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Article Changes in Lactate Production, Genes Expression and DNA Methylation in Response to Tamoxifen Resistance Development in MCF-7 Cell Line

Lama Hamadneh 1,* , Lara Al-Lakkis 1, Ala A. Alhusban 1, Shahd Tarawneh 2, Bashaer Abu-Irmaileh 3, Sokiyna Albustanji 1 and Abdel Qader Al-Bawab 1

1 Faculty of Pharmacy, Al-Zaytoonah University of Jordan, Amman 11733, Jordan; [email protected] (L.A.-L.); [email protected] (A.A.A.); [email protected] (S.A.); [email protected] (A.Q.A.-B.) 2 Faculty of Science, Mutah University, Karak 61710, Jordan; [email protected] 3 Hamdi Mango Center for Scientific Research, The University of Jordan, Amman 11942, Jordan; [email protected] * Correspondence: [email protected]; Tel.: +96-277-777-1900

Abstract: Lactate dehydrogenase (LDH) is a key enzyme in the last step of glycolysis, playing a role in the pyruvate-to-lactate reaction. It is associated with the prognosis and metastasis of many cancers,   including breast cancer. In this study, we investigated the changes in LDH expression and lactate concentrations in the culture media during tamoxifen resistance development in the MCF-7 Citation: Hamadneh, L.; Al-Lakkis, L.; Alhusban, A.A.; Tarawneh, S.; cell line, and examined LDHB promoter methylation levels. An upregulation of 2.9 times of LDHB Abu-Irmaileh, B.; Albustanji, S.; was observed around the IC50 concentration of tamoxifen in treated cells, while Al-Bawab, A.Q. Changes in Lactate fluctuation in LDHA gene expression levels was found. Furthermore, morphological changes in the Production, Lactate Dehydrogenase cell shape accompanied the changes in gene expression. Bisulfate treatment followed by sequencing Genes Expression and DNA of the LDHB promoter was performed to track any change in methylation levels; hypomethylation of Methylation in Response to CpG areas was found, suggesting that gene expression upregulation could be due to methylation Tamoxifen Resistance Development level changes. Changes in LDHA and LDHB gene expression were correlated with the increase in in MCF-7 Cell Line. Genes 2021, 12, lactate concentration in the culture media of treated MCF-7 cells. 777. https://doi.org/10.3390/ genes12050777 Keywords: LDHB hypomethylation; breast cancer; lactate; LDHA and LDHB gene expression; tamox- ifen resistance Academic Editors: Tiziana Angrisano and Michele Longo

Received: 26 March 2021 1. Introduction Accepted: 18 May 2021 Published: 19 May 2021 Breast cancer remains a major health problem in most parts of the world, despite the advances achieved in the field [1]. Female breast cancer incidence has exceeded lung cancer

Publisher’s Note: MDPI stays neutral as the most common cancer in 2020, with an estimated 2.3 million new cases [2]. Although with regard to jurisdictional claims in the mortality rate for women with an already confirmed diagnosis has been declining [3], published maps and institutional affil- it remains the leading cause of cancer deaths among women [2]. iations. Tamoxifen treatment in estrogen receptor-positive patients reduced recurrence up to 9 years after acquiring cancer. Also, breast cancer mortality rates were significantly reduced by about one third through the first 15 years of follow up among tamoxifen treated patients [4,5]. However, even in the presence of many therapeutic options, drug resistance

Copyright: © 2021 by the authors. remains a challenging issue in cancer treatment, as approximately a quarter of breast cancer Licensee MDPI, Basel, Switzerland. cases treated with tamoxifen for 5 years displayed tamoxifen resistance (TamR) [4,6]. This article is an open access article The mechanisms underlying tamoxifen resistance are complex and many of them distributed under the terms and remain unknown [7]. The alteration of gene expression and signaling pathways was conditions of the Creative Commons reported to have a role in inducing TamR [8], in which, significant degradation of estro- Attribution (CC BY) license (https:// gen receptors (ER) was noticed in TamR cancer cells [9]. Additionally, activation of the creativecommons.org/licenses/by/ mitogen-activated kinase (MAPK) signaling pathway and the phosphatidylinositol 4.0/).

