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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 9  |  Page : 324-330

Synergistic effect of thymoquinone and melatonin against breast cancer implanted in mice


Department of Clinical Pharmacy and Therapeutics, Applied Science University, Amman, Jordan

Date of Web Publication29-Jun-2018

Correspondence Address:
Wamidh H Talib
Department of Clinical Pharmacy and Therapeutics, Applied Science University, Amman
Jordan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.235349

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 > Abstract 

Aim: To test the anticancer potential of a combination of thymoquinone (TQ) and melatonin (MLT) against breast cancer implanted in mice.
Materials and Methods: The antiproliferative activity of TQ, MLT, and their combination was tested against mouse epithelial breast cancer cell line (EMT6/P) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The combination index (CI) was calculated using isobolographic method. Balb/C mice were transplanted with EMT6/P cell line and in vivo antitumor activity was assessed for TQ, MLT, and their combination. Changes in tumor size were measured for each treatment. Histological examination of tumor sections was performed using standard hematoxylin/eosin staining protocol and TUNEL colorimetric assay was used to test the apoptosis induction ability for all treatments. Immunohistochemical staining was used to detect vascular endothelial growth factor (VEGF) expression in tumor section and ELISA was used to measure serum levels of interferon gamma (INF-γ) and interleukin-4. Serum levels of the liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were used as biomarkers of hepatotoxicity of the combination therapy.
Results: Synergistic anticancer effect was observed between TQ and MLT with CI value of 0.552. The combination of TQ and MLT caused a significant decrease in tumor size with a percentage cure of 60%. The combination therapy induced extensive necrosis, increased apoptosis rate, and decreased VEGF expression in tumor sections. Serum levels of INF-γ were increased in mice treated with combination therapy and AST and ALT levels were close to their normal values.
Conclusions: The combination TQ and MLT act synergistically to inhibit breast cancer implanted in mice. The anticancer effect of this combination is mediated by induction of apoptosis, angiogenesis inhibition, and activation of T helper 1 anticancer immune response.

Keywords: Apoptosis induction, combination anticancer therapy, melatonin, natural products, thymoquinone


How to cite this article:
Odeh LH, Talib WH, Basheti IA. Synergistic effect of thymoquinone and melatonin against breast cancer implanted in mice. J Can Res Ther 2018;14:324-30

How to cite this URL:
Odeh LH, Talib WH, Basheti IA. Synergistic effect of thymoquinone and melatonin against breast cancer implanted in mice. J Can Res Ther [serial online] 2018 [cited 2018 Nov 19];14:324-30. Available from: http://www.cancerjournal.net/text.asp?2018/14/9/324/235349


 > Introduction Top


Breast cancer is one of the common cancers worldwide. Recent estimates showed a continuous rise in the number of patients living with this type of cancer with more than 1 million new cases diagnosed every year.[1]

Emphasis on breast cancer is due to being the most common invasive malignancy in women.[2] Difficulty in its treatment rises from the high risk of disease recurrence and the negative impact it has on long-term survival rate and quality of life.[3]

Traditional cancer therapies including chemotherapy, radiation and surgery exhibited limited efficiency in treating cancer and the high mortality rate among cancer patients is an indication of such limitation.[4] In addition, these therapies are always accompanied by severe side-effects such as: Nausea, vomiting, anemia, weight loss, hair loss, fever, fatigue, excessive sweating, pain, lymphedema, and bone marrow suppression.[5]

Thymoquinone (TQ) is the active ingredient isolated from Nigella sativa and several in vitro and in vivo studies reported anti-inflammatory, anti-oxidant, and anticancer activities of this compound. The anticancer effect of TQ is mediated mainly by cell cycle arrest, induction of apoptosis, and synergism with other therapies.[6],[7]

Since cancer development depends on multiple mechanisms, it is thought that effectiveness of anticancer agents may be increased when multiple agents are used in optimal combinations.[8] Low toxicity and improved anticancer activity were observed in combinations consisting of TQ and diosgenin.[9] Another combination of TQ and tamoxifen showed high ability to induce apoptosis in breast cancer cells.[10] Moreover, a combination consisting of TQ and the anticancer drugs CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) caused enhanced anticancer activity and reduced liver toxicity in breast cancer bearing mice.[11]

