Identification of SLC22A17 DNA methylation hotspot as a potential biomarker in cutaneous melanoma.

BACKGROUND: Cancer onset and progression are driven by genetic and epigenetic alterations leading to oncogene activation and the silencing of tumor suppressor genes. Among epigenetic mechanisms, DNA methylation (methDNA) is gaining growing interest in cancer. Promoter hypomethylation is associated with oncogene activation while intragenic methDNA can be involved in transcriptional elongation, alternative spicing, and the activation of cryptic start sites. Several genes involved in the modulation of the tumor microenvironment are regulated by methDNA, including the Solute Carrier Family 22 Member 17 (SLC22A17), which is involved in iron trafficking and extracellular matrix remodeling cooperating with the Gelatinase-Associated Lipocalin (NGAL) ligand. However, the exact role of intragenic methDNA in cancer has not been fully investigated. Therefore, the aim of the present study is to explore the role of methDNA in the regulation of SLC22A17 in cutaneous melanoma (CM), used as a tumor model. METHODS: Correlation and differential analyses between SLC22A17 expression and methDNA were performed using the data contained in The Cancer Genome Atlas and Gene Expression Omnibus databases. Functional studies on melanoma cell lines treated with 5-Azacytidine (5-Aza) were conducted to assess the correlation between methDNA and SLC22A17 expression. A validation study on the diagnostic potential of the in silico-identified SLC22A17 methDNA hotspot was finally performed by analyzing tissue samples obtained from CM patients and healthy controls. RESULTS: The computational analyses revealed that SLC22A17 was significantly downregulated in CM, and its expression was related to promoter hypomethylation and intragenic hypermethylation. Moreover, SLC22A17 overexpression and hypermethylation of two intragenic methDNA hotspots were associated with a better clinical outcome in CM patients. The correlation between SLC22A17 methDNA and expression was confirmed in 5-Aza-treated cells. In agreement with in silico analyses, the SLC22A17 promoter methylation hotspot showed higher methDNA levels in CM samples compared to nevi. In addition, the methDNA levels of this hotspot were positively correlated with advanced CM. CONCLUSIONS: The SLC22A17 methDNA hotspot could represent a promising biomarker for CM, highlighting the regulatory role of methDNA on SLC22A17 expression. These results pave the way for the identification of novel epigenetic biomarkers and therapeutic targets for the management of CM patients.

Glioblastoma mesenchymal subtype enhances antioxidant defence to reduce susceptibility to ferroptosis.

Glioblastoma (GBM) represents an aggressive brain tumor, characterized by intra- and inter-tumoral heterogeneity and therapy resistance, leading to unfavourable prognosis. An increasing number of studies pays attention on the regulation of ferroptosis, an iron-dependent cell death, as a strategy to reverse drug resistance in cancer. However, the debate on whether this strategy may have important implications for the treatment of GBM is still ongoing. In the present study, we used ferric ammonium citrate and erastin to evaluate ferroptosis induction effects on two human GBM cell lines, U-251 MG, with proneural characteristics, and T98-G, with a mesenchymal profile. The response to ferroptosis induction was markedly different between cell lines, indeed T98-G cells showed an enhanced antioxidant defence, with increased glutathione levels, as compared to U-251 MG cells. Moreover, using bioinformatic approaches and analysing publicly available datasets from patients' biopsies, we found that GBM with a mesenchymal phenotype showed an up-regulation of several genes involved in antioxidant mechanisms as compared to proneural subtype. Thus, our results suggest that GBM subtypes differently respond to ferroptosis induction, emphasizing the significance of further molecular studies on GBM to better discriminate between various tumor subtypes and progressively move towards personalized therapy.

Benefits and concerns of probiotics: an overview of the potential genotoxicity of the colibactin-producing Escherichia coli Nissle 1917 strain.

