Iron withdrawal strategies fail to prevent the growth of SiHa-induced tumors in mice

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Gynecologic Oncology 90 (2003) 91–95

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Iron withdrawal strategies fail to prevent the growth of SiHa-induced tumors in mice Thierry Simonart,a,* Johan R. Boelaert,b Graciela Andrei,c Erik De Clercq,c and Robert Snoecka b

a Department of Dermatology, Erasme University Hospital, Brussels, Belgium Unit of Renal and Infectious Diseases, Algemeen Ziekenhuis Sint Jan, Brugge, Belgium c Rega Institute for Medical Research, Katholieke Univeristeit Leuven, Belgium

Received 7 August 2002

Abstract Objective. Cervical carcinoma is a human papillomavirus (HPV)-associated cancer for which treatment options still mainly rely on surgical procedures, with or without adjuvant radiotherapy and chemotherapy. We have previously shown that the chemically unrelated iron chelators desferrioxamine and deferiprone inhibit the growth and induce the apoptosis of HPV-positive cervical carcinoma cell lines, suggesting that iron chelators may represent a potential therapeutic approach for the treatment of cervical carcinoma. The present study was designed to investigate the effect of iron deprivation on the growth of human cervical carcinoma xenografts in athymic nude mice. Methods. Nude mice (nu/nu) of BALB/c background were treated with iron chelators [desferrioxamine (DFO), deferiprone (L1), or starch-DFO conjugate] or were fed with an iron-poor diet 6 weeks prior to subcutaneous injection of Si-Ha cells. These treatments were continued for 5 weeks after injection of the tumor cells. Results. Treatment with the maximum tolerated doses of DFO, L1, or starch-DFO conjugate induced no significant iron deprivation in non-iron-overloaded mice, while an iron-poor diet led to a dramatic decrease in serum iron, transferrin iron saturation, and ferritin levels. However, neither iron chelators nor an iron-poor diet could significantly inhibit tumor growth. Conclusion. Despite a potent antitumor effect in vitro, iron chelators fail to prevent the growth of cervical carcinoma xenografts in mice. On the basis of these results, clinical trials with iron chelators in patients with cervical carcinoma appear inappropriate. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Desferrioxamine; Deferiprone

Introduction Cervical carcinoma ranks second in cancer mortality in women worldwide [1–3]. However, despite major advance in the knowledge of the pathogenesis of the disease, treatment options still mainly rely on surgical procedures, with or without adjuvant radiotherapy and chemotherapy [4 – 6]. In patients with disseminated (extrapelvic) disease at the time of presentation, the goal of

* Corresponding author. Dr. Thierry Simonart, Department of Dermatology, Erasme University Hospital, Route de Lennik 808, B-1070 Brussels, Belgium. Fax: ⫹32-2-555-49-69. E-mail address: [email protected] (T. Simonart).

therapy is palliative and the principal modality is chemotherapy [4,6]. Several clinical observations have been made linking cellular iron content to the development of viral infections and cancers in humans. Iron is essential for viruses to replicate and can also impair defense cell functions and increase oxidative stress [7,8]. In addition, the metal is carcinogenic due to its catalytic effect on the formation of hydroxyl radicals and promotion of cancer cell proliferation [9,10]. Based on the probable role of iron in tumor development, iron withdrawal strategies are currently investigated in the management of human tumors [10]. More particularly, we have recently shown that iron chelators inhibit the growth of human papilloma virus (HPV)-positive

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carcinoma cell lines in vitro [11]. The inhibition of cell growth was associated with a decrease in the expression of proliferating cell nuclear antigen and of Ki-67, a proliferation marker indicative of reduced survival in patients with uterine cervical carcinoma [12,13]. Iron chelators also induce the apoptosis of these cells [11], suggesting that iron chelation may represent a potential therapeutic approach for the treatment of cervical carcinoma. In the present study, we investigated the effect of iron deprivation on the growth of human cervical carcinoma xenografts in athymic nude mice.

