Specific MRP4 Inhibitor Ceefourin-1 Enhances Apoptosis Induced by 6-Mercaptopurine in Jurkat Leukemic Cells, but Not in Normal Lymphoblast Cell Line CRL-1991

Background and objectives: The multidrug resistance protein 4 (MRP4) is a member of the ABC transporter, which has been extensively related to many types of cancer including leukemia. MRP4 overexpression and activity over the efflux of some chemotherapeutic drugs are the main causes of chemoresistance. 6-mercaptopurine (6-MP) is a chemotherapeutic drug widely used in the consolidation and maintenance phases of leukemia treatment. However, 6-MP is a substrate of MRP4, which decreases its chemotherapeutic efficacy. Current research is focused on the development of MRP4 inhibitors to combat chemoresistance by allowing the accumulation of the drug substrates inside the cells. To date, the only specific MRP4 inhibitor that has been developed is ceefourin-1, which has been reported to inhibit MRP4 in many cancer cells and which makes it an excellent candidate to enhance the activity of 6-MP in a combined treatment in vitro of leukemic cells. Materials and methods: in the present work, we determined the enhancing activity of ceefourin-1 on the antiproliferative and apoptotic effect of 6-MP in leukemic Jurkat cells by trypan blue assay and flow cytometry. Besides, we determined the 6-MP and ceefourin-1 binding sites into MRP4 by molecular docking and molecular dynamics. Results: ceefourin-1 enhanced the apoptotic activity of 6-MP in Jurkat cells, while in CRL-1991 cells both antiproliferative and apoptotic effect were significantly lower. Ceefourin-1 additively cooperates with 6-MP to induce apoptosis in leukemic cells, but normal lymphoblast CRl-1991 showed resistance to both drugs. Conclusion: ceefourin-1 and 6-MP cooperates to trigger apoptosis in leukemic Jurkat cells, but the full mechanism needs to be elucidated in further works. In addition, our perspective is to test the cooperation between ceefourin-1 and 6-MP in samples from patients and healthy donnors.


Introduction
Multidrug resistance protein 4 (MRP4) is encoded by the ABCC4 gene located on chromosome 13. MRP4 transports mainly organic anions and glucuronide conjugates [1]. Since MRP4 is present in multiple organs, the cellular exposure to drugs or metabolites can be modified leading to metabolic dysregulation. The MRP4 expression in most cancer cells is pivotal for the treatment because the protein can efflux many anticancer drugs or interfere with physiopathological processes.
The relation between MRP4 overexpression in 6-mercaptopurine (6-MP) resistance was reported in T-lymphoblastic leukemia (T-ALL) cells (CEM cells), where MRP4 was up-regulated in tumorous cells but influx transporters were down-regulated, leading to a high efflux of 6-MP and survival of resistant cells [2]. In addition, the overexpression

Proliferation Assay and Viability
To determine the effect of ceefourin-1 and 6-MP over the cellular proliferation and viability, 1 × 10 6 cells/mL were treated with the concentrations listed in Table 1 and counted after 24 h. The cell counting was performed in a CytoSmart cell counter, cloud-based image processing to perform cell counting from Corning (Cat number 6749), following the trypan blue protocol [16].

Apoptosis Assay
The apoptosis assay was performed with the FITC Annexin V Apoptosis Detection Kit I (No. 556547), according to the manufacturer's instructions. Briefly, 1 × 10 6 cells were treated with different 6-MP and ceefourin-1 concentrations of the molecules (Table 1). After 24 h of drug treatment, the cells were washed twice with cold phosphate-buffered saline (PBS) and then resuspended in binding buffer at a concentration of 1 × 10 6 cells/mL. An amount of 100 µL (1 × 10 5 cells) was transferred to a 5 mL culture tube, and the cells were then stained with 5 µL of FITC Annexin V and 5 µL of propidium iodide. The cell suspension was gently mixed with a vortex and incubated for 15 min at room temperature. After incubation, 400 µL of 1× Binding Buffer was added to each tube, and the cells were analyzed by flow cytometry in a BD FACSVerse TM system (BD Biosciences, MH, México city, México) and BD FACSuite TM software, within 1 h.
Data were normalized to the maximum signal, and concentration-response curves were fitted with a three-parameter logistic model by non-linear regression using Prism 8 to determine the concentration that inhibits 20% and 40% of proliferation and the concentration that induces 20% and 40% of cell death. To determine significant differences between groups are represented as different letters (p-value ≤ 0.05), according to two-way ANOVA and Bonferroni s multiple comparison test.

