Advancing precision oncology through systematic germline and tumor genetic analysis: The oncogenetic point of view on findings from a prospective multicenter clinical trial of 666 patients.

INTRODUCTION: With the emergence of targeted therapies, there is a need to accurately identify more tumor biomarkers. The EXOMA trial was designed to offer tumor and germline exome sequencing (ES) to patients with solid malignant tumors and facing therapeutic failure. As hereditary cancer predispositions could be identified, with genetic counseling and health management implications, a genetic consultation was systematically established. This design needs to be discussed as genetic human resources are limited and indication of theranostic tests will increase. METHODS: Genetic counseling was conducted within 15 days following inclusion in the study for patients recruited between December 2015 and July 2019. In silico analyses from theranostic ES were limited to 317 genes involved in oncogenesis, from both tumor and blood DNA. RESULTS: Six hundred and sixty six patients had a genetic consultation before ES. In 65/666 patients, 66 germline pathogenic or likely pathogenic (P/LP) variants were identified in 16 actionable genes and seven non-actionable genes according to French guidelines. 24/65 patients had previously received genetic analysis for diagnostic purposes, and for 17 of them, a P/LP variant had already been identified. Among the 48/65 remaining cases for which the EXOMA protocol revealed a previously unknown P/LP variant, only 19 met the criteria for genetic testing for inherited cancer risk after familial survey. These criteria had not been identified by the oncologist in 10 cases. In 21/65 cases, the variant was considered incidental. DISCUSSION: In 7.4% of patients, an undiagnosed hereditary genetic predisposition was identified, whether or not related to the clinical presentation, and germline analysis impacted oncological management for only 6.3% of the cohort. This low percentage should be weighed against the burden of systematic genetic consultation and urgent circuits. Information or training tools to form oncologists to the prescription of germline genetic analyses should be explored, as well as information supports and patient preferences.

Patient satisfaction, experience and preferences in the implementation of genetics teleconsultations in the North-eastern region of France.

INTRODUCTION: In France, few centres per region offer genetics consultations. Consequently, each centre covers a large area, often requiring patients to take a day off to travel long distances. In certain situations, genetic counselling in particular, a physical exam is not required. In these cases, teleconsultations between medical professional and patients, at the patient's location of choice, are an interesting offer. The COVID-19 pandemic has accelerated the implementation and the use of this type of consultation. With the aim of developing teleconsultation for certain types of referrals, a study of patient satisfaction, experience and preferences has been set up in our region. METHODS: 2307 patients who had a teleconsultation by phone or videoconferencing with professionals from one of five genetic centres in North-eastern France between March and December 2020 were asked by e-mail or by post to answer an online survey. RESULTS: 20% of the patients (n = 465) responded to the survey (80% women, 55% over 40 years old). In 64% of the cases (n = 299), the teleconsultation replaced a physical consultation due to the pandemic. In 56% of cases (n = 217), the consultations were conducted by videoconference. The teleconsultation involved the disclosure of results in 56% of cases (n = 260), a first consultation in 30% of cases (n = 138), and a follow-up consultation in 14% of cases (n = 67). The satisfaction rate was 96% (n = 447), with a rating of "excellent" in 72% of responses (n = 290) and "good" in 24% of responses (n = 157). Only 22% of the patients (n = 103), particularly patients who lived near the hospital or who were older than 70 years, would have preferred a physical consultation. Half of respondents (n = 232) declared that they avoided more than 1.5 h of transport, and 69% (n = 321) avoided taking a work day off. Patients were less often accompanied by a relative than if the consultation had taken place face-to-face (43%; n = 201 vs. 61%; n = 285). There was no change in responses during or after lock-down. CONCLUSION: This collection of feedback and analysis of patients' preferences has validated the long-term implementation of medical genetics teleconsultations in certain circumstances and indications, for patients who prefer this approach.

