A549 human lung cancer cells from the American Type Culture Collection were maintained in RPMI-1640 medium (Lonza Group, Ltd.) supplemented with 10% FBS (Serana Europe GmbH) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂ in a humidified atmosphere. Cells were sub-cultured twice a week and used at passages <15. The cells were confirmed to be mycoplasma free and authenticated by the Samsung Medical Center institutional research support center. All experiments were performed at cell confluence of 70–80%.

Genipin, N-acetylcysteine (NAC) and tert-butylhydroperoxide (TBHP) were obtained from Sigma-Aldrich; Merck KGaA. Treatment concentrations of NAC and TBHP were 10 mM and 200 µM, respectively, and were co-treated with elesclomol and genipin at 37°C for 24 or 48 h. Elesclomol was obtained from Biorbyt. Elesclomol and genipin stocks were dissolved in DMSO at 20 and 250 mM, respectively, and further diluted with DMSO. 5-(And-6)-Chloromethyl-2′, 7′-dichlorodihydrofluoresscein diacetate acetyl ester (CM-H₂DCFDA), MitoSOX Red and Lipofectamine^® LTX were obtained from Invitrogen; Thermo Fisher Scientific, Inc. Non-targeting small interfering RNA (siRNA), siRNA targeting UCP2 and the anti-β-actin antibody (cat. no. 18470) were obtained from Santa Cruz Biotechnology, Inc. Primary anti-UCP2 (cat. no. 89326), anti-cleaved caspase-3 (cat. no. 9661) and anti-PARP (cat. no. 9542), and secondary anti-rabbit (cat. no. 7074) and anti-mouse IgG (cat. no. 7076) antibodies were obtained from Cell Signaling Technology, Inc.

After the culture medium was changed to that containing 5% FBS without antibiotics, 80% confluent A549 cells were transfected with 40 nM UCP2-specific or non-targeted siRNA mixed with Lipofectamine^® RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) in opti-MEM (Gibco; Thermo Fisher Scientific, Inc.). At 24 h, the medium was replaced with fresh medium containing 10% FBS and antibiotics, and the cells were incubated for another 24 h prior to subsequent experiments.

A549 cells treated with elesclomol and genipin on 100 mm plates were washed twice with 4°C cold PBS, harvested by trypsinization and seeded in 6-well plates at 500 cells/well. After 9 days, the cells were fixed with 100% methanol at room temperature (RT) for 2 min and washed twice with cold PBS. The cells were then stained with crystal violet solution (Sigma-Aldrich; Merck KGaA) at RT for 2 min and washed twice with cold PBS. The number of colonies containing ≥50 cells were finally manually counted in each well using an inverted phase contrast microscopy (magnification, ×4; Olympus Corporation).

Cell survival was evaluated by SRB assay in 1×10⁴ cells/well seeded overnight in a 96-well plate. Following treatment for 24 and 48 h, A549 cells were fixed in 10% (wt/vol) trichloroacetic acid at 4°C for 2 h. After the cells were stained with SRB dye at RT for 30 min, excess dye was removed by repeated washing with 1% (v/v) acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution, and absorbance at 510 nm was measured using a VERSA max microplate reader (Molecular Devices, LLC).

A549 cells were seeded in a 12-well plate at a density of 2×10⁵ cells/well and genipin and elelsclomol treated the following day for 24 h. LDH activity was measured in 500 µl cell culture medium for released LDH and 20 µg cell lysate for intracellular LDH using a Cobas c501 assay kit (cat. no. c501; Roche Diagnostics) according to the manufacturer's instructions. Tumor tissues were homogenized with T-PER™ tissue protein extraction reagent (Thermo Fisher Scientific, Inc.) on ice and centrifuged for 10 min at 10,000 × g at 4°C. Subsequently, 20 µg of supernatants were assayed for LDH activity as above.

A549 cells were seeded in a black 96-well plate at a density of 4×10⁴ cells/well and genipin and elesclomol treated the following day for 24 h. The culture medium was replaced with 100 µl phenol red-free RPMI-1640 medium containing 2% FBS and 500 nM MitoTracker™ Red FM (Invitrogen; Thermo Fisher Scientific, Inc.), a fluorescent dye that stains the mitochondria of live cells as a function of MMP The cells were incubated for 30 min at 37°C in 5% CO₂ and washed with 100 µl cold PBS per well. The fluorescence in each well was measured using a microplate reader (Mithras LB 940; Titertek-Berthold) using 594 nm excitation and 642 nm emission wavelengths.

A549 cells at density of 5×10⁵ cells/ml were genipin and elesclomol treated for 24 h, and apoptotic rates were determined using an Annexin V-FITC Apoptosis kit (BD Biosciences) according to the manufacturer's instructions. Annexin V for early apoptotic cells and propidium iodide (PI) for late apoptotic cells were used. A total of 10,000 cells per sample underwent FACS analysis on a Calibur flow-cytometer (BD Biosciences) using CellQuest software version 5.1 (Becton-Dickinson and Company).

