ART (cat. no. MB7316), SOR (cat. no. MB1666), and Cell Counting Kit-8 (CCK-8; cat. no. MA0218) were purchased from Dalian Meilun Biology Technology Co., Ltd. Erastin (Era; cat. no. HY-15763), ferrostatin-1 (Fer; cat. no. HY-100579) and rapamycin (Rapa; cat. no. HY-10219) were purchased from MedChemExpress. Monodansylcadaverine (MDC; cat.no. 30432) was purchased from Sigma-Aldrich; Merck KGaA. BODIPY-C11 581/591 (cat. no. RM02821) was purchased from ABclonal Biotech Co., Ltd. According to the manufacturers' protocols, products (ART, SOR, Era, Fer, Rapa, MDC and BODIPY-C11) were dissolved in DMSO and stored at −20°C. Plasmids (pLKO.1-Neo, cat. no. SHC001, originally from Sigma-Aldrich; Merck KGaA; psPAX2, cat. no. 12260, originally from Addgene, Inc.; and pMD2.G, cat. no. 12259, originally from Addgene, Inc.) were obtained from the Institute of Hematology, West China Hospital, Sichuan University (Chengdu, China). The short hairpin RNAs (shRNAs) [shRNA (sh)-signal transducer and activator of transcription 3 (STAT3), 5′-CCGGGCACAATCTACGAAGAATCAACTCGAGTTGATTCTTCGTAGATTGTGCTTTTTG-3′; non-targeting negative control, 5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′] were synthesized by TsingKe Biological Technology.

Human lymphoma cell lines (U2932, cat. no. ACC 633; SU-DHL4, cat. no. CRL-2957; SU-DHL6, cat. no. CRL-2959; and Jurkat, cat. no. CRL-2898), a mouse lymphoma cell line (EL4, cat. no. TIB-39) and 293T (cat. no. CRL-11268) cells were obtained from the Institute of Hematology, West China Hospital, Sichuan University, and provided by Professor Yongqian Jia. U2932 was originally from the Leibniz Institute DSMZ and the other cell lines were originally from the ATCC. U2932, SU-DHL4 and SU-DHL6 cells were cultured in RPMI-1640 medium (cat. no. SH30809; HyClone; Cytiva) supplemented with 20% FBS (cat. no. 900-108; Gemini Bio Products); Jurkat and EL4 cells were cultured in RPMI-1640 medium supplemented with 10% FBS; and 293T cells were cultured in DMEM/high glucose medium (cat. no. SH30243.01; HyClone; Cytiva) supplemented with 10% FBS. All cells were treated with 1% penicillin/streptomycin (cat. no. SV30010; HyClone; Cytiva) and maintained at 37°C in an atmosphere containing 5% CO₂. U2932, SU-DHL4 and SU-DHL6 are B-cell lymphoma cell lines, and Jurkat is T-cell lymphoma cell line. Not all lymphoma cell lines were used in each experiment.

The primary antibodies used were as follows: Anti-cleaved poly (ADP-ribose) polymerase (PARP; cat. no. A5034; 1:1,000), anti-p62 (cat. no. A5180; 1:1,000), anti-LC3B-I/II (cat. no. A5202; 1:1,000), anti-ERK1/2 (ERK; cat. no. A5029; 1:1,000), anti-phosphorylated (p)-ERK (cat. no. A5036; 1:1,000), anti-MEK1/2 (MEK; cat. no. A5606; 1:1,000), anti-p-MEK (cat. no. A5191; 1:1,000), anti-glutathione peroxidase 4 (GPX4; cat. no. A5569; 1:1,000), anti-ATG5 (cat. no. A5745; 1:1,000) and anti-ferritin heavy chain 1 (FTH1; cat. no. A5654; 1:1,000) (all from Bimake). In addition, anti-STAT3 (cat. no. 4904; 1:2,000), anti-p-STAT3 (Tyr727; cat. no. 9134; 1:1000), anti-AKT (cat. no. 2938; 1:1,000), anti-p-AKT (cat. no. 9018; 1:1,000) and anti-Bcl-XL (cat. no. 2764; 1:1,000) were purchased from Cell Signaling Technology, Inc. Anti-cleaved caspase-3 (cat. no. WL02117; 1:500), P70S6K (cat. no. WL03839; 1:500) and anti-p-P70S6K (cat. no. WL04213; 1:500) antibodies were purchased from Wanleibio Co., Ltd. Anti-CD31 (cat. no. GB11063; 1:500) was purchased from Wuhan Servicebio Technology Co., Ltd., and anti-β-actin (cat. no. TA-09; 1:2,000), HRP-conjugated anti-rabbit IgG (cat. no. ZB-2306) and HRP-conjugated anti-mouse IgG (cat. no. ZB-2305) secondary antibodies were purchased from OriGene Technologies, Inc.

