6‐Shogaol induces apoptosis in acute lymphoblastic leukaemia cells by targeting p53 signalling pathway and generation of reactive oxygen species

Abstract Combination therapies, using medicinal herbs, are broadly recommended to attenuate the chemotherapy adverse effects. Based on our previous findings considering the anti‐leukaemic effects of ginger extract on acute lymphoblastic leukaemia (ALL) cells, the present study was aimed to investigate the anti‐cancer role of this pharmaceutical plant on ALL mice models. Moreover, we worked towards identifying the most anti‐leukaemic derivative of ginger and the mechanism through which it may exert its cytotoxic impact. In vivo experiments were performed using five groups of six C57BL/6 nude mice, and the anti‐leukaemic activity of ginger extract alone or in combination with methotrexate (MTX) was examined. Results showed increased survival rate and reduced damages in mice brain and liver tissues. Subsequently, MTT assay demonstrated synergistic growth inhibitory effect of 6‐shogaol (6Sh) and MTX on ALL cell lines and patients primary cells. Eventually, the molecular anti‐neoplastic mechanism of 6Sh was evaluated using Bioinformatics. Flow cytometry illustrated 6Sh‐mediated apoptosis in Nalm‐6 cells confirmed by Western blotting and RT‐PCR assays. Further analyses exhibited the generation of reactive oxygen species (ROS) through 6Sh. The current study revealed the in vivo novel anti‐leukaemic role of ginger extract, promoted by MTX. Moreover, 6‐shogaol was introduced as the major player of ginger cytotoxicity through inducing p53 activity and ROS generation.

are major polyphenols in fresh and dried ginger, respectively. These compounds are responsible for different bioactivities of ginger. 12 Investigating the cytotoxic effect of gingerols and shogaols on human A549, SK-OV-3, SK-MEL-2, HCT15 and PC3 tumour cells has shown that 6-shogaol (6Sh) possesses the most potent inhibitory effect among these derivatives. 13,14 In addition, different studies indicated that growth inhibitory effect of 10-gingerol is more than other gingerols in cancer cells, including melanoma, promyelocytic leukaemia, breast, lung, ovarian and colon cancers. 13,[15][16][17][18] However, none of these derivatives are proved to have impact on ALL patients. Several pathways are suggested through which ginger derivatives may apply their anti-cancer effects. There are published data indicating the role of ginger derivatives in increasing the expression levels of the tumour suppressor protein p53. 19,20 Moreover, 6Sh is claimed to target cell-cycle arrest by decreasing cyclinD1/3, survivin and cMyc in prostate and non-small cell lung cancers. 21,22 Interestingly, it has been shown that 10-gingerol may induce apoptosis and inhibit proliferation through PI3K/AKT, AMPK, mTOR and p38MAPK in cervical and breast cancer cells, respectively. 20,23 Fatty acid synthase (FASN) is another key regulator which may be responsible for leukaemia drug resistance. According to our previous study FASN expression levels are upregulated in ALL drug resistant children, and they may be reduced by cells treating with ginger extract. 24 It would be interesting to know which ginger derivative could play role in the FASN molecular pathway. Moreover, it is shown that FASN overexpression may inhibit TNFα expression, caspase 8 and NF-κB activation in several cancer cells. 25 However, the exact plant derivative which may influence its apoptotic effect is yet to be identified.
The anti-cancer effect of ginger extract was documented in ALL cell lines and primary cells by our group. 26 However, its inhibitory effect on ALL animal models was remained unknown. In the current study, we examined the anti-leukaemic activity of ginger extract, as a single agent or combined with MTX, on the cell linederived xenograft mouse models. Additionally, the cell growth inhibitory effects of 6-shogaol and 10-gingerol were compared.
Subsequently, the most anti-leukaemic ginger derivative was selected, and combined therapies with MTX were investigated on the Nalm-6 cell line and ALL primary cells. Finally, bioinformatics and in vitro assays were performed to identify the molecular mechanisms through which, 6-shogaol may exert its cytotoxic impact on leukaemic cells. The rationale for selecting MTX in this project was its broad clinical use in every stages of ALL treatment and to validate our interesting in-vitro results of anti-MTX resistant effects of ginger extract 26 in ALL mice models.

