Radiation-induced H3K9 methylation on E-cadherin promoter mediated by ROS/Snail axis : Role of G9a signaling during lung epithelial-mesenchymal transition.

Lung cancer patients who have undergone radiotherapy developed severe complications such as pneumonitis and fibrosis. Upon irradiation, epithelial cells acquire mesenchymal phenotype via a process called epithelial to mesenchymal transition (EMT), which plays a vital role in organ fibrosis. Several mechanisms have been studied on EMT, however, the correlation between radiation-induced EMT and epigenetic changes are not well known. In the present study, we investigated the role of histone methyltransferase G9a on radiation-induced EMT signaling. There was an increase in total global histone methylation level in irradiated epithelial cells. Western blot analysis on irradiated cells showed an increased expression of H3K9me2/3. The pre-treatment of G9a inhibitor enhanced E-cadherin expression and decreased the mesenchymal markers like N-cadherin, vimentin in the radiated group. Surprisingly, radiation-induced ROS generation and pERK1/2 levels were also inhibited by G9a inhibitor BIX01294, which is showing its antioxidant potential. The ChIP-qPCR analysis on the E-cadherin promoter suggested that G9a and Snail might have formed complex to enrich suppressive marker H3K9me2/3. On the whole, our present study suggested that 1] ROS could modify H3K9 methylation via G9a and promote radiation-induced lung EMT in Beas2B and A549 cells 2] E-cadherin promoter enrichment with heterochromatin mark H3K9me2 expression upon irradiation could be modified by regulating G9a methyltransferase.

Lead Generation for Human Mitotic Kinesin Eg5 Using Structure-based Virtual Screening and Validation by In-vitro and Cell-based Assays.

BACKGROUND: Human mitotic kinesins play a crucial role in mitotic cell division. Targeting the spindle separation phase of mitosis has gained much attention pharmaceutically in cancer chemotherapy. Spindle segregation is carried out mainly by Eg5 kinesin, and currently, it has many inhibitors in different phases of clinical trials. All the current drug candidates bind un-competitively with ATP/ADP at allosteric site 1 (formed by loop L5, helix α2 and helix α3). Recent experiments show that inhibitors that bind to the site 2 (formed by helix α4 and helix α6) are either competitive or uncompetitive to ATP/ADP. OBJECTIVES: To identify suitable lead compounds that target the mitotic kinesin Eg5, using in silico screening and their validation using in vitro and cell-based assays. METHODOLOGY: Potential inhibitors were screened for human Eg5 (kinesin-5) through structurebased virtual screening and the top-scoring compounds were validated using steady-state ATPase assay, differential scanning fluorimetry, and microscale thermophoresis. The anti-cancer activity of the compounds was evaluated in the epithelial (A549) and chronic myelogenous leukemia (K562) cancer cell lines. A known strong binding inhibitor, S-trityl-L-cystine, is used as a reference compound. RESULTS: Out of the many compounds tested, MM01 and MM03 showed good cell-based activity against the cancer cell lines A549 and K562 and can be further studied in animal models. CONCLUSION: In this study, a structure-based approach was used to identify the potential inhibitors and validate them using different in-vitro and cell-based assays.

Literature review on virus and host response proteins in COVID-19: pathobiology, management, diagnosis and treatment.

Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome caused by a novel strain of coronavirus (SARS-CoV-2) which was declared by WHO as a cause of global pandemic. By human-to-human transmission it caused severe damage to mankind with increased mortality rate worldwide. Coronavirus is a spherical enveloped virus with single stranded positive-sense RNA with a size of ~30 kilobases encoding various structural, non-structural and accessory proteins. The entry of coronavirus into the host cells is mediated by spike proteins. SARS-CoV-2 efficiently replicates in host cell and by evading immune surveillance, like innate and adaptive immune responses, in the host cells ultimately leads to increased virulence and disease outcome. In the current review, we highlighted the molecular insights of SARS-CoV-2 and its infection mechanism in the host cell via host-viral protein interactions based on currently available data up to 16thMay 2020 using various research literature databases. The diagnostics of SARS-CoV-2 is mainly done by RT-qPCR and serological tests. There is no effective treatment for COVID-19, however, few methods like plasma therapy and remdesivir treatment are reported to show promising results in improving patient's health and decreasing mortality rate. Keywords: SARS-CoV; spike protein; nucleocapsid; COVID-19; interferon.

Neuromyelitis optica spectrum disorder: an overview.

