Peroxisome Proliferator-Activated Receptor-γ Agonist Rosiglitazone– Induced Apoptosis in Leukemia K562 Cells and Its Mechanisms of Action

Abstract

This study investigates the ability of a synthetic PPAR-γ agonist, rosiglitazone (RGZ), to induce apoptosis in leukemia K562 cells. The results revealed that RGZ (>40 mmol/L) inhibits the growth of K562 cells and causes apoptosis in a time and dose-dependent manner. Apoptosis is observed clearly by Hoechst 33258 staining. Western blotting analysis demonstrates the cleavage of caspase-3 zymogen protein with the appearance of its 17-kD subunit and a dose-dependent cleavage of poly (ADP-ribose) polymerase. Furthermore, RGZ treatment down-regulates anti-apoptotic protein Bcl-2 and up-regulates pro-apoptotic protein Bax in a dosedependent manner after the cells are treated for 48 hours. Telomerase activity is decreased concurrently in a dosedependent manner. We therefore conclude that RGZ induces apoptosis in K562 cells in vitro, and that RGZ-induced apoptosis in K562 cells is highly correlated with activation of caspase-3, decreasing telomerase activity, down-regulation of the anti-apoptotic protein Bcl-2, and up-regulation of the pro-apoptotic protein Bax.

Peroxisome proliferator-activated receptor-γ (PPAR-γ), one of the best characterized nuclear hormone receptors in the superfamily of ligand-activated transcriptional factors, is predominantly expressed in adipose tissue and plays an important regulatory role in lipid and glucose metabolism, adipocyte differentiation, and energy homeostasis. ^1,2 In common with other members of the nuclear receptor gene family, the PPARs function as ligand-activated transcription factors forming heterodimer with the retinoid X receptor (RXR). Upon ligand binding, the complex of PPAR and RXR binds to specific recognition sites on DNA, the peroxisome proliferator response elements, and regulates transcription of specific genes. ^3,4 Recently a number of studies have demonstrated that PPAR-γ is expressed in a variety of types of cancer cells and has crucial roles in suppressing cancer cell growth. ^5,6 Previous studies have shown that PPAR-γ expression can be detected in a number of hematologic cells. ^7–9 Furthermore, many human leukemia cells, including myeloid and lymphoid blasts, have been reported to express PPAR-γ. ^10,11 PPAR-γ can be activated not only by a naturally occurring arachidonic acid metabolite, 15-deoxy-δ (12,14)-prostaglandin J2 (15d-PGJ2), but also by synthetic ligands such as those belonging to the antidiabetic thiazolidinedione (TZD) class of compounds. ^12,13 Recent studies have shown that activation of PPAR-γ by TZDs or 15d-PGJ2 leads to either inhibition of cell growth or apoptosis on leukemia cells. ^14,15

Apoptosis, or programmed cell death, plays an important role in the prevention of leukemia development, and impaired apoptosis is now recognized to be a key step in the pathogenesis of leukemia. ¹⁶ Activation of apoptosis pathways is a key mechanism by which cytotoxic drugs kill leukemia cells, and defects in apoptosis signaling contribute to leukemic cell drug resistance. ^17,18 Thus, induction of apoptosis has now been considered as an important method of assessment for the clinical effectiveness of many anti-leukemia drugs and a significant index for the selection of new drugs in clinical practice. ^19,20

Chronic myeloid leukemia (CML), of which the median duration of the chronic phase is 3 to 4 years, is a hematological malignant disease, occurring predominantly in younger populations, and has been found to have a higher morbidity in eastern countries, especially in China, than in western countries. ²¹ Even with modern treatment protocols, many CML patients eventually die of the subsequent blast crisis. Therefore, efforts are ongoing to find new anti-leukemia drugs and effective therapies for the clinical treatment of CML. ^22,23

K562 cell is leukemia cell line derived from a patient with CML in 1975. ²⁴ This CML cell line represents a unique source of CML cells with meaningful indicators of malignancy for clinical and experimental studies. ²⁵ Studies ^26,27 have shown that the expression level of PPAR-γ was much higher in leukemia K562 and THP-1 (acute monocytic leukemia cell line) cells than that in other leukemia cells such as MOLT-4 (acute lymphoblastic leukemia cell line), HL60 (acute myeloid leukemia cell line), and SO4 (adult T-cell leukemia cell line) and that treatment by 15d-PGJ2 or synthetic dual PPAR-α/γ ligand TZD18 might trigger apoptosis in these leukemia cells via different signal pathways.

