1. Introduction
The Epstein–Barr virus (EBV), which belongs to the human gamma-herpesvirus family (HHV-4), consist of a ~170-kb double-stranded linear DNA virus [
1]. EBV easily infects close family members through salivary transmission during infancy or childhood. Most EBV infections remain asymptomatic, given their ability to establish lifelong latency [
2]. EBV is among the most common viruses, with over 95% of world population having been infected and approximately 143,000 deaths having been related to EBV-associated malignancies in 2010 [
3]. The EBV virus was the first human tumor virus discovered in Burkitt’s lymphoma cells in 1964 [
4]. Thereafter, studies have revealed that EBV infection could cause several different malignancies in lymphoid and epithelial cells. Each EBV-associated malignancy has a unique type of EBV latency phase that exhibits a distinctive pattern of EBV gene expression [
5]. Infectious mononucleosis and lymphoproliferative disorder both occur during EBV latency phase III, whereas Burkitt’s lymphoma and gastric carcinoma occur during EBV latency phase I.
Most EBV latent genes are expressed during EBV latency III, whereas only limited viral genes, such as EBERs and EBNA1, are expressed during EBV latency I [
6]. Together with other immune evasion strategies, limitations in viral gene expression have been known to help EBV escape the host’s immune system. T cell responses to EBV lytic antigens represent a dominant fraction of EBV-specific T cells generated during primary EBV infection or EBV latency III. Although these EBV-specific T cells aggressively respond to EBV lytic cycle antigens, they barely respond to EBV latent cycle antigens [
7]. Moreover, some latent genes, such as
BNLF2a,
BGLF5, and
BILF1, have been reported to interfere with HLA-I antigen presentation from EBV-infected cells. Thus, the switch from latency to lytic infection is a major requisite for the treatment of EBV-associated malignancies [
8].
EBV-associated gastric carcinoma (EBVaGC), first discovered in 1992, accounts for 10% of all gastric carcinomas worldwide [
9]. This malignancy exhibits male predominance (approximately twice as many as in females), occurs preferably in the proximal stomach or remnant stomach, and has a favorable overall and disease-free survival [
10,
11]. EBVaGC has been known to feature epigenetic characteristics, such as
PIK3CA mutation;
CDKN2A silencing; JAK2, PD-L1, and PD-L2 overexpression; immune cell signaling enrichment; and CpG island methylator phenotype (CIMP) [
12]. In particular, EBVaGC has unique hypermethylation phenotypes that allow for higher proportions of DNA methylation than any other gastric cancer [
12]. These characteristics help explain why EBVaGC exhibits more distinct phenotypes than EBV-negative gastric carcinoma (EBVnGC). Epigenetic processes, including chromatin remodeling, histone acetylation, and DNA methylation, can regulate gene expression without altering DNA sequences [
13,
14]. DNA methylation is a well-known epigenetic alteration that occurs on cytosine residues [
15] wherein methyl groups (-CH
3) are added at 5′-carbon of the cytosine ring, subsequently forming 5′-methylcytosine (5
mC). These methylations frequently occur at cytosine–guanine dinucleotides (CpGs), with condensed CpG regions, called CpG islands. Promoter CpG islands are one of the major regions in which DNA methylation controls gene transcription [
16]. Aside from promoter regions, methylation of the untranslated region (UTR), gene body, and exon 1 can also control gene transcription in several cancers [
17,
18].
Most platinum-based anticancer drugs used as first-line chemotherapeutic treatment for several types of cancer patients are genotoxic [
19]. Accordingly, they can selectively bind with genomic DNA and form DNA cross-links within or between DNA strands [
20]. DNA cross-linking interferes with DNA replication and transcription and triggers a DNA damage response, leading to cell death [
21]. Cisplatin, approved for clinical chemotherapy in 1971 as the first platinum analog, is currently being broadly used as a first-line cancer treatment, alone or in combination with other chemotherapeutics [
22]. However, cisplatin-based treatments have shown limitations in some patients owing to side effects, including nephrotoxicity, lack of therapeutic effects, and development of tumor resistance to cisplatin [
20]. For instance, one study showed that metastatic nasopharyngeal carcinoma (NPC) usually developed resistance after six cycles of cisplatin-based chemotherapy [
23]. Numerous molecular mechanisms have been suggested to have promoted resistance in the NPC cases. Another study showed that EBV latency modulates the
p53 gene to produce chemoresistance in EBV-carrying cells [
24]. Dysregulated epigenetic machineries are able to disturb the normal expression of tumor-suppressor genes and oncogenes, resulting in tumorigenesis [
25].