Resveratrol Interferes with IL1-ß-Induced Pro-Inflammatory Paracrine Interaction between Primary Chondrocytes and Macrophages.

State of the art. Osteoarthritis (OA) is a chronic articular disease characterized by cartilage degradation and osteophyte formation. OA physiopathology is multifactorial and involves mechanical and hereditary factors. So far, there is neither preventive medicine to delay cartilage breakdown nor curative treatment. Objectives. To investigate pro-inflammatory paracrine interactions between human primary chondrocytes and macrophages following interleukin-1-ß (IL-1ß) treatment; to evaluate the molecular mechanism responsible for the inhibitory effect of resveratrol. Results. The activation of NF-κB in chondrocytes by IL-1ß induced IL-6 secretion. The latter will then activate STAT3 protein in macrophages. Moreover, STAT3 was able to positively regulate IL-6 secretion, as confirmed by the doubling level of IL-6 in the coculture compared to macrophage monoculture. These experiments confirm the usefulness of the coculture model in the inflammatory arthritis-linked process as a closer biological situation to the synovial joint than separated chondrocytes and macrophages. Il also demonstrated the presence of an inflammatory amplification loop induced by IL-1ß. Resveratrol showed a strong inhibitory effect on the pro-inflammatory marker secretion. The decrease of IL-6 secretion is dependent on the NFκB inhibition in the chondrocytes. Such reduction of the IL-6 level can limit STAT3 activation in the macrophages, leading to the interruption of the inflammatory amplification loop. Conclusion. These results increase our understanding of the anti-inflammatory actions of resveratrol and open new potential approaches to prevent and treat osteoarthritis.

Anti-Oxidant, Anti-Inflammatory and Anti-Angiogenic Properties of Resveratrol in Ocular Diseases.

Resveratrol (3,4',5 trihydroxy-trans-stilbene) is one of the best known phytophenols with pleiotropic properties. It is a phytoalexin produced by vine and it leads to the stimulation of natural plant defenses but also exhibits many beneficial effects in animals and humans by acting on a wide range of organs and tissues. These include the prevention of cardiovascular diseases, anti-cancer potential, neuroprotective effects, homeostasia maintenance, aging delay and a decrease in inflammation. Age-related macular degeneration (AMD) is one of the main causes of deterioration of vision in adults in developed countries This review deals with resveratrol and ophthalmology by focusing on the antioxidant, anti-inflammatory, and anti-angiogenic effects of this molecule. The literature reports that resveratrol is able to act on various cell types of the eye by increasing the level of natural antioxidant enzymatic and molecular defenses. Resveratrol anti-inflammatory effects are due to its capacity to limit the expression of pro-inflammatory factors, such as interleukins and prostaglandins, and also to decrease the chemo-attraction and recruitment of immune cells to the inflammatory site. In addition to this, resveratrol was shown to possess anti-VEGF effects and to inhibit the proliferation and migration of vascular endothelial cells. Resveratrol has the potential to be used in a range of human ocular diseases and conditions, based on animal models and in vitro experiments.

Exploring new ways of regulation by resveratrol involving miRNAs, with emphasis on inflammation.

This review presents recent evidence implicating microRNAs (miRNAs) in the beneficial effects of resveratrol (trihydroxystilbene), a nonflavonoid plant polyphenol, with emphasis on its anti-inflammatory effects. Many diseases and pathologies have been linked, directly or indirectly, to inflammation. These include infections, injuries, atherosclerosis, diabetes mellitus, obesity, cancer, osteoarthritis, age-related macular degeneration, demyelination, and neurodegenerative diseases. Resveratrol can both decrease the secretion of proinflammatory cytokines (e.g., IL-6, IL-8, and TNF-α) and increase the production of anti-inflammatory cytokines; it also decreases the expression of adhesion proteins (e.g., ICAM-1) and leukocyte chemoattractants (e.g., MCP-1). Resveratrol's primary targets appear to be the transcription factors AP-1 and NF-κB, as well as the gene COX2. Although no mechanistic link between any particular miRNA and resveratrol has been identified, resveratrol effects depend at least in part upon the modification of the expression of a variety of miRNAs that can be anti-inflammatory (e.g., miR-663), proinflammatory (e.g., miR-155), tumor suppressing (e.g., miR-663), or oncogenic (e.g., miR-21).

