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Effectiveness of 1-Hydroxyvitamin D₂ in Inhibiting Tumor Growth in a Murine Transgenic Pigmented Ocular Tumor Model

Daniel M. Albert, MD, MS; Amit Kumar, MD; Stephen A. Strugnell, PhD; Soesiawati R. Darjatmoko, MS; Janice M. Lokken; Mary J. Lindstrom, PhD; Christine M. Damico, BS; Sarit Patel, MD

Arch Ophthalmol. 2004;122:1365-1369.

ABSTRACT
Objective  To study the effectiveness of the vitamin D analogue 1-hydroxyvitamin D₂ (1-OH-D₂) in inhibiting ocular tumor growth in transgenic "Tyr-Tag" mice that developed pigmented ocular tumors produced with the simian virus 40 T and t antigens under the control of the mouse tyrosinase gene. These animals develop pigmented intraocular tumors primarily from the retinal pigment epithelium that closely resemble the histologic features and growth pattern of human choroidal melanoma.

Methods  A total of 73 Tyr-Tag transgenic mice between 6 and 7 weeks old were randomly assigned by sex and litter to 3 treatment groups to receive 0.05 µg/d, 0.1 µg/d, or 0.2 µg/d of 1-OH-D₂; a control group received vehicle (coconut oil). The drug was administered by oral gavage 5 times a week for 5 weeks. The animals were then euthanized and their eyes were enucleated and processed histologically. Three serial sections from each eye were examined microscopically and the mean tumor area measured using Optimus software version 6.5 (Media Cybernetics LP, Silver Spring, Md). Toxic adverse effects were assessed on the basis of mortality, weight loss, and serum calcium levels.

Results  The mean tumor size in the 0.1-µg/d and 0.2-µg/d dose groups was smaller than in the controls (P<.001). No significant difference was seen between the 0.05-µg/d dose group and the control group (P = .64). Survival for the 0.1-µg/d and 0.2-µg/d dose groups was lower than for the controls (95% in the controls vs 85.7% and 73.7%, respectively; P<.01).

Conclusion  In the Tyr-Tag transgenic mouse, 1-OH-D₂ inhibits pigmented ocular tumor growth at moderate drug levels with relatively low mortality.

Clinical Relevance  Vitamin D analogues merit further preclinical study in the treatment of ocular melanoma.

INTRODUCTION

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In a recent "Perspective" on changing concepts in the treatment of choroidal melanoma, Robertson concluded, "Gains in our ability to manage choroidal melanoma will likely be modest at best until effective systemic therapies can be identified."^1(p161) The present study was carried out to evaluate the effectiveness of a vitamin D analogue, 1-hydroxyvitamin D₂ (1-OH-D₂), in inhibiting tumor growth in a transgenic mouse ocular pigmented tumor model.

The role of vitamin D as a regulator of calcium and phosphate metabolism has been known since early in the 20th century.² More recently it has been demonstrated that vitamin D analogues have important biological actions besides those related to mineral metabolism.^3-6 The classic signaling pathway, as with other steroid molecules, is through a nuclear receptor, the vitamin D receptor that is a transcription factor. Studies during the past 20 years have shown vitamin D receptors in a wide range of tissues,⁷ as well as in numerous types of malignant cells.^8-10 Vitamin D analogues have been shown to inhibit cellular proliferation, induce the differentiation of both normal and malignant cells, act as an antiangiogenic agent, and cause apoptosis.^11-17 Cutaneous melanoma cells were one of the first malignant cells in which the antiproliferative and prodifferentiation effects of vitamin D compounds were demonstrated.^18-20 Calcitriol and various analogues have also been demonstrated to significantly enhance the antitumor efficacy of other anticancer drugs in vitro and in vivo.^{11, 21-22} The clinical potential of older agents has been limited by the induction of hypercalcemia. However, recent analogues have been developed with decreased calcemic activity, but potent antineoplastic and differentiating activity; these analogues have therapeutic promise.²³ One such compound, 1-OH-D₂, was used in the present study.

METHODS

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All research using mouse models of melanoma conform to the guidelines set by the Research Animal Resources Center, University of Wisconsin, Madison, and the Association of Vision Research in Ophthalmology statement on the Use of Animals in Ophthalmic and Vision Research.