DNA methylation is one of epigenetic alterations associated with tumorigenesis. EBV infection clearly affects the DNA methylation that subsequently leads to the development of different cancers. For example, DNA hypermethylation of tumor-related genes is more frequent in EBV-positive Hodgkin’s lymphoma (HL) cases than EBV-negative HL cases [
26]. This hypermethylation is observed in NPC and EBVaGC as well [
27,
28], suggesting the distinct role of EBV in DNA methylation for tumorigenesis. Other exemplary studies have shown that alterations in DNA methylation patterns are strongly associated with not only disease prognosis but also patient survival after anticancer therapy [
29]. The altered DNA methylation patterns have been used as key biomarkers for predicting disease prognosis and the efficacy of anticancer therapy [
30].
Thus, we hypothesized that EBVaGC with a hypermethylated DNA phenotype would have quite different therapeutic responses to anticancer drugs compared to EBVnGC. In support of this hypothesis, one study showed that EBVaGC was more resistant to docetaxel and 5-fluorouracil-based chemotherapies than EBVnGC [
31]. However, differences in responses to platinum-based chemotherapy between EBVaGC and EBVnGC still remain unclear due to a lack of presenting molecular mechanisms. The current study therefore (1) evaluated the bioactive effects of a combination of cisplatin and 5-Azacytidine (5-AZA) on EBV and EBVaGC, (2) determined the epigenetic mechanisms used by the drug combination to overcome tumor resistance, and (3) presented a novel therapeutic approach for EBVaGC.
3. Discussion
DNA methylation can be an attractive approach to epigenetic modulation favorable for tumor development and related chemotherapeutic resistance [
16]. DNA methyltransferases (DNMTs) responsible for DNA methylation have been known to be overexpressed in cancer cells. Moreover, their upregulation has been strongly associated with the loss of tumor suppressor genes (TSG), with the resultant TSG suppression being a good biomarker for a positive prognosis in patients with cancer [
29]. In fact, DNA hyper- or hypomethylations have been strongly correlated with chemoresistance and survival rate in gastric carcinoma [
44]. Therefore, the biological roles of DNA methylation in the development of EBV-associated cancer should be further understood to establish better therapeutic approaches. The current study determined (1) the anticancer effects of platinum-based anticancer drugs, (2) epigenetic mechanism used for cisplatin-mediated activities, and (3) synergistic anti-EBV effects of cisplatin and 5′-azacytidine.
Firstly, our study showed that EBV infection induces the upregulation of
DNMT3A depending on types of EBVaGC. EBV infection could upregulate
DNMT3A in MKN1 cells through cisplatin treatment. Similarly, EBVaGC SNU719 cells highly overexpressed
DNMT3A with cisplatin while EBVaGC YCCLE1 cells did not. Cisplatin treatment slightly increased
BZLF1 proteins while reducing
DNMT3A proteins in YCCLE1 cells. We could infer some reasons why these cellular differences occur in responding to cisplatin treatment. A possible reason is related to the difference in the ability to induce EBV lytic reactivation. A conventional TPA treatment was not able to induce the EBV lytic cycle in YCCLE1 cells, but was enough for EBV lytic reactivation in SNU719 cells [
45]. There was no detection of BZLF1, BRLF1, BMRF1, or BHRF1 in YCCLE1. However, these proteins were detectable in SNU719 cells. DNA methylation is included in the epigenetic alterations associated with EBV-associated tumorigenesis. In cells with EBV type I latency, the promoters of EBV lytic genes are intensively repressed and suppressed by DNA methylation [
46]. Thus, previous studies led to speculation that DNMT3A in YCCLE1 cells might not play a role as a key regulator for expressing EBV lytic genes, which might be associated with a loss of cisplatin-mediated
DNMT3A upregulation. In contrast, DNMT3A might keep epigenetic control of the expression of EBV lytic genes in SNU719 cells and MKN1–EBV cells. The maintenance of DNMT3A in epigenetic control might contribute to cisplatin-mediated
DNMT3A upregulation. That is, cisplatin was likely to unsystematically induce EBV lytic reactivation and DNMT3A might be overexpressed to suppress the EBV lytic reactivation. This is a reasonable inference, yet further studies are necessary to have a clear understanding of the loss of DNMT3A upregulation in YCCLE1 cells.
