EZH2 Inhibition and Cisplatin as a Combination Anticancer Therapy: An Overview of Preclinical Studies.

Anticancer monotherapies are often insufficient in eradicating cancer cells because cancers are driven by changes in numerous genes and pathways. Combination anticancer therapies which aim to target several cancer traits at once represent a substantial improvement in anticancer treatment. Cisplatin is a conventional chemotherapy agent widely used in the treatment of different cancer types. However, the shortcomings of cisplatin use include its toxicity and development of resistance. Therefore, from early on, combination therapies that include cisplatin were considered and used in a variety of cancers. EZH2, an epigenetic regulator, is frequently upregulated in cancers which, in general, potentiates cancer cell malignant behavior. In the past decade, numerous EZH2 inhibitors have been explored for their anticancer properties. In this overview, we present the studies that discuss the joint action of cisplatin and EZH2 inhibitors. According to the data presented, the use of cisplatin and EZH2 inhibitors may be beneficial in the treatment of lung, ovarian, and breast cancers, since there is a substantial amount of published evidence that suggests their concerted action. However, in testicular germ cell tumors, such a combination would not be recommended because cisplatin resistance seems to be associated with decreased expression of EZH2 in this tumor type.

COVID-19: Are We Facing Secondary Pellagra Which Cannot Simply Be Cured by Vitamin B3?

Immune response to SARS-CoV-2 and ensuing inflammation pose a huge challenge to the host's nicotinamide adenine dinucleotide (NAD+) metabolism. Humans depend on vitamin B3 for biosynthesis of NAD+, indispensable for many metabolic and NAD+-consuming signaling reactions. The balance between its utilization and resynthesis is vitally important. Many extra-pulmonary symptoms of COVID-19 strikingly resemble those of pellagra, vitamin B3 deficiency (e.g., diarrhoea, dermatitis, oral cavity and tongue manifestations, loss of smell and taste, mental confusion). In most developed countries, pellagra is successfully eradicated by vitamin B3 fortification programs. Thus, conceivably, it has not been suspected as a cause of COVID-19 symptoms. Here, the deregulation of the NAD+ metabolism in response to the SARS-CoV-2 infection is reviewed, with special emphasis on the differences in the NAD+ biosynthetic pathway's efficiency in conditions predisposing for the development of serious COVID-19. SARS-CoV-2 infection-induced NAD+ depletion and the elevated levels of its metabolites contribute to the development of a systemic disease. Acute liberation of nicotinamide (NAM) in antiviral NAD+-consuming reactions potentiates "NAM drain", cooperatively mediated by nicotinamide N-methyltransferase and aldehyde oxidase. "NAM drain" compromises the NAD+ salvage pathway's fail-safe function. The robustness of the host's NAD+ salvage pathway, prior to the SARS-CoV-2 infection, is an important determinant of COVID-19 severity and persistence of certain symptoms upon resolution of infection.

Nicotinamide N-Methyltransferase in Acquisition of Stem Cell Properties and Therapy Resistance in Cancer.

The activity of nicotinamide N-methyltransferase (NNMT) is tightly linked to the maintenance of the nicotinamide adenine dinucleotide (NAD+) level. This enzyme catalyzes methylation of nicotinamide (NAM) into methyl nicotinamide (MNAM), which is either excreted or further metabolized to N1-methyl-2-pyridone-5-carboxamide (2-PY) and H2O2. Enzymatic activity of NNMT is important for the prevention of NAM-mediated inhibition of NAD+-consuming enzymes poly-adenosine -diphosphate (ADP), ribose polymerases (PARPs), and sirtuins (SIRTs). Inappropriately high expression and activity of NNMT, commonly present in various types of cancer, has the potential to disrupt NAD+ homeostasis and cellular methylation potential. Largely overlooked, in the context of cancer, is the inhibitory effect of 2-PY on PARP-1 activity, which abrogates NNMT's positive effect on cellular NAD+ flux by stalling liberation of NAM and reducing NAD+ synthesis in the salvage pathway. This review describes, and discusses, the mechanisms by which NNMT promotes NAD+ depletion and epigenetic reprogramming, leading to the development of metabolic plasticity, evasion of a major tumor suppressive process of cellular senescence, and acquisition of stem cell properties. All these phenomena are related to therapy resistance and worse clinical outcomes.

Nutritional Stress in Head and Neck Cancer Originating Cell Lines: The Sensitivity of the NRF2-NQO1 Axis.