COMPOUND PREPARATION

Pure crystalline 1-OH-D₂ (Bone Care International, Middleton, Wis) was prepared for administration and the drug concentrations were confirmed in the manner previously described.²⁴ A solution of the drug was diluted in coconut oil to concentrations of 0.05 µg per 0.1 mL; 0.1 µg per 0.1 mL; and 0.2 µg per 0.1 mL. The effectiveness and toxicity of these concentrations have previously been described in dose-response studies in retinoblastoma animal models.^23-25

TREATMENT OF Tyr-Tag TRANSGENIC MICE

A total of 73 Tyr-Tag transgenic mice²⁶ between 6 and 7 weeks old were randomly assigned by sex and litter to 1 of 3 1-OH-D₂ treatment groups: (1) those that received 0.05 µg/d (approximately 2.5 µg/kg), (2) those that received 0.1 µg/d (approximately 5.0 µg/kg), and (3) those that received 0.2 µg/d (approximately 10 µg/kg). In a serial study of the development of the ocular tumors, all mice were found to have small tumors at this age.²⁶ A control group received 0.1 mL of vehicle (coconut oil). The treatment was administered by oral gavage 5 times a week for 5 weeks. The mice were maintained on a vitamin D and calcium–restricted diet (vitamin D deficient and calcium deficient, catalog 23980 PD; Purina Mills Inc, St Louis, Mo). Body weights were measured twice a week and just prior to euthanization on the last day of treatment.

TUMOR SIZE DETERMINATION

The mice were euthanized on day 35 of treatment. Their eyes were then enucleated and placed in a 10% neutral-buffered formalin solution. Four serially sectioned 5-µm-thick sections were cut from each of the superior, middle, and inferior areas of the globe in the manner previously described^23-24,27 and stained with hematoxylin-eosin. All 4 of the sections from each globe area were examined under a microscope and the section with the largest area of tumor was used for measurement. The outline of the tumor was traced on a microscopically digitized image and the tumor area measured using Optimus software version 6.5 (Media Cybernetics, Silver Spring, Md). Three measurements from each tumor representation were averaged to obtain the mean tumor measurement. These methods have been described elsewhere.^23-24,27

TOXICITY ASSESSMENT

Toxicity assessment for 1-OH-D₂ was based on analyses of survival, changes in body weight, and serum calcium levels. Mice that died prior to the end of the study were excluded from the body weight, and serum calcium level. Autopsies were performed on all mice.

Serum calcium levels were measured in representative samples of mice from each group selected at random (control group, 7 samples; 0.05-µg/d dose group, 5 samples; 0.1-µg/d dose group, 6 samples; and 0.2-µg/d dose group, 5 samples). Blood was drawn from axillary veins just prior to euthanization, and calcium levels were analyzed by an independent commerical laboratory (Marshfield Laboratories Inc, Marshfield, Wis).

STATISTICAL ANALYSIS

The effect of drug dose on tumor area, animal weight, and serum calcium level was assessed using 1-way analysis of variance. The tumor area was transformed to the log scale before calculating the mean area. Serum calcium data were not transformed. Differences were considered statistically significant at P<.05.

RESULTS

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TUMOR SIZE

The mean tumor area (x10⁴ µm²) of the various groups was as follows: control, 219.77; 0.05 µg/d of 1-OH-D₂, 200.71; 0.1 µg/d of 1-OH-D₂, 87.67; and 0.2 µg/d of 1-OH-D₂, 63.40 (Table 1 and Figure 1). A dose-dependent curve is apparent with higher-dose groups exhibiting a greater degree of tumor growth inhibition. The 0.1-µg/d and 0.2-µg/d dose groups both showed significant tumor growth inhibition compared with the control group (P<.001) and with the 0.05-µg/d dose group (P<.0011 and .0001, respectively). No primary cutaneous tumors or metastases were found in the control or treatment groups.

