Antiproliferative and apoptotic interaction between azathioprine and N-acetylcysteine in acute lymphoblastic leukemia Jurkat cells

Journal Pre-proof Antiproliferative and apoptotic interaction between azathioprine and N-acetylcysteine in acute lymphoblastic leukemia Jurkat cells Edgardo Becerra, Laura C. Berumen, T. Garc´ıa-Gasca, Jesica Escobar, Ulisses Moreno C, Guadalupe Garc´ıa-Alcocer

PII:

S2213-7130(18)30038-5

DOI:

https://doi.org/10.1016/j.synres.2019.100061

Reference:

SYNRES 100061

To appear in:

Synergy

Received Date:

23 November 2018

Revised Date:

20 November 2019

Accepted Date:

21 November 2019

Please cite this article as: Becerra E, Berumen LC, Garc´ıa-Gasca T, Escobar J, Moreno C U, Garc´ıa-Alcocer G, Antiproliferative and apoptotic interaction between azathioprine and N-acetylcysteine in acute lymphoblastic leukemia Jurkat cells, Synergy (2019), doi: https://doi.org/10.1016/j.synres.2019.100061

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

1 Antiproliferative and apoptotic interaction between azathioprine and Nacetylcysteine in acute lymphoblastic leukemia Jurkat cells -Interaction of azathioprine and N-acetylcysteine on Jurkat cellsEdgardo Becerra 1, Laura C. Berumen1, García-Gasca T2, Jesica Escobar1, Ulisses Moreno C2 and Guadalupe García-Alcocer1

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1- Laboratorio de Investigación Genética, Posgrado en Ciencias Químico

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Biológicas, Universidad Autónoma de Querétaro, Facultad de Química, Querétaro, México

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Corresponding author email:

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2- Laboratorio de investigación “Biología Celular, molecular y cultivos,” Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro

Guadalupe García-Alcocer: [email protected] Posgrado en Ciencias Químico Biológicas, Facultad de Química, Universidad

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Autónoma de Querétaro, Centro Universitario, Querétaro, CP 76010, México

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Graphical abstract

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Abstract T cell acute lymphoblastic leukaemia is a type of cancer that develops from lymphoid progenitors, and chemotherapy is corner stone of the treatment. Thiopurine drugs, consisting of 6-mercaptopurine, 6-thioguanine, and azathioprine (Aza), can effectively treat this disease. To be activated, Aza must first be biotransformed to 6mercaptopurine by thiol groups in glutathione. However, glutathione is decreased in

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cancer cells due to high levels of reactive oxygen species (ROS). N-acetylcysteine could provide thiol groups for glutathione synthesis to biotransform Aza. Using flow cytometry, the ability of N-acetylcysteine to increase the antiproliferative and apoptotic effects of Aza without increasing ROS was tested in Jurkat cells. Individually, Aza 1.0 and 2.0 µM, as well as N-acetylcysteine 3.0 mM, induced apoptosis and cell cycle arrest. Together, Aza + N-acetylcysteine significantly reduced proliferation compared to that obtained with the individual drugs.

3 Combination of N-acetylcysteine 3.0 mM with Aza 1.0 µM was as effective at inducing apoptosis as Aza 2.0 µM alone. The combination of N-acetylcysteine 3.0 mM + Aza 1.0 µM increased cell cycle arrest at the G2/M phase. We found that Aza 1.0 or 2.0 µM induced a significant increase in ROS compared to that in untreated cells, while N-acetylcysteine 3.0 mM and N-acetylcysteine 3.0 mM + Aza 1.0 µM kept ROS at control levels; the latter drugpairing represents a favourable combination to reduce oxidative stress in the presence of Aza. In conclusion, Nacetylcysteine augments antiproliferative and apoptotic effects of Aza without

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increasing ROS in vitro.

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Keywords: leukaemia, chemotherapy, apoptosis, oxidative stress, proliferation

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1. Introduction T cell acute lymphoblastic leukaemia (T-ALL) is a type of cancer that develops from

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lymphoid progenitors and is characterized as a cell differentiation disorder [1]. The blockage of lymphoid differentiation drives aberrant cell proliferation and survival [2].

