British Journal of Radiology 75 (2002),909-912 © 2002 The British Institute of Radiology
A study of hypoxic cell radiosensitizer applied to Ehrlich ascite tumour: a comparison of FC43 emulsion and pentoxyfilline
T Wakasa, DDS, DDSc,
N Kawai, DDS,
Y Yanagi, DDS,
Y Hayase, DDS and
K Kishi, DDS, MD
Department of Oral and Maxillofocial Rodiology, Okayama University Graduate School of Medicine and Dentistry, 700-8525, 2-5-1 Shikata-cho, Okayama City, Japan
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Abstract
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In this study, we examined the effects of various combinations of treatments involving radiation, injections of perfluorochemicals (FC-43 emulsion) and injections of pentoxifylline on the growth delay of Ehrlich ascite tumours. Ehrlich ascite tumour cells were transplanted into the legs of ddY-strain mice. Tumour-bearing mice were divided into seven groups: group 1, no treatment; group 2, irradiated only; group 3, injected with FC-43 emulsion and kept in a carbogen atmosphere; group 4, injected with pentoxifylline and nicotinamide; group 5, injected with FC-43 emulsion, kept in a carbogen atmosphere and irradiated; group 6, injected with pentoxifylline and nicotinamide and irradiated; and group 7, injected with FC-43 emulsion, pentoxifylline and nicotinamide, kept in a carbogen atmosphere and irradiated. When 20 Gy irradiation was applied, tumour growth delay was 11 days in group 2, 20 days in group 5, 22 days in group 6, and 24 days in group 7. For a growth delay of 20 days, the dose modifying factor was 1.95±0.04 (standard deviations) in group 5, 1.97±0.09 standard deviations in group 6, and 2.01±0.07 standard deviations in group 7. It was concluded that FC-43 emulsion and pentoxifylline did not have an interactive effect.
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Introduction
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It is well known that oxygen is a potent radiosensitizing agent. Solid tumours may contain areas of avascular necrosis that are surrounded by viable cells, and which are subjected to chronic environmental insufficiencies [1]. Owing to these vascular deficiencies, solid tumours are expected to contain cells that exist in a state of hypoxia and are therefore resistant to radiation [1, 2]. It is thus likely that the efficacy of radiotherapy would be improved if the hypoxic cells were reoxygenated. There have been many attempts to increase radiation sensitivity of hypoxic cells, and it is likely that the efficacy of radiotherapy would be improved if hypoxic cells were reoxygenated.
It has been demonstrated that perfluorochemicals are able to absorb a great amount of oxygen when environmental oxygen pressure (pO2) is high, and can rapidly release oxygen when environmental pO2 is low. A perfluorochemical emulsion treatment in combination with inspiration of atmospheres comprising 95% oxygen has been shown to enhance the response of solid rodent tumours to radiation treatment [3–7]. Conversely, pentoxifylline is thought to increase the oxygen density of local areas by increasing the plasticity of red blood cells [8]. Like other methylxanthines, at relatively high concentrations pentoxifylline has been shown to enhance the cytotoxicity of radiation in cell cultures [9, 10]. In addition, there are few side effects associated with both of these treatments [8, 11].
In this study, we examined the effects of various combinations of treatments involving radiation, injections of perfluorochemicals (FC-43 emulsion) and injections of pentoxifylline on the growth delay of Ehrlich ascite tumours.
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Materials and methods
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Tumours and animals
4-week-old, male ddY-strain mice were used. Ehrlich ascite tumour cells were maintained in the abdominal cavity of ddY mice. After a tumour cell count, 8.0 x 106 cells were transplanted into the abdominal cavity of ddY mice and incubated for 8–9 days. The incubated cells reached the exponential growth phase and 5.5 x 105 cells per 0.01 ml were subcutaneously transplanted into the dorsal side of the thigh region of the right legs of the mice. When the tumour volume reached approximately 200 mm3, the mice were used for experiments. Tumour volume was calculated by means of the formula V=0.4 x a(b2), where a and b are the longer and shorter diameters of the tumours, respectively, as measured with calipers. The mice were killed under anesthesia (sodium pentobarbital injection) after the experiment, in accordance with the ethical standards of the supervising committee on animal experimentation of Okayama University Institute.