Previously, we and others proved that synthetic PPAR-γ agonists such as troglitazone and pioglitazone could inhibit K562 cell growth by induction of apoptosis. ^14,15 Rosiglitazone (RGZ), another important synthetic PPAR-γ agonist approved by the US Food and Drug Administration (FDA) in 1999 and by the centralized process of the European Medicines Agency (EMEA) in 2000, ²⁸ is now widely used clinically in the treatment of diabetes. No reports are available about the effects of RGZ on leukemic K562 cells. Because data have proven that many synthetic PPAR-γ agonists have antiproliferation effects on leukemia cells, it is reasonable that RGZ may also demonstrate growth inhibition effects on leukemia K562 cells. To verify this hypothesis and investigate the anti-leukemia mechanisms of RGZ, we tested the apoptotic effects of various concentrations of RGZ on K562 cells in vitro.

Materials and Methods

Main Reagents

Rosiglitazone (RGZ) was purchased from Cayman Chemicals (St Louis, Missouri). RGZ was dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. Hoechst 33258 was purchased from Sigma Company. Antibodies against Bcl-2, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP), and β-actin were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Caspase-3 colorimetric assay kit was purchased from R&D Systems Inc (Minneapolis, Minnesota). Telomerase polymerase chain reaction (PCR)–enzyme-linked immunosorbent assay (ELISA) kit was obtained from Boehringer-Mannheim (Mannheim, Germany).

Cell Culture

Human leukemia cell line K562 cells (purchased from Shanghai Rui-jin Hospital) were cultured in RPMI-1640 medium supplemented with 10% heatinactivated calf serum and 100 U/mL penicillin in a humidified incubator at 37°C with 5% CO₂. Cells were passaged twice weekly and routinely examined for mycoplasma contamination.

Cell Growth Inhibitory Rate

Cell growth inhibitory rate was assayed using the microculture tetrazolium method. Briefly, 2 × 10⁵ cells/well were dispensed within 96-well culture plates in 100-μL volumes. Then different concentrations of RGZ (20, 40, 60, and 80 μmol/L) were put in different wells (pilot studies showed that these concentrations could inhibit cell growth). Each of these concentrations was regarded as 1 treated group, whereas the control group contained no RGZ. Each treated or control group consisted of 6 parallel wells. Culture plates were incubated for 0, 24, 48, and 72 hours, and then 20 μL of MTT working solution was added and incubated continuously for 4 hours. All culture medium supernatant was removed from each well after centrifugation and replaced with 100 μL of DMSO. Following thorough solubilization, the absorbance (A value) of each well was measured using a microculture plate reader at 570 nm. Cell inhibitory rate was calculated according to the following formula: inhibitory rate = 100 × (A value of control group – A value of treated group)/A value of control group.

Flow Cytometric (FCM) Analysis for Cell Apoptosis

For FCM analysis, 2 × 10⁶ cells treated with different concentrations of RGZ were collected, pelleted, washed with phosphate-buffered saline (PBS), and fixed in 75% ethanol at −20°C overnight. Prior to analysis, cells were washed again with PBS, resuspended, and treated with RNase 200 mg/L for 30 minutes at 37°C; cells were then incubated with 20 mg/L Propidium Iodide (PI) in the dark for 15 minutes. Then the suspension was passed through a nylon mesh filter and analyzed using flow cytometry (FACSort; Becton-Dickinson, Franklin Lakes, New Jersey). All data were collected, stored, and analyzed by LYSIS II software (Becton-Dickinson). The experiments were repeated 3 times, and the results are presented as mean ± standard deviation (SD).

Hoechst 33258 Staining

Hoechst 33258 staining was used to observe the apoptotic morphology of K562 cells. Cells were fixed with 4% formaldehyde in PBS) for 10 minutes, stained by Hoechst 33258 (10 mg/L) for 1 hour, and then examined by fluorescence microscopy.

Caspase-3 Activity Assay

The activity of caspase-3 was determined by a caspase colorimetric assay kit, according to the manufacturer’s protocol. Briefly, RGZ-treated cells were washed with ice-cold PBS and lysed in a lysis buffer. The cell lysates were tested for protease activity using a caspase-specific peptide, conjugated to the color reporter molecule p-nitroanaline. The chromophore p-nitroanaline, cleaved by caspases, was quantitated with a spectrophotometer at a wavelength of 405 nm. The caspase enzymatic activities in cell lysates are directly proportional to the color reaction.