Recently, the mounting integration of probiotics into human health strategies has gathered considerable attention. Although the benefits of probiotics have been widely recognized in patients with gastrointestinal disorders, immune system modulation, and chronic-degenerative diseases, there is a growing need to evaluate their potential risks. In this context, new concerns have arisen regarding the safety of probiotics as some strains may have adverse effects in humans. Among these strains, Escherichia coli Nissle 1917 (EcN) exhibited traits of concern due to a pathogenic locus in its genome that produces potentially genotoxic metabolites. As the use of probiotics for therapeutic purposes is increasing, the effects of potentially harmful probiotics must be carefully evaluated. To this end, in this narrative review article, we reported the findings of the most relevant in vitro and in vivo studies investigating the expanding applications of probiotics and their impact on human well-being addressing concerns arising from the presence of antibiotic resistance and pathogenic elements, with a focus on the polyketide synthase (pks) pathogenic island of EcN. In this context, the literature data here discussed encourages a thorough profiling of probiotics to identify potential harmful elements as done for EcN where potential genotoxic effects of colibactin, a secondary metabolite, were observed. Specifically, while some studies suggest EcN is safe for gastrointestinal health, conflicting findings highlight the need for further research to clarify its safety and optimize its use in therapy. Overall, the data here presented suggest that a comprehensive assessment of the evolving landscape of probiotics is essential to make evidence-based decisions and ensure their correct use in humans.

Extracellular RNAs from Whole Urine to Distinguish Prostate Cancer from Benign Prostatic Hyperplasia.

RNAs, especially non-coding RNAs (ncRNAs), are crucial players in regulating cellular mechanisms due to their ability to interact with and regulate other molecules. Altered expression patterns of ncRNAs have been observed in prostate cancer (PCa), contributing to the disease's initiation, progression, and treatment response. This study aimed to evaluate the ability of a specific set of RNAs, including long ncRNAs (lncRNAs), microRNAs (miRNAs), and mRNAs, to discriminate between PCa and the non-neoplastic condition benign prostatic hyperplasia (BPH). After selecting by literature mining the most relevant RNAs differentially expressed in biofluids from PCa patients, we evaluated their discriminatory power in samples of unfiltered urine from 50 PCa and 50 BPH patients using both real-time PCR and droplet digital PCR (ddPCR). Additionally, we also optimized a protocol for urine sample manipulation and RNA extraction. This two-way validation study allowed us to establish that miRNAs (i.e., miR-27b-3p, miR-574-3p, miR-30a-5p, and miR-125b-5p) are more efficient biomarkers for PCa compared to long RNAs (mRNAs and lncRNAs) (e.g., PCA3, PCAT18, and KLK3), as their dysregulation was consistently reported in the whole urine of patients with PCa compared to those with BPH in a statistically significant manner regardless of the quantification methodology performed. Moreover, a significant increase in diagnostic performance was observed when molecular signatures composed of different miRNAs were considered. Hence, the abovementioned circulating ncRNAs represent excellent potential non-invasive biomarkers in urine capable of effectively distinguishing individuals with PCa from those with BPH, potentially reducing cancer overdiagnosis.

Baseline Association between Healthy Eating Index-2015 and Health-Related Quality of Life in Breast Cancer Patients Enrolled in a Randomized Trial.

Health-related quality of life (HRQoL) represents one of the most concerning aspects for cancer patients. The Healthy Eating Index (HEI) is an a priori diet quality index directly associated with health outcomes and HRQoL in cancer survivors in North American populations. We evaluated, in a Mediterranean population, the baseline associations between HEI-2015 and HRQoL in 492 women with breast cancer recruited in a DEDiCa lifestyle trial. Dietary data were obtained from 7-day food records; HRQoL was assessed through the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (EORTC QLQ C30) and the C30 Summary Score (SumSc). Analysis of variance and multivariable linear and log-gamma regression models were performed. Mean and standard deviation for HEI-2015 score was 68.8 ± 11.2; SumSc was 81.5 ± 12.9. Women with lower HEI-2015 score had higher BMI, were more frequently exposed to tobacco smoke and had fewer years of education. Patients with a HEI-2015 score greater than 68.7 (median value) showed a significant increase in SumSc of 4% (p = 0.02). HEI-2015 components also associated with SumSc were beans and greens (ß = 1.04; p = 0.02). Weak associations were found for total vegetables and saturated fats. Higher diet quality in breast cancer survivors was associated with higher overall HRQoL in this cross-sectional analysis.

Role of Oral Microbiota Dysbiosis in the Development and Progression of Oral Lichen Planus.

