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NEOPLASIA
From the Division of Hematology and Oncology,
Weill Medical College of Cornell University, New York, NY; Memorial
Sloan Kettering Cancer Center, New York, NY.
Arsenic trioxide (As2O3) has recently
been used successfully in the treatment of acute promyelocytic leukemia
and has been shown to induce partial differentiation and apoptosis of
leukemic cells in vitro. However, the mechanism by which
As2O3 exerts its antileukemic effect remains
uncertain. Emerging data suggest that the endothelium and angiogenesis
play a seminal role in the proliferation of liquid tumors, such as
leukemia. We have shown that activated endothelial cells release
cytokines that may stimulate leukemic cell growth. Leukemic cells, in
turn, can release endothelial growth factors, such as vascular
endothelial growth factor (VEGF). On the basis of these observations,
we hypothesized that As2O3 may interrupt a
reciprocal loop between leukemic cells and the endothelium by direct
action on both cell types. We have shown that treatment of
proliferating layers of human umbilical vein endothelial cells (HUVECs)
with a variety of concentrations of As2O3
results in a reproducible dose- and time-dependent sequence of events
marked by change to an activated morphology, up-regulation of
endothelial cell adhesion markers, and apoptosis. Also, treatment with
As2O3 caused inhibition of VEGF
production in the leukemic cell line HEL. Finally, incubation of
HUVECs with As2O3 prevented capillary
tubule and branch formation in an in vitro endothelial cell-differentiation assay. In conclusion, we believe that
As2O3 interrupts a reciprocal stimulatory loop
between leukemic cells and endothelial cells by causing apoptosis of
both cell types and by inhibiting leukemic cell VEGF production.
(Blood. 2000;96:1525-1530) Arsenic has been used for centuries to treat a wide
variety of illnesses. Most recently, arsenic trioxide
(As2O3) has been used successfully in the
treatment of patients with acute promyelocytic leukemia (APL) who
relapsed after initial therapy with chemotherapy and all-trans retinoic
acid.1-6 As2O3 has been shown to
induce dose- and time-dependent apoptosis in vitro in a variety of
malignant myeloid,7-13 lymphoid,14-18 and
megakaryocytic19 cells. Recent investigations have also
found that treatment with As2O3 induces apoptosis in plasma cell lines and primary myeloma
cells,20 esophageal carcinoma cells,21 and
immortalized human cervical epithelial cells.22 In vivo,
arsenic appears to induce apoptosis of APL cells without significant
myelosuppression or serious systemic toxicity.2,3
The mechanism by which arsenic exerts its antileukemic effect remains
uncertain. In APL cells, arsenic targets PML onto nuclear bodies and induces degradation of the PML/RAR- Emerging evidence suggests that the endothelium and angiogenesis may be
critical for the proliferation of liquid tumors, such as
leukemia.26-28 It is well established that angiogenesis,
the formation of new capillaries from preexisting blood vessels, plays a critical role in the growth of solid tumors by facilitating delivery
of nutrients to and removal of waste products from the tumor
bed.29 Tumor growth also requires expansion of existing endothelium; an increase in tumor mass requires an equivalent increase
in endothelial cell mass. The ultimate endothelial mass is dependent
upon a delicate balance between proliferative and apoptotic signals
within the tumor microenvironment. A similar system may be required for
the development of liquid tumors. Our laboratory and others have shown
that activated endothelial cells can release a variety of cytokines
that may stimulate leukemic cell growth.30,31 Leukemic
cells, in turn, have the capacity to release endothelial growth
factors, such as vascular endothelial growth factor (VEGF), and may
also express growth factor receptors on their surface, such as VEGF
receptor-2 (VEGFR-2).31,32 Therefore, leukemic blasts may
be able to support their own development both by promoting
neoangiogenesis and via an autocrine loop.31,32
On the basis of these observations, we speculated that arsenic may
interrupt a reciprocal loop between leukemic cells and the endothelium
by direct action on both cell types. We have found that
As2O3 causes dose- and time-dependent
activation and apoptosis of human endothelial cells, but not
fibroblasts, in vitro. Also, As2O3 inhibits
VEGF-induced capillary tubule formation and decreases leukemic cell
VEGF production. These data suggest that As2O3
may exert its antileukemic effect in part through inhibition of angiogenesis.
