Evaluation of the activity of cardiac glycosides on RORγ and RORγT nuclear receptors.

Cardiac glycosides, derived from plants and animals, have been recognized since ancient times. These substances hinder the function of the sodium-potassium pump within eukaryotic cells. Many reports have shown that these compounds influence the activity of nuclear receptors. Thus, we assessed the effects of various cardiac glycosides at nontoxic concentrations on RORγ and RORγT. RORγT is a crucial protein involved in the differentiation of Th17 lymphocytes. Sixteen analyzed cardiac glycosides exhibited varying toxicities in HepG2 cells, all of which demonstrated agonistic effects on RORγ, as confirmed in the RORγ-HepG2 reporter cell line. The overexpression of both the RORγ and RORγT isoforms intensified the effects of these compounds. Additionally, these glycosides induced the expression of G6PC, a gene regulated by RORγ, in HepG2 cells. Subsequently, the effects of two endogenous cardiac glycosides (marinobufagenin and ouabain) and the three most potent glycosides (bufalin, oleandrin, and telecinobufagenin) were evaluated in Th17 primary lymphocytes. All of these compounds increased the expression of the IL17A, IL17F, IFNG, and CXCL10 genes, but they exhibited varying effects on GZMB and CCL20 expression. Molecular docking analysis revealed the robust binding affinity of cardiac glycosides for the ligand binding domain of the RORγ/RORγT receptors. Thus, we demonstrated that at nontoxic concentrations, cardiac glycosides have agonistic effects on RORγ/RORγT nuclear receptors, augmenting their activity. This potential can be harnessed to modulate the phenotype of IL17-expressing cells (e.g., Th17 or Tc17 lymphocytes) in adoptive therapy for combating various types of cancer.

Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity.

RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives.

Chlorpromazine, a Clinically Approved Drug, Inhibits SARS-CoV-2 Nucleocapsid-Mediated Induction of IL-6 in Human Monocytes.

The COVID-19 pandemic, caused by the rapidly spreading SARS-CoV-2 virus, led to the unprecedented mobilization of scientists, resulting in the rapid development of vaccines and potential pharmaceuticals. Although COVID-19 symptoms are moderately severe in most people, in some cases the disease can result in pneumonia and acute respiratory failure as well as can be fatal. The severe course of COVID-19 is associated with a hyperinflammatory state called a cytokine storm. One of the key cytokines creating a proinflammatory environment is IL-6, which is secreted mainly by monocytes and macrophages. Therefore, this cytokine has become a target for some therapies that inhibit its biological action; however, these therapies are expensive, and their availability is limited in poorer countries. Thus, new cheaper drugs that can overcome the severe infections of COVID-19 are needed. Here, we show that chlorpromazine inhibits the expression and secretion of IL-6 by monocytes activated by SARS-CoV-2 virus nucleocapsid protein and affects the activity of NF-κB and MEK/ERK signaling. Our results, including others, indicate that chlorpromazine, which has been used for several decades as a neuroleptic, exerts antiviral and immunomodulatory activity, is safe and inexpensive, and might be a desirable drug to support the therapy of patients with COVID-19.

Targeting SIRT2 Sensitizes Melanoma Cells to Cisplatin via an EGFR-Dependent Mechanism.

Melanoma cells are resistant to most anticancer chemotherapeutics. Despite poor response rates and short-term efficacy, chemotherapy remains the main approach to treating this cancer. The underlying mechanisms of the intrinsic chemoresistance of melanoma remain unclear, but elucidating these mechanisms is important to improve the efficacy of chemotherapy regimens. Increasing evidence suggests that sirtuin 2 (SIRT2) plays a key role in the response of melanoma cells to chemotherapeutics; thus, in the present study, we evaluated the impact of shRNA-mediated and pharmacological inhibition of SIRT2 on the sensitivity of melanoma cells to cisplatin, which is used in several regimens to treat melanoma patients. We found that cells with SIRT2 inhibition revealed increased sensitivity to cisplatin and exhibited increased accumulation of γ-H2AX and reduced EGFR-AKT-RAF-ERK1/2 (epidermal growth factor receptor-protein B kinase-RAF kinase-extracellular signal-regulated kinase 1/2) pathway signaling compared to control cells. Thus, our results show that sirtuin 2 inhibition increased the in vitro efficacy of cisplatin against melanoma cells.

