The Effect of Epigallocatechin gallate on Cross-talk Between Autophagy and Apoptosis in NALM-6 Cell Line

Background: Acute lymphoblastic leukemia is a prevalent hematological malignancy in 2-5-year-old children. Chemotherapy, as the most common treatment for ALL, is not usually responsive. Epigallocatechin gallate (EGCG), a small molecule extracted from green tea, has signicant effects on tumor cells through different mechanisms, such as DNA damage, cell cycle arrest, oxidative stress, apoptosis, and autophagy. In this study, we investigated the impact of EGCG on autophagy and apoptosis in NALM-6 cell line. Methods and results: Cell viability and apoptosis were assessed by MTT and Trypan blue exclusion assay, and ow cytometry. It was shown that EGCG remarkably inhibited proliferation, reduced cell viability, and induced apoptosis in NALM-6 cell line (P<0.05). In addition, real-time PCR and western blot analysis were used to examine autophagy. It was observed that EGCG resulted in a 4-fold increase in LC3 protein level (P<0.05) while reducing the mRNA expression level of LC3B, P62/SQSTM1, and Atg2B genes (P<0.01). It also caused around 1.3-fold increase in DRAM1 mRNA expression level (P<0.05). Finally, it was indicated that the inhibition of autophagy affects apoptosis neither in untreated nor treated cells with EGCG. Conclusion: These results show that EGCG can induce apoptosis and autophagy in NALM-6 cell line while inhibition of autophagy cannot affect apoptosis in this cell line.


Introduction
Acute lymphoblastic leukemia (ALL) is a hematological malignancy disorder, involving B or T lymphoid precursors with various biological and clinical manifestations [1]. Typically, different genetic changes in the lymphoid precursors lead to maturation and differentiation arrest, or excessive proliferation; hence, by creating a clone of immature lymphoid cells in the bone marrow, normal hematopoiesis is suppressed [2].
Precursor B-ALL accounts for approximately 88% of leukemia cases amongst children [3]. Despite new studies on the use of speci c transcription factor inhibitors, immunotherapy, and epigenetic methods, the current treatment modality of ALL is based on the chemotherapy principle, bone marrow transplantation, or CNS prophylaxis through radiation therapy in some cases [4]. Given that there are signi cant number of cases without any appropriate prognosis and responsiveness to treatment, there is a need to investigate new methods and approaches [2]. Today, it is well known that autophagy and apoptosis play an important role in the physiology and pathophysiology of various diseases such as cancer. Apoptosis or programmed cell death is one of the most crucial modulators in cancer [5]. In addition, autophagy acts as a tumor suppressor, which protects normal cells from genetic damage, oxidative stress, and stem cell proliferation or progenitors associated with malignancy. However, with respect to the cancer stage or patient's therapeutic condition, it can differently affect cancer cells that are heavily stressed by starvation, hypoxia, and chemotherapy or radiotherapy [6]. Cross-talk between autophagy and other types of cell death such as apoptosis has been extensively studied. In fact, autophagy, also known as type II cell death, can act in parallel to apoptosis or lead to cell survival by suppressing it or appearing as a pre-requisite for this type of cell death [7]. It was also observed that autophagy and apoptosis play a crucial role in ALL recurrence. Bcl2/Bax ratio, as one of the most important apoptosis regulators, is associated with relapse in ALL. In addition, ALL recurrence is related to some defects in signaling pathways such as PI3K/Akt/mTORC1 and Notch1 that are regulating autophagy [8].
Several studies have shown that natural compounds play a signi cant role in cancer therapy by inducing various types of cell death, including apoptosis and autophagy [9]. Epigallocatechin Gallate (EGCG) is a polyphenol derived from green tea, and the most important catechin leading to cell cycle arrest, apoptosis, proliferation arrest, and autophagy induction or inhibition in cancer cells through different pathways. It also disrupts the angiogenesis and sensitizes tumor cells, which are normally resistant to chemo-and radiotherapy [10]. In addition, several in vitro and in vivo studies have found that this compound does not damage healthy cells [11][12][13].
Although there is evidence showing that inhibition of autophagy doesn't affect cell viability in NALM-6 cell line [14], some observations have concluded that inhibition of autophagy leads to cell death [15,16].
There are other studies in which a decrease in cell death due to the inhibition of autophagy has been implicated in this speci c cell line [17,18]. These different ndings and the importance of the role of apoptosis, autophagy, and their cross-talk in the treatment of ALL and also lack of studies showing this interaction make it necessary to investigate the effect of EGCG on autophagy, apoptosis, and their crosstalk in NALM-6, a pre-B-ALL cell line.