Intracellular ROS levels were quantified using the cell permeant indicator CM-H₂DCFDA. Briefly, A549 cells were seeded at a density of 4×104 cells/well in a black 96-well plate. After 24 h, the culture medium was changed to 10% FBS-containing phenol red-free RPMI-1-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) and cells were treated with genipin and elesclomol or transfected with UCP2 siRNA. Following incubation with 10 µM CM-H₂DCFDA at 37°C for 30 min, fluorescence was measured using a GloMax^®-Multi Detection System microplate reader (Promega Corporation) at 490 nm excitation and 510–570 nm emission wavelengths.

Mitochondrial ROS was measured using MitoSOX Red with dihydroethidium conjugated to a mitochondria-localization tag. A549 cells were seeded at a density of 4×10⁴ cells/well in a black 96-well plate, and the culture medium was changed the next day to 10% FBS-containing phenol red-free RPMI-1640 medium. Following genipin and elesclomol treatment, the culture medium was removed, and MitoSOX dye in HBSS buffer (Lonza Group, Ltd.) containing 2% bovine serum albumin (Bovogen Biologicals Pvt. Ltd.) was added. Following incubation at 37°C for 10 min, the buffer was changed to HBSS without dye, and fluorescence was measured on a Mithras LB 940 microplate reader at 510 nm excitation and 580 nm emission wavelengths.

A549 cells in 24-well plates at a densities of 5×10⁴ cells/well were incubated for 40 min with 100–250 kBq ¹⁸F-fluorodeoxyglucose (FDG) (provided by Samsung Medical Centre) added to the culture medium at 37°C in 5% CO₂. The cells were rapidly washed twice with cold PBS and lysed with 0.05 N NaOH, and cell-associated radioactivity was measured on a Wallac high-energy γ-counter (PerkinElmer, Inc.). Glucose uptake levels were corrected for cell protein content as assessed by Bradford assay.

Immunoblotting was performed in A549 cells and tumor tissues lysed with cold PRO-PREP™ protein extraction solution (iNtRON Biotechnology, Inc.) and T-PER™ tissue protein extraction reagent (Thermo Fisher Scientific, Inc.), respectively, with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Cell experiments were performed by obtaining cell lysates following 24-h incubation with 250 µM genpin and 0.1 µM elesclomol Following Bradford assays, 30 µg of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline and 0.1% Tween-20 for 1 h at room temperature and incubated overnight at 4°C with primary antibodies (1:1,000 dilution), followed by 1 h incubation at room temperature with secondary antibodies (1:2,000 dilution). Immunoreactive protein was detected by chemiluminescence (Pierce ECL plus western blotting substrate; Pierce; Thermo Fisher Scientific, Inc.), and band intensities were quantified using a GS-800 densitometer with Quantity One software version 4.6.6 (Bio-Rad Laboratories, Inc.).

Animal experiments were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Samsung Biomedical Research Institute at Samsung Medical Center (approval no. 20200413001). The tumor model was established in 5-week-old male BALB/c-nude mice weighing 20–25 g (Orient Bio, Inc.) by subcutaneous injection of 5×10⁶ A549 cells into the right shoulder. After two weeks, when tumors reached a diameter of ~0.5 cm, the animals were treated with intraperitoneal injections of DMSO control (n=6), 60 mg/kg genipin (n=6), 30 mg/kg elesclomol (n=7) or 60 mg/kg genipin plus 30 mg/kg elesclomol (n=7) three times per week for a total of four weeks. The dosages of genipin and elesclomol were based on previous studies (23,24). The mice were weighed and the tumor size was measured with a caliper before each injection. The experiment was repeated twice for a final total of 6–7 animals per treatment group.

Positron emission tomography (PET) imaging was performed on living tumor-bearing mice on the day following the final drug injection. At 1 h post-injection with 7.4 MBq of FDG via the tail vein, the animals were anesthetized by isoflurane inhalation, and PET images were acquired on an Inveon micro-PET scanner (Siemens Healthineers). Non-attenuation-corrected tomographic images were analyzed on an Inveon Research Workplace workstation version 4.2 (Siemens Healthineers). Polygonal regions of interest were manually drawn with care to include all tumor mass while excluding adjacent tissue. After PET imaging, the anesthetized mice were sacrificed by cervical dislocation, and the tumor tissues were excised.

Data are presented as mean ± standard deviation or the mean ± standard error of the mean. Significant differences between groups were analyzed by paired Student's t-test for two groups and one-way ANOVA with Bonferroni post hoc test for multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Western blot analysis demonstrated that A549 cells exhibited high protein expression levels of UCP2. Transfection with UCP2-specific siRNA effectively knocked down UCP2 expression to 34.8±8.0% of that in the non-targeted siRNA-transfected control cells (Fig. 1A).

Silencing of UCP2 expression with siRNA slightly suppressed FDG uptake to 88.5±3.3% of that in control cells (Fig. 1B. ROS production was significantly enhanced by UCP2 knockdown to 174.7±9.6% of that in controls (Fig. 1C). SRB assays results revealed that UCP2 silencing significantly decreased A549 cell viability to 60.8±7.9% of that in the control group (Fig. 1D).

