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Rhabdomyosarcoma (RMS) is a malignant soft tissue cancer that develops mostly in children and young adults. With regard to histopathology, four rhabdomyosarcoma types are distinguishable: embryonal, alveolar, pleomorphic and spindle/sclerosing. Currently, increased amounts of evidence indicate that not only gene mutations, but also epigenetic modifications may be involved in the development of RMS. Epigenomic changes regulate the chromatin architecture and affect the interaction between DNA strands, histones and chromatin binding proteins, thus, are able to control gene expression. The main aim of the study was to assess the role of protein arginine methyltransferases (PRMT) in the cellular biology of rhabdomyosarcoma. In the study we used two pan-inhibitors of PRMT, called AMI-1 and SAH, and evaluated their effects on proliferation and apoptosis of RMS cells. We observed that AMI-1 and SAH reduce the invasive phenotype of rhabdomyosarcoma cells by decreasing their proliferation rate, cell viability and ability to form cell colonies. In addition, microarray analysis revealed that these inhibitors attenuate the activity of the PI3K-Akt signaling pathway and affect expression of genes related to it.

As a therapeutic approach, epigenetic modifiers have the potential to enhance the efficacy of chemotherapeutic agents. Protein arginine methyltransferase 5 (PRMT5), highly expressed in lung adenocarcinoma, was identified to be involved in tumorigenesis. In the current study, we examined the potential antineoplastic activity of PRMT5 inhibitor, arginine methyltransferase inhibitor 1 (AMI-1), and cisplatin on lung adenocarcinoma. Bioinformatic analyses identified apoptosis, DNA damage, and cell cycle progression as the main PRMT5-associated functional pathways, and survival analysis linked the increased PRMT5 gene expression to worse overall survival in lung adenocarcinoma. Combined AMI-1 and cisplatin treatment significantly reduced cell viability and induced apoptosis. Cell cycle arrest in A549 and DMS 53 cells was evident after AMI-1, and was reinforced after combination treatment. Western blot analysis showed a reduction in demethylation histone 4, a PRMT5- downstream target, after treatment with AMI-1 alone or in combination with cisplatin. While the combination approach tackled lung cancer cell survival, it exhibited cytoprotective abilities on HBEpC (normal epithelial cells). The survival of normal bronchial epithelial cells was not affected by using AMI-1. This study highlights evidence of novel selective antitumor activity of AMI-1 in combination with cisplatin in lung adenocarcinoma cells.

Protein arginine methyltransferase 1 (PRMT1), which primarily causes asymmetric arginine methylation of histone and nonhistone proteins, has been found to activate gene expression and mediate multiple pathological processes. Its role in renal fibrosis, however, remains unclear. In the present study, we observed that PRMT1 and its specific epigenetic marker, asymmetric di-methylated histone 4 arginine 3 (H4R3Me2a), were highly expressed in cultured renal interstitial fibroblasts. Treatment of PRMT1 with AMI-1, a selective inhibitor of PRMT1, or silencing PRMT1 with siRNA inhibited serum-induced and transforming growth factor (TGF)-β 1 -induced expression of α-smooth muscle actin (α-SMA) and collagen type I, two hallmarks of renal fibroblast activation, in a dose-dependent and time-dependent manner. In a murine model of renal fibrosis induced by unilateral ureteral obstruction, PRMT1 expression and H4R3Me2a were also upregulated, which was coincident with increased expression of α-SMA, collagen type I, and fibronectin. Administration of AMI-1 reduced PRMT1 and H4R3Me2a expression, attenuated extracellular matrix protein deposition, and inhibited renal fibroblast activation and proliferation. Moreover, AMI-1 treatment inhibited Smad3 phosphorylation and TGF-β receptor I expression but prevented Smad7 downregulation both in the kidney after unilateral ureteral obstruction injury and in cultured renal interstitial fibroblasts exposed to TGF-β 1 . Collectively, these results demonstrate that PRMT1 may mediate renal fibroblast activation and renal fibrosis development through activation of the TGF-β/Smad3 signaling pathway. They also suggest that PRMT1 inhibition may be a potential therapeutic approach for the treatment of fibrotic kidney disease.

