Epigenetics Meets CAR-T-Cell Therapy to Fight Cancer.

Based on the impressive success of Car-T-cell therapy in the treatment of hematological malignancies, a broad application for solid tumors also appears promising. However, some important hurdles need to be overcome. One of these is certainly the identification of specific target antigens on cancer cells. Hypomethylation is a characteristic epigenetic aberration in many tumor entities. Genome-wide screenings for consistent DNA hypomethylations in tumors enable the identification of aberrantly upregulated transcripts, which might result in cell surface proteins. Thus, this approach provides a new perspective for the discovery of potential new Car-T-cell target antigens for almost every tumor entity. First, we focus on this approach as a possible treatment for prostate cancer.

Unraveling the immune response of the spleen in sepsis using green-synthesized silver nanoparticles from pomegranate peel extracts.

Sepsis is a serious disease characterized by an inappropriate host response to infection, resulting in widespread inflammation and systemic organ failure. The aim of this research is to investigate the possibility of pomegranate peel-derived silver nanoparticles (PGNP) as a potential alternative therapy for sepsis. Characterization using transmission electron microscopy revealed 10-30 nm spherical nanoparticles. In a rat model of sepsis, PGNP treatment improved spleen health, histology, and immune response as compared with septic rats. In rats treated with PGNP during sepsis, significant alterations in oxidative stress markers (p < .01) were observed. These included elevated levels of glutathione (0.63 ± 0.08 mmol/mg protein), reduced concentrations of nitric oxide (8.7 ± 0.8 µ mol/mg protein) and malondialdehyde (2.2 ± 0.3 nmol/mg protein), as well as increased activity of superoxide dismutase (159 ± 33 U/mg protein). Following PGNP administration, gene expression analysis revealed a decrease in spleen IL-1ß, IL-6, and TNF-α, highlighting its anti-inflammatory potential. Furthermore, PGNP effectively controlled apoptosis-related genes (Bax, Bcl-2, and Casp3), indicating its role in cellular survival pathways. This study sheds light on the immunological regulation of the spleen during sepsis using PGNP, demonstrating its potential as a new effective treatment approach. The study emphasizes the necessity of continuing to investigate and develop alternative medicines, particularly in light of antibiotic resistance and the global impact of sepsis. RESEARCH HIGHLIGHTS: The study explored the potential medicinal benefits of pomegranate peel-derived silver nanoparticles (PGNP) in the treatment of sepsis. PGNP suppressed pro-inflammatory cytokines and enhanced the immune response. The study recommends PGNP as a viable substitute treatment.

CD133-Dependent Activation of Phosphoinositide 3-Kinase /AKT/Mammalian Target of Rapamycin Signaling in Melanoma Progression and Drug Resistance.

Melanoma frequently harbors genetic alterations in key molecules leading to the aberrant activation of PI3K and its downstream pathways. Although the role of PI3K/AKT/mTOR in melanoma progression and drug resistance is well documented, targeting the PI3K/AKT/mTOR pathway showed less efficiency in clinical trials than might have been expected, since the suppression of the PI3K/mTOR signaling pathway-induced feedback loops is mostly associated with the activation of compensatory pathways such as MAPK/MEK/ERK. Consequently, the development of intrinsic and acquired resistance can occur. As a solid tumor, melanoma is notorious for its heterogeneity. This can be expressed in the form of genetically divergent subpopulations including a small fraction of cancer stem-like cells (CSCs) and non-cancer stem cells (non-CSCs) that make the most of the tumor mass. Like other CSCs, melanoma stem-like cells (MSCs) are characterized by their unique cell surface proteins/stemness markers and aberrant signaling pathways. In addition to its function as a robust marker for stemness properties, CD133 is crucial for the maintenance of stemness properties and drug resistance. Herein, the role of CD133-dependent activation of PI3K/mTOR in the regulation of melanoma progression, drug resistance, and recurrence is reviewed.

