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Heart failure (HF) represents the terminal stage of cardiovascular diseases, with limited therapeutic options currently available. Calotropin (CAL), a cardenolide isolated from Calotropis gigantea, exhibits a similar chemical structure and inhibitory effect on Na+/K+-ATPase to digoxin, a positive inotropic drugs used in heart failure treatment. However, the specific effect of calotropin in ischemic HF (IHF) remains unknown. The objective of this study is to assess the anti-HF effect and clarify its underlying mechanisms. The left anterior descending (LAD) artery ligation on Male Sprague-Dawley (SD) rats was used to construct ischemic HF model. Daily administration of CAL at 0.05 mg/kg significantly enhanced ejection fraction (EF) and fractional shortening (FS), while inhibiting cardiac fibrosis in IHF rats. CAL reduced the OGD/R-induced H9c2 cell injury. Furthermore, CAL upregulated the expression of SERCA2a and SIRT1. The cardioprotective effect of CAL against IHF was abrogated in the presence of the SIRT1 inhibitor EX527. Notably, we identified FOXD3 as a pivotal transcription factor mediating CAL-induced SERCA2a regulation. CAL promoted the deacetylation and nuclear translocation of FOXD3 in a SIRT1-dependent manner. In conclusion, our study explores a novel mechanism of calotropin for improving cardiac dysfunction in ischemic heart failure by regulating SIRT1/FOXD3/SERCA2a pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Lowering the levels of the cellular prion protein (PrPC) is widely considered a promising strategy for the treatment of prion diseases. Building on work that established immediate spatial proximity of PrPC and Na+, K+-ATPases (NKAs) in the brain, we recently showed that PrPC levels can be reduced by targeting NKAs with their natural cardiac glycoside (CG) inhibitors. We then introduced C4'-dehydro-oleandrin as a CG with improved pharmacological properties for this indication, showing that it reduced PrPC levels by 84% in immortalized human cells that had been differentiated to acquire neural or astrocytic characteristics. Here we report that our lead compound caused cell surface PrPC levels to drop also in other human cell models, even when the analyses of whole cell lysates suggested otherwise. Because mice are refractory to CGs, we explored guinea pigs as an alternative rodent model for the preclinical evaluation of C4'-dehydro-oleandrin. We found that guinea pig cell lines, primary cells, and brain slices were responsive to our lead compound, albeit it at 30-fold higher concentrations than human cells. Of potential significance for other PrPC lowering approaches, we observed that cells attempted to compensate for the loss of cell surface PrPC levels by increasing the expression of the prion gene, requiring daily administration of C4'-dehydro-oleandrin for a sustained PrPC lowering effect. Regrettably, when administered systemically in vivo, the levels of C4'-dehydro-oleandrin that reached the guinea pig brain remained insufficient for the PrPC lowering effect to manifest. A more suitable preclinical model is still needed to determine if C4'-dehydro-oleandrin can offer a cost-effective complementary strategy for pushing PrPC levels below a threshold required for long-term prion disease survival., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: GS and DW declare that they are co-inventors on patent applications named ‘Compounds for altering levels of one or more NKA alpha subunits and their use in treating prion diseases or brain diseases associated with cellular prion protein’ filed in the US and Canada as well as a European patent application named ‘Compounds and methods to treat prion and related diseases’ based on a PCT international filing (PCT/CA2022/050358). These patent applications describe the use of CGs for the purpose of reducing steady-state levels of PrPC. This potential conflict-of-interest does not affect the adherence of all authors to journal policies on the sharing of data and materials. This does not alter our adherence to PLoS One policies on sharing data and materials., (Copyright: © 2024 Eid et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The treatment of non-small cell lung cancer (NSCLC) is known as a significant level of unmet medical need in spite of the progress in targeted therapy and personalized therapy. Overexpression of the Na + /K + -ATPase contributes to NSCLC progression, suggesting its potentiality in antineoplastic approaches. Epi-reevesioside F, purified from Reevesia formosana, showed potent anti-NSCLC activity through inhibiting the Na + /K + -ATPase, leading to internalization of α1- and α3-subunits in Na + /K + -ATPase and suppression of Akt-independent mTOR-p70S6K-4EBP1 axis. Epi-reevesioside F caused a synergistic amplification of apoptosis induced by gefitinib but not cisplatin, docetaxel, etoposide, paclitaxel, or vinorelbine in both NCI-H460 and A549 cells. The synergism was validated by enhanced activation of the caspase cascade. Bax cleavage, tBid formation, and downregulation of Bcl-xL and Bcl-2 contributed to the synergistic apoptosis induced by the combination treatment of epi-reevesioside F and gefitinib. The increase of membrane DR4 and DR5 levels, intracellular Ca 2+ concentrations, and active m-calpain expression were responsible for the caspase-8 activation and Bax cleavage. The increased α-tubulin acetylation and activation of MAPK (i.e., p38 MAPK, Erk, and JNK) depending on cell types contributed to the synergistic mechanism under combination treatment. These signaling pathways that converged on profound c-Myc downregulation led to synergistic apoptosis in NSCLC. In conclusion, the data suggest that epi-reevesioside F inhibits the Na + /K + -ATPase and displays potent anti-NSCLC activity. Epi-reevesioside F sensitizes gefitinib-induced apoptosis through multiple pathways that converge on c-Myc downregulation. The data support the inhibition of Na + /K + -ATPase as a switch-on mechanism to sensitize gefitinib-induced anti-NSCLC activity., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The present study aimed to examine the effect of Calotropis procera (Aiton) and its major cardenolides; calotropin and gomphogenin on ovariectomy-induced osteoporosis in rats. Osteoporotic rats were orally treated with C. procera alcoholic extract (100 mg/kg), calotropin (CLT; 100 μg/kg) and gomphogenin (GPG; 100 μg/kg) for 14 consecutive days. Bone resorption/formation biomarkers; bone specific alkaline phosphatase (BALP), osteoprotegerin (OPG) and nuclear factor-κβ ligand (RANKL) as well as serum calcium and phosphorus were assessed 24 h after last doses of treatments. Serum levels of estradiol (E2) and catalase were also measured. Oral treatment with C. procera extract, CLT and GPG caused E2 restoration to normal level with a marked regulation in the RANKL/OPG axis. Serum phosphorus and calcium were up-leveled whereas BALP was downregulated. Histopathological examination, bone histomorphometric analysis and immunohistochemical staining for osteopontin (OPN) inspection further emphasized the aforementioned outcomes. The results revealed the superiority of CLT and to a lesser extent GPG osteoporotic effect over C. procera extract. Molecular docking of the two compounds on ER-α and RANKL/OPG complex showed noteworthy binding affinities which also confirmed the supremacy of CLT due to the additional hydrogen bonding of the hydroxyl groups of the sugar moiety with RANKL/OPG complex. Finally, it is concluded that CLT and GPG from C. procera hinder bone turnover by decreasing osteoclastic bone cells activity and increasing calcium mineralization thus suppressing bone remodeling and preventing bone infirmity in OVX osteoporotic rats directly via binding to RANKL/OPG complex and ER-α and indirectly through elevating level of E2., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Despite active clinical trials on the use of Oleandrin alone or in combination with other drugs for the treatment of solid tumors, the potential synergistic effect of Oleandrin with radiotherapy remains unknown. This study reveals a new mechanism by which Oleandrin targets ATM and ATR kinase-mediated radiosensitization in lung cancer. Various assays, including clonogenic, Comet, immunofluorescence staining, apoptosis and Cell cycle assays, were conducted to evaluate the impact of oleandrin on radiation-induced double-strand break repair and cell cycle distribution. Western blot analysis was utilized to investigate alterations in signal transduction pathways related to double-strand break repair. The efficacy and toxicity of the combined therapy were assessed in a preclinical xenotransplantation model. Functionally, Oleandrin weakens the DNA damage repair ability and enhances the radiation sensitivity of lung cells. Mechanistically, Oleandrin inhibits ATM and ATR kinase activities, blocking the transmission of ATM-CHK2 and ATR-CHK1 cell cycle checkpoint signaling axes. This accelerates the passage of tumor cells through the G2 phase after radiotherapy, substantially facilitating the rapid entry of large numbers of inadequately repaired cells into mitosis and ultimately triggering mitotic catastrophe. The combined treatment of Oleandrin and radiotherapy demonstrated superior inhibition of tumor proliferation compared to either treatment alone. Our findings highlight Oleandrin as a novel and effective inhibitor of ATM and ATR kinase, offering new possibilities for the development of clinical radiosensitizing adjuvants., (© 2024 John Wiley & Sons Ltd.)

