Introduction

Medicine International

2754-3242 2754-1304

D.A. Spandidos

MI-2-5-00052

10.3892/mi.2022.52

Articles

Na+/K+‑ATPase subunit α3 expression is associated with the efficacy of digitoxin treatment in pancreatic cancer cells

Lindholm

Heléne

Ejeskär

Katarina

Szekeres

Ferenc

Department of Biomedicine, Translational Medicine, School of Health Sciences, University of Skövde, 54145 Skövde, Sweden

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

09 2022

02 09 2022

2 5

20 06 2022 01 09 2022

2020

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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.

pancreatic cancer digitoxin Na⁺/K⁺-ATPase Na⁺/K⁺-ATPase alpha subunits 1 and 3 calcium

Funding: No funding was received.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of <5% (1,2). Novel complementary treatments are warranted since current treatments are often inefficient and are associated with severe side-effects (3). Data from epidemiological studies have suggested positive therapeutic effects using cardiac glycosides in the treatment of various types of cancer, and may thus prove to be promising as a complementary treatment to current PDAC therapeutics (4-7).

Cardiac glycosides are natural compounds most well known for their cardiovascular effects. Digitoxin is one of the most extensively used cardiac glycosides and it inhibits the membrane receptor Na⁺/K⁺-ATPase by binding to its α-subunit (8). The Na⁺/K⁺-ATPase consists of three parts, the β-subunit β1-3, the α-subunit with its four isoforms α1-4 (ATP1A1-4), and accessory proteins FXYD 1-7(9). The α- and β-subunits are differentially expressed depending on the type of tissue (10). ATP1A1 is expressed in almost all tissues (11), ATP1A2 is mainly expressed in the heart, brain and skeletal muscles, ATP1A3 mostly in nervous and muscle tissue, and ATP1A4 is only expressed in spermatozoa (12,13). Binding experiments with various cardiac glycosides have demonstrated that ATP1A2 and ATP1A3 have a higher affinity to digitoxin than ATP1A1 (9,10,14). A higher affinity indicates a more effective blockage by digitoxin. The values for digitoxin binding constants, dissociation constant (K_D; inverse to affinity), are 38 nM for ATP1A1 and 14 nM for ATP1A3(9).

The blockage of the Na⁺/K⁺-ATPase pumping function leads to an increase in the intracellular concentration of sodium ions which, in turn, forces the Na⁺/Ca²⁺-exchanger (NCX) to transport Na⁺ out of the cell and Ca²⁺ into the cell (15). As a result, the intracellular Ca²⁺ concentrations increase (16). In non-excitable cells under basal conditions, the intracellular concentrations of Ca²⁺ are maintained at a low homeostatic level, with a balance of influx and efflux of Ca²⁺ (17). Maintaining a low intracellular concentration of calcium (Ca²⁺) is essential for the majority of eukaryotic cells, since a high persistent intracellular concentrations of Ca²⁺ is harmful and trigger apoptosis (18).

A deregulation of the α-subunits is often observed in cancer cells, with an upregulation of ATP1A3 and the downregulation of ATP1A1, such as in colorectal cancer and renal cell carcinoma (13,19). Cancer cells have been shown to be more sensitive to digitoxin than normal cells, where nanomolar concentrations of digitoxin induce the apoptosis of cancer cells (8,20-22). It was thus hypothesized that the increased sensitivity of cancer cells is dependent on the altered expression of the α-subunits of Na⁺/K⁺-ATPase.

To investigate this hypothesis, the present study we used three well-characterized commercially available pancreatic cancer cell lines (AsPC-1, Panc-1 and CFPAC-1). The basal gene expression of the three α-subunit isoforms of Na⁺/K⁺-ATPase, ATP1A1, ATP1A2 and ATP1A3, as well as the protein levels of ATP1A1 and ATP1A3 were measured. The effects of digitoxin treatment at 1-100 nM (therapeutic concentrations, 10-40 nM) were evaluated by examining the changes in the expression of ATP1A1 of ATP1A3, intracellular Ca²⁺ levels and cell viability.

