Promising Preclinical Activity of 2-Methoxyestradiol in Multiple Myeloma¹

MM³ is a malignant neoplasm of terminally differentiated plasma cells residing in the bone marrow. In 2002, approximately 14,600 new cases of myeloma will be diagnosed in the United States, and over 10,800 patients will die of the disease (1). Current treatment strategies including alkylating agents, corticosteroids, stem cell transplantation, and thalidomide are not curative, and the median survival is approximately 3–4 years (2, 3, 4); therefore, there is a need for new agents to combat this disease.

2ME2 is a natural metabolite of the endogenous estrogens 17β-estradiol and 2-hydroxyestradiol (5). It has shown promising antitumor effects in preclinical studies (6, 7). 2ME2 directly induces tumor cell apoptosis, in addition to its potent antiangiogenic properties (6, 7, 8, 9). Because angiogenesis is increased in myeloma (10, 11), and given the unique antitumor and antiangiogenic properties of 2ME2, we hypothesized that the agent may be of therapeutic benefit in this disease. In this study, we report the results of our laboratory studies with 2ME2 against a variety of myeloma cell lines and primary cells in vitro. In addition, we also studied its effects on myeloma xenograft growth in a mouse model.

2ME2 (a generous gift of Entremed, Rockville, MD) was dissolved in DMSO as a 30 mm stock solution and diluted in S-10 (RPMI 1640, 10% FCS, penicillin/streptomycin, and l-glutamine) for the in vitro experiments. DMSO control was used for the in vitro studies. The solubility of 2ME2 in water is very low. To ensure uniform absorption of the drug, a liposomal preparation of 2ME2 dissolved in 1,2-dioleoyl-SN-glycero-3-phosphocholine (Avanti Polar Lipids, Alabaster, AL), a gift from Glenn Swartz (Entremed), was used for the in vivo xenograft experiments. Empty liposomes and untreated animals were used as controls for the xenograft studies. In cell culture media, the drug is not free but is complexed with serum proteins that enhance its solubility, and hence a liposomal preparation was not needed.

The MM cell lines KAS-6/1, ANBL6 and ARH9 were a generous gift of Diane F Jelinek (Mayo Clinic, Rochester, MN). RMPI 8226 and My-5 were purchased from the American Type Culture Collection. RMPI 8226, My-5, and ARH9 were maintained in S-10 medium. KAS-6/1 and ANBL6 were maintained in S-10 supplemented with interleukin 6 (R&D, Minneapolis, MN) at a concentration of 1 ng/ml.

Apoptosis was detected as described previously (12). In brief, bone marrow samples were obtained from waste bone marrow aspirates taken from patients with myeloma. All patients provided signed informed consent for the research use of the waste marrow, and this study was approved by the Institutional Review Board of the Mayo Clinic/Foundation. RBCs in the bone marrow were lysed with ACK (0.155 m NH₄Cl and 0.1 m KHCO₃), and the nucleated cells were washed twice in PBS and cultured in S-10 with and without 2ME2 at 37°C. After 48 h, cells were washed with PBS and stained with the monoclonal antibodies CD45-FITC (Becton Dickinson) and CD38 phycoerythrin (CD38-PE; Becton Dickinson, San Jose, CA). To differentiate viable, apoptotic, and dead cells, the DNA dye 7-AAD (Calbiochem) was also used as the third color. Samples were analyzed on a FACScan (Becton Dickinson) within 30 min of staining.

A two-color flow cytometry assay using annexin V-FITC (Caltag, Burlingame, CA) and PI (Sigma) was used to differentiate viable, apoptotic, and dead cells after 48 h of incubation with 2ME2. Briefly, 1 × 10⁶ cells were taken from each well, washed twice in PBS, and resuspended in 100 μl of annexin V binding buffer (Ca²⁺). Five μl of annexin V were added and incubated at 4°C for 15 min. Samples were then washed in 3 ml of binding buffer, resuspended in 500 μl of binding buffer with 5 μg/ml PI, and then analyzed on a flow cytometer within 30 min. Cells that take up PI are dead, whereas cells that are positive for annexin V but negative for PI have undergone apoptosis. The flow cytometry data were analyzed using the Cell Quest software package (Becton Dickinson).

Cell proliferation assays were performed using tritiated thymidine uptake. Cells (50,000) were seeded in triplicate in 96-well plates, and 2.5 μCi of tritiated thymidine were added followed by incubation at 37°C for 4 h. The cells were then washed, and retained activity was measured using a beta counter (Beckman, Fullerton, CA).