Methods And Materials
Cell culture and treatment NALM-6 cell line was purchased from Pasteur Institute (Iran, Tehran) and maintained in RPMI1640 media (Cell Biotechnology Saba Arna, Iran, Tehran) supplemented with fetal bovine serum 10% v/v and Penicillin/Streptomycin 100 IU/ml (Gibco Life Technologies, USA, Waltham, MA) and L-glutamine 2 mM (Shell Max, China) at 37 ℃ in an environment of 5% CO 2 and humidi ed atmosphere of 95%.

MTT assay
MTT assay was performed to indicate the impact of EGCG on NALM-6 cell line. The cells were seeded into 96-well cell culture plates at a 1×10 4 density in 100 µL of growth medium and incubated for at least 2 hours. EGCG (Abcam, UK, Cambridge) dissolved in DMSO (Shell Max, China) and the cells were treated with different concentrations of it (2-110 µM) for 24 and 48 hours. Cell viability was evaluated, using 10 µL of 5 mg/ml sterile 3-4, 5-dimethylthiazol-2-yl-2, 5-diphenyl-tetrazolium bromide solution (Melford, UK. Ipswich) that was added to each well. Spectrometric absorbance at 545 nm was measured by a microplate photometer (Stat Fax 2100, USA). Three independent experiments were performed in quadruplicate.
Trypan blue exclusion assay Trypan blue exclusion assay was carried out to evaluate the effect of EGCG on the viability of NALM-6 cells. Brie y, the cells were seeded into a 24-well plate at a 1 × 10 5 density in 700 µL of growth medium and incubated for at least 2 hours. They were then treated with different concentrations of EGCG (2-110 µM) for 24 and 48 hours. The cells were then washed and resuspended in 1X PBS containing 0.4% trypan blue. They were counted, using a hemocytometer, according to standard protocol. Two independent experiments were performed in duplicate.

Western blotting
Five million NALM-6 cells were grown to 70-80% con uency, followed by treatment with EGCG 20 µM and 45 µM for 36 hours. NH 4 CL (Merck, Germany, Darmstadt) 10 mM, dissolved in sterile deionized water, was added in the last 12 hours of incubation to inhibit autophagy. The cells were washed once with phosphate-buffered saline and then lysed, using 1X ice-cold lysis buffer (5X formulation: Tris-HCl 0.06 M, pH 6.8, SDS 2%, Bromophenol blue 0.2%, glycerol 20%) supplemented with protease phosphatase inhibitor cocktail (Sigma-Aldrich, Germany, Darmstadt). Protein collection was performed by centrifugation at 4℃ for 30 minutes. Bradford protein assay was used to determine protein concentration. Equal aliquots of 20 µg proteins were boiled at 90℃ for 5 minutes in 5X loading buffer (Tris-HCl 0.06 M, pH 6.8, SDS 2%, Bromophenol blue 0.2%, glycerol 20%), and resolved by SDS-PAGE (12% gels). Then, samples were transferred to Polyvinylidene Fluoride (PVDF) (Roche, Switzerland, Basal) for 1 h. The blots were blocked for 1 hour with 5% skim milk in TBS-T and probed with rabbit anti-LC3 A/B antibody (Cell Signaling Technology, USA, Boston, MA) (1:1000) and mouse anti-β-Actin antibody (Santa Cruz, USA, Dallas, Texas) (1:1000) in 5% non-fat skim milk in TBS-T at 4°C overnight. After washing with TBS-T, the blots were treated with horseradish peroxidase-conjugated anti-rabbit IGg (Sigma-Aldrich, Germany, Darmstadt) and anti-mouse IGg (Bio-Rad, USA, Hercules, California) for 1 hour and were subsequently washed with TBS-T three times for 5 minutes each. Proteins were detected by the enhanced chemiluminescence system (ChemiDoc™ MP System, Bio-Rad, USA). Data were analyzed by Image J software.

