Correction: Sallam et al. DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy. Int. J. Mol. Sci. 2022, 23, 16214.

Radia Tamarat and Susana Constantino Rosa Santos were not included as authors in the original publication [...].

The role of connexin proteins and their channels in radiation-induced atherosclerosis.

Radiotherapy is an effective treatment for breast cancer and other thoracic tumors. However, while high-energy radiotherapy treatment successfully kills cancer cells, radiation exposure of the heart and large arteries cannot always be avoided, resulting in secondary cardiovascular disease in cancer survivors. Radiation-induced changes in the cardiac vasculature may thereby lead to coronary artery atherosclerosis, which is a major cardiovascular complication nowadays in thoracic radiotherapy-treated patients. The underlying biological and molecular mechanisms of radiation-induced atherosclerosis are complex and still not fully understood, resulting in potentially improper radiation protection. Ionizing radiation (IR) exposure may damage the vascular endothelium by inducing DNA damage, oxidative stress, premature cellular senescence, cell death and inflammation, which act to promote the atherosclerotic process. Intercellular communication mediated by connexin (Cx)-based gap junctions and hemichannels may modulate IR-induced responses and thereby the atherosclerotic process. However, the role of endothelial Cxs and their channels in atherosclerotic development after IR exposure is still poorly defined. A better understanding of the underlying biological pathways involved in secondary cardiovascular toxicity after radiotherapy would facilitate the development of effective strategies that prevent or mitigate these adverse effects. Here, we review the possible roles of intercellular Cx driven signaling and communication in radiation-induced atherosclerosis.

DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy.

Radiation-Induced CardioVascular Disease (RICVD) is an important concern in thoracic radiotherapy with complex underlying pathophysiology. Recently, we proposed DNA methylation as a possible mechanism contributing to RICVD. The current study investigates DNA methylation in heart-irradiated rats and radiotherapy-treated breast cancer (BC) patients. Rats received fractionated whole heart X-irradiation (0, 0.92, 6.9 and 27.6 Gy total doses) and blood was collected after 1.5, 3, 7 and 12 months. Global and gene-specific methylation of the samples were evaluated; and gene expression of selected differentially methylated regions (DMRs) was validated in rat and BC patient blood. In rats receiving an absorbed dose of 27.6 Gy, DNA methylation alterations were detected up to 7 months with differential expression of cardiac-relevant DMRs. Of those, SLMAP showed increased expression at 1.5 months, which correlated with hypomethylation. Furthermore, E2F6 inversely correlated with a decreased global longitudinal strain. In BC patients, E2F6 and SLMAP exhibited differential expression directly and 6 months after radiotherapy, respectively. This study describes a systemic radiation fingerprint at the DNA methylation level, elucidating a possible association of DNA methylation to RICVD pathophysiology, to be validated in future mechanistic studies.

Protective effect of N-acetylcysteine on fenitrothion-induced toxicity: The antioxidant status and metabolizing enzymes expression in rats.

The existence of fenitrothion (FNT) in the soil, water, and food products has harmful effects on non-target organisms. Therefore, this study was conducted to evaluate the hepatotoxic, nephrotoxic and neurotoxic effects of FNT and the possible ameliorative effect of N-acetylcysteine (NAC), a precursor of intracellular GSH, on FNT-induced toxicity. For this purpose, thirty-two adult male albino rats were allocated into control group and groups treated with NAC (200 mg/kg), FNT (10 mg/kg) and FNT + NAC via gastric tube daily for 28 days. FNT intoxication significantly reduced food intake, water intake, body weight, and body weight gain and altered the expression of phase I and phase II xenobiotic-metabolizing enzymes-cytochrome P450 (CYP1A1) and glutathione S-transferase (GSTA4-4). In hepatic, renal and brain tissues, FNT induced oxidative stress, hepatopathy, nephropathy, and encephalopathy, and significantly increased pro-inflammatory cytokines. Furthermore, FNT exposure significantly elevated the level of hepatic and renal injury biomarkers and significantly inhibited the brain acetylcholinesterase activity. Co-administration of NAC with FNT modulated most of these altered biochemical, oxidative and inflammatory markers and restored the xenobiotic-metabolizing enzymes expression and histological structures. Our study indicated the involvement of oxidative damage, inflammation, and alteration of xenobiotic-metabolizing enzymes expression in FNT-induced toxicity and revealed that they were significantly improved by NAC co-treatment. These findings suggest that NAC administration might protect against FNT-induced toxicity in non-target organisms, including humans.