Secondly, our study showed that cisplatin alone could not induce EBV lytic reactivation but did dose-dependently upregulate
DNMT3A. Interestingly, cisplatin-mediated
DNMT3A upregulation had adverse effects on the expression of EBV lytic genes in SNU719 cells. Moreover, cisplatin was able to stabilize
DNMT3A proteins from proteasome degradation in SNU719 cells. These results imply that DNMTs were related to the cytotoxicity of cisplatin on SNU719 cells. After further investigating the role of DNMT1 or DNMT3A on EBV lytic reactivation, we found that knockdown of either DNMT1 or DNMT3A altered EBV lytic reactivation in SNU719 cells. In particular, cisplatin treatment greatly upregulated the EBV immediate-early (IE) lytic gene, BZLF1, and accelerated apoptotic cell death in SNU719-shDNMT3A cells. These results suggest that DNMT3A plays a key role in suppressing EBV lytic reactivation through de novo methylation. Consistently, several clinical studies have reported that DNA hypermethylation occurred more frequently in EBVaGC than in EBVnGC [
44,
47]. Moreover, EBV infection in B cells has been shown to cause DNA hypermethylation in host genes responsible for the cell cycle, DNA damage repair, and apoptosis [
35]. Therefore, we can reasonably infer a mutual regulation between DNMT3A and EBV lytic reactivation.
Thirdly, we determined how DNMT3A plays a key role in controlling the EBV lifecycle. Based on several previous studies, ATM kinase activity was selected for further investigation, given that the ATM kinase orchestrates the DNA damage response and induces EBV lytic reactivation in EBV-infected B cells and LCLs [
48]. Interestingly, we observed that
ATM protein and mRNA were considerably upregulated in the absence of DNMT3A, while the 5′-UTR of
ATM was significantly less methylated after DAC treatment. These findings implied that DNMT3A likely hypermethylated the
ATM 5′-UTR, with the resultant
ATM downregulation possibly preventing both a DNA damage response and EBV lytic reactivation. Consistent with our results, studies have shown that the DNA damage response could be considered a cellular stress similar to hypoxia and that differentiation could effectively turn on EBV lytic reactivation in epithelial cells [
33]. ATM has been considered one of the key transducers for the DNA damage response [
49] and plays an important role in the signaling pathways commonly activated during EBV lytic reactivation [
41,
50]. Moreover, the
BGLF4 protein, one of EBV’s early lytic proteins, had previously been shown to phosphorylate topoisomerase II and TIP60 to activate the DNA damage response [
51]. Therefore, EBV lytic reactivation is closely linked to DNA damage response.
Fourthly, the current study further investigated how the ATM signaling pathway was involved in EBV lytic reactivation. Previous studies have reported that chloroquine induced EBV lytic reactivation by activating TRIM28 phosphorylation at serine 824 in Burkitt’s lymphoma cells, with the resultant ATM-dependent TRIM28 phosphorylation being required for the upregulation of
BZLF1 and
EA-D in Burkitt’s lymphoma and lymphoblastoid cells [
41]. Aside from EBV, human cytomegalovirus has also been shown to require ATM-dependent TRIM28 phosphorylation for the biological switch from the latency to the lytic phase [
52]. Thus, we determined whether cisplatin phosphorylated TRIM28 and whether the resultant phosphorylation was involved in ATM-mediated EBV lytic reactivation. Accordingly, our results showed that cisplatin treatment in the absence of DNMT3A facilitated (1)
ATM upregulation, (2) ATM-dependent TRIM28 phosphorylation, and (3) EBV lytic reactivation. Furthermore, our study demonstrated that
ATM knockdown SNU719 cells found cisplatin less cytotoxic than control cells, suggesting the disabling of EBV lytic reactivation due to the loss of ATM kinase activity. Consistent with previous studies, all data presented herein support the notion that the ATM-TRIM28 pathway plays a key role in suppressing EBV lytic reactivation during cisplatin treatment.