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T-ALL accounts for 15% of childhood and 25% of adult leukaemia and has multiple drug resistance, leading to a poor prognosis [3]. Due to their efficacy, thiopurine drugs are the most important and utilized drugs to treat T-ALL, especially in the maintenance stage. Thiopurines, such as 6-mercaptopurine (6-MP), 6-thioguanine

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(6-TG), and azathioprine (Aza), are widely used as chemotherapeutic and immunosuppressant agents [4]. All thiopurines are prodrugs, and their metabolism forms thioguanine nucleotides (TGNs) [5]. TGNs are incorporated into DNA and replace guanine, leading to cell cycle arrest and apoptosis by triggering the activation of mismatch repair systems

4 [6, 7]. Alternatively, one of the TGNs, 6-thioguanine triphosphate (TGTP), inhibits Rac-1 protein signalling, leading to an apoptotic mitochondrial pathway of T cells [8]. Particularly, Aza must be converted by a non-enzymatic reaction, mediated by thiol (SH) groups, to release 6-MP; 6-MP is then biotransformed to DNA precursors [9]. Therefore, the anticancer efficacy of Aza depends, in part, on the bioavailability of SH groups, which is decreased in cancer cells. SH groups of glutathione (GSH) and

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cysteine can interact with Aza to release 6-MP. However, GSH and cysteine are

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depleted in T-ALL, due to the high levels of reactive oxygen species (ROS) produced by altered cellular metabolism [10]. In fact, Aza increases ROS levels, favouring

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GSH depletion and increasing cytotoxicity and the generation of oxidized thiopurine

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metabolites that are mutagenic [11].

N-acetylcysteine (NAC), an aminothiol and synthetic precursor of intracellular

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cysteine and GSH, is considered an important antioxidant and a well-tolerated drug and is used in the treatment of acetaminophen intoxication. NAC can reduce ROS

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and provide SH groups [12]. NAC increases GSH levels via free radical scavenging properties NAC is also a reducing agent with thiol-disulfide exchange activity [13]. However, the use of antioxidants in chemotherapy is not well accepted because of

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their potential to protect cancer cells and promote proliferation and survival. During cancer treatment, antioxidants are used only to protect the body from excessive cytotoxicity at later stages of chemotherapy. Despite this fact, NAC can exert an antineoplastic effect by modulating the mitogen-activated protein kinase (MAPK) pathway. This modulation involves directly interacting with the thiol groups on target proteins, such as Raf1, MEK, and extracellular signal-regulated kinase

5 (ERK), via thiol-disulfide exchange, resulting in cell cycle arrest [14]. NAC was tested in Jurkat-T cells, in which cell cycle arrest and apoptosis were promoted [15]. Because of the antiproliferative, apoptotic, and antioxidant actions of NAC, the effect of Aza could be enhanced by NAC co-administration, leading to an increase in its antineoplastic effect. This augmentation can reduce the required dose of common chemotherapeutics, lowering toxicity and helping to minimize the occurrence of

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treatment-related deaths. [16]

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In this study, we aimed to test different combinations of NAC, considering that the

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observed IC25 and IC50 of NAC are reported to be well tolerated by human patients [17] and Aza concentrations to determine their effect on cell cycle arrest, apoptosis,

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and ROS levels in Jurkat cells. Previous reports indicate that NAC and Aza induce caspase activity [18, 19]. On the other hand, the increasing of ROS levels play an

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important role in drug-mediated oxidative cell death [20]. We hypothesized that a treatment containing NAC would reduce ROS, which would enhance the metabolic

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activation of Aza, increasing its cytotoxic efficacy. As a result, lower doses of Aza would be required, which would be expected to attenuate side effects in patients during the maintenance phase of chemotherapy.

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2. Methods

2.1 Cell culture Jurkat cells (ATCC, No. TIB-152) immortalized human T-lymphocyte cell line used to study T cell leukemia, were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown at 37°C in a humidified 95% air, 5% CO2 atmosphere, with RPMI 1640 medium (ATCC, No. 30-2001)

6 containing 10% foetal bovine serum (FBS, ATCC, No. 30-2020) and 2% antibiotics (penicillin and streptomycin sulphate). 2.2 Drug preparations NAC and Aza were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Canada Drugs (TPW, Canada), respectively. Stock solutions of NAC and Aza were freshly made in culture media at concentrations of 50 mM and 200 µM,

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respectively. The stock solutions were stored at 4°C for a maximum of four days

concentrations.