Materials and methods <sec> <title>Cells and cell culture

The present study used three pancreatic cancer cell lines, AsPC-1 (CRL-1682), Panc-1 (CRL-1469) and CFPAC-1 (CRL-1918) (ATCC; LGC Standards GmbH). The AsPC-1 cells were grown in RPMI-1640 medium supplemented with 1% HEPES and 1% sodium pyruvate, the Panc-1 cells in Dulbecco's modified Eagle's medium (DMEM) with 1% L-glutamine, and the CFPAC-1 cells in DMEM. Media were also supplemented with 10% FBS and 1% penicillin-streptomycin (PEST). All cell culture media and reagents were purchased from MilliporeSigma. All incubations were performed at 37˚C in 5% CO₂. PCR was used to test the cell lines for mycoplasma infection (using the LookOut^® Mycoplasma PCR Detection kit; cat. no. MP0035, MilliporeSigma).

Seeding and treatment with digitoxin

For digitoxin (cat. no. D5878, MilliporeSigma) treatment, 5,000 cells were seeded in 100 µl complete growth medium in 96-well plates and incubated at 3˚C with 5% CO₂ for 20 h to a sub-confluent monolayer. Following 20 h of incubation, new media (100 µl) containing digitoxin was added to the cells. The control cells only received new media. The cells were further incubated at 37˚C with 5% CO₂ for 48 h. The concentrations of digitoxin used in all experiments were as follows: 0 nM (controls), and 1, 10, 25, 40 and 100 nM (human therapeutic range, 10-40 nM).

Cell viability assay

The analysis of cell viability was performed using the colorimetric method CellTiter 96^® AQueous One Solution Cell Proliferation assay (MTS) assay (Promega Corporation). Following 48 h of incubation at 37˚C with 5% CO₂ with digitoxin, 20 µl MTS tetrazolium were added to each well and incubated for 1 h at 37˚C with 5% CO₂. Viable cells metabolize tetrazolium to formazan, which was measured at 490 nm (FLUOstar Omega, BMG Labtech). The obtained value is directly proportional to the number of viable cells in the cell culture.

Intracellular Ca2+ assay

Fluo-4 assay (Ca²⁺; Abcam) was used to determine the intracellular Ca²⁺concentrations following treatment with digitoxin. The cells were seeded and treated for 48 h with digitoxin at 37˚C with 5% CO₂ in triplicate. Following treatment, 100 µl of Fluo-4 AM dye-loading solution were added to each well and incubated for 1 h. The fluorescence intensity was measured at Ex/Em 485/520 (FLUOstar Omega, BMG Labtech).

RNA-extraction and reverse transcription-quantitative PCR

The cells were seeded at a density of 1.5x10⁵ in 2.4 ml medium in six-well plates and incubated at 37˚C in 5% CO₂ to a sub-confluent monolayer. After 20 h, the old medium was removed and new medium with digitoxin was added. The control cells only received new media. Following 48 h of treatment, RNA was extracted using a RNeasy Mini kit (cat. no. 74104, Qiagen GmbH), according to the supplier's protocol. A total 1 µg RNA from each sample was used for cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit Reagent (25˚C for 10 min, 37˚C for 120 min, 85˚C for 5 min) according to the manufacturer's manual (Thermo Fisher Scientific, Inc.).

Complementary DNA (cDNA) corresponding to 5 ng RNA was used in each qPCR reaction performed in duplicate for the following TaqMan target transcripts: ATP1A1 (Hs00167556_m1), ATP1A2 (Hs00265131_m1) and ATP1A3 (Hs00958036_m1) using TaqMan™ Gene Expression Master Mix (4369016, Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: initial denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 1 min. Quantitative gene expression data were normalized to the expression level of the human reference gene, phosphomannomutase 1 (PMM1; Hs00963626_m1). The ΔCq values was used to analyze the relative expression between the genes ATP1A1 and ATP1A3 for each cell line (23). All qPCR reactions were performed on a Pikoreal qPCR System (Thermo Fisher Scientific, Inc.).