Finally, in vivo experiments were conducted to confirm the in vitro synergistic effect of the anticancer drug and epigenetic modulator on cytotoxicity. While monotreatment with either cisplatin or 5′-Azacitidine (5-AZA) partially reduced tumor development, cotreatment of both drugs synergistically suppressed tumor development. Our results show that 5-AZA could indeed upregulate ATM by demethylating the ATM promoter, which consequently increased the vulnerability of EBV-infected cells to EBV lytic reactivation. Cotreatment of cisplatin and 5-AZA also accelerated apoptotic cell death in SNU719 cells compared to monotreatment. Both cisplatin and 5-AZA synergistically worked to remove in vitro EBV-infected cells and suppress in vivo tumor development. However, this synergistic effect in tumorigenesis was undermined by the loss of functional ATM. Interestingly, cotreatment of cisplatin and 5-AZA stimulated the development of the xenograft tumor in mice even though this stimulatory effect was insignificant. The loss of ATM clearly led to different cellular responses to cisplatin and 5-AZA, whose mechanism is left for further study.
The current study evaluated the clinical utility of cisplatin and 5-AZA cotreatment for EBVaGC. Our in vitro and in vivo experiments showed that DNA methylation inhibitors, such as 5-AZA, can work synergistically with cisplatin to maximize the effectiveness of cisplatin-based chemotherapy (
Figure 14). These synergistic effects may help the development of a novel therapeutic approach for the treatment of EBVaGC. Several papers have been published that show that 5-AZA enhances the efficacy of cisplatin chemotherapy in lung and ovarian cancers [
53,
54], but not in gastric cancer. Thus, this study is the first to show the potential for cisplatin and 5-AZA cotreatment as an effective anti-gastric cancer approach in the near future.
4. Materials and Methods
4.1. Cell Lines and Reagents
Both gastric carcinoma cell lines SNU719 (EBVaGC) and MKN1 (EBVnGC) were purchased from Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI 1640 (Hyclone, Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Marlborough, MA, USA), antibiotics/antimycotics (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco, Waltham, MA, USA) at 37 °C with 5% CO
2 and 95% humidity. EBV BART
+ bacmid was obtained from Dr. Teru Kanda [
55]. MKN1 cells were transfected with EBV BART
+ bacmid and selected with 30 μg/mL hygromycin B (Wako, Osaka, Japan) for at least 2 weeks. Resultant MKN1 cells were named MKN1–EBV, cultured in RPMI 1640, and supplemented with 10% FBS, antibiotics/antimycotics, and GlutaMAX. YECCL1 cells were cultured in EMEM (Lonza, Basel, Switzerlan) supplemented with 10% fetal bovine serum (FBS, Hyclone, Marlborough, MA, USA), antibiotics/antimycotics (Gibco, Waltham, MA, USA) and GlutaMAX (Gibco, Waltham, MA, USA) at 37 °C with 5% CO
2 and 95% humidity.
4.2. Cytotoxicity Assay
The cytotoxic effects of cisplatin and 5′-Azacitidine (5-AZA) on SNU719 cells, MKN1 cells, MKN1–EBV cells, and YECCL1 cells were evaluated using Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). Briefly, 100 μL of cell suspension (2 × 10
4 cells/well) was seeded into a 96-well plate. The following day, cisplatin or 5-AZA was applied at various concentrations: 0.00–66.66 or 0.00–83.50 μM of cisplatin and 0.00–3275.9 μM of 5-AZA. After 48 h of treatment, 10 μL of CCK-8 solution was added to each sample. After incubating the samples for another 3 h, the absorbance of each cell suspension was measured at 450 nm using an enzyme-linked immunosorbent assay reader. All steps of the manufacturer’s recommended protocol were followed. Approximately 50% cytotoxicity (CD
50) was determined as previously described [
56]. Briefly, the middle absorbance between the highest and lowest absorbance was first calculated. Secondly, the concentration of the compound was evaluated by assigning a corresponding concentration to the middle absorbance. Thirdly, this compound concentration was identified as the CD
50 concentration. In subsequent experiments, cells were treated with compounds at CD
50 concentration for 48 h, after which old media containing mostly dead cells were removed, cells were further washed with phosphate-buffered saline at least twice to remove clearly dead cells, and finally 90% of live cells on average were harvested for analysis.
4.3. Luciferase Assay
SNU719 cells carrying the EBV BHLF1 promoter-luciferase construct were established and named SNU719-BHLF1 for subsequent luciferase assays [
57]. SNU719-BHLF1 cells were treated with either cisplatin alone (0.00~21.10 μM) or in combination with 5-AZA (0.00~1.32 μM for cisplatin, 0.00~134.9 μM for 5-AZA) in various concentrations for 48 h. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega, Madison, WI, USA) according to the manufacturer’s protocol.
