Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism

In cancer cells, global genomic hypomethylation is found together with localized hypermethylation of CpG islands within the promoters and regulatory regions of silenced tumor suppressor genes. Demethylating agents may reverse hypermethylation, thus promoting gene re-expression. Unfortunately, demethylating strategies are not efficient in solid tumor cells. DNA demethylation is mediated by ten-eleven translocation enzymes (TETs). They sequentially convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is associated with active transcription; 5-formylcytosine; and finally, 5-carboxylcytosine. Although α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, the major n-3 polyunsaturated fatty acids, have anti-cancer effects, their action, as DNA-demethylating agents, has never been investigated in solid tumor cells. Here, we report that EPA demethylates DNA in hepatocarcinoma cells. EPA rapidly increases 5hmC on DNA, inducing p21Waf1/Cip1 gene expression, which slows cancer cell-cycle progression. We show that the underlying molecular mechanism involves TET1. EPA simultaneously binds peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), thus promoting their heterodimer and inducing a PPARγ-TET1 interaction. They generate a TET1-PPARγ-RXRα protein complex, which binds to a hypermethylated CpG island on the p21 gene, where TET1 converts 5mC to 5hmC. In an apparent shuttling motion, PPARγ and RXRα leave the DNA, whereas TET1 associates stably. Overall, EPA directly regulates DNA methylation levels, permitting TET1 to exert its anti-tumoral function.-Ceccarelli, V., Valentini, V., Ronchetti, S., Cannarile, L., Billi, M., Riccardi, C., Ottini, L., Talesa, V. N., Grignani, F., Vecchini, A., Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism

Liquid biopsy in early breast cancer. A preliminary report

Background: Liquid biopsy (LB) is a technique that utilizes circulating biomarkers from cancer patients to provide information regarding the genetic landscape of the cancer. LB is emerging as an alternative and complementary diagnostic and prognostic tool to surgical biopsy and is expected to provide the tool for the implementation of precision oncology in clinical settings. In fact, it may contribute to enhance understanding of tumor heterogeneity and permitting the dynamic monitoring of treatment responses and genomic variations. Thus, LB is a promising method for the management of cancer, including breast cancer (BC), whose incidence in Italy is progressively increasing. Previous studies focused mainly on patients with advanced-stage BC. In the present study we evaluated the number of circulating tumor cells (CTCs), the quantity of cell free tumor DNA (cftDNA) and the analysis of the mutational profile of DNA from CTCs (ctcDNA) and cftDNA in early stage BC patients. Methods: Matched pre- and post-surgery blood samples were collected from 47 early stage BC patients. CTCs enumeration was done using Isoflux system, molecular profile of ctcDNA and cftDNA was performed with the Spotlight 59 Panels kit on a MiSeq Illumina instrument. Results: Eighty percent of samples was CTCs-positive, while healthy controls were all CTCs-negative. Forty-four patients provided a pre-surgery and 21 post-surgery sample. By comparing the number of CTCs post-surgery with that of pre-surgery, we found that 66% of patients showed a decreased number of CTCs, 14% of patients continued to have the same number of CTCs, while, interestingly, 19% of patients showed an increased number of CTCs. Next Generation Sequencing (NGS) of ctcDNA and cftDNA showed that 52% of samples had mutations in 9 genes (TP53, CDKN2A, FBXW7, PTPN11, KRAS, NRAS, BRAF, IDH1, ALK) and in 5 genes (PIK3CA, APC ALK, KRAS, TSC1), respectively, with KRAS and ALK overlapping and TP53 being the most frequently mutated gene in ctcDNA analysis. Conclusions: LB could facilitate early detection of minimal residual disease, aiding in the initiation of adjuvant therapy to prevent recurrence and progression towards metastasis, enhance individualized treatment and longitudinal screening, thus improving the clinical management and outcome of patients with early BC

Alteration of glyoxalase genes expression in response to testosterone in LNCaP and PC3 human prostate cancer cells.

