All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) unless specified otherwise. RPMI-1640 and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). ENERGI-F706 was a proprietary compound generously provided by Energenesis-Biomedical Co., Ltd. (New Taipei, Taiwan). Antibodies against caspase-3 (cat. no. 9668), caspase-8 (cat. no. 9496), caspase-9 (cat. no. 2876), B-cell lymphoma 2 (Bcl-2; cat. no. 2876), Bcl-2-associated X protein (Bax; cat. no. 2774), phospho-(p-) AMPK (T172) (cat. no. 2531), AMPK (cat. no. 2532), p-p53 (S15) (cat. no. 9286), p53 (cat. no. 9282) and poly(adenosine diphosphate ribose) polymerase (PARP; cat. no. 9541) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Antibodies against GAPDH (cat. no. G8795) were obtained from Sigma-Aldrich. Horseradish peroxidase (HRP)-conjugated secondary antibodies against mouse immunoglobulin (Ig)G (cat. no. ab6789) and rabbit IgG (cat. no. ab97051) were purchased from Abcam Inc. (Cambridge, UK).

The human RCC cell line 786-O (ATCC^® CRL-1932™) and HK-2 human renal cell line (ATCC^® CRL-2190™) was obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in RPMI-1640 with 10% FBS at 37°C in a humidified atmosphere containing 5% CO₂. Cells were trypsinized upon confluency and propagated to passage two prior to being sub-cultured into six-well plates for further treatments.

For treatments, cells were seeded in six-well culture plates at an initial density of 1×10⁵ cells/ml and grown to 80% confluence. All treatments were performed using serum-free RPMI-1640. For cell viability and cell cycle distribution, the cells were treated with ENERGI-F706 (0.05, 0.1, 0.2, 0.4 and 0.6 mg/ml) for 24 h. To determine the involvement of AMPK in the suppression of cell viability, the cells were pre-incubated with or without 5 µM compound C (Sigma-Aldrich) for 4 h, and then treated with ENERGI-F706 (0.05, 0.1, 0.2, 0.4 and 0.6 mg/ml) for 24 h. After the treatments, the cells were washed with phosphate-buffered saline (PBS; 25 mM sodium phosphate; 150 mM NaCl; pH 7.2) and collected for the subsequent analyses.

Cell viability was determined using the MTT assay (12) in the absence or presence of ENERGI-F706. After 24- or 48-h treatments, the culture medium was aspirated and cells were incubated with MTT (0.5 mg/ml) at 37°C for 4 h. The viable cell number was directly proportional to the production of formazan, which was dissolved in isopropanol and determined by measuring the absorbance at 570 nm using a microplate reader (SpectraMAX 360 pc; Molecular Devices, Sunnyvale, CA, USA).

Cells were synchronized at G₀ phase by serum starvation for 16 h and then incubated in fresh serum-containing medium to allow for cell-cycle progression. At various time-points after release from G₀ arrest, cells were analyzed by flow cytometry to determine the cell cycle distribution. At the end of the treatment, cells were collected, fixed with 1 ml ice-cold 70% ethanol, incubated at −20°C for 24 h and centrifuged at 380 ×g for 5 min at room temperature. Cell pellets were treated with l ml cold staining solution containing 20 µg/ml propidium iodide (PI), 20 µg/ml RNase A and 1% Triton X-100, and incubated for 15 min in the dark at room temperature. Subsequently, the samples were analyzed in a FACS Calibur system (BD Biosciences, Franklin Lakes, NJ, USA) using CellQuest version 2.0 software. Representative results were acquired from three independent experiments.

Cells were washed with PBS and then lysed with lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride and 1 mM NaF) containing complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). The crude extracts were collected and protein contents were quantitated using the bicinchoninic acid (BCA) method. Crude proteins (20 µg per lane) were subjected to 12.5% SDS-PAGE and then transferred onto a nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking with 5% w/v skimmed milk/PBS, the membrane was incubated with optimal diluted first antibodies (1:1,000) for 2 h at room temperature, and then with HRP-conjugated secondary antibodies (1:2,000) for 1 h at room temperature. Development was performed using an enhanced chemiluminescence reagent (Millipore). The luminescent signal was acquired and quantitated using the LAS-4000 image analysis system with LAS-4000mini analysis software (Fuji Film, Tokyo, Japan).

Values are expressed as the mean ± standard error of the mean of the three independent experiments. Statistical significance was determined by using one-way analysis of variance followed by Dunnett's test for multiple comparisons with the control. Statistical analysis was conducted using SigmaStat version 3.5 (Systat Software, Inc., San Jose, CA, USA). P<0.05 was considered to indicate a statistically significant difference between values.

