Cardiac glycosides: Looking beyond heart failure and atrial fibrillation

Abstract

Cardiac glycosides, historically used for managing heart failure and atrial fibrillation, have demonstrated significant pharmacological versatility extending into oncology, virology, and immunotherapy. By targeting Na⁺/K⁺-ATPase, these compounds regulate ionic homeostasis and initiate signalosome formation, influencing key pathways such as MAPK/ERK and PI3K/Akt. Beyond their cardiovascular effects, cardiac glycosides exhibit potent immunomodulatory and senolytic properties, particularly in cancer therapy. They induce immunogenic cell death by releasing damage-associated molecular patterns, which enhance tumor antigen presentation and activate cytotoxic T lymphocytes. In addition, their ability to selectively eliminate senescent tumor cells through Na⁺/K⁺-ATPase inhibition reduces inflammation and improves therapeutic outcomes in synergistic cancer treatments. Furthermore, their antiviral activities have been explored against SARS-CoV-2 and Ebola virus infections, with mechanisms involving the disruption of viral entry, replication, and protein synthesis. Despite their promise, concerns about cardiotoxicity and a narrow therapeutic window persist, necessitating precise dosing and novel derivatives with improved safety profiles. This review consolidates current insights into the mechanisms, therapeutic applications, and limitations of cardiac glycosides, highlighting their potential as a cornerstone for future drug development in oncology and infectious diseases. Advancing pharmacogenomic approaches and clinical trials will further define their role in precision medicine.

Introduction

The use of medicinal plants for the treatment of diseases predates current conventional medicine. Medicinal plants and herbs due to their rich phytochemicals or plant chemicals are used extensively for the development of newer pharmaceuticals and therapies. Cardiac glycosides are naturally occurring steroids found in various plant species including (1) Digitalis purpurea and Digitalis lanata (foxgloves), (2) Nerium oleander (oleander), (3) Thevetia peruviana (yellow oleander), (4) Convallaria majalis (lily of the valley), (5) Urginea maritima and Urginea indica (squill), (6) Strophanthus gratus (ouabain), (7) Apocynum cannabinum (dogbane), (8) Cheiranthus cheiri (wallflower), and (9) Cerbera odollam and Cerbera manghas (bintaro - ”Indian suicide tree”). In addition, it is found in the venom gland of the cane toad (Bufo marinus).[1,2,3] Structurally, cardiac glycosides irrespective of their source (plants, animals, and humans) are made up of a steroid core, a lactone ring, and a sugar moiety. They are further classified into two major groups: (1) the 5-membered lactone ring cardiac glycosides called cardenolides and (2) the 6-membered lactone ring cardiac glycosides called bufadienolides.[4] Therapeutic agents including digoxin, marinobufagenin, digitoxin, oleandrin, bufalin, aerobufagenin, ouabain, and telocinobufagin[2,3] have been isolated for the treatment of heart failure and atrial fibrillation. These compounds exert profound effects on cardiac contractility and electrical activity. Due to their cardiovascular system (CVS) effects, digitalis therapy was considered the gold standard for the treatment of heart failure at the beginning of the 20^th century.[5,6]

Recently, the biosynthesis, exact physiological, and pathophysiological mechanisms of cardiac glycosides have been debated.[7] Following results from observational and randomized clinical trials and meta-analyses conducted toward the end of the 20^th century, the safety and potency of cardiac glycosides for the treatment of heart failure have been challenged and questioned.[8,9,10,11,12,13,14] Despite their long history of use, cardiac glycosides are associated with a narrow therapeutic index, posing significant challenges in their clinical application. Furthermore, the availability of alternative agents for CVS disorders, especially those with broader therapeutic index and safety profiles has further decreased the use of cardiac glycosides. However, they have been recently considered for antineoplastic, anti-inflammatory, senolytic, antiviral, hormonal, immunomodulatory, and neuroprotective potentials. Thus, understanding the physiological, pharmacological, and toxicological mechanisms of this class of molecules is crucial for drug discovery and development and strategies to prevent and/or manage cardiac glycoside-induced toxicities. This review aims to explore the physiological mechanisms underlying the action of cardiac glycosides, their therapeutic potential in modern medicine, and the toxicity concerns associated with their use, providing insights that could inform clinical practice and future research.

Pharmacological Mechanisms and Cardiac Glycosides

The primary mechanism by which cardiac glycosides exert their effects is by inhibiting the Na⁺/K⁺-ATPase enzyme.[15,16] Specifically, cardiac glycosides bind to the α-subunit of the sodium-potassium pump, blocking its activity. This pump is an antiport transporter of sodium and potassium ions energized by adenosine triphosphate (ATP) and it is essential for the preservation of the plasma membrane potential of the cell. The inhibitory effect of cardiac glycosides on this pump results in the build-up of Na⁺ ions in the cytosol,[17] which subsequently affects the sodium–calcium exchanger (NCX). Instead of calcium efflux in exchange for sodium, the NCX begins to import calcium into the cell, leading to increased intracellular calcium levels [Figure 1]. Calcium ions are crucial players in the contractility of the cardiac muscle. The elevated calcium concentration enhances myocardial contractility (positive inotropic effect). Their interaction involves binding and modifying the structure of troponin, which, when the muscle is in a “relaxed condition”, inhibits the contraction of actin–myosin filaments.[4,18] Therefore, Ca²⁺ facilitates the interaction between actin and myosin filaments, which enhances the contractile force of the cardiac muscle. This mechanism is particularly beneficial in heart failure, where the heart’s ability to pump blood is compromised. In addition, cardiac glycosides modulate autonomic nervous system activity, leading to vagomimetic effects, which include increased vagal tone and decreased sympathetic activity [Figure 1]. This results in a reduction in heart rate (negative chronotropy) and slowed conduction through the atrioventricular (AV) node, making these drugs useful in managing atrial fibrillation with rapid ventricular response. As reported by Škubník et al.,[17] when cardiac glycosides bind to the sodium–potassium ATPase, several transduction cascades are indirectly activated, including epidermal growth factor receptors (EGFR)-mediated phosphorylation and coupling of the “Son of Sevenless” protein (Sos) and subsequent Ras/Raf/MEK/ERK signaling cascade activation, known to be involved in cell cycle progression and cellular proliferation. Furthermore, EGFRs are implicated in the regulation of other physiological processes including the regulation of phosphatidylinositol 3-kinase activity, G protein-coupled receptors, and cytokines.[4,17]

Figure 1.

