D/F12 medium and fetal bovine serum (FBS) were purchased from Hyclone; GE Healthcare Life Sciences (Logan, UT, USA) and Biological Industries (Kibbutz Beit-Haemek, Israel), respectively. The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Trypsin, dimethyl sulfoxide (DMSO), and Hoechst 33258 were purchased from Sigma-Aldrich; Merck Millipore (Darmstadt, Germany). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining kit, DNA content quantitation assay kit, 5,5,6,6-tetra-chloro-1,1,3,3-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) dye and caspase-3 activity assay kit were purchased from Nanjing KeyGen Biotech. Co., Ltd. (Nanjing, China). Glutathione (GSH; cat. no. CEA294Ge) and N-acetyl-β-D-Glucosaminidase (NAG; cat. no. CSB-E07444 m) ELISA kits were purchased from Uscn Life Science, Inc. (Wuhan, China) and CUSABIO Biotech. Co., Ltd. (Wuhan, China), respectively. Radioimmunoprecipitation assay (RIPA) lysis buffer and enhanced chemiluminescence (ECL) kit were purchased from Biomiga, Inc. (San Diego, CA, USA) and Beyotime Institute of Biotechnology (Haimen, China), respectively. The bicinchoninic acid (BCA) protein assay kit was purchased from BioTeke Corporation (Beijing, China). Fas cell surface death receptor (Fas; dilution, 1:4,000; cat. no. ab133619), B-cell lymphoma 2 apoptosis regulator (Bcl-2; dilution, 1:4,000; cat. no. ab182858), Bcl-2 associated protein X apoptosis regulator (Bax; dilution, 1:4,000; cat. no. ab32503) and β-actin (dilution, 1:4,000; cat. no. ab16039) antibodies were purchased from Abcam (Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (dilution, 1:80,000; cat. no. IH-0011) was obtained from Boster Systems, Inc. Pleasanton. CA, USA. All other chemicals were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). POA was provided by the South China Sea Institute of Oceanology (Guangzhou, China). The structure of POA was determined by infrared, nuclear magnetic resonance and mass spectrometry and its purity of >98% was determined by high performance liquid chromatography. POA was dissolved in DMSO and phosphate buffer saline (PBS) to obtain stock solutions (40 mM), which were stored at −20°C. Prior to use in an experiment, the stock solution was diluted to the indicated concentrations with culture medium. During the experiments, the DMSO content in the medium never exceeded 0.5% (v/v).

HK-2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were grown in D/F12 supplemented with 10% FBS in a humidified incubator at 37°C in the presence of 5% CO₂. The culture medium was changed every 2 days. Cells for assays were detached by a solution of 0.25% trypsin and 0.02% EDTA.

HK-2 cell viability was evaluated by the CCK-8 assay. Briefly, HK-2 cells (1×10⁴cells/well) were seeded in 96-well microplates and then cultured in D/F12 growth medium for 24 h. Subsequently, the medium was replaced with D/F12 growth medium containing 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 µM POA. Cells containing equal volumes of cell culture medium but no POA (0 µM), were used as a control in each experiment throughout the study. Following exposure to POA for 24, 48 or 72 h, 10 µl of the CCK-8 assay solution was added into each well, followed by incubation of the microplates at 37°C in 5% CO₂/95% air for 2 h. Finally, absorption was measured at 450 nm using a microplate reader (PerkinElmer, Inc., Waltham, MA, USA), with a reference wavelength of 650 nm (7). Three different experiments were performed and the average value was calculated.

Morphological changes in the HK-2 cells were evaluated by phase contrast optical microscopy (Leica Microsystems Gmbh, Wetzlar, Germany). Morphological changes of the cell nuclei were evaluated by fluorescent visualization with Hoechst 33258 staining. Briefly, cells (4×10⁴ cells/well) cultured on slides were treated with 0, 20 or 40 µM POA for 24 h. Following treatment, cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min and then incubated for 5 min with 5 mg/ml Hoechst 33258 fluorescent dye. The cells were then washed, dried, and photographed using a fluorescence microscope.