Glyoxalase system, a ubiquitous detoxification pathway protecting against cellular damage caused by potent cytotoxic metabolites, is involved in the regulation of cellular growth. Aberrations in the expression of glyoxalase genes in several human cancers have been reported. Recently, we described a possible regulatory effect by estrogens on glyoxalase genes in human breast cancer cell lines. This result, along with those ones regarding changes in glyoxalases activity and expression in other human hormone-regulated cancers, such as prostate cancer, has prompted us to investigate whether also androgens, whose functional role in prostate cancer pathogenesis is well known, could modulate glyoxalases gene expression. Therefore, we treated LNCaP androgen-responsive and PC3 androgen-independent human prostate cancer cell lines with testosterone at the concentrations of 1 nM and 100 nM. After a two days treatment, glyoxalases mRNA levels as well as cell proliferation were evaluated by real-time RT-PCR analysis and [3H]thymidine incorporation, respectively. Results pointed out that testosterone affects the expression of glyoxalase system genes and cell proliferation in a different manner in the two cell lines. The possibility that modulation of glyoxalase genes expression by testosterone is due to glyoxalases-mediated intracellular response mechanisms to the androgen-induced oxidative stress or to the presence of androgen response elements (ARE) in glyoxalase promoters are discussed. Knowledge regarding the regulation of glyoxalases by testosterone may provide insights into the importance of these enzymes in human prostate carcinomas in vivo

Data in support of sustained upregulation of adaptive redox homeostasis mechanisms caused by KRIT1 loss-of-function

This article contains additional data related to the original research article entitled “KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: implication for Cerebral Cavernous Malformation disease” (Antognelli et al., 2017) [1].Data were obtained by si-RNA-mediated gene silencing, qRT-PCR, immunoblotting, and immunohistochemistry studies, and enzymatic activity and apoptosis assays. Overall, they support, complement and extend original findings demonstrating that KRIT1 loss-of-function induces a redox-sensitive and JNK-dependent sustained upregulation of the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), and a drop in intracellular levels of AP-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that sensitizes cells to oxidative DNA damage and apoptosis.In particular, immunoblotting analyses of Nrf2, Glo1, AP-modified Hsp70 and Hsp27 proteins, HO-1, phospho-c-Jun, phospho-ERK5, and KLF4 expression levels were performed both in KRIT1-knockout MEF cells and in KRIT1-silenced human brain microvascular endothelial cells (hBMEC) treated with the antioxidant Tiron, and compared with control cells. Moreover, immunohistochemistry analysis of Nrf2, Glo1, phospho-JNK, and KLF4 was performed on histological samples of human CCM lesions. Finally, the role of Glo1 in the downregulation of AP-modified Hsp70 and Hsp27 proteins, and the increase in apoptosis susceptibility associated with KRIT1 loss-of-function was addressed by si-RNA-mediated Glo1 gene silencing in KRIT1-knockout MEF cells. Keywords: Cerebrovascular disease, Cerebral cavernous malformations, CCM1/KRIT1, Oxidative stress, Antioxidant defense, Adaptive redox homeostasis, Redox signaling, Nuclear factor erythroid 2-related factor 2 (Nrf2), c-Jun, Glyoxalase 1 (Glo1), Heme oxygenase-1 (HO-1), Argpyrimidine-modified heat-shock proteins, Oxidative DNA damage and apoptosi

KRIT1 loss-of-function induces a chronic Nrf2-mediated adaptive homeostasis that sensitizes cells to oxidative stress: Implication for Cerebral Cavernous Malformation disease

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3â0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies

Expression and biological-clinical significance of hTR, hTERT and CKS2 in washing fluids of patients with bladder cancer

<p>Abstract</p> <p>Background</p> <p>at present, pathogenesis of bladder cancer (BC) has not been fully elucidated. Aim of this study is to investigate the role of human telomerase RNA (<it>hTR</it>), human telomerase reverse transcriptase (<it>hTERT</it>) and CDC28 protein kinase regulatory subunit 2 (<it>CKS2</it>) in bladder carcinogenesis and their possible clinical significance;</p> <p>Methods</p> <p>the transcript levels of <it>hTR</it>, <it>hTERT </it>and <it>CKS2 </it>were quantified by Real time reverse transcriptase chain reaction in exfoliated cells from bladder washings of 36 patients with BC and 58 controls. The statistical significance of differences between BC bearing patients and control groups, in the general as well as in the stratified analysis (superficial or invasive BC), was assessed by Student's t test. Non parametric Receiver Operating Characteristics analysis (ROC) was performed to ascertain the accuracy of study variables to discriminate between BC and controls. The clinical value of concomitant examination of <it>hTR</it>, <it>hTERT </it>and <it>CKS2 </it>was evaluated by logistic regression analysis;</p> <p>Results</p> <p>a significant decrease in <it>hTR </it>and a significant increase in <it>hTERT </it>or <it>CKS2 </it>gene expression were found between BC bearing patients and controls, as well as in the subgroups analysis. The area under the curve (AUC) indicated an average discrimination power for the three genes, both in the general and subgroups analysis, when singularly considered. The ability to significantly discriminate between superficial and invasive BC was observed only for <it>hTR </it>transcript levels. A combined model including <it>hTR </it>and <it>CKS2 </it>was the best one in BC diagnosis;</p> <p>Conclusions</p> <p>our results, obtained from a sample set particularly rich of exfoliated cells, provide further molecular evidence on the involvement of <it>hTR, hTERT </it>and <it>CKS2 </it>gene expression in BC carcinogenesis. In particular, while <it>hTERT </it>and <it>CKS2 </it>gene expression seems to have a major involvement in the early stages of the disease, <it>hTR </it>gene expression, seems to be more involved in progression. In addition, our findings suggest that the studied genes have a clinical role in discriminating between BC and controls in the general as well as in the stratified analysis, when singularly considered. A combined model improved over the single marker BC diagnosis.</p