Tissue remodeling of subepithelial mesenchymal cells is a major pathologic condition of chronic obstructive pulmonary disease and asthma. Fibroblasts contribute to fibrotic events and inflammation in both airway diseases. Recent mechanistic studies established a link between mitochondrial dysfunction or aberrant biogenesis leading to tissue remodeling of the airway wall in asthma. Protein arginine methyltransferase-1 (PRMT1) participated in airway wall remodeling in pulmonary inflammation. This study investigated the mechanism by which PRMT1 regulates mitochondrial mass in primary human airway wall fibroblasts. Fibroblasts from control or asthma patients were stimulated with TGF-β for up to 48 h, and the signaling pathways controlling PRMT1 expression and mitochondrial mass were analyzed. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI-1. The SMAD2/3 pathway was blocked by SB203580 and C/EBPβ by small interference RNA treatment. The data obtained from unstimulated cells showed a significantly higher basal expression of PRMT1 and mitochondrial markers in asthmatic compared with control fibroblasts. In all cells, TGF-β significantly increased the expression of PRMT1 through SMAD2/3 and C/EBPβ. Subsequently, PRMT1 upregulated the expression of the mitochondria regulators PGC-1α and heat shock protein 60. Both the inhibition of the SAMD2/3 pathway or PRMT1 attenuated TGF-β-induced mitochondrial mass and C/EBPβ and α-SMA expression. These findings suggest that the signaling sequence controlling mitochondria in primary human lung fibroblasts is as follows: TGF-β→SMAD2/3→C/EBPβ→PRMT1→PGC-1α. Therefore, PRMT1 and C/EBPβ present a novel therapeutic and diagnostic target for airway wall remodeling in chronic lung diseases., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Protein arginine methyltransferase 5 (PRMT5), a type II PRMT, is highly expressed in several types of tumors including cervical cancer. Arginine methyltransferase inhibitor 1 (AMI-1) inhibits solid tumors by targeting PRMT5. However, the effect of AMI-1 on cervical cancer is still unknown. In this study, we provided the first evidence that AMI-1 reduced cervical cancer cell proliferation, colony formation and promoted cell apoptosis in vitro. Suppression of tumorigenicity was also confirmed in vivo. Mechanistic studies revealed that AMI-1 significantly reduced PRMT5 level in cells and mice xenografts model of cervical cancer. These results suggest that AMI-1 inhibits cervical cancer by type II PRMT5.

Posttranslational modifications of histone tails can alter chromatin structure and regulate gene transcription. While recent studies implicate the lysine/arginine protein methyltransferases in the regulation of genes for endothelial metabolism, the role of AMI-1 and AMI-5 compounds in angiogenesis remains unknown. Here, we show that global inhibition of arginine and lysine histone methyltransferases (HMTs) by AMI-5 induced an angiostatic profile in human microvascular endothelial cells and human umbilical vein endothelial cells. Based on FACS analysis, we found that inhibition of HMTs significantly affects proliferation of endothelial cells, by suppressing cell cycle progression in the G 0 /G 1 phase. Immunofluorescent studies of the endothelial cells replication pattern by 5-ethynyl-2'-deoxyuridine incorporation disclosed that AMI-5, and the arginine methyltransferase inhibitor AMI-1, induced heterochromatin formation and a number of nuclear abnormalities, such as formation of micronuclei (MNs) and nucleoplasmic bridges (NPBs), which are markers of chromosomal instability. In addition to the modification of the cell cycle machinery in response to AMIs treatment, also endothelial cells migration and capillary-like tube formation processes were significantly inhibited, implicating a stimulatory role of HMTs in angiogenesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Protein arginine methyltransferase 5 (PRMT5), a type II PRMT, is highly expressed in some tumors, but its role in hepatocellular carcinoma (HCC) is still unknown., Methods: PRMT5 level in HCC specimens was determined by immunohistochemical staining and the association with clinicopathologic features was evaluated. PRMT5 was inhibited by AMI-1 (a small molecule inhibitor of PRMTs) or small interference RNA (siRNA). The proliferation of HCC cells was tested by Cell Counting Kit-8, cell migration was evaluated by Transwell assay and cell cycle and apoptosis were analyzed by flow cytometry. The effect of AMI-1 on HCC in vivo was examined by mouse xenograft model., Results: PRMT5 expression was markedly upregulated in HCC tissues, and correlated inversely with overall patient survival. Knockdown of PRMT5 significantly reduced the proliferation of HCC cells, but did not affect the growth of normal liver cells. Furthermore, β-catenin was identified as a target of PRMT5. Silencing PRMT5 significantly down-regulated the expression of β-catenin and the downstream effector Cyclin D1 in HCC cells. AMI-1 strongly inhibited HCC growth in vivo, increased the ratio of Bax/Bcl-2, and led to apoptosis and loss of migratory activity in several HCC cells. Meanwhile, AMI-1 decreased the expression levels of symmetric dimethylation of H4 (H4R3me2s), a histone mark of PRMT5., Conclusions: PRMT5 plays an important role in HCC. PRMT5 may be a promising target for HCC therapy.