Oral lichen planus (OLP) is a chronic inflammatory autoimmune disease of the oral cavity with malignant potential affecting 1.01% of the worldwide population. The clinical patterns of this oral disorder, characterized by relapses and remissions of the lesions, appear on buccal, lingual, gingival, and labial mucosa causing a significant reduction in the quality of life. Currently, there are no specific treatments for this disease, and the available therapies with topical and systemic corticosteroids only reduce symptoms. Although the etiopathogenesis of this pathological condition has not been completely understood yet, several exogenous and endogenous risk factors have been proposed over the years. The present review article summarized the underlying mechanisms of action involved in the onset of OLP and the most well-known triggering factors. According to the current data, oral microbiota dysbiosis could represent a potential diagnostic biomarker for OLP. However, further studies should be undertaken to validate their use in clinical practice, as well as to provide a better understanding of mechanisms of action and develop novel effective intervention strategies against OLP.

Methylation­sensitive restriction enzyme­droplet digital PCR assay for the one­step highly sensitive analysis of DNA methylation hotspots.

DNA methylation is an epigenetic modification that plays a key role in several cellular processes mediating the fine regulation of gene expression. Aberrant DNA methylation is observed in a wide range of pathologies, including cancer. Since these DNA modifications are transferred to the cell progenies and are stable over the time, the analysis of DNA methylation status has been proposed for diagnostic and prognostic purposes in cancer. Currently, DNA bisulfite conversion is the gold standard method for the high­throughput analysis of DNA methylation alterations. However, bisulfite treatment induces DNA fragmentation affecting its quality for the downstream analyses. In this field, it is mandatory to identify novel methods to overcome the limits of conventional approaches. In the present study, the Methylation­Sensitive Restriction Enzyme­droplet digital PCR (MSRE­ddPCR) assay was developed as a novel sensitive method for the analysis of DNA methylation of short genomic regions, combining the MSRE assay with the high­sensitivity ddPCR and using an exogenous methylation sequence as control. Setup and validation experiments were performed analyzing a methylation hotspot of the Solute Carrier Family 22 Member 17 in DNA samples derived from melanoma cell lines as well as from tissues and serum samples obtained from patients with melanoma and healthy controls. Compared with the standard MSRE approaches, the MSRE­ddPCR assay is more appropriate for the analysis of DNA methylation (methDNA) in samples with low amounts of DNA (up to 0.651 ng) showing a greater sensitivity. These findings suggested the potential clinical application of MSRE­ddPCR paving the way to the analysis of other methDNA hotspots in different tumors.

Ketogenic Diet and Breast Cancer: Recent Findings and Therapeutic Approaches.

Breast cancer (BC), a complex disease with several influencing factors, is significantly impacted by dietary habits. The ketogenic diet (KD), characterized by high fat and low carbohydrate intake, has gained attention as a potential therapeutic approach, but its effects on BC remain unclear. This review seeks to summarize the current knowledge on the principles of the KD, its metabolic influence on BC cells, and the findings of recent clinical trials, in order to elucidate the potential therapeutic role of the KD in BC management. For these purposes, a comprehensive literature review was conducted selecting preclinical and clinical studies that investigate the relationship between the KD and BC. The selection criteria prioritized studies exploring the KD's metabolic effects on BC cells and current clinical trials involving the KD in BC management. The reviewed studies provide a diverse range of findings, with some suggesting potential benefits of the KD in inhibiting tumor growth and improving treatment response. However, robust clinical trials providing clear evidence of the KD's efficacy as a standalone therapeutic approach in BC are still lacking. There are also significant concerns regarding the safety and long-term effects of sustained ketosis in cancer patients. The therapeutic potential of the KD in BC remains an area of active research and debate. While preliminary findings are promising, definitive conclusions are hindered by inconsistent results and limited human trial data. Future research, specifically well-structured, large-scale clinical trials, is necessary to provide a comprehensive understanding of the role of the KD in BC treatment. Until then, caution should be exercised in its application, and patients should continue prioritizing evidence-based, standard-of-care treatments.

Prebiotics and the Risk of Upper Digestive Tract and Stomach Cancers: The PrebiotiCa Study.

