Abstract. Background: Since MAP kinases represent an important pathway of transducing external stimuli to internal signals in cells, determining their possible role in cancer cells may offer a promising way for the treatment and prognosis of malignant diseases. Our previous experiments have shown that a flavonoid-rich solution, Flavin7, was able to diminish kidney tumor growth in vivo. Materials and Methods: Effects of Flavin7 on the MAPK signaling pathway were determined in immortalized mouse proximal tubule cells by determining cell viability, flow cytometric analysis, luciferase assays and Western blots. Results: At a nontoxic dose, Flavin7 markedly reduced phosphorylation of ERK and inhibited activity of its downstream targets such as Elk1 and CREB via inhibition of the ERK-kinase MEK1. Conclusion: Because of its ability to temporarily inhibit kidney tumor growth and activation of the MEK1/ERK pathway in vitro, further in vivo investigations may determine the potential role of Flavin7 in the treatment of malignancies. The mitogen-activated protein kinase (MAPK) cascade is a major signaling system by which cells transduce extracellular signals into intracellular responses. Many steps of this cascade are conserved, and homologs have been discovered in different species (1). The first three mammalian MAP kinases, ERK1, ERK2 and ERK3 were cloned in the early 1990s, facilitating the development of reagents for their study. It has become clear that ERK1 and ERK2 are among the protein kinases most commonly activated in signal transduction pathways. They have particularly been linked to cell proliferation, but have important roles in many other events (2-4). In mammalian cells, ERK1 and ERK2, often referred to as p44 and p42 MAP kinases, are the archetypal members of the MAPK family. Therefore, determining the possible role of MAPKs in cancer cells may offer a promising way for treatment and prognosis of cancerous diseases. According to recent results, activation of the ERK pathway is a frequent event in tumorigenesis. ERK has been implicated in cell initiation, tumor promotion and progression, invasion, metastasis, and regulation of apoptosis and angiogenesis, events that are essential for successful completion of developing a metastatic tumor (reviewed in 5). On the other hand, ERK activation is not unambiguously an advantage or a disadvantage for patients with cancerous diseases, since it has been shown to trigger cell proliferation and survival in normal cells, as well as in tumor cells. Flavonoids, found in great quantity in fruit extracts, are secondary metabolites of superior plants exhibiting antitumor effects. They are known to exert antioxidant and antiproliferative effects on tumor cells (6). Recent studies have speculated that the classical antioxidant activity of flavonoids is unlikely to be the sole explanation for their cellular effects. This hypothesis is based on several lines of reasoning: i) flavonoids are extensively metabolized in vivo, thus, their redox potentials are significantly altered (7), and ii) the concentrations of flavonoids and their metabolites accumulated in vivo are lower than those of small of antioxidant nutrients (8). Investigations have indicated that flavonoids may selectively interact with the MAPK signaling pathway due to their ability to inhibit tyrosine kinase activity (9, 10). A natural compound, Flavin7 (F7), composed of the extracts from seven different fruits, was investigated in our kidney tumor animal model (11). Ne/De tumor cells were transplanted underneath the renal capsule of 6- to 8-weekold Fisher344 rats and animals were treated with human 871 Correspondence to: Edit Nádasi, MD, Ph.D., Department of Public Health and Preventive Medicine, University of Pécs, 7624 Pécs, Szigeti u. 12, Hungary. Tel: +36 72 536394, Fax: +36 72 536395, e-mail: email@example.com Key Words: Flavin7 extract, plant-derived natural compounds, mitogen-activated protein kinase pathway, anticarcinogenic effect. in vivo 21: 871-876 (2007) Effect of a Plant-derived Natural Compound, Flavin7, on the ERK Signaling Pathway in Immortalized Mouse Proximal Tubule Cells EDIT NÁDASI1,2, ISTVÁN EMBER2 and ISTVÁN ARANY1 1Department of Internal Medicine, University of Arkansas for Medical Sciences and Central Arkansas Veteran HealthCare System, Little Rock, AR 72205, U.S.A.; 2Department of Public Health and Preventive Medicine, University of Pécs, 7624 Pécs, Hungary 0258-851X/2007 $2.00+.40 dose-equivalent F7 solution according to the manufacturer’s instructions. After two weeks of treatment rats were sacrificed and tumor growth was determined. F7 significantly (p<0.05) reduced tumor growth in vivo. Accordingly, the aim of this study was to determine whether F7 influences the ERK signaling pathway in immortalized mouse renal proximal tubule cells.
