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.
Highly metastatic B16 melanoma (B16M)-F10 cells, as compared with the low metastatic B16M-F1 line, have higher GSH content and preferentially overexpress BCL-2. In addition to its anti-apoptotic properties, BCL-2 inhibits efflux of GSH from B16M-F10 cells and thereby may facilitate metastatic cell resistance against endothelium-induced oxidative/nitrosative stress. Thus, we investigated in B16M-F10 cells which molecular mechanisms channel GSH release and whether their modulation may influence metastatic activity. GSH efflux was abolished in multidrug resistance protein 1 knock-out (MRP؊/؊1) B16M-F10 transfected with the Bcl-2 gene or in MRP؊/؊1 B16M-F10 cells incubated with L-methionine, which indicates that GSH release from B16M-F10 cells is channeled through MRP1 and a BCL-2-dependent system (likely related to an L-methionine-sensitive GSH carrier previously detected in hepatocytes). The BCL-2-dependent system was identified as the cystic fibrosis transmembrane conductance regulator, since monoclonal antibodies against this ion channel or H-89 (a protein kinase A-selective inhibitor)-induced inhibition of cystic fibrosis transmembrane conductance regulator gene expression completely blocked the BCL-2-sensitive GSH release. By using a perifusion system that mimics in vivo conditions, we found that GSH depletion in metastatic cells can be achieved by using Bcl-2 antisense oligodeoxynucleotide-and verapamil (an MRP1 activator)-induced acceleration of GSH efflux, in combination with acivicin-induced inhibition of ␥-glutamyltranspeptidase (which limits GSH synthesis by preventing cysteine generation from extracellular GSH). When applied under in vivo conditions, this strategy increased tumor cytotoxicity (up to ϳ90%) during B16M-F10 cell adhesion to the hepatic sinusoidal endothelium.B16 melanoma (B16M) 1 cells with high glutathione (GSH; ␥-glutamylcysteinylglycine) content show higher metastatic activity in the liver than those with low GSH content (1). The liver is a common site for metastasis development, and we demonstrated that GSH protects B16M cells against nitrosative and oxidative stress in the murine hepatic microvasculature (2, 3). The concept that high GSH content status is an important factor for metastasis progression was strongly supported by the fact that metastatic B16M cell survival and growth can be enhanced by directly increasing their GSH content with GSH ester, which readily enters the cell and delivers free GSH (4, 5). In consequence, the maintenance of high intracellular levels of GSH appears critical for metastatic cells to survive intravascularly and to progress extravascularly.Multidrug and/or radiation resistance, which are characteristic features of malignant tumors, frequently associate with high GSH content in the cancer cells (6). Efflux of GSH and GSH S-conjugates from different mammalian cells is mediated by multidrug resistance proteins (MRP), among which MRP1 and MRP2 have been characterized at the functional level as ATP-dependent pumps with broad specificity for GSH and glucuro...