BACKGROUND: Fiber intake may lower digestive tract cancer risk, possibly by modulating the composition of gut microbiota. However, no data are available about the role of specific fiber fractions with prebiotic activity (e.g., inulin-type fructans (ITFs), fructo-oligosaccharides (FOSs) and galactooligosaccharides (GOSs)) on the risk lower digestive tract cancers. OBJECTIVE: The objective was to assess the association between prebiotic intake and the risk of cancers of the upper digestive tract and stomach. DESIGN: Within the PrebiotiCa study, data were derived from a network of Italian case-control studies conducted between 1992 and 2009. Participants' usual diet was assessed using a food frequency questionnaire. ITFs, and selected FOSs (nystose, kestose, and 1F-ß-fructofuranosylnystose) and GOSs (raffinose and stachyose) were quantified in several food products via laboratory analyses. Participants' prebiotic intake was calculated by multiplying food frequency questionnaire intake by the prebiotic content of each food item. PARTICIPANTS/SETTING: Cases were patients admitted to major hospitals with incident histologically confirmed cancers; there were 946 cases of cancer of the oral cavity/pharynx, 198 of the nasopharynx, 304 of the esophagus, 230 of the stomach. More than 4,000 patients admitted to the same hospitals for acute nonneoplastic and not diet-related conditions were selected as control subjects. MAIN OUTCOME MEASURES: The outcomes were oral and pharyngeal, nasopharyngeal, esophageal, and stomach cancers. STATISTICAL ANALYSES PERFORMED: The odds ratios and corresponding 95% CIs of the various cancers were derived using logistic regression models adjusted for major confounders and energy intake. RESULTS: No association was observed between intake of prebiotics and risk of cancers of the oral cavity and pharynx, nasopharynx, and esophagus. High raffinose intake reduced stomach cancer risk (odds ratio for the third vs the first tertile 0.6, 95% CI 0.3 to 0.9); no other prebiotic was associated with stomach cancer. CONCLUSIONS: The current study does not support a major role of prebiotic fibers on selected upper digestive tract cancers. The association between high raffinose intake and reduced stomach cancer risk needs further investigation.

Cancer Resistance Is Mediated by the Upregulation of Several Anti-Apoptotic Gene Products via the Inducible Nitric Oxide Synthase/Nitric Oxide Pathway: Therapeutic Implications.

Significance: Several therapeutic strategies for cancer treatments have been developed with time, and significant milestones have been achieved recently. However, with these novel therapies, not all cancer types respond and in the responding cancer types only a subset is affected. The failure to respond is principally the result that these cancers develop several mechanisms of resistance. Thus, a focus of current research investigations is to unravel the various mechanisms that regulate resistance and identify suitable targets for new therapeutics. Recent Advances: Hence, many human cancer types have been reported to overexpress the inducible nitric oxide synthase (iNOS) and it has been suggested that iNOS/nitric oxide (NO) plays a pivotal role in the regulation of resistance. We have postulated that iNOS overexpression or NO regulates the overexpression of pivotal anti-apoptotic gene products such as B-cell lymphoma 2 (Bcl-2), B-cell lymphoma extra large (Bcl-xL), myeloid cell leukemia-1 (Mcl-1), and survivin. In this report, we describe the various mechanisms, transcriptional, post-transcriptional, and post-translational, by which iNOS/NO regulates the expression of the above anti-apoptotic gene products. Critical Issues: The iNOS/NO-mediated regulation of the four gene products is not the same with both specific and overlapping pathways. Our findings are, in large part, validated by bioinformatic analyses demonstrating, in several cancers, several direct correlations between the expression of iNOS and each of the four examined anti-apoptotic gene products. Future Directions: We have proposed that targeting iNOS may be highly efficient since it will result in the underexpression of multiple anti-apoptotic proteins and shifting the balance toward the proapoptotic gene products and reversal of resistance. Antioxid. Redox Signal. 39, 853-889.

In silico analysis of the solute carrier (SLC) family in cancer indicates a link among DNA methylation, metabolic adaptation, drug response, and immune reactivity.

Introduction: The oncogenic transformation is driven by genetic and epigenetic alterations influencing cancer cell fate. These alterations also result in metabolic reprogramming by modulating the expression of membrane Solute Carrier (SLC) transporters involved in biomolecules trafficking. SLCs act as tumor suppressors or promoters influencing cancer methylome, tumor growth, immune-escape, and chemoresistance. Methods: This in silico study aimed to identify the deregulated SLCs in various tumor types compared to normal tissues by analyzing the TCGA Target GTEx dataset. Furthermore, the relationship between SLCs expression and the most relevant tumor features was tackled along with their genetic regulation mediated by DNA methylation. Results: We identified 62 differentially expressed SLCs, including the downregulated SLC25A27 and SLC17A7, as well as the upregulated SLC27A2 and SLC12A8. Notably, SLC4A4 and SLC7A11 expression was associated with favorable and unfavorable outcome, respectively. Moreover, SLC6A14, SLC34A2, and SLC1A2 were linked to tumor immune responsiveness. Interestingly, SLC24A5 and SLC45A2 positively correlated with anti-MEK and anti-RAF sensitivity. The expression of relevant SLCs was correlated with hypo- and hyper-methylation of promoter and body region, showing an established DNA methylation pattern. Noteworthy, the positive association of cg06690548 (SLC7A11) methylation with cancer outcome suggests the independent predictive role of DNA methylation at a single nucleotide resolution. Discussion: Although our in silico overview revealed a wide heterogeneity depending on different SLCs functions and tumor types, we identified key SLCs and pointed out the role of DNA methylation as regulatory mechanism of their expression. Overall, these findings deserve further studies to identify novel cancer biomarkers and promising therapeutic targets.