Melatonin (MLT) is a hormone produced by the pineal gland and is responsible for the biological clock. This hormone has oncostatic and anticancer activities through different mechanisms including the stimulation of the immune system and shifting the immune response toward cancer inhibition mechanisms. MLT showed effectiveness in reducing tumor growth and cell proliferation as well as inhibition of angiogenesis against breast cancer.[12] Previous reports have described a decrease in the production of vascular endothelial growth factor (VEGF) protein induced by MLT in human pancreatic carcinoma cells.[13] Suppression of tumor angiogenesis and diminished formation of capillaries were also reported in mice bearing renal adenocarcinoma treated with MLT.[14]

Targeting multiple steps in cancer progression is essential to provide more effective therapies. Therefore in this study, we employed a combination therapy consisting of MLT and TQ to target breast cancer implanted in mice. We hypothesized that MLT and TQ may work synergistically against breast cancer implanted in mice using different mechanisms including induction of apoptosis, angiogenesis inhibition and activation of T helper 1 (Th1) anticancer immune response.


 > Materials and Methods Top


Preparation of thymoquinone and melatonin working solutions

Stock solutions of 1 M TQ (Sigma, USA) and 6 mM MLT (Sigma, USA), were dissolved in dimethyl sulfoxide (AZ Chem., Canada), stored at 4°C, and diluted in fresh medium just before use.

Animals

This study was carried out according to standard ethical guidelines, and all of the experimental protocols were approved by the Research and Ethical Committee of the Faculty of Pharmacy, Applied Science University.

The study was carried out on 40 Balb/C female mice ranging between 4 and 6 weeks old (weight 21–25 g/mouse). Mice were kept in separate cages with wooden shavings as bedding. The environmental parameters were: Temperature around 25°C, 50–60% humidity, with alternating 12-h light/dark cycles and continuous air ventilation.

Cell line and culture conditions

EMT6/P cell line was purchased from the European Collection of Cell Cultures. Cells were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, 0.1% gentamycin, and 1% penicillin-streptomycin solution. Cultured cells were incubated at 37°C in 5% CO2 and 95% humidity.

Antiproliferative assay

Cells were harvested, washed and suspended in tissue culture media. Cells were dispensed (100 μl/well) into 96-well tissue culture plates (flat bottom) at an optimized concentration of 10,000 cells/well in a complete medium. After 24 h, cells were treated in triplicates with different concentration of TQ (10–800 μm), MLT (0.1–5 mm), and with different combinations of TQ + MLT resulting in a total volume of 200 μl, and incubated for 48 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Where 100 μl of culture media were removed from each well and replaced with 10 μl of thiazolyl blue tetrazolium solution (Sigma, USA) and incubated at 37°C for an additional 3 h. MTT solubilization solution (Sigma, USA) was added (100 μl/well), mixed and incubated for another hour. Absorbance was measured at 595 nm by ELISA Microplate reader (Biotek, USA). Cell viability (%) was calculated for all groups compared to control sample. Untreated cells were used as a negative control and cells treated with vincristine sulfate were used as a positive control.

Calculation of combination index

The interaction between TQ and MLT was assessed using isobolographic method and the combination index (CI) was determined for combinations of TQ and MLT against EMT6/P cells (14):

CI = (D) 1/(Dx) 1+ (D) 2/(Dx) 2+ α (D) 1 (D) 2/(Dx) 1 (Dx) 2

Where: (Dx) 1 = Dose of drug 1 to produce 50% cell kill alone;

(D) 1 = Dose of drug 1 to produce 50% cell kill in combination with (D) 2; (Dx) 2 = Dose of drug 2 to produce 50% cell kill alone;

(D) 2 = Dose of drug 2 to produce 50% cell kill in combination with (D) 1;

α = 0 for mutually exclusive or 1 for mutually nonexclusive modes of drug action.

