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Attenuated JNK signaling in multidrug-resistant leukemic cells. Dual role of MAPK in cell survival
Cellular Signalling 30 (2017) 162–170
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Attenuated JNK signaling in multidrug-resistant leukemic cells. Dual role of MAPK in cell survival☆ David Cerezo 1, Antonio J. Ruiz-Alcaraz 1, Miriam Lencina-Guardiola, Manuel Cánovas, Pilar García-Peñarrubia, Inmaculada Martínez-López, Elena Martín-Orozco ⁎ Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, Murcia Biohealth Research Institute-University of Murcia (IMIB-UMU), Regional Campus of International Excellence “Campus Mare Nostrum”, 30100 Murcia, Spain
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Article history: Received 1 August 2016 Received in revised form 28 November 2016 Accepted 5 December 2016 Available online 07 December 2016 Keywords: Leukemic cells MDR P-glycoprotein Cold-stress Collateral sensitivity MAPK
a b s t r a c t Having found previously that leukemic cells with multidrug resistant (MDR) phenotype, but not their sensitive counterparts, exhibit collateral sensitivity to cold stress in a P-gp-dependent manner, our aim was to study the signaling pathways involved in this phenomenon in sensitive (L1210) and resistant cells (L1210R and CBMC6). It was observed that the acquisition of MDR phenotype by leukemic cells or their transfection with the extrussion pump, P-gp, modifies the activation profile and regulation of Mitogen-Activated Protein Kinases (MAPK) in cells exposed to low temperatures. More specifically, cold stress provoked the activation of c-Jun Nterminal kinase (JNK) in sensitive cells, while attenuated JNK signaling was observed in MDR cells. This effect was also observed, although with less intensity, in P-gp-transfected cells. Using pharmacological inhibitors to determine the role of MAPK in leukemic cell survival in physiological conditions or under cold stress, a dual temperature-dependent role was observed for JNK in MDR cell survival. At 37 °C JNK is necessary for the survival of parental, resistant and P-gp-transfected cells; however, the use of inhibitors of either extracellular signal-regulated protein kinase (ERK) or JNK significantly counteracts cold-induced death of resistant and P-gp-transfected cells, supporting a role for ERK and JNK in cold-stress induced cell death. Finally, a connectivity model concerning MAPK is proposed, summarizing how cold stress and MDR-1 might trigger apoptosis in resistant cell lines. These findings on MDR cells may assist in the design of specific therapeutic strategies to complement current chemotherapy. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The acquisition of MDR phenotype by tumor cells is a multifactorial phenomenon that includes the overexpression of drug extrusion pumps such as P-glycoprotein (MDR-1, ABCB-1), increased activity of DNA repair mechanisms and alterations of the signaling pathways involved in apoptosis control. Cross-regulation among the different mechanisms involved and the increased resistance to die is the final result of such a complex event. Whether or not a given cell dies through apoptosis
☆ Grants: This work was supported by the following Grants: 03112/PI/05 from Fundación Séneca-CARM, PI060006 from the Instituto de Salud Carlos III, BIO-201454411-C2-1-R from Ministry of Science and Innovation, and partially by Seneca Foundation CARM 19236/PI/14 and FEDER funds. ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, Murcia Biohealth Research Institute-University of Murcia (IMIB-UMU), Regional Campus of International Excellence “Campus Mare Nostrum”, P.O. Box 4021, Murcia, Spain. E-mail address: [email protected] (E. Martín-Orozco). 1 Equal contributors.
http://dx.doi.org/10.1016/j.cellsig.2016.12.003 0898-6568/© 2016 Elsevier Inc. All rights reserved.
depends on a balance among several factors. Modulation of an individual component or event involved in apoptotic cell death through targeted treatments may produce other alterations in signaling molecules that could shift the regulation of apoptosis. As a result, MDR cells may show paradoxical hypersensitivity to certain stimuli such as therapeutic molecules which induce survival/death transduction pathways other than those modified by the antineoplastic drug. This phenomenon is known as collateral sensitivity (CS) and has been described as the Achilles heel of MDR cells [1,2,3]. Numerous stimuli may act as potential apoptotic stressors in both susceptible and resistant cancer cells. For example, we have shown that the acquisition of MDR phenotype by murine and human leukemic cells exhibit collateral sensitivity to cold stress and we have also described the involvement of P-gp in this cell-death process in our murine model [4,5]. Early events of apoptotic signaling affect, among others, members of the mitogen-activated protein kinase (MAPK) family. The c-Jun NH2terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2 are important members of three different MAPK pathways that can be activated by growth factors, DNA damage, cytokines,
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2. Materials and methods 2.1. Tumor cells The L1210 leukemic cell line from DBA/2 mice was used as a tumor model. A DNM-resistant (L1210R) subline (~ 160-fold resistant to DNM) that overexpresses P-gp was selected from parental L1210 cells [10], and maintained in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 10 U/ml penicillin G and 10 μg/ml streptomycin sulphate (Gibco). The P-gp-transfected cell line CBMC-6 was obtained by transfecting L1210 cells with the plasmid pcDNA 3-mpgp containing the mouse mdr1a P-gp cDNA under the control of the CMV promoter [10]. 2.2. Cell culture, cellular extracts and MAPK inhibitors. To prepare cellular extracts, cells were plated at 3 × 105 cells/ml and incubated at 4 °C or at 37 °C for 0 to 48 h. The total cells were collected, washed with phosphate buffer saline (PBS) and resuspended in Cell Signaling lysis buffer (Cell Signaling Technologies, Beverly, MA) following the manufacturer's instructions. For experiments involving signaling inhibitors, cells were pre-treated for 1 h at 37 °C with 50 μM of the following inhibitors: SB203580 (p38 MAPK Inhibitor), PD098059 (MEK1/ERK Inhibitor), and SP600125 (JNK Inhibitor II). All inhibitors were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Fig. 1. Cell survival in L1210, L1210R and CBMC-6 cells incubated at 37 °C or at 2–4 °C. Cells (3 × 105 cells/ml) were incubated at 37 °C or at 2–4 °C for up to 20 h. Cell survival/death was determined by cell staining with propidium iodide and Annexin-V-FITC and analyzed by flow cytometry. (A) First row histograms show results after incubating cells for 20 h at 37 °C. Second row histograms show results after maintaining cells for 20 h at 4 °C. Figure shows a representative dot-plot for each condition out of the three performed. (B) Bar graphs showing results for cell survival of the three cell lines incubated at 37 °C (left hand graph) or at 4 °C (right hand graph). Data show means ± SEM from at least three independent experiments. Asterisks represent statistical significance with respect to control cells (Cells incubated at 37 °C) (*p b 0.05, **p b 0.01, ***p b 0.001, t-test).
2.3. Measurement of cell-death Leukemic cells were cultured at 3 × 105/ml/well on 24-well plates and maintained at 37 °C or at 4 °C for 20–24 h. Then, cell death was evaluated by adding propidium iodide (BD Pharmingen, Cary, NC, USA) or a mix of propidium iodide and Annexin V-FITC (Biotool, Houston, TX, USA) according to the manufacturer's instructions. The analysis was performed in a Flow cytometer (Becton Dickinson) argon laser of 15 mW at 488 nm. Ten thousand events were collected and analyzed using CellQuest software (Beckton Dickinson). 2.4. Antibodies and western blotting assays
oxidant stress, UV light, anticancer drugs, or osmotic shock [6]. All three MAPK pathways play key roles in survival, proliferation, and apoptosis. A large body of evidence has accumulated to show that antitumor agents alter the activity of MAPK subgroups in many cancer cell lines [7]. Importantly, pharmacological or molecular modulation of MAPK signaling has been shown in many cases to influence the apoptotic response to antitumor agents [8], which suggests that MAPK may mediate in the destructive and/or protective responses to these drugs. However, the roles played by MAPK tend to be strongly context-dependent, influenced by the cell type, drug concentration and the duration of exposure, and on the type of assay used to monitor apoptosis or cell survival [7,9]. In addressing this question, report that the acquisition of MDR phenotype by leukemic cells attenuates the JNK activation profile under cold-stress. Furthermore, MAPK seem to be key elements of leukemic cell apoptosis/survival processes and exert opposite roles at physiological temperatures and under cold-stress conditions.
Cell extract proteins (10–15 μg/lane) were subjected to polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking (2% BSA-TBST), the membranes were incubated with each primary antibody and then with a horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized using the ECL detection system (Amersham Biosciences, Buckinghamshire, UK). The following antibodies were used: anti-ERK1/2 pAb [11], anti-phosphoThr202/Tyr204 ERK1/2 pAb [12], anti-SAP/JNK pAb [13], anti-phospho-Thr183/Tyr185 SAP/JNK pAb [14], anti-p38 pAb [15], anti-phospho Thr180/Tyr182 p38 pAb [16], anti-phospho Thr334 MAPKAPK-2 mAb (27B7) [17] (Cell signaling Technologies), anti-c-Jun pAb, anti-phospho Ser63 cJun mAb (KM-1) [18] anti-GAPDH pAb [19] (Santa Cruz Biotechnologies, Dallas, TX, USA). Quantitation was carried out using Scion Image or Image J Software, normalizing to the respective loading control.
