Human endometrial carcinoma RL95-2 (ATCC^®CRL-1671^™) and HEC-1-A (ATCC^®HTB-112^™) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The RL95–2 cells were cultivated in Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F-12 (DMEM/F12). The HEC-1-A cells were cultivated in McCoy’s 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). N-acetyl cysteine (NAC), copper (II) chloride (CuCl₂), 2′,7-dichlorofluorescein diacetate (DCFH-DA), disulfiram (DSF), Erastin, hydrogen peroxide (H₂O₂), propidium iodide (PI), and thiazolyl blue tetrazlium bromide (MTT) were obtained from Sigma Aldrich (St. Louis, MO, USA). Copper (I) chloride (CuCl) was obtained from Alfa Aesar (Tewksbury, MA, USA).

The RL95-2 (8x10³) and HEC-1-A (5 × 10³) cells were plated in 96-well plates and cultured in the presence of the indicated drugs. The cells were then incubated with MTT solution (0.5 mg/mL in PBS) for 1 h at 37°C, after which dimethyl sulfoxide (100 μl) was added, and the absorbances at 570 nm and 650 nm were measured using a plate reader (Multiskan EX, Thermo, Waltham, MA, USA). The combination index (CI) was calculated using CalcuSyn (Biosoft, Cambridge, UK) to generate the isobologram as previously described (39). In general, CI < 1 indicates a synergistic combination effect, and CI > 1 indicates an antagonistic combination effect (40).

Early- and late-stage apoptotic cells were evaluated using a fluorescein phycoerythrin (PE) Annexin V Apoptosis Detection Kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. RL95-2 (6x10⁵) and HEC-1-A (3x10⁵) cells were plated in 6-well plates and cultured in the presence of either DSF, CuCl, CuCl₂, or a combination of DSF with CuCl or CuCl₂. The cells were stained with 5 μl PE Annexin V and 5 μl 7-amino-actinomycin (7-AAD) (5 μg/mL), which enabled identification of the early apoptotic cells. The viable cells were PE Annexin V- and 7-AAD-negative; the early apoptotic cells were PE Annexin V-positive and 7-AAD-negative; and the late apoptotic and dead cells were both PE Annexin V- and 7-AAD-positive.

The cell cycle profiles were evaluated based on cellular DNA content using FACSCalibur flow cytometry. RL95-2 (6x10⁵) and HEC-1-A (3x10⁵) cells were plated in 6-well plates and cultured in the presence of either DSF, CuCl, CuCl₂, or a combination of DSF with CuCl or CuCl₂. The cells were fixed in 70% ice-cold ethanol and stored at −30°C overnight, after which they were washed twice with ice-cold PBS supplemented with 1% FBS and stained with propidium iodide (PI) solution (5 μg/mL PI in PBS, 0.5% Triton x-100, and 0.5 μg/mL RNase A) for 30 min at 37 °C in the dark. The cell-cycle distribution was then evaluated using FACS, based on cellular DNA content.

The fluorescent markers DCFH-DA and MitoSOX™ Red (Invitrogen, Carlsbad, CA, USA) were used to determine the intracellular and mitochondrial ROS levels, respectively. RL95-2 (6x10⁵) and HEC-1-A (3x10⁵) cells were plated in 6-well plates and cultured in the presence of either DSF, CuCl, CuCl₂, or a combination of DSF with CuCl or CuCl₂. The cells were incubated for the indicated times with different combinations of DSF–Cu⁺/Cu²⁺ complexes. The living cells were then stained with DCFH-DA (10 μM) and MitoSOX™ (5 μM) Red for 30 min at 37 °C and harvested. After washing the cells once with PBS, they were evaluated using a FACSCalibur flow cytometer and Cell Quest Pro software (BD Biosciences, Franklin Lakes, NJ, USA).

The lipid peroxidation levels were measured using C11-BODIPY dye (D3861, ThermoFisher Scientific, Waltham, MA, USA) with flow cytometry, following the manufacturer’s instructions. RL95-2 (6x10⁵) and HEC-1-A (3x10⁵) cells were plated in 6-well plates and cultured in the presence of either DSF, CuCl, CuCl₂, or a combination of DSF with CuCl or CuCl₂. The cells were seeded and treated with the indicated drug in six-well plates for the indicated time. Subsequently, the medium was replaced with 10 µM C11-BODIPY-containing medium for 1 hour. After incubation, the cells were harvested using trypsin, washed three times with ice-cold PBS, and then re-suspended in PBS with 1% BSA. The lipid peroxidation levels within the cells were assessed using a FACSCalibur flow cytometer and Cell Quest Pro software.

The autophagy flux was measured using DALGreen (D675, Dojindo Molecular Technologies, Kumamoto, Japan). RL95-2 (6x10⁵) and HEC-1-A (3x10⁵) cells were plated in 6-well plates and cultured in the presence of either DSF, CuCl, CuCl₂, or a combination of DSF with CuCl or CuCl₂. After drug treatment, the cells were washed three times with PBS and then incubated with 1 μM DALGreen in the culture medium for 1 hour at 37°C. After washing three times with PBS, the fluorescence intensity was evaluated using a FACSCalibur flow cytometer and Cell Quest Pro software.

Immunocytochemical analysis was performed with RL95-2 (1.5x10⁵) and HEC-1-A (8x10⁴) cells adhered to cover slips in 24-well plates. After treatment with the indicated drug, the cells were fixed for 10 min in 4% formaldehyde, incubated for 10 min in 0.1% Triton X-100 solution, washed 3 times in PBS, and treated for 1 h at room temperature with 1% BSA. Thereafter, the cells were incubated with anti-TOM20 antibody at 4°C overnight. The next day, the cells were washed with PBS and incubated for 1 h with FITC-conjugated secondary antibody. The cell nuclei were stained with DAPI. The mitochondrial morphology was observed using a THUNDER Imager microscope equipped with a 100× objective (Leica, Wetzlar, Germany).

The RL95-2 (6x10⁵) and HEC-1-A (3x10⁵) cells were seeded into 6-well plates and cultured in the presence of either DSF, CuCl, CuCl₂, or a combination of DSF with CuCl or CuCl₂. The following day, the cells were treated with the indicated drugs in fresh medium for the specified durations. After treatment, both the dead and viable cells were collected, washed with PBS, and incubated with 1× binding buffer containing the MMP-sensitive fluorescent dye JC-1 for 30 min at 37°C in the dark. Subsequently, the cells were then washed twice with PBS, resuspended in 500 μl of 1× binding buffer, and analyzed using a FACSCalibur flow cytometer and Cell Quest Pro software (BD Biosciences, Franklin Lakes, NJ, USA). The cell-volume gating strategy involved forward scatter height (FSC-H) and side scatter height (SSC-H), and the median fluorescence intensity of the vehicle was used as the starting point for M2 gating. Mitochondrial depolarization was determined from a decrease in the red/green fluorescence intensity ratio.

The drug-treated RL95-2 (6x10⁵) and HEC-1-A (3x10⁵) cells were lysed in RIPA buffer (100 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% SDS, and 1% Triton 100) at 4°C. The proteins in the resultant lysates were separated using 12% SDS-PAGE, transferring to PVDF membrane, blocked for 1 hour with 5% non-fat milk in 1x TBST, and incubating the membrane with primary antibodies (all at a dilution of 1:1000) at 4°C overnight. The primary antibodies included against mtTFA, PGC-1α, β-actin, p53, p21, CyclinB1, ATF3, p62 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CHOP, LC3BI/II, PARP (Cell Signaling, Danvers, MA, USA), cyclin D1, γH2AX, (Abcam, Cambridge, UK), and HO-1 (Enzo Life Sciences, Farmingdale, NY, USA). The blots were subsequently incubated with HRP-conjugated secondary antibodies (all at a dilution of 1:10000) (anti-mouse IgG, AP192P; and anti-rabbit IgG, AP132P, Merck-Millipore). The immunoreactive proteins were detected using the ECLTM Western Blotting Detection Reagent and Amersham Hyperfilm™ ECL (GE Healthcare, USA), as previously described (39).

The values are expressed as the mean ± SD of at least three independent experiments. All comparisons between groups were made using Student’s t-tests. Statistical significance was set at p < 0.05.