Protein extraction and western blot analysis

The cells were seeded at a quantity of 5x10⁵ cells per 75 cm² flask and incubated for 20 h in 37˚C in 5% CO₂. Thereafter, the old medium was removed and new medium with digitoxin was added. The controls only received new, complete medium. Following 48 h of treatment, protein extraction was performed and the cells were lysed in lysis buffer (cat. no. FNN0011, Thermo Fisher Scientific, Inc.) supplemented with phenylmethylsulphonyl fluoride (PMSF; cat. no. 36978, Thermo Fisher Scientific, Inc.) and protein inhibitor cocktail (cat. no. P2714, MilliporeSigma). The protein concentration was determined using the Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Inc.).

From each sample, 10 µg of total protein were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis in 8-16%, stain free gel (Bio-Rad Laboratories Inc.), Proteins were later blotted onto PVDF membranes, blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) with 5% milk in 20˚C for 1 h, incubated with either ATP1A1 and ATP1A3 primary antibodies (1:1,000; cat. nos. MA1-16731 and MA3-915, Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h in room temperature. After washing with TBST three times, the membranes were further incubated with the secondary antibody, Alexa Fluor Plus 555 (1:2,500; cat. no. A32727, Invitrogen; Thermo Fisher Scientific, Inc.). The protein expression was measured using the ChemiDoc System (Bio-Rad Laboratories Inc.), densitometric analysis of single and total protein expression was performed using Image Lab Software ver. 6.1 (Bio-Rad Laboratories, Inc.). Protein expression was normalized to the total protein for each sample, total protein normalization (Fig. S1, Fig. S2, Fig. S3 and Fig. S4).

Statistical analysis

The used assays are based on absorbance and fluorescence in microplate format and RT-qPCR. Biological and technical replicates were three or more, (two technical replicates for RT-qPCR ). Statistical analysis was performed using IBM SPSS Statistics 27 software (IBM Corp.) and one-way analysis of variance (ANOVA) followed by the Bonferroni correction, to confirm significant differences between treated vs. untreated cells (i.e., control) in each assay. A P-value <0.05 was considered to indicate a statistically significant difference.

Results <sec> <title>Digitoxin within the therapeutic range exerts an effect on cell viability and intracellular Ca2+ concentrations in Panc-1 and CFPAC-1 cells, but not in AsPC-1 cells

The AsPC-1 cell line was less affected by digitoxin compared to the Panc-1 and CFPAC-1 cells, with only a significant effect observed on cell viability in the AsPC-1 treated with 25 and 100 nM digitoxin (Fig. 1A). Digitoxin at concentrations ranging from 10-100 nM decreased the viability of the Panc-1 cells following 48 h of treatment and also that of the CFPAC-1 cells at concentrations ranging from 1-100 nM. For the Panc-1 cells treated with digitoxin at the concentration of 10 nM, the number of viable cells decreased (number of unviable cells, 12.6±1.15%, P<0.001) compared to the control cells. Within the therapeutic range in humans (25-40 nM), the viable cell number was decreased even further (number of unviable cells at 40 nM: Panc-1 cells, 43.8±1.15%, P<0.001; CFPAC-1 cells, 23.0±1.16%; P<0.001) compared to the controls (Fig. 1B and C).

The intracellular Ca²⁺ concentrations increased in a concentration-dependent manner following 48 h of treatment with digitoxin in the Panc-1 cells. Digitoxin at a concentration of 25 nM led to a 2-fold increase (±0.04, P<0.001) in intracellular Ca²⁺ levels in the Panc-1 cells. The digitoxin concentrations of 40 and 100 nM increased the intracellular Ca²⁺ levels 2.6-fold (±0.04, P<0.001) and 3.4-fold (±0.04, P<0.001), respectively in Panc-1 cells (Fig. 1B). In the Panc-1 cells, a marked increase in intracellular Ca²⁺ levels were observed, while the CFPAC-1 cells only exhibited a significant increase in intracellular Ca²⁺ levels following treatment with digitoxin at 100 nM (±0.03, P<0.001) (Fig. 1C). In the AsPC-1 cells, the concentration of intracellular Ca²⁺ was not markedly altered following treatment with digitoxin (Fig. 1A).

