Calmodulin Triggers Activity-Dependent rRNA Biogenesis via Interaction with DDX21.

Protein synthesis in response to neuronal activity, known as activity-dependent translation, is critical for synaptic plasticity and memory formation. However, the signaling cascades that couple neuronal activity to the translational events remain elusive. In this study, we identified the role of calmodulin (CaM), a conserved Ca2+-binding protein, in ribosomal RNA (rRNA) biogenesis in neurons. We found the CaM-regulated rRNA synthesis is Ca2+-dependent and necessary for nascent protein synthesis and axon growth in hippocampal neurons. Mechanistically, CaM interacts with nucleolar DEAD (Asp-Glu-Ala-Asp) box RNA helicase (DDX21) in a Ca2+-dependent manner to regulate nascent rRNA transcription within nucleoli. We further found CaM alters the conformation of DDX21 to liberate the DDX21-sequestered RPA194, the catalytic subunit of RNA polymerase I, to facilitate transcription of ribosomal DNA. Using high-throughput screening, we identified the small molecules batefenterol and indacaterol that attenuate the CaM-DDX21 interaction and suppress nascent rRNA synthesis and axon growth in hippocampal neurons. These results unveiled the previously unrecognized role of CaM as a messenger to link the activity-induced Ca2+ influx to the nucleolar events essential for protein synthesis. We thus identified the ability of CaM to transmit information to the nucleoli of neurons in response to stimulation.

O-GlcNAcylation promotes topoisomerase IIα catalytic activity in breast cancer chemoresistance.

DNA topoisomerase IIα (TOP2A) plays a vital role in replication and cell division by catalytically altering DNA topology. It is a prominent target for anticancer drugs, but clinical efficacy is often compromised due to chemoresistance. In this study, we investigate the role of TOP2A O-GlcNAcylation in breast cancer cells and patient tumor tissues. Our results demonstrate that elevated TOP2A, especially its O-GlcNAcylation, promotes breast cancer malignant progression and resistance to adriamycin (Adm). O-GlcNAcylation at Ser1469 enhances TOP2A chromatin DNA binding and catalytic activity, leading to resistance to Adm in breast cancer cells and xenograft models. Mechanistically, O-GlcNAcylation-modulated interactions between TOP2A and cell cycle regulators influence downstream gene expression and contribute to breast cancer drug resistance. These results reveal a previously unrecognized mechanistic role for TOP2A O-GlcNAcylation in breast cancer chemotherapy resistance and provide support for targeting TOP2A O-GlcNAcylation in cancer therapy.

The triglyceride-glucose index, ventricular arrhythmias and major cardiovascular events in patients at high risk of sudden cardiac death.

BACKGROUND: The association between the triglyceride-glucose (TyG) index and ventricular arrhythmias (VAs) is unclear. This study aimed to investigate the relationship between the TyG index, VAs, and major cardiovascular events in patients at high risk of sudden cardiac death (SCD). METHODS: We enrolled 1046 patients at high risk of SCD with an indication for implantable cardioverter-defibrillator (ICD) implantation at the Chinese National Center for Cardiovascular Diseases. The primary outcome was VAs, defined as sustained ventricular tachycardia and ventricular fibrillation documented by the ICD. The secondary outcomes were cardiac mortality, heart transplantation, and rehospitalization for heart failure. RESULTS: The mean (± SD) age was 59.6 ± 14.0 years old, and 25.7% were female. During the mean follow-up of 36.1 months, 342 (32.7%) patients had VAs, and 185 (17.7%) patients had major cardiovascular events. The mean fasting glucose and triglyceride levels were 111.9 ± 42.7 mg/dL and 140.0 ± 95.4 mg/L, respectively, with a TyG index range of 6.96-11.8. In the Fine-Gray subdistribution hazard model analysis, an increase in the TyG index was associated with a significant increase in the VAs (per 1 TyG index, hazard ratio [HR] 2.95; 95% confidence interval [CI], 2.29-3.80) and secondary outcome (HR 2.84; 95% CI 1.86-4.34). When stratified into tertiles, the risk of VAs was significantly higher in the highest tertile (HR 4.08; 95% CI, 2.81-5.92) than in the lowest tertile. Analysis of the secondary outcome revealed similar findings (HR 3.18; 95% CI, 1.73-5.85). CONCLUSIONS: In our cohort, the pre-operational TyG index is significantly associated with VAs and major cardiovascular events for patients with high risk of SCD.

