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Na+/K+‑ATPase subunit α3 expression is associated with the efficacy of digitoxin treatment in pancreatic cancer cells
University of Skövde, School of Health Sciences. University of Skövde, Digital Health Research (DHEAR). (Translational Medicine TRIM)ORCID iD: 0000-0003-2462-0178
University of Skövde, School of Health Sciences. University of Skövde, Digital Health Research (DHEAR). (Translational Medicine TRIM)ORCID iD: 0000-0001-8962-0860
University of Skövde, Digital Health Research (DHEAR). University of Skövde, School of Health Sciences. (Translational Medicine TRIM)ORCID iD: 0000-0003-0943-7797
2022 (English)In: Medicine International, ISSN 2754-3242, Vol. 2, no 5, article id 27Article in journal (Refereed) Published
Abstract [en]

The alpha subunits (ATP1A1-3) of Na+/K+-ATPase binds digitoxin with varying affinity. The expression levels of these subunits dictate the anticancer effects of digitoxin. In the present study, three pancreatic cancer cell lines, AsPC-1, Panc-1 and CFPAC-1, were used to investigate the effects of digitoxin in relation to the expression of the subunits ATP1A1 and ATP1A3. Cell viability and intracellular calcium concentrations was measured in relation to the gene and protein expression of ATP1A1 and ATP1A3. Digitoxin was used to treat the cells at concentrations of 1-100 nM, and the intracellular calcium concentrations increased in a concentration-dependent manner in the Panc-1 and in the CFPAC-1 cells with treatment at 100 nM. In the AsPC-1 cells only the supraphysiological concentration of digitoxin (100 nM) resulted in a decrease in the number of viable cells (unviable cells increased to 22%), whereas it had no effect on intracellular calcium levels. The number of viable Panc-1 and CFPAC-1 cells decreased after digitoxin treatment at 25-100 nM (unviable Panc-1 cells increased to 33-59%; unviable CFPAC-1 cells increased to 22-56%). Digitoxin treatment also affected the transcriptional expression of the ATP1A1 and ATP1A3 subunits. In Panc-1 cells, ATP1A3 gene expression was negatively associated with the digitoxin concentration (25-100 nM). In the AsPC-1 and CFPAC-1 cells, the expression of the ATP1A1 gene increased in the cells treated with the 100 nM digitoxin concentration. The protein expression of ATP1A1 and ATP1A3 was not altered with digitoxin treatment. The basal protein expression of ATP1A1 was high in the AsPC-1 and CFPAC-1 cells, compared to the Panc-1 cells, in contrast to the basal expression of ATP1A3, which was higher in the Panc-1 cells, compared to the other pancreatic cancer cells used. On the whole, the present study demonstrates that the high expression of ATP1A3 renders pancreatic cancer cells more susceptible to digitoxin-induced cell death. The findings suggest that the expression of ATP1A3 may be used as a marker for tumor sensitivity to digitoxin treatment, where a high expression of ATP1A3 is favorable for the anticancer effects of digitoxin.

Place, publisher, year, edition, pages
Spandidos Publications , 2022. Vol. 2, no 5, article id 27
Keywords [en]
Na+/K+-ATPase, Na+/K+-ATPase alpha subunits 1 and 3, calcium, digitoxin, pancreatic cancer
National Category
Clinical Laboratory Medicine Cell and Molecular Biology Cell Biology Cancer and Oncology
Research subject
Translational Medicine TRIM
Identifiers
URN: urn:nbn:se:his:diva-22471DOI: 10.3892/mi.2022.52PubMedID: 36698913OAI: oai:DiVA.org:his-22471DiVA, id: diva2:1753454
Note

CC BY-NC-ND 4.0

Correspondence to: Dr Ferenc Szekeres, Department of Biomedicine, Translational Medicine, School of Health Sciences, University of Skövde, Högskolevägen 1, 54145 Skövde, Sweden E‑mail: ferenc.szekeres@his.se

No funding was received.