Interpreted as: CI >1.3 antagonism; CI 1.1–1.3 moderate antagonism; CI 0.9–1.1 additive effect; CI 0.8–0.9 slight synergism; CI 0.6–0.8 moderate synergism; CI 0.4–0.6 synergism; CI 0.2–0.4 strong synergism.

Antitumor activity assay

Exponentially growing EMT6/P cells were harvested by trypsinization, centrifuged, washed and re-suspended in complete MEM media at a density of 2 × 106/ml. Cell viability was assessed using trypan blue exclusion method and a tumorigenic dose of 500,000 cells in 0.1 ml was injected subcutaneously in the abdominal area of each mouse. Tumors were allowed to grow for 7 days, and all tumors were measured using digital calipers. Then tumor volume was calculated using the formula (A × B 2 × 0.5). Where A was the length of the longest aspect of the tumor and B was the length of the aspect perpendicular to A. The mice were then randomly divided into the four groups so that the average tumor volume for all groups was closely matched; Group I: Control group (10 mice) were injected IP with vehicle (phosphate buffered saline) 0.1 ml daily. Group II: TQ (10 mice) were injected IP with 10 mg/kg/day of TQ. Group III: MLT (10 mice) were injected IP with 1 mg/kg twice daily of MLT (Early in the morning and before the sun set). Group IV: Combination (10 mice) were injected IP with 10 mg/kg/day TQ + 1 mg/kg twice daily of MLT.

Tumors were measured again at the end of 2 weeks after which mice were sacrificed, tumors extracted, weighed and stored in 10% formalin.

Histological examination of tumor sections

Formalin fixed specimens were gradually dehydrated and embedded to prepare paraffin blocks. Sections (5 μm thick) were prepared using microtome, and standard hematoxylin and eosin (H and E) procedure was used to stain different section. A light microscope (Zeiss, Germany) equipped with a computer-controlled digital camera (Canon, Taiwan) was used to visualize images on the slides.

Immuno-histochemical staining of vascular endothelial growth factor in tumor sections

Tumor-paraffin sections were deparaffinized by immersion in xylene then gradually hydrated using serial concentrations of ethanol. The slides were heated in citrate buffer solution (pH = 6.0) at 90°C for 20 min for heat-induced epitope retrieval. The slides were then cooled and treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity then incubated with 5% bovine serum albumin (Sigma-Aldrich, USA) for 20 min. Afterward the slides were incubated for 60 min at room temperature with an anti-VEGF primary antibody, followed by a biotinylated secondary antibody (Sigma, USA) conjugated to horseradish peroxidase (Sigma, USA) for 1 h. Specimens were stained with 3,3' diaminobenzidine (DAB) solution (Sigma, USA) for 20 min and counterstained with Meyer's hematoxylin. Then mounted with glycerol and examined under the microscope for visualization of the images.

Apoptosis detection in tumor sections

The degree of apoptosis induced by each treatment was detected using the DeadEnd TUNEL Colorimetric Apoptosis Detection System from (Promega, USA). Paraffin-embedded sections were deparaffinized using xylene then rehydrated by immersing through serial concentrations of ethanol. Slides were washed in 0.85% NaCl and fixed in 10% buffered formalin for 15 min. 20 μg/ml proteinase K solution was added to each slide and incubated for 20 min at room temperature. Sections were re-fixed using 10% buffered formalin then covered with equilibration buffer for 5–10 min at room temperature. The end-labeling reaction occurs through the even distribution of rTdT reaction mixture on the sections while incubating for 60 min at 37°C in a humidified chamber. Termination of the reaction occurs when slides are immersed in 2X SSC termination solvent. Endogenous peroxidases were blocked by 0.3% hydrogen peroxide. Horseradish peroxidase-labeled streptavidin was then added to bound to the biotinylated nucleotides for 30 min at room temperature followed by incubation with DAB for 20 min in the dark. Afterward slides were mounted with glycerol and observed under the light microscope.