Fig. 2. Differential phosphorylation of MAPK on L1210R and CBMC-6 compared with L1210 cells in response to cold stress. Cells (3 × 105 cells/ml) were incubated at 2–4 °C for 0.5 to 8 h. Cells were harvested and whole cell lysates were obtained (10 μg of each) and subjected to western blotting using the indicated phospho-specific and total protein antibodies to detect activation and total relative expression of kinases. (A) Graph showing results on JNK phosphorylation. (B) Representative western blotting showing JNK phosphorylation under cold stress. (C) Graph showing results on ERK phosphorylation. (D) Representative western blotting showing ERK phosphorylation under cold stress. (E) Graph showing results on p38 MAPK phosphorylation (F) Representative western blotting showing p38 MAPK phosphorylation under cold stress. This figure shows a representative western blot for each kinase out of the four performed with similar results. Control cells (cultured at 37 °C) are indicated by an arrow. Data show means ±SEM from at least four independent assays. Asterisks (*) represent statistical significance for each kinase with respect to control cells (37 °C), a represents statistical significance of L1210R cells relative to CBMC-6 cells, b represents statistical significance of L1210 cells relative to L1210R cells, c represents statistical significance of L1210 cells relative to CBMC-6 cells; (*/a/b/c p b 0.05, **/aa/bb/cc p b 0.01, ***/aaa/bbb/ccc p b 0.001, t-test).
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2.5. Statistical analysis Data are represented as the mean ± SEM. Data were analyzed with Student's t-test by using SPSS 19.0 software package (IBM, Armonk, NY, USA). Statistical significance was defined as p b 0.05 (*), p b 0.01 (**) or p b 0.001 (***). 3. Results 3.1. Cold stress induces collateral sensitivity in MDR leukemic cells (L1210R and CBMC-6) compared with parental cells (L1210). As a model of MDR tumor cells, we used the mouse leukemic cell line L1210, its daunomycin-resistant (DNM-resistant) subline L1210R, which is characterized by expression of the ATP-Binding-Cassette (ABC) transporter P-gp (MDR-1), and the P-gp-transfected cell line CBMC-6, obtained as described in Materials and methods (Section 2.1). First, as previously reported [4,5], the effect of extreme hypothermia was studied in the three cell lines (Fig. 1A). To quantify cell death, the three cell lines were maintained for 20–24 h at 37 °C or at 4 °C, and the extent of cell death/survival was evaluated by counting the number of viable cells by flow cytometry analysis after staining with propidium iodide and Annexin V-FITC. Fig. 1 shows that only 11% of the parental cells died after 20 h at 4 °C, while survival of the resistant (L1210R) and P-gp-transfected (CBMC-6) cell population decreased drastically (up to 70% cell death). These data confirm previously published results [4,5] demonstrating that cold stress induces collateral sensitivity in MDR leukemic cells. 3.2. JNK phosphorylation is attenuated in cold-incubated MDR leukemic cells compared with their sensitive counterparts. As shown in Fig. 2, the signaling profile of MAPK differs between resistant and sensitive cells cultured at 37 °C or under cold conditions. As regards the activation of JNK (pJNK) (Fig. 2A and B), basal phosphorylation levels detected in L1210R cells cultured at 37 °C were low compared with the levels of these MAPK in L1210 and CBMC-6 cells. Furthermore, JNK activation in L1210 and CBMC-6 cells increased after 30 min of cold exposure, reaching a maximum after 2 h under hypothermia (4.5-fold and 3-fold higher than the control in L1210 and CBMC-6 cells, respectively). JNK activation then remained almost constant for several hours (up to 8 h), before starting to decrease slightly after 24 h (data not shown). However, in L1210R cells, there was no increase in JNK phosphorylation (compared with the control at 37 °C) after short cold exposure times, and the phosphorylation levels were much less intense than in sensitive or P-gp-transfected cells. A slight increase in JNK activation was observed after 8 h of cold exposure, which lasted for up to 12 h of cold incubation, but was undetectable after 24 h, as in the control cells (data not shown). Regarding ERK phosphorylation (pERK) no significant differences were observed in CBMC-6 cells compared with L1210 cells (Fig. 2C and D). Nevertheless, there was a decrease, although not statistically significant, in ERK2 activation in L1210R cells compared with the other two cell lines after 0.5 h of cold exposure. This activation returned to previous levels after 4 h of incubation at 4 °C, reaching similar values in all three leukemic cell lines. Phosphorylation levels of p38 MAPK (pp38) were similar in the three cell lines cultured at 37 °C, and remained similar, with no significant differences, after 8 h of cold exposure (Fig. 2E and F).
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3.3. MAP kinases play different roles in the survival of sensitive and MDR leukemic cells under physiological conditions. Initially, we used specific inhibitors to evaluate the individual roles of ERK, p38 MAPK and JNK in cell survival at physiological temperature. The three cell lines were treated with one of the inhibitors or vehicles (DMSO) and, following 20 h incubation at 37 °C, survival/apoptosis was measured as specified in Materials and methods (Section 2.3). The ERK inhibitor by itself did not affect survival in any cell type (Fig. 3A and C). Treatment with p38 MAPK inhibitor appeared to lower survival rates in all three cell lines although the decrease was only statistically significant in the case of L1210 cells. Finally, treatment with the JNK inhibitor alone strongly decreased the viability of the three cell lines cultured for 20 h at 37 °C, the L1210R cell line being most affected by this inhibitor. When a combination of two or three of the MAPK inhibitors was used a statistically significant decrease was observed in the survival of L1210, L1210R and CBMC-6 cell lines (Fig. 3A and C). Thus, we conclude that the use of the JNK inhibitor either alone, or in combination with the ERK and/or with p38 MAPK inhibitors, strongly affected the viability of the leukemic cells used, at 37 °C. The decrease in survival induced by MAPK inhibitors was similar in L1210 and CBMC-6 cells, but more drastic in L1210R cells (Fig. 3A and C). 3.4. Pharmacological inhibition of ERK and JNK phosphorylation partially blocks cold stress-induced collateral sensitivity in MDR and P-gptransfected cells. ERK and/or JNK inhibitors improved the viability of MDR cells maintained at 4 °C. Thus, the ERK inhibitor attenuated cold-induced celldeath in L1210R (20%) and CBMC-6 cells (25–30%), while there was no change in L1210 cell death levels relative to untreated cells. Moreover, the JNK inhibitor induced an increase of 20–30% in the viability of L1210R and CBMC-6 cells incubated under cold stress conditions, while no effect was observed in the viability of cold-treated L1210 cells. The combination of both ERK and JNK inhibitors had a partially additive effect on the MDR cell lines cultured at 4 °C, producing a significant rescue from cold-induced death in L1210R cells (up to 30%) and CBMC-6 cells (up to 40%) compared with negative controls (Fig. 3B and D). Conversely, no differences in cell survival were found when the MDR cell lines cultured at 4 °C were treated with the p38 MAPK inhibitor compared with the same cells treated only with DMSO under the same conditions (negative control) although a 10% decrease in cell survival was observed in L1210 cells treated with p38 MAPK inhibitor (Fig. 3B and D). 3.5. Differences in MAPK cross-talk among resistant and parental cells under cold-stress. When the phosphorylation levels of MAPK were studied in the presence of their specific pharmacological inhibitors (Fig. 4), p38 MAPK and JNK inhibitors were seen to increase ERK phosphorylation in sensitive and resistant cells (Fig. 4A, C and E). No difference was observed in JNK phosphorylation in sensitive cells cultured in the presence of p38 MAPK inhibitor (Fig. 4A), but in the presence of ERK inhibitor, JNK phosphorylation decreased in sensitive cells (Fig. 4A) and increased in resistant sublines compared with control cells (Fig. 4C and E)). No differences were found in p38 phosphorylation levels in the three cell lines treated with the different inhibitors compared with control cells (Fig. 4A, C and E). The above results led us to propose a cross-talk
Fig. 3. Effect of MAPKs inhibitors on L1210, L1210R and CBMC-6 cold-induced cell-death. Leukemic cell lines (3 × 105 cells/ml) were incubated for 1 h at 37 °C in complete culture medium or in the presence of 1% DMSO (Negative control) or the following inhibitors: p38 MAPK inhibitor (SB203580), MEK1/ERK inhibitors (PD098059) or JNK inhibitor II (SP600125). Then, the cells were incubated for an additional 20 h at 37 °C or at 4 °C. Cell death was determined by cell staining with propidium iodide ± Annexin-V-FITC and analyzed by flow cytometry. (A) Dotplot results after incubating cells with or without inhibitors for 20 h at 37 °C. (B) Dot-plot results after maintaining cells with or without inhibitors for 20 h at 2–4 °C. Figure shows a representative dot-plot for each condition out of three that were performed with similar results. (C) Bar graphs representation of relative cell survival after incubating cells with or without inhibitors for 20 h at 37 °C. (D) Bar graphs representation of relative cell survival after maintaining cells with or without inhibitors for 20 h at 2–4 °C. Data show means ±SEM from at least three independent experiments. Asterisks represent statistical significance with respect to control cells (w/o inhibitors) (*p b 0.05, **p b 0.01, ***p b 0.001, t-test).
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Fig. 4. MAPK cross-talk at 4 °C. Cells (3 × 105 cells/ml) were incubated at 2–4 °C for 20–24 h in the presence of 1% DMSO (Negative control) or the following inhibitors: p38 MAPK inhibitor (SB203580), MEK1/ERK inhibitors (PD098059) or JNK inhibitor II (SP600125). Cells were harvested and whole cell lysates were obtained (10 μg of each) and subjected to western blotting using the indicated phospho-specific and reference protein antibodies (total protein or GAPDH) to detect activation and total relative expression of kinases. (A, C, E) Graphs showing western blotting results on JNK, ERK, p38 MAPK, MAPKAPK-2 and c-Jun phosphorylation in L1210, L1210R and CBMC-6 cells, respectively. The figure shows a representative western blotting for each kinase out of the three performed with similar results. (B, D, F) Qualitative interaction graph with three nodes that represent the three MAPK in their activated (phosphorylated) form. Positive (arrows) and negative (blunt-ended lines) edges represent activating or inhibiting influences between proteins.