Cels (1 × 10⁶) were washed twice in PBS, resuspended in 2 ml of lysolecithin, and incubated on ice for 30 min. The cells were washed again twice in PBS and resuspended in 0.5 ml of PBS with RNase (30 units/ml). After a 30-min incubation at 37°C, 10 μl of PI (1 mg/ml) were added, and the cells were stored at 4°C and run on a flow cytometer (Becton Dickinson).

Four-week-old female SCID mice (CB17) were purchased from Harlan Sprague Dawley (Madison, WI). The mice were maintained in a pathogen-free environment at the institutional animal care facility and cared for using accepted standards. The mice were given 250 cGy of total body irradiation, and 24 h later, the mice received injection of 1 × 10⁷ KAS-6/1 cells in the right flank. The cells were counted and assessed for viability by trypan blue exclusion and washed three times in PBS (Life Technologies, Inc.) before injection. Two weeks after implantation of the cells, therapy was initiated. One group of mice did not receive any therapy (control group), the second group was injected i.p. with empty liposomes, and the third group was treated i.p. with liposomal 2ME2 at a dose of 150 mg/kg. This dose was based on studies performed at Entremed and represented a well-tolerated dose that showed efficacy in other tumor models; there are no reported maximum tolerated dose or LD₁₀ for this drug.⁴ The mice were treated with the drug daily for 2 weeks and observed for another 3 weeks after cessation of therapy. Tumor xenografts were measured in two dimensions, three times a week, using calipers. Tumor volumes were calculated using the following formula: volume = (a²b)/2, where a is the smaller diameter. The protocol was approved by the Institutional Animal Care and Use Committee at Mayo Foundation in accordance with federal regulations.

Five different myeloma cell lines (ANBL6, ARH9, KAS-6/1, My-5, and RPMI 8226) were incubated in growth medium with the addition of 2ME2 at three different concentrations (0.1, 1, and 10 μm). Tritiated thymidine uptake was measured as a marker of DNA replication and cell proliferation. 2ME2 inhibited myeloma cell proliferation in a dose-dependent manner in all cell lines tested (Fig. 1). Exposure of plasma cell lines to 2ME2 resulted in cell cycle arrest at the G₂-M phase (Fig. 2, M3; 18–38.6%) with a concomitant decrease in the number of cells in G₀-G₁ phase (Fig. 2, M1; 36–9.4%). As expected, the number of cells in the sub-G₁ phase increased from 5.6% to 10% (Fig. 2, M4).

2ME2 induced a dose-dependent increase in the number of cells undergoing apoptosis in three (ANBL6, KAS-6/1, and My-5) of the five cell lines tested (Figs. 3 and 4). Accompanying this effect on apoptosis (annexin V positive, PI negative), there was an increase in the number of dead cells (PI positive) in all cell lines tested. The proportion of dead cells increased with higher concentrations of 2ME2, suggesting a drug-induced effect (Fig. 5).

Plasma cells are identified by their strong CD38 expression and dim to absent CD45 expression (Fig. 6; Ref. 12). This method identifies cells that are in excess of 95% pure myeloma cells. These cells were selected and analyzed for 7-AAD positivity as a measure of apoptosis (12). Apoptotic cells are permeable to 7-AAD, allowing it to bind to DNA. In two of the patient samples (patients 1 and 4), there was a dose-dependent increase in the number of cells undergoing apoptosis compared with controls (Fig. 7). However, in two of the primary myeloma cell samples tested, 2ME2 inhibited apoptosis. Thus, it appears that, similar to other drugs used for myeloma, only a fraction of the samples may be sensitive to the drug.

Measurable tumor xenografts developed in all mice (30 mice; 10 mice/group). All tumors were measured in two dimensions three times a week, and tumor volumes were estimated as described in “Materials and Methods.” 2ME2 induced a rapid and complete regression of tumor growth, whereas the tumors continued to grow in both the control and placebo-treated mice (Fig. 8). All of the mice had a postmortem looking for small foci of disease, and there were none in the treated group. Mice treated with 2ME2 developed weight loss after 2 weeks of therapy; therefore, 2ME2 was discontinued, and all mice were observed. The mice regained the weight without the reappearance of the tumors during the remaining observation period.