Neuromyelitis optica also known as Devic's syndrome is a rare autoimmune disorder that predominantly targets the optic nerves and the spinal cord. It is a debilitating disorder that damages a person's health. Initially it was considered as a variant of multiple sclerosis (MS). But, in 2004, a water channel protein associated antibody was found to be responsible for the disease. This helped in distinguishing the disease from multiple sclerosis. Multiple molecular mechanisms like complement dependent cytotoxicity, antibody­dependent cellular cytotoxicity etc. contribute to the disease. Certain environmental and genetic factors have been identified as risk factors of the disease. Initially, the disease was thought to affect only the optic nerves and the spinal cord. But certain regions of the brain have also been found to be attacked during the course of the disease. A small proportion of the patients have been found to be seronegative for the AQP4­IgG. Recently, the term neuromyelitis optica spectrum disorder has been framed to include all the features of the disease. The disease remains incurable despite the availability of various treatment modalities. This review presents critical information obtained from prior studies regarding the disease and also raise several questions to understand the research gaps in this field.

Radiation-induced H3K9 tri-methylation in E-cadherin promoter during lung EMT: in vitro and in vivo approaches using vanillin.

Radiotherapy is an important treatment regime for lung cancer, worldwide. However, radiation-induced pneumonitis and fibrosis are the treatment-limiting toxicities among patients who have undergone radiotherapy. The epithelial cells via epithelial to mesenchymal transition [EMT] acquires mesenchymal phenotype, which ultimately leads to fibrosis. Many investigations are focussed on understanding the signalling pathways mediating in EMT, however, the role of histone methylation is less understood in radiation-induced lung EMT. In the present study, we analysed the effect of vanillin, an antioxidant, on histone methylation during radiation-induced EMT. The thoracic region of Wistar rats was irradiated with a fractionated dose of X-ray (3 Gy/day) for two weeks (total of 30 Gy). The irradiated animals were sacrificed at the 8th and 16th weeks and tissues were used for analyses. Our data showed that radiation decreased the level of antioxidant enzymes such as SOD, catalase and reduced glutathione that would ultimately enhance oxidative stress in the tissues. Histopathological analysis revealed that radiation increased the infiltration of inflammatory cells to the tissue injury site. Total global histone methylation was increased upon irradiation, which was effectively prevented by vanillin administration. Vanillin enhanced E-cadherin expression and decreased the mesenchymal markers N-cadherin and vimentin in the irradiated lung tissue. The ChIP-qPCR analysis suggested that snail expression in the nucleus might involve in the enrichment of suppressive marker H3K9me3 on the E-cadherin promoter. Finally, we suggested that vanillin administration decreased radiation-induced oxidative stress and EMT expression. Additionally, irradiation increased the H3K9 methylation status with nuclear translocation of snail during lung EMT.

Host transcriptome-guided drug repurposing for COVID-19 treatment: a meta-analysis based approach.

BACKGROUND: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a pandemic by the World Health Organization, and the identification of effective therapeutic strategy is a need of the hour to combat SARS-CoV-2 infection. In this scenario, the drug repurposing approach is widely used for the rapid identification of potential drugs against SARS-CoV-2, considering viral and host factors. METHODS: We adopted a host transcriptome-based drug repurposing strategy utilizing the publicly available high throughput gene expression data on SARS-CoV-2 and other respiratory infection viruses. Based on the consistency in expression status of host factors in different cell types and previous evidence reported in the literature, pro-viral factors of SARS-CoV-2 identified and subject to drug repurposing analysis based on DrugBank and Connectivity Map (CMap) using the web tool, CLUE. RESULTS: The upregulated pro-viral factors such as TYMP, PTGS2, C1S, CFB, IFI44, XAF1, CXCL2, and CXCL3 were identified in early infection models of SARS-CoV-2. By further analysis of the drug-perturbed expression profiles in the connectivity map, 27 drugs that can reverse the expression of pro-viral factors were identified, and importantly, twelve of them reported to have anti-viral activity. The direct inhibition of the PTGS2 gene product can be considered as another therapeutic strategy for SARS-CoV-2 infection and could suggest six approved PTGS2 inhibitor drugs for the treatment of COVID-19. The computational study could propose candidate repurposable drugs against COVID-19, and further experimental studies are required for validation.

The epigenetics of brain tumors and its modulation during radiation: A review.

