Mechanism of action of cisplatin on Na+/K+ ATPase of Caco-2 colon cells in vitro

Rida Mourad; Rawad Hodeify; Sawsan Kreydiyyeh

doi:10.1371/journal.pone.0342707

Abstract

Cisplatin is a chemotherapeutic agent that reverts cancerous cells to the apoptotic route. A decrease in the activity of the Na⁺/K⁺ ATPase is one of the hallmarks of apoptosis and a major causative factor of the drug-induced nephrotoxicity. Whether cisplatin targets also the colonic ATPase is a question that was addressed in this work using Caco-2 cells as a model. ATPase activity was measured via inorganic phosphate release with and without ouabain, and protein expression was assessed by Western blot. Cisplatin reduced the activity of the Na⁺/K⁺ ATPase, an effect that was dependent on the transmembrane chloride gradient but had no effect on the purified enzyme, suggesting an indirect action. Fluorescence imaging showed a decrease in the membrane ATPase abundance. Cisplatin was shown to act by increasing intracellular calcium, triggering a sequential activation of p38MAPK and PKA that results in JNK inhibition and a decrease in the Na⁺/K⁺ ATPase activity.

Citation: Mourad R, Hodeify R, Kreydiyyeh S (2026) Mechanism of action of cisplatin on Na⁺/K⁺ ATPase of Caco-2 colon cells in vitro. PLoS One 21(2): e0342707. https://doi.org/10.1371/journal.pone.0342707

Editor: Zu Ye, Zhejiang Cancer Hospital, CHINA

Received: October 19, 2025; Accepted: January 27, 2026; Published: February 24, 2026

Copyright: © 2026 Mourad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting information files.

Funding: S.K received a fund from the University Research Board, American University of Beirut The funders did not play any role in the study or the preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Cancer remains a pressing global health concern, affecting millions worldwide. Among the common chemotherapeutic agents, cisplatin—also known as cis-diaminedichloroplatinum (CDDP)—is one of the most widely used. This platinum-based coordination compound features two chlorides and two ammonias as ligands and has proven effective in combating various cancers. The mechanism of cisplatin’s transport across cell membranes remains however contentious. Research supports both passive and active transport processes, with evidence pointing to the involvement of copper transporters as well as endocytosis and transport via organic cation transporters [1]. Once inside the cell, cisplatin undergoes aquation. Its chloride ligands are replaced by water molecules generating reactive platinum species that bind DNA, inflicting damage on cancerous cells and ultimately triggering apoptosis [2]. Cisplatin can also induce apoptosis and tissue necrosis by disrupting calcium homeostasis, which leads to mitochondrial dysfunction and depletion of adenosine triphosphate (ATP) [3,4].

Despite its efficacy, cisplatin use is marred by challenges, particularly the emergence of resistance. Mechanisms of resistance include altered drug transport across cell membranes, defects in apoptotic signaling pathways, or a higher capacity of cancer cells to mend DNA damage. Furthermore, cisplatin is associated with numerous side effects, with diarrhea being a common complication [5]. This side effect stems from reduced sodium and fluid absorption in the intestines [6] a process regulated by the Na + /K + ATPase, or Na + /K+ pump. Situated in the basolateral membrane of intestinal epithelial cells, this ATPase maintains the sodium gradient essential for electrolytes and secondary active transport processes. Notably, it has been identified as a contributor to cancer initiation, and a reduction in its activity has been recognized as a hallmark of apoptosis [7–9] and a causative agent of the cisplatin induced nephrotoxicity [10–12]. Since the ATPase plays overlapping roles in the kidney and colon, maintaining ion gradients, epithelial polarity, and barrier integrity, the observed cisplatin-induced disruption in its activity in renal tissue may plausibly extend to colonic epithelium, affecting electrolyte and water absorption and epithelial homeostasis. In fact, whether cisplatin modulates the activity of colonic Na ⁺ /K ⁺ -ATPase remains till now an open question, and data on this topic are still scarce. This gap in knowledge forms the foundation of the present research, which employs Caco-2 cells as a model to investigate whether cisplatin directly or indirectly targets colonic Na⁺/K⁺ ATPase. Additionally, this study aims to delineate the underlying mechanisms involved, providing insights that may alleviate some of the drug’s adverse effects by targeting relevant mediators.

Materials and methods

Materials

Cisplatin and the p38 MAPK inhibitor, doramapimod, were purchased from Abcam, Cambridge, UK. The primary mouse monoclonal antibody for GAPDH (cat #sc-47724 used at a dilution of 1:150) and the Goat anti-mouse horseradish peroxidase (HRP) conjugated secondary IgG (cat # sc-516102 used at a dilution of 1:4000) were purchased from Santa Cruz Biotechnology, CA, USA. Biorad protein reagent, nitrocellulose membranes, and western blotting luminol reagent were obtained from Biorad, California, USA. The JNK inhibitor (SP600125) and RpcAMP were purchased from Tocris Bioscience (Abingdon,Oxford, UK).The ERK inhibitor, PD98059, and the calcium chelator, BAPTA-AM (CAS-126150-97-8), were procured from Calbiochem, CA, USA.

Ouabain, Dulbecco’s Minimal Essential Medium (DMEM) with 4500 mg/L glucose and pyridoxine HCL, trypsin-EDTA, Penicillin/Streptomycin, Fetal Bovine Serum (FBS), 10x Phosphate Buffered Saline (PBS) without magnesium and calcium, Adenosine 5′-triphosphate disodium salt (ATP), 2’-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt (dbcAMP), a purified Adenosine Triphosphatase (ATPase) from porcine cerebral cortex and the mouse monoclonal anti-Na⁺/K⁺ ATPase α1 (cat # ZMS1029, used at 1:5000 dilution) were procured all from Sigma, Chemical Co, St Louis Missouri, USA.

The Caco-2 cell line (male human colonic adenocarcinoma cell line, RRID: CVCL_0025) was obtained in 2021 from the American Type Culture Collection (ATCC). Cells were authenticated by ATCC and certified to be mycoplasma-free upon receipt. Later on, routine mycoplasma screening was conducted every 6 weeks in our lab.

All other chemicals were purchased from Sigma, Chemical Co, St Louis Missouri, USA.

Methods

Culture and collection of Caco-2 cells.

Caco-2 cells, at passages 32–50, were cultured in DMEM supplemented with 1% penicillin, 1% streptomycin, and 10% fetal bovine serum (FBS). Cells were seeded in six-well plates at a density of 150,000 cells per well and maintained in a humidified incubator (95% O₂, 5% CO₂) at 37°C. Treatments were performed when cells reached 85–90% confluence following overnight starvation. After treatment, cells were washed with PBS buffer (pH 7.4) and collected using lysis buffer containing protease inhibitors. The lysates were homogenized and centrifuged at 20,000g for 30 minutes at 4°C. Proteins in the supernatant were quantified using the Bradford assay. These protein samples were then utilized for assessing Na⁺/K⁺ ATPase activity and conducting western blot analysis.

Cell viability test.