Molecular Docking
To study the binding site and the intermolecular interactions of 6-MP and ceefourin-1 in MRP4, molecular docking studies were carried out using the AUTODOCK 4.2.6 software (The Scripps Research Institute, San Diego, CA, USA) [17]. The MRP4 structure, previously modeled [18], was used as an input for the AUTOGRID 4.2.6. The maps were calculated with 0.375 Å spacing between grid points. The center of the grid box was defined as follows: [−0.65 8.78 28.26], corresponding to Glu103, Gly359, Arg362, and Ser328 according to previous reports [19]. The dimensions of the active site box were set at 40 × 60 × 80 points. Rigid-flexible docking was performed for the compounds and each docked system was built using AUTODOCK, with 25 runs using Lamarckian genetic algorithm with 5 million maximum energy evaluations per run. to determine the differences in the intermolecular interactions to MRP4 related to chemical structure between each group.

Umbrella Sampling
In order to determine the binding energy of ceefourin-1 and 6-MP into MRP4, umbrella sampling simulations were performed by using the MRP4 C1 of the previous 25 ns AA-MD trajectory [18], the system was built under the previously mentioned conditions. Once the system was obtained and relaxed using the AA-MD relaxation protocol, a 10 ns (100 frames) MD simulation was performed in the Metadynamics module of Desmond using the protein and ligand center of mass distance as the collective variable, with 0.3 kcal/mol height and 0.1 kcal/mol width as the Gaussian parameters for the umbrella protocol, on an NPT ensemble at 310.15 K and 1.01325 bar. Finally, the analysis was performed in the Metadynamics Analysis module of Desmond.

Effect of Ceefourin-1 and 6-MP on the Cellular Proliferation
Different concentrations of 6-MP were tested to determine the concentrations that inhibit 20% and 40% of proliferation at 24 h in Jurkat cells. Such concentrations for 6-MP were 4.25 µM and 8.5 µM, respectively. The analyst error was avoided by using the auto-matic cell counter CytoSmart by Corning, to obtain better results. As observed in Figure 1, the effect of 6-MP over the Jurkat cells proliferation is concentration-dependent, but not linear. Trypan blue was used for the cell counting, so, the cells observed could be alive or dead, according to the principle of the proliferation test. As is shown in Figure 1, all the 6-MP concentrations impact Jurkat proliferation, but at this point, we did not determine the mechanisms involved in such an effect. In addition, four concentrations of ceefourin-1 were also tested in Jurkat cells to determine if this MRP4 inhibitor interferes with cellular proliferation to further evaluation in combination with 6-MP. As can be appreciated in Figure 2, the anti-proliferative effect of ceefourin-1 is concentration-dependent, as well as 6-MP, and all the concentrations tested interfere with Jurkat proliferation. The concentrations of ceefourin-1 (Ceef1) that inhibit 20% and 40% of proliferation were 1.5 µM and 12 µM, respectively.

Effect of Ceefourin-1 and 6-MP on the Cellular Proliferation
Different concentrations of 6-MP were tested to determine the concentrations that inhibit 20% and 40% of proliferation at 24 h in Jurkat cells. Such concentrations for 6-MP were 4.25 μM and 8.5 μM, respectively. The analyst error was avoided by using the automatic cell counter CytoSmart by Corning, to obtain better results. As observed in Figure  1, the effect of 6-MP over the Jurkat cells proliferation is concentration-dependent, but not linear. Trypan blue was used for the cell counting, so, the cells observed could be alive or dead, according to the principle of the proliferation test. As is shown in Figure 1, all the 6-MP concentrations impact Jurkat proliferation, but at this point, we did not determine the mechanisms involved in such an effect. In addition, four concentrations of ceefourin-1 were also tested in Jurkat cells to determine if this MRP4 inhibitor interferes with cellular proliferation to further evaluation in combination with 6-MP. As can be appreciated in Figure 2, the anti-proliferative effect of ceefourin-1 is concentration-dependent, as well as 6-MP, and all the concentrations tested interfere with Jurkat proliferation. The concentrations of ceefourin-1 (Ceef1) that inhibit 20% and 40% of proliferation were 1.5 μM and 12 μM, respectively.