Background & objectives Despite advancements in antiretroviral therapy, drug-resistant strains of HIV (human immunodeficiency virus) remain a global health concern. Natural compounds from medicinal plants offer a promising avenue for developing new HIV-1 PR (protease) inhibitors. This study aimed to explore the potential of compounds derived from Calotropis procera, a medicinal plant, as inhibitors of HIV-1 PR. Methods This in silico study utilized natural compound information and the crystal structure of HIV-1 PR. Molecular docking of 17 steroidal cardenolides from Calotropis procera against HIV-1 PR was performed using AutoDock 4.2 to identify compounds with higher antiviral potential. A dynamic simulation study was performed to provide insights into the stability, binding dynamics, and potential efficacy of the top potential antiviral compound as an HIV-1 therapeutic. Results We found that all tested cardenolides had higher binding affinities than Amprenavir, indicating their potential as potent HIV-1 PR inhibitors. Voruscharin and uscharidin displayed the strongest interactions, forming hydrogen bonds and hydrophobic interactions with HIV-1 PR. Voruscharin showed improved stability with lower RMSD (Root Mean Square Deviation) values and reduced fluctuations in binding site residues but increased flexibility in certain regions. The radius of gyration analysis confirmed a stable binding pose between HIV-1 PR and Voruscharin. Interpretation & conclusions These findings suggest that Calotropis procera could potentially be a source of compounds for developing novel HIV-1 PR inhibitors, contributing to the efforts to combat HIV. Further studies and clinical trials are needed to evaluate the safety and efficacy of these compounds as potential drug candidates for the treatment of HIV-1 infection.

Plants have evolved a diverse arsenal of defensive secondary metabolites in their evolutionary arms race with insect herbivores. In addition to the bottom-up forces created by plant chemicals, herbivores face top-down pressure from natural enemies, such as predators, parasitoids and parasites. This has led to the evolution of specialist herbivores that do not only tolerate plant secondary metabolites but even use them to fight natural enemies. Monarch butterflies (Danaus plexippus) are known for their use of milkweed chemicals (cardenolides) as protection against vertebrate predators. Recent studies have shown that milkweeds with high cardenolide concentrations can also provide protection against a virulent protozoan parasite. However, whether cardenolides are directly responsible for these effects, and whether individual cardenolides or mixtures of these chemicals are needed to reduce infection, remains unknown. We fed monarch larvae the four most abundant cardenolides found in the anti-parasitic-milkweed Asclepias curassavica at varying concentrations and compositions to determine which provided the highest resistance to parasite infection. Measuring infection rates and infection intensities, we found that resistance is dependent on both concentration and composition of cardenolides, with mixtures of cardenolides performing significantly better than individual compounds, even when mixtures included lower concentrations of individual compounds. These results suggest that cardenolides function synergistically to provide resistance against parasite infection and help explain why only milkweed species that produce diverse cardenolide compounds provide measurable parasite resistance. More broadly, our results suggest that herbivores can benefit from consuming plants with diverse defensive chemical compounds through release from parasitism., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Phytochemical investigations on the fruits of Cascabela thevetia (L.) Lippold led to obtain three new cardenolides ( 1 - 3 ) and five known analogues ( 4 - 7 ). Their structures were elucidated by means of UV, IR, HR-ESI-MS, 1D and 2D NMR spectroscopic data analysis. Compounds 1 and 2 represent the first examples of naturally occurring cardenolides with 19- nor -5(10)-ene group and α - l -3-demethyl-thevetose, respectively. Compound 3 is a rare C -nor- D-homocardenolide in nature. All isolated cardenolides ( 1 - 7 ) were evaluated for their cytotoxic activities against four human cancer cell lines (MCF-7, HCT-116, HeLa and HepG2), and the results indicated the compounds with sugar units ( 1 , 2 , 4 , and 5 ) exhibited stronger cytotoxic activities with IC 50 values ranging between 0.022 and 0.308  μ M.

The discovery that Na/K-ATPase acts as a signal transducer led us to investigate the structural diversity of cardiotonic steroids and study their ligand effects. By applying Na/K-ATPase activity assay-guided fractionation, we isolated a total of 20 cardiotonic steroids from Streptocaulon juventas , including an undescribed juventasoside B (10: ) and 19 known cardiotonic steroids. Their structures have been elucidated. Using our platform of purified Na/K-ATPase and an LLC-PK1 cell model, we found that 10: , at a concentration that induces less than 10% Na/K-ATPase inhibition, can stimulate the Na/K-ATPase/Src receptor complex and selectively activate downstream pathways, ultimately altering prostate cancer cell growth. By assessing the ligand effect of the isolated cardiotonic steroids, we found that the regulation of cell viability by the isolated cardiotonic steroids was not associated with their inhibitory potencies against Na/K-ATPase activity but reflected their ligand-binding affinity to the Na/K-ATPase receptor. Based on this discovery, we identified a unique active cardiotonic steroid, digitoxigenin (1: ), and verified that it can protect LLC-PK1 cells from hypoxic injury, implicating its potential use in ischemia/reperfusion injury and inducing collagen synthesis in primary human dermal fibroblast cells, and implicating that compound 2: is the molecular basis of the wound healing activity of S. juventas ., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The biosynthesis of cardiac glycosides, broadly classified as cardenolides and bufadienolides, has evolved repeatedly among flowering plants. Individual species can produce dozens or even hundreds of structurally distinct cardiac glycosides. Although all cardiac glycosides exhibit biological activity by inhibiting the function of the essential Na + /K + -ATPase in animal cells, they differ in their level of inhibitory activity. For within- and between-species comparisons of cardiac glycosides to address ecological and evolutionary questions, it is necessary to not only quantify their relative abundance, but also their effectiveness in inhibiting the activity of different animal Na + /K + -ATPases. Here we describe protocols for characterizing the amount and toxicity of cardenolides from plant samples and the degree of insect Na + /K + -ATPase tolerance to inhibition: (1) an HPLC-based assay to quantify the abundance of individual cardenolides in plant extracts, (2) an assay to quantify inhibition of Na + /K + -ATPase activity by plant extracts, and (3) extraction of insect Na + /K + -ATPases for inhibition assays., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cardiac glycosides are a large class of secondary metabolites found in plants. In the genus Asclepias , cardenolides in milkweed plants have an established role in plant-herbivore and predator-prey interactions, based on their ability to inhibit the membrane-bound Na + /K + -ATPase enzyme. Milkweed seeds are eaten by specialist lygaeid bugs, which are the most cardenolide-tolerant insects known. These insects likely impose natural selection for the repeated derivatisation of cardenolides. A first step in investigating this hypothesis is to conduct a phytochemical profiling of the cardenolides in the seeds. Here, we report the concentrations of 10 purified cardenolides from the seeds of Asclepias curassavica . We report the structures of new compounds: 3- O - β -allopyranosyl coroglaucigenin ( 1 ), 3-[4'- O - β -glucopyranosyl- β -allopyranosyl] coroglaucigenin ( 2 ), 3'- O - β -glucopyranosyl-15- β -hydroxycalotropin ( 3 ), and 3- O - β -glucopyranosyl-12- β -hydroxyl coroglaucigenin ( 4 ), as well as six previously reported cardenolides ( 5 - 10 ). We test the in vitro inhibition of these compounds on the sensitive porcine Na + /K + -ATPase. The least inhibitory compound was also the most abundant in the seeds-4'- O - β -glucopyranosyl frugoside ( 5 ). Gofruside ( 9 ) was the most inhibitory. We found no direct correlation between the number of glycosides/sugar moieties in a cardenolide and its inhibitory effect. Our results enhance the literature on cardenolide diversity and concentration among tissues eaten by insects and provide an opportunity to uncover potential evolutionary relationships between tissue-specific defense expression and insect adaptations in plant-herbivore interactions.

