Li JE et al / Acta Pharmacol Sin 2002 Nov; 23 (11): 991-996

Apoptotic effect of As2S2 on K562 cells and its mechanism1

LI Jun-E, WU Wei-Li, WANG Zhen-Yi, SUN Guan-Lin2

Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China

1 Supported in part by Grants from the National Natural Science and Technology Foundation of 9th Quinquennial Program of China (969060117), National Natural Science Foundation of China(No 39870773), and Clyde Wu Foundation of Shanghai Institute of Hematology.

2 Correspondence to Prof SUN Guan-Lin. Phn 86-21-6437-0045, ext 610705. E-mail junelijune@yahoo.com

Received 2001-11-14 Accepted 2002-07-11

KEY WORDS arsenicals; chronic myeloid leukemia; apoptosis; Bcr-Abl fusion proteins

ABSTRACT

AIM: To investigate the apoptotic effect of As2S2 on K562 cells and its mechanism. METHODS: The effect of As2S2 on proliferation of K562 cells was determined by counting the number of cells. Apoptosis was assessed by flow cytometry, DNA fragmentation analysis, and morphology observation. Expression of protein was determined by Western blot. RT-PCR was used to evaluate changes in gene expression. RESULTS: As2S2 greatly inhibited the proliferation and induced apoptosis of K562 cells in a concentration- and time-dependent manner at the concentration range of 1-5 µmol/L during 24-72 h. Viable cells were decreased to approximately 71 % of control at the concentration of 5 µmol/L after 48-h incubation, 31.4 % after 72-h incubation, and 45.4 % at 3 µmol/L after 72-h incubation. At 3 µmol/L for 72 h, 5 µmol/L for 48 h, and 5 µmol/L for 72 h, the apoptosis rate were 34.4 %, 21.8 %, and 46 % of the treated-cells, respectively. As2S2 decreased the Bcr-Abl fusion protein and protein tyrosine kinase (PTK) activity of c-abl and Bcr-Abl, but it did not change the transcription of bcr-abl assayed. As2S2 also induced apoptosis in fresh mononuclear cells derived from chronic myelogenous leukemia (CML) patients. CML Ph+ leukemia cells were more sensitive to the apoptotic effect of As2S2 than Ph- mononuclear cells (P<0.05). CONCLUSION: As2S2 inhibited the proliferation and induced apoptosis in K562 and fresh CML mononuclear cells. The decline of the Bcr-Abl protein and its PTK activity may play an important role in the apoptotic effect of As2S2. As2S2 may be a useful agent for the treatment of CML.

INTRODUCTION

At present, chronic myelogenous leukemia (CML) therapies are mostly consisted of using chemotherapies, interferon (IFN-) and bone marrow transplantation. Recently, STI571 was introduced in the treatment of CML as an effective agent [1,2], because it is a specific inhibitor of Bcr-Abl tyrosine kinase. It was reported that this agent was very effective in the treatment of CML at chronic phase, and the adverse effects were mild. However, according to the results of clinical trials, relapse of the disease usually occurs after suspending the treatment, because the malignant colony of the CML cannot be completely eradicated[3,4]. Therefore, it is very important to find other effective agents for CML therapy.

During recent years, it was demonstrated that As2O3 is very effective for the treatment of acute promyelocytic leukemia (APL)[5]. In 1970s, it was reported by Shanghai Leukemia Research Cooperation Group that a compound from Chinese traditional medicine named Niuhuang JieduPian containing As2S2 was shown effective for CML treatment.

In this study, we investigated the effects of As2S2 on K562 cells, a human CML cell line from a CML patient in blast crisis, as well as fresh mononuclear cells (MNC) derived from CML patients, and its mechanism.

MATERIALS AND METHODS

Reagents As2S2 (AlfaAesar Company) was dissolved in NaOH 1 mol/L. The final concentration was 1.5 mmol/L and HCl was used to adjust the pH to 7.35-7.45.

Cell culture Human CML cell line K562 cells were cultured in RPMI-1640 medium supplemented with 10 % heat-inactivated fetal calf serum. Bone marrow mononuclear cells were obtained from CML patients or idiopathic thrombocytopenic purpura (ITP) patients and were cultured in Iscove's modified Dulbecco's medium supplemented with 10 % heat-inactivated fetal calf serum (Gibco/BRL, Grand Island NY). Diagnosis of CML was performed according to the standard criteria. All cells were cultured in a humidified atmosphere of 5 % CO2 at 37 ºC.