Reagents
Cell culture
Flow cytometry To characterize the activated endothelial cells, the following fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies were used: intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), E-selectin, and immunoglobulin (Ig)G isotype control (Immunotech, Marcella, France). For flow cytometry, 100 000 cells were incubated with the respective antibodies, fixed with 1% formalin, washed, and analyzed by means of a Coulter Elite flow cytometer.Annexin-V staining Cells were incubated with FITC-conjugated Annexin-V according to the recommendations of the manufacturer (Immunotech). Nuclei were counterstained with propidium iodide (PI) and screened by flow cytometry (approximately 10 000 events). Early apoptosis was estimated by the relative amount of FITC+PI cell
populations. FITC+PI+ cells represented either
secondary necrotic or postapoptotic populations.
Protein extraction and Western blotting Total cell extracts from HUVECs were obtained by lysing the cells in cold RIPA buffer (50 mmol/L Tris, 5 mmol/L EDTA, 1% Triton X-114, 0.4% sodium cacodylate, and 150 mmol/L NaCl) in the presence of protease inhibitors (1 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mmol/L -glycerophosphate, and 1 mmol/L phenylmethylsulfonyl fluoride). After centrifugation to remove cell debris,
supernatants were subjected to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (using 10% acrylamide gels) under reducing
conditions (in the presence of 250 mmol/L -mercaptoethanol).
Proteins were subsequently blotted onto a nitrocellulose membrane
(Schleicher and Schuell, Keene, NH) following conventional protocols.
The membranes were stained with 0.2% ponceau S red to assure equal
protein loading. Finally, blots were blocked in 1% bovine serum
albumin/PBS with 1% Tween-20 for 1 hour at room temperature
and incubated with primary and secondary antibodies. Monoclonal
antibodies against human Bcl-2 (Calbiochem, Cambridge, MA) and Bax
(Santa Cruz Biotechnology, Santa Cruz, CA) were used at
dilutions of 1:500 and 1:1000, respectively. The ECL chemiluminescence
(Amersham Pharmacia Biotech, Piscataway, NJ) detection system and ECL
film (Amersham Pharmacia Biotech) were used to visualize the presence
of specific proteins in the nitrocellulose blots.
TUNEL assay HUVECs were grown to 70% confluence on poly-D-lysine-coated coverslips and incubated for 24 hours with varying concentrations of As2O3. The TUNEL (terminal deoxynucleotidyl transferase--mediated deoxyuridine triphosphate nick end labeling) assay was performed with the use of the In Situ Cell Death Detection Kit, AP (Boehringer Mannheim, Mannheim, Germany), according to the manufacturer's instructions. Fast red tablets (Boehringer Mannheim) were used as the chromogenic substrate.Matrigel-induced capillary tube formation The Matrigel assay has been widely used as an in vitro measurement of endothelial cell differentiation and was performed as described previously.34,35 Briefly, 24-well plates (Costar, Cambridge, MA) were coated with 300 µL/well growth-factor-reduced Matrigel (Becton Dickinson, San Jose, CA) and allowed to stand for 30 minutes at 37°C to form a gel layer. After gel formation, HUVECs (1 × 105 cells) in a medium containing X-Vivo (Bio-Whittaker) and endothelial cell growth supplement were applied to each well in the absence or presence of VEGF165 20 ng/mL and various concentrations of As2O3. Each treatment was performed in triplicate wells. The plates were incubated at 37°C for 48 hours with 5% CO2 and photographed with the use of inverted phase-contrast microscopy (Nikon, Tokyo, Japan).Enzyme-linked immunosorbent assay for VEGF The VEGF concentration in samples of conditioned medium was measured by means of a human VEGF immunoassay that recognizes the soluble isoforms VEGF121 and VEGF165 (R&D Systems). For cell culture supernatants, a sensitivity of 5 pg/mL could be achieved.Statistical analysis All experiments were performed in triplicate unless otherwise noted; results are expressed as mean ± standard deviation. Comparison of means was performed by means of the analysis of variance procedure (Dunnett's t test, SAS for Windows version 6.12, SAS Institute, Cary, NC).