The cytotoxicity of copper and the DSF–Cu⁺/Cu²⁺ complexes has previously been documented (25, 41, 42). It was necessary to investigate the cytotoxic effects of Cu⁺, Cu²⁺, DSF, or the combination of Cu⁺ or Cu²⁺ with DSF in HEC-1-A and RL95–2 cells. Isobologram analysis, which has been mathematically proven and widely used to evaluate drug interactions, was employed (43). This analysis assesses the dose at which two drugs, when used alone, have the same efficacy; this is generally expressed as the half-effective dose (ED₅₀). We designed combinations of concentrations and calculated the combination index (CI) between CuCl (Cu⁺) or CuCl₂ (Cu²⁺) with DSF in HEC-1-A and RL95–2 cells. We first calculated the value of ED₅₀ for the treatment of CuCl, CuCl₂, and DSF alone from the isobologram in HEC-1-A and RL95–2 cells (Figures 1A, B). The ED₅₀ of DSF was 63 nM in the HEC-1-A cells and 57 nM in the RL95–2 cells. The ED₅₀ of Cu⁺ was 909 µM in the HEC-1 cells and 1.4x10⁷ µM in the RL95–2 cells; the ED₅₀ of Cu²⁺ was 4.6x10⁷ µM in the HEC-1 cells and 6.4x10²⁴ µM in the RL95–2 cells, suggesting that Cu⁺ and Cu²⁺ both had no therapeutic effect on either of the cell lines. The definition of CI<1 as a synergistic effect was observed when the HEC-1-A and RL95–2 cells were treated with the Cu²⁺/DSF complex. The CI<1 of Cu⁺/DSF complex was measured in the RL95–2 cells, but not in the HEC-1-A cells. The combination of Cu⁺ and Cu²⁺ with DSF apparently decreased the ED₅₀ of Cu⁺ (170 nM in the HEC-1-A cells and 80 nM in the RL95–2 cells) and Cu²⁺ (50 nM in the HEC-1-A cells and 100 nM in the RL95–2 cells), along with the decrease in the DSF’s ED₅₀ from 63 nM to 20 nM (Cu²⁺) and from 57 nM to 32 nM (Cu⁺) and to 41 nM (Cu²⁺) in the HEC-1-A and RL95–2 cells, respectively.

Elevated copper concentrations have been found to correlate with the cancer stage and/or progression in various types of tumors (44). The cytotoxicity of copper has been observed under both physiological and pathological conditions. For instance, the IC₅₀ of copper sulfate was found to be 225–300 μM in HeLa cells (45). However, as demonstrated in Figure 1, Cu⁺ and Cu²⁺ had no discernible therapeutic effect in the HEC-1-A and RL95–2 cells. Several studies have supported the varying cytotoxic effects of different concentrations of DSF and Cu⁺/Cu²⁺ in cancer cells (33, 46). Hence, we examined the differential combinations of DSF and Cu⁺/Cu²⁺ using the Annexin V-apoptotic analysis (Figure 2). Firstly, we examined the induction of apoptosis with 0.5 μM DSF complexed with 5 μM Cu⁺ or 200 μM Cu²⁺ in the HEC-1-A and RL95–2 cells (Figure 2, top panels). With the exception of Cu²⁺ in the HEC-1-A cells, the total apoptotic populations were only induced by the combination of DSF and Cu⁺ or Cu²⁺ in both cell lines in the 5 h treatment. Next, the combinations of relatively lower concentrations of DSF and Cu⁺ or Cu²⁺ were applied in the Annexin V-apoptotic analysis (Figure 2, bottom panels). The induction of total apoptotic populations was observed in the HEC-1-A cells treated with 0.2 μM DSF complexed with 1 μM Cu⁺ or 0.2 μM Cu²⁺and the RL95–2 cells treated with 0.1 μM DSF complexed with 0.1 μM Cu²⁺ for 48 h.

