Transcriptome Profiling of Cardiac Glycoside Treatment Reveals EGR1 and Downstream Proteins of MAPK/ERK Signaling Pathway in Human Breast Cancer Cells.

Cardiac glycosides (CGs) constitute a group of steroid-like compounds renowned for their effectiveness in treating cardiovascular ailments. In recent times, there has been growing recognition of their potential use as drug leads in cancer treatment. In our prior research, we identified three highly promising CG compounds, namely lanatoside C (LC), peruvoside (PS), and strophanthidin (STR), which exhibited significant antitumor effects in lung, liver, and breast cancer cell lines. In this study, we investigated the therapeutic response of these CGs, with a particular focus on the MCF-7 breast cancer cell line. We conducted transcriptomic profiling and further validated the gene and protein expression changes induced by treatment through qRT-PCR, immunoblotting, and immunocytochemical analysis. Additionally, we demonstrated the interactions between the ligands and target proteins using the molecular docking approach. The transcriptome analysis revealed a cluster of genes with potential therapeutic targets involved in cytotoxicity, immunomodulation, and tumor-suppressor pathways. Subsequently, we focused on cross-validating the ten most significantly expressed genes, EGR1, MAPK1, p53, CCNK, CASP9, BCL2L1, CDK7, CDK2, CDK2AP1, and CDKN1A, through qRT-PCR, and their by confirming the consistent expression pattern with RNA-Seq data. Notably, among the most variable genes, we identified EGR1, the downstream effector of the MAPK signaling pathway, which performs the regulatory function in cell proliferation, tumor invasion, and immune regulation. Furthermore, we substantiated the influence of CG compounds on translational processes, resulting in an alteration in protein expression upon treatment. An additional analysis of ligand-protein interactions provided further evidence of the robust binding affinity between LC, PS, and STR and their respective protein targets. These findings underscore the intense anticancer activity of the investigated CGs, shedding light on potential target genes and elucidating the probable mechanism of action of CGs in breast cancer.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g., whether it involves cell differentiation, remains largely unexplored. METHODS: Digoxin, a user-designed digitoxigenin-α-L-rhamnoside (D6-MA), and ouabain were tested against various human AML-derived cells with different maturation phenotypes. Herein, we established two study models to specifically determine the effects of cardiac glycosides on LSC death and differentiation-one allowed change in dynamics of LSCs and leukemic progenitor cells (LPCs), while another maintained their undifferentiated status. Regulatory mechanisms underlying cardiac glycoside-induced cytotoxicity were investigated and linked to cell cycle distribution and apoptotic machinery. RESULTS: Primitive AML cells containing CD34+ LSCs/LPCs were very responsive to nanomolar concentrations of cardiac glycosides, with ouabain showing the greatest efficiency. Ouabain preferentially induces caspase-dependent apoptosis in LSCs, independent of its cell differentiation status, as evidenced by (i) the tremendous induction of apoptosis by ouabain in AML cells that acquired less than 15% differentiation and (ii) the higher rate of apoptosis in enriched LSCs than in LPCs. We sorted LSCs and LPCs according to their cell cycle distribution into G0/G1, S, and G2/M cells and revealed that G0/G1 cells in LSCs, which was its major subpopulation, were the top ouabain responders, indicating that the difference in ouabain sensitivity between LSCs and LPCs involved both distinct cell cycle distribution and intrinsic apoptosis regulatory mechanisms. Further, Mcl-1 and c-Myc, which were differentially expressed in LSCs and LPCs, were found to be the key apoptosis mediators that determined ouabain sensitivity in AML cells. Ouabain induces a more rapid loss of Mcl-1 and c-Myc in LSCs than in LPCs via the mechanisms that in part involve an inhibition of Mcl-1 protein synthesis and an induction of c-Myc degradation. CONCLUSIONS: Our data provide new insight for repurposing cardiac glycosides for the treatment of relapsed/refractory AML through targeting LSCs via distinct cell cycle and apoptosis machinery. Video Abstract.

The mechanistic role of cardiac glycosides in DNA damage response and repair signaling.