At 60% confluence, Caco-2 cells were starved overnight and treated the next day with 8µM cisplatin for 24 hrs. The cells were washed with PBS, trypsinized, suspended in 1 ml DMEM, and spun at 206g and 4°C for 5 minutes. The pelleted cells were then re-suspended in 1 ml DMEM. Equal volumes of cell suspension and trypan blue were mixed and aliquots were used to count the cells (viable/dead) using a hemocytometer.

Na⁺/K⁺ ATPase activity assay.

The activity of the Na⁺/K⁺ ATPase was assayed as described by Esmann [13] by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain, a specific inhibitor of the ATPase. Following protein quantification in the supernatant obtained by centrifugation of the cell lysate (as previously described), protein concentrations were adjusted to 0.5 μg/μl using histidine buffer (pH 7.4, 150 mM). Samples containing phosphatase inhibitors (2.7 mM pyrophosphate and 2.7 mM glycerophosphate) were incubated with 0.2% saponin at room temperature for 30 minutes. Subsequently, aliquots were taken and incubated for 10 minutes at 37 °C in histidine buffer supplemented with NaCl (121.5 mM), KCl (19.6 mM), and MgCl₂ (3.92 mM), both in the presence and absence of ouabain (1.47 mM). Adenosine triphosphate (ATP; 10 mM) was then added (final concentration 2.94 mM), and the samples were incubated for an additional 30 minutes. The reaction was terminated by adding an equal volume of 50% trichloroacetic acid, followed by centrifugation at 3000 g for 5 minutes. Inorganic phosphate released into the supernatant was quantified colorimetrically at 750 nm, according to the method described by Taussky & Shorr [14]. The results are reported as a percentage of the control values.

Preparation of cells with GFP-tagged Na^+/K⁺ ATPase.

Caco-2 cells were stably transfected with GFP-tagged Na⁺/K⁺ ATPase α-1 using a plasmid generously provided by the Sznajder Laboratory at Northwestern University and the membrane marker mCherry-Mem (Addgene plasmid #55779), kindly gifted by Catherine Berlot [15]. The wheat germ agglutinin Alexa Fluor ^TM 594 was acquired from Invitrogen, OR, USA.

Treatment of Caco2 cells.

In all treatments with cisplatin, an equal volume of the vehicle, 0.9% NaCl, was added to the control.

Dose and time-response study on the effect of cisplatin on the Na⁺/K⁺ ATPase activity: Following an overnight starvation period, Caco-2 cells were treated with various concentrations of cisplatin (2, 4, 6, 8, and 10 µM) for 12 or 24 hours and the activity of Na⁺/K⁺ ATPase was subsequently measured. Changes in activity at 24 hours mirrored those observed at 12 hours. Notably, the highest enzymatic change was observed at 8 µM. Consequently, this concentration and a treatment period of 24 hours were adopted for all subsequent experiments.

The influence of the Na-K-2Cl cotransporter (NKCC) and the coupled Na⁺/H⁺ and Cl^-/HCO3^- exchangers on the effect of cisplatin on the Na⁺/K⁺ ATPase: Cisplatin-resistant cells were found to feature higher intracellular chloride concentrations. To test whether changes in intracellular chloride levels could affect the cisplatin-induced changes in the ATPase activity, Caco-2 cells were treated with 600µM furosemide, an inhibitor of the NKCC cotransporter, or with 100µM amiloride, an inhibitor of the exchangers Na⁺/H⁺.

Investigating a possible direct effect of cisplatin on the sodium-potassium ATPase: The effect of cisplatin (8µM) on the activity of a purified porcine cerebral cortex Na⁺/K⁺ ATPase (50 µg) was assayed as described before by directly incubating the enzyme in the presence of the drug for 20 minutes at 37°C in a histidine buffer containing all the substrates described before.

The effect of cisplatin on the membrane abundance of the Na⁺/K⁺ ATPase: Caco-2 cells expressing GFP-tagged Na⁺/K⁺ ATPase (50,000 cells) were seeded onto Poly-D-Lysine-coated glass-bottomed dishes and incubated for 24 hours in DMEM supplemented with FBS, penicillin, and streptomycin. After an overnight starvation period, the cells were treated with 8 µM cisplatin for 24 hours. Following treatment, the cells were washed with PBS buffer (pH 7.4), fixed with 500 µL of 4% paraformaldehyde, and stained with Alexa Fluor™ Wheat Germ Agglutinin (WGA) dye to label the plasma membrane.

Imaging was performed using an inverted immunofluorescence microscope equipped with a × 40 objective lens. Specific excitation wavelengths for GFP and WGA were used (FS 14 and FS 10, respectively). The images were captured and scaled using AxioVision software, and subsequent image processing was carried out with ImageJ software. Cells displaying GFP and WGA expression were analyzed, and the green-to-red fluorescence ratio was calculated.

Western blot analysis.

Fifty micrograms of cell homogenate proteins were run on 10% SDS polyacrylamide gels transferred to nitrocellulose membranes and then cut into two strips containing each the Na⁺/K⁺ ATPase or the GAPDH proteins. They were then incubated overnight at 4°C with a primary anti-Na⁺/K⁺ ATPase α1 antibody, or an anti-GAPDH followed by an incubation with a goat anti-mouse HRP conjugated IgG secondary antibody for 1 hour at room temperature. The signal was detected using the ChemiDoc™ MP Imaging System with the Clarity™ ECL Substrate (luminol-based).

Determination of the components of the signaling cascade.

Involvement of JNK, ERK, p38 MAPK, PKA and calcium in the effect of cisplatin on the Na⁺/K⁺ ATPase activity: Caco-2 cells were treated for 24 hrs and after an overnight starvation with 8µM cisplatin in presence and absence of inhibitors of JNK (SP600125,50µM), ERK (PD98059, 50µM), p38 MAPK (doramapimod, 40nM), or PKA (RpcAMP, 30µM). All inhibitors were added 30 min before cisplatin.

The involvement of JNK was further investigated by treating the cells with cisplatin in the presence of anisomycin (250 ng/ml), a JNK activator. Anisomycin was added 30 minutes prior to cisplatin treatment.

To investigate any possible role of calcium in this pathway, the cells were pre-treated, 30 minutes before cisplatin, with BAPTA-AM (10µM) a calcium chelator, or with ionomycin (6µM), a calcium ionophore.

Locating the involved molecules with respect to each other: The position of p38 MAPK with respect to PKA was determined by treating the cells with 8µM cisplatin after a 30 min simultaneous pre-treatment with an inhibitor of p38 MAPK (doramapimod, 40nM), and an activator of PKA (dibutyryl cyclic adenosine mono phosphate, dbcAMP, 10µM). The Caco-2 cells were then incubated for 24 hours, and processed for the ATPase activity assay.

The relationship between PKA with JNK was determined by studying the effect of cisplatin (8µM, 24 hrs) when PKA and JNK were inhibited simultaneously with respectively RpcAMP (30µM) and SP600125 (50µM). The inhibitors were added 30 min before cisplatin.