The brain tumor is the abnormal growth of heterogeneous cells around the central nervous system and spinal cord. Most clinically prominent brain tumors affecting both adult and pediatric are glioblastoma, medulloblastoma, and ependymoma and they are classified according to their origin of tissue. Chemotherapy, radiotherapy, and surgery are important treatments available to date. However, these treatments fail due to multiple reasons, including chemoresistance and radiation resistance of cancer cells. Thus, there is a need of new therapeutic designs to target cell signaling and molecular events which are responsible for this resistance. Recently epigenetic changes received increased attention because it helps in understanding chromatin-mediated disease mechanism. The epigenetic modification alters chromatin structure that affects the docking site of many drugs which cause chemo-resistance of cancer therapy. This review centers the mechanism of how epigenetic changes affect the transcription repression and activation of various genes including Polycomb gene, V-Myc avian myelocytomatosis viral oncogene (MYCN). This review also put forth the pathway of radiation-induced reactive oxygen species generation and its role in epigenetic changes in the cellular level and its impact on tissue physiology. Additionally, there is a strong relationship between the behavior of an individual and environment-induced epigenetic regulation of gene expression. The review also discusses Transcriptome heterogeneity and role of tumor microenvironment in glioblastoma. Overall, this review emphasis important and novel epigenetic targets that could be of therapeutic benefit, which helps in overcoming the unsolved chromatin alteration in brain cancer.

Radiation-induced lung injury: impact on macrophage dysregulation and lipid alteration - a review.

Lung cancer continues to be the leading cause of cancer deaths and more than one million lung cancer patients will die every year worldwide. Radiotherapy (RT) plays an important role in lung cancer treatment, but the side effects of RT are pneumonitis and pulmonary fibrosis. RT-induced lung injury causes damage to alveolar-epithelial cells and vascular endothelial cells. Macrophages play an important role in the development of pulmonary fibrosis despite its role in immune response. These injury activated macrophages develop into classically activated M1 macrophage or alternative activated M2 macrophage. It secretes cytokines, interleukins, interferons, and nitric oxide. Several pro-inflammatory lipids and pro-apoptotic proteins cause lipotoxicity such as LDL, FC, DAG, and FFA. The overall findings in this review conclude the importance of macrophages in inducing toxic/inflammatory effects during RT of lung cancer, which is clinically vital to treat the radiation-induced fibrosis.

Radiation-Induced Pulmonary Epithelial-Mesenchymal Transition: A Review on Targeting Molecular Pathways and Mediators.

BACKGROUND: Radiotherapy is the most widely used treatment method for average and advanced lung cancer patients. Moreover, the clinical toxicities caused by radiotherapy are categorized into acute radiation pneumonitis and late pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) is a complex physiological process involves many signaling molecules and proteins like adaptor proteins, and transcriptional factors. It was identified as a significant mechanism for fibrosis, wound healing and also cancer. A variety of biomarkers have appeared in radiation-induced lung EMT, some of which are acquired (N-cadherin, vimentin and fibronectin, etc.) and some of which are repressed during the transition of epithelial cells (E-cadherin, zona occludens-1). OBJECTIVE: In the current review, we highlighted the radiation-induced lung EMT signaling pathway and their mediators. We also discuss the EMT in cancer, fibrosis and its epigentics. RESULTS: Radiation-induced lung EMT is controlled by numerous signaling pathways like MAPK, NF-kB, Wnt, microRNAs and histone modifications. Transcriptional factors such as Snail, slug, twist, ZEB1 (Zinc finger E-box binding-1) and ZEB2 (Zinc finger E-box binding-2) proteins are inducers linking radiation-induced EMT and fibrosis. Epigenetic modulations are heritable changes in the structure and function of the genome that occurs without any change in the sequence. Several approaches showed the role of epigenetic modifications and its inhibitors in controlling fibrosis and cancer. Only limited reports are focused on understanding the epigenetic regulations of radiation-induced lung EMT. CONCLUSION: The current review focused on recent findings regarding radiation-induced lung fibrosis and EMT, thus provides some information on important signaling pathways, its subsequent expression of genes and proteins involved in EMT. This review also discussed various inhibitors that could be used to treat EMT related diseases, i.e., fibrosis, cancer.

Trichostatin A inhibits radiation-induced epithelial-to-mesenchymal transition in the alveolar epithelial cells.