Protein arginine methyltransferases (PRMTs) plays critical roles in cancer. PRMT5 has been implicated in several types of tumors. However, the role of PRMT5 in cancer development remains to be fully elucidated. Here, we provide evidence that PRMT5 is overexpressed in colorectal cancer (CRC) cells and patient-derived primary tumors, correlated with increased cell growth and decreased overall patient survival. Arginine methyltransferase inhibitor 1 (AMI-1)strongly inhibited tumor growth, increased the ratio of Bax/Bcl-2, and induced apoptosis in mouse CRC xenograt model. AMI-1 also induced apoptosis and decreased the migratory activity in several CRC cells. In CRC xenografts AMI-1 significantly decreased symmetric dimethylation of histone 4 (H4R3me2s), a histone mark of type II PRMT5, but not the expression of H4R3me2a, a histone mark of type I PRMTs. These results suggest that the inhibition of PRMT5 contributes to the antitumor efficacy of AMI-1. Chromatin immunoprecipitation (ChIP) identified FGFR3 and eIF4E as two key genes regulated by PRMT5. PRMT5 knockdown reduced the levels of H4R3me2s and H3R8me2s methylation on FGFR3 and eIF4E promoters, leading to decreased expressions of FGFR3 and eIF4E. Collectively, our findings provide new evidence that PRMT5 plays an important role in CRC pathogenesis through epigenetically regulating arginine methylation of oncogenes such as eIF4E and FGFR3.