Nutritional stress disturbs the cellular redox-status, which is characterized by the increased generation of reactive oxygen species (ROS). The NRF2-NQO1 axis represents a protective mechanism against ROS. Its strength is cell type-specific. FaDu, Cal 27 and Detroit 562 cells differ with respect to basal NQO1 activity. These cells were grown for 48 hours in nutritional conditions (NC): (a) Low glucose-NC2, (b) no glucose, no glutamine-NC3, (c) no glucose with glutamine-NC4. After determining the viability, proliferation and ROS generation, NC2 and NC3 were chosen for further exploration. These conditions were also applied to IMR-90 fibroblasts. The transcripts/transcript variants of NRF2 and NQO1 were quantified and transcript variants were characterized. The proteins (NRF2, NQO1 and TP53) were analyzed by a western blot in both cellular fractions. Under NC2, the NRF2-NQO1 axis did not appear activated in the cancer cell lines. Under NC3, the NRF2-NQO1axis appeared slightly activated in Detroit 562. There are opposite trends with respect to TP53 nuclear signal when comparing Cal 27 and Detroit 562 to FaDu, under NC2 and NC3. The strong activation of the NRF2-NQO1 axis in IMR-90 resulted in an increased expression of catalytically deficient NQO1, due to NQO1*2/*2 polymorphism (rs1800566). The presented results call for a comprehensive exploration of the stress response in complex biological systems.

Polycomb repressive complex's evolutionary conserved function: the role of EZH2 status and cellular background.

When assembled in multiprotein polycomb repressive complexes (PRCs), highly evolutionary conserved polycomb group (PcG) proteins epigenetically control gene activity. Although the composition of PRCs may vary considerably, it is well established that the embryonic ectoderm development (EED) 1, suppressor of zeste (SUZ) 12, and methyltransferase enhancer of zeste (EZH2)-containing complex, PRC2, which is abundant in highly proliferative cells (including cancer cells), establishes a repressive methylation mark on histone 3 (H3K27me3). From the perspective of molecular cancer pathogenesis, this effect, when directed towards a promoter of tumor suppressor genes, represents pro-tumorigenic effect. This mode of action was shown in several cancer models. However, EZH2 function extends beyond this scenario. The highly specific cellular background, related to the origin of cell and numerous external stimuli during a given time-window, may be the trigger for EZH2 interaction with other proteins, not necessarily histones. This is particularly relevant for cancer. This review provides a critical overview of the evolutional importance of PRC and discusses several important aspects of EZH2 functioning within PRC. The review also deals with mutational studies on EZH2. Due to the existence of several protein (and messenger RNA (mRNA)) isoforms, these mutations were stratified, using the protein sequence which is considered canonical. This approach showed that there is an urgent need for the uniformed positioning of currently known EZH2 mutations (somatic-in tumors, as well as germline mutations in the Weaver's syndrome). Finally, we discuss EZH2 function with respect to amount of trimethylated H3K27, in a specific cellular milieu, through presenting the most recent data related to EZH2-H3K27m3 relationship in cancer. All these points are significant in considering EZH2 as a therapeutic target.

Pyridine nucleotides in regulation of cell death and survival by redox and non-redox reactions.

Changes of the level and ratios of pyridine nucleotides determine metabolism- dependent cellular redox status and the activity of poly(ADP-ribose) polymerases (PARPs) and sirtuins, thereby influencing several processes closely related to cell survival and death. Pyridine nucleotides participate in numerous metabolic reactions whereby their net cellular level remains constant, but the ratios of NAD+/NADP+ and NADH/NADPH oscillate according to metabolic changes in response to diverse stress signals. In non-redox reactions, NAD+ is degraded and quickly, afterward, resynthesized in the NAD+ salvage pathway, unless overwhelming activation of PARP-1 consumes NAD+ to the point of no return, when the cell can no longer generate enough ATP to accommodate NAD+ resynthesis. The activity of PARP-1 is mandatory for the onset of cytoprotective autophagy on sublethal stress signals. It has become increasingly clear that redox status, largely influenced by the metabolism-dependent composition of the pyridine nucleotides pool, plays an important role in the synthesis of pro-apoptotic and anti-apoptotic sphingolipids. Awareness of the involvement of the prosurvival sphingolipid, sphingosine-1-phosphate, in transition from inflammation to malignant transformation has recently emerged. Here, the participation of pyridine nucleotides in redox and non-redox reactions, sphingolipid metabolism, and their role in cell fate decisions is reviewed.

Possible prognostic value of BORIS transcript variants ratio in laryngeal squamous cell carcinomas - a pilot study.

BORIS is a paralog of a highly conserved, multi-functional chromatin factor CTCF. Unlike CTCF, which has been shown to possess tumor-suppressive properties, BORIS belongs to the "cancer/testis antigen" family normally expressed only in germ cells and aberrantly activated in a variety of tumors. The consequences of BORIS expression, relative abundance of its isoforms, and its role in carcinogenesis have not been completely elucidated. It activates transcription of hTERT and MYC, genes relevant for laryngeal carcinoma progression. In this study, BORIS expression has been analyzed at the transcriptional level by RT-PCR and protein level by semi-quantitative immunohistochemistry in 32 laryngeal squamous cell carcinomas and adjacent non-tumorous tissue. BORIS was detected in 44 % (14/32) laryngeal squamous cell carcinoma samples, while it was detected only in one normal, tumor-adjacent tissue sample. Tree based survival analysis, using the recursive partitioning algorithm mvpart, extracted the ratio of relative abundance of BORIS transcript variants containing exon 7 (BORIS 7+) and those lacking exon 7 (BORIS 7-) as an independent prognostic factor associated with disease relapse during a 5-year follow-up period. Patients having BORIS 7+/BORIS 7- ratio ≥1 had a higher rate of disease relapse than patients with BORIS 7+/BORIS 7- ratio <1. Hazard ratio for that group, based on Cox Proportional Hazard Regression, was 3.53. This is the first study analyzing expression of BORIS protein and transcript variants in laryngeal squamous cell carcinoma relative to its possible prognostic value for recurrence and overall survival.