There is growing interest in exploring Digitalis cardenolides as potential antiviral agents. Hence, we herein investigated the influence of structural features and lipophilicity on the antiherpes activity of 65 natural and semisynthetic cardenolides assayed in vitro against HSV-1. The presence of an α,β-unsaturated lactone ring at C-17, a β-hydroxy group at C-14 and C-3β-OR substituents were considered essential requirements for this biological activity. Glycosides were more active than their genins, especially monoglycosides containing a rhamnose residue. The activity enhanced in derivatives bearing an aldehyde group at C-19 instead of a methyl group, whereas inserting a C-5β-OH improved the antiherpes effect significantly. The cardenolides lipophilicity was accessed by measuring experimentally their log P values (n-octanol-water partition coefficient) and disclosed a range of lipophilicity (log P 0.75±0.25) associated with the optimal antiherpes activity. In silico studies were carried out and resulted in the establishment of two predictive models potentially useful to identify and/or optimize novel antiherpes cardenolides. The effectiveness of the models was confirmed by retrospective analysis of the studied compounds. This is the first SAR study addressing the antiherpes activity of cardenolides. The developed computational models were able to predict the active cardenolides and their log P values., (© 2022 The Authors. Chemistry & Biodiversity published by Wiley-VHCA AG, Zurich, Switzerland.)

Oleandrin, a cardiac glycoside isolated from the leaves of Nerium oleander, has known effects on the heart. Evidence from recent studies have highlighted its potential for anticancer properties. Therefore, we aimed to investigate the effects of oleandrin on cancer cell proliferation, viability and apoptosis in vitro and in vivo. We performed a systematic search in six electronic databases up to Jan 2022. We extracted information about the effects of oleandrin on cell proliferation, cell viability, apoptosis and/or cell cycle arrest in in vitro studies, and the effects on tumor size and volume in animal experimental models. We have retrieved 775 scientific studies. 14 studies met the inclusion criteria. They investigated the effects of oleandrin on breast, lung, pancreatic, colon, prostate, colorectal, oral, ovarian, glioma, melanoma, glioblastoma, osteosarcoma, and histiocytic lymphoma cancers. Overall, in vitro studies demonstrated that oleandrin was able to inhibit cell proliferation, decrease cell viability, and induce apoptosis and/or cell cycle arrest. In addition, oleandrin had an effect on reducing mean tumor size and volume in animal studies. Oleandrin, as a cytotoxic agent, demonstrated antitumor effects in different types of cancers, however important clinical limitations remain a concern. These results encourage future studies to verify the applicability of oleandrin in antineoplastic therapeutic protocols human and veterinary medicine, the investigation of antimetastatic properties, as well as the potential increase in patient survival and the decrease of tumor markers., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pergularia tomentosa L., a milkweed tropical plant belonging to the family Asclepiadaceae, is a rich source of unusual cardiac glycosides, characterised by transfused A/B rings and a sugar moiety linked by a double link, generating a dioxanoid structure. In the present report, five cardenolides isolated from the aerial parts of the plant (calactin, calotropin, 12β-hydroxycalactin, 12β,6'-dihydroxycalotropin, and 16α-hydroxycalotropin) were investigated for their biological effects on a human hepatocarcinoma cell line. Cell viability was monitored by an MTT assay. The occurrence of apoptosis was evaluated by detecting caspase-3 activation and chromatin fragmentation. The ability of these compounds to induce autophagy was analysed by monitoring two markers of the autophagic process, LC3 and p62. Our results indicated that all cardenolides had cytotoxic effects, with IC 50 ranging from 0.127 to 6.285 μM. All compounds were able to induce apoptosis and autophagy, calactin being the most active one. Some of them also caused a reduction in cell migration and a partial block of the cell cycle into the S-phase. The present study suggests that selected cardenolides from aerial parts of P. tomentosa , particularly calactin, possess potentially desirable properties for further investigation as anticancer agents.

Na + /K + -ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several cardiotonic steroids (CTS) has been associated with the cytotoxic effect on cancer cell lines of phytochemicals such as ouabain and digitoxin. This study evaluated the inhibitory capacity of cardenolides calotropin and corotoxigenin 3-O-glucopyranoside (C3OG) from Asclepias subulata over the Na + /K + -ATPase activity in vitro and silico . The inhibitory assays showed that calotropin and C3OG decreased the Na + /K + -ATPase activity with IC 50 values of 0.27 and 0.87 μM, respectively. Furthermore, the molecules presented an uncompetitive inhibition on Na + /K + -ATPase activity, with K i values of 0.2 μM to calotropin and 0.5 μM to C3OG. Furthermore, the molecular modeling indicated that calotropin and C3OG might interact with the Thr 797 and Gln 111 residues, considered essential to the interaction with the Na + /K + -ATPase. Besides, these cardenolides can interact with amino acid residues such as Phe 783 , Leu 125 , and Ala 323 , to establish hydrophobic interactions on the binding site. Considering the results, these provide novel evidence about the mechanism of action of cardenolides from A. subulata , proposing that C3OG is a novel cardenolide that deserves further consideration for in vitro cellular antiproliferative assays and in vivo studies as an anticancer molecule., Competing Interests: Rogerio R. Sotelo-Mundo is an Academic Editor for PeerJ., (© 2022 Meneses-Sagrero et al.)

Background: Several studies have shown that active compounds of Asclepias subulata (cardenolides) have antiproliferative effect on human cancer cells. Cardenolides isolated from A. subulata can be used as active chemical markers to elaborate phytopharmaceutical preparations. The aim of this work was to evaluate the antiproliferative effect of a standardized extract of the aerial parts, based on Asclepias subulata cardenolides. Methods: Four standardized extracts were prepared by HPLC-DAD depending on the concentration of calotropin and the antiproliferative activity was measured for the MTT assay, on the A549, MCF-7, HeLa, PC3 and ARPE cell lines. The concentrations of calotropin used for the standardization of the extracts were 10, 7.6, 5 and 1 mg/dL. Results: Standardization of the A. subulata extract based on calotropin at 7.6 mg/g dry weight was achieved and the antiproliferative activity was evaluated over A549, HeLa and MCF-7 cell lines, obtaining proliferation percentages of 3.8 to 13.4% . Conclusions: The standardized extracts of A. subulata at different concentrations of calotropin showed antiproliferative activity against all the cell lines evaluated. The greatest effect was observed against the HeLa cell line., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 González Gutiérrez FH et al.)

Background: Several studies have shown that active compounds of Asclepias subulata (cardenolides) have antiproliferative effect on human cancer cells. Cardenolides isolated from A. subulata can be used as active chemical markers to elaborate phytopharmaceutical preparations. To evaluate the antiproliferative effect of a standardized extract of the aerial parts, based on Asclepias subulata cardenolides. Methods: Four standardized extracts were prepared by HPLC-DAD depending on the concentration of calotropin and the antiproliferative activity was measured for the MTT assay, on the A549, MCF-7, HeLa, PC3 and ARPE cell lines. The concentrations of calotropin used for the standardization of the extracts were 10, 7.6, 5 and 1 mg/dL. Results: Standardization of the A. subulata extract based on calotropin at 7.6 mg/g dry weight was achieved and the antiproliferative activity was evaluated over A549, HeLa and MCF-7 cell lines, obtaining proliferation percentages of 3.8 to 13.4% . Conclusions: The standardized extracts of A. subulata at different concentrations of calotropin showed antiproliferative activity against all the cell lines evaluated. The greatest effect was observed against the HeLa cell line., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 González Gutiérrez FH et al.)

Two cardenolide glycosides, corotoxigenin 3-O-[β-D-glucopyranosyl-(1→4)-6-deoxy-β-D-glucopyranoside] (1) and coroglaucigenin 3-O-[β-D-glucopyranosyl-(1→4)-6-deoxy-β-D-glucopyranoside] (2), were isolated from the seed fairs of Asclepias curassavica. The structures of 1-2 were determined based on the combination of the analysis of their MS, NMR spectroscopic data and acid hydrolysis. The inhibitory effects of compounds 1 and 2 on human colorectal carcinoma cells (HCT116), non-small cell lung carcinoma cells (A549) and hepatic cancer cells (SMMC-7721) were evaluated. The results showed that both compounds 1 and 2 significantly inhibited the viability, proliferation, and migration of A549, HCT116 and SMMC-7721 cells, suggesting that compounds 1 and 2 can be applied in the treatment of lung, colon and liver cancers in clinical practice. This study may not only provide a scientific basis for clarifying the active ingredients in A. curassavica, but also help to understand its antitumor activity, which can promote the application of A. curassavica in clinical treatment of various cancers., (Copyright © 2022 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.)