X-ray exposure
An X-ray unit (KXC-19; Toshiba, Tokyo, Japan) was used for irradiation. The legs with tumours were locally irradiated with various graded doses of 10 Gy, 20 Gy and 30 Gy of 200 kVp X-rays in a single exposure at 0.66 Gy min-1. Radiation factors were 25 mA with added filtration of 0.5 mmCu+0.5 mmAl. The focus-to-skin distance (FSD) was 50 cm.
Perfluorochemical emulsion and pentoxifylline
We used perfluorochemicals (FC-43 emulsion; Green Cross Corp., Osaka, Japan) and pentoxifylline (Sigma, Stlouis, MO) in the experiments. FC-43 emulsion is an emulsified preparation of a mixture of perfluorochemicals consisting of 14% (weight by volume) perfluorodecalin, 6% perfluorotripropylamine and other components used as emulsifiers and to adjust the osmotic pressure in Krebs–Ringer bicarbonate solution.
12 ml kg-1 FC-43 emulsion was administered via intraperitoneal injection. The animals were then placed in a plastic box and continuously gassed with carbogen through a hole in the cover. After being made to breathe carbogen for 45 min, the mice were anesthetized with 50 mg kg-1 of sodium pentobarbital injected into the abdominal cavity and during radiation, were kept in the carbogen-filled box. Pentoxifylline (100 mg kg-1) and nicotinamide (500 mg kg-1) were injected intravenously at a volume of 0.01 ml g-1 body weight.
Tumour-bearing mice were randomly divided into seven groups. Group 1 was the control group and the mice received no treatment of any type. Group 2 received only irradiation. Groups 3 and 4 received no irradiation; in group 3, mice were injected with FC-43 emulsion and kept in a carbogen atmosphere for 1 h, and in group 4, mice were injected with pentoxifylline and nicotinamide. Group 5 mice were injected with FC-43 emulsion and kept in a carbogen atmosphere before and during irradiation. In group 6, mice were injected with pentoxifylline and nicotinamide, and were irradiated. Finally, group 7 mice were injected with FC-43 emulsion, pentoxifylline and nicotinamide, and were kept in a carbogen atmosphere before and during irradiation. Each experimental group contained 7–12 mice. Tumour size was measured twice weekly.
The dose modifying factor (DMF) for a growth delay of 20 days was defined as the radiation dose required to induce a growth delay of 20 days for groups 5, 6 and 7 divided by the radiation dose needed for the same extent of growth delay for group 2.
Statistical comparisons were made using the Student's t-test.
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Results
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Figure 1
shows mean tumour volume changes as a function of time after various treatments following irradiation of 20 Gy. Each point represents a mean of 7–12 tumours. The untreated control tumours of 200 mm2 grew to 800 mm2, four times the initial volume, in approximately 7 days. When tumours were not exposed to X-irradiation, growth of control tumours (group 1) was not significantly different from that of those treated with the FC-43 emulsion plus carbogen (group 3), or of those treated with only pentoxifylline plus nicotinamide (group 4) (p<0.05). When 20 Gy of X-irradiation was applied (group 2), the tumour volumes increased by four times the initial volume in approximately 18 days. When mice were treated with the FC-43 emulsion plus carbogen, and tumours were exposed to 20 Gy of X-irradiation (group 5), the time required to reach a four-fold increase in the initial tumour volume was approximately 27 days. With a treatment of pentoxifylline plus nicotinamide, and exposure to 20 Gy of X-irradiation (group 6), the four-fold increase in tumour volume took approximately 29 days. Finally, when mice were treated with the FC-43 emulsion plus carbogen, pentoxifylline and nicotinamide, and tumours were exposed to 20 Gy of X-irradiation (group 7), the four-fold increase in tumour volume took approximately 31 days.