Analysis of hsa-miR-19a-3p and hsa-miR-19b-3p modulation and phosphodiesterase type 5 expression in the vaginal epithelium of premenopausal women with genital arousal disorder.

BACKGROUND: Few studies have investigated the role of the phosphodiesterase type 5A (PDE5A) isoenzyme in female genital tissue disorders, exclusively taken from cadavers, as well as the epigenetic mechanisms responsible for the regulation of PDE5A levels. AIM: The aim was to study the in vivo association between microRNA (miRNA) expression and the expression levels of PDE5A in women with female genital arousal disorder (FGAD) compared with healthy women. METHODS: Premenopausal women affected by FGAD (cases) and sexually healthy women (control group) underwent microbiopsy of the periclitoral anterior vaginal wall for the collection of tissue samples. Computational analyses were preliminarily performed in order to identify miRNAs involved in the modulation of PDE5A by using miRNA-messenger RNA interaction prediction tools. Differences in the expression levels of miRNAs and PDE5A were finally investigated in cases and control subjects by using the droplet digital polymerase chain reaction amplification system and stratifying women considering their age, number of pregnancies, and body mass index. OUTCOMES: Expression levels of miRNAs were able to target PDE5A and the tissue expression in women with FGAD compared with healthy women. RESULTS: The experimental analyses were performed on 22 (43.1%) cases and 29 (56.9%) control subjects. Two miRNAs with the highest interaction levels with PDE5A, hsa-miR-19a-3p (miR-19a) and hsa-miR-19b-3p (miR-19b), were identified and selected for validation analyses. A reduction of the expression levels of both miRNAs was observed in women with FGAD compared with the control subjects (P < .05). Moreover, PDE5A expression levels were higher in women with FGAD and lower in women without sexual dysfunctions (P < .05). Finally, a correlation between body mass index and the expression levels of miR-19a was found (P < .01). CLINICAL IMPLICATIONS: Women with FGAD had higher levels of PDE5 compared with control subjects; therefore, the administration of PDE5 inhibitors (PDE5 inhibitors) could be useful in women with FGAD. STRENGTHS AND LIMITATIONS: The strength of the current study was to analyze genital tissue obtained in vivo from premenopausal women. A limitation was to not investigate other factors, including endothelial nitric oxide synthetases, nitric oxide, and cyclic guanosine monophosphate. CONCLUSION: The results of the present study indicate that the modulation of selected miRNAs could influence PDE5A expression in genital tissues in healthy women or in those with FGAD. Such findings further suggest that treatment with PDE5 inhibitors, as a modulator of PDE5A expression, could be indicated for women with FGAD.

Cancer resistance via the downregulation of the tumor suppressors RKIP and PTEN expressions: therapeutic implications.

