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Keywords:

  • arsenic trioxide;
  • apoptosis;
  • proliferation;
  • cancer therapy;
  • chemotherapy resistance

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Summary. Clinical efficacy of As2O3 has been shown in patients with relapsed acute promyelocytic leukaemia (APL). There is evidence that the effects of As2O3 are not restricted to events specific for APL. As2O3 might target mechanisms involved in the pathogenesis of other malignancies. We assessed susceptibility to induction of apoptosis by As2O3 and cytostatics in 22 myeloid and non-myeloid malignant cell lines. As2O3 was used in concentrations of 0·01–10 µmol/l. Cell lines displayed different kinetics of response and different sensitivity to As2O3. The minimum concentration of As2O3 for induction of apoptosis was 0·1 µmol/l. High concentrations of As2O3 (5 µmol/l) induced apoptosis in a large proportion of cells in all cell lines tested. Low (1 µmol/l As2O3) concentrations induced apoptosis in NB-4, HL-60, U-937, CEM, HL-60, KG-1a, PBL-985, ML-2 and MV-4–11, but not in HEL, K-562, KG-1 and Jurkat up to 35 d of incubation. However, the non-apoptotic population of 1 µmol/l As2O3-treated HEL, K-562, K-562 (0·02), K-562(0·1) and Jurkat showed reduced proliferation. CEM as well as its' multidrug-resistant derivatives were sensitive to 1 µmol/l As2O3. In summary, these data demonstrate that As2O3-induced apoptosis is not restricted to cell lines with t(15;17). Apoptosis was induced in vitro by As2O3 concentrations that are achievable in vivo after infusion of well-tolerated As2O3 doses. Thus, As2O3 might be a suitable therapeutic agent for malignancies other than APL provided the adequate dose and duration of As2O3 treatment are used.

Several reports have demonstrated the efficacy of As2O3 in the treatment of relapsed or refractory acute promyelocytic leukaemia (APL) with complete remission rates of > 72% (Shen et al, 1997; Soignet et al, 1998; Warrell et al, 1998; Niu et al, 1999; Spencer & Firkin, 1999; Jiong et al, 2000). As2O3 was found to be a potent alternative in treatment of all-trans retinoic acid (ATRA)-resistant APL (Chen et al, 1997; Kitamura et al, 1997). To date, the clinical use of As2O3 in leukaemia treatment has been restricted to APL because it was thought that As2O3 triggered the degradation of the t(15;17)-specific fusion protein PML-RARα (Chen et al, 1996).

However, recent reports show that As2O3 can exert PML-RARα-independent effects (Wang et al, 1998; Huang, C. et al, 1999; Huang, X.J. et al, 1999). Moreover, the potency of As2O3 to induce apoptosis was shown for non-APL leukaemia and lymphoma cell lines (Konig et al, 1997; Wang et al, 1998; Lu et al, 1999; Zhu et al, 1999; Puccetti et al, 2000; Walter et al, 2000) as well as other tumour cell lines (Akao et al, 1999; Huang, C. et al, 1999; Zhang et al, 1999; Deng et al, 2000; Chen, F. et al, 2001; Jiang et al, 2001). The necessity of the PML-RARα fusion protein present in APL-cell lines for As2O3-induced apoptosis thus became questionable. As2O3 can induce activation of caspases (Akao et al, 1999; Huang, X.J. et al, 1999; Kitamura et al, 2000; Jiang et al, 2001), downregulation of Bcl-2 (Chen et al, 1996; Deng et al, 2000; Perkins et al, 2000; Puccetti et al, 2000), modulation of p53 (Jiang et al, 2001), as well as the uncoupling of the mitochondrial potential (Dai et al, 1999; Jing et al, 1999; Kroemer & de The, 1999; Larochette et al, 1999; Cai et al, 2000). It is speculated that these effects are responsible for programmed cell death, whereas the induction of cyclin-dependent kinase inhibitors seems to play a role in As2O3-mediated cell cycle arrest and inhibition of cell proliferation (Eguchi et al, 1997; Seol et al, 1999; Park et al, 2000).

In this paper, we address the question of whether clinically achievable concentrations of As2O3 (Shen et al, 1997; Ni et al, 1998) are able to induce both apoptosis and growth inhibition in a broad spectrum of leukaemia and lymphoma cell lines without t(15;17). We therefore tested 22 cell lines that represent various stages of lympho-haemopoietic differentiation, including six cell lines resistant to different cytostatic agents. The focus is set on induction of apoptosis in long-term incubation experiments. Incubation time and As2O3 concentrations were derived from treatment duration and serum As2O3 concentrations in successful clinical trials of As2O3 in APL.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Cell culture.  Cell lines 697, CCRF-CEM, DOHH-2, HL-60, Jurkat, K-562, KG-1a, LOUCY, ML-2, MV-4–11, NB-4, NC-NC and PBL-985 were purchased from The German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), KG-1 cells from the European Collection of Cell Cultures (Salisbury, UK), CEM/C1, CEM/C2, HEL, HL-60/MX1, HL-60/MX-2 and U-937 from the American Type Culture Collection (Manassas, USA). The doxorubicin-resistant cell lines K-562(0·02) and K-562(0·1) were established in our laboratory. K-562(0·02) cell line is resistant to up to 0·02 µg/ml doxorubicin, K-562(0·1) is resistant to up to 0·1 µg/ml doxorubicin. Cells were grown according to the providers' recommendations.

