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Strophanthidin (SPTD), one of the cardiac glycosides, is refined from traditional Chinese medicines such as Semen Lepidii and Antiaris toxicaria, and was initially used for the treatment of heart failure disease in clinic. Recently, SPTD has been shown to be a potential anticancer agent, but the underlying mechanism of action is poorly understood. Herein, we explored the molecular mechanism by which SPTD exerts anticancer effects in A549 human lung adenocarcinoma cells by means of mass spectrometry-based quantitative proteomics in combination with bioinformatics analysis. We revealed that SPTD promoted the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2, or DR5) in A549 cells to activate caspase 3/6/8, in particular caspase 3. Consequently, the activated caspases elevated the expression level of apoptotic chromatin condensation inducer in the nucleus (ACIN1) and prelamin-A/C (LMNA), ultimately inducing apoptosis via cooperation with the SPTD-induced overexpressed barrier-to-autointegration factor 1 (Banf1). Moreover, the SPTD-induced DEPs interacted with each other to downregulate the p38 MAPK/ERK signaling, contributing to the SPTD inhibition of the growth of A549 cells. Additionally, the downregulation of collagen COL1A5 by SPTD was another anticancer benefit of SPTD through the modulation of the cell microenvironment. [ABSTRACT FROM AUTHOR]

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

Strophanthidin (SPTD), one of the cardiac glycosides, is refined from traditional Chinese medicines such as Semen Lepidii and Antiaris toxicaria, and was initially used for the treatment of heart failure disease in clinic. Recently, SPTD has been shown to be a potential anticancer agent, but the underlying mechanism of action is poorly understood. Herein, we explored the molecular mechanism by which SPTD exerts anticancer effects in A549 human lung adenocarcinoma cells by means of mass spectrometry-based quantitative proteomics in combination with bioinformatics analysis. We revealed that SPTD promoted the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2, or DR5) in A549 cells to activate caspase 3/6/8, in particular caspase 3. Consequently, the activated caspases elevated the expression level of apoptotic chromatin condensation inducer in the nucleus (ACIN1) and prelamin-A/C (LMNA), ultimately inducing apoptosis via cooperation with the SPTD-induced overexpressed barrier-to-autointegration factor 1 (Banf1). Moreover, the SPTD-induced DEPs interacted with each other to downregulate the p38 MAPK/ERK signaling, contributing to the SPTD inhibition of the growth of A549 cells. Additionally, the downregulation of collagen COL1A5 by SPTD was another anticancer benefit of SPTD through the modulation of the cell microenvironment.

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

Cardenolides, a group of cardiac glycosides are potent inhibitors of Na+/K+ ATPase pump in mammals, animals including insects. Some insects can circumvent the toxicity of cardenolides by mechanisms like target site resistance and metabolic resistance resulting in enhanced tolerance or adaptation. In this paper, we report an intriguing observation of a polyphagous feeder feeding gregariously on the leaves of Calotropis gigantea (L.) without any apparent adverse effect. No choice feeding assay showed higher larval biomass and reduced number of days to develop on C. gigantea leaves compared to Ricinus and banana. We found the activity of GST higher in C. gigantea fed larva and HR LC-MS analysis of Olepa sps. revealed the presence of glutathione-strophanthidin conjugate in larval body tissue. In silico molecular simulation results confirmed strong interaction between delta variant GST and glutathione-strophanthidin complex. The sequestration site and cost benefit of glutathione-strophanthidin sequestration in body tissues of Olepa sps. needs further investigation.