Cells (1×10⁴/well; U2932, SU-DHL4, SU-DHL6 and Jurkat) were inoculated in a 96-well plate with 100 µl medium, and were treated with ART (1, 2, 3, 4 and 5 µM for SU-DHL4 and SU-DHL6; 1, 5, 10, 15 and 20 µM for U2932 and Jurkat,) and SOR (2.5, 5, 10, 15 and 20 µM) at different concentrations for 48 h at 37°C, or combined with or without Rapa (5 µM)/Era (1 µM)/Fer (5 µM) for 24 h at 37°C. Subsequently, CCK-8 (10 µl) was added to each well and incubated for 2 h at 37°C. Subsequently, the optical density (OD) of each well was detected at a wavelength of 450 nm (SpectraMax 190; Molecular Devices, LLC). Cell viability was calculated using the following equation: Cell viability (%)=[(OD _treated-OD _blank)/(OD _control-OD _blank)] ×100. The combination indices were analyzed using CompuSyn software (v1.0; ComboSyn, Inc.).

Cells, cultured in a 6-well plate (1×10⁵/well; U2932, SU-DHL4, SU-DHL6 and Jurkat), were harvested and detected using the Annexin V-647/PI Cell Apoptosis Detection kit (cat. no. Y6026; Suzhou Yuheng Biotechnology Co., Ltd). According to the manufacturer's instructions, each sample was resuspended in 300 µl binding buffer with 2 µl Annexin V and 1 µl PI, and then incubated for 15 min at room temperature in the dark. Sample acquisition and analysis were performed using a flow cytometer (Cytoflex; Beckman Coulter, Inc.) and FlowJo software 10.0 (FlowJo LLC).

Cells, cultured in a 6-well plate (1×10⁵/well; U2932, SU-DHL4 and Jurkat), were collected and stained with 5 µM diluted dichlorodihydrofluorescein diacetate (cat. no. S0033S; Beyotime Institute of Biotechnology), followed by incubation for 30 min in the dark at 37°C according to the manufacturer's instructions. Cell acquisition and analysis were performed using a flow cytometer, as aforementioned.

Mitochondrial membrane potential (MMP) was measured using a JC-1 Kit (cat. no. M8650; Beijing Solarbio Science & Technology Co., Ltd.). According to the manufacturer's instructions, cells, cultured in a 6-well plate (1×10⁵/well; U2932, SU-DHL4 and Jurkat), were stained with JC-1 dye and incubated for 30 min in the dark at 37°C. The fluorescence intensity was measured and analyzed using a flow cytometer, as aforementioned.

Autophagic vacuoles in cells were detected using MDC staining. Briefly, cells, cultured in a 6-well plate (1×10⁵/well; U2932, SU-DHL4 and Jurkat), were collected and stained with 50 µM MDC in 1X PBS at 37°C for 30 min. MDC-stained cells were detected and analyzed using a flow cytometer, as aforementioned.

Changes in lipid peroxidation were detected using BODIPY-C11 581/591. Cells, cultured in a 6-well plate (1×10⁵/well; U2932, SU-DHL4 and Jurkat), were harvested, washed, stained with 50 µM C11-BODIPY and incubated at 37°C for 1 h in the dark. Subsequently, cells were detected and the mean fluorescence intensity was quantified using a flow cytometer, as aforementioned.

The relative levels of MDA were qualitatively detected using a Lipid Peroxidation MDA Assay kit (cat. no. S0131S; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Briefly, proteins were extracted from U2932 and Jurkat cells (1×10⁷) using lysis buffer, and supernatants were mixed with TBA detection solution, heated for 15 min in a boiling water bath, cooled to room temperature and centrifuged (1,000 × g for 10 min at room temperature). Subsequently, the supernatants were transferred to a 96-well plate. The optical density was detected at a wavelength of 532 nm, and MDA content was calculated according to a standard curve and normalized to total protein levels.

The levels of GSH were evaluated using a GSH assay kit (cat. no. BC1175; Beijing Solarbio Science & Technology Co., Ltd) according to the manufacturer's protocol. Briefly, cells (1×10⁷; U2932 and Jurkat) were collected and homogenized in solution I, followed by centrifugation. The supernatants were added into a 96-well plate, then incubated at room temperature for 2 min with solution II and solution III, along with standards. The optical density was detected at a wavelength of 412 nm (SpectraMax 190; Molecular Devices, LLC). GSH levels were calculated according to the standard curve and normalized to total protein levels.

