Original Articles

Antineoplastic Effects of Gallium Nitrate on Human Medulloblastoma in Vivo H a r r y T. W h e l a n , MD*% M e i c H . S c h m i d t , B A * , G r e g o r y S. A n d e r s o n t , B r y a n K. Chan% K e l l i e Hunter% D a w n K u b a c k i , B S * , A n n e t t e D. S e g u r a , MD~, a n d

C h r i s t o p h e r R. C h i t a m b a r , M D ~

Gallium nitrate possesses antineoplastic activity against certain solid tumors and has been demonstrated to be an effective agent in reducing cell proliferation and DNA synthesis in the medulloblastoma Daoy cell line in vitro. In prior studies, gallium inhibited the cellular uptake of 59Fe by brain tumor cells; however, this block in 59Fe uptake was variable and closely paralleled the inhibitory effects of gallium on cell growth. In vivo trials now have been conducted and have yielded some promising results. Nude mice received intradermal injections of medulloblastoma Daoy and then allowed to grow tumors. When the mice had developed at least one tumor between 9 to I0 mm in diameter, a 10-day course of intraperitoneal gallium nitrate injections was initiated. Gallium nitrate treatment reduced overall tumor growth rate and reduced actual tumor size. Nephrotoxicity was severe, but may be preventable by continuous gallium infusion and use of diuretics and hyperhydration. Whelan HT, Schmidt MH, Anderson GS, Chan BK, Hunter K, Kubacki D, Segura AD, Chitambar CR. Antineoplastic effects of gallium nitrate on human medulloblastoma in vivo. Pediatr Neurol 1992;8:323-7.

Introduction Medulloblastoma is the most common malignant brain tumor in children. It represents approximately 20-25% of all pediatric central nervous system neoplasms in the posterior fossa [1-7]. Despite advances in surgery, radiation therapy, and adjuvant chemotherapy, survival rates for medulloblastoma have increased only slowly. Currently, survival rates range from 50-70% at 5 years and 40-50% at 10 years [3,4,6-9]; therefore, there is a need for continued evaluation of new chemotherapeutic agents for the treatment of this malignancy. Gallium nitrate may be such an agent [10]. It possesses significant antineoplastic activ-

From the Departments of *Neurology, tPediatrics, CPathology, and §Medicine; Medical College of Wisconsin; Milwaukee, Wisconsin.

ity in vitro and in vivo. In addition, it is currently being evaluated in phase H I clinical trials for those with refractory lymphomas, bladder cancer, and other malignancies. The mechanism by which gallium blocks tumor cell growth involves an inhibition in the cellular uptake of iron as well as a direct effect of gallium on the enzyme ribonucleotide reductase [ 11,12]. The high-affinity binding of gallium to transferrin results in the formation of transferrin-gallium complexes that can be incorporated into ceils through transferrin receptor-mediated endocytosis [1316]. Medulloblastoma cells in both cell lines and surgical biopsies have been demonstrated to express high numbers of transferrin receptors [17]; therefore, these cells appear to be good targets for treatment with gallium. Prior to our studies, gallium had not been evaluated for effects on brain tumors. Previously, we reported that gallium nitrate suppresses the in vitro proliferation of medulloblastomas [18,19]. In this study, we conducted in vivo trials in which nude mice were inoculated with medulloblastoma Daoy and subsequently were treated with gallium nitrate which produced a marked reduction of tumor growth in these animals.

Methods Nude mice were obtained from Harlen Sprague Dawly (Indianapolis, IN). Gallium nitrate was purchased from Alfa Products (Danvers, MA). Trypsin, fetal bovine serum, and Dulbecco's modified eagle's medium (DMEM) were acquired from Sigma Chemical Co. (St. Louis, MO). Azostix® reagent strips for estimating urea nitrogen in whole blood were purchased from Miles Inc. (Elkhart, WI). Tissue Culture and Tumor Inoculation. Medulloblastoma Daoy cells were grown in DMEM supplemented with 10% fetal bovine serum and then harvested from 18 individual P-100 dishes using trypsin. Cells were collected by centrifugation. The supernatant was removed and cells resuspended in 2.5 ml of serum-free DMEM media. Cells were counted using a hemocytometer. Mice were injected in each flank (2 injections per mouse) intradermally with 1.25 × 106 cells (50 laL) per injection using a 25 gauge straight needle. Progressive tumor growth became apparent in the nude mice approximately 1 month post-injection. Tumor size then was assessed biweekly using calipers. Within 2-3 months, tumor size increased to about

Communications should be addressed to: Dr. Whelan; Pediatric Neurology; MACC Fund Research Center; Medical College of Wisconsin; 8701 Watertown Plank Road; Milwaukee, WI 53226. Received March 31, 1992; accepted June 15, 1992.