Induction of apoptosis with arsenic(III)trioxide (As2O3) and cytostatic drugs.  Apoptosis was induced with an aqueous stock solution of 1 mmol/l arsenic(III)-oxide (Sigma, Deisenhofen, Germany) in phosphate-buffered saline (PBS) without Ca2+/Mg2+ (Gibco Life Technologies, Karlsruhe, Germany), 50 mmol/l stock solution of etoposide (Sigma) in dimethyl sulphoxide (DMSO; Merck, Darmstadt, Germany), 10 mmol/l mitoxantrone (Sigma) or 10 mmol/l doxorubicin (Sigma) in 70% ethanol. Cells were seeded at an initial density according to their optimal growth condition, given by the provider. The medium was changed every second day. Cells were seeded again at the original density. Inductors of apoptosis and 10% of d 2-conditioned medium were added.

Detection of apoptosis using flow cytometry.  The percentage of apoptotic cells was determined with two-colour fluorescence using an Annexin V-fluorescein isothiocyanate (FITC) and 7-amino-actinomycin D (7-AAD) system. Cells (5 × 105−1 × 106) were used for each staining. All incubation and wash steps were performed in Annexin V binding buffer (ABB: 10 mmol/l Hepes, pH 7·4; 140 mmol/l NaCl; 2·5 mmol/l CaCl2). Cells were washed once and stained with 5 µl of Annexin V-FITC (BD Pharmingen, Heidelberg, Germany) in 100 µl of ABB for 20 min in the dark at room temperature, washed once and counterstained with a final concentration of 20 µg/ml 7-AAD (Sigma) for 20 min in the dark at 4°C (Philpott et al, 1996), washed twice and immediately analysed by flow cytometry. Whenever possible, fluorescence data of 50 000 cells was acquired using fluorescence channels FL-1 and FL-3 of a FACScan (Becton Dickinson, Heidelberg, Germany). Cells that showed a breakdown of symmetry of the cell membrane leading to an exposure of phosphatidylserines in the outer membrane layer were referred to as ‘Annexin V-positive cells’.

PKH26-proliferation assays.  Reduction of proliferation rate was measured using the PKH26 red fluorescent cell linker kit (Sigma) according to the manufacturers protocol. In order to achieve a sufficient intensity of staining, we had to use double the suggested amount of fluorescent dye and a longer incubation time for cell lines HEL, K-562, K-562(0·02), K-562(0·1), HL-60, HL-60/MX1 and HL-60/MX2. To compensate for the effects of the prolonged exposure to diluent C reagent, unstained control cells were also treated with this reagent. Diluent C was added containing the same amount of 70% ethanol as in stained cells but without fluorescent membrane dye PKH26. Before taking stained cells in culture, they were blocked with fetal bovine serum and washed three times in the same way as stained cells, according to the standard protocol.

Analysis of cell cycle by propidium iodide (PI) staining.  Pro- pidium iodide staining was performed as described by Lee et al (2001) with the following exceptions: digest with DNase-free RNase (Sigma) and staining with PI (Molecular Probes/MoBiTec, Göttingen, Germany) was performed at room temperature in the dark for 30 min. Flow cytometry was performed using a FACScan (Becton Dickinson).

Ki67 expression.  Cells (5 × 105−1 × 106) were washed with PBS without Ca2+/Mg2+ (Gibco Life Technologies), fixed and permeabilized for staining using the IntrastainKit from DAKO (Hamburg, Germany). Detection of the cytoplasmatic protein Ki67 was performed by flow cytometry analysis after incubation of fixed and permeabilized cells using a mouse anti-human anti-Ki67-FITC antibody from DAKO. An isotype control was performed for each sample.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Leukaemia and lymphoma cell lines differ in their sensitivity towards As2O3-induced apoptosis

Several leukaemia and lymphoma cell lines representing different stages of lympho-haemopoietic differentiation were tested for their sensitivity towards induction of apoptosis by arsenic trioxide at clinically achievable concentrations. All 22 cell lines tested were sensitive towards prolonged incubation with As2O3; however, the incubation time and concentration of As2O3 necessary to induce apoptosis differed. A classification based on the response for As2O3 (class A) reveals three different groups of sensitivity (see Table I and Fig 1). For LOUCY and DOHH-2 cell lines, As2O3 concentrations of 0·1 µmol/l were sufficient to induce apoptosis (Fig 1A), but prolonged incubation was necessary for DOHH-2 cells. After 6 d, 50% of LOUCY cells were Annexin V positive, whereas an incubation time of over 24 d was necessary to obtain the same percentage of Annexin V-positive cells for DOHH-2. The second group of cell lines (Fig 1B), consisting of CEM, ML-2, MV-4–11, KG-1a, HL-60, NC-NC, PBL985, 697 and KG-1, as well as APL-cell line NB-4, was sensitive to 1 µmol/l As2O3 (class B). However, the kinetics of apoptosis induction by 1 µmol/l As2O3 differed in this group. Time to induce apoptosis in at least 50% of cells was 4 d for CEM, 6 d for NB-4, MV-4–11 and NC-NC, 8 d for ML-2, 10 d for KG-1a, 16 d for HL-60 and 28 d for PBL985 respectively. In the 697 and KG-1 cell lines the percentage of apoptotic cells was < 50%, even after prolonged periods of incubation up to 35 d in 1 µmol/l As2O3.