Calcium, oxidative stress and connexin channels, a harmonious orchestra directing the response to radiotherapy treatment?

Although radiotherapy is commonly used to treat cancer, its beneficial outcome is frequently hampered by the radiation resistance of tumor cells and adverse reactions in normal tissues. Mechanisms of cell-to-cell communication and how intercellular signals are translated into cellular responses, have become topics of intense investigation, particularly within the field of radiobiology. A substantial amount of evidence is available demonstrating that both gap junctional and paracrine communication pathways can propagate radiation-induced biological effects at the intercellular level, commonly referred to as radiation-induced bystander effects (RIBE). Multiple molecular signaling mechanisms involving oxidative stress, kinases, inflammatory molecules, and Ca2+ are postulated to contribute to RIBE. Ca2+ is a highly versatile and ubiquitous second messenger that regulates diverse cellular processes via the interaction with various signaling cascades. It furthermore provides a fast system for the dissemination of information at the intercellular level. Channels formed by transmembrane connexin (Cx) proteins, i.e. hemichannels and gap junction channels, can mediate the cell-to-cell propagation of increases in intracellular Ca2+ by ministering paracrine and direct cell-cell communication, respectively. We here review current knowledge on radiation-induced signaling mechanisms in irradiated and bystander cells, particularly focusing on the contribution of oxidative stress, Ca2+ and Cx channels. By illustrating the tight interplay between these different partners, we provide a conceptual framework for intercellular Ca2+ signaling as a key player in modulating the RIBE and the overall response to radiation.

Synthesis and docking studies of novel antitumor benzimidazoles.

In this work, the benzimidazole-pyrrole conjugates 6a-h and benzimidazole-tetracycles conjugates 12-14 were prepared. The cytotoxicity of the compounds 3, 4a-h, 6a-h, 8, 10 and 12-14 was tested against lung cancer cell line A549. Compound 6b exhibited higher activity than the bis-benzoxazole natural product (UK-1), the standard. The tested 4g,h, 6a-h, 10 and 12-14 exhibited remarkable cytotoxicity activity against breast cancer cell line MCF-7 with higher activity than tamoxifen. Furthermore, compound 4h was found to be also more potent than doxurubicin. The antitumor promotion activity of synthesized compounds 4g,h, 6a-h, 10 and 12-14 has been estimated by studying their possible inhibitory effects on EBV-EA activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds, the inhibitory activities of compounds 8, 13 and 14 demonstrated strong inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation without showing any cytotoxicity on the Raji cells and their effects being stronger than that of a representative control, oleanolic acid. Moreover, the molecular docking of the new compounds into plasminogen activator (uPA) receptor has been in correlation with the antitumor activity. All synthesized compounds 3, 4a-h, 6a-h, 8, 10 and 12-14 were docked into same groove of the binding site of the native co-crystalized (4-iodobenzo[b]thiophene-2-carboxamidine) ligand (PDB code:1c5x) for activity explaination. Compounds 4h, 6b and 13, giving the best docking results, were further studied to estimate their effect on the level of uPA using AssayMax human urokinase (uPA) ELISA kit. In case of A549 cell line, compound 6 exhibited similar activity to MMC, and for MCF-7 cell line, compound 4h exhibited similar activity to doxorubicin, in inhibiting the expression of uPA.

Meta-Analysis of RNA-Seq Datasets Identifies Novel Players in Glioblastoma.