Clinical Characteristics and Comparative Proteomics Analysis of COVID-19-Related Atrioventricular Block.

Background: Atrioventricular block (AVB) is thought to be a rare cardiovascular complication of the coronavirus disease 2019 (COVID-19), though limited data are available beyond case reports. We aim to describe the baseline characteristics, proteomics profile, and outcomes for patients with COVID-19-related AVB. Methods: We prospectively recruited patients diagnosed with COVID-19-related AVB between November 2022 and March, 2023. Inclusion criteria were hospitalization for COVID-19 with the diagnosis of AVB. A total of 24 patients diagnosed with COVID-19 without AVB were recruited for control. We analyzed patient characteristics and outcomes and performed a comparative proteomics analysis on plasma samples of those patients and controls. Results: A total of 17 patients diagnosed with COVID-19-related AVB and 24 individuals diagnosed with COVID-19 infection without AVB were included. Among patients with COVID-19-related AVB, the proportion of concurrent pneumonia was significantly higher than controls (7/17 versus 2/24, p < 0.05). All 17 AVB patients (9 of permanent AVB, 8 of paroxysmal AVB) received permanent pacemaker implantation. No procedural-related complication occurred. In laboratory tests, the level of biomarkers indicating myocardial damage were substantially higher than controls, including high-sensitivity cardiac troponin-I (median 0.005 versus 0.002 ng/mL, p < 0.05), myoglobulin (median 39.0 versus 27.6 ng/mL, p < 0.05), and MB isoenzyme of creatine kinase (median 1.2 versus 0.8 U/L, p < 0.05). The level of N-terminal pro-b-type natriuretic peptide (median 241.0 versus 33.5 pg/mL, p < 0.05), C-reactive protein (median 4.8 versus 2.0 mg/L, p < 0.05), D-dimer (median 1.2 versus 0.2 µg/mL, p < 0.05), left ventricular end-diastolic diameter (median 49.3 versus 45.7 mm, p < 0.05) in patients with COVID-19-related AVB were significantly higher than controls. The level of albumin (median 41.9 versus 44.5 g/L, p < 0.05) was significantly lower than controls. In comparative proteomics analysis, we identified 397 human proteins. Several significantly altered plasma proteins related to inflammatory response (Serum amyloid A protein, C-reactive protein, Protein Adenosine 5'-monophosphate-activated protein kinase (AMPK), Alpha-2-macroglobulin), complement and coagulation cascades (Tetranectin, haptoglobin), and immune response (Neutrophil defensin 3, Fibrinogen beta chain) may contribute to the pathogenesis of COVID-19-related AVB. Conclusions: Patients with COVID-19-related AVB are more prone to have myocardial damage and concurrent pneumonia. Through laboratory tests and comparative proteomics analysis, we identified several differential expressed proteins (Serum amyloid A protein, Tetranectin, Neutrophil defensin 3) releated to the inflammatory response, complement and coagulation cascades, and immune response, which provides evidence of potential biomarkers and sheds light on the pathogenesis of COVID-19-related AVB.

Developing a Semi-Supervised Approach Using a PU-Learning-Based Data Augmentation Strategy for Multitarget Drug Discovery.