Physiological mechanisms of cardiac glycosides. TNC = Troponin C, NCX = sodium-calcium exchanger

Cardiac glycoside-mediated signalosome formation

It is important to note that the inhibition of the sodium–potassium ATPase by cardiac glycosides occurs at high concentrations. At low concentrations, they form a complex cascade called sodium–potassium ATPase-signalosome which has been thoroughly discussed by Paula et al.,[19] Pratt et al.,[20] and Lopina et al.[21] A signalosome is a substantial protein complex that enhances the local concentration and signaling activity of its constituents. Signalosomes consist of distinct combinations of signaling pathway components that are localized to specific regions within the cell. The primary functions of this complex are (1) signal transduction, where the colocalization of pathway components facilitates efficient signal transmission between adjacent molecules;[22] (2) prevention of cross-talk, as signalosomes inhibit inappropriate interactions with other cellular pathways; and (3) regulation of protein degradation, with the COP9 signalosome (CSN) potentially playing a role in this process. The CSN is a protein complex essential for the development of multicellular organisms.[23] At low ouabain concentrations, Na⁺/K⁺ ATPase forms a signalosome, a multiprotein complex involving Src kinase and other signaling proteins. This complex regulates pathways critical for cell proliferation, survival, and apoptosis. For example, interactions between Na⁺/K⁺ ATPase and Src kinase initiate cascades such as MAPK/ERK, promoting cellular responses independent of ion transport.[24,25] El-Mallakh et al.[26] reported the concentrations of endogenous ouabain in body fluids to be in the pico to the nanomolar range, arguing that at these relatively low concentrations, ouabain rather than inhibition actually increases sodium pump activity. Similarly, the interactions with signaling proteins such as Src kinase and caveolin-1 specifically enable the formation of the signalosome complex by directly binding to the Na⁺/K⁺-ATPase α1 subunit. This interaction activates downstream pathways such as MAPK/ERK and PI3K/Akt, promoting cell survival, proliferation, and apoptosis inhibition in cancer cells.[27,28] Such pathways highlight the therapeutic potential of targeting the signalosome in disease contexts. Further exploration of its molecular components and their functional roles enhances our understanding of its therapeutic significance.

Summarily, the binding of cardiac glycosides to sodium–potassium ATPase results in a variety of signaling pathways, many of which have a direct bearing on the development, division, and death of cells.[28] Besides their primary inhibition of the Na⁺/K⁺ ATPase, this class of molecules can function through a variety of other mechanisms. Thus far, both targeted and nontargeted mechanisms of action have been identified. The nontargeted effects refer to the consequences that occur as a result of an initial action or event, rather than being a direct outcome. These effects can influence various systems or processes in complex ways, often requiring careful analysis to fully understand their implications. For instance, these cardiotonic steroids (CS) interact with nuclear receptors, which function as transcription factors that regulate various cellular functions. These interactions may influence hormonal regulation, immune function, bodily defenses, and the process of carcinogenesis. Through mimicry, they can exert the same effects of steroid hormones or other hormones on several signaling pathways. For example, they can elevate cholesterol levels by stimulating 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the primary enzyme involved in cholesterol synthesis,[29] or changing the fluidity of the cell plasma membrane.[30] This gives insight into the intricate cellular responses associated with cardiac glycoside therapy, which could lead to fairly unpredictable results. Apart from the intricate mode of actions exhibited by this class of molecules, the Na⁺/K⁺-ATPase itself contributes significantly to the variety of their biological activities. Since diverse tissues express distinct isoforms of Na⁺/K⁺-ATPase subunits (α-1, α-2, and α-3),[18] cardiac glycosides have varying affinities for these isoforms. Digoxin and digitoxin, for instance, are more selective to NKA isoforms α-2 and α-3 than to Na⁺/K⁺-ATPase isoform α-1. Conversely, ouabain is more selective for the α-1 Na⁺/K⁺-ATPase isoform.[31] However, there exist some variations in certain species. For instance, Wansapura et al.[32] reported the resistance of α-1 Na⁺/K⁺-ATPase isoform to ouabain in rats and mice. The variability of the biological roles of the different isoforms of Na⁺/K⁺-ATPase contributes to the notable variations in the physiological responses elicited by cardiac glycosides. While Na⁺/K⁺-ATPase isoform α-1 is concerned with pumping and signaling functions, the α-2 isoform is involved in Ca² + signaling and positive inotropy, whereas the α-3 isoform is connected to cardiac hypertrophy.[4,33]

Therapeutic Potentials of Cardiac Glycosides

The use of cardiac glycosides extends beyond heart failure and atrial fibrillation. Recent studies have explored their potential in cancer therapy, where they are believed to exert anticancer effects through mechanisms such as the induction of apoptosis, inhibition of cell proliferation, and modulation of immune responses.[4,17] However, these applications are still in the experimental stages, and research is ongoing to establish their efficacy and safety in oncology. In heart failure, cardiac glycosides remain a cornerstone of therapy, particularly in patients with reduced ejection fraction. By increasing cardiac output and reducing symptoms such as dyspnea and fatigue, these drugs improve the quality of life in heart failure patients. However, their use is often limited by the risk of toxicity, necessitating careful monitoring of drug levels and patient response. In atrial fibrillation, particularly in cases with rapid ventricular response, cardiac glycosides are used to control heart rate. Their ability to enhance vagal tone and slow AV nodal conduction makes them effective in managing this arrhythmia, although they are often used in combination with other antiarrhythmic agents.