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2.3 Determination of inhibitory concentrations

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and were then filtered and diluted with appropriate media to the final, working

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To determine the 25 and 50% inhibitory concentrations (IC25 and IC50), 2.5 × 105 cells were incubated for 24 h without treatment. After preincubation, the cells

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were exposed to Aza at concentrations ranging from 0.039 to 40 mM or to NAC ranging from 0.008 to 100 µM, for 24 h; the cells were then counted in a Neubauer

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chamber. GraphPad Prism 6 software was used to calculate the IC 25 and IC50 values using logarithmic dose-response curves. 2.4 Cell cycle assay

Jurkat cells, in exponential growth phase, were seeded into a 12-well plate at 2.5 5

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× 10 cells/mL in RPMI 1640 medium with 10% FBS and were cultured for 24 h. After, the cells were treated for another 24 h with Aza and NAC as indicated in Table 1. The cells were harvested from the plate, transferred to 50 mL conical tubes, and centrifuged (400×g) at room temperature for 5 min. The pellet was lysed with lysis buffer (trypsin in a spermine tetrahydrochloride detergent buffer),

7 and the isolated nuclei were stained with propidium iodide. Flow cytometry data were acquired with the BD FACSVerseTM system (BD Biosciences, MH, México) and BD FACSuiteTM software, using the BD CycletestTM Plus DNA Reagent Kit (No. 340242). 2.5 Apoptosis

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An apoptosis assay was performed with the FITC Annexin V Apoptosis Detection Kit I (No. 556547), according to manufacturer’s instructions. Briefly, 1 × 106 cells

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were treated with different Aza and NAC concentrations as indicated in Table 1.

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After 24h of drug treatment, the cells were washed twice with cold phosphatebuffered saline (PBS) and then resuspended in 1× binding buffer at a

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concentration of 1 × 106 cells/mL. One hundred microlitres of the solution (1 × 105 cells) was transferred to a 5 mL culture tube. The cells were then stained with

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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

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incubation, 400 μL of 1× Binding Buffer was added to each tube, and the cells were analysed by flow cytometry in a BD FACSVerseTM system (BD Biosciences, MH, México) and BD FACSuiteTM software, within 1 h.

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2.6 Multicaspase assay

Suspension cells (3 × 105) were treated as indicated in Table 1. Subsequently, 50 L was sampled from each cell suspension and transferred to a conical tube. Then, 5 L of Multicaspase (caspases 1, 3, 4, 5, 6, 7, 8, and 9) and Muse® reagents (Muse® Multicaspase Assay Kit, No. MCH100109, EMD Millipore) were

8 added. The cell suspension and reagents were mixed by gentle pipetting, and the mixtures were incubated for 30 min at 37°C. After incubation, 150 L of Muse caspase 7-ADD reagent was added, mixed by gentle pipetting, and incubated for 5 min, protected from light. Following this second incubation, samples were immediately analysed on the flow cytometer, Muse ® Cell Analyzer. The results

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were calculated as the percentage of multicaspase activity. 2.7 ROS assay

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ROS were measured using the 2’-7’-dichlorofluorescin diacetate (DCFDA)

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Cellular ROS Detection Assay Kit (Abcam, Monterrey, México, ab113851). Jurkat cells were grown to at least 1.5 × 104 cells per experimental condition (per well),

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and then transferred into conical test tubes. To ensure a single-cell suspension, cells were gently pipetted up and down. After, the cells were stained in culture

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media with 20 μM DCFDA and then incubated for 30 min at 37°C. Once the incubation was completed, the cells were treated as described in Table 2 for 4 h.

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After incubation, the cell suspension was gently pipetted up and down and then analysed on a BD FACSVerseTM Flow Cytometer (BD, MH, México) at excitation and emission fluorescence wavelength settings of 488 and 535 nm, respectively.

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All of the experiments were performed with three independent samples.

3. Results 3.1 Antiproliferative assay for Aza and NAC Dose-response curves were used to determine the ICs of Aza and NAC to evaluate the inhibitory effect on proliferation against the Jurkat leukaemia cell

9 line. The antiproliferative effects of Aza are shown in Figure 1-A. Aza (0.08 to 100 μM) significantly inhibited proliferation compared to that of the control cells; the IC25 and IC50 were 1.03 and 2.06 M, respectively (figure 1-B). As shown in Figure 1-C, 0.156 to 40 mM NAC significantly inhibited proliferation compared to that of untreated control cells. We found that low doses of NAC are

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antiproliferative in Jurkat cells. The IC25 and IC50 for NAC were 3.0 and 6 mM, respectively (figure 1-D). Both drugs inhibit cell proliferation in a dose-dependent

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manner. We found that the doses of NAC from 0.156 mM and higher inhibited proliferation. When NAC and Aza were combined at their IC25 concentrations, the

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inhibition of cell proliferation was significantly higher than that of each individual

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drug, even more than that of Aza at its IC50 concentration.