Effect of As2O3 on viability and proliferation of HUVECs To investigate the effects of As2O3 on the survival and proliferation of HUVECs, 70% to 80% confluent monolayers of rapidly proliferating cells were grown for 1 to 6 days in the presence of endothelial cell growth supplement, 20% serum, FGF-2 (5 ng/mL), VEGF (5 ng/mL), and various concentrations of As2O3, ranging from 2.5 × 107
mol/L to 5 × 105 mol/L. Each condition was performed
and counted in triplicate. As shown in Figure
1, As2O3
significantly decreased cell viability and proliferation at all
concentrations tested in comparison with control cells. The curves were
reproducible using several different cell passages, from number 1 to 3 (data not shown). At the highest concentrations tested
(5 × 105 mol/L and 5 × 106 mol/L),
As2O3 superseded the effects of the endothelial
growth factors and prevented cell proliferation, causing a rapid
decline in the number of viable cells; after 6 days, no viable cells
remained in either condition. At intermediate doses of
As2O3 (2.5 × 106 mol/L and
1 × 106 mol/L), cell proliferation was completely
inhibited after 48 hours exposure, and the number of viable cells
steadily decreased to approximately 30% of control cells by day 6. At
low concentrations of As2O3
(5 × 107 mol/L and 2.5 × 107 mol/L),
cell proliferation was initially increased compared with controls, but
subsequently declined, leaving approximately 50% fewer viable cells
than controls at day 6.
HUVECs appeared not to develop resistance to the effects of arsenic:
more than 75% of cells treated with low or intermediate concentrations
of As2O3 for 2 to 3 days were able to recover
and proliferate if exposure to arsenic was discontinued, and they were
subsequently susceptible to its effects upon retreatment (data not
shown). Even at low concentrations, all cells eventually died after
continuous exposure. In contrast, human foreskin fibroblasts and MRC-5
embryonic fibroblasts were minimally affected even by high
concentrations of As2O3; after longer than 12 days of continuous exposure to 5 × 10 Treatment of HUVECs with As2O3 increases apoptosis in a dose- and time-dependent manner To show whether the growth inhibition on endothelial cells by As2O3 was caused by induction of apoptosis, rapidly proliferating, early passage HUVECs were incubated with different concentrations of As2O3. All conditions were performed in triplicate. The cells were dual-stained with Annexin-V-FITC and PI and analyzed by flow cytometry. After 72 hours, there was a dose-dependent increase in early and late apoptotic cells (Figure 2). At the highest concentration tested (5 × 106 mol/L), nearly 40% of
the cells showed signs of early apoptosis, and approximately 80% of
the population was undergoing either early or late apoptosis after 72 hours, compared with 12% of the control cells (growth-factor treated),
18% of the 5 × 107 mol/L, and 34% of the
2.5 × 106 mol/L groups.
To further characterize the apoptosis observed in arsenic-treated
HUVECs, we assessed Bcl-2 and Bax expression using Western blotting
(Figure 3) and DNA strand breaks using
the TUNEL assay (Figure 4). HUVECs
constitutively express Bcl-2, even under serum-free conditions. As
expected, Bcl-2, an antiapoptotic signal, is increased after treatment
with VEGF, the most potent growth and survival factor for endothelium.
