Human leukemia K562 cells were obtained from the Affiliated Hospital of Guiyang Medical University (Guiyang, China). Cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin at 37°C in an incubator containing 5% CO₂ and humidified air. The study was approved by the ethics committee of Hospital affiliated to Guiyang medical college (Guiyang, China).

Two different methods were used to evaluate the cytotoxic effects of PEITC under various experimental conditions. Cell viability was determined using the MTT assay. Briefly, cells were seeded at a density of 20,000 cells/well on 96-well plates, treated with various concentrations of PEITC and then cultured for 24, 48 and 72 h at 37°C with 5% CO₂. Following culture, the medium was replaced with 200 μl fresh medium containing MTT at a final concentration of 250 μg/ml. Subsequent to incubation at 37°C for 4 h, the reaction was terminated with the addition of 150 μl dimethyl sulfoxide (DMSO)/well. The solution was then agitated for 10 min to dissolve the crystals. The absorbance of each sample well was read at 492 nm using an automated 96-well plate reader. All assays were performed in triplicate. The inhibition ratio (I%) was calculated using the following formula:

Cell death was determined using flow cytometric analysis, subsequent to the cells being double-stained with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Cells were incubated for 48 h following the addition of 5, 10, 15 and 20 mol/l PEITC. The cells (10⁵/sample) were then washed twice with cold phosphate-buffered saline (PBS) and suspended in 500 μl 1X binding buffer (PBS solution included 10 mM HEPES, 140 mM NaCl and 2.5 mM CaCl₂, pH 7.4). A total of 5 μl PI was then added subsequent to the addition of 5 μl Annexin-V-FITC. The reagents were mixed gently and the samples were incubated in the dark for 15 min at room temperature. The samples were then analyzed by flow cytometry using the BD FACSCalibur™ flow cytometer with BD CellQuest™ software (Becton, Dickinson and Company, Franklin Lakes, NJ USA).

Total RNA was extracted using TRIzol^® Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA was reverse transcribed into cDNA using a reverse transcription kit (Promega Corp., Madison, WI, USA), which was stored at −20°C. The primers (Takara Bio Inc., Dalian, China), shown in Table I, were designed using Primer Express 3.0 Software (Applied Biosystems, Foster City, CA, USA). qPCR was performed using a real-time PCR thermocycler (Applied Biosystems) and quantified using SYBR^® Green PCR Master Mix (Applied Biosystems) using 1 μl cDNA in a final reaction volume of 20 μl. The reactions were cycled 40 times subsequent to an initial denaturation step of 95°C for 10 min. The PCR cycle conditions were as follows: Denaturation, 95°C for 15 sec; annealing, 60°C for 10 sec and extension, 72°C for 10 sec. Following the PCR, the temperature was increased from 60 to 95°C at a rate of 2°C/min, and the fluorescence was measured every 15 sec to construct the melting curve. GAPDH was used as an internal control. SDS 2.2.1 software (Applied Biosystems) was used to perform relative quantification of the target genes using the ΔΔCt method.

Cells were washed three times with ice-cold PBS and then lysed in Tris-buffered saline (TBS; 50 mmol/l Tris-HCl, pH 8.0, 150 mmol/l NaCl, 100 μg/ml phenylmethylsulfonyl fluoride and 1% Triton X-100) for 30 min on ice. Aliquots of cell lysates containing 20 μg protein were separated using 12% SDS-PAGE and transferred to nitrocellulose membranes. Subsequent to being blocked with 5% skimmed milk in TBS, membranes were incubated with polyclonal antibodies against HO-1 (1:200), cytochrome c (1:200), caspase-3 (1:100), caspase-8 (1:500), caspase-9 (1:500), Fas (1:1,000), FasL (1:1,000) and β-actin (1:500) overnight. The membranes were then washed in 0.1% Tween 20 in TBS and incubated with secondary antibodies (Cell signaling technology, Boston, MA, USA) at room temperature for 1 h. Immunoreactive signals were visualized by exposing the membrane to Kodak X-ray film (Carestream Health, Inc., Xiamen, China), and quantified using Image J 1.44p (National Institutes of Health, Bethesda, MD, USA).

