Development of potent isoflavone-based formyl peptide receptor 1 (FPR1) antagonists and their effects in gastric cancer cell models.

Formyl peptide receptor-1 (FPR1) is a G protein-coupled chemoattractant receptor that plays a crucial role in the trafficking of leukocytes into the sites of bacterial infection and inflammation. Recently, FPR1 was shown to be expressed in different types of tumor cells and could play a significant role in tumor growth and invasiveness. Starting from the previously reported FPR1 antagonist 4, we have designed a new series of 4H-chromen-2-one derivatives that exhibited a substantial increase in FPR1 antagonist potency. Docking studies identified the key interactions for antagonist activity. The most potent compounds in this series (24a and 25b) were selected to study the effects of the pharmacological blockade of FPR1 in NCl-N87 and AGS gastric cancer cells. Both compounds potently inhibited cell growth through a combined effect on cell proliferation and apoptosis and reduced cell migration, while inducing an increase in angiogenesis, thus suggesting that FPR1 could play a dual role as oncogene and onco-suppressor.

VEGFA Status as a Predictive Marker of Therapy Outcome in Metastatic Gastric Cancer Patients Following Ramucirumab-Based Treatment.

Metastatic gastric cancer (mGC) often has a poor prognosis and may benefit from a few targeted therapies. Ramucirumab-based anti-angiogenic therapy targeting the VEGFR2 represents a milestone in the second-line treatment of mGC. Several studies on different cancers are focusing on the major VEGFR2 ligand status, meaning VEGFA gene copy number and protein overexpression, as a prognostic marker and predictor of response to anti-angiogenic therapy. Following this insight, our study aims to examine the role of VEGFA status as a predictive biomarker for the outcome of second-line therapy with Ramucirumab and paclitaxel in mGC patients. To this purpose, the copy number of the VEGFA gene, by fluorescence in situ hybridization experiments, and its expression in tumor tissue as well as the density of micro-vessels, by immunohistochemistry experiments, were assessed in samples derived from mGC patients. This analysis found that amplification of VEGFA concomitantly with VEGFA overexpression and overexpression of VEGFA with micro-vessels density are more represented in patients showing disease control during treatment with Ramucirumab. In addition, in the analyzed series, it was found that amplification was not always associated with overexpression of VEGFA, but overexpression of VEGFA correlates with high micro-vessel density. In conclusion, overexpression of VEGFA could emerge as a potential biomarker to predict the response to anti-angiogenic therapy.

Targeting Angiogenesis in the Era of Biliary Tract Cancer Immunotherapy: Biological Rationale, Clinical Implications, and Future Research Avenues.

Although biliary tract cancers are traditionally considered rare in Western countries, their incidence and mortality rates are rising worldwide. A better knowledge of the genomic landscape of these tumor types has broadened the number of molecular targeted therapies, including angiogenesis inhibitors. The role of immune checkpoint inhibitors (ICIs) could potentially change the first-line therapeutic approach, but monotherapy with ICIs has shown disappointing results in CCA. Several clinical trials are evaluating combination strategies that include immunotherapy together with other anticancer agents with a synergistic activity. The tumor microenvironment (TME) composition plays a pivotal role in the prognosis of BTC patients. The accumulation of immunosuppressive cell types, such as tumor-associated macrophages (TAMs) and regulatory T-cells, together with the poor infiltration of cytotoxic CD8+ T-cells, is known to predispose to a poor prognosis owing to the establishment of resistance mechanisms. Likewise, angiogenesis is recognized as a major player in modulating the TME in an immunosuppressive manner. This is the mechanistic rationale for combination treatment schemes blocking both immunity and angiogenesis. In this scenario, this review aims to provide an overview of the most recent completed or ongoing clinical trials combining immunotherapy and angiogenesis inhibitors with/without a chemotherapy backbone.

Corrigendum: Variations in circulating levels of angiopoietin-2 over time are predictive of ramucirumab-paclitaxel therapy outcome in advanced gastric cancer: results of prospective study.

[This corrects the article DOI: 10.3389/fonc.2022.862116.].

The multiple combination of Paclitaxel, Ramucirumab and Elacridar reverses the paclitaxel-mediated resistance in gastric cancer cell lines.