Compared binding properties between resveratrol and other polyphenols to plasmatic albumin: consequences for the health protecting effect of dietary plant microcomponents.

Phytophenols are considered to have beneficial effects towards human physiology. They are food microcomponents with potent chemopreventive properties towards the most three frequent contemporary human diseases, e.g., cardiovascular alterations, cancer and neurodegenerative pathologies. Related to this, the plasmatic form and plasmatic level of plant polyphenols in the body circulation are crucial for their efficiency. Thus, determinations of the binding process of resveratrol and of common flavonoids produced by major edible plants, berries and fruits to plasma proteins are essential. The interactions between resveratrol and albumin, a major plasma protein, were compared with those already published, involving curcumin, genistein, quercetin and other well-known food-containing polyphenols. The approaches used are usually intrinsic fluorescence intensity changes, quenching of protein intrinsic fluorescence and infrared spectroscopy. It appears that: (1) all of the studied polyphenols interact with albumin; (2) while most of the studied polyphenols interact at one albumin binding site, there are two different types of resveratrol binding sites for bovine serum albumin, one with the highest affinity (apparent KD of 4 µM) with a stoichiometry of one per monomer and a second with a lower affinity (apparent KD of 20 µM) with also a stoichiometry of one per monomer; (3) at least one binding site is in the vicinity of one tryptophanyl residue of bovine serum albumin; and (4) resveratrol binding to bovine serum albumin produces a very small structural conformation change of the polypeptide chain. These results support a role played by polyphenols-albumin interactions in the plasma for the bio-activities of these food microcomponents in the body.

Effects of dietary phytophenols on the expression of microRNAs involved in mammalian cell homeostasis.

Besides synthesizing nutritive substances (proteins, fats and carbohydrates) for energy and growth, plants produce numerous non-energetic so-called secondary metabolites (mainly polyphenols) that allow them to protect themselves against infections and other types of hostile environments. Interestingly, these polyphenols often provide cells with valuable bioactive properties for the maintenance of their functions and homeostasis (signaling, gene regulation, protection against acquired or infectious diseases, etc.) both in humans and animals. Namely, from a nutritional point of view, and based on epidemiological data, it is now well accepted that the regular consumption of green vegetables, fruits and fibers has protective effects against the onset of cancer as well as of inflammatory, neurodegenerative, metabolic and cardiovascular diseases, and consequently increases the overall longevity. In particular, grapevine plants produce large amounts of a wide variety of polyphenols. The most prominent of those-resveratrol-has been shown to impair or delay cardiovascular alterations, cancer, inflammation, aging, etc. Until recently, the molecular bases of the pleiotropic effects of resveratrol remained largely unclear despite numerous studies on a variety of signaling pathways and the transcriptional networks that they control. However, it has been recently proposed that the protective properties of resveratrol may arise from its modulation of small non-coding regulatory RNAs, namely microRNAs. The aim of this review is to present up-to-date data on the control of microRNA expression by dietary phytophenols in different types of human cells, and their impact on cell differentiation, cancer development and the regulation of the inflammatory response.

Resveratrol initiates differentiation of mouse skeletal muscle-derived C2C12 myoblasts.