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3.2 Apoptosis induced by Aza and NAC

Our next goal was to evaluate the apoptotic effects of the individual and

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combined drugs. Jurkat cells were treated as indicated in Table 1, and each treatment induced significantly higher apoptosis than the control group. There was no significant difference among individual drug treatments. However, when 3 mM NAC and 1.0 µM Aza were used simultaneously, the apoptotic effect

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significantly increased compared to that of the individual drugs, except for that of 2.0 µM Aza. This finding suggests that a half-dose of Aza and a safe dose of NAC are as efficient as a full-dose of Aza (Figure 2). In addition, we aimed to determine if the drugs induced apoptosis or necrosis. In Figure 3, the apoptosis graphs show that apoptosis was more predominant than necrosis was. These

10 results suggest that the Aza and NAC drug combination would minimize side effects because necrosis can promote unwanted reactions, such as inflammation.

3.3 Multicaspase activity We determined by flow cytometry the multicaspase (1, 3, 4, 5, 6, 7, 8, and 9)

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activity induced by the drugs, individually and combined. Figure 5 shows the

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multicaspase activity percentage for each experiment. Drug treatments induced significantly higher multicaspase activity compared to that of control cells.

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Individually, Aza (1.0 μM) and NAC (3 mM) produced a similar effect on caspase

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activity. Moreover, 2.0 µM Aza and the combination of 3 mM NAC and 1.0 µM Aza significantly increased multicaspase activity compared to that of 3 mM NAC

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and 1.0 µM Aza individually. Caspase activation is related to apoptosis, which is reflected in the similar trends obtained between the numbers of apoptotic cells

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and the multicaspase activities (Figure 3 vs. Figure 4). These assays allowed us to characterize the type of controlled cell death as mostly apoptosis.

3.3 Cell cycle analysis

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Aza induces cell cycle arrest at G0/G1 and G2/M, with predominance at G2/M. Based on this fact, we tested the effect of NAC plus Aza and NAC individually on cell cycle progression. As shown in Figure 5, all treatments induced cell cycle arrest at G0/G1, which is consistent with previous reports for Aza. NAC increased the cell population at G0/G1 similar to the effect of 1.0 µM Aza. Aza (2.0 μM) was the most effective at inducing cell cycle arrest at G0/G1, while 3 mM NAC plus

11 1.0 µM Aza had the same effect as the individual drugs did. However, 3 mM NAC plus 1.0 µM Aza significantly increased the cell population at G 2/M compared to that of all groups. Aza and NAC complement each other in the antiproliferative effect.

3.4 Aza increases and NAC prevents ROS

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Previous reports have indicated that increased ROS levels play an important role

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in drug-mediated oxidative cell death. Therefore, decreased ROS levels could protect cancer cells. We evaluated the effect of Aza and NAC on ROS production

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to determine if NAC exerts antiproliferative and apoptotic effects by increasing

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ROS levels. ROS levels are presented as fold changes relative to control. We determined that 1.0 and 2.0 µM Aza increased ROS levels higher than control

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and 3 mM NAC. NAC (3 mM) showed no change compared to that of control, but, when used together with 1.0 µM Aza, NAC prevented the increase of ROS

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(Figure 6). These findings suggest that NAC exerts antiproliferative and apoptotic effects without increasing ROS, but preventing the induction of ROS by Aza.

4. Discussion

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The thiopurine Aza is a common treatment in the clinic for T-ALL. However, its use is limited because of its adverse effects. One of the reasons for the increased systemic toxicity of Aza compared to that of other thiopurines is that Aza requires more biotransformation reactions to form its active, thioguanine nucleotide metabolites, necessitating higher doses [21]. Biotransformation of Aza requires SH groups present in GSH or cysteine [22], which are diminished in cancer cells due to

12 oxidative stress that increases the production of free radicals [23]. Thus, Aza biotransformation in cancer cells is limited. The use of antioxidants containing SH groups, such as NAC, which also favours GSH synthesis, is limited because it could protect cancer cells and reduce the chemotherapeutic effect.

