(‐)‐Epigallocatechin‐3‐gallate induced apoptosis by dissociation of c‐FLIP/Ku70 complex in gastric cancer cells

Abstract Anti‐cancer properties of (‐)‐epigallocatechin‐3‐gallate (EGCG) are mediated via apoptosis induction, as well as inhibition of cell proliferation and histone deacetylase. Accumulation of stabilized cellular FLICE‐inhibitory protein (c‐FLIP)/Ku70 complex in the cytoplasm inhibits apoptosis through interruption of extrinsic apoptosis pathway. In this study, we evaluated the anti‐cancer role of EGCG in gastric cancer (GC) cells through dissociation of c‐FLIP/Ku70 complex. MKN‐45 cells were treated with EGCG or its antagonist MG149 for 24 h. Apoptosis was evaluated by flow cytometry and quantitative RT‐PCR. Protein expression of c‐FLIP and Ku70 was analysed using western blot and immunofluorescence. Dissociation of c‐FLIP/Ku70 complex as well as Ku70 translocation were studied by sub‐cellular fractionation and co‐immunoprecipitation. EGCG induced apoptosis in MKN‐45 cells with substantial up‐regulation of P53 and P21, down‐regulation of c‐Myc and Cyclin D1 as well as cell cycle arrest in S and G2/M check points. Moreover, EGCG treatment suppressed the expression of c‐FLIP and Ku70, decreased their interaction while increasing the Ku70 nuclear content. By dissociating the c‐FLIP/Ku70 complex, EGCG could be an alternative component to the conventional HDAC inhibitors in order to induce apoptosis in GC cells. Thus, its combination with other cancer therapy protocols could result in a better therapeutic outcome.


| INTRODUC TI ON
Gastric cancer (GC) is the fifth most common cancer with the fourth highest mortality rate in the world. 1 GC patients often present several symptoms, including dyspepsia and reflux.
Advanced stages of GC are usually characterized by progressive signs such as dysphagia, loss of weight, gastrointestinal bleeding and anaemia. 2 Surgery combined with chemotherapy is still the only available treatment method for non-metastatic GC. Despite the advances in early diagnosis and chemotherapy protocols, current therapeutic approaches for GC have still low efficacy due to multidrug resistance. 3,4 Preventive methods aimed at limiting the risk factors and accelerating early detection of precancerous lesions. 5 Accordingly, discovering new treatment strategies is an essential and remarkable priority. 6,7 For instance, novel classification of GC based on different molecular subtypes and specific biomarkers resulted in a more precise personalized therapy. 2 Understanding tumour biology will also lead to the development of effective targeted therapies based on the molecular aetiology of cancer. 8,9 Chemoprevention for GC using natural, synthetic or biological agents is another therapeutic approach under evaluation. 10,11 Combined anticancer therapy based on molecular targets and using approved natural components could be an innovative strategy to induce anti-apoptotic pathways. 12 In many solid tumours and hematologic malignancies, abnormalities in histone acetylation and/or histone deacetylases (HDACs) may contribute to carcinogenesis. 13 The overexpression of HDACs induces tumour cell growth and resulted in poor prognosis for various cancers. 14,15 Also, HDACs are implicated in the proliferation, apoptosis, invasion and migration of a variety of cancer cells, including GC cells. 16 Therefore, HDACs were considered as potential targets in the therapeutic approaches developed for different cancers. 17 Although HDACs have the leading role in the deacetylation of histones, they also play an important role in the deacetylation of multiple nonhistone proteins and modulation of their activity, cell localisation of proteins and protein-protein interactions. 17 Ku70 is one of the non-histone proteins which is targeted by HDACs. This protein evolved in tumorigenesis through regulating cell death. 18 Ku70 is a nuclear DNA repair factor that forms a heterodimer with Ku80 and binds to double-strand DNA (dsDNA). Ku70/Ku80 dimer formation is necessary for DNA repair via the classical non-homologous endjoining (c-NHEJ) pathway. 19,20 In cancer cells, Ku70 can translocate from nucleus to the cytoplasm because of the increased levels of HDACs. 21 The function of cytosolic deacetylated Ku70 as a cell death regulator has already been investigated because of its association with apoptotic proteins. 22 One of the critical anti-apoptotic proteins in cancer cells is cellular FLICE-inhibitory protein (c-FLIP).
This protein could be a potential anti-cancer target in order to induce apoptosis via blocking caspase 8 activation and binding to the FAS-associated death domain (FADD). 4,23 c-FLIP and Ku70 interact and bond together in the cytoplasm of cancer cells. Ku70 stabilizes c-FLIP protein via its C-terminal region and prevents its degradation in the cytoplasm. Furthermore, acetylation of Ku70 inhibits its interaction with c-FLIP and results in c-FLIP degradation by the ubiquitin proteasome system. 4,24 Thus, HDAC inhibition induces dissociation of c-FLIP/Ku70 complex after acetylation of cytosolic Ku70. This dissociation restores apoptosis through the extrinsic pathway by decreasing cytosolic c-FLIP levels. 17 Therefore, novel therapies have focused on HDAC proteins as potential targets and an alternative approach in cancer treatment by using various components such as histone deacetylases inhibitors (HDACi).
Suberoylanilide hydroxamic acid (SAHA) was FDA approved in 2006 and is one of the effective HDACi for the treatment of cutaneous T-cell lymphoma (CTCL). 25

