OCI-LY3 and OCI-LY10 cells were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Hyclone, USA) with, 10% fetal bovine serum (Gibco, USA), 1% penicillin/streptomycin (Beyotime, Shanghai), and 0.5% Glutamine (Beyotime, Shanghai) in 5% CO2 at 37°C. All cells were used within 20 passages.

OCI-LY3 and OCI-LY10 cells were kindly provided by professor Wenbin Qian of Zhejiang University (Zhejiang, China). Z-VAD-FMK and SB203580 were obtained from Selleck Chemicals (Houston, TX). CCK-8 Assay kit was purchased from Meilunbio (Dalian, China). N-acetylcysteine (NAC) and glutathione (GSH) were obtained from Abcam (Cambridge, MA). The Cell Apoptosis detection kit and Cell Cycle Staining Kit were purchased from MultiSciences (Hangzhou, China). Primary antibodies, including anti-cleaved-PARP (ab191217), anti-cleaved-Caspase 3(ab32042), anti-Bax (ab32503), anti-Bad(ab32445), anti-Bcl2(ab32124), anti-β-Actin(ab179467), anti-p21(ab109520), anti-p27(ab32034), anti-p53 (ab179477), anti-p38(ab170099), anti-chk1(ab40866), anti-p-chk2, and anti-γ-H2AX, were purchased from abcam (Cambridge, MA). Antibodies including anti-p-p38(9215S), anti-chk2(3440S), anti-p-chk1(2348P) were purchased from Cell Signaling Technology (Massachusetts, USA). Secondary antibodies, including goat anti-rabbit and goat anti-mouse IgG-HRP were obtained from Beyotome (Shanghai, China).

Cell viability was detected by Cell Counting Kit 8. OCI-LY3 and OCI-LY10 cells were seeded onto 96-well plates at a density of 4 × 10⁴ cells/well with 100 µl Serum-free medium, and treated with increasing concentrations of TEOA (5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 µM) for indicated time. Then, 10 µl cell counting kit-8 was added to each well and incubated for 2 h at 37°C. The absorbance was measured at 450 nm using the microplate reader.

Cell apoptosis was determined by the Annexin V-FITC/PI apoptosis kit. The OCI-LY3 and OCI-LY10 cells were cultured in 6-well plates at a density of 1.5 × 10⁶ cells/well, and treated with the indicated concentrations of TEOA for 12 h. Then, the cells were collected, washed once with PBS, and mixed with 500 µl of binding buffer. After incubating with 5 µl Annexin V-FITC and 10 µl PI for 5 min at room temperature in the dark, the cell apoptosis was assessed by flow cytometry (ACEA NovoCyte, USA).

A soft agar colony assay was used to detect the long-term effects of TEOA in OCI-LY3 and OCI-LY10 cells. Cells were seeded into 6-well plates at 2,000 cells/well, with 0.48% top agar layer and 1% bottom agar layer (Agarose, Sigma-Aldrich), and treated with different concentrations of TEOA in RPMI-1640 medium with 10% fetal bovine serum (FBS). The cell colonies were formed following incubation for 2 weeks. Thereafter, they were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) for 15 min, and stained with crystal violet (Beyotime, China) for 15 min at room temperature.

Cells were treated with the indicated concentrations of TEOA for 12 h. Then, the cells were collected and washed once with ice-cold PBS and lysed in RIPA lysis buffer (Beyotime, China) for 10 min on ice. The mixture was centrifuged at 14,000 rpm/min, and 4°C (Thermo Fisher, USA) for 10 min. The supernatant was removed, and the cellular protein concentration was measured using the bicinchoninic acid protein assay kit (Thermo Fisher, USA). For western blot analysis, 10 µg protein was loaded into each lane and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto Polyvinylidene fluoride (PVDF) membranes (Bio-Rad, USA). After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were incubated overnight with the primary antibodies at 4°C. They were subsequently washed with TBST three times, and the membranes were incubated with secondary antibodies at room temperature for 1 h. The expression of target bands was detected using an enhanced chemiluminescence system (Bio-Rad, USA). The working concentration of primary antibodies was 1:1,000, except where illustrated. The primary antibodies used in this experiment: anti-cleaved-PARP, anti-Bax, anti-Bad, anti-cleaved-Caspase 3, anti-Bcl2, anti-p21, anti-p27, anti-p53, anti-p-p38, anti-p38, anti-p-chk1, anti-chk1, anti-p-chk2, anti-chk2, anti-γ-H2AX(1:2,000), and anti-β-Actin(1:6,000). The secondary antibodies were goat anti-rabbit IgG (H+L) (1:2,000) and goat anti-mouse IgG (H+L) (1:2,000). β-actin served as the loading control. Statistical analysis was performed using Image J software.