Genes 2021, 12, 777. https://doi.org/10.3390/genes12050777 https://www.mdpi.com/journal/genes Genes 2021, 12, 777 2 of 7

3-kinase/protein kinase B (PI3K/AKT) pathway have been reported to have roles in cell proliferation, regrowth, autophagy, and endocrine resistance [10,11]. Many cancer cells convert most of the pyruvate to lactate, whether there is oxygen or not, in a phenomenon called the Warburg effect [12]. In breast cancer, lactate is produced mainly by the activity of (LDHA), and it was studied to be used as a predictive marker for prognosis and overall survival in patients [13]. On the other hand, some studies reported that (LDHB) gene expression was found to be reduced in many commonly used breast cancer cell lines due to the hypermethylation of the promoter area leading to gene silencing [14], while other researchers reported that upregulated gene and protein expression are seen in triple-negative cells in comparison to luminal breast cancer cells [15]. In this study, lactate dehydrogenase A and B gene expression levels were determined during tamoxifen resistance development in MCF-7 cell lines and correlated with the concentration of lactate secreted to the culture media.

2. Materials and Methods Cell culturing and tamoxifen treatment MCF-7 (HTB-22™) cells (ATCC, Manassas, VA, USA) of passage 9 were cultured in RPMI medium (EuroClone S.p.A., Via Figino, Italy) containing 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, and sodium pyruvate (EuroClone S.p.A., Via Figino, ◦ Italy). Cells were grown in a humidified incubator under 5% CO2 at 37 C. Growth medium was routinely replaced. When cells were 70% confluent, they were treated with low concentrations of tamoxifen starting with a concentration of 10 nM, incubated for 3 days, then fresh media with no tamoxifen was added and the cells were allowed to grow until 70% confluency before the next tamoxifen concentration was added. Tamoxifen concentrations were gradually increased up to 40 µM to induce resistance. Gene expression and DNA methylation analysis DNA and RNA from treated cells were extracted using the innuPREP DNA/RNA Mini Kit (Analytik Jena, Jena, Germany) according to the manufacturer’s protocols. DNA and RNA were quantified to be used in DNA methylation and gene expression analysis, respectively. After quantification and a PCR integrity check, total mRNA samples were used to synthesize cDNA using the SuperScript® VILO™ cDNA Synthesis Kit (Life Technologies, Grand Island, NY, USA), and gene expression analysis of lactate dehydrogenase A and lactate dehydrogenase B was performed using the following primers: LDHA F: 50 CTCTGGCAAAGTGGATATCTTGAC 30 and R: 50 GGTAACGGAATCGGCTGAA 30; LDHB F: 50 CTCTCCTGGTAGGTTTCGGC 30 and R: 50 GCCGGATGCTCAGAGCTAAA 30. DNA methylation analysis: DNA samples were bisulfate-treated using the EZ DNA Methylation-Gold Kit (ZYMO Research Corp., Irvine, CA, USA) according to the kit’s protocols. Treated DNA samples were used to determine the DNA methylation levels of lactate dehydrogenase B promotor. PCR amplification followed by sequencing was performed using two sets of primers to sequence LDHB promoter (set 1 and set 2). The sequences of set 1 were previously used by Leiblich et al. [16] and gave 197bp by PCR with 14 CpG sites, while set 2 was used by Maekawa et al. [17] and gave 282bp with 14 CpG sites. All primers were ordered from Integrated DNA Technologies, Inc. (IDT, Coralville, IA, USA) Set 1 F: 50 TTTGGTTTATAGGTAAGTTTGATGG 30 and R: 50 ACTACTACCCTCTACCTTCTACTCCTC 30; Set 2 F: 50 AGGGAGTGTGTATATTTGAGTT 30 and R: 50 TCAAACTTACCTATAAACCAAA 30. Lactate detection using capillary electrophoresis—conductivity detector (CE-C4D): Genes 2021, 12, 777 3 of 7 Genes 2021, 12, 777 3 of 7