To determine if calcium is upstream or downstream PKA the activity of the ATPase was examined in cells treated 30 min before cisplatin with a calcium chelator BAPTA-AM (10µM), and a PKA activator dbcAMP (10µM). Cells were also treated with cisplatin in the simultaneous presence of the calcium ionophore ionomycin (6µM) and the p38 MAPK inhibitor doramapinod (40nM)

Statistical analysis.

The data were tested for statistical significance using a t- test or a one-way analysis of variance followed by a Tukey-Kramer multiple comparison test using GraphPad InStat3. The results are reported as mean ± SEM.

Results

Dose- and time-dependent inhibition of Na ⁺ /K ⁺ -ATPase by Cisplatin

Treating Caco-2 cells for 12 or 24 hours with various sub-toxic concentrations of cisplatin revealed a dose-dependent inhibition of the Na⁺/K⁺ ATPase that appeared at 4 µM and was maintained at 6, 8, and 10µM across both treatment periods, although the highest inhibitory effect was observed at 8µM and 24 hrs with a residual activity of around 37% (Fig 1A). Consequently, this concentration and time period were adopted in all subsequent experiments.

Download:

Fig 1. A) Dose- and time-dependent effects of cisplatin on Na⁺/K⁺ pump activity in cells treated for 12 or 24 hours. Data are presented as means ± SEM from 3 observations.

Statistical significance compared to the control ***P < 0.001, *p < 0.01, determined by ANOVA followed by Tukey’s test. B) Cell viability following treatment with 8 µM cisplatin for 24 hours. Data are shown as means ± SEM from 3 observations. Bars with the same letter on top are not significantly different from each other, as determined by an unpaired t-test. p < 0.001. C) Effect of cisplatin on purified Na⁺/K⁺ ATPase. Data are presented as means ± SEM from 3 observations. Bars with the same letter on top are not significantly different from each other, as determined by an unpaired t-test. p < 0.01.

https://doi.org/10.1371/journal.pone.0342707.g001

Cisplatin does not affect cell viability

To confirm that the cells remained alive throughout the 24-hour treatment period and to rule out the possibility that reduced activity was due to cell death, a viability test was performed. The results revealed comparable cell viability in the presence and absence of 8 µM cisplatin after 24 hours (Fig 1B).

Cisplatin does not act directly on the Na⁺/K⁺ ATPase

To determine whether cisplatin directly interacts with the enzyme, its effect on purified Na ⁺ /K ⁺ -ATPase was evaluated. No change in activity was detected, suggesting that cisplatin does not inhibit the enzyme via direct binding (Fig 1C).

Cisplatin induces a decrease in the abundance of Na⁺/K⁺ ATPase units at the plasma membrane

To assess whether the reduction in the activity of the Na⁺/K⁺ ATPase is due to a decrease in its membrane abundance, fluorescence imaging was performed in Caco-2 cells expressing GFP-tagged Na ⁺ /K ⁺ -ATPase α1. The plasma membrane was labeled using wheat germ agglutinin Alexa Fluor ^TM 594. The relative quantification of GFP/ Alexa Fluor ^TM 594 showed a significant cisplatin-induced decrease in the ATPase residence at the membrane (Fig 2A).

Download:

Fig 2. A) Fluorescence images of Caco-2 cells co-expressing GFP-tagged Na + /K + α1 subunits and mCherry-membrane marker after treatment with cisplatin for 24 hours.

Statistical differences from the control were tested using a t-test (p < 0.01). B) Protein expression of the Na⁺/K⁺ ATPase in homogenates of cells treated with 8 µM cisplatin for 24 hours. The blots are representative of an experiment repeated 4 times. Values are normalized to GAPDH. Bars not sharing a common letter are considered significantly different from each other at p < 0.01 as indicated by a t- test.

https://doi.org/10.1371/journal.pone.0342707.g002

Na⁺/K⁺ ATPase molecules are internalized but not degraded

The reduced membrane abundance of Na⁺/K⁺-ATPase may result from either decreased protein expression or increased degradation. To investigate these possibilities, western blot analysis of whole-cell homogenates was performed. No significant change in total Na⁺/K⁺-ATPase levels was observed, indicating that the pumps were internalized and not degraded (Fig 2B).

The Na-K-2Cl cotransporter and Na⁺/H⁺ exchanger modulate Cisplatin’s effect on Na⁺/K⁺ ATPase

To explore the involvement of the Na-K-2Cl cotransporter (NKCC) and Na + /H+ exchanger in the cisplatin’s effect, cells were co-treated with furosemide (NKCC inhibitor) or amiloride (Na ⁺ /H⁺ exchanger inhibitor). Both treatments further reduced Na ⁺ /K ⁺ -ATPase activity by respectively 40% and 35% (Fig 3A & B).

Download:

Fig 3. Effect of A) furosemide (600µM), and B) amiloride (100 µM) on the activity of the Na⁺/K⁺ ATPase in cells treated with 8µM cisplatin for 24 hrs.

Data are presented as means ± SEM from 3 observations. Bars sharing the same letter are not significantly different as determined by ANOVA followed by Tukey’s test. p < 0.01.

https://doi.org/10.1371/journal.pone.0342707.g003

Mediators of cisplatin effect on the Na⁺/K⁺ ATPase

Cisplatin interacts with multiple signaling pathways involving mediators such as calcium and various kinases like the Mitogen-Activated Protein Kinases (MAPKs), p38 MAPK, c-Jun N-terminal Kinase (JNK), as well as Protein Kinase A (PKA). These interactions significantly influence cisplatin’s effects on both cancerous and normal tissues. As a result, the specific roles and impacts of each of these signaling molecules were investigated.

Calcium Signaling Mediates Cisplatin-Induced Inhibition: Calcium chelation with BAPTA-AM (10µM) abolished cisplatin’s effect, indicating its involvement in the induced inhibition of the ATPase (Fig 4A). Treating the cells with the calcium ionophore ionomycin (6µM) reduced the activity of the Na⁺/K⁺ ATPase down to around 27% of its control value. No further reduction was observed in the simultaneous presence of cisplatin (Fig 4B) suggesting that calcium is along the pathway activated by cisplatin.

Download:

Fig 4. Effect of cisplatin in presence of A) the calcium chelator BAPTA-AM (10µM) and B) the calcium ionophore ionomycin (6µM) on the activity of the Na + /K + ATPase. Values are means ± SEM of 3 observations.

Bars not sharing the same letter are considered significantly different from each other at p < 0.001, as indicated by ANOVA followed by Tukey test.

https://doi.org/10.1371/journal.pone.0342707.g004

C. effect of cisplatin on the ATPase in presence of PD98059 an ERK inhibitor. Values are means ± SEM of 3 observations. Bars not sharing a common letter are considered significantly different from each other at p < 0.01, as indicated by ANOVA followed by Tukey test.