Multifactorial diseases demand therapeutics that can modulate multiple targets for enhanced safety and efficacy, yet the clinical approval of multitarget drugs remains rare. The integration of machine learning (ML) and deep learning (DL) in drug discovery has revolutionized virtual screening. This study investigates the synergy between ML/DL methodologies, molecular representations, and data augmentation strategies. Notably, we found that SVM can match or even surpass the performance of state-of-the-art DL methods. However, conventional data augmentation often involves a trade-off between the true positive rate and false positive rate. To address this, we introduce Negative-Augmented PU-bagging (NAPU-bagging) SVM, a novel semi-supervised learning framework. By leveraging ensemble SVM classifiers trained on resampled bags containing positive, negative, and unlabeled data, our approach is capable of managing false positive rates while maintaining high recall rates. We applied this method to the identification of multitarget-directed ligands (MTDLs), where high recall rates are critical for compiling a list of interaction candidate compounds. Case studies demonstrate that NAPU-bagging SVM can identify structurally novel MTDL hits for ALK-EGFR with favorable docking scores and binding modes, as well as pan-agonists for dopamine receptors. The NAPU-bagging SVM methodology should serve as a promising avenue to virtual screening, especially for the discovery of MTDLs.

Etiology Distribution, Clinical Characteristics, and Suboptimal Pacing Outcome of Atrioventricular Block in Young Patients.

Background: The causes of atrioventricular block (AVB) are different and diverse young patients, as compared to the old. However, little is known about the etiology distribution and clinical characteristics of AVB in the young group. Methods: We retrospectively analyzed clinical information for AVB patients under 50 years of age. We summarized clinical phenotypes for patients with undetermined AVB etiology, according to AVB type and cardiac-structural change, whereas those who received pacing therapy were followed up for suspected heart failure events (HFEs). Results: AVB etiology was identified in only 289 (61.4%) patients, while 38.6% still have undertermined etiology for AVB. Non-ischemic cardiomyopathy (16.6%) and complication of cardiac surgery (13.4%) were the top two etiologies. In addition, four distinct phenotypes were identified in AVB patients with undetermined etiology, of which the severe phenotype (both borderline/elevated left ventricular diameter or abnormal left ventricular ejection fraction and advanced AVB) accounted for 17%. Notably, 80.7% of patients with severe phenotype received pacing therapy. Based on a median follow-up time of 17.5 months, we found the occurrence of 16 suspected HFEs in 110 pacemaker receivers (12 were lost to follow up). Notably, the severe phenotype was associated with a higher risk of heart failure (HF) symptoms. Conclusions: AVB etiology in young patients under 50 years of age is complex and underdiagnosed. In patients with undetermined etiology, severe phenotype featuring advanced AVB and abnormal Left ventricle (LV) structure/function is associated with a higher rate of HF symptoms even after pacing therapy.

Left Bundle Branch Area Pacing Contributes to a Greater Acute Blood Pressure Reduction Compared to Right Ventricular Pacing.

Background: Several previous studies have explored the potential arterial blood pressure (BP) changes in patients undergoing right ventricular pacing (RVP), however, the relationship between left bundle branch area pacing (LBBAP) and BP variations remains unknown. This study aimed to examine the acute BP variations following LBBAP and RVP implantation in patients with bradycardia. Methods: We conducted a single-center retrospective study including all patients who underwent de-novo dual-chamber pacemaker implantation between January 2019 and June 2021. Patients were divided into two groups, LBBAP and RVP, and propensity score-matching (PSM) was used to balance confounding factors. Three time periods were defined according to the timing of the implant: baseline (within 24 hours before implantation), hyper-acute period (0-24 hours post-implantation), and acute period (24-48 hours post-implantation). BP was measured at least three times per period using an arm pressure cuff and then averaged for analysis, which allowed us to determine the acute impact of pacemaker implantation on BP. Results: From a cohort of 898 patients, 193 LBBAP receivers were matched to 193 RVP receivers. A significant decrease in systolic BP (SBP) after the implantation was observed in the study cohort, from baseline 137.3 ± 9.2 mmHg to the acute period of 127.7 ± 9.4 mmHg (p < 0.001). The LBBAP group exhibited a greater SBP reduction than the RVP group ( Δ 11.6 ± 6.2 mmHg vs. Δ 7.6 ± 5.8 mmHg, p < 0.001). In further subgroup analysis, LBBAP receivers who had high baseline SBP (p < 0.001) and those without using anti-hypertensive drugs (p = 0.045) appeared to have a higher magnitude of SBP reduction. Conclusions: Permanent pacemaker implantation may contribute to an acute decrease in systolic BP, which was more pronounced in LBBAP receivers. Future experimental and clinical investigations are necessary to explore the underlying mechanisms and the long-term hemodynamic effects of LBBAP versus RVP.