Characterization of avian thymic hormone and chicken parvalbumin 3 target cells.

Avian thymic hormone (ATH) is a ß-parvalbumin produced by epithelial cells in the thymic cortex and in the eyes of chickens. Chicken parvalbumin 3 (CPV3) is a homologous protein produced in the thymus and in hair cells of the chicken ear. ATH circulates in the blood on a five-day cycle and stimulates cell-mediated immunity when administered to young chickens. We report the identification of target cells for ATH and CPV3 and the immunophenotype of target cells for ATH. Newly hatched chicks were injected intracoelomically with ATH and killed 5, 10, 15 or 20 min later. Naïve chickens also were killed at 1, 7 and 14 days of age. Various tissues were examined by EM for the presence of either ATH or CPV3 using colloidal gold labeling. Gold particles were initially present on plasma membranes of lymphocytes in T cell areas of spleen and cecal tonsils from the chicks injected with ATH, internalized within 10 min, and accumulated in nuclei by 20 min. Immunofluorescence staining also identified the presence of ATH in T cell areas of spleen and cecal tonsils. Target cells labeled for ATH were immunophenotyped by double labeling. They were positive for CD3, CD8 and the lymphocyte receptor TCR-1, a phenotype characteristic of cytotoxic γδ T cells. Some of the target cells in the spleen were TCR-3 positive. Targeting of lymphocytes by CPV3 indicated that it may also be an immunomodulating hormone.

Loss of imprinting of IGF2 and H19, loss of heterozygosity of IGF2R and CTCF, and Helicobacter pylori infection in laryngeal squamous cell carcinoma.

Imprinting analyses of IGF2 and H19, loss of heterozygosity (LOH) analyses of IGF2R and CTCF and Helicobacter pylori detection, were performed on 35 human laryngeal squamous cell carcinomas (LSCC). Forty-six percent of the tumors were heterozygous for IGF2, and 54% were informative for the H19. Biallelic expression of IGF2 was observed in 33% (5 out of 15) of the tumors and in 27% (4 out of 15) of adjacent non-tumorous laryngeal tissues. H19 loss of imprinting (LOI) was observed in 24% (4 out of 17) of the tumors. For IGF2R and CTCF, 71% (25 out of 35) and 50% (17/34), respectively, of the samples were heterozygous, and LOH was detected in 12% (3 out of 25) and 6% (1 out of 17), respectively, of the tumors. H. pylori was found in 26% (9/35) of these tumors. Among them, four were informative for the imprinting analysis. The presence of H. pylori had no effect on IGF2/H19 imprinting. Only the H. pylori detection was further broadened with an additional 47 laryngeal tumors, resulting in a total final positivity of close to 16% (13 out of 82). This study represents the largest comprehensive IGF2/H19 imprinting study done to date on well-defined samples of human laryngeal carcinomas and corresponding non-tumorous tissue. For the first time, the analyses of IGF2/H19 imprinting have been broadened with LOH analyses of IGF2R and CTCF, with both of these genes acting as modulators of IGF2 and H19 activity. Although there were indications that H. pylori may be present in LSCC, we are the first to show its presence in LSCC by two direct techniques: Giemsa staining and nested-PCR.

Curcumin downregulates H19 gene transcription in tumor cells.

Curcumin (diferuloymethane), a natural compound used in traditional medicine, exerts an antiproliferative effect on various tumor cell lines by an incompletely understood mechanism. It has been shown that low doses of curcumin downregulate DNA topoisomerase II alpha (TOP2A) which is upregulated in many malignances. The activity of TOP2A is required for RNA polymerase II transcription on chromatin templates. Recently, it has been reported that CTCF, a multifunctional transcription factor, recruits the largest subunit of RNA polymerase II (LS Pol II) to its target sites genome-wide. This recruitment of LS Pol II is more pronounced in proliferating cells than in fully differentiated cells. As expression of imprinted genes is often altered in tumors, we investigated the potential effect of curcumin treatment on transcription of the imprinted H19 gene, located distally from the CTCF binding site, in human tumor cell lines HCT 116, SW 620, HeLa, Cal 27, Hep-2 and Detroit 562. Transcription of TOP2A and concomitantly H19 was supressed in all tumor cell lines tested. Monoallelic IGF2 expression was maintained in curcumin-treated cancer cells, indicating the involvement of mechanism/s other than disturbance of CTCF insulator function at the IGF2/H19 locus. Curcumin did not alter H19 gene transcription in primary cell cultures derived from normal human tissues.