Effect of Ceefourin-1 and 6-MP on the Cellular Proliferation
Different concentrations of 6-MP were tested to determine the concentrations that inhibit 20% and 40% of proliferation at 24 h in Jurkat cells. Such concentrations for 6-MP were 4.25 μM and 8.5 μM, respectively. The analyst error was avoided by using the automatic cell counter CytoSmart by Corning, to obtain better results. As observed in Figure  1, the effect of 6-MP over the Jurkat cells proliferation is concentration-dependent, but not linear. Trypan blue was used for the cell counting, so, the cells observed could be alive or dead, according to the principle of the proliferation test. As is shown in Figure 1, all the 6-MP concentrations impact Jurkat proliferation, but at this point, we did not determine the mechanisms involved in such an effect. In addition, four concentrations of ceefourin-1 were also tested in Jurkat cells to determine if this MRP4 inhibitor interferes with cellular proliferation to further evaluation in combination with 6-MP. As can be appreciated in Figure 2, the anti-proliferative effect of ceefourin-1 is concentration-dependent, as well as 6-MP, and all the concentrations tested interfere with Jurkat proliferation. The concentrations of ceefourin-1 (Ceef1) that inhibit 20% and 40% of proliferation were 1.5 μM and 12 μM, respectively.   In order to determine if Ceef1 cooperates or enhances the 6-MP activity over the proliferation of Jurkat cells in vitro, we treated the cells with different combinations of 6-MP and Ceef1, as indicated in Figure 3. The concentration 0.1 µM of Ceef1 induced slightly but significant higher antiproliferative effect with respect to negative control, while 1.5 µM of Ceef1 represents the 50% of MRP4 inhibition as reported by Fusaku Usuki and coworkers [20]. We decided to test 1.5 µM Ceef1 to determine if such a concentration enhances the antiproliferative and apoptotic effect of 6-MP. On the other hand, we also tested the same 6-MP and Ceef1 combinations in CRL-1991 cells with the aim of studying if both cell lines present different responses to the treatments, considering that they are metabolic and genetically different, even though they are derived from the same lineage. In Jurkat cells, all the treatments significantly reduced cell proliferation compared to the control group, where the combined treatment 4. In order to determine if Ceef1 cooperates or enhances the 6-MP activity over th liferation of Jurkat cells in vitro, we treated the cells with different combinations of and Ceef1, as indicated in Figure 3. The concentration 0.1 μM of Ceef1 induced sl but significant higher antiproliferative effect with respect to negative control, wh μM of Ceef1 represents the 50% of MRP4 inhibition as reported by Fusaku Usuk coworkers [20]. We decided to test 1.5 μM Ceef1 to determine if such a concentratio hances the antiproliferative and apoptotic effect of 6-MP. On the other hand, w tested the same 6-MP and Ceef1 combinations in CRL-1991 cells with the aim of stu if both cell lines present different responses to the treatments, considering that th metabolic and genetically different, even though they are derived from the same lin In Jurkat cells, all the treatments significantly reduced cell proliferation compared control group, where the combined treatment 4.25 μM 6-MP + 0.1 μM Ceef1 reduce proliferation with no significant difference with respect to 4.25 μM 6-MP alone, indi that Ceef1 at 0.1 μM did not enhance the effect of 6-MP, at least at 24 h of treatmen combined treatment 4.25 μM 6-MP + 1.5 μM Ceef1 significantly reduces cell prolife compared to 4.25 μM 6-MP alone. In addition, 4.25 μM 6-MP + 10 μM Ceef1 had an tive effect at reducing cell proliferation in the same way that the sum of the indiv effects of 4.25 μM 6-MP and 10 μM Ceef1. Moreover, 4.25 μM 6-MP + 10 μM Ceef1 s icantly reduced cell proliferation greater than 8.5 μM 6-MP, which represents a h concentration and possibly toxic effects.