Changes in cell viability in response to ENERGI-F706 were determined using an MTT assay. As shown in Fig. 1A, the viability of HK-2 cells was reduced to 90.1±7.2 and 79.6±6.9% of the control following treatment with 0.4 and 0.6 mg/ml ENERGI-F706, respectively (P<0.05). In addition, the viability of 786-O cells was reduced to 76.3±3.9, 62.4±4.2 and 36.4±2.4% of that of the control following treatment with 0.2, 0.4 and 0.6 mg/ml ENERGI-F706, respectively (P<0.05) (Fig. 1B). These findings demonstrated that ENERGI-F706 treatment (>0.2 mg/ml) significantly inhibited the viability of 786-O cells.

To explore the effects of ENERGI-F706 on the cell cycle and apoptosis of 786-O cells, they were assessed using flow cytometric assays. As shown in Fig. 2, 24-h ENERGI-F706 treatment increased the sub-G₁-phase population in a dose-dependent manner by up to 28.6±1.7% (0.6 mg/ml; P<0.01 vs. control). In addition, the S-phase population was also increased in response to ENERGI-F706 at low doses (0.05, 0.1 and 0.2 mg/ml; P<0.05 as compared to control). By contrast, the G₀/G₁-phase population was decreased in response to ENERGI-F706 treatment (0.05–0.6 mg/ml; P<0.05 as compared to control). Collectively, these results demonstrated that ENERGI-F706 treatment remarkably altered the cell cycle distribution of 786-O cells and induced apoptosis.

To further investigate the apoptosis induced by ENERGI-F706, apoptotic cascades were examined. As shown in Fig. 3, the levels of anti-apoptotic Bcl-2 were dose-dependently decreased in 786-O cells exposed to ENERGI-F706. By contrast, levels of apoptotic proteins, including Bax, cleaved caspase-9 (35 kDa), cleaved caspase-3 (17 kDa) and cleaved PARP (89 kDa) were significantly elevated in 786-O cells treated with ENERGI-F706. These findings showed that ENERGI-F706 triggered activation of caspase cascades, including suppression of anti-apoptotic Bcl-2 and enhancement of apoptotic signaling cascades.

A previous study has reported that ENERGI-F704, another purine analogue similar to ENERGI-F706, possesses anti-inflammatory activity, which was attributed to the activation of AMPK signaling and subsequent inhibition of nuclear factor-κB signaling in vascular endothelial cells (13). Accordingly, the effects of ENERGI-F706 on AMPK and the associated signaling were investigated in the present study. After 24 h of treatment, ENERGI-F706 enhanced the phosphorylation of AMPKα (T172) and p53 (S15) (Fig. 4A).

The involvement of AMPK (T172) phosphorylation in ENERGI-F706-induced p53 (S15) phosphorylation and apoptotic signaling cascades was subsequently examined. The results revealed that AMPK (T172) phosphorylation induced by ENERGI-F706 was inhibited by pre-treatment with AMPK inhibitor compound C (dorsomorphin). In parallel to inhibition of AMPK (T172) phosphorylation, p53 (S15) phosphorylation and cleavage of caspase-9 as well as PARP induced by ENERGI-F706 were also diminished by pre-treatment with compound C (Fig. 4B). In contrast to the apoptotic signals, the levels of anti-apoptotic Bcl-2 was restored by pre-treatment with compound C as compared to those following treatment with ENERGI-F706 alone (Fig. 4B). Collectively, these findings revealed that ENERGI-F706 activated AMPK and consequently triggered apoptotic signaling cascades in 786-O cells.

The involvement of AMPK activation in the suppression of cell viability in response to ENERGI-F706 was next investigated. As shown in Fig. 5, treatment with ENERGI-F706 alone at 0.2, 0.4 and 0.6 mg/ml for 24 h decreased the cell viability to 67.3±4.2, 55.2±3.4, and 40.8±4.7% of that of the control, respectively (P<0.05 as compared to control). By contrast, after pre-treatment with 5 mM compound C for 2 h followed by treatment with ENERGI-F706 at 0.2, 0.4 and 0.6 mg/ml for 24 h, the cell viability was restored to 72.9±5.9, 67.1±3.1, and 53.8±2.3% of that of the control, respectively (P<0.05 as compared to ENERGI-F706 alone). These results revealed that AMPK activation has an important role in the suppression of 786-O cell viability in response to ENERGI-F706.