Introduction: Paclitaxel (PTX) interferes with microtubule architecture by binding to ß-tubulin, thereby blocking progression at the G2/M phase and inducing apoptosis. This study aimed to investigate molecular processes underlying PTX-mediated resistance in gastric cancer (GC) cells. Methods: PTX-mediated resistance involves many processes, and in this work some of the factors involved in the resistance mechanism were identified by comparing two GC lines with PTX induced resistance to their sensitive counterparts. Results: Thus, the key feature of PTX-resistant cells was the overexpression of pro-angiogenic factors such as VEGFA, VEGFC, and Ang2, known to support tumor cell growth. A second relevant change detected in PTX-resistant lines was the elevated level of TUBßIII, a tubulin isoform that opposes microtubule stabilization. A third identified factor contributing to PTX-resistance was P-glycoprotein (P-gp), a transporter responsible for chemotherapy efflux from the cells, highly expressed in PTX-resistant lines. Discussion: These findings were in line with a greater sensitivity of resistant cells to treatment with both Ramucirumab and Elacridar. Ramucirumab significantly reduced the expression of angiogenic molecules and TUBßIII, while Elacridar restored the access of chemotherapy, recovering its anti-mitotic and pro-apoptotic effects. Finally, this study highlighted the role played by exosomes in spreading factors responsible for resistance in the tumor microenvironment.

Durvalumab in advanced cholangiocarcinoma: is someone knocking down the door?

Following the practice-changing results observed in several hematological and solid tumors, immunotherapy with immune checkpoint inhibitors (ICIs) has been tested in cholangiocarcinoma (CCA) patients. However, ICI monotherapy has had disappointing results in CCA, and phase I-III clinical trials have assessed whether combinatorial strategies including immunotherapy plus other anticancer agents may have a synergistic activity. The TOPAZ-1 trial has recently highlighted improved survival in CCA patients receiving first-line durvalumab plus gemcitabine-cisplatin compared with gemcitabine plus cisplatin alone, and several guidelines consider adding durvalumab to the reference doublet as standard of care. This article provides an overview of durvalumab pharmacology, safety and efficacy in CCA, highlighting current and future research directions in this setting.

Several treatments have been recently tested for cholangiocarcinoma patients. Among these, interesting results have been reported for immunotherapy with durvalumab, and the combination of immunotherapy plus chemotherapy represents a novel and important option in this setting.

Variations in Circulating Levels of Angiopoietin-2 Over Time Are Predictive of Ramucirumab-Paclitaxel Therapy Outcome in Advanced Gastric Cancer: Results of Prospective Study.

The combination of paclitaxel and ramucirumab is the second-line therapy of choice in the treatment of advanced gastric cancer. To date, no biomarkers are available in gastric cancer to predict the outcome of antiangiogenic therapy. The present prospective study included 35 patients undergoing second-line therapy with ramucirumab and paclitaxel. Serum samples were systematically collected from the beginning of therapy and at each cycle until disease progression. Multiplex analysis of a panel of angiogenic factors identified markers for which the changes at defined time intervals were significantly different in patients with progression-free survival ≤3 (Rapid Progression Group) compared to those with progression-free survival >3 (Control Disease Group). Comparative analysis revealed significantly different results in the two groups of patients for VEGFC and Angiopoietin-2, both involved in angiogenesis and lymphangiogenesis. VEGFC increased in the progressive-disease group, while it decreased in the control-disease group. This decrease persisted beyond the third cycle, and it was statistically significant compared to the basal level in patients with longer progression-free survival. Angiopoietin-2 decreased significantly after 2 months of therapy. At progression time, there was a significant increase in VEGFC and Angiopoietin-2, suggesting the activation pathways counteracting the blockade of VEGFR2 by ramucirumab. Overall results showed that a greater change in VEGFC and Angiopoietin-2 levels measured at the beginning of the third cycle of therapy corresponded to a lower risk of progression and thus to longer progression-free survival.

Nonalcoholic Fatty Liver Disease: Focus on New Biomarkers and Lifestyle Interventions.