Table I.  Classification of 22 leukaemia and lymphoma cell lines according to their sensitivity to As2O3.
Cell lineDescription and originClass
LOUCYHuman T-cell leukaemia, Peripheral bloodA
DOHH-2Human B-cell lymphoma, pleural effusionA
CEMAcute lymphoblastic leukaemia, peripheral bloodB
CEM/C1MDR acute lymphoblastic leukaemia, peripheral bloodB
CEM/C2MDR acute lymphoblastic leukaemia, peripheral bloodB
NB-4Human acute promyelocytic leukaemia (APL), bone marrowB
ML-2Human acute myelomonocytic leukaemia, peripheral bloodB
MV-4–11Biphenotypic B-myelomonocytic leukaemia, peripheral bloodB
NC-NCHuman B-lymphoblastoid cells, peripheral bloodB
HL-60Acute promyelocytic leukaemia, peripheral bloodB
KG-1aAcute myelogenous leukaemia, bone marrowB
PBL-985Acute myeloid leukaemia, peripheral bloodB
697Human B-cell precursor leukaemia, bone marrowB
KG-1Human bone marrow myelogenous leukaemia, bone marrowB
HELHuman erythroleukaemia, bone marrowC
HL-60/MX1MDR acute promyelocytic leukaemia, peripheral bloodC
HL-60/MX2MDR acute promyelocytic leukaemia, peripheral bloodC
JurkatHuman T-cell leukaemia, tumourC
K-562Chronic myelogenous leukaemia (CML), bone marrowC
K-562 (0·02)MDR chronic myelogenous leukaemia (CML), bone marrowC
K-562 (0·1)MDR chronic myelogenous leukaemia (CML), bone marrowC
U-937Histocytic lymphoma, pleural effusionC
image

Figure 1. Classification of leukaemia and lymphoma cell lines according to their sensitivity towards As2O3-induced apoptosis. Apoptosis was measured by flow cytometric analysis as the percentage of Annexin V-positive cells after incubation of the indicated cell lines for 35 d without (control, □) or with 0·1 µmol/l (◆), 1 µmol/l (▵) and 5 µmol/l (●) As2O3. Three different profiles for sensitivity and time course were observed (A, B and C). (A) The LOUCY and DOHH-2 cell lines were sensitive to concentrations down to 0·1 µmol/l As2O3 (◆), whereas for (B) CEM, NB-4, MV-4–11, KG-1a, HL-60 and 697 cells a concentration of 1 µmol/l As2O3 (▵) and for the cell lines (C) Jurkat and K-562 concentrations of 5 µmol/l As2O3 (●) were necessary to induce apoptosis.

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In the third group of cell lines (Jurkat, U937, HEL and K-562), higher concentrations of As2O3 (5 µmol/L) were necessary to induce apoptosis (Fig 1C) whereas 1 µmol/L As2O3 did not induce apoptosis even after incubation for 35 d (class C). Several titration experiments were repeated (data not shown). Slight interassay variability in the percentage of apoptotic cells could be observed. However, the general response pattern described above was reproducible. Measurement of apoptosis by Annexin V-FITC and 7-AAD staining in a total number of 987 samples revealed a correlation of r = 0989 (P < 0,0001).

As2O3 induces apoptosis in cell lines resistant to cytostatic agents

In order to analyse As2O3 sensitivity of cell lines resistant to cytostatic drugs, we compared cell lines CEM, HL-60 and K-562 and their resistant derivates CEM/C1, CEM/C2, HL-60/MX1, HL-60/MX2, K-562(0·02) and K-562(0·1) respectively (Fig 2).

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Figure 2. As2O3-induced apoptosis in cell lines resistant to cytostatic drugs. Induction of apoptosis by 1 µmol/l (▵) and 5 µmol/l (●) As2O3 in (A) CEM (□, control; ○, 50 nmol/l camptothecin; ▴, 100 nmol/l camptothecin; ◊, 500 nmol/l camptothecin), (B) K-562 (□, control; ○, 0·02 μg/ml doxorubicin; ▴, 0·05 μg/ml doxorubicin; ◊, 0·1 μg/ml doxorubicin) and (C) HL-60 cells (□, control; ○, 20 nmol/l mitoxantrone; ▴, 50 nmol/l mitoxantrone; ◊, 75 nmol/l mitoxantrone) was compared with apoptosis in their cytostatic resistant counterparts CEM/C1, CEM/C2, K-562(0·02), K-562(0·1), HL60/MX1 and HL-60/MX2. Cells were incubated for 20 d with the drugs indicated. The percentage of apoptotic cells was determined by flow cytometric analysis as the percentage of Annexin V-positive cells.

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All concentrations of As2O3 (1 µmol/l and 5 µmol/l) and camptothecin (50–500 nmol/l) tested induced apoptosis in more than 90% of cells in the parental cell line CEM (Fig 2A). In contrast, CEM/C1 was completely resistant to 50 nmol/l camptothecin and cell line CEM/C2 to 500 nmol/l camptothecin. The percentage of apoptotic cells after incubation with 5 µmol/l As2O3 was not reduced in camptothecin-resistant cell lines CEM/C1 and CEM/C2 compared with the parental CEM cell line. Low doses of As2O3 (1 µmol/l) were equally effective in CEM and CEM/C1; however, low doses induced a lower proportion of apoptotic cells in the high-resistant CEM/C2 cell line.