Glioblastoma is a devastating grade IV glioma with poor prognosis. Identification of predictive molecular biomarkers of disease progression would substantially contribute to better disease management. In the current study, we performed a meta-analysis of different RNA-seq datasets to identify differentially expressed protein-coding genes (PCGs) and long non-coding RNAs (lncRNAs). This meta-analysis aimed to improve power and reproducibility of the individual studies while identifying overlapping disease-relevant pathways. We supplemented the meta-analysis with small RNA-seq on glioblastoma tissue samples to provide an overall transcriptomic view of glioblastoma. Co-expression correlation of filtered differentially expressed PCGs and lncRNAs identified a functionally relevant sub-cluster containing DANCR and SNHG6, with two novel lncRNAs and two novel PCGs. Small RNA-seq of glioblastoma tissues identified five differentially expressed microRNAs of which three interacted with the functionally relevant sub-cluster. Pathway analysis of this sub-cluster identified several glioblastoma-linked pathways, which were also previously associated with the novel cell death pathway, ferroptosis. In conclusion, the current meta-analysis strengthens evidence of an overarching involvement of ferroptosis in glioblastoma pathogenesis and also suggests some candidates for further analyses.

Application of radiation omics in the development of adverse outcome pathway networks: an example of radiation-induced cardiovascular disease.

BACKGROUND: Epidemiological studies have indicated that exposure of the heart to doses of ionizing radiation as low as 0.5 Gy increases the risk of cardiac morbidity and mortality with a latency period of decades. The damaging effects of radiation to myocardial and endothelial structures and functions have been confirmed radiobiologically at high dose, but much less are known at low dose. Integration of radiation biology and epidemiology data is a recommended approach to improve the radiation risk assessment process. The adverse outcome pathway (AOP) framework offers a comprehensive tool to compile and translate mechanistic information into pathological endpoints which may be relevant for risk assessment at the different levels of a biological system. Omics technologies enable the generation of large volumes of biological data at various levels of complexity, from molecular pathways to functional organisms. Given the quality and quantity of available data across levels of biology, omics data can be attractive sources of information for use within the AOP framework. It is anticipated that radiation omics studies could improve our understanding of the molecular mechanisms behind the adverse effects of radiation on the cardiovascular system. In this review, we explored the available omics studies on radiation-induced cardiovascular disease (CVD) and their applicability to the proposed AOP for CVD. RESULTS: The results of 80 omics studies published on radiation-induced CVD over the past 20 years have been discussed in the context of the AOP of CVD proposed by Chauhan et al. Most of the available omics data on radiation-induced CVD are from proteomics, transcriptomics, and metabolomics, whereas few datasets were available from epigenomics and multi-omics. The omics data presented here show great promise in providing information for several key events (KEs) of the proposed AOP of CVD, particularly oxidative stress, alterations of energy metabolism, extracellular matrix (ECM), and vascular remodeling. CONCLUSIONS: The omics data presented here shows promise to inform the various levels of the proposed AOP of CVD. However, the data highlight the urgent need of designing omics studies to address the knowledge gap concerning different radiation scenarios, time after exposure, and experimental models. This review presents the evidence to build a qualitative omics-informed AOP and provides views on the potential benefits and challenges in using omics data to assess risk-related outcomes.

A systems radiation biology approach to unravel the role of chronic low-dose-rate gamma-irradiation in inducing premature senescence in endothelial cells.

PURPOSE: The aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence. MATERIALS AND METHODS: Different datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence. RESULTS: Simultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario. CONCLUSIONS: This study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.

X­irradiation induces acute and early term inflammatory responses in atherosclerosis­prone ApoE­/­ mice and in endothelial cells.