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model of MAPK for sensitive and resistant cells (Fig. 4B, D and F). In parental cells (L1210) pERK cross-activate pJNK signaling (Fig. 4A and B), while in resistant cells (L1210R and CBMC-6) these kinases negatively regulated each other's activity (Fig. 4C, D, E and F). Since the p38 MAPK and the JNK inhibitors specifically inhibit p38 MAPK and JNK activity by competitively binding to the ATP binding pockets, thus preventing the phosphorylation of proteins downstream, their use does not result in decreased phosphorylation levels in either p38 MAPK or JNK [18]. Thus, to assess whether the doses of the ERK1/ 2, JNK and p38 MAPK inhibitors assayed were sufficient to exert their specific inhibitory effects, additional control experiments were designed by analyzing the phosphorylation levels of MAPKAPK-2 (p38 substrate) and c-Jun (ERK and JNK substrate) (Fig. 4A, C and E). 4. Discussion Using a model characterized by the hypersensitivity of resistant and P-gp-transfected leukemic cells to cold stress [4,5], key elements of the MAPK activation profile in sensitive versus resistant cells incubated under cold stress were studied, observing that cold treatment activates SAPK/JNK very efficiently in parental cells but to a much reduced extent
in resistant cells. The above results suggest that the SAPK/JNK kinase activation profile is related with the acquisition of DNM resistance and, it might be speculated that its attenuation is involved in protection against DNM in resistant cells. This result agrees with those obtained by Brozovic et al. [20], who observed that cells that acquired cisplatin (cDDP) resistance showed a reduced activation of SAPK/JNK and p38 MAPK when incubated in the presence of cDDP compared with the parental line. Moreover, there are several other lines of evidence indirectly suggesting that enhancement of the JNK pathway down-regulates P-gp and reverses P-gp-mediated MDR in cancer cells [21,22,23]. In contrast to these findings and ours, other researchers [24] showed a positive correlation between JNK activity and MDR-1 gene expression, reflecting the complex nature of MDR-1/P-gp regulation by JNK and the involvement of other regulators. All three cell lines studied manifested a dependence on JNK phosphorylation for survival at 37 °C. By contrast, at 4 °C resistant cells are partially rescued from cell death by the inhibition of JNK and/or ERK phosphorylation. These results suggest that exposure to low temperatures switches the anti-apoptotic role of JNK observed under physiological conditions to a pro-apoptotic one, since the death of resistant cells under cold stress is both JNK- and ERK-dependent. In this sense, it has
Fig. 5. Proposed mechanism of MAPK-mediated cold-stress-induced apoptosis. The qualitative interaction graph model is based on our experimental data and supported by the literature. (A) Interaction graph for L1210 cells. (B) Interaction graph for L1210R cells (C) Interaction graph for CBMC-6 cells. Acquisition of MDR phenotype induces an increase in MDR-1 expression and functionality, which, in turn, attenuates JNK expression and phosphorylation. Cold stress increases JNK phosphorylation in sensitive cells and, with less intensity, in resistant cells. There is a cross-talk among kinases, since p38 MAPK and JNK negatively regulates ERK activity. Additionally, ERK negatively regulates JNK activity in resistant cells (DNM-treated and P-gp-transfected cells) but has the opposite effect in sensitive cells. Furthermore, JNK phosphorylation inhibits cold stress-induced apoptosis in sensitive cells but plays a part in cold stress-induced apoptosis in resistant cells. Finally, we and others propose a role for MDR-1 in cell survival. Arrows represent activating (positive) interactions, blunt-ended lines represent inhibitory (negative) interactions and question marks represent an unknown link. The thickness of the arrows represents the strength of these effects. The letter size of the molecules represents the expression levels in each cell type. MAPK inhibitors are represented by their initial capital letters.
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been described that the growth factor-induced transient activation of JNK promotes cell survival and proliferation, whereas stress-induced sustained JNK activity promotes growth arrest and cell death [7]. Additionally, Xiao et al. [25] have shown that JNK can be activated by thermotherapy, ultimately leading to cell apoptosis. Furthermore, Muscarella et al. [26] have shown that two major types of cell stress responses, global proteotoxic damage induced by arsenic or heat stress and microtubule damage induced by vincristine, result in JNK pathway activation and the induction of apoptosis in B-cells. However, JNK-pathway activation was required for apoptosis only in the case of vincristineinduced microtubule inhibition. Cold-induced death of L1210R and CBMC-6 cells was significantly avoided in the presence of ERK inhibitor. In fact, the involvement of the MEK1/2–ERK1/2-pathway in apoptosis induced by cytotoxic compounds has been demonstrated previously [27]. In this regard, it has been described that the duration and intensity of MAPK activation is a determinant factor in cell fate, namely, growth arrest versus apoptosis [28,29]. JNK and ERK cooperate as cell-death controllers at low temperatures in resistant cells (Fig. 3). This partially additive effect suggests that both molecules activate common control pathways to regulate apoptosis in MDR cells. In fact, both kinases have been described as being responsible for modulating the expression and/or phosphorylation of common substrates, which ultimately promotes the fate of the cell through an apoptotic or survival pathway [30,31,32]. As regards MAPK cross-talk, our results suggest that p38 and JNK negatively regulate ERK phosphorylation (Fig. 4). In the case of JNK, the results differed between sensitive and resistant cells. Thus, the use of the ERK inhibitor in parental cells induced the inhibition of JNK phosphorylation while, in resistant cells, which exhibit low JNK activation at 4 °C, there was a slight increase in JNK phosphorylation compared with control cells. In this sense, the role for the JNK pathway in the negative regulation of the ERK pathway was greatly strengthened by RNAi in Drosophila S2 cells [33]. This study indicated that other signaling pathways, such as JNK and Akt/Tor, negatively affect ERK signaling. Moreover, Drosophila AP-1 transcription factors were identified as negative regulators of ERK pathway activity [33], which agrees with the results of others [34]. Finally, SB203580 has also been shown to enhance the phosphorylation of ERK in HL-60 and ML-1 cells [35]. Complex networks have been studied in the field of apoptosis, and network motifs have been defined [36,37]. In Figs. 4 and 5 a connectivity graph based on qualitative units of three nodes of interaction concerning p38, ERK and JNK is depicted, indicating how cold stress and MDR-1 might trigger apoptosis in resistant cell lines. We present a dynamic model featuring a JNK-positive feedback loop that generates a proliferative apoptotic switch and the factors controlling it, with a particular focus on feedback loops and cross-talk (Fig. 5). First, the acquisition of MDR phenotype induces an increase in MDR-1 expression; second, the expression of MDR-1 attenuates JNK phosphorylation; third, cold stress induces JNK phosphorylation; fourth, p38 MAPK inhibits ERK activity and fifth, ERK inhibits JNK activity in resistant cells but has the opposite effect in sensitive cells. Finally, JNK phosphorylation inhibits cold stress-induced apoptosis in sensitive cells but has a pro-apoptotic role in cold stress-induced apoptosis in resistant cells.
5. Conclusions This work demonstrates that MDR phenotype and P-gp expression modify the activation profile and regulation of Mitogen-Activated Protein Kinases (MAPK) in cells exposed to low temperatures. Furthermore, JNK and ERK cooperate as cell-death controllers at low temperatures in resistant cells. Finally, we propose a connectivity model based on qualitative units, integrating the cross-talk of the different molecules that participate in cold stress-induced apoptosis in our leukemic model, indicating how cold stress and MDR-1 might trigger apoptosis in resistant cell lines.