Oleandrigenin-3-O-β-D-diginoside (a derivative of odoroside A), isolated and purified by our group, has seldom been explored for its pharmacological activity. This study aimed at clarifying the mechanisms towards the leukaemia-suppressive role of odoroside A (compound #1) and its derivative, oleandrigenin-3-O-β-D-diginoside (compound #2) isolated from Nerium oleander. Viability and nuclear morphology change were assessed by CCK-8 assay and fluorescence microscope, respectively. Then, the cell apoptosis and autophagy induced by the compounds were detected by flow cytometry and Western blot. Xenograft model of nude mice was also applied to measure the leukaemia-suppressive effects of compound #2 in vivo. The result displayed that compound #1 and compound #2 inhibited the proliferation of HL60 and K562 cells and stronger effects were found in HL60 than K562 cells. Both of the compounds induced a dose-dependent apoptosis and autophagy in HL60 cells, where compound #2 was more potent than compound #1. Compound #2 also demonstrated a time-dependent apoptosis and autophagy in HL60 cells. Furthermore, ROS generation and JNK phosphorylation occurred in a dose-dependent manner in the cells treated with compound #2. Mitochondria also played critical role, proved by the decrease of Bcl-2, the release of cyto c to cytosol and the activation of caspase-3 and caspase-9. Moreover, the antitumour effects of compound #2 were validated in the nude mouse xenograft model in vivo. Odoroside A and its derivative inhibited the growth of leukaemia by inducing apoptosis and autophagy through the activation of ROS/JNK pathway. These results suggest that the compounds can serve as potential antitumour agents against leukaemia, especially acute myeloid leukaemia (AML)., (© 2021 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society). Published by John Wiley & Sons Ltd.)

Background: The herbivores Danaus plexippus (Lepidoptera), Oncopeltus fasciatus, and Aphis nerii (Hemiptera) are special insects that feed on Calotropis procera (Apocynaceae) (Sodom Apple). At least 35 chemically distinct cardenolides have been reported in C. procera., Objective: We aimed to evaluate the interaction between cardenolides and Na + /K + ATPases from herbivores., Methods: The Na + /K + ATPases from these insects were modeled, and docking studies were performed involving cardenolides from C. procera., Results: The replacement of serine in sensitive Na + /K + ATPase by histidine, phenylalanine, and tyrosine in the structures examined suggested spatial impairment caused by interaction, probably making the herbivorous insects resistant against the cardenolides of C. procera. In addition, the ability of the insects to avoid cardenolide toxicity was not correlated with cardenolide polarity. Therefore, the plant fights predation through molecular diversity, and the insects, regardless of their taxonomy, face this molecular diversity through amino acid replacements at key positions of the enzyme targeted by the cardenolides., Conclusion: The results show the arsenal of chemically distinct cardenolides synthesized by the C. procera., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In the present study, we investigated the cardioactive glycosides oleandrin and ouabain, and compared them to digoxin in a model of cardiotoxicity induced by doxorubicin. Adult rats were distributed into four experimental groups. Each group was challenged with a single intraperitoneal application of doxorubicin at a dose of 12 mg/kg. Then, they were treated with saline solution and the glycosides oleandrin, ouabain, and digoxin at a dose of 50 µg/kg, for 7 days. They underwent echocardiography, electrocardiography, hematologic, biochemical tests, and microscopic evaluation of the heart. All animals presented congestive heart failure, which was verified by a reduction in the ejection fraction. Oleandrin and digoxin were able to significantly reduce (p < 0.05) the eccentric remodeling caused by doxorubicin. Oleandrin and digoxin were significantly lower (p < 0.05) than the control group in maintaining systolic volume and left ventricular volume in diastole. Other parameters evaluated did not show significant statistical differences. All animals showed an increase in erythrocyte count, and an increase in the duration of the QRS complex on the ECG and myocardial necrosis at the histopathological analysis. It is concluded that the glycosides oleandrin, ouabain, and digoxin in the used dosage do not present therapeutic potential for the treatment of congestive heart failure caused by doxorubicin., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV). and bovine coronavirus (BCV) threaten the productivity of cattle worldwide. Development of therapeutics that can control the spread of these viruses is an unmet need. The present research was designed to explore the in vitro antiviral activity of the Nerium oleander derived cardiac glycoside oleandrin and a defined N. oleander plant extract (PBI-05204) containing oleandrin., Methods: Madin Darby Bovine Kidney (MDBK) cells, Bovine Turbinate (BT) cells, and Human Rectal Tumor-18 (HRT-18) cells were used as in vitro culture systems for BVDV, BRSV and BCV, respectively. Cytotoxicity was established using serial dilutions of oleandrin or PBI-05204. Noncytotoxic concentrations of each drug were used either prior to or at 12 h and 24 h following virus exposure to corresponding viruses. Infectious virus titers were determined following each treatment., Results: Both oleandrin as well as PBI-05204 demonstrated strong antiviral activity against BVDV, BRSV, and BCV, in a dose-dependent manner, when added prior to or following infection of host cells. Determination of viral loads by PCR demonstrated a concentration dependent decline in virus replication. Importantly, the relative ability of virus produced from treated cultures to infect new host cells was reduced by as much as 10,000-fold at noncytotoxic concentrations of oleandrin or PBI-05204., Conclusions: The research demonstrates the potency of oleandrin and PBI-05204 to inhibit infectivity of three important enveloped bovine viruses in vitro . These data showing non-toxic concentrations of oleandrin inhibiting infectivity of three bovine viruses support further investigation of in vivo antiviral efficacy.

Bioactivity-guided isolation of the CHCl 3 -soluble fraction of the roots of Calotropis gigantea was carried out to obtain a new cardenolide glycoside, caloside G. Its absolute structure was elucidated based on NMR and ECD spectroscopic data interpretation. Caloside G showed noteworthy cytotoxicity against the PANC-1 human pancreatic and HeLa human cervical carcinoma cell lines, with the submicromolar IC 50 values of 0.038 and 0.09  µ M, respectively.

Sixteen cardiac glycosides, including five previously undescribed compounds, were extracted and purified from whole plants of Calotropis gigantea (L.). Spectroscopic data and electronic circular dichroism (ECD) analyses were used to determine their structures. Calogiganin C is the first naturally occurring example of a cardenolide containing a 7-membered lactone in ring A. The cytotoxic activities of these compounds against A172, U251, AGS, PANC-1, HepG2, HCT116 and NCI-H226 cell lines were evaluated. Four of them exhibited the most potent growth inhibitory activity against a panel of human cancer cell lines, including A172, U251, AGS, PANC-1 and HCT116. Notably, uscharidin and calotropin showed pronounced cytotoxicities at low nanomolar concentrations against A172 and U251 cells, and possible cell death mechanism studies manifested that these two compounds induced G2/M cell cycle arrest, which demonstrated promising anticancer potential., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Phytochemical analysis of Thevetia neriifolia seeds resulted in the isolation of one new ( 1 ) and 23 known ( 2 － 24 ) cardenolide glycosides. The structure of 1 was determined based on one- and two-dimensional NMR spectroscopic analysis and acid hydrolytic cleavage reaction. The effect of the cytotoxic activity of 1 － 24 on three human oral carcinoma cell lines was assessed. The cell lines included Ca9-22 human gingival carcinoma cells, HSC-2 human mouth carcinoma cells, HSC-4 human tongue carcinoma cells, and HGF human gingival fibroblast cells. The isolated compounds had a cytotoxic effect on the carcinoma cells with IC 50 values ranging from 0.004 μM to 64.9 μM. The structure-activity relationship is also discussed.

Since the beginning, natural products have represented an important source of bioactive molecules for cancer treatment. Among them, cardenolides attract the attention of different research groups due to their cardiotonic and antitumor activity. The observed biological activity is closely related to their Na + /K + -ATPase inhibition potency. Currently, the discovery of new compounds against cancer is an urgent need in modern pharmaceutical research. Thus, the aim of this work is to determine the physicochemical properties and substituent effects that module the antiproliferative activity of cardenolides on the human lung cancer cell line A549. We build and curate a library with results obtained from literature; molecular descriptors were calculated in PaDEL software, and SAR/QSAR analysis was performed. The SAR results showed that cardenolides were sensitive to modifications in C and D steroidal ring and required substituent groups with the function of hydrogen bond acceptor at the C3 position. QSAR models to doubly linked-type cardenolides indicated that properties as lipoaffinity and atoms with the capacity to be hydrogen bond acceptors are involved in the increment of antiproliferative activity on A549 cell line. In contrast, the presence and position of very electro-negative atoms on the molecule decreased the antiproliferative effect on A549 cells. These results suggest that the antiproliferative capacity of cardenolides on the cell line A549 is strongly related to substituent groups on the C3 position, which must not be carbohydrate. Additionally, the steroidal rings C and D must remain without modifications., (© 2020. Springer Nature Switzerland AG.)