Apoptotic Interaction between Ceefourin-1 and 6-MP
The evaluation of the viability induction by 6-MP and Ceef1 was carried out in the same way as the proliferation assay: with the same time, same concentrations, and same cell lines, and 6.75 µM 6-MP and 13.5 µM 6-MP induced cell death around 20% and 40%, respectively. In addition, 1.5 µM Ceef1 and 12.5 µM Ceef1 induced cell death around 20% and 40%, respectively. The graph in Figure 4 indicated that the effect of 6-MP on cell death induction was concentration-dependent, being the concentration 0.1 µM statistically same with respect to the negative control. The minimum concentration of 6-MP required to induce apoptosis significantly different with respect to the negative control was 1.0 µM. It was observed that the concentrations of 6-MP and Ceef1 required to induce both cell death and inhibition of cell proliferation were quite similar. In the same way, the treatments 8.5 μM 6-MP and 4.25 μM 6-MP + 1.5 μM Ceef1 had the same effect on the inhibition of CRL-1991 cells proliferation, but 8.5 μM 6-MP induced higher inhibition on Jurkat cells proliferation compared to 4.25 μM 6-MP + 1.5 μM Ceef1. The combined treatment 4.25 μM 6-MP + 10 μM Ceef1 exerted the highest inhibition on cell proliferation in both cell lines.

Apoptotic Interaction between Ceefourin-1 and 6-MP
The evaluation of the viability induction by 6-MP and Ceef1 was carried out in the same way as the proliferation assay: with the same time, same concentrations, and same cell lines, and 6.75 μM 6-MP and 13.5 μM 6-MP induced cell death around 20% and 40%, respectively. In addition, 1.5 μM Ceef1 and 12.5 μM Ceef1 induced cell death around 20% and 40%, respectively. The graph in Figure 4 indicated that the effect of 6-MP on cell death induction was concentration-dependent, being the concentration 0.1 μM statistically same with respect to the negative control. The minimum concentration of 6-MP required to induce apoptosis significantly different with respect to the negative control was 1.0 μM. It was observed that the concentrations of 6-MP and Ceef1 required to induce both cell death and inhibition of cell proliferation were quite similar. Ceef1 induced cell death in a concentration-dependent manner ( Figure 5). The concentration 0.1 μM of Ceef1 induced the same percentage of cell death as the negative control, and the 1.5 μM concentration was the minimum concentration required to induce cell death, which is significantly different with respect to the negative control. Ceef1 50 μM induced cell death around 56%, which was 8% higher than 6-MP at the same concentration.  Ceef1 induced cell death in a concentration-dependent manner ( Figure 5). The concentration 0.1 µM of Ceef1 induced the same percentage of cell death as the negative control, and the 1.5 µM concentration was the minimum concentration required to induce cell death, which is significantly different with respect to the negative control. Ceef1 50 µM induced cell death around 56%, which was 8% higher than 6-MP at the same concentration. To test if Ceef1 enhances the apoptotic effect by 6-MP, the Jurkat cells were treated as indicated in Figure 6. All the treatments with 6-MP, Ceef1, and combined significantly induced a higher percentage of apoptosis than the negative control, and in all the treatments significant differences were observed. Even though 0.1 μM Ceef1 did not induce apoptosis greater than the negative control, it enhances the apoptotic effect of 6.75 μM 6-MP. Moreover, 1.5 μM Ceef1 together with 6.75 μM 6-MP induced apoptosis 2.5 higher To test if Ceef1 enhances the apoptotic effect by 6-MP, the Jurkat cells were treated as indicated in Figure 6. All the treatments with 6-MP, Ceef1, and combined significantly induced a higher percentage of apoptosis than the negative control, and in all the treatments significant differences were observed. Even though 0.1 µM Ceef1 did not induce apoptosis greater than the negative control, it enhances the apoptotic effect of 6.75 µM 6-MP. Moreover, 1.5 µM Ceef1 together with 6.75 µM 6-MP induced apoptosis 2.5 higher than 6.75 µM 6-MP and significantly higher than 13.5 µM 6-MP. It could be suggested that 6.75 µM 6-MP together with 1.5 µM Ceef1 exert synergistic cooperation rather than additive cooperation at inducing apoptosis. In addition, 6.75 µM 6-MP together with 10 µM Ceef1 induced the highest apoptotic percentage at around 75%. On the other hand, the same 6-MP and Ceef1 treatments significantly induced lower apoptosis in CRL-1991 cells than those observed in Jurkat cells. The combination of 6.75 µM 6-MP and 0.1 µM Ceef1 significantly induced lower apoptosis than 6.75 µM 6-MP alone, and 13.5 µM 6-MP significantly induced higher apoptosis than 6.75 µM 6-MP combined with 1.5 µM Ceef1. The combination of 6.75 µM 6-MP with 10 µM Ceef1 promoted apoptosis of CRL-1991 cells, similar than 13.5 µM 6-MP alone, which indicates that Ceef1 does not enhance apoptosis in the significant way it does for Jurkat cells.  The comparison of the apoptotic effect induced by each treatment in both cell indicated that 6-MP and Ceef1 alone or in combination, significantly induced lower a tosis on CRL-1991 cells compared with Jurkat cells, which suggests that both mole exerted cytotoxic effects in leukemic cells greater than in normal cells, at least in vitro 6.75 μM 6-MP combined with 1.5 μM Ceef1 and 6.75 μM 6-MP combined with 1 Ceef1 induced apoptosis higher in Jurkat cells than in CRL-1991 cells.