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Conflict of interest statement None declared. Contributors D. Cerezo and A.J. Ruiz-Alcaraz performed the experiments, analyzed the data and critically read the manuscript. M. Lencina-Guardiola and I. Martínez-López performed the experiments. P. Garcia-Peñarrubia and M. Canovas sponsored the study, proposed experiments and critically read the manuscript. E. Martin-Orozco designed and performed the experiments, analyzed data and wrote the paper. Aknowledgments and role of the funding source We thank Dr. Manuel Sánchez Angulo for his critical reading of the manuscript and helpful suggestions. This work was supported by the following Grants: 03112/PI/05 from Fundación Séneca-CARM, PI060006 from the Instituto de Salud Carlos III, BIO-2014-54411-C2-1R from Ministry of Science and Innovation, and partially by Seneca Foundation CARM 19236/PI/14 and FEDER funds. References [1] H. Zahreddine, K.L.B. Borden, Mechanisms and insights into drug resistance in cancer, Front. Pharmacol. 4 (28) (2013) 1–8, http://dx.doi.org/10.3389/fphar.2013.00028. [2] R.M. Mohammad, I. Muqbil, L. Lowe, C. Yedjou, H.Y. Hsu, L.T. Lin, et al., Broad targeting of resistance to apoptosis in cancer, Semin. Cancer Biol. 35 (2015) S78–103, http://dx.doi.org/10.1016/j.semcancer.2015.03.001 (Suppl). [3] G. Szakacs, M.D. Hall, M.M. Gottesman, A. Boumendjel, R. Kachadourian, B.J. Day, H. Baubichon-Cortay, A. Di Pietro, Targeting the Achilles heel of multidrug-resistant cancer by exploiting the fitness cost of resistance, Chem. Rev. 114 (11) (2014) 5753–5774, http://dx.doi.org/10.1021/cr4006236. [4] D. Cerezo, M. Lencina, A.J. Ruiz-Alcaraz, J.A. Ferragut, M. Saceda, M. Sánchez, M. Cánovas, P. García-Peñarrubia, E. Martin-Orozco, Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress, J. Cell. Biochem. 113 (4) (2012) 1416–1425, http://dx. doi.org/10.1002/jcb.24016. [5] D. Cerezo, M. Cánovas, P. García-Peñarrubia, E. Martín-Orozco, Collateral sensitivity to cold stress and differential BCL-2 family expression in new daunomycin-resistant lymphoblastoid cell lines, Exp. Cell Res. 331 (1) (2015) 11–20, http://dx.doi.org/10. 1016/j.yexcr.2014.11.017. [6] M. Qi, E.A. Elion, MAP kinase pathways, J. Cell Sci. 118 (2005) 3569–3572, http://dx. doi.org/10.1242/jcs.02470. [7] D. Fey, D.R. Croucher, W. Kolch, B.N. Kholodenko, Crosstalk and signaling switches in mitogen-activated protein kinases cascade, Front. Physiol. 3 (355) (2012) 1–21, http://dx.doi.org/10.3389/fphys.2012.00355. [8] Y. Ohsaka, S. Ohgiya, T. Hoshino, K. Ishizaki, Phosphorylation of c-Jun N-terminal kinase in human hepatoblastoma cells is transiently increased by cold exposure and further enhanced by subsequent warm incubation of the cells, Cell. Physiol. Biochem. 12 (2002) 111–118 (http://dx.doi.org/63787). [9] M. Krishna, H. Narang, The complexity of mitogen-activates protein kinases (MAPKs) made simple, Cell. Mol. Life Sci. 65 (2008) 3525–3544, http://dx.doi.org/ 10.1007/s00018-008-8170-7. [10] M.D. Castro-Galache, J.A. Ferragut, V.M. Barbera, E. Martin-Orozco, J.M. GonzalezRos, P. Garcia-Morales, et al., Susceptibility of multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors, Int. J. Cancer 104 (5) (2003) 579–586, http://dx.doi.org/10.1002/ijc.10998. [11] A.M. Domina, J.H. Smith, R.W. Craig, Myeloid cell leukemia 1 is phosphorylated through two distinct pathways, one associated with extracellular signal-regulated kinase activation and the other with G2/M accumulation or protein phosphatase 1/2A inhibition, J. Biol. Chem. 275 (28) (2000) 21688–21694, http://dx.doi.org/10. 1074/jbc.M000915200. [12] H. Xu, M. Goldfarb, Multiple effector domains within SNT1 coordinate ERK activation and neuronal differentiation of PC12 cells, J. Biol. Chem. 276 (16) (2001) 13049–13056, http://dx.doi.org/10.1074/jbc.M009925200. [13] E.G. Lee, D.L. Boone, S. Chai, S.L. Libby, M. Chien, J.P. Lodolce, et al., Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice, Science 289 (5488) (2000) 2350–2354. [14] A. Shukla, C.R. Timblin, A.K. Hubbard, J. Bravman, B.T. Mossman, Silica-induced activation of c-Jun-NH2-terminal amino kinases, protracted expression of the activator protein-1 proto-oncogene, fra-1, and S-phase alterations are mediated via oxidative stress, Cancer Res. 6 (5) (2001) 1791–1795. [15] A. Rossi, P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin, et al., Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase, Nature 403 (6765) (2000) 103–108, http://dx.doi.org/10.1038/47520.
170
D. Cerezo et al. / Cellular Signalling 30 (2017) 162–170
[16] E. Patrucco, A. Notte, L. Barberis, G. Selvetella, A. Maffei, M. Brancaccio, et al., PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and –independent effects, Cell 118 (3) (2004) 375–387, http://dx.doi.org/10.1016/j.cell.2004.07.017. [17] M. Tesker, S.E. Selamat, J. Beenstock, R. Hayouka, O. Livnah, D. Engelberg, Tighter αC-helix-αL16-helix interactions seem to make p38α less prone to activation by autophosphorylation than Hog1, Biosci. Rep. 36 (2) (2016)http://dx.doi.org/10. 1042/BSR20160020 (pii: e00324). [18] C. Xu, X. Wang, Y. Zhu, X. Dong, C. Liu, H. Zhang, et al., Rapamycin ameliorates cadmium-induced activation of MAPK pathway and neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A, Neuropharmacology 105 (2016) 270–284, http://dx.doi.org/10.1016/j.neuropharm.2016.01.030. [19] X. Bai, Y. Zhang, H. Jiang, P. Yang, H. Li, Y. Zhang, et al., Effects of maslinic acid on the proliferation and apoptosis of A549 lung cancer cells, Mol. Med. Rep. 13 (1) (2016) 117–122, http://dx.doi.org/10.3892/mmr.2015.4552. [20] A. Brozovic, G. Fritz, M. Christmann, J. Zisowsky, U. Jaehde, M. Osmak, B. Kaina, Longterm activation of SAPK/JNK, p38 kinase and Fas-l expression by cisplatin is attenuated in human carcinoma cells that acquired drug resistance, Int. J. Cancer 112 (2004) 974–985, http://dx.doi.org/10.1002/ijc.20522. [21] Z.H. Miao, J. Ding, Transcription factor c-Jun activation represses mdr-1 Gene expression, Cancer Res. 63 (2003) 4527–4532. [22] A. Brozovic, M. Osmak, Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance, Cancer Lett. 251 (2007) 1–16, http://dx.doi. org/10.1016/j.canlet.2006.10.007. [23] D.E. Muscarella, S.E. Bloom, The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses, Toxicol. Appl. Pharmacol. 228 (1) (2008) 93–104, http://dx.doi.org/10.1016/j.taap.2007.11.032. [24] J. Zhou, M. Liu, R. Aneja, R. Chandra, H. Lage, H.C. Joshi, Reversal of P-glycoprotein– mediated multidrug resistance in cancer cells by the c-Jun NH2-terminal kinase, Cancer Res. 66 (1) (2006) 445–452, http://dx.doi.org/10.1158/0008-5472.CAN-051779. [25] H. Sui, S. Zhou, Y. Wang, X. Liu, L. Zhou, P. Yin, et al., COX-2 contributes to P-glycoprotein-mediated multidrug resistance via phosphorylation of c-jun at Ser63/73 in colorectal cancer, Carcinogenesis 32 (5) (2011) 667–675, http://dx.doi.org/10. 1093/carcin/bgr016. [26] F. Xiao, B. Liu, Q.X. Zhu, C-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells, World J. Gastroenterol. 18 (48) (2012) 7348–7356, http://dx.doi.org/10.3748/wjg.v18.i48.7348.
[27] A. Calcabrini, J.M. Garcia-Martinez, L. Gonzalez, M.J. Tendero, M.T. Ortuño, P. Crateri, et al., Inhibition of proliferation and induction of apoptosis in human breast cancer cells by lauryl gallate, Carcinogenesis 27 (2006) 1699–1712, http://dx.doi.org/10. 1093/carcin/bgl044. [28] W. Yu, Q.Y. Liao, F.M. Hantash, B.G. Sanders, K. Kline, Activation of extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells, Cancer Res. 2061 (2001) 6569–6576. [29] M. Ebisuya, K. Kondoh, E. Nishida, The duration, magnitude and compartmentalization of ERK MAP kinase activity: mechanisms for providing signaling specificity, J. Cell Sci. 118 (2005) 2997–3002, http://dx.doi.org/10.1242/jcs.02505. [30] M. Fan, M. Goodwin, T. Vu, C. Brantley-Finley, W.A. Gaarde, T.C. Chambers, Vinblastine-induced phosphorylation of Bcl-2 and Bcl-xL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade, J. Biol. Chem. 275 (39) (2000) 29980–29985, http://dx.doi.org/10.1074/jbc.M003776200. [31] R.K. Barr, M.A. Bogoyevitch, The c-Jun N-terminal protein kinase family of mitogenactivated protein kinases (JNK MAPKs), Int. J. Biochem. Cell Biol. 233 (11) (2001) 1047–1063. [32] R. Dai, W. Frejtag, B. He, Y. Zhang, N.F. Mivechi, c-Jun NH2-terminal kinase targeting and phosphorylation of heat shock factor-1 suppress its transcriptional activity, J. Biol. Chem. 275 (24) (2000) 18210–18218, http://dx.doi.org/10.1074/jbc. M000958200. [33] A. Friedman, N. Perrimon, A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signaling, Nature 444 (2006) 230–234, http://dx.doi.org/10. 1038/nature05280. [34] M.R. Junttila, S.P. Li, J. Westermarck, Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival, FASEB J. 22 (2008) 954–965, http://dx.doi.org/10.1096/fj.06-7859rev. [35] M. Hirosawa, M. Nakahara, R. Otosaka, A. Imoto, T. Okazaki, S. Takahashi, The p38 pathway inhibitor SB202190 activates MEK/MAPK to stimulate the growth of leukemia cells, Leuk. Res. 33 (2009) 693–699, http://dx.doi.org/10.1016/j.leukres.2008. 09.028. [36] R. Milo, S. Shen-Orr, S. Itzkovitz, N. Kashtan, D. Chklovskii, U. Alon, Network motifs: simple building blocks of complex networks, Science 298 (2002) 824–827, http:// dx.doi.org/10.1126/science.298.5594.824. [37] S. Shen-Orr, R. Milo, S. Mangan, U. Alon, Network motifs in the transcriptional regulation network of Escherichia coli, Nat. Genet. 31 (2002) 64–68, http://dx.doi.org/ 10.1038/ng881.
