|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on January 16, 2003; DOI 10.1182/blood-2002-08-2512.
NEOPLASIA
From the Departments of Molecular Pathology,
Experimental Therapeutics, and Leukemia, The University of Texas
M. D. Anderson Cancer Center, Houston, TX.
2-Methoxyestradiol (2-ME), a new anticancer agent currently in
clinical trials, has been demonstrated to inhibit superoxide dismutase
(SOD) and to induce apoptosis in leukemia cells through a free
radical-mediated mechanism. Because the accumulation of superoxide
(O2 2-Methoxyestradiol (2-ME) is an endogenous
metabolite of 17 Several mechanisms of action have been proposed for the anticancer
activity of 2-ME. These pharmacologic actions include the antiangiogenetic effect,3,8,11,12 inhibition of
microtubule polymerization,2,13-17 induction of
G2/M phase arrest,18-21 accumulation of
proapoptotic protein p53,10,22-28 and inhibition of
superoxide dismutase (SOD).10,29 Among these pharmacologic
actions, inhibition of SOD by 2-ME and the subsequent accumulation of
superoxide radical is likely a major mechanism responsible for the
cytotoxic action, because forced overexpression of SOD by transfection
or exogenous antioxidants are able to protect cells from the toxic
effect of 2-ME.10 The p53 accumulation and
G2/M cell cycle arrest likely reflect the cellular
responses to free radical-mediated DNA damage, caused by 2-ME
inhibition of SOD, the key enzyme responsible for metabolic elimination
of superoxide radical in the cells. Our recent studies demonstrated
that inhibition of SOD and accumulation of O2 Mitochondrial respiration is the major biochemical pathway by which
O2 Chemicals and reagents
Isolation of CLL cells
Cytotoxicity assays Cytotoxicity was determined using multiple assays. Cell survival was determined by a 3-(4,5-dimethyl thiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously.46 The cell viability was determined after CLL cells were treated with various concentrations of 2-ME, ATO, and their combination for 72 hours. The potency of drug activity was expressed as IC50, defined as the drug concentration that induced 50% loss of cell viability. Median-effect analysis and combination index (CI) were used to evaluate the combination effect of 2-ME and ATO in CLL cells. The detail drug incubation conditions and computer-assisted analysis are described in the respective tables and figure legends.To examine the apoptotic change in cell morphology, the control and drug-treated cells were centrifuged (550 rpm, 5 minutes) onto glass slides, using a Shandon-Elliot cytospin (London, United Kingdom). The slides were fixed with 100% methanol for 45 minutes, air-dried, and then stained with Wright-Giemsa stain solution (Biochemical Sciences, Swedesboro, NJ). Cells were examined for morphologic changes characteristic of apoptosis. Microscopic photographs were taken using a × 60 objective (Nikon, Tokyo, Japan). Drug-induced apoptosis was further confirmed by a DNA fragmentation assay. After treatment with 2-ME, approximately 5 × 106 cells were collected, and the cell pellets were digested in 1 mL buffer containing 10 mM Tris (tris(hydroxymethyl)aminomethane)-HCl, pH 7.8, 100 mM NaCl, 25 mM EDTA (ethylenediaminetetraacetic acid), 0.5% SDS (sodium dodecyl sulfate), and 10 µL proteinase K (1 mg/mL, added fresh) at 45°C overnight. The samples were analyzed on a 1.8% agarose gel in 1 × TBE buffer (100 mM Tris-borate, pH 8.3, 2 mM EDTA). After electrophoresis, the gel was incubated overnight in 400 mL of 0.1 × TBE buffer containing 20 µL RNase (500 µg/mL) and then photographed. DNA bands were quantitated using the ChemiImager 4400 imager system (Alpha Innotech Corporation, San Leandro, CA). Apoptosis of CLL cells was quantified by a double staining with annexin V-FITC and propidium iodide (PI). Briefly, approximately 1 × 106 cells were washed with 1 × binding buffer (10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl), incubated with annexin V-FITC for 15 minutes at room temperature, and then washed once with 1 × binding buffer. The samples were then labeled with PI and analyzed by flow cytometry, using a FACSCalibur (Becton Dickinson, Mountain View, CA). The flow cytometry data were analyzed using the Becton Dickinson CellQuest Pro software package. Measurement of cellular superoxide Intracellular O2 contents were
measured by flow cytometry analysis using hydroethidine as described
previously.10 This fluorescent dye is commonly used for
detection of O2, although it may also react
with other oxidants to a certain degree.47,48 CLL cells
(2 × 106 per sample in 1 mL) were incubated with
hydroethidine (50 ng/mL) for 1 hour, washed once with 2 mL PBS, and
resuspended in 1 mL PBS. The samples were analyzed by flow cytometry
using a FACSCalibur (Becton Dickinson). The flow cytometry data were
analyzed using the Becton Dickinson CellQuest Pro software package.
