Association between pterostilbene and quercetin inhibits metastatic activity of B16 melanoma

Abstract

Inhibition of cancer growth by resveratrol (trans-3,5,4'-trihydroxystilbene; RESV), a phytoalexin present in many plant species, is limited by its low bioavailability. Pterostilbene (3,5-dimethoxy-4'-hydroxystilbene; PTER) and quercetin (3,3',4',5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols, show longer half-life in vivo. In vitro growth of highly malignant B16 melanoma F10 cells (B16M-F10) is inhibited (56%) by short-time exposure (60 min/day) to PTER (40 microm) and QUER (20 microm) (approximate mean values of plasma concentrations measured within the first hour after intravenous administration of 20 mg/kg each polyphenol). Intravenous administration of PTER and QUER (20 mg/kg per day) to mice inhibits (73%) metastatic growth of B16M-F10 cell in the liver, a common site for metastasis development. The anti-metastatic mechanism involves: 1) a PTER-induced inhibition of vascular adhesion molecule 1 expression in the hepatic sinusoidal endothelium, which consequently decreases B16M-F10 cell adhesion to the endothelium through very late activation antigen 4; and 2) a QUER- and PTER-induced inhibition of Bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. Our findings demonstrate that the association of PTER and QUER inhibits metastatic melanoma growth and extends host survival.

Figure 1

Plasma levels and half-life of pterostilbene and quercetin after their intravenous administration to mice. Animals were treated with 20 mg of t-PTER or QUER per kilogram body weight. Plasma levels were determined as explained under Materials and Methods section. Results are mean ± SD for five to six mice per time point.

Figure 2

In vitro inhibition of B16M-F10 cell growth by short-time exposure to resveratrol, pterostilbene, and/or quercetin. B16M-F10 cells were cultured for 72 hours. t-RESV (12 µM), t-PTER (40 µM), and QUER (20 µM) were added at 6, 30, and 54 hours of culture time, and were present each time for only 60 minutes. After the 60-minute period in the presence of polyphenols, the culture flasks were washed out (three times with PBS) and the medium was renewed. All points are mean ± SD for five to six independent experiments. *P < .05, **P < .01 compared to control values.

Figure 3

Flow cytometric analysis of VCAM-1 on HSE cells. Flow cytometry was performed (see the legend to Table 4) on cultured endothelial cells incubated in the absence or in the presence of t-PTER (40 µM). Black histogram: control cells. Grey histogram: t-PTER-treated cells. Results shown are representative of five different experiments.

Figure 4

Western blot analysis of Bcl-2 in B16M-F10 cells. B16M-F10 cells were cultured x 72 hours in the absence or in the presence of t-PTER (40 µM) or QUER (20 µM) (see the legend to Table 6). Lane a, QUER; lane b, t-PTER; lane c, control untreated cells.