Lactate detection using capillary electrophoresis—conductivity detector (CE-C4D): TheThe supernatant media media from from treated treated MCF-7 MCF-7 cells cells were were collected, collected, and 1 mL and was 1 mL trans- was transferredferred into 2 into mL 2 of mL Milli-Q of Milli-Q water water in a glass in a vial glass to vial form to a form diluted a diluted working working solution solution with with1:2 in 1:2 ratio in ratioand mixed and mixed very well, very well,then filtered then filtered with syringe with syringe filters filtersof 45 µM of 45poreµM size. pore The size. Theanalysis analysis operation operation was wasperformed performed using using the optimized the optimized method method of an ofin-house an in-house built CE- built CE-CC4D as4D in as [18,19] in [18 at,19 room] at room temperature, temperature, and the and flow the rate flow was rate also was optimized also optimized for best for anal- best analysisysis to separate to separate each each peak peak of analyt of analyteses without without overlapping overlapping or broadening. or broadening. TheThe standardstandard solution of lactate (Sigma-A (Sigma-Aldrich,ldrich, St. St. Louis, Louis, MO, MO, USA) USA) was was prepared prepared byby dissolvingdissolving the powdered lactate in in 10 10 mL mL of of Milli-Q Milli-Q water water to to prepare prepare 200 200 mM. mM. Then, Then, a a serialserial dilutiondilution was applied to to prepare prepare the the wo workingrking solutions solutions with with different different concentrations concentrations ofof lactatelactate asas follows:follows: 0, 0.5, 1 and and 2 2 mM. mM.

3.3. ResultsResults MorphologicalMorphological changes of of breast breast cancer cancer ce cellll line line MCF-7 MCF-7 were were observed observed during during the the processprocess ofof tamoxifentamoxifen resistance development as as reported reported previously previously [20] [20 and] and are are shown shown inin FigureFigure1 1.. Cells Cells treatedtreated withwith 3030 µMµM tamoxife tamoxifenn started started to to lose lose their their epithelial-like epithelial-like shape shape andand becamebecame round.round. AtAt concentrationconcentration 4040 µµM,M, thethe cellscells startedstarted toto aggregate,aggregate, andand cells’cells’ raterate of growthof growth increased. increased.

Figure 1. Morphological changes accompanied tamoxifen resistance development; (A) untreated Figure 1. Morphological changes accompanied tamoxifen resistance development; (A) untreated control MCF-7 cells, (B) MCF-7 cells treated with 30 µM tamoxifen and (C) MCF-7 cells treated control MCF-7 cells, (B) MCF-7 cells treated with 30 µM tamoxifen and (C) MCF-7 cells treated with with 40 µM tamoxifen. Arrows indicate the change in cell shape in comparison to the epithelial- 40likeµM shape tamoxifen. of MCF-7 Arrows cells. indicateImages were the change taken usin in cellg ZOE shape Fluorescent in comparison Cell Imager to the epithelial-like(Bio-Rad, Hercu- shape ofles, MCF-7 CA USA) cells. (scale Images bar were100 µm). taken Experime using ZOEnts were Fluorescent repeated Cell at Imager3 different (Bio-Rad, times. Hercules, CA USA) (scale bar 100 µm). Experiments were repeated at 3 different times. Gene expression analysis of LDHA and LDHB is presented in Figure 2. LDHA gene expressionGene expression showed fluctuation analysis ofbutLDHA was mostlyand LDHB downregulatedis presented with in Figureincreased2. LDHAtamoxifengene expressiondoses. LDHB showed downregulation fluctuation was but maintained was mostly until downregulated 30 µM (−1.6), withafter increasedwhich a significant tamoxifen doses.changeLDHB was seendownregulation in MCF-7 cell wass treated maintained with 35 untilµM, and 30 µ theM( upregulation−1.6), after which was maintained a significant changein cellswas treated seen with in MCF-7 40 µM, cells where treated the with fold 35 changeµM, and was the 2.9 upregulation in both treated was maintainedcells. These in cellschanges treated in LDHB with 40 geneµM, expression where the foldwere change accompanied was 2.9 by in bothpromoter treated hypomethylation cells. These changes in intheseLDHB cellsgene after expression sequencing were of bisulfate-treated accompanied by DNA promoter samples hypomethylation (Figure S1). Promoter in these hy- cells Genes 2021, 12, 777 4 of 7 afterpomethylation sequencing was of bisulfate-treatedobserved as seen DNA in Figure samples 3. (Figure S1). Promoter hypomethylation was observed as seen in Figure3.

Figure 2. FoldFigure changes 2. Fold in gene changes expression in geneof (A) expressionLDHA and (B of) LDHB (A) LDHA from MCF-7and (cellsB) LDHBtreated fromwith increasing MCF-7 cells doses treatedof with tamoxifen.increasing Gene expression doses concentration of tamoxifen. results Gene were expressed expression as mean concentration ± SD (n = 3 runs results for each were sample). expressed as mean ± SD (n = 3 runs for each sample).