To avoid overly stressful cellular conditions, we further applied 0.2 μM DSF complexed with 1 μM Cu⁺ or 0.2 μM Cu²⁺ to the HEC-1-A cells and 0.1 μM DSF complexed with 0.1 μM Cu²⁺ to the RL95–2 cells for 48 h to address their effects on the cell cycle profile, cytosolic ROS, mitochondrial ROS, and lipid peroxide and ICC analysis. The PI cell cycle profiles were performed using flow cytometry analysis. Our data showed that the combination of DSF with Cu⁺ or Cu²⁺ increased the populations of the subG1 and G2/M phase and significantly decreased the population of the G1 phase in the HEC-1-A and RL95–2 cells (Figure 3).

We examined the effect of DSF–Cu⁺/Cu²⁺ complexes on cytosolic ROS using flow cytometry analysis with DCFH-DA dye in HEC-1-A and RL95–2 cells (Figures 4A, E). Compared with the hydrogen peroxide positive control, the Cu²⁺ and DSF–Cu⁺/Cu²⁺ complexes elevated cytosolic ROS in the HEC-1-A cells, and the Cu⁺, Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes decreased cytosolic ROS in the RL95–2 cells. We examined the effect of DSF–Cu⁺/Cu²⁺ complexes on mitochondrial ROS using MitoSOX dye in HEC-1-A and RL95–2 cells (Figures 4B, F). Compared with the hydrogen peroxide positive control, the Cu⁺, Cu²⁺, and DSF–Cu⁺/Cu²⁺ complexes significantly elevated mitochondrial ROS in the HEC-1-A cells, and the Cu⁺, Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes decreased cytosolic ROS in the RL95–2 cells. We further examined the effect of DSF–Cu⁺/Cu²⁺ complexes on the level of lipid peroxide using C-11 BODIPY dye in HEC-1-A and RL95–2 cells (Figures 4C, G). Compared with the Erastin positive control, the DSF and DSF–Cu⁺/Cu²⁺ complexes elevated the level of lipid peroxide in the HEC-1-A cells, and the Cu⁺, Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes all elevated the level of lipid peroxide in the RL95–2 cells. The fluorescence of DALGreen intensifies at acidic pH, making it suitable for monitoring the degradation phase of autophagy, specifically autophagolysosomes (Figures 4D, H). Our data showed that the DSF and DSF–Cu⁺/Cu²⁺ complexes induced the formation of autophagosomes in the HEC-1-A and RL95–2 cells. Cu⁺ was able to form autophagolysosomes in the HEC-1-A cells.

Furthermore, studies have found that mitochondrial dysfunction contributes to the resistance of these tumors to treatment (47–50). It has been demonstrated that mitochondrial dynamics and bioenergetic metabolism play a role in cisplatin resistance. ICC analysis was used to evaluate the effects of Cu⁺, Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes on the fusion–fission status of mitochondria in HEC-1-A and RL95–2 cells (Figure 5). In the HEC-1-A cells, we applied NAC (reduced status) and hydrogen peroxide (oxidized status) to monitor the mitochondrial dynamics via the mitochondrial outer membrane protein TOM20 (Figure 5A). The DSF and DSF–Cu⁺/Cu²⁺ complexes favored the oxidized status (fission status), and the others favored the reduced status (fusion status). In the RL95–2 cells, we applied fusion- and fission-specific drugs, Mdivi-1 (fusion status) and MYLS22 (fission status), to compare the effects of Cu⁺, Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes (Figure 5B). Our data showed that the DSF–Cu⁺/Cu²⁺ complexes favored the fusion status and that the others favored the fission status.

Based on the change in the mitochondrial fusion–fission cycle caused by the Cu⁺, Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes, we applied JC-1 dye to measure the mitochondrial membrane potential in HEC-1-A and RL95–2 cells (Figure 6). JC-1 dimers form in mitochondria with red fluorescence, and JC-1 monomers form in the cytoplasm with green fluorescence to measure mitochondrial depolarization, as indicated by an increase in green fluorescence intensity. Our JC-1 data showed that the DSF and DSF–Cu⁺/Cu²⁺ complexes increased the green fluorescence intensity in the HEC-1-A and RL95–2 cells, whereas Cu⁺ and Cu²⁺ decreased the green fluorescence intensity in the HEC-1-A and RL95–2 cells (Figures 6A–D). Mitochondrial transcription factor A (mtTFA) and peroxisome-proliferator-activated receptor γ co-activator-1α (PGC-1α) are two key mitochondrial respiratory and biogenic factors for mitochondrial respiratory functions (51, 52). Our Western blotting data showed that the Cu⁺, Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes decreased the mtTFA proteins in the RL95–2 cells and that the Cu²⁺, DSF, and DSF–Cu⁺/Cu²⁺ complexes decreased the PGC-1α proteins in the HEC-1-A cells (Figures 6E, F).