Emerging applications in oncology

Recent research has highlighted the potential anticancer properties of cardiac glycosides.[4,15,17,33,34] These compounds have been shown to inhibit the proliferation of cancer cells, induce apoptosis, and modulate immune responses. The exact mechanisms are still being investigated, but it is believed that the inhibition of the Na⁺/K⁺-ATPase enzyme plays a role in disrupting cancer cell metabolism and signaling pathways. Table 1 summarizes some of these compounds with established anticancer properties. Fürst et al.[5] explored the potential use of cardiac glycosides in cancer treatment, focusing on inflammation-driven cancers, thus offering therapeutic promise in oncology. However, the clinical application of cardiac glycosides in oncology is still in its infancy, with concerns regarding toxicity being a significant barrier to their widespread use.

Table 1.

Anticancer activities of some cardiac glycosides

Compound	Mechanism	Cancer type	Reference
Digoxin	ICD via calreticulin release	Breast cancer	Li et al.[33]
Lanatoside C	G2/M cell cycle arrest Inhibition of the PI3K/AKT/mTOR signalling pathway DNA damage and ROS-mediated apoptosis	Breast, lung and liver cancers	Reddy et al.[35]
Convallotoxin	ROS-mediated apoptosis	Skin (HaCaT keratinocytes)	Jiang et al.[36]
Ouabain	Na+/K+-ATPase (sodium pump) independent STAT3 downregulation	Human NSCLC (A549 and H460) cells Colorectal carcinoma (HCT116) cells Pancreatic cancer (PANC1) cells Cervical cancer (HeLa) cells	Du et al.[37]
Ouabain	DNA damage and altered DNA repair-associated proteins	Osteosarcoma	Yang et al.[38]
Digoxin	DNA damage and altered DNA repair-associated proteins	Lung cancer	Lee et al.[39]
Oleandrin	ER stress-mediated PERK/eIF2α/ATF4/CHOP pathway activation and cytotoxicity	Breast cancer	Li et al.[33]
Digitoxin	G2/M cell cycle arrest and apoptosis	HeLa cells	Gan et al.[40]
R=β-D-gal-(1→4)-α-L-thevetose	G2/M phase cell cycle arrest and apoptosis (selective cytotoxicity)	Human lung cancer (P15) cells Human stomach cancer (MGC-803) cells Human pancreatic cancer (SW1990) cells	Cheng et al.[41]
(5α) R=β-D-glc-(1→4)-α-L-thevetose	G2/M phase cell cycle arrest and apoptosis	Human lung cancer (P15) cells Human stomach cancer (MGC-803) cells Human pancreatic cancer (SW1990) cells	Cheng et al.[41]
Thevetioside C	G2/M phase cell cycle arrest and apoptosis (selective cytotoxicity)	Human lung cancer (P15) cells Human stomach cancer (MGC-803) cells Human pancreatic cancer (SW1990) cells	Cheng et al.[41]
Oleandrin and oleandrigenin (Anvirzel™)	Inhibition of GSK-3, NOS and HIF1-α expressions as well as activation of ERK	U87 human glioblastoma cells	Terzioglu-Usak et al.[42]
Oleandrin and oleandrigenin (Anvirzel™)	Activation of calcineurin and NFAT via the Fas ligand; Inhibition of Na+/K+-ATPase and the export of FGF-2	Breast, colon, lung, prostate, melanoma and pancreatic cancer cell lines	Apostolou et al.[43]

ICD=Immunogenic cell death, ROS=Reactive oxygen species, NFAT=Nuclear factor of activated T-cells, ER=Endoplasmic reticulum, FGF-2=Fibroblast growth factor, NSCLC=Nonsmall cell lung cancer, GSK-3=Glycogen synthase kinase-3, NOS=Nitric oxide synthase, HIF1a=Hypoxia-inducible factor 1a

Cardiac glycosides as immunomodulators

Cardiac glycosides are known to induce programmed cell death. Cell death is an important phenomenon that is crucial for the maintenance of homeostasis. This can occur both physiologically and in response to external stress stimuli.[4,17] Programmed cell death also known as regulated cell death (RCD) is governed by specific molecular mechanisms and signaling pathways. It is often a physiological process, allowing the body to maintain homeostasis, remove damaged cells, and respond to various stimuli. Classically, RCD includes apoptosis, necroptosis, pyroptosis, and ferroptosis. Each has distinct pathways and molecular signals. While apoptosis involves caspases and is characterized by DNA fragmentation, membrane blebbing, and cell shrinkage, necroptosis involves activating receptor-interacting protein kinases, leading to a more inflammatory form of cell death than apoptosis. On the other hand, ferroptosis is an iron-dependent cell death driven by lipid peroxidation. Under physiological conditions, RCD, particularly apoptosis is noninflammatory and nonimmunogenic. It helps in tissue remodeling,[44] development,[45] and eliminating cells without triggering immune responses unless dysregulated.

Sixty-eight naturally isolated cardiac glycosides were systematically examined to explore their structure-activity relationship and mechanism of action. The study found that specific structural elements such as the orientation of key functional groups directly impact their ability to inhibit cancer cell survival. Cardiac glycosides induce autophagy followed by apoptosis of cancer cells.[46] Notably, autophagy – a cellular process involved in the degradation and recycling of cellular components is first activated as a protective mechanism, but eventually, the cells undergo programmed death. The compounds induce cancer cell death by targeting the Na⁺/K⁺-ATPase. The inhibition of this pump is central to the activation of these pathways, though it suggests other unidentified mechanisms may also contribute to cancer cell death. According to Geng et al.,[46] data from RNA sequencing illustrate how cardiac glycosides reprogram cellular gene expression, leading to the sequential activation of the death pathways. In addition, mouse xenograft models confirm the anticancer efficacy of cardiac glycosides in vivo, further supporting their potential as therapeutic agents.[46]