4.4. Western Blot Assay
To assess the regulatory effects of cisplatin and 5-AZA on EBV protein synthesis, Western blotting was performed using SNU719 cells, MKN1 cells, MKN1–EBV cells, and YECCL1 cells treated with either cisplatin and 5-AZA alone or in combination. Treated cells were harvested using trypsin 48 h after treatment. Cells (10 × 106) were lysed using 200 μL of radioimmunoprecipitation assay (RIPA) lysis buffer (Tris-HCl (50 mM, pH 8.0)), NaCl (150 mM), ethylenediaminetetraacetic acid (EDTA; 2 mM, pH 8.0), 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) and supplemented with proteinase inhibitor (PI, Sigma, St. Louis, MO, USA) and phenylmethylsulfonyl fluoride (PMSF, Sigma, St. Louis, MO, USA). Xenograft tissue samples were lysed using 500 μL of RIPA lysis buffer with PI and PMSF and then homogenized using pestles. Cell or tissue lysates were further fractionated using a Bioruptor Sonicator set to provide 30-s on/off pulses for 5 min (Cosmo Bio, Tokyo, Japan). Protein in the cell lysates was measured using the Bradford assay. Equivalent quantities of protein were separated in 10% SDS polyacrylamide electrophoresis gel and transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). Membranes were probed with antibodies against EBV and cellular proteins. Thereafter, BZLF1 (Santa Cruz Biotechnology (SCB), Santa Cruz, CA, USA), EA-D (SCB), DNMT1 (Cell Signaling Technology (CST), Danvers, MA, USA), DNMT3A (CST), ATM (CST), TRIM28 (CST), and phospho-TRIM28 (Ser 824, CST) were detected using GAPDH (CST) and β-actin (SCB) as internal controls. goat-anti-mouse IgG-HRP (Genetex, Irvine, CA, USA) and goat-anti-rabbit IgG-HRP (Genetex, Irvine, CA, USA) were used as secondary antibodies. Antibody-bound proteins were visualized using an enhanced chemiluminescent detection reagent (GE Healthcare, Chicago, IL, USA). Membranes were stripped and reprobed with other antibodies.
4.5. Protein Stability Assay
To determine protein stability, SNU719 cells were treated with 100 μg/mL cycloheximide (CHX, Sigma, St. Louis, MO, USA) and 21.10 μM cisplatin for 0, 6, 12, 24, and 48 h. Treated cells were then harvested and prepared for Western blot assay to analyze DNMT3A stability. Intensity bands were analyzed using Image J software
https://imagej.nih.gov/ij/docs/index.html (accessed on 12 May 2021)).
4.6. Lentiviral Transduction
pLKO.1 vector-based shRNA constructs for DNA methyltransferase 1 (TRCN 0000021891, TRCN 0000021892, and TRCN 0000021893 for DNMT1) and DNA methyltransferase 3A (TRCN 0000035756, TRCN 0000035757, and TRCN 0000035758 for DNMA3A) were purchased (Sigma, St. Louis, MO, USA). Control shRNA (shCTL) were generated in the pLKO.1 vector with the target sequence 5′-TTA TCG CGC ATA TCA CGC G-3′. Lentiviruses were produced using envelope and packaging vectors pMD2.G and pSPAX2 as described previously. SNU719 cells were infected with lentivirus stocks carrying pLKO.1-puro vectors by overlaying the lentivirus stock on SNU719 cells for 24 h. Thereafter, the lentivirus stocks were replaced with fresh RPMI medium and treated with 2.0 μg/mL puromycin 48 h after infection. The RPMI medium with 1.0 μg/mL puromycin was replaced every 2 to 3 days. Cells were selected using puromycin for at least 14 days. Resultant SNU719 cells were named as followed then subjected to further analyses: SNU719-pLKO.1 cells, SNU719-shDNMT1 cells, and SNU719-shDNMT3A cells.