Induction of superoxide accumulation and apoptosis by 2-ME in CLL cells Because 2-ME is able to inhibit SOD, we first determined its effect on cellular O2 content in CLL cells.
When freshly isolated CLL cells were incubated with 2-ME, a
substantial accumulation of cellular O2
was observed within 24 hours, as evidenced by a right-shift of the
ethidium fluorescent signal (Figure 1A).
This free radical accumulation was associated with morphologic and
biochemical changes typical of apoptosis. As illustrated in Figure 1B,
cell shrinkage and nuclear condensation were seen 24 hours after CLL
cells were incubated with 10 or 30 µM 2-ME. Fragmented nuclei
and blebbing cell membranes were apparent at 48 hours. Electrophoresis
of DNA isolated from 2-ME-treated cells showed a typical DNA
fragmentation associated with apoptosis (Figure 1C). In the sample
treated with 30 µM 2-ME for 72 hours, no intact DNA remained in the
well (lane 3). When the 2-ME-treated cells were analyzed for
reactivity with annexin V (an indication of phosphatidylserine exposure
on the cell surface during apoptosis), a significant increase of the apoptotic signal was detected 24 hours after 2-ME incubation (Figure 1D). We frequently observed various degrees of spontaneous apoptosis in
CLL cells, ranging from 5% to 20% among different patients. This
finding is consistent with previous observations.49,50 Nevertheless, 2-ME was able to cause a significant increase of apoptosis in CLL cells that showed either high spontaneous cell death
(patient A) or low spontaneous apoptosis (patient B).
CLL cells from patients with prior chemotherapy possess high levels
of endogenous O2 is a
critical event in 2-ME-induced apoptosis in leukemia
cells,10 we hypothesized that the endogenous cellular
production of O2 might be an important factor
that affects the antileukemia activity of 2-ME. We determined the basal
O2 contents in primary leukemia cells
isolated from 50 patients with CLL and evaluated their correlation with
patient's sex, Rai stages, and clinical treatment status. As shown in
Table 1, the O2
radical levels were significantly higher in CLL cells isolated from
patients previously treated with various therapeutic agents (fludarabine, cyclophosphamide, etc) than the CLL cells from previously untreated patients (P = .0056). Interestingly, CLL cells
from male patients appeared to have a higher superoxide content
(24.5 ± 16.1) than CLL cells from female patients (17.4 ± 8.8),
although such difference is not statistically significant
(P = .09). A multivariable analysis by analysis of
variance (ANOVA) indicates that there was no statistically
significant correlation between cellular O2
contents and the Rai stage (P = .32). The seemingly higher
superoxide contents in CLL cells from stage 4 patients were not
statistically different from other disease stages.
Correlation between basal O2 in
CLL cells suggest that leukemia cells from different patients with CLL
have different rates of O2 generation. This
heterogeneity provided a possibility to investigate the relationship
between the basal cellular O2 levels and the
sensitivity of CLL cells to 2-ME. Each of the freshly isolated CLL
samples from the 50 patients was incubated with various concentration
of 2-ME, and the sensitivity of the leukemia cells to 2-ME was
determined by the MTT assay. The IC50 values were then
plotted as a function of the basal O2
contents to reveal the possible correlation between these 2 parameters. As shown in Figure 2, there was an
inverse correlation between the IC50 values and the
cellular basal O2 contents. Statistical
analysis indicates that this correlation was significant
(P = .011). These data suggest that the CLL cells that
were active in O2 generation (and thus had a
high cellular O2 content) were more sensitive
to 2-ME treatment (low IC50) than the CLL cells with lower
basal O2 contents. This finding is consistent
with the hypothesis that cells with an active metabolic production of
O2 radicals are highly dependent on SOD for
elimination of the toxic chemical species and are more vulnerable to
inhibition of SOD by 2-ME.
Induction of O2 accumulation in leukemia
cells.10 The present study further evaluated the
relationship between 2-ME-induced increase of
O2 and the sensitivity of CLL cells to 2-ME.
Cellular O2 contents were determined before
and after 2-ME incubation (10 and 30 µM, 24 hours), and the
percentage of superoxide increase was calculated. 2-ME-induced loss of
cell viability was determined by the MTT assay and was plotted as a
function of 2-ME-induced accumulation of superoxide. As shown in
Figure 3, the induction of superoxide
accumulation by 2-ME and cell death were heterogeneous among the 50 patient samples (2 drug concentrations each). However, there was a
highly significant correlation between the loss of cell viability and
free radical increase (P = .000088). In general, a higher
accumulation of superoxide was associated with a greater loss of cell
viability. Thus, the cellular generation of
O2 may play an important role in the
cytotoxic action of 2-ME in CLL cells.