Mechanisms of Melanoma Progression and Treatment Resistance: Role of Cancer Stem-like Cells.

Melanoma is the third most common type of skin cancer, characterized by its heterogeneity and propensity to metastasize to distant organs. Melanoma is a heterogeneous tumor, composed of genetically divergent subpopulations, including a small fraction of melanoma-initiating cancer stem-like cells (CSCs) and many non-cancer stem cells (non-CSCs). CSCs are characterized by their unique surface proteins associated with aberrant signaling pathways with a causal or consequential relationship with tumor progression, drug resistance, and recurrence. Melanomas also harbor significant alterations in functional genes (BRAF, CDKN2A, NRAS, TP53, and NF1). Of these, the most common are the BRAF and NRAS oncogenes, with 50% of melanomas demonstrating the BRAF mutation (BRAFV600E). While the successful targeting of BRAFV600E does improve overall survival, the long-term efficacy of available therapeutic options is limited due to adverse side effects and reduced clinical efficacy. Additionally, drug resistance develops rapidly via mechanisms involving fast feedback re-activation of MAPK signaling pathways. This article updates information relevant to the mechanisms of melanoma progression and resistance and particularly the mechanistic role of CSCs in melanoma progression, drug resistance, and recurrence.

Nephroprotective Efficacy of Echinops spinosus against a Glycerol-Induced Acute Kidney Injury Model.

Nephroprotection or renal rescue is to revive and restore kidney function after damage, with no need for further dialysis. During acute kidney injury (AKI), sudden and recent reductions in kidney functions occur. Causes are multiple, and prompt intervention can be critical to diminish or prevent morbidity. Echinops spinosus (ES) is a curative plant with proven pharmacological and biological effects including anti-inflammatory, antioxidant, and antibacterial competencies. The principal goal of this research is to scrutinize the nephroprotective features of E. spinosa extract (ESE) against glycerol-induced AKI. Male Wistar albino rats were equally divided into five separated groups: negative control rats (vehicle-injected), ESE control rats (ESE-treated rats), positive control rats, glycerol-induced AKI-model rats (single IM injection of 50% glycerol), and 2 groups of diseased rats but pretreated with different concentrations of ESE for 7 days (ESE150 + AKI rats and ESE250 + AKI rats). Kidney tissues were collected and used for histopathology analysis. The relative kidney weight percentage was assessed. ESE effects were investigated via scanning several biomarkers, such as serum urea and creatinine, as kidney function biomarkers. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities were examined as rhabdomyolysis (RM) indicators. Kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) were also examined to investigate kidney injury. Enzymatic and nonenzymatic oxidative stress markers were analyzed, namely, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), malondialdehyde (MDA), nitric oxide (NO), and reduced glutathione GSH. Proinflammatory cytokine [tumor necrosis factor-α (TNF-α) and interleukin-1 ß (IL-1ß)] and the renal proapoptotic protein (Bax) and antiapoptotic protein (Bcl-2) levels were evaluated. Statistical analysis for the resulting data revealed that ESE pretreatment turned AKI-induced biological antioxidant levels to an extent comparable to normal results. Furthermore, ESE decreased kidney function markers and RM-related biomarkers (LDH, CK, Kim-1, and NGAL) compared to those in untreated AKI-model rats. ESE treatment dropped the apoptotic renal Bax levels, enhanced antiapoptotic Bcl-2 manufacture, and disallowed the release of IL-1ß and TNF-α. This study revealed the protective effect of ESE as therapeutic medicine against AKI-encouraged oxidative stress, inflammation, and apoptosis. It can be effectively used as adjuvant therapy, helping in renal rescue, and for kidney healing in cases with risk factors of AKI.

Tumor Microenvironment as a Therapeutic Target in Melanoma Treatment.