In K-562 cells and the doxorubicin-resistant cell lines K-562(0·02) and K-562(0·1) apoptosis could be induced with 5 µmol/l As2O3 (Fig 2B). A concentration of 20 ng/ml doxorubicin induced apoptosis in the K-562 cell line, whereas higher concentrations were necessary for apoptosis induction in cell lines K-562(0·02) (50 ng/ml) and K-562(0·1) (100 ng/ml). Apoptosis induction by 5 µmol/l As2O3 was not different in resistant cell lines K-562(0·02) and K-562(0·1) compared with their parental K-562 cell line.

In contrast to their parental cell line HL-60, the mitoxantrone-resistant cell lines HL-60/MX1 and HL-60/MX2 showed resistance towards 1 µmol/l As2O3 (Fig 2C). All three cell lines were sensitive to 5 µmol/l As2O3. Whereas even low concentrations (20 nmol/l) of mitoxantrone induced apoptosis in HL-60 cells, both HL-60/MX1 and HL-60/MX2 were resistant to 20 nmol/l mitoxantrone. HL-60/MX2 was resistant up to 75 nmol/l mitoxantrone, showing a slight increase in the percentage of apoptotic cells after 6 d. However, the percentage of apoptotic cells decreased and converged with the percentage of controls after 16 d of incubation.

As2O3-induced growth inhibition is independent from apoptosis induction

We were interested to analyse whether As2O3 could inhibit proliferation in non-apoptotic cells. Therefore, proliferation of those cell lines, in which 1 µmol/l As2O3 did not induce apoptosis within 10 d, was assessed by staining the cell membrane with the stable fluorescent membrane dye PKH26. We incubated PKH26-stained cell lines that showed different sensitivities to As2O3-induced apoptosis with 1 µmol/l As2O3 up to 14 d. After various incubation periods we performed Annexin V-FITC staining which allowed separate analysis of PKH26 staining (i.e. proliferative activity) in apoptotic and non-apoptotic subpopulations respectively. For these experiments, we used cell lines in which 1 µmol/l As2O3 did not induce apoptosis [HEL, K-562, K-562(0·02), K-562(0·1), HL-60/MX1, HL-60/MX2, see Fig 1B] or cell lines in which 1 µmol/l As2O3 induced apoptosis only after prolonged incubation (HL-60, 697, KG-1, see Fig 1C). Histogram plots show the reduction of fluorescence intensity in untreated 697 cells owing to dilution of PKH26 as a consequence of cell divisions (Fig 3). In contrast, decrease of fluorescence intensity was substantially reduced by incubation with 1 µmol/l As2O3 for 12 d. Counterstaining of cells with Annexin V-FITC allowed separate analysis of PKH26 fluorescence in apoptotic and non-apoptotic cells (Fig 4). The maximum percentage of Annexin V-positive cells (data not shown) was less than 10% for HEL, K-562, HL-60/MX-1 and HL-60/MX-2, about 25% for KG-1 and HL-60, < 40% for 697 and K-562(0·02), and < 42% for K-562(0·1). Mean fluorescence channel of unstained cells was subtracted from mean fluorescence channel of PKH26 stained cells. Comparison of ‘Δ mean fluorescence’ for untreated cells and As2O3-treated cells showed less decline in As2O3-treated apoptotic cells as well as non-apoptotic cells for all cell lines tested except mitoxantrone-resistant cell lines HL-60/MX1 and HL-60/MX2. The higher Δ mean fluorescence values for non-apoptotic untreated than for As2O3-treated cell lines reflected an apoptosis-independent growth reduction.

image

Figure 3. As2O3-induced growth inhibition in 697 cells. Flow cytometric analysis of the PKH26 cell proliferation assay for cell line 697. Cells were stained with the stable membrane dye PKH26. After incubation for various periods with 1 µmol/l As2O3, cells were analysed for PKH26 staining as a marker for cell proliferation.

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image

Figure 4. As2O3-induced growth inhibition is independent from apoptosis induction. Flow cytometric analysis of the PKH26 cell proliferation assay for cell lines HEL, 697, KG-1, Jurkat, PBL-985, U937, K-562, K-562(0·02), K-562(0·1), HL-60, HL-60/MX1 and HL-60/MX2. Cells were stained using the stable membrane dye PKH26. After incubation for various periods with 1 µmol/l As2O3, cells were stained with Annexin V-fluorescein isothiocyanate (FITC). Simultaneous measurement of PKH26 staining as a marker for cell proliferation and Annexin V-FITC as a marker for apoptotic cells was performed. The decline in ‘Δ mean fluorescence channel’ of PKH26 staining for apoptotic (▮) and non-apoptotic (□) populations of cells after incubation without As2O3 or for apoptotic (●) and non-apoptotic (○) populations incubated with 1 µmol/l As2O3 is shown. The value for mean fluorescence channel of control experiments with unstained cells was substracted from the value for fluorescence of PKH26-stained cells to obtain Δ mean fluorescence channel.

