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Involvement of cardiac glycosides targeting Na/K-ATPase in their inhibitory effects on c-Myc expression via its transcription, translation, and proteasomal degradation

ABSTRACT: Cardiac glycosides (CGs) have been used for decades to treat heart failure and arrhythmic diseases. Recent non-clinical and epidemiological findings have suggested that CGs exhibit anti-tumor activities. Therefore, CGs may be repositioned as drugs for the treatment of cancer. A detailed understanding of the anticancer mechanisms of CGs is essential for their application to the treatment of targetable cancer types. To elucidate the factors associated with the anti-tumor effects of CGs, we performed transcriptome profiling on human multiple myeloma AMO1 cells treated with periplocin, one of CGs. Periplocin significantly down-regulated the transcription of MYC (c-Myc), a well-established oncogene. Periplocin also suppressed c-Myc expression at the protein levels. This repression of c-Myc was also observed in several cell lines. To identify target proteins for the inhibition of c-Myc, we generated CG-resistant (C9) cells using a sustained treatment with digoxin. We confirmed that C9 cells acquired resistance to the inhibition of c-Myc expression and cell proliferation by CGs. Moreover, the sequencing of genomic DNA in C9 cells revealed the mutation of D128N in α1-Na/K-ATPase, indicating the target protein. These results suggest that CGs suppress c-Myc expression in cancer cells via α1-Na/K-ATPase, which provides further support for the anti-tumor activities of CGs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE227301 | GEO | 2023/04/07

REPOSITORIES: GEO

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Project description:The Na/K-ATPase is the specific receptor for cardiotonic steroids (CTS) such as ouabain and digoxin. At pharmacological concentrations used in the treatment of cardiac conditions, CTS inhibit the ion-pumping function of Na/K-ATPase. At much lower concentrations, in the range of those reported for endogenous CTS in the blood, they stimulate hypertrophic growth of cultured cardiac myocytes through initiation of a Na/K-ATPase-mediated and reactive oxygen species (ROS)-dependent signaling. To examine a possible effect of endogenous concentrations of CTS on cardiac structure and function in vivo, we compared mice expressing the naturally resistant Na/K-ATPase α1 and age-matched mice genetically engineered to express a mutated Na/K-ATPase α1 with high affinity for CTS. In this model, total cardiac Na/K-ATPase activity, α1, α2 and β1 protein content remained unchanged, and the cardiac Na/K-ATPase dose-response curve to ouabain shifted to the left as expected. In males aged 3–6 months, increased α1 sensitivity to CTS resulted in a significant increase of cardiac carbonylated protein content, suggesting that ROS production was elevated. A moderate but significant increase of about 15% of the heart-weight-to-tibia-length ratio, accompanied by an increase of myocyte cross-sectional area was detected. Echocardiographic analyses did not reveal any change in cardiac function, and there was no fibrosis or re-expression of the fetal gene program. RNA sequencing analysis indicated that pathways related to energy metabolism were upregulated, while those related to extracellular matrix organization were downregulated. Consistent with a functional role of the latter, an angiotensin-II challenge that triggered fibrosis in the α1r/rα2s/s mouse failed to do so in the α1s/sα2s/s. Taken together, these results are indicative of a link between circulating CTS, Na/K-ATPase α1, ROS, and physiological cardiac hypertrophy in mice under baseline laboratory conditions.

Project description:Background: Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary fluid, which is related to inflammatory cell infiltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Methods: Using an unbiased, discovery and quantitative proteomic approach, we discovered the differentially expressed proteins binding to Na, K-ATPase α1 between LPS-A549 cells and Control-A549 cells. These Na, K-ATPase α1 interacting proteins were screened by co-immunoprecipitation (Co-IP) technology. Among them, some of the differentially expressed proteins with significant performance were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The protein interaction network was constructed by the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Several differentially expressed proteins were validated by Western blot. Results: Of identified 1598 proteins, 89 were differentially expressed proteins between LPS-A549 cells and Control-A549 cells. Intriguingly, protein-protein interaction network showed that there were 244 significantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 significant enriched interactions among 29 proteins. The related GO and KEGG analysis found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Conclusions: In conclusion, our proteomic approach revealed targets related to the occurrence and development of ARDS, being the first study to investigate significant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS.

Project description:We used gene expression accompanied by physical characteristics and gill Na+/K+-ATPase activity to analyze physiological differences associated with two life history variations of juvenile fall Chinook Salmon in the Snake River basin. Subyearlings originating in the Snake River typically migrate seaward as subyearlings, whereas many subyearlings from the Clearwater River delay seaward migration during summer and complete seaward migration the following spring as yearlings. We examined gill Na+/K+-ATPase activity and gene expression of subyearlings at different times during rearing and seaward emigration. Natural-origin Snake River subyearlings rearing under an increasing photoperiod and seasonally increasing temperatures showed a typical increasing pattern of parr to smolt gill Na+/K+-ATPase activity development, which then declined into autumn. In contrast, Clearwater River subyearlings that had experienced cooler temperatures showed no pattern of increasing gill Na+/K+-ATPase activities and were not different from parr. Liver transcription of genes involved in DNA repair and binding, the cell cycle, metabolism (steroid, fatty acid and other metabolic pathways) iron homeostasis, heme and oxygen binding, the immune response, and male sexual development were enriched amongst genes differentially expressed between Snake River parr versus smolts. Gene expression results confirmed that Clearwater River subyearlings were parr-like in their physiological status. By autumn, subyearlings had low gill Na+/K+-ATPase activities despite their large size and external smolt characteristics. We suggest that environmental factors like temperature and photoperiod influence subyearling physiological status in each river that ultimately dictates juvenile life history pathways. Non-migrating and migrating natural subyearling fall Chinook salmon were collected from the Snake River. Non-migrating natural subyearling fall Chinook salmon were collected from the Clearwater River. Twelve fish were collected at each of four different time points for a total of 48 fish. Total RNA was extracted from the liver of each fish. Equal amounts of RNA from three fish were pooled to create four pools of RNA per time point. Each RNA pool was hybridized to an array for a total of 16 arrays with four arrays per time point.

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Involvement of cardiac glycosides targeting Na/K-ATPase in their inhibitory effects on c-Myc expression via its transcription, translation, and proteasomal degradation

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