Whelan et al: Gallium and Medulloblastoma in Vivo

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were calculated in control and gallium-treated tumor-bearing mice intervals with calipers. The rate of tumor growth was measured in in tumor size. Positive growth rates represent actual tumor growth. which was significant in the gallium-treated mice at the end of the

10 mm in diameter. At this stage, the nude mice began 10 days of gallium chemotherapy. When the mice died or were sacrificed, their tumors were fixed in formalin and sectioned for histologic examination with hematoxylin and eosin stain. Gallium Nitrate Chemotherapy. A solution of gallium nitrate (10 mg/ml) was prepared in nanopure water. The solution was filtersterilized and stored at 4°C. Each mouse was treated with gallium nitrate (50 mg/kg/day) for 10 days. The solution of gallium was administered intraperitoneally with a 26 gauge 3/8-inch needle. During the injection, all animals were anesthetized. Control animals with tumors received no treatment. Tumor Growth Evaluation. Control and gallium-treated mice were monitored for tumor growth. Measurements were conducted twice a week at 3- and 4-day intervals using calipers. The rate of tumor growth was measured in millimeters per day by calculating the overall change in tumor size during the 3-4 days of growth. Positive growth rates represented actual tumor growth. Negative growth denoted tumor shrinkage. Treated tumor data represented mean + S.E.; n ranges from 3 to 16, due to the high mortality rate among the treated mice. Untreated tumor data represented mean + S.E.; n = 4. Pathology. Control tumors (no treatment), treated tumors, and posttreatment tumors were fixed in formalin. Representative sections of the tumors were processed for light microscopy using hematoxylin and eosin stain. Mitotic counts were obtained for each representative section. Assessment of Gallium on Renal Function. Five nude mice without tumors received gallium nitrate (50 mg/kg/day) for 10 days. Blood urea nitrogen levels of these mice were measured before, during, and after gallium nitrate treatment. Measurements were obtained every 3-4 days for a total of 50 days using Azostix® reagent strips. Results D u r i n g the pretreatment period, the tumors of all mice exhibited similar growth rates (Fig 1). For the a n i m a l s that received gallium, the tumor growth rate decreased significantly d u r i n g the 10-day treatment period. After the gall i u m c h e m o t h e r a p y was discontinued, the t u m o r growth rates o f these a n i m a l s returned to pretreatment levels. In

324 PEDIATRIC NEUROLOGY Vol. 8 No. 5

contrast, control mice had no significant change in tumor growth. Microscopic tumor characteristics were similar in our experiments to those previously described for the transplanted m e d u l l o b l a s t o m a Daoy line [20]. P l u m p spindleshaped to elongated tumor cells were arranged in short. intersecting fascicles and alternated with scattered, larger polygonal-shaped cells c o n t a i n i n g large nuclei, prominent nucleoli, and moderately a b u n d a n t eosinophilic cytoplasm (Fig 2). Areas of necrosis and mitoses were present. The mitotic rates o f tumors from control and gallium-treated animals are illustrated in Figure 3. T h e n u m b e r o f mitotic figures in tumors from animals during g a l l i u m treatment was significandy decreased compared to control and posttreatment tumors. There was no significant difference in mitotic rates b e t w e e n control and gallium-treated tumors after g a l l i u m therapy was completed (post-treatment). The group of mice receiving gallium exhibited a substantial mortality. Four days after the completion of gallium chemotherapy, 18% of the tumor mice were still alive; therefore, the overall mortality was 82%. Due to the significant mortality during treatment, we assessed blood urea nitrogen (BUN) levels in order to evaluate possible renal toxicity of gallium. The results of B U N experiments are illustrated in Figure 4. Normal range of B U N levels for mice is 13.9-28.3 mg/dl [21]. B U N levels increased significantly o n day 3 and day 14 but were stable b e t w e e n these 2 days. O n e a n i m a l (Mouse 2) died on day 14. In general, B U N levels r e m a i n e d elevated and fluctuated from 30-40 mg/dl and 50-80 mg/dl. Discussion