With continued expansion of the coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome 2 (SARS-CoV-2), both antiviral drugs as well as effective vaccines are desperately needed to treat patients at high risk of life-threatening disease. Here, we present in vitro evidence for significant inhibition of SARS-CoV-2 by oleandrin and a defined extract of N. oleander (designated as PBI-06150). Using Vero cells, we found that prophylactic (pre-infection) oleandrin (as either the pure compound or as the active principal ingredient in PBI-06150) administration at concentrations as low as 0.05 µg/ml exhibited potent antiviral activity against SARS-CoV-2, with an 800-fold reduction in virus production, and a 0.1 µg/ml concentration resulted in a greater than 3000-fold reduction in infectious virus production. The half maximal effective concentration (EC 50 ) values were 11.98 ng/ml when virus output was measured at 24 h post-infection, and 7.07 ng/ml measured at 48 h post-infection. Therapeutic (post-infection) treatment up to 24 h after SARS-CoV-2 infection of Vero cells also reduced viral titers, with 0.1 µg/ml and 0.05 µg/ml concentrations causing greater than 100-fold reduction as measured at 48 h, and the 0.05 µg/ml concentration resulting in a 78-fold reduction. Concentrations of oleandrin up to 10 µg/ml were well tolerated in Vero cells. We also present in vivo evidence of the safety and efficacy of defined N. oleander extract (PBI-06150), which was administered to golden Syrian hamsters in a preparation containing as high as 130 µg/ml of oleandrin. In comparison to administration of control vehicle, PBI-06150 provided a statistically significant reduction of the viral titer in the nasal turbinates (nasal conchae). The potent prophylactic and therapeutic antiviral activities demonstrated here, together with initial evidence of its safety and efficacy in a relevant hamster model of COVID-19, support the further development of oleandrin and/or defined extracts containing this molecule for the treatment of SARS-CoV-2 and associated COVID-19 disease and potentially also for reduction of virus spread by persons diagnosed early after infection., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Acovenoside A (Acov-A) and acobioside A (Acob-A) were isolated from Acokanthera oblongifolia . Their anticancer properties were explored regarding, antiproliferative and antiangiogenic activities. The study included screening phase against six cancer cell lines followed by mechanistic investigation against HepG2 cancer cell line. The sulforhodamine-B (SRB) was used to determine their growth inhibitory power. In the other hand, flow cytometry techniques were recorded the cell death type and cell cycle analysis. The clonogenic (colony formation) and wound healing assays, enzyme-linked immunosorbent assay (ELISA) and molecular docking, were performed to evaluate the antiangiogenesis capability. Both compounds were strongly, inhibited four cancer cell lines at GI 50 less than 100 nM. The in vitro mechanistic investigation against HepG2 resulted in cell accumulations at G2M phase and induction of apoptosis upon treating cells separately, with 400 nM Acov-A and 200 nM Acob-A. Interestingly, the same concentrations were able to activate caspase-3 by 7.2 and 4.8-fold, respectively. Suppressing the clonogenic capacity of HepG2 cells (20 and 40 nM) and inhibiting the migration of the colon Caco-2 cancer cells were provoke the results of vascular endothelial growth factor receptor2 (VEGFR2) kinase enzyme inactivation. The docked study was highly supportive, to the antiangiogenic approach of both cardenolides. The isolated cardenolides could orchestrate pivotal events in fighting cancer., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

Microbial biotransformation is an important tool in drug discovery and for metabolism studies. To expand our bioactive natural product library via modification and to identify possible mammalian metabolites, a cytotoxic cardenolide (gitoxigenin) was biotransformed using the endophytic fungus Alternaria eureka 1E1BL1. Initially, oleandrin was isolated from the dried leaves of Nerium oleander L. and subjected to an acid-catalysed hydrolysis to obtain the substrate gitoxigenin (yield; ~25%). After 21 days of incubation, five new cardenolides 1 , 3 , 4 , 6 , and 8 and three previously- identified compounds 2 , 5 and 7 were isolated using chromatographic methods. Structural elucidations were accomplished through 1D/2D NMR, HR-ESI-MS and FT-IR analysis. A. eureka catalyzed oxygenation, oxidation, epimerization and dimethyl acetal formation reactions on the substrate. Cytotoxicity of the metabolites were evaluated using MTT cell viability method, whereas doxorubicin and oleandrin were used as positive controls. Biotransformation products displayed less cytotoxicity than the substrate. The new metabolite 8 exhibited the highest activity with IC 50 values of 8.25, 1.95 and 3.4 µM against A549, PANC-1 and MIA PaCa-2 cells, respectively, without causing toxicity on healthy cell lines (MRC-5 and HEK-293) up to concentration of 10 µM. Our results suggest that A. eureka is an effective biocatalyst for modifying cardenolide-type secondary metabolites.

Cardiac glycosides (CGs) are useful drugs to treat cardiac illnesses and have potent cytotoxic and anticancer effects in cultured cells and animal models. Their receptor is the Na + ,K +  ATPase, but other plasma membrane proteins might bind CGs as well. Herein, we evaluated the short- and long-lasting cytotoxic effects of the natural cardenolide glucoevatromonoside (GEV) on non-small-cell lung cancer H460 cells. We also tested GEV effects on Na + ,K +  -ATPase activity and membrane currents, alone or in combination with selected chemotherapy drugs. GEV reduced viability, migration, and invasion of H460 cells spheroids. It also induced cell cycle arrest and death and reduced the clonogenic survival and cumulative population doubling. GEV inhibited Na + ,K + -ATPase activity on A549 and H460 cells and purified pig kidney cells membrane. However, it showed no activity on the human red blood cell plasma membrane. Additionally, GEV triggered a Cl-mediated conductance on H460 cells without affecting the transient voltage-gated sodium current. The administration of GEV in combination with the chemotherapeutic drugs paclitaxel (PAC), cisplatin (CIS), irinotecan (IRI), and etoposide (ETO) showed synergistic antiproliferative effects, especially when combined with GEV + CIS and GEV + PAC. Taken together, our results demonstrate that GEV is a potential drug for cancer therapy because it reduces lung cancer H460 cell viability, migration, and invasion. Our results also reveal a link between the Na + ,K + -ATPase and Cl -  ion channels.