Dietary γ-mangostin triggers immunogenic cell death and activates cGAS signaling in acute myeloid leukemia.

Immunogenic cell death (ICD), one of cell-death types through release of damage-associated molecular patterns from dying tumor cells, activates tumor-specific immune response and elicits anti-tumor immunity by traditional radiotherapy and chemotherapy. However, whether natural products could induce ICD in leukemia is not elucidated. Here, we report dietary γ-mangostin eradicates murine primary leukemic cells and prolongs the survival of leukemic mice. As well, it restrains primary leukemic cells and CD34+ leukemic progenitor cells from leukemia patients. Strikingly, γ-mangostin attenuates leukemic cells by inducing ICD as characterized by expression of HSP90B1, ANXA1 and IL1B. Additionally, γ-mangostin accelerates cytoplasmic chromatin fragments generation, promoting DNA damage response, and enhances cGAS activation, leading to up-regulation of chemokines. Meanwhile, it induces HDAC4 degradation and acetylated histone H3 accumulation, which promotes chemokines transcription. Ultimately, CD8+ T cell is activated and recruited by γ-mangostin-induced chemokines in the microenvironment. Our study identifies γ-mangostin triggers ICD and activates cGAS signaling through DNA damage response and epigenetic modification. Therefore, dietary γ-mangostin would act as a potential agent to provoke anti-tumor immunity in the prevention and treatment of leukemia.

Cardiac magnetic resonance-derived myocardial scar is associated with echocardiographic response and clinical prognosis of left bundle branch area pacing for cardiac resynchronization therapy.

AIMS: Left bundle branch area pacing (LBBAP) is a novel approach for cardiac resynchronization therapy (CRT), but the impact of myocardial substrate on its effect is poorly understood. This study aims to assess the association of cardiac magnetic resonance (CMR)-derived scar burden and the response of CRT via LBBAP. METHODS AND RESULTS: Consecutive patients with CRT indications who underwent CMR examination and successful LBBAP-CRT were retrospectively analysed. Cardiac magnetic resonance late gadolinium enhancement was used for scar assessment. Echocardiographic reverse remodelling and composite outcomes (defined as all-cause death or heart failure hospitalization) were evaluated. The echocardiographic response was defined as a ≥15% reduction of left ventricular end-systolic volume. Among the 54 patients included, LBBAP-CRT resulted in a 74.1% response rate. The non-responders had higher global, septal, and lateral scar burden (all P < 0.001). Global, septal, and lateral scar percentage all predicted echocardiographic response [area under the curve (AUC): 0.857, 0.864, and 0.822; positive likelihood ratio (+LR): 9.859, 5.594, and 3.059; and negative likelihood ratio (-LR): 0.323, 0.233, and 0.175 respectively], which was superior to QRS morphology criteria (Strauss left bundle branch abnormality: AUC: 0.696, +LR 2.101, and -LR 0.389). After a median follow-up time of 20.3 (11.5-38.7) months, higher global, lateral and septal scar burdens were all predictive of the composite outcome (hazard ratios: 4.996, 7.019, and 4.741, respectively; P's < 0.05). CONCLUSION: Lower scar burden was associated with higher response rate of LBBAP-CRT. The pre-procedure CMR scar evaluation provides further useful information to identify potential responders and clinical outcomes.