The Dichotomous Nature of AZ5104 (an EGFR Inhibitor) Towards RORγ and RORγT.

The RORC (RAR related orphan receptor C) gene produces two isoforms by alternative promoter usage: RORγ (nuclear receptor ROR-gamma isoform 1) and RORγT (nuclear receptor ROR-gamma isoform 1). Both proteins have distinct tissue distributions and are involved in several physiological processes, including glucose/lipid metabolism and the development of Th17 lymphocytes. Previously, we developed a stably transfected reporter cell line and used it to screen a library of kinase inhibitors. We found that AZ5104 acts as an RORγ agonist at low micromolar concentrations. Molecular docking analysis showed that this compound occupies the ligand binding domain of the receptor with a significant docking score. However, analysis of the biological activity of this compound in Th17 cells revealed that it downregulates RORγT expression and Th17-related cytokine production via inhibition of SRC-ERK-STAT3 (SRC proto-oncogene - extracellular regulated MAP kinase - signal transducer and activator of transcription 3). We thus identified a compound acting as an agonist of RORγ that, due to the inhibition of downstream elements of EGFR (epidermal growth factor receptor) signaling, exerts different biological activity towards a Th17-specific isoform. Additionally, our results may be relevant in the future for the design of treatments targeting signaling pathways that inhibit Th17-related inflammation in certain autoimmune disorders.

Upstream stimulating factors regulate the expression of RORγT in human lymphocytes.

Retinoic acid-related orphan receptor γT (RORγT) is the orphan nuclear receptor that regulates the development of Th17 cells and the expression of IL-17. The differentiation of Th17 cells is associated with the upregulation of RORγT mRNA, and the mechanisms regulating that process in human cells are not well understood. We investigated the transcriptional regulation of RORγT in a human lymphocytic cell line and Th17 differentiated from naive CD4+ cells from human peripheral blood. A series of experiments, including 5' deletion and in situ mutagenesis analysis of the human RORγT promoter, chromatin immunoprecipitation, and overexpression of selected transcription factors, revealed that the transcription factors upstream stimulatory factor 1 (USF-1) and USF-2 are indispensable for the transcription of RORγT in human lymphocytes. There was also upregulation of USF-1 and USF-2 during the differentiation of Th17 cells from naive CD4+ cells. In this article, we report the first analysis, to our knowledge, of the human RORγT promoter and demonstrate the role of the USF-1 and USF-2 transcription factors in regulating the expression of RORγT in human lymphocytes. Thus, USFs are important for the molecular mechanisms of Th17 differentiation, and possible changes in the expression of USFs might be of interest for inflammatory conditions with a Th17 component. Furthermore, these observations suggest a possible link between metabolic disorders in which the role of glucose-induced USF expression has already been established and autoimmune diseases in which the upregulation of RORγT is frequently detected.

Epigenetic regulation of CD34 and HIF1A expression during the differentiation of human mast cells.