Cancer is one of the main causes of death in the world and thus a global public health problem. Among the treatments available for cancer are surgery, radiotherapy, and chemotherapy. Currently, there is increased interest in the combination of two or more antitumor agents to achieve a synergistic effect in cancer therapy. Doxorubicin (DOX), a chemotherapeutic which has a potent antineoplastic action, has been used in the treatment of various tumors. However, the use of DOX is limited, mainly due to the cardiotoxicity. Therefore, nanostructured systems, such as liposomes, have been developed to carry this drug and target the tumor region, since tumor tissues present enhanced permeability and retention for nanosystems. Cardiac glycosides, such as digitoxin, have recently shown great antitumor potential despite the low therapeutic index which may limit their use. Furthermore, some compounds of this class have low water solubility, which makes their in vivo administration difficult. In this context, liposomes represent a valid strategy to carry simultaneously antitumor drugs allowing their intravenous administration. In this study, liposomes loaded with glucoevatromonoside containing peracetylated glucose hydroxyl groups (GEVPG) and DOX at molar ratio of 1:1 (SpHL-GEVPG:DOX 1:1) were developed, and their chemical and physicochemical properties were evaluated. This formulation presented a combination index (CI) lower than 1 at inhibitory concentration of 90 % growth (IC 90 ) for three human breast tumor lines evaluated (0.52 ± 0.39 for MDA-MB-231, 0.19 ± 0.13 for MCF-7, and 0.99 ± 0.09 for SKBR-3). These results indicate a synergistic cytotoxic effect of the GEVPG and DOX combination encapsulated in liposomes. In addition, SpHL-GEVPG:DOX 1:1 presented selectivity towards these cancer cells. Long-term in vitro cytotoxicity studies demonstrated that MDA-MB-231 surviving cells after treatment with SpHL-GEVPG:DOX 1:1 did not recover proliferation capacity after 21 d. From the studies of cell cycle and death pathway evaluation, it was observed that SpHL-GEVPG:DOX 1:1 arrested the cell cycle in the G2/M phase and similarly induced apoptosis and necrosis. However, SpHL-GEVPG:DOX at molar ratio of 1:1 showed lower induction of both apoptotic and necrotic pathways compared to free DOX and SpHL-DOX, suggesting that the mechanism of death involved may not be related to necrosis or apoptosis. Lastly, SpHL-GEVPG:DOX 1:1 showed a good storage stability for 90 d at 4 °C. Therefore, the results of the present work indicate the potential use of SpHL-GEVPG:DOX 1:1 as a new anticancer formulation., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Six new non-classical cardenolides (1-6), and seventeen known ones (7-23) were isolated from Calotropis gigantea. All cardenolides showed inhibitory effect on hypoxia inducible factor-1 (HIF-1) transcriptional activity with IC 50 of 8.85 nM-16.69 µM except 5 and 7. The novel 19-dihydrocalotoxin (1) exhibited a comparable HIF-1 inhibitory activity (IC 50 of 139.57 nM) to digoxin (IC 50 of 145.77 nM), a well-studied HIF-1 inhibitor, and 11, 12, 14, 16 and 19 presented 1.4-15.4 folds stronger HIF-1 inhibition than digoxin. 1 and 11 showed a dose-dependent inhibition on HIF-1α protein, which led to their HIF-1 suppressing effects. Compared with LO2 and H9c2 normal cell lines, both 1 and 11 showed selective cytotoxicity against various cancer cell lines including HCT116, HeLa, HepG2, A549, MCF-7, A2780 and MDA-MB-231. Moreover, a comprehensive structure-activity relationship was concluded for these non-classical cardenolides as HIF-1 inhibitors, which may shed some light on the rational design and development of cardenolide-based anticancer drugs., (Copyright © 2021 Elsevier Inc. All rights reserved.)

From remaining aqueous fraction of the roots of Streptocaulon juventas , one new cardiac glycoside named periplogenin 3- O - β -gentiobioside ( 1 ) together with six known ones ( 2-7 ) were isolated. Their relative structures were elucidated based on NMR spectroscopic analysis. Compound 1 showed moderate cytotoxicity against non-small cell lung carcinoma NCI-H460 and ovarian cancer HeLa cells. Moreover, compounds 2 and 3 exhibited remarkable cytotoxicity against NCI-H460 cell with the IC 50 values of 0.34 and 0.068  μ M, respectively.

Column chromatography (CC) analysis of methanol and butanol extracts of the aerial parts of Calortopis procera as well as the methanol extract of its latex, led to the isolation of 8 cardenolides, of which the structures were elucidated by NMR and HRESIMS spectroscopy. They also revealed several triterpenes and flavonoid glycoside. Based on the antiproliferative activity reported for cardenolides, the activity of calotropin and calotoxin was tested against two common cancer cell lines, human triple-negative breast cancer cell line (MDA-MB-231) and human lung adenocarcinoma cell line (A549). The high toxicity of the latex also encouraged performing the same test on the same cancer cell lines. The anti-proliferative activity of calotropin and calotoxin was compared to the methanol extract and the wax of the latex. The results showed that calotropin and calotoxin have significant cytotoxicity against MDA-MB-231 and A549 cell lines ranging from 0.046 to 0.072 μM compared to the methanol extract and the wax of its latex ranging from 0.47 to 58.41 μM. Moreover, the results showed lower toxicity of all treatments to the human skin fibroblasts compared to the toxicity to both MDA-MB-231 and A549 cancer cells lines except the higher toxicity of Methanolic extracts of C. procera latex to the MDA-MB-231 cells. In conclusion, C. procera is a medicinal plant with a wide spectrum of cardinolides including calotropin and calotoxin, which are promising agents for targeted cancer phytotherapy., (© 2021 Walter de Gruyter GmbH, Berlin/Boston.)

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.

Pentacyclic triterpenes and cardenolides were isolated from Acokanthera oblongifolia leaves. Their chemical structures were determined based on comprehensive 1D and 2D NMR spectroscopy. Their MIC was determined against 12 microorganisms. Their exerted cytotoxicity on the immortalized normal cells, hTERT-RPE1 was assessed by the sulforhodamine-B assay. The viral inhibitory effects of compounds against Newcastle disease virus (NDV) and H5N1 influenza virus IV were evaluated. Four in vitro antioxidant assays were performed in comparison with BHT and trolox and a weak activity was exhibited. Acovenoside A was with potent against H5N1-IV and NDV with IC 50  ≤ 3.2 and ≤ 2.1 μg/ml and SI values of 93.75 and 95.23%, respectively, in comparison to ribavirin. Its CC 50 record on Vero cells was > 400 and 200 μg/ml, respectively. Acobioside A was the most active compound against a broad range of microbes while Pseudomonas aeruginosa was the most sensitive. Its MIC (0.07 μg/ml) was 1/100-fold of the recorded CC 50 (7.1 μg/ml/72 h) against hTERT-RPE1. The molecular docking of compounds on human DNA topoisomerase I (Top1-DNA) and IV glycoprotein hemagglutinin were studied using MOE program. This study has introduced the cardenolides rather than triterpenoids with the best docking score and binding interaction with the active site of the studied proteins., (© 2020 Walter de Gruyter GmbH, Berlin/Boston.)

Purpose: Angiogenesis related abnormalities underlie several life-threatening disorders. Despite approved therapies, scientists have yet to develop highly efficient, low cost approaches with minimal side effects., Methods: We evaluated the antiangiogenic activity of 50% hydroalcoholic extracts of Pergularia tomentosa L. root and aerial parts along with their EtOAc and water fractions, in vivo and in vitro. Transgenic zebrafish line Tg(fli1:EGFP) was used for in vivo assay and human umbilical vein endothelial cell (HUVEC) migration test along with possibility of tube formation were performed as in vitro tests. Furthermore, microvasculature in chicken chorioallantoic membrane (CAM) was assessed under P. tomentosa treatment. The fractionation of the 50% hydroalcoholic extracts was led to the identification of the best active fraction in this study. The metabolite profiling of the active fraction was also carried out using LC-HRESIMS analysis., Results: Pergularia tomentosa markedly inhibited intersegmental vessel (ISV) formation at 48 h post-fertilization (hpf) embryos in zebrafish. The water fraction of root hydroalcoholic extract (PtR2), showed strong antiangiogenic effect with minimal adverse viability impacts. Over 80% of embryos showed more than 50% inhibition in their ISV development at 20 and 40 μg/mL. PtR2 at 20 μg/mL substantially reduced human umbilical vein endothelial cell (HUVEC) migration up to 40%, considerable destruction of the formed tubes in the tube formation and microvasculature in CAM assays. Immunocytochemistry showed a marked reduction in vascular endothelial cadherin (VE-cadherin) abundance at cell junctions concurrent with substantial reduction of phospho-Akt (p-Akt) and β-catenin protein expressions. Phytochemical profile of PtR2 showed a rich source of cardenolide structures, including ghalakinoside, calactin and calotropin derivatives., Conclusion: Thus, the P. tomentosa cardenolide-rich fraction (PtR2) may hold a considerable promise for an antiangiogenic impact by impairment of endothelial cell (EC) migration and viability. Graphical abstract.

Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs. In this work, two semisynthetic derivatives of digitoxigenin, namely C10 (3β-(( N -(2-hydroxyethyl)aminoacetyl)amino-3-deoxydigitoxigenin) and C11 (3β-(hydroxyacetyl)amino-3-deoxydigitoxigenin), showed anti-influenza A virus activity by affecting the expression of viral proteins at the early and late stages of replication cycle, and altering the transcription and synthesis of new viral proteins, thereby inhibiting the formation of new virions. Such antiviral action occurred due to the interference in the assembly of viral polymerase, resulting in an impaired polymerase activity and, therefore, reducing viral replication. Confirming the in vitro results, a clinically relevant ex vivo model of influenza virus infection of human tumor-free lung tissues corroborated the potential of these compounds, especially C10, to completely abrogate influenza A virus replication at the highest concentration tested (2.0 µM). Taken together, these promising results demonstrated that C10 and C11 can be considered as potential new anti-influenza drug candidates.

