The inhibition of lung colonization of B16-F10 melanoma cells in EFA-deficient animals is related to enhanced apoptosis and reduced angiogenesis

Abstract

Previous studies conducted in our laboratory showed that the reproduction of spontaneous and experimental metastases was reduced in host animals deprived of essential fatty acids (EFA). In the present study, we have explored the possibility whether apoptosis, proliferation, and angiogenesis might be involved in the antimetastatic effect of EFA deficiency. To this aim, in pulmonary colonies developed from B16-F10 cells in EFA-deficient animals or in animals fed a 5% corn oil diet, we performed an immunohistochemical analysis of bcl-2/bax proteins, PCNA, and VEGF and von Willebrand Factor (vWF), typical markers of apoptosis, proliferation, and angiogenesis, respectively. Apoptosis was also evaluated by detecting DNA fragments in metastatic cells. We found that the reduction of pulmonary colonies grown in EFA-deficient animals was associated with a high expression of apoptotic activity as revealed by the presence of apoptotic nuclei and a high immunoreactivity for bax. Cell proliferation seemed not to be influenced by EFA deficiency in view of the observation that PCNA was highly expressed in pulmonary colonies of control as well as EFA-deficient animals. Pulmonary colonies developed in EFA- deficient animals showed a lower expression of VEGF and a decreased microvessel density, indicating that a reduced angiogenesis contributes to the antimetastatic effects of EFA deficiency. Our analysis of the results invokes the possibility that a relationship between angiogenesis and apoptosis may account for the diminution of the development of experimental metastases in the lungs of EFA-deficient animals.

An enhanced apoptosis and a reduced angiogenesis are associated with the inhibition of lung colonisation in animals fed ann-3 polyunsaturated fatty acid-rich diet injected with a highly metastatic murine melanoma line

“…Total lipids were extracted by the method of Folch et al ( 38 ) from liver homogenates. Phosphatidylcholine was separated from the other lipid classes by HPLC following the conditions reported in a previous paper ( 7 ) . Phosphatidylcholine, solubilised in 0·2 ml benzene, was submitted to transmethylation with 5 % H 2 SO 4 in methanol, at 80°C, overnight.…”

“…Immunoreactivities of PCNA, bax/bcl-2, VEGF and vWF were determined in 5 μm thick sections of formalin-fixed paraffin-embedded metastatic colonies, according to a procedure described in a previous paper ( 7 ) . Monoclonal antibodies against PCNA, bax/bcl-2 and VEGF were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA), while the polyclonal antibody against mouse vWF was supplied by Dako (Glostrup, Denmark).…”

“…Animal studies provide evidence that a high content of linoleic acid (18 : 2 n -6) in the diet influences the development of carcinomas of the breast, colon and prostate, by acting at the promotional stage of carcinogenesis (for a review, see Carroll & Khor ( 3 ) ). The reproduction of experimental metastasis has also been shown to be favoured by 18 : 2 n -6-rich diets ( 4 – 7 ) . These experimental data prompted interest in 18 : 2 n -6 as a possible factor promoting the development of human tumours, in view of the fact that 18 : 2 n -6 is the most abundant fatty acid of the n -6 series in vegetable oils which are the major lipid component in the diets of the Western world.…”