The intracellular ROS level was detected using an oxidation sensitive fluorescent probe (DCF-DA) (Beyotime, China) according to the manufacturer’s instructions. DCF-DA can be hydrolyzed by intracellular esterase to form DCFH; it can be oxidized by reactive oxygen to produce the fluorescent compound, 2′,7′-dichlorofluorescein (DCF), which is a stable fluorescent ROS-sensitive compound that can readily permeate into cells. OCI-LY10 cells were incubated with the indicated concentrations of TEOA in serum-free medium for 12 h. Then, cells were harvested and washed thrice with serum-free medium. Thereafter, cells were resuspended in 1640 medium and stained with DCF-DA (4 µM) for 30 min at 37°C. Lastly, cells were washed thrice with serum-free medium and fluorescence intensity was measured by flow cytometry.

The cells were seeded into 96-well plates at a density of 4 × 10⁴ cells/well, and treated with the indicated concentrations of TEOA for 12 h. Then, PI was added to each well at a final concentration of 10 µg/ml and incubated at 37°C for 10 min in the dark. Images were obtained using a fluorescence microscope (Nikon, Germany).

The cells were seeded into 24-well plates at a density of 2 × 10⁵ to 3 × 10⁵ cells/well and treated with the indicated concentrations of TEOA for 12 h. Cells were collected and stained with Hoechst 33258 for 5 min, then washed thrice with PBS. The blue nuclei fluorescence was detected by confocal laser scanning microscopy (Leica, Germany).

The formation of γ-H2AX was detected by immunocytochemistry. The cell slides were treated with polylysine for 24 h. Simultaneously, OCI-LY10 cells were seeded into 6-well plates and treated with the indicated concentrations of TEOA for 12 h. Then cells were harvested and seeded on cell slides and incubated for 2 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 1% Triton X-100 for 5 min at room temperature. After being blocked with 5% BSA, the cells were stained with 1:200 of γ-H2AX primary antibody and 1:500 of FITC-labeled Goat Anti-Rabbit IgG (H+L) secondary antibody (Beyotime, USA). DAPI was used for the staining of the nucleus. Images were acquired using confocal microscopy (Leica, Germany).

After indicated treatment for12 h, cells were harvested and fixed in ice-cold 70% ethanol overnight at 4°C. Next day, the ethanol was removed by centrifugation, cells were washed with PBS and re-suspended in PBS containing PI (50 mg/ml) and RNase A (10 mg/ml) and then incubated at 37°C for 30 min in the dark. After washing and filtration, the single-cell suspensions were subjected to the flow cytometry (ACEA NovoCyte, USA) and the percentage of cells at G0/G1, S, or G2/M phase were quantified.

The therapeutic effect of TEOA in combination with vindesine or cyclophosphamide was measured by the CCK-8 assay. 15 µM TEOA treated with increasing concentrations of CTX(0.5, 1, 2 µM)or Vindesine(16, 24, 32 µg/ml)for 12 h and combination index was calculated through the formula: Q= E(A+B)/(E A +E B -E A ×E B). EA and EB represent the inhibition ratio of drug A and drug B on cells respectively, and E(A+B) is the combined inhibition effect of drug A and B. Q>1.15, = 0.85~1.15 and <0.85 indicate synergistic effect, additive effect and antagonistic effect, respectively.

The effects of various in vitro drug treatments were compared by Student’s t-test using GraphPrism.5. All results were presented as the mean  ±  standard deviation (SD). Differences with p < 0.05 were considered statistically significant.