The Raf kinase inhibitor protein (RKIP) has been reported to be underexpressed in many cancers and plays a role in the regulation of tumor cells' survival, proliferation, invasion, and metastasis, hence, a tumor suppressor. RKIP also regulates tumor cell resistance to cytotoxic drugs/cells. Likewise, the tumor suppressor, phosphatase and tensin homolog (PTEN), which inhibits the phosphatidylinositol 3 kinase (PI3K)/AKT pathway, is either mutated, underexpressed, or deleted in many cancers and shares with RKIP its anti-tumor properties and its regulation in resistance. The transcriptional and posttranscriptional regulations of RKIP and PTEN expressions and their roles in resistance were reviewed. The underlying mechanism of the interrelationship between the signaling expressions of RKIP and PTEN in cancer is not clear. Several pathways are regulated by RKIP and PTEN and the transcriptional and post-transcriptional regulations of RKIP and PTEN is significantly altered in cancers. In addition, RKIP and PTEN play a key role in the regulation of tumor cells response to chemotherapy and immunotherapy. In addition, molecular and bioinformatic data revealed crosstalk signaling networks that regulate the expressions of both RKIP and PTEN. These crosstalks involved the mitogen-activated protein kinase (MAPK)/PI3K pathways and the dysregulated nuclear factor-kappaB (NF-κB)/Snail/Yin Yang 1 (YY1)/RKIP/PTEN loop in many cancers. Furthermore, further bioinformatic analyses were performed to investigate the correlations (positive or negative) and the prognostic significance of the expressions of RKIP or PTEN in 31 different human cancers. These analyses were not uniform and only revealed that there was a positive correlation between the expression of RKIP and PTEN only in few cancers. These findings demonstrated the existence of signaling cross-talks between RKIP and PTEN and both regulate resistance. Targeting either RKIP or PTEN (alone or in combination with other therapies) may be sufficient to therapeutically inhibit tumor growth and reverse the tumor resistance to cytotoxic therapies.

Clinical Relevance of Targeted Therapy and Immune-Checkpoint Inhibition in Lung Cancer.

Lung cancer (LC) represents the second most diagnosed tumor and the malignancy with the highest mortality rate. In recent years, tremendous progress has been made in the treatment of this tumor thanks to the discovery, testing, and clinical approval of novel therapeutic approaches. Firstly, targeted therapies aimed at inhibiting specific mutated tyrosine kinases or downstream factors were approved in clinical practice. Secondly, immunotherapy inducing the reactivation of the immune system to efficiently eliminate LC cells has been approved. This review describes in depth both current and ongoing clinical studies, which allowed the approval of targeted therapies and immune-checkpoint inhibitors as standard of care for LC. Moreover, the present advantages and pitfalls of new therapeutic approaches will be discussed. Finally, the acquired importance of human microbiota as a novel source of LC biomarkers, as well as therapeutic targets to improve the efficacy of available therapies, was analyzed. Therapy against LC is increasingly becoming holistic, taking into consideration not only the genetic landscape of the tumor, but also the immune background and other individual variables, such as patient-specific gut microbial composition. On these bases, in the future, the research milestones reached will allow clinicians to treat LC patients with tailored approaches.

Proton boron capture therapy (PBCT) induces cell death and mitophagy in a heterotopic glioblastoma model.

Despite aggressive therapeutic regimens, glioblastoma (GBM) represents a deadly brain tumor with significant aggressiveness, radioresistance and chemoresistance, leading to dismal prognosis. Hypoxic microenvironment, which characterizes GBM, is associated with reduced therapeutic effectiveness. Moreover, current irradiation approaches are limited by uncertain tumor delineation and severe side effects that comprehensively lead to unsuccessful treatment and to a worsening of the quality of life of GBM patients. Proton beam offers the opportunity of reduced side effects and a depth-dose profile, which, unfortunately, are coupled with low relative biological effectiveness (RBE). The use of radiosensitizing agents, such as boron-containing molecules, enhances proton RBE and increases the effectiveness on proton beam-hit targets. We report a first preclinical evaluation of proton boron capture therapy (PBCT) in a preclinical model of GBM analyzed via µ-positron emission tomography/computed tomography (µPET-CT) assisted live imaging, finding a significant increased therapeutic effectiveness of PBCT versus proton coupled with an increased cell death and mitophagy. Our work supports PBCT and radiosensitizing agents as a scalable strategy to treat GBM exploiting ballistic advances of proton beam and increasing therapeutic effectiveness and quality of life in GBM patients.

Coevolutionary analysis of Forkhead box protein P3 and its physical binary interactors E3 ubiquitin-protein ligase CHIP, Zfp-90, and nuclear receptor ROR-α.

Forkhead box protein P3 (FOXP3) is known to orchestrate the development and maintenance of T regulatory cells, a cell population specialized in immune suppression and peripheral immune tolerance. FOXP3 activity is fine-tuned through its interaction with several protein-binding partners. By using IntAct database, we retrieved three physical binary interactors: E3 ubiquitin-protein ligase CHIP, Zfp-90, and nuclear receptor ROR-α. Coevolution clusters between FOXP3 and its interactors were identified with the use of iBIS2 algorithm, the iterative version of BIS/BIS2. Most of the coevolving pairs came from some species of monotremes and marsupials, as well as from a group of bats, thus suggesting that protein interactions of FOXP3 with its partners may be changed and/or modulated during mammalian speciation. Furthermore, our analysis would suggest the occurrence of a determinant role of FOXP3 in suppressing pregnancy alloreactions in placental mammals. Similarly, FOXP3, through its interaction with different protein interaction mechanisms, would explain the unique control of inflammatory response to infections in bats. By identifying several inter-protein clusters between the different protein pairs, our findings may provide a guide for new therapeutic approaches to modulate T regulatory suppression and/or enhance immune tolerance.

