Combining Sulfonylureas with Anticancer Drugs: Evidence of Synergistic Efficacy with Doxorubicin In Vitro and In Vivo

Abstract

Sulfonylureas (SUs)-a class of drugs primarily used to treat type 2 diabetes-have recently attracted interest for their potential anticancer properties. While some studies have explored the chemical modification or design of new SU derivatives, our work instead centers on biological evaluations of all commercially available SUs in combination with doxorubicin (DOXO). These antidiabetic agents act by stimulating insulin secretion via K_ATP channel inhibition, and because K_ATP channels share structural features with ATP-binding cassette (ABC) transporters involved in multidrug resistance (e.g., P-glycoprotein, MRP1, and MRP2), SUs may also reduce cancer cell drug efflux. In this study, we systematically examined each commercially available SU for potential synergy with DOXO in a panel of human cancer cell lines. Notably, combining DOXO with glimepiride (GLIM), the newest SU, results in a 4.4-fold increase in cytotoxicity against MCF-7 breast cancer cells relative to DOXO alone. Mechanistic studies suggest that the observed synergy may arise from increased intracellular accumulation of DOXO. Preliminary in vivo experiments support these findings, showing that DOXO (5 mg/kg, i.v.) plus GLIM (4 mg/kg, i.p.) is more effective at inhibiting 4T1 tumor growth in mice than DOXO alone. Additionally, we show that adding a small amount of the surfactant Tween-80 to culture media affects SU binding to bovine serum albumin (BSA), potentially unmasking anticancer effects of SUs that strongly bind to proteins. Overall, these results underscore the potential of repurposing existing SUs to enhance standard chemotherapy regimens.

Keywords: combination index; combination therapy; doxorubicin; drug repurposing; sulfonylureas; synergistic cytotoxicity.

Figure 1

Effect of drug concentrations and Tween-80 on particle size distribution. Experiments were conducted in PBS (pH 7.4) at 37 °C, with 0.5% DMSO and 4.5 mg/mL BSA. This study examined 100–500 µM GLIM without Tween-80 (a,b); 300 µM GLIM with 0.0125–0.1% Tween-80 (c,d); and 100–500 µM CHLO with 0.05% Tween-80 (e,f). Size distribution histograms for GLIM samples in PBS alone and with 0.05% Tween-80, along with detailed DLS results, are provided in the Supplementary Materials (Figures S3–S5). Concentrations reported are expected concentrations (C_exp), based on the total drug added. This preliminary experiment was conducted once (n = 1) to evaluate feasibility; additional replicates are needed to confirm findings and ensure reproducibility.

Figure 2

Impact of BSA and Tween-80 on observed drug concentrations (C_obs) determined by HPLC using different separation techniques. C_obs of GLIM under varying conditions (a–d); C_obs of CHLO (e,f). In each panel, Tween-80(–) indicates the absence and Tween-80(+) the presence of the surfactant, while BSA(–) denotes the absence and BSA(+) the presence of the protein. The results are expressed as mean ± SD (n = 3).

Figure 3

Effect of Tween-80 on the viability of MCF-7 breast cancer cells (a) and on the cytotoxicity of co-administered drugs (b). Cells were exposed to the compounds for 72 h. Results are presented as means ± SD (n = 3). *, p < 0.05 vs. Tween-80 alone; ***, p < 0.001 vs. Tween-80 alone.

Figure 4

Heat maps illustrate the effects of combined treatment on the survival of MCF-7 cells (a) and the synergistic effects observed for these treatments (b). In the CI heat map, green indicates very strong synergy (CI < 0.1), strong synergy (0.1 ≤ CI < 0.3), and synergy (0.3 ≤ CI < 0.9), white indicates additive effects (0.9 ≤ CI < 1.1) and red indicates weak antagonism (1.1 ≤ CI < 1.2), moderate antagonism (1.2 ≤ CI < 1.45), and antagonism (CI > 1.45). Detailed information, including median effect plots and CI values, can be found in the Supplementary Materials (Figures S9–S12).

Figure 5

Heat maps illustrate selected drug combinations’ effects on cell panel viability (a–c) and the synergistic effects observed for these treatments (d–f). For CHLO, which did not achieve an IC₅₀ below 500 µM, three successive drug dilutions were applied. In the CI heat maps, green indicates very strong synergism (CI < 0.1), strong synergism (CI < 0.3), and synergism (CI < 0.9). White indicates additive effects (0.9 < CI < 1.1), while red indicates weak antagonism (1.1 < CI < 1.2), moderate antagonism (1.2 < CI < 1.45), antagonism (1.45 < CI < 3.3), strong antagonism (3.3 < CI < 10), and very strong antagonism (CI > 10). Cells marked in yellow indicate dose combinations selected for the cell cycle studies discussed in the next section. Additional information on cytotoxicity and CI values can be found in the Supplementary Materials (Figures S13–S15).