The Cytotoxic Cardiac Glycoside (-)-Cryptanoside A from the Stems of Cryptolepis dubia and Its Molecular Targets.

A cardiac glycoside epoxide, (-)-cryptanoside A (1), was isolated from the stems of Cryptolepis dubia collected in Laos, for which the complete structure was confirmed by analysis of its spectroscopic and single-crystal X-ray diffraction data, using copper radiation at a low temperature. This cardiac glycoside epoxide exhibited potent cytotoxicity against several human cancer cell lines tested, including HT-29 colon, MDA-MB-231 breast, OVCAR3 and OVCAR5 ovarian cancer, and MDA-MB-435 melanoma cells, with the IC50 values found to be in the range 0.1-0.5 µM, which is comparable with that observed for digoxin. However, it exhibited less potent activity (IC50 1.1 µM) against FT194 benign/nonmalignant human fallopian tube secretory epithelial cells when compared with digoxin (IC50 0.16 µM), indicating its more selective activity toward human cancer versus benign/nonmalignant cells. (-)-Cryptanoside A (1) also inhibited Na+/K+-ATPase activity and increased the expression of Akt and the p65 subunit of NF-κB but did not show any effects on the expression of PI3K. A molecular docking profile showed that (-)-cryptanoside A (1) binds to Na+/K+-ATPase, and thus 1 may directly target Na+/K+-ATPase to mediate its cancer cell cytotoxicity.

The catalytic mechanism of direction-dependent interactions for 2,3-dihydroxybenzoate decarboxylase.

Benzoic acid decarboxylases offer an elegant alternative to CO2 fixation by reverse reaction-carboxylation, which is named the bio-Kolbe-Schmitt reaction, but they are unfavorable to carboxylation. Enhancing the carboxylation efficiency of reversible benzoic acid decarboxylases is restricted by the unexplained carboxylation mechanisms. The direction of reversible enzyme catalytic reactions depends on whether catalytic residues at the active center of the enzyme are protonated, which is subjected by the pH. Therefore, the forward and reverse reactions could be separated at different pH values. Reversible 2,3-dihydroxybenzoate acid decarboxylase undergoes decarboxylation at pH 5.0 and carboxylation at pH 8.6. However, it is unknown whether the interaction of enzymes with substrates and products in the forward and reverse reactions can be exploited to improve the catalytic activity of reversible enzymes in the unfavorable direction. Here, we identify a V-shaped tunnel of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae (2,3-DHBD_Ao) through which the substrate travels in the enzyme, and demonstrate that the side chain conformation of a tyrosine residue controls the entry and exit of substrate/product during reversible reactions. Together with the kinetic studies of the mutants, it is clarified that interactions between substrate/product traveling through the enzyme tunnel in 2,3-DHBD_Ao are direction-dependent. These results enrich the understanding of the interactions of substrates/products with macromolecular reversible enzymes in different reaction directions, thereby demonstrating a possible path for engineering decarboxylases with higher carboxylation efficiency. KEY POINTS: • The residue Trp23 of 2,3-DHBD_Ao served as a switch to control the entry and exit of catechol • A V-shaped tunnel of 2,3-DHBD_Ao for decarboxylation and carboxylation reactions was identified • The results provide a promising strategy for engineering decarboxylases with direction-dependent residues inside the substrate/product traveling tunnel of the enzyme.

A Targetless Method for Simultaneously Measuring Three-Degree-of-Freedom Angular Motion Errors with Digital Speckle Pattern Interferometry.