The early apoptosis rate was measured using Annexin V-FITC/PI double staining and a Accuri™ C6 FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) with BD CFlow Software v.264.15 (BD Biosciences). Following treatment with 0, 20 or 40 µM POA for 24 h, 5×10⁵ HK-2 cells were harvested by centrifugation at 800 × g for 5 min at 4°C, washed twice with ice-cold PBS and resuspended in 500 µl binding buffer, followed by the addition of 5 µl Annexin V-FITC conjugate and 5 µl PI buffer, according to the manufacturer's protocol. Following incubation in the dark for 15 min at room temperature, the cells were analyzed by flow cytometry. Each determination is based on the acquisition of 10,000 events (8).

Following treatment with 0, 20 or 40 µM POA for 24 h, 5×10⁵ cells were collected by centrifugation at 800 × g for 5 min at 4°C, washed twice with PBS, and then fixed with 70% chilled ethanol for 12 h. Following fixation, cells were washed twice with PBS and incubated in PBS containing 50 mg/ml PI, 1 mg/ml RNase A and Triton X-100 (0.5%) at 4°C for 30 min in the dark. The fluorescence emitted from the PI-DNA complex was measured using Accuri™ C6 FACScan flow cytometry (BD Biosciences) with BD CFlow Software v.264.15 (BD Biosciences). The cells with nuclei with sub-G1 content were considered apoptotic cells (9).

A caspase 3 activity assay kit was used to measure caspase 3 activity, as previously described (10). In brief, 1×10⁶ cells were treated with 0, 20, 40, or 80 µM POA for 24 h, then cells were harvested by centrifugation at 800 × g for 5 min at 4°C, washed twice with ice-cold PBS, resuspended in lysis buffer and left on ice for 60 min. The lysate was centrifuged at 12,000 × g at 4°C for 5 min. The cell supernatant was incubated with the enzyme specific colorimetric substrate acetyl-Asp-Gla-Val-Asp-phosphorylated nitroanilide (Ac-DEVD-pNA) in assay buffer for 2 h at 37°C. The concentration of pNA from the Ac-DEVD-pNA substrate was determined by the optical absorbance at 405 nm using a microplate reader (PerkinElmer, Inc., Waltham, MA, USA).

For assays of GSH, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide (NO), N-acetyl-β-D-glucosaminidase (NAG), lactate dehydrogenase (LDH) and reactive oxygen species (ROS), HK-2 cells (1×10⁶ cells/well) were seeded in 6-well plates and then cultured in D/F12 growth medium for 24 h. Subsequently, the medium was replaced with D/F12 growth medium containing 0, 20, 40, or 80 µM POA and incubated for 24 h at 37°C. Following treatment, 1×10⁶ cells were harvested by centrifugation at 800 × g for 5 min at 4°C, then lysed on ice for 30 min using lysis buffer (50 mmol Tris-HCl, 1.0 mmol/l EDTA, 150 mmol/l NaCl and 0.1% SDS), and centrifuged once more at 5,000 × g for 10 min at 4°C. The supernatant was used for the enzymatic assays. For assessment of extracellularly released molecules, the culture medium was analyzed following the 24 h POA treatment.

The antioxidant enzyme GSH was measured using a GSH ELISA kit, as per the manufacturer's instructions. GSH concentration was determined by measurement of the absorbance at 450 nm using an ELISA Reader (PerkinElmer, Inc., Waltham, MA, USA).

SOD is a scavenger of superoxide. SOD activity was detected using the xanthine/xanthine oxidase method based on the production of O²⁻ anions (11). HK-2 cells and lysates were prepared as described above, but in 25 cm² culture flasks at 1×10⁶ cells/flask. The SOD activity in the cell lysates was determined using a formula calculation based on absorbance values at 550 nm (11). Activity of SOD is expressed as units per mg of cellular protein (U/mg prot).

