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]

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. However, the potential anticancer properties of t-RESV are strongly limited because of its low bioavailability (5). Thus, structural modifications of the t-RESV molecule appeared to be necessary to increase its bioavailability while preserving its biological activity. Recently, we found that trans-pterostilbene (t-PTER; trans-3,5-dimethoxy-4Ј-hydroxystilbene) and quercetin (QUER; 3,3Ј,4Ј,5,6-pentahydroxyflavone) have longer in vivo half-lives compared with t-RESV (6). In vitro growth of highly malignant B16 melanoma F10 (B16M-F10) cells is inhibited (56%) by short-term exposure (60 min/day) to t-PTER (40 M) and QUER (20 M) (6). Intravenous administration of t-PTER and QUER (20 mg/kg/day) to mice inhibits (73%) metastatic growth of B16M-F10 cells in the liver, a common site for metastasis development (6). The antimetastatic mechanism involves (a) t-PTER-induced inhibition of vascular adhesion molecule 1 (VCAM-1) expression in the hepatic sinusoidal endothelium (HSE), which decreases B16M-F10 cell adhesion to the endothelium via very late activation antigen 4, and (b) QUER-and t-PTER-induced inhibition of bcl-2 expression in metastatic cells, which sensitizes them to vascular endothelium-induced cytotoxicity (6).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-x L (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), thus suggesting that Bcl-2 by itself plays a critical role in regulating B16M-F10 resistance against vascular endothelium-in-

Thioredoxin 1 (Trx1) is a key regulator of cellular redox balance and participates in cellular signaling events. Recent evidence from yeast indicates that members of the Trx family interact with the 20S proteasome, indicating redox regulation of proteasome activity. However, there is little information about the interrelationship of Trx proteins with the proteasome system in mammalian cells, especially in the nucleus. Here, we have investigated this relationship under various cellular conditions in mammalian cells. We show that Trx1 levels and its subcellular localization (cytosol, endoplasmic reticulum, and nucleus) depend on proteasome activity during the cell cycle in NIH3T3 fibroblasts and under stress conditions, when proteasomes are inhibited. In addition, we also studied in these cells how the main cellular antioxidant systems are stimulated when proteasome activity is inhibited. Finally, we describe a reduction in Trx1 levels in Lafora disease fibroblasts and demonstrate that the nuclear colocalization of Trx1 with 20S proteasomes in laforin-deficient cells is altered compared with control cells. Our results indicate a close relationship between Trx1 and the 20S nuclear proteasome and give a new perspective to the study of diseases or physiopathological conditions in which defects in the proteasome system are associated with oxidative stress.

BackgroundBcl-2 is believed to contribute to melanoma chemoresistance. However, expression of Bcl-2 proteins may be different among melanomas. Thus correlations among expression of Bcl-2-related proteins and in vivo melanoma progression, and resistance to combination therapies, was investigated.MethodsHuman A375 melanoma was injected s.c. into immunodeficient nude mice. Protein expression was studied in tumor samples obtained by laser microdisection. Transfection of siRNA or ectopic overexpression were applied to manipulate proteins which are up- or down-regulated, preferentially, during melanoma progression. Anti-bcl-2 antisense oligonucleotides and chemoradiotherapy (glutathione-depleting agents, paclitaxel protein-binding particles, daunorubicin, X rays) were administered in combination.ResultsIn vivo A375 cells down-regulated pro-apoptotic bax expression; and up-regulated anti-apoptotic bcl-2, bcl-xl, and mcl-1, however only Bcl-2 appeared critical for long-term tumor cell survival and progression in vivo. Reduction of Bcl-2, combined with partial therapies, decreased melanoma growth. But only Bcl-2 targeting plus the full combination of chemoradiotherapy eradicated A375 melanoma, and led to long-term survival (> 120 days) without recurrence in 80% of mice. Tumor regression was not due to immune stimulation. Hematology and clinical chemistry data were within accepted clinical toxicities.ConclusionStrategies to target Bcl-2, may increase the effectiveness of antitumor therapies against melanomas overexpressing Bcl-2 and likely other Bcl-2-related antiapoptotic proteins.