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Attenuated JNK signaling in multidrug-resistant leukemic cells. Dual role of MAPK in cell survival☆ David Cerezo 1, Antonio J. Ruiz-Alcaraz 1, Miriam Lencina-Guardiola, Manuel Cánovas, Pilar García-Peñarrubia, Inmaculada Martínez-López, Elena Martín-Orozco ⁎ Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, Murcia Biohealth Research Institute-University of Murcia (IMIB-UMU), Regional Campus of International Excellence “Campus Mare Nostrum”, 30100 Murcia, Spain
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Article history: Received 1 August 2016 Received in revised form 28 November 2016 Accepted 5 December 2016 Available online 07 December 2016 Keywords: Leukemic cells MDR P-glycoprotein Cold-stress Collateral sensitivity MAPK
a b s t r a c t Having found previously that leukemic cells with multidrug resistant (MDR) phenotype, but not their sensitive counterparts, exhibit collateral sensitivity to cold stress in a P-gp-dependent manner, our aim was to study the signaling pathways involved in this phenomenon in sensitive (L1210) and resistant cells (L1210R and CBMC6). It was observed that the acquisition of MDR phenotype by leukemic cells or their transfection with the extrussion pump, P-gp, modifies the activation profile and regulation of Mitogen-Activated Protein Kinases (MAPK) in cells exposed to low temperatures. More specifically, cold stress provoked the activation of c-Jun Nterminal kinase (JNK) in sensitive cells, while attenuated JNK signaling was observed in MDR cells. This effect was also observed, although with less intensity, in P-gp-transfected cells. Using pharmacological inhibitors to determine the role of MAPK in leukemic cell survival in physiological conditions or under cold stress, a dual temperature-dependent role was observed for JNK in MDR cell survival. At 37 °C JNK is necessary for the survival of parental, resistant and P-gp-transfected cells; however, the use of inhibitors of either extracellular signal-regulated protein kinase (ERK) or JNK significantly counteracts cold-induced death of resistant and P-gp-transfected cells, supporting a role for ERK and JNK in cold-stress induced cell death. Finally, a connectivity model concerning MAPK is proposed, summarizing how cold stress and MDR-1 might trigger apoptosis in resistant cell lines. These findings on MDR cells may assist in the design of specific therapeutic strategies to complement current chemotherapy. © 2016 Elsevier Inc. All rights reserved.
1. Introduction The acquisition of MDR phenotype by tumor cells is a multifactorial phenomenon that includes the overexpression of drug extrusion pumps such as P-glycoprotein (MDR-1, ABCB-1), increased activity of DNA repair mechanisms and alterations of the signaling pathways involved in apoptosis control. Cross-regulation among the different mechanisms involved and the increased resistance to die is the final result of such a complex event. Whether or not a given cell dies through apoptosis
☆ Grants: This work was supported by the following Grants: 03112/PI/05 from Fundación Séneca-CARM, PI060006 from the Instituto de Salud Carlos III, BIO-201454411-C2-1-R from Ministry of Science and Innovation, and partially by Seneca Foundation CARM 19236/PI/14 and FEDER funds. ⁎ Corresponding author at: Department of Biochemistry and Molecular Biology B and Immunology, School of Medicine, Murcia Biohealth Research Institute-University of Murcia (IMIB-UMU), Regional Campus of International Excellence “Campus Mare Nostrum”, P.O. Box 4021, Murcia, Spain. E-mail address: [email protected] (E. Martín-Orozco). 1 Equal contributors.
http://dx.doi.org/10.1016/j.cellsig.2016.12.003 0898-6568/© 2016 Elsevier Inc. All rights reserved.
depends on a balance among several factors. Modulation of an individual component or event involved in apoptotic cell death through targeted treatments may produce other alterations in signaling molecules that could shift the regulation of apoptosis. As a result, MDR cells may show paradoxical hypersensitivity to certain stimuli such as therapeutic molecules which induce survival/death transduction pathways other than those modified by the antineoplastic drug. This phenomenon is known as collateral sensitivity (CS) and has been described as the Achilles heel of MDR cells [1,2,3]. Numerous stimuli may act as potential apoptotic stressors in both susceptible and resistant cancer cells. For example, we have shown that the acquisition of MDR phenotype by murine and human leukemic cells exhibit collateral sensitivity to cold stress and we have also described the involvement of P-gp in this cell-death process in our murine model [4,5]. Early events of apoptotic signaling affect, among others, members of the mitogen-activated protein kinase (MAPK) family. The c-Jun NH2terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK) 1/2 are important members of three different MAPK pathways that can be activated by growth factors, DNA damage, cytokines,
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2. Materials and methods 2.1. Tumor cells The L1210 leukemic cell line from DBA/2 mice was used as a tumor model. A DNM-resistant (L1210R) subline (~ 160-fold resistant to DNM) that overexpresses P-gp was selected from parental L1210 cells [10], and maintained in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% FBS (Gibco), 2 mM L-glutamine, 10 U/ml penicillin G and 10 μg/ml streptomycin sulphate (Gibco). The P-gp-transfected cell line CBMC-6 was obtained by transfecting L1210 cells with the plasmid pcDNA 3-mpgp containing the mouse mdr1a P-gp cDNA under the control of the CMV promoter [10]. 2.2. Cell culture, cellular extracts and MAPK inhibitors. To prepare cellular extracts, cells were plated at 3 × 105 cells/ml and incubated at 4 °C or at 37 °C for 0 to 48 h. The total cells were collected, washed with phosphate buffer saline (PBS) and resuspended in Cell Signaling lysis buffer (Cell Signaling Technologies, Beverly, MA) following the manufacturer's instructions. For experiments involving signaling inhibitors, cells were pre-treated for 1 h at 37 °C with 50 μM of the following inhibitors: SB203580 (p38 MAPK Inhibitor), PD098059 (MEK1/ERK Inhibitor), and SP600125 (JNK Inhibitor II). All inhibitors were purchased from Sigma (Sigma-Aldrich, St. Louis, MO). Fig. 1. Cell survival in L1210, L1210R and CBMC-6 cells incubated at 37 °C or at 2–4 °C. Cells (3 × 105 cells/ml) were incubated at 37 °C or at 2–4 °C for up to 20 h. Cell survival/death was determined by cell staining with propidium iodide and Annexin-V-FITC and analyzed by flow cytometry. (A) First row histograms show results after incubating cells for 20 h at 37 °C. Second row histograms show results after maintaining cells for 20 h at 4 °C. Figure shows a representative dot-plot for each condition out of the three performed. (B) Bar graphs showing results for cell survival of the three cell lines incubated at 37 °C (left hand graph) or at 4 °C (right hand graph). Data show means ± SEM from at least three independent experiments. Asterisks represent statistical significance with respect to control cells (Cells incubated at 37 °C) (*p b 0.05, **p b 0.01, ***p b 0.001, t-test).
2.3. Measurement of cell-death Leukemic cells were cultured at 3 × 105/ml/well on 24-well plates and maintained at 37 °C or at 4 °C for 20–24 h. Then, cell death was evaluated by adding propidium iodide (BD Pharmingen, Cary, NC, USA) or a mix of propidium iodide and Annexin V-FITC (Biotool, Houston, TX, USA) according to the manufacturer's instructions. The analysis was performed in a Flow cytometer (Becton Dickinson) argon laser of 15 mW at 488 nm. Ten thousand events were collected and analyzed using CellQuest software (Beckton Dickinson). 2.4. Antibodies and western blotting assays
oxidant stress, UV light, anticancer drugs, or osmotic shock [6]. All three MAPK pathways play key roles in survival, proliferation, and apoptosis. A large body of evidence has accumulated to show that antitumor agents alter the activity of MAPK subgroups in many cancer cell lines [7]. Importantly, pharmacological or molecular modulation of MAPK signaling has been shown in many cases to influence the apoptotic response to antitumor agents [8], which suggests that MAPK may mediate in the destructive and/or protective responses to these drugs. However, the roles played by MAPK tend to be strongly context-dependent, influenced by the cell type, drug concentration and the duration of exposure, and on the type of assay used to monitor apoptosis or cell survival [7,9]. In addressing this question, report that the acquisition of MDR phenotype by leukemic cells attenuates the JNK activation profile under cold-stress. Furthermore, MAPK seem to be key elements of leukemic cell apoptosis/survival processes and exert opposite roles at physiological temperatures and under cold-stress conditions.
Cell extract proteins (10–15 μg/lane) were subjected to polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking (2% BSA-TBST), the membranes were incubated with each primary antibody and then with a horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized using the ECL detection system (Amersham Biosciences, Buckinghamshire, UK). The following antibodies were used: anti-ERK1/2 pAb [11], anti-phosphoThr202/Tyr204 ERK1/2 pAb [12], anti-SAP/JNK pAb [13], anti-phospho-Thr183/Tyr185 SAP/JNK pAb [14], anti-p38 pAb [15], anti-phospho Thr180/Tyr182 p38 pAb [16], anti-phospho Thr334 MAPKAPK-2 mAb (27B7) [17] (Cell signaling Technologies), anti-c-Jun pAb, anti-phospho Ser63 cJun mAb (KM-1) [18] anti-GAPDH pAb [19] (Santa Cruz Biotechnologies, Dallas, TX, USA). Quantitation was carried out using Scion Image or Image J Software, normalizing to the respective loading control.
Fig. 2. Differential phosphorylation of MAPK on L1210R and CBMC-6 compared with L1210 cells in response to cold stress. Cells (3 × 105 cells/ml) were incubated at 2–4 °C for 0.5 to 8 h. Cells were harvested and whole cell lysates were obtained (10 μg of each) and subjected to western blotting using the indicated phospho-specific and total protein antibodies to detect activation and total relative expression of kinases. (A) Graph showing results on JNK phosphorylation. (B) Representative western blotting showing JNK phosphorylation under cold stress. (C) Graph showing results on ERK phosphorylation. (D) Representative western blotting showing ERK phosphorylation under cold stress. (E) Graph showing results on p38 MAPK phosphorylation (F) Representative western blotting showing p38 MAPK phosphorylation under cold stress. This figure shows a representative western blot for each kinase out of the four performed with similar results. Control cells (cultured at 37 °C) are indicated by an arrow. Data show means ±SEM from at least four independent assays. Asterisks (*) represent statistical significance for each kinase with respect to control cells (37 °C), a represents statistical significance of L1210R cells relative to CBMC-6 cells, b represents statistical significance of L1210 cells relative to L1210R cells, c represents statistical significance of L1210 cells relative to CBMC-6 cells; (*/a/b/c p b 0.05, **/aa/bb/cc p b 0.01, ***/aaa/bbb/ccc p b 0.001, t-test).