Enhancement of 2-ME activity against CLL cells by arsenic trioxide in vitro Because cellular O2 content was an
important factor that affected the antileukemia activity of 2-ME, we
further tested the possibility of using an exogenous ROS-generating
agent to enhance the activity of 2-ME. ATO, a
trivalent arsenite capable of causing ROS generation,37-39
was used in combination with 2-ME to evaluate the activity against CLL
cells. Primary leukemia cells freshly isolated from 25 patients with
CLL were treated with 10, 30 µM 2-ME, alone or in combination with
0.5 µg/mL or 1.0 µg/mL ATO for 72 hours, and cell viability was
determined by MTT assay. As shown in Figure
4A, ATO at the concentrations of 0.5 and
1.0 µg/mL alone reduced cell survival to 60% and 30% of the
control, respectively. Addition of these concentrations of ATO to the
2-ME-treated CLL cells significantly enhanced the cytotoxic activity
(P < .05). This combination effect appears to be
additive, consistent with the notion that both compounds act through a
free radical-mediated mechanism. In contrast, no significant
enhancement of the cytotoxic activity when 2-ME was combined with
fludarabine in 3 cases of CLL cells tested (data not shown). The
IC50 values of 2-ME, used alone or in combination with ATO,
in 25 patient samples tested are shown in Table
2.
We measured the cellular O2 Because arsenic trioxide was able to enhance the activity of 2-ME in
CLL cells, we further tested if ATO could increase the cytotoxic
effect of 2-ME in CLL cells that were resistant to 2-ME alone. We
identified 10 CLL patient samples that were resistant to treatment with
2-ME alone (defined as IC50 > 30 µM), and 8 CLL
patient samples that were sensitive to treatment with 2-ME alone
(defined as IC50 < 11 µM). As shown in Figure
5A, the 2-ME-resistant cells showed
significantly lower basal cellular O2
The data shown in Figures 4 and 5 suggest that the combination of 2-ME
and ATO may have additive or more than additive activity. We further
tested this possibility by a formal median-effect analysis and
determination of CI in 6 CLL patient samples, which were each incubated with various concentrations of 2-ME and ATO, alone or in
combinations. Figure 6A-B illustrates the
results of such analysis in one of the patient samples. The CI values
for all 6 patient samples (mean ± SD) under various drug
combination conditions are summarized in Table
3. A CI value of 1.0 indicates an
additive effect; CI greater than 1.0 indicates antagonism, whereas CI
less than 1.0 indicates synergism. It is clear that under most
combination conditions, 2-ME and ATO produced more than additive
effect. The 2 compounds showed additive activity at a lower 2-ME/ATO
ratio (Table 3).
ROS are potentially toxic "byproducts" of cellular metabolism that are generated during the production of adenosine triphosphate (ATP) by aerobic metabolism in the mitochondria, where electrons escaping from the respiratory complexes I and III may react with molecular oxygen to form oxygen radicals.52-55 ROS play important roles in cell proliferation, aging, and cancer development. Because of their reactive chemical properties, ROS may cause various types of tissue injury in a wide range of human diseases, and an excessive amount of ROS can lead to cell death by apoptosis or by necrosis.56,57 The ability of ROS to damage cellular components and cause cell death suggests a possibility to exploit this chemical property for killing cancer cells through a free radical-mediated mechanism. This strategy is of therapeutic relevance, because most cancer cells are active in metabolic production of ROS and are intrinsically under increased oxidative stress, and thus more susceptible to exogenous free radical insults.31 This provides a biochemical basis to preferentially kill cancer cells. Previous studies demonstrated that 2-ME is able to inhibit SOD, causes
a substantial accumulation of O2 Among the several mechanisms of actions proposed for 2-ME
(antiangiogenetic effect,3,11,12 interruption microtubule
assembling,2,13-17 interfering of cell cycle
progression,18-21 and inhibition of
SOD10,29), inhibition of SOD and the subsequent
accumulation of superoxide radical is likely a major mechanism of the
drug action. It has been previously observed that overexpression of SOD
protects cells from the toxic effect of 2-ME, whereas decreased SOD
expression by antisense oligos enhanced 2-ME toxicity.10
In a separate set of experiments, we demonstrated that 2-ME caused a
significant accumulation of superoxide well before apoptosis
became apparent and that addition of antioxidants such as
N-acetyl-L-cysteine (NAC) effectively prevented the
2-ME-induced increase of O2 The reason for the high degree of heterogeneity in basal superoxide contents in primary CLL cells is not yet clear. Several possibilities may contribute to this variation. First, because superoxide is produced mainly in the mitochondria during oxidative phosphorylation for ATP generation, CLL cells at different disease stages may have different metabolic activities and thus produce various levels of superoxide radicals, depending on the energy requirement by the leukemia cells. Second, the mitochondrial oxidative phosphorylation coupling efficiency directly affects the production of superoxide. An inefficient electron transport through the respiratory chain tends to cause a "leak" of electrons from the transport chain (especially complex I and III) and generates superoxide when the escaping electrons react with molecular oxygen.58 Because the mitochondrial DNA (mtDNA) encodes 13 important protein components of the electron transport complexes, certain mutations in the mtDNA are likely to affect the electron transport and lead to an increase of superoxide formation in CLL cells. Third, the patients' individual variations such as intake of antioxidants and other medication might also contribute to the variation in free radical contents observed in CLL cells from different patients. It is of interest to note that the CLL cells from patients with prior
chemotherapy exhibited significantly higher basal superoxide contents
than the CLL cells from previously untreated patients. Drug-induced
mutations in mtDNA may be a possible reason for the increased
O2 The observations that CLL cells from patients with a history of prior
chemotherapy contain significantly higher levels of superoxide and are
more sensitive to 2-ME suggest a promising possibility to use 2-ME for
the treatment of patients with CLL who failed prior therapy with other
agents. This possibility warrants further investigation in both
preclinical and clinical settings. However, the heterogeneity of
superoxide content in CLL cells (and likely in other types of cancer
cells) also presents a challenge to the use of 2-ME for treating
patients with CLL, because the CLL cells with low endogenous superoxide
content are less sensitive to 2-ME (Figures 2 and 5A). One strategy to
overcome such drug resistance is to use exogenous free
radical-generating agents in combination with 2-ME. The rationale is
to use exogenous ROS-generating agents to enhance intracellular
O2 Arsenic trioxide appears to be a logical choice for combination with 2-ME. ATO has been used in the clinical treatment of relapsed/refractory acute promyelocytic leukemia (APL).40-44 Oxidative damage has been suggested to be a key mechanism by which arsenic trioxide causes cell death.44 ATO-induced apoptosis has been shown to be associated with the generation of ROS in several experimental models.37-39,61 Antioxidants and free radical scavengers are able to inhibit apoptosis induced by ATO.38 On the basis of the ability of ATO to cause free radical generation in cells, one would expect that its combination with 2-ME should enhance the cytotoxic activity. In fact, the present study showed that combination of ATO with 2-ME caused an additive accumulation of superoxide in CLL cells and resulted in additive or more than additive activity against the leukemia cells. Importantly, this combination was effective in killing CLL cells that were resistant to 2-ME alone. These results suggest that it is indeed possible to combine 2-ME with ROS-generating agents to enhance therapeutic activity and overcome drug resistance. Previous studies showed that 2-ME selectively kills leukemia cells without a significant toxic effect on healthy lymphocytes.10 It would be important to further test the effect of 2-ME and its combination with other ROS-generating agents such as ATO on a variety of nontumor cells, including healthy bone marrow residents and other healthy cells such as macrophages and neutrophils that produce ROS. Studies along this line would provide important information on the relative selectivity of this therapeutic strategy. Although such a study would require the development of appropriate methods for isolation/purification of large number of the healthy cells without artificially affecting their metabolic production of ROS, its potential therapeutic implications seem to justify future research efforts in this area. In summary, we demonstrated that the cellular production of
O2
Submitted August 16, 2002; accepted January 4, 2003.
Prepublished online as Blood First Edition Paper, January 16, 2003; DOI 10.1182/blood-2002-08-2512.
Supported in part by research grants CA77339, CA85563, and CA81534 from the National Cancer Institute, National Institutes of Health, and by a Cancer Center Support Grant P30 CA16672 from the National Cancer Institute.
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: Peng Huang, Department of Molecular Pathology, Box 089, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: phuang{at}mdanderson.org.
1. Gelbke HP, Knuppen R. The excretion of five different 2-hydroxyoestrogen monomethyl ethers in human pregnancy urine. J Ster Biochem. 1976;7:457-463[CrossRef][Medline] [Order article via Infotrieve].
2.
D'Amato RJ, Lin CM, Flynn E, Folkman J, Hamel E.
2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at the colchicine site.
Proc Natl Acad Sci U S A.
1994;91:3964-3968 3. Fotsis T, Zhang Y, Pepper MS, et al. The endogenous oestrogen metabolite 2-methoxyestradiol inhibits angiogenesis and suppresses tumour growth. Nature. 1994;368:237-239[CrossRef][Medline] [Order article via Infotrieve]. 4. Attalla H, Makela TP, Adlercreutz H, Anderson LC. 2-Methoxyestradiol arrests cells in mitosis without depolymerizing tubulin. Biochem Biophys Res Commun. 1996;228:467-473[CrossRef][Medline] [Order article via Infotrieve].
