MCF-7, a human BC cell line, was purchased from Procell Life Science & Technology Co., Ltd. and cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (100 U/ml; Cytiva) at 37°C in a humidified incubator with 5% CO₂ atmosphere. Cells were routinely screened for mycoplasma contamination. Que (Beijing Solarbio Science & Technology Co., Ltd.) was dissolved in 100% DMSO to prepare a 100 mM stock solution, which was then diluted in DMEM such that the final concentration of DMSO was maintained at 0.1% (v/v) across all working concentrations of Que (30, 60, 90 and 120 µmol/l). The pLVX-Puro-control (Plvx-con) and pLVX-Puro-human-PTEN (Plvx-PTEN, NM_000314.6) plasmids were sourced from Xiamen Life Internet Technology Co., Ltd. and LY294002, Verapamil and Rhodamine 123 was purchased from MedChemExpress.

A total of 1×10⁶ MCF-7 cells were transfected with 5 µg Plvx-con or Plvx-PTEN with the Lipo8000^™ Transfection Reagent (Beyotime Biotechnology) in 6-cm dishes, strictly according the manufacturer's protocol. Following transfection, the cells were treated with 60 µmol/l Que for 24 h at 37°C before subsequent experiments.

For each treatment group, 5×10³ MCF-7 cells were plated in triplicate into 96-well plates and allowed to adhere for 24 h at 37°C in a CO₂ incubator. Cells were treated with increasing concentrations of Que (0, 30, 60, 90 and 120 µmol/l) for 24 or 48 h at 37°C. For combination treatments, cells were transfected with 0.2 µg Plvx-con or 0.2 µg Plvx-PTEN for 24 h at 37 °C, followed by treatment with 60 µmol/l Que for another 24 h at 37°C. After that, 10 µl of CCK-8 solution (Beijing Lablead Trading Co., Ltd.) was added to each well and the cells were incubated for 2 h at 37°C. The absorbance was measured at 450 nm using an Infinite M1000 Pro microplate reader (Tecan Trading Ag).

A total of 1×10⁵ MCF-7 cells were cultured in six-well plates and treated with Que (0, 30, 60, 90 or 120 µmol/l) for 24 h at 37°C. In a separate experiment, cells were treated with 60 µmol/l Que, 25 µmol/l LY294002 or a combination of both for 24 h at 37°C. Cells were then trypsinized, and harvested via centrifugation at 800 × g (4°C), and stained with 10 µl of Annexin V-FITC and 5 µl of propidium iodide using an apoptosis detection kit (Beyotime Biotechnology). Cells were analyzed on a Beckman Coulter (BC) FC 500 flow cytometer using the CXP software (version 2.2) for data acquisition and analysis.

MCF-7 cells were lysed in cold RIPA lysis buffer (Beyotime Biotechnology) supplemented with protease and phosphatase inhibitors (cat. no. C0104, Beijing Lanbolide Trading Co., Ltd.). The protein concentration was determined using the BCA assay according to the manufacturer's instructions (Dalian Meilun Biology Technology Co., Ltd.), and 30 µg protein samples were separated by 8–12% SDS-PAGE, transferred to PVDF membranes (Millipore Sigma) and blocked at room temperature with blocking buffer (cat. no. BM10-100; Energenesis Biomedical Co., Ltd.). The membranes were incubated with primary antibodies overnight at 4°C. Primary antibodies included PTEN (1:5,000; cat. no. ab267787), Bcl-2 (1:6,000; cat. no. ab196495), Bax (1:6,000; cat. no. ab32503) and ABCG2 (1:5,000; cat. no. ab108312) from Abcam; p-PI3K (p85) (1:2,000; cat. no. 4228), p-AKT (Ser473) (1:2,000; cat. no. 4060), cleaved caspase-3 (1:5,000; cat. no. 9664), ABCC2 (1:1,000; cat. no. 12559) and ABCB1 (1:2,000; cat. no. 13342) from Cell Signaling Technology, Inc.; and GAPDH (1:12,000; cat. no. 60004-1-Ig), PI3K (1:3,000; cat. no. 20584-1-AP), AKT (1:3,000; 10176-2-AP), Caspase-3 (1:3,000; 19677-1-AP) and β-actin (1:10,000; cat. no. 66009-1-Ig) from Proteintech Group, Inc. After incubation with HRP-conjugated secondary antibodies (1:10,000; cat. no. SA00001-1 and SA00001-2; Proteintech Group, Inc.), protein bands were visualized using an ECL reagent (Beijing Lanbolide Trading Co., Ltd.) and imaged with a ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc.).

Total RNA was extracted from MCF-7 cells using TRIzol^® (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequently, cDNA was synthesized from the isolated RNA using a commercial reverse transcription kit (cat. no. F0202; Beijing Lanbolide Trading Co., Ltd.). qPCR was carried out using SYBR Green master mix (Beijing Lanbolide Trading Co., Ltd.) on a Roche LightCycler^® 96 System (Roche Diagnostics). The thermal cycling protocol consisted of an initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 30 sec. GAPDH was used as the internal control and relative gene expression was calculated using the 2^−ΔΔCq method (14). Primer sequences are listed in Table I.

Transfected and Que-treated MCF-7 cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with Hoechst 33342 solution (Beijing Solarbio Science & Technology co., Ltd.) for 30 min at room temperature. Cell nuclei were visualized and imaged under an inverted fluorescence microscope (Leica Microsystems GmbH).