Expression of ATP1A3 is high in Panc-1 and CFPAC-1 cells, but not in AsPC-1 cells

The basal transcriptional expression of ATP1A1 was low in AsPC-1 and Panc-1 cells, and 6-fold higher in the CFPAC-1 cells compared to the AsPC-1 cells (Fig. 2A). The ATP1A3 subunit in Panc-1 cells was transcriptionally expressed >50-fold higher (P<0.001) compared to the AsPC-1 cells, and 4-fold times higher than the CFPAC-1 cells (Fig. 2B). At the protein level, the AsPC-1 cells exhibited a significant ~7-fold higher expression of ATP1A1 compared to the Panc-1 cells (±0.01, P=0.002, AsPC-1 cells) (Fig. 2C). The Panc-1 cells exhibited a significantly higher expression (4-fold) of ATP1A3 compared to the AsPC-1 cells (±0.01, P=0.022), while the CFPAC-1 cells had a 2-fold higher protein expression of ATP1A3 compared to the AsPC-1 cells (Fig. 2D).

Digitoxin treatment affects the gene and protein expression of ATP1A1 and ATP1A3 in Panc-1 cells

A one-way between-groups ANOVA was conducted to explore the effects of digitoxin treatment on ATP1A1 and ATP1A3 gene expression levels. Significant differences were found for ATP1A expression in the AsPC-1 cells [F(5, 12)=14.71, P<0.001] and CFPAC-1 cells [F(5, 11)=7.16, P=0.003]. No effects on ATP1A1 gene expression were observed in the Panc-1 cells; instead a change in the expression of the ATP1A3 gene was observed with digitoxin treatment. A decrease in ATP1A3 expression with the increasing digitoxin concentration was confirmed in the Panc-1 cells [F(5, 12)=5.51, P=0.007]. In order to analyze the effects of each digitoxin concentration on ATP1A1 and ATP1A3 expression separately, post hoc comparisons using the Bonferroni correction were made. The analysis revealed a significant difference in ATP1A1 expression in the AsPC-1 cells with digitoxin treatment at 100 nM (±0.172, P<0.001) (Fig. 3A). In the CFPAC-1 cells, treatment with digitoxin at 100 nM led to a significant increase in ATP1A1 gene expression (±3.474, P=0.011) (Fig. 3A). In the Panc-1 cells, no significant effects on ATP1A1 gene expression were observed for the individual digitoxin concentrations; however, there was a significant decrease in ATP1A3 gene expression with various digitoxin concentrations (25 nM: ±0.059, P=0.034; 40 nM: ±0.059, P=0.024; 100 nM: ±0.059, P=0.018) (Fig. 3B). ATP1A3 expression was not markedly altered by digitoxin either in the AsPC-1 or in the CFPAC-1 cells (Fig. 3B).

Western blot analysis of ATP1A1 and ATP1A3 was performed following treatment with digitoxin to evaluate the effects at the protein level (Fig. 4). A one-way between-groups ANOVA was conducted to explore the effects of digitoxin treatment on ATP1A1 and ATP1A3 protein expression. The results revealed slightly increased protein levels of ATP1A1, and decreased protein levels of ATP1A3 in the Panc-1 cells following treatment, although with no significant differences (Fig. 4).

Discussion

The cardiac glycoside, digitoxin, is an extensively used drug in the treatment of cardiovascular diseases (15), and when used within the therapeutic range of 25-40 nM, no major side-effects have been observed in normal cells (24,25). The present study investigated a potentially novel use of this already established drug for the treatment of pancreatic cancer, a disease which remains very difficult to cure with current treatments (26,27). Previous studies have demonstrated a reduction in cell viability in several cancer cell lines following treatment with cardiac glycosides (20,27), and the results from the present study with digitoxin treatment in the pancreatic cancer cell lines, AsPC-1, Panc-1 and CFPAC-1, confirm that digitoxin may be useful as a potential treatment for subgroups of patients with pancreatic cancer.