Western Blotting Analysis

For Western blotting, cells were washed with ice-cold PBS twice and lysed for 30 minutes at 4°C; then debris was removed by centrifugation for 15 minutes at 15 000 × g at 4°C, and equivalent amounts of protein were separated by 10% SDS-PAGE and transferred onto nitrocellulose filters. The filters were first stained with 10% Ponceau S solution for 2 minutes to confirm uniform transfer of all samples and then incubated in blocking solution for 2 hours at room temperature. The filters were first incubated with the primary antibodies at 4°C overnight, followed by washes with PBS twice and Tris-buffered saline Tween-20 (TBST) twice. Filters were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour, washed with TBST, and developed using the Super Signal West Pico Kit.

Telomerase Activity Assay

Telomerase activity was measured quantitatively by the telomere repeat amplification protocol (TRAP)– ELISA based on the modified TRAP. The sequences of the primers were CX 5′-CCCTTACCCTTACCCTTACCCTTA- 3′ and TS 5′-AATCCGTCGAGCAGAGTT- 3′. Cell extracts were prepared according to TRAP. Telomerase added telomeric repeats TTGGG to the 3′-end of the biotin-labeled synthetic P1-TS primer. These elongation products were then amplified by PCR using the primers P1-TS and P2-CX to generate PCR products with the telomerase-specific 6-nucleotide increments. Then an aliquot of the PCR product was denatured and hybridized to a dioxigenin- DIG-labeled, telomeric repeat-specific detection probe. The final product was immobilized to a streptavidin-coated microtiter plate via the biotin-labeled primer and detected with an antibody against dioxigenin (anti-DIGPOD) that was conjugated to peroxidase. The probe was visualized by virtue of peroxidase metabolizing 3,3′,5,5′ tetramethylbenzidine (TMB) to form a colored reaction product. Finally, the absorbance of the samples was measured at 450 nm (with a reference wave length of approximately 690 nm) using a microtiter plate (ELISA) reader within 30 minutes after addition of the stop reagent. Absorbance values were reported as the A_{450 nm} – A_{690 nm}.

Statistical Analysis

All experiments were performed in triplicates. The results are expressed as mean ± SD. For statistical analysis, Student’s t tests were performed using SAS 6.12 software (SAS Institute, Cary, North Carolina). Statistical significance was accepted at the level of P < .05.

Results

Cell Growth Inhibition Caused by RGZ

RGZ inhibited the growth of K562 cells significantly at concentrations greater than 40 μmol/L. The inhibitory rate caused by 80 μmol/L RGZ was much higher than that of lower concentrations of RGZ (P < .01) (Figure 1).

Cell Apoptotic Rate Detected by FCM

As shown in Figure 2, RGZ (>40 μmol/L) induced apoptosis when cultured with K562 cells for 24 to 72 hours. The percentage of apoptotic cells was gradually increased in a dose-dependent manner and was between 40% and 50% when cells were cultured with RGZ for 48 hours (RGZ >40 μmol/L). RGZ at 80 μmol/L caused an apoptotic rate that was much higher (>50%) than that for lower concentrations of RGZ (P < .01).

Hoechst 33258 Staining

After treatment with different concentrations of RGZ for 72 hours, K562 cells were harvested and visualized by Hoechst staining. No remarkable apoptotic morphology was observed after the cells were treated with 20 μmol/L (Figure 3A). Apoptotic cells gradually increased when cells were treated with 40 μmol/L RGZ (Figure 3B). RGZ at 60 μmol/L and 80 μmol/L caused typical cell apoptosis (Figures 3C and 3D). Marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found clearly using Hoechst 33258 staining. Apoptotic cells gradually increased in a dose-dependent manner in RGZ-treated K562 cells.

Western Blotting of Bcl-2 and Bax Expression

To determine whether apoptosis-related gene expression might play a role in RGZ-induced apoptosis, we detected the protein levels of Bcl-2 and Bax after the cells were treated by RGZ for 48 hours. Western blotting analysis revealed that Bcl-2 expression was down-regulated, whereas Bax expression was up-regulated concomitantly (Figure 4).

Variation of Caspase-3 Activity

To understand the activation of the caspase cascade during RGZ-induced apoptosis in K562 cells, we investigated caspase-3 activity after the cells were treated with RGZ for 24, 48, and 72 hours. As shown in Figure 5, caspase-3 activity was increased in a time-and dose-dependent manner, with the maximal response at 80 μmol/L (P < .01).

