Abstract

   m^6A modification is a crucial epigenetic regulatory mechanism in
   diffuse large B-cell lymphoma (DLBCL). Low-dose cardiotonic drugs have
   been shown to induce apoptosis in DLBCL cells through epigenetic
   modulation. However, the involvement of the cardiotonic drug ouabain in
   the malignant progression of DLBCL remains unclear. Our study revealed
   that ouabain indeed contributes to the malignant progression of DLBCL
   through m^6A modification. Through qPCR analysis, we observed a
   negative correlation between ouabain concentration and the expression
   levels of the demethylase ALKBH5 and the m^6A-binding protein IGF2BP2
   in DLBCL cells. Furthermore, high expression levels of ALKBH5 and
   IGF2BP2 were identified in both the GEO database and DLBCL patient
   tissue samples. Notably, elevated ALKBH5 and IGF2BP2 promoted cell
   proliferation both in vitro and in vivo. Inhibition of their expression
   rendered DLBCL cells more sensitive to ouabain treatment, resulting in
   significant suppression of cell proliferation, G1/S phase cell cycle
   arrest, and increased apoptosis. In summary, our results clarify that
   the demethylase ALKBH5 and the m^6A-binding protein IGF2BP2 are
   involved in the malignant progression of DLBCL, and that the
   cardiotonic drug ouabain can inhibit the proliferation of DLBCL cells
   by inhibiting the expression of ALKBH5 and IGF2BP2, which provides new
   insights into the targeted treatment of DLBCL.

   Keywords: m^6A, ALKBH5, IGF2BP2, ouabain, DLBCL

1 Introduction

   Diffuse large B-cell lymphoma (DLBCL) is an aggressive malignancy and
   ranks as one of the most common non-Hodgkin’s lymphomas worldwide
   ([46]Takahara et al., 2023). The current standard treatment for DLBCL
   consists primarily of R-CHOP therapy, which includes rituximab,
   cyclophosphamide, doxorubicin, vincristine, and prednisone ([47]Sehn
   and Gascoyne, 2015). Approximately 60% of patients achieve complete
   remission with R-CHOP therapy ([48]Sehn et al., 2021), but owing to the
   high heterogeneity of DLBCL, approximately 40% of patients experience
   posttreatment relapses, leading to unfavorable treatment outcomes
   ([49]Sarkozy and Sehn, 2018). Therefore, there is an urgent need to
   develop a new combination therapy to address this clinical challenge.
   It is now widely accepted among clinicians that adding new drugs
   (preferably less toxic) to the standard R-CHOP regimen is necessary for
   treating relapsed and refractory DLBCL patients ([50]Chiappella et al.,
   2017). Consequently, the discovery of targeted drugs for first-line
   DLBCL treatment and the identification of new molecular targets are
   crucial for improving treatment efficacy.

   As a representative cardiotonic drug, ouabain is commonly used to treat
   heart failure and atrial fibrillation. Although there have been
   attempts to treat tumors with cardiac glycosides ([51]Shen et al.,
   2019), their use has been limited because of the difficulty in
   determining the effective therapeutic dose and the lack of
   understanding of their mechanism of action ([52]Prassas and Diamandis,
   2008).In the 1970s, Stenkvist reported that patients with breast cancer
   who took cardiac glycosides (digoxin) for heart disease presented more
   benign changes in tumor tissues than those who did not take digoxin.
   The recurrence rate and mortality rate of patients with breast cancer
   after surgery are also significantly lower among those taking digoxin
   ([53]Stenkvist et al., 1980). These findings led researchers to
   investigate the potential tumor-suppressing effects of cardiac
   glycosides. In 2001, Haux et al. investigated whether patients taking
   digitoxin for cardiac disease have a different cancer incidence than
   does the general population. They reported that as the blood
   concentration of digitalis increased, the incidence of leukemia,
   lymphoma, and urinary system tumors decreased ([54]Haux et al., 2001).
   Previous studies have shown that ouabain can inhibit proliferation in
   acute myeloid leukemia (AML), and promote tumor cell apoptosis in acute
   T lymphoblastic leukemia ([55]Valdes et al., 2007; [56]Tailler et al.,
   2011). These findings suggest that ouabain may play a role in various
   hematologic tumors including DLBCL, suggesting further exploration into
   its possible function and mechanism in DLBCL is needed.