The role of the tumor microenvironment in tumor growth and therapy has recently attracted more attention in research and drug development. The ability of the microenvironment to trigger tumor maintenance, progression, and resistance is the main cause for treatment failure and tumor relapse. Accumulated evidence indicates that the maintenance and progression of tumor cells is determined by components of the microenvironment, which include stromal cells (endothelial cells, fibroblasts, mesenchymal stem cells, and immune cells), extracellular matrix (ECM), and soluble molecules (chemokines, cytokines, growth factors, and extracellular vesicles). As a solid tumor, melanoma is not only a tumor mass of monolithic tumor cells, but it also contains supporting stroma, ECM, and soluble molecules. Melanoma cells are continuously in interaction with the components of the microenvironment. In the present review, we focus on the role of the tumor microenvironment components in the modulation of tumor progression and treatment resistance as well as the impact of the tumor microenvironment as a therapeutic target in melanoma.

Induction of Antimicrobial Protein S100A15 Expression by Oral Microbial Pathogens Is Toll-like Receptors-Dependent Activation of c-Jun-N-Terminal Kinase (JNK), p38, and NF-κB Pathways.

The antimicrobial protein S100A15 belongs to the S100 family, which is differentially expressed in a variety of normal and pathological tissues. Although the function of S100A15 protein has been discussed in several studies, its induction and regulation in oral mucosa, so far, are largely unknown. In this study, we demonstrate that S100A15 is induced by the stimulation of oral mucosa with gram- or gram+ bacterial pathogens, as well as with the purified membrane components, namely lipopolysaccharides (LPS) and lipoteichoic acid (LTA). The stimulation of the human gingival fibroblast (GF) and the human mouth epidermal carcinoma (KB) cell lines with either gram- or gram+ bacterial pathogens or their purified membrane components (LPS and LTA) results in the activation of NF-κB, apoptosis-regulating kinase1 (ASK1), and MAP kinase signaling pathways including, c-Jun N-terminal kinase (JNK) and p38 together with their physiological substrates AP-1 and ATF-2, respectively. Inhibition of S100A15 by antibodies-mediated Toll-like receptor 4 (TLR4) or Toll-like receptor 2 (TLR2) neutralization reveals the induction of S100A15 protein by LPS/gram- bacterial pathogens to be TLR4- dependent mechanism, whereas induction by LTA/gram+ bacterial pathogens to be TLR2- dependent mechanism. Pre-treatment of GF and KB cells with JNK (SP600125), p38 (SB-203580), or NF-κB (Bay11-7082) specific inhibitors further demonstrates the importance of JNK, p38 and NF-κB pathways in the regulation of gram-/gram+ bacterial pathogen-induced S100A15 expression. Our data provide evidence that S100A15 is induced in cancer and non-cancer oral mucosa-derived cell lines by gram-/gram+ bacterial pathogens and provide insight into the molecular mechanisms by which gram- and gram+ bacterial pathogens induce S100A15 expression in the oral mucosa.

Differential DNA Methylation of THOR and hTAPAS in the Regulation of hTERT and the Diagnosis of Cancer.

BACKGROUND: Although DNA methylation in the gene promoters usually represses gene expression, the TERT hypermethylated oncological region (THOR) located 5' of the hTERT gene is hypermethylated when hTERT is expressed in diverse cancer types, including urothelial cancer (UC). METHODS: Comprehensive MeDIP and DNA methylation array analyses complemented by the technically independent method of bisulfite genomic sequencing were applied on pathologically reviewed and classified urothelial carcinoma specimens and healthy urothelial tissue samples to reveal the methylation status of THOR in detail. RESULTS: The detailed DNA methylation profiles reveal the exact positions of differentially methylated CpG dinucleotides within THOR in urothelial cancer and provide evidence ofa diverging role of methylation of these CpGs in the regulation of hTERT. In particular, our data suggest a regulating mechanism in which THOR methylation acts on hTERT expression through epigenetic silencing of the lncRNA hTERT antisense promoter-associated (hTAPAS), which represses hTERT. CONCLUSIONS: These findings precisely define the most differentially methylated CpGs of THOR in early urothelial cancer, enabling optimal design of Methylation-Specific PCR (MSPCR) primers to reliably probe these methylation differences for diagnostic and prognostic purposes. In addition, this strategy presents a prime example that is also applicable to many other malignancies. Finally, the first evidence for the underlying epigenetic mechanism regulating hTERT expression through the methylation status of THOR is provided.