4.7. Quantitative Reverse-Transcription Polymerase Chain Reaction Assay
Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays were performed to quantify transcripts of interesting genes in SNU719 cells, MKN1 cells, MKN1–EBV cells, and YECCL1 cells. RNA was isolated from the cells using the RNeasy mini kit (Qiagen, Germantown, MD, USA). Purified RNA was converted into cDNA using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Diluted RT products were analyzed using real-time PCR (LightCycler 96, Roche, Basel, Switzerland). mRNA levels of actin or GAPDH in each sample were used as the internal control. qRT-PCR with non-reverse-transcribed RNA was conducted to serve as the negative control in each reaction. Gene primer sets specific to qRT-PCR were as follows: DNMT3A (forward: CCT GTG GGA GCC TCA ATG TTA, reverse: CTT GCA GTT TTG GCA CAT TCC) and ATM (forward: TTT ACC TAA CTG TGA GCT GTC TCC AT, reverse: ACT TCC GTA AGG CAT CGT AAC AC)
4.8. EBV Promoter Usage Assay
RNA was isolated from SNU719 cells using an RNeasy Mini Kit (Qiagen, Germantown, MA, USA), after which purified RNA was synthesized into cDNA using SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). Resultant cDNA was subjected to semi-quantitative PCR assay for the detection of EBV promoter activity. The effects of cisplatin on EBV promoter activity were then examined. Primer sequences for β-actin, EBV Qp, and EBV Fp were similar to those previously published. cDNA was amplified in a 25 μL reaction solution containing 2.5 μL of 10× reaction mix, 2.5 μL of TuneUp solution, 0.25 μL of Taq Plus polymerase, and 2.5 μL of 10 pmol forward/reverse primer. The following cycle conditions were used: 95 °C for 3 min; 30 cycles of 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s; and 72 °C for 10 min. Primers specific to the EBV promoters were as follows: Qp (F: 5′-GTG CGC TAC CGG ATG GC-3′, R: 5′-CAT GAT TCA CAC TTA AAG GAG ACG G-3′), and Fp (F: 5′-GGG TGA GGC CAC GCT TT-3′, R: 5′-CAG GTC TAC TGG CGG TCT ATG AT-3′). PCR reactions were subjected to a TaKaRa PCR Thermal Cycler (TaKaRa, Kyoto, Japan) and run on a 1.2% agarose/TBE gel.
4.9. Cell Viability Assay
Cell viability was determined using a MUSE count and viability kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s protocol. Serval cells (1 × 106) including SNU719 cells and MKN1–EBV cells were seeded into 6-well plates and treated with cisplatin or 5′ azacytidine for 24 or 48 h. A negative control sample was treated with the same volume of Dulbecco’s PBS (DPBS) for 24 or 48 h. Treated cells were trypsinized, harvested, and resuspended at 106–107 cells/mL of fresh serum. The cell suspension (20 μL) was mixed with 380 μL of the MUSE count and viability reagent and then incubated at 25 °C for 5 min. Cell viability was measured using MUSE cell analyzer (Millipore, Burlington, MA, USA).
4.10. Annexin V and Dead Cell Assay
To determine apoptotic cell death according to cisplatin treatment, a MUSE annexin V and dead cells assay kit (Merck Millipore, Darmstadt, Germany) was used. Serval cells (1 × 106), such as SNU719-pLKO.1, SNU719-shDNMT1, and SNU719-shDNMT3A, were seeded into a 6-well plate and treated for 48 h with 1.32 μM and 21.1 μM of cisplatin for 48 h. A negative control sample was treated with the same volume of PBS for 48 h. Treated cells were trypsinized, harvested, and resuspended at 106/mL in RPMI medium with FBS. The cell suspension (100 μL) was mixed with 100 μL of MUSE annexin V and dead cell reagent for 20 min at room temperature. Apoptosis was measured using the MUSE cell analyzer (Millipore, Burlington, MA, USA) according to the manufacturer’s protocol.