Cardiac glycosides (CGs) are a class of bioactive organic compounds well-known for their application in treating heart disease despite a narrow therapeutic window. Considerable evidence has demonstrated the potential to repurpose CGs for cancer treatment. Chemical modification of these CGs has been utilized in attempts to increase their anti-cancer properties; however, this has met limited success as their mechanism of action is still speculative. Recent studies have identified the DNA damage response (DDR) pathway as a target of CGs. DDR serves to coordinate numerous cellular pathways to initiate cell cycle arrest, promote DNA repair, regulate replication fork firing and protection, or induce apoptosis to avoid the survival of cells with DNA damage or cells carrying mutations. Understanding the modus operandi of cardiac glycosides will provide critical information to better address improvements in potency, reduced toxicity, and the potential to overcome drug resistance. This review summarizes recent scientific findings of the molecular mechanisms of cardiac glycosides affecting the DDR signaling pathway in cancer therapeutics from 2010 to 2022. We focus on the structural and functional differences of CGs toward identifying the critical features for DDR targeting of these agents.

New Insights on the Role of Marinobufagenin from Bench to Bedside in Cardiovascular and Kidney Diseases.

Marinobufagenin (MBG) is a member of the bufadienolide family of compounds, which are natural cardiac glycosides found in a variety of animal species, including man, which have different physiological and biochemical functions but have a common action on the inhibition of the adenosine triphosphatase sodium-potassium pump (Na+/K+-ATPase). MBG acts as an endogenous cardiotonic steroid, and in the last decade, its role as a pathogenic factor in various human diseases has emerged. In this paper, we have collated major evidence regarding the biological characteristics and functions of MBG and its implications in human pathology. This review focused on MBG involvement in chronic kidney disease, including end-stage renal disease, cardiovascular diseases, sex and gender medicine, and its actions on the nervous and immune systems. The role of MBG in pathogenesis and the development of a wide range of pathological conditions indicate that this endogenous peptide could be used in the future as a diagnostic biomarker and/or therapeutic target, opening important avenues of scientific research.

The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells.

Overcoming multidrug resistance (MDR) represents a major obstacle in cancer chemotherapy. Cardiac glycosides (CGs) are efficient in the treatment of heart failure and recently emerged in a new role in the treatment of cancer. ZINC253504760, a synthetic cardenolide that is structurally similar to well-known GCs, digitoxin and digoxin, has not been investigated yet. This study aims to investigate the cytotoxicity of ZINC253504760 on MDR cell lines and its molecular mode of action for cancer treatment. Four drug-resistant cell lines (P-glycoprotein-, ABCB5-, and EGFR-overexpressing cells, and TP53-knockout cells) did not show cross-resistance to ZINC253504760 except BCRP-overexpressing cells. Transcriptomic profiling indicated that cell death and survival as well as cell cycle (G2/M damage) were the top cellular functions affected by ZINC253504760 in CCRF-CEM cells, while CDK1 was linked with the downregulation of MEK and ERK. With flow cytometry, ZINC253504760 induced G2/M phase arrest. Interestingly, ZINC253504760 induced a novel state-of-the-art mode of cell death (parthanatos) through PARP and PAR overexpression as shown by western blotting, apoptosis-inducing factor (AIF) translocation by immunofluorescence, DNA damage by comet assay, and mitochondrial membrane potential collapse by flow cytometry. These results were ROS-independent. Furthermore, ZINC253504760 is an ATP-competitive MEK inhibitor evidenced by its interaction with the MEK phosphorylation site as shown by molecular docking in silico and binding to recombinant MEK by microscale thermophoresis in vitro. To the best of our knowledge, this is the first time to describe a cardenolide that induces parthanatos in leukemia cells, which may help to improve efforts to overcome drug resistance in cancer. A cardiac glycoside compound ZINC253504760 displayed cytotoxicity against different multidrug-resistant cell lines. ZINC253504760 exhibited cytotoxicity in CCRF-CEM leukemia cells by predominantly inducing a new mode of cell death (parthanatos). ZINC253504760 downregulated MEK1/2 phosphorylation and further affected ERK activation, which induced G2/M phase arrest.

Toxic effects of Tripterygium glycoside tablets on the reproductive system of male rats by metabolomics, cytotoxicity, and molecular docking.