Mast cells differentiate from circulating pluripotent hematopoietic progenitors. During this differentiation, the progenitor cells are exposed to changes in oxygen availability. HIF1A is the major sensor of oxygen concentration in mammalian cells. We investigated the expression of HIF1A during the in vitro differentiation of peripheral blood-derived progenitors into human mast cells. In a series of experiments, we determined the changes in CD34 expression, selected mast cell markers, and HIF1A in human mast cell cultures. While the expression of CD34 dramatically decreased, the expression of mast cell-specific genes, including FCER1A, MS4A2, TPSB2, and CMA1, steadily increased. HIF1A expression similarly increased during mast cell differentiation, reaching its maximum level at five weeks of culture. The analysis of the promoter methylation status showed decreasing levels of methylation at the HIF1A promoter, increasing levels of methylation at the CD34 promoter, and no significant changes in other genes. In silico analysis of the promoter regions of these genes revealed large CpG islands in close proximity to the HIF1A and CD34 transcription initiation sites, but not in other investigated genes. In conclusion, in vitro mast cell differentiation was associated with decreased CD34 expression and increased HIF1A expression. These changes were paralleled with changes in the methylation status of the respective promoters, suggesting that DNA methylation-dependent epigenetic regulation mediates the gene expression changes involved in maintaining the phenotype of hematopoietic stem cells and mature mast cells. Therefore, the baseline expression of HIF1A is epigenetically regulated in a cell type- and differentiation stage-specific fashion.

Anti-hepatocellular carcinoma activity of the cyclin-dependent kinase inhibitor AT7519.

Hepatocellular carcinoma (HCC) is one of the most common cancerous tumors and one of the leading causes of death among cancer-related disorders. Chemotherapy is ineffective in HCC patients, and the number of drugs that are in use is limited. Thus, new molecules are needed that could increase the effectiveness of anti-HCC regimens. Here, we show that AT7519, a CDK inhibitor, exerts positive effects on HCC cells: it inhibits proliferation, migration and clonogenicity. Detailed analysis of the transcriptomes of cells treated with this compound indicated that AT7519 affects a substantial portion of genes that are associated with HCC development and progression. Moreover, we showed that the concomitant use of AT7519 with gefitinib or cabozantinib sensitized HCC cells to these drugs. Thus, our research indicates that AT7519 is worth considering in monotherapy for hepatocellular carcinoma patients or in combination with other drugs, e.g., gefitinib or cabozantinib.

Nucleocapsid and Spike Proteins of the Coronavirus SARS-CoV-2 Induce IL6 in Monocytes and Macrophages-Potential Implications for Cytokine Storm Syndrome.

The pandemic of the new coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has led to the deaths of more than 1.5 million people worldwide. SARS-CoV-2 causes COVID-19, which exhibits wide variation in the course of disease in different people, ranging from asymptomatic and mild courses to very severe courses that can result in respiratory failure and death. Despite the rapid progression of knowledge, we still do not know how individual cells of the immune system interact with the virus or its components, or how immune homeostasis becomes disrupted, leading to the rapid deterioration of a patient's condition. In the present work, we show that SARS-CoV-2 proteins induce the expression and secretion of IL-6 by human monocytes and macrophages, the first line cells of antiviral immune responses. IL-6 may play a negative role in the course of COVID-19 by inhibiting Th1-dependent immunity and stimulating Th17 lymphocytes, thus leading to an increased probability of a cytokine storm.

SARS-CoV-2 Proteins Induce IFNG in Th1 Lymphocytes Generated from CD4+ Cells from Healthy, Unexposed Polish Donors.

The outbreak of the SARS-CoV-2 virus in December 2019 has caused the deaths of several hundred thousand people worldwide. Currently, the pathogenesis of COVID-19 is poorly understood. During the course of COVID-19 infection, many patients experience deterioration, which might be associated with systemic inflammation and cytokine storm syndrome; however, other patients have mild symptoms or are asymptomatic. There are some suggestions that impaired cellular immunity through a reduction in Th1 response and IFNG (interferon gamma) expression, as well as cross-reactivity with common cold coronaviruses, might be involved in the differential COVID-19 course. Here, we show that CD4+ cells isolated from unexposed healthy donors that were differentiated towards the Th1 lineage in the presence of SARS-CoV-2 proteins exhibited induction of IFNG. Interestingly, the same cells induced to differentiate towards a Th17 lineage did not exhibit changes in IFNG expression or Th17-related cytokines. This suggests the cellular response to SARS-CoV-2 viral proteins is primarily associated with Th1 lymphocytes and may be dependent on past infections with common cold coronaviruses or vaccinations that induce unspecific cellular responses, e.g., BCG (Bacillus Calmette-Guérin). Thus, our results might explain the high variability in the course of COVID-19 among populations of different countries.