5.
Kataoka M, Schumacher G, Cristiano RJ, et al.
An agent that increases tumor suppressor transgene product coupled with systemic transgene delivery inhibits growth of metastatic lung cancer in vivo.
Cancer Res.
1998;58:4761-4765 6. Reiser F, Bernas M, Witte M, Witte C. Inhibition of normal and experimental angiotumor endothelial cell proliferation and cell cycle progression by 2-methoxyestradiol. Proc Soc Exp Biol Med. 1998;219:211-216[CrossRef][Medline] [Order article via Infotrieve].
7.
Schumacher G, Kataoka M, Roth JA, Mukhopadhyay T.
Potent antitumor activity of 2-methoxyestradiol in human pancreatic cancer cell lines.
Clin Cancer Res.
1999;5:493-499 8. Pribluda VS, Gubish ER, LaValle TM, Treston A, Swartz GM, Green SJ. 2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate. Cancer Metastasis Rev. 2000;19:173-179[CrossRef][Medline] [Order article via Infotrieve]. 9. Kinuya S, Kawashima A, Yokoyama K, et al. Anti-angiogenic therapy and radioimmuno-therapy in colon cancer xenografts. Eur J Nucl Med. 2001;28:1306-1312[CrossRef][Medline] [Order article via Infotrieve]. 10. Huang P, Feng L, Hileman EO, Keating MJ, Plunkett W. Superoxide dismutase as a target for the selective killing of cancer cells. Nature. 2000;407:390-395[CrossRef][Medline] [Order article via Infotrieve].
11.
Yue TL, Wang X, Louden CS, et al.
2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression.
Mol Pharmacol.
1997;51:951-962 12. Banerjeei SK, Zoubine MN, Sarkar DK, Weston AP, Shah JH, Campbell DR. 2-Methoxy-estradiol blocks estrogen-induced rat pituitary tumor growth and tumor angiogenesis: possible role of vascular endothelial growth factor. Anticancer Res. 2000;20:2641-2645[Medline] [Order article via Infotrieve]. 13. Cushman M, He HM, Katzenellenbogen JA, Lin CM, Hamel E. Synthesis, antitubulin and antimitotic activity, and cytotoxicity of analogs of 2-methoxyestradiol, an endogenous mammalian metabolite of estradiol that inhibits tubulin polymerization by binding to the colchicine binding site. J Med Chem. 1995;38:2041-2049[CrossRef][Medline] [Order article via Infotrieve]. 14. Attalla H, Makela TP, Adlercreutz H, Anderson LC. 2-Methoxyestradiol arrests cells in mitosis without depolymerizing tubulin. Biochem Biophys Res Commun. 1996;228:467-473[CrossRef][Medline] [Order article via Infotrieve].
15.
Klauber N, Parangi S, Flynn E, Hamel E, D'Amato RJ.
Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol.
Cancer Res.
1997;57:81-86
16.
Verdier-Pinard P, Wang Z, Mohanakrishnan AK, Cushman M, Hamel E.
A steroid derivative with paclitaxel-like effects on tubulin polymerization.
Mol Pharmacol.
2000;57:568-575 17. Brueggemeier RW, Bhat AS, Lovely CJ, et al. 2-Methoxymethylestradiol: a new 2-methoxy estrogen analog that exhibits antiproliferative activity and alters tubulin dynamics. J Steroid Biochem Mol Biol. 2001;78:145-156[CrossRef][Medline] [Order article via Infotrieve]. 18. Kumar AP, Garcia GE, Slaga TJ. 2-methoxyestradiol blocks cell-cycle progression at G(2)/M phase and inhibits growth of human prostate cancer cells. Mol Carcinog. 2001;31:111-124[CrossRef][Medline] [Order article via Infotrieve]. 19. Lin HL, Liu TY, Wu CW, Chi CW. 2-Methoxyestradiol-induced caspase-3 activation and apoptosis occurs through G(2)/M arrest dependent and independent pathways in gastric carcinoma cells. Cancer. 2001;92:500-509[CrossRef][Medline] [Order article via Infotrieve]. 20. Qadan LR, Perez-Stable CM, Anderson C, et al. 2-Methoxyestradiol induces G2/M arrest and apoptosis in prostate cancer. Biochem Biophys Res Commun. 2001;285:1259-1266[CrossRef][Medline] [Order article via Infotrieve]. 21. Schumacher G, Neuhaus P. The physiological estrogen metabolite 2-methoxyestradiol reduces tumor growth and induces apoptosis in human solid tumors. J Cancer Res Clin Oncol. 2001;127:405-410[CrossRef][Medline] [Order article via Infotrieve]. 22. Seegers JC, Lottering M, Grobler CJS, et al. The mammalian metabolite, 2-methoxyestradiol, affects p53 levels and apoptosis induction in transformed cells but not in normal cells. J Steroid Biochem Mol Biol. 1997;62:253-267[CrossRef][Medline] [Order article via Infotrieve]. 23. Mukhopadhyay T, Roth JA. Induction of apoptosis in human lung cancer cells after wild-type p53 activation by methoxyestradiol. Oncogene. 1997;14:379-384[CrossRef][Medline] [Order article via Infotrieve]. 24. Mukhopadhyay T, Roth JA. Superinduction of wild-type p53 protein after 2-methoxyestradiol treatment of Ad5p53-transduced cells induces tumor cell apoptosis. Oncogene. 1998;17:241-246[CrossRef][Medline] [Order article via Infotrieve].