An unprecedented biological function of natural cardenolides independent of their membrane target Na + /K + -ATPase is disclosed. Previously, we reported that cardenolides impart anti-transmissible gastroenteritis coronavirus (anti-TGEV) activity through the targeting of Na + /K + -ATPase and its associated PI3K&#95;PDK1&#95;RSK2 signaling. Swine testis cells with Na + /K + -ATPase α1 knocked down exhibited decreased susceptibility to TGEV infectivity and attenuated PI3K&#95;PDK1&#95;RSK2 signaling. Herein, we further explored a Na + /K + -ATPase-independent signaling axis induced by natural cardenolides that also afforded significant anti-coronaviral activity for porcine TGEV and human HCoV-OC43. Using pharmacological inhibition and gene silencing techniques, we found that this anti-TGEV or anti-HCoV-OC43 activity was caused by JAK1 proteolysis and mediated through upstream activation of Ndfip1/2 and its effector NEDD4. This study provides novel insights into the pharmacological effects of natural cardenolides, and is expected to inform their future development as antiviral agents., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Oleandrin, the main component of Nerium oleander L. extracts, is a cardiotoxic glycoside with multiple pharmacological implications, having potential anti-tumoral and antiviral characteristics. Although it is accepted that the main mechanism of oleandrin action is the inhibition of Na + /K + -ATPases and subsequent increase in cell calcium, many aspects which determine oleandrin cytotoxicity remain elusive. In this study, we used the model Saccharomyces cerevisiae to unravel new elements accounting for oleandrin toxicity. Using cells expressing the Ca 2+ -sensitive photoprotein aequorin, we found that oleandrin exposure resulted in Ca 2+ influx into the cytosol and that failing to pump Ca 2+ from the cytosol to the vacuole increased oleandrin toxicity. We also found that oleandrin exposure induced Mn 2+ accumulation by yeast cells via the plasma membrane Smf1 and that mutants with defects in Mn 2+ homeostasis are oleandrin-hypersensitive. Our data suggest that combining oleandrin with agents which alter Ca 2+ or Mn 2+ uptake may be a way of controlling oleandrin toxicity.

Natural cardiac-active principles built upon the 14,16β-dihydroxy-5β,14β-androstane core and bearing a heterocyclic substituent at 17β, in particular, a cardenolide - oleandrin and a bufadienolide - bufotalin, are receiving a great deal of attention as potential anticancer drugs. The densely substituted and sterically shielded ring D is the particular structural feature of these compounds. The first synthesis of oleandrigenin from easily available steroid starting material is reported here. Furthermore, selected 17β-(4-butenolidyl)- and 17β-(3-furyl)-14,16β-dihydroxy-androstane derivatives were en route synthesized and examined for their Na + /K + -ATP-ase inhibitory properties as well as cytotoxic activities in normal and cancer cell lines. It was found that the furyl-analogue of oleandrigenin/bufatalin (7) and some related 17-(3-furyl)- derivatives (19, 21) show remarkably high Na + /K + -ATP-ase inhibitory activity as well as significant cytotoxicity in vitro. In addition, oleandrigenin 2 compared to derivatives 21 and 25 induced strong apoptosis in human cervical carcinoma HeLa cells after 24 h of treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Masson SAS.)

Nerium oleander, a member of family Apocynaceae, is commonly known as Kaner in various countries of Asia and Mediterranean region. This plant has been renowned to possess significant therapeutic potential due to its various bioactive compounds which have been isolated from this plant e.g., cardiac glycosides, oleandrin, α-tocopherol, digitoxingenin, urosolic acid, quercetin, odorosides, and adigoside. Oleandrin, a saponin glycoside is one of the most potent and pharmacologically active phytochemicals of N. oleander. Its remarkable pharmacotherapeutic potential have been interpreted as anticancer, anti-inflammatory, anti-HIV, neuroprotective, antimicrobial and antioxidant. This particular bioactive entity is known to target the multiple deregulated signaling cascades of cancer such as NF-κB, MAPK, and PI3K/Akt. The main focus of the current study is to comprehend the action mechanisms of oleandrin against various pathological conditions. The current review is a comprehensive summary to facilitate the researchers to understand the pharmacological position of the oleandrin in the arena of drug discovery, representing this compound as a new drug candidate for further researches. Moreover, in vivo and in silico based studies are required to explore the mechanistic approaches regarding the pharmacokinetics and biosafety profiling of this compound to completely track its candidature status in natural drug discovery., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Triple-negative breast cancers (TNBC) are aggressive and heterogeneous cancers that lack targeted therapies. We implemented a screening program to identify new leads for subgroups of TNBC using diverse cell lines with different molecular drivers. Through this program, we identified an extract from Calotropis gigantea that caused selective cytotoxicity in BT-549 cells as compared to four other TNBC cell lines. Bioassay-guided fractionation of the BT-549 selective extract yielded nine cardenolides responsible for the selective activity. These included eight known cardenolides and a new cardenolide glycoside. Structure-activity relationships among the cardenolides demonstrated a correlation between their relative potencies toward BT-549 cells and Na + /K + ATPase inhibition. Calotropin, the compound with the highest degree of selectivity for BT-549 cells, increased intracellular Ca 2+ in sensitive cells to a greater extent than in the resistant MDA-MB-231 cells. Further studies identified a second TNBC cell line, Hs578T, that is also highly sensitive to the cardenolides, and mechanistic studies were conducted to identify commonalities among the sensitive cell lines. Experiments showed that both cardenolide-sensitive cell lines expressed higher mRNA levels of the Na + /Ca 2+ exchanger NCX1 than resistant TNBC cells. This suggests that NCX1 could be a biomarker to identify TNBC patients that might benefit from the clinical administration of a cardiac glycoside for anticancer indications.

A series of oleandrin-4'-yl ester derivatives were designed, synthesized, and evaluated for their proliferation inhibition activities against tumor cell lines. Cytotoxicity data revealed that the C4' moiety had an important influence on cytotoxic activity. Several compounds that we designed and synthesized exhibit significant in vitro antiproliferative activity against the tested tumor cell lines. Among the derivatives of OL, 4b-HCl not only had good anti-tumor activity but also had good water solubility. Furthermore, 4b-HCl can significantly inhibit tumor growth by 96.4% at a dose of 6 mg/kg/d by ip., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Human herpesviruses are among the most prevalent pathogens worldwide and have become an important public health issue. Recurrent infections and the emergence of resistant viral strains reinforce the need of searching new drugs to treat herpes virus infections. Cardiac glycosides are used clinically to treat cardiovascular disturbances, such as congestive heart failure and atrial arrhythmias. In recent years, they have sparked new interest in their potential anti-herpes action. It has been previously reported by our research group that two new semisynthetic cardenolides, namely C10 (3β-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin) and C11 (3β-(hydroxyacetyl)amino-3-deoxydigitoxigenin), exhibited potential anti-HSV-1 and anti-HSV-2 with selectivity index values > 1,000, comparable with those of acyclovir. This work reports the mechanism investigation of anti-herpes action of these derivatives. The results demonstrated that C10 and C11 interfere with the intermediate and final steps of HSV replication, but not with the early stages, since they completely abolished the expression of the UL42 (β) and gD (γ) proteins and partially reduced that of ICP27 (α). Additionally, they were not virucidal and had no prophylactic effects. Both compounds inhibited HSV replication at nanomolar concentrations, but cardenolide C10 was more active than C11 and can be considered as an anti-herpes drug candidate including against acyclovir-resistant HSV-1 strains.