4.11. Methylated DNA Precipitation (MeDIP) Assay
A quantity of2’-deoxy-5′-azacytidine (DAC) was used to treat SNU719, SNU719-pLKO.1, and SNU719-shDNMT3A cells for 72 h. These cells were then subjected to genomic DNA (gDNA) isolation as follows: gDNA was isolated using the proteinase K and phenol/chloroform extraction protocol. Accordingly, 8 μg of gDNA was resuspended in up to 450 μL of 1× TE buffer (50 mM Tris buffer (pH 8.0) and 10 mM EDTA buffer) and denaturized by heating at 95 °C for 10 min followed by rapid chilling on ice. Thereafter, 1 μg of DNA was extracted to be used as the input control DNA, after which 45 μL of 10× immunoprecipitation buffer (100 mM sodium phosphate buffer (pH 7.0), 1.4 M NaCl, and 0.5% Triton X-100) was added. DNA samples were immunoprecipitated with 2 μg of anti-5mC (Active Motif, Carlsbad, USA) or 2 μg of mouse isotype anti-IgG control (Sigma-Aldrich) for 2 h at 4 °C with rotation. The antibody-bound DNA was captured with Dynabeads Protein G (ThermoFisher Scientific, Grand Island, NY, USA) for 2 h at 4 °C with rotation. The DNA captured beads were washed three times with 1× IP buffer for 10 min at room temperature with rotation and eluted in 250 μL of proteinase K digestion buffer (50 mM Tris buffer (pH 8.0), 10 mM EDTA buffer, 0.5% SDS, and 70 μg of proteinase K) at 50 °C for 3 h using a Thermomix C (Eppendorf, Hamburg, Germany) at 800 rpm. Eluted DNA samples were isolated with phenol/chloroform DNA purification and precipitated using 2 volumes of 100% EtOH, 400 mM NaCl, and 20 μg glycogen overnight at 4 °C. Precipitated DNA was measured using Nanodrop, and PCR was performed on 5 ng of DNA to determine ATM methylation, with EBV Cp and Qp as methylation controls. Conventional PCR assays were performed using a HelixAmp Taq Polymerase Kit (Nanohelix, Deajeon, Korea). The PCR cycle conditions and primer sets used are available upon request.
4.12. Bisulfite Conversion and Pyrosequencing Analysis
To analyze ATM 5′-UTR II methylation, SNU719 cells were treated with either DPBS or 1.32 μM cisplatin and SNU719-shDNMT3A cells were treated with 1.32 μM cisplatin for 48 h. gDNA was then isolated from these cells using the procedure described above. Resultant gDNA was further subjected to bisulfite modification using an EZ DNA methylation kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s protocol. A total of 2 μg of gDNA was denatured by adding 5.5 μL of 2M NaOH at 37 °C for 10 min, after which 30 μL of 10 mM hydroquinone and 520 μL of 3M sodium bisulfite was added. The mixture was incubated at 50 °C for 17 h, desalted using the Wizard DNA Purification Resin Kit (Promega, Madison, WI, USA), and desulfonated by adding 5.5 μL of 3 M NaOH for 5 min. The modified DNA was precipitated with ethanol and resuspended in 35 μL of nuclease-free water. The PCR for the ATM 5′-UTR II was performed using the Solg™ h-Taq DNA Polymerase Kit (Solgent, Daegeon, Korea) with the following primer and cycle conditions: F: 5′-TGT TGT TTA GGT TGG AGT ATA GT-3′, R: 5′-biotin-ACC AAC ATA AAA CCC TAT CTC T-3′, sequencing primer: 5′-TTT TGA GTA GTT GGG ATT A-3′, and 95 °C for 15 min; 40 cycles of 95 °C for 55 s, 60 °C for 55 s, 72 °C for 60 s, and finally 72 °C for 10 min. PCR products were confirmed using agarose gel electrophoresis, while pyrosequencing analysis was performed using the PyroMark Q24 (Qiagen, Hilden, Germany). Methylated and unmethylated DNA were used as methylation and unmethylation controls, respectively.
4.13. Depletion of ATM Proteins via CRISPR/Cas9 Genomic Editing
To knock out
ATM in SNU719 cells and MKN1–EBV cells, the CRISPR/Cas9 system was applied to these cells. A single-guide (sg) RNA sequence was designed using the web tool of the Centre for Organismal Studies, Heidelberg University (CCTop,
https://crispr.cos.uni-heidelberg.de/ (accessed on 7 August 2021)). The following oligo set was used as sgRNA to target the
ATM: forward oligo 5′-AAAc TGA GTC TAG TAC TTA ATG ATC-3′, reverse oligo 5′-CACCg ATC ATT AAG TAC TAG ACT CA-3′. The sgRNA targeting human ATM (NG_009830.1) was designed and cloned into the LentiCRISPRv2 vector (Addgene number 52961). The LentiCRISPRv2 vector or LentiCRISPRv2-ATM sgRNA vector was co-transfected with the lentivirus packaging plasmids (psPAX2, pMD2.G) into HEK 293T cells for 72 h. The viral supernatants were then harvested, filtered, and used to infect SNU 719 cells and MKN1–EBV cells for 24 h. Thereafter, the viral supernatants were replaced with fresh RPMI medium and treated with 0.5 μg/mL puromycin every 2 to 3 days, after which knockout efficiency was confirmed through protein expression. Finally, resultant SNU719 cells were named SNU719-C/Cas9 and SNU719-ATM(-) cells, and the resultant MKN1–EBV cells named MKN1–EBV-C/Cas9 and MKN1–EBV-ATM(-) cells.