Molecular Docking and Umbrella Sampling
The binding sites of MRP4 substrates and inhibitors may be located at differe gions of the transmembrane domains (TMDs) of the protein. Based on the previous ment, molecular docking simulations were performed between 6-MP-MRP4 and C The comparison of the apoptotic effect induced by each treatment in both cell lines indicated that 6-MP and Ceef1 alone or in combination, significantly induced lower apoptosis on CRL-1991 cells compared with Jurkat cells, which suggests that both molecules exerted cytotoxic effects in leukemic cells greater than in normal cells, at least in vitro. The 6.75 µM 6-MP combined with 1.5 µM Ceef1 and 6.75 µM 6-MP combined with 10 µM Ceef1 induced apoptosis higher in Jurkat cells than in CRL-1991 cells.

Molecular Docking and Umbrella Sampling
The binding sites of MRP4 substrates and inhibitors may be located at different regions of the transmembrane domains (TMDs) of the protein. Based on the previous statement, molecular docking simulations were performed between 6-MP-MRP4 and Ceef1-MRP4 complexes, to determine if both molecules share a similar binding site. In Figure 7 the ligand interaction diagram (LID) of 6-MP into MRP4 is shown. 6-MP exerts H-bonds interactions with leucine 836 and threonine 839 into a hydrophobic pocket binding where 6-MP did not perfectly fit. Interestingly, Ceef1 interacts in the same binding site as 6-MP but did not exert H-bonds ( Figure 8). Nevertheless, Ceef1 seems to perfectly fit in its hydrophobic pocket binding, where its interactions are mainly hydrophobic, and those lead to the lower binding energy. The Docking Scores (DSs) for 6-MP and Ceef1 were −4.79 kcal/mol and −7.63 kcal/mol, respectively ( Table 2). The DS indicated that Ceef1 conformations require lower energy than 6-MP conformations to interact and bind to MRP4.       9 and 10 show the binding sites of 6-MP and Ceef1, respectively. For both molecules the binding site is located in the TMD2, close to the inner cavity. The structure of Ceef1 is bigger than the 6-MP structure and interacts with more residues fitting better in the hydrophobic binding site. For the proximity of both 6-MP and Ceef1 binding sites into MRP4, umbrella sampling simulations were performed to determine their theoretical affinity to MRP4, expressed as free binding energy (∆G), and according to the results presented in Table 2, Ceef1 has significantly higher affinity than 6-MP. Such results suggested that the Ceef1-MRP4 complex is more stable than the 6-MP-MRP4 complex, which is interesting regarding the efflux by MRP4 considering that 6-MP and Ceef1 would be in the cell at the same time.   Figures 9 and 10 show the binding sites of 6-MP and Ceef1, respectively. For both molecules the binding site is located in the TMD2, close to the inner cavity. The structure of Ceef1 is bigger than the 6-MP structure and interacts with more residues fitting better in the hydrophobic binding site. For the proximity of both 6-MP and Ceef1 binding sites into MRP4, umbrella sampling simulations were performed to determine their theoretical affinity to MRP4, expressed as free binding energy (ΔG), and according to the results presented in Table 2, Ceef1 has significantly higher affinity than 6-MP. Such results suggested that the Ceef1-MRP4 complex is more stable than the 6-MP-MRP4 complex, which is interesting regarding the efflux by MRP4 considering that 6-MP and Ceef1 would be in the cell at the same time.