Determination of interleukin-4 and IF-γ in serum samples

At the end of treatment, serum samples were collected from mice of different treatments. Serum levels of interleukin-4 (IL-4) and IFN-γ were measured using commercially available ELISA kits according to the manufacturer instructions (Quantikine ELISA, R and D systems, USA). The concentrations were determined in triplicates for each treatment.

Assessment of liver function

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum levels from each group were measured using commercial kits, AST (Biosystems, Spain) and ALT (Biosystems, Spain), respectively, using a spectrophotometer set at 340 nm.

Statistical analysis

Data are presented using mean ± standard error from three independent experiments. The statistical significance among the groups was determined by using one-way analysis of variance. A P < 0.05 was considered significant. The IC50 values obtained with the different concentrations of TQ and/or MLT were calculated using nonlinear regression in Statistical Package for the Social Sciences (SPSS Inc. Released 2009. PASW Statistics for Windows, Version 18.0. Chicago, IL, US).


 > Results Top


Antiproliferative assay and combination index

Treatment of EMT6/P cell line with serial concentrations of TQ (5–800 μm) caused a dose-dependent inhibition of cell growth and proliferation with IC50 value of 339.5 μM. MLT inhibited this cell line at IC50 value of 4.51 Mm. Testing different combinations of TQ and MLT revealed a clear synergistic effect of these two agents against EMT6/P cell line with a CI value of 0.552 [Table 1].
Table 1: The IC50 values and CI for TQ, MLT and their combination against EMT6/P cell line

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Antitumor effect of thymoquinone, melatonin, and their combination against breast cancer implanted in mice

Treatment of tumor-bearing mice with 10 mg/kg/day TQ showed significant (P < 0.05) reduction in tumor growth with a percentage change in tumor size of (−48.31%), compared to untreated mice (+52.25%). A greater reduction in tumor growth was observed in tumor-bearing mice treated with 2 mg/kg/day of MLT with a change in tumor size of (−56.68%). Although the combined treatment showed a reduction in tumor size (−41.69%), this reduction was lower than the reductions observed in single agent treatment. Remarkably, this combination treatment caused the highest percentage of cured mice (60%) compared with the single treatments that produced cure percentages of 30% and 40% for TQ and MLT, respectively [Table 2]. In addition, the combination treatment and MLT single treatment were able to reduce percentage death from 20% in the untreated mice to 0%. On the other hand, TQ single treatment caused a slight reduction in the percentage death to 10%.
Table 2: Effect of different treatments on tumor size, tumor weight, and survival rates

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Histological examination of tumor sections

To further understand how the different agents affected the tumors implanted in mice, tumors of similar sizes were sectioned and stained using standard H and E staining procedure and were examined for the presence of necrotic areas. It was observed that tumors treated with MLT had more necrotic areas compared to control group where no necrotic areas were detected [Figure 1]. As for tumors treated with TQ, larger necrotic regions were demonstrated in contrast to tumors treated with MLT. However, most dramatic effect was shown with the combination of TQ and MLT which resulted in extensive necrosis and more frequent necrotic areas than other treatments [Figure 1].
Figure 1: H and E staining of tumors treated with vehicle (a), 1 mg/kg twice daily of melatonin (b), 10 mg/kg thymoquinone (c), and a combination of thymoquinone and melatonin (d). N: Necrotic area. Extensive necrosis was evident in tumors treated with a combination of thymoquinone and melatonin (d)

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Induction of apoptotic DNA fragmentation in tumor sections

In order to gain more insight into the mechanism of action of our combination, the ability of TQ, MLT, and their combination to induce apoptosis in EMT6/P cells implanted in mice was tested using a TUNEL colorimetric assay which detects DNA fragmentation during programmed cell death. Both TQ and MLT caused an increase in the number of apoptotic cells with fragmented DNA compared with untreated cells. The highest degree of apoptosis was detected in tumor sections treated with a combination of TQ and MLT [Figure 2].
Figure 2: Colorimetric TUNEL assay for detection of apoptosis in tumor sections treated with vehicle (a), thymoquinone 10 mg/kg (b), melatonin 1 mg/kg (c) and a combination of thymoquinone 10 mg/kg and melatonin 1 mg/kg (d). Apoptotic nuclei are stained dark brown