Material and methods Compounds Desferrioxamine (DFO) was purchased as its commercially available mesylate salt. Deferiprone (L1) was from Duchefa Farma (Haarlem, the Netherlands). 25SD04 starchDFO conjugate was kindly provided by Dr. Dragsten (Biomedical Frontiers, Minneapolis, MN). Iron-poor diet (6.7 mg of iron/kg) and diet with normal iron content (132.9 mg of iron/kg) were purchased from Pavan Carfil Quality (Oud Turnhout, Belgium). Iron was the only element that the low-iron diet contained in an abnormally low quantity. The diet contained all of the nutritional elements recommended by the Nutritional Research Council Subcommittee on nutritional requirements for normal growing mice. Cell culture SiHa cells were grown without additional growth factors in Dulbecco’s modified Eagle’s medium (Gibco, Paisly, Scotland) containing 10% fetal calf serum (Gibco), 1% glutamine, and 1% nonessential amino acids. Nude mice and induction of cervical carcinoma xenografts Nude mice (nu/nu) of BALB/c background were housed in a controlled environment in filtered cages, given sterile water, and fed ad libitum. Cages were changed twice weekly. Male mice 6 – 8 weeks of age were used. All procedures were carried out in a laminar flow hood. Cervical carcinoma xenografts were induced as previously reported [14]. Briefly, cervical carcinoma cells were removed from tissue culture flasks with trypsin. The cells were washed twice in phosphate-buffered saline (PBS), and cell clumps were dissociated by gentle pipetting with a Pasteur pipette. Cell number was determined with a Coulter Counter; 0.2 ml of cell suspensions of 1.5 ⫻ 107 cells/ml was injected subcutaneously through a 25-gauge needle into the right flank of the mice. These procedures were carried out in accordance with the Interdisciplinary Principles and Guide-

lines for the Use of Animals in Marketing and Education (New York Academy of Sciences, NY). Treatment Mice were allocated to cohorts for various treatments. The starting doses and route of administration of iron chelators were determined by a review of the literature [15,16]. The maximum tolerated dose (MTD) was defined as the dose level at which 20% or more of a cohort died or lost weight in excess of 20%; 200 ␮l of each compound was injected intraperitoneally with a 22-gauge needle three times a week. Controls were injected with identical volumes of PBS over the same duration. Dietary iron deficiency was induced by placing mice on a purified diet with low iron content and demineralized water. These treatments, aiming at inducing iron deprivation, were initiated 6 weeks prior to the injection of tumor cells and were continued for 5 weeks following injection. In a next series of experiments, DFO (400 mg/kg/dose) or PBS were injected intratumorally in a volume of 50 ␮l, once the tumors were established (approximately 20 –30 days after tumor injection). Monitoring Animals were weighed once weekly, and tumor growth was monitored weekly by measurement with calipers. Five weeks after tumor injection, the mice were killed. Blood was taken for hematocrit, ferritin, iron, transferrin iron saturation, and total iron-binding capacity determination. Tumors and livers were fixed in formalin and processed as described in the next section. Terminal deoxynucleotidyl transferase (TdT)-mediated desoxyuridinetriphosphate (dUTP) nick end-labeling (TUNEL) assay TdT assay for strand breaks was performed as previously reported [11]. Briefly, formalin-fixed samples were incubated with 20 ␮g/ml proteinase K (Sigma) for 15 min at room temperature to strip the nuclei from proteins. The samples were then washed four times in double-distilled water for 2 min. Endogenous peroxidase was inactivated by covering the slides with 2% (vol/vol) H2O2 in PBS for 30 min at room temperature. The preparations were rinsed with double-distilled water and immersed in terminal TdT labeling buffer (30 mM Trisma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride). TdT (0.3 e.u./␮l), dATP, and biotinylated dUTP in TdT buffer were then added to cover the cytospin preparations in a humid atmosphere at 37°C for 60 min. The reaction was terminated by transferring the slides to terminating buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min at room temperature. The slides were then rinsed with double-distilled water, covered with a 2% aqueous solution of bovine serum