The metabolite profiling of white foamy substance local white cultivar of Cucumber (Cucumis sativus L.) was performed using HR-LCMS. Prominent compounds with MS spectra, peak list, and compound structure were studied. Which pertains to Cucurbitaceae. The cytotoxic activity was studied on onion root tip squash and microscopic evaluation was done. The principal component analysis (PCA) of the analytical data showed the presence of Triterpenes: Cucurbitacin-C (0.8ppm), Cucurbitacin-E (5.53ppm) and Cardiac glycosides: Gitoxigenin (7.76ppm), Strophanthidin (13.79ppm), and Digitoxigenin (2.13ppm) in 10-l of the loaded sample.100 other compounds were identified by accurate mass of Q-TOF/MS and verified by database compounds (IRM calibration). Strophanthidin in the highest concentration in the foamy extract is a significant finding. This study reports the traditional significance of removing white foamy substances for bitterness in cucumber before slicing. The cytotoxic study on onion root tips by photomicrograph study indicates reduction in root growth and chromosomal aberrations, disturbed telophase, anaphase, abnormal cell shape, dissolved chromosomes, membrane damage and nuclear lesions. [ABSTRACT FROM AUTHOR]

The Na+-K+-ATPase (Na+-K+ pump) is essential for setting resting membrane potential and restoring transmembrane Naþ and Kþ gradients after neuronal firing, yet its roles in developing neurons are not well understood. This study examined the contribution of the Na+-K+ pump to resting membrane potential and membrane excitability of developing CA1 and CA3 neurons and its role in maintaining synchronous network bursting. Experiments were conducted in postnatal day (P)9 to P13 rat hippocampal slices using whole cell patch-clamp and extracellular field-potential recordings. Blockade of the Na+-K+ pump with strophanthidin caused marked depolarization (23.1 mV) in CA3 neurons but only a modest depolarization (3.3 mV) in CA1 neurons. Regarding other membrane properties, strophanthidin differentially altered the voltage-current responses, input resistance, action-potential threshold and amplitude, rheobase, and input-output relationship in CA3 vs. CA1 neurons. At the network level, strophanthidin stopped synchronous epileptiform bursting in CA3 induced by 0 Mg2+ and 4-aminopyridine. Furthermore, dual whole cell recordings revealed that strophanthidin disrupted the synchrony of CA3 neuronal firing. Finally, strophanthidin reduced spontaneous excitatory postsynaptic current (sEPSC) bursts (i.e., synchronous transmitter release) and transformed them into individual sEPSC events (i.e., nonsynchronous transmitter release). These data suggest that the Na+-K+ pump plays a more profound role in membrane excitability in developing CA3 neurons than in CA1 neurons and that the pump is essential for the maintenance of synchronous network bursting in CA3. Compromised Naþ-Kþ pump function leads to cessation of ongoing synchronous network activity, by desynchronizing neuronal firing and neurotransmitter release in the CA3 synaptic network. These findings have implications for the regulation of network excitability and seizure generation in the developing brain. [ABSTRACT FROM AUTHOR]

Lung cancer is the most prevalent in cancer-related deaths, while breast carcinoma is the second most dominant cancer in women, accounting for the most number of deaths worldwide. Cancers are heterogeneous diseases that consist of several subtypes based on the presence or absence of hormone receptors and human epidermal growth factor receptor 2. Several drugs have been developed targeting cancer biomarkers; nonetheless, their efficiency are not adequate due to the high reemergence rate of cancers and fundamental or acquired resistance toward such drugs, which leads to partial therapeutic possibilities. Recent studies on cardiac glycosides (CGs) positioned them as potent cytotoxic agents that target multiple pathways to initiate apoptosis and autophagic cell death in many cancers. In the present study, our aim is to identify the anticancer activity of a naturally available CG (strophanthidin) in human breast (MCF-7), lung (A549), and liver cancer (HepG2) cells. Our results demonstrate a dose-dependent cytotoxic effect of strophanthidin in MCF-7, A549, and HepG2 cells, which was further supported by DNA damage on drug treatment. Strophanthidin arrested the cell cycle at the G2/M phase; this effect was further validated by checking the inhibited expressions of checkpoint and cyclin-dependent kinases in strophanthidin-induced cells. Moreover, strophanthidin inhibited the expression of several key proteins such as MEK1, PI3K, AKT, mTOR, Gsk3α, and β-catenin from MAPK, PI3K/AKT/mTOR, and Wnt/β-catenin signaling. The current study adequately exhibits the role of strophanthidin in modulating the expression of various key proteins involved in cell cycle arrest, apoptosis, and autophagic cell death. Our in silico studies revealed that strophanthidin can interact with several key proteins from various pathways. Taken together, this study demonstrates the viability of strophanthidin as a promising anticancer agent, which may serve as a new anticancer drug.