Colorectal cancer is one of the most common malignancies worldwide. The treatment of advanced colorectal cancer with chemotherapy and radiation has two major problems: development of tumor resistance to therapy and nonspecific toxicity towards normal tissues. Different plant-derived polyphenols show anticancer properties and are pharmacologically safe. In vitro growth of human HT-29 colorectal cancer cells is inhibited (f56%) by bioavailable concentrations of trans-pterostilbene (trans-3,5-dimethoxy-4 ¶-hydroxystilbene; t-PTER) and quercetin (3,3 ¶,4 ¶,5,6-pentahydroxyflavone; QUER), two structurally related and naturally occurring small polyphenols. I.v. administration of t-PTER and QUER (20 mg/kg  day) inhibits growth of HT-29 xenografts (f51%). Combined administration of t-PTER + QUER, FOLFOX6 (oxaliplatin, leucovorin, and 5-fluorouracil; a first-line chemotherapy regimen), and radiotherapy (X-rays) eliminates HT-29 cells growing in vivo leading to long-term survival (>120 days). Gene expression analysis of a Bcl-2 family of genes and antioxidant enzymes revealed that t-PTER + QUER treatment preferentially promotes, in HT-29 cells growing in vivo, (a) superoxide dismutase 2 overexpression (f5.7-fold, via specificity protein 1-dependent transcription regulation) and (b) down-regulation of bcl-2 expression (f3.3-fold, via inhibition of nuclear factor-KB activation). Antisense oligodeoxynucleotides to human superoxide dismutase 2 and/or ectopic bcl-2 overexpression avoided polyphenols and chemoradiotherapy-induced colorectal cancer elimination and showed that the mangano-type superoxide dismutase and Bcl-2 are key targets in the molecular mechanism activated by the combined application of t-PTER and QUER. [Mol Cancer Ther 2008;7(10):3330 -42]
B16 melanoma (B16M) cells with high GSH contentshow high metastatic activity. However, the molecular mechanisms linking GSH to metastatic cell survival are unclear. The possible relationship between GSH and the ability of Bcl-2 to prevent cell death was studied in B16M cells with high (F10) and low (F1) metastatic potential. Analysis of a Bcl-2 family of genes revealed that B16M-F10 cells, as compared with B16M-F1 cells, overexpressed preferentially Bcl-2 (ϳ5.7-fold). Hepatic sinusoidal endothelium-induced B16M-F10 cytotoxicity in vitro increased from ϳ19% (controls) to ϳ97% in GSH-depleted B16M-F10 cells treated with an antisense Bcl-2 oligodeoxynucleotide (Bcl-2-AS). L-Buthionine (S,R)-sulfoximine-induced GSH depletion or Bcl-2-AS decreased the metastatic growth of B16M-F10 cells in the liver. However, the combination of L-buthionine (S,R)-sulfoximine and Bcl-2-AS abolished metastatic invasion. Bcl-2-overexpressing B16M-F1/Tet-Bcl-2 and B16M-F10/TetBcl-2 cells, as compared with controls, showed an increase in GSH content, no change in the rate of GSH synthesis, and a decrease in GSH efflux. Thus, Bcl-2 overexpression may increase metastatic cell resistance against oxidative/nitrosative stress by inhibiting release of GSH. In addition, Bcl-2 availability regulates the mitochondrial GSH (mtGSH)-dependent opening of the permeability transition pore complex. Death in B16M-F10 cells was sharply activated at mtGSH levels below 30% of controls values. However, this critical threshold increased to ϳ60% of control values in Bcl-2-AS-treated B16M-F10 cells. GSH ester-induced replenishment of mtGSH levels (even under conditions of cytosolic GSH depletion) prevented cell death. Our results indicate that survival of B16M cells with high metastatic potential can be challenged by inhibiting their GSH and Bcl-2 synthesis.The majority of metastatic cells entering microvascular beds are killed within the first hours and do not generate colonies. This failure, termed "metastatic inefficiency" (1), is due to mechanical trauma produced by blood flow (2), the inability of cancer cells to withstand deformation (3), cytotoxicity of locally released reactive oxygen and nitrogen species (4), and the lytic action of lymphocytes and macrophages (5). The B16 melanoma (B16M) 1 is a model widely used to study metastatic spread and tissue invasion (6). The liver is a common site for metastasis development, and we recently reported that GSH (␥-glutamylcysteinyl-glycine) protects B16M-F10 cells (with high metastatic potential) against nitrosative and oxidative stress in the hepatic microvasculature (4, 6). In fact, multidrug and/or radiation resistance, which are characteristic features of malignant tumors, frequently associate with high GSH content in the cancer cells (7). B16M-F10 cell resistance to the HSE-induced cytotoxicity is highly dependent on GSH and GSH peroxidase (4). However, B16M-F10 cells cultured to low density (LD), with high GSH content, were more resistant to NO and H 2 O 2 than B16M cultured to high density (with ϳ25% of th...
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