Nonalcoholic fatty liver disease (NAFLD) is considered a hepatic manifestation of metabolic syndrome, characterized from pathological changes in lipid and carbohydrate metabolism. Its main characteristics are excessive lipid accumulation and oxidative stress, which create a lipotoxic environment in hepatocytes leading to liver injury. Recently, many studies have focused on the identification of the genetic and epigenetic modifications that also contribute to NAFLD pathogenesis and their prognostic implications. The present review is aimed to discuss on cellular and metabolic alterations associated with NAFLD, which can be helpful to identify new noninvasive biomarkers. The identification of accumulated lipids in the cell membranes, as well as circulating cytokeratins and exosomes, provides new insights in understanding of NAFLD. This review also suggests that lifestyle modifications remain the main prevention and/or treatment for NAFLD.

Autumn Royal and Egnatia Grape Extracts Differently Modulate Cell Proliferation in Human Colorectal Cancer Cells.

OBJECTIVE: Polyphenols extracted by table grape have been demonstrated to decrease cell proliferation in vitro and to exert anti-atherosclerotic and antithrombotic activities, regulating cell functions. A grape polyphenolic profile is affected by climate as well as a grape cultivar. This study was aimed to characterize the berry skin polyphenolic composition, antioxidant activity and antiproliferative properties of two black grape cultivars, Autumn Royal and Egnatia. METHODS: The phenolic composition of Grape Skin Extracts (GSEs) was determined by HPLC analyses. The antioxidant activity was determined using DPPH, ABTS and ORAC tests. Caco2, HT29 and SW480 human colon cancer cell lines were used to test the effects of GSEs in vitro. Cell proliferation and cell cycle were assessed with the MTT method and a Muse cell analyzer, respectively. qPCR and Western Blotting analysis were used to evaluate gene and protein expression, respectively. RESULTS: The total polyphenolic content and the total antioxidant capacity were significantly higher in Autumn Royal than in Egnatia. However, table grape Egnatia showed greater ability to affect cell proliferation and apoptosis, as well as to exert a growth arrest in the S phase of the cell cycle, particularly in the Caco2 cell line. CONCLUSION: These data suggest that the new grape variety Egnatia is an interesting source of phenolic compounds that could be of interest in the food and pharmaceutical industries.

Molecular mechanisms of synergistic action of Ramucirumab and Paclitaxel in Gastric Cancers cell lines.

Ramucirumab is approved both as monotherapy and in combination with Paclitaxel for advanced gastric cancer in patients with disease progression after chemotherapy. In tumor cells, the VEGFA-VEGFR2 binding activates autocrine survival and migration signaling in angiogenesis independent manner. The present in vitro study investigated the effects of single and combined treatments with Ramucirumab and Paclitaxel on cell growth and migration highlighting the mechanisms underlying the interaction between the two drugs in gastric cancer cells. Cell growth and motility were investigated in human gastric cancer cell lines characterized by different tumorigenicity. The inhibitory effect on cell growth exerted by both drugs was potentiated by their combination and was synergistic. Ramucirumab was able to enhance the inhibitory effect exerted by Paclitaxel on cell cycle progression. A synergistic action was also observed in the expression of proteins crucial for cell motility, microtubule organization and epithelial-mesenchymal transition. Furthermore, synergistic inhibition of VEGFR2 expression was obtained by the drug combination. These findings highlighted the importance of the combined treatment to strongly inhibit all the main molecules of both PI3K/Akt/mTOR and MAPK pathways thus preventing possible reactivations due to cross-talk phenomena. The combined treatment with Ramucirumab seems to be a promising option to overcome the Paclitaxel resistance.

Inflammatory Mechanisms of HCC Development.

HCC (hepatocellular carcinoma) is the second leading cause of cancer deaths worldwide, with several etiologic causes, mostly inflammation-associated. Different inflammatory responses in the liver can be triggered by different etiological agents. The inflammatory process can be resolved or be persistent, depending on the etiology and multiple other factors. Chronic inflammation, tissue remodeling, genetic alterations, and modifications in cellular signaling are considered to be key processes promoting immunosuppression. The progressive immunosuppression leads to the inactivation of anti-tumor immunity involved in HCC carcinogenesis and progression. Tumor cellular processes including DNA damage, necrosis, and ER (endoplasmic reticulum) stress can affect both immune-surveillance and cancer-promoting inflammation, supporting a mutual interdependence. Here, we review the current understanding of how chronic liver injury and inflammation is triggered and sustained, and how inflammation is linked to HCC. The identification of many hepatic microenvironmental inflammatory processes and their effector molecules, has resulted in extensive translational work and promising clinical trials of new immunomodulatory agents.

