endothelial growth factor (VEGF) on HCC and CCA spheroids to uncover posUsible common ways of response. Four cell lines were used: Hep3B and HepG2 (HCC), HuCC-T1 (iCCA), and EGI-1 (extrahepatic CCA). We treated the spheroids with recombinant human (rh) ANG-2 and/or VEGF and then observed the changes at the baseline, after 24 h, and again after 48 h. Proangiogenic stimuli increased migration and invasion capability in HCC- and iCCA-derived spheroids and were associated with a modification in EMT phenotypic markers (a decrease in E-cadherin and an increase in N-cadherin and Vimentin), especially at the migration front. Inhibitors targeting ANG-2 (Trebananib) and the VEGF (Bevacizumab) effectively blocked the migration ability of spheroids that had been stimulated with rh-ANG-2 and rh-VEGF. Overall, our findings highlight the critical role played by ANG-2 and the VEGF in enhancing the ability of HCC- and iCCA-derived spheroids to migrate and invade, which are key processes in cancer progression.

Abstract Background Intrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell–cell and cell–matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway. Methods THY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown THY1, respectively. Results CD90 co-localized with EPCAM, showing its epithelial origin. In vitro, NOTCH1 silencing triggered HES1 and THY1 down-regulation. RBPJ, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the THY1 promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of NOTCH1/HES1/THY1 displayed a significantly shorter survival. Conclusions iCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA.

Grapes represent a significant source of phenolic compounds known for their health-promoting properties, such as antioxidant capacity on normal cells and prooxidant activity on tumor cells. The genotype highly affects the polyphenolic composition in grapes and, consequently, the nutritional quality of berries. This work aimed to characterize the phenolic composition, the antioxidant, and anticancer activity of grape skin extracts (GSEs) of nine new table grape genotypes selected from a breeding program to obtain new cultivars of seedless table grapes, well adapted to the climatic change and with higher nutraceutical properties. The grape polyphenolic profile was characterized by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry analysis. GSE antioxidant activity was determined by the ABTS, DPPH, and ORAC assays; GSE cell growth inhibition test was carried out in the Caco2 human cancer cell line. The nine GSEs showed different flavonoid and non-flavonoid profiles, and all possessed antioxidant activity, with the ‘Aika N.’, ‘Turese N.’, and ‘Egnatia N.’ the most active. As anticancer activity against the tested cancer cell line, ‘Daunia N.’ and ‘Apenestae N.’ showed the EC50 after 24 h of 35.60 µg/mL and 150.91 µg/mL, respectively. The relationship between polyphenolic profile and the antioxidant and anticancer activity of GSE was also investigated. Interestingly, among the different classes of polyphenolics, flavan-3-ols e proanthocyanidins showed the highest positive correlation with the anticancer activity of extracts. These findings can be helpful for the preparation of new extracts for the pharmaceutical and nutraceutical industry and geneticists working in vine breeding programs.

Grape pomace (GP)—the major by-product of winemaking processes—still contains bioactive molecules with known beneficial properties for human health, such as an antiradical scavenging activity or an antiproliferative activity of tumors. In vitro studies have demonstrated that GP polyphenols specifically influence colon cancer cell proliferation. In addition to previously published work, we tested the phenolic compounds of Aglianico GP following an in vitro simulated gastrointestinal digestion on colorectal cancer cell lines at different degrees of differentiation. Our experiments, using HT29 and SW480 cells, confirmed the anti-proliferative effect of GP gastrointestinal digested extract and provided intriguing insights on the way it influences the cancer cell features (i.e., viability, proliferation, and apoptosis). We observed that Aglianico GP extract showed a great ability to affect cell proliferation and apoptosis. Interestingly, both HT29 and SW480 cells produced a significant increase in Bax, and a significant increase in the Bax/Bcl-2 ratio and caspase-3. The gastrointestinal digested GP extract was previously characterized both for antioxidant activity and phenolic composition. As a result, the TPC and the antioxidant activity reached high values in the Aglianico GP digested extract, and the main compounds assessed by UHPLC-DAD were anthocyanins, phenolic acids, and flavonoids. This work shed light on the use of digested GP extract as a dietary ingredient, a very sustainable source of nutritional compounds with potential health benefits for colon cancer cell proliferation.