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As2O3 does not cause cell cycle arrest

In order to assess whether the treatment with 1 µmol/l As2O3 of the malignant cell lines HL-60, HL-60/MX2, Jurkat, K-562, K-562(0·1) and U937 leads to a cell cycle arrest, PI-staining was performed every second day. A concentration of 1 µmol/l As2O3 was used because it induced apoptosis only in a small proportion of cells of the cell lines used for these experiments after incubation up to 14 d (see Figs 1 and 2). As shown in Fig 5 for the HL-60 and K-562 cell lines, incubation with 1 µmol/l As2O3 had no effect on cell cycle after 14 d. No change in the relation of cells in G0/G1 and G2/M phase could be detected compared with untreated cells. All other cell lines tested [data not shown for HL-60/MX2, Jurkat, K-562(0·1) and U937] showed a similar pattern. Expression of the proliferation marker Ki67 was analysed for the cell lines 697, Jurkat, KG-1a, HL-60, HL-60/MX-1, HL-60/MX2, K-562, K-562(0·02) and K-562(0·1). No significant change (P > 0·05, Mann–Whitney U-test) in mean expression of Ki67 was observed over a period of 14 d incubation with 1 µmol/l As2O3 compared with untreated cells (data not shown).

image

Figure 5. As2O3 does not cause cell cycle arrest. Flow cytometric analysis of the propidium iodide (PI) cell cycle assay for cell lines HL-60 and K-562. After incubation for 14 d with 1 µmol/l As2O3, no change of relative proportion of cells in G0/G1 and G2/M phase could be observed compared with untreated cells for both HL-60 and K-562.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

As2O3 is being increasingly used as a successful treatment of APL (Chen, Z. et al, 2001). At first it was thought that the effects of As2O3, e.g. reduction of cell division rates and induction of apoptosis, were specific for APL cell lines. In APL, the t(15;17) translocation results in a fusion of PML and RARα. It has recently been shown that As2O3 triggers the degradation of both PML-RARα and wild-type PML (Muller et al, 1998a,b; Sternsdorf et al, 1999). The ubiquitin-like protein SUMO-1 can covalently bind to the PML and PML-RARα. This post-transcriptional modification of PML leads to a delocalization of the protein from nucleus into compartments, in which the protein is degraded. As2O3 modulates the SUMO-1 binding to PML and PML-RARα. Several reports showed evidence that the activation of caspases (Akao et al, 1999; Huang, X.J. et al, 1999; Kitamura et al, 2000; Jiang et al, 2001), modulation of p53 (Jiang et al, 2001) and uncoupling of the mitochondrial potential (Dai et al, 1999; Jing et al, 1999; Kroemer & de The, 1999; Larochette et al, 1999; Cai et al, 2000) are involved in As2O3-induced apoptosis.

These cellular effects suggest that As2O3 may induce apoptosis not only in APL cell lines. Therefore, we performed a systematic analysis of the effect of As2O3 in a great variety of non-APL cell lines representing a broad range of lympho-haemopoietic differentiation. In our report, the analysis of 22 different leukaemia and lymphoma cell lines clearly shows that induction of apoptosis as well as reduction of proliferation rate are not restricted to APL cell lines. We were able to classify 22 leukaemia and lymphoma cell lines into three groups according to their sensitivity to As2O3-induced apoptosis. All cell lines responded to As2O3 at concentrations that were shown to be achievable by administration of well-tolerated doses of As2O3 in clinical studies (Shen et al, 1997; Ni et al, 1998). As2O3 at a concentration of 0·1 µmol/l was sufficient to induce apoptosis in group A cell lines; group B cell lines became apoptotic with As2O3 concentrations ≥ 1 µmol/l. An As2O3 concentration of 5 µmol/l was necessary to induce apoptosis in group C cell lines.

The findings of apoptosis-inducing effects of As2O3 in non-APL cells are in agreement with several reports about apoptotic effects of As2O3 even in non-haemopoietic cells such as the cervical cancer cell line SiHa (Deng et al, 2000), neuroblastoma cell lines (Akao et al, 1999), head neck cancer cell line (Seol et al, 1999), gastric cell line MGC-803 (Zhang et al, 1999), hepatocarcinoma cell lines (Liu et al, 2000), prostate and ovary cancer cell lines (Uslu et al, 2000).

The incubation time necessary to induce apoptosis shows broad variation. Notably, some cell lines (e.g. HL-60, K-562, HEL, Jurkat) became apoptotic only after prolonged incubation. This should be considered when designing schedules for treatment of leukaemia or lymphomas with As2O3. In clinical trials of As2O3 in refractory APL, the median time to remission during As2O3 therapy ranged from 31 d (Niu et al, 1999) to 47 d (Soignet et al, 1998).

Patients refractory to conventional acute myeloid leukaemia (AML) treatment might be the first group considered for clinical trials of As2O3 in non-APL leukaemias. In this context, it is important to exclude the possibility that resistance to, for example, anthracyclines or topoisomerase inhibitors is also associated with resistance to As2O3. Therefore, we studied As2O3 sensitivity in cell lines resistant to camptothecin, doxorubicin and mitoxantrone.

Apoptosis could be induced in four out of six cell lines resistant to conventional cytostatic drugs in the same range as in their parental cytostatic-sensitive cell lines. Only in the case of mitoxantrone-resistant HL-60/MX1 and HL-60/MX2 cell lines was the parental cell line HL-60 more sensitive to As2O3-induced apoptosis. Thus, our in vitro results suggest that As2O3 might be effective in AML refractory to conventional cytostatic drugs.