Integrated immune gene expression signature and molecular classification in gastric cancer: New insights.

Gastric cancer (GC) is characterized by extreme heterogeneity due to histopathological differences, molecular characteristics, and immune gene expression signature. Until recently, several targeted therapies failed due to this complexity. The recent immunotherapy resulted in more effective and safe approaches in several malignancies. All tumors could be considered potentially immunogenic and the new knowledge regarding the interactions among tumor cells, immune cells, and tumor microenvironment (TME) allowed to reverse possible immune resistance. The immune response is a complex multisteps process that finely regulates the balance between the recognition of non-self and the prevention of autoimmunity. Cancer cells can use these pathways to suppress tumor immunity as a major mechanism of immune resistance. The recent molecular classifications of GCs by The Cancer Genome Atlas (TCGA) and by the Asian Cancer Research (ACRG) networks, together with the identification of multiple biomarkers, open new perspectives for stratification of patients who might benefit from a long-term immune checkpoint therapy. One of the major processes that contribute to an immunosuppressive microenvironment is represented by tumor angiogenesis. The cellular mechanisms inducing both angiogenesis and immunosuppressive responses are often reached by the same cell types and soluble factors, such as vascular endothelial growth factor A (VEGFA). Recent studies point out that combinatorial strategies should be adapted as useful therapeutic approach to reverse the immunosuppressive status of microenvironment occurring in a relevant percentage of gastric tumors.

Ramucirumab and GSK1838705A Enhance the Inhibitory Effects of Low Concentration Sorafenib and Regorafenib Combination on HCC Cell Growth and Motility.

Several new multikinase inhibitors have recently been introduced into clinical practice for hepatocellular carcinoma (HCC) therapy. Small increases in survival were reported as well as considerable toxicity. There is thus a need for effective therapies with lower toxicities. We examined whether a combination of sorafenib and regorafenib might also be effective at very low concentrations, with resulting potential for lessened clinical toxicity. MTT test, clonogenic assay, Ki67 staining and cell cycle analysis were assessed for cell proliferation and Annexin V and western blotting analysis relative to the expression of cleaved Caspase-3 and BID for cell apoptosis. In these experimental conditions cell growth and migration were potently inhibited and apoptosis induced even in HCC cells producing high alpha fetoprotein (AFP) levels (clinically worse prognosis). The combination also inhibited levels of the two HCC biomarkers, AFP and des gamma carboxy prothrombin (DCP). Additional inhibition of Vascular Endothelial Growth Factor Receptor (VEGFR) or Insulin-like Growth Factor 1 Receptor (IGF1R) enhanced effects on AFP and DCP levels, cell growth inhibition and MAPK and PI3K/Akt signaling inhibition due to sorafenib/regorafenib combination. These combinations have the potential for decreased toxicity while simultaneously enhancing therapeutic effects. This potential decrease in toxicity is being explored in ongoing studies.

News about non-secretory exocytosis: mechanisms, properties, and functions.

The fusion by exocytosis of many vesicles to the plasma membrane induces the discharge to the extracellular space of their abundant luminal cargoes. Other exocytic vesicles, however, do not contain cargoes, and thus, their fusion is not followed by secretion. Therefore, two distinct processes of exocytosis exist, one secretory and the other non-secretory. The present review deals with the knowledge of non-secretory exocytosis developed during recent years. Among such developments are the dual generation of the exocytic vesicles, initially released either from the trans-Golgi network or by endocytosis; their traffic with activation of receptors, channels, pumps, and transporters; the identification of their tethering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes that govern membrane fusions; the growth of axons and the membrane repair. Examples of potential relevance of these processes for pathology and medicine are also reported. The developments presented here offer interesting chances for future progress in the field.

News about the Role of the Transcription Factor REST in Neurons: From Physiology to Pathology.