Cell proliferation and viability assay K562 cells 2×105 in logarithmic growth phase were collected and seeded in RPMI-1640 medium and were treated with As2S2 . At the indicated time points, viable cells were counted by Trypan blue exclusion method. Each assay was triplicated, then, a cell growth curve was drawn.

Flow cytometric analysis for apoptosis K562 cells 1×106 were washed with PBS and fixed in 75 % ethanol at -20 ºC overnight. Prior to analysis, cells were washed again with PBS and resuspended and treated with RNase 200 mg/L for 30 min at 37 ºC, then, cells were incubated in propidium iodide (PI) solution containing PI 20 mg/L in the darkness for 15 min. The suspension was then passed through a nylon mesh filter and analyzed on flow cytometry (Becton Dickson). Apoptosis induced by As2S2 in fresh mononuclear cells was detected by annexin V binding assay according to the manufacture's instruction (Clontech). All data were collected and analyzed by lysis II software (Becton Dickson). The experiments were repeated three times and the results were presented as mean±SD.

Morphological examination for apoptosis Cells (2×104) were prepared by cytospin centrifugation (250×g for 5 min at room temperature) and stained with Wright's staining.

DNA fragmentation assay Apoptosis was also confirmed by detection of fragmentation of chromo somal DNA with the classic DNA ladder method with slight modification. Briefly, cells (2×106) were immersed in cytolysis buffer (Tris-HCl 1 mmol/L pH 8.0, edetic acid 10 mmol/L pH 8.0, proteinase K 200 mg/L, 0.5 % SDS) and incubated for 3 h at 50 ºC. DNA was extracted with phenol-chloroform, precipitated in 1/10 volume of NaAc 2 mol/L and 2 volumes of ethanol at _20 ºC overnight, recovered by centrifugation at 1000×g for 30 min at 4 ºC, and then resuspended in TE buffer. RNase A was then added at a concentration of 200 mg/L, then the treated extract was incubated at 37 ºC for 30 min and electrophoresed for 2 h on a 1.2 % agarose gel.

Western blot for protein expression Cells were lysed by RIPA-buffer, homogenized by sonication, centrifuged at 10 000×g for 10 min and the supernatant was collected. Samples containing 100 mg of total protein were resolved by SDS-PAGE gel, transferred onto a nitrocellulose membrane by electroblotting, and probed with anti c-abl polyclonal antibody. The blot was developed by ECL chemiluminescence reaction kit (Amersham).

Immunoprecipitation and PTK activity assay C-abl and Bcr-Abl in samples containing 100 µg of total protein were obtained using immunoprecipitation. Immunoprecipitates were washed with RIPA-buffer and then resuspended in the assay buffer (Tris-HCl 50 mmol/L pH 7.4, MgCl2 40 mmol/L, sodium vanadate 50 µmol/L, DTT 2 mmol/L, MnCl2 1 mmol/L) and centrifuged. After removal of the supernatant , the precipitated protein was used directly for the protein tyrosine kinase (PTK) assay performed according to the protocol described by the kit (Boehring Mannhein). Each group contained 6 repeated tubes with blank and positive control, and the PTK activity was expressed by OD value measured at 405 nm (reference wavelength 490 nm).

RT-PCR for detection of bcr-abl gene Total cellular RNA was isolated with Trizol reagent (Gibco). RT-PCR was performed according to the protocol described by TaKaRa RT-PCR kit. Human housekeeping gene GAPDH was used to normalize for equal cDNA. The primers for bcr-abl were synthesized as flollows: 5'CTT CTC CCT GAC ATC CGT GG3', 5'CAT GTG GGC CAT GAG GTC CAC CAC 3'; the primers for GAPDH were synthesized as follows: 5'AGATGCTACTGGCCG CTGAA 3', 5'TGA AGG TCG GAG TCA ACG GAT TTG GT3'. The denaturing was performed for 3 min at 95 ºC, annealing for 30 min at 56 ºC, extension for 45 min at 72 ºC, and PCR for 32 cycles.

Statistical analysis All experiments were performed at least in triplicate and the results were expressed as mean±SD. Statistical analysis were performed with t-test using SAS6.12 software.