BACKGROUND: Tripterygium glycoside tablets (TGT) is the most common preparation from Tripterygium wilfordii Hook F, which is widely used in clinical for treating rheumatoid arthritis (RA) and other autoimmune diseases. However, its serious reproductive toxicity limits its application. PURPOSE: This study aimed to elucidate the toxic effects of TGT on the reproductive system of male RA rats and its potential toxic components and mechanism. METHODS: Collagen-induced arthritis (CIA) rat model was established, and TGT suspension was given at low, medium, and high doses. Gonadal index, pathological changes, and the number of spermatogenic cells were used to evaluate the toxic effects of TGT on the reproductive system. Non-targeted metabolomics of testicular tissue was conducted by UHPLC-QTOF/MS. Combined with network toxicology, the key targets of TGT-induced reproductive toxicity were screened and RT-qPCR was used to validation. In vitro toxicity of 19 components of TGT was evaluated using TM3 and TM4 cell lines. Molecular docking was used to predict the interaction between toxic components and key targets. RESULTS: TGT reduced testicular and epididymis weight. Pathology analysis showed a lot of deformed and atrophic spermatogenic tubules. The number of spermatogenic cells decreased significantly (P<0.0001). A total of 58 different metabolites including platelet-activating factor (PAF), lysophosphatidylcholine (Lyso PC), phosphatidylinositol (PI), glutathione (GSH), and adenosine monophosphate (AMP) were identified by testicular metabolomics. Glycerophospholipid metabolism, ether lipid metabolism, and glutathione metabolism were key pathways responsible for the reproductive toxicity of TGT. Ten key reproductive toxicity targets were screened by network toxicology. The cytotoxicity test showed that triptolide, triptonide, celastrol, and demethylzeylasteral could significantly reduce the viability of TM3 and TM4 cells. Alkaloids had no apparent toxic effects. Molecular docking showed that the four toxic components had a good affinity with 10 key targets. All binding energies were less than -7 kcal/mol. The RT-qPCR results showed the Cyp19a1 level was significantly up-regulated. Pik3ca and Pik3cg levels were significantly down-regulated. CONCLUSION: Through testicular metabolomics, we found that TGT may cause reproductive toxicity through CYP19A1, PIK3CA, and PIK3CG three target, which was preliminarily revealed. This study laid the foundation for elucidating the toxicity mechanism of TGT and evaluating its safety and quality.

Multi-component immune knockout: A strategy for studying the effective components of traditional Chinese medicine.

Periploca forrestii Schltr., a traditional Chinese medicine (TCM), is commonly used to treat autoimmune diseases such as rheumatoid arthritis (RA). However, its mechanism, involving a variety of cardiac glycosides, remains largely unknown. The immune knockout strategy can highly selectively deplete target components by immunoaffinity chromatography (IAC). We aimed to identify the common structural features of cardiac glycosides in P. forrestii and design IAC to specifically recognize these features to achieve the multi-component knockout of potential active substances from the extracts of P. forrestii. A content detection experiment confirmed that the content of a compound with periplogenin structure (CPS) in the extract of P. forrestii was reduced by 45% by IAC of periplogenin. The immunosuppressive ability of the extract on H9 human T lymphocytic cells was weakened after CPS knockout from P. forrestii extract. Molecular biology experiments showed that mRNA expression of interferon-γ (IFN-γ), interleukin-2 (IL-2), and interleukin-6 (IL-6) in H9 cells was up-regulated after CPS knockout, while no significant changes in the expression of interleukin-4 (IL-4) were found. CPS knockout from P. forrestii extract did not cause significant changes in the proliferation of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells incubated with this extract. These results indicate that CPS exhibited immunosuppressive effects via inhibiting the T helper 1 (Th1) cell immune response and not via the anti-inflammatory components in P. forrestii. This is the first use of IAC to achieve multi-component knockout in TCM extracts for identifying effective compounds. This method is effective and reliable and warrants further exploration.

Localized Microsphere/Hydrogel for Tumor Immunotherapy of Cardiac Glycoside with Minimal Toxicity.