Figure 3. Promoter hypomethylation seen in bisulfate-treated DNA samples from cells treated with (B) 35 and (C) 40 µM tamoxifen in comparison with untreated control cells (A). Unmethyl- ated cytosine was replaced with thymine after bisulfate treatment in cells treated with 35 and 40 µM tamoxifen.

To detect the changes in lactate concentrations during tamoxifen resistance develop- ment in MCF-7 cells, the electrophoretic analysis was prepared for RPMI-1640 media alone as presented in Figure 4A. The calibration curve of lactate by CE was calculated by spiking RPMI-1640 media with four different concentrations of lactate (0, 0.5, 1 and 2 mM), then the AUC of lactate peaks were calculated and corrected with chloride peaks as an internal standard, as shown in Figure 4B. Based on the calculated equation from the cali- bration curve, the analysis was made for the developed tamoxifen-resistant MCF-7 cells, and the lactate concentration was measured in the acquired supernatant media. The con- centration of lactate was measured from the AUC in electropherograms after correction with the chloride AUC as an internal standard, as shown in Figure 5.

Figure 4. (A) Electropherogram of analyzed anions in prepared RPMI-1640 cell culture media. Conditions were as follow: 90 cm × 50 µm I.D. fused silica capillary coated with HDMB/PSS/HDMB. BGE: 30 mM TRIS/30 mM CHES, pH 8.4 with 0.025% PEI; +30 kV applied to Genes 2021, 12, 777 4 of 7

Genes 2021, 12, 777 4 of 7

GenesFigure2021, 12 2., 777 Fold changes in gene expression of (A) LDHA and (B) LDHB from MCF-7 cells treated with increasing doses of 4 of 7 tamoxifen.Figure 2. FoldGene changes expression in gene concentration expression results of (A )were LDHA expressed and (B) LDHBas mean from ± SD MCF-7 (n = 3 cellsruns treatedfor each with sample). increasing doses of tamoxifen. Gene expression concentration results were expressed as mean ± SD (n = 3 runs for each sample).

Figure 3. Promoter hypomethylation seen in bisulfat e-treated DNA samples from cells treated withFigure (B) 353. Promoterand (C) 40 hypomethylation µM tamoxifen in seen comparison in bisulfat withe-treated untreated DNA control samples cells from (A). cells Unmethyl- treated Figure 3. Promoter hypomethylation seen in bisulfate-treated DNA samples from cells treated with atedwith cytosine (B) 35 andwas ( replacedC) 40 µM with tamoxifen thymine in comparisonafter bisulfate with treatment untreated in cellcontrols treated cells with (A). Unmethyl-35 and 40 (B) 35 and (C) 40 µM tamoxifen in comparison with untreated control cells (A). Unmethylated cytosine µMated tamoxifen. cytosine was replaced with thymine after bisulfate treatment in cells treated with 35 and 40 µ wasµM replacedtamoxifen. with thymine after bisulfate treatment in cells treated with 35 and 40 M tamoxifen. To detect the changes in lactate concentrations during tamoxifen resistance develop- To detect the changes in lactate concentrations during tamoxifen resistance develop- ment inTo MCF-7detect thecells, changes the electrophoretic in lactate concentrat analysisions was during prepared tamoxifen for RPMI-1640resistance develop- media ment in MCF-7 cells, the electrophoretic analysis was prepared for RPMI-1640 media alone alonement as in presented MCF-7 cells, in Figure the electrophoretic 4A. The calibration analysis curve was of lactateprepared by CEfor was RPMI-1640 calculated media by as presented in Figure4A. The calibration curve of lactate by CE was calculated by spiking spikingalone as RPMI-1640 presented media in Figure with 4A. four The different calibration concentrations curve of lactate of lactate by CE(0, 0.5,was 1 calculated and 2 mM), by RPMI-1640 media with four different concentrations of lactate (0, 0.5, 1 and 2 mM), then spiking RPMI-1640 media with four different concentrations of lactate (0, 0.5, 1 and 2 mM), thenthe AUC the AUC of lactate of lactate peaks peaks were calculatedwere calculat anded corrected and corrected with chloride with chloride peaks as peaks an internal as an then the AUC of lactate peaks were calculated and corrected with chloride peaks as an internalstandard, standard, as shown as inshown Figure in4 FigureB. Based 4B. on Based the calculatedon the calculated equation equation from the from calibration the cali- internal standard, as shown in Figure 4B. Based on the calculated equation from the cali- brationcurve, thecurve, analysis the analysis was made was for made the developedfor the developed tamoxifen-resistant tamoxifen-resistant MCF-7 cells,MCF-7 and cells, the bration curve, the analysis was made for the developed tamoxifen-resistant MCF-7 cells, andlactate the concentrationlactate concentration was measured was measured in the acquired in the acquired supernatant supernatant media. The media. concentration The con- centrationofand lactate the lactate wasof lactate measured concentration was measured from was the measured AUCfrom th ine electropherograms AUCin the in acquired electropherograms supernatant after correction after media. correction with The con- the withchloridecentration the chloride AUC of lactate as AUC an internal was as an measured standard,internal standard,from as shown the AUCas in shown Figure in electropherograms in5. Figure 5. after correction with the chloride AUC as an internal standard, as shown in Figure 5.