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Measurement of vascular endothelial growth factor expression in tumor sections

To test whether angiogenesis inhibition may contribute in the observed antitumor effect; the expression of VEGF was measured in tumor sections and indicated by the presence of yellow to brown cytoplasm. VEGF was highly expressed in the tumor sections obtained from untreated mice. Lower degree of expression was observed in tumor sections obtained from mice treated with TQ. On the other hand, VEGF was barely detectable in tumor sections treated with MLT. The lowest VEGF expression was observed in tumor sections treated with a combination of TQ and MLT [Figure 3].
Figure 3: Vascular endothelial growth factor stained tumor sections treated with vehicle (a), thymoquinone 10 mg/kg (b), melatonin 1 mg/kg (c) and a combination of thymoquinone 10 mg/kg and melatonin 1 mg/kg (d). Yellow to brown cytoplasm indicates vascular endothelial growth factor expression

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Serum levels of interleukin-4 and IFN-γ

In order to investigate the changes in the immune response associated with each treatment, serum levels of IL-4 and IFN-γ were detected using standard ELISA kits [Table 3]. Our results suggest that mice treated with TQ, MLT, and their combination had high levels (more than 50 pg/ml) of IFN-γ compared with the negative control group (17.92 pg/ml). However, the combination therapy caused the highest increase in serum IFN-γ compared with other treatments. For serum IL-4 levels, all groups showed low levels of IL-4 (2.9–3.94) except the negative control group which had IL-4 level of 21.25 pg/ml.
Table 3: Levels of INF-γ and IL-4 for different treatments

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Serum levels of aspartate aminotransferase and alanine aminotransferase

To evaluate the possible toxicity associated with our combination, serum levels of ALT and AST were measured for all treatments. High levels of both enzymes were detected in the serum of untreated tumor-bearing mice. Different treatments caused a significant reduction in the levels of these enzymes and the highest reduction was observed in the combination treatments [Table 4].
Table 4: Serum levels of AST and ALT for different treatments

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 > Discussion Top


Combination therapy has been proposed as a method of treatment for different types of cancer to overcome the diversity in how cancer works as well as the severity of medication side-effects. It was emphasized by Komarova and Boland that combination therapy is more effective as well as preventative of drug resistance in cancer therapy.[15] Our in vitro results showed a clear antiproliferative effect of TQ against EMT6/P cell line. The antiproliferative activity of TQ was further confirmed by its ability to reduce tumor size, induce apoptosis, and enhance necrosis in EMT6/P implanted in mice. These results are consistent with the previous findings that reported the antiproliferative and apoptosis induction effects of TQ against different cancer cell lines including adenocarcinoma, prostate cancer cells, gastric cancer cells, and osteosarcoma cells.[4],[16],[17],[18]

As for MLT, our study has shown a potent antiproliferative response against EMT6/P cell line in vitro with high ability to reduce tumor size, induce apoptosis and necrosis in vivo. Our findings are supported by previous studies that explained the anticancer effect of MLT. Cos et al. in 2002 explained that MLT exerted its antiproliferative effect through modulation of cell cycle length.[19] This response was further explained by Yang et al. where they indicated that MLT inhibited cell growth and increased cell apoptotic activity of rat pituitary tumor.[20] In addition, MLT was identified as a novel inhibitor of Sirt1, a NAD(+)-dependent histone deacetylase overexpressed in prostate cancer; imparting an antiproliferative response.[21]

In our study, the combination of TQ and MLT has proved to be more powerful in producing an antiproliferative response against the tested cell lines as compared with single treatments. This combination acts synergistically to inhibit EMT6/P cells. Our in vivo results also confirmed the synergistic effect of TQ and MLT that induced extensive necrosis and apoptosis in EMT6/P cells implanted in mice. Our results are consistent with the previous studies that reported a successful combination of TQ with other therapeutic agents such as docetaxel, cisplatin, and zoledronic acid.[22],[23],[24]