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2.5. Statistical analysis Data are represented as the mean ± SEM. Data were analyzed with Student's t-test by using SPSS 19.0 software package (IBM, Armonk, NY, USA). Statistical significance was defined as p b 0.05 (*), p b 0.01 (**) or p b 0.001 (***). 3. Results 3.1. Cold stress induces collateral sensitivity in MDR leukemic cells (L1210R and CBMC-6) compared with parental cells (L1210). As a model of MDR tumor cells, we used the mouse leukemic cell line L1210, its daunomycin-resistant (DNM-resistant) subline L1210R, which is characterized by expression of the ATP-Binding-Cassette (ABC) transporter P-gp (MDR-1), and the P-gp-transfected cell line CBMC-6, obtained as described in Materials and methods (Section 2.1). First, as previously reported [4,5], the effect of extreme hypothermia was studied in the three cell lines (Fig. 1A). To quantify cell death, the three cell lines were maintained for 20–24 h at 37 °C or at 4 °C, and the extent of cell death/survival was evaluated by counting the number of viable cells by flow cytometry analysis after staining with propidium iodide and Annexin V-FITC. Fig. 1 shows that only 11% of the parental cells died after 20 h at 4 °C, while survival of the resistant (L1210R) and P-gp-transfected (CBMC-6) cell population decreased drastically (up to 70% cell death). These data confirm previously published results [4,5] demonstrating that cold stress induces collateral sensitivity in MDR leukemic cells. 3.2. JNK phosphorylation is attenuated in cold-incubated MDR leukemic cells compared with their sensitive counterparts. As shown in Fig. 2, the signaling profile of MAPK differs between resistant and sensitive cells cultured at 37 °C or under cold conditions. As regards the activation of JNK (pJNK) (Fig. 2A and B), basal phosphorylation levels detected in L1210R cells cultured at 37 °C were low compared with the levels of these MAPK in L1210 and CBMC-6 cells. Furthermore, JNK activation in L1210 and CBMC-6 cells increased after 30 min of cold exposure, reaching a maximum after 2 h under hypothermia (4.5-fold and 3-fold higher than the control in L1210 and CBMC-6 cells, respectively). JNK activation then remained almost constant for several hours (up to 8 h), before starting to decrease slightly after 24 h (data not shown). However, in L1210R cells, there was no increase in JNK phosphorylation (compared with the control at 37 °C) after short cold exposure times, and the phosphorylation levels were much less intense than in sensitive or P-gp-transfected cells. A slight increase in JNK activation was observed after 8 h of cold exposure, which lasted for up to 12 h of cold incubation, but was undetectable after 24 h, as in the control cells (data not shown). Regarding ERK phosphorylation (pERK) no significant differences were observed in CBMC-6 cells compared with L1210 cells (Fig. 2C and D). Nevertheless, there was a decrease, although not statistically significant, in ERK2 activation in L1210R cells compared with the other two cell lines after 0.5 h of cold exposure. This activation returned to previous levels after 4 h of incubation at 4 °C, reaching similar values in all three leukemic cell lines. Phosphorylation levels of p38 MAPK (pp38) were similar in the three cell lines cultured at 37 °C, and remained similar, with no significant differences, after 8 h of cold exposure (Fig. 2E and F).
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3.3. MAP kinases play different roles in the survival of sensitive and MDR leukemic cells under physiological conditions. Initially, we used specific inhibitors to evaluate the individual roles of ERK, p38 MAPK and JNK in cell survival at physiological temperature. The three cell lines were treated with one of the inhibitors or vehicles (DMSO) and, following 20 h incubation at 37 °C, survival/apoptosis was measured as specified in Materials and methods (Section 2.3). The ERK inhibitor by itself did not affect survival in any cell type (Fig. 3A and C). Treatment with p38 MAPK inhibitor appeared to lower survival rates in all three cell lines although the decrease was only statistically significant in the case of L1210 cells. Finally, treatment with the JNK inhibitor alone strongly decreased the viability of the three cell lines cultured for 20 h at 37 °C, the L1210R cell line being most affected by this inhibitor. When a combination of two or three of the MAPK inhibitors was used a statistically significant decrease was observed in the survival of L1210, L1210R and CBMC-6 cell lines (Fig. 3A and C). Thus, we conclude that the use of the JNK inhibitor either alone, or in combination with the ERK and/or with p38 MAPK inhibitors, strongly affected the viability of the leukemic cells used, at 37 °C. The decrease in survival induced by MAPK inhibitors was similar in L1210 and CBMC-6 cells, but more drastic in L1210R cells (Fig. 3A and C). 3.4. Pharmacological inhibition of ERK and JNK phosphorylation partially blocks cold stress-induced collateral sensitivity in MDR and P-gptransfected cells. ERK and/or JNK inhibitors improved the viability of MDR cells maintained at 4 °C. Thus, the ERK inhibitor attenuated cold-induced celldeath in L1210R (20%) and CBMC-6 cells (25–30%), while there was no change in L1210 cell death levels relative to untreated cells. Moreover, the JNK inhibitor induced an increase of 20–30% in the viability of L1210R and CBMC-6 cells incubated under cold stress conditions, while no effect was observed in the viability of cold-treated L1210 cells. The combination of both ERK and JNK inhibitors had a partially additive effect on the MDR cell lines cultured at 4 °C, producing a significant rescue from cold-induced death in L1210R cells (up to 30%) and CBMC-6 cells (up to 40%) compared with negative controls (Fig. 3B and D). Conversely, no differences in cell survival were found when the MDR cell lines cultured at 4 °C were treated with the p38 MAPK inhibitor compared with the same cells treated only with DMSO under the same conditions (negative control) although a 10% decrease in cell survival was observed in L1210 cells treated with p38 MAPK inhibitor (Fig. 3B and D). 3.5. Differences in MAPK cross-talk among resistant and parental cells under cold-stress. When the phosphorylation levels of MAPK were studied in the presence of their specific pharmacological inhibitors (Fig. 4), p38 MAPK and JNK inhibitors were seen to increase ERK phosphorylation in sensitive and resistant cells (Fig. 4A, C and E). No difference was observed in JNK phosphorylation in sensitive cells cultured in the presence of p38 MAPK inhibitor (Fig. 4A), but in the presence of ERK inhibitor, JNK phosphorylation decreased in sensitive cells (Fig. 4A) and increased in resistant sublines compared with control cells (Fig. 4C and E)). No differences were found in p38 phosphorylation levels in the three cell lines treated with the different inhibitors compared with control cells (Fig. 4A, C and E). The above results led us to propose a cross-talk
Fig. 3. Effect of MAPKs inhibitors on L1210, L1210R and CBMC-6 cold-induced cell-death. Leukemic cell lines (3 × 105 cells/ml) were incubated for 1 h at 37 °C in complete culture medium or in the presence of 1% DMSO (Negative control) or the following inhibitors: p38 MAPK inhibitor (SB203580), MEK1/ERK inhibitors (PD098059) or JNK inhibitor II (SP600125). Then, the cells were incubated for an additional 20 h at 37 °C or at 4 °C. Cell death was determined by cell staining with propidium iodide ± Annexin-V-FITC and analyzed by flow cytometry. (A) Dotplot results after incubating cells with or without inhibitors for 20 h at 37 °C. (B) Dot-plot results after maintaining cells with or without inhibitors for 20 h at 2–4 °C. Figure shows a representative dot-plot for each condition out of three that were performed with similar results. (C) Bar graphs representation of relative cell survival after incubating cells with or without inhibitors for 20 h at 37 °C. (D) Bar graphs representation of relative cell survival after maintaining cells with or without inhibitors for 20 h at 2–4 °C. Data show means ±SEM from at least three independent experiments. Asterisks represent statistical significance with respect to control cells (w/o inhibitors) (*p b 0.05, **p b 0.01, ***p b 0.001, t-test).
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Fig. 4. MAPK cross-talk at 4 °C. Cells (3 × 105 cells/ml) were incubated at 2–4 °C for 20–24 h in the presence of 1% DMSO (Negative control) or the following inhibitors: p38 MAPK inhibitor (SB203580), MEK1/ERK inhibitors (PD098059) or JNK inhibitor II (SP600125). Cells were harvested and whole cell lysates were obtained (10 μg of each) and subjected to western blotting using the indicated phospho-specific and reference protein antibodies (total protein or GAPDH) to detect activation and total relative expression of kinases. (A, C, E) Graphs showing western blotting results on JNK, ERK, p38 MAPK, MAPKAPK-2 and c-Jun phosphorylation in L1210, L1210R and CBMC-6 cells, respectively. The figure shows a representative western blotting for each kinase out of the three performed with similar results. (B, D, F) Qualitative interaction graph with three nodes that represent the three MAPK in their activated (phosphorylated) form. Positive (arrows) and negative (blunt-ended lines) edges represent activating or inhibiting influences between proteins.