Cardiac glycosides with target at direct and indirect interactions with nuclear receptors.

Cardiac glycosides are compounds isolated from plants and animals and have been known since ancient times. These compounds inhibit the activity of the sodium potassium pump in eukaryotic cells. Cardiac glycosides were used as drugs in heart ailments to increase myocardial contraction force and, at the same time, to lower frequency of this contraction. An increasing number of studies have indicated that the biological effects of these compounds are not limited to inhibition of sodium-potassium pump activity. Furthermore, an increasing number of data have shown that they are synthesized in tissues of mammals, where they may act as a new class of steroid hormones or other hormones by mimicry to modulate various signaling pathways and influence whole organisms. Thus, we discuss the interactions of cardiac glycosides with the nuclear receptor superfamily of transcription factors activated by low-weight molecular ligands (including hormones) that regulate many functions of cells and organisms. Cardiac glycosides of endogenous and exogenous origin by interacting with nuclear receptors can affect the processes regulated by these transcription factors, including hormonal management, immune system, body defense, and carcinogenesis. They can also be treated as initial structures for combinatorial chemistry to produce new compounds (including drugs) with the desired properties.

Identification of Novel Molecular Markers of Human Th17 Cells.

Th17 cells are important players in host defense against pathogens such as Staphylococcus aureus, Candida albicans, and Bacillus anthracis. Th17 cell-mediated inflammation, under certain conditions in which balance in the immune system is disrupted, is the underlying pathogenic mechanism of certain autoimmune disorders, e.g., rheumatoid arthritis, Graves' disease, multiple sclerosis, and psoriasis. In the present study, using transcriptomic profiling, we selected genes and analyzed the expression of these genes to find potential novel markers of Th17 lymphocytes. We found that APOD (apolipoprotein D); C1QL1 (complement component 1, Q subcomponent-like protein 1); and CTSL (cathepsin L) are expressed at significantly higher mRNA and protein levels in Th17 cells than in the Th1, Th2, and Treg subtypes. Interestingly, these genes and the proteins they encode are well associated with the function of Th17 cells, as these cells produce inflammation, which is linked with atherosclerosis and angiogenesis. Furthermore, we found that high expression of these genes in Th17 cells is associated with the acetylation of H2BK12 within their promoters. Thus, our results provide new information regarding this cell type. Based on these results, we also hope to better identify pathological conditions of clinical significance caused by Th17 cells.

SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib.

Malignant melanoma is the most aggressive skin cancer and can only be cured if detected early. Unfortunately, later stages of the disease do not guarantee success due to the rapid rate of melanoma cell metastasis and their high resistance to applied therapies. The search for new molecular targets and targeted therapy may represent the future in the development of effective methods for combating this cancer. SIRT2 is a promising target; thus, we downregulated SIRT2 expression in melanoma cells in vertical growth and metastatic phases and demonstrated that sirtuin acts as regulator of the basic functions of melanoma cells. A detailed transcriptomic analysis showed that SIRT2 regulates the expression of multiple genes encoding the tyrosine kinase pathways that are molecular targets of dasatinib. Indeed, cells with low SIRT2 expression were more susceptible to dasatinib, as demonstrated by multiple techniques, e.g., neutral red uptake, 3/7 caspase activity, colony formation assay, and in vitro scratch assay. Furthermore, these cells showed an altered phosphorylation profile for proteins playing roles in the response to dasatinib. Thus, our research indicates new, previously unknown SIRT2 functions in the regulation of gene expression, which is of key clinical significance.