ERK signaling is not involved in cisplatin’s effect: To evaluate ERK’s role, the activity of the pump was measured in Caco-2 cells treated with cisplatin in presence of PD98059 (a MEK/ERK inhibitor). The ATPase activity was not significantly different from that of the control. ERK’s inhibition did not negate the inhibitory effect of cisplatin (Fig 4C), inferring that cisplatin acts independently of ERK.

p38 MAPK and PKA mediate cisplatin’s inhibitory effect: In presence of doramapimod (40nM), or RpcAMP (30µM), respective inhibitors of p38 MAPK and PKA, cisplatin did not induce its inhibitory effect on the ATPase (Figs 5 A & B) indicating their participation in the signaling cascade.

Download:

Fig 5. Effect of cisplatin in presence of A) doramapimod (40nM), a p38 MAPK inhibitor; B) RpcAMP (30µM), a PKA inhibitor; C) SP600125, a JNK inhibitor; D) anisomycin, a JNK activator; Values are means ± SEM of 3 observations.

Bars not sharing the same letter are considered significantly different from each other at p < 0.01, as indicated by ANOVA followed by Tukey test.

https://doi.org/10.1371/journal.pone.0342707.g005

Cisplatin inhibits the Na⁺/K⁺ ATPase by inhibiting JNK: The inhibition of JNK alone with SP600125(50 µM) induced, like cisplatin, a decrease in the activity of the Na⁺/K⁺ ATPase that was maintained in the simultaneous presence of the drug (Fig 5C). The effect of cisplatin was not manifested in presence of anisomycin, a JNK activator (Fig 5D). The results suggest that cisplatin inhibits the Na⁺/K⁺ ATPase by suppressing JNK activity.

Signaling hierarchy: calcium, PKA, and JNK: To define the signaling hierarchy, cells were co-treated with BAPTA-AM and dbcAMP. While cisplatin could not exert any effect on the ATPase in presence of the calcium chelator BAPTA-AM (10 µM), the simultaneous addition of dbcAMP (10µM), a PKA activator, restored the inhibitory effect of cisplatin, implying that PKA is downstream of calcium (Fig 6A).

Download:

Fig 6. Effect of cisplatin on the Na⁺/K⁺ ATPase in A) the simultaneous presence of BAPTA-AM(10 µM) and dbcAMP (10 µM); B) the simultaneous presence of an inhibitor of JNK (SP600125) and an inhibitor of PKA (Rp cAMP).

Values are means ± SEM of 3 observations. Bars not sharing the same letter are considered significantly different from each other at p < 0.05, as indicated by ANOVA followed by Tukey test.

https://doi.org/10.1371/journal.pone.0342707.g006

Inhibition of PKA with RpcAMP blocked the effect of cisplatin. This effect reappeared upon the simultaneous addition of SP600125, an inhibitor of JNK, indicating that PKA is upstream JNK, and inhibits it (Fig 6B).

Discussion

Cisplatin induces cell damage and triggers apoptosis [3]. Since apoptosis is known to reduce the activity of the Na⁺/K⁺ ATPase [16], we opted to study the effect of sub-toxic levels of the drug on the activity of the enzyme and to determine, if such an effect exists, the mediators involved. At 6–10 µM, cisplatin significantly inhibited pump activity without compromising cell viability, supporting the reported link between cisplatin’s apoptotic effects and ATPase dysfunction. These results suggest that gastrointestinal side-effects of cisplatin may stem in part from colonic ATPase dysfunction, opening avenues for gut-protective interventions. These results may offer insight into some of the drug’s undesirable side effects, such as diarrhea and constipation, which are associated with dysregulation of the pump [5].

Cisplatin uptake remains debated, with evidence for both passive diffusion and copper transporter-mediated entry [3]. Its short-lived efficacy is often attributed to resistance mechanisms, including reduced cytoplasmic accumulation, which depends on intracellular chloride levels [17]. Salerno et al. [17] reported that cisplatin uptake is enhanced under conditions of reduced intracellular Cl ⁻ . Intracellular Cl⁻ levels are maintained primarily by NKCC1 together with the coupled Cl ⁻ /HCO₃⁻ and Na ⁺ /H⁺ exchangers. Pharmacological inhibition of these transporters using furosemide and amiloride, which lowers intracellular Cl⁻ and increases the Cl transmembrane gradient, potentiated further cisplatin’s inhibitory effect on the Na ⁺ /K ⁺ -ATPase. This observation is consistent with increased cellular cisplatin uptake driven by the Cl⁻ gradient, thereby attributing the greater ATPase inhibition to enhanced drug accumulation.

To determine whether cisplatin acts directly on the ATPase, we tested its effect on the purified enzyme. No change in activity was observed, indicating an indirect mechanism. Although the literature reports an interaction of cisplatin with five cysteine residues (C452, C456, C457, C577, and C656) within the intracellular loop connecting transmembrane helices 4 and 5 [10,11], these findings were based on studies using the isolated C45 loop, rather than the full enzyme which may influence the accessibility and reactivity of these residues, limiting the physiological relevance of these observations.

The observed inhibition of the Na + /K + ATPase could result from reduced membrane abundance, a decreased specific activity, or both. Imaging of GFP-tagged ATPase revealed fewer membrane-localized units while western blot analysis showed unchanged total protein levels. The results infer that the ATPase units were internalized but did not undergo degradation.

Cisplatin’s apoptotic effects involve kinases such as ERK, JNK, p38 MAPK [18–21], and PKA [22], as well as calcium signaling [23]. We examined their roles in ATPase regulation.

ERK inhibition with PD98059 did not alter cisplatin’s effect, suggesting that ERK is not involved. Notably, ERK’s role in apoptosis is context-dependent, promoting survival in some cell lines (e.g., SK-OV-3 [24]) and apoptosis in others (e.g., osteosarcoma, neuroblastoma [25,26]).

In contrast, inhibition of p38 MAPK and PKA blocked cisplatin’s effect, confirming their involvement [22,27–29]. However, JNK inhibition, even in the absence of cisplatin, significantly reduced the pump’s activity, suggesting that JNK is constitutively active and plays a basal role in maintaining the basal activity of the Na + /K + ATPase. This finding was further supported by the stimulation observed in cells treated with the JNK activator, anisomycin. Furthermore, JNK inhibition in the presence of cisplatin did not lead to a greater inhibition, implying that JNK operates along the pathway activated by cisplatin. A greater inhibition would have been expected, had JNK been acting independently of this pathway.

The relationship between JNK and cisplatin-induced apoptosis is context-dependent. Recent studies describe JNK as a double-edged sword, playing a crucial role in promoting cell death while also contributing to enhanced resistance to cisplatin by activating cell survival mechanisms [30].

The effect of cisplatin was abolished by p38 MAPK inhibition, yet restored upon co‑administration of dbcAMP, indicating that PKA functions downstream of p38 MAPK. Consistently, inhibition of PKA with RpcAMP abrogated the cisplatin response, but this effect was reversed when JNK was simultaneously inhibited, supporting a model in which JNK operates downstream of PKA. Together, these observations delineate a signaling cascade in which cisplatin engages p38 MAPK, leading to PKA activation and subsequent modulation of JNK activity.