   In the past decade, many epigenetic modifiers have been developed and
   applied in the clinical treatment of patients with hematological tumors
   ([57]Yankova et al., 2021; [58]Weng et al., 2022; [59]Chen et al.,
   2023a), but little has been explored for the treatment of DLBCL with
   epigenetic modifications ([60]Sermer et al., 2019; [61]Uddin et al.,
   2021). Through RNA sequencing and detection of m^6A-modifying enzymes,
   we demonstrated that the cardiac glycoside drug ouabain affects DLBCL
   by influencing m^6A methylation. m^6A RNA methylation is the most
   common internal modification of mammalian mRNAs and plays an important
   biological role by regulating important cellular processes ([62]Sun et
   al., 2019; [63]Uddin et al., 2021). m^6A modifications are dynamically
   regulated by methyltransferases, demethylases, and binding proteins,
   thereby determining mRNA fate by regulating mRNA stability, transport,
   translation, and degradation progression ([64]Li H.-B. et al., 2017).
   qPCR further demonstrated that ouabain downregulated the m^6A
   demethylase ALKBH5 and the m^6A-binding protein IGF2BP2 in
   ouabain-treated DLBCL cells. ALKBH5 is a demethylase that affects
   nuclear RNA output, metabolism, and gene expression ([65]Zhu et al.,
   2019). Human insulin-like growth Factor 2(IGF2) mRNA binding protein
   2(IGF2BP2/IMP2) is an RNA-binding protein that regulates various
   biological processes ([66]Bell et al., 2012). They are involved in the
   development of cancer by communicating with different RNAs such as
   microRNAs (miRNAs) ([67]Li X. et al., 2017), messenger RNAs (mRNAs)
   ([68]Dai et al., 2017), and long noncoding RNAs (lncRNAs) ([69]Wu et
   al., 2018; [70]Wang et al., 2021). Increasing evidence shows that
   ALKBH5 and IGF2BP2 are closely related to tumor growth, proliferation,
   and survival. Inhibition of ALKBH5-mediated m^6A modification decreases
   USP1 expression, which can ameliorate glucocorticoid resistance in
   T-ALL ([71]Ye et al., 2023). As m^6A-binding proteins, IGF2BP2 and
   IGF2BP3 increase the stability of DDX21 in a m^6A-dependent manner,
   leading to the progression of AML ([72]Liu et al., 2022). However, the
   role of ALKBH5 and IGF2BP2 in DLBCL tumorigenesis and their molecular
   mechanisms remain unclear.

   It has been reported that ouabain regulates tumor suppressor genes via
   epigenetic silencing ([73]Raynal et al., 2016). Ouabain can also
   suppress the ability of a cancerous lesion to spontaneously shed CTC
   clusters by inhibiting Na^+/K^+ ATPase in vivo, leading to a remarkable
   reduction in metastasis seeding ability ([74]Gkountela et al., 2019).
   These findings undoubtedly reveal the possibility of an antitumor
   effect of ouabain through the regulation of epigenetic factors.
   Therefore, this study aimed to investigate the downregulation of the
   m^6A demethylase ALKBH5 and m^6A-binding protein IGF2BP2 induced by
   treatment with the cardiac glycoside drug ouabain, which inhibits the
   proliferation and promotes apoptosis of DLBCL cells. Based on these
   findings, the key effective molecules of ouabain in the treatment of
   DLBCL through epigenetic modification were identified, which provides a
   theoretical and experimental basis for clinical guidance for the
   combination of DLBCL with other drugs to improve poor patient
   prognosis.

2 Materials and methods

2.1 Cell lines and reagents

   The human cell lines SU-DHL4, OCI-Ly3, SU-DHL2, and U2932 were cultured
   in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL
   penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA,
   United States) under an atmosphere with 5% CO[2] at 37°C. Ouabain was
   purchased from Sigma-Aldrich (St. Louis, MO, United States).

2.2 Tissue samples

   A total of 7 tissue samples from DLBCL patients and 3 tissue samples
   from patients with lymph node hyperplasia were obtained from Nanjing
   Drum Tower Hospital. All the tissues were immediately stored at −80°C.
   Informed consent was obtained from each patient, and all samples were
   collected with informed consent and approved by the Medical Ethics
   Committee of the Affiliated Hospital of Nanjing University Medical
   School.

2.3 RNA interference and lentiviral infection

   The ALKBH5, shALKBH5, or empty vector lentiviral plasmids were
   purchased from GeneChem (Shanghai, China) and the IGF2BP2, shIGF2BP2,
   empty vector lentiviral plasmidsandsmall interfering RNAs (siRNAs) were
   purchased from GenePharma (Suzhou, China). 48 h after transfection,
   cell lines were treated with 2 μg/mL puromycin for 2 weeks and screened
   for cell lines stably overexpressing or silencing ALKBH5/IGF2BP2. The
   primer sequences are shown in [75]Supplementary Table S1.