Cells were collected and lysed in RIPA lysis buffer (cat. no. HY-K1001; MedChemExpress) supplemented with protease inhibitors (cat. no. P1260; Beijing Solarbio Science & Technology Co., Ltd.) and PMSF (cat. no. P0100; Beijing Solarbio Science & Technology Co., Ltd) on ice for 30 min. Protein concentration was measured using a BCA protein assay kit (cat. no. CW0014S; CoWin Biosciences). Protein samples (20–30 µg/lane) were separated by SDS-PAGE on 8–12% gels and transferred onto nitrocellulose membranes (cat. no. BSP0161; Pall Life Sciences). Membranes were subsequently blocked with 5% (w/v) skimmed milk at room temperature for 1 h and were incubated with primary antibodies at different dilutions according to the manufacturers' protocols overnight at 4°C. After primary antibody incubation, the membranes were washed with 1X TBS-0.1% Tween-20 three times and were incubated with HRP-conjugated secondary antibodies (1:5,000) at room temperature for 1 h. After further washing, signals were visualized using an Ultra High Sensitivity ECL kit (cat. no. HY-K1005; MedChemExpress) on a Tanon 5200 chemiluminescence image analysis system (Tanon Science and Technology Co., Ltd).

Tumor tissues and organs were extracted from mice and fixed in 4% paraformaldehyde for 24 h at room temperature. IHC staining was performed using an immunohistochemistry kit (cat. no. G1215; Wuhan Servicebio Technology Co., Ltd.). Briefly, after dehydrating in a gradient alcohol series and permeabilizing with xylene, tissues were embedded in paraffin and cut into 5-µm sections. After dewaxing and hydration, organ tissue sections were stained using a hematoxylin/eosin staining kit (cat. no. G1120; Beijing Solarbio Science & Technology Co., Ltd.). The tumor tissue sections were also dewaxed and rehydrated, and antigen retrieval was performed in boiled saline sodium citrate buffer for 10 min. The tissue sections were then blocked with 5% FBS at 37°C for 15 min and stained with an anti-CD31 antibody (1:1,000) overnight at 4°C. Subsequently, sections were detected using an IHC detection kit; the slides were incubated with secondary antibody (1:200) at 37°C for 1 h, and processed with 3,3′-diaminobenzidine. Slides were observed under an optical microscope (Olympus Corporation) and analyzed using Image-Pro plus software (version 6.0; Media Cybernetics, Inc.)

According to the manufacturer's protocol, briefly, 293T cells were seeded in 10 cm dishes at a cell density of 60–70% and cultured overnight. The medium was replaced 6 h before transfection. Then pLKO.1-shRNA (10 µg), psPAX2 (7.5 µg) and pMD2.G (2.5 µg) plasmids, mixed with CaCl₂ (2 M; 62.5 µl; cat. no.C4901; Merck KGaA) and HBSS (2X HBSS; 500 µl; cat. no. 14065056; Thermo Fisher Scientific, Inc.) were co-transfected into 293T cells for 48 h using calcium phosphate co-precipitation at 37°C. Viral supernatants were centrifuged at 1,000 × g for 10 min at 4°C, filtered through a 0.45-µm filter. For infection, U2932 and SU-DHL4 cells (2×10⁵/well) were seeded in a 6-well plate overnight and 1 ml of viral supernatant was added, then cells were cultured at 37°C for 48 h. Next cells were selected with 1 µg/ml puromycin (cat. no. P8230; Beijing Solarbio Science & Technology Co., Ltd.). Infection efficiency was evaluated by western blotting. The concentration of puromycin used for maintenance was also 1 µg/ml.

To establish lymphoma xenograft models, a total of 20 female C57/BL/6 mice (weight, 20±2 g; age, 6–8 weeks) were purchased from Beijing Huafukang Biotechnology Co., Ltd. Mice were maintained in the West China Animal Experiment Center of Sichuan University. All mice had free access to food and water, and were maintained in stress-free, hygienic and animal-friendly conditions (temperature, 22°C; humidity, 60%) under a 12-h light/dark cycle. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sichuan University (ethics approval no. 20211284A). Mice were injected subcutaneously with 5×10⁵ EL4 cells resuspended in 100 µl serum-free medium (n=5 mice/group). Tumor size was measured using electronic digital calipers, and the tumor volume (mm³) was calculated as (length × width²)/2. When the tumor volume reached 50–100 mm³ after 5 days, mice were randomly divided into four groups and administered normal saline (NS, the control), ART (90 mg/kg), SOR (30 mg/kg) or the combined treatment of both ART and SOR (ART 90 mg/kg and SOR 30 mg/kg). Drugs were injected intraperitoneally every day until tumors reached 2 cm in diameter or had ulcerated, and mice were euthanized using cervical dislocation. Tumor tissues, livers and spleens were extracted from mice. Death of the mice was verified by cessation of heartbeat and breathing. The duration of the animal experiment was 2 weeks and no mice died during the experiment. The body weight, fur condition and general survival status were observed and recorded daily.