It has been reported that cardiac glycosides (CGs) commonly used in clinical practice can inhibit tumor growth by inducing immunogenic cell death (ICD), and their positive benefits have been documented in several clinical trials of drug combinations. However, the inherent cardiogenic side effects need to be addressed before CGs can be truly applied in clinical antitumor therapy. In this study, a dual controlled release microsphere/hydrogel platform (OL-M/Gel) was constructed to precisely control the output of oleandrin (OL, one of the representative CGs) in situ in tumors. With the help of this intelligent drug release platform, OL can be released in vitro and in vivo in a sustained and stable manner. The ability of OL to induce ICD and the subsequent antigen presentation and cytotoxic T-cell cascades was first stated, which resulted in potent tumor growth suppression without significant side effects. In addition, the inhibition of autologous tumor recurrence and metastasis by OL-M/Gel was also revealed. This study is expected to break through the inherent bottleneck of CGs and promote their clinical transformation in the field of antitumor treatment.

Identification of a Cardiac Glycoside Exhibiting Favorable Brain Bioavailability and Potency for Reducing Levels of the Cellular Prion Protein.

Several strands of investigation have established that a reduction in the levels of the cellular prion protein (PrPC) is a promising avenue for the treatment of prion diseases. We recently described an indirect approach for reducing PrPC levels that targets Na,K-ATPases (NKAs) with cardiac glycosides (CGs), causing cells to respond with the degradation of these pumps and nearby molecules, including PrPC. Because the therapeutic window of widely used CGs is narrow and their brain bioavailability is low, we set out to identify a CG with improved pharmacological properties for this indication. Starting with the CG known as oleandrin, we combined in silico modeling of CG binding poses within human NKA folds, CG structure-activity relationship (SAR) data, and predicted blood-brain barrier (BBB) penetrance scores to identify CG derivatives with improved characteristics. Focusing on C4'-dehydro-oleandrin as a chemically accessible shortlisted CG derivative, we show that it reaches four times higher levels in the brain than in the heart one day after subcutaneous administration, exhibits promising pharmacological properties, and suppresses steady-state PrPC levels by 84% in immortalized human cells that have been differentiated to acquire neural or astrocytic characteristics. Finally, we validate that the mechanism of action of this approach for reducing cell surface PrPC levels requires C4'-dehydro-oleandrin to engage with its cognate binding pocket within the NKA α subunit. The improved brain bioavailability of C4'-dehydro-oleandrin, combined with its relatively low toxicity, make this compound an attractive lead for brain CG indications and recommends its further exploration for the treatment of prion diseases.

Evaluation of the Sensitivity of Breast Cancer Cell Lines to Cardiac Glycosides Unveils ATP1B3 as a Possible Biomarker for the Personalized Treatment of ERα Expressing Breast Cancers.

The molecular classification of breast cancer (BC) dictates pharmacological treatment. Estrogen receptor α (ERα) expressing tumors are treated with 4OH-tamoxifen or fulvestrant, which inhibits the receptor, or with aromatase inhibitors (i.e., anastrozole, letrozole, and exemestane) that reduce the 17ß-estradiol (E2) circulating blood levels. Besides such endocrine therapy (ET) drugs, ERα-positive BCs can be treated with epidermal growth factor receptor (EGF-R) inhibitors (i.e., gefitinib, erlotinib, and lapatinib) according to HER2 expression. Notwithstanding these anti-BC drugs, novel personalized approaches for BC treatment are required because prolonged administration of those pharmaceutics determines resistant phenotypes, which result in metastatic BC. We have recently reported that the cardiac glycoside (CG) (i.e., Na/K ATPase inhibitor) ouabain could be repurposed for ERα-positive primary and metastatic BC treatment as it induces ERα degradation and kills BC cells. Here, we evaluated if other CGs could represent additional treatment options for ERα-positive BCs and if the Na/K ATPase could be considered a biomarker for ERα-positive BC treatment. The results indicate that the ATP1B3 Na/K ATPase isoform can educate the choice for the personalized treatment of ERα-positive BC with CGs and that CGs could be more efficacious if they are administered in association with gefitinib.

Chemistry and anticancer activity of cardiac glycosides: A review.

The objective of this review is an attempt to emphasize the development in the chemistry and to display review of diverse therapeutic actions of cardiac glycosides. Anticancer activity of cardiac glycosides is the main activity as discussed in this review. The aim of the review is to gather the recent researches on cardiac glycosides. The present manuscript gives the platform for the researcher to have complete literature on the topic.