Resveratrol is one of the most widely studied bio-active plant polyphenols. While its effect on endothelial blood vessel cells, cancer cells, inflammatory processes and neurodegenerative events is well documented, little is known about the implication of this phytophenol in differentiating processes, particularly in skeletal muscle cells. Here, we report the effects of resveratrol on mouse skeletal muscle-derived cells (C2C12) in either a nondifferentiated (myoblasts) or differentiated state (myotubes) by evaluating resveratrol uptake, cell proliferation, changes in cell shape, and the expression of genes encoding muscle-specific transcription factors or contractile proteins. Resveratrol: (1) rapidly accumulates within cells through passive and facilitated processes; (2) does not strongly affect cell viability, cell cycle and apoptosis; (3) behaves as a pro-differentiating agent as shown by the lengthening of cells, leading to a myotube phenotype; (4) upregulates muscular pro-differentiation markers and transcription factors (myogenin, Scrp3) starting after 12h of exposure and strongly increases heavy chain myosin content after 18h of exposure to resveratrol; (5) increases the Srf transcription factor's transcript level, a target mRNA of the miRNA-133b, which is itself downregulated by this polyphenol. These results put forward new pro-differentiating regulatory properties of resveratrol on skeletal muscles at least partly via modulation of specific miRNAs.

Control of MicroRNA expression as a new way for resveratrol to deliver its beneficial effects.

Grapes produce large amounts of polyphenols. Many of them accumulate in the skin, pulp, and seeds and are consequently found in wine. The health benefits of a moderate consumption of wine have been attributed at least in part to grape's polyphenols. Among them, resveratrol (3,5,4'-trihydroxystilbene) is a phytoalexin that stimulates plant cell defenses against infections and also plays protective roles in humans, where it delays cardiovascular alterations and exerts anticancer and anti-inflammatory effects. Despite numerous studies, the molecular mechanisms of resveratrol action are only partially understood. Given its pleiotropic effects, it was previously suggested that resveratrol protective properties may arise from its modulation of the expression of microRNAs. Therefore, this review will focus on the effects of resveratrol on microRNA populations in humans and human cell lines, especially emphasizing the microRNAs that have been implicated in resveratrol effects on inflammation, cancer, metabolism, and muscle differentiation.

Effects of endocrine disruptors on genes associated with 17beta-estradiol metabolism and excretion.

In order to provide a global analysis of the effects of endocrine disruptors on the hormone cellular bioavailability, we combined 17beta-estradiol (E2) cellular flow studies with real-time PCR and Western blot expression measurements of genes involved in the hormone metabolism and excretion. Three endocrine disruptors commonly found in food were chosen for this study, which was conducted in the estrogen receptor (ER) negative hepatoblastoma HepG2 cell line: bisphenol A (BPA), genistein (GEN) and resveratrol (RES). We showed that 24 h after a single dose treatment with genistein, resveratrol or bisphenol A, the expression of ATP-binding cassette transporters (the multidrug resistance or MDR, and the multidrug resistance associated proteins or MRP) uridine diphosphate-glucuronosyltransferases (UGT) and/or sulfotransferases (ST) involved in 17beta-estradiol elimination process were significantly modulated and that 17beta-estradiol cellular flow was modified. Resveratrol induced MDR1 and MRP3 expressions, bisphenol A induced MRP2 and MRP3 expressions, and both enhanced 17beta-estradiol efflux. Genistein, on the other hand, inhibited ST1E1 and UGT1A1 expressions, and led to 17beta-estradiol cellular retention. Thus, we demonstrate that bisphenol A, genistein and resveratrol modulate 17beta-estradiol cellular bioavailability in HepG2 and that these modulations most probably involve regulations of 17beta-estradiol phase II and III metabolism proteins. Up to now, the estrogenicity of environmental estrogenic pollutants has been based on the property of these compounds to bind to ERs. Our results obtained with ER negative cells provide strong evidence for the existence of ER-independent pathways leading to endocrine disruption.

Antiproliferative activities of resveratrol and related compounds in human hepatocyte derived HepG2 cells are associated with biochemical cell disturbance revealed by fluorescence analyses.