The hypothesis behind the present study relies on the fact that the membrane receptor Na⁺/K⁺-ATPase isoform ATP1A3 has a higher affinity for digitoxin than the ATP1A1 isoform, which suggests that a higher expression of ATP1A3 compared to ATP1A1 would provide a more effective blockage of the Na⁺/K⁺-ATPase by digitoxin (14). A change in expression of the α-subunits ATP1A1 and ATP1A3 is observed in certain tumors compared to normal tissue. In a study on human colorectal carcinoma, colorectal cancer tissue was compared with normal mucosa, and it was found that ATP1A1 was transcriptionally downregulated in cancer tissues and that ATP1A3 expression was upregulated (13). Banerjee et al (28) also demonstrated a decreasing expression of ATP1A1 in primary tumors and metastases compared to the normal tissue. The altered expression of these two isoforms is probably the reason for an increased sensitivity to digitoxin treatment, considering the higher affinity of digitoxin to the ATP1A3 isoform (9). In another study, an overall downregulation in Na⁺/K⁺-ATPase activity was observed in colorectal cancer cells following digitoxin treatment (13), and an unregulated increase in intracellular Ca²⁺ levels induced the apoptosis of HeLa cells (29).

In the present study, the data suggested a close association between the fraction of unviable cells and intracellular Ca²⁺ concentrations in all three pancreatic cancer cell lines. However, there was a major difference in the response to digitoxin within the therapeutic range (25-40 nM) between the different cell lines, where the Panc-1 cells exhibited a marked response, with a notable decrease in viability and a marked increase in the intracellular Ca²⁺ levels, while AsPC-1 cell viability was only slightly affected by digitoxin at these concentrations.

There is a notable difference in characteristics between the cell lines in the present study with regards to their basal levels of ATP1A1 and ATP1A3 gene and protein expression. Since the Panc-1 cells had a very high basal expression of ATP1A3, and a lower expression of ATP1A1 compared to the other two cell lines, it was hypothesized that the blockage of Na⁺/K⁺-ATPase by digitoxin in this cell line was dependent on the relative high expression of ATP1A3. The more effective blockage of ATP1A3 by digitoxin led to a notable increase of the intracellular Ca²⁺concentrations in the Panc-1 cells. The CFPAC-1 cells with a relatively higher expression of ATP1A1 and a lower expression of ATP1A3 compared to the Panc-1 cells exhibited only a significant increase in intracellular Ca²⁺ levels with the supratherapeutic concentration of digitoxin (100 nM). No change in the intracellular Ca²⁺concentrations was found in the AsPC-1 cells. The AsPC-1 cell line had a high ATP1A1 expression and a low ATP1A3 expression, which further underlines the importance of the expression levels of these subunits.

The Panc-1 cells exhibited a response to digitoxin affecting the gene expression of the Na⁺/K⁺-ATPase α-subunit ATP1A3, which decreased in the cells treated with 25-100 nM digitoxin. This effect is possibly a protective cell survival mechanism. In the AsPC-1 and CFPAC-1 cells, only the gene expression of ATP1A1 was affected (increased expression) by treatment with 100 nM digitoxin. Cells are heavily dependent on the function of Na⁺/K⁺-ATPases for the intracellular ion homeostasis, blocking these pumps with drugs or siRNA trigger the cells to produce more Na⁺/K⁺-ATPases to compensate (28).

The sensitivity to digitoxin may be explained by this difference in the expression of the Na⁺/K⁺-ATPase α-subunit isoforms between the cell lines. A high ATP1A3 and low ATP1A1 expression corresponded with a high sensitivity to digitoxin treatment, considering the decrease in cell viability. An increase in ATP1A1 expression and/or a decrease in ATP1A3 expression may be a way for the cells to rescue the blockage of the ATP1A3 subunits in the Na⁺/K⁺-ATPase by digitoxin. These results are correlative and further studies on the mechanisms behind the difference in responses of the different ATP1A isoforms are warranted.

In conclusion, the present study demonstrated the potential of digitoxin as an anticancer agent for a subset of pancreatic cancers. A very high anticancer efficacy of digitoxin was observed in pancreatic cancer cells with a high expression of the ATP1A3 subunit of the Na⁺/K⁺-ATPase, compared to the cells that had a low expression of the ATP1A3 Na⁺/K⁺-ATPase subunit. Thus, this may be useful as a marker for effective digitoxin treatment.