Treatment of A549 cells for 24 h with 250 µM genipin, which blocks UCP2 function without reducing its expression (16), did not affect the protein expression levels of UCP2 (Fig. 2A). FDG uptake was unaffected by 24-h treatment with 0.1 µM elesclomol alone, but was significantly suppressed by 250 µM genipin alone or by 250 µM genipin plus 0.1 µM elesclomol to 72.0±7.1 and 65.8±4.6% of that in the control group, respectively (Fig. 2B).

Intracellular ROS production was also unaffected by 0.1 µM elesclomol alone. However, it was increased to 274.9±1.8% of that in the control group following 6-h treatment with 250 µM genipin, and was further increased to 404.3±21.4% of that in the control group when elesclomol was also present (Fig. 2C). Similarly, mitochondrial ROS production, which was unaffected by elesclomol alone, increased to 169.3±24.5% of that in the control group in the cells treated with 250 µM genipin and further increased to 256.9±36% of that in the control group following co-treatment with genipin and elesclomol (Fig. 2D).

Following UCP2 knockdown, the effects of genipin and elesclomol were more pronounced compared with those in cells transfected with the scrambled control. FDG uptake was decreased to 58.6±2.6% of that in the control group by genipin plus elesclomol in the scrambled siRNA-transfected group, and it was further reduced to 42.4±1.8% when UCP2 was knocked down (Fig. 2E). Similarly, cytosolic and mitochondrial ROS production levels, which were increased to 252.3±14.8 and 186.0±4.5% of those in controls by genipin plus elesclomol, respectively, were further increased to 406.8±35.0 and 253.2±27.9%, respectively, when UCP2 was knocked down (Fig. 2F and G).

The anticancer effects of co-treatment with genipin and elesclomol were analyzed in vitro. Clonogenic assays demonstrated that, when used alone, 0.1 µM elesclomol and 250 µM genipin exerted limited effects on the colony formation capacity of A549 cells. However, combining the two drugs reduced cell colony numbers to 50.6±7.4% of those in the control group following 48-h treatment (Fig. 3A).

Similarly, the results of the SRB assay demonstrated that 48-h treatment with 0.1 µM elesclomol or 250 µM genipin alone suppressed cell survival to 78.9±5.2 and 76.9±7.1% of that in the control group, respectively. However, combining the two drugs significantly reduced cell survival to 42.0±3.4% of controls (Fig. 3B, middle). Furthermore, LDH release, assessed as a measure of cytotoxicity, was unaffected by 24-h treatment with 0.1 µM elesclomol or 250 µM genipin alone, but was increased to 128.8±1.6% of that in the control group by co-treatment (Fig. 3C), accompanied by decreased intracellular LDH to 63.8±1.3% of that in the control group (data not shown).

The role of ROS on the cytotoxic effects of the two drugs was assessed in the presence of the exogenous ROS inducer TBHP and ROS scavenger NAC. The results demonstrated that the cytotoxic effects of genipin, elesclomol and their combination were enhanced by TBHP and reduced by NAC; the cell survival in the elesclomol and genipin co-treated cells at 48 h was decreased to 22.3±1.9% of that in the control group by TBHP (Fig. 3D) and was partially recovered to 57.5±4.8% of that in the control group by NAC (Fig. 3E).

The present study next evaluated whether the antitumor effect of combining elesclomol with genipin involved the induction of apoptosis. The results of the MitoTracker Red assay demonstrated that co-treatment with elesclomol and genipin for 24 h significantly reduced A549 cell MMP to 70.4±2.5% of that in the control group (Fig. 4A). This was accompanied by a 6-fold increase in the percentage of Annexin V-positive apoptotic cells (Fig. 4B). In addition, the co-treatment induced a marked increase in the levels of cleaved PARP to 837.6±221.9% and of cleaved caspase-3 to 495.9±68.6% of those in untreated controls (Fig. 4C and D).

Treatment of A549 tumor xenografts in mice with genipin or elesclomol led to similar magnitudes of modest retardation in tumor growth (280.1±130.8 and 310.1±114.6 mm³ on day 31, respectively) compared with that in the vehicle-treated control mice (584.2±482.5 mm³ on day 31; Fig. 5A). By contrast, combination therapy with elesclomol plus genipin resulted in near complete suppression of tumor growth (Fig. 5A); the tumor volume in the co-treatment group (84.0±27.8 mm³ on day 31) was significantly lower compared with that in the control group No significant differences were observed in animal weight during treatment (Fig. 5A).

PET/CT performed after the final treatment revealed significantly smaller tumors with lower FDG uptake in animals co-treated with elesclomol and genipin compared with those in animals treated with control or with elesclomol or genipin alone (Fig. 5B).

Western blots analysis of UCP2 protein expression in the xenograft tumor tissues demonstrated that UCP2 expression levels in the tumors isolated from genipin-treated mice was reduced to 56.4±12.0% of those in the tissues from the untreated control group (Fig. 5C). In addition, the LDH levels were lower in the tumor tissue of mice treated with genipin plus elesclomol compared with the control groups (Fig. 5D).