Figure 5

Heat maps illustrate selected drug combinations’ effects on cell panel viability (a–c) and the synergistic effects observed for these treatments (d–f). For CHLO, which did not achieve an IC₅₀ below 500 µM, three successive drug dilutions were applied. In the CI heat maps, green indicates very strong synergism (CI < 0.1), strong synergism (CI < 0.3), and synergism (CI < 0.9). White indicates additive effects (0.9 < CI < 1.1), while red indicates weak antagonism (1.1 < CI < 1.2), moderate antagonism (1.2 < CI < 1.45), antagonism (1.45 < CI < 3.3), strong antagonism (3.3 < CI < 10), and very strong antagonism (CI > 10). Cells marked in yellow indicate dose combinations selected for the cell cycle studies discussed in the next section. Additional information on cytotoxicity and CI values can be found in the Supplementary Materials (Figures S13–S15).

Figure 6

Cell cycle phase distribution analysis in A549 (a), HepG2 (b), MCF-7 (c), and U-87MG (d) cells following treatment with single drugs and combinations for 72 h, assessed by propidium iodide staining and flow cytometry. Results are expressed as the mean ± SD (n = 3). *, p < 0.05 vs. CTRL; **, p < 0.01 vs. CTRL; ***, p < 0.001 vs. CTRL; #, p < 0.05 vs. 0.25 IC₅₀ DOXO; ##, p < 0.01 vs. 0.25 IC₅₀ DOXO. Detailed data can be found in the Supplementary Materials (Figure S16).

Figure 7

Effects of selected SUs on intracellular DOXO accumulation in A549 (a), HepG2 (b), HMEC-1 (c), HUH7 (d), MCF-7 (e), and U-87MG cells (f). Cells were treated with 50 µM DOXO and various concentrations of SUs (from 0.78 to 50 µM) for 3 h. Intracellular DOXO levels were measured by fluorometry and are expressed as a percentage relative to the control (DOXO alone). The bars represent the mean ± SD (n = 3). *, p < 0.05 vs. control; **, p < 0.01 vs. control; ***, p < 0.001 vs. control.

Figure 8

Percentage of hemolysis caused by SUs, naïve DOXO, and their combinations after 4 h of incubation at RT under open-air conditions (a). Microscopic images showing the impact of the drugs and their combinations on RBCs after 15 min of incubation at RT in open-air conditions (b–f). The negative control (CTRL−) is untreated blood in PBS, while the positive control (CTRL+) is blood incubated in deionized water. Each experimental sample contains 0.5% DMSO, except for the control samples. The bars represent the mean ± SD (n = 3). ***, p < 0.0001 vs. CTRL− ###, p < 0.0001 vs. 0.5% DMSO.

Figure 9

Therapeutic efficacy of DOXO alone and in combination with GLIM against 4T1 breast cancer in vivo. Tumor growth curves (a) and body weight changes (b) in BALB/c mice bearing 4T1 tumors. Beginning nine days after tumor inoculation, mice with well-developed tumors were treated with either monotherapies or combination therapy. GLIM was administered intraperitoneally (i.p.) at 4 mg/kg, and DOXO was administered intravenously (i.v.) at 5 mg/kg. Both treatments were given simultaneously on days 9, 11, 14, 17, and 21, as indicated by the arrows in the figure. Tumor volume and body weight were measured daily for the first 8 days and then three times per week. Data are presented as mean ± SD (n = 5–6). *, p < 0.05 vs. CTRL; **, p < 0.01 vs. CTRL; ***, p < 0.001 vs. CTRL; #, p < 0.05 vs. DOXO; ##, p < 0.01 vs. DOXO 5 mg/kg (i.v.); ###, p < 0.001 vs. DOXO 5 mg/kg (i.v.).

Figure 10

Effects of Tween-80 on GLIM aggregation in the absence and presence of BSA, and potential in vitro implications. Panel (a) illustrates aggregation parameters as a function of mixture composition, with initial and final values indicated. ^a Changes observed within the range of 0–500 µM GLIM; ^b changes observed within the range of 100–500 µM GLIM; ^c at GLIM concentrations exceeding 200 µM, the contribution of the BSA fraction became negligible; ^d initial and maximum values on the curve are provided. INT%—the relative contribution of specific size distributions to the total scattered light intensity in the sample. Panel (b) illustrates the potential implications of these findings for in vitro studies.