RE-1 silencing transcription factor (REST) (known also as NRSF) is a well-known transcription repressor whose strong decrease induces the distinction of neurons with respect to the other cells. Such distinction depends on the marked increased/decreased expression of specific genes, accompanied by parallel changes of the corresponding proteins. Many properties of REST had been identified in the past. Here we report those identified during the last 5 years. Among physiological discoveries are hundreds of genes governed directly/indirectly by REST, the mechanisms of its neuron/fibroblast conversions, and the cooperations with numerous distinct factors induced at the epigenetic level and essential for REST specific functions. New effects induced in neurons during brain diseases depend on the localization of REST, in the nucleus, where functions and toxicity occur, and in the cytoplasm. The effects of REST, including cell aggression or protection, are variable in neurodegenerative diseases in view of the distinct mechanisms of their pathology. Moreover, cooperations are among the mechanisms that govern the severity of brain cancers, glioblastomas, and medulloblastomas. Interestingly, the role in cancers is relevant also for therapeutic perspectives affecting the REST cooperations. In conclusion, part of the new REST knowledge in physiology and pathology appears promising for future developments in research and brain diseases.

High degradation and no bioavailability of artichoke miRNAs assessed using an in vitro digestion/Caco-2 cell model.

Although the cross-kingdom transfer of vegetable miRNAs (miRNAs) in mammalian species, including humans, is still controversial, recent studies have rejected this theory. Based on these recent studies, we hypothesized that artichoke-derived miRNAs (cca-miRNAs) are not adsorbed into human intestinal cells after cooking and in vitro digestion. In order to test this hypothesis, we evaluated miRNA (cca-miRNAs) in the edible part of globe artichokes (head portion), after cooking and digestion by an in vitro digestion system. The cca-miRNA levels were analyzed by real-time PCR (RT-qPCR), and those that withstood cooking and digestion conditions were further analyzed for their bioavailability using an in vitro system (Caco-2/TC7 cell clone). We detected 20 cca-miRNAs after cooking, 5 of which were statistically down-regulated in comparison with uncooked samples. Only 4 cca-miRNAs were found after in vitro digestion. By using scanning electron microscopy (SEM), we also evaluated the extracellular vesicles (EVs) in homogenized artichoke as possible miRNA transporters. However, approximately 81% were degraded after cooking, while the remaining EVs had changed shape from round to elliptical. Finally, we detected no cell-free cca-miRNAs, miRNAs bound to protein complex, and no cca-miRNAs encapsulated in EVs inside Caco-2 cells or in basolateral medium after bioavailability experiments. In conclusion, the data from the present study agrees with recent findings that the human small intestine does not uptake dietary miRNAs from raw or cooked artichoke heads.

Cannabinoid Receptor-1 Up-regulation in Azoxymethane (AOM)-treated Mice After Dietary Treatment with Quercetin.

BACKGROUND/AIM: The expression of cannabinoid receptor-1 (CB1-R) seems to be modulated by bioactive natural components such as the flavonoid quercetin. The aim of this study was to determine in an animal model of induced-colon cancer, whether quercetin inhibits colon carcinogenesis through changes in the expression of CB1-R. MATERIALS AND METHODS: C57BL/6J male mice were randomly assigned to standard diet or experimental diet supplemented with 0.5% quercetin. Azoxymethane (AOM) (10 mg/kg body weight) or saline solution (PBS) was intraperitoneally injected, once weekly for 6 weeks. RESULTS: The diet supplemented with quercetin induced CB1-R gene expression and protein, inhibiting the protein levels of STAT3 and p-STAT3 (both mediators of cell proliferation). Dietary quercetin also caused a significant increase in Bax/Bcl2 ratio protein expression. CONCLUSION: The anti-proliferative and pro-apoptotic effects of quercetin in AOM-treated mice are mediated by induction of the protein and gene expression levels of CB1-R.

Chlorogenic Acid Improves the Regorafenib Effects in Human Hepatocellular Carcinoma Cells.