Supplementary Material

Basal protein expression of ATP1A1 and ATP1A3. Protein standard (Precision Plus Protein™ All Blue Prestained Protein Standards; cat. no. 1610373, Bio-Rad Laboratories, Inc.). (A) Total protein for normalization of protein expression of ATP1A1, (B) Total protein for normalization of protein expression of ATP1A3. (C) Protein expression of ATP1A1, and (D) protein expression of ATP1A3. ATP1A1 and ATP1A3, Na+/K+-ATPase alpha subunits 1 and 3.

Protein expression of ATP1A1 and ATP1A3 in AsPC-1 cells and the control with digitoxin treatment. Protein standard (Precision Plus Protein™ All Blue Prestained Protein Standards; cat. no. 1610373, Bio-Rad Laboratories, Inc.). (A) Total protein for normalization of protein expression of ATP1A1, (B) Total protein for normalization of protein expression of ATP1A3, (C) protein expression of ATP1A1, and (D) protein expression of ATP1A3. ATP1A1 and ATP1A3, Na+/K+-ATPase alpha subunits 1 and 3.

Protein expression of ATP1A1 and ATP1A3 in Panc-1 cells and the control and with digitoxin treatment. Protein standard (Precision Plus Protein™ All Blue Prestained Protein Standards; cat. no. 1610373, Bio-Rad Laboratories, Inc.). (A) Total protein for normalization of protein expression of ATP1A1, (B) Total protein for normalization of protein expression of ATP1A3, (C) protein expression of ATP1A1, and (D) protein expression of ATP1A3. ATP1A1 and ATP1A3, Na+/K+-ATPase alpha subunits 1 and 3.

Protein expression of ATP1A1 and ATP1A3 in CFPAC-1 cells and the control and with digitoxin treatment. Protein standard (Precision Plus Protein™ All Blue Prestained Protein Standards; cat. no. 1610373, Bio-Rad Laboratories, Inc.). (A) Total protein for normalization of protein expression of ATP1A1, (B) Total protein for normalization of protein expression of ATP1A3, (C) protein expression of ATP1A1, and (D) protein expression. Of ATP1A3. ATP1A1 and ATP1A3, Na+/K+-ATPase alpha subunits 1 and 3.

Acknowledgements

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HL has performed the experiments, statistical analysis and drafted the manuscript. HL and FS designed the study and performed the data interpretation. FS contributed with manuscript writing and critical editing. KE contributed to the study methodology, manuscript writing and critical editing. HL and FS confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References 1

Ansari

Gustafsson

Andersson

Update on the management of pancreatic cancer: Surgery is not enough

World J Gastroenterol21315731652015

25805920

10.3748/wjg.v21.i11.3157

Becker

Hernandez

Frucht

Lucas

Pancreatic ductal adenocarcinoma: Risk factors, screening, and early detection

World J Gastroenterol2011182111982014

25170203

10.3748/wjg.v20.i32.11182

Pereira

Corrêa

Pancreatic cancer: Treatment approaches and trends

J Cancer Metastasis Treat4302018

Frankel

Eskiocak

Gill

Yuan

Ramesh

Froehlich

Ahn

Morrison

Digoxin plus trametinib therapy achieves disease control in BRAF wild-type metastatic melanoma patients

Neoplasia192552602017

28278423

10.1016/j.neo.2017.01.010

Coleman

Gray

Stephens

Scott

Cardelli

Repurposed drug screen identifies cardiac glycosides as inhibitors of TGF-β-induced cancer-associated fibroblast differentiation

Oncotarget732200322092016

27058757

10.18632/oncotarget.8609

Kepp

Menger

Vacchelli

Adjemian

Martins

Sukkurwala

Michaud

Galluzzi

Zitvogel

Kroemer

Anticancer activity of cardiac glycosides: At the frontier between cell-autonomous and immunological effects

Oncoimmunology1164016422012

23264921

10.4161/onci.21684

Haux

Digitoxin has specific properties for potential use to treat cancer and inflammatory diseases. Research and Reviews on Healthcare: Open Access Journal 2: 2018.

