Polyhemoglobin–Tyrosinase, an Oxygen Carrier with Murine B16F10 Melanoma Suppression Properties: A Preliminary Report

Abstract

Melanoma now represents the fifth most common cancer in North America and it has increased dramatically in the past decade. One of the approaches shows that lowering of tyrosine level can inhibit the growth of melanoma in cell culture and in mice bearing B16BL6 melanoma. However, human cannot tolerate the tyrosine restricted diets for lowering tyrosine due to nausea, vomiting, and severe body weight loss. We therefore prepare a novel soluble polyhemoglobin-tyrosinase complex. Our studies show that this preparation can lower systemic tyrosine level in normal animals. This preparation also prevents the native tyrosinase from having adverse effects and from rapid removal after injection. In cell culture study, we find that this preparation inhibits the growth of murine B16F10 melanoma culture. Furthermore, in animal studies we observe that daily intravenous injection of this polyhemoglobin-tyrosinase preparation significantly delays the growth of B16F10 melanoma in mice, without causing adverse effects or changes in the growth of the treated animals.

• B16F10 murine melanoma
• Polyhemoglobin–tyrosinase
• Systemic tyrosine
• Tyrosinase
• Cross-linking

Introduction

Melanoma is a common tumor which accounts for an incidence of 10% of all malignancies. It is most commonly found on the skin but 10% arise in the eye (Borden, [[2002]]). At least 20% of people diagnosed with melanoma progress to advanced disease and die within 5 years of diagnosis (Beahers et al., [[1992]]). At present, surgical excision is the standard option for operable local-regional disease. Nonsurgical treatments for advanced or recurrent melanoma include radiation therapy, chemotherapy, and thermochemotherapy (Burd et al., [[2003]]).

Tyrosine is an important amino acid in the metabolic cycle of melanoma (Letellier et al., [[1999]]; Potterf and Hearing, [[1998]]). Previous research done by Meadows’ group has shown that malignant melanoma requires higher concentrations of tyrosine for growth. Their research also indicates that lowering of systemic tyrosine level by tyrosine and phenylalanine restricted diet can inhibit the growth of malignant melanoma in vitro and in vivo (Pelayo et al., [[1999]], [[2001]]). However, human cannot well tolerate this low tyrosine diets due to severe body weight loss and other adverse effects (Lorincz et al., [[1969]]). In our study, we prepare a soluble solution polyhemoglobin-tyrosinase (PolyHb-tyrosinase) complex by crosslinking tyrosinase with hemoglobin to lower systemic tyrosine level. It is well documented that PolyHb is safe and effective as blood substitutes (Chang, [[1997]], [[2000]], [[2003]]; Squires, [[2002]]). It is in phase III clinical trial with up to 10 L infused to replace lost blood in trauma surgery (Gould, [[2002]]; Sprung et al., [[2002]]). Furthermore, our previous in vitro enzyme studies show that longer crosslinking time results in more PolyHb-tyrosinase in higher molecular weight (Yu and Chang, [[2004b]]). The crosslinking time from 3.5 to 24 h has no adverse effect on the tyrosinase activity. In addition, PolyHb-tyrosinase possesses similar oxygen transport characteristics as that of hemoglobin solution (Yu and Chang, [[2004a]]). When incubated at 37°C, PolyHb-tyrosinase shows higher stability than tyrosinase in free form. Our studies also show that intravenous PolyHb-tyrosinase can effectively lower systemic tyrosine level in rats (Yu and Chang, [[2004b]]). In the present study, we investigate the effects of our preparation of PolyHb-tyrosinase on the growth of B16F10 melanoma cell culture. In animal experiments, we first carry out the study of intravenous injection of PolyHb-tyrosinase on normal mice. This is followed by further study in B16F10 melanoma bearing mice model. This is a preliminary report and a detailed article will be published elsewhere later (Yu and Chang [[2004c]]).

Materials and Methods

Materials

Glutaraldehyde (25%) was obtained from Polysciences (Warrington, PA, USA). l-Lysine (monohydrochloride, SigmaUltra >99%), l-tyrosine (98% TLC), tyrosinase from mushroom (EC 1.14.18.1, 3000 U/mg stated activity) were purchased from Sigma-Aldrich (Ontario, Canada). Purified bovine hemoglobin was purchased from Biopure Biopharmaceuticals (Cambridge, MA, USA). All other reagents were of analytical grade.