Protein arginine methyltransferase (PRMT)1, methylating both histones and key cellular proteins, has emerged as a key regulator of various cellular processes. This study aimed to identify the mechanism that regulates PRMT1 in chronic Ag-induced pulmonary inflammation (AIPI) in the E3 rat asthma model. E3 rats were challenged with OVA for 1 or 8 wk to induce acute or chronic AIPI. Expression of mRNAs was detected by real-time quantitative PCR. PRMT1, TGF-β, COX2, and vascular endothelial growth factor protein expression in lung tissues was determined by immunohistochemistry staining and Western blotting. In the in vitro study, IL-4-stimulated lung epithelial cell (A549) medium (ISEM) with or without anti-TGF-β Ab was applied to human fibroblasts from lung (HFL1). The proliferation of HFL1 was determined by MTT. AMI-1 (pan-PRMT inhibitor) was administered intranasally to chronic AIPI rats to determine PRMT effects on asthmatic parameters. In lung tissue sections, PRMT1 expression was significantly upregulated, mainly in epithelial cells, in acute AIPI lungs, whereas it was significantly upregulated mainly in fibroblasts in chronic AIPI lungs. The in vitro study revealed that ISEM elevates PRMT1, COX2, and vascular endothelial growth factor expressions, and it promoted fibroblast proliferation. The application of anti-TGF-β Ab suppressed COX2 upregulation by ISEM. AMI-1 inhibited the expression of COX2 in TGF-β-stimulated cells. In the in vivo experiment, AMI-1 administered to AIPI rats reduced COX2 production and humoral immune response, and it abrogated mucus secretion and collagen generation. These findings suggested that TGF-β-induced PRMT1 expression participates in fibroblast proliferation and chronic airway inflammation in AIPI., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The enzymatic activities of protein methyltransferases serve to write covalent modifications on histone and non-histone proteins in the control of gene transcription. Here, we describe gene expression changes in human endothelial cells caused by treatment with methyltransferase inhibitors 7,7'-carbonylbis (azanediyl) bis(4-hydroxynaphthalene-2 -sulfonic acid (AMI-1) and disodium-2-(2,4,5,7- tetrabromo-3-oxido-6-oxoxanthen-9-yl) benzoate trihydrate (AMI-5). Deep sequencing of mRNA indicated robust change on transcription following AMI-5 treatment compared with AMI-1. Functional annotation analysis revealed that both compounds suppress the expression of genes associated with translational regulation, suggesting arginine methylation by protein arginine methyltransferases (PRMTs) could be associated with regulation of this pathway. Interestingly, AMI-5 but not AMI-1 was found to decrease methylation of H3 histones at lysine 4 and down-regulate gene expression associated with interleukin-6 (IL-6) and activator protein-1 (AP-1) signaling pathways. These results imply that inhibition of protein methylation by AMI-1 and AMI-5 can differentially regulate specific pathways with potential to interrupt pathological signaling in the vascular endothelium., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein arginine methyltransferases (PRMTs), catalyzing methylation of both histones and other cellular proteins, have emerged as key regulators of various cellular processes. This study aimed to identify key PRMTs involved in Ag-induced pulmonary inflammation (AIPI), a rat model for asthma, and to explore the role of PRMT1 in the IL-4-induced eosinophil infiltration process. E3 rats were i.p. sensitized with OVA/alum and intranasally challenged with OVA to induce AIPI. The expressions of PRMT1-6, eotaxin-1, and CCR3 in lungs were screened by real-time quantitative PCR. Arginine methyltransferase inhibitor 1 (AMI-1, a pan-PRMT inhibitor) and small interfering RNA-PRMT1 were used to interrupt the function of PRMT1 in A549 cells. In addition, AMI-1 was administrated intranasally to AIPI rats to observe the effects on inflammatory parameters. The results showed that PRMT1 expression was mainly expressed in bronchus and alveolus epithelium and significantly upregulated in lungs from AIPI rats. The inhibition of PRMTs by AMI-1 and the knockdown of PRMT1 expression were able to downregulate the expressions of eotaxin-1 and CCR3 with the IL-4 stimulation in the epithelial cells. Furthermore, AMI-1 administration to AIPI rats can also ameliorate pulmonary inflammation, reduce IL-4 production and humoral immune response, and abrogate eosinophil infiltration into the lungs. In summary, PRMT1 expression is upregulated in AIPI rat lungs and can be stimulated by IL-4. Intervention of PRMT1 activity can abrogate IL-4-dependent eotaxin-1 production to influence the pulmonary inflammation with eosinophil infiltration. The findings may provide experimental evidence that PRMT1 plays an important role in asthma pathogenesis.

The methylation of proteins is an important post-translational mechanism that has been established to influence the activity of nuclear and nucleic acid binding proteins. Much less is known about the importance of protein methylation in the regulation of cytosolic proteins. Increased methylation of proteins is observed in cardiovascular disease and occurs in conjunction with elevated production of reactive oxygen species. However, the nature of the relationship between reactive oxygen species and protein methylation is poorly understood. Therefore, the goal of the current study was to determine whether protein methylation influences the catalytic activity of the NADPH oxidases (Nox), which are a family of enzymes responsible for the generation of superoxide. We found that the selective inhibitor of protein arginine methyltransferases 7,7'-carbonylbis(azanediyl)bis(4-hydroxynaphthalene-2-sulfonic acid (AMI-1) was a potent antagonist of Nox-derived superoxide production. However, structurally and mechanistically dissimilar inhibitors of protein methylation and coexpression of protein arginine methyltransferase 1 did not influence Nox activity. Rather, the effect of AMI-1 was rapidly reversible and could be demonstrated in an assay using chemically synthesized superoxide. We conclude that protein methylation does not regulate the activity of NADPH-oxidases and that AMI-1 is a potent antioxidant with a greater potency than 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) and 4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl (Tempol).

Protein arginine methyltransferases (PRMTs) play important roles in both normal physiology and human diseases. Deregulation of PRMT activity has been linked to several pathological states such as cancer and cardiovascular disorders. Herein, we report our work of designing and using new fluorescent reporters to perform single-step analysis of substrate binding and methylation by PRMT1. Both fluorescence intensity and anisotropy of the two reporters, R4-FL and H4-FL, were shown to effectively manifest enzyme-substrate interactions, highlighting their application in investigating PRMT inhibitors. In particular, the methylation process of R4-FL can be directly studied using fluorescence intensity readout. By combining the fluorescent measurement with radioactive analysis, we determined that AMI-1 inhibits PRMT1 activity through the mechanism of blocking peptide substrate binding.