Radiation-induced pneumonitis and fibrosis are major complications following thoracic radiotherapy. Epithelial-to-mesenchymal transition (EMT) plays an important role in tissue injury leading to organ fibrosis, including lung. Our previous studies have reported that radiation can induce EMT in the type II alveolar epithelial cells in both in vitro and in vivo. HDAC inhibitors are a new family of anti-cancer agents currently being used in several clinical trials. In addition to their intrinsic anti-tumor properties, HDAC inhibition is also important in other human diseases, including fibrosis and radiation-induced damage. In this study, we evaluated the effect of Trichostatin A (TSA), a HDAC inhibitor, on radiation-induced EMT in type II alveolar epithelial cells (RLE-6TN). Pre-treatment of RLE-6TN cells with TSA inhibited radiation-induced EMT-like morphological alterations including elevated protein level of α-SMA and Snail, reduction of E-cadherin expression, enhanced phosphorylation of GSK3ß and ERK1/2, increased generation of ROS. Radiation enhanced the protein level of TGF-ß1, which was blocked by N-acetylcysteine, an antioxidant. Treating cells with SB-431542, TGF-ß1 type I receptor inhibitor, diminished radiation-induced alterations in the protein levels of p-GSK-3ß, Snail-1 and α-SMA, suggesting a regulatory role of TGF-ß1 in EMT. Pre-incubation of cells with TSA showed significant decrease in the level of TGF-ß1 compared to radiation control. Collectively, these results demonstrate that i] radiation-induced EMT in RLE-6TN cells is mediated by ROS/MEK/ERK and ROS/TGF-ß1 signaling pathways and ii] the inhibitory role of TSA in radiation-induced EMT appears to be due, at least in part, to its action of blocking ROS and TGF-ß1 signaling.

Effect of Trichostatin A on radiation induced epithelial-mesenchymal transition in A549 cells.

Radiotherapy is used to treat tumors of different origins and nature, but often lead to development of radioresistance and metastasis of cells. Interestingly, radiation induces epithelial-mesenchymal transition (EMT), a process by which epithelial cells undergo mesenchymal phenotype and stimulates tumor progression capability. Our study investigated the effect of Trichostatin A (TSA), a natural derivate isolated from Streptomyces, upon radiation-induced lung EMT and we tried to understand the role of signaling molecules in irradiated lung cancer cells (A549). The cells were categorized into four groups: untreated control, radiation alone (R; 8Gy, X-ray), radiation combined with TSA (R + T) and TSA (100nM). Radiation-induced lung EMT were evidenced by decreased expression of epithelial marker like E-cadherin, Zona occluden1 (ZO-1) and increased expression of N-cadherin and Vimentin. The Snail protein, a master regulator of EMT, was observed to be elevated after radiation treatment. In addition, TGF-ß1 signaling (smad2, 3, and 4) proteins were activated upon irradiation. Western blot data were supported by the altered m-RNA expression of E-cadherin, TGF-ß and Snail genes and this effect were reversed by TSA treatment. In addition to this, as supportive evidence, we performed docking studies between snail protein and TSA using Auto docking software and results suggested that less binding energy was needed for the putative binding of TSA on C-terminal domain of Snail protein. Based on our report, we suggest that TSA can effectively inhibit radiation-induced EMT (i) by altering epithelial and mesenchymal markers (ii) by modulating signaling molecules of TGFß1 pathway (iii) by inhibiting cancer cell migratory potential in A549 cells (iv)by effectively binding to Snail which is an enhancer of EMT.

All-Trans Retinoic Acid supplementation prevents cardiac fibrosis and cytokines induced by Methylglyoxal.

Methylglyoxal (MG), a metabolic intermediate of glycolysis is a precursor for endogeneous production of advanced glycation end-products. The increased production of MG have negative influence over the structure and function of different biomolecules and thus plays an important role in the pathogenesis of diabetic cardiac complications. Retinoic acid (RA), an active metabolite of vitamin A, has a major role in preventing cardiac remodeling and ventricular fibrosis. Hence, the objective of the present study was to determine whether rats administered with all-trans retinoic acid (RA) could attenuate MG induced pathological effects. Wistar rats were divided into 4 groups. Group 1 rats were kept as control; Group 2 rats were administrated with MG (75 mg/kg/day) for 8 weeks. Group 3 rats were given RA (Orally, 1.0 mg/kg/day) along with MG; Group 4 rats received RA alone. Cardiac antioxidant status, induction of fibrosis, AGE receptor (RAGE) and cytokines expression was evaluated in the heart tissues. Administration of MG led to depletion of antioxidant enzymes, induction of fibrosis (p < 0.001), up-regulated expression of RAGE (3.5 fold), TGF-ß (4.4 fold), SMAD2 (3.7 fold), SMAD3 (6.0 fold), IL-6 (4.3 fold) and TNF-α (5.5 fold) in the heart tissues compared to control rats. Moreover, the exogenous administration of MG caused significant (p < 0.001) increase in the circulating CML levels. Whereas, RA treatment prevented the induction of fibrosis and restored the levels of cytokines and RAGE expression. Methylglyoxal-induced fibrosis can lead to pathological effects in the heart tissues. RA attenuates the effects of MG in the heart, suggesting that it can be of added value to usual diabetic therapy.