Preparation of PolyHb and PolyHb-Tyrosinase

Reaction mixtures were prepared containing hemoglobin (10 g/dL), tyrosinase (6000 U/mL) in 0.1 M potassium phosphate buffer, pH 7.6. In PolyHb mixtures, an equivalent volume of buffer replaced enzyme condition. Prior to the start of crosslinking, 1.3 M lysine was added at a molar ratio of 7:1 lysine/hemoglobin. Crosslinking reaction was started with the addition of glutaraldehyde (5%) at molar ratio of 16:1 glutaraldehyde/hemoglobin. Glutaraldehyde was added in four equal aliquots over a period of 15 min. After 3.5–24 h at 4°C under aerobic conditions with constant stirring, reaction was stopped with 2.0 M lysine at a molar ratio of 200:1 lysine/hemoglobin. Solutions were dialyzed in physiological saline solution and passed through sterile 0.45 µM filter. Aliquots (500 µL) of the 16:1 crosslinked preparation were concentrated using 100 KD microconcentrators (Millipore Corporation, Ontario, Canada). Samples were centrifuged at 2500g for 55 min at 23°C. Then, retentate was collected. Hemoglobin concentration was determined by cyanomethemoglobin at 540 nm.

Tumor Cells and Culture Conditions

B16F10 murine melanoma cells were obtained from American Type Tissue Collection (Manassas, VA, USA). The tumor cells were routinely cultured in DMEM (Life Technologies, Invitrogen Canada) supplemented with 10% FBS. Cells were passaged every 2–3 days. For experiment, melanoma cells were cultured in complete DMEM until they became 30–40% confluent. Then, appropriate aliquots of different samples (0.57 mL sample per 10 mL medium) were added to the medium. The cell viability was followed up to 4 days thereafter (Rodriguez-ayerbe and Smith-zubiaga, [[2000]]; Kayaga et al., [[1999]]). Tumor cells were routinely monitored by phase microscopy. Cell counts were obtained daily with a hemacytometer. Cell viability was determined by trypan blue exclusion.

Intravenous Injection of PolyHb-Tyrosinase in Normal Mice

BD2F1 female mice (C57BL/6 × DBA/2F1) at age 57–63 days were purchased from Charles River Canada (St. Constant, Quebec, Canada). All animals were housed and cared for according to the regulations of McGill University on Animal Care. Mice were kept at a 12-h light interval and fed conventional food and water ad lib. All mice were acclimatized for at least 7 days prior to use. Two groups of mice (5–6 mice per group) were studied. Intravenous injection of saline at 0.1 mL for control group every day, 0.1 mL of PolyHb-tyrosinase solution was injected to test group every day and took blood from lateral saphenous vein after each injection for both groups.

B16F10 Melanoma Bearing Mice Model

B16F10 melanoma cells prepared at 1 × 10⁶ in 0.1 mL of HBSS were injected subcutaneously into shaved lateral flank of the mice. The sizes of primary tumors were measured every 2 days using calipers. Tumor volume is calculated using the formula V = (A × B²)/2, where V is volume (mm³), A is long diameter (mm), and B is short diameter (mm) (Barthelmes et al., [[2001]]). We started intravenous injection at day 9 after tumor implantation when the tumors reached an average size of 125 mm³. End point of study is when the tumor in any group of animal reaches 10% of the body weight. This is based on the regulations of the ethics committee of the Faculty of Medicine, Animal Care Committee of McGill University. Statistical analysis was performed using Student's t-test within ANOVA and considered significant at P < 0.05.

Results

Studies of Melanoma Cell Culture

As described under Methods, we culture B16F10 melanoma cells in DMEM and add one of the following four solutions to the culture medium: (1) saline solution (0.9 g/dL NaCl); (2) free tyrosinase solution; (3) PolyHb solution; and (4) PolyHb-tyrosinase solution. Table 1 shows that PolyHb by itself does not have any effects on the growth of the B16F10 cells when compared to saline. Tyrosinase in the complex has the similar inhibition effects as the free tyrosinase on the growth of the melanoma cell culture (Table 1). Unlike tyrosinase in the free form (Fu et al., [[1999]]; Gili et al., [[2000]]; Jimbow, [[1998]]), tyrosinase in PolyHb-tyrosinase is covered and protected from being exposed to the body by PolyHb with a hemoglobin:tyrosinase molar ratio of 100:2.

Table 1. B16F10 melanoma cell numbers (1 × 10⁵) in cell culture after the addition of saline, PolyHb, free tyrosinase or PolyHb-tyrosinase to the medium

Time (Day)	Saline	PolyHb	Free tyrosinase	PolyHb-tyrosinase
0	0.7 ± 0.1	0.7 ± 0.04	0.8 ± 0.05	0.8 ± 0.1
4	29.5 ± 1.7	28.6 ± 3.9	0.4 ± 0.06	0.3 ± 0.03

Study of Intravenous Injection of PolyHb-Tyrosinase in Normal Mice

Daily intravenous injection of PolyHb-tyrosinase in the test group, reduces plasma tyrosine levels rapidly to 0.19 ± 0.09 mg/dL on day 2, compared to 1.44 ± 0.16 mg/dL in the control group that received daily injected of 0.1 mL saline. The systemic tyrosine concentration remains at low level by continuing with this daily injection of PolyHb-tyrosinase. Since one of the adverse effects of the use of tyrosine restricted diet is nausea, vomiting, and weight loss, we also follow this in the mice. Daily measurements of body weight show no difference between the control group and the test group. Furthermore, we have not observed any nausea or vomiting in these mice.