Data are presented as the mean ± standard deviation of three independent experiments. GraphPad Prism 8 (Dotmatics) was used for statistical analysis. A one-way ANOVA with Tukey's post hoc test was used for multi-group comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the inhibitory effects of ART and SOR on lymphoma cell lines, the cells were treated with ART and SOR at different concentrations for 48 h. As shown in Fig. 1A and B, cell viability decreased in a dose-dependent manner when exposed to ART or SOR in U2932, SU-DHL4, SU-DHL6 and Jurkat cells. Subsequently, the present study aimed to examine whether ART and SOR exhibited synergistic effects on NHL cells. As shown in Fig. 1C, lymphoma cells treated with a combination of ART and SOR displayed a significant reduction in cell viability compared with that in groups treated with either ART or SOR alone. In addition, the combination indices were <1 in U2932 and Jurkat cells, which revealed the synergistic inhibitory effect of ART and SOR on the viability of NHL cells (Fig. S1A).

To further determine the mechanism underlying the synergistic anti-lymphoma effects of ART and SOR, cell apoptosis induced by ART and SOR was assessed using flow cytometry. As shown in Fig. 2A, ART or SOR treatment induced the early and late apoptosis of lymphoma cells, but this was not obvious. Moreover, the combined treatment of ART and SOR significantly promoted the apoptosis of NHL cells compared with ART or SOR treatment alone. Notably, western blot analysis was performed to further verify these results. As shown in Fig. 2B, the expression levels of cleaved caspase-3 and PARP were increased, whereas Bcl-XL and MCL-1 expression levels were markedly decreased in the combined treatment group compared with those in the groups treated with either ART or SOR alone. These findings suggested that ART promoted SOR-induced apoptosis in NHL cells.

The results of a previous study demonstrated that ART inhibits tumor cell proliferation via regulation of the AKT signaling pathway (29). Subsequently, whether the combined treatment of ART and SOR affected AKT and MEK/ERK pathways was assessed in U2932, SU-DHL4 and Jurkat cells. The results of western blot analysis demonstrated that the protein expression levels of p-AKT, p-P70S6K, p-MEK and p-ERK were reduced following ART or SOR treatment alone; however, these reductions were minor. Notably, the expression levels of the aforementioned proteins were more markedly reduced following the combined treatment of ART and SOR in lymphoma cells (Fig. 3). These results suggested that the combined treatment of ART and SOR exerted inhibitory effects on AKT and MEK/ERK pathways in NHL cells.

The results of our previous study demonstrated that ART induced autophagy in DLBCL cell lines (29). Subsequently, whether the combined treatment of ART and SOR synergistically induced autophagy in lymphoma cells was investigated in the present study. As shown in Fig. 4A, the expression levels of LC3B-I/II and ATG5 were increased, whereas those of p62 were decreased following the combined treatment of ART and SOR compared with those in groups treated with ART or SOR alone. In addition, the MDC staining assay was performed to detect the levels of acidic vesicular organelles (30) following exposure to ART and/or SOR treatment for 24 h. Consistent with the aforementioned results, the fluorescence intensity of MDC was increased following ART or SOR treatment, but this was not significant, and a greater increase was observed in the combined treatment group compared with that in groups treated with ART or SOR alone (Fig. 4B). Notably, Rapa, an inducer of autophagy, enhanced the inhibitory effects of ART or SOR on the viability of U2932, SU-DHL4 and Jurkat cells (Fig. 4C). These results indicated that the synergistic inhibition induced by ART and SOR partially involved autophagy in lymphoma cells.