Resveratrol is a well known polyphenol largely produced in grapevine. It is a strong antioxidant and a free radical scavenger. It exhibits several beneficial effects for health including cancer. Resveratrol antioxidant activity is essential in the prevention of chemical-induced cancer by inhibiting initiation step of carcinogenesis process but it is also considered to inhibit cancer promotion and progression steps. While the effects of resveratrol on cancer cells are widely described, the data available on the antiproliferative potential of resveratrol derivatives remain weak. Nevertheless, resveratrol analogs could exhibit stronger potentials than the parent molecule. So, we compared the cellular effects of trans-resveratrol, trans-epsilon-viniferin and their respective acetate derivatives, as well as a polyphenol mixture extracted from grapevine shoots, called vineatrol. We studied their abilities to interfere with cell proliferation, their uptake and their effects on parameters of cellular state in human hepatoma cells (HepG2). Cell growth experiments show that resveratrol triacetate presents a slightly better antiproliferative potential than resveratrol. The dimer epsilon-viniferin,as well as its pentaacetate analog, is less powerful than resveratrol, although a similar uptake kinetics in cells. Interestingly, among the tested polyphenols, vineatrol is the most potent solution, indicating a possible synergistic effect of both resveratrol and epsilon-viniferin. We took advantage of the fluorescence properties of these compounds to evidence cellular uptake by using flow cytometry. In addition, by competition assay, we demonstrate that resveratrol triacetate enters in hepatic HepG2 cells by the same way as resveratrol. By autofluorescence in situ measurement we observed that resveratrol and related compounds induce deep changes in cells activity. These changes occur mainly by increasing NADPH cell content and the number of green fluorescent cytoplasmic granular structures which may be related to an induction of detoxifying enzyme mechanisms.

Resveratrol in human hepatoma HepG2 cells: metabolism and inducibility of detoxifying enzymes.

trans-Resveratrol is a polyphenol present in several plant species. Its chemopreventive properties against several diseases have been largely documented. To validate a model for the study of the factors influencing its biological fate at the hepatic level, the metabolism and the efflux of resveratrol were studied in the human hepatoblastoma cell line, HepG2. Comparative high-performance liquid chromatography analysis of cell culture media before and after deconjugation showed that resveratrol was rapidly conjugated; at the concentration of 10 microM, it was entirely metabolized at 8 h of incubation. Two main resveratrol metabolites, monosulfate and disulfate, were identified by atmospheric pressure chemical ionization-mass spectrometry, thanks to their quasi-molecular ion and their characteristic fragmentation. To correlate with the auto-induction of resveratrol metabolism evidenced in HepG2 cells after a pretreatment for 48 h with 10 microM resveratrol, the inducibility of phase II enzymes by resveratrol was studied by real-time quantitative reverse transcriptase-polymerase chain reaction and flow cytometry. Observed, in particular, were an increase in mRNA expression levels of three metabolizing enzymes, two isoforms of UDP-glucuronosyltransferases, UGT1A1 and UGT2B7 (5-fold increased), and a sulfotransferase, ST1E1, in cells pretreated for 24 h with 10 microM resveratrol. These results were correlated with an increase in protein expression, especially after 48 h of treatment. On the other hand, the intracellular resveratrol retention in cells treated with MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), a multidrug resistance-associated protein inhibitor, strongly suggests the involvement of this ABC transporter family in the efflux of resveratrol conjugates from human liver.

Resveratrol as a chemopreventive agent: a promising molecule for fighting cancer.