The measurement of intracellular ROS was based on the ROS-mediated conversion of non-fluorescent dichlorofluorescin diacetate (DCFH-DA) into DCFH. Briefly, K562 cells were treated by PEITC for an additional 48 h. Cells were then centrifuged and resuspended in 1X PBS containing a final concentration of 10 μM DCFH-DA and incubated at 37°C for 30 min. Cells were subsequently washed with 1X PBS and analyzed using the FITC channel in a flow cytometer.

Data are expressed as the mean ± standard deviation. Analysis of variance was performed using SPSS 13.0 (SPSS, Inc., Chicago, IL USA). A value of P<0.05 was considered to indicate a statistically significant difference.

The effect of treatment with various concentrations of PEITC (0, 2.5, 5, 10, 15 and 20 μmol/l) for various time intervals (24, 48 and 72 h) on K562 cell growth was investigated. As shown in Fig. 1A, PEITC inhibited cell proliferation in a dose- and time-dependent manner. Therefore, the inhibition of K562 cell proliferation using high concentrations of PEITC for a long duration may be cytotoxic. The apoptosis-inducing effect of PEITC on K562 cells was determined using flow cytometry. Following treatment with 5, 10, 15 and 20 μmol/l PEITC for 48 h, apoptosis was induced in 21.45±1.68, 30.06±1.94, 68.51±1.95 and 66.09±2.01% of K562 cells, respectively (Fig. 1B).

To investigate the effect of PEITC on the expression of apoptosis-associated genes, K562 cells were exposed to 5, 10, 15 and 20 μmol/l PEITC for 48 h and gene expression levels were analyzed using qPCR. As shown in Fig. 2, exposure to PEITC significantly increased the mRNA expression of cytochrome c, the key enzyme responsible for caspase-9 activation (P=0.024). The mRNA expression of the apoptosis-related genes caspase-3 and -8, Fas and FasL was also observed to be increased by PEITC.

Following treatment with the aforementioned concentrations of PEITC for 48 h, K562 cell extracts were collected for western blot analysis. As shown in Fig. 3, PEITC treatment was found to increase the protein expression of cytochrome c, caspase-9, -3 and -8, Fas and FasL. These results are in accordance with those of the qPCR analysis.

To investigate whether the PEITC-induced cytotoxic effects in K562 cells involved ROS, ROS levels were measured using flow cytometry. As shown in Fig. 4, PEITC concentrations of 5, 10, 15 and 20 μmol/l were found to promote ROS production in K562 cells.

In the present study, PEITC was observed to induce apoptosis and elevate ROS production in K562 cells; therefore, the impact of HO-1 on cell survival was investigated. To assess the effect of HO-1 expression on PEITC-induced apoptosis, PEITC was combined with the HO-1 inhibitor ZnPPIX and the HO-1 inducer hemin in order to decrease and increase the expression of HO-1 in K562 cells, respectively. Cells were cultured with 20 μmol/l hemin and 10 μmol/l ZnPPIX for 48 h, and the expression levels of the apoptosis-related genes cytochrome c, caspase-9, -3 and -8, Fas and FasL were detected and compared with those of the cells treated solely with PEITC (Figs. 5 and 6). HO-1 was observed to protect K562 cells from PEITC-induced apoptosis.

HO-1 is involved in regulating the redox equilibrium in human cells; therefore, K562 cells were incubated with PEITC, hemin and PEITC plus hemin for 48 h. As shown in Fig. 7, ROS production, which was facilitated by PEITC in a dose-dependent manner, was downregulated with high HO-1 expression. Furthermore, fluorescence-activated cell sorting analysis revealed that PEITC significantly elevated ROS production (P=0.017), and that this elevation was attenuated upon addition of hemin, indicating that the decreased ROS production originated from high HO-1 expression.