Periplocymarin alleviates pathological cardiac hypertrophy via inhibiting the JAK2/STAT3 signalling pathway.

Pathological cardiac hypertrophy is the most important risk factor for developing chronic heart failure. Therefore, the discovery of novel agents for treating pathological cardiac hypertrophy remains urgent. In the present study, we examined the therapeutic effect and mechanism of periplocymarin (PM)-mediated protection against pathological cardiac hypertrophy using angiotensinII (AngII)-stimulated cardiac hypertrophy in H9c2 cells and transverse aortic constriction (TAC)-induced cardiac hypertrophy in mice. In vitro, PM treatment significantly reduced the surface area of H9c2 cells and expressions of hypertrophy-related proteins. Meanwhile, PM markedly down-regulated AngII-induced translocation of p-STAT3 into the nuclei and enhanced the phosphorylation levels of JAK2 and STAT3 proteins. The STAT3 specific inhibitor S3I-201 or siRNA-mediated depleted expression could alleviate AngII-induced cardiac hypertrophy in H9c2 cells following PM treatment; however, PM failed to reduce the expressions of hypertrophy-related proteins and phosphorylated STAT3 in STAT3-overexpressing cells, indicating that PM protected against AngII-induced cardiac hypertrophy by modulating STAT3 signalling. In vivo, PM reversed TAC-induced cardiac hypertrophy, as determined by down-regulating ratios of heart weight to body weight (HW/BW), heart weight to tibial length (HW/TL) and expressions of hypertrophy-related proteins accompanied by the inhibition of the JAK2/STAT3 pathway. These results revealed that PM could effectively protect the cardiac structure and function in experimental models of pathological cardiac hypertrophy by inhibiting the JAK2/STAT3 signalling pathway. PM is expected to be a potential lead compound of the novel agents for treating pathological cardiac hypertrophy.

A suicide attempt by ingestion of oleander leaves and treatment with digoxin-specific Fab antibody fragments.

Natural cardiac glycosides have positive inotropic heart effects but at high, toxic doses they can cause life-threatening cardiac arrhythmias. Here we present the first Croatian case of a 16-year-old girl who attempted suicide by eating dried oleander leaves, which contain natural cardiac glycosides, and her treatment with a specific antidote. The girl presented with an oedema of the uvula indicating local toxicity, severe bradycardia, first-degree atrioventricular block, drowsiness, and vomiting. Having taken her medical history, we started treatment with atropine, intravenous infusion of dextrose-saline solution and gastroprotection, but it was not successful. Then we introduced digoxin-specific Fab antibody fragments and within two hours, the patient's sinus rhythm returned to normal. Cases of self-poisoning with this oleander are common in South-East Asia, because it is often used as a medicinal herb, and digoxin-specific Fab fragments have already been reported as effective antidote against oleander poisoning there. Our case has taught us that it is important to have this drug in the hospital pharmacy both for digitalis and oleander poisoning.

Emergence of Cardiac Glycosides as Potential Drugs: Current and Future Scope for Cancer Therapeutics.

Cardiac glycosides are natural sterols and constitute a group of secondary metabolites isolated from plants and animals. These cardiotonic agents are well recognized and accepted in the treatment of various cardiac diseases as they can increase the rate of cardiac contractions by acting on the cellular sodium potassium ATPase pump. However, a growing number of recent efforts were focused on exploring the antitumor and antiviral potential of these compounds. Several reports suggest their antitumor properties and hence, today cardiac glycosides (CG) represent the most diversified naturally derived compounds strongly recommended for the treatment of various cancers. Mutated or dysregulated transcription factors have also gained prominence as potential therapeutic targets that can be selectively targeted. Thus, we have explored the recent advances in CGs mediated cancer scope and have considered various signaling pathways, molecular aberration, transcription factors (TFs), and oncogenic genes to highlight potential therapeutic targets in cancer management.

Repurposing Cardiac Glycosides: Drugs for Heart Failure Surmounting Viruses.