Lactobacillus rhamnosus GG cell-free supernatant as a novel anti-cancer adjuvant.

BACKGROUND: Gut microbiota modulation has been demonstrated to be effective in protecting patients against detrimental effects of anti-cancer therapies, as well as to improve the efficacy of certain anti-cancer treatments. Among the most characterized probiotics, Lactobacillus rhamnosus GG (LGG) is currently utilized in clinics to alleviate diarrhea, mucositis or intestinal damage which might be associated with several triggers, including Clostridium difficile infections, inflammatory gut diseases, antibiotic consumption, chemotherapy or radiation therapy. Here, we investigate whether LGG cell-free supernatant (LGG-SN) might exert anti-proliferative activity toward colon cancer and metastatic melanoma cells. Moreover, we assess the potential adjuvant effect of LGG-SN in combination with anti-cancer drugs. METHODS: LGG-SN alone or in combination with either 5-Fuorouracil and Irinotecan was used to treat human colon and human melanoma cancer cell lines. Dimethylimidazol-diphenyl tetrazolium bromide assay was employed to detect cellular viability. Trypan blue staining, anti-cleaved caspase-3 and anti-total versus anti-cleaved PARP western blots, and annexin V/propidium iodide flow cytometry analyses were used to assess cell death. Flow cytometry measurement of cellular DNA content (with propidium iodide staining) together with qPCR analysis of cyclins expression were used to assess cell cycle. RESULTS: We demonstrate that LGG-SN is able to selectively reduce the viability of cancer cells in a concentration-dependent way. While LGG-SN does not exert any anti-proliferative activity on control fibroblasts. In cancer cells, the reduction in viability is not associated with apoptosis induction, but with a mitotic arrest in the G2/M phase of cell cycle. Additionally, LGG-SN sensitizes cancer cells to both 5-Fluorouracil and Irinotecan, thereby showing a positive synergistic action. CONCLUSION: Overall, our results suggest that LGG-SN may contain one or more bioactive molecules with anti-cancer activity which sensitize cancer cells to chemotherapeutic drugs. Thus, LGG could be proposed as an ideal candidate for ground-breaking integrated approaches to be employed in oncology, to reduce chemotherapy-related side effects and overcome resistance or relapse issues, thus ameliorating the therapeutic response in cancer patients.

DNA Damage and Apoptosis as In-Vitro Effect Biomarkers of Titanium Dioxide Nanoparticles (TiO2-NPs) and the Food Additive E171 Toxicity in Colon Cancer Cells: HCT-116 and Caco-2.

This study investigated the DNA damage and apoptosis in colon cancer cells HCT-116 and Caco-2 induced by engineered titanium dioxide nanoparticles (TiO2-NPs) (60 nm) and titanium dioxide food additive E171. MTT assays showed that both chemical forms significantly reduced cancer cell viability in a dose-dependent manner. In particular the food additive E171 induced a pronounced inhibitory effect on the growth of HCT-116 and Caco-2 cell lines (E171 IC50: 3.45 mg/L for HTC-116 and 1.88 mg/L Caco-2; TiO2-NPs 60 nm IC50: 41.1 mg/L for HTC-116 and 14.3 mg/L for Caco-2). A low level of genotoxicity was observed in Caco-2 cells, especially when treated with TiO2 60 nm. Western blot analysis showed that HCT116 and Caco-2 treated cells did not overexpress apoptotic markers such as cleaved Caspase 3 and cleaved Parp. Moreover, further analysis by quantitative real-time PCR (qRT-PCR) showed that TiO2-NPs and E171 did not promote the expression of Bax or downregulation of Bcl-2, nor did they increase the Bax/Bcl-2 ratio. The assay data provide clear evidence that TiO2 can cause DNA damage but does not induce apoptosis or decrease long-term cell proliferation. In addition, the results show that E171 has a slightly higher level of cytotoxicity and genotoxicity. This suggests that exposure to E171 may be hazardous to health and that further research on biological effects is needed to promote safer practices in the use of this compound.