The cardenolides strophanthidin, digoxigenin and dihydroouabain act as activators of the human RORγ/RORγT receptors.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential.

Digoxin, an Overlooked Agonist of RORγ/RORγT.

Digoxin was one of the first identified RORγT receptor inverse agonists inhibiting the differentiation of Th17 cells. However, this compound exhibits inhibitory activity at relatively high concentrations that mediate cytotoxic effects. We previously identified several cardenolides that are structurally similar to digoxin that were able to induce RORγ/RORγT-dependent transcription. These observations encouraged us to reanalyze the effects of digoxin on RORγ/RORγT-dependent transcription at low, noncytotoxic concentrations. Digoxin induced RORγ/RORγT-dependent transcription in HepG2 and Th17 cells. Furthermore, analysis of the transcriptomes of Th17 cells cultured in the presence of digoxin revealed the induction of the expression of numerous Th17-specific genes, including IL17A/F, IL21, IL22, IL23R, CCR4, and CCR6. Thus, our study, which includes data obtained from intact cells, indicates that digoxin, similar to other cardenolides, is a potent RORγ/RORγT receptor activator and that its structure may serve as a starting point for the design of dedicated molecules that can be used in the development of adoptive cell therapy (ACT).

Differentiation stage-specific effect of histone deacetylase inhibitors on the expression of RORγT in human lymphocytes.

The role of epigenetic mechanisms in the regulation of the human RORγT gene, which encodes a Th17 lymphocyte signature transcription factor, remains largely unknown. We investigated the effect of histone deacetylase (HDAC) inhibition on RORγT and RORγT-dependent gene expression in human T lymphocytes. We found that, in Jurkat T cells and in in vitro-differentiated Th17 cells, treatment with 2 HDAC inhibitors, butyrate and apicidin, led to the induction of the RORγT gene, which was associated with an increase in histone H4 acetylation near the RORγT proximal promoter. In contrast, when the same inhibitors were added to naive CD4+ cells differentiating in vitro to Th17 cells, they mediated the down-regulation of RORγT expression. In conclusion, HDAC inhibitor-mediated H4 acetylation is involved in the epigenetic regulation of RORγT expression in Th17 cells. However, that epigenetic mechanism was observed only at a specific stage of T cell differentiation, suggesting a complex interaction with additional mechanisms that sequentially regulate RORγT expression. These observations may be relevant to the development of applications for HDAC inhibitors for diseases in which Th17 cells have a role in pathogenic mechanisms, such as some types of cancer or autoimmunologic disorders, to prevent unwanted side effects.

Functional Analysis of the rs774872314, rs116171003, rs200231898 and rs201107751 Polymorphisms in the Human RORγT Gene Promoter Region.

RAR-related orphan receptor gamma RORγT, a tissue-specific isoform of the RORC gene, plays a critical role in the development of naive CD4+ cells into fully differentiated Th17 lymphocytes. Th17 lymphocytes are part of the host defense against numerous pathogens and are also involved in the pathogenesis of inflammatory diseases, including autoimmune disorders. In this study, we functionally examined four naturally occurring polymorphisms located within one of the previously identified GC-boxes in the promoter region of the gene. The single nucleotide polymorphisms (SNPs) rs774872314, rs116171003 and rs201107751 negatively influenced the activity of the RORγT promoter in a gene reporter system and eliminated or reduced Sp1 and Sp2 transcription factor binding, as evidenced by the electrophoretic mobility shift assay (EMSA) technique. Furthermore, we investigated the frequency of these SNPs in the Polish population and observed the presence of rs116171003 at a frequency of 3.42%. Thus, our results suggest that polymorphisms within the RORγT promoter occurring at significant rates in populations affect promoter activity. This might have phenotypic effects in immune systems, which is potentially significant for implicating pathogenetic mechanisms under certain pathological conditions, such as autoimmune diseases and/or primary immunodeficiencies (e.g., immunoglobulin E (IgE) syndrome).