Effect of Intravenous Injection of PolyHb-Tyrosinase on Melanoma Mice Model

As described under Methods, we inoculate B16F10 cells subcutaneously into mice and when the tumor volume reaches an average of 125 mm³ on day 9, we start the following studies: (1) Sham control group receiving no intravenous injections; (2) Saline group receiving daily intravenous injection of 0.1 mL saline; (3) PolyHb-tyrosinase group receiving 0.1 mL of PolyHb-tyrosinase solution. End point of this study is based on the Faculty of Medicine Animal Care Committee's regulation that tumor burden should not exceed 10% of the animal's normal body weight. Table 2 shows that there is no significant difference in tumor size between the sham control group and the saline group. On the other hand, 4 days after the daily intravenous injection of PolyHb-tyrosinase, the tumor volume is significantly lower than the saline group. Six days after the daily intravenously injection, the tumor volume in the PolyHb-tyrosinase group was only 53 ± 14% of that in the saline group. Nineteen days after the inoculation of the B16F10 melanoma cells, the tumor volume of the control has reached the maximal of 10% of body weight allowed by the Animal Care Committee and we have to terminate the study. At this time, the tumor size in the PolyHb-tyrosinase group was only 45 ± 10% of the saline group. Therefore, our results suggest that PolyHb-tyrosinase retards the growth of B16F10 melanoma in mice. We also follow the body weight of these three groups of mice and there is no significant difference in weight gain.

Table 2. Tumor growth (mm³) of B16F10 melanoma in mice. Sham control group: no intravenous injection; saline group: 0.1 mL intravenous saline daily; (PolyHb-tyrosinase group: 0.l mL intravenous PolyHb-tyrosinase daily. All values are represented as mean ± SEM

Time (Day)	Sham control (mm^3)	Saline (mm^3)	PolyHb-tyrosinase (mm^3)
9	123 ± 33	125 ± 24	124 ± 35
19	3263 ± 259	3190 ± 367	1444 ± 322

Discussion

Melanoma is an increasingly common fatal skin cancer. Despite extensive research, at present there is no practical method for this malignant disease. Meadows’ group has carried out important studies showing that lowering of systemic tyrosine can inhibit the growth of melanoma in mice (Fu et al., [[1999]]; Pelayo et al., [[1999]], [[2001]]). Unfortunately, tyrosine restriction diet is not well tolerated in human resulting in weight loss, nausea, and vomiting in the patients who are already severely ill from their melanoma. Thus, it is not possible to carry out any meaningful clinical trials. We have reported here a possible biotechnological solution to this problem based on intravenous injection of PolyHb-tyrosinase. In this form, the enzyme is covered by hemoglobin molecules at a ratio of hemoglobin:tyrosinase 100:2. This prevents the tyrosinase from having any adverse effect. PolyHb-tyrosinase decreases the systemic tyrosine levels in mice to 13% of the original level without adverse effects of nausea, vomiting, or weight loss. This is compared to tyrosine restricted diet that lowers the tyrosine levels only to 67% of the original level but with adverse effects of nausea, vomiting, or weight loss (Meadows et al., [[1982]]). In our in vitro study, PolyHb-tyrosinase inhibits the growth of B16F10 cells. When injected intravenously into B16F10 melanoma bearing mice, PolyHb-tyrosinase also delays the growth of the melanoma when compared to the control group. Furthermore, the presence of a high concentration of oxygen is important in radiotherapy for cancer cells (Cooper, [[1998]]). Our earlier study shows that there was no significant difference in the oxygen dissociation curves between polyhemoglobin-tyrosinase solution and free bovine hemoglobin solution (Yu and Chang, [[2004a]]). Based on the analysis of oxygen saturation curves, the P₅₀ values for noncrosslinked hemoglobin and PolyHb-tyrosinase were 23 mmHg and 21 mmHg respectively. Thus, polymerization of hemoglobin with tyrosinase did not alter oxygenation transport characteristics of the hemoglobin. In addition to removing tyrosine, this preparation has the potential advantages of being able to more easily perfuse the melanoma to supply more oxygen needed for more effective radiotherapy. These results encourages further studies to optimize this further; to investigate the combine use of this approach with other methods for treating malignant melanoma.

Acknowledgments

TMSC gratefully acknowledges the operating grants from the Medical Research Council of Canada and Canadian Institutes of Health Research. BY gratefully acknowledges the studentship award from Medical Research Council of Canada and the Canadian Institutes of Health Research.