The mainstay of therapy for MM for many years has been a combination of melphalan and prednisone, which produces a response rate of 50% (13). However, complete responses are uncommon (<10%), and only one-fourth of patients treated with this combination survive up to 5 years (13). Recently, many centers have been offering high-dose therapy with autologous stem cell transplantation to patients with MM. High-dose therapy produces response rates of 75–90% (14, 15) with complete responses in 20–40% and a 5-year survival of 52% in one series (16). However, transplantation does not cure the disease, and the survival curves do not achieve a plateau. Patients who relapse after high-dose therapy can be treated with thalidomide, and approximately 25–35% of patients will respond (17).

2ME2, a natural endogenous product of estradiol metabolism, has been shown to have activity against tumor cell lines in preclinical models (6). In this study, 2ME2 induced apoptosis and cell death in all myeloma cell lines tested and had significant activity against primary plasma cells in two of the four patient samples studied. This compares favorably with other agents currently used for therapy of myeloma. Other studies have shown that 2ME2 induced apoptosis both by stabilizing wild-type p53 and by inducing phosphorylation of Bcl-2 leading to its inactivation (18, 19). However, these mechanisms are not universal in all cells assessed (6).

In our in vitro studies, the percentage of myeloma cells undergoing apoptosis after a 48-h incubation with 2ME2 was relatively small (<25%) compared with what has been observed with pancreatic cancer cell lines (30–90%; Ref. 20). This difference may be due to differences in sensitivity to 2ME2-induced apoptosis between the cell lines. However, myeloma cell viability was low at 48 h. Our studies show that 2ME2 also inhibits myeloma cell proliferation by inducing cell cycle arrest at the G₂-M phase. This supports similar observations with the drug against pancreatic carcinoma cell lines, prostate cancer cells, and lymphoblastic cell lines (20, 21, 22). In addition, 2ME2 has been shown to arrest cells in mitosis without inhibiting tubulin depolymerization (23). Our data are in keeping with other reports that 2ME2 blocks the cell cycle and induces apoptosis (22, 23, 24, 25, 26). The concentrations of 2ME2 that are required to effect apoptosis are much higher in patient samples compared with myeloma cell lines, probably because the latter proliferate slowly and are typically resistant to most forms of therapy compared with cell lines. However, given the anticipated low toxicity of 2ME2, we fully expect to achieve adequate tissue concentrations in clinical trials.

Finally, our in vivo studies show that 2ME2 can suppress myeloma xenograft growth in SCID mice. 2ME2 was reasonably tolerated by the mice. Transient weight loss was observed, but this resolved rapidly with cessation of therapy. However, the tumors did not reappear, suggesting that the effect of the drug may be prolonged even after therapy is stopped.

Angiogenesis is essential for tumor growth because tumor cells require a constant supply of nutrients and growth factors to sustain their growth. Indeed, without new vessel formation, tumors cannot reach diameters beyond 1 mm because diffusion will not be able to keep up with their demands for nutrients (11, 24). Angiogenesis is increased in myeloma, and bone marrow microvessel density correlates with prognosis. Patients with lower levels of new vessel formation have an improved survival compared with those with higher levels of new vessel formation (25). Treatment of patients with MM using high-dose therapy does not lead to a significant change in marrow microvessel density, and this may possibly be a factor in relapse of the disease (26). A number of studies have shown that 2ME2 can inhibit angiogenesis (8, 27, 28, 29). The postulated mechanisms include inhibition of endothelial cell proliferation and migration (8), induction of endothelial cell apoptosis (28), and down-regulation of vascular endothelial growth factor expression (29). Vascular endothelial growth factor is a survival factor for myeloma cells; therefore, its down-regulation could add to the effects of the drug against myeloma cells (30). Targeting blood vessels in myeloma may lead to improved responses or decreased risk of relapse after high-dose therapy and stem cell transplantation. The antiangiogenic properties of 2ME2 and the importance of angiogenesis in myeloma provide additional rationale for the study of 2ME2 in myeloma. However, we have no data indicating that 2ME2 inhibits myeloma angiogenesis at present, and we are planning such studies in the context of clinical trials.

Recently, Phase I clinical trials with 2ME2 have been conducted in breast and prostate cancer. Based on the preclinical activity seen in this study, the antiangiogenic properties of the drug, and the limited options available to patients with advanced myeloma, we believe translational clinical trials to test the efficacy of 2ME2 are warranted in the disease. We have thus initiated a multi-institutional Phase II clinical trial with 2ME2 for patients with relapsed and plateau-phase myeloma, and accrual to this study is ongoing.

We thank Entremed for providing 2ME2 for this study and Victor Pribluda and Glen Swartz of Entremed for helpful suggestions on the manuscript.