Discussion
The combination of two or more drugs has been used to treat cancer for many years, and it represents both the first line of chemotherapy and the main source of drug-derived toxicity [21]. The design of combined chemotherapy relies on the pharmacological activity   Figures 9 and 10 show the binding sites of 6-MP and Ceef1, respectively. For both molecules the binding site is located in the TMD2, close to the inner cavity. The structure of Ceef1 is bigger than the 6-MP structure and interacts with more residues fitting better in the hydrophobic binding site. For the proximity of both 6-MP and Ceef1 binding sites into MRP4, umbrella sampling simulations were performed to determine their theoretical affinity to MRP4, expressed as free binding energy (ΔG), and according to the results presented in Table 2, Ceef1 has significantly higher affinity than 6-MP. Such results suggested that the Ceef1-MRP4 complex is more stable than the 6-MP-MRP4 complex, which is interesting regarding the efflux by MRP4 considering that 6-MP and Ceef1 would be in the cell at the same time.

Discussion
The combination of two or more drugs has been used to treat cancer for many years, and it represents both the first line of chemotherapy and the main source of drug-derived toxicity [21]. The design of combined chemotherapy relies on the pharmacological activity

Discussion
The combination of two or more drugs has been used to treat cancer for many years, and it represents both the first line of chemotherapy and the main source of drug-derived toxicity [21]. The design of combined chemotherapy relies on the pharmacological activity of the drugs, where their action must be complementary to each other-drug A enhances the action of drug B. In the present work, the antiproliferative and apoptotic effect of Ceef1 was tested in both Jurkat cells and CRL-1991 cells to further determine if it enhances the action of the 6-MP.
The concentration 0.1 µM of 6-MP was not able to interfere with the proliferation nor the significant apoptosis induction on Jurkat cells. Such results are consistent with previous reports, and no researcher has reported antiproliferative nor apoptotic effect at concentrations lower than 1 µM [12,22]. In addition, 6-MP is metabolized into their active metabolites also called tioguanine nucleotides (TGN), which are also substrates of MRP4 [23] leading to an even lower concentration of active metabolites than 0.1 µM. Any 6-MP concentration once inside the cell will be decreased by MRP4 activity. Nevertheless, it was observed that at least 1 µM of 6-MP is necessary to significantly interfere with cell proliferation, taking in consideration that a fraction of 6-MP has been previously metabolized and effluxed. Even though the antiproliferative effect of 6-MP is concentrationdependent, it is not linear because the more intracellular levels of 6-MP the more activity of metabolizing enzymes and MRP4 would be [24]. 6-MP acts by numerous mechanisms to induce antiproliferative and apoptotic effects, which includes its incorporation into the DNA chains promoting cell cycle arrest and further apoptosis. Additionally, 6-MP inhibits de novo purine synthesis, which is more important for lymphocyte proliferation than the salvage pathway [25], and there is evidence suggesting that thiopurine drugs might regulate metabolic checkpoints that promote reprogramming in normal and leukemic T cells as Jurkat cells. Such checkpoints include mTOR, AMP-activated kinase (AMPK), Myc, and HIF-1α [26], and those previous actions of 6-MP require higher concentration than 0.1 µM to get significant effects over cell proliferation and apoptosis.
In this line, Ceef1 was tested at various concentrations to determine if its previous tested ability to inhibit MRP4 also induces apoptosis or interferes with cellular proliferation [12]. In concordance with 6-MP, Ceef1 at 0.1 µM did not induce a significant antiproliferative effect on Jurkat cells, possibly because such concentration is not sufficient to regulate the activity of MRP4. The main action attributed to Ceef1, a benzothiazole-containing compound, is the selective inhibition of MRP4 among all the ABC transporters. However, the benzothiazole derivatives are known by their anticancer, anti-inflammatory, antiviral, anticonvulsant, antidiabetic, and more activities [27], which means that Ceef1 interacts with other targets, but the antiproliferative effect was dimly observed at 0.1 µM. Moreover, the more Ceef1 concentration was increased the more significant antiproliferative effect was observed, but not linearly. In the same manner, the apoptotic by Ceef1 was concentrationdependent and it was significant from 1.5 µM to 50 µM. According to previous evidence, the inhibition of MRP4 leads to increased cAMP levels to further intrinsic apoptosis activation once cAMP activated PKA [5,28], being this the most probable mechanism by which Ceef1 induces apoptosis. Nevertheless, the main question is how Ceef1 enhanced 6-MP-induced apoptosis. If 6-MP is an MRP4 substrate, and its intracellular concentration depends on the MRP4 activity, the most adequate explanation of the enhanced action of 6-MP would be that Ceef1 inhibits MRP4, and thus, blocking the efflux of 6-MP. When we treated the cells with 13.5 µM 6-MP alone, a certain amount of 6-MP must be effluxed by MRP4, but once we added Ceef1, a lower amount of 6-MP could be effluxed leading to more bioavailability inside the cell. The measure of intracellular 6-MP must be performed to demonstrate our hypothesis.