Apart from the apoptosis-inducing effect, As2O3 seems to inhibit proliferation of all leukaemia and lymphoma cell lines tested. Cell lines such as 697, Jurkat, HL-60, U937, PBL-987, K-562, KG-1 and HEL that were less sensitive or insensitive to 1 µmol/l As2O3 showed a clear reduction of proliferation rate after 10 d at 1 µmol/l As2O3. Growth inhibition could even be observed in all derivates of K562, HL-60 which are resistant to different cytostatic drugs. As demonstrated by DNA histograms, As2O3 at concentrations of 1 µmol/l did not lead to cell cycle arrest. A potential action of As2O3 that could explain the effects on proliferation could be the inhibition of aggregation of tubulin (Li & Broome, 1999). Our cell cycle studies are in accordance with recently published data reporting that arsenic(III)-oxide in concentrations lower than 2 µmol/l does not induce G2/M growth arrest in U937 cells (Park et al, 2001).

In a murine model, the antitumour effect of As2O3 on experimental liver cancer was reported. As2O3 showed an obvious antitumour effect, caused by apoptosis induction with reduction of Bcl-2 expressing cells in the tumour mass (Chen et al, 2000). The in vivo and in vitro effects of As2O3 on a neuroblastoma mouse model were described (Ora et al, 2000). As2O3 inhibited the tumour growth and induced apoptosis.