On the other hand, MLT when combined with doxorubicin significantly increased the effects of cell growth inhibition and apoptosis in hepatoma cells.[25] MLT was also found to potentiate the antiproliferative and pro-apoptotic effects of ursolic acid in colon cancer cells through modulation of multiple signaling pathways.[26] Nevertheless, the synergistic effect of MLT with several chemotherapeutic agents such as vincristine and ifosfamide was also effective against sarcoma cell line.[27]

Compared with other combinations, our study is the first that combine the anticancer effects of TQ with a relatively nontoxic hormonal therapy (MLT). Other combination therapies depended mainly on combinations of TQ with chemotherapeutic agents that exhibit high toxicity limiting the potential efficiency of such combinations.

A principle step in cancer progression is angiogenesis. Our study has confirmed the substantial reduction in tumor size accompanied by the highest percentage of cured mice in the combination treatment of TQ and MLT. This was consistent with the barely expressed VEGF in stained tumor sections treated with the combination. On the other hand, each agent exhibited partial inhibition of VEGF expression.

The antiangiogenic activity of TQ and MLT was described in previous studies. TQ antiangiogenic activity was observed in human prostate cancer cells.[28] This inhibition of tumor angiogenesis and growth was also verified by Peng et al.in 2013 through the suppression of nuclear factor-kappa B and its regulated molecules in osteosarcoma.[29] MLT regulated tumor microenvironment through decreasing the secretion and expression of VEGF by malignant epithelial cells in human breast cancer which supports its antiangiogenic activity in tumor tissue.[30] The potent inhibition of VEGF in our combination therapy seems to be a result of combined antigiogenic mechanisms mediated by TQ and MLT.

IFN-γ is the signature cytokine of Th1 immune response. This type of immune response is characterized by high activity of anticancer natural killer cells and cytotoxic T lymphocytes. On the other hand, increased levels of IL-4 are an indication of T helper 2 immune response which has limited anticancer activity. Evaluation of the immune response in treated mice showed high levels of IFN-γ in all treatments compared with the negative control. However the highest levels were observed in tumor-bearing mice treated with the combination of TQ and MLT suggesting a shift in immune response toward Th1 anticancer immune response. On the other hand, IL-4 levels experienced a decrease with all treatments except for control.

Extensive studies have demonstrated the immuno-modulatory property of single treatment of TQ and MLT against different tumors implanted in mice.[31],[32],[33],[34],[35] Our results are consistent with these studies as significantly high levels of IFN-γ were detected in the combination therapy compared with single treatments. The increase in IFN-γ levels caused a shift in the immune response toward Th1 anticancer response which participates directly in the observed regression in the tumor size and enhanced cell death.

To investigate the safety of this combination, liver functions were assessed through measuring AST and ALT serum levels. AST and ALT serum levels were found normal in the combination therapy group indicating the safety of this combination. The low toxicity in the combination therapy group is a result of low toxicity of each agent used in this combination.

The low toxicity of TQ at a concentration of 10 mg/kg/day was showed in previous studies.[36] In spite of the fact that there is no long-term safety data on MLT administration, there is no evidence of toxicological effects when MLT is administered to humans or animals and is sold over the counter nationwide.[37] In addition, another study concluded the absence of MLT toxicity and side-effects, even over long period consumption.[38]


 > Conclusions Top


A combination of MLT and TQ can act synergistically to inhibit breast cancer implanted in mice. The combination acts mainly by apoptosis induction, inhibition of angiogenesis, and shifting the immune response toward Th1 anticancer response. This combination deserves further investigations to be considered as a possible therapeutic option to treat breast cancer.

Acknowledgments

The authors are grateful to the Applied Science Private University, Amman, Jordan, for the full financial support granted to this research project.

Financial support and sponsorship

Applied Science University, Amman, Jordan.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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