Flow cytometry for Apoptosis
The cells were seeded on a 24-well plate at a 1 × 10 5 density in 700 µL growth medium and incubated for at least 2 hours and treated with different concentrations of EGCG (2-110 µM) for 24 and 48 hours. In addition, they were treated with EGCG 45 µM for 36 hours and NH 4 CL 10 mM was added in the last 12 hours of incubation to inhibit autophagy in combined treatments. Accessory apoptosis was determined, using PE-Annexin V/7-AAD Apoptosis Detection kit I (BD Biosciences, USA, San Jose, CA). The cells were washed with 1X PBS and 1×10 5 cells were resuspended in 100 µL of 1X Annexin V binding buffer. The cells were stained with PE-Annexin V and 7-AAD for 15 minutes in a dark room. They were then topped with 400 µL of Annexin V binding buffer and analyzed by FACS Calibur ow cytometer (BD Biosciences) within an hour. Data were analyzed by FlowJo TreeStar LLC software.

Real-time PCR
To determine the effect of EGCG on the expression of P62, Atg2B, LC3B, and DRAM1 genes, the cells were seeded into 6-well plates at a 1.2×10 6 density in 3.3 ml of complete medium and were then treated with EGCG 45 µM for 36 hours. Total RNA was extracted, using TRI Reagent (Sigma-Aldrich, Germany, Darmstadt) according to the manufacturer's instructions. The concentration of the extracted RNA was determined, using a Nanodrop instrument (Hellma, NY, China).
Complementary DNA (cDNA) was synthesized by reverse transcription, using 500 ng of total RNA and Prime Script TM RT reagent Kit (Yektatajhiz Azma, Iran). Quantitative real-time PCR was performed, using a SYBR Premix Ex Taq (Yektatajhiz azma, Iran) in 20 µL total volume. (8 µL DNase free water, 0.5 µL of 10 pM forward and reverse primers, 10 µL of SYBR Premix Ex Taq and 1 µL of cDNA template) on a Rotor gene system (Qiagen, USA) based on the following program in Table 1. Relative quanti cation of gene expression was performed, using the β-2 microglobulin gene as the internal control. The qRT-PCR also included a no-template sample as a negative control. Three independent experiments were performed in triplicate and comparative relative quanti cation of gene expression was performed based on the Pfa method. The used primers, listed in Table 2, were designed by allele ID software.

Statistical analysis
All analyses were performed, using the Graph Pad Prism 8.4.3 software (Graph Pad Software, Inc. La Jolla, CA) and represented as the mean± SEM. The data were analyzed, using Ordinary One-way ANOVA, two-way ANOVA, Kruskal-Wallis, and unpaired t-test. They were considered signi cant if the P-value was <0.05.