Chlorogenic acid (CGA) is a polyphenol present in many human dietary foods. Several studies indicated a beneficial role of CGA in the prevention of cancer and an enhancement of chemotherapy when combined with CGA in the treatment of human hepatocarcinoma (HCC). Drug toxicity, resistance and subsequent disease progression represent a problem in HCC management, although treatment with the multikinase inhibitor Regorafenib improved overall survival. This study focused on the evaluation of the effects of combined treatment using both low Regorafenib concentrations and CGA as natural compound in HCC cells. The analysis of cell proliferation by Ki67 staining and cell cycle progression showed that CGA enhanced Regorafenib-mediated cell growth inhibition. Moreover, CGA potentiated the apoptotic effect of Regorafenib by the activation of the pro-apoptotic Annexin V, Bax and Caspase 3/7 and the inhibition of anti-apoptotic Bcl2 and Bcl-xL. Combined treatments were also effective in inhibiting cell motility. The mechanisms underlying the positive effects of combining CGA and Regorafenib were also addressed and an increased inhibition of MAPK (mitogen-activated protein kinase)and PI3K/Akt/mTORC (phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling was observed. Overall, these data demonstrated that co-treatment with Regorafenib and CGA enhanced Regorafenib action, reducing its cytotoxicity in HCC cells. In conclusion, this drug combination could be considered as a safe and more effective approach in HCC therapy.

Strong enhancement by IGF1-R antagonists of hepatocellular carcinoma cell migration inhibition by Sorafenib and/or vitamin K1.

PURPOSE: Emerging evidence indicates that combining Sorafenib with vitamin K1 (VK1) may result in a synergistic inhibition of hepatocellular carcinoma (HCC) cell migration and proliferation. Despite this synergy, its benefits may be limited due to drug resistance resulting from cross-talk with the tumor microenvironment. Insulin-like growth factor-1 (IGF1) signaling acts as an important modulator of HCC cell growth, motility and drug resistance. Therefore, we aimed to explore the effects of Sorafenib in combination with VK1 and/or IGF1-R antagonists on HCC cells. METHODS: Scratch wound migration assays were performed to assess the motility of HCC-derived PLC/PRF/5, HLF and Hep3B cells. The synergistic, additive or antagonistic effects of Sorafenib, VK1 and IGF1-R antagonists on HCC cell motility were assessed using CompuSyn software. The effects mediated by these various compounds on HCC cytoskeleton organization were evaluated using DyLight 554 Phalloidin staining. Proliferation and migration-associated signaling pathways were analyzed in PLC/PRF/5 cells using Erk1/2 and Akt activation kits and Western blotting (Mek, JNK, Akt, Paxillin and p38), respectively. RESULTS: The effects of the IGF1-R antagonists GSK1838705A and OSI-906 on HCC cell migration inhibition after Sorafenib and/or VK1 administration, individually or in combination, were evaluated. We found a synergistic effect in PLC/PRF/5, HLF and Hep3B cells for combinations of fixed doses of GSK1838705A or OSI-906 together with different doses of Sorafenib and/or VK1. The levels of synergy were found to be stronger at higher Sorafenib and/or VK1 concentrations and lower or absent at lower concentrations, with some variation among the different cell lines tested. In addition, we found that in PLC/PRF/5 and HLF cells IGF1-R blockage strongly enhanced the reduction and redistribution of F-actin induced by Sorafenib and/or VK1 through alterations in the phosphorylation levels of some of the principal proteins involved in the MAPK signaling cascade, which is essential for cell migration. CONCLUSIONS: Our results indicate that modulation of the efficacy of Sorafenib through combinations with VK1 and/or IGF1-R antagonists results in synergistic inhibition of HCC cell migration.

IGF-1R tyrosine kinase inhibitors and Vitamin K1 enhance the antitumor effects of Regorafenib in HCC cell lines.

The recent RESORCE trial showed that treatment with Regorafenib after Sorafenib failure provided a significant improvement in overall survival in HCC patients. Preclinical and clinical trial data showed that Regorafenib is a more potent drug than Sorafenib. In this study we aimed at improving Regorafenib actions and at reducing its toxicity, by targeting parallel pathways or by combination with Vitamins K (VKs). We investigated the effects of Regorafenib administrated at low concentrations and in combination with either VK1 and/or with GSK1838705A or OSI-906, two IGF1-R inhibitors, on HCC cell growth and motility. Our results showed that both IGF1-R inhibitors potentiated the antiproliferative and pro-apoptotic effects of Regorafenib and/or VK1 in HCC cell lines. Moreover we provide evidence that the combined treatment with IG1-R antagonists and Regorafenib (and/or VK1) also caused a significant reduction and depolymerization of actin resulting in synergistic inhibition exerted on cell migration. Thus, simultaneous blocking of MAPK and PI3K/Akt cascades with IGF1-R inhibitors plus Regorafenib could represent a more potent approach for HCC treatment.