SAHA disrupts any complex between
Ku70 and other proteins through Ku70 acetylation. 4 In addition, several studies showed that this dissociation causes cell death through induction of extrinsic apoptosis pathway by decreasing the level of c-FLIP in cancer cells. 4,26 Although SAHA is a potential HDACi, the use of natural components with the same mechanisms of action could be more convenient and probably with less side effects in cancer.

| Cell culture
The study and related experiments were all approved by the competent ethical committee (IRC98-0335). MKN-45 cells were obtained from Royan Institute Cell Bank (Tehran, Iran), and cultured in RPMI 1640 supplemented with 5% hPL, 1% NEAA, 1% L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin in a humidified 5% CO 2 incubator at 37°C. The medium was changed every day. Twenty-four hours after seeding, the cell culture media were replaced by medium supplemented with 5% hPL. In all assays, the cells were incubated with appropriate concentrations of EGCG and MG149 for 24 h.

| Evaluation of the effective Dose
The effect of EGCG and MG149 on apoptosis rate of MKN-45 cells was analysed using Annexin V/PI double staining analyses.

| Apoptosis analysis
MKN-45 cells were seeded in 6-well tissue culture plates (2 × 10 5 cells/well). After starvation and treatment with 100 μM EGCG and 1 μM MG149 for 24 h, the floating cells in the medium were collected.
The adherent cells were detached using 0.05% trypsin-EDTA. Then, the fresh culture medium containing hPL was used to inactivate the enzyme. After gently homogenising the suspension, the cells were centrifuged for 5 min at 500 rpm. The cells were stained with Annexin V-FITC and PI according to the manufacturer's instructions.
Untreated cells were used as controls. The number of dead cells was analysed by FACSCalibur (BD, Franklin Lakes). The data were analysed using FlowJo 9.0 software (Tree Star).

| Quantitative real time-PCR assay
After seeding 8 × 10 5 cells/well (6-well plate) and treatment with 100 μM EGCG and 1 μM MG149, total mRNA was extracted from each group according to the manufacturer's protocol. RNA concentrations were measured at 260 nm, and the mRNA purity was spectrophotometrically evaluated at 260 and 280 nm. Two micrograms of RNA was used as a template for complementary DNA (cDNA) synthesis. The primer sequences used in the study are listed in Table 1.

Real-time PCR was performed with Taq DNA Polymerase Master
Mix and the StepOnePlus™ Real-Time PCR System. Data were analysed and presented using the comparative CT method (2 −ΔΔCt ). We checked at least six images with the same magnification (scale bar = 100 μm) from different sections in each group.
After treatment with 100 μM EGCG and 1 μM MG149, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors.
Sonication was used with the frequency of 60 kHz, three pulses for the 30 s, and then the cell lysate centrifuged (16,

| Sub-cellular fractionation technique
Cytoplasm and nuclei of the treated cells were separated, as described in Nabbi and Riabowol's protocol. 34  The whole-cell proteins and nuclear fraction were sonicated with the 20 kHz frequency, two pulses for 16 s. Finally, to quantify the target proteins, immunoblotting assay was used.

| Co-immunoprecipitation assay
To assess c-FLIP/Ku70 and the effect of EGCG treatment on this complex, co-immunoprecipitation (Co-IP) assay was performed

| Statistical analyses
Data were presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 8.4.3 (GraphPad). Differences between groups were compared using anova and t-test followed by LSD test. The p < 0.05 was considered as statistically significant (n = 3).

| EGCG treatment induces apoptosis and upregulates the expression of related genes in MKN-45 cells
Several studies have proposed 100 μM EGCG could be an optimal concentration to treat cancer cells. 35 In addition, we did analyse the mRNA expression of P53 and P21 by using quantitative RT-PCR after treatment with 100 μm EGCG and 1 μm MG149 (n = 3). Our results showed that P53 (p < 0.05) and P21 (p < 0.01) mRNAs were significantly upregulated (2 times and 1.5 times respectively) in EGCG-treated cells as compared to the non-treated control group ( Figure 1C). when treated with 1 μm MG149, only the expression level of P53 was significantly increased (two times) as compared to the control group (p < 0.05), while no significant effect was measured for P21 mRNA expression ( Figure 1C).

| EGCG treatment induces G2/M cell cycle arrest and decreases the number of cells in S phase
Our data demonstrated that EGCG treatment of MKN-45 cells Cyclin D1 (p < 0.05) ( Figure 1F).