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model of MAPK for sensitive and resistant cells (Fig. 4B, D and F). In parental cells (L1210) pERK cross-activate pJNK signaling (Fig. 4A and B), while in resistant cells (L1210R and CBMC-6) these kinases negatively regulated each other's activity (Fig. 4C, D, E and F). Since the p38 MAPK and the JNK inhibitors specifically inhibit p38 MAPK and JNK activity by competitively binding to the ATP binding pockets, thus preventing the phosphorylation of proteins downstream, their use does not result in decreased phosphorylation levels in either p38 MAPK or JNK [18]. Thus, to assess whether the doses of the ERK1/ 2, JNK and p38 MAPK inhibitors assayed were sufficient to exert their specific inhibitory effects, additional control experiments were designed by analyzing the phosphorylation levels of MAPKAPK-2 (p38 substrate) and c-Jun (ERK and JNK substrate) (Fig. 4A, C and E). 4. Discussion Using a model characterized by the hypersensitivity of resistant and P-gp-transfected leukemic cells to cold stress [4,5], key elements of the MAPK activation profile in sensitive versus resistant cells incubated under cold stress were studied, observing that cold treatment activates SAPK/JNK very efficiently in parental cells but to a much reduced extent
in resistant cells. The above results suggest that the SAPK/JNK kinase activation profile is related with the acquisition of DNM resistance and, it might be speculated that its attenuation is involved in protection against DNM in resistant cells. This result agrees with those obtained by Brozovic et al. [20], who observed that cells that acquired cisplatin (cDDP) resistance showed a reduced activation of SAPK/JNK and p38 MAPK when incubated in the presence of cDDP compared with the parental line. Moreover, there are several other lines of evidence indirectly suggesting that enhancement of the JNK pathway down-regulates P-gp and reverses P-gp-mediated MDR in cancer cells [21,22,23]. In contrast to these findings and ours, other researchers [24] showed a positive correlation between JNK activity and MDR-1 gene expression, reflecting the complex nature of MDR-1/P-gp regulation by JNK and the involvement of other regulators. All three cell lines studied manifested a dependence on JNK phosphorylation for survival at 37 °C. By contrast, at 4 °C resistant cells are partially rescued from cell death by the inhibition of JNK and/or ERK phosphorylation. These results suggest that exposure to low temperatures switches the anti-apoptotic role of JNK observed under physiological conditions to a pro-apoptotic one, since the death of resistant cells under cold stress is both JNK- and ERK-dependent. In this sense, it has
Fig. 5. Proposed mechanism of MAPK-mediated cold-stress-induced apoptosis. The qualitative interaction graph model is based on our experimental data and supported by the literature. (A) Interaction graph for L1210 cells. (B) Interaction graph for L1210R cells (C) Interaction graph for CBMC-6 cells. Acquisition of MDR phenotype induces an increase in MDR-1 expression and functionality, which, in turn, attenuates JNK expression and phosphorylation. Cold stress increases JNK phosphorylation in sensitive cells and, with less intensity, in resistant cells. There is a cross-talk among kinases, since p38 MAPK and JNK negatively regulates ERK activity. Additionally, ERK negatively regulates JNK activity in resistant cells (DNM-treated and P-gp-transfected cells) but has the opposite effect in sensitive cells. Furthermore, JNK phosphorylation inhibits cold stress-induced apoptosis in sensitive cells but plays a part in cold stress-induced apoptosis in resistant cells. Finally, we and others propose a role for MDR-1 in cell survival. Arrows represent activating (positive) interactions, blunt-ended lines represent inhibitory (negative) interactions and question marks represent an unknown link. The thickness of the arrows represents the strength of these effects. The letter size of the molecules represents the expression levels in each cell type. MAPK inhibitors are represented by their initial capital letters.
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been described that the growth factor-induced transient activation of JNK promotes cell survival and proliferation, whereas stress-induced sustained JNK activity promotes growth arrest and cell death [7]. Additionally, Xiao et al. [25] have shown that JNK can be activated by thermotherapy, ultimately leading to cell apoptosis. Furthermore, Muscarella et al. [26] have shown that two major types of cell stress responses, global proteotoxic damage induced by arsenic or heat stress and microtubule damage induced by vincristine, result in JNK pathway activation and the induction of apoptosis in B-cells. However, JNK-pathway activation was required for apoptosis only in the case of vincristineinduced microtubule inhibition. Cold-induced death of L1210R and CBMC-6 cells was significantly avoided in the presence of ERK inhibitor. In fact, the involvement of the MEK1/2–ERK1/2-pathway in apoptosis induced by cytotoxic compounds has been demonstrated previously [27]. In this regard, it has been described that the duration and intensity of MAPK activation is a determinant factor in cell fate, namely, growth arrest versus apoptosis [28,29]. JNK and ERK cooperate as cell-death controllers at low temperatures in resistant cells (Fig. 3). This partially additive effect suggests that both molecules activate common control pathways to regulate apoptosis in MDR cells. In fact, both kinases have been described as being responsible for modulating the expression and/or phosphorylation of common substrates, which ultimately promotes the fate of the cell through an apoptotic or survival pathway [30,31,32]. As regards MAPK cross-talk, our results suggest that p38 and JNK negatively regulate ERK phosphorylation (Fig. 4). In the case of JNK, the results differed between sensitive and resistant cells. Thus, the use of the ERK inhibitor in parental cells induced the inhibition of JNK phosphorylation while, in resistant cells, which exhibit low JNK activation at 4 °C, there was a slight increase in JNK phosphorylation compared with control cells. In this sense, the role for the JNK pathway in the negative regulation of the ERK pathway was greatly strengthened by RNAi in Drosophila S2 cells [33]. This study indicated that other signaling pathways, such as JNK and Akt/Tor, negatively affect ERK signaling. Moreover, Drosophila AP-1 transcription factors were identified as negative regulators of ERK pathway activity [33], which agrees with the results of others [34]. Finally, SB203580 has also been shown to enhance the phosphorylation of ERK in HL-60 and ML-1 cells [35]. Complex networks have been studied in the field of apoptosis, and network motifs have been defined [36,37]. In Figs. 4 and 5 a connectivity graph based on qualitative units of three nodes of interaction concerning p38, ERK and JNK is depicted, indicating how cold stress and MDR-1 might trigger apoptosis in resistant cell lines. We present a dynamic model featuring a JNK-positive feedback loop that generates a proliferative apoptotic switch and the factors controlling it, with a particular focus on feedback loops and cross-talk (Fig. 5). First, the acquisition of MDR phenotype induces an increase in MDR-1 expression; second, the expression of MDR-1 attenuates JNK phosphorylation; third, cold stress induces JNK phosphorylation; fourth, p38 MAPK inhibits ERK activity and fifth, ERK inhibits JNK activity in resistant cells but has the opposite effect in sensitive cells. Finally, JNK phosphorylation inhibits cold stress-induced apoptosis in sensitive cells but has a pro-apoptotic role in cold stress-induced apoptosis in resistant cells.
5. Conclusions This work demonstrates that MDR phenotype and P-gp expression modify the activation profile and regulation of Mitogen-Activated Protein Kinases (MAPK) in cells exposed to low temperatures. Furthermore, JNK and ERK cooperate as cell-death controllers at low temperatures in resistant cells. Finally, we propose a connectivity model based on qualitative units, integrating the cross-talk of the different molecules that participate in cold stress-induced apoptosis in our leukemic model, indicating how cold stress and MDR-1 might trigger apoptosis in resistant cell lines.