| Animal model and treatments
Four-to six-week-old female athymic nude mice (C57BL/6 nude) were purchased from Pasteur Institute of Iran. Animals were housed in the Specific-Pathogen-Free Animal Laboratory, Department of Biology, University of Isfahan. The protocol was approved by the university's Ethics Committee on Animals Handling (Permission number: IR.UI.REC.1396.056). Mice were kept in the laboratory for two weeks without testing for acclimation to the new environment. Transplantation was initialized by giving mice 300 mg/kg cyclophosphamide intraperitoneally. Three days later, they were injected with 15 × 10 6 CCRF-CEM cells in 100 μL FBS, subcutaneously. In order to confirm leukaemia engraftment, flow cytometry was performed on mice blood samples by using antibodies against CCRF-CEM cell-line CD markers. Separate treatment protocols were started the day after engraftment. Mice were divided into four groups of six. Two groups were injected intraperitoneally with 80 mg/kg ginger extract five times a week or 5 mg/kg MTX once a week. The third group was given both of the aforementioned regimens and the fourth group was treated with vehicle. Mice were sacrificed two months post-transplantation.
Liver, brain and bone marrow were collected following slaughter.
Liver and brain tissues were stained with H&E, and bone marrow samples were stained with Wright-Giemsa stains along with the conventional methods.

| Cell lines
CCRF-CEM (T-ALL) and Nalm-6 (B-ALL) human cell lines were obtained from Pasteur Institute. R-CCRF-CEM (a T-ALL subline resistant to MTX) (National patent number: 98824) and RN95 (a B-ALL cell line derived from an Iranian female child with relapsed ALL) (National patent number: 100281) cell lines were developed in-house. Cells were maintained in RPMI1640 containing 10% heat-inactivated FBS and 1% penicillin/streptomycin. For Nalm-6 and R-CCRF-CEM cell lines, culture medium was additionally supplemented with 1% l-glutamine and 1.2 μmol/L MTX, respectively. Cells were incubated at 37°C and an atmosphere of 5% CO 2 .

| Cell treatment
Cell lines were cultured in 96-well tissue culture plates at a density  26

| In vitro viability/proliferation assays
To determine cell proliferation and viability, MTT assay was used. At the end of the incubation time, 10 μL of MTT solution (5 mg/mL) was added to wells. Following incubation at 37°C for 3hours, supernatant was removed and formazan crystals were solubilized by adding 100 μL DMSO into each well. Absorbance was recorded at 492 nm by using a stat fax-2100 microplate reader (Awareness Technology, Inc).

| Functional enrichment and pathway analysis
Sufficient literature mining was performed to identify all the genes whose expressions were significantly affected by 6Sh. Subsequently, gene-enrichment and functional annotation analyses were performed using Functional Annotation Tool (the visualization and integrated discovery (DAVID) Bioinformatics database (https://david.ncifc rf.gov/) to gather pathways related to the target genes and proteins.

| Apoptosis assay
The FITC Annexin-V Apoptosis Detection Kit with PI was used to de-

| Western blot analysis
Western blot analysis was performed to investigate the potential role of p53 and p21 proteins in the cytotoxic effects of 6Sh. Lysis buffer was used to extract total proteins from cells, and Bradford protein assay was done to determine the concentration of proteins.
30 μg of protein extracts were separated on a gradient polyacrylamide gel and transferred from the gel to a nitrocellulose membrane.
Membrane was blocked with 5% skimmed milk in 1 × TBS/Tween solution for 1 hour followed by incubation with primary antibodies for 1.5 hour at room temperature and overnight at 4°C. Mouse anti-p53, anti-p21 and antiβ actin monoclonal primary antibodies were used at 1:250, 1:100 and 1:250 dilutions, respectively. After washing with 1 × TBS/Tween solution, membrane was incubated with secondary antibody goat anti-mouse immunoglobulins/HRP (1:1000) at room temperature for 1 hour. Finally protein detection was performed using sensitive radiology films in a dark room using ECL Prime Western Blotting Detection Reagent.

