n-3 polyunsaturated fatty acids decrease mucosal/epidermal reactions and enhance antitumour effect of ionising radiation with inhibition of tumour angiogenesis

Abstract

The purpose of this study was to evaluate the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs) on normal tissue (lip mucosa) and tumour growth when combined with ionising radiation. The oral region (snout) of C57 black mice was irradiated with 16.5 Gy and n-3 PUFAs (100 microl) were injected intravenously for 2 weeks. After exposure to irradiation, the degree and duration of the acute reactions decreased significantly when mice were treated with n-3 PUFAs as compared to the control group. Interestingly, the range of the reactions in the n-3 PUFAs-treated group compared favourably to the group receiving amifostine (27.5 mg/kg i.v.). the effect of n-3 PUFAs was further evaluated in HEP-2 human carcinoma xenograft transplanted in nude mice. An inhibition of tumour growth was observed when mice were treated with n-3 PUFAs alone and this effect was maximal when combined with irradiation. Similar results were obtained using eicosapentaenoic acid. The effect of n-3 PUFAs was associated with inhibition of angiogenesis and tumour proliferation, and significantly decreased expression of cyclooxygenase-2. In conclusion, n-3 PUFAs administration decrease mucosal response, while moderately enhancing the antitumour effect of irradiation. The magnitude of the differential effect suggests that n-3 PUFAs need to be further investigated in the clinic.

Figure 1

Epidermal/mucosal lip reaction scores in mice, which were the mean of two experiments for irradiation alone (16.5 Gy) and for irradiation plus Intralipid (soja oil) and irradiation plus n-3 PUFAs.

Figure 2

Epidermal/mucosal lip reaction scores in mice; P=0.0002 for irradiation alone and irradiation plus amifostine; P<0.0001 for irradiation alone and irradiation plus n-3 PUFAs.

Figure 3

Change in tumour volume of tumour xenografts (HEP-2) in nude mice irradiated (RT) to a total dose of 15 Gy in two fractions of 7.5 Gy. Values at each point are the mean ratios of the observed tumour volume at a considered time divided by the initial tumour volume. Error bars represent the mean±the standard error of the ratio.

Figure 4

Tumour control probability at 90 days for 8 mm diameter HEP-2 tumours according to the radiation dose for animals treated with irradiation alone (dark triangles), or irradiation and n-3 PUFA (dark squares). Clear triangles and clear square, respectively, represent the dose curing 50% of the animals for irradiation alone and irradiation plus n-3 PUFA; error bars show the 95% confidence interval.

Figure 5

Detection of vessels within the tumour of HEP-2 tumour xenograft: Pecam-1 endothelial immunostaining, magnification × 100. (A) Control group, highly vascularised tumour with thick and large vascular channels. Presence of large vessels located at the periphery of the tumour. (B) Irradiation alone, moderately vascularised tumours. (C) n-3 PUFA alone, moderately vascularised tumours. (D) Irradiation combined with n-3 PUFA, marked inhibition in tumour vascularisation.

Figure 6

Evaluation of the degree of tumour vascularisation in HEP-2 tumour xenograft according to the scoring of HEP-2 tumours from 20 animals that were either untreated, treated by radiation alone, n-3 PUFA alone or both. The diagram shows the mean scoring for each group, error bars represent the standard error. Differences between the irradiation group and irradiation plus n-3 PUFA were statistically significant, P<0.05.

Figure 7

Representative Western blot expression of COX-2 and protein in HEP-2 cells were treated with EPA (pure n-3 PUFA) 100 μg ml⁻¹ and/or irradiated with 5 Gy. The interval for combined treatment was 24 h. All cells were harvested at 48 h treatment. Lane 1: control, lane 2: n-3 PUFAs 100 μg ml⁻¹, lane 3: irradiation with 5 Gy plus n-3 PUFAs 100 μg ml⁻¹, lane 4: 5 Gy irradiation.

Figure 8

Expression of COX-2 mRNA in HEP-2. HEP-2 cells were treated with EPA 100 μg ml⁻¹ and/or irradiated with 5 Gy. The interval for combined treatment was 24 h. All cells were harvested at 48 h after treatment. Lane 1: control, lane 2: n-3 PUFAs 100 μg ml⁻¹, lane 3: n-3 PUFAs 100 μg ml⁻¹+5 gy, lane 4: 5 Gy irradiation alone.