In recent years, nutraceuticals have gained great popularity, owing to their physiological and potential health effects, such as anti-inflammatory, anti-cancer, antioxidant, and prebiotic effects, and their regulation of lipid metabolism. Since the Mediterranean diet is a nutritionally recommended dietary pattern including high-level consumption of nutraceuticals, this review aimed to summarize the main results obtained by our in vitro and in vivo studies on the effects of the major constituents of the Mediterranean diet (i.e., extra virgin olive oil compounds, polyunsaturated fatty acids, and fruit components). Based on experimental studies, the therapeutic purpose of nutraceuticals depends on their bioavailability, solubility, toxicity, and delivery system. This review provides more in-depth knowledge on the effects linked to nutraceuticals administration on human health, focusing the gastrointestinal tract and suggesting specific dietary components for personalized adjuvant therapies.

The administration of a ketogenic diet (KD) has been considered therapeutic in subjects with irritable bowel syndrome (IBS). This study aimed to investigate the molecular mechanisms by which a low-carbohydrate diet, such as KD, can improve gastrointestinal symptoms and functions in an animal model of IBS by evaluating possible changes in intestinal tissue expression of endocannabinoid receptors. In rats fed a KD, we detected a significant restoration of cell damage to the intestinal crypt base, a histological feature of IBS condition, and upregulation of CB1 and CB2 receptors. The diet also affected glucose metabolism and intestinal membrane permeability, with an overexpression of the glucose transporter GLUT1 and tight junction proteins in treated rats. The present data suggest that CB receptors represent one of the molecular pathways through which the KD works and support possible cannabinoid-mediated protection at the intestinal level in the IBS rats after dietary treatment.

Functional alterations in irritable bowel syndrome have been associated with defects in bioenergetics and the mitochondrial network. Effects of high fat, adequate-protein, low carbohydrate ketogenic diet (KD) involve oxidative stress, inflammation, mitochondrial function, and biogenesis. The aim was to evaluate the KD efficacy in reducing the effects of stress on gut mitochondria. Newborn Wistar rats were exposed to maternal deprivation to induce IBS in adulthood. Intestinal inflammation (COX-2 and TRL-4); cellular redox status (SOD 1, SOD 2, PrxIII, mtDNA oxidatively modified purines); mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, mtDNA content); and autophagy (Beclin-1, LC3 II) were evaluated in the colon of exposed rats fed with KD (IBD-KD) or standard diet (IBS-Std), and in unexposed controls (Ctrl). IBS-Std rats showed dysfunctional mitochondrial biogenesis (PPAR-γ, PGC-1α, COX-4, and mtDNA contents lower than in Ctrl) associated with inflammation and increased oxidative stress (higher levels of COX-2 and TLR-4, SOD 1, SOD 2, PrxIII, and oxidatively modified purines than in Ctrl). Loss of autophagy efficacy appeared from reduced levels of Beclin-1 and LC3 II. Feeding of animals with KD elicited compensatory mechanisms able to reduce inflammation, oxidative stress, restore mitochondrial function, and baseline autophagy, possibly via the upregulation of the PPAR-γ/PGC-1α axis.

Grapes contain many flavonoid and non-flavonoid compounds with anticancer effects. In this work we fully characterized the polyphenolic profile of two grape skin extracts (GSEs), Autumn Royal and Egnatia, and assessed their effects on Polyunsaturated Fatty Acid (PUFA) membrane levels of Caco2 and SW480 human colon cancer cell lines. Gene expression of 15-lipoxygenase-1 (15-LOX-1), and peroxisome proliferator-activated receptor gamma (PPAR-γ), as well as cell morphology, were evaluated. The polyphenolic composition was analyzed by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) analysis. PUFA levels were evaluated by gas chromatography, and gene expression levels of 15-LOX-1 and PPAR-γ were analyzed by real-time Polymerase Chain Reaction (PCR). Morphological cell changes caused by GSEs were identified by field emission scanning electron microscope (FE-SEM) and photomicrograph examination. We detected a different profile of flavonoid and non-flavonoid compounds in Autumn Royal and Egnatia GSEs. Cultured cells showed an increase of total PUFA levels mainly after treatment with Autumn Royal grape, and were richer in flavonoids when compared with the Egnatia variety. Both GSEs were able to affect 15-LOX-1 and PPAR-γ gene expression and cell morphology. Our results highlighted a new antitumor mechanism of GSEs that involves membrane PUFAs and their downstream pathways.