Cardiotonic steroids-mediated immunomodulation in cancer

Cardiac glycosides have demonstrated immunomodulatory effects, specifically within the context of cancer, by inducing immunogenic cell death (ICD). ICD is a specific subtype of RCD that activates the immune system.[47,48] ICD activates an antitumor immune response through the release of danger-associated molecular patterns (DAMPs), such as (1) calreticulin exposure (on the cell surface), (2) release of ATP (which serves as a chemoattractant for dendritic cells), (3) release of high-mobility group box 1 protein (HMGB1) (which promotes antigen presentation), and (4) type I interferon (IFN) signaling, which recruit and activate dendritic cells and cytotoxic T lymphocytes. These responses enhance tumor antigen presentation and promote adaptive immunity. Thus, ICD is particularly relevant in cancer immunotherapy, where the goal is to induce the immune system to recognize and destroy tumor cells [Figure 2]. In cancer cells, the apoptotic properties of cardiac glycosides can be triggered through various pathways, including reactive oxygen species (ROS) production, inhibition of Na⁺/K⁺-ATPase, and mitochondrial dysfunction. This leads to caspase activation and controlled cell death. For example, oleandrin has been shown to induce endoplasmic reticulum stress and trigger the PERK/eIF2α/ATF4/CHOP pathway [Figure 3], enhancing CD8+ T-cell-mediated cytotoxicity in breast cancer models.[33] Lanatoside C has been reported to exert dose-dependent and cell-specific antiproliferative and apoptotic effects on breast (MCF-7), lung (A549), and liver (HepG2) cancer cell lines.[35] Specifically, lanatoside C inhibits the MAPK/Wnt/PAM signaling pathways to arrest the G2/M phase of the cell cycle; it induces apoptosis by inducing DNA damage and inhibiting PI3K/AKT/mTOR signaling pathways.[35]

Figure 2.

Molecular mechanisms for cardiotonic steroids-mediated immunogenic cell death in cancer. ICD = Immunogenic cell death, MOMP = Mitochondrial outer membrane permeabilization. Created in https://BioRender.com

Figure 3.

Cardiotonic steroids-mediated apoptosis via PERK/elF2α/ATF4/CHOP pathway. Created in https://BioRender.com

In some contexts, cardiac glycosides can also induce necroptosis, a more inflammatory form of RCD, depending on the cellular environment and specific stimuli. In tumorigenesis, necroptosis can be a double-edged sword. While it can promote tumor suppression by activating immune responses, chronic necroptosis can lead to inflammation, which may aid tumor growth. Convallatoxin (CMT) – a cardiac glycoside derived primarily from the Lily of the Valley (Convallaria majalis) plant was reported to be a potent inhibitor of the human HaCaT keratinocytes cell viability by inducing HaCaT cell death by necroptosis[36,37] and is mediated by oxidative stress. By distorting the ionic balance, CMT destroyed the membrane integrity of the HaCaT cells, leading to cellular lysis. Ouabain exerts anticancer activity through the downregulation of signal transducer and activator of transcription 3 (STAT3).[37] STAT3 is a transcription factor that modulates gene expression and participates in various cellular processes. It regulates cellular proliferation and division, cellular motility, and apoptosis. Therefore, when downregulated, STAT3 can adversely affect tumor-promoting processes, including tumor cell viability and invasiveness.

Furthermore, cardiac glycosides have been shown to convert nonimmunogenic forms of RCD into ICDs, especially in cancer cells. This happens by promoting the release of DAMPs such as ATP and HMGB1, which help recruit and activate immune cells. This also involves the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome. The NLRP3 inflammasome is a multiprotein complex that plays a key role in the body’s innate immune system by detecting harmful stimuli and triggering inflammation. NLRP3 pathway activation is critical to the maturation and release of the inflammatory cytokine IL-1β, apoptosis, and pyroptosis [Figure 2]. The ability of cardiac glycosides to induce ICD has significant implications for cancer immunotherapy. They can enhance the immune system’s ability to recognize and eliminate cancer cells when used alongside chemotherapy. Nevertheless, cardiac glycosides promote the exposure of calreticulin on the surface of dying cancer cells, which acts as an “eat me” signal to dendritic cells (DCs) and promotes antigen presentation to T cells, thereby initiating an immune response. Li et al.[33] demonstrated that oleandrin provoked breast cancer cell ICD through the induction and promotion of calreticulin exposure on the cell surface and the release of HMGB1, heat shock protein 70/90 (HSP70/90), and ATP.

Cardiac glycosides as DNA repair inhibitors

The therapeutic potential of cardiac glycosides has expanded beyond their traditional utilization for heart diseases. They have been identified to impact DNA damage response (DDR) pathways, which are critical to maintaining genomic stability by regulating DNA repair, cell cycle checkpoints, and cell death mechanisms. Ouabain, oleandrin, and digitoxin have demonstrated the ability to induce DNA damage [Table 1], particularly by generating ROS and activating DDR-related proteins such as ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia mutated and Rad3-related (ATR) kinase, and γ-H2A histone family member X (γ-H2AX). Yang et al.[38] reported the ability of ouabain to induce DNA breaks and fragmentation. Notably, ouabain was associated with the increased phosphorylation and expression of p53, poly-ADP ribose polymerase (PARP), γ-H2AX, ATM, ATR, and mediator of DNA damage checkpoint 1 (MDC-1). As reported by Lee et al.,[39] following ionization therapy, digoxin potentiated enhanced DNA damage and reduced levels of DNA repair proteins including protein phosphatase 2A. These actions result in cell cycle arrest, apoptosis, and autophagy-dependent cell death in cancer cells. Furthermore, the structural features of cardiac glycosides, such as their steroid core, lactone ring, and sugar groups influence their lipid solubility and anticancer activity. Understanding their structure-activity relationships can aid in developing more potent derivatives with fewer side effects.