Cisplatin failed to exert its effect under calcium chelation, highlighting calcium’s role in Na ⁺ /K ⁺ -ATPase inhibition and the activation of PKA and p38 MAPK. Consistently, treatment with BAPTA-AM abolished the drug’s activity, which reappeared upon co-administration of dbcAMP. Likewise, the inhibitory action of the calcium ionophore ionomycin was suppressed in the presence of the p38 MAPK inhibitor doramapimod.

These findings suggest that PKA is downstream of calcium and p38 MAPK, with all three mediators acting along the same signaling pathway.

Calcium regulates apoptosis by modulating intracellular levels through channels, pumps, exchangers, and binding proteins [23,31–33]. Calcium-induced activation of p38 MAPK has been linked to CaMKII and ASK1/MAP3K5, which trigger apoptosis under stress [34–36].

It can be concluded that cisplatin inhibits Na ⁺ /K ⁺ -ATPase via a signaling cascade involving regulators of apoptosis. PKA and p38 MAPK are activated, while JNK is inhibited. JNK’s dual role in apoptosis and resistance is well-documented, and p38 MAPK knockout studies show increased JNK activity [37,38]. Our findings suggest that PKA mediates JNK inhibition, consistent with reports of cAMP reducing JNK phosphorylation via CREB-induced c-FLIP(L) and MKP-1 [39,40]. JNK’s stimulatory effect on Na ⁺ /K ⁺ -ATPase has also been observed in LLC-PK1 cells [41].

We propose that cisplatin uptake is driven by the chloride gradient. Once inside the cell, it elevates intracellular calcium, activates p38 MAPK and PKA, and inhibits JNK, leading to reduced Na ⁺ /K ⁺ -ATPase activity. This pathway is summarized in Fig 7.

Download:

Fig 7. The signaling pathway mediating the effect of cisplatin on the Na + /K + ATPase.

https://doi.org/10.1371/journal.pone.0342707.g007

Supporting information

S1 Fig. Uncropped raw western blot images showing protein expression of Na + /K + ATPase and GAPDH.

https://doi.org/10.1371/journal.pone.0342707.s001

(PDF)

S2 File. Raw data for all experiments.

https://doi.org/10.1371/journal.pone.0342707.s002

(XLSX)