Promising results of the clinical trials in APL, our in vitro results on As2O3-mediated apoptosis and cell proliferation, and the promising results from mouse models suggest the use of As2O3 also for refractory non-APL leukaemia. In one patient with secondary acute leukaemia we have induced partial remission by using As2O3 (unpublished observations). Thus, As2O3 might be a suitable therapeutic agent for myeloid malignancies other than APL and non-myeloid malignancies, provided an adequate dose and duration of As2O3 treatment are used. We plan a pilot study for patients with refractory AML who are not eligible for allogeneic stem cell transplantation.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  • Akao, Y., Nakagawa, Y. & Akiyama, K. (1999) Arsenic trioxide induces apoptosis in neuroblastoma cell lines through the activation of caspase 3 in vitro. FEBS Letters, 455, 5962.DOI: 10.1016/S0014-5793(99)00841-8
  • Cai, X., Shen, Y.L., Zhu, Q., Jia, P.M., YuY., Zhou, L., Huang, Y., Zhang, J.W., Xiong, S.M., Chen, S.J., Wang, Z.Y., Chen, Z. & Chen, G.Q. (2000) Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia. Leukemia, 14, 262270.
  • Chen, F., Lu, Y., Zhang, Z., Vallyathan, V., Ding, M., Castranova, V. & Shi, X. (2001) Opposite effect of NF-kB and c-Jun-N-terminal kinase on p53-independent GADD45 induction by arsenite. Journal of Biological Chemistry , 276, 1141411419.
  • Chen, G.Q., Zhu, J., Shi, X.G., Ni, J.H., Zhong, H.J., Si, G.Y., Jin, X.L., Tang, W., Li, X.S., Xong, S.M., Shen, Z.X., Sun, G.L., Ma, J., Zhang, P., Zhang, T.D., Gazin, C., Naoe, T., Chen, S.J., Wang, Z.Y. & Chen, Z. (1996) In vitro studies on cellular and molecular mechanisms of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia: As2O3 induces NB4 cell apoptosis with downregulation of Bcl-2 expression and modulation of PML-RAR alpha/PML proteins. Blood, 88, 10521061.
  • Chen, H., Qin, S. & Pan, Q. (2000) Antitumor effect of arsenic trioxide on mice experimental liver cancer. Chung-Hua Kan Tsang Ping Tsa Chih, 8, 2729.
  • Chen, Z., Wang, Z.Y. & Chen, S.J. (1997) Acute promyelocytic leukemia: cellular and molecular basis of differentiation and apoptosis. Pharmacology and Therapeutics, 76, 141149.
  • Chen, Z., Chen, G.Q., Shen, Z.X., Chen, S.J. & Wang, Z.Y. (2001) Treatment of acute promyelocytic leukemia with arsenic compounds: in vitro and in vivo studies. Seminars in Hematology, 38, 2636.
  • Dai, J., Weinberg, R.S., Waxman, S. & Jing, Y. (1999) Malignant cells can be sensitized to undergo growth inhibition and apoptosis by arsenic trioxide through modulation of the glutathione redox system. Blood, 93, 268277.
  • Deng, Y., Lin, C., Zheng, J., Fu, M., Liang, X., Chen, J., Xiao, P. & Wu, M. (2000) Overexpression of Bcl-2 partly inhibits apoptosis of human cervical cancer SiHa cells induced by arsenic trioxide. Chinese Medical Journal, 113, 8488.
  • Eguchi, N., Kuroda, K. & Endo, G. (1997) Metabolites of arsenic induced tetraploids and mitotic arrest in cultured cells. Archives of Environmental Contamination and Toxicology, 32, 141145.
  • Huang, C., Ma, W.Y., Li, J. & Dong, Z. (1999) Arsenic induces apoptosis through a c-Jun NH2-terminal kinase-dependent, p53-independent pathway. Cancer Research, 59, 30533058.
  • Huang, X.J., Wiernik, P.H., Klein, R.S. & Gallagher, R.E. (1999) Arsenic trioxide induces apoptosis of myeloid leukemia cells by activation of caspases. Medical Oncology, 16, 5864.
  • Jiang, X.H., Chun, Yu, Wong, B., Yuen, S.T., Jiang, S.H., Cho, C.H., Lai, K.C., Lin, M.C., Kung, H.F. & Lam, S.K. (2001) Arsenic trioxide induces apoptosis in human gastric cancer cells through up-regulation of p53 and activation of caspase-3. International Journal of Cancer, 91, 173179.
  • Jing, Y., Dai, J., Chalmers-Redman, R.M., Tatton, W.G. & Waxman, S. (1999) Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Blood, 94, 21022111.
  • Jiong, H., Zhixiang, S., Wen, W., Xiusong, L., Guanlin, S. & Zhenyi, W. (2000) Long-term survey of outcome in acute promyelocytic leukemia. Chinese Medical Journal, 113, 107110.
  • Kitamura, K., Kiyoi, H., Yoshida, H., Tobita, T., Takeshita, A., Ohno, R. & Naoe, T. (1997) New retinoids and arsenic compounds for the treatment of refractory acute promyelocytic leukemia: clinical and basic studies for the next generation. Cancer Chemotherapy and Pharmacology, 40, S36S41.DOI: 10.1007/s002800051059
  • Kitamura, K., Minami, Y., Yamamoto, K., Akao, Y., Kiyoi, H., Saito, H. & Naoe, T. (2000) Involvement of CD95-independent caspase 8 activation in arsenic trioxide-induced apoptosis. Leukemia, 14, 17431750.
  • Konig, A., Wrazel, L., Warrell, Jr, R.P., Rivi, R., Pandolfi, P.P., Jakubowski, A. & Gabrilove, J.L. (1997) Comparative activity of melarsoprol and arsenic trioxide in chronic B-cell leukemia lines. Blood, 90, 562570.
  • Kroemer, G. & De The, H. (1999) Arsenic trioxide, a novel mitochondriotoxic anticancer agent? Journal of the National Cancer Institute, 91, 743745.DOI: 10.1093/jnci/91.9.743
  • Larochette, N., Decaudin, D., Jacotot, E., Brenner, C., Marzo, I., Susin, S.A., Zamzami, N., Xie, Z., Reed, J. & Kroemer, G. (1999) Arsenite induces apoptosis via a direct effect on the mitochondrial permeability transition pore. Experimental Cell Research, 249, 413421.DOI: 10.1006/excr.1999.4519
  • Lee, S.H., Choi, J.G. & Cho, M.H. (2001) Apoptosis, bcl2 expression, and cell cycle analyses in nickel (II)-treated normal rat kidney cells. Journal of Korean Medical Science, 16, 165168.
  • Li, Y.M. & Broome, J.D. (1999) Arsenic targets tubulins to induce apoptosis in myeloid leukemia cells. Cancer Research, 59, 776780.
  • Liu, L., Qin, S., Chen, H., Wang, J., Chen, H., Ma, J. & Liu, W. (2000) An experimental study on arsenic trioxide-selectively induced human hepatocarcinoma cell lines apoptosis and its related genes. Chung-Hua Kan Tsang Ping Tsa Chih, 8, 367369.