Gan et al.[40] reported digitoxin potentials to induce and activate γ-H2AX before apoptotic chromosomal degradation in HeLa cervical cancer cells. Similar to ouabain, digitoxin enhanced the phosphorylation of ATM and ATR, indicating the activation of the DDR pathway. The altering of the cell cycle in the G₂/M phase, and activating the DDR pathway indicate the activation of the mitochondrial apoptosis in the mechanism of action of digitoxin. The ability of cardiac glycosides to inhibit critical DNA repair pathways makes them effective and suitable for combination with other DNA-damaging cancer treatments, such as cisplatin or radiotherapy. Overtly, such combinations will enhance the therapeutic effect and overcome drug resistance.

Cardiac glycosides as broad-spectrum senolytic and senostatic agents

Senolytics are a class of drugs or compounds that specifically target and eliminate senescent cells. These cells are characterized by a state of permanent growth arrest in response to damage or stress and play a dual role in health and disease cells.[49] While senescent cells can act as tumor suppressors by halting the division of damaged cells, their accumulation in tissues over time contributes to aging, chronic inflammation, and the progression of age-related diseases. The selective removal of senescent cells has been shown to improve tissue function, enhance lifespan, and reduce the burden of several diseases, making senolytics a promising therapeutic approach in the emerging field of geroscience. Senolytics work by selectively inducing apoptosis in senescent cells. Unlike normal cells, senescent cells resist apoptosis due to the upregulation of survival pathways such as BCL-2, PI3K/AKT, and p53/p21. Senolytics target these pathways, making senescent cells more vulnerable to cell death. The mechanisms vary based on the type of senolytic agent but generally involve:

1. Inhibition of antiapoptotic pathways

2. Induction of proapoptotic proteins

3. Inhibition of cellular pumps and channels.

Senescence and senolytic properties in cancer

While senolytic properties are relevant to various age-related conditions, their role in cancer treatment is particularly significant. The induction of senescence in tumor therapy presents a dual challenge; generally, it substantially aids in the therapeutic suppression of tumor growth, however, it simultaneously sets the stage for relapse with potentially increased tumorigenicity.[50,51] In other words, the induction of senescence restricts cancer advancement and enhances therapeutic efficacy; however, persistent senescent cells promote progression, recurrence, and metastasis [Figure 4]. Senescence could be grouped as (1) stress-mediated oncogene-induced senescence (SOIS). In normal cells, the overexpression of oncogenes initiates oncogene-induced senescence through replicative stress and serves as a significant natural tumor suppression mechanism,[52] and (2) treatment-induced senescence (TIS). For instance, DNA-damaging therapies have been reported to provoke both apoptosis and senescence in cancer cells.[52] Similarly, chemotherapy and radiotherapy initiate senescence in cancer cells. At low doses, chemotherapy induces cellular senescence and typically causes programmed cell death at high doses.[53,54] Many senolytics block pathways that allow senescent cells to resist apoptosis, such as the BCL-2 family of proteins. Some agents increase the expression of proapoptotic proteins, such as Noxa, which directly leads to cell death.[55] Cardiac glycosides, such as digoxin and ouabain, eliminate senescent tumor cells by disrupting ion homeostasis through Na⁺/K⁺-ATPase inhibition [Figure 4]. This induces apoptosis selectively in senescent cells, reducing protumorigenic inflammation mediated by the senescence-associated secretory phenotype (SASP) factors such as cytokines, chemokines, and extracellular matrix proteases. As senolytics, cardiac glycosides exert their effects in cancer and noncancer disease models. For instance, Triana-Martínez et al.[49] demonstrated the senolytic effect of digoxin in a mouse model of lung fibrosis induced by intratracheal administration of senescent human cells. Furthermore, the coadministration of digoxin and gemcitabine elicited potent synergistic effects against lung adenocarcinoma A549 and melanoma SK-MEL-103 compared to when administered singly.[49] The duo reportedly resulted in tremendous tumor reduction. Similarly, the coadministration of digoxin and doxorubicin provoked drastic antitumor effects, especially by preventing cell survival.[49] This suggests that cardiac glycosides may be exploited as combinations in the chemotherapy of cancer. The combination of a senescence-inducing agent with a senolytic agent to respectively halt tumor cells irreversibly and provoke cell death is more effective than monotherapy with traditional anticancer drugs.

Figure 4.

Senescence and senolytic properties of cardiac glycosides. (a) Molecular sites and targets. (b) Mechanisms of action. OIS = Oncogene-induced senescence, TIS = Treatment-induced senescence, SASP = Senescence-associated secretory phenotype, CGs = Cardiac glycosides. Created in https://BioRender.com

Berendes et al.[55] explored the role of cardiac glycosides as agents that selectively eliminate senescent cells. The study identified ouabain as a potent senolytic that kills senescent cells via apoptosis. Using drug screens and experiments in cellular models, the authors demonstrate that ouabain, digitoxin, and digoxin, selectively kill senescent cells across different models of senescence, such as SOIS and TIS. Importantly, ouabain triggers apoptosis in senescent cells by activating proapoptotic proteins, including Noxa, and disrupting cellular ion balance by inhibiting Na⁺/K⁺-ATPase. Furthermore, ouabain was reported to enhance the effects of anticancer therapies by eliminating senescent cells induced by chemotherapy or radiotherapy, which may reduce cancer recurrence and therapy side effects. Nevertheless, in mouse models, ouabain reduced the number of senescent cells, improved metabolic parameters, and decreased chronic inflammation, indicating its potential for broader therapeutic use in age-related diseases and cancer.[18,55]

Unlike senolytics, senostatic agents do not eliminate senescent cells; rather, they impede paracrine signaling, thereby obstructing the accumulation of senescence caused by the bystander effect [Figure 4]. The latter (i.e. the bystander effect) is a process where senescent cells release stress signals resulting in ROS-mediated and NF-κB-activated DNA damage of nearby cells, thereby making them senescent too. Antioxidants or NF-κB inhibitors can serve as effective senostatic agents, and there is evidence suggesting that various flavonoids, polyphenols, and other phytochemicals may exhibit senostatic properties.[56] Cardiac glycosides can inhibit the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a signaling pathway that regulates inflammation and immune responses. Oleandrin,[57] lanatoside C,[58] digitoxin, and structurally-related cardiac glycosides[59] exert their anticancer effects via the inhibition of this pathway [Figure 3].

