Growth Suppression of Ovarian Cancer Xenografts in Nude Mice by Vitamin D Analogue EB1089 Free

Ovarian cancer is the leading cause of death among gynecologic malignancies, which results in more deaths than all other gynecologic cancers combined (1). In contrast to declining death rates for cervical and uterus cancers, the annual report of ovarian cancer mortality has risen by 250% since 1930 (2). The prognosis is generally poor for ovarian cancer patients of whom only ∼32% survive for >5 years. The dismal response of advanced ovarian cancer to current treatments necessitates the development of alternative therapies to curb this deadly disease.

The active form of vitamin D, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], has been shown to regulate differentiation and proliferation of multiple human cancer cell lines and thus is a potential therapeutic agent for cancers. A suppressive role for 1,25(OH)₂D₃ in ovarian cancer has been speculated based on the inverse correlation between ovarian cancer incidence and mortality rates and sunlight exposure (3, 4). In addition, a case-control study in Mexico reported an inverse association between dietary vitamin D content and risk for ovarian cancer (5). Consistent with these epidemiologic analyses, recent studies in our laboratories have shown that 1,25(OH)₂D₃ inhibits the growth of multiple ovarian cancer cell lines, including OVCAR3, CAOV3, A2008, etc. (6), by causing cell cycle arrests at G₁-S (6) and G₂-M (7) checkpoints. These findings provide a molecular foundation for further investigation of the in vivo growth-suppressing effects of 1,25(OH)₂D₃ compounds.

Because the concentration that induces the growth suppression of ovarian cancer cell lines is expected to cause hypercalcemia in vivo, 1,25(OH)₂D₃ cannot be directly used clinically to treat ovarian cancer patients. Chemical modifications of the 1,25(OH)₂D₃ have yielded several analogues that display enhanced tumor-suppressing but less calcemic activity (8). Among these is EB1089, a deltanoid derivative generated by Leo Pharmaceutical Products (Ballerup, Denmark) through structural alteration of the side chain of 1,25(OH)₂D₃ (9). EB1089 has been shown to suppress the growth of prostate (10, 11), breast (12), colon (13), and retinoblastoma (14) tumors, but the in vivo response of human ovarian cancer xenografts to EB1089 has not been examined.

In this article, we report that EB1089 inhibits the growth of OVCAR3 xenograft tumors in nude mice without causing hypercalcemia. Further analysis indicates that the suppression of the tumor growth in vivo is due to the combined effect of EB1089 in inhibiting cell proliferation as well as inducing apoptosis.

Materials. 1,25(OH)₂D₃ was purchased from Calbiochem (La Jolla, CA). EB1089 (seocalcitol) and corresponding placebo were generously provided by Leo Pharmaceutical Products. Anti-human Ki-67 antibody was purchased from BD PharMingen, Inc. (San Diego, CA).

Cell Culture and Assays for Growth, Apoptosis, and Transcriptional Activation of the GADD45 Reporter. OVCAR3 human ovarian cancer cells (HTB-161, obtained from American Type Culture Collection, Rockville, MD) were cultured in RPMI 1640 supplemented with 15% fetal bovine serum (Life Technologies, Grand Island, NY), 2 mmol/L l-glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and 10 μg/mL bovine insulin. BG-1 cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum.

To determine cell numbers, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were done as described (15). Absorption at 595 nm (A_{595 nm}) was measured on a MRX microplate reader (Dynex Technologies, Chantilly, VA). Cell numbers were calculated based on a standard curve. Apoptosis was determined by flow cytometry analysis after cells were stained with Annexin V-FITC and propidium iodide following manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, CA). Transcriptional activation of the vitamin D receptor was done as described (6). Briefly, 4 hours post-transfection with GADD45Luc and pCMVgal, OVCAR3 cells were treated with vehicle, EB1089, or 1,25(OH)₂D₃ in fresh medium for 36 hours. Cells were harvested and luciferase and β-galactosidase activities were determined as described (6).

Nude Mouse Studies of OVCAR3 Tumor Xenografts. All mice were handled according to the Guide for the Care and Use of Laboratory Animals. Mouse studies were carried out following the procedures approved by the Institutional Animal Care and Use Committee at University of South Florida. For inoculation into nude mice, OVCAR3 cells were washed with PBS, digested with trypsin, resuspended in RPMI 1640 containing fetal bovine serum, and pooled. After centrifugation, cells were resuspended in Matrigel (BD Biosciences Discovery Labware, Bedford, MA)-RPMI 1640 (1:1) at a concentration of 5× 10⁶ cells per 100 μL. Cell/Matrigel mixture of 100 μL was injected s.c. into 6-week-old female athymic nu/nu mice (Harlan Sprague-Dawley, Indianapolis, IN) on the dorsal surface. The mice were fed a vitamin D–deficient diet supplemented with 0.47% calcium (Harlan Teklad, Madison, WI) for the duration of the study. Tumor volumes were monitored by caliper measurement of the length and width and calculated using the formula of length × width × 0.5 the greater of length or width. Treatment was begun when tumors reached volumes of ∼150 mm³ in average, which took ∼4 weeks. Mice were randomized and treated daily by gavage with placebo or EB1089 at 0.3 or 1.0 μg/kg body weight in a volume of 20 μL. Tumor volumes and body weights were monitored every 5 days over the course of treatment. Mice were sacrificed after 30 days of treatments and tumors were removed and fixed in 10% neutral buffered formalin for histologic and immunohistochemical analyses.