The chemical structure of TEOA is indicated in Figure 1A. To examine the inhibitory effect of TEOA on cell viability, OCI-LY3 and OCI-LY10 cells were seeded in 96-well plates and treated with increasing concentrations of TEOA (0, 5, 15, 20, 25, 30, 35, 40, 45, or 50 µM) for indicated times. Cell viability was measured by the CCK-8 assay. Results showed that TEOA significantly inhibited cell viability in a dose and time-dependent manner (Figures 1C, D). The half-maximal inhibitory concentration (IC50) of TEOA at 12 h, 24 h, and 36 h were calculated and shown in Figure 1B. Further, we observed morphological changes by phase-contrast microscopy and found the cells were shattered, metamorphous and multidirectional after TEOA treatment. Moreover, the number of PI-positive cells was increased in a dose-dependent manner (Figure 1E). The soft agar clone formation assay was performed to determine the long-term growth inhibitory effect of TEOA. The OCI-LY10 cells were treated with increasing concentrations of TEOA (0, 15, 20, and 25 µM) in 0.48% agarose with 10% FBS for 14 days; the results revealed that TEOA significantly inhibited clone formation (Figure 1F). The clones were counted and corresponded quantification histograms were shown on the right. In addition, the effect of TEOA on non-cancerous cell lines was also detected and the results shown that TEOA exhibited lower toxicity on mouse embryonic fibroblast and immortalized lymphocyte cells (Figure S1A). To determine whether TEOA decreased cell viability by affecting the cell cycle distribution or not. The cell cycle distribution was performed and revealed that cells were arrested at G0/G1 phase and the proportion was increased in a dose-dependent manner (Figures S1D). In addition, TEOA inhibited cell migration rate by approximately 30% and 40% at the doses of 20 and 25μM, respectively (Figure S1E). Taken together, these results suggest that TEOA reduced the viability and inhibited cell proliferation of DLBCL cells.

To further determine whether TEOA decreased cell viability through the regulation of apoptosis. OCI-LY10 cells were treated with TEOA at concentrations of 0, 15, 20, 25, and 30 μM, respectively, and followed by apoptosis detection and Hoechst 33258 staining. The results showed that, after treatment with TEOA overnight, the percentage of apoptotic cells significantly increased in a concentration-dependent manner, accompanied by nuclear fragmentation and condensation (Figure 2A, white arrow). As shown in Figures 2B, C, we observed that TEOA induced significant apoptosis in DLBCL cells, detected by Annexin-V and PI staining followed by flow cytometry assays. Furthermore, the apoptotic rate significantly increased with the increasing concentration of TEOA. Meanwhile, to determine whether apoptosis induced by TEOA was caspase-dependent, the cells were pretreated with the pan-caspase inhibitor, ZVAD-FMK, (5 μM) for 1 h. Importantly, we found that the rate of apoptosis was significantly decreased by ZVAD-FMK pretreatment, especially in late apoptosis (Figure 2C). Moreover, the results of PI staining also demonstrated that ZVAD-FMK rescued TEOA-induced apoptosis (Figure 2D). These findings indicate that TEOA-induced apoptosis in DLBCL is dependent on the caspase pathway. Next, we investigated the expression of apoptosis-associated proteins by western blot. The results showed TEOA treatment induced the cleavage of caspase-3 and PARP, up-regulated the expression of Bad, and Bax, and down-regulated the expression of Bcl2 (Figures 3A, B). Taken together, our findings suggest that TEOA promotes apoptosis in DLBCL cells.

Accumulating evidence indicates that apoptosis is mediated by some pro-apoptotic genes, such as p53. Therefore, we detected the expression of p53 and found that TEOA upregulated the level of p53 protein. Simultaneously, we observed that the expression of P21, P27, and γ-H2AX was also increased (Figures 3C, D). As a transcription factor, p53 could not only active apoptosis but also induces cell cycle arrest in response to DNA damage. γ-H2AX is a product of DNA fragmentation, which can be detected in the early stages of DNA damage. Additionally, we observed that TEOA increased the co-staining of DAPI and γ-H2AX foci in OCI-LY10 cells (Figure 3E). And the level of 8-hydroxy-2’-deoxyguanosine (8-OHdG), which is a modified nucleoside base and one of the most commonly studied byproduct of DNA damage, was also elevated under the treatment of TEOA (Figure S2). Thus, these results suggest that TEOA could induce DNA damage in DLBCL cells.