The polyphenolic compounds present in grape extracts have chemopreventive and anticancer properties. Here, we studied the ability of two grape skin extracts (GSEs), Autumn Royal and Egnatia, to influence the cell motility and membrane fluidity regulated by the enzyme Stearoyl-CoA desaturase-1 (SCD1) which increases with the cancer aggressiveness. Caco2 and SW480 human colon cancer cell lines were treated with increasing concentrations of GSEs to evaluate cell proliferation and motility. SCD1 levels were evaluated in both treated cell lines, by membrane lipidomic analysis conducted by gas chromatography. The expression levels of SCD1 and other factors involved in the reorganization of the cytoskeleton and focal adhesions were assessed by Real-time PCR, Western Blotting, and Immunofluorescence staining. High-performance liquid chromatography (HPLC) analyses were performed to determine the phenolic composition in the GSEs, finding them more expressed in Autumn Royal than in Egnatia. Both treatments reduced the levels of SCD1, phospho-Rac1/Cdc42/Rac1/Cdc42 ratio, Cofilin, Vimentin, and phospho-Paxillin especially in Caco2 compared to SW480, showing a different behavior of the two cell lines to these natural compounds. Our findings show that GSEs block the cell migration and membrane fluidity through a new mechanism of action involving structural cellular components.

Celiac disease (CD) presents as chronic low-grade inflammation of the small intestine often characterized by psychiatric comorbidities. The brain-derived neurotrophic factor (BDNF), which we have shown to be reduced in the serum of CD patients, acts as the bridge between immune activation and the nervous system adaptive response. Since Lactobacillus has been shown to upregulate BDNF, this study aimed to evaluate whether the administration of Lactobacillus rhamnosus GG (L.GG) could positively affect the brain BDNF system in rats mimicking the CD lesions. Data have shown that the administration of pepsin-trypsin digested gliadin (PTG) and L.GG alter the levels of mature BDNF (mBDNF), as evaluated by Western blotting. PTG provoked a reduction of mBDNF compared to controls, and a compensatory increase of its receptor TrkB. L.GG induced a slight positive effect on mBDNF levels under normal conditions, while it was able to rescue the PTG-induced reduced expression of mBDNF. The curative effect of L.GG was finely tuned, accompanied by the reduction of TrkB, probably to avoid the effect of excessive BDNF.

CD99 is a transmembrane glycoprotein expressed in physiological conditions by cells of different tissues, including osteoblasts (OBs). High or low CD99 levels have been detected in various pathological conditions, and the supernatant of some carcinoma cell lines can modulate CD99 expression in OB-like cells. In the present work we demonstrate for the first time that two different human myeloma cell lines (H929 and U266) and, in a less degree, their conditioned media significantly downregulate CD99 expression in normal human OBs during the differentiation process. In the same experimental conditions the OBs display a less differentiated phenotype as demonstrated by the decreased expression of RUNX2 and Collagen I. On the contrary, when CD99 was activated by using a specific agonist antibody, the OBs become more active as demonstrated by the upregulation of Alkaline Phosphatase, Collagen I, RUNX2, and JUND expression. Furthermore, we demonstrate that the activation of CD99 is able to induce the phosphorylation of ERK 1/2 and AKT intracellular signal transduction molecules in the OBs.

Periodontal disease (PD), or periodontitis, is defined as a bacterially induced disease of the tooth-supporting (periodontal) tissues. It is characterized by inflammation and bone loss; therefore understanding how they are linked would help to address the most efficacious therapeutic approach. Bacterial infection is the primary etiology but is not sufficient to induce the disease initiation or progression. Indeed, bacteria-derived factors stimulate a local inflammatory reaction and activation of the innate immune system. The innate response involves the recognition of microbial components by host cells, and this event is mediated by toll-like receptors (TLRs) expressed by resident cells and leukocytes. Activation of these cells leads to the release of proinflammatory cytokines and recruitment of phagocytes and lymphocytes. Activation of T and B cells initiates the adaptive immunity with Th1 Th2 Th17 Treg response and antibodies production respectively. In this inflammatory scenario, cytokines involved in bone regulation and maintenance have considerable relevance because tissue destruction is believed to be the consequence of host inflammatory response to the bacterial challenge. In the present review, we summarize host factors including cell populations, cytokines, and mechanisms involved in the destruction of the supporting tissues of the tooth and discuss treatment perspectives based on this knowledge.