4.14. Anti-Tumor Assay in a Xenograft Mouse Model
Nude mice (female, 5-week-old; Raonbio Co., Ltd., Seoul, Korea) were used to establish xenograft animal models to assess anti-tumor effects. Mice were individually housed in a pathogen-free controlled environment (23–27 °C under a 12-h light/dark cycle) and provided food and water ad libitum. At first, we evaluated the synergistic anti-tumor effect of a combination therapy of cisplatin and 5-AZA. To this end, 2.5 × 106 MKN1–EBV cells were subcutaneously implanted into the dorsum next to the right hind leg of the mice (n = 35). After 14 days, xenograft mice were randomly divided into four groups that received control (PBS, n = 8), cisplatin (0.5 mg/kg, n = 9), 5-AZA (2 mg/kg, n = 9), and combination (0.5 mg/kg cisplatin and 2 mg/kg 5-AZA, n = 9) for 43 days. Animals in each group received the same volume of PBS, cisplatin, 5-AZA, and combination therapy via weekly intraperitoneal injection. Tumors were identified and measured every other day using a standard caliper, while tumor size was calculated using the following formula: [tumor length (mm) × tumor width (mm)2]/2. After the tumor size had reached 1000 mm3, the animals were euthanized, and tumors were harvested. The animal experiments were conducted in accordance with the National Research Council’s Guide (IACUC, Seoul, Korea) for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Experiments Committee of Duksung Women’s University (permit number: 2019-003-004).
Subsequently, we investigated the necessity of functional ATM for producing a synergistic anti-tumor effect in combination therapy with cisplatin and 5-AZA. To this aim, 2.5 × 106 MKN1–EBV-ATM(-) cells were subcutaneously implanted into the dorsum next to the right hind leg of the mice (n = 32). After 14 days, xenograft mice were randomly divided into four groups that received control (PBS, n = 8), cisplatin (0.5 mg/kg, n = 8), 5-AZA (2 mg/kg, n = 8), and combination (0.5 mg/kg cisplatin and 2 mg/kg 5-AZA, n = 8) for 33 days. Similarly to the previous anti-tumor assay using MKN1–EBV cells, animals in each group received the same volume of PBS, cisplatin, 5-AZA, and combination via weekly intraperitoneal injection. Tumors were identified and measured every other day by same methods. After the tumor size had reached approximately 1000 mm3, the animals were euthanized, and tumors were harvested. The animal experiments were conducted in accordance with the National Research Council’s Guide (IACUC, Seoul, Korea) for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Experiments Committee of Duksung Women’s University (permit number: 2020-007-004).
4.15. IHC Analysis
The tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Subsequently, paraffin sections were performed according to the manufacturer’s protocol (Abcam, Cambridge, UK). Deparaffinized and rehydrated 7-μm sections were immunostained with the antibodies against EBNA1 (Santa Cruz). After development with a peroxidase reagent, diaminobenzidine (Vector, Linaris, Germany) and counterstaining with hematoxylin, stained tumor tissues were visualized by light microscopy.
4.16. Kaplan–Meier Analysis
The 5-year overall survival (OS,
n = 881) rates were analyzed for gastric cancer cases from the Kaplan–Meier plotter [
43]. A total of 320 patients were analyzed from the following datasets: GSE14210, GSE15459, GSE22377, GSE29272, GSE51105, and GSE62254. Input genes were DNMT3A (Affy id: 222640_at) and ATM (Affy id: 212672_at). These analyses were restricted to Lauren classification intestinal (
n = 336) types of gastric cancers. Other subtypes included all patients (gender, perforation, treatment, and HER2 status).
4.17. Statistical Analysis
Statistical analyses were conducted using a two-tailed Student’s t-test (Microsoft, Redmond, WA, USA) and a one-way analysis of variance (ANOVA) and re-verified thorough Dunnett’s multiple comparison test using GraphPad Prism (San Diego, CA, USA).