References

1. Elmorsy EA, Saber S, Hamad RS, Abdel-Reheim MA, El-Kott AF, AlShehri MA, et al. Advances in understanding cisplatin-induced toxicity: Molecular mechanisms and protective strategies. Eur J Pharm Sci. 2024;203:106939. pmid:39423903
- View Article
- PubMed/NCBI
- Google Scholar
2. Salazar-Barrientos JI, Guevara-Vela JM, García-Revilla MA, Francisco E, Gallegos M, Rocha-Rinza T, et al. Effect of Explicit Hydration on the Cisplatin Reaction Mechanism with Adenine and Guanine. Molecules. 2025;30(3):510. pmid:39942615
- View Article
- PubMed/NCBI
- Google Scholar
3. Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol. 2014;740:364–78. pmid:25058905
- View Article
- PubMed/NCBI
- Google Scholar
4. Zhu S, Tang M, Chen J, Li S, Xue R. Mitophagy Protects Against Cisplatin-Induced Injury in Granulosa Cells. Toxics. 2025;13(5):332. pmid:40423411
- View Article
- PubMed/NCBI
- Google Scholar
5. Kris MG, Gralla RJ, Clark RA, Tyson LB, Groshen S. Control of chemotherapy-induced diarrhea with the synthetic enkephalin BW942C: a randomized trial with placebo in patients receiving cisplatin. J Clin Oncol. 1988;6(4):663–8. pmid:3282034
- View Article
- PubMed/NCBI
- Google Scholar
6. Bearcroft CP, Domizio P, Mourad FH, André EA, Farthing MJ. Cisplatin impairs fluid and electrolyte absorption in rat small intestine: a role for 5-hydroxytryptamine. Gut. 1999;44(2):174–9. pmid:9895375
- View Article
- PubMed/NCBI
- Google Scholar
7. Durlacher CT, Chow K, Chen X-W, He Z-X, Zhang X, Yang T, et al. Targeting Na⁺/K⁺ -translocating adenosine triphosphatase in cancer treatment. Clin Exp Pharmacol Physiol. 2015;42(5):427–43. pmid:25739707
- View Article
- PubMed/NCBI
- Google Scholar
8. Honisch S, Alkahtani S, Kounenidakis M, Liu G, Alarifi S, Al-Yahya H, et al. A steroidal Na+/K+ ATPase inhibitor triggers pro-apoptotic signaling and induces apoptosis in prostate and lung tumor cells. Anticancer Agents Med Chem. 2014;14(8):1161–8. pmid:24942548
- View Article
- PubMed/NCBI
- Google Scholar
9. Bejček J, Spiwok V, Kmoníčková E, Rimpelová S. Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation. Molecules. 2021;26(7):1905. pmid:33800655
- View Article
- PubMed/NCBI
- Google Scholar
10. Šeflová J, Čechová P, Štenclová T, Šebela M, Kubala M. Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase. J Enzyme Inhib Med Chem. 2018;33(1):701–6. pmid:29577756
- View Article
- PubMed/NCBI
- Google Scholar
11. Kubala M, Geleticova J, Huliciak M, Zatloukalova M, Vacek J, Sebela M. Na(+)/K(+)-ATPase inhibition by cisplatin and consequences for cisplatin nephrotoxicity. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2014;158(2):194–200. pmid:24781046
- View Article
- PubMed/NCBI
- Google Scholar
12. Tadini-Buoninsegni F, Sordi G, Smeazzetto S, Natile G, Arnesano F. Effect of cisplatin on the transport activity of PII-type ATPases. Metallomics. 2017;9(7):960–8. pmid:28636017
- View Article
- PubMed/NCBI
- Google Scholar
13. Esmann M. ATPase and phosphatase activity of Na+,K+-ATPase: molar and specific activity, protein determination. Methods Enzymol. 1988;156:105–15. pmid:2835596
- View Article
- PubMed/NCBI
- Google Scholar
14. Taussky HH, Shorr E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953;202(2):675–85. pmid:13061491
- View Article
- PubMed/NCBI
- Google Scholar
15. Yost EA, Mervine SM, Sabo JL, Hynes TR, Berlot CH. Live cell analysis of G protein beta5 complex formation, function, and targeting. Mol Pharmacol. 2007;72(4):812–25. pmid:17596375
- View Article
- PubMed/NCBI
- Google Scholar
16. Wang XQ, Xiao AY, Sheline C, Hyrc K, Yang A, Goldberg MP, et al. Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress. J Cell Sci. 2003;116(Pt 10):2099–110. pmid:12679386
- View Article
- PubMed/NCBI
- Google Scholar
17. Salerno M, Yahia D, Dzamitika S, de Vries E, Pereira-Maia E, Garnier-Suillerot A. Impact of intracellular chloride concentration on cisplatin accumulation in sensitive and resistant GLC4 cells. J Biol Inorg Chem. 2009;14(1):123–32. pmid:18797938
- View Article
- PubMed/NCBI
- Google Scholar
18. Achkar IW, Abdulrahman N, Al-Sulaiti H, Joseph JM, Uddin S, Mraiche F. Cisplatin based therapy: the role of the mitogen activated protein kinase signaling pathway. J Transl Med. 2018;16(1):96. pmid:29642900
- View Article
- PubMed/NCBI
- Google Scholar
19. St Germain C, Niknejad N, Ma L, Garbuio K, Hai T, Dimitroulakos J. Cisplatin induces cytotoxicity through the mitogen-activated protein kinase pathways and activating transcription factor 3. Neoplasia. 2010;12(7):527–38. pmid:20651982
- View Article
- PubMed/NCBI
- Google Scholar
20. Yang F, Li M, Xu D, Jiang Z, Jiang H, Xiao Y, et al. Inhibition of JNK/c-Jun-ATF2 Overcomes Cisplatin Resistance in Liver Cancer through down-Regulating Galectin-1. Int J Biol Sci. 2023;19(8):2366–81. pmid:37215991
- View Article
- PubMed/NCBI
- Google Scholar
21. Lin H, Lin Q, Liu M, Lin Y, Wang X, Chen H, et al. PKA/Smurf1 signaling-mediated stabilization of Nur77 is required for anticancer drug cisplatin-induced apoptosis. Oncogene. 2014;33(13):1629–39. pmid:23584473
- View Article
- PubMed/NCBI
- Google Scholar
22. Abdel-Wahab BA, Walbi IA, Albarqi HA, Ali FEM, Hassanein EHM. Roflumilast protects from cisplatin-induced testicular toxicity in male rats and enhances its cytotoxicity in prostate cancer cell line. Role of NF-κB-p65, cAMP/PKA and Nrf2/HO-1, NQO1 signaling. Food Chem Toxicol. 2021;151:112133. pmid:33757793
- View Article
- PubMed/NCBI
- Google Scholar
23. Kouba S, Hague F, Ahidouch A, Ouadid-Ahidouch H. Crosstalk between Ca2+ Signaling and Cancer Stemness: The Link to Cisplatin Resistance. Int J Mol Sci. 2022;23(18):10687. pmid:36142596
- View Article
- PubMed/NCBI
- Google Scholar
24. Kielbik M, Krzyzanowski D, Pawlik B, Klink M. Cisplatin-induced ERK1/2 activity promotes G1 to S phase progression which leads to chemoresistance of ovarian cancer cells. Oncotarget. 2018;9(28):19847–60. pmid:29731988
- View Article
- PubMed/NCBI
- Google Scholar
25. Woessmann W, Chen X, Borkhardt A. Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines. Cancer Chemother Pharmacol. 2002;50(5):397–404. pmid:12439598
- View Article
- PubMed/NCBI
- Google Scholar
26. Lu Y, Liu B, Liu Y, Yu X, Cheng G. Dual effects of active ERK in cancer: A potential target for enhancing radiosensitivity. Oncol Lett. 2020;20(2):993–1000. pmid:32724338
- View Article
- PubMed/NCBI
- Google Scholar
27. Goel Y, Argueta DA, Peterson K, Lomeli N, Bota DA, Gupta K. Neuronal p38 MAPK Signaling Contributes to Cisplatin-Induced Peripheral Neuropathy. Antioxidants (Basel). 2025;14(4):445. pmid:40298791
- View Article
- PubMed/NCBI
- Google Scholar
28. Yuan P, Feng A, Wei Y, Li S, Fu Y, Wang X, et al. Indole-3-carboxaldehyde alleviates cisplatin-induced acute kidney injury in mice by improving mitochondrial dysfunction via PKA activation. Food Chem Toxicol. 2024;186:114546. pmid:38408633
- View Article
- PubMed/NCBI
- Google Scholar
29. Rodríguez-García ME, Quiroga AG, Castro J, Ortiz A, Aller P, Mata F. Inhibition of p38-MAPK potentiates cisplatin-induced apoptosis via GSH depletion and increases intracellular drug accumulation in growth-arrested kidney tubular epithelial cells. Toxicol Sci. 2009;111(2):413–23. pmid:19578154
- View Article
- PubMed/NCBI
- Google Scholar
30. Yan D, An G, Kuo MT. C-Jun N-terminal kinase signalling pathway in response to cisplatin. J Cell Mol Med. 2016;20(11):2013–9. pmid:27374471
- View Article
- PubMed/NCBI
- Google Scholar
31. Shen L, Wen N, Xia M, Zhang YU, Liu W, Xu YE, et al. Calcium efflux from the endoplasmic reticulum regulates cisplatin-induced apoptosis in human cervical cancer HeLa cells. Oncol Lett. 2016;11(4):2411–9. pmid:27073489
- View Article
- PubMed/NCBI
- Google Scholar
32. Hodeify R, Siddiqui SS, Matar R, Vazhappilly CG, Merheb M, Al Zouabi H, et al. Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells. Heliyon. 2021;7(1):e06041. pmid:33532651
- View Article
- PubMed/NCBI
- Google Scholar
33. Meng X, Zhang Y, Ma Y, Du Y, Gan C, Feng J, et al. Transient receptor potential ankyrin 1 mediates cisplatin-induced apoptosis in renal tubular cells via calcium-dependent signaling pathway. Ann Palliat Med. 2021;10(8):9025–38. pmid:34488389
- View Article
- PubMed/NCBI
- Google Scholar
34. Jiang Y, Fang Y, Ye Y, Xu X, Wang B, Gu J, et al. Anti-Cancer Effects of 3, 3’-Diindolylmethane on Human Hepatocellular Carcinoma Cells Is Enhanced by Calcium Ionophore: The Role of Cytosolic Ca2+ and p38 MAPK. Front Pharmacol. 2019;10:1167. pmid:31649538
- View Article
- PubMed/NCBI
- Google Scholar
35. Kashiwase K, Higuchi Y, Hirotani S, Yamaguchi O, Hikoso S, Takeda T, et al. CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy. Biochem Biophys Res Commun. 2005;327(1):136–42. pmid:15629441
- View Article
- PubMed/NCBI
- Google Scholar
36. Takeda K, Matsuzawa A, Nishitoh H, Tobiume K, Kishida S, Ninomiya-Tsuji J, et al. Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation. EMBO Rep. 2004;5(2):161–6. pmid:14749717
- View Article
- PubMed/NCBI
- Google Scholar
37. Caballero-Franco C, Choo M-K, Sano Y, Ritprajak P, Sakurai H, Otsu K, et al. Tuning of protein kinase circuitry by p38α is vital for epithelial tissue homeostasis. J Biol Chem. 2013;288(33):23788–97. pmid:23836897
- View Article
- PubMed/NCBI
- Google Scholar
38. Pereira L, Igea A, Canovas B, Dolado I, Nebreda AR. Inhibition of p38 MAPK sensitizes tumour cells to cisplatin-induced apoptosis mediated by reactive oxygen species and JNK. EMBO Mol Med. 2013;5(11):1759–74. pmid:24115572
- View Article
- PubMed/NCBI
- Google Scholar
39. Hewer RC, Sala-Newby GB, Wu Y-J, Newby AC, Bond M. PKA and Epac synergistically inhibit smooth muscle cell proliferation. J Mol Cell Cardiol. 2011;50(1):87–98. pmid:20971121
- View Article
- PubMed/NCBI
- Google Scholar
40. Zhang J, Wang Q, Zhu N, Yu M, Shen B, Xiang J, et al. Cyclic AMP inhibits JNK activation by CREB-mediated induction of c-FLIP(L) and MKP-1, thereby antagonizing UV-induced apoptosis. Cell Death Differ. 2008;15(10):1654–62. pmid:18566605
- View Article
- PubMed/NCBI
- Google Scholar
41. Ramia N, Kreydiyyeh SI. TNF-alpha reduces the Na+/K+ ATPase activity in LLC-PK1 cells by activating caspases and JNK and inhibiting NF-kappaB. Cell Biol Int. 2010;34(6):607–13. pmid:20222869
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Elmorsy EA, Saber S, Hamad RS, Abdel-Reheim MA, El-Kott AF, AlShehri MA, et al. Advances in understanding cisplatin-induced toxicity: Molecular mechanisms and protective strategies. Eur J Pharm Sci. 2024;203:106939. pmid:39423903