Other pharmacologic potentials of cardiac glycosides

Antiviral potentials

Cardiac steroids such as lanatoside C, ouabain, digoxin, and digitoxin have been repurposed for virus-mediated diseases, including the most recent SARS-COV-2 (COVID-19) infection. They disrupt (1) the early stages of the viral life cycle, (2) synthesis and RNA processing, and (3) synthesis and release of viral proteins.[15,17,60,61] Their primary target is the life cycle of the virus. Although the exact antiviral mechanisms of this class of drug differ across viral species, in general, they can disrupt both the early and late stages of the life cycle. They induce viral cell arrest by disrupting the ion balance, and autophagy of the host cell, or triggering various signaling cascades including the nonreceptor tyrosine kinase (c-Src) pathway.[15,17] Their interaction with the Na⁺-K⁺-ATPase causes the pump to undergo conformational changes leading to the phosphorylation of c-Src and corresponding interaction with and phosphorylation of EGFR. This interaction results in the activation of various cascade proteins, such as Sos, growth factor receptor-bound protein 2, ras, and Src homology 2 domain-containing transforming protein 1. The hallmark of this complex signaling pathway is ROS-mediated apoptosis or autophagy.[17] Burkard et al.[62] investigated the effect of cardiac glycosides in a coronavirus infection model and reported the role of the α-1 subunit of Na⁺-K⁺-ATPase in the Src-mediated viral entry. The authors further highlighted that by preventing viral entry, cardiac glycosides potentiated the accumulation of virions close to the cell surface and a drastic reduction of plasma membrane fusion.[62]

In transmissible gastroenteritis coronavirus (TGEV), ouabain exerts its antiviral actions by activating the phosphatidylinositol 3-kinase/3phosphoinositide-dependent protein kinase 1/p90 ribosomal S6 kinase (PI3K/PDK1/RSK2) pathway.[63] These cardiac steroids have also been reported to provoke their antiviral effects via mechanisms other than the inhibition of the Na⁺-K⁺-ATPase [Table 2]. For instance, Yang et al.[64] demonstrated the antiviral mechanisms of cardiac glycosides to involve the down-regulation and proteolysis of the Janus kinase 1 (JAK1). This proteolysis is initiated by the activation of neural precursor cells of the downregulated protein 4 E3 ubiquitin-protein ligase family and is activated by ouabain and mediated by membrane surface proteins other than the sodium-potassium pump. Similarly, digoxin has been reported to suppress the viral replication of coronaviruses using the same mechanism.[64] Cardiac glycosides have also been tried against the Ebola virus. Digitoxin, lanatoside C, and strophanthidin have been reported to exert potent antiviral actions against the Ebola virus.[65] This confirms the fact that the antiviral actions of cardiac glycosides are not restricted to only positive-sense ssRNA viruses (e.g., coronaviruses), but also negative-sense ssRNA viruses (e.g., Ebola virus). Similarly, ouabain and digoxin have been reported to show potent antiviral properties against the Ebola virus, including the inhibition of viral entry into the host cells.[66]

Table 2.

Antiviral activities of some cardiac glycosides

Compound	Mechanism	Target virus	Reference
Ouabain and bufalin	Na+/K+-ATPase inhibition and ATP1A1-mediated Src tyrosine kinase phosphorylation	Coronaviruses	Burkard et al.[62]
Ouabain	Na+/K+-ATPase inhibition and PI3K/PDK1/RSK2 pathway activation	SARS-CoV-2	Lingemann et al.[63]
Digoxin	Na+/K+-ATPase (sodium pump) independent JAK1 down-regulation and proteolysis	Coronaviruses	Yang et al.[64]
Digitoxin, lanatoside C, and strophanthidin	Inhibition of viral replication and entry	Coronaviruses and Ebola virus	Edwards et al.[65]
Ouabain and digitoxin	Inhibition of mRNA and viral protein expression of SARS-CoV-2	SARS-CoV-2	Cho et al.[60]
Ouabain and digoxin	Inhibition of viral replication and entry	Ebola virus	Dowall et al.[66]
Digitoxigenin, digoxin, digitoxin, digoxigenin, and ouabain	Inhibition of ACE2 binding to the SARS-CoV-2 spike proteins	SARS-CoV-2	Caohuy et al.[67]
Digoxin	Inhibition of ACE2 binding to the SARS-CoV-2 spike proteins	SARS-CoV-2	Ardiana et al.[68]
Digoxin	Inhibition of NFκB activation and prevention of cytokine storms	Influenza (A/Wuhan/H3N2/359/95 strain)	DeDiego et al.[69]
Ouabain	Inhibition of NFκB and NFAT resulting in the blockage of TNFα, GRO/KC, MCP1, MIP2, IL-1β, TGFβ, and IFNγ	SARS-CoV-2	DeDiego et al.[69]

NFAT=Nuclear factor of activated T-cells, IFNγ=Interferon-gamma, TNFα=Tumor necrosis factor-alpha, JAK1=Janus kinase 1, IL=Interleukin

Anti-inflammatory potentials

Recent research has expanded the role of cardiac glycosides beyond heart failure. Notably, they exhibit significant anti-inflammatory actions in various animal models of both acute and chronic inflammation.[5,70] They modulate NF-κB signaling, a key pathway involved in inflammation, and inhibit the release of proinflammatory cytokines (e.g., TNF-α, IL-6, and IL-1β), reduce leukocyte infiltration, and modulate cellular immune responses.[71,72,73] This includes their influence on TH17 cell differentiation, a critical element in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis, and their impact on inflammation-driven cancer.[5,74] Preclinical evidence shows the effectiveness of cardiac glycosides in inflammation-related conditions such as sepsis, ischemia-reperfusion injury,[75] and colitis.[5]

