Flavonoids are a class of polyphenolic compounds widely distributed in the plant kingdom, which display a variety of biological activities, including chemoprevention and tumor growth inhibition. Our aim was to investigate the effects of several polyphenols on the growth and metastatic potential of B16-BL6 melanoma cells in vivo. Intraperitoneal administration of quercetin, apigenin, (-)-epigallocathechin-3-gallate (EGCG), resveratrol, and the anti-estrogen tamoxifen, at the time of i.m. injection of B16-BL6 cells into syngeneic mice, resulted in a significant, dose-dependent delay of tumor growth, without toxicity. The relative descending order of potency was EGCG > apigenin ‫؍‬ quercetin ‫؍‬ tamoxifen > resveratrol > control. Furthermore, polyphenols significantly potentiated the inhibitory effect of a non-toxic dose of cisplatin. When tested for the ability to inhibit lung colonization, quercetin, apigenin, and tamoxifen (but not EGCG or resveratrol) significantly decreased the number of B16-BL6 colonies in the lungs in a dose-dependent manner, with quercetin and apigenin being more effective than tamoxifen. Interestingly, quercetin, apigenin, and tamoxifen (but not EGCG or resveratrol) significantly decreased the invasion of B16-BL6 cells in vitro, with quercetin and apigenin being more effective than tamoxifen. This suggests that anti-invasive activity is one of the mechanisms underlying inhibition of lung colonization by quercetin and apigenin. In conclusion, quercetin and apigenin inhibit melanoma growth and invasive and metastatic potential; therefore, they may constitute a valuable tool in the combination therapy of metastatic melanoma.

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A 2. We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34 ؉ hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34 ؉ cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34 ؉ cells synthesized more PGE 2 than TXB2 (214 ؎ 50 vs. 30 ؎ 10 pg͞10 6 cells), whereas the reverse was true in mature megakaryocytes (TXB 2 8,440 ؎ 2,500 vs. PGE 2 906 ؎ 161 pg͞10 6 cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE 2 and TXB2 to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE 2 and TXA2 may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

n-3 Polyunsaturated fatty acids (PUFAs) inhibit the development of microvessels in mammary tumors growing in mice. Human colorectal tumors produce vascular endothelial growth factor (VEGF) whose expression is up-regulated in tumor cells by both cyclooxygenase-2 (COX-2) and PGE(2) and directly correlated to neoangiogenesis and clinical outcome. The goal of this study was to examine the capability of n-3 PUFAs to regulate VEGF expression in HT-29 human colorectal cells in vitro and in vivo. Constitutive VEGF expression was augmented in cultured HT-29 cells by serum starvation and the effects of eicosapentaenoic (EPA) or docosahexaenoic acid (DHA) on VEGF, COX-2, phosphorylated extracellular signal-regulated kinase (ERK)-1 and -2 and hypoxia-inducible-factor 1-alpha (HIF-1alpha) expression and PGE(2) levels were assessed. Tumor growth, VEGF, COX and PGE(2) analysis were carried out in tumors derived from HT-29 cells transplanted in nude mice fed with either EPA or DHA. Both EPA and DHA reduced VEGF and COX-2 expression and PGE(2) levels in HT-29 cells cultured in vitro. Moreover, they inhibited ERK-1 and -2 phosphorylation and HIF-1alpha protein over-expression, critical steps in the PGE(2)-induced signaling pathway leading to the augmented expression of VEGF in colon cancer cells. EPA always showed higher efficacy than DHA in vitro. Both fatty acids decreased the growth of the tumors obtained by inoculating HT-29 cells in nude mice, microvessel formation and the levels of VEGF, COX-2 and PGE(2) in tumors. The data provide evidence that these n-3 PUFAs are able to inhibit VEGF expression in colon cancer cells and suggest that one possible mechanism involved may be the negative regulation of the COX-2/PGE(2) pathway. Their potential clinical application as anti-angiogenic compounds in colon cancer therapy is proposed.