View this table: [in this window] [in a new window] Summary of Data Analyzed in Study of 1-Hydroxyvitamin D2 in 73 Tyr-Tag Transgenic Mice

View larger version (24K): [in this window] [in a new window] Figure 1. Tumor area in Tyr-Tag transgenic mice treated with 1-hydroxyvitamin-D2 for 5 weeks (group mean [SE]).

TOXICITY ASSESSMENT

The survival rates for different treatment groups are shown in Table 1 and Figure 2. The mortality was significant in the 0.1- and 0.2-µg/d dose groups compared with the control group (P<.01 for both groups).

View larger version (31K): [in this window] [in a new window] Figure 2. Survival of Tyr-Tag transgenic mice treated with 1-hydroxyvitamin-D2 for 5 weeks.

The serum calcium levels increased with the increased dose of 1-OH-D₂ (Table 1). The difference was significant for the 0.1- and 0.2-µg/d dose groups compared with the control group (P<.003 and P<.001, respectively). The 0.2-µg/d dose group was also significant compared with the 0.05-µg/d dose group (P = .008)

There was no significant difference in mean body weight change—measured as the difference between body weight at the start and at the end of the study—between various treatment groups. There was no significant difference between the 0.05-µg/d dose group and the control group for any of the factors analyzed.

COMMENT

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The incidence of melanoma in the white US population has increased from 1 case per 100 000 in 1935 to 15 per 100 000 in 1996.²⁸ The skin is the most common site of melanoma development; the eye is the second most likely site, with ocular melanoma constituting the most common primary intraocular malignancy in adults. In the white population, ocular melanoma has an average annual incidence of 6 cases per 1 million with approximately 1200 cases diagnosed each year in the United States.^29-30 Enucleation and radiation are the principal means of treatment. The recent Collaborative Ocular Melanoma Study (COMS) demonstrated that enucleation of large choroidal tumors, the principal method of treatment, was made neither more nor less effective when preceded by external irradiation.³¹ The medium-sized tumor trial of COMS demonstrated that mortality rates following brachytheraphy were similar to mortality rates following enucleation for up to 12 years after treatment.³² Robertson¹ points out that in large tumors the estimated 5-year tumor-specific mortality rate is approximately 33% for large tumors and 10% for medium-sized melanomas, and continues to increase when followed for up to 20 years. In addition, no effective treatment has been demonstrated for metastatic uveal melanoma. Although there is a trend toward earlier treatment of small melanomas, there continues to be controversy regarding the indications for treatment as well as the choice of specific therapy.¹ Clearly there is a need for improved methods of treatment.

Much more information is available about the genetics and response to treatment of cutaneous melanoma than for ocular melanoma, but while these tumors have many similarities, they also have some significant differences.³³ The genetics of uveal melanoma are less well defined than cutaneous melanoma, and it appears that p16 (CDKN2A) mutations do not play a role in ocular melanoma as they do in certain cases of familial cutaneous melanoma.³³ In addition, patients with systemic ocular melanoma respond less well to chemotherapy and immunotherapy than do patients with systemic cutaneous melanoma.^34-35

A major deficiency in attempting to develop new methods of therapy for human uveal melanoma has been the absence of an ideal model animal for the human condition.³⁶ Although several animal models of human uveal melanoma have been used, each of these models has unique advantages and disadvantages.³⁶ Spontaneous uveal melanoma rarely occurs in other species and its occurrence is unpredictable. Chemical- or irradiation-induced intraocular pigmented tumors may originate from the retinal pigment epithelium. Both feline leukemia–sarcoma virus and simian virus 40–induced uveal tumors failed to metastasize. The Greene hamster melanoma and the B16 murine melanoma cells lines are virus-containing tumors that grow more aggressively than human uveal melanomas. Their biological behavior after intraocular injection into various animal species is, however, predictable, allowing study of the mechanisms of growth and metastasis. Human cell lines can be grown in the eyes of immune-deficient mice, but the lack of an intact immune system is a significant artifact. Transgenic murine models have been developed using the promoter region of the tyrosinase gene to target expression of oncogenes in melanin-producing cells.^{26, 37-42} Spontaneous intraocular pigmented tumors and distant metastases may occur, although these tumors develop primarily from the retinal pigment epithelium.