25.
Kataoka M, Schumacher G, Cristiano RJ, Atkinson EN, Roth JA, Mukhopadhyay T.
An agent that increases tumor suppressor transgene product coupled with systemic transgene delivery inhibits growth of metastatic lung cancer in vivo.
Cancer Res.
1998;58:4761-4765 26. Wang SH, Myc A, Koenig RJ, Bretz JD, Arscott PL, Baker JR. 2-Methoxyestradiol, an endo-genous estrogen metabolite, induces thyroid cell apoptosis. Mol Cell Endocrinol. 2000;165:163-172[CrossRef][Medline] [Order article via Infotrieve]. 27. Kumar AP, Garcia GE, Slaga TJ. 2-methoxyestradiol blocks cell-cycle progression at G2/M phase and inhibits growth of human prostate cancer cells. Mol Carcinog. 2001;31:111-124[CrossRef][Medline] [Order article via Infotrieve]. 28. Maran A, Zhang M, Kennedy AM, et al. 2-methoxyestradiol induces interferon gene expression and apoptosis in osteosarcoma cells. Bone. 2002;30:393-398[Medline] [Order article via Infotrieve]. 29. Wood L, Leese MR, Leblond B, et al. Inhibition of superoxide dismutase by 2-methoxyoestradiol analogues and oestrogen derivatives: structure-activity relationships. Anticancer Drug Des. 2001;16:209-215[Medline] [Order article via Infotrieve]. 30. Devi GS, Prasad MH, Saraswathi I, Raghu D, Rao DN, Reddy PP. Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias. Clin Chim Acta. 2000;293:53-62[CrossRef][Medline] [Order article via Infotrieve]. 31. Hileman EO, Achanta G, Huang P. Superoxide dismutase: an emerging chemotherapeutic target. Expert Opin Ther Targets. 2001;5:697-710[CrossRef][Medline] [Order article via Infotrieve]. 32. Ivanova R, Lepage V, Loste MN, et al. Mitochondrial DNA sequence variation in human leukemic cells. Int J Cancer. 1998;76:495-498[CrossRef][Medline] [Order article via Infotrieve].
33.
Fliss MS, Usadel H, Caballero OL, et al.
Facile detection of mitochondrial DNA mutations in tumors and bodily fluids.
Science.
2000;287:2017-2019
34.
Tan DJ, Bai RK, Wong LJ.
Comprehensive scanning of somatic mitochondrial DNA mutations in breast cancer.
Cancer Res.
2002;62:972-976 35. Miyazono F, Schneider PM, Metzger R, et al. Mutations in the mitochondrial DNA D-Loop region occur frequently in adenocarcinoma in Barrett's esophagus. Oncogene. 2002;21:3780-3783[CrossRef][Medline] [Order article via Infotrieve]. 36. Singh KK, Russell J, Sigala B, Zhang Y, Williams J, Keshav KF. Mitochondrial DNA determines the cellular response to cancer therapeutic agents. Oncogene. 1999;18:6641-6646[CrossRef][Medline] [Order article via Infotrieve]. 37. Wang TS, Kuo CF, Jan KY, Huang H. Arsenite induces apoptosis in chinese hamster ovary cells by generation of reactive oxygen species. J Cell Physiol. 1996;169:256-268[CrossRef][Medline] [Order article via Infotrieve]. 38. Chen Y-C, Lin-Shiau S-Y, Lin J-K. Involvement of reactive oxygen species and caspase 3 activation in arsenite-induced apoptosis. J Cell Physiol. 1998;177:324-333[CrossRef][Medline] [Order article via Infotrieve]. 39. Iwama K, Nakajo S, Aiuchi T, et al. Apopotsis induced by arsenic trioxide in leukemia U937 cells is dependent on activation of p38, inactivation of ERK and the Ca2+-dependent production of superoxide. Int J Cancer. 2001;92:518-526[CrossRef][Medline] [Order article via Infotrieve].
40.