As a guide rail is the basic motion unit of precision equipment, the measurement of and compensation for its motion errors are important preconditions for precision machining and manufacturing. A targetless and simultaneous measurement method of three-degree-of-freedom (3-DOF) angular motion errors using digital speckle pattern interferometry (DSPI) is introduced in this paper. Based on the analysis of the sensitivity mechanism of DSPI to DOF errors and the formation mechanism of the phase fringes, the relationship between the angular motion errors and the distribution of the interferometric phases was established, and a new simultaneous measurement model of 3-DOF angular motion errors was further proposed. An optical setup based on a three-dimensional spatial-carrier DSPI with a right-angle symmetrical layout was used in the measurement system. Furthermore, repetitive tests, noise tests, and precision analysis were carried out to verify the performance of the system. The test results showed that the measurement resolution of the system was <1 µrad, which is capable of measuring the pitch angle, yaw angle, and roll angle at the submicron arc level simultaneously without target mirrors. The method has the advantages of no need to install cooperative targets and high measurement resolution, showing broad application prospects in many fields, including mechanical manufacturing, laser detection, aerospace, etc.

Variations in surface functional groups, carbon chemical state and graphitization degree during thermal deactivation of diesel soot particles.

The thermal deactivation of diesel soot particles exerts a significant influence on the control strategy for the regeneration of diesel particulate filters (DPFs). This work focused on the changes in the surface functional groups, carbon chemical state, and graphitization degree during thermal treatment in an inert gas environment at intermediate temperatures of 600°C, 800°C, and 1000°C and explore the chemical species that were desorbed from the diesel soot surface during thermal treatment using a thermogravimetric analyser coupled with a gas-chromatograph mass spectrometer (TGA-GC/MS). The surface functional groups and carbon chemical state were characterized using Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS). The graphitization degree was evaluated by means of Raman spectroscopy (RS). The concentrations of aliphatic C-H, C-OH, C=O, and O-C=O groups are reduced for diesel soot and carbon black when increasing the thermal treatment temperature, while the sp2/sp3 hybridized ratio and graphitization degree enhance. These results provide comprehensive evidence of the decreased reactivity of soot samples. Among oxygenated functional groups, the percentage reduction during thermal treatment is the largest for the O-C=O groups owing to its worst thermodynamic stability. TGA-GC/MS results show that the aliphatic and aromatic chains and oxygenated species would be desorbed from the soot surface during 1000°C thermal treatment of diesel soot.

ppGalNAc-T4-catalyzed O-Glycosylation of TGF-ß type â ¡ receptor regulates breast cancer cells metastasis potential.

GalNAc-type O-glycosylation, initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), is one of the most abundant and complex posttranslational modifications of proteins. Emerging evidence has proven that aberrant ppGalNAc-Ts are involved in malignant tumor transformation. However, the exact molecular functions of ppGalNAc-Ts are still unclear. Here, the role of one isoform, ppGalNAc-T4, in breast cancer cell lines was investigated. The expression of ppGalNAc-T4 was found to be negatively associated with migration of breast cancer cells. Loss-of-function studies revealed that ppGalNAc-T4 attenuated the migration and invasion of breast cancer cells by inhibiting the epithelial-mesenchymal transition (EMT) process. Correspondingly, transforming growth factor beta (TGF-ß) signaling, which is the upstream pathway of EMT, was impaired by ppGalNAc-T4 expression. ppGalNAc-T4 knockout decreased O-GalNAc modification of TGF-ß type Ⅰ and Ⅱ receptor (TßR Ⅰ and Ⅱ) and led to the elevation of TGF-ß receptor dimerization and activity. Importantly, a peptide from TßR Ⅱ was identified as a naked peptide substrate of ppGalNAc-T4 with a higher affinity than ppGalNAc-T2. Further, Ser31, corresponding to the extracellular domain of TßR Ⅱ, was identified as the O-GalNAcylation site upon in vitro glycosylation by ppGalNAc-T4. The O-GalNAc-deficient S31 A mutation enhanced TGF-ß signaling activity and EMT in breast cancer cells. Together, these results identified a novel mechanism of ppGalNAc-T4-catalyzed TGF-ß receptors O-GalNAcylation that suppresses breast cancer cell migration and invasion via the EMT process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment.

Targeting SOST using a small-molecule compound retards breast cancer bone metastasis.