Lung cancer is the most prevalent in cancer-related deaths, while breast carcinoma is the second most dominant cancer in women, accounting for the most number of deaths worldwide. Cancers are heterogeneous diseases that consist of several subtypes based on the presence or absence of hormone receptors and human epidermal growth factor receptor 2. Several drugs have been developed targeting cancer biomarkers; nonetheless, their efficiency are not adequate due to the high reemergence rate of cancers and fundamental or acquired resistance toward such drugs, which leads to partial therapeutic possibilities. Recent studies on cardiac glycosides (CGs) positioned them as potent cytotoxic agents that target multiple pathways to initiate apoptosis and autophagic cell death in many cancers. In the present study, our aim is to identify the anticancer activity of a naturally available CG (strophanthidin) in human breast (MCF-7), lung (A549), and liver cancer (HepG2) cells. Our results demonstrate a dose-dependent cytotoxic effect of strophanthidin in MCF-7, A549, and HepG2 cells, which was further supported by DNA damage on drug treatment. Strophanthidin arrested the cell cycle at the G2/M phase; this effect was further validated by checking the inhibited expressions of checkpoint and cyclin-dependent kinases in strophanthidin-induced cells. Moreover, strophanthidin inhibited the expression of several key proteins such as MEK1, PI3K, AKT, mTOR, Gsk3α, and β-catenin from MAPK, PI3K/AKT/mTOR, and Wnt/β-catenin signaling. The current study adequately exhibits the role of strophanthidin in modulating the expression of various key proteins involved in cell cycle arrest, apoptosis, and autophagic cell death. Our in silico studies revealed that strophanthidin can interact with several key proteins from various pathways. Taken together, this study demonstrates the viability of strophanthidin as a promising anticancer agent, which may serve as a new anticancer drug. [ABSTRACT FROM AUTHOR]

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

Cardiac glycosides are the efficacious groups of naturally occurring compounds that influences the vital blood pumping mechanism. They are constituted of two parts i.e. steroidal aglycon part and the glycon. The glycon part may contain a monosaccharide or di to oligosaccharide. In this present paper we have made glycosyl donars of glucose, mannose, galactose and gentiobiose a disaccharide, glycosyl donars of these mono and disaccaharide were prepared, further they were glycosidically linked to two cardenolides i.e. digoxigenin and strophanthidin by TMS-OTF method. The structure of synthetically prepared six monoglycosides and two diglycosides of digoxigenin and strophanthidin were confirmed by 1HNMR. [ABSTRACT FROM AUTHOR]

Strophanthus extracts containing cardioactive cardenolides are still applied in European complementary medicine for the treatment of heart diseases. However, the cardenolide profile and the fate of individual compounds during extraction and storage are not well understood. Therefore, the objective of the present study was to characterize the cardenolide compound pattern in extracts of different polarity and their structural changes upon storage in aqueous fermented preparations. For this purpose, individual cardenolides were quantitated by a UHPLC-DAD validated method using an internal standard. Three different extraction protocols were compared: hydroethanolic extraction under reflux with and without previous defatting of the seed material and ultrasonic-assisted extraction at ambient temperature. Reflux extraction of non-defatted seeds showed maximum cardenolide yields. Differences in the cardenolide contents of seeds of the different origins Zimbabwe and Malawi were observed. The cardenolide profile and metabolization of individual compounds upon fermentation and storage of S. kombé seed extracts revealed that predominant cardenolides, mainly strophanthidin glycosides, changed upon storage over 12 months. Cardenolides exhibiting two or three saccharide moieties were degraded presumably by β-glucosidase activities, originating from the plant material or lactobacilli, releasing the corresponding monoglycosides. The latter were further degraded into the corresponding aglycones probably by acid hydrolysis as a result of lactic acid accumulation. [ABSTRACT FROM AUTHOR]