Calcium Measurements. To determine the effect of active vitamin D compounds on serum calcium, nude mice without tumor xenografts were treated with placebo, EB1089, or 1,25(OH)₂D₃. Blood samples were obtained from the retro-orbital sinus at 0, 15th, and 30th days of the treatment. Blood samples from three mice were used for each group. Serum calcium was measured as a paid service by Antech Diagnostics (Southaven, MS) using a colorimetric method on Hitachi 747 analyzer. The measurement was repeated twice.

Tumor Histology and Immunohistochemical Analysis. Tumors were embedded in paraffin, sectioned at 5 μm, and stained with H&E. Cell proliferation was assessed by quantification with Ki-67 immunohistochemistry. The anti-human Ki-67 antibody was 1:400 diluted and immunostaining was done on the DAKO Autostainer using the Chemicon Mouse-to-Mouse Peroxidase Detection kit (Serologicals, Inc., Temecula, CA). Ki-67-positive cells were scored by visual examination of 40 randomly selected fields of ×400 magnification containing at least 2,000 cells.

Tissue Terminal Deoxynucleotide Transferase–Mediated dUTP Nick End Labeling Assay. Histologic analysis of DNA fragmentation was used to identify apoptotic cells in paraffin sections of the xenograft tumors. In situ terminal deoxynucleotide transferase–mediated dUTP nick end labeling (TUNEL) was done using the Apoptag Peroxidase In situ Apoptosis Detection Kit (Serologicals). TUNEL-positive cells were quantified in 40 randomly selected high-power fields (×400) of each tissue section.

EB1089 Suppresses the Growth of Ovarian Cancer Cells and Transcriptionally Activates the GADD45 Reporter Gene at Concentrations Lower Than 1,25(OH)₂D₃. Before investigating the in vivo response of ovarian tumor xenografts to EB1089, we first examined the activity of EB1089 in suppressing the growth of ovarian cancer cells in vitro using 1,25(OH)₂D₃ as a control. OVCAR3 and BG-1 cells were treated with vehicle, 1,25(OH)₂D₃, or EB1089 at indicated concentrations and the cell growth was determined in MTT assays, As shown in Fig. 1A and B. 1,25(OH)₂D₃ at 10⁻⁷ mol/L caused a significant decrease in the growth of OVCAR3 and BG-1 cells after treatment for 6 days (43.9 ± 7.8% for OVCAR3 and 62.4 ± 17.7% for BG-1) and 9 days (59.8 ± 5.3% for OVCAR3 and 71.1 ± 14.2% for BG-1). The inhibition was not observed for 1,25(OH)₂D₃ at 10⁻⁸ or 10⁻⁹ mol/L, confirming our data from previous studies (7). In contrast to 1,25(OH)₂D₃, EB1089 at 10⁻⁸ mol/L inhibited the growth by 35.9 ± 9.6% and 43.1 ± 5.0% for OVCAR3 and by 55.4 ± 23.6% and 66.3 ± 14.7% for BG-1 after 6- and 9-day treatments, respectively (Fig. 1A), which is comparable with the inhibition by 1,25(OH)₂D₃ at 10⁻⁷ mol/L. The data suggest that EB1089 is 10 times more active than 1,25(OH)₂D₃. EB1089 at 10⁻⁷ mol/L inhibited growth by 39.1 ± 4.1% and 49.4 ± 6.9% for OVCAR3 and by 57.6 ± 29.1% and 63.4 ± 18.6% for BG-1 for 6- and 9-day treatments, respectively. The magnitude of the growth inhibition did not surpass that induced by 1,25(OH)₂D₃ at 10⁻⁷ mol/L or EB1089 at 10⁻⁸ mol/L, suggesting the existence of a saturation point for 1,25(OH)₂D₃ compounds in suppressing the growth of ovarian cancer cells. Treatment with EB1089 for short time at all tested concentrations did not cause significant growth suppression, suggesting that the growth suppression by EB1089, similar to 1,25(OH)₂D₃, is a chronic process, although its effective concentration is lower than the 1,25(OH)₂D₃.