RESULTS

As2S2 inhibited the proliferation and viability of K562 cells To investigate the effect of As2S2 on proliferation and viability of K562 cells, the cells were treated with various concentrations of As2S2 ranging from 1 µmol/L to 5 µmol/L for 24_72 h. Significant concentration- and time-dependent inhibition of cell growth was observed. As shown in Fig 1, viable cells decreased to approximately 71 % of the control cells at 5 µmol/L for 48 h, 31.4 % for 72 h, and 45.4 % at 3 µmol/L for 72 h.

Fig 1. The effect of As2S2 on growth curve of K562 cells. n=3. Mean±SD.

As2S2 induced apoptosis of K562 cells To show whether the growth inhibition induced by As2S2 in K562 cells was caused by induction of apoptosis, the cells were stained with PI and analyzed by flow cytometry. The percentage of sub-G1 cells (apoptotic pick) in K562 cells was increased in a time- and concentration-dependent manner. At 1 µmol/L, sub-G1 pick was not observed. When the cells were treated with As2S2 3 µmol/L for 72 h, the percentage of sub-G1 cells was 34.4 %. At 5 µmol/L for 48 h and 72 h, the percentage of sub-G1 cells reached 21.8 % and 46 %, respectively (Fig 2). The apoptosis of the cells was confirmed by morphological examination. Typical changes of apoptosis represented by plasma membrane blebbing, chromatin condensation, and fragmentation of nuclei were shown (Fig 3).

Fig 2. The percentage of sub-G1 cells in K562 cells treated with As2S2 at different concentrations for different times n=3. Mean±SD.

Fig 3. Morphological changes of K562 cells and CML mononuclear cells treated with As2S2 (Wright's staining, ×1000).A: Untreated K562 cells; B: K562 cells treated with As2S2 at 5 µmol/L for 72 h: C: Untreated CML mononuclear cells; D: CML mononuclear cells treated with As2S2 at 5 µmol/L for 72 h.

As2S2 decreased Bcr-Abl fusion protein in K562 cells without bcr-abl fusion gene alteration To explore the mechanism by which As2S2 induced apoptosis in K562 cells, we detected the changes of Bcr-Abl protein in K562 cells treated with As2S2 at concentration of 1 µmol/L, 3 µmol/L, and 5 µmol/L, respectively for 72 h and at 5 µmol/L for 24 h, 48 h, and 72 h, respectively using Western-blot. We found that the content of Bcr-Abl protein in treated K562 cells was decreased in a time- and concentration-dependent manner. However, the mRNA level of bcr-abl fusion gene was not changed in any sample assayed using RT-PCR method (Fig 4).

Fig 4. The changes of bcr-abl in K562 cells treated with As2S2. (A) Western-blot for Bcr-Abl protein content in K562 cells treated with As2S2 at different concentrations for different times. (B) RT-PCR for bcr-abl in K562 cells treated with As2S2 at different concentrations for different time using GAPDH as internal standard.

As2S2 decreased the activity of protein tyrosine kinase of c-Abl and Bcr-Abl in K562 cells Correlating with the changes of the content of c-Abl and Bcr-Abl, the PTK activity of c-Abl and bcr-abl in K562 cells was decreased in a time- and concentration-dependent manner (Fig 5).

Fig 5. PTK activity of c-Abl and Bcr-Abl in K562 cells treated with As2S2 at different concentrations for different time. (A) PTK activity of c-Abl and Bcr-Abl in K562 cells treated with As2S2 5 mmol/L for different time. (B) PTK activity of c-Abl and Bcr-Abl in K562 cells treated with As2S2 at different concentrations for 72 h. n=6. Mean±SD.

As2S2 induced the apoptosis in fresh CML mononuclear cells CML mononuclear cells treated with As2S2 were dual-stained with Annexin-V-FITC and PI and analyzed by flow cytometry. A concentration-dependent increase in apoptotic cells was shown when the cells were treated with As2S2 1 µmol/L to 5 µmol/L, and a time-dependent increase in apoptotic cells was shown when the cells were treated with As2S2 at 5 µmol/L for 24-72 h. At different concentration, at As2S2 1 µmol/L for 72 h, (3.4±0.6) % cells underwent apoptosis and (8.6±1.5) % and (23±4) % cells showed signs of apoptosis at 3 µmol/L for 72 h and 5 µmol/L for 72 h, respectively. In the different time group, (8.2±0.5) %, (9.6±1.5) %, and (23±4) % cells showed signs of apoptosis at 5 µmol/L for 24 h, 48 h, and 72 h, respectively. Apoptosis induced by As2S2 was verified by the morphology (Fig 3) and a typical DNA "ladder" was shown by gel electrophoresis (Fig 6).