Thoracic radiotherapy is an effective treatment for many types of cancer; however it is also associated with an increased risk of developing cardiovascular disease (CVD), appearing mainly ≥10 years after radiation exposure. The present study investigated acute and early term physiological and molecular changes in the cardiovascular system after ionizing radiation exposure. Female and male ApoE­/­ mice received a single exposure of low or high dose X­ray thoracic irradiation (0.1 and 10 Gy). The level of cholesterol and triglycerides, as well as a large panel of inflammatory markers, were analyzed in serum samples obtained at 24 h and 1 month after irradiation. The secretion of inflammatory markers was further verified in vitro in coronary artery and microvascular endothelial cell lines after exposure to low and high dose of ionizing radiation (0.1 and 5 Gy). Local thoracic irradiation of ApoE­/­ mice increased serum growth differentiation factor­15 (GDF­15) and C­X­C motif chemokine ligand 10 (CXCL10) levels in both female and male mice 24 h after high dose irradiation, which were also secreted from coronary artery and microvascular endothelial cells in vitro. Sex­specific responses were observed for triglyceride and cholesterol levels, and some of the assessed inflammatory markers as detailed below. Male ApoE­/­ mice demonstrated elevated intercellular adhesion molecule­1 and P­selectin at 24 h, and adiponectin and plasminogen activator inhibitor­1 at 1 month after irradiation, while female ApoE­/­ mice exhibited decreased monocyte chemoattractant protein­1 and urokinase­type plasminogen activator receptor at 24 h, and basic fibroblast growth factor 1 month after irradiation. The inflammatory responses were mainly significant following high dose irradiation, but certain markers showed significant changes after low dose exposure. The present study revealed that acute/early inflammatory responses occurred after low and high dose thoracic irradiation. However, further research is required to elucidate early asymptomatic changes in the cardiovascular system post thoracic X­irradiation and to investigate whether GDF­15 and CXCL10 could be considered as potential biomarkers for the early detection of CVD risk in thoracic radiotherapy­treated patients.

Connexin43 Hemichannel Targeting With TAT-Gap19 Alleviates Radiation-Induced Endothelial Cell Damage.

BACKGROUND: Emerging evidence indicates an excess risk of late occurring cardiovascular diseases, especially atherosclerosis, after thoracic cancer radiotherapy. Ionizing radiation (IR) induces cellular effects which may induce endothelial cell dysfunction, an early marker for atherosclerosis. In addition, intercellular communication through channels composed of transmembrane connexin proteins (Cxs), i.e. Gap junctions (direct cell-cell coupling) and hemichannels (paracrine release/uptake pathway) can modulate radiation-induced responses and therefore the atherosclerotic process. However, the role of endothelial hemichannel in IR-induced atherosclerosis has never been described before. MATERIALS AND METHODS: Telomerase-immortalized human Coronary Artery/Microvascular Endothelial cells (TICAE/TIME) were exposed to X-rays (0.1 and 5 Gy). Production of reactive oxygen species (ROS), DNA damage, cell death, inflammatory responses, and senescence were assessed with or without applying a Cx43 hemichannel blocker (TAT-Gap19). RESULTS: We report here that IR induces an increase in oxidative stress, cell death, inflammatory responses (IL-8, IL-1ß, VCAM-1, MCP-1, and Endothelin-1) and premature cellular senescence in TICAE and TIME cells. These effects are significantly reduced in the presence of the Cx43 hemichannel-targeting peptide TAT-Gap19. CONCLUSION: Our findings suggest that endothelial Cx43 hemichannels contribute to various IR-induced processes, such as ROS, cell death, inflammation, and senescence, resulting in an increase in endothelial cell damage, which could be protected by blocking these hemichannels. Thus, targeting Cx43 hemichannels may potentially exert radioprotective effects.

Single and fractionated ionizing radiation induce alterations in endothelial connexin expression and channel function.