2.4 Quantitative PCR

   Total RNA from cells or frozen tissues was isolated via TRIzol reagent
   (Invitrogen, Carlsbad, CA, United States) and used as a template to
   synthesize cDNA via a reverse transcription kit (Takara, Dalian,
   China). Quantitative PCR (qPCR) was performed via SYBR Green reagents
   (Vazyme, Nanjing, China), with β-actin used as an internal control. The
   primer sequences are shown in [76]Supplementary Table S2.

2.5 Western blot analysis

   The protein concentration of the cell lysates was quantified via a
   bicinchoninic acid protein assay kit (Beyotime, Jiangsu, China). Equal
   amounts of protein were analyzed by immunoblotting with an anti-ALKBH5
   antibody (#D264377, BBI), an anti-IGF2BP2 antibody (#D120813, BBI), an
   anti-SF3B4 antibody (Catalog #10482-1-AP, Proteintech), and an
   anti-YWHAG antibody (12381-1-AP, Proteintech),with an anti-β-actin
   antibody (Sigma-Aldrich, United States) used as an internal control.

2.6 Cell viability and proliferation assays

   To calculate the IC50 values of ouabain, SU-DHL4, OCI-Ly3, SU-DHL2, and
   U2932 cells were seeded into 96-well plates at a density of 3 × 10^4
   per well, treated with different concentrations of ouabain (0, 30, 60,
   90, 120, 150 or 180 nM) and cultured for 24 h. Cell viability was
   measured by adding Cell Counting Kit-8 (CCK-8; Vazyme, Nanjing, China)
   to each well. The absorbance was measured at 450 nM by
   spectrophotometry after incubation for 3 h at 37°C.

   Cell proliferation was evaluated via a Cell Counting Kit-8 (CCK-8;
   Vazyme, Nanjing, China). The cells (3 × 10^4 per well) were treated
   with ouabain (SU-DHL4 at 80 nM; OCI-LY3 at 140 nM; U2932 at 140 nM) in
   triplicate wells of a 96-well plate and incubated at 37°C. CCK-8
   reagent was added to the wells at 0 h, 24 h, 48 h, and 72 h.
   Subsequently, the absorbance values of the samples were measured at
   450 nm.

2.7 Cell cycle analysis

   After 48 h of transfection, the cells were first harvested, washed
   twice with PBS and incubated in PBS containing 0.02% Triton X-100,
   0.1 mg/mL RNase (Sigma-Aldrich), and 10 μg/mL propidium iodide (PI,
   40%, Sigma-Aldrich) for 30 min at 37°C. The cell cycle distribution was
   detected via a FACScan flow cytometer (Becton Dickinson & Co., San
   Jose, CA, United States).

2.8 Apoptosis assay

   The cells (5 × 10^5 cells per well) were seeded in 24-well plates and
   incubated with ouabain at the IC50 for 24 h. Apoptosis was examined via
   flow cytometry on a FACScan flow cytometer (Becton Dickinson & Co., San
   Jose, CA, United States) after the cells were stained with Annexin V-PE
   and 7-AAD.

2.9 Bioinformatic analysis

   The gene expression profiles were obtained from Gene Expression
   Profiling Interactive Analysis (GEPIA) and the Gene Expression Omnibus
   (GEO, [77]GSE12453; GEO, [78]GSE83632).

2.10 RNA-sequencing analysis

   RNA sequencing analysis of the differentially expressed enriched genes
   of OCI-LY3 cells treated with ouabain was performed via GO and KEGG
   analyses. RNA sequencing was performed by Lc-Bio Technologies
   (Hangzhou, China).

2.11 Statistical analysis

   Student’s t-test (two-tailed) was performed to evaluate differences
   between two groups. One-way analysis of variance (ANOVA) was used to
   evaluate differences between more than two groups. A minimum of three
   replicates were conducted in all the experimental trials, and the
   results are reported as the means ± standard deviations (SDs).
   Differences were considered significant when the P-value was <0.05 (*p
   < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Statistical analyses
   were performed via GraphPad Prism 8.0.

3 Results

3.1 Establishment of the IC50 for each cell line treated with ouabain

   To investigate the impact of ouabain on DLBCL cell viability, we
   exposed DLBCL cells to varying concentrations of ouabain. The IC50
   values of ouabain in OCI-LY3, U2932, SU-DHL4, and SU-DHL2 cells were
   141.8 nM, 144.3 nM, 81.76 nM and 91.37 nM, respectively ([79]Figure
   1A). Furthermore, upon treating four DLBCL cell lines with ouabain at
   the IC50, we observed significant induction of cell apoptosis
   ([80]Figure 1B). To further assess the ability of ouabain to
   effectively prevent the proliferation of DLBCL cells, we treated DLBCL
   cells with ouabain and observed their proliferation for three
   consecutive days ([81]Figure 1C). Our findings revealed substantial
   suppression of DLBCL cell proliferation under ouabain treatment,
   confirming the efficacy of ouabain in inhibiting DLBCL cell
   proliferation and promoting apoptosis at low concentrations.