BACKGROUND: Breast cancer metastasis to the bone can be exacerbated by osteoporosis, is associated with poor long-term survival, and has limited therapeutic options. Sclerostin (SOST) is an endogenous inhibitor of bone formation, and an attractive target for treatment of osteoporosis. However, it is unclear whether SOST can be used as a therapeutic target for bone metastases of breast cancer, and whether small molecule compounds that target SOST in breast cancer cells can inhibit breast cancer bone metastasis. METHODS: SOST expression in 442 breast cancer tissues was characterized by immunohistochemistry and statistically analyzed for the association with breast cancer bone metastases. Bone metastatic breast cancer SCP2 cells were induced for SOST silencing or overexpression and their bone metastatic behaviors were tested in vitro and in vivo. To identify potential therapeutics, we screened inhibitors of the interaction of SOST with STAT3 from a small chemical molecule library and tested the inhibitory effects of one inhibitor on breast cancer growth and bone metastasis in vitro and in vivo. RESULTS: We found that up-regulated SOST expression was associated with breast cancer bone metastases and worse survival of breast cancer patients. SOST silencing significantly reduced the bone metastatic capacity of SCP2 cells. SOST interacted with STAT3 to enhance the TGF-ß/KRAS signaling, increasing both tumor growth and bone metastasis. Treatment with one lead candidate, S6, significantly inhibited the growth of breast-cancer organoids and bone metastasis in mice. CONCLUSIONS: Our findings highlight a new class of potential therapeutics for treatment of bone metastasis in breast cancer.

Structural Insights into the Interactions of Digoxin and Na+/K+-ATPase and Other Targets for the Inhibition of Cancer Cell Proliferation.

Digoxin is a cardiac glycoside long used to treat congestive heart failure and found recently to show antitumor potential. The hydroxy groups connected at the C-12, C-14, and C-3'a positions; the C-17 unsaturated lactone unit; the conformation of the steroid core; and the C-3 saccharide moiety have been demonstrated as being important for digoxin's cytotoxicity and interactions with Na+/K+-ATPase. The docking profiles for digoxin and several derivatives and Na+/K+-ATPase were investigated; an additional small Asn130 side pocket was revealed, which could be useful in the design of novel digoxin-like antitumor agents. In addition, the docking scores for digoxin and its derivatives were found to correlate with their cytotoxicity, indicating a potential use of these values in the prediction of the cancer cell cytotoxicity of other cardiac glycosides. Moreover, in these docking studies, digoxin was found to bind to FIH-1 and NF-κB but not HDAC, IAP, and PI3K, suggesting that this cardiac glycoside directly targets FIH-1, Na+/K+-ATPase, and NF-κB to mediate its antitumor potential. Differentially, digoxigenin, the aglycon of digoxin, binds to HDAC and PI3K, but not FIH-1, IAP, Na+/K+-ATPase, and NF-κB, indicating that this compound may target tumor autophagy and metabolism to mediate its antitumor propensity.

Interaction of (+)-Strebloside and Its Derivatives with Na+/K+-ATPase and Other Targets.

Docking profiles for (+)-strebloside, a cytotoxic cardiac glycoside identified from Streblus asper, and some of its derivatives and Na+/K+-ATPase have been investigated. In addition, binding between (+)-strebloside and its aglycone, strophanthidin, and several of their other molecular targets, including FIH-1, HDAC, KEAP1 and MDM2 (negative regulators of Nrf2 and p53, respectively), NF-κB, and PI3K and Akt1, have been inspected and compared with those for digoxin and its aglycone, digoxigenin. The results showed that (+)-strebloside, digoxin, and their aglycones bind to KEAP1 and MDM2, while (+)-strebloside, strophanthidin, and digoxigenin dock to the active pocket of PI3K, and (+)-strebloside and digoxin interact with FIH-1. Thus, these cardiac glycosides could directly target HIF-1, Nrf2, and p53 protein-protein interactions, Na+/K+-ATPase, and PI3K to mediate their antitumor activity. Overall, (+)-strebloside seems more promising than digoxin for the development of potential anticancer agents.