| EGCG reduced cytoplasmic localisation c-FLIP and induced nuclear translocation of Ku70
To explore the effect of EGCG treatment on the c-FLIP level in the cytoplasm and the level of Ku70 in both cytoplasm and nucleus, the subcellular fractionation analysis was performed. First, the pools of cytoplasmic and nucleus proteins were extracted and c-FLIP and Ku70 protein levels assessed in each fraction. As shown in Figure 3A,B, the total c-FLIP protein level in whole-cell lysate was  Figure 3B). In the MG149-treated group, we did not notice any significant change in the c-FLIP content after treatment compared to the non-treated control group ( Figure 3B).

| DISCUSS ION
Recently, development of components inducing apoptosis, especially natural components, has gained remarkable interest in cancer therapy. 37,38 In our study, treatment of the MKN-45 cells by EGCG, a F I G U R E 1 Analysis of cell cycle and apoptosis induction after EGCG treatment. (A, B) EGCG induces dose-dependent apoptosis in MKN-45 cells at the early apoptosis phase. Cells were treated with EGCG and MG149 for 24 h. Apoptotic cells were stained with Annexin V/PI kit and determined by flow cytometry analysis. The cells were analysed using Graph Pad Prism-8 software. Data are shown as mean ± SD (n = 3). Statistical analysis was performed using one-way anova (* p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). (C) P53 and P21 gene expression in EGCG and MG149 treated groups was compared to the non-treated control group. Data were normalized to GAPDH and were shown as mean ± SD (n = 3). Statistical analysis was performed using unpaired two-tailed Student's t-tests. * p < 0.05 and ** p < 0.01. (D, E) Flow cytometry analysis of cell cycle progression after EGCG and MG149 treatment and its regulatory mechanism. Cell cycle assays revealed that EGCG treatment significantly increased the ratio of cells in the G2 phase and decreased the ratio of cells in the S phase after 24 h (n = 5). (F) The expression of c-Myc and Cyclin D1 genes after treatment with EGCG and MG149 was compared to the non-treated control group. Data were normalized to GAPDH and are shown as mean ± SD (n ≥ 3). reported that acetylation of Ku70 in lysine K542 by SIRT6 disrupts its interaction with Bax, which finally resulted in Bax translocation.
In the current study for the first time we showed that EGCG treatment induced dissociation of c-FLIP/Ku70 complex. 45 To the best F I G U R E 3 Evaluation of c-FLIP/Ku70 complex localization. (A, B) Evaluation of c-FLIP and Ku70 localization before and after treatment with 100 μM EGCG by the subcellular fractionation method. The expression level of c-FLIP and Ku70 was normalized with their corresponding markers. GAPDH was used as a cytoplasmic control marker, and histone H3 as a nucleus control marker. (C) The interaction between c-FLIP and Ku70 was evaluated before and after treatment with EGCG by Co-IP assay. After treatment with EGCG, we observed that the c-FLIP/ Ku70 complex was decreased.
of our knowledge, no specific report has described the potential of EGCG to induce apoptosis through dissociation of c-FLIP/Ku70 complex in GC cells.
We addressed this concern in our study by using EGCG at a dose of 100 μM that has been reported to potentially induce apoptosis in GC cells. 45,46 Our data confirmed the efficiency of this dose to induce apoptosis by increasing the number of early apoptotic cells ( Figure 1A,B). As already mentioned in some studies, EGCG can increase the number of cells in early apoptosis phase of different cancers. 32 This result is supported by the enhanced expression of P53 and P21, two key-apoptotic genes, observed in the EGCG-treated cells as compared to the control group ( Figure 1C). Furthermore, we demonstrated that the protein expression of c-FLIP was reduced after EGCG treatment which was compatible with previous studies (Figure 2A,B,E,F). 47,48 The translocation of acetylated Ku70 from the cytoplasm to the nucleus after treatment with EGCG has been described in previous studies. 49,50 According to our IF ( Figure 2C,D) and subcellular fractionation analyses ( Figure 3A,B), we demonstrated that Ku70 protein was translocated from cytoplasm to the nucleus after treatment with EGCG. Based on these findings, we suggested that treatment with EGCG as an HDACi could induce Ku70 acetylation. In addition, numerous studies showed that inducing apoptosis can cause G2/M arrest in treated cells 51  Based on IF and Sub-cellular fractionation results, we speculate that following the dissociation of this complex, Ku70 will return to the nucleus where it exerts its normal function as a nuclear DNA repair factor and the cytoplasmic level of c-FLIP will be decreased.
Altogether, our data showed that EGCG, a natural HDAC in- writing -review and editing (equal).

ACK N O WLE D G E M ENTS
We would like to express our sincere gratitude to our colleagues at Royan institute, Liver research group and Institute of Experimental and Clinical Research, UCLouvain.

FU N D I N G I N FO R M ATI O N
This study was funded by grants from Bahar Tashkhis Teb Co, (R.98005) and Royan Institute (R1398-0335).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.