FIGURE 1.

   [82]FIGURE 1
   [83]Open in a new tab

   The viability of the four DLBCL cell lines was determined following
   treatment with different concentrations of ouabain. (A) The viability
   of four DLBCL cancer cell lines treated with different concentrations
   of ouabain was measured via a Cell Counting Kit 8 assay (CCK-8). IC50,
   50% inhibitory concentration. (B) Representative flow cytometric images
   of apoptosis in four DLBCL cell lines after treatment with ouabain.
   Apoptosis was detected by Annexin V-PE and 7-AAD. (C) A Cell Counting
   Kit 8 assay was utilized to assess the proliferation of DLBCL cells
   (OC-LY3, U2932, SU-DHL4, and SU-DHL2) treated with ouabain. The data
   are presented as the means ± SDs; n = 3. *p < 0.05, **p < 0.01, ***p <
   0.001, ****p < 0.0001 (Student’s t-test).

3.2 Involvement of ouabain in RNA methylation progression in DLBCL

   To clarify the role of ouabain in DLBCL, we first added ouabain to
   OCI-LY3 cells and conducted RNA-sequencing (RNA-seq) analysis to
   analyze the differentially expressed enriched genes ([84]Figure 2A).
   KEGG pathway enrichment analysis revealed a close association between
   DLBCL cells and RNA degradation following ouabain treatment ([85]Figure
   2B). m^6A methylation of RNAplays a crucial role in the degradation
   process. For example, YTHDF2 mediates mRNA degradation of tumor
   suppressors and induces AKT phosphorylation in an
   N^6-methyladenosine-dependent way in prostate cancer ([86]Li et al.,
   2020). As we all know, the miRNA-dependent mechanism also plays an
   important role in RNA degradation. The circular RNA circular RNA sponge
   for miR-7 (ciRS-7) inhibits the expression of UCHL1 to promote APP and
   BACE1 degradation by inhibiting the translation of NF-κB and inducing
   its cytoplasmic localization ([87]Shi et al., 2017). Therefore, we
   aimed to explore whether ouabain-induced RNA degradation was caused by
   m^6A modification or miRNA-dependent mechanism.

FIGURE 2.

   [88]FIGURE 2
   [89]Open in a new tab

   Ouabain is involved in the RNA degradation progression of DLBCL. (A)
   Heatmap displaying the differentially expressed genes between the
   control group and the ouabain treatment group. (B) KEGG Pathway
   analysis revealed that the RNA degradation signaling pathway was
   significantly enriched after ouabain treatment.

3.3 Effect of ouabain on RNA m^6A methylation-related enzyme classes

   To explore whether ouabain induces RNA degradation through m^6A
   modification or through miRNA-dependent mechanisms, we detected changes
   in RNA m^6A methyltransferases, demethylases, and reader enzymes in
   response to different concentrations of ouabain ([90]Figure 3). The RNA
   demethylase ALKBH5 and the m^6A-binding protein IGF2BP2 were most
   significantly regulated by ouabain, and their mRNA expression levels
   gradually decreased with increasing concentrations of ouabain,
   indicating an obvious gradient dependent relationship ([91]Figures 3G,
   H). microRNA-induced silencing complex (miRISC) is a multi-protein
   assembly that uses microRNAs (miRNAs) to identify mRNAs targeted for
   degradation. The miRISCs contain the Argonaute (AGO) family protein
   AGO2, which plays an important role in the function of miRNA. To
   further explore whether AGO2 is involved in the mRNA degradation of
   ouabain-treated DLBCL cells, we performed mRNA half-life on LSM1 and
   DCP1A, which were enriched in the RNA degradation pathway after
   treatment with ouabain. We found that the half-life of LSM1 and DCP1A
   did not change significantly compared with the control after
   interference with AGO2 ([92]Supplementary Figure S1A, B).On the other
   hand, it has been reported that miR-7-5p and miR-132-3p play RNA
   degradation roles in DLBCL([93]Morales-Martinez et al., 2020;
   [94]Mansoor et al., 2023), so we further examined whether ouabain
   affects the RNA degradation process by regulating miRNAs in DLBCL. The
   experimental results showed that ouabain had no effect on the
   expression of miR-7-5p and miR-132-3p ([95]Supplementary Figure S1C,
   D). These results showed that the miRNA-dependent mechanism did not
   play a role in ouabain-mediated RNA degradation. Therefore, we suggest
   that ouabain is involved in the progression of DLBCL by mediating m^6A
   modification leading to RNA degradation, and ALKBH5/IGF2BP2 is a target
   molecule regulated by ouabain.