In this study we used Tyr-Tag transgenic mice that were produced with the simian virus 40T and antigens under the control of the mouse tyrosinase gene.²⁶ These tumors were studied sequentially and found to develop primarily from retinal pigment epithelium, but their growth and histologic appearance closely resemble that of human choroidal melanoma.²⁶ Tumor development begins between birth and 1 week of age, with lateral infiltration along Bruch's membrane noted at 6 weeks, choroidal invasion beginning at 7 weeks, and retinal invasion by 10 weeks.²⁶ Metastases occurred between 12 and 40 weeks with the most common sites being subcutaneous tissues, lungs, retroperitoneal space, and brain.²⁶ The immunohistochemistry and electron microscopic features of these tumors were studied in detail, as was their response to chemotherapy and irradiation.²⁶

The antiproliferative-prodifferentiation effects of vitamin D compounds have been shown in cultured human melanocytes, human melanoma cells, and melanoma xenograft models.^18-20 A MEDLINE search and review of the literature between 1982 and 2004 revealed more than 40 additional articles regarding the response of cutaneous melanoma to vitamin D. The literature indicates that all human melanoma cell lines tested express the vitamin D receptor. This research has been carried out almost exclusively with calcitriol and has been limited to preclinical studies by the toxic effects associated with the induced hypercalcemia.

In the past several years, new vitamin D analogues have been produced with reduced calcium effect but with equivalent or enhanced antineoplastic effect compared with calcitriol or vitamin D₂.²³ The compound studied in the present experiments was 1-OH-D₂. This has been demonstrated to induce low levels of hypercalcemia while providing effective systemic serum drug levels for tumor treatment.^43-44 An Investigational New Drug application for 1-OH-D₂ as a cancer treatment was submitted to the Food and Drug Administration in 1996. 1-Hydroxyvitamin D₂ has received Food and Drug Administration approval in both oral and intravenous formulations for the treatment of elevated parathyroid hormone levels secondary to renal failure. In phase 1 trials for treatment of prostate cancer, 1-OH-D₂ caused tumor growth suppression or stabilization in some patients with low but reversible toxic adverse effects.⁴⁵ This has progressed to a phase 2 study.⁴⁶ This compound also proved effective in in vivo studies of treatment of retinoblastoma in the LHbeta-Tag transgenic mouse.^24-25 The mechanism of action was demonstrated to be apoptosis associated with the up-regulation of both p53 and p2147 and inhibition of angiogensis.^47-48

In the present study 1-OH-D₂ produced a statistically significant decrease in intraocular tumor size in the 0.1- and 0.2-µg/d doses. A reduction in tumor area was also seen with the 0.05-µg/d dose, but this was not statistically significant. As in previous experiments, the effective doses produce a significant elevation in the level of serum calcium with associated decreased survival.

These results suggest that further investigation of vitamin D analogues as a possible therapy of melanoma is warranted. Its usefulness as a therapeutic agent both given individually and in combination therapy should be examined in xenograft models using human melanoma cell lines as well as in other animal models for this tumor.

AUTHOR INFORMATION

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Correspondence: Daniel M. Albert, MD, MS, Department of Ophthalmology and Visual Sciences, F4/344 Clinical Science Center, 600 Highland Ave, Madison, WI 53792-3284 (dalbert{at}wisc.edu).

Submitted for publication November 13, 2003; final revision received January 30, 2004; accepted February 2, 2004.

This study was supported by grant EY01917 from the National Eye Institute, Bethesda, Md; and an unrestricted grant from Research to Prevent Blindness, New York, NY.

We thank Bone Care International for providing the pure crystalline 1a-OH-D₂.

We acknowledge the assistance of Daniel Dawson, MD, and Joel Gleiser, MD, in treating the animals and measuring tumor size. Kirsten Hope contributed to preparation of the manuscript.

From the Department of Ophthalmology and Visual Sciences (Drs Albert, Kumar, and Patel, and Mss Darjatmoko, Lokken, and Damico) and the Department of Biostatistics and Medical Informatics (Dr Lindstrom), University of Wisconsin Medical School, Madison; and Bone Care International, Middleton, Wis (Dr Strugnell). The authors have no relevant financial interest in this article.

REFERENCES

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