Methyl Group Metabolism in Differentiation, Aging, and Cancer.

Methyl group metabolism belongs to a relatively understudied field of research. Its importance lies in the fact that methyl group metabolic pathways are crucial for the successful conversion of dietary nutrients into the basic building blocks to carry out any cellular methylation reaction. Methyl groups play essential roles in numerous cellular functions such as DNA methylation, nucleotide- and protein biosynthesis. Especially, DNA methylation is responsible for organizing the genome into transcriptionally silent and active regions. Ultimately, it is this proper annotation that determines the quality of expression patterns required to ensure and shape the phenotypic integrity and function of a highly specialized cell type. Life is characterized by constantly changing environmental conditions, which are addressed by changes in DNA methylation. This relationship is increasingly coming into focus as it is of fundamental importance for differentiation, aging, and cancer. The stability and permanence of these metabolic processes, fueling the supplementation of methyl groups, seem to be important criteria to prevent deficiencies and erosion of the methylome. Alterations in the metabolic processes can lead to epigenetic and genetic perturbations, causative for diverse disorders, accelerated aging, and various age-related diseases. In recent decades, the intake of methyl group compounds has changed significantly due to, e.g., environmental pollution and food additives. Based on the current knowledge, this review provides a brief overview of the highly interconnected relationship between nutrition, metabolism, changes in epigenetic modifications, cancer, and aging. One goal is to provide an impetus to additionally investigate changes in DNA methylation as a possible consequence of an impaired methyl group metabolism.

Phyloepigenetics.

Traditionally, phylogenetic interspecies relationships are estimated based on genetic diversity, since it is assumed that the more recently diverged a species, with comparable constancy of development, the more similar their genetic material and proteins should be. However, occasional controversies in the field may reflect limited resolution and accuracy of this approach. Epigenetics has, meanwhile, provided significant evidence that CpG dinucleotides (CpGs) within genetic material are of particular importance for the annotation and function of the genome and the formation of the phenotype, which is continuously shaped by evolutionary interaction with environmental factors. Based on this, it can be concluded that CpGs follow a distinct rate of evolution, compared to all other nucleotide positions. Evidence is provided that supports this conclusion. Therefore, using CpGs to fathom evolutionary relationships between species could turn out to be a valuable approach to achieve, in some cases, an improved understanding of evolutionary development.

Epigenetics in the Diagnosis and Therapy of Malignant Melanoma.

Epigenetic mechanisms are fundamentally important for cancer initiation and development. However, a survey of the literature reveals that, to date, they appear less comprehensively investigated in melanoma than in many other cancers, e.g., prostate, breast, and colon carcinoma. The aim of this review is to provide a short summary of epigenetic aspects of functional relevance for melanoma pathogenesis. In addition, some new perspectives from epigenetic research in other cancers with potential for melanoma diagnosis and therapy are introduced. For example, the PrimeEpiHit hypothesis in urothelial carcinoma, which, similarly to malignant melanoma, can also be triggered by a single exogenous noxa, states that one of the first steps for cancer initiation could be epigenetic changes in key genes of one-carbon metabolism. The application of such insights may contribute to further progress in the diagnosis and therapy of melanoma, a deadly type of cancer.

Consistent DNA Hypomethylations in Prostate Cancer.

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15. These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS).

Aging-associated distinctive DNA methylation changes of LINE-1 retrotransposons in pure cell-free DNA from human blood.