FIGURE 3.

   [96]FIGURE 3
   [97]Open in a new tab

   Effect of ouabain on RNA m^6A methylation-related enzymes. (A) Effects
   of different concentrations of ouabain on the expression of the RNA
   m^6A methylase WTAP were detected via q-PCR. (B) Effects of different
   concentrations of ouabain on the RNA m^6A methylase METTL14 were
   detected via q-PCR. (C) Effects of different concentrations of ouabain
   on the RNA m^6A methylase METTL3 were detected via q-PCR. (D) Effects
   of different concentrations of ouabain on the RNA m^6A-binding protein
   IGF2BP1 were detected via q-PCR. (E) Effects of different
   concentrations of ouabain on the RNA m^6A-binding protein IGF2BP3 were
   detected via q-PCR. (F) Effects of different concentrations of ouabain
   on the RNA m^6A demethylase FTO were detected via q-PCR. (G) Effects of
   different concentrations of ouabain on the RNA m^6A demethylase ALKBH5
   were detected via q-PCR. (H) Effects of different concentrations of
   ouabain on the RNA m^6A-binding protein IGF2BP2 were detected via
   q-PCR. The data are presented as the means ± SDs; n = 3. *p < 0.05, **p
   < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

3.4 Gene expression analysis of ALKBH5 and IGF2BP2 in DLBCL tissues and cells

   To further investigate the expression of ALKBH5 and IGF2BP2, we
   searched the GEO database ([98]GSE12453, [99]GSE83632) and found that
   ALKBH5 and IGF2BP2 were highly expressed ([100]Figures 4A, D). In
   addition, we collected 3 examples of normal lymph node tissues and 7
   examples of diffuse large B-cell lymphoma tissues. qPCR confirmed that
   ALKBH5 and IGF2BP2 are highly expressed in DLBCL patients, and are
   associated with poor prognosis ([101]Figures 4B, E). To select the
   appropriate cell model for each experiment, we detected the relative
   expression of ALKBH5 and IGF2BP2 in four DLBCL cell lines SU-DHL4,
   OCI-LY3, U2932, and SU-DHL2 ([102]Figures 4C, F). Our findings revealed
   that ALKBH5 was relatively low in SU-DHL4 cells but relatively high in
   OCI-LY3 cells, whereas IGF2BP2 was relatively low in SU-DHL4 cells but
   relatively high in U2932 cells.

FIGURE 4.

   [103]FIGURE 4
   [104]Open in a new tab

   Gene expression analysis of ALKBH5 and IGF2BP2 in DLBCL tissues and
   cells. (A) The expression level of ALKBH5 in diffuse large B-cell
   lymphoma (DLBCL) was analyzed in the [105]GSE12453 cohort. (B) Gene
   expression analysis of ALKBH5 in DLBCL tissues. (C) Gene expression
   analysis of ALKBH5 in DLBCL cells. (D) The expression level of IGF2BP2
   in diffuse large B-cell lymphoma (DLBCL) samples from the [106]GSE83632
   cohort was analyzed. (E) Gene expression analysis of IGF2BP2 in DLBCL
   tissues. (F) Gene expression analysis of IGF2BP2 in DLBCL cells. The
   data are presented as the means ± SDs; n = 3. *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001 (Student’s t-test).