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Table 1 Effect of treatment with DFO, L1, 25SD04, and iron-poor diet on survival, weight, and parameters of iron statusa

Survival Weight (g/mice) Hematocrit (%) Serum iron (␮g/dl) TIBC (␮g/dl) Transferrin iron saturation (%) Ferritin (␮g/l)

Control

DFO

L1

25SD04

Iron-poor diet

28/30 31 ⫾ 3b 36 ⫾ 4 298 ⫾ 70 441 ⫾ 85 72 ⫾ 18 157 ⫾ 78

27/30 31 ⫾ 3 35 ⫾ 3 305 ⫾ 78 437 ⫾ 97 77 ⫾ 20 169 ⫾ 94

26/30 32 ⫾ 3 35 ⫾ 4 274 ⫾ 65 460 ⫾ 71 70 ⫾ 15 158 ⫾ 86

25/30 30 ⫾ 2 34 ⫾ 4 270 ⫾ 66 490 ⫾ 91 67 ⫾ 22 143 ⫾ 70

26/30 32 ⫾ 4 33 ⫾ 3c 101 ⫾ 40d 677 ⫾ 143d 13 ⫾ 4d 82 ⫾ 71d

a

DFO, desferrioxamine; L1, deferioprone; TIBC, total iron-binding capacity. Mean ⫾ SE. c P ⬍ 0.01; d P ⬍ 0.001, compared with control. b

albumin for 10 min at room temperature, rinsed in doubledistilled water, and immersed in PBS for 5 min. Peroxidase activity was developed with 3,3⬘-diaminobenzidine and H2O2. Statistical analysis The Student’s t test (two-tailed) was used to determine differences between the cohorts.

Results and discussion Based on a possible role of iron in tumor development, several studies have suggested a potential antitumor activity of iron chelation [9,10]. Promising in vitro antitumor effects of iron chelators have been achieved on a wide range of neoplastic cell types, obtained from patients with hepatoma, neuroblastoma, melanoma, leukemia, and Kaposi’s sarcoma [17–21]. Recently, we found that the chemically unrelated iron chelators DFO and L1 could inhibit the growth and induce the apoptosis of human cervical carcinoma cells in vitro [11]. However, there are so far few studies that have investigated the effect of iron chelation on in vivo tumor growth. Moreover, the results of these studies appear to be cell type dependent. Iron chelators such as DFO have been shown to reduce tumor growth of hepatocellular carcinoma xenografts in athymic nude mice [22] and in mammary adenocarcinoma in rats [23]. By contrast, no reduction in tumor growth was achieved for neuroblastoma xenografts [24], and even tumor enhancement was observed for Kaposi’s sarcoma xenografts [25]. The hypothesis that iron chelation may affect tumor growth has been evaluated here in an animal model of human cervical carcinoma. To examine the effects of iron deprivation on the outcome of SiHa-induced tumors in the murine xenograft model, mice were fed with an iron-poor diet or treated intraperitoneally with DFO (100, 400, or 1000 mg/kg/dose), L1 (150 or 400 mg/kg/dose), or 25SD04 starch-DFO conjugate (600 or 1200 mg/kg/dose). Preliminary experiments in nonxenografted animals determined the

MTD of iron chelator to be used. The MTD of DFO, L1, and 25SD04 were 400, 150, and 600 mg/kg/dose, respectively. The effects of these treatments on survival, weight, hematocrit, and serum iron are shown in Table 1. Treatment with the MTD of DFO, L1, or 25SD04 starch-DFO conjugate induced no significant change in hematological parameters, while iron-poor diet led to a dramatic decrease in serum iron, transferrin iron saturation, and ferritin levels (Table 1). However, neither iron chelation nor iron-poor diet significantly inhibited SiHa-induced tumor growth (Fig. 1). Combined treatment with iron-poor diet and DFO also failed to reduce tumor growth and did not significantly modify the iron status compared to the diet alone (data not shown). Preliminary experiments performed on mice fed with an iron-poor diet or treated intraperitoneally with DFO (400 mg/kg/dose) and injected with HeLa cells led to similar results. We then evaluated the effect of intratumoral injections of DFO (400 mg/kg/dose), administered three or five times per week for 1 month, on the growth of SiHa-induced tumors. As seen with intraperitoneal injections, DFO did not significantly modify tumor growth at any of the treatment schedules (Fig. 2). As we previously found that iron chelation could induce