One-year nutrition counselling in the context of a Mediterranean diet reduced the dietary inflammatory index in women with breast cancer: a role for the dietary glycemic index.

Background: the Mediterranean diet, the low dietary glycemic index (GI) and the dietary inflammation index (DII®) have been associated with lower risk of breast cancer (BC) incidence and mortality. Objective: to investigate whether one-year nutrition counselling in the context of a Mediterranean diet, with or without low-GI carbohydrates counselling, may influence the DII in women with BC. Methods: data were obtained from participants of DEDiCa trial randomized to a Mediterranean diet (MD, n = 112) or a Mediterranean diet with low-GI carbohydrates (MDLGI, n = 111). The diet-derived DII and GI were calculated from 7-day food records while Mediterranean diet adherence from PREDIMED questionnaire. Differences between study arms were evaluated through Fisher's exact test or Mann-Whitney test and associations with multivariable regression analyses. Results: Mediterranean diet adherence significantly increased by 15% in MD and 20% in MDLGI with no difference between arms (p < 0.326). Dietary GI significantly decreased from 55.5 to 52.4 in MD and 55.1 to 47.6 in MDLGI with significant difference between arms (p < 0.001). DII significantly decreased by 28% in MD and 49% in MDLGI with no difference between arms (p < 0.360). Adjusting for energy intake (E-DII) did not change the results. Higher Mediterranean diet adherence and lower dietary GI independently contributed to DII lowering (ß-coefficient -0.203, p < 0.001; 0.046, p = 0.003, respectively). Conclusions: DII and E-DII scores decreased significantly after one-year with 4 nutrition counselling sessions on the Mediterranean diet and low GI. Increased adherence to the Mediterranean diet and low GI independently contributed to the DII changes. These results are relevant given that lowering the inflammatory potential of the diet may have implications in cancer prognosis and overall survival.

Bioinformatic analysis of the LCN2-SLC22A17-MMP9 network in cancer: The role of DNA methylation in the modulation of tumor microenvironment.

Several features of cancer cells such as proliferation, invasion, metastatic spreading, and drug resistance are affected by their interaction with several tumor microenvironment (TME) components, including neutrophil gelatinase-associated lipocalin (NGAL), solute carrier family 22 member 17 (SLC22A17), and matrix metallopeptidase 9 (MMP9). These molecules play a key role in tumor growth, invasion, and iron-dependent metabolism of cancer cells. However, the precise epigenetic mechanisms underlying the gene regulation of Lipocalin 2 (LCN2), SLC22A17, and MMP9 in cancer still remain unclear. To this purpose, computational analysis was performed on TCGA and GTEx datasets to evaluate the expression and DNA methylation status of LCN2, SLC22A17, and MMP9 genes in different tumor types. Correlation analysis between gene/isoforms expression and DNA methylation levels of LCN2, SLC22A17, and MMP9 was performed to investigate the role of DNA methylation in the modulation of these genes. Protein network analysis was carried out using reverse phase protein arrays (RPPA) data to identify protein-protein interactions of the LCN2-SLC22A17-MMP9 network. Furthermore, survival analysis was performed according to gene expression and DNA methylation levels. Our results demonstrated that LCN2 and MMP9 were mainly upregulated in most tumor types, whereas SLC22A17 was largely downregulated, representing a specific hallmark signature for all gastrointestinal tumors. Notably, the expression of LCN2, SLC22A17, and MMP9 genes was negatively affected by promoter methylation. Conversely, intragenic hypermethylation was associated with the overexpression of SLC22A17 and MMP9 genes. Protein network analysis highlighted the role of the LCN2-SLC22A17-MMP9 network in TME by the interaction with fibronectin 1 and claudin 7, especially in rectal tumors. Moreover, the impact of expression and methylation status of LCN2, SLC22A17, and MMP9 on overall survival and progression free interval was tumor type-dependent. Overall, our analyses provide a detailed overview of the expression and methylation status of LCN2, SLC22A17, and MMP9 in all TCGA tumors, indicating that the LCN2-SLC22A17-MMP9 network was strictly regulated by DNA methylation within TME. Our findings pave the way for the identification of novel DNA methylation hotspots with diagnostic and prognostic values and suitable for epi-drug targeting.