In this study, we determined that NAC has an antiproliferative effect on Jurkat cells.

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This finding is consistent with the study by Mansour et al. in 2014, which determined

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that NAC inhibited proliferation in the same cell line [17]. Once we verified the antiproliferative effect of NAC, the effect of 1.0 µM Aza + 3 mM NAC was evaluated.

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The combination of both drugs exerted a significantly higher antiproliferative effect

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compared to that of 2.0 µM Aza; therefore, NAC does not exert a protective effect on cancer cells, but it interacts with Aza additively. The antiproliferative mechanism

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of this interaction may be twofold. The first is due to the antiproliferative effect of Aza, previously shown by Elion in 1989, which results from the inhibition of cyclin-

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dependent kinases 1 and 4 and favours cell cycle arrest at G 2/M and G0/G1 phases [19]. The second is the incorporation of thioguanine nucleotides into DNA, preventing replication and inhibiting purine synthesis [21]. NAC can decrease the expression of cyclins A, B, and D [18], thereby exerting an antiproliferative effect. In addition, NAC

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provides SH groups and also favours the synthesis of GSH, which is necessary for initial Aza conversion to 6-MP. Furthermore, ROS can oxidize active metabolites of Aza and diminish its action; however, because NAC is an antioxidant capable of reducing ROS in Jurkat cells [24], it favours the protection of active Aza metabolites and maintenance of the antiproliferative effect. A ROS decrease does not affect the action of Aza because Aza does not use oxidative cell death as its primary

13 mechanism, as most chemotherapeutic drugs do.

Apoptosis is an event of paramount importance for the cell, allowing the elimination of potentially dangerous or dysfunctional cells [25]. However, cancer cells utilize mechanisms that allow them to continue to proliferate and evade cell death. Chemotherapeutic agents act through different mechanisms to induce apoptosis. In

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the present study, we observed that both drugs, Aza and NAC, exerted an apoptotic

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effect when used separately and when combined. Aza acts by two previously elucidated mechanisms. The first is by induction of DNA mismatch repair (MMR),

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which induces apoptosis when the mismatches are significant, such as GC → AT

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transitions [21]. The active Aza metabolite, TGTP, inhibits the small GTPase Rac1, which is involved in anti-apoptotic signalling pathways, such as those using Akt.

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When this pathway is blocked, the cells are not capable of inhibiting apoptotic stimuli [8]. Moreover, the anticancer activity of antioxidants has received little study, as

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many reports have indicated that antioxidants protect cancer cells and promote their survival. The mechanism of the apoptotic effect of NAC observed in our study was not fully elucidated, though we propose that NAC acidifies the medium and triggers apoptosis by promoting executor caspase 3 activity. In 2002, Hentze reported that

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depleted GSH inhibits apoptosis in Jurkat cells; 80% of cells that had normal levels of GSH entered the apoptotic process and increased the activity of initiator and executor caspases, thus indicating the importance of GSH in initiating this process of cell death [26]. NAC is a precursor of GSH. Therefore, when administering NAC, GSH levels increase [18], and the cell can initiate apoptosis when a stimulus is applied. This suggests that NAC plus Aza complement each on inducing a G2/M

14 arrest, which is favourable for the induction of apoptosis because the cell has already synthesized genetic material and is unable to repair further injuries. Both drugs favoured the induction of apoptosis with minimal necrosis. This result was determined by measuring multicaspase activities (1, 3, 4, 5, 6, 7, 8, and 9) by flow cytometry. The percentage of apoptosis induced by each drug is similar to the percentage of multicaspase activity, corroborating the type of cell death as

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apoptosis. This finding is important because a drug that promotes necrosis would be

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harmful due to the release of cell contents, including pro-inflammatory cytokines that propagate inflammation. When Aza and NAC are administered simultaneously, the

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combination acts through different mechanisms to induce apoptosis. This interaction

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involves nucleophilic attack of NAC on Aza to generate the active metabolite, 6-MP, and thus enhance its bioavailability. In addition, NAC neutralizes ROS, protecting

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Aza and its metabolites from oxidation.