  • Lu, M., Levin, J., Sulpice, E., Sequeira-Le Grand, A., Alemany, M., Caen, J.P. & Han, Z.C. (1999) Effect of arsenic trioxide on viability, proliferation, and apoptosis in human megakaryocytic leukemia cell lines. Experimental Hematology, 27, 845852.
  • Muller, S., Matunis, M.J. & Dejean, A. (1998a) Conjugation with the ubiquitin-related modifier SUMO-1 regulates the partitioning of PML within the nucleus. EMBO Journal, 17, 6170.
  • Muller, S., Miller, Jr, W.H. & Dejean, A. (1998b) Trivalent antimonials induce degradation of the PML-RAR oncoprotein and reorganization of the promyelocytic leukemia nuclear bodies in acute promyelocytic leukemia NB4 cells. Blood, 92, 43084316.
  • Ni, J., Chen, G., Shen, Z., Li, X., Liu, H., Huang, Y., Fang, Z., Chen, S., Wang, Z. & Chen, Z. (1998) Pharmacokinetics of intravenous arsenic trioxide in treatment of acute promyelocytic leukemia. Chinese Medical Journal, 111, 11071110.
  • Niu, C., Yan, H., Yu, T., Sun, H.P., Liu, J.X., Li, X.S., Wu, W., Zhang, F.Q., Chen, Y., Zhou, L., Li, J.M., Zeng, X.Y., Yang, R.R., Yuan, M.M., Ren, M.Y., Gu, F.Y., Cao, Q., Gu, B.W., Su, X.Y., Chen, G.Q., Xiong, S.M., Zhang, T., Waxman, S., Wang, Z.Y. & Chen, S.J. (1999) Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients. Blood, 94, 33153324.
  • Ora, I., Bondesson, L., Jonsson, C., Ljungberg, J., Porn-Ares, I., Garwicz, S. & Pahlman, S. (2000) Arsenic trioxide inhibits neuroblastoma growth in vivo and promotes apoptotic cell death in vitro. Biochemical and Biophysical Research Communications, 277, 179185.
  • Park, J.W., Choi, Y.J., Jang, M.A., Baek, S.H., Lim, J.H., Passaniti, T. & Kwon, T.K. (2001) Arsenic trioxide induces g2/m growth arrest and apoptosis after caspase-3 activation and bcl-2 phosphorylation in promonocytic u937 cells. Biochemical and Biophysical Research Communications, 286, 726734.
  • Park, W.H., Seol, J.G., Kim, E.S., Hyun, J.M., Jung, C.W., Lee, C.C., Kim, B.K. & Lee, Y.Y. (2000) Arsenic trioxide-mediated growth inhibition in MC/CAR myeloma cells via cell cycle arrest in association with induction of cyclin-dependent kinase inhibitor, p21, and apoptosis. Cancer Research, 60, 30653071.
  • Perkins, C., Kim, C.N., Fang, G. & Bhalla, K.N. (2000) Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-x (L). Blood, 95, 10141022.
  • Philpott, N.J., Turner, A.J., Scopes, J., Westby, M., Marsh, J.C., Gordon-Smith, E.C., Dalgleish, A.G. & Gibson, F.M. (1996) The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques. Blood, 87, 22442251.
  • Puccetti, E., Guller, S., Orleth, A., Bruggenolte, N., Hoelzer, D., Ottmann, O.G. & Ruthardt, M. (2000) BCR-ABL mediates arsenic trioxide-induced apoptosis independently of its aberrant kinase activity. Cancer Research, 60, 34093413.
  • Seol, J.G., Park, W.H., Kim, E.S., Jung, C.W., Hyun, J.M., Kim, B.K. & Lee, Y.Y. (1999) Effect of arsenic trioxide on cell cycle arrest in head and neck cancer cell line PCI-1. Biochemical and Biophysical Research Communications, 265, 400404.
  • Shen, Z.X., Chen, G.Q., Ni, J.H., Li, X.S., Xiong, S.M., Qiu, Q.Y., Zhu, J., Tang, W., Sun, G.L., Yang, K.Q., Chen, Y., Zhou, L., Fang, Z.W., Wang, Y.T., Ma, J., Zhang, P., Zhang, T.D., Chen, S.J., Chen, Z. & Wang, Z.Y. (1997) Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL). II. Clinical efficacy and pharmacokinetics in relapsed patients. Blood, 89, 33543360.
  • Soignet, S.L., Maslak, P., Wang, Z.G., Jhanwar, S., Calleja, E., Dardashti, L.J., Corso, D., DeBlasio, A., Gabrilove, J., Scheinberg, D.A., Pandolfi, P.P. & Warrell, Jr, R.P. (1998) Complete remission after treatment of acute promyelocytic leukemia with arsenic trioxide. New England Journal of Medicine, 339, 13411348.
  • Spencer, A. & Firkin, F. (1999) Arsenic trioxide treatment of relapsed acute promyelocytic leukaemia: initial Australian experience. Australian and New Zealand Journal of Medicine, 29, 385386.
  • Sternsdorf, T., Puccetti, E., Jensen, K., Hoelzer, D., Will, H., Ottmann, O.G. & Ruthardt, M. (1999) PIC-1/SUMO-1-modified PML-retinoic acid receptor alpha mediates arsenic trioxide- induced apoptosis in acute promyelocytic leukemia. Molecular and Cellular Biology, 19, 51705178.
  • Uslu, R., Sanli, U.A., Sezgin, C., Karabulut, B., Terzioglu, E., Omay, S.B. & Goker, E. (2000) Arsenic trioxide-mediated cytotoxicity and apoptosis in prostate and ovarian carcinoma cell lines. Clinical Cancer Research, 6, 49574964.
  • Walter, R., Schoedon, G., Bachli, E., Betts, D.R., Hossle, J.P., Calandra, T., Joller-Jemelka, H.I., Fehr, J. & Schaffner, A. (2000) Establishment and characterization of an arsenic-sensitive monoblastic leukaemia cell line (SigM5). British Journal of Haematology, 109, 396404.DOI: 10.1046/j.1365-2141.2000.02013.x
  • Wang, Z.G., Rivi, R., Delva, L., Konig, A., Scheinberg, D.A., Gambacorti-Passerini, C., Gabrilove, J.L., Warrell, Jr, R.P. & Pandolfi, P.P. (1998) Arsenic trioxide and melarsoprol induce programmed cell death in myeloid leukemia cell lines and function in a PML and PML-RARalpha independent manner. Blood, 92, 14971504.
  • Warrell, Jr, R.P., Soignet, S.L., Maslak, P., Scheinberg, D.A., Gabrilove, J., Calleja, E., Chen, Y.-W. & Pandolfi, P.P. (1998) Initial Western study of arsenic trioxide in acute promyelocytic leukemia. Journal of Clinical Oncology, 17, 19.
  • Zhang, T.C., Cao, E.H., Li, J.F., Ma, W. & Qin, J.F. (1999) Induction of apoptosis and inhibition of human gastric cancer MGC-803 cell growth by arsenic trioxide. European Journal of Cancer, 35, 12581263.
  • Zhu, X.H., Shen, Y.L., Jing, Y.K., Cai, X., Jia, P.M., Huang, Y., Tang, W., Shi, G.Y., Sun, Y.P., Dai, J., Wang, Z.Y., Chen, S.J., Zhang, T.D., Waxman, S., Chen, Z. & Chen, G.Q. (1999) Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations. Journal of the National Cancer Institute, 91, 772778.