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Figure 1. Effects of each treatment regime (groups 1–7) on the tumouricidal effect of 20 Gy X-irradiation. The changes in volume of Ehrlich ascite tumour in the leg of ddY-strain mice are shown as a function of days after various treatments. Means of 7–12 tumours and standard errors are shown.
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Figure 2 shows the growth delay in each group. For a growth delay of 20 days, the DMF±standard deviation for the FC-43 emulsion plus carbogen treatment (group 5) was 1.95±0.04, that for pentoxifylline plus nicotinamide treatment (group 6) was 1.97±0.09 and that for the FC-43 emulsion plus carbogen, pentoxifylline and nicotinamide treatment (group 7) was 2.01±0.07.
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Figure 2. Growth delays for the Ehrlich ascite tumour of ddY-strain mice by various treatments. Means of 7–12 tumours and standard errors are shown. The lines of best fit are obtained by the least square method.
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There were no significant differences in DMF among groups 5, 6 and 7 (p<0.05).
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Discussion
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Misonidazole and its derivatives have been applied in clinical use because they reoxygenate hypoxic cells in tumours. However, these treatments have clinical side effects that strongly affect the central nervous system [12]. Perfluorochemicals or pentoxifylline, on the other hand, have no known side effects. Furthermore, Dion et al [8], who applied pentoxifylline to early and late radiation injuries following fractionated irradiation in C3H mice, reported that pentoxifylline had no effect on the acute radiation injury, but significantly reduced the late injury in a test group compared with a control group.
In studies concerning radiosensitization of Lewis lung tumours, Teicher and Rose [13, 14] reported that in the group treated with perfluorochemicals, carbogen and 20 Gy irradiation, tumour growth delay (TGD) was 28.5 days; in the group receiving 20 Gy irradiation only, TGD was 6.2 days. Song et al [15] applied perfluorochemicals plus carbogen and 20 Gy irradiation to RIF1 tumours and found a TGD of 32 days, while in the group receiving 20 Gy irradiation only, TGD was 13 days. In a study carried out by Lee et al [11], perfluorochemicals plus carbogen and 20 Gy irradiation were applied to SCK tumours with a resultant TGD of 21 days, while a TGD of 16.7 days was found for the group that received 20 Gy irradiation only. The TGD results in this study are therefore almost equal to those previously reported.
In studies of radiosensitization in which pentoxifylline was applied to FSa II tumours, Lee et al [16] found a TGD of 32 days for the group treated with pentoxifylline plus nicotinamide and 20 Gy irradiation; in the group receiving 20 Gy irradiation only TGD was 14 days. The present study demonstrates that mice under the former treatment regimen (group 6) showed a TGD of 29 days, while the TGD in mice treated with 20 Gy irradiation only (group 2) was 18 days. Again, these results are consistent with those of previous reports.
Both Song et al [15] and Lee et al [17] demonstrated that an intravenous injection of perfluorochemicals together with carbogen inhalation increased the intratumour pO2. Additionally, Sperduto et al [10] and Lee et al [16] showed that pentoxifylline alone increased intratumour pO2, while a combination of pentoxifylline and nicotinamide increased it further.
The DMF for the carbogen plus Fluosol-DA treatment was found to be 1.96±0.09 by Song et al [14], while Lee et al [15] reported it to be 2.10±0.01; our DMF data for the carbogen plus the FC-43 emulsion treatment were similar. For the pentoxifylline plus nicotinamide treatment, Lee et al [16] reported a DMF of approximately 2.5; our results for this treatment were a little lower.
In this study, both the perfluorochemicals plus carbogen treatment and the pentoxifylline plus nicotinamide treatment enhanced the effects of X-irradiation. There were, however, no significant differences between the DMFs of these two treatments. Furthermore, a treatment of carbogen plus a combination of perfluorochemicals, pentoxifylline and nicotinamide also enhanced the effects of X-irradiation. However, again there was no significant difference in the DMF among the three groups (groups 5, 6 and 7). We therefore concluded that FC-43 emulsion and pentoxifylline have no interactive effects.