The prior in vitro studies by W h e l a n et al. [ 18,19] examined the effects o f gallium nitrate on m e d u l l o b l a s t o m a

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Figure 2. (A) Cellular undifferentiated tumor with zone of necrosis infiltrating subcutaneous tissue of nude mouse (hematoxylin and eosin stain, original magnification xlO0). (B) Plump, spindle-shaped cells surround a large, pale-stained polygonal cell containing large nucleus and prominent nucleolus (hematoxylin and eosin stain, original magnification x400).

cells. Our study demonstrated that medulloblastoma Daoy is also inhibited by gallium in vivo. Of particular interest, the rate of tumor growth slowed, followed by tumor shrinkage, as evidenced by the negative tumor growth

rates of the treated tumors during the 10-day treatment period. It is assumed that these antiproliferative effects are due to the ability of gallium nitrate to interfere with iron uptake and DNA synthesis. In addition, our finding that

Whelan et al: Galliumand Medulloblastomain Vivo 325

Of concern was the high mortality rate (82%) in the mice treated with gallium. Although the precise causc of these deaths is unknown, previous reports indicated thal renal or hepatic damage may be responsible [23,24[. Animal and clinical studies demonstrated that while gallium nitrate is taken up in tumors and its concentration reaches nearly the same levels as normal tissue, the liver may have levels up to 28 times the tumor levels, and the kidneys may be as high as 130 times the tumor levels. Comparable levels of gallium nitrate, like another heavy metal salt, cis-diamminedichloroplatinum (II) (CDDP), have been found to cause renal tubular damage. Gallium is different from CDDP in that it also may precipitate in the renal tubules and can cause hepatic lesions, as well as some depression of lymphoid cells; however, there was no observable bone marrow toxicity [23]. Our BUN measurements demonstrated significant renal toxicity which probably contributed to mortality and morbidity in our mice. Through the use of animal and clinical trials 123,24], it has been found that these toxic levels of gallium in the liver and kidneys can be controlled by diuretics. The use of mannitol diuresis and isosorbide diuresis is effective in lowering the toxicity of gallium in doses up to 750 mg/m 2. At very high doses, osmotic diuresis cannot dilute the gallium concentrations enough to prevent toxicity. It was calculated that the animals in this experiment received 213 mg/m2/day for 10 days. Toxicity at these levels should be controllable through osmotic diuresis without changing the serum levels in the tumor. Furthermore, clinical studies demonstrated that administration of gallium nitrate by continuous infusion rather than by bolus injection appears to protect against nephrotoxicity [25]. We are currently con-

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Figure 3. The mitotic rates of tumors from control and galliumtreated tumor-bearing animals demonstrate a marked decrease in the number of mitotic cells per 10 high-power fields during the treatment period. The number of mitotic fgures in tumors from animals during gallium treatment was significantly decreased compared to control and post-treatment tumors.

tumor growth returns to the pretreatment growth rate soon after gallium treatment has ended suggests that the effects of gallium on DNA synthesis are reversible/cytostatic. The antiproliferative, growth inhibitory effect of gallium may be potentiated by other agents given in combination; we are currently conducting experiments to address this potential for synergism.

Effect Of Gallium On Renal Function Gallium Nitrate ( 6 0 l m g / k g ) was administered to n o n - t u m o r bearing mice.

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ducting experiments in which subcutaneous saline hyperhydration is administered to tumor-bearing mice concurrently with gallium nitrate therapy. Although other studies have investigated the uptake of gallium in several tumor cell lines, the uptake of gallium has never been measured on medulloblastoma Daoy. Although it is assumed that medulloblastoma Daoy possesses similar gallium nitrate uptake properties to those tumors, further studies will be needed to establish this quality.