This study demonstrates that the flavonoid quercetin (Q), a plant-derived compound with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of Adriamycin (ADR) on MCF-7 ADR-resistant human breast cancer cells. The effect of Q was dose-dependent at concentrations ranging between 1 and 10 microM. Since ADR resistance in these cells is associated with the expression of high levels of P-glycoprotein (Pgp), we evaluated the effect of Q and related flavonoids of Pgp activity in cytofluorographic efflux experiments with the fluorescent dye rhodamine 123 (Rh 123). Our results indicate that Q and 3-OMe Q (3',4',7-trimethoxyquercetin) but not the 3-rhamnosylglucoside of Q (rutin) inhibit the Pgp pump-efflux activity in a dose-related manner. Moreover, 10 microM Q reduces the expression of the immunoreactive Pgp in MCF-7 ADR-resistant cells as evaluated by cytofluorimetric assay. In conclusion, these findings provide a further biological basis for the potential therapeutic application of Q as an anti-cancer drug either alone or in combination with ADR in multidrug-resistant breast tumor cells.

The assessment of COX-2 status could provide additional information in order to identify ovarian cancer patients with a poor chance of response to chemotherapy and potentially candidates for more individualised treatments.

Several evidences suggest that cancer cells have abnormal cholesterol biosynthetic pathways and prenylation of small guanosine triphosphatase proteins. Tomato lycopene has been suggested to have beneficial effects against certain types of cancer, including that of prostate, although the exact molecular mechanism(s) is unknown. We tested the hypothesis that lycopene may exert its antitumor effects through changes in mevalonate pathway and in Ras activation. Incubation of the Ras-activated prostatic carcinoma LNCaP cells with a 24 h lycopene treatment (2.5-10 μM) dose dependently reduced intracellular total cholesterol by decreasing 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase expression and by inactivating Ras, as evidenced by its translocation from cell membranes to cytosol. Concomitantly, lycopene reduced the Ras-dependent activation of nuclear factor-kappaB (NF-κB). Such a reduction was parallel to an inhibition of reactive oxygen species production and to a decrease in the phosphorylation ofc-jun N-terminal kinase, extracellular signal-regulated kinase 1/2 and p38. These effects were also accompanied by an arrest of cell cycle progression and by apoptosis induction, as evidenced by a decrease in cyclin D1 and phospho-AKT levels and by an increase in p21, p27 and p53 levels and in Bax:Bcl-2 ratio. The addition of mevalonate prevented the growth-inhibitory effects of lycopene as well as its increase in Ras cytoplasmatic accumulation and the subsequent changes in NF-κB. The ability of lycopene in inhibiting HMG-CoA reductase expression and cell growth and in inactivating Ras was also found in prostate PC-3, colon HCT-116 and HT-29 and lung BEN cancer cells. These findings provide a novel mechanistic insight into the growth-inhibitory effects of lycopene in cancer.

Morphologic findings of thymuses from 32 anti-acetylcholine receptor (AChR)-negative myasthenia gravis patients, 12 with and 20 without antibodies against the muscle-specific kinase (MuSK), were compared with those from 30 AChR-positive subjects. In contrast with the high frequency of thymic hyperplastic changes in AChR-positive patients, in MuSK-positive subjects histologic alterations were minimal, arguing against an intrathymic disease pathogenesis. Since hyperplastic changes were seen in 35% of MuSK-negative patients, the thymus could be involved in some of these cases.

Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of ⌬ m after stimulation of the respiratory state. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A (A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.Taxanes are natural products derived from trees of the genus Taxoidaceae. The first taxane introduced in cancer therapy was paclitaxel, firstly isolated from Taxus brevifolia (Schiff et al., 1979). The clinical success of taxanes is dependent on the excellent response rate in second-line treatment of relapsing/resistant cancers and on the efficacy of taxanes in the multichemotherapeutic approach of ovarian and breast cancer (Verweij et al., 1994).In earlier studies, the microtubule network appeared as the main target of paclitaxel (Schiff et al., 1979;Manfredi et al., 1982). In fact, taxanes bind to ␤-tubulin subunits, thereby disrupting normal turnover of the microtubules. The final consequence is the arrest of the cell cycle in M phase with formation of aberrant mitosis and the activation of cell death pathways (Jordan et al., 1993). Along with arrest in M phase of the cell cycle, taxanes have also been reported to induce post-translational serine phosphorylation of the Bcl-2 protein (Haldar et al., 1995). The BCL2 gene is the homologous of the nematode CED-9 gene product (Hengartner and Horvitz, 1994) and is capable of prolonging cell survival by inhibiting apoptotic cell death. Overexpression of Bcl-2 has been observed in follicular lymphoma, where this protein is deregulated by chromosomal translocation, and in a large number of human tumors, including breast, lung, and prostate cancer.Disagreement exists on the levels of Bcl-2 and resistance to taxanes. A strong suggestion for a direct role of Bcl-2 in mediating paclitax...