Fig 6. DNA ladder of CML MNC treated with As2S2 at 3 µmol/L for different time.

Effect of As2S2 on fresh Ph_ mononuclear cells To compare the sensitivity of Ph+ cells to the apoptotic effect of As2S2 with that of Ph_ cells, as the representative of Ph_ cells, the fresh mononuclear cells were collected respectively from bone marrow of ITP patients. Compared with CML Ph+ leukemia cells, Ph_ mononuclear cells were less sensitive to As2S2 at any concentration mentioned previously (P<0.05). At As2S2 1 µmol/L, 3 µmol/L, and 5 µmol/L for 72 h, (4.5± 1.3) %, (5.5±0.8) %, and (9.5±1.5) % cells showed signs of apoptosis, respectively. At As2S2 5 µmol/L for 24 h, 48 h, and 72 h, (3.2±1.8) %, (6.8±2.4) %, and (9.5± 1.5) % cells were underwent apoptosis, respectively (P<0.05, Tab 1).

Tab 1. The apoptotic effect of As2S2 on CML Ph+ MNC and Ph_ MNC. n=3. Mean±SD. bP<0.05 vs Ph_ MNC.

DISCUSSION

CML is a leukemia characterized cytogenetically by the presence of Philadelphia chromosome (Ph+) resulting in the generation of bcr-abl chimeric gene that encodes Bcr-Abl protein with elevated PTK activity. It accounts for about 20 % in all leukemias in adults[6-8]. One of the clinical features of CML is the presence of excessive number of mature myeloid cells in peripheral blood. The percentage of myeloblasts and promyelocytes is generally less than 10 % and the progenitors show no greater proliferative potency than normal. So, it is presumed that CML arises from apoptosis suppression rather than abnormal proliferation and differentiation[9,10]. Bcr-Abl is considered as a crucial factor in the pathogenesis of CML. Therefore, induction of apoptosis through targeting Bcr-Abl may be an effective therapy for CML.

Recently, it was reported that NB4 cells exposed to As4S4 could be induced to apoptosis. Induction of apoptosis was one of the major mechanisms of therapeutic effect of As4S4 for APL at therapeutic concentration. In 1970s, a compound containing As2S2 from Chinese traditional medicine termed Niuhuang JieduPian was shown effective for the treatment of CML by Shanghai Leukemia Research Cooperation Group and other clinician of the northern part of China, suggesting that As2S2 may be effective agent for the treatment of CML.

In this study, we found that K562 cells, a CML cell line with Bcr-Abl fusion protein could be induced to apoptosis by As2S2. In the studies on the mechanism of As2S2-induced apoptosis in K562 cells, we found that c-abl and Bcr-Abl protein were reduced during the process of apoptosis in K562 cells treated with As2S2 at concentration from 1 µmol/L to 5 µmol/L and the changes of PTK activity of c-Abl and Bcr-Abl was corresponded with that of protein content. However, the bcr-abl gene expression was unchanged, suggesting that Bcr-Abl was down-regulated by As2S2 at post-transcription level and the decline of PTK activity of Bcr-Abl might resulted from the decline of c-abl and Bcr-Abl protein. Furthermore, the time when the significant decline of Bcr-Abl occured was the time when apoptosis occurred, suggesting that the decline of Bcr-Abl protein played a role in the apoptosis induced by As2S2 in Ph+ cells.

Similar apoptotic effect of As2S2 was also observed on primary CML mononuclear cells (Ph+), and compared with CML mononuclear cells, fresh Ph- mononuclear cells showed less sensitivity to As2S2 under the same condition. These data supported our presumption about the mechanism by which As2S2 induces apoptosis in K562 cells. It was reported[11] that Bcr-Abl mediated As2O3-induced apoptosis in Ph+ CML and Ph+ ALL (acute lymphocytic leukemia). We consider that the effect of As2S2 on Ph+ CML is analogous to that of As2O3.

In summary, the results of the present studies suggest that As2S2-induced apoptosis in K562 was probably realized through degradation of Bcr-Abl protein and it could be used as a new therapeutic agent for CML.

REFERENCES