Resveratrol (3,4',5 tri-hydroxystilbene) is a phytoalexin produced in hudge amount in grapevine skin in response to infection by Bothrytis cinerea. This production of resveratrol blocks the proliferation of the pathogen, thereby acting as a natural antibiotic. Numerous studies have reported interesting properties of trans-resveratrol as a preventive agent against important pathologies i.e. vascular diseases, cancers, viral infection or neurodegenerative processes. Moreover, several epidemiological studies have revealed that resveratrol is probably one of the main microcomponents of wine responsible for its health benefits such as prevention of vaso-coronary diseases and cancer. Resveratrol acts on the process of carcinogenesis by affecting the three phases: tumor initiation, promotion and progression phases and suppresses the final steps of carcinogenesis, i.e. angiogenesis and metastasis. It is also able to activate apoptosis, to arrest the cell cycle or to inhibit kinase pathways. Interestingly, resveratrol does not present any cytotoxicity in animal models. Moreover, concentrations of resveratrol in blood seem to be sufficient for anti-invasive activity. The enterohepatic recirculation may contribute to a delayed elimination of the drug from the body and bring about a prolonged effect. By its binding to plasmatic proteins, resveratrol also exhibits a prolonged effect. Interestingly, low doses of resveratrol can sensitize to low doses of cytotoxic drugs and so provide an innovative strategy to enhance the efficacy of anticancer therapy in various human cancers. By these properties, resveratrol appears to be a good candidate in chemopreventive or chemotherapeutic strategies and is believed to be a novel weapon for new therapeutic strategies.

Transport of resveratrol, a cancer chemopreventive agent, to cellular targets: plasmatic protein binding and cell uptake.

Resveratrol produced by several plants, berries and fruits, including grapes, is one of the best known natural food microcomponents with potent chemopreventive properties towards the most severe contemporary human diseases: cardiovascular sickness, cancer and neurodegenerative pathologies. Demonstration of its mechanism of action also implies the elucidation of the steps of bioavailability and bioabsorption in cells and tissues. In order to estimate the relationships between the amounts of resveratrol taken up by food or drink intake, and the several possible benefits illustrated from in vitro/in vivo experiments and from epidemiological studies, it is essential to demonstrate step by step the route of resveratrol from plasma to the cell active site. In plasma, resveratrol was shown to interact with lipoproteins. This commentary also contains previously unpublished results about interactions between resveratrol and albumin and the enhancement of this binding in presence of fatty acids. We have previously described that resveratrol uptake by hepatic cells involves two processes--a passive one and a carrier-mediated one. Thanks to this last process, resveratrol, while tightly bound to blood proteins, could be largely delivered to body tissues. The intracellular proteic targets of resveratrol remain to be identified.

Preferential induction of 20S proteasome subunits during elicitation of plant defense reactions: towards the characterization of "plant defense proteasomes".

Plants have evolved efficient mechanisms to resist pathogens. The earliest defense response is the hypersensitive response (HR) considered as the main step leading to plant systemic acquired resistance (SAR) that protects the whole plant against a large spectrum of pathogens. We showed previously that elicitation of defense reactions in tobacco cells by cryptogein, a proteinaceous elicitor of plant defense reactions, leads to a rapid and differential accumulation of transcripts corresponding to genes encoding defense-induced (din) subunits of 20S proteasome: beta1din, alpha3din and alpha6din.Here, expression of these three subunits was investigated by Northern blotting and by Western blotting using specific antibodies synthesized against two peptides deduced from the beta1din, alpha3din or alpha6din encoding sequence. Kinetics of mRNA and protein accumulation in various defense models showed a simultaneous accumulation of beta1din, alpha3din and alpha6din corresponding mRNAs and proteins only in plants developing a systemic acquired resistance. Inhibition by diphenyleneiodonium of the oxidative burst induced in defense reactions blocked the expression of beta1din, alpha3din and alpha6din. Using 2D gel electrophoresis and Western blotting, we showed multiple spots for each induced subunit suggesting the possible existence of multigenic families confirmed by genomic DNA analysis. These results suggest a complex regulation of induced subunits tightly correlated with the activation of plant defense reactions. beta1din, alpha3din and alpha6din subunits could probably replace the corresponding constitutive subunits in 20S proteasome leading to "plant defense proteasomes" which could play an important role in plant defense reactions.