Lopez-Lazaro

Digitoxin as an anticancer agent with selectivity for cancer cells: Possible mechanisms involved

Expert Opin Ther Targets11104310532007

17665977

10.1517/14728222.11.8.1043

Katz

Lifshitz

Bab-Dinitz

Kapri-Pardes

Goldshleger

Tal

Karlish

SJD

Selectivity of digitalis glycosides for isoforms of human Na,K-ATPase

J Biol Chem28519582195922010

20388710

10.1074/jbc.M110.119248

Newman

Yang

Pawlus

Block

Cardiac glycosides as novel cancer therapeutic agents

Mol Interv836492008

18332483

10.1124/mi.8.1.8

Mijatovic

Kiss

Cardiotonic steroids-mediated Na+/K+-ATPase targeting could circumvent various chemoresistance pathways

Planta Med791891982013

23412992

10.1055/s-0032-1328243

Clausen

Hilbers

Poulsen

The structure and function of the Na,K-ATPase isoforms in health and disease

Front Physiol83712017

28634454

10.3389/fphys.2017.00371

Sakai

Suzuki

Maeda

Takahashi

Horikawa

Minamimura

Tsukada

Takeguchi

Up-regulation of Na+,K+-ATPase α3-isoform and down-regulation of the α1-isoform in human colorectal cancer

FEBS Lett5631511542004

15063740

10.1016/S0014-5793(04)00292-3

Noël

Fagoo

Godfraind

A comparison of the affinities of rat (Na+ + K+)-ATPase isozymes for cardioactive steroids, role of lactone ring, sugar moiety and KCl concentration

Biochem Pharmacol40261126161990

2175607

10.1016/0006-2952(90)90578-9

Arispe

Diaz

Simakova

Pollard

Heart failure drug digitoxin induces calcium uptake into cells by forming transmembrane calcium channels

Proc Natl Acad Sci105261026152008

18272488

10.1073/pnas.0712270105

Menger

Vacchelli

Kepp

Eggermont

Tartour

Zitvogel

Kroemer

Galluzzi

Trial watch: Cardiac glycosides and cancer therapy

Oncoimmunology2e230822013

23525565

10.4161/onci.23082

Contreras

Drago

Zampese

Pozzan

Mitochondria: The calcium connection

Biochim Biophys Acta17976076182010

20470749

10.1016/j.bbabio.2010.05.005

Brini

Carafoli

The plasma membrane Ca²+ ATPase and the plasma membrane sodium calcium exchanger cooperate in the regulation of cell calcium

Cold Spring Harb Perspect Biol3a0041682011

21421919

10.1101/cshperspect.a004168

Zhang

Yang

Wang

Yang

Zhu

Liang

Downregulation of ATP1A1 promotes cancer development in renal cell carcinoma

Clin Proteomics14152017

28484360

10.1186/s12014-017-9150-4

Calderón-Montaño

Burgos-Morón

Orta

Mateos

López-Lázaro

A hydroalcoholic extract from the leaves of Nerium oleander inhibits glycolysis and induces selective killing of lung cancer cells

Planta Med79101710232013

23824549

10.1055/s-0032-1328715

Yang

Dalgard

Eidelman

Jozwik

Pollard

Srivastava

Pollard

Digitoxin induces apoptosis in cancer cells by inhibiting nuclear factor of activated T-cells-driven c-MYC expression

J Carcinog1282013

23858296

10.4103/1477-3163.112268

Lindholm

Ejeskär

Szekeres

Digitoxin affects metabolism, ROS production and proliferation in pancreatic cancer cells differently depending on the cell phenotype

Int J Mol Sci2382372022

35897809

10.3390/ijms23158237

Livak

Schmittgen

Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method

Methods254024082001

11846609

10.1006/meth.2001.1262

López-Lázaro

Pastor

Azrak

Ayuso

Cortés

Austin

Digitoxin, at concentrations commonly found in the plasma of cardiac patients, antagonizes etoposide and idarubicin activity in K562 leukemia cells