Our previous studies have identified GADD45 as a primary target gene for 1,25(OH)₂D₃, which specifically mediates the inhibitory effect of the hormone on cell cycle progression through the G₂-M checkpoint (6). The induction of GADD45Luc by 1,25(OH)₂D₃ is mediated through an exonic vitamin D response element that binds the vitamin D receptor (6). Thus, GADD45Luc provides a measurement for the transcriptional activation of vitamin D receptor. To test whether EB1089 regulates the transcriptional activation of the vitamin D receptor more effectively than 1,25(OH)₂D₃, the activation of the GADD45Luc reporter by the natural and synthetic 1,25(OH)₂D₃ compounds was compared in transient transfection assays. Consistent with the growth analyses, the reporter activation by EB1089 was more efficient than 1,25(OH)₂D₃ at most concentrations, but no difference was detected for the maximal activation induced at the saturating concentration (Fig. 1C).

Besides cell cycle arrests at G₁-S and G₂-M checkpoints, our studies showed that treatment of OVCAR3 cells with 1,25(OH)₂D₃ for 6 days induces apoptosis (Fig. 1D). To determine whether EB1089 also induces apoptosis in ovarian cancer cells, the apoptotic index of OVCAR3 cells treated with vehicle or EB1089 was determined by flow cytometry after staining for Annexin V. As shown in Fig. 1D, EB1089 induced apoptosis after 6-day treatment, whereas no increase in apoptosis was observed at earlier time points. Treatment for >6 days did not further increase the rate of apoptosis. Because the apoptotic analyses used EB1089 and 1,25(OH)₂D₃ at the saturating concentration, the magnitude of apoptotic induction by EB1089 is comparable with that induced by 1,25(OH)₂D₃.

Overall, the above analyses show that EB1089 is transcriptionally more active than 1,25(OH)₂D₃, inhibits the growth of ovarian cancer cells at lower concentrations, and induces apoptosis in a similar way as 1,25(OH)₂D₃. The analyses argue strongly for further investigation of its in vivo effects in whole animal experiments.

EB1089 Inhibits the Growth of OVCAR3 Tumor Xenografts in Nude Mice without Causing Hypercalcemia. After the growth-suppressing activity of EB1089 was verified in vitro using OVCAR3 and BG-1 cells, we next investigated its in vivo effect on the growth of OVCAR3 tumor xenografts as described in MATERIALS AND METHODS. As shown in Fig. 2A, the size of OVCAR3 tumor xenografts treated with the placebo increased proportionally during the treatment. Treatment with EB1089 at 0.3 or 1 μg/d/kg body weight almost completely suppressed the growth of the tumors. In comparison with the placebo controls, the tumor suppression by EB1089 at both concentrations after 30 days reached statistical significance (P<0.01). In contrast, no significant difference was observed between the size of the tumors treated with EB1089 at 0.3 and 1.0μg/d/kg for 30 days (P = 0.246). Due to the heterogeneity of the tumor sizes to begin with, there is a significant variation among the tumor volumes in each group, but the suppressive effect of EB1089 on the tumor growth is obvious when the representative tumors from the placebo and treated groups were placed side-by-side to compare their size (Fig. 2B).

Mice treated with EB1089 at both doses did not show signs of morbidity during the treatment. Their body weight was not affected by the treatment with EB1089 either (data not shown). Because active vitamin D compounds are known to increase the level of blood calcium causing hypercalcemia, we next examined the effects of EB1089 and 1,25(OH)₂D₃ on blood calcium. Separate groups of nude mice was treated with 1,25(OH)₂D₃ and EB1089 and the serum calcium was measured at 0, 15th, and 30th days of the treatment. As shown in Fig. 2C, although EB1089 increased the level of serum calcium in a dose-dependent manner, it did not cause clinical hypercalcemia. In contrast, 1,25(OH)₂D₃ at 1.0 μg/d/kg induced hypercalcemia as expected.

EB1089 Alters Tumor Histology and Decreases Proliferation but Increases Apoptosis of Ovarian Tumor Cells In vivo. EB1089 has been shown to change the histology of prostate tumor xenografts (11). To investigate the effect of EB1089 on the histology of OVCAR3 xenografts, tumor sections taken from mice treated with placebo or EB1089 at 0.3 or 1.0 μg/d/kg were stained with H&E. As shown in Fig. 3, the histologic appearance of tumors from mice treated with placebo (Fig. 3A) is quite different from that of mice treated with EB1089 (Fig. 3C). Tumors from placebo controls were composed primarily of epithelial cells (Fig. 3A). Mitotic figures are frequently visible (Fig. 3C) and apoptotic cells are rare. In contrast, mice treated with EB1089 at 0.3 μg/d/kg (data not shown) and 1.0 μg/d/kg (Fig. 3B) shared a histology that showed an increased proportion of noncellular stroma components. Compared with placebo controls, mitotic figures were fewer, whereas apoptotic cells with condensed or pyknotic nuclei and eosinophilic cytoplasm were more frequently seen (Fig. 3D).

