The fatty acid synthase inhibitor orlistat reduces experimental metastases and angiogenesis in B16-F10 melanomas

Abstract

Background: Fatty acid synthase (FASN) is overexpressed and associated with poor prognosis in several human cancers. Here, we investigate the effect of FASN inhibitors on the metastatic spread and angiogenesis in experimental melanomas and cultured melanoma cells.

Methods: The lung colonisation assay and cutaneous melanomas were performed by the inoculation of mouse melanoma B16-F10 cells in C57BL6 mice. Blood vessel endothelial cells (RAEC and HUVEC) were applied to determine cell proliferation, apoptosis, and the formation of capillary-like structures. Vascular endothelial growth factor A (VEGFA) expression was evaluated by quantitative RT-PCR and ELISA in B16-F10, human melanoma (SK-MEL-25), and human oral squamous carcinoma (SCC-9) cells. Conditioned media from these cancer cell lines were used to study the effects of FASN inhibitors on endothelial cells.

Results: B16-F10 melanoma-induced metastases and angiogenesis were significantly reduced in orlistat-treated mice. Fatty acid synthase inhibitors reduced the viability, proliferation, and the formation of capillary-like structures by RAEC cells, as well as the tumour cell-mediated formation of HUVEC capillary-like structures. Cerulenin and orlistat stimulated the production of total VEGFA in B16-F10, SK-MEL-25, and SCC-9 cells. Both drugs also enhanced VEGFA(121), (165), (189,) and (165b) in SK-MEL-25 and SCC-9 cells.

Conclusion: FASN inhibitors reduce metastasis and tumour-induced angiogenesis in experimental melanomas, and differentially modulate VEGFA expression in B16-F10 cells.

Figure 1

Orlistat inhibits lung colonisation by B16-F10 cells and peritumoral blood vessels in experimental melanomas. (A) Macroscopic aspects of the lungs from control mice, with many black B16-F10 colonies at the surface, which were clearly reduced in orlistat-treated animals (B). (C and D) Histological sections showing B16-F10 metastatic colonies at the periphery and central portion of the lung (arrows). Representative images from three independent experiments (original magnification: C: × 25 and D: × 100, H&E staining). (E–H) Representative images of the mouse ventral skin showing blood vessels at the periphery of the tumour. (E) Blood vessels directed to the tumour of a control animal (arrows) and (F) detail of the small peritumoral blood vessels of the same animal shown in E. (G) Animal treated with orlistat and (H) detail of G. Orlistat-treated mice showed a significant reduction in both length (I) and area (J) of peritumoral blood vessels in comparison with the controls treated with the vehicle ethanol. The total length of the more calibrous blood vessels (E and G) and the area of small blood vessels (represented by the dashed lines in F and H) were estimated with the aid of a computer programme. All measurements were normalised by the area of the tumour (C: ethanol control; orl: orlistat; *P=0.024; **P=0.002, Mann–Whitney test; original magnification: E and G: × 8, F and H: × 20).

Figure 2

FASN is essential for rabbit aortic endothelial cell (RAEC) growth and survival. (A and B) 0.75 μg ml⁻¹ of cerulenin (A) or 100 μℳ of orlistat (B) strongly reduced the proliferation of RAEC cells cultured in standard conditions (10% of FBS; -▴-), in comparison with the respective controls (-▪-; *P<0.001, Mann–Whitney test). (C and D) Cell cycle analysis by flow cytometry demonstrated that the incubation of RAEC cells with 0.75 μg ml⁻¹ of cerulenin for 48 h or 100 μℳ of orlistat for 96 h enhances the G0–G1 population and reduces the number of cells in the S phase (▪ G0/G1; □ S ; G2/M; *P<0.05, t-test). (E and F) 3 (4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide experiments showing that the viability of RAEC cells is reduced by cerulenin or orlistat in a dose–response manner (*P<0.05, t-test). (G and H) The percentage of B16-F10 cells in apoptosis was significantly enhanced following the treatment with 0.75 μg ml⁻¹ of cerulenin or 100 μℳ of orlistat for 48 h (*P<0.05, t-test). (I–L) The length of capillary-like structures formed by RAEC cells in matrigel was significantly reduced by both drugs (C: control with dimethyl sulfoxide for cerulenin or ethanol for orlistat; cer: cerulenin; orl: orlistat; original magnification in K: × 40).

Figure 3

FASN inhibitors enhance VEGFA expression. Quantitative RT–PCR analysis of total mouse VEGFA in B16-F10 cells (A and B) and total human VEGFA, VEGFA ₁₈₉, ₁₆₅, _121, and _165b transcripts in SK-MEL-25 (C and D) and SCC-9 cells (E and F) after 24 h of treatment with cerulenin (cer) or orlistat (orl). Fatty acid synthase inhibitors at different concentrations increased both total VEGFA and VEGFA isoforms, in comparison with the respective controls (C: control with dimethyl sulfoxide for cerulenin or ethanol for orlistat; expression levels in control cells=1; *P<0.05, t-test).

Figure 4

(A) Total VEGFA protein levels in B16-F10-conditioned medium is higher than in RAEC-conditioned medium after 24 and 48 h. The growth of RAEC (B) and HUVEC (C and D) cells is stimulated by the incubation with culture medium previously conditioned by B16-F10 (CM-B16-F10), SK-MEL-25 (CM-SK-MEL-25), or SCC-9 (CM-SCC-9) cells, in contrast with medium conditioned by RAEC (CM-RAEC) or HUVEC (CM-HUVEC) cells (*P<0.05, t-test).

Figure 5

Total VEGFA production by B16-F10 and SK-MEL-25 melanoma cells is stimulated by the treatment with orlistat (orl) for 48 h (A and E) or FASN knockdown with specific siRNAs (C and G). Western blotting reactions confirming FASN knockdown in the siRNAs transfected B16-F10 (B) and SK-MEL-25 (F) cell lysates. (H) The incubation with 300 μℳ of orlistat for 48 h enhances the secretion of VEGF_165b by SK-MEL-25 cells. (D) Conditioned medium from orlistat-treated B16-F10 cells does not affect the growth of RAEC cells after 24 h. At 48 h, RAEC cells were well preserved in the presence of medium from orlistat-treated B16-F10 cells, while cell death was observed with control medium. (I) Conditioned medium from orlistat-treated SK-MEL-25 cells significantly decreased the growth of HUVEC cells after 48 h (*P<0.05, t-test). (J) The proliferation of HUVEC cells in conditioned medium from orlistat-treated SK-MEL-25 cells is restored in the presence of 10 μg ml⁻¹ of neutralising anti-VEGFA_165b monoclonal antibodies. C: ethanol or nonspecific siRNA oligos.

Figure 6

Effect of orlistat on the formation of capillary-like structures by HUVEC cells. (A and B) 3 (4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide experiments showing that the viability of HUVEC cells is not affected by the presence of cerulenin (cer) or orlistat (orl). (C and D) Orlistat alone (300 μℳ) does not change the formation of capillary-like structures by HUVECs in matrigel. However, conditioned media (CM) from orlistat-treated (300 μℳ) SK-MEL-25 or SCC-9 cells significantly decrease the length of capillary-like structures, in comparison with the respective controls (E–H). C: ethanol control.