Chen GQ, Zhu J, Shi XG, et al.
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
41.
Shen ZX, Chen GQ, Ni JH, et al.
Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL). II. Clinical efficacy and pharmacokinetics in relapsed patients.
Blood.
1997;89:3354-3360
42.
Shao W, Fanelli M, Ferrara FF, et al.
Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR
43.
Niu C, Yan H, Yu T, et al.
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.
1999;94:3315-3324
44.
Miller WH Jr.
Molecular targets of arsenic trioxide in malignant cells.
Oncologist.
2002;7(suppl 1):14-19 45. Huang P, Sandoval A, Van Den NE, Keating MJ, Plunkett W. Inhibition of RNA transcription: a biochemical mechanism of action against chronic lymphocytic leukemia cells by fludarabine. Leukemia. 2000;14:1405-1413[CrossRef][Medline] [Order article via Infotrieve]. 46. Zhou Y, Achanta G, Pelicano H, Gandhi V, Plunkett W, Huang P. Action of (E)-2'-deoxy-2'-(fluoromethylene) cytidine on DNA metabolism: incorporation, excision, and cellular response. Mol Pharmacol. 2001;61:222-229.
47.
Fujimura M, Morita-Fujimura Y, Kawase M, et al.
Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice.
J Neurosci.
1999;19:3414-3422
48.
Zhang DX, Zou AP, Li PL.
Ceramide reduces endothelium-dependent vasodilation by increasing superoxide production in small bovine coronary arteries.
Circ Res.
2001;88:824-831 49. Robertson LE, Plunkett W. Apoptotic cell death in chronic lymphocytic leukemia. Leuk Lymphoma. 1993;11(suppl 2):71-74.
50.
Robertson LE, Chubb S, Meyn RE, et al.
Induction of apoptotic cell death in chronic lymphocytic leukemia by 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosyl-2-fluoroadenine.
Blood.
1993;81:143-150 51. Chou TC, Hayball MP. CalcuSyn: window software for dose effect analysis. Cambridge United Kingdom: Biosoft; 1996. 52. Beyer R. An analysis of the role of coenzyme Q in free radical generation and as an oxidant. Biochem Cell Biol. 1991;70:390-343. 53. Boveris A, Chance B. Mitochondrial production of superoxide radical and hydrogen peroxide. In: Reivich M,Coburn R,Lahiri S,Chance B, eds. Tissue Hypoxia and Ischemia. New York: Plenum Press; 1997:67-82. 54. Du G, Mouithys-Mickalad A, Sluse-Goffart CM, Droy-Lefaix MT, Sluse FE. Generation of superoxide anion by mitochondria and impairment of their functions during anoxia and reoxygenation in vitro. Free Radic Biol Med. 1998;25:1066-1074[CrossRef][Medline] [Order article via Infotrieve].
55.
Sankarapandi S, Zweier JL.
Evidence against the generation of free hydroxyl radicals from the interaction of copper, zinc superoxide dismutase and hydrogen peroxide.
J Biol Chem.
1999;274:34576-34583 56. Hampton MB, Fadeel B, Orrenius S. Redox regulation of the caspases during apoptosis. Ann N Y Acad Sci. 1998;854:328-335[CrossRef][Medline] [Order article via Infotrieve]. 57. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 3rd ed. Oxford, United Kingdom: Oxford University Press; 1999. 58. Staniek K, Gille L, Kozlov AV, Nohl H. Mitochondrial superoxide radical formation is controlled by electron bifurcation to the high and low potential pathways. Free Radic Res. 2002;36:381-387[CrossRef][Medline] [Order article via Infotrieve]. 59. Huang P, Siciliano MJ, Plunkett W. Gene deletion, a mechanism of induced mutation by arabinosyl nucleosides. Mutation Res. 1989;210:291-301. 60. Sanderson BJ, Shield AJ. Mutagenic damage to mammalian cells by therapeutic alkylating agents. Mutat Res. 996;355:41-57.
61.
Jing Y, Dai J, Chalmers-Redman RME, Tatton WG, Waxman S.
Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway.
Blood.