Leuk Res308958982006

16387358

10.1016/j.leukres.2005.11.005

Bavendiek

Berliner

Dávila

Schwab

Maier

Philipp

Rieth

Westenfeld

Piorkowski

Weber

Rationale and design of the DIGIT-HF trial (DIGitoxin to Improve ouTcomes in patients with advanced chronic Heart Failure): A randomized, double-blind, placebo-controlled study

Eur J Heart Fail216766842019

30892806

10.1002/ejhf.1452

Orth

Metzger

Gerum

Mayerle

Schneider

Belka

Schnurr

Lauber

Pancreatic ductal adenocarcinoma: Biological hallmarks, current status, and future perspectives of combined modality treatment approaches

Radiat Oncol141412019

31395068

10.1186/s13014-019-1345-6

Varbanov

Kuttler

Banfi

Turcatti

Dyson

Repositioning approved drugs for the treatment of problematic cancers using a screening approach

PLoS One12e01710522017

28166232

10.1371/journal.pone.0171052

Banerjee

Cui

Cai

Jia

Wang

Gao

Xie

Na/K-ATPase Y260 phosphorylation-mediated src regulation in control of aerobic glycolysis and tumor growth

Sci Rep8123222018

30120256

10.1038/s41598-018-29995-2

Gan

Chan

Leung

Lei

Liu

Xue

Liu

Digitoxin inhibits HeLa cell growth through the induction of G2/M cell cycle arrest and apoptosis in vitro and in vivo

Int J Oncol575625732020

32468057

10.3892/ijo.2020.5070

Figure 1

Cell viability and intracellular Ca²⁺ levels following treatment with digitoxin in (A) AsPC-1, (B) Panc-1, and (C) CFPAC-1 cells. The cells were incubated for 48 h with digitoxin at concentrations of 1-100 nM; the control cells only received complete cell medium. The number of viable cells was measured using MTS assay. The decrease in viable cells ‘unviable cells’, was normalized to the control (left axis) and intracellular Ca²⁺ values (right axis) control. Error bars represent the means ± SEM. Data were analyzed using one-way analysis of variance (ANOVA) followed by the Bonferroni correction, to confirm significant differences between treated vs. untreated cells (i.e., control). ^***P<0.001, significant differences in cell viability; ^{^}P<0.05; and ^{^^^}P<0.001, significant differences in intracellular calcium levels.

Figure 2

Gene expression for the control (basal) samples, 2^-ΔCq for (A) ATP1A1 and (B) ATP1A3. Protein expression of (C) ATP1A1 and (D) ATP1A3, control (basal) protein expression of the subunits ATP1A1 and ATP1A3 normalized to total protein in each sample. Error bars represent the means ± SEM. Data were analyzed using one-way between-groups ANOVA. ^*P<0.05; ^**P<0.01 and ^***P<0.001. Protein expression was normalized to the total protein for each sample (Fig. S1). ATP1A1 and ATP1A3, Na⁺/K⁺-ATPase alpha subunits 1 and 3.

Figure 3

Gene expression, 2^-ΔCq for (A) ATP1A1 and (B) ATP1A3 in untreated cells (control) compared to cells treated with digitoxin 1-100 nM for 48 h. Error bars represent the means ± SEM. Data were analyzed using one-way between-groups ANOVA. ^{*, ^, †} <0.05, significant differences for the Panc-1 (*), AsPC-1 (^) and CFPAC-1 (†) cells. ATP1A1 and ATP1A3, Na⁺/K⁺-ATPase alpha subunits 1 and 3.

Figure 4

Protein expression of (A) ATP1A1 and (B) ATP1A3, normalized to total protein for each sample, in AsPC-1, Panc-1 and CFPAC-1 cells. Untreated cells (Control) compared to cells treated with digitoxin 1-100 nM for 48 h. Error bars represent the means ± SEM. Data were analyzed using one-way between-groups ANOVA. No significant differences between controls and treatments were found. Protein expression was normalized to the total protein for each sample (Fig. S2, Fig. S3 and Fig. S4). ATP1A1 and ATP1A3, Na⁺/K⁺-ATPase alpha subunits 1 and 3.