Expression of human gene coding RORγT receptor depends on the Sp2 transcription factor.

Th17 cells are involved in the immune response against pathogens, autoimmunity, and tumor progression. The differentiation of human Th17 cells requires the upregulation of RORγT, which in human cells is still not well understood. We identified 2 putative binding motifs for specificity protein transcription factors from the specificity protein/Kruppel-like factor family in the promoter of human RORγT and investigated the involvement of specificity proteins in the transcriptional regulation of this gene. To this end, a human lymphocytic cell line and in vitro-differentiated Th17 cells were used in promoter activity assays, in situ mutagenesis, chromatin immunoprecipitation, and real-time RT-PCR assays. In some experiments, specificity protein expression and activity was inhibited by siRNA and mithramycin A. The results showed that the transcription factor specificity protein 2 recognized binding motifs in the human RORγT promoter, which was critical for maintaining expression. Furthermore, specificity protein 2 was necessary for maximum IL-17 expression in in vitro-differentiated Th17 cells. These observations demonstrate the significant role of specificity protein 2 in the regulation of the Th17 signature transcription factor RORγT and the maintenance of the Th17 phenotype. The findings also suggest that specificity protein 2 plays a role in Th17-dependent physiologic and pathologic immune responses and might serve as a potential novel target for their modulation.

Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus.

Exotoxin A (PE) from Pseudomonas aeruginosa is a bacterial ADP-ribosyltransferase, which can permanently inhibit translation in the attacked cells. Consequently, this toxin is frequently used in immunotoxins for targeted cancer therapies. In this study, we propose a novel modification to PE by incorporating the NLS sequence at its C-terminus, to make it a selective agent against fast-proliferating cancer cells, as a nucleus-accumulated toxin should be separated from its natural substrate (eEF2) in slowly dividing cells. Here, we report the cytotoxic activity and selected biochemical properties of newly designed PE mutein using two cellular models: A549 and HepG2. We also present a newly developed protocol for efficient purification of recombinant PE and its muteins with very high purity and activity. We found that furin cleavage is not critical for the activity of PE in the analyzed cell lines. Surprisingly, we observed increased toxicity of the toxin accumulated in the nucleus. This might be explained by unexpected nuclease activity of PE and its potential ability to cleave chromosomal DNA, which seems to be a putative alternative intoxication mechanism. Further experimental investigations should address this newly detected activity to identify catalytic residues and elucidate the molecular mechanism responsible for this action.

Aflatoxins upregulate CYP3A4 mRNA expression in a process that involves the PXR transcription factor.

Pregnane X receptor (PXR) is a member of the nuclear hormone receptor (NHR) superfamily, which regulates xenobiotic and endobiotic metabolism in the liver. This transcription factor is activated by structurally diverse ligands, including drugs and environmental pollutants. PXR regulates the expression of numerous genes that function in biotransformation and the disposition of xenobiotics upon binding to an AG(G/T)TCA DNA motif in target promoter regions. We performed a screen of mycotoxins that pose a known environmental threat to human and animal health for the ability to activate PXR function in a human hepatocyte cell line, HepG2. We found that aflatoxins B1, M1, and G1 activated PXR. This activation was associated with upregulation of CYP3A4 expression and increased occupancy of PXR protein on the CYP3A4 promoter. Using a microarray approach, we also found that aflatoxin B1 upregulated the expression of multiple genes involved in xenobiotic metabolism, including genes known to be regulated in a PXR-dependent fashion. We also observed an effect of aflatoxin B1 on the expression in other functional groups of genes, including the downregulation of genes involved in cholesterologenesis. The results of this study indicate that aflatoxin B1 is able to activate PXR, a known regulator of liver xenobiotic metabolism, in human hepatocytes, and it can upregulate the expression of PXR-dependent genes responsible for aflatoxin B1 biotransformation, including CYP3A4.