3.5 Knockdown of ALKBH5 reduced cell proliferation, and cell cycle arrest,
and increased apoptosis in DLBCL

   ALKBH5 has been reported to be upregulated in a variety of cancers
   including breast ([107]Zhang et al., 2016), lung ([108]Sun et al.,
   2022), and epithelial ovarian cancers ([109]Nie et al., 2021), where it
   plays an oncogenic role in tumor progression. To assess the effect of
   ALKBH5 on DLBCL cells under the influence of ouabain, we overexpressed
   ALKBH5 in SU-DHL4 cells and downregulated ALKBH5 in OCI-LY3 cells
   ([110]Figure 5A). These two cell lines were subsequently treated with
   ouabain. Proliferation experiments were conducted via a CCK-8 assay for
   the following groups: control group (Lv-NC), ouabain treatment group
   (Ouabain), ALKBH5 knockdown group (Lv-shALKBH5), combination of ouabain
   treatment and control group (Lv-NC + Ouabain), and combination of
   ouabain treatment and ALKBH5 knockdown group (Lv-shALKBH5+Ouabain)
   ([111]Figure 5B left). As depicted in [112]Figure 5B, ouabain decreased
   the viability of OCI-LY3 and SU-DHL4 cells when administered alone.
   Additionally, the overexpression of ALKBH5 significantly increased cell
   viability. Furthermore, the groups treated with combinations of ouabain
   and Lv-shALKBH5 exhibited a greater reduction in proliferation than did
   the group treated with ouabain alone. Flow cytometry analysis revealed
   that the cell cycle was significantly inhibited in the G1/S phase
   ([113]Figure 5C). On the other hand, apoptosis was significantly
   greater in the Lv-shALKBH5+Ouabain group than in the other groups
   ([114]Figure 5E). In contrast, when ouabain was applied to cells
   overexpressing ALKBH5, the CCK-8 method revealed that the inhibition of
   cell proliferation and cell cycle arrest was reduced in the
   Lv-oeALKBH5+Ouabain group, and cell apoptosis was reduced compared with
   that in the Lv-NC + Ouabain group ([115]Figure 5B right, [116]Figures
   5D, F). These results suggest that downregulated ALKBH5 enhances the
   chemosensitivity of DLBCL cells to ouabain.

FIGURE 5.

   [117]FIGURE 5
   [118]Open in a new tab

   ALKBH5 affects the sensitivity of DLBCL cell lines to ouabain. (A) The
   knockdown effect of ALKBH5 in OCI-LY3 cells and the overexpression
   effect of ALKBH5 in SU-DHL4 cells were assessed via qPCR and protein
   expression of western blotting analysis (WB). (B) A Cell Counting Kit 8
   assay was utilized to assess the proliferation of OCI-LY3 and SU-DHL4
   cells treated with ouabain. (C) ALKBH5 knockdown combined with ouabain
   treatment significantly induced cell cycle arrest at the G1/S phase in
   OCI-LY3 cells, as assessed by flow cytometry. (D) ALKBH5 overexpression
   inhibited ouabain-induced cell cycle arrest at the G1/S phase in
   SU-DHL4 cells, as assessed by flow cytometry. (E) ALKBH5 knockdown
   combined with ouabain treatment significantly promoted apoptosis in
   OCI-LY3 cells, as assessed by flow cytometry. (F) ALKBH5 overexpression
   inhibited ouabain-induced apoptosis in SU-DHL4 cells, as assessed by
   flow cytometry. The data are presented as the means ± SDs; n = 3. *p <
   0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

3.6 Knockdown of IGF2BP2 reduced cell proliferation, and cell cycle arrest,
and increased apoptosis in DLBCL

   IGF2BP2 plays an important role as a m^6A-modified reading protein in
   regulating cellular mRNA localization, stability, and translation
   ([119]Wang et al., 2021). To investigate the effect of IGF2BP2 on DLBCL
   cells under the influence of ouabain, we overexpressed IGF2BP2 in
   SU-DHL4 cells and downregulated IGF2BP2 in U2932 cells ([120]Figure
   6A). We then treated these two cell lines with ouabain Cell
   proliferation in the Lv-shIGF2BP2+Ouabain group was significantly
   inhibited according to the results of the CCK-8 assay ([121]Figure 6B).
   Flow cytometry analysis revealed that the cell cycle of the
   Lv-shIGF2BP2+Ouabain group was inhibited considerably in the G1/S phase
   ([122]Figure 6C). Furthermore, IGF2BP2 knockdown considerably increased
   the degree of apoptosis induced by ouabain treatment ([123]Figure 6E).
   Conversely, when ouabain was administered to the IGF2BP2-overexpressing
   cell lines, the CCK-8 method resulted in reduced inhibition of cell
   proliferation and cell cycle arrest in the Lv-oeIGF2BP2+Ouabain group,
   along with decreased levels of apoptosis compared with those observed
   in the Lv-NC + Ouabain group ([124]Figure 6B right, [125]Figures 6D,
   F). These results suggest that knockdown of IGF2BP2 enhances the
   sensitivity of DLBCL cells to ouabain.

FIGURE 6.