Available from: 2023-04-19 Created: 2023-04-27 Last updated: 2023-07-14Bibliographically approved
In thesis
1. Repurposing digitoxin in the treatment of pancreatic ductal adenocarcinoma: genotypic and phenotypic features as biomarkers for digitoxin sensitivity in vitro
Open this publication in new window or tab >>Repurposing digitoxin in the treatment of pancreatic ductal adenocarcinoma: genotypic and phenotypic features as biomarkers for digitoxin sensitivity in vitro
2023 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The incidence of pancreatic ductal adenocarcinoma (PDAC) is increasing worldwide. The dismal prognosis and lack of effective treatments urges for increased research efforts in developing new treatment regimens. Since the development of new cancer treatments are expensive and time consuming, repurposing drugs is preferable when possible. This strategy will also be of great value for people in low income countries, to increase the availability of effective cancer treatments to affordable costs. Digitoxin, a cardiac glycoside, has been shown to have anti-cancer effects. It binds to the α subunit of the Na+/K+-ATPase, leading to increased concentrations of intracellular calcium and eventually cell death. There seems also to be other mechanisms elicited in cancer cells by digitoxin. The α subunit occurs in three isoforms, of which α3 has the highest affinity to digitoxin and frequently found over-expressed in tumor cells compared to normal cells.

PDAC cancer cells, both cell lines and tumors, differ in their genotype and in the metabolic subtype, proliferation rate and inflammatory status. To be able to individualize treatment regimens it is important to be aware of the specific vulnerabilities (genotypic or phenotypic characteristics increasing the sensitivity to digitoxin) of each PDAC tumor/cell line. This research aims to investigate the potential of using digitoxin as an anti-cancer treatment in PDAC, and analyze its effects on cell viability, metabolism and inflammatory status in PDAC cell lines in vitro with the goal to find biomarkers for digitoxin sensitivity.

The analyses of the effects of digitoxin was performed in five cell lines derived from PDAC tumors, either from primary tumors or metastases. Cell lines derived from PDAC are sensitive to digitoxin treatment to different degrees. High expression of α3 seems to be indicative for digitoxin sensitivity, as do a high proliferation rate seen in cell lines derived from primary tumors. Both subunit expression and proliferation rate should be further evaluated in PDAC tumors to confirm their potential to be used as biomarkers clinically.

In the hunt for the working mechanism behind the anti-cancer effects of digitoxin, the choline pathway, a pathway commonly affected in tumors was enlightened in the metabolomics study, affected in all five cell lines tested. Choline metabolites are important for maintaining the cell membrane and are involved in energy metabolism using lipids. Digitoxin induced an up-regulation of choline and glycerophosphocholine, which rendered us to propose a novel theory about possible interactions between two functional complexes in the cell membrane, the Na+/K+-ATPase/EGFR/c-Src and the EGFR/c-Src – Chkα. The hypothesis is that when digitoxin binds to the Na+/K+-ATPase it leads to inactivation of Chkα with a subsequent decrease in the synthesis of phosphocholine and phosphatidylcholine. Since cancer cells rely on abundance of choline metabolites, we believe an inhibition of this pathway to be deleterious for these cells.

Finally, we conclude that digitoxin has great potential as an anti-cancer treatment for some patients with pancreatic ductal adenocarcinoma. To optimize treatment results, a thorough investigation of the tumor genotype and phenotype must be done for each patient. To further increase treatment success, combination of digitoxin with other treatments for synergistic effects could be beneficial. 

Place, publisher, year, edition, pages
Jönköping: Jönköping University, School of Health and Welfare, 2023. p. 107
Series
Dissertation Series. School of Health and Welfare, ISSN 1654-3602 ; 124
Keywords
pancreatic ductal adenocarcinoma, Na+/K+-ATPase, digitoxin, cardiac glycoside, metabolomics, choline
National Category
Clinical Laboratory Medicine Cancer and Oncology
Research subject
Translational Medicine TRIM
Identifiers
urn:nbn:se:his:diva-22470 (URN)978-91-88669-23-0 (ISBN)978-91-88669-24-7 (ISBN)
Public defence
2023-05-12, G110, University of Skövde, Skövde, 13:00 (Swedish)
Opponent
Supervisors
Note

"This thesis is based on research conducted at the University of Skövde during the enrollment as a PhD-student at the School of Health and Welfare at Jönköping University."

Två av fyra delarbeten (övriga se rubriken Delarbeten/List of papers):

Paper III

Lindholm H, Herring M, Faresjö M, Haux J, Szekeres F, Ejeskär K. Inflammasomes can be formed in pancreatic cancer cells, and NLRP3 inflammasome associated genes are up-regulated in primary pancreatic tumors. (Submitted)

Paper IV

Lindholm H, Ulfenborg B, Faresjö M, Haux J, Ejeskär K, Szekeres F. Digitoxin treatment affects choline metabolism in pancreatic cancer cells (Manuscript)

Available from: 2023-04-27 Created: 2023-04-27 Last updated: 2023-04-27Bibliographically approved

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