EGCG decreases cell viability of NALM-6 cell line
The effect of different concentrations of EGCG (2-110 µM) on proliferation and cell viability was evaluated by the MTT assay. We observed that EGCG caused a reduction in cell viability in a dosedependent manner. After 24 hours, cell viability was signi cantly reduced by 5.4%, 40.8%, 57.4%, and 68.2%, when exposed to EGCG 2, 20, 45, and 110 µM, respectively ( Fig. 1A) . 2C and D).
To determine the effect of EGCG on apoptosis in NALM-6 cell line, ow cytometry was performed, using EGCG induces autophagy in NALM-6 cell line To investigate the effect of EGCG on autophagy, NALM-6 cells were treated with EGCG 20 µM and 45 µM for 36 hours. LC3 protein level, a common marker of autophagy, was monitored, using Western Blot analysis. We showed that EGCG 45 µM, in comparison with 20 µM, caused more conversion of LC3-I to LC3-II within 36 hours (Fig. S.1). The cells were subsequently treated with NH 4 Cl to investigate whether the increase in LC3 level is due to an impairment of autophagic ux or activation of autophagy. There was a signi cant accumulation of LC3-II after 12 hours of treatment with NH 4 CL 10 mM. LC3-II expression in the cells treated with EGCG 45 µM was twice as much as the untreated cells (P<0.05), and when NH 4 CL was added, it showed more than a four-fold change, thus suggesting that activation of autophagy, rather than ux impairment, is responsible for the increased levels of LC3-II upon EGCG treatment (P<0.01) (Fig. 3). Hence, EGCG 45 µM proved to be the optimal concentration to be used for the rest of the experiments.

EGCG affects the expression of the autophagy genes
To further investigate whether the EGCG treatment affected the autophagy at a transcriptional level, realtime PCR was performed to evaluate the effect of EGCG 45µM treatment on autophagy-related genes such as LC3B, P62/SQSTM1, Atg2B, and DRAM1. We detected that treatment of the cells with EGCG 45 µM led to a signi cant reduction of the expression of LC3B, P62, Atg2B, and genes, 33.3% (P<0.01), 46.5% (0.0001), and 45.5% (P<0.0001), respectively. We also assessed the impact of EGCG 45 µM on DRAM1 expression and observed a 1.3-fold increase in its expression level (P<0.05) (Fig. 4).

Inhibition of Autophagy does not interfere with apoptosis in NALM-6 cell line
To determine the effect of autophagy on apoptosis in NALM-6 cells, FACS analysis was performed, using FITC-Annexin V/PI Apoptosis Detection Kit I. The cells were treated with EGCG 45 µM for 36 hours in the presence and absence of NH 4 CL 10 mM in the last 12 hours of incubation. As shown in Figure 5, autophagy inhibition by NH 4 CL did not affect the rate of apoptosis in untreated and treated cells.