Reversible inhibitor of CRM1 sensitizes glioblastoma cells to radiation by blocking the NF-κB signaling pathway.

BACKGROUND: Activation of nuclear factor-kappa B (NF-κΒ) through DNA damage is one of the causes of tumor cell resistance to radiotherapy. Chromosome region 1 (CRM1) regulates tumor cell proliferation, drug resistance, and radiation resistance by regulating the nuclear-cytoplasmic translocation of important tumor suppressor proteins or proto-oncoproteins. A large number of studies have reported that inhibition of CRM1 suppresses the activation of NF-κΒ. Thus, we hypothesize that the reversible CRM1 inhibitor S109 may induce radiosensitivity in glioblastoma (GBM) by regulating the NF-κΒ signaling pathway. METHODS: This study utilized the cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and colony formation assay to evaluate the effect of S109 combined with radiotherapy on the proliferation and survival of GBM cells. The therapeutic efficacy of S109 combined with radiotherapy was evaluated in vivo to explore the therapeutic mechanism of S109-induced GBM radiosensitization. RESULTS: We found that S109 combined with radiotherapy significantly inhibited GBM cell proliferation and colony formation. By regulating the levels of multiple cell cycle- and apoptosis-related proteins, the combination therapy induced G1 cell cycle arrest in GBM cells. In vivo studies showed that S109 combined with radiotherapy significantly inhibited the growth of intracranial GBM and prolonged survival. Importantly, we found that S109 combined with radiotherapy promoted the nuclear accumulation of IκΒα, and inhibited phosphorylation of p65 and the transcriptional activation of NF-κΒ. CONCLUSION: Our findings provide a new therapeutic regimen for improving GBM radiosensitivity as well as a scientific basis for further clinical trials to evaluate this combination therapy.

Cytotoxic and non-cytotoxic cardiac glycosides isolated from the combined flowers, leaves, and twigs of Streblus asper.

A new non-cytotoxic [(+)-17ß-hydroxystrebloside (1)] and two known cytotoxic [(+)-3'-de-O-methylkamaloside (2) and (+)-strebloside (3)] cardiac glycosides were isolated and identified from the combined flowers, leaves, and twigs of Streblus asper collected in Vietnam, with the absolute configuration of 1 established from analysis of its ECD and NMR spectroscopic data and confirmed by computational ECD calculations. A new 14,21-epoxycardanolide (3a) was synthesized from 3 that was treated with base. A preliminary structure-activity relationship study indicated that the C-14 hydroxy group and the C-17 lactone unit and the established conformation are important for the mediation of the cytotoxicity of 3. Molecular docking profiles showed that the cytotoxic 3 and its non-cytotoxic analogue 1 bind differentially to Na+/K+-ATPase. Compound 3 docks deeply in the Na+/K+-ATPase pocket with a sole pose, and its C-10 formyl and C-5, C-14, and C-4' hydroxy groups may form hydrogen bonds with the side-chains of Glu111, Glu117, Thr797, and Arg880 of Na+/K+-ATPase, respectively. However, 1 fits the cation binding sites with at least three different poses, which all depotentiate the binding between 1 and Na+/K+-ATPase. Thus, 3 was found to inhibit Na+/K+-ATPase, but 1 did not. In addition, the cytotoxic and Na+/K+-ATPase inhibitory 3 did not affect glucose uptake in human lung cancer cells, against which it showed potent activity, indicating that this cardiac glycoside mediates its cytotoxicity by targeting Na+/K+-ATPase but not by interacting with glucose transporters.

Na+/K+-ATPase-Targeted Cytotoxicity of (+)-Digoxin and Several Semisynthetic Derivatives.

(+)-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)-ß-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-12ß, HO-14ß, 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.