To quantitatively compare the proliferation and apoptotic index of tumors treated with placebo and EB1089, the tumor sections were stained for expression of Ki-67 and DNA fragmentation by tissue TUNEL assays, respectively. As shown in Fig. 4A, treatment with EB1089 decreases the number of Ki-67-positive cells in tumors (Fig. 4A,(2)) compared with placebo controls (Fig. 4A,(1)). Based on the counting of 40 randomly selected microscopic fields, the proliferation index was decreased from 82.4 ± 5.5% in the placebo controls to 45.6 ± 3.9% in EB1089-treated tumors (Fig. 4A,(3)). In contrast to the proliferation index, EB1089-treated tumors compared with placebo controls (Fig. 4B,(1)) showed increased numbers of apoptotic cells with condensed and irregularly shaped nuclei, staining positively for TUNEL (Fig. 4B,(2)). Based on the counting of randomly selected microscopic fields, the number of apoptotic cells was increased from 18 ± 5 per 10 high-power microscopic fields in placebo controls to 57 ± 6 in EB1089-treated tumors (Fig. 4B (3)). The changes in both proliferation and apoptosis indexes were statistically significant (P < 0.001 and P = 0.001, respectively).

In the present studies, we have shown for the first time that the synthetic 1,25(OH)₂D₃ analogue EB1089 significantly suppresses the growth of ovarian cancer in vivo in nude mice without causing hypercalcemia. We also showed that the natural and synthetic 1,25(OH)₂D₃ compounds induce apoptosis of human ovarian cancer cells in vitro and in nude mice. In combination with the data from our earlier studies indicating that 1,25(OH)₂D₃ induces cell cycle arrest (6, 7) and apoptosis (16) in ovarian cancer cell lines, the current observation that EB1089 decreases the proliferation index and increases apoptosis index in the tumor xenografts makes it reasonable to conclude that both decreased cell proliferation and increased cell death contribute to the antitumor activity of EB1089 against OVCAR3 tumor xenografts in vivo. The fact that EB1089 induces apoptosis and transcriptionally activates GADD45, a mediator for 1,25(OH)₂D₃-induced cell cycle arrest at G₂-M checkpoint (6), suggests that EB1089 acts through the genomic effect of the vitamin D receptor to suppress the growth of ovarian cancer.

Our cell growth assays and reporter gene analysis showed that EB1089 is more potent than 1,25(OH)₂D₃ (Fig. 1), which concurs with data recently reported for OVCAR3 cells (17). The fact that the growth suppression of OVCAR3 xenografts by EB1089 was observed at 0.3 μg/d/kg, which is lower than the dose (0.5-1.0 μg/d/kg) used for other tumors (10, 11, 12, 13, 14), suggests that ovarian cancer might be more sensitive than other cancers to the growth suppressive effect of EB1089. Because the use of EB1089 at a higher dose such as 1.0 μg/d/kg did not further enhance the tumor-suppressing effects but caused an increase in serum calcium, it is critical to determine the lowest dose of EB1089 that is effective in suppressing the growth of ovarian cancer but with minimal effect on the level of blood calcium.

Although EB1089 was found to induce apoptosis in vitro and in vivo, it did not cause regression of OVCAR3 tumor xenografts during a 4-week treatment as judged by the measurement of tumor volumes. Histologic examination showed that EB1089-treated tumors contained more prominent a cellular stroma compared with placebo controls, suggesting that the tumors may have undergone actual regression that was not reflected by simply measuring the tumor volume. Because a chemopreventive role of 1,25(OH)₂D₃ compounds has been suggested, the early application of EB1089 during tumor development may have allowed the detection of more dramatic in vivo antitumor effects or may have prevented the formation of tumors altogether. Furthermore, the combination of EB1089 with other therapeutic agents may clinically achieve more desirable therapeutic effects.

In summary, our study strongly argues for further investigation of EB1089 and other synthetic 1,25(OH)₂D₃ analogues in the therapeutic treatment of ovarian cancer patients. In addition, EB1089 has been reported to regulate genomic stability (18, 19), to suppress angiogenesis (20), and to inhibit in vivo tumor metastasis. Further studies may reveal more significant applications for synthetic 1,25(OH)₂D₃ compounds in the clinical management of ovarian cancer.

We thank Dr. L. Binderup (Leo Pharmaceutical Products) for providing the placebo, EB1089, and 1,25(OH)₂D₃ compounds formulated for animal studies and Paul Byvoet for the critical reading of the article.