Next, we investigated intracellular signaling events upon TEOA-induced DNA damage and apoptosis. We detected intracellular ROS levels using a DCF-DA probe. ROS was significantly activated by the treatment of TEOA (Figures 4A, B). The use of ROS scavenger, NAC, and GSH could markedly decrease TEOA-induced cell death, as shown by the CCK-8 assay (Figures 4C, D), demonstrating that the intracellular signaling events induced by TEOA were ROS dependent. To further confirm whether increased ROS promotes apoptosis, we detected apoptosis by flow cytometry (Figures 4F, G). Compared with the control group, the apoptosis rate with TEOA was significantly increased, while adding ROS scavenger NAC significantly inhibited apoptosis of OCI-LY10 cells, suggesting that TEOA-induced apoptosis of OCI-LY10 cells was related to ROS. And this phenomenon was further confirmed by PI staining (Figure 4E). These data indicate that the TEOA-induced ROS accumulation might be a potential mechanism underlying the anti-DLBCL activity.

P38 kinase is a member of the mitogen-activated protein kinase (MAPK) family, and plays a pivotal role in apoptosis (Yuan et al., 2016). In addition to DNA damage, we analyzed the changes in the MAPK pathway after treatment with TEOA, and examined the relationship between p38 MAPK activation and DNA damage induced by TEOA. Interestingly, we found that TEOA could activate the p38 MAPK pathway by increasing the phosphorylation of p38, CHK1, and CHK2, and improve the expression of γ-H2AX in a dose-dependent manner in OCI-LY10 (Figures 5A, B) and OCI-LY3 cells (Figures 5C, D). These results indicate that TEOA activates the p38 MAPK pathway and induces DNA damage repair. Furthermore, the active oxygen scavenger, NAC, significantly downregulated the expression of p-p38 and decreased DNA damage (Figure 6A). The p38 phosphorylation inhibitor SB203580 could block TEOA-induced DNA damage and reduce the expression of p-p38 (Figure 6B). Further, immunofluorescence was used to detect the effect of SB203580 on reversing DNA damage. Results showed that TEOA-induced γ-H2AX accumulation were indeed blocked in the treated cells with p38 inhibitor (Figure 6C). And p38 inhibitor SB203580 could recuse cell viability under challenge by TEOA (Figure S3). Thus, these results suggest that ROS mediated activation of the p38 MAPK signal pathway plays an important role in initiating TEOA-induced DNA damage.

Due to the long-term exposure of drugs, drug resistance is inevitable in the treatment of diffuse large B-cell lymphoma (Noble et al., 2017). Important chemotherapeutic drugs of standard chemotherapy regimens, vindesine and cyclophosphamide, were commonly used in patients with DLBCL. Considering the resistance to vindesine and cyclophosphamide, we detected the therapeutic effect of TEOA in combination with vindesine or cyclophosphamide by the CCK-8 assay. Therefore, OCI-LY10 cells were treated with a certain dose of TEOA with or without vindesine or cyclophosphamide. As shown in Figure 7A, the viability of OCI-LY10 cells was decreased with TEOA (15 µM) or vindesine (16 µg/ml, 24 µg/ml, 32 µg/ml) treatment alone, but the viability was decreased dramatically when cells were treated with a combination of both. Similar results were also obtained for cyclophosphamide (0.5 mM, 1 mM, 1.5 mM) (Figure 7B). In addition, combination index was calculated and revealed that significant synergies between TEOA and chemotherapeutic drugs (Q>1.15) in both cell lines. Further, TEOA exhibits no synergistic effect with vindesine and CTX on immortalized lymphocyte cells (Figure S4). and mouse embryonic fibroblast. These results suggest that TEOA may be a promising agent when used in combination with vindesine or cyclophosphamide for the treatment of DLBCL.