FigureFigure 4. 4. (A)(A Electropherogram) Electropherogram of analyzed of analyzed anions anions in prepared in prepared RPMI-1640 RPMI-1640 cell culture cell culture media. media. ConditionsConditionsFigure 4. (A) were were Electropherogram as follow: 90 cm ×of× 50 50analyzed µmµm I.D. anions fused silica silicain prepared capillarycapillary RPMI-1640 coated coated with with cell HDMB/PSS/HDMB. culture media. HDMB/PSS/HDMB.BGE:Conditions 30 mM TRIS/30were as BGE: follow: mM 30 CHES, 90mM cm pHTRIS/30 × 8.450 µm with mM I.D. 0.025% CHES, fused PEI; pHsilica +30 8.4 capillary kVwith applied 0.025% coated to PEI; outlet with +30 vial kV while applied interface to wasHDMB/PSS/HDMB. grounded. Signal BGE: was obtained30 mM TRIS/30 using a mM Trace CHES, DEC conductivitypH 8.4 with 0.025% detector PEI; C4 D+30 positioned kV applied 10 to cm from the outlet. (B) Calibration curve of lactate by CE-C4D, using 4 different concentrations.

Genes 2021, 12, 777 5 of 7

Genes 2021, 12, 777 5 of 7 outlet vial while interface was grounded. Signal was obtained using a Trace DEC conductivity detector C4D positioned 10 cm from the outlet. (B) Calibration curve of lactate by CE-C4D, using 4 different concentrations.