The dynamics of different ionic currents shape the bursting activity of neurons and networks that control motor output. Despite being ubiquitous in all animal cells, the contribution of the Na+/K+ pump current to such bursting activity has not been well studied. We used monensin, a Na+/H+ antiporter, to examine the role of the pump on the bursting activity of oscillator heart interneurons in leeches. When we stimulated the pump with monensin, the period of these neurons decreased significantly, an effect that was prevented or reversed when the h-current was blocked by Cs+. The decreased period could also occur if the pump was inhibited with strophanthidin or K+- free saline. Our monensin results were reproduced in model, which explains the pump's contributions to bursting activity based on Na+ dynamics. Our results indicate that a dynamically oscillating pump current that interacts with the h-current can regulate the bursting activity of neurons and networks. [ABSTRACT FROM AUTHOR]

Cardenolides, a group of cardiac glycosides are potent inhibitors of Na + /K + ATPase pump in mammals, animals including insects. Some insects can circumvent the toxicity of cardenolides by mechanisms like target site resistance and metabolic resistance resulting in enhanced tolerance or adaptation. In this paper, we report an intriguing observation of a polyphagous feeder feeding gregariously on the leaves of Calotropis gigantea (L.) without any apparent adverse effect. No choice feeding assay showed higher larval biomass and reduced number of days to develop on C. gigantea leaves compared to Ricinus and banana. We found the activity of GST higher in C. gigantea fed larva and HR LC-MS analysis of Olepa sps. revealed the presence of glutathione-strophanthidin conjugate in larval body tissue. In silico molecular simulation results confirmed strong interaction between delta variant GST and glutathione-strophanthidin complex. The sequestration site and cost benefit of glutathione-strophanthidin sequestration in body tissues of Olepa sps . needs further investigation., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Abstract: Two new strophanthidol cardenolides, toxicarioside N (1) and toxicarioside O (2), together with three known ones, were isolated from the seeds of Antiaris toxicaria. The structures of the new compounds were identified as strophanthidol-3-O-β-d-glucopyranosyl-(1→4)-6-deoxy-β-d-allopyranoside (1) and strophanthidol-3-O-β-d-glucopyranoside (2), respectively. The cytotoxic test showed that compounds 1 and 2 exhibited significant cytotoxicity against SMMC-7721 and K562. [Copyright &y& Elsevier]

Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of ouabain play a crucial role. In the present study, a series of ouabain analogues were tested on baculovirus-expressed Na,K-ATPase and an ouabain-sensitive mutant of non-gastric H,K-ATPase (D312E/ S319G/ A778P/ I795L/ F802C). For each analogue, the results obtained by measuring ATPase inhibition and [3H]ouabain replacement agreed rather well. In Na,K-ATPase, strophanthidin had a 7–10 times higher and digoxin a 4–12 times lower affinity than ouabain. The results of the non-gastric H,K-ATPase mutant were rather similar to that of Na,K-ATPase with exception of dihydro-ouabain that showed a much lower affinity with the non-gastric H,K-ATPase mutant. Docking studies showed that all analogues bind to the same pocket in Na,K-ATPase. However, the amino acids to which hydrogen bonds were formed differed and depended on the availability of hydroxyl or keto groups in the ouabain analogues. [ABSTRACT FROM AUTHOR]