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Salazar-Barrientos JI, Guevara-Vela JM, García-Revilla MA, Francisco E, Gallegos M, Rocha-Rinza T, et al. Effect of Explicit Hydration on the Cisplatin Reaction Mechanism with Adenine and Guanine. Molecules. 2025;30(3):510. pmid:39942615
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Dasari S, Tchounwou PB. Cisplatin in cancer therapy: molecular mechanisms of action. Eur J Pharmacol. 2014;740:364–78. pmid:25058905
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Zhu S, Tang M, Chen J, Li S, Xue R. Mitophagy Protects Against Cisplatin-Induced Injury in Granulosa Cells. Toxics. 2025;13(5):332. pmid:40423411
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Kris MG, Gralla RJ, Clark RA, Tyson LB, Groshen S. Control of chemotherapy-induced diarrhea with the synthetic enkephalin BW942C: a randomized trial with placebo in patients receiving cisplatin. J Clin Oncol. 1988;6(4):663–8. pmid:3282034
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Bearcroft CP, Domizio P, Mourad FH, André EA, Farthing MJ. Cisplatin impairs fluid and electrolyte absorption in rat small intestine: a role for 5-hydroxytryptamine. Gut. 1999;44(2):174–9. pmid:9895375
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Durlacher CT, Chow K, Chen X-W, He Z-X, Zhang X, Yang T, et al. Targeting Na⁺/K⁺ -translocating adenosine triphosphatase in cancer treatment. Clin Exp Pharmacol Physiol. 2015;42(5):427–43. pmid:25739707
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Honisch S, Alkahtani S, Kounenidakis M, Liu G, Alarifi S, Al-Yahya H, et al. A steroidal Na+/K+ ATPase inhibitor triggers pro-apoptotic signaling and induces apoptosis in prostate and lung tumor cells. Anticancer Agents Med Chem. 2014;14(8):1161–8. pmid:24942548
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Bejček J, Spiwok V, Kmoníčková E, Rimpelová S. Na+/K+-ATPase Revisited: On Its Mechanism of Action, Role in Cancer, and Activity Modulation. Molecules. 2021;26(7):1905. pmid:33800655
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Šeflová J, Čechová P, Štenclová T, Šebela M, Kubala M. Identification of cisplatin-binding sites on the large cytoplasmic loop of the Na+/K+-ATPase. J Enzyme Inhib Med Chem. 2018;33(1):701–6. pmid:29577756
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Kubala M, Geleticova J, Huliciak M, Zatloukalova M, Vacek J, Sebela M. Na(+)/K(+)-ATPase inhibition by cisplatin and consequences for cisplatin nephrotoxicity. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2014;158(2):194–200. pmid:24781046
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Tadini-Buoninsegni F, Sordi G, Smeazzetto S, Natile G, Arnesano F. Effect of cisplatin on the transport activity of PII-type ATPases. Metallomics. 2017;9(7):960–8. pmid:28636017
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Esmann M. ATPase and phosphatase activity of Na+,K+-ATPase: molar and specific activity, protein determination. Methods Enzymol. 1988;156:105–15. pmid:2835596
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Taussky HH, Shorr E. A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem. 1953;202(2):675–85. pmid:13061491
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Yost EA, Mervine SM, Sabo JL, Hynes TR, Berlot CH. Live cell analysis of G protein beta5 complex formation, function, and targeting. Mol Pharmacol. 2007;72(4):812–25. pmid:17596375
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Wang XQ, Xiao AY, Sheline C, Hyrc K, Yang A, Goldberg MP, et al. Apoptotic insults impair Na+, K+-ATPase activity as a mechanism of neuronal death mediated by concurrent ATP deficiency and oxidant stress. J Cell Sci. 2003;116(Pt 10):2099–110. pmid:12679386
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Salerno M, Yahia D, Dzamitika S, de Vries E, Pereira-Maia E, Garnier-Suillerot A. Impact of intracellular chloride concentration on cisplatin accumulation in sensitive and resistant GLC4 cells. J Biol Inorg Chem. 2009;14(1):123–32. pmid:18797938
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Achkar IW, Abdulrahman N, Al-Sulaiti H, Joseph JM, Uddin S, Mraiche F. Cisplatin based therapy: the role of the mitogen activated protein kinase signaling pathway. J Transl Med. 2018;16(1):96. pmid:29642900
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. St Germain C, Niknejad N, Ma L, Garbuio K, Hai T, Dimitroulakos J. Cisplatin induces cytotoxicity through the mitogen-activated protein kinase pathways and activating transcription factor 3. Neoplasia. 2010;12(7):527–38. pmid:20651982
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Yang F, Li M, Xu D, Jiang Z, Jiang H, Xiao Y, et al. Inhibition of JNK/c-Jun-ATF2 Overcomes Cisplatin Resistance in Liver Cancer through down-Regulating Galectin-1. Int J Biol Sci. 2023;19(8):2366–81. pmid:37215991
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Lin H, Lin Q, Liu M, Lin Y, Wang X, Chen H, et al. PKA/Smurf1 signaling-mediated stabilization of Nur77 is required for anticancer drug cisplatin-induced apoptosis. Oncogene. 2014;33(13):1629–39. pmid:23584473
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Abdel-Wahab BA, Walbi IA, Albarqi HA, Ali FEM, Hassanein EHM. Roflumilast protects from cisplatin-induced testicular toxicity in male rats and enhances its cytotoxicity in prostate cancer cell line. Role of NF-κB-p65, cAMP/PKA and Nrf2/HO-1, NQO1 signaling. Food Chem Toxicol. 2021;151:112133. pmid:33757793
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Kouba S, Hague F, Ahidouch A, Ouadid-Ahidouch H. Crosstalk between Ca2+ Signaling and Cancer Stemness: The Link to Cisplatin Resistance. Int J Mol Sci. 2022;23(18):10687. pmid:36142596
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Kielbik M, Krzyzanowski D, Pawlik B, Klink M. Cisplatin-induced ERK1/2 activity promotes G1 to S phase progression which leads to chemoresistance of ovarian cancer cells. Oncotarget. 2018;9(28):19847–60. pmid:29731988
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Woessmann W, Chen X, Borkhardt A. Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines. Cancer Chemother Pharmacol. 2002;50(5):397–404. pmid:12439598
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Lu Y, Liu B, Liu Y, Yu X, Cheng G. Dual effects of active ERK in cancer: A potential target for enhancing radiosensitivity. Oncol Lett. 2020;20(2):993–1000. pmid:32724338
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Goel Y, Argueta DA, Peterson K, Lomeli N, Bota DA, Gupta K. Neuronal p38 MAPK Signaling Contributes to Cisplatin-Induced Peripheral Neuropathy. Antioxidants (Basel). 2025;14(4):445. pmid:40298791
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Yuan P, Feng A, Wei Y, Li S, Fu Y, Wang X, et al. Indole-3-carboxaldehyde alleviates cisplatin-induced acute kidney injury in mice by improving mitochondrial dysfunction via PKA activation. Food Chem Toxicol. 2024;186:114546. pmid:38408633
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Rodríguez-García ME, Quiroga AG, Castro J, Ortiz A, Aller P, Mata F. Inhibition of p38-MAPK potentiates cisplatin-induced apoptosis via GSH depletion and increases intracellular drug accumulation in growth-arrested kidney tubular epithelial cells. Toxicol Sci. 2009;111(2):413–23. pmid:19578154
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Yan D, An G, Kuo MT. C-Jun N-terminal kinase signalling pathway in response to cisplatin. J Cell Mol Med. 2016;20(11):2013–9. pmid:27374471
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Shen L, Wen N, Xia M, Zhang YU, Liu W, Xu YE, et al. Calcium efflux from the endoplasmic reticulum regulates cisplatin-induced apoptosis in human cervical cancer HeLa cells. Oncol Lett. 2016;11(4):2411–9. pmid:27073489
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Hodeify R, Siddiqui SS, Matar R, Vazhappilly CG, Merheb M, Al Zouabi H, et al. Modulation of calcium-binding proteins expression and cisplatin chemosensitivity by calcium chelation in human breast cancer MCF-7 cells. Heliyon. 2021;7(1):e06041. pmid:33532651
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref33] 33. Meng X, Zhang Y, Ma Y, Du Y, Gan C, Feng J, et al. Transient receptor potential ankyrin 1 mediates cisplatin-induced apoptosis in renal tubular cells via calcium-dependent signaling pathway. Ann Palliat Med. 2021;10(8):9025–38. pmid:34488389
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref34] 34. Jiang Y, Fang Y, Ye Y, Xu X, Wang B, Gu J, et al. Anti-Cancer Effects of 3, 3’-Diindolylmethane on Human Hepatocellular Carcinoma Cells Is Enhanced by Calcium Ionophore: The Role of Cytosolic Ca2+ and p38 MAPK. Front Pharmacol. 2019;10:1167. pmid:31649538
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref35] 35. Kashiwase K, Higuchi Y, Hirotani S, Yamaguchi O, Hikoso S, Takeda T, et al. CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy. Biochem Biophys Res Commun. 2005;327(1):136–42. pmid:15629441
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref36] 36. Takeda K, Matsuzawa A, Nishitoh H, Tobiume K, Kishida S, Ninomiya-Tsuji J, et al. Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation. EMBO Rep. 2004;5(2):161–6. pmid:14749717
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref37] 37. Caballero-Franco C, Choo M-K, Sano Y, Ritprajak P, Sakurai H, Otsu K, et al. Tuning of protein kinase circuitry by p38α is vital for epithelial tissue homeostasis. J Biol Chem. 2013;288(33):23788–97. pmid:23836897
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref38] 38. Pereira L, Igea A, Canovas B, Dolado I, Nebreda AR. Inhibition of p38 MAPK sensitizes tumour cells to cisplatin-induced apoptosis mediated by reactive oxygen species and JNK. EMBO Mol Med. 2013;5(11):1759–74. pmid:24115572
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref39] 39. Hewer RC, Sala-Newby GB, Wu Y-J, Newby AC, Bond M. PKA and Epac synergistically inhibit smooth muscle cell proliferation. J Mol Cell Cardiol. 2011;50(1):87–98. pmid:20971121
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref40] 40. Zhang J, Wang Q, Zhu N, Yu M, Shen B, Xiang J, et al. Cyclic AMP inhibits JNK activation by CREB-mediated induction of c-FLIP(L) and MKP-1, thereby antagonizing UV-induced apoptosis. Cell Death Differ. 2008;15(10):1654–62. pmid:18566605
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref41] 41. Ramia N, Kreydiyyeh SI. TNF-alpha reduces the Na+/K+ ATPase activity in LLC-PK1 cells by activating caspases and JNK and inhibiting NF-kappaB. Cell Biol Int. 2010;34(6):607–13. pmid:20222869
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Materials