Figure 5. Calculated concentration by CE-C4D of produced lactate from MCF-7 cell supernatant Figuremedia normalized 5. Calculated with cell concentration density after treatm byent CE-C4D with tamoxifen of produced in gradual lactate increased from doses. MCF-7 cell supernatant mediaStatistical normalized significance was with calculated cell density by one-way after ANOVA treatment followed with by Tukey tamoxifen post hoc intest gradualin increased doses. StatisticalGraphPad prism significance 8.0 software, was considering calculated the statis bytical one-way significance ANOVA as follows: followed * significant by at Tukey P ≤ post hoc test in 0.05; results were expressed as mean ± SD (n = 3 runs for each sample). GraphPad prism 8.0 software, considering the statistical significance as follows: * significant at P4. ≤Discussion0.05; results were expressed as mean ± SD (n = 3 runs for each sample). Breast cancer cells’ production of lactate under aerobic conditions contribute to their 4.proliferation, Discussion angiogenesis, and aggressive behavior [21]. Lactate was also found to have an oncometabolicBreast cancer effect, cells’ where production a transcriptional of lactate increase under in the PIK3/AKT/mTOR aerobic conditions signal- contribute to their ing pathway accompanying lactate exposure in MCF-7 cells was seen [22]. proliferation,The role of angiogenesis,lactate dehydrogenases and aggressive in lactate metabolism behavior [has21]. been Lactate studied was exten- also found to have an oncometabolicsively in breast cancer, effect, and where many have a transcriptional reported that LDHA increase overexpression in the PIK3/AKT/mTOR is seen under signaling pathwayhypoxic conditions accompanying and is associated lactate with exposure c-MYC gene in overexpression MCF-7 cells wasand glutaminolysis seen [22]. [23]. ItThe was rolealso found of lactate to be dehydrogenasesoverexpressed in Taxol-resistant in lactate metabolism breast cancer hascells been[24]. On studied extensively inthe breast other hand, cancer, LDHB and was many reported have to be reported silenced in that the LDHAestrogenoverexpression and progesterone-posi- is seen under hypoxic tive MCF-7 cell line due to promoter hypermethylation [14,25]. However, LDHB was conditionsfound to be overexpressed and is associated in triple-negative with c- MYCbreast cancergene cell overexpression lines and was correlated and glutaminolysis [23]. Itwith was poor also prognosis found among to be breast overexpressed cancer patients in [15]; Taxol-resistant it was also found breast to be cancer overex- cells [24]. On the otherpressed hand, in highlyLDHB glycolytic,was mesenchymal reported to breast be silenced cancer cell in lines the [26]. estrogen and progesterone-positive MCF-7In this cell study, line we due correlated to promoter the changes hypermethylation in lactate levels in MCF-7 [14,25 culture]. However, media withLDHB was found to gene expression of LDHA and LDHB during the process of tamoxifen resistance develop- bement. overexpressed Gradual increase in in triple-negative TAM doses was accompanied breast cancer with cell downregulation lines and wasof LDHA correlated with poor prognosisgene expression among and a significant breast cancer increase patients in LDHB gene [15 ];expression, it was also parallel found with demeth- to be overexpressed in highlyylation of glycolytic, certain CpG mesenchymalsites in the promoter breast region. cancer Interestingly, cell lines these [26 changes]. in LDHA and LDHBIn this gene study, expression we were correlated in parallel the with changes our reported in lactate finding levelsthat there in was MCF-7 no culture media withc-MYC gene gene overexpression, expression of withLDHA a significantand LDHB increaseduring in glutamine the production process of during tamoxifen resistance TamR development [20]. development. Gradual increase in TAM doses was accompanied with downregulation of LDHA gene expression and a significant increase in LDHB gene expression, parallel with demethylation of certain CpG sites in the promoter region. Interestingly, these changes in LDHA and LDHB gene expression were in parallel with our reported finding that there was no c-MYC gene overexpression, with a significant increase in glutamine production during TamR development [20]. Different methods of tamoxifen resistance development in the MCF-7 cell line could affect gene expression differently [20]. Using the gradual increase in tamoxifen concentra- tions reported in this study, we showed that the increase in LDHB gene expression but not LDHA was correlated with lactate concentration increase in the media during the process of TamR development. Lactate overproduction is linked to the Warburg effect in different types of cancer cells. The Warburg effect is also more favorable in these cells than oxidative phosphorylation for energy production, despite the presence of an adequate level of oxygen, and consequently results in the conversion of pyruvate into lactate [27]. Accumulation of lactate in the Genes 2021, 12, 777 6 of 7

tumor cell microenvironment was reported to have a key role in carcinogenesis and tumor invasion [28] and could serve as a possible resistance biomarker and drug target.

5. Conclusions Lactate participation in tumorigenesis is well documented and its role as a metabolic marker and therapeutic target has been explored, with most reports focused on the role of LDHA in breast cancer resistance to treatment, and few focused on LDHB. In this study, we report the potential involvement of lactate dehydrogenase B in breast cancer resistance to treatment that could provide a molecular marker to detect early resistance to tamoxifen among patients.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/genes12050777/s1, Figure S1: DNA sequencing of bisulfate-treated samples file. Author Contributions: Conceptualization, L.H. and A.A.A.; Methodology, L.A.-L., S.A. and B.A.-I.; formal analysis, L.H. and L.A.-L.; Resources, L.H. and A.A.A.; Data curation, L.A.-L., B.A.-I.; Writing— original draft preparation, L.H., S.T. and A.Q.A.-B.; Writing—review and editing, L.H., S.T. and A.Q.A.-B.; Supervision, L.H., A.A.A., and A.Q.A.-B.; Project administration, L.H. and A.A.A.; fund- ing acquisition, L.H. and A.A.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Al-Zaytoonah University of Jordan, grant numbers: (2019- 2018/18/06) and (2020-2019/23/06). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Mariam Hasan for the technical support. Conflicts of Interest: The authors declare no conflict of interest.

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