Discussion
Although new strategies have signi cantly improved chemotherapy outcomes in pediatric ALL patients, the prognosis is still poor amongst adults and infants [19]. Identifying a natural compound with minimum toxicity and high e cacy will help develop a novel therapy, which might improve the response to currently available therapies. There are numerous studies regarding the effect of EGCG on cell viability or apoptosis in leukemic cell lines while its effect on autophagy or cross-talk between autophagy and apoptosis in leukemia has not been previously explored. Here, we focused on B lymphoblastic leukemia and studied the effect of EGCG on autophagy, apoptosis, and their interaction in NALM-6 cell line.
The anti-proliferative and cytotoxic effect of EGCG has been investigated in different cancer cell lines, animal models, as well as clinical trials [20]. It has been stated that this compound inhibits proliferation and induces DNA fragmentation in different human leukemic cell lines in a dose-dependent manner [21].
It is now well established that apoptosis induction is a crucial approach in cancer therapy. Cornwall Cull, et al. have indicated that EGCG results in apoptosis in B-CLL and T-CLL cells in a dose-dependent manner, while it does not affect normal B and T cells [22]. Furthermore, it has been observed that EGCG induces apoptosis in Jurkat cell line through the expression of Fas and increasing Caspase 3 levels [23]. It has been also shown that EGCG prompts apoptosis in leukemic cells such as KG-1, TH-P1, and PML/RARα leukemic mice [24,25]. The results of our study also showed that EGCG treatment results in apoptosis in NALM-6 cell line in a dose-dependent manner, which is in accordance with the study of Fatih M. Uckun (Fig. 2), the concentration which also caused a signi cant rate of proliferation arrest (Fig. 1).
Since autophagy and apoptosis induction pathways are related [27] and autophagy plays a dual role in the pathophysiology of cancer [6], several studies have been conducted to investigate the effect of EGCG treatment on both apoptosis and autophagy mechanisms and their cross-talk. It has been reported that EGCG induces apoptosis and autophagy in ve different mesothelium cell lines while treating these cells with EGCG in the presence of chloroquine, an autophagy inhibitor, leads to cell death [28]. Besides, it has been shown that EGCG antagonizes proteasome inhibitors bortezomib toxicity by autophagy induction and protecting PC3 cells from death [29]. However, there is evidence that has demonstrated that activation of autophagic ux by EGCG decreases TRAIL-induced apoptosis in HCT-116, a colon-cancer cell line, by downregulation of death receptors [30]. Interestingly, Jiao Meng, et al. have shown that cotreatment of A549, a non-small cell lung cancer cell line, with EGCG and ge tinib can sensitize them to this drug by autophagy inhibition; hence, this resistance might be due to the induction of cytoprotective autophagy in this cell line [31]. In the present study, we examined the effect of EGCG on autophagy and apoptosis in the presence and absence of NH 4 CL, an autophagy inhibitor, to investigate how autophagy affects NALM-6 cell survival. Western blot analysis for LC3-II, one of the most important markers of autophagy, showed that EGCG 45 µM signi cantly induces autophagy in NALM-6 cell line (P<0.05). However, autophagy inhibition by NH 4 CL didn't affect apoptosis in the presence or absence of EGCG. In fact, our data are in line with Wong, J et al. that have shown that inhibition of autophagy doesn't affect apoptosis in NALM-6 cell line, contrary to others who either revealed that inhibition of autophagy increases cell death [15,16] or showing that inhibition of autophagy can be cytoprotective for this cell line [18,14].
Recently, researches have shown that an entire network of transcription factors like FOXO, STAT1, STAT3, NF-κB, and TP53 are involved in the modulation of autophagy. Indeed, several transcription factors including TP53, STAT3, and NF-kB play a dual role in autophagy regulation, through both transcriptional (nuclear interaction) and transcriptional-independent (cytoplasmic interaction) mechanisms, acting as both activators and repressors. In addition, autophagy is regulated by post-transcriptional effectors, including microRNAs, siRNAs, lncRNAs, or post-translational mechanisms such as phosphorylation, ubiquitination, acetylation, as well as histones acetylation [32,33]. Some pieces of evidence have shown that EGCG can affect the expression of downstream autophagy genes by inhibiting Sp1, NF-κB, AP-1, STAT1, STAT3, and FOXO1 or activation of Nrf2 and TP53 transcription factors [34]. It has been also indicated that EGCG participates in gene expression regulation through interfering with the methylation or expression level of miRNAs [35].