LINE-1 hypomethylation of cell-free DNA has been described as an epigenetic biomarker of human aging. However, in the past, insufficient differentiation between cellular and cell-free DNA may have confounded analyses of genome-wide methylation levels in aging cells. Here we present a new methodological strategy to properly and unambiguously extract DNA methylation patterns of repetitive, as well as single genetic loci from pure cell-free DNA from peripheral blood. Since this nucleic acid fraction originates mainly in apoptotic, senescent and cancerous cells, this approach allows efficient analysis of aged and cancerous cell-specific DNA methylation patterns for diagnostic and prognostic purposes. Using this methodology, we observe a significant age-associated erosion of LINE-1 methylation in cfDNA suggesting that the threshold of hypomethylation sufficient for relevant LINE-1 activation and consequential harmful retrotransposition might be reached at higher age. We speculate that this process might contribute to making aging the main risk factor for many cancers.

Melanoma stem cell maintenance and chemo-resistance are mediated by CD133 signal to PI3K-dependent pathways.

Melanoma stem cells (MSCs) are characterized by their unique cell surface proteins and aberrant signaling pathways. These stemness properties are either in a causal or consequential relationship to melanoma progression, treatment resistance and recurrence. The functional analysis of CD133+ and CD133- cells in vitro and in vivo revealed that melanoma progression and treatment resistance are the consequences of CD133 signal to PI3K pathway. CD133 signal to PI3K pathway drives two downstream pathways, the PI3K/Akt/MDM2 and the PI3K/Akt/MKP-1 pathways. Activation of PI3K/Akt/MDM2 pathway results in the destabilization of p53 protein, while the activation of PI3K/Akt/MKP-1 pathway results in the inhibition of mitogen-activated protein kinases (MAPKs) JNK and p38. Activation of both pathways leads to the inhibition of fotemustine-induced apoptosis. Thus, the disruption of CD133 signal to PI3K pathway is essential to overcome Melanoma resistance to fotemustine. The pre-clinical verification of in vitro data using xenograft mouse model of MSCs confirmed the clinical relevance of CD133 signal as a therapeutic target for melanoma treatment. In conclusion, our study provides an insight into the mechanisms regulating MSCs growth and chemo-resistance and suggested a clinically relevant approach for melanoma treatment.

Basic Hallmarks of Urothelial Cancer Unleashed in Primary Uroepithelium by Interference with the Epigenetic Master Regulator ODC1.

Urothelial carcinoma (UC) is a common disease causing significant morbidity and mortality as well as considerable costs for health systems. Extensive aberrant methylation of DNA is broadly documented in early UC, contributing to genetic instability, altered gene expression and tumor progression. However the triggers initiating aberrant methylation are unknown. Recently we discovered that several genes encoding key enzymes of methyl group and polyamine metabolism, including Ornithine Decarboxylase 1 (ODC1), are affected by DNA methylation in early stage UC. In this study, we investigated the hypothesis that these epigenetic alterations act in a feed-forward fashion to promote aberrant DNA methylation in UC. We demonstrate that siRNA-mediated knockdown of ODC1 expression elicits genome-wide LINE-1 demethylation, induction of LINE-1 transcripts and double-strand DNA breaks and decreases viability in primary cultured uroepithelial cells. Similarly, following siRNA-mediated knockdown of ODC1, UC cells undergo double-strand DNA breaks and apoptosis. Collectively, our findings provide evidence that ODC1 gene hypermethylation could be a starting point for the onset of genome-wide epigenetic aberrations in urothelial carcinogenesis. Furthermore, LINE-1 induction enabled by ODC1 interference provides a new experimental model to study mechanisms and consequences of LINE-1 activation in the etiology and progression of UC as well as presumably other cancers.

Evaluation of nanoselenium and nanogold activities against murine intestinal schistosomiasis.