| RNA isolation and real-time PCR
Extraction of total RNA, DNase treatment and cDNA synthesis were performed using TRIzol reagent and PrimeScript™ RT reagent Kit F I G U R E 1 The outcome of ginger extract treatment, alone or in combination with MTX, in the mouse models of ALL. After 14 d of adaptation, C57BL/6 nude mice were intraperitoneally injected with cyclophosphamide (300 mg/kg). 72 h later, CCRF-CEM cell transplantation was performed subcutaneously and leukaemia engraftment was authenticated by flow cytometry (Data not shown). One group of six remained untransplanted (UT). ALL mice were divided into four groups of six. Treatment was administered by intraperitoneal injections of 80 mg/kg ginger extract (AG), 5 mg/kg MTX (AM) and a combination of 80 mg/kg ginger extract and 5 mg/kg MTX (AGM). The fourth group was treated with vehicle (A). 60 d after transplantation, mice were sacrificed. (A,B,C) Mice treated with combined concentrations of ginger extract and MTX showed significant decrease in the number of bone marrow (BM) blasts, and their survival rate was increased compared with those which got single treatments. There was no significant difference in percentage body weight between different groups of transplanted mice. (D) Brain and liver tissue sections of drug-and vehicle-treated ALL mice models were fixed in 10% formalin, paraffin-embedded and stained with haematoxylin and eosin dyes. Results showed that single and combined treatment of the ALL mice models with ginger extract showed decreased histopathological damages in the brain and liver tissues. Some areas of damage are marked by arrows. For statistical analysis, Image J software was used. Results were consistent with pathological interpretation of the tissues. Three sections were examined in each mouse. Values are mean ± SEM, *P < .05, **P < .01, ***P < .001 (Takara) in accordance with the manufacturer's protocols, respectively. Gene expression was determined using Ampliqon RealQ Plus Master Mix Green high ROX™. All PCR reactions were done in triplicates during two independent experiments by a Chromo4™ system (Bio-Rad). The sequences of the primers utilized in the current study are mentioned in Table 1. Relative quantifications of gene expressions were calculated by 2 −ΔΔCt method.

| Detection of the ROS
In order to detect ROS production, cells (5 × 10 5 cell/well) were incubated with 500 μL FBS-free medium containing 40 μmol/L DCFH-DA in the dark. 15 minute later, 500 μL ice-cold PBS were added to each well and the fluorescence was monitored by a Partec CyFlow ML Flow Cytometer supported by FloMax software in the FL-1 channel.
Data were plotted on histograms using FlowJo software version 10.

| Combination index
Drug interaction was measured using the combination index (CI), where CI˂0.9, 0.9 ˂CI<1.1, and CI>1.1 show synergistic, additive and antagonistic effects, respectively. CI value was calculated according to the equation stated below: (D x ) com1 (or (D x ) com2 ) was the drug concentration in the combination treatment that inhibited×% proliferation, and (D x ) 1 (or (D x ) 2 ) was the drug concentration 1 (or 2) alone, that triggered the same percentage inhibition.

| Statistical analysis
Image J software was used for histomorphometric analyses. In this software, haematoxylin and eosin pathological images were converted to RBG format and the percentage of tissue damages were calculated by determining a specific threshold. All statistical analyses including one-way ANOVA and t-tests were performed using the GraphPad Prism version 6 software (GraphPad Prism Inc). All data were reported as mean ± standard error of mean (SEM). P ˂ .05 was considered to be statistically significant.