In addition, studies have stated that the molecular mechanism of autophagy involves several conserved genes such as MAP1LC3, SQSTM1/P62, Atg2B, and DRAM1 [36]. It has been shown that MAP1LC3 gene expression, with an important role in autophagy induction, may change differently and it is regulated by numerous transcription factors such as ATF4, C/EBPβ, FOXO1, FOXO3, GATA1, and TFEB [37]. SQSTM1/P62 gene also codes an important protein involved in autophagy, transcribed by NF-B, βcatenin, and TFEB [38]. In addition, Atg2B, an essential protein in the autophagy process localizes on the autophagy membrane or the surface of lipid droplets [39]. DRAM1 also known as DNA damage-regulated autophagy modulator 1, transcribed by P53, modulates autophagy and apoptosis [40,41]. It plays an essential role in apoptosis regulation involving BAX and lysosomes [42] and genotoxic stress-induced autophagy [43].
Tumenjin Enkhbat, et al. have shown that treatment of HCT-116 cell line, with EGCG 12.5 µM for 48 h in combination with 2 Gy radiation up-regulated LC3 mRNA. While treatment with EGCG alone caused about a 4-fold change, radiation itself led to an 11-fold change and when they were combined, a change over 15-fold was observed [44]. In contrast, Li, et al. showed that EGCG 20 µM did not alter LC3 mRNA expression in primary hepatocytes, whereas in the pretreated animal models with concanavalin A, a plant lectin that used to induce acute hepatitis, EGCG resulted in a signi cant decrease in LC3 mRNA level in liver tissue [45]. We also observed that treatment of NALM-6 with EGCG 45 µM for 36 hours led to a signi cant decrease in the LC3 mRNA level compared to untreated cells (P<0.01), which is in line with the Sainan Li study. Decreasing the LC3 mRNA level may be related to the FOXO1 transcription factor which has been previously shown to be inhibited by EGCG [46].
Regarding P62 expression, Zhong, L. et al. have investigated the effect of EGCG 25 µM and 50 µM on P62 protein and mRNA level in HepG2, a hepatocarcinoma cell line. They observed that EGCG led to the downregulation of the P62 protein level but it did not affect the P62 mRNA level signi cantly [47]. Li, et al. also have found that EGCG 20 µM did not affect P62 mRNA expression in primary hepatocytes [45]. Nonetheless, it has been reported that the treatment of DIV8 primary rat cortical neurons with EGCG 50 µM for 24 hours caused a signi cant increase in mRNA levels of P62 compared to DMSO control (P<0.05) [48]. In the present study, we revealed that EGCG 45 µM caused a signi cant decrease in the P62/SQSTM1 mRNA level (P<0.0005). P62 is one of the Nf-B target genes that can itself be inhibited by EGCG, thus decreasing its mRNA levels [34].
In contrast to LC3B and SQSTM1/P62 genes, there is no evidence concerning the effect of EGCG on Atg2B and DRAM1 genes. We observed that ATG2B mRNA expression level reduces signi cantly in response to the treatment of NALM-6 with EGCG 45µM after 36 h (P<0.0001). However, it was reported that Atg2B gene expression remained constant in MEFs (mouse embryonic broblast cells), in response to starvation after 2, 4, 6, and 8 hours, while, an early upregulation was followed by a later reduction in other autophagy genes and protein markers [49]. Moreover, it has been observed that starvation induces DRAM-1 mediated autophagy in 7702, HepG2, Hep3B, and Huh7 cells. These cell lines were starved for 48 h and DRAM1 mRNA and protein levels had increased signi cantly [50]. We also observed that treatment of NALM-6 cell line with EGCG 45 µM results in a slight but signi cant increase in DRAM1 gene expression (P<0.05). Therefore, our data suggest that the reduction of the mRNA levels of autophagy genes and increasing the mRNA levels of DRAM-1 might be related to apoptosis induction with EGCG 45 µM. Nonetheless, observing the decreased expression of autophagy genes, while observing the induction of autophagy upon EGCG treatment in NALM-6 cells, might be due to post-transcriptional or posttranslational alterations. Therefore, more investigations are required to nd the exact mechanism by which EGCG affects the expression of these genes.
In conclusion, we observed that EGCG decreases the cell viability and induces apoptosis in NALM-6 cell line in a time-and dose-dependent manner. It also results in autophagy induction dose-dependently, and also an alteration in LC3B, P62, Atg2B, and DRAM1 gene expression. Furthermore, inhibition of autophagy does not interfere with the apoptotic cell death induced by EGCG. Considering the importance of autophagy as a secondary cell death mechanism, it would be of great importance to further study the possible involvement of EGCG in inducing autophagic cell death in cells in which the apoptotic pathway is impaired. This compound can also be used in combination with chemotherapeutic drugs to study leukemic cell lines or animal models. Investigating the effect of this combination on autophagy and cell death or proliferation could be bene cial in cancer therapy.

Consent to participate
This was an in vitro study and we did not present any patient information; therefore, there was no need to obtain informed consent.
Consent to publish assay experiments performed in quadruplicate. In addition, data represented in graphs C and D as mean± SEM of two independent trypan blue exclusion assay experiments performed in duplicate. MTT, 3-(4, 5dimethylthiazol-2-yl) -2, 5 -diphenyl -tetrazolium bromide. Ordinary one-way ANOVA was used to analyze.

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