Expression of Na⁺/K⁺-ATPase subunits in cancer tissues

Numerous alterations in Na⁺/K⁺-ATPase expression have been noted in cancer cells, including increased activity as malignant cells transform. The α1-subunit or isoform is overexpressed in aggressive cancers, promoting cell proliferation; whereas the α2/α3-subunits are implicated in altered calcium signaling in certain tumor types. In this regard, differences in the expression of the various subunits of the enzyme in comparison to normal tissues have been reported.[76] For example, renal carcinoma cells, nonsmall cell lung cancer, and gliomas have been shown to exhibit elevated expression of the α1-isoform. Furthermore, PBI-05204 (oleandrin) reportedly inhibits the α-3 subunit Na⁺/K⁺-ATPase pump. The relative expression of the α-3 subunit in tumor cells also correlates with proliferation. The expression of the α3-isoform is elevated in colon carcinoma, whereas that of the α1-isoform is decreased in prostate carcinoma.[77] It is unclear how the various isoforms are distributed in pathological circumstances like cancer; however, the α-subunit is thought to be a target for novel anticancer treatments. The expression of the α1-isoform is generally high in the early stages of carcinogenesis and low in the later stages, with an increase in the expression of the α3-isoform.[76,78] As a result, Na⁺/K⁺-ATPase might serve as both a molecular target and biomarker for cancer treatment. In this regard, the therapeutic potential of Na⁺/K⁺-ATPase modulators, including cardiac glycosides has been assessed. For instance, as earlier stated, ouabain and other CS can induce apoptosis, cell cycle arrest, or even autophagy[78,79] in various cancer cell types. Elevated levels of endogenous CS, such as ouabain, in the blood of cancer patients have been reported, suggesting potential roles as biomarkers for the disease prognosis.

Clinical trials of cardiac glycosides in cancer

Despite being primarily known for the treatment of heart failure and certain types of cardiac arrhythmias, CS have also been shown to have antiproliferative effects on various cancer cells, including breast, lung, prostate, colon, and leukemia. Several CSs have been tested in several phases of clinical trials to determine their safety profile, efficacy and mechanism of action, dose-limiting toxicities, and maximum tolerated dose. For example, the safety profile of two CS, Anvirzel™ and PBI-02504 have been completed in a phase 1 clinical trial. CSs may exert their antiproliferative and/or anticancer effects by: (1) inhibiting DNA topoisomerase II, (2) activating the Src kinase pathway, (3) inducing or inhibiting autophagy, (4) inhibiting autosis, and (5) sensitizing drug-resistant glioblastoma cells to necroptosis.[80] For instance, oleandrin inhibits FGF-2 export, NF-Kβ activation, Akt phosphorylation, p70S6K, and decreases mTOR activity.[81] Anvirzel™ provoked a time-and dose-dependent growth inhibition of U87 cells. Furthermore, the antitumor effects of Anvirzel™ were associated with the inhibition of GSK-3, NOS, and HIF1-α expressions and activation of ERK in U87 cells compared to the control.[82] The antileukemic activity of UNBS1450,[80] a hemisynthetic cardenolide has been demonstrated. Phase II trials of digoxin in breast and colorectal cancer have shown improved survival rates when combined with standard chemotherapy. Preclinical models of ouabain highlight its potential in reducing tumor growth, with trials exploring its safety and efficacy in humans. Some of these molecules are in ongoing trials while others have completed their trials. These are presented in Tables 3 and 4.

Table 3.

Completed clinical studies on cardiotonic steroids in cancer patients

Compound	Cancer type	Study types (number total subjects)	Main findings	Trial ID	Reference
Digoxin + Rosuvastatin + Enzalutamide	Newly diagnosed metastatic prostate cancer or disease progression	Phase I single-arm trial n=24	Reported that the oral androgen receptor inhibitor (enzalutamide) mildly inhibited P-glycoprotein, thereby increasing the plasma concentration (Cmax) and AUC of digoxin by 17% and 29%, respectively Low potential for drug–drug interactions between digoxin, rosuvastatin, and enzalutamide, and requires no dose adjustment	NCT04094519	Poondru et al.[83]
Cinobufacini +Pt-based chemotherapy	Advanced NSCLC	Meta-analysis of 19 randomized controlled trials obtained from English and Chinese database n=1564 (control [n=768]; treatment [n=796])	Combination therapy showed a significant increase in objective response rate, 1- and 2-year survival times, and percentage of CD3+, CD4+ and CD4+/CD8+. Also reported was a significant decrease in therapy-induced emesis, thrombocytopenia and leukocytopenia	NA	Peng et al.[84]
Digoxin	Prostate cancer	8 cohort, 2 case-control, and one systematic review n=92,633 (digoxin users, n=15,835)	The authors reported a decreased risk of prostate cancer among digoxin users and an increased prostate cancer-specific mortality among digoxin users	NA	Zhao et al.[85]
Oleandrin (PBI-05204)	Stage IV metastatic pancreatic adenocarcinoma	Phase II, multicenter n=38	The administration of PBI-05204 capsules (0.225 mg/kg/day) twice daily on a 28-day cycle was associated with an overall 26.3% survival at 4.5 months, a median progression-free survival of 56 days, an objective response rate (n=1), and a disease control rate (n=8) Recorded AE were nausea, emesis, fatigue, reduced appetite, abdominal pain, and diarrhea 55.3% of the population had Grade 3 AE (anemia, nausea, emesis, and dehydration)	NCT02329717	Roth et al.[86]
Digoxin	All types of cancer	Retrospective cohort obtained from the Taiwan National Health Insurance Database, 1998–2010 n=1516 (digoxin, n=758)	Digoxin versus β-blocker was associated with increased cancer incidence at 4-year (HR=1.99, 95% CI 1.22–3.01, P=0.006), and 8-year (HR=1.46, 95% CI 1.01–2.15, P=0.054)	NA	Tai et al.[87]
Cinobufacini +Chemotherapy (FOLFOX or CAPOX)	Stage III or high-risk stage II CRC	Phase II n=250	The primary endpoint was 3-year DFS, and the secondary endpoints were 3-year OS and toxicity. Co-administration of cinobufacini with chemotherapy significantly increased 3-year DFS (median 31.6 months) compared to the chemotherapy group (median 28.7 months, P=0.027) The study reported decreased rates of diarrhea, leukopenia, and neutropenia with no grade 4 AE	Unspecified	Li et al.[88]
Digoxin	Prostate cancer	Retrospective cohort obtained from the Taiwan National Health Insurance 1998–2003. n=6387 (digoxin users, n=2154)	Significantly decreased risk of prostate cancer in digoxin versus nonusers. Prolonged use was associated with decreased risk for high-grade prostate cancer	NA	Lin et al.[89]