The concentration of MDA, an end product generated from lipid peroxidation, was measured using an MDA detection kit according to the manufacturer's instructions (12). Briefly, MDA reacts with thiobarbituric acid (TBA) at 95–100°C in acidic conditions and the reaction produces a pink MDA-TBA conjugate, which can be measured using an EnSpire multi-mode microplate reader (PerkinElmer, Inc., Waltham, MA, USA) at 532 nm. The cellular MDA concentration was expressed as nmol/mg of cellular protein (13).

NO, a potentially toxic molecule, is a labile, diffusible product of mammalian cells. It serves as a short-lived messenger molecule involved in diverse biological phenomena such as cytotoxicity (14). NO levels were estimated by measuring the accumulation of nitrite in the culture medium, which is the resulting byproduct of NO metabolites. The culture medium samples were tested with a NO detection kit, according to the manufacturer's instruction. Absorbance at 550 nm was measured with a microtiter plate reader (PerkinElmer, Inc., Waltham, MA, USA). A range of sodium nitrite concentrations were used to generate the standard curve. The cellular NO content was expressed as µmol/g of cellular protein.

NAG is a lysosomal enzyme that is present in proximal tubular cells. The activity of NAG is generally low in HK-2 cells and increases as a consequence of the breakdown of renal tubular cells (15). The culture medium samples were tested for NAG by ELISA (16), according to the manufacturer's instructions. Absorbance at 450 nm was measured with a microtiter plate reader, and NAG concentration was determined based on a standard curve.

LDH is a cytoplasmic oxidoreductase, that is important in maintaining cell membrane integrity. When cells are damaged, LDH leaks into the culture medium. LDH leakage was measured by testing the culture medium samples with an LDH ELISA, according to the manufacturer's instructions. Absorbance at 450 nm was measured with a microtiter plate reader and the concentration of LDH was determined based on a standard curve.

ROS are oxygenic free radicals which can alter the balance of endogenous protective systems, such as glutathione and enzymatic antioxidant defense systems (17). ROS production in cells was detected by a standard, cell permeable, fluorescent dye method, 2,7-dichlorofluorescin diacetate (DCFH-DA). Intracellular esterases hydrolyze DCFH-DA to 2,7-dichlorofluorescin (DCFH), which is then oxidized by ROS to dichlorofluorescein (DCF), and therefore amount of fluorescence reflects ROS production in cells. Fluorescence intensity was measured by Accuri™ C6 flow cytometry (BD Biosciences) with BD CFlow Software v.264.15 (BD Biosciences) at excitation and emission wavelengths of 485 nm and 530 nm, respectively (18).

Flow cytometry following JC-1 staining was used to evaluate loss of MMP (19,20). HK-2 cells (5×10⁵ cells/well) in 6-well culture plates were treated with 0, 20, 40 or 80 µM POA for 24 h. Cells were trypsinized, washed twice with ice-cold PBS, resuspended in PBS and labeled with 5 µg/ml JC-1 cationic dye for 30 min in the dark at 37°C. Following labeling, cells were washed twice with PBS, resuspended in PBS at a concentration of 1 to 5×10⁶ cells/ml and analyzed by Accuri™ C6 flow cytometry (BD Biosciences) with BD CFlow Software v.264.15 (BD Biosciences).

HK-2 cells cultured at a density of 1×10⁶ cells/well in 6-well microplates were treated with 0, 20 or 40 µM POA for 24 h. The treated cells were lysed in RIPA lysis buffer containing 1% phenylmethane sulfonyl fluoride and incubated on ice for 30 min. Lysates were centrifuged at 8,000 × g for 8 min at 4°C, and the protein concentration in the supernatant was determined by BCA assay. A total of 60 µg/lane protein was separated by 12% SDS-PAGE and then electrotransferred to a polyvinylidene fluoride membrane. The membrane was incubated in 5% skim milk in TBST (0.1% Tween-20) buffer for 2 h, followed by incubation with the primary antibody (diluted in TBST with 5% skim milk) overnight at 4°C. The incubated membrane was washed 3 times with TBST prior to incubation for 1 h at 25°C with the secondary antibody which was diluted in TBST with 5% skim milk. Following 3 washes with TBST, the immune complexes were detected using an ECL kit.