CYP17, GSTP1, PON1 and GLO1 gene polymorphisms as risk factors for breast cancer: an Italian case-control study

<p>Abstract</p> <p>Background</p> <p>Estrogens, environmental chemicals with carcinogenic potential, as well as oxidative and carbonyl stresses play a very important role in breast cancer (BC) genesis and progression. Therefore, polymorphisms of genes encoding enzymes involved in estrogen biosynthesis pathway and in the metabolic activation of pro-carcinogens to genotoxic intermediates, such as cytochrome P450C17α (CYP17), endogenous free-radical scavenging systems, such as glutathione S-transferase (GSTP1) and paraoxonase 1 (PON1), and anti-glycation defenses, such as glyoxalase I (GLO1), could influence individual susceptibility to BC. In the present case-control study, we investigated the possible association of CYP17 A1A2, GSTP1 ILE105VAL, PON1 Q192R or L55M, and GLO1 A111E polymorphisms with the risk of BC.</p> <p>Methods</p> <p>The above-said five polymorphisms were characterized in 547 patients with BC and in 544 healthy controls by PCR/RFLP methods, using DNA from whole blood. To estimate the relative risks, Odds ratios and 95% confidence intervals were calculated using unconditional logistic regression after adjusting for the known risk factors for BC.</p> <p>Results</p> <p>CYP17 polymorphism had no major effect in BC proneness in the overall population. However, it modified the risk of BC for certain subgroups of patients. In particular, among premenopausal women with the A1A1 genotype, a protective effect of later age at menarche and parity was observed. As to GSTP1 and PON1 192 polymorphisms, the mutant Val and R alleles, respectively, were associated with a decreased risk of developing BC, while polymorphisms in PON1 55 and GLO1 were associated with an increased risk of this neoplasia. However, these findings, while nominally significant, did not withstand correction for multiple testing.</p> <p>Conclusion</p> <p>Genetic polymorphisms in biotransformation enzymes CYP17, GSTP1, PON1 and GLO1 could be associated with the risk for BC. Although significances did not withstand correction for multiple testing, the results of our exploratory analysis warrant further studies on the above mentioned genes and BC.</p

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p &lt; 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p &lt; 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p &lt; 0.0001) or urgent (20.4% vs. 38.5%; p &lt; 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p &lt; 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice

Glyoxalases in Urological Malignancies

Urological cancers include a spectrum of malignancies affecting organs of the reproductive and/or urinary systems, such as prostate, kidney, bladder, and testis. Despite improved primary prevention, detection and treatment, urological cancers are still characterized by an increasing incidence and mortality worldwide. While advances have been made towards understanding the molecular bases of these diseases, a complete understanding of the pathological mechanisms remains an unmet research goal that is essential for defining safer pharmacological therapies and prognostic factors, especially for the metastatic stage of these malignancies for which no effective therapies are currently being used. Glyoxalases, consisting of glyoxalase 1 (Glo1) and glyoxalase 2 (Glo2), are enzymes that catalyze the glutathione-dependent metabolism of cytotoxic methylglyoxal (MG), thus protecting against cellular damage and apoptosis. They are generally overexpressed in numerous cancers as a survival strategy by providing a safeguard through enhancement of MG detoxification. Increasing evidence suggests that glyoxalases, especially Glo1, play an important role in the initiation and progression of urological malignancies. In this review, we highlight the critical role of glyoxalases as regulators of tumorigenesis in the prostate through modulation of various critical signaling pathways, and provide an overview of the current knowledge on glyoxalases in bladder, kidney and testis cancers. We also discuss the promise and challenges for Glo1 inhibitors as future anti-prostate cancer (PCa) therapeutics and the potential of glyoxalases as biomarkers for PCa diagnosis