Drug repositioning is a successful approach in medicinal research. It significantly simplifies the long-term process of clinical drug evaluation, since the drug being tested has already been approved for another condition. One example of drug repositioning involves cardiac glycosides (CGs), which have, for a long time, been used in heart medicine. Moreover, it has been known for decades that CGs also have great potential in cancer treatment and, thus, many clinical trials now evaluate their anticancer potential. Interestingly, heart failure and cancer are not the only conditions for which CGs could be effectively used. In recent years, the antiviral potential of CGs has been extensively studied, and with the ongoing SARS-CoV-2 pandemic, this interest in CGs has increased even more. Therefore, here, we present CGs as potent and promising antiviral compounds, which can interfere with almost any steps of the viral life cycle, except for the viral attachment to a host cell. In this review article, we summarize the reported data on this hot topic and discuss the mechanisms of antiviral action of CGs, with reference to the particular viral life cycle phase they interfere with.

Cardiac Glycosides as Immune System Modulators.

Cardiac glycosides (CGs) are natural steroid compounds occurring both in plants and animals. They are known for long as cardiotonic agents commonly used for various cardiac diseases due to inhibition of Na+/K+-ATPase (NKA) pumping activity and modulating heart muscle contractility. However, recent studies show that the portfolio of diseases potentially treatable with CGs is much broader. Currently, CGs are mostly studied as anticancer agents. Their antiproliferative properties are based on the induction of multiple signaling pathways in an NKA signalosome complex. In addition, they are strongly connected to immunogenic cell death, a complex mechanism of induction of anticancer immune response. Moreover, CGs exert various immunomodulatory effects, the foremost of which are connected with suppressing the activity of T-helper cells or modulating transcription of many immune response genes by inhibiting nuclear factor kappa B. The resulting modulations of cytokine and chemokine levels and changes in immune cell ratios could be potentially useful in treating sundry autoimmune and inflammatory diseases. This review aims to summarize current knowledge in the field of immunomodulatory properties of CGs and emphasize the large area of potential clinical use of these compounds.

Identification of key regulators responsible for dysregulated networks in osteoarthritis by large-scale expression analysis.

BACKGROUND: Osteoarthritis (OA) is a worldwide musculoskeletal disorder. However, disease-modifying therapies for OA are not available. Here, we aimed to characterize the molecular signatures of OA and to identify novel therapeutic targets and strategies to improve the treatment of OA. METHODS: We collected genome-wide transcriptome data performed on 132 OA and 74 normal human cartilage or synovium tissues from 7 independent datasets. Differential gene expression analysis and functional enrichment were performed to identify genes and pathways that were dysregulated in OA. The computational drug repurposing method was used to uncover drugs that could be repurposed to treat OA. RESULTS: We identified several pathways associated with the development of OA, such as extracellular matrix organization, inflammation, bone development, and ossification. By protein-protein interaction (PPI) network analysis, we prioritized several hub genes, such as JUN, CDKN1A, VEGFA, and FOXO3. Moreover, we repurposed several FDA-approved drugs, such as cardiac glycosides, that could be used in the treatment of OA. CONCLUSIONS: We proposed that the hub genes we identified would play a role in cartilage homeostasis and could be important diagnostic and therapeutic targets. Drugs such as cardiac glycosides provided new possibilities for the treatment of OA.

Periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation in mice.