The cleavage of procaspase-3 was also detected by Western blotting. The results revealed that caspase-3 was activated, as measured by the loss of caspase-3 proenzyme (32 kD) and the appearance of its 17-kD subunit, after the cells had been exposed to RGZ (>40 μmol/L) (Figure 6A). Caspase-3 activation was found to increase concomitantly with increased concentration of RGZ treatment. To confirm RGZ induced activation of caspase-3, the cleavage of PARP, a known substrate of caspase-3, was also examined by Western blotting. As shown in Figure 6B, RGZ treatment caused a dose-dependent cleavage of PARP, with the appearance of an 89-kD fragment and disappearance of the intact 116-kD PARP (Figure 6B). These results indicated that activation of caspase-3 was involved in RGZ-induced apoptosis in K562 cells.

Telomerase Activity Caused by RGZ

Telomerase activity in K562 cells decreased significantly when exposed to RGZ (>40 μmol/L, P < .01 vs control group); the higher the RGZ concentration, the lower the telomerase activity of K562 cells, especially at 72 hours, when the telomerase activity is approximately zero (RGZ concentration was 80 μmol/L) (Figure 7).

Discussion

In this study, we found that RGZ could inhibit cell growth and induce apoptosis in leukemia K562 cells at RGZ concentrations of more than 40 μmol/L. Western blot analysis revealed down-regulation of Bcl-2 and up-regulation of Bax protein as well as activation of caspase-3. Telomerase activity was gradually down-regulated along with the increased concentration of RGZ. These results verified our hypothesis that RGZ would induce apoptosis in leukemia K562 cells. Previously, we found that troglitazone (TGZ) could significantly inhibit K562 cell growth when TGZ concentrations were greater than 50 μmol/L, ¹⁴ whereas 15d-PGJ2 induced apoptosis in K562 cells at lower concentrations (>20 μmol/L). These results agree with previous reports that the growth inhibition induced by 15d-PGJ₂ was much more pronounced than growth inhibition induced by the synthetic PPAR-γ ligands PGZ and RGZ. ²⁹ To date, no reports are available regarding the in vivo antileukemia effects of RGZ, indicating that the antileukemia effects of RGZ need further investigation.

The Bcl-2 family consists of about 20 homologues of important pro- and anti-apoptotic regulators of programmed cell death. Bcl-2 protein is first presented and functions in preventing programmed cell death. ³⁰ Bax is a 21-kDa protein that shares homology with Bcl-2 clustered in conserved regions including BH1 and BH2. ^31,32 When Bax was overexpressed in cells, apoptotic death in response to death signals was accelerated, earning its designation as a death agonist. When Bcl-2 was overexpressed, it heterodimerized with Bax and cell death was repressed; thus, the ratio of Bcl-2 to Bax is important in determining susceptibility to apoptosis. ^33,34 Therefore, down-regulation of Bcl-2 and up-regulation of Bax expression may play an important role in chemotherapeutic drug-induced apoptosis.

Telomerase is a reverse transcriptase that adds nucleotide repeats to telomeres by using an RNA template providing karyotype stability and compensating for the loss of DNA. ^35,36 Studies have shown that telomerase activity may be detected in more than 80% of human tumor cells and telomerase activity is detected in almost all cancer cell lines. ³⁷ In malignant hematological diseases, high telomerase activity almost always correlates with disease severity, and the activity of telomerase is a very useful index for the diagnosis and clinical staging in hematologic malignancies. ³⁸ Recent studies suggest that telomerase inhibition can promote apoptosis in many hematologic malignancies such as myeloma and that inhibition of telomerase results in telomere shortening, repressed proliferation, and altered cell cycles that result in apoptosis in cancer cells. ^39,40 This indicates that anti-telomerase therapy not only can enhance apoptosis in tumor cells but also can be one of the most important and effective markers for the selection of new anti-tumor drugs. ⁴¹ Our results agree with these findings. We found in our study that K562 cells express high levels of telomerase activity, and telomerase activity was down-regulated remarkably when apoptosis occurred.

Molecular mechanisms of apoptosis involve release of cytochrome c from mitochondria to the cytosol, thereby initiating a cascade of proteolytic events. ⁴² The caspases are a family of intracellular cysteine proteases with specificity for aspartic acid residues. ^42,43 Two of these groups, termed initiator and effector caspases, play important roles in the apoptotic process. ^44,45 Caspases-3 is one of the most important executioners and is capable of cleaving many important cellular substrates. Caspase-3-mediated cell death plays an important role in pathogenesis and therapy of a variety of cancers. ^45,46 Our results showed that RGZ may trigger apoptosis of K562 cells via activation of caspase-3 and variation of protein levels of apoptotic related genes.

Conclusions

Our results indicate that RGZ has apoptosis-inducing effects on K562 cells in vitro and that RGZ-induced apoptosis in K562 cells is mainly related to activation of caspase-3 and decrease of telomerase activity as well as down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax.