The anticancer drug XR5944 was originally developed as a topoisomerase inhibitor and was subsequently shown to be a transcription inhibitor. It has shown exceptional anticancer activity both in vitro and in vivo and was significantly more potent than traditional topoisomerase inhibitors. The solution structure of the XR5944/DNA complex recently obtained in our laboratory indicates that XR5944 bis-intercalates at the 5′-(TpG):(CpA) site of duplex DNA, which is found in the consensus DNA-binding site of estrogen receptor (ER). Thus, we tested the ability of XR5944 to inhibit ER activity both in vitro and in cultured cells. In electrophoretic mobility shift assays, it is seen that the DNA binding of recombinant ERα protein, as well as ER from nuclear extracts, is inhibited by XR5944 in a dose-dependent manner. In luciferase reporter assays, XR5944 inhibited the reporter gene expression from an estrogen response element–containing promoter but not from a basal promoter sequence that lacks any cis-acting elements. In contrast, the RNA polymerase inhibitor actinomycin D inhibits the transcription from both the above-mentioned promoters. The specificity of XR5944 activity is displayed by a separate reporter assay in which the transactivation of reporter gene expression by Sp1 proteins was not inhibited by XR5944. Collectively, these data suggest that XR5944 is capable of specifically inhibiting the binding of ER to its consensus DNA sequence and its subsequent activity. This represents a novel mechanism of ER inhibition, which may allow the development of agents capable of overcoming resistance to current antiestrogens. [Mol Cancer Ther 2007;6(1):213–9]

In women, breast cancer is the most frequent type of cancer and represents the second leading cause of cancer death. Although there have been major advances in the treatment of breast cancer in the last 10 to 15 years, it remains a disease for which improved treatments are still urgently needed. Targeting signal transduction pathways in breast cancer chemotherapy has continued to develop as a compelling approach over time.

The link between human breast cancer and estrogen was first identified over a century ago and more recent epidemiologic data show clear associations of serum estrogen concentrations (1), use of estrogenic hormone replacement therapies (2, 3), and oral contraceptives (4) with an increased risk of breast cancer. Thus, antagonizing the biological effects of estrogens is an effective therapeutic strategy for women with breast cancer. Since the discovery of estrogen receptor (ER) as the modulator of estrogen action (5), ER has evolved to be one of the most effective targets for breast cancer therapy (6). Binding of estrogen to ER initiates a series of events that include dissociation of heat shock proteins and receptor dimerization. Such dimerization facilitates the binding of ER to specific estrogen response elements (ERE) that are located upstream of estrogen-regulated genes. Currently available methods for interfering with estrogen action are composed of “antiestrogens” that either compete with estrogens for binding to ER or reduce the circulating ER levels, and “aromatase inhibitors” that inhibit estrogen biosynthesis. The antiestrogen tamoxifen was the hormonal treatment of choice for ER-expressing breast cancers. Although it still remains so for premenopausal women, it is gradually being replaced by third-generation aromatase inhibitors in treatment of postmenopausal women with ER-expressing breast cancers (7). However, despite being the ideal treatment of choice for patients with ER-expressing breast cancer, these current ER-targeting therapies have a main limitation related with resistance. Half of the breast cancers expressing hormone receptors are inherently resistant to the current endocrine therapy (8), and most initially responsive breast tumors acquire resistance. Hence, there is an urgent need for new and effective agents that lack cross-resistance with existing treatments.

The anticancer drug XR5944 (Fig. 1A) is a bis-phenazine in phase I clinical trials (9). It originated from a rational drug design program aimed at developing potential topoisomerase inhibitors (10). The parent compounds of XR5944, phenazinecarboxamides, as well as closely related acridinecarboxamides, are both dual DNA topoisomerase I/II inhibitors (1113). Although initial reports showed that XR5944 could bind strongly to DNA and that it may interfere with the normal function of topoisomerase I and II in vitro (14), recent studies have indicated that the primary mechanism of XR5944 action is independent of topoisomerases and, furthermore, that it is related with transcription inhibition (9, 15).

Figure 1.

A, the chemical structure of XR5944 with nitrogen protonations as observed in the DNA-bound conformation. B, the XR5944-DNA complex structure. The CPK model of XR5944 shows the two phenazine rings intercalated with DNA parallel to the DNA base pairs and the amino alkyl linker lying in the major grove. C, schematic representations of the three reporter vectors used in this study. Top, the EREv-tk-Luc vector containing the vitellogenin A2 ERE and the thymidine kinase promoter upstream of the luciferase gene. Middle, the pGL3-promoter vector (Promega) containing the SV40 promoter sequence upstream of the luciferase gene. Bottom, the pGAGC6 plasmid containing multiple Sp1 binding sites and the adenovirus major late initiator TATA box upstream of the luciferase gene.

Figure 1.

A, the chemical structure of XR5944 with nitrogen protonations as observed in the DNA-bound conformation. B, the XR5944-DNA complex structure. The CPK model of XR5944 shows the two phenazine rings intercalated with DNA parallel to the DNA base pairs and the amino alkyl linker lying in the major grove. C, schematic representations of the three reporter vectors used in this study. Top, the EREv-tk-Luc vector containing the vitellogenin A2 ERE and the thymidine kinase promoter upstream of the luciferase gene. Middle, the pGL3-promoter vector (Promega) containing the SV40 promoter sequence upstream of the luciferase gene. Bottom, the pGAGC6 plasmid containing multiple Sp1 binding sites and the adenovirus major late initiator TATA box upstream of the luciferase gene.

Close modal

XR5944 has shown exceptional activity against human and murine tumor models both in vitro and in vivo and was significantly more potent than well-known topoisomerase inhibitors such as doxorubicin, topotecan, and TAS-103 (14). This potency translates well to ex vivo systems where XR5944 was 40- to 300-fold more potent than the other cytotoxic agents tested (16). Recent data show the in vitro and in vivo additive potency of XR5944 in combination with carboplatin or doxorubicin in non–small-cell lung carcinoma, and with 5-fluorouracil and irinotecan in colon cancer cell lines (17, 18).

Despite the progress in understanding the activity and function of XR5944, its precise mechanism of action is yet to be revealed. A major contribution to the understanding of the mechanism of action is the structure of the DNA-XR5944 complex recently determined by us (19). According to this structure, XR5944 has a novel DNA-binding mode using a DNA oligomer of sequence d(ATGCAT). XR5944 bis-intercalates at the 5′-(TpG):(CpA) site, with the carboxamide aminoalkyl linker lying in the major groove of the DNA (Fig. 1B). Such binding in the major groove is significant because most DNA-binding drugs bind in the minor groove, whereas transcription factors generally bind in the major groove. The DNA binding of XR5944 seems to be highly specific as shown by parallel studies of drug binding with other DNA sequences. The same dinucleotide sequence is found in the consensus binding site of the ER, which has a base sequence of GGTCAnnnTGACC with palindromic half sites (20, 21). Thus, we speculate that XR5944 can inhibit the DNA binding of ERs. Following up on previous findings that XR5944 inhibits DNA transcription (9) and that it inhibits DNA binding of activator protein 1 transcription factors in vitro to a consensus sequence that contains 5′-TG-3′ (19), we tested the ability of XR5944 to inhibit the DNA binding of ER and its transcriptional activation function. Our data show that XR5944 specifically inhibits the DNA binding, and the subsequent activity of ERs both in vitro and in cell systems. Furthermore, it is shown that the activity of XR5944 in inhibition of transcription is different from that of the RNA polymerase inhibitor-actinomycin D. Thus, XR5944 is likely to have a mechanism of action that has not been seen before with other clinically used ER inhibitors. Our data warrant the development of specific ER inhibitors that use a DNA-binding mode and a mechanism of action similar to XR5944, which may be useful in breast cancer therapeutics.

Cells and Reagents

MCF-7 cells were originally obtained from the American Type Culture Collection (Manassas, VA) and periodically tested negative for Mycoplasma. Cells were cultured in DMEM (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum and 100 IU/mL penicillin/streptomycin at 37°C and 5% CO2 in air. XR5944 was provided by Xenova Ltd. (Slough, United Kingdom). The powder was dissolved in deionized water at a concentration of 10 mmol/L and stored at 4°C. Actinomycin D was purchased from Sigma (St. Louis, MO), dissolved at a concentration of 1 mg/mL in DMSO solution (Sigma), and stored at −20°C.

Electrophoretic Mobility Shift Assay

Binding of ER to the ERE was done using either MCF-7 nuclear extract (Active Motif, Carlsbad, CA) or recombinant ERα (Affinity BioReagents, Golden, CO). ER gel shift oligonucleotide of sequence 5′-GGATCTAGGTCACTGTGACCCCGGATC-3′ (the consensus half sites are underlined) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and 32P 5′-end-labeled using T4 polynucleotide kinase and [γ-32P]ATP (3,000 Ci/mmol). Approximately 8 μg nuclear extract or 0.3 μg ERα were mixed with indicated amounts of XR5944 and a 1× solution of Gel Shift Binding Buffer (Promega Corporation, Madison, WI) containing poly(deoxyinosinic-deoxycytidylic acid) in a total volume of 20 μL. The mixture was incubated at room temperature for 20 min and the binding reaction was initiated by adding ∼40,000 cpm DNA per reaction. Following incubation for further 15 min, the samples were analyzed in a pre-electrophoresed 6% polyacrylamide native gel in 0.5× Tris-borate EDTA (Sigma). After 1 h of electrophoresis at 240 V, the gel was vacuum dried and subjected to autoradiography. The binding of ER and ERE was observed by Phosphorimaging analysis.

Plasmids

The plasmid EREv-tk-Luc, which was used to monitor the ER transactivation, contains the vitellogenin A2 ERE sequence from −336 to −310 inserted upstream of the herpes simplex virus thymidine kinase promoter and has been described previously (22, 23). The pGL3-promoter vector was purchased from Promega and contains an SV40 promoter of 202 bp upstream of the luciferase gene with no transcription factor binding sites. The plasmid pGAGC6, containing Sp1 binding sites upstream of the adenovirus TATA box, was a gift from Dr. A.S. Kraft (Medical University of South Carolina, Charleston, SC) and has been described previously (24).

Transient Transfection Assays

MCF-7 cells were plated at 2 × 105 to 3 × 105 per well in six-well plates. The following day, cells were transfected separately with 1 μg of EREv-tk-Luc plasmid, pGL3-promoter plasmid, or pGAGC6 plasmid per well using Fugene6 transfection reagent (Roche, Indianapolis, IN). Seven hours after transfection, indicated amounts of XR5944 or actinomycin D were added into the medium and incubated for a further 24 h. Cells were harvested in 50 μL of Reporter Lysis Buffer (Promega) per well. Following one freeze-thaw cycle, the protein concentration in each sample was quantitated using a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA). A volume of cellular extract corresponding to 20 μg of total protein per sample was used to measure the luminescence using the Luciferase Assay System (Promega). Variation in cell number and transfection efficiency between samples were compensated for by using a fixed amount of total protein and by carrying out each treatment in triplicate and repeating each experiment separately at least thrice.

XR5944 Directly Inhibits the ER Binding to ERE In vitro

To examine the effect of XR5944 on ER binding to DNA, we first carried out electrophoretic mobility shift assay (EMSA) experiments using an MCF-7 cell nuclear extract and an ERE consensus oligonucleotide. MCF-7 is an ER-expressing cell line; therefore, the nuclear extract is a good source for observing the binding of ER in the presence of other cellular components. As shown in Fig. 2, incubation of radiolabeled oligonucleotide with nuclear extract resulted in two significant bands on a polyacrylamide gel subsequent to electrophoresis. Free DNA electrophoresed more rapidly compared with DNA bound to ER protein that has decreased mobility. The DNA binding of ER was significantly inhibited by XR5944 in a dose-dependent manner. Specifically, the ER DNA binding was clearly inhibited by XR5944 at 1.25 μmol/L concentration and was completely blocked by the drug at 25 μmol/L concentration (Fig. 2A). Whereas the EC50 for XR5944 in EMSA was ∼1.25 μmol/L, the recorded IC50 value for this drug in cell culture systems is in the range of 0.4 to 4 nmol/L (14). This change in effective concentrations between in vitro (in test tube) and in vivo (cell culture) data is assumed to be due to the low sensitivity of in vitro assays and also due to the significantly different conditions between the two assays, such as incubation times with drug and incubation temperatures. Similar discrepancies between effective concentrations in vitro and in vivo have previously been seen with a number of cytotoxic drugs, e.g., topotecan, a topoisomerase I inhibitor that has an IC50 of 12 nmol/L in cells (25), but is used at 1 to 50 μmol/L concentrations in in vitro assays.

Figure 2.

EMSAs of the ER binding to the ERE in the absence and presence of XR5944. A, XR5944 effect on the DNA binding of ERs from an MCF-7 nuclear extract. B, XR5944 effect on the DNA binding of recombinant ERα in the presence of E2. The bands of the free DNA probe and the ER-ERE complex are labeled. The concentrations of XR5944 used are as indicated. The phosphorimage shown is one of several repeats.

Figure 2.

EMSAs of the ER binding to the ERE in the absence and presence of XR5944. A, XR5944 effect on the DNA binding of ERs from an MCF-7 nuclear extract. B, XR5944 effect on the DNA binding of recombinant ERα in the presence of E2. The bands of the free DNA probe and the ER-ERE complex are labeled. The concentrations of XR5944 used are as indicated. The phosphorimage shown is one of several repeats.

Close modal

To confirm the inhibition of ER binding to DNA by XR5944, we carried out similar gel shift assays with recombinant ERα instead of the nuclear extract used above. Using 25 μmol/L XR5944, we show that the DNA binding of recombinant ERα in the presence of E2 is completely inhibited by the drug, similar to its inhibition of ER DNA binding from the nuclear extract (Fig. 2B).

XR5944 Inhibits ER-Mediated Transcription

The effect of XR5944 on transcriptional activity of ER in cell culture systems was determined using transient transfection assays with a luciferase reporter plasmid construct containing an ERE inserted upstream of an herpes simplex virus thymidine kinase promoter (EREv-tk-Luc; Fig. 1C). These plasmids were transfected into MCF-7 cells that were subsequently treated with XR5944 at concentrations ranging from 0.75 to 10 nmol/L. The luciferase activity in each sample was measured to determine the level of ER transactivation. The samples were standardized by using a constant amount of total cell lysate protein for each measurement. Our data indicate that the transcriptional activity of ER is significantly inhibited by XR5944, even at 0.75 nmol/L concentration (Fig. 3A). This inhibition by XR5944 seems to be dose dependent, with the maximum inhibition observed at a concentration of 4 nmol/L drug. It seems that the observed inhibition of ER transactivation by XR5944 is not due to cell kill by the drug because no significant cell death is observed even in cultures treated with 10 nmol/L XR5944 for 24 h. In addition, the luciferase activity is measured using a constant amount of total protein of both control and treated samples, which normalizes the measurements to exclude cell death.

Figure 3.

Effects of XR5944 (A) and actinomycin D (B) on ER transactivation in MCF-7 cells. Various concentrations of XR5944 and actinomycin D used are as indicated. Columns, mean of three independent experiments; bars, SE. *, P < 0.01, statistically significant difference.

Figure 3.

Effects of XR5944 (A) and actinomycin D (B) on ER transactivation in MCF-7 cells. Various concentrations of XR5944 and actinomycin D used are as indicated. Columns, mean of three independent experiments; bars, SE. *, P < 0.01, statistically significant difference.

Close modal

The same luciferase reporter system was used to test the activity of the universal transcription inhibitor actinomycin D as a positive control for transcription inhibition. Actinomycin D is an antibiotic that binds to DNA and inhibits RNA synthesis by interfering with RNA polymerase II activity (26, 27). Therefore, it is expected to inhibit the transcription from any transcriptionally active reporter construct, including the EREv-tk-Luc reporter used here. As expected, actinomycin D inhibits the transcription of the reporter gene at the standard concentrations (0.08–0.8 μmol/L) used in cellular assays (Fig. 3B).

The Effect of XR5944 Is Observed Only in the Inducible ER Transactivating Function but not in the Constitutive Promoter Activity

It was shown by our previously discussed data that XR5944 inhibits the transcriptional activity of the reporter vector containing the ERE element. This reporter plasmid also contains a basal herpes simplex virus thymidine kinase promoter in addition to the cis-acting ERE element. To verify that the inhibition of transactivation is due to the specific interference of ER binding to DNA by the drug, but not due to inhibition of basal promoter activity, we carried out similar transactivation assays with a reporter system (pGL3-promoter) containing only an SV40 basal promoter sequence but no specific cis-acting ERE elements (Fig. 1C). Unlike the ERE-containing luciferase reporter, the luciferase gene expression of this system was not significantly affected by XR5944 at the 0.75 to 10 nmol/L concentrations that it inhibited the transactivation of ERE-containing reporter (Fig. 4A). This result suggests that XR5944 specifically inhibit the activity of the ER transcription factor but not that of a basal promoter.

Figure 4.

Effects of XR5944 (A) and actinomycin D (B) on luciferase reporter gene expression by the pGL3-promoter vector. This vector contains an SV40 minimal promoter upstream of the luciferase coding sequence. Columns, mean of three independent experiments; bars, SE. *, P < 0.01, statistically significant difference.

Figure 4.

Effects of XR5944 (A) and actinomycin D (B) on luciferase reporter gene expression by the pGL3-promoter vector. This vector contains an SV40 minimal promoter upstream of the luciferase coding sequence. Columns, mean of three independent experiments; bars, SE. *, P < 0.01, statistically significant difference.

Close modal

XR5944 Preferentially Inhibits Transcription Initiation

We have also carried out a similar study using the reporter containing the SV40 basal promoter sequence (pGL3-promoter) for actinomycin D. Actinomycin D inhibits RNA synthesis by interfering with the RNA elongation activity of RNA polymerase II (26, 27); therefore, it is expected to inhibit the transcription from the pGL3-promoter vector. As expected, actinomycin D was capable of inhibiting reporter gene expression from this vector (Fig. 4B). The fact that no such inhibition was observed with XR5944 (Fig. 4A) at the concentrations at which it inhibited ER activity suggests that, in the luciferase gene expression systems used, XR5944 does not act through inhibiting the RNA elongation or the basal promoter activities.

XR5944 Displays a Certain Degree of Specificity In Action

Our previous data (19) and the data presented here indicate that XR5944 inhibits the DNA binding of transcription factors activator protein 1 and ER, both of which contain 5′-TG-3′ in their consensus binding sites. To determine whether inhibition is specific to such transcription factors, whose consensus DNA binding sites contain 5′-TG-3′ motifs, we carried out similar reporter transactivation assays using the pGAGC6 plasmid (Fig. 1C) that contains tandem repeats of the consensus binding site (5′-GGGGCGGGGC-3′) for the Sp1 transcription factor. Sp1 binds to a DNA sequence containing multiple guanines and cytosines. Moreover, based on the preferred DNA-binding sequence of XR5944 revealed by structural information, the drug should have less affinity for the Sp1 binding site than for ER or activator protein 1 binding sites and therefore should inhibit the Sp1 activity to a lesser extent. Indeed, Sp1 transactivation is not significantly inhibited by XR5944 at the same concentrations at which it inhibits ER transactivation (Fig. 5A). Thus, the inhibition of transcription factor activity by XR5944 seems to be DNA sequence specific. In contrast, the RNA polymerase inhibitor actinomycin D still inhibits the transcription from this Sp1-containing promoter, as expected (Fig. 5B).

Figure 5.

Effect of XR5944 on Sp1 transactivation in MCF-7 cells. The vector contains multiple Sp1 binding sites upstream of the adenovirus major late-initiator TATA box. Columns, mean of three independent experiments; bars, SE. *, P < 0.01, statistically significant difference.

Figure 5.

Effect of XR5944 on Sp1 transactivation in MCF-7 cells. The vector contains multiple Sp1 binding sites upstream of the adenovirus major late-initiator TATA box. Columns, mean of three independent experiments; bars, SE. *, P < 0.01, statistically significant difference.

Close modal

Here, we have investigated the potential of XR5944 to inhibit the DNA binding and the subsequent transcriptional activity of the ER protein. Using in vitro and cell-based assays, we have shown that XR5944 specifically inhibits the DNA-binding and transactivation functions of ER. The potency of XR5944 in inhibiting the ER activity in cultured cells is reflected by its effective concentrations in luciferase reporter assays in which the drug is active at concentrations as low as 0.75 to 4 nmol/L following 24 h of incubation. It needs to be noted that these effective XR5944 concentrations observed by us in luciferase assays, and by Di Nicolantonio et al. (16) in ex vivo experiments, are significantly lower than the XR5944 concentrations used to obtain ER inhibition in EMSA. This difference in effective concentrations is likely due to the low sensitivity of in vitro EMSA and to the significantly different conditions between EMSA and cell culture–related assays, such as incubation times with drug and incubation temperatures. Similar discrepancies between effective concentrations in vitro and in cells have previously been seen with a number of cytotoxic drugs (25).

XR5944 does not seem to inhibit the basal promoter activity of reporter vectors, as shown by the assay with the vector containing an SV40 promoter. In contrast, the RNA polymerase inhibitor actinomycin D is capable of inhibiting the transcription from the same SV40 promoter-reporter vector. Hence, it is suggested that, unlike actinomycin D, XR5944 acts through inhibition of the DNA binding of transcription factors rather than through nonspecific inhibition of RNA elongation in these systems. It is of significance to note that XR5944 exhibits some degree of specificity in its action, as it does not inhibit the reporter gene transactivation by Sp1 transcription factors. Although the remarkable degree of cytotoxic potency exhibited by XR5944 may also suggest direct DNA damage by the drug, indirect evidences such as its effect being independent of p53, which is a major marker for DNA damage (15), and its inhibition of RNA synthesis occurring at a much lower concentration than inhibition of DNA synthesis (9), indicate that the primary action of XR55944 is likely to be related with its transcriptional inhibition. In addition, such potent inhibition of transcription factors has not been observed for other DNA-damaging agents, including DNA major groove binders.

The concept of ligand-DNA interactions with regard to hormones has been a subject of significant research and discussions (2832). The “ligand insertion hypothesis” was a result of such observations, in which hormonal activation is hypothesized to involve a step in which the ligand is inserted between DNA base pairs, facilitated by receptor proteins (33, 34). A few estrogen agonists and antagonists have recently been designed de novo based on this hypothesis (3537). This area of research, in which inhibition of ER binding to DNA is attempted by the use of DNA intercalators, is still in its early stages. It is thus quite significant that XR5944 has been shown to be capable of inhibiting the DNA binding of ER, both in vitro and in vivo. Together, these data render importance to XR5944 as a compound with an attractive mechanism of ER inhibition that may be used in the development of potential therapeutic agents for treatment of ER-expressing breast cancers.

Currently, endocrine therapy is the treatment of choice for patients with ER-expressing breast cancers. Only about half of metastatic breast cancers expressing ER respond to endocrine therapy, and, of these, only 50% reduction is seen in the development of recurrent disease (8). These statistics reflect the limitations of the use of endocrine therapy in treatment of ER-expressing breast cancers, where half of the cases seem to be de novo resistant to endocrine therapy and a significant proportion tend to develop acquired resistance. A major reason for the observed de novo or acquired resistance is likely to be the estrogen-independent receptor activation that continues to control the expression of estrogen-regulated genes. Such estrogen-independent receptor activation has been shown to occur as a result of various mechanisms, including phosphorylation of ER by different protein kinases (38, 39) and mutations in ER (40, 41). A significant feature of these resistance mechanisms is that they all still involve the DNA binding of ERs. Thus, a DNA intercalator that binds and occupies the ER binding site, and thereby inhibits the DNA binding of ER transcription factors, will be a useful breast cancer therapeutic agent that would overcome the resistance to the existing endocrine therapies.

In addition to the drug resistance, other disadvantages of endocrine therapy include the increased risks of endometrial cancer and thromboembolic disease seen with selective estrogen modulators like tamoxifen that act as estrogen agonists in bone, liver, and uterus (42, 43). Unlike tamoxifen, which binds to ER, drugs targeting ERE are less likely to display ER agonist activity and subsequent side effects. Moreover, this approach of targeting ERE may also be used in conjunction with other endocrine agents for management of ER-expressing breast cancers.

Previous data has suggested that while XR5944 may be a general inhibitor of transcription, it may also inhibit specific genes at low concentrations (9). We speculate that the specific transcription factor(s) inhibited by XR5944 in different cancer cells are likely to display a similar sequence specificity in their DNA binding. Our data presented here indicate that ER is specifically inhibited by XR5944 in MCF-7 breast cancer cells. Due to the high levels of ER activity observed, it is likely that ER is the major target for XR5944 action in these cells. Interestingly, it has been found that breast and gynecologic malignancies are very sensitive to XR5944 in ex vivo assays (16), possibly owing to the high levels of ER activity in these cancers.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Key T, Appleby P, Barnes I, Reeves G. The Endogenous Hormones and Breast Cancer Collaborative Group. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies.
J Natl Cancer Inst
2002
;
94
:
606
–16.
2
Magnusson C, Baron JA, Correia N, Bergstrom R, Adami HO, Persson I. Breast-cancer risk following long-term oestrogen- and oestrogen-progestin-replacement therapy.
Int J Cancer
1999
;
81
:
339
–44.
3
Schairer C, Gail M, Byrne C, et al. Estrogen replacement therapy and breast cancer survival in a large screening study.
J Natl Cancer Inst
1999
;
91
:
264
–70.
4
Berger T, Brigl M, Herrmann JM, et al. The apoptosis mediator mDAP-3 is a novel member of a conserved family of mitochondrial proteins.
J Cell Sci
2000
;
113
:
3603
–12.
5
Jensen EV, Jacobson HI. Basic guides to the mechanism of estrogen action.
Recent Prog Horm Res
1962
;
18
:
387
–414.
6
Pearce ST, Jordan VC. The biological role of estrogen receptors α and β in cancer.
Crit Rev Oncol Hematol
2004
;
50
:
3
–22.
7
Dixon JM, Bundred N. Aromatase inhibitors for early breast cancer therapy: a choice of effective treatment strategies.
Eur J Surg Oncol
2006
;
32
:
123
–5.
8
Kurebayashi J. Resistance to endocrine therapy in breast cancer.
Cancer Chemother Pharmacol
2005
;
56
:
s39
–46.
9
Byers SA, Schafer B, Sappal DS, Brown J, Price DH. The antiproliferative agent MLN944 preferentially inhibits transcription.
Mol Cancer Ther
2005
;
4
:
1260
–7.
10
Gamage SA, Spicer JA, Finlay GJ, et al. Dicationic bis(9-methylphenazine-1-carboxamides): relationships between biological activity and linker chain structure for a series of potent topoisomerase targeted anticancer drugs.
J Med Chem
2001
;
44
:
1407
–15.
11
Finlay GJ, Riou JF, Baguley BC. From amsacrine to DACA (N-[2-(dimethylamino)ethyl]acridine-4-carboxamide): selectivity for topoisomerases I and II among acridine derivatives.
Eur J Cancer
1996
;
32A
:
708
–14.
12
Mistry P, Stewart AJ, Dangerfield W, et al. In vitro and in vivo characterization of XR11576, a novel, orally active, dual inhibitor of topoisomerase I and II.
Anticancer Drugs
2002
;
13
:
15
–28.
13
Vicker N, Burgess L, Chuckowree IS, et al. Novel angular benzophenazines: dual topoisomerase I and topoisomerase II inhibitors as potential anticancer agents.
J Med Chem
2002
;
45
:
721
–39.
14
Stewart AJ, Mistry P, Dangerfield W, et al. Antitumor activity of XR5944, a novel and potent topoisomerase poison.
Anticancer Drugs
2001
;
12
:
359
–67.
15
Sappal DS, McClendon AK, Fleming JA, et al. Biological characterization of MLN944: a potent DNA binding agent.
Mol Cancer Ther
2004
;
3
:
47
–58.
16
Di Nicolantonio F, Knight LA, Whitehouse PA, et al. The ex vivo characterization of XR5944 (MLN944) against a panel of human clinical tumor samples.
Mol Cancer Ther
2004
;
3
:
1631
–7.
17
Harris SM, Scott JA, Brown JL, Charton PA, Mistry P. Preclinical anti-tumor activity of XR5944 in combination with carboplatin or doxorubicin in non-small-cell lung carcinoma.
Anticancer Drugs
2005
;
16
:
945
–51.
18
Harris SM, Mistry P, Freathy C, Brown JL, Charlton PA. Antitumour activity of XR5944 in vitro and in vivo in combination with 5-fluorouracil and irinotecan in colon cancer cell lines.
Br J Cancer
2005
;
92
:
722
–8.
19
Dai J, Punchihewa C, Mistry P, Ooi AT, Yang D. Novel DNA bis-intercalation by MLN944, a potent clinical bisphenazine anticancer drug.
J Biol Chem
2004
;
279
:
46096
–103.
20
Beato M, Chalepakis G, Schauer M, Slater EP. DNA regulatory elements for steroid hormones.
J Steroid Biochem Mol Biol
1989
;
32
:
737
–47.
21
Lannigan DA, Notides AC. Estrogen regulation of transcription.
Prog Clin Biol Res
1990
;
322
:
187
–97.
22
Nunez SB, Medin JA, Braissant O, et al. Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.
Mol Cell Endocrinol
1997
;
127
:
27
–40.
23
Sawatsri S, Samid D, Malkapuram S, Sidell N. Inhibition of estrogen-dependent breast cell responses with phenylacetate.
Int J Cancer
2001
;
93
:
687
–92.
24
Biggs JR, Kudlow JE, Kraft AS. The role of the transcription factor Sp1 in regulating the expression of the WAF1/CIP1 gene in U937 leukemic cells.
J Biol Chem
1996
;
271
:
901
–6.
25
Mi ZH, Malak H, Burke TG. Reduced albumin-binding promotes the stability and activity of topotecan in human blood.
Biochemistry
1995
;
34
:
13722
–8.
26
Reich E, Goldberg IH. Actinomycin and nucleic acid function.
Prog Nucleic Acid Res Mol Biol
1964
;
3
:
183
–234.
27
Sentenac A, Simon EJ, Fromageot P. Initiation of chains by RNA polymerase and the effects of inhibitors studied by a direct filtration technique.
Biochim Biophys Acta
1968
;
161
:
299
–308.
28
Nutter LM, Ngo EO, Abul-Hajj YJ. Characterization of DNA damage induced by 3,4-estrone-o-quinone in human cells.
J Biol Chem
1991
;
266
:
16380
–6.
29
Telang NT, Suto A, Wong GY, Osborne MP, Bradlow HL. Induction by estrogen metabolite 16 α-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells.
J Natl Cancer Inst
1992
;
84
:
634
–8.
30
Liehr JG, Gladek A, Macatee T, Randerath E, Randerath K. DNA adduct formation in liver and kidney of male Syrian hamsters treated with estrogen and/or α-naphthoflavone.
Carcinogenesis
1991
;
12
:
385
–9.
31
DeSombre ER, Shafii B, Hanson RN, Kuivanen PC, Hughes A. Estrogen receptor-directed radiotoxicity with Auger electrons: specificity and mean lethal dose.
Cancer Res
1992
;
52
:
5752
–8.
32
Snyder RD, Brown JE. Evidence for and role of the dimethylamino group in tamoxifen DNA intercalation in intact Chinese hamster V79 cells.
Drug Chem Toxicol
2002
;
25
:
473
–9.
33
Hendry LB, Mahesh VB. A putative step in steroid hormone action involves insertion of steroid ligands into DNA facilitated by receptor proteins.
J Steroid Biochem Mol Biol
1995
;
55
:
173
–83.
34
Hendry LB, Bransome ED, Jr., Mahesh VB. The ligand insertion hypothesis in the genomic action of steroid hormones.
J Steroid Biochem Mol Biol
1998
;
65
:
75
–89.
35
Mahesh VB, Lewis WR, Cannady WE, et al. Gene based pharmacophores for drug design.
Med Chem Res
2001
;
10
:
440
–55.
36
Hendry LB, Chu CK, Copland JA, Mahesh VB. Antiestrogenic piperidinediones designed prospectively using computer graphics and energy calculations of DNA-ligand complexes.
J Steroid Biochem Mol Biol
1994
;
48
:
495
–505.
37
Sidell N, Tanmahasamut P, Ewing DE, Hendry LB. Transcriptional inhibition of the estrogen response element by antiestrogenic piperidinediones correlates with intercalation into DNA measured by energy calculations.
J Steroid Biochem Mol Biol
2005
;
96
:
335
–45.
38
Michalides R, Griekspoor A, Balkenende A, et al. Tamoxifen resistance by a conformational arrest of the estrogen receptor α after PKA activation in breast cancer.
Cancer Cell
2004
;
5
:
597
–605.
39
Campbell RA, Bhat-Nakshatri P, Patel NM, Constantinidou D, Ali S, Nakshatri H. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor α: a new model for anti-estrogen resistance.
J Biol Chem
2001
;
276
:
9817
–24.
40
Weis KE, Ekena K, Thomas JA, Lazennec G, Katzenellenbogen BS. Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein.
Mol Endocrinol
1996
;
10
:
1388
–98.
41
Daffada AA, Johnston SR, Smith IE, Detre S, King N, Dowsett M. Exon 5 deletion variant estrogen receptor messenger RNA expression in relation to tamoxifen resistance and progesterone receptor/pS2 status in human breast cancer.
Cancer Res
1995
;
55
:
288
–93.
42
Bergman L, Beelen ML, Gallee MP, Hollema H, Benraadt J, van Leeuwen FE; Comprehensive Cancer Centres' ALERT Group. Risk and prognosis of endometrial cancer after tamoxifen for breast cancer. Assessment of Liver and Endometrial cancer Risk following Tamoxifen.
Lancet
2000
;
356
:
881
–7.
43
Sismondi P, Biglia N, Volpi E, Giai M, de Grandis T. Tamoxifen and endometrial cancer.
Ann N Y Acad Sci
1994
;
734
:
310
–21.