Radiotherapy is an effective treatment for most tumor types. However, emerging evidence indicates an increased risk for atherosclerosis after ionizing radiation exposure, initiated by endothelial cell dysfunction. Interestingly, endothelial cells express connexin (Cx) proteins that are reported to exert proatherogenic as well as atheroprotective effects. Furthermore, Cxs form channels, gap junctions and hemichannels, that are involved in bystander signaling that leads to indirect radiation effects in non-exposed cells. We here aimed to investigate the consequences of endothelial cell irradiation on Cx expression and channel function. Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells were exposed to single and fractionated X-rays. Several biological endpoints were investigated at different time points after exposure: Cx gene and protein expression, gap junctional dye coupling and hemichannel function. We demonstrate that single and fractionated irradiation induce upregulation of proatherogenic Cx43 and downregulation of atheroprotective Cx40 gene and protein levels in a dose-dependent manner. Single and fractionated irradiation furthermore increased gap junctional communication and induced hemichannel opening. Our findings indicate alterations in Cx expression that are typically observed in endothelial cells covering atherosclerotic plaques. The observed radiation-induced increase in Cx channel function may promote bystander signaling thereby exacerbating endothelial cell damage and atherogenesis.

Part III: Novel checkpoint kinase 2 (Chk2) inhibitors; design, synthesis and biological evaluation of pyrimidine-benzimidazole conjugates.

Recently a dramatic development of the cancer drug discovery has been shown in the field of targeted cancer therapy. Checkpoint kinase 2 (Chk2) inhibitors offer a promising approach to enhance the effectiveness of cancer chemotherapy. Accordingly, in this study many pyrimidine-benzimidazole conjugates were designed and twelve feasible derivatives were selected to be synthesized to investigate their activity against Chk2 and subjected to study their antitumor activity alone and in combination with the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). The results indicated that the studied compounds inhibited Chk2 activity with high potency (IC50 = 5.56 nM - 46.20 nM). The studied candidates exhibited remarkable antitumor activity against MCF-7 (IG50 = 6.6  µM - 24.9 µM). Compounds 10a-c, 14 and 15 significantly potentiated the activity of the studied genotoxic drugs, whereas, compounds 9b and 20-23 antagonized their activity. Moreover, the combination of compound 10b with cisplatin revealed the best apoptotic effect as well as combination of compound 10b with doxorubicin led to complete arrest of the cell cycle at S phase where more than 40% of cells are in the S phase with no cells at G2/M. Structure-activity relationship was discussed on the basis of molecular modeling study using Molecular modeling Environment program (MOE).

Design, synthesis and structure-activity relationship of novel quinoxaline derivatives as cancer chemopreventive agent by inhibition of tyrosine kinase receptor.

The cancer chemopreventive activity of quinoxaline derivatives 1-20 has been evaluated by studying the inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation. The quinoxaline derivatives 1-20 showed inhibitory effect on EBV-EA activation without cytotoxicity on Raji cells. All compounds exhibited dose dependent inhibitory activities, most of them showed significant activity at 1000 mol ratio/12-O-tetradecanoylphorbol-13-acetate (TPA). Compounds 7 and 9 exhibited stronger inhibitory effects on the EBV-EA activation than that of the representative control, oleanolic acid, at the highest measured concentration. In addition, compounds 7-10 showed potent and selective inhibition of human tyrosine kinase (TRK) in liver cancer HepG2 and breast cancer MCF-7 cell lines similar to the positive control, doxorubicin.

Part I: Synthesis, cancer chemopreventive activity and molecular docking study of novel quinoxaline derivatives.

The reaction of o-phenylene diamine and ethyl oxamate is reinvestigated and led to 3-aminoquinoxalin-2(1H)-one rather than benzimidazole-2-carboxamide as was previously reported. The structure of the obtained quinoxaline has been confirmed by X-ray. The anti-tumor activity of synthesized quinoxalines 1-21 has been evaluated by studying their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Among the studied compounds 1-21, compounds 12, 8, 13, 18, 17 and 19, respectively, demonstrated strong inhibitory effects on the EBV-EA activation without showing any cytotoxicity and their effects being stronger than that of a representative control, oleanolic acid. Furthermore, compound 12 exhibited a remarkable inhibitory effect on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and TPA as a promoter. The result of the present investigation indicated that compound 12 might be valuable as a potent cancer chemopreventive agent. Moreover, the molecular docking into PTK (PDB: 1t46) has been done for lead optimization of the aforementioned compounds as potential PTK inhibitors.