It has been reported that both ART and SOR are ferroptosis inducers (31). Thus, the present study aimed to investigate whether ART and SOR induced ferroptosis in lymphoma cells. As shown in Fig. 5A, the expression levels of GPX4 and FTH1 were decreased following exposure to ART or SOR alone; however, these reductions were minor. Notably, a more marked reduction in the expression levels of the aforementioned proteins was observed following treatment with ART and SOR in combination compared with those in groups treated with ART or SOR alone. Levels of lipid peroxidation were detected using BODIPY-C11, and the results demonstrated that the combined treatment of ART and SOR induced a higher production of lipid peroxidation than groups treated with ART or SOR alone (Fig. 5B and C). Moreover, the levels of ROS accumulation were markedly increased to various extents following ART or SOR treatment, and ROS generation was significantly higher in the combined treatment group compared with that in groups treated with ART or SOR alone (Fig. S1B and C). Notably, the contents of MDA were significantly higher following the combined administration of ART and SOR compared with that in groups treated with ART or SOR alone (Fig. S1D). However, the levels of GSH were significantly lower in the combined treatment group compared with those in groups treated with ART or SOR alone (Fig. S1E). In addition, MMP was decreased following exposure to ART or SOR in lymphoma cells, but this was not significant; notably, more monomers were detected, which indicated a lower ratio of JC-1 aggregates and monomers occurred in drug-treated cells. A markedly lower level of MMP was also observed in the combined treatment group compared with that in groups treated with ART or SOR alone (Fig. 5D).

To further investigate the role of ferroptosis induced by ART and SOR, cells were co-treated with or without a ferroptosis inducer or inhibitor. As shown in Fig. 6A, Era further inhibited cell viability following treatment with ART or SOR. Moreover, co-treatment with Era markedly increased the levels of apoptosis in ART- or SOR-treated cells (ART: 5 µM, SOR: 5 µM) (Fig. 6B). By contrast, ferrostatin-1 partially reduced apoptosis induced by ART or SOR (ART: 10 µM, SOR: 10 µM; higher concentration used to ensure effect) in SU-DHL4 cells (Fig. 6C). These results suggested that ART and SOR synergistically induced ferroptosis, and ferroptosis may contribute to apoptosis induced by ART and SOR in NHL cells.

The results of previous studies have demonstrated that STAT3 regulates ferroptosis (32,33). Therefore, whether STAT3 was involved in the inhibitory effects induced by a combination of ART and SOR was investigated in the present study. The results of the western blot analysis demonstrated that the combined treatment of ART and SOR inhibited p-STAT3 protein expression compared with that in groups treated with ART or SOR alone (Fig. 7A). Subsequently, to further validate the role of STAT3 in ART- and SOR-induced ferroptosis in lymphoma cells, the expression of STAT3 was inhibited using RNA interference (Fig. S1F), as previously described (29). As shown in Fig. 7B, the expression levels of MCL-1 and GPX4 were markedly decreased following ART or SOR treatment in cells transfected with sh-STAT3 compared with those in the sh-Control cells. In addition, the apoptotic rates induced by ART and SOR were higher in the sh-STAT3 groups than those in the sh-Control groups in SU-DHL4 cells, whereas there was no significant difference in apoptosis between cells treated with ART/SOR and the control group (Fig. 7C). Moreover, the production of lipid peroxidation was increased in cells treated with ART or SOR and transfected with sh-STAT3 compared with that in the sh-Control groups (Fig. 7D). Collectively, these results suggested that STAT3 regulated ART- and SOR-induced apoptosis and ferroptosis in lymphoma cells.

Subsequently, the anti-lymphoma effects of ART and SOR were investigated in vivo. EL4 cells were subcutaneously injected into C57BL/6 mice. In Fig. S2, the in vitro results shown that ART/SOR could also inhibit cell viability, induce apoptosis, ROS and lipid peroxidation accumulation in EL4 cells. After the tumors were palpable, mice were randomly divided into four groups and treated intraperitoneally with NS (control), ART, SOR or ART + SOR. As shown in Fig. 8A and B, tumor growth was suppressed following ART and SOR administration. In addition, compared with groups treated with ART or SOR alone, tumor volumes in the ART + SOR treatment group were significantly reduced. Moreover, compared with that in groups treated with ART or SOR alone, the weight of tumors was also smaller in the combined treatment group (Fig. 8C). Consistent with the in vitro results, the expression levels of GPX4, FTH1 and p-STAT3 were reduced in the ART + SOR treatment group compared with those in the groups treated with ART or SOR alone (Fig. 8D). In addition, IHC staining was performed to measure the effect of ART and SOR on angiogenesis. Reduced CD31-positive areas were observed in the ART or SOR groups compared with those in the NS group. Moreover, ART + SOR exhibited a markedly synergistic effect on inhibiting tumor angiogenesis compared with either ART or SOR monotherapy (Fig. 8E and F). However, the body weights of mice did not change significantly among the four groups (Fig. 8G). Finally, the liver and spleen tissues were removed from the mice and stained with hematoxylin and eosin. There were no marked changes among the four groups, which indicated that the combination treatment did not lead to apparent toxicity (Fig. 8H). Hence, the results demonstrated that the combination of ART and SOR performed synergistic effects and was well tolerated in vivo.