Abstract
Intravenous administration to mice of trans-pterostilbene (t-PTER; 3,5-dimethoxy-4'-hydroxystilbene) and quercetin (QUER; 3,3',4',5,6-pentahydroxyflavone), two structurally related and naturally occurring small polyphenols, inhibits metastatic growth of highly malignant B16 melanoma F10 (B16M-F10) cells. t-PTER and QUER inhibit bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity. However, the molecular mechanism(s) linking polyphenol signaling and bcl-2 expression are unknown. NO is a potential bioregulator of apoptosis with controversial effects on Bcl-2 regulation. Polyphenols may affect NO generation. Short-term exposure (60 min/day) to t-PTER (40 microM) and QUER (20 microM) (approximate mean values of the plasma concentrations measured within the first hour after intravenous administration of 20 mg of each polyphenol/kg) down-regulated inducible NO synthetase in B16M-F10 cells and up-regulated endothelial NO synthetase in the vascular endothelium and thereby facilitated endothelium-induced tumor cytotoxicity. Very low and high NO levels down-regulated bcl-2 expression in B16M-F10 cells. t-PTER and QUER induced a NO shortage-dependent decrease in cAMP-response element-binding protein phosphorylation, a positive regulator of bcl-2 expression, in B16M-F10 cells. On the other hand, during cancer and endothelial cell interaction, t-PTER- and QUER-induced NO release from the vascular endothelium up-regulated neutral sphingomyelinase activity and ceramide generation in B16M-F10 cells. Direct NO-induced cytotoxicity and ceramide-induced mitochondrial permeability transition and apoptosis activation can explain the increased endothelium-induced death of Bcl-2-depleted B16M-F10 cells.
Highlights
Analysis of the bcl-2 family of genes revealed that B16M-F10 cells, compared with B16M-F1 cells, overexpress bcl-2 preferentially [7]. t-PTER increases expression of pro-death bax (ϳ2.2-fold) and decreases expression of anti-death bcl-2 (ϳ2.0-fold) [6], whereas QUER increases expression of different pro-death genes and decreases expression of all anti-death genes analyzed (bcl-2 (ϳ7.3-fold), bcl-w (ϳ1.5-fold), and bcl-xL (ϳ2-fold)) [6]. bcl-2 overexpression prevents the QUER- and t-PTER-dependent increase in metastatic B16M-F10 cell death caused by the HSE in vivo [6], suggesting that Bcl-2 by itself plays a critical role in regulating B16M-F10 resistance against vascular endothelium-in
Reverse transcription-PCR expression analysis revealed that the t-PTER- and/or QUER-induced decrease in NO generation by B16M-F10 cells was associated with inhibition of inducible nitric-oxide synthetase (iNOS) gene expression (Fig. 1)
PTER/QUER-induced bcl-2 downregulation was reversed by a guanylate cyclase activator (YC-1, 3-(5Ј-hydroxymethyl-2Ј-furyl)-1-benzylindazole, Yung Shin Pharmaceutical Industry Co., Ltd., Taichung, Taiwan) (Fig. 5). These results prove the direct connection of PTER/QUER to cAMPresponse element-binding protein (CREB) and to bcl-2 expression. bcl-2 expression and phosphoCREB levels returned to control values in surviving B16M-F10 cells after 12 h of co-culture with endothelial cells, in agreement with the data showing that the effect of t-PTER and QUER could be reversed by NO directly (Table 4)
Summary
Analysis of the bcl-2 family of genes revealed that B16M-F10 cells (high metastatic potential), compared with B16M-F1 cells (low metastatic potential), overexpress bcl-2 preferentially [7]. t-PTER increases expression of pro-death bax (ϳ2.2-fold) and decreases expression of anti-death bcl-2 (ϳ2.0-fold) [6], whereas QUER increases expression of different pro-death genes (bax, bak, bad, and bid; 1.5–2.5-fold) and decreases expression of all anti-death genes analyzed (bcl-2 (ϳ7.3-fold), bcl-w (ϳ1.5-fold), and bcl-xL (ϳ2-fold)) [6]. bcl-2 overexpression prevents the QUER- and t-PTER-dependent increase in metastatic B16M-F10 cell death caused by the HSE in vivo [6], suggesting that Bcl-2 by itself plays a critical role in regulating B16M-F10 resistance against vascular endothelium-in-. Bcl-2 overexpression prevents the QUER- and t-PTER-dependent increase in metastatic B16M-F10 cell death caused by the HSE in vivo [6], suggesting that Bcl-2 by itself plays a critical role in regulating B16M-F10 resistance against vascular endothelium-in-. These apparently controversial facts suggest that different intracellular NO levels may likely determine opposite effects Whether natural polyphenols such as t-PTER and QUER cause reduction in iNOS gene expression in metastatic cells (and a decrease in their intracellular NO levels) and whether this is linked to changes in bcl-2 expression is unknown. Our results show that this polyphenolic association decreases NO production in isolated B16M-F10 cells and increases NO release from the vascular endothelium during B16M-F10/endothelial cell interaction At both steps, changes in NO levels trigger Bcl-2 down-regulation and activation of death mechanisms in metastatic B16M-F10 cells
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