Methods

Culture and collection of Caco-2 cells.

Cell viability test.

Na+/K+ ATPase activity assay.

Preparation of cells with GFP-tagged Na+/K+ ATPase.

Treatment of Caco2 cells.

Western blot analysis.

Determination of the components of the signaling cascade.

Statistical analysis.

Results

Dose- and time-dependent inhibition of Na ⁺ /K ⁺ -ATPase by Cisplatin

Cisplatin does not affect cell viability

Cisplatin does not act directly on the Na+/K+ ATPase

Cisplatin induces a decrease in the abundance of Na+/K+ ATPase units at the plasma membrane

Na+/K+ ATPase molecules are internalized but not degraded

The Na-K-2Cl cotransporter and Na+/H+ exchanger modulate Cisplatin’s effect on Na+/K+ ATPase

Mediators of cisplatin effect on the Na+/K+ ATPase

Discussion

Supporting information

S1 Fig. Uncropped raw western blot images showing protein expression of Na + /K + ATPase and GAPDH.

S2 File. Raw data for all experiments.

References

Na⁺/K⁺ ATPase activity assay.

Preparation of cells with GFP-tagged Na^+/K⁺ ATPase.

Cisplatin does not act directly on the Na⁺/K⁺ ATPase

Cisplatin induces a decrease in the abundance of Na⁺/K⁺ ATPase units at the plasma membrane

Na⁺/K⁺ ATPase molecules are internalized but not degraded

The Na-K-2Cl cotransporter and Na⁺/H⁺ exchanger modulate Cisplatin’s effect on Na⁺/K⁺ ATPase

Mediators of cisplatin effect on the Na⁺/K⁺ ATPase