1999;94:2102-2111
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  A. M. McElligott, E. N. Maginn, L. M. Greene, S. McGuckin, A. Hayat, P. V. Browne, S. Butini, G. Campiani, M. A. Catherwood, E. Vandenberghe, et al. The Novel Tubulin-Targeting Agent Pyrrolo-1,5-Benzoxazepine-15 Induces Apoptosis in Poor Prognostic Subgroups of Chronic Lymphocytic Leukemia Cancer Res., November 1, 2009; 69(21): 8366 - 8375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Trachootham, H. Zhang, W. Zhang, L. Feng, M. Du, Y. Zhou, Z. Chen, H. Pelicano, W. Plunkett, W. G. Wierda, et al. Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism Blood, September 1, 2008; 112(5): 1912 - 1922. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. Whibley, K. L. McPhail, R. A. Keyzers, M. F. Maritz, V. D. Leaner, M. J. Birrer, M. T. Davies-Coleman, and D. T. Hendricks Reactive oxygen species mediated apoptosis of esophageal cancer cells induced by marine triprenyl toluquinones and toluhydroquinones Mol. Cancer Ther., September 1, 2007; 6(9): 2535 - 2543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Sharma, C. Joseph, S. Ghosh, A. Agarwal, M. K. Mishra, and E. Sen Kaempferol induces apoptosis in glioblastoma cells through oxidative stress Mol. Cancer Ther., September 1, 2007; 6(9): 2544 - 2553. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ge, I. Montano, G. Rustici, W. J. Freebern, C. M. Haggerty, W. Cui, D. Ponciano-Jackson, G. V. R. Chandramouli, E. R. Gardner, W. D. Figg, et al. Selective leukemic-cell killing by a novel functional class of thalidomide analogs Blood, December 15, 2006; 108(13): 4126 - 4135. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zou, M. Adachi, K. Imai, M. Hareyama, K. Yoshioka, S. Zhao, and Y. Shinomura 2-Methoxyestradiol, an Endogenous Mammalian Metabolite, Radiosensitizes Colon Carcinoma Cells through c-Jun NH2-Terminal Kinase Activation. Clin. Cancer Res., November 1, 2006; 12(21): 6532 - 6539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lemarie, C. Morzadec, D. Merino, O. Micheau, O. Fardel, and L. Vernhet Arsenic Trioxide Induces Apoptosis of Human Monocytes during Macrophagic Differentiation through Nuclear Factor-{kappa}B-Related Survival Pathway Down-Regulation J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 304 - 314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Carew, S. T. Nawrocki, Y. V. Krupnik, K. Dunner Jr, D. J. McConkey, M. J. Keating, and P. Huang Targeting endoplasmic reticulum protein transport: a novel strategy to kill malignant B cells and overcome fludarabine resistance in CLL Blood, January 1, 2006; 107(1): 222 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hu, D. G. Rosen, Y. Zhou, L. Feng, G. Yang, J. Liu, and P. Huang Mitochondrial Manganese-Superoxide Dismutase Expression in Ovarian Cancer: ROLE IN CELL PROLIFERATION AND RESPONSE TO OXIDATIVE STRESS J. Biol. Chem., November 25, 2005; 280(47): 39485 - 39492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. E. Sutherland, M. Schuliga, T. Harris, B. L. Eckhardt, R. L. Anderson, L. Quan, and A. G. Stewart 2-Methoxyestradiol Is an Estrogen Receptor Agonist That Supports Tumor Growth in Murine Xenograft Models of Breast Cancer Clin. Cancer Res., March 1, 2005; 11(5): 1722 - 1732. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. Shanafelt, Y. K. Lee, N. D. Bone, A. K. Strege, V. L. Narayanan, E. A. Sausville, S. M. Geyer, S. H. Kaufmann, and N. E. Kay Adaphostin-induced apoptosis in CLL B cells is associated with induction of oxidative stress and exhibits synergy with fludarabine Blood, March 1, 2005; 105(5): 2099 - 2106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Qian, K. J. Liu, Y. Chen, D. C. Flynn, V. Castranova, and X. Shi Cdc42 Regulates Arsenic-induced NADPH Oxidase Activation and Cell Migration through Actin Filament Reorganization J. Biol. Chem., February 4, 2005; 280(5): 3875 - 3884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Pelicano, L. Feng, Y. Zhou, J. S. Carew, E. O. Hileman, W. Plunkett, M. J. Keating, and P. Huang Inhibition of Mitochondrial Respiration: A NOVEL STRATEGY TO ENHANCE DRUG-INDUCED APOPTOSIS IN HUMAN LEUKEMIA CELLS BY A REACTIVE OXYGEN SPECIES-MEDIATED MECHANISM J. Biol. Chem., September 26, 2003; 278(39): 37832 - 37839. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-estradiol that is present in human urine and blood.
2-ME is produced by sequential 2-hydroxylation and
O-methylation.
) or 30 (
) µM 2-ME for 24 hours. The
O2
and # indicate a
statistically significant difference when compared with the control
cells (P < .05); *, a statistically significant
difference when compared with cells exposed to 2-ME alone
(P < .05). Error bars indicate SD of the means.
, ATO; and
, 2-ME+0.5 µg/mL ATO. Dashed line at CI = 1.0 indicates
an additive effect. If a CI value is below this line, it indicates
synergistic activity, whereas CI values above this line suggest an
antagonist effect.
/PML proteins.
Blood.
1996;88:1052-1061