| Effects of ginger extract, alone or in combination with methotrexate on ALL mouse models
After a two-week adaptation period, mice were injected with cyclophosphamide and then with CCRF-CEM cells. Transplantation was confirmed using flow cytometry (Data not shown). Subsequently, mice were treated with ginger extract and MTX, alone or in combination.
Two months after transplantation, mice were sacrificed. The malignant cells percentage invasion in bone marrow was calculated using Wright-Giemsa Stain protocols. Results showed significant decrease in bone marrow blasts when mice were treated with ginger extract/MTX compared with the vehicle-treated mice [0.45% ± 0.17% vs 4.33% ± 1.07% (mean ± SEM), respectively, P < .05] ( Figure 1A). Moreover, combined treatment with ginger extract and MTX prolonged significantly the ALL mice percentage survival rate compared with the single treatments (50% vs 20% and 25%, respectively, P < .001) ( Figure 1B). Loss of body weight was not evident in any of the animal groups ( Figure 1C).

| Effects of ginger derivatives on ALL cell lines viability
Two T-ALL cell lines; one purchased, CCRF-CEM; and one in-house generated MTX-resistant subline, R-CCRF-CEM, and two B-ALL cell lines; one purchased, Nalm-6; and one patient-derived in-house generated cell line, RN95, were chosen to evaluate the impact of the ginger derivatives: 6-shogaol and 10-gingerol on ALL cell viability.
Plates were coated with 2 × 10 4 cells per well in triplicates. Cell viability was assessed using MTT assays after 72 hours (for CCRF-CEM, R-CCRF-CEM and RN95) and 96 hours (for Nalm-6). Incubation times were chosen according to the cell lines doubling times (Figure 2A,B).
The half-maximal concentration of proliferation inhibition (IC50) for each of the abovementioned derivatives is presented in Table 2.
Since Nalm-6 was the most vulnerable cell line to ginger derivatives at high concentrations (Figure 2A,B), and B-ALL is the most frequent   Figure 3A).
The calculated combination index (CI) from dose-effect data of single and combination treatments, indicated synergistic effect for 6Sh and MTX on Nalm-6 (CI = 0.76).

| Effect of 6Sh/MTX combination treatment on patient primary cells
The anti-proliferative effect of 6Sh/MTX combination was studied on the fresh primary malignant samples collected from eight de novo and two relapsed patients with childhood ALL ( Table 3). Isolation of mononuclear cells from patients' whole blood/bone marrow, at diagnosis, was accomplished by using density gradient Ficoll media.

| Pathway analysis through DAVID database
The 6-shogaol regulated target genes and proteins were identified by studying literature reviews and analysed using DAVID Bioinformatics database to find 6Sh related pathways. Results demonstrated that 6Sh induces apoptosis through different pathways including intrinsic and extrinsic apoptosis pathways, calcium signalling pathway, protein processing in endoplasmic reticulum and p53 signalling pathways (Table 4, Figure 4).

| Effect of 6-shogaol on inducing apoptosis
To evaluate the given information by bioinformatics data analysis and investigate whether 6Sh could trigger apoptosis in Nalm-6, cells
Results of the performed trypan blue assay to quantify live cells by labelling dead cells were also consistent with results of the apoptosis assay (Data not shown).
To determine the possible targets through which 6Sh may exert its effects on cell-cycle arrest and apoptosis, Nalm-6 cells were seeded into 6-well plates and treated with 200 µmol/L 6Sh for 48 hours or 96 hours, respectively. Cells total protein was extracted, and Western blot analysis was performed using p53 and p21 primary antibodies. Increased expression levels of p53 and p21 were observed in 6Sh-treated cells compared with the vehicletreated cells. However, Western blot analysis demonstrated that 6Sh did not increase p53 protein expression in MNCs ( Figure 5B).
On the other hand, total RNA was isolated, cDNA was synthesized and the expression profile of genes involved in apoptosis, including PUMA and FASN, were investigated using real-time PCR. The expression level of PUMA gene was increased in the treated compared with the vehicle-treated cells by 1.70 ± 0.03 fold (P < .01).