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Conflict of interest statement None declared. Contributors D. Cerezo and A.J. Ruiz-Alcaraz performed the experiments, analyzed the data and critically read the manuscript. M. Lencina-Guardiola and I. Martínez-López performed the experiments. P. Garcia-Peñarrubia and M. Canovas sponsored the study, proposed experiments and critically read the manuscript. E. Martin-Orozco designed and performed the experiments, analyzed data and wrote the paper. Aknowledgments and role of the funding source We thank Dr. Manuel Sánchez Angulo for his critical reading of the manuscript and helpful suggestions. This work was supported by the following Grants: 03112/PI/05 from Fundación Séneca-CARM, PI060006 from the Instituto de Salud Carlos III, BIO-2014-54411-C2-1R from Ministry of Science and Innovation, and partially by Seneca Foundation CARM 19236/PI/14 and FEDER funds. References [1] H. Zahreddine, K.L.B. Borden, Mechanisms and insights into drug resistance in cancer, Front. Pharmacol. 4 (28) (2013) 1–8, http://dx.doi.org/10.3389/fphar.2013.00028. [2] R.M. Mohammad, I. Muqbil, L. Lowe, C. Yedjou, H.Y. Hsu, L.T. Lin, et al., Broad targeting of resistance to apoptosis in cancer, Semin. Cancer Biol. 35 (2015) S78–103, http://dx.doi.org/10.1016/j.semcancer.2015.03.001 (Suppl). [3] G. Szakacs, M.D. Hall, M.M. Gottesman, A. Boumendjel, R. Kachadourian, B.J. Day, H. Baubichon-Cortay, A. Di Pietro, Targeting the Achilles heel of multidrug-resistant cancer by exploiting the fitness cost of resistance, Chem. Rev. 114 (11) (2014) 5753–5774, http://dx.doi.org/10.1021/cr4006236. [4] D. Cerezo, M. Lencina, A.J. Ruiz-Alcaraz, J.A. Ferragut, M. Saceda, M. Sánchez, M. Cánovas, P. García-Peñarrubia, E. Martin-Orozco, Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress, J. Cell. Biochem. 113 (4) (2012) 1416–1425, http://dx. doi.org/10.1002/jcb.24016. [5] D. Cerezo, M. Cánovas, P. García-Peñarrubia, E. Martín-Orozco, Collateral sensitivity to cold stress and differential BCL-2 family expression in new daunomycin-resistant lymphoblastoid cell lines, Exp. Cell Res. 331 (1) (2015) 11–20, http://dx.doi.org/10. 1016/j.yexcr.2014.11.017. [6] M. Qi, E.A. Elion, MAP kinase pathways, J. Cell Sci. 118 (2005) 3569–3572, http://dx. doi.org/10.1242/jcs.02470. [7] D. Fey, D.R. Croucher, W. Kolch, B.N. Kholodenko, Crosstalk and signaling switches in mitogen-activated protein kinases cascade, Front. Physiol. 3 (355) (2012) 1–21, http://dx.doi.org/10.3389/fphys.2012.00355. [8] Y. Ohsaka, S. Ohgiya, T. Hoshino, K. Ishizaki, Phosphorylation of c-Jun N-terminal kinase in human hepatoblastoma cells is transiently increased by cold exposure and further enhanced by subsequent warm incubation of the cells, Cell. Physiol. Biochem. 12 (2002) 111–118 (http://dx.doi.org/63787). [9] M. Krishna, H. Narang, The complexity of mitogen-activates protein kinases (MAPKs) made simple, Cell. Mol. Life Sci. 65 (2008) 3525–3544, http://dx.doi.org/ 10.1007/s00018-008-8170-7. [10] M.D. Castro-Galache, J.A. Ferragut, V.M. Barbera, E. Martin-Orozco, J.M. GonzalezRos, P. Garcia-Morales, et al., Susceptibility of multidrug resistance tumor cells to apoptosis induction by histone deacetylase inhibitors, Int. J. Cancer 104 (5) (2003) 579–586, http://dx.doi.org/10.1002/ijc.10998. [11] A.M. Domina, J.H. Smith, R.W. Craig, Myeloid cell leukemia 1 is phosphorylated through two distinct pathways, one associated with extracellular signal-regulated kinase activation and the other with G2/M accumulation or protein phosphatase 1/2A inhibition, J. Biol. Chem. 275 (28) (2000) 21688–21694, http://dx.doi.org/10. 1074/jbc.M000915200. [12] H. Xu, M. Goldfarb, Multiple effector domains within SNT1 coordinate ERK activation and neuronal differentiation of PC12 cells, J. Biol. Chem. 276 (16) (2001) 13049–13056, http://dx.doi.org/10.1074/jbc.M009925200. [13] E.G. Lee, D.L. Boone, S. Chai, S.L. Libby, M. Chien, J.P. Lodolce, et al., Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice, Science 289 (5488) (2000) 2350–2354. [14] A. Shukla, C.R. Timblin, A.K. Hubbard, J. Bravman, B.T. Mossman, Silica-induced activation of c-Jun-NH2-terminal amino kinases, protracted expression of the activator protein-1 proto-oncogene, fra-1, and S-phase alterations are mediated via oxidative stress, Cancer Res. 6 (5) (2001) 1791–1795. [15] A. Rossi, P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin, et al., Anti-inflammatory cyclopentenone prostaglandins are direct inhibitors of IkappaB kinase, Nature 403 (6765) (2000) 103–108, http://dx.doi.org/10.1038/47520.
170
D. Cerezo et al. / Cellular Signalling 30 (2017) 162–170
[16] E. Patrucco, A. Notte, L. Barberis, G. Selvetella, A. Maffei, M. Brancaccio, et al., PI3Kgamma modulates the cardiac response to chronic pressure overload by distinct kinase-dependent and –independent effects, Cell 118 (3) (2004) 375–387, http://dx.doi.org/10.1016/j.cell.2004.07.017. [17] M. Tesker, S.E. Selamat, J. Beenstock, R. Hayouka, O. Livnah, D. Engelberg, Tighter αC-helix-αL16-helix interactions seem to make p38α less prone to activation by autophosphorylation than Hog1, Biosci. Rep. 36 (2) (2016)http://dx.doi.org/10. 1042/BSR20160020 (pii: e00324). [18] C. Xu, X. Wang, Y. Zhu, X. Dong, C. Liu, H. Zhang, et al., Rapamycin ameliorates cadmium-induced activation of MAPK pathway and neuronal apoptosis by preventing mitochondrial ROS inactivation of PP2A, Neuropharmacology 105 (2016) 270–284, http://dx.doi.org/10.1016/j.neuropharm.2016.01.030. [19] X. Bai, Y. Zhang, H. Jiang, P. Yang, H. Li, Y. Zhang, et al., Effects of maslinic acid on the proliferation and apoptosis of A549 lung cancer cells, Mol. Med. Rep. 13 (1) (2016) 117–122, http://dx.doi.org/10.3892/mmr.2015.4552. [20] A. Brozovic, G. Fritz, M. Christmann, J. Zisowsky, U. Jaehde, M. Osmak, B. Kaina, Longterm activation of SAPK/JNK, p38 kinase and Fas-l expression by cisplatin is attenuated in human carcinoma cells that acquired drug resistance, Int. J. Cancer 112 (2004) 974–985, http://dx.doi.org/10.1002/ijc.20522. [21] Z.H. Miao, J. Ding, Transcription factor c-Jun activation represses mdr-1 Gene expression, Cancer Res. 63 (2003) 4527–4532. [22] A. Brozovic, M. Osmak, Activation of mitogen-activated protein kinases by cisplatin and their role in cisplatin-resistance, Cancer Lett. 251 (2007) 1–16, http://dx.doi. org/10.1016/j.canlet.2006.10.007. [23] D.E. Muscarella, S.E. Bloom, The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses, Toxicol. Appl. Pharmacol. 228 (1) (2008) 93–104, http://dx.doi.org/10.1016/j.taap.2007.11.032. [24] J. Zhou, M. Liu, R. Aneja, R. Chandra, H. Lage, H.C. Joshi, Reversal of P-glycoprotein– mediated multidrug resistance in cancer cells by the c-Jun NH2-terminal kinase, Cancer Res. 66 (1) (2006) 445–452, http://dx.doi.org/10.1158/0008-5472.CAN-051779. [25] H. Sui, S. Zhou, Y. Wang, X. Liu, L. Zhou, P. Yin, et al., COX-2 contributes to P-glycoprotein-mediated multidrug resistance via phosphorylation of c-jun at Ser63/73 in colorectal cancer, Carcinogenesis 32 (5) (2011) 667–675, http://dx.doi.org/10. 1093/carcin/bgr016. [26] F. Xiao, B. Liu, Q.X. Zhu, C-Jun N-terminal kinase is required for thermotherapy-induced apoptosis in human gastric cancer cells, World J. Gastroenterol. 18 (48) (2012) 7348–7356, http://dx.doi.org/10.3748/wjg.v18.i48.7348.
[27] A. Calcabrini, J.M. Garcia-Martinez, L. Gonzalez, M.J. Tendero, M.T. Ortuño, P. Crateri, et al., Inhibition of proliferation and induction of apoptosis in human breast cancer cells by lauryl gallate, Carcinogenesis 27 (2006) 1699–1712, http://dx.doi.org/10. 1093/carcin/bgl044. [28] W. Yu, Q.Y. Liao, F.M. Hantash, B.G. Sanders, K. Kline, Activation of extracellular signal-regulated kinase and c-Jun-NH2-terminal kinase but not p38 mitogen-activated protein kinases is required for RRR-alpha-tocopheryl succinate-induced apoptosis of human breast cancer cells, Cancer Res. 2061 (2001) 6569–6576. [29] M. Ebisuya, K. Kondoh, E. Nishida, The duration, magnitude and compartmentalization of ERK MAP kinase activity: mechanisms for providing signaling specificity, J. Cell Sci. 118 (2005) 2997–3002, http://dx.doi.org/10.1242/jcs.02505. [30] M. Fan, M. Goodwin, T. Vu, C. Brantley-Finley, W.A. Gaarde, T.C. Chambers, Vinblastine-induced phosphorylation of Bcl-2 and Bcl-xL is mediated by JNK and occurs in parallel with inactivation of the Raf-1/MEK/ERK cascade, J. Biol. Chem. 275 (39) (2000) 29980–29985, http://dx.doi.org/10.1074/jbc.M003776200. [31] R.K. Barr, M.A. Bogoyevitch, The c-Jun N-terminal protein kinase family of mitogenactivated protein kinases (JNK MAPKs), Int. J. Biochem. Cell Biol. 233 (11) (2001) 1047–1063. [32] R. Dai, W. Frejtag, B. He, Y. Zhang, N.F. Mivechi, c-Jun NH2-terminal kinase targeting and phosphorylation of heat shock factor-1 suppress its transcriptional activity, J. Biol. Chem. 275 (24) (2000) 18210–18218, http://dx.doi.org/10.1074/jbc. M000958200. [33] A. Friedman, N. Perrimon, A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signaling, Nature 444 (2006) 230–234, http://dx.doi.org/10. 1038/nature05280. [34] M.R. Junttila, S.P. Li, J. Westermarck, Phosphatase-mediated crosstalk between MAPK signaling pathways in the regulation of cell survival, FASEB J. 22 (2008) 954–965, http://dx.doi.org/10.1096/fj.06-7859rev. [35] M. Hirosawa, M. Nakahara, R. Otosaka, A. Imoto, T. Okazaki, S. Takahashi, The p38 pathway inhibitor SB202190 activates MEK/MAPK to stimulate the growth of leukemia cells, Leuk. Res. 33 (2009) 693–699, http://dx.doi.org/10.1016/j.leukres.2008. 09.028. [36] R. Milo, S. Shen-Orr, S. Itzkovitz, N. Kashtan, D. Chklovskii, U. Alon, Network motifs: simple building blocks of complex networks, Science 298 (2002) 824–827, http:// dx.doi.org/10.1126/science.298.5594.824. [37] S. Shen-Orr, R. Milo, S. Mangan, U. Alon, Network motifs in the transcriptional regulation network of Escherichia coli, Nat. Genet. 31 (2002) 64–68, http://dx.doi.org/ 10.1038/ng881.