Background: Breast cancer is the most common malignant tumor in women. Due to limited treatment outcome and high rate of metastasis, the prognosis is especially poor for triple-negative breast cancer. It is urgent to discover and develop novel agents for treatment of breast cancer. Herein, we investigated the potential mechanisms of Oleandrin's (a cardiac glycoside) cytotoxic activity against breast cancer cells., Methods: Cell proliferation was assessed by xCELLigence Real-Time Cell Analyzer (RTCA)-MP system. Apoptotic cells were detected by using Annexin V/PI staining and nuclear fragments observation. The effect of oleandrin on ATP1B3 expression and markers of ER stress were determined by western blot. A primary cell sensitivity assay was performed via a collagen gel droplet-embedded culture drug sensitivity method (CD-DST)., Results: Oleandrin suppressed cell proliferation and colony formation in the three breast cancer cell lines but did not affect normal mammary epithelial cells. Additionally, the expression of ATP1B3 was higher in the three breast cancer cell lines compared to MCF10A cells. Treatment with oleandrin increased the number of apoptotic cells and led to nuclear pyknosis, fragmentation, and apoptotic body formation in breast cancer cells. Furthermore, oleandrin treatment increased expression of Bax and Bim but decreased that of Bcl-2. Treatment with oleandrin also upregulated the expression of endoplasmic reticulum stress associated proteins, including eIF2α, ATF4, and CHOP, but not PERK. oleandrin treatment also induced the phosphorylation of PERK and eIF2α. Of note, oleandrin exhibited antitumor effects on patient-derived breast cancer cells under three-dimensional culture conditions., Conclusions: Taken together, our results suggest that oleandrin induces mitochondrial-mediated apoptosis by activating endoplasmic reticulum stress in breast cancer. Moreover, oleandrin may be an effective strategy for the treatment of breast cancer., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2020. Published by Elsevier Masson SAS.)

(+)-Digoxin ( 1 ) is a well-known cardiac glycoside long used to treat congestive heart failure and found more recently to show anticancer activity. Several known cardenolides ( 2 - 5 ) and two new analogues, (+)-8(9)-β-anhydrodigoxigenin ( 6 ) and (+)-17- epi -20,22-dihydro-21α-hydroxydigoxin ( 7 ), were synthesized from 1 and evaluated for their cytotoxicity toward a small panel of human cancer cell lines. A preliminary structure-activity relationship investigation conducted indicated that the C-12 and C-14 hydroxy groups and the C-17 unsaturated lactone unit are important for 1 to mediate its cytotoxicity toward human cancer cells, but the C-3 glycosyl residue seems to be less critical for such an effect. Molecular docking profiles showed that the cytotoxic 1 and the noncytotoxic derivative 7 bind differentially to Na + /K + -ATPase. The HO-12β, HO-14β, and HO-3'aα hydroxy groups of (+)-digoxin ( 1 ) may form hydrogen bonds with the side-chains of Asp121 and Asn122, Thr797, and Arg880 of Na + /K + -ATPase, respectively, but the altered lactone unit of 7 results in a rotation of its steroid core, which depotentiates the binding between this compound and Na + /K + -ATPase. Thus, 1 was found to inhibit Na + /K + -ATPase, but 7 did not. In addition, the cytotoxic 1 did not affect glucose uptake in human cancer cells, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na + /K + -ATPase but not by interacting with glucose transporters.

Background: Cardiac glycosides, which inhibit Na + /K + -ATPase, display inotropic effects for the treatment of congestive heart failure and cardiac arrhythmia. Recent studies have suggested signaling downstream of Na + /K + -ATPase action in the regulation of cell proliferation and apoptosis and have revealed the anticancer activity of cardiac glycosides. The study aims to characterize the anticancer potential of ascleposide, a natural cardenolide, and to uncover its primary target and underlying mechanism against human castration-resistant prostate cancer (CRPC)., Methods: Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay, carboxyfluorescein succinimidyl ester staining assay and clonogenic examination. Flow cytometric analysis was used to detect the distribution of cell cycle phase, mitochondrial membrane potential, intracellular Na + and Ca 2+ levels, and reactive oxygen species production. Protein expression was examined using Western blot analysis. Endocytosis of Na + /K + -ATPase was determined using confocal immunofluorescence microscopic examination., Results: Ascleposide induced an increase of intracellular Na + and a potent antiproliferative effect. It also induced a decrease of G1 phase distribution while an increase in both G2/M and apoptotic sub-G1 phases, and downregulated several cell cycle regulator proteins, including cyclins, Cdk, p21, and p27 Cip/Kip proteins, Rb and c-Myc. Ascleposide decreased the expression of antiapoptotic Bcl-2 members (eg, Bcl-2 and Mcl-1) but upregulated proapoptotic member (eg, Bak), leading to a significant loss of mitochondrial membrane potential and activation of both caspase-9 and caspase-3. Ascleposide also dramatically induced tubulin acetylation, leading to inhibition of the catalytic activity of Na + /K + -ATPase. Notably, extracellular high K + (16 mM) significantly blunted ascleposide-mediated effects. Furthermore, ascleposide induced a p38 MAPK-dependent endocytosis of Na + /K + -ATPase and downregulated the protein expression of Na + /K + -ATPase α1 subunit., Conclusion: Ascleposide displays antiproliferative and apoptotic activities dependent on the inhibition of Na + /K + -ATPase pumping activity through p38 MAPK-mediated endocytosis of Na + /K + -ATPase and downregulation of α1 subunit, which in turn cause tubulin acetylation and cell cycle arrest. Cell apoptosis is ultimately triggered by the activation of caspase cascade attributed to mitochondrial damage through the downregulation of Bcl-2 and Mcl-1 protein expressions while upregulation of Bak protein levels. The data also suggest the potential of ascleposide in anti-CRPC development., (© 2020 Wiley Periodicals, Inc.)

Na + /K + -ATPase α1 was reported to directly interact with and recruit FGF2 (fibroblast growth factor 2), a vital cell signaling protein implicated in angiogenesis, to the inner plasma membrane for subsequent secretion. Cardenolides, a class of cardiac glycosides, were reported to downregulate FGF2 secretion upon binding to Na + /K + -ATPase α1 in a cell system with ectopically expressed FGF2 and Na + /K + -ATPase α1. Herein, we disclose that the cardenolides ouabain and reevesioside A significantly enhance the secretion/release of FGF2 and the phosphorylation of FGFR1 (fibroblast growth factor receptor 1) in a time- and dose-dependent manner, in A549 carcinoma cells. A pharmacological approach was used to elucidate the pertinent upstream effectors. Only the ERK1/2 inhibitor U0126 but not the other inhibitors examined (including those inhibiting the unconventional secretion of FGF2) was able to reduce ouabain-induced FGF2 secretion and FGFR1 activation. ERK1/2 phosphorylation was increased upon ouabain treatment, a process found to be mediated through upstream effectors including ouabain-induced phosphorylated EGFR and a reduced MKP1 protein level. Therefore, at least two independent lines of upstream effectors are able to mediate ouabain-induced ERK1/2 phosphorylation and the subsequent FGF2 secretion and FGFR1 activation. These finding constitute unprecedent insights into the regulation of FGF2 secretion by cardenolides., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aim: To investigate the cytotoxic effect of Peruvoside and mechanism of action in human cancers., Main Methods: Cell viability was measured by MTT assay and the cell cycle arrest was identified by FACS. Real-time qPCR and western blotting studies were performed to identify important gene and protein expressions in the different pathways leading to apoptosis. Immunofluorescence was performed to understand protein localization and molecular docking studies were performed to identify protein-ligand interactions., Key Findings: Peruvoside showed significant anti-proliferative activities against human breast, lung, and liver cancer cells in dose-dependent manner. The anti-cancer mechanism was further confirmed by DNA damage and cell cycle arrest at the G0/G1 phase. Dysregulation of Wnt/β-catenin signaling with Peruvoside treatment resulted in inhibition of cyclin D1 and c-Myc also observed in this study. Furthermore, we identified that Peruvoside can inhibit autophagy by PI3K/AKT/mTOR signaling and through downregulating MEK1. Moreover, Peruvoside has the ability to modulate the expressions of key proteins from the cell cycle, MAPK, NF-kB, and JAK-STAT signaling. In silico studies revealed that Peruvoside has the ability to interact with crucial proteins from different biochemical signaling pathways., Significance: Our results demonstrated that Peruvoside has the ability to inhibit cancer cell proliferation by modulating the expression of various key proteins involved in cell cycle arrest, apoptosis, and autophagic cell death. Clinical data generated from the present study might provide a novel impetus for targeting several human cancers. Conclusively, our findings suggest that the Peruvoside possesses a broad spectrum of anticancer activity in breast, lung, and liver cancers, which provides an impetus for further investigation of the anticancer potentiality of this biomolecule., (Copyright © 2019 Elsevier Inc. All rights reserved.)