The role of alveolar epithelium in radiation-induced lung injury.

Pneumonitis and fibrosis are major lung complications of irradiating thoracic malignancies. In the current study, we determined the effect of thoracic irradiation on the lungs of FVB/N mice. Survival data showed a dose-dependent increase in morbidity following thoracic irradiation with single (11-13 Gy) and fractionated doses (24-36 Gy) of (137)Cs γ-rays. Histological examination showed a thickening of vessel walls, accumulation of inflammatory cells, collagen deposition, and regional fibrosis in the lungs 14 weeks after a single 12 Gy dose and a fractionated 30 Gy dose; this damage was also seen 5 months after a fractionated 24 Gy dose. After both single and fractionated doses, i] aquaporin-5 was markedly decreased, ii] E-cadherin was reduced and iii] prosurfactant Protein C (pro-SP-c), the number of pro-SP-c(+) cells and vimentin expression were increased in the lungs. Immunofluorescence analysis revealed co-localization of pro-SP-c and α-smooth muscle actin in the alveoli after a single dose of 12 Gy. These data suggest that, i] the FVB/N mouse strain is sensitive to thoracic radiation ii] aquaporin-5, E-cadherin, and pro-SP-c may serve as sensitive indicators of radiation-induced lung injury; and iii] the epithelial-to-mesenchymal transition may play an important role in the development of radiation-induced lung fibrosis.

ERK/GSK3ß/Snail signaling mediates radiation-induced alveolar epithelial-to-mesenchymal transition.

Radiotherapy is one of the major treatment regimes for thoracic malignancies, but can lead to severe lung complications including pneumonitis and fibrosis. Recent studies suggest that epithelial-to-mesenchymal transition (EMT) plays an important role in tissue injury leading to organ fibrosis. To investigate whether radiation can induce EMT in lung epithelial cells and also to understand the potential mechanism(s) associated with this change, rat alveolar type II lung epithelial RLE-6TN cells were irradiated with 8 Gy of (137)Cs γ-rays. Western blot and immunofluorescence analyses revealed a time-dependent decrease in E-cadherin with a concomitant increase in α-smooth muscle actin (α-SMA) and vimentin after radiation, suggesting that the epithelial cells acquired a mesenchymal-like morphology. Protein levels and nuclear translocation of Snail, the key inducer of EMT, were significantly elevated in the irradiated cells. Radiation also induced a time-dependent inactivation of glycogen synthase kinase-3ß (GSK3ß), an endogenous inhibitor of Snail. A marked increase in phosphorylation of ERK1/2, but not JNK or p38, was observed in irradiated RLE-6TN cells. Silencing ERK1/2 using siRNAs and the MEK/ERK inhibitor U0126 attenuated the radiation-induced phosphorylation of GSK3ß and altered the protein levels of Snail, α-SMA, and E-cadherin in RLE-6TN cells. Preincubating RLE-6TN cells with N-acetylcysteine, an antioxidant, abolished the radiation-induced phosphorylation of ERK and altered protein levels of Snail, E-cadherin, and α-SMA. These findings reveal, for the first time, that radiation-induced EMT in alveolar type II epithelial cells is mediated by the ERK/GSK3ß/Snail pathway.

Dose-response effect of ferulic acid against nicotine-induced tissue damage and altered lipid levels in experimental rats: a pathohistological evaluation.

In the present study, ferulic acid (FA), a naturally occurring nutritional compound, was investigated for its protective effect against nicotine-induced toxicity in a dose dependent manner. The toxicity was induced by subcutaneous injection of nicotine at a dose of 2.5 mg/kg body weight (b.w.) for 22 weeks in female albino Wister rats. Simultaneously, rats were treated with FA at three different doses (10, 20 and 40 mg/kg b.w.) via intragastric intubations for 22 weeks. At the end of the experiment, circulatory marker enzymes (i.e. lactate dehydrogenase and alkaline phosphatase) and tissue (lung, liver and kidney) lipid levels (i.e. cholesterol, free fatty acids, triglycerides and phospholipids) were analysed, which were significantly increased in the nicotine-treated group, whereas FA treatment positively modulated these levels. FA at a dose of 20 mg/kg b.w. was found to be more effective when compared with the other two doses. The lung, liver and kidney excised from the different groups were fixed and stained to perform histological assessments. The protective effect of FA on histological observations confirms that 20 mg/kg b.w. of FA modulates the deleterious effects of nicotine. The results suggest that FA exerts its inhibitory action against nicotine-induced tissue damage through its antioxidant and antihyperlipidemic properties.