| Effect of 6-shogaol on generating reactive oxygen species
To further explore the mechanism by which 6Sh may exert its impact on cell apoptosis, ROS levels were calculated upon the treatment of Nalm-6 cells by this pharmaceutical drug. The generated ROS level, as assessed by 2′,7′-Dichloro-fluorescin diacetate (DCFH-DA) staining, was particularly higher in 6Sh-treated cells than their relative controls [1.49 ± 0.02 vs 1 (mean ± SEM; n = 2), P < .05] ( Figure 6A).
This effect was reversed by the addition of N-acetyl cysteine as an ROS scavenger ( Figure 6B).

| D ISCUSS I ON
The current study revealed increased anti-cancer effects of MTX when combined with ginger extract in ALL mice models compared with single treatment. This finding was supported by our previously published in vitro experiments. 26 Moreover, results demonstrated that combination of ginger extract with MTX may reduce the brain and liver damages attributed to chemotherapy (Figure 1). It was currently illustrated that co-administration of ginger extract and doxorubicin in breast cancer mouse models may increase the mice survival rate and decrease the tumour volume compared with single treatment with doxorubicin. 27 On the other hand, the healing effects of ginger extract, as a single pharmaceutical drug, were confirmed for non-cancer pathological conditions including the neuropathological damages produced in diabetics 28 and liver lesions induced by cytotoxic drugs. 29,30 In order to identify the specific derivate of ginger playing the most effective anti-leukaemic role of this herbal medicine, two of its most active anti-cancer derivatives were selected according to the review literature 31  To investigate the signalling pathway through which 6-shogaol could exert its cytotoxic impact, bioinformatics analysis using DAVID Functional Annotation Tool was performed. Results determined that 6-shogaol may contribute to cell death through targeting apoptosis and activating p53 (Figure 4) in the Nalm-6 cells, where p53 was previously shown to be wild type (unpublished data). Followed by the in vitro quantitative real-time PCR assays and Western blotting, it was shown that the tumour suppressor, p53, upregulates genes involved in cell-cycle arrest and apoptosis including p21 and PUMA, respectively ( Figure 5). A balance between p21 and PUMA has been recently known in response to exogenous p53 expression in human colorectal cancer cells. 33 Moreover, ROS assay showed that 6Sh may generate reactive oxygen species in the leukaemic cell line ( Figure 6). It was previously declared that ROS may cause DNA damage, contributing to p53 activation followed by apoptosis. 34,35 Interestingly, FASN overexpression is recently introduced by our group as a negative prognostic factor for paediatric ALL. 24 Additionally, the association of FASN with resistance to chemotherapeutic drugs was previously determined. 36 Supportingly, published data revealed that FASN inhibitors may block proliferation and induce apoptosis in CML cell lines. 37  Additionally, DNA damage leads to the activation of p53, which, as a transcription factor, upregulates the expression levels of the apoptotic genes and those involved in the cell-cycle arrest, including PUMA and p21, respectively. = activation, = inhibition, = induction, = suppression, = gene expression resistance in paediatric ALL. However, mechanistic studies are required to confirm the causative impact of 6Sh on FASN.
Taken together, the current study is the first research demonstrating the anti-cancer effect of ginger extract on ALL mice models.
Moreover, the specific anti-leukaemic derivative of this pharmaceutical medicine is identified. 6-Shogaol is introduced as a novel naturally occurring small molecule, which may selectively and synergistically amplify the cytotoxicity of MTX on the malignant lymphoblasts.
6Sh may exert its anti-neoplastic effects through activation of p53 and generation of ROS, leading to apoptosis and cell-cycle arrest, or downregulation of fatty acid synthesis, respectively (Figure 7). Since cancer cells often use specific signalling pathways for growth and proliferation, identifying 6-shogaol as the most effective derivative of ginger and illuminating its molecular anti-leukaemic mechanism can be an important step in targeted therapy.

ACK N OWLED G M ENT
Authors would like to thank Dr. Jamal Moshtaghian for his generous help with animal experiments; Dr. Ardeshir Talebi for his insight and expertise with the analysis of the pathological data and all patients and their parents who participated in the present study.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during the current study are included in this article. Supplementary and raw data are available if required.