FOLFOX=Folinic acid, fluorouracil and oxaliplatin, CAPOX=Capecitabine and oxaliplatin, CRC=Colorectal cancer, NA=Not applicable, NSCLC=Nonsmall cell lung cancer, DFS=Disease-free survival, OS=Overall survival, AE=Adverse event, HR=Hazard ratio, AUC=Area under the curve, CI=Confidence interval

Table 4.

Cardiotonic steroids in ongoing clinical trials for cancer therapy

Compound	Cancer type	The trial phase and outcome measured (number total subjects)	Current status	Trial ID
Cinobufacini	Primary liver cancer	Open-label phase IV (n=90)	Not known	NCT03843229
		Tumor size, tumor marker (carcinoembryonic antigen, alpha-fetoprotein), AE, QoL; the number of immune cells (CD4+, CD8+, T cell), and cytokines (IL-2, IL-4, IL-6, TNF-a, INF-c) in the blood
Digoxin	Recurrent/refractory metastatic advanced pancreatic cancer	Single-arm Phase IIA trial (n=25)	Recruitment ended in December 2024	NCT06030622
		Safety and tolerability
		Tumour biomarkers: BIRC5, CA19-9, CEA
Cinobufacini	Gastrointestinal cancer	Open-label phase IV (n=120)	Not known	NCT02860429
		Control (FOLFOX)	Study results yet to be publicly available
		Chemotherapy-induced peripheral neuropathy, QoL, AE, nerve toxicity
		Biomarkers: IL-6, TNF, number of immune cells, and level of stress hormone in the blood
Digoxin	Kaposi’s classic Kaposi’s endemic Kaposi’s sarcoma	Phase II (n=17) Tumour response, time-to-progression, AE	Not known	NCT02212639
Cinobufacini	Unresectable hepatocellular carcinoma	Phase III (n=261)	Ongoing recruitment till August 2025	NCT05594927
		Aim: To examine the efficacy of icaritin versus cinobufacini
		OS time, time-to-progression, progression-free survival, objective response rate, disease control rate, and treatment-associated AE
Digoxin	Resectable pancreatic cancer	Single-arm phase IIA trial (n=20)	Ongoing recruitment till February 2025	NCT04141995
		Number of patients who had resection surgery, treatment response, progression-free survival, OS, circulating free DNA tumour, and treatment-associated AE (severe diarrhoea and thrombocytopenia)
Cinobufacini	Diffuse large B cell lymphoma	Multi-center phase II/III (n=316)	Not known	NCT02871869
		Progression-free survival, objective response rate, OS, treatment-associated AE, and relating cinobufacini action with Na+/K+-
		ATPase α3 subunit
Digoxin	Prostate cancer	Phase I (n=18)	Not known	NCT04621669
		Determination of the pharmacokinetic parameters of digoxin, metformin, and rosuvastatin
Cinobufacini	Gastrointestinal cancer with ascites	Phase I (n=48)	Not known	NCT02530398
		Treatment-associated AE
Digitoxin (CP4071)	Advanced sarcoma	Phase II (n=25 patients, estimated)	Not known	NCT00017446
		Response rate, safety profile
Cinobufacini	esophageal squamous cell carcinoma	Open-label phase II trial (n=134)	Not known	NCT02647125
		Local control rate, overall survival, progression-free survival
Digoxin	Metastatic breast cancer	Phase I (n=9)	Ongoing recruitment	NCT03928210
		CTC cluster assessment (size, number, and dissolution time)
Cinobufacini	Advanced hepatocellular carcinoma	Phase II (n=120)	Not known	NCT01715532
		Aim: Correlating prognosis and expression of Na+/K+-ATPase α3 subunit
		Disease progression, free and OS

FOLFOX=Folinic acid, fluorouracil and oxaliplatin, IL-6=Interleukin-6, TNF=Tumor necrosis factor, QoL=Quality of life, INF=Interferon, CTC=Circulating tumor cell, OS=Overall survival, AE=Adverse event

Conclusion

This manuscript provides a comprehensive review of the physiological mechanisms, pharmacological potentials, and mechanisms of actions of cardiac glycosides – an old class of drug currently repurposed for an array of medical conditions, especially neoplastic and viral diseases. Cardiac glycosides continue to play an essential role in the management of heart failure and atrial fibrillation, offering unique benefits due to their positive inotropic and vagomimetic effects. However, their narrow therapeutic window and potential for toxicity necessitate careful patient selection, dosing, and monitoring. Emerging research suggests potential new applications in oncology, although these remain experimental. As our understanding of these molecules appreciates, future developments may lead to safer and more effective use of cardiac glycosides in clinical practice. Research into the development of safer cardiac glycosides with a wider therapeutic index is ongoing. In addition, the exploration of their role in cancer therapy offers a promising new avenue, albeit one that requires further investigation. Advances in pharmacogenomics may also provide insights into individual susceptibility to cardiac glycoside toxicity, paving the way for personalized medicine approaches in their use.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.