Nanomedicine is one of the most important methods used to treat human diseases including parasitic diseases. Schistosomiasis is a major parasitic disease that affects human health in tropical regions. Whilst Praziquantel is the main classic antischistosomal drug, new drugs are required due to the poor effect of the drug on the parasite juveniles and immature worms, and the emergence of drug resistant strains of Schistosoma. The present study aimed to examine the curative roles of both gold and selenium nanoparticles on jejunal tissues of mice infected with Schistosoma mansoni. Transmission electron microscopy was used for characterization of nanoparticles. Gold nanoparticles of 1 mg/kg mice body weight and selenium nanoparticles 0.5 mg/kg body weight were inoculated separately into mice infected with S. mansoni. The parasite induced a significant decrease in glutathione levels; however, the levels of nitric oxide and malondialdehyde were significantly increased. Additionally, the parasite introduced deteriorations in histological architecture of the jejunal tissue. Treatment of mice with metal nanoparticles reduced the levels of body weight changes, oxidative stress and histological impairment in the jejunal tissue significantly. Therefore, our results revealed the protective role of both selenium and gold nanoparticles against jejunal injury in mice infected with S. mansoni.

Tumor necrosis factor-α triggers opposing signals in head and neck squamous cell carcinoma and induces apoptosis via mitochondrial- and non-mitochondrial-dependent pathways.

Head and neck squamous cell carcinoma (HNSCC) remains one of the most common malignancies worldwide. Although the treatment outcomes of HNSCC have improved in recent years, the prognosis of patients with advanced-stage disease remains poor. Current treatment strategies for HNSCC include surgery as a primary therapy, while radio-, chemo-, and biotherapeutics can be applied as second-line therapy. Although tumor necrosis factor-α (TNF-α) is a potent tumor suppressor cytokine, the stimulation of opposing signals impairs its clinical utility as an anticancer agent. The aim of this study was to elucidate the mechanisms regulating TNF-α­induced opposing signals and their biological consequences in HNSCC cell lines. We determined the molecular mechanisms of TNF-α-induced opposing signals in HNSCC cells. Our in vitro analysis indicated that one of these signals triggers apoptosis, while the other induces both apoptosis and cell survival. The TNF-α-induced survival of HNSCC cells is mediated by the TNF receptor-associated factor 2 (TRAF2)/nuclear factor (NF)-κB-dependent pathway, while TNF-α-induced apoptosis is mediated by mitochondrial and non-mitochondrial-dependent mechanisms through FADD-caspase-8-caspase-3 and ASK-JNK-p53-Noxa pathways. The localization of Noxa protein to both the mitochondria and endoplasmic reticulum (ER) was found to cause mitochondrial dysregulation and ER stress, respectively. Using inhibitory experiments, we demonstrated that the FADD­caspase-8­caspase-3 pathway, together with mitochondrial dysregulation and ER stress-dependent pathways, are essential for the modulation of apoptosis, and the NF-κB pathway is essential for the modulation of anti-apoptotic effects/cell survival during the exposure of HNSCC cells to TNF-α. Our data provide insight into the mechanisms of TNF-α-induced opposing signals in HNSCC cells and may further help in the development of novel therapeutic approaches with which to minimize the systemic toxicity of TNF-α.

Genome-wide hypomethylation of LINE-1 and Alu retroelements in cell-free DNA of blood is an epigenetic biomarker of human aging.

Aging associated DNA hypomethylation of LINE-1 and Alu retroelements may be a crucial determinant of loss of genomic integrity, deterioration and cancer. In peripheral blood LINE-1 hypomethylation has been reported to increase during aging, but other studies did not observe significant changes. We hypothesized that these apparently inconsistent reports might relate to differences between cellular and cell-free DNA. Using the technique of idiolocal normalization of real-time methylation-specific PCR (IDLN-MSP) for genetic imbalanced DNA specimens we obtained evidence that LINE-1 hypomethylation in cell-free DNA, but not cellular DNA from peripheral blood is an epigenetic biomarker for human aging. Furthermore, hypomethylation of cell-free DNA is more extensive in smokers, suggesting that it might be used as a surrogate marker for monitoring the improvement of smoking-induced adverse effects after cancelling smoking.

Author Correction: Aberrant methylated key genes of methyl group metabolism within the molecular etiology of urothelial carcinogenesis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.