Data are presented as the mean ± standard deviation of at least 3 independent experiments. Statistical differences between means among multiple groups were analyzed with one way analysis of variance followed by either the Bonferroni or Tamhane's T2 post hoc test when appropriate. Statistical analysis was performed using SPSS v.16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

CCK-8 assays were used to determine the effect of POA on the growth of HK-2 cells. As presented in Fig. 2, cell growth became markedly inhibited from dose concentrations of 70, 50 and 50 µM POA at 24, 48, or 72 h, respectively. Increased cytotoxicity was observed as time and POA concentration increased (Fig. 2). DMSO, which was the vehicle control for the treatments, did not exhibit any inhibitory effect on the cell viability (0 µM POA; Fig. 2).

Using a phase contrast microscope, the untreated HK-2 cells appeared to assume a common spindle cell shape with intact nuclei (Fig. 3A). In the presence of 20 µM POA for 24 h, there were slight morphological alterations when compared with the untreated HK-2 cells, however, in the presence of 40 µM POA for 24 h, the HK-2 cells contracted and became rounded and detached from the substrate (Fig. 3A). HK-2 cells were stained with the nuclear dye Hoechst 33258, which also serves as marker of apoptosis. Control nuclei did not exhibit distinct changes (Fig. 3B). In the presence of 40 µM POA, the cells' nuclei revealed brighter Hoechst 33258 staining, karyopyknosis and fragmented chromatin, indicating that they might be undergoing apoptosis. By contrast, there were no obvious alterations in the nuclei with the 20 µM POA treatment (Fig. 3B).

The apoptotic effect of POA on HK-2 cells was further investigated by flow cytometric analysis. Annexin V-FITC/PI double-labeling was used for the detection of phosphatidylserine (PS) externalization, a characteristic of early apoptosis (21,22). As demonstrated in Fig. 4A, a significant increase in early apoptotic cells was observed in cells treated with 20, 40 or 80 µM POA for 24 h (P<0.05), when compared with untreated cells (Annexin V⁺/PI⁻ cells, as measured in the Q4 quadrant of the plots; Fig. 4A). To gain insights into the mechanism of the growth inhibition activity of POA in HK-2 cells, its effect on cell cycle distribution was examined by flow cytometry. The number of apoptotic cells in each treatment group was determined by observing the SubG1 apoptotic peaks, which are attributed to the reduced DNA content. As demonstrated in Fig. 4B, treatment with POA for 24 h resulted in a significant, dose-dependent, increase in the number of apoptotic cells and a significant increase in the proportion of cells in the S and G2/M phases compared with untreated cells. By contrast, a significant reduction of the proportion of cells in the G1 phase was observed in POA-treated cells compared with untreated cells (Fig. 4B). In conclusion, the results from the Annexin V-FITC/PI double-labeling assay and the cell-cycle analysis indicated that POA-mediated growth inhibition was accompanied by a dose-dependent increase in apoptosis.

Caspase 3 is a key regulator of the terminal phase of apoptosis (23). As demonstrated in Fig. 5, a significant, dose-dependent increase in caspase 3 activity was observed in HK-2 cells treated with 20, 40 or 80 µM POA for 24 h when compared with control untreated cells (P<0.001).

The content of the crucial, cellular, non-enzymatic antioxidant GSH was determined by ELISA. Treatment with 40 and 80 µM POA induced a significant decrease in cellular GSH content (Fig. 6A). The level of GSH in the control cells was 3.85 µg/ml, and this was reduced to 3.71 (P>0.05 vs. control), 3.19 (P<0.01 vs. control) and 2.81 (P<0.01 vs. control) µg/ml with 20, 40 and 80 µM POA treatment, respectively (Fig. 6A).