Bcl-2 expression decreased in a dose-dependent manner after treatment
with As2O3. Bax expression was not affected by
treatment with As2O3 as compared with control
cells, but the Bcl-2/Bax ratio decreased after treatment, thus
correlating with the observations of increased Annexin-V staining and
apoptosis. TUNEL staining of HUVECs was markedly increased after only
24 hours of treatment with 5 × 10
Treatment of HUVECs with As2O3 results in endothelial cell activation and increased binding of leukemic cells Prior to undergoing apoptosis, HUVECs treated with endotoxin-free As2O3 were observed to attain the spindle-shaped morphology of activated endothelium, as opposed to the characteristic cobblestone appearance of resting HUVECs grown in tissue culture (Figure 5). FACS analysis was performed to determine whether this activated morphology corresponded with up-regulation of 3 molecular markers of endothelial cell activation: E-selectin, ICAM, and VCAM. There was significantly increased expression of all 3 markers after 5 days' incubation with 5 × 107 mol/L As2O3 (Figure
5). To investigate whether leukemic cells would demonstrate increased
binding to arsenic-activated endothelial cells, 1 × 105
HEL cells were added to HUVECs that had become spindle-shaped in the
presence of 2 different concentrations of
As2O3; HUVECs activated with interleukin
(IL)-1 were used as a positive control. All conditions were
performed in triplicate. The coculture system was allowed to incubate
for 1 hour, and the adherent population was quantitated with the use of
phase-contrast microscopy. Treatment with As2O3
or IL-1 resulted in an approximately 15-fold increase in the binding
of HEL cells (Figure 6).
Effect of As2O3 on leukemic cell VEGF production We investigated the effects of As2O3 treatment on VEGF production by 2 different leukemia cell lines: HL60, which is refractory to the effects of As2O3,19,24 and HEL, which undergoes dose- and time-dependent apoptosis upon exposure to As2O3.19 In 2 independent experiments, treatment of HEL cells with 5 × 106 mol/L
As2O3 for 48 hours resulted in a 34% reduction
in leukemic cell VEGF (Figure 7). In
contrast, VEGF production by HL60 cells was unaffected under the same
treatment conditions (data not shown).
Effect of As2O3 on endothelial cell differentiation in vitro To observe the effects of As2O3 on tubule formation in vitro, HUVECs were plated onto growth-factor-reduced Matrigel in the presence and absence of additional VEGF165 and varying concentrations of As2O3. All conditions were performed in triplicate. The HUVECs adhered to the Matrigel surface within 18 to 24 hours and formed a branching, anastomosing network of capillarylike tubules with multicentric junctions over 24 to 48 hours, consistent with published observations.34 The largest number of tubules was produced with the addition of exogenous VEGF165 (Figure 8). As2O3 inhibited VEGF-induced tubule and branch formation in a dose-dependent manner, as shown in Figure 8.
Emerging data suggest that the proliferation of both solid and
liquid tumors may be dependent upon angiogenesis. In the present work,
we sought to investigate the potential antiangiogenic activity of
As2O3. Treatment of HUVECs with
As2O3 at a variety of different concentrations
results in a reproducible sequence of events marked by a change to an
activated morphology, up-regulation of endothelial cell adhesion
markers, and apoptosis, as measured by increased Annexin-V staining and
a decreased Bcl-2-to-Bax ratio. This sequence may be unique to
vascular endothelial cells, as As2O3 did not similarly affect fibroblasts treated with identical concentrations. The
activation of HUVECs was not caused by endotoxin, as both the
endothelial cell medium and the arsenic preparations were free of
endotoxin. Also, treatment with As2O3 induced
up-regulation of adhesion molecules 48 hours after exposure, whereas
endotoxin-mediated up-regulation of adhesion molecules should take
place within a few hours. It is possible that
As2O3-induced apoptosis of endothelial cells
results in the release of inflammatory cytokines, such as IL-1 Our data show that As2O3 induces dose- and time-dependent apoptosis of HUVECs. There is ample evidence that As2O3 probably does not simply cause nonspecific cytotoxicity. The drug has few immediate toxicities at clinically effective doses, suggesting that it does not induce generalized apoptosis or necrosis. In vitro, it causes minimal apoptosis of fibroblasts and is not active against a number of malignant cell lines, even at high concentrations. Pharmacokinetic analyses of clinical samples have shown peak plasma arsenic concentrations of 5.5 to 7.3 µmol/L per liter, and the steady state is believed to be between 1 and 2 µmol/L.4 The concentrations of As2O3 achieved in bone marrow may be significantly higher than those in plasma, as accumulation of As2O3 is greatest in tissues rich in sulfhydryl-group-containing proteins, such as bone marrow.4 Arsenic is also known to bind hemoglobin and can be distributed rapidly, especially to highly vascular organs. These properties, in combination with the observations described in this work, suggest that As2O3 may exert its antileukemic effect through direct action against both leukemic cells and the endothelium. Perez-Atayde et al26 have noted increased
blood vessel density in the bone marrow of children with acute
lymphoblastic leukemia. Data from our laboratory also show increased
vascularity in bone marrow biopsy specimens from adult patients with
different types of acute leukemia (manuscript in preparation). It is
known that impaired production or expression of VEGF will disrupt
angiogenesis.37 We believe that
As2O3 interrupts a reciprocal stimulatory loop between leukemic cells and endothelial cells by causing apoptosis of
both cell types and by inhibiting leukemic cell VEGF production (Figure
9). Release of VEGF by leukemic blasts
may be an important stimulus for neoangiogenesis in the bone marrow.