Fig. 1. Effect of intraperitoneal injection of iron chelators and of iron-poor diet on the growth of SiHa xenografts in nude mice. 1, control mice (n ⫽ 28); 2, DFO (400 mg/kg/dose)-treated mice (n ⫽ 27); 3, L1 (150 mg/kg/ dose)-treated mice (n ⫽ 26); 4, 25SD04 starch-DFO conjugate (600 mg/ kg/dose)-treated mice (n ⫽ 25); 5, low iron diet-fed mice (n ⫽ 26). DFO, desferrioxamine; L1, deferiprone.

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the apoptosis of SiHa cells [11], we performed TUNEL assays on tumors obtained from the different cohorts. Tumors from 10 mice from each cohort were randomly collected for microscopic evaluation. Microscopic examination of the tumors revealed no significant difference between the lesions obtained from the different cohorts. There was also no difference between the mitotic and apoptotic index of the lesions obtained from the different cohorts (Fig. 3). A possible explanation for this lack of in vivo antitumor activity of iron chelators could be related to their failure to induce significant iron depletion in non-iron-overloaded mice. The hematologic data obtained in the present study confirm the findings of others, indicating that the tested iron chelators, as opposed to iron-restricted diet, are unable to remove iron from its storage sites in the absence of iron excess [16,26,27]. This may be due to a different intracellular iron location in iron-normal versus excess of iron status. However, induction of significant iron depletion by an iron-poor diet was also ineffective at causing tumor shrinkage. Another possible explanation for the discrepancy between in vitro and in vivo results could be that iron withdrawal strategies may not be as effective in vivo as in vitro. This could be related to the relative iron-deficient environment and absence of human transferrin in the cell culture medium. The addition of an iron chelator is lethal in a closed system, where no alternate source of iron is available. In vivo, however, iron can be mobilized from the macrophages and hepatocytes, in an attempt to maintain the integrity of essential functions. It may not be possible to deplete sufficiently an organism in iron to affect the growth of malignant cells without causing major toxicity to the host. In conclusion, we have shown that despite potent antitumor effect in vitro, iron chelators fail to prevent the growth of cervical carcinoma xenografts in mice. On the basis of these results, clinical trials using iron chelators in cervical carcinoma cannot be recommended.

Fig. 2. Effect of intratumoral injection of DFO (400 mg/kg/dose) on the growth of SiHa xenografts in nude mice. 1, three times weekly PBS-treated mice (n ⫽ 9); 2, three times weekly DFO-treated mice (n ⫽ 9); 3, five times weekly PBS-treated mice (n ⫽ 9); 4, five times weekly DFO-treated mice (n ⫽ 8). DFO, desferrioxamine; PBS, phosphate-buffered saline.

Fig. 3. Effect of intraperitoneal injection of iron chelators and of iron-poor diet on mitotic and apoptotic indexes in SiHa xenografts. The mitotic index (black columns) and the TUNEL-based apoptotic index index (white columns) were calculated as the number of positive cells per 1000 counted cells. 1, control mice (n ⫽ 10); 2, DFO (400 mg/kg/dose)-treated mice (n ⫽ 10); 3, L1 (150 mg/kg/dose)-treated mice (n ⫽ 10); 4, 25SD04 starchDFO conjugate (600 mg/kg/dose)-treated mice (n ⫽ 10); 5, low iron diet-fed mice (n ⫽ 10). TUNEL, TdT-mediated dUTP nick end-labeling; DFO, desferrioxamine; L1, deferiprone.

Acknowledgments The authors are indebted to W. Zeegers and C. Degraef for their expert technical assistance.

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