HK-2 cells treated with 40 or 80 µM POA demonstrated a significant decrease in SOD activity compared with control (Fig. 6B). The SOD activity of the control cells was 10.93 U/ml, which was reduced to 10.55 (P>0.05 vs. control), 9.47 (P<0.001 vs. control) and 9.12 (P<0.001 vs. control) U/ml in cells treated with 20, 40 and 80 µM POA, respectively (Fig. 6B).

MDA is a well-studied intermediate of oxidative stress (24). HK-2 cells treated with 20, 40 and 80 µM POA exhibited a significant increase in MDA levels (Fig. 6C). MDA levels in control cells were 0.40 nmol/mg of total protein, and these levels were increased to 0.72 (P<0.01 vs. control), 1.22 (P<0.001 vs. control) and 1.65 nmol/mg (P<0.001 vs. control) with 20, 40 and 80 µM POA, respectively (Fig. 6C).

A significant effect of POA was observed on NO production in HK-2 cells: In control cells, the concentration of NO released in the culture medium was 11.42 µmol/l, and this was significantly decreased to 10.20, 7.25 and 4.42 µmol/l with 20, 40 and 80 µM POA treatment, respectively (P<0.001 vs. control; Fig. 6D).

NAG is commonly regarded as a marker of proximal tubular cell integrity, and measurement of its release is used as an indicator of early kidney cells injury (25). As demonstrated in Fig. 6E, the NAG release level in HK-2 cells was significantly increased to 1.19-(P<0.01 vs. control), 1.40-(P<0.001 vs. control) and 1.70-fold (P<0.001 vs. control) relative to the control cells, with 20, 40 and 80 µM POA, respectively.

Cellular LDH leakage in the culture medium was measured as a key signal of damaged cells. LDH activity was significantly increased to 1.48-(P<0.01 vs. control), 2.02-(P<0.01 vs. control) and 3.15-fold (P<0.001 vs. control) relative to control cells with 20, 40 and 80 µM POA, respectively (Fig. 6F).

ROS is a critical regulator of cellular homeostasis, and overproduction of ROS induces apoptosis and cell death (26). The results presented in Fig. 7 indicated that POA treatment significantly promoted ROS accumulation in HK-2 cells, in a dose-dependent manner. Treatment of HK-2 cells with 20, 40 or 80 µM POA for 24 h increased the % of cells that exhibit ROS accumulation to 3.8, 6.2 and 15.2% of total, respectively, compared with 0.9% of total in the control (Fig. 7).

Disruption of mitochondrial integrity is one of the early events leading to apoptosis (27). Accumulation of the cationic dye JC-1 was assessed by flow cytometry to evaluate the effect of POA on MMP. Following exposure of HK-2 cells to 20, 40 or 80 µM POA for 24 h, MMP disruption was detected in 14.8, 16.2 and 26.5% of total cells, respectively, compared with 12.3% of total cells in the untreated control group (Fig. 8).

Bcl-2 is a Bcl-2 family anti-apoptotic protein, which helps cells to prevent apoptosis. Bax is a Bcl-2 family pro-apoptotic protein, that translocates from the internal to the outer mitochondrial membrane, inducing the release of pro-apoptotic factors, and resulting in apoptosis. The expression levels of Fas, Bax and Bcl-2 were analyzed by western blot in HK-2 cells treated with 0, 20 or 40 µM POA for 24 h (Fig. 9). POA treatment significantly increased protein expression levels of Fas and Bax compared with control (Fig. 9). By contrast, the expression of Bcl-2 was significantly decreased with POA treatment, compared with control (Fig. 9). These results indicated that POA treatment resulted in an upregulation of the pro-apoptotic proteins Fas and Bax and a downregulation of the anti-apoptotic protein Bcl-2.