   [126]FIGURE 6
   [127]Open in a new tab

   IGF2BP2 affects the sensitivity of DLBCL cell lines to ouabain. (A) The
   effect of IGF2BP2 knockdown in U2932 cells and the effect of IGF2BP2
   overexpression in SU-DHL4 cells were assessed through quantitative PCR
   (qPCR) and western blotting (WB). (B) A Cell Counting Kit-8 assay was
   utilized to assess the proliferation of U2932 and SU-DHL4 cells treated
   with ouabain. (C) IGF2BP2 knockdown combined with ouabain treatment
   significantly induced cell cycle arrest at the G1/S phase in U2932
   cells, as assessed by flow cytometry. (D) IGF2BP2 overexpression
   inhibited ouabain-induced cell cycle arrest at the G1/S phase in
   SU-DHL4 cells, as assessed by flow cytometry. (E) IGF2BP2 knockdown
   combined with ouabain treatment significantly promoted apoptosis in
   U2932 cells, as assessed by flow cytometry. (F) IGF2BP2 overexpressed
   inhibits ouabain-induced apoptosis in SU-DHL4 cells, as assessed by
   flow cytometry. The data are presented as the means ± SDs; n = 3. *p <
   0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

4 Discussion

   DLBCL is a monoclonal proliferative malignant tumor with heterogeneity
   and aggressiveness, and its incidence is increasing year annually
   ([128]Chapuy et al., 2018). Epigenetic modifications are important
   drivers of the development of hematologic malignancies. Among them,
   N6-methyladenosine (m^6A) methylation is a key epigenetic modification
   that controls a variety of fundamental biological processes ([129]Wang
   et al., 2015). For example, high expression of IGF2BP3 is associated
   with a proliferative phenotype of pediatric B-cell acute lymphoblastic
   leukemia (B-ALL) at the mRNA and protein levels and portends favorable
   survival in high-risk B-ALL patients ([130]Mäkinen et al., 2021).
   ALKBH5 is a demethylase and increasing evidence has shown that ALKBH5
   is closely related to tumor growth, proliferation, and survival.
   Studies have shown that ALKBH5 regulates the PI3K/AKT pathway by
   regulating the stability of AXL mRNA ([131]Wang et al., 2020).In
   addition, high expression of ALKBH5 is associated with poor prognosis
   in AML patients. IGF2BP2 is a carcinomatous fetal gene that is
   expressed at lower levels in normal adult tissues than in fetal liver
   tissues and is overexpressed in various types of cancer, such as
   glioblastoma, liver cancer, and breast cancer, thus making it a
   promising therapeutic target in cancer ([132]Hammer et al., 2005;
   [133]Weng et al., 2022). In particular, [134]Weng et al. (2022)
   reported the oncogenic role and therapeutic targeting of the reading
   protein IGF2BP2 in AML ([135]Weng et al., 2022). Research on m^6A
   modification is important in the study of DLBCL pathogenesis. Our study
   revealed that ALKBH5 and IGF2BP2 may serve as novel therapeutic targets
   for the treatment of DLBCL.

   Cardiac glycosides can not only inhibit the growth of some cancer cells
   but also reduce their recurrence and metastasis ([136]Mijatovic et al.,
   2007; [137]Laverdière et al., 2018; [138]Zheng et al., 2021). Ouabain
   is a cardiotonic steroid drug derived from digitalis, that is mainly
   used to treat heart failure ([139]Xiao et al., 2017). In recent years,
   researchers have reported that ouabain can promote the apoptosis of
   lung cancer ([140]Chanvorachote and Pongrakhananon, 2013), acute
   myeloid leukemia ([141]Valdes et al., 2007; [142]Tailler et al., 2011),
   liver cancer ([143]Ozdemir et al., 2012), and other tumor cells. These
   findings suggest that ouabain may be effective against a variety of
   hematological tumors, including DLBCL. In our previous study, the
   addition of cardiac glycosides (digoxin) to 3 patients with DLBCL was
   found to prolong the survival of patients. This led us to explore the
   potential mechanism of action of cardiac glycosides, such as ouabain,
   in patients with DLBCL. To clarify the effect of ouabain on DLBCL
   progression, we treated OCI-LY3 cells with ouabain and performed RNA
   sequencing, which revealed that differentially expressed genes
   associated with cell proliferation, and apoptosis were significantly
   enriched after ouabain treatment. Further verification experiments
   revealed that ouabain significantly inhibited the proliferation and
   accelerated the apoptosis of DLBCL cells. These results suggest that
   ouabain may be a candidate for the treatment of DLBCL. Therefore,
   elucidating the target of action where ouabain may act in the treatment
   of DLBCL has become a critical issue that needs to be urgently
   addressed.