Endothelium is exquisitely sensitive to the dose of VEGF to which it is
exposed, and it is possible that the 34% reduction in VEGF production
observed after treatment with arsenic is physiologically significant.
In our in vitro endothelial cell differentiation assay, used as a measure of angiogenesis, treatment with As2O3
prevents capillary tubules and branch formation even in the presence of
additional VEGF.
A combination of direct toxicity against leukemic cells with antiangiogenic activity may be the optimum characteristic of a successful antileukemic agent. Future experiments are planned to establish the role of the endothelium in leukemic cell proliferation using coculture systems and to combine As2O3 with other specific antiangiogenic factors, such as neutralizing monoclonal antibodies to VEGF and VEGFR-2. Finally, we plan to evaluate the role of As2O3 in inhibiting angiogenesis in murine leukemia models.
We thank Alice Hafner, MS, for her help with statistical analysis, and Barbara Ferris for expert technical assistance.
Submitted November 19, 1999; accepted April 15, 2000.
S.R. supported by the American Heart Association Grant-In-Aid; NHLBI grants R01-HL-58707 and R01-HL-61849; the Dorothy Rodbell Foundation for Sarcoma Research; and the Rich Foundation. G.J.R. supported by the Susan Murphy Fellowship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Shahin Rafii, Division of Hematology and Oncology, Department of Medicine, Weill Medical College of Cornell University, 1300 York Ave, Rm C-606, New York, NY 10021; e-mail: srafii{at}mail.med.cornell.edu.
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G. Mufti, A. F. List, S. D. Gore, and A. Y.L. Ho Myelodysplastic Syndrome Hematology, January 1, 2003; 2003(1): 176 - 199. [Abstract] [Full Text] [PDF]
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T. Hayashi, T. Hideshima, M. Akiyama, P. Richardson, R. L. Schlossman, D. Chauhan, N. C. Munshi, S. Waxman, and K. C. Anderson Arsenic Trioxide Inhibits Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment Mol. Cancer Ther., August 1, 2002; 1(10): 851 - 860. [Abstract] [Full Text] [PDF]
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 W. H. Miller Jr., H. M. Schipper, J. S. Lee, J. Singer, and S. Waxman Mechanisms of Action of Arsenic Trioxide Cancer Res., July 15, 2002; 62(14): 3893 - 3903. [Abstract] [Full Text] [PDF]
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S. Dias, S. V. Shmelkov, G. Lam, and S. Rafii VEGF165 promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition Blood, April 1, 2002; 99(7): 2532 - 2540. [Abstract] [Full Text] [PDF]
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 M. A. Hussein Nontraditional Cytotoxic Therapies for Relapsed/Refractory Multiple Myeloma Oncologist, April 1, 2002; 7(90001): 20 - 29. [Abstract] [Full Text] [PDF]
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 B. Noel Regarding "Cannabis Arteritis Revisited--Ten New Case Reports" Angiology, July 1, 2001; 52(7): 505 - 506. [PDF]
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S. Waxman and K. C. Anderson History of the Development of Arsenic Derivatives in Cancer Therapy Oncologist, April 1, 2001; 6(90002): 3 - 10. [Abstract] [Full Text]
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N. C. Munshi Arsenic Trioxide: An Emerging Therapy for Multiple Myeloma Oncologist, April 1, 2001; 6(90002): 17 - 21. [Abstract] [Full Text]
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Top
Abstract
Introduction
fusion
protein.