Periplocymarin is an effective component of Periplocae Cortex, which was wildly used as an ingredient in Traditional Chinese Medicine. Our group previously reported that periplocymarin exerted cardiotonic role via promoting calcium influx. However, its exact role in the pathogenesis of myocardial fibrosis has not been elucidated yet. The present study was aimed at determining the potential effect and underlying mechanism of periplocymarin in isoproterenol (ISO)-induced myocardial fibrosis. C57BL/6 mice were subcutaneously injected with ISO (5 mg/kg/day) or saline for 1 week. The early-to-atrial wave ratio (E/A ratio) measured by echocardiography revealed that ISO-induced heart stiffness was remarkably reversed by administration of periplocymarin (5 mg/kg/day). Masson trichrome staining exhibited that treatment of periplocymarin reduced the excessive deposition of extracellular matrix (ECM). Further investigations employing real-time PCR and western blot demonstrated that periplocymarin suppressed the expression of fibrosis related genes (Col1a1, Col3a1, Acta2 and Tgfb1) and proteins (Collagen I, Collagen III, α-SMA and TGF-ß1) induced by ISO. Metabolomics analysis demonstrated that periplocymarin ameliorated the disorders triggered by ISO and many of the differential metabolic substances were involved in amino acid, glucose and lipid metabolism. Further analysis using network pharmacology revealed that three key genes, namely NOS2, NOS3 and Ptgs2, may be the potential targets of periplocymarin and responsible for the disorders. Validation using heart tissues showed that the mRNA expression of NOS3 was decreased while Ptgs2 was increased upon ISO treatment, which were reversed by periplocymarin. Moreover, the expression of COX-2 (Ptgs2 encoded protein) was consistent with the aspect of Ptgs2 mRNA, while eNOS (NOS3 encoded protein) expression was unchanged. In vitro studies exhibited that periplocymarin exerts anti-fibrotic function via regulating at least eNOS and COX-2 in cardiomyocyte. Taken together, periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation, the potential mechanism was that periplocymarin targeted on, at least eNOS and COX-2, to improve the metabolic processes of cardiomyocyte and thus attenuated the myocardial fibrosis. Our study highlighted that periplocymarin is a potential therapeutic agent for the prevention of myocardial fibrosis.

Oleandrin, a cardiac glycoside, induces immunogenic cell death via the PERK/elF2α/ATF4/CHOP pathway in breast cancer.

Chemotherapeutic agents have been linked to immunogenic cell death (ICD) induction that is capable of augmenting anti-tumor immune surveillance. The cardiac glycoside oleandrin, which inhibits Na+/K+-ATPase pump (NKP), has been shown to suppress breast cancer growth via inducing apoptosis. In the present study, we showed that oleandrin treatment triggered breast cancer cell ICD by inducing calreticulin (CRT) exposure on cell surface and the release of high-mobility group protein B1 (HMGB1), heat shock protein 70/90 (HSP70/90), and adenosine triphosphate (ATP). The maturation and activation of dendritic cells (DCs) were increased by co-culturing with the oleandrin-treated cancer cells, which subsequently enhanced CD8+ T cell cytotoxicity. Murine breast cancer cell line EMT6 was engrafted into BALB/c mice, and tumor-bearing mice were administered with oleandrin intraperitoneally every day. Oleandrin inhibited tumor growth and increased tumor infiltrating lymphocytes including DCs and T cells. Furthermore, the differential mRNA expression incurred by oleandrin was investigated by mRNA sequencing and subsequently confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Mechanistically, oleandrin induced endoplasmic reticulum (ER) stress-associated, caspase-independent ICD mainly through PERK/elF2α/ATF4/CHOP pathway. Pharmacological and genetic inhibition of protein kinase R-like ER kinase (PERK) suppressed oleandrin-triggered ICD. Taken together, our findings showed that oleandrin triggered ER stress and induced ICD-mediated immune destruction of breast cancer cells. Oleandrin combined with immune checkpoint inhibitors might improve the efficacy of immunotherapy.

Preeclampsia: Cardiotonic Steroids, Fibrosis, Fli1 and Hint to Carcinogenesis.

Despite prophylaxis and attempts to select a therapy, the frequency of preeclampsia does not decrease and it still takes the leading position in the structure of maternal mortality and morbidity worldwide. In this review, we present a new theory of the etiology and pathogenesis of preeclampsia that is based on the interaction of Na/K-ATPase and its endogenous ligands including marinobufagenin. The signaling pathway of marinobufagenin involves an inhibition of transcriptional factor Fli1, a negative regulator of collagen synthesis, followed by the deposition of collagen in the vascular tissues and altered vascular functions. Moreover, in vitro and in vivo neutralization of marinobufagenin is associated with the restoration of Fli1. The inverse relationship between marinobufagenin and Fli1 opens new possibilities in the treatment of cancer; as Fli1 is a proto-oncogene, a hypothesis on the suppression of Fli1 by cardiotonic steroids as a potential anti-tumor therapeutic strategy is discussed as well. We propose a novel therapy of preeclampsia that is based on immunoneutralization of the marinobufagenin by monoclonal antibodies, which is capable of impairing marinobufagenin-Na/K-ATPase interactions.