Regarding the previous statement, the more Ceef1 levels inside the cell the more levels of 6-MP and their active TGN metabolites would be bioavailable, due to the inhibition of MRP4 by Ceef1. However, is MRP4 really inhibited or how Ceef1 regulate its activity? A theoretical explanation of the inhibitory activity of Ceef1 can be addressed through the molecular docking and umbrella sampling simulations performed in this work. The molecular docking simulations indicated that both Ceef1 and 6-MP share the same binding site into MRP4, and it can be inferred that they compete for such binding site, where Ceef1 fits better than 6-MP. However, the free binding energy of Ceef1 was significantly lower than the free binding energy of 6-MP, which indicates that Ceef1 is a competitive antagonist that interferes with the binding of 6-MP into MRP4.
Furthermore, 6-MP can indirectly regulate MRP4 activity by depleting ATP. It has been determined that 6-MP induces rapid ATP depletion in Jurkat cells, even though the biochemical basis for such mechanism remains unestablished [26], and such ATP depletion may affect the function of MRP4 due to it requires ATP to start the transport cycle [29]. ATP depletion promotes metabolic stress, and it could be a possible mechanism to inhibit cell proliferation and perhaps apoptosis. Such metabolic stress might be higher in Jurkat cells because they have high intracellular ATP, and they could be more sensitive to changes in ATP levels [30].
The inhibition of cell proliferation and the induction of apoptosis were significantly higher in Jurkat cells than in CRL-1991 cells, and it may occur due to the action of cAMP and the sensibility of each cell line to higher levels of cAMP. More than 2000 published articles have demonstrated the relation between cAMP and apoptosis or inhibition of cell proliferation, and it was reported that such effects would depend on the cell type. In this regard, the increase of intracellular cAMP is reported to be pro-apoptotic in leukemic cells and anti-apoptotic in normal hematopoietic cells as CRL-1991, being the reason why some cancer treatments use cAMP analogs, phosphodiesterase E inhibitors, and adenyl cyclase activators [31]. In addition, the most effective mechanism to increase intracellular cAMP levels is through MRP4 inhibition, and studies in S49 lymphoma cells have determined cAMP triggers apoptosis via PKA leading intrinsic, mitochondriadependent mechanism [32]. Another factor regarding selective apoptosis is the selective involvement of the PKA isozymes I and II, where cAMP analogs selective for type I regulatory subunit inhibit natural killer-mediated cytotoxicity. However, expression of type II regulatory subunit modulates apoptosis of fibroblasts. Deregulation of the balance between these isozymes have been linked to various cancers, and the manipulations of such isozymes may increase or decrease cAMP-mediated apoptosis [31]. Concerning CRL-1991 cells, it was reported that the guanine nucleotide exchange factor Epac, also a second effector of cAMP an, mediates the anti-apoptotic effect once cAMP levels are increased [33]. In addition, the phosphorylation of dynamin-related protein 1 by PKA is reported to protect against apoptosis [34]. Such events may explain the lower sensitivity of CRL-1991 to 6-MP and Ceef1. The approaches focused on the inhibition of MRP4 to induce cAMP-mediated apoptosis shall be extensively studied in each cell type, to elucidate the mechanisms involved in the selectivity between cancer and normal cells.

Conclusions
The antiproliferative and apoptotic effect by Ceef1 was evaluated for the first time in Jurkat and CRL-1991 cells, as the first step to further evaluate its enhancing activity on the 6-MP chemotherapeutic drug. Once we determined that Ceef1 enhanced the activity of 6-MP, the next goal is determining the mechanisms involved in such additive cooperation. We are currently working on genetic engineering techniques and in silico experiments to better understand the architecture and function of MRP4 and the interaction with Ceef1 and other molecules, as basic research for gene therapy. We also conclude that the MRP4 specific inhibitor Ceef1 induces apoptosis per se, and it could be tested in vivo and in primary cell cultures to determine if they can be used in combined chemotherapy protocols.

Conflicts of Interest:
The authors declare no conflict of interest.