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Due to their chemotherapeutic responses, ROS play important roles in the development of cancer. Maintaining redox homeostasis is important, especially in leukaemia cells, which have significantly higher levels of ROS than normal cells do because of alterations in the pro-oxidant and antioxidant pathways. These high

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levels of ROS induce genomic instability, survival, growth, and motility [23]. The major source of ROS is the mitochondria; once mutations in mitochondrial DNA have occurred, the expressions of electron transport chain proteins and antioxidant enzymes decrease. This favours a decoupling of electron transport and a sustained increase in ROS levels [20]. This process is also the mechanism by which Aza significantly increases ROS levels, by damaging mitochondrial DNA due to the

15 incorporation of thioguanine nucleotides. NAC-treated cells generated similar levels of ROS as control cells, which did not exacerbate cytotoxicity but favoured redox homeostasis. When used in combination with Aza, the antioxidant effect of NAC predominated to reduce the generation of Aza-mediated ROS. As noted above, Aza and NAC act together to inhibit cell proliferation and induce apoptosis, suggesting that they are not acting by oxidative

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mechanisms. Reduction of ROS levels leads to a decrease in signalling pathways

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related to proliferation and survival. This reduction is possible because ROS act as second messengers, facilitating signalling involving kinases, phosphatases, and

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transcription factors [27]. According to our results, the administration of NAC should

the systemic cytotoxicity of Aza.

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allow for the use of lower chemotherapeutic doses of Aza and thus should diminish

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The use of antioxidants is taboo in cancer treatment, and it remains unclear if they are beneficial to cancer cells or favour their death or cell cycle arrest. This should be

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validated in each type of cancer and with each drug. In this study, the antiproliferative effect of NAC in Jurkat cells was determined and represents a system to test its adjuvant effect with chemotherapeutics in vitro. NAC and Aza interact and exert an antiproliferative effect by inducing apoptosis and cell cycle arrest, in addition to

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reducing ROS. This work sets a precedent to continue studying the mechanisms of antioxidant-chemotherapeutic interactions including the quantification of potential synergism or additivism. It presents a new approach in the development of treatments for T-ALL which should be investigated further for the sake of reducing chemotherapy-related mortality.

16 Conflict of Interest

Thank you for the opportunity to resend our manuscript entitle: Antiproliferative and apoptotic interaction between azathioprine and N-acetylcysteine in acute lymphoblastic leukaemia Jurkat cells, and we like to infom you about the not

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conflict of interest in our manuscript.

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Acknowledgements

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We thank FOFI-UAQ FCQ-2016-05 for financial support and Peter Karran, PhD for

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19 Tables

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Table 1. Drug treatment design for cell cycle, inhibitory concentrations, ROS measurement, multicaspase and apoptosis assays Control Group 1 Group 2 Group 3 Group 4 Aza IC50 ----+ ------------Aza IC25 --------+ ----+ NAC IC25 ------------+ +

20 Figure Captions

Figure 1. Antiproliferative effect of NAC and Aza. A) Decreased proliferation of Jurkat cells induced by Aza, after 24 h incubation. B) Logarithmic dose-response curve of Aza. C) Reduction in cell proliferation induced by NAC, after 24 h incubation. D)

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Logarithmic dose-response curve of NAC. * Significant difference compared to the

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control, Dunnett, p <0.05.

21 Figure 2. Apoptotic effect of Aza and NAC, alone and combined. Percentage of apoptosis induction is indicated for each group. Different letters indicate significant

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differences between groups, ANOVA and Tukey post hoc, p <0.05.

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Figure 3. Flow cytometry apoptotic graphs. Each quadrant of the graph indicates the type of apoptosis, as well as necrosis. A) Control, B) Aza 1.0 μM, C) Aza 2.0 μM, D) NAC 3 mM, E) Aza 1.0 μM + NAC 3 mM. All treatments induced apoptosis predominantly.

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Figure 4. Multicaspase activity percentage induced by different treatments. All treatments significantly favoured multicaspase activity compared to control. Significant differences are presented as different letters. Aza 1.0 M + NAC 3.0um and Aza 2.0 M are significant different from all the other groups. Aza 1.0M and NAC 3.0 mM are not different each other. ANOVA and post hoc Tukey, p <0.05.

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Figure 5. Cell cycle phase analysis. Graphs show the effect of different treatments on the G0/G1 and G2/M phases of the cell cycle. Percentages of cells arrested in

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each phase are presented. Significant differences between groups are represented as different letters, Tukey, p <0.05.

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Figure 6. Aza induces elevated levels of ROS while NAC maintains the levels, similar to the negative control. Data are presented as fold change relative to control.

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Different letters indicate significant difference, ANOVA and post hoc Tukey, p <0.05.