The authors thank Therese Lundgren for help in preparation of this manuscript. This study was supported by the American Cancer Society Clinical Oncology Career Development Award No. 89-119 and grants from the Cancer Research Foundation of America, Children's Hospital Foundation, the family of Ingrid Haagensen, and the MACC Fund (Midwest Athletes Against Childhood Cancer Fund) to Dr. Whelan and by the USPHS Grant No. R01 CA41740 from the National Cancer Institute to Dr. Chitambar.

References

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[9] Finlay JL, Goins SC. Brain tumors in children III: Advances in chemotherapy. Am J Pediatr Hematol Oncol 1987;9:264-71. [10] Foster BJ, Clagett-Carr K, Hoth D, Leyland-Jones B. Gallium nitrate: The second metal with clinical activity. Cancer Treat Rep 1986; 70:1311-9. [11] Chitambar CR, Matthaeus WG, Antholine WE, Graft K, O'Brien WJ. Inhibition of leukemic HL60 cell growth by transferringallium: Effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea. Blood 1988;72:1930-6. [12] Chltambar CR, Narasimhan J, Guy J, Sere DS, O'Brien WJ. Inhibition of ribonucleotide reductase by gallium in murine leukemic 11210 cells. Cancer Res 1991;51:6199-201. [13] Larson SM, Rasey JS, Allen DR, et al. Common pathway for tumor cell uptake of gallium-67 and iron-59 via a transferrin receptor. J Natl Cancer Inst 1980;64:41-53. [14] Chitambar CR, Zivkovic Z. Uptake of gallium-67 by human leukemic cells: Demonstration of transferrin receptor-dependent and transferrin-independent mechanisms. Cancer Res 1987;47:3929-34. [15] Chitambar CR, Zivkovic-Gilgenbach Z. Role of the acidic receptosome in the uptake and retention of 67Ga by human leukemic HL60 cells. Cancer Res 1990;50:1484-7. [16] Vallabhajosula SR, Harwig JF, Siemsen JK, Wolf W. Radiogallium localization in tumors: Blood binding and transport and the role of transferrin. J Nucl Met 1980;21:650-6. [17] Zoviekian J, Johnson VG, Yonle RJ. Patent and specific killing of human malignant brain tumor cells by anti-transferrin receptor antibody-ricin immunotoxin. J Neurosurg 1987;66:850-61. [18] Whelan HT, Przybylski C, Chitambar CR. Differential effects of gallium nitrate on proliferation of brain tumor cells in vitro. Pediatr Neurol 1991;7:23-7. [19] Whelan HT, Przybylski C, Chitambar CR. Alteration of DNA synthesis in human brain tumor cells by gallium nitrate in vitro. Pediatr Neurol 1991;7:352-4. [20] Jacobsen PF, Jenkin DJ, Papadimitriou JM. Establishment of a human medulloblastoma cell line and its hetero-transplantation into nude mice. J Neuropathol Exp Neurol 1985;44:472-85. [21] Mitruka BM, Rawnsley HM. Clinical, biochemical and hematological reference values in normal experimental animals. New York: Masson Publishing USA, 1977;118. [22] Chitambar CR, Seligman PA. Effects of different transferrin forms on transferrin receptor expression, iron uptake, and cellular proliferation of human leukemic HL60 cells. Mechanisms responsible for the specific cytotoxicity of transferrin-gallium. J Clin Invest 1986;78: 1538-46. [23] Krakoff IH, Newman RA, Goldberg RS. Clinical toxicologic and pharmacologic studies of gallium nitrate. Cancer 1979;44:1722-7. ]24] Newman RA, Brody AR, Krakoff IH. Gallium nitrate (NSC15200) induced toxicity in the rat. Cancer 1978;44:1728-40. [25] Warrell RP, Coonley CJ, Straus DJ, Young CW. Treatment of patients with malignant lymphoma using gallium nitrate administered as a seven-day continuous infusion. Cancer 1983;51:1982-7.

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Antineoplastic effects of gallium nitrate on human medulloblastoma in vivo.

Gallium nitrate possesses antineoplastic activity against certain solid tumors and has been demonstrated to be an effective agent in reducing cell pro...
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