We further examined cell cycle and cellular stress proteins using Western blotting analysis (Figure 7). We first examined proteins related to the cell cycle profile, including p53, p21, cyclin D1, and cyclin B1. Our data showed that p53 proteins were decreased by the DSF and DSF–Cu⁺/Cu²⁺ complexes; p21 proteins were increased by the DSF–Cu⁺/Cu²⁺ complexes; and cyclin D1 and cyclin B1 proteins were decreased by the DSF and DSF/Cu²⁺ complex in the HEC-1-A cells. In the RL95–2 cells, p53 proteins were increased by the DSF and DSF–Cu⁺/Cu²⁺ complexes, and p21 proteins were decreased by Cu²⁺, DSF, and the DSF/Cu⁺ complex. These cellular stress proteins were examined, including the DNA damage biomarker (γH2AX), oxidative stress biomarkers (ATF3 and HO-1), ER stress biomarker (CHOP), autophagy biomarkers (p62 and LC3BII), and apoptosis biomarker (cleaved PARP). In the HEC-1-A cells, γH2AX, CHOP, p62, and LC3BII proteins were increased by the DSF and the DSF–Cu⁺/Cu²⁺ complexes, and ATF3 and HO-1 proteins were increased by the DSF–Cu⁺/Cu²⁺ complexes. In the RL95–2 cells, the γH2AX, HO-1, CHOP, p62, and cleaved PARP proteins were increased by the DSF and DSF–Cu⁺/Cu²⁺ complexes.

As presented in Figure 1, our study demonstrates the synergistic effect on the cytotoxicity of human endometrial cancer cells when DSF is combined with Cu⁺/Cu²⁺. Combination chemotherapy is proposed as a way to achieve synergistic effects and minimize drug doses, thereby enhancing treatment specificity for endometrial cancer patients and combating chemotherapeutic drug resistance (4). We conducted an examination to determine whether two commonly used chemotherapeutic drugs, cisplatin and doxorubicin, acted synergistically with DSF–Cu⁺/Cu²⁺ complexes in HEC-1-A and RL95–2 cells. Based on the data of Figure 1, we set the DSF/Cu⁺ (1:1) complex and DSF/Cu²⁺ (1:1) complex to measure whether they acted synergistically with cisplatin and doxorubicin in HEC-1-A and RL95–2 cells. Our combination index analysis showed that only the DSF/Cu⁺ complex acted synergistically with doxorubicin in the RL95–2 cells (Figures 8A–D). The CI values of the other combinations were above 1. By comparing Figures 1, 8, we further examined the relationship between Cu⁺, Cu²⁺, or DSF and cisplatin and doxorubicin in HEC-1-A and RL95–2 cells. With the exception of the combination of Cu⁺ and cisplatin or doxorubicin in the RL95–2 cells, the combinations expressed the synergistic effects (Figures 9A–D). In the HEC-1-A cells, the ED50 values of cisplatin were reduced from 134 µM to 5.3 µM, 8.7 µM, and 14.6 µM by DSF, Cu⁺, and Cu²⁺, respectively; the ED50 values of doxorubicin were reduced from 1.4 µM to 0.16 µM, 0.6 µM, and 0.29 µM by DSF, Cu⁺, and Cu²⁺, respectively (Figures 9A, B). In the RL95–2 cells, the ED50 values of cisplatin were reduced from 5.5 µM to 2.7 µM and 2.3 µM by DSF and Cu²⁺, respectively; the ED50 values of doxorubicin were reduced from 0.81 µM to 0.25 µM and 0.66 µM by DSF and Cu²⁺, respectively (Figures 9C, D).