Aim: To determine whether different Na+/K+ -ATPase signal transduction pathways have positive inotropic effects on normal ventricular myocytes (NC) and failing ventricular myocytes (FC), and are involved in an increase of [Ca2+]i induced by strophanthidin (Str). Methods: A guinea pig model of congestive heart failure was made by constricting descending aorta. The left ventricular myocytes were enzymatically isolated. The effects of 25 μmol/L Str with different signal-transducing inhibitors on contractility and the calcium transient of NC or FC from guinea pigs were simultaneously assessed and compared with those in the 25 μmol/L Stronly group by a video-based, motion-edge detection system. Results: Str at 1, 10, and 25 μmol/L in NC and Str at 0.1, 1, 10, and 25 μmol/L) in FC elevated the calcium transient amplitude and increased the positive inotropic effects in a concentration-dependent manner, respectively. At the same concentration, the effects of Str were more potent in FC than in NC. In FC, both the mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) signal transduction pathway of Na+/K+-ATPase were involved in the increase of the calcium transient induced by Str, but only activation of the MAPK pathway increased the calcium transient in NC. However, only the ROS pathway was involved in positive inotropic effects both in NC and FC. Conclusion: The present study suggests that Na+/K+-ATPase signaling pathways involved in the inotropic effects of Str in NC and FC are consistent, and Na+/K+-ATPase signaling pathways involved in the increase of [Ca2+]i by Str in NC and FC are different. [ABSTRACT FROM AUTHOR]

Abstract: Rotenone is a pesticide that produces a rodent model of Parkinson''s disease. Although much evidence suggests that oxidative stress mediates the toxicity of rotenone on dopamine neurons, rotenone can also potentiate glutamate excitotoxicity. We used whole-cell patch pipettes to investigate actions of rotenone on currents evoked by N-methyl-d-aspartate (NMDA) in dopamine neurons in slices of rat midbrain. After superfusing the slice for 20–30min, rotenone (100nM) caused a 162% increase in the average amplitude of inward current evoked by 30μM NMDA. This effect of rotenone was mimicked by the sodium pump inhibitor strophanthidin (10μM) and was abolished when pipettes contained an ATP regeneration solution. Although strophanthidin also significantly increased the amplitude of inward currents evoked by (±)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10μM), rotenone failed to potentiate AMPA currents. Because rotenone potentiated NMDA- but not AMPA-dependent currents, this suggests that rotenone acts selectively to augment NMDA receptor function. Furthermore, the failure of rotenone to mimic strophanthidin suggests that rotenone does not inhibit sodium pump activity. Our results suggest that an excitotoxic mechanism might contribute to rotenone neurotoxicity. [Copyright &y& Elsevier]

Nitric oxide (NO) has been demonstrated to have several effects on the heart. Through the stimulation of guanylate cyclase, NO increases cyclic GMP and decreases intracellular calcium. The purpose of this study was to evaluate the effects of NO on ventricular arrhythmia induced by strophanthidin in guinea pigs and dogs. In experiment 1, after strophanthidin-induced ventricular tachycardia, guinea pigs received different doses of L-arginine (0, 25, 50, 100, 200, and 400 mg/kg; n = 10 for each dose), 200 mg/kg L-arginine combined with 100 mg/kg N[sup G] -nitro-L-arginine methylester (L-NAME, n = 10), or 200 mg/kg D-arginine (n = 10). In experiment 2, after strophanthidin-induced ventricular tachycardia, dogs (n = 7) received 200 mg/kg L-arginine. By 12-lead ECG, monophasic action potentials in left and right ventricles were recorded throughout the study. In experiment 1, guinea pigs which received 200 mg/kg or 400 mg/kg L-arginine had greater incidences of ventricular tachycardia termination (60 and 80%, respectively) than those which received 0, 25, 50, and 100 mg/kg L-arginine (0, 0, 20, and 30%, respectively), those which received L-arginine with L-NAME (0%), and those which received D-arginine (0%). In experiment 2, 5 (71%) of the dogs had successful termination of ventricular tachycardia. These findings suggest that L-arginine was effective in treating strophanthidin-induced ventricular tachycardia in vivo and that the underlying mechanism is through a NO pathway.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]