Given that carbogen inhalation is time consuming and the treatment difficult to perform, a pentoxifylline plus nicotinamide treatment might be both easier and more suitable for sensitization of tumour cells to irradiation.
Received for publication January 16, 2001.
Revision received October 1, 2001.
Accepted for publication June 26, 2002.
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References
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- Gray LH. Radiobiologic basis of oxygen as a modifying factor in radiation therapy. Am J Radiol 1961;85:803–15.
- Moulder JE, Rockwell S. Hypoxic fractions of solid tumours: experimental techniques, methods of analysis and a survey of existing data. Int J Radiat Oncol Biol Phys 1984;10:695–712.[Medline]
- Herman TS, Teicher BA, Holden SA, Pfeffer MR, Jones SM. Addition of 2-nitroimidazole radiosensitizers to trimodality therapy (cis-diamminedicloroplatinum II/hyperthermia/radiation) in the murine Fsa II C fibrosarcoma. Cancer Res 1990;50:2734–40.[Abstract/Free Full Text]
- Rockwell S. Use of perfluorochemical emulsion to improve oxygenation in a solid tumour. Int J Radiat Oncol Biol Phys 1985;11:97–103.[Medline]
- Teicher BA, Lazo ZS, Sartorelli AC. Classification of antineoplastic hypoxic tumour cells. Cancer Res 1981;41:73–81.[Abstract/Free Full Text]
- Teicher BA, Rose CM. Sensitization of solid mouse tumour to X-ray treatment by oxygen carrying perfluorochemical emulsion. Cancer Res 1984;44:4285–8.[Abstract/Free Full Text]
- Teicher BA, McIntosh-Lowe NL, Rose CM. Effect of various oxygenation conditions and Fluosol-DA on cancer chemotherapeutic agents. Biomater Artif Cells Artif Organs 1988;16:533–46.[Medline]
- Dion MW, Hussey DH, Osborne JW. The effect of pentoxifylline on early and late radiation injury following fraction fractionated irradiation in C3H mice. Int J Radiat Oncol Biol Phys 1989;17:101–7.[Medline]
- Lee I, Levitt SH, Song CW. Improved tumour oxygenation and radiosensitization by combination with nicotinamide and pentoxifylline. Int J Radiat Biol 1993;64:237–44.[Medline]
- Sperduto PW, Song CW, Levitt SH. The in vitro radiosensitization of human glioblastoma with pentoxifylline. Am J Clin Oncol 1993;16:407–11.[Medline]
- Lee I, Levitt SH, Song CW. Effects of Fluosol DA 20% and carbogen on the radioresponse of SCK tumors and skin of A/J mice. Radiat Res 1987;112:173–82.[Medline]
- Brown JM, Yu N, Brown DM, Lee WW. SR-2508: a 2-nitro-imidazole amide which should be superior to misonidazole as a radiosensitizer for clinical use. Int J Radiat Oncol Biol Phys 1985;7:695–703.
- Teicher BA, Rose CM. Perfluorochemical emulsions can increase tumour radiosensitivity. Science 1984;223:934–6.[Abstract/Free Full Text]
- Teicher BA, Rose CM. Oxygen-carrying perfluorochemical emulsion as an adjuvant to radiation therapy in mice. Cancer Res 1984;44:4285–8.
- Song CW, Zhan WL, Pence DM, Lee IL, Levitt SH. Increased radiosensitivity of tumours by perfluorochemicals and carbogen. Int J Radiat Oncol Biol Phys 1985;11:1833–6.[Medline]
- Lee I, Levitt SH, Song CW. Improved tumour oxygenation and radiosensitization by combination with nicotinamide and pentoxifylline. Int J Radiat Biol 1993;64:237–44.
- Lee I, Levitt SH, Song CW. Radiosensitization of Murine tumours by Fluosol-DA 20%. Radiat Res 1990;122:275–9.[Medline]
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Abstract
Introduction