   After RNA sequencing of the DLBCL cell line OCI-LY3 and detection of
   m^6A-modifying enzymes, we observed that, compared with the control
   group, the ouabain treatment group was significantly involved in the
   progression of RNA degradation, which was affected by m^6A methylation.
   m^6A methylation is associated with the development of multiple tumors,
   and ouabain has been shown to regulate tumor suppressor genes through
   epigenetic mechanisms ([144]Raynal et al., 2016; [145]Yang et al.,
   2020; [146]Song et al., 2022). Epigenetic m^6A isomerization is
   regulated by regulators such as methyltransferases, demethylases, and
   reading enzymes ([147]Zhang et al., 2020; [148]Oerum et al., 2021). For
   example, the m^6A-binding protein IGF2BP2 is highly expressed in acute
   myeloid leukemia (AML) and is associated with poor prognosis ([149]Weng
   et al., 2022). N^6-methyladenosine modification of TRERNA1 mediated by
   the m^6A demethylase ALKBH5 promotes DLBCL proliferation by
   downregulating p21 ([150]Song et al., 2022). In addition, Chen et al.
   found that YTHDF2 is involved in the progression and poor prognosis of
   DLBCL. By enhancing the stability and expression of ACER2, YTHDF2
   triggers endogenous ceramide catabolism, increases S1P levels, and
   activates the PI3K/AKT and ERK pathways. This study highlights the
   potential of YTHDF2 as a therapeutic target and predictor of DLBCL
   ([151]Chen et al., 2023b).Some studies have shown that m^6A shows
   different heterogeneity in most tumors. Cancer type-specific m^6A
   levels regulate the expression of different cancer-related genes in
   different cancer types ([152]Lin et al., 2024). We treated DLBCL cells
   with different concentrations of ouabain and detected the expression
   levels of m^6A-related modification enzymes. Our findings revealed a
   positive correlation between the expression levels of ALKBH5 and
   IGF2BP2 and increasing concentrations of ouabain.This interesting
   phenomenon leads us to think about how exactly ouabain regulates the
   expression of ALKBH5 and IGF2BP2. Subsequent analysis of RNA-seq data
   revealed the downregulation of two regulatory factors, SF3B4 and YWHAG,
   following ouabain treatment. SF3B4 and YWHAG have been identified as
   regulators of m^6A ([153]An et al., 2020). Therefore, we treated
   OCI-LY3 cells with IC50 concentration of ouabain and verified that the
   expression of SF3B4 and YWHAG was indeed downregulated after ouabain
   treatment Knockdown of SF3B4 resulted in decreased expression levels of
   ALKBH5 and IGF2BP2.However, no such effect was observed for YWHAG
   knockdown. Further validation at the protein level confirmed that SF3B4
   indeed influences the expression of ALKBH5 and IGF2BP2
   ([154]Supplementary Figure S4). Notably, recent research suggests that
   H3K36me3 histone modification may guide the installation of classically
   enriched m^6A modifications near stop codons by directly recruiting
   METTL14 ([155]Huang et al., 2019). Therefore, it is hypothesized that
   ouabain affects the expression of ALKBH5 and IGF2BP2 through modulation
   of SF3B4-mediated m^6A regulation. Next, we aimed to explore whether
   ALKBH5 and IGF2BP2 are involved in the development of DLBCL. Through
   analysis via the GEO database and examination of tissues from DLBCL
   patients, it was evident that both ALKBH5 and IGF2BP2 were highly
   expressed in DLBCL patients compared with normal controls.
   Subsequently, stable cell lines which ALKBH5 and IGF2BP2 were
   overexpressed or knocked downwere constructed via lentivirus technology
   which demonstrated that overexpression significantly promoted cell
   proliferation, whereas knockdown resulted in a significant decrease in
   cell viability. In conclusion, these results suggest important roles
   for ALKBH5 and IGF2BP2 in DLBC development and highlight their
   potential as therapeutic targets for further investigation. The same
   dose of ouabain had a more pronounced inhibitory effect on the
   proliferation and cell cycle of cells in which ALKBH5 or IGF2BP2 was
   knocked down. In addition, ALKBH5 and IGF2BP2 upregulation
   significantly reversed ouabain-induced promotion of apoptosis.
   Conversely, when ALKBH5 and IGF2BP2 were knocked down, the
   apoptosis-inducing effect of ouabain on DLBCL cells was significantly
   enhanced. These results indicate that ouabain affects the malignant
   progression of DLBCL by influencing the m^6A demethylase ALKBH5 and the
   m^6A-binding protein IGF2BP2, suggesting that ALKBH5 and IGF2BP2 may be
   potential targets of ouabain action.

   Taken together, these results suggest that ouabain mediates epigenetic
   regulation for the treatment of DLBCL through the effects of the m^6A
   demethylase ALKBH5 and the m^6A-binding protein IGF2BP2, which can be
   used as novel biomarkers for the diagnosis of DLBCL. This study
   provides new theoretical support and targeted therapeutic strategies
   for the treatment of DLBCL.

Acknowledgments