A total of 1×10⁵ cells were seeded into 3.5-cm confocal dishes, transfected and subsequently treated with 60 µmol/l Que and/or 25 µmol/l LY294002 for 24 h at 37°C. After washing with PBS, cells were fixed with 4% paraformaldehyde for 30 min at room temperature, blocked with 2% BSA (Beijing Lanbolide Trading Co., Ltd.) for 1 h at room temperature and incubated overnight at 4°C with primary antibody against ABCG2 (1:400). Cells were then incubated with Alexa Fluor^® 488-conjugated secondary antibody (1:1,000; cat. no. ab150077; Abcam) at room temperature for 1 h, and the nuclei was stained with DAPI-containing mounting medium (cat. no. ab104139; Abcam) for 10 min at room temperature. Fluorescence images were captured using a Leica SP8 confocal microscope (Leica Microsystems GmbH).

MCF-7 cells were treated as following groups: i) Control group, ii) 100 µmol/l Verapamil group, iii) 60 µmol/l Que group, iv) 25 µmol/l LY294002 group and v) 25 µmol/l LY294002 + 60 µmol/l Que group. MCF-7 cells were treated with the indicated agents and cultured for 24 h at 37°C. Notably, Verapamil (100 µmol/l) was added to the corresponding group 1 h prior to Rhodamine 123 staining. Following the 24 h treatment, the cells were washed three times with Hanks' balanced salt solution (HBSS; Beijing Solarbio Science & Technology co., Ltd.) and incubated with 100 µmol/l Rhodamine 123 for 15 min at 37°C (No fixation or other staining steps were performed). After three additional washed with HBSS, fluorescence images were acquired using an inverted fluorescence microscope (Leica Microsystems GmbH) and fluorescence intensity was quantified to assess ABC transporter activity.

Statistical analyses were performed using the GraphPad Prism 6.0 (Dotmatics). Data are presented as the mean ± SEM from at least three independent experiments. Comparisons between groups were conducted using an unpaired Student's t-test or one-/two-way ANOVA, as appropriate. For two-way ANOVA, Bonferroni's post-hoc test was used to evaluate multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

As assessed by the CCK-8 assay, Que treatment caused a marked suppression of MCF-7 cell viability, and the inhibitory effect was dependent on both the concentration and duration of exposure (Fig. 1A). Furthermore, Que downregulated the mRNA and protein expression of ABCB1, ABCC2 and ABCG2 (Fig. 1B-E). It also promoted apoptosis, as evidenced by decreased Bcl-2 and increased Bax and cleaved caspase-3 expression (Fig. 1C-D), as well as an increased percentage of apoptotic cells in flow cytometry analysis (Fig. 1F and G). Based on these results, 60 µmol/l Que was selected for subsequent experiments.

PTEN inactivation or mutation is implicated in various types of cancer, including BC (15–18). Patients with PTEN mutations have a higher risk of primary and secondary types of BC (19). The present study verified successful PTEN overexpression, which significantly reduced the mRNA and protein levels of ABCB1, ABCC2 and ABCG2 (Fig. 2A-E). It also reduced the anti-apoptotic protein Bcl-2 and increased the level of the pro-apoptotic protein Bax (Fig. 2C and E), mirroring the effects of Que.

The combination of PTEN overexpression and Que treatment resulted in a greater suppression of cell viability compared with Que treatment alone (Fig. 3A). This combination also led to a more pronounced upregulation of PTEN and Bax and downregulation of Bcl-2 at the gene and protein level (Fig. 3B, C, E-H). Hoechst 33342 staining analysis revealed a marked reduction in viable cells in the combination group (Fig. 3D).

Clinical studies have shown that overexpression of ABC efflux transporters, such as ABCB1, ABCC2 and ABCG2, contributes to MDR in cancer by reducing intracellular drug concentrations (20,21). Therefore, the present study examined whether PTEN expression influences Que-mediated regulation of ABC transporters. Analysis revealed that PTEN overexpression potentiates Que-induced downregulation of ABCB1, ABCC2 and ABCG2 mRNA and protein (Fig. 4A-E). Concurrently, the combination treatment led to a significant reduction in the levels of phosphorylated PI3K and AKT (Fig. 4D, F and G), indicating inhibition of the PI3K/AKT signaling pathway.

The PI3K/AKT signaling pathway is involved in cell proliferation and the expression of drug-resistant proteins, including ABC transporters (22–24). To explore the relationship between Que and PI3K/AKT signaling in regulating apoptosis and ABC transporters, MCF-7 cells were treated with LY294002 (a PI3K inhibitor), Que or a combination of both. Treatment with LY294002 alone enhanced the effects of cell apoptosis and inhibited ABC transporter expression. The combination of Que and LY294002 resulted in the most substantial reduction of ABCB1, ABCC2 and ABCG2 mRNA and protein, as well as the strongest inhibition of PI3K/AKT signaling (Fig. 5A-F). Immunofluorescence analysis corroborated the downregulation of ABCG2 protein (Fig. 5G) and flow cytometry revealed that the highest rate of apoptosis occurred in the combination group (Fig. 5H and I).

Rhodamine 123, a substrate for ABC transporters, is used to assess transporter activity (25). The present study investigated the functional consequence of ABC transporter downregulation using the Rhodamine 123 accumulation assay. Analysis revealed that both Que and LY294002 increased intracellular Rhodamine 123 retention, with the combination treatment showing the most significant effect (Fig. 6A and B), comparable to the positive control Verapamil. These results indicate that both Que and LY294002 inhibit the efflux activity of ABC transporters. The observed increase in fluorescent dye accumulation is consistent with a decrease in ABC transporter protein expression, as confirmed in Figs. 1B and C and 5A and B. This suggests that the downregulation of ABC transporters by Que and LY294002 may enhance the intracellular accumulation and efficacy of co-administered therapeutic agents.