Few studies have investigated the effects produced by combinations of polysaccharides and chemotherapeutic drugs in cancer treatment. We hypothesized that a combination of polysaccharides (COP) from Lentinus edodes and Tricholoma matsutake would improve the efficacy of 5-fluorouracil (5-FU)-mediated inhibition of H22 cell growth.
Mice were injected H22 cells and then treated with either 5-FU, polysaccharides from Tricholoma matsutake (PTM), polysaccharides from Lentinus edodes (PL), PTM+PL, 5-FU+PTM, 5-FU+ PL, or 5-FU + COP. The tumor weight and volume, and splenic CD4 + and CD8 + T cell frequencies, were determined. Additionally, splenic natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activities were assessed and the serum levels of tumor necrosis factor-alpha (TNF-alpha), Interleukin-2 (IL-2), and Interferon-gamma (IFN-gamma) were measured.
Compared with mice from the control, 5-FU, PL, PTM, PTM + PL, 5-FU + PL, and 5-FU + PTM groups, mice treated with 5-FU + COP showed: (a) significantly reduced tumor weight and volume (P < 0.05); (b) significantly higher serum levels of TNF-alpha, IL-2, and IFN-gamma (P < 0.05); (c) significantly increased CD4+ and CD8+ T cell frequencies in the spleen (P < 0.05); and (d) significantly increased splenic NK cell and CTL activities (P < 0.05). The tumor weight and volume in mice treated with 5-FU+PL or 5-FU+PTM were significantly reduced compared with mice treated with 5-FU alone (P < 0.05). Serum levels of TNF-alpha, IL-2, and IFN-gamma, frequencies of CD4 + and CD8+ T cells in the spleen, and splenic NK and CTL activities were also significantly increased in mice treated with 5-FU+PL or 5-FU+PTM compared with mice treated with 5-FU alone (P < 0.05).
Polysaccharides from Lentinus edodes and Tricholoma matsutake could enhance the efficacy of 5-FU-mediated H22 cell growth inhibition.
Prevention of cardiovascular disease through nutritional supplements is growing in popularity throughout the world. Multiple epidemiologic studies found that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases (CHD). Chronic inflammation and atherosclerosis associated with CHD culminate in aberrant intravascular expression of tissue factor (TF), which triggers blood coagulation leading to thrombosis, a major cause for heart attack. We showed earlier that two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells. In the present study, we investigated efficacy of seven resveratrol derivatives, which were shown to be effective in regulating cancer cell growth in vitro at much lower concentrations than the parent compound resveratrol, in inhibiting TF induction in peripheral blood mononuclear cells (PBMCs). We also tested possible synergistic effects of resveratrol and quercetin with the other major red wine phenolics in suppression of lipopolysaccharide-induced TF expression in human PBMCs. We found that several resveratrol derivatives were 2- to 10-fold more efficient than resveratrol in inhibiting TF induction. Our study found no evidence for synergism among red wine polyphenolics. These data suggest that structural alterations of resveratrol can be effective in producing potent antithrombotic agents that will have therapeutic potential in the improvement of cardiovascular health and prevention of CHD. Among major red wine phenolics, quercetin appears to be the predominant suppressor of TF induction.
The currently available chemotherapeutic regimens against gastric cancer are not very effective, leading to high recurrence and poor survival. Resveratrol is a naturally occurring polyphenol with potent apoptosis-inducing activity. However, the mechanism underlying its actions remains unknown. In the present study, human gastric adenocarcinoma SGC7901 cells were treated with resveratrol (0, 25, 50, 100 and 200 μmol/L) for 48 h, and cellular apoptosis DNA damage were determined. In certain experiments, cells were incubated with superoxide dismutase (100 U/mL), catalase (300 U/mL) or sirtinol (10 μmol/L) to determine the role of reactive oxygen species (ROS) and sirtuin1 in resveratrol-induced cellular apoptosis. Treatment with resveratrol (50-200 μmol/L) for 48 h significantly induced apoptosis and DNA damage in human gastric cancer SGC7901 cells. This was due to the increased generation of ROS following resveratrol treatment because incubation of cells with superoxide dismutase (100 U/mL) or catalase (300 U/mL) attenuated resveratrol-induced cellular apoptosis. Interestingly, treatment with resveratrol (25-200 μmol/L) did not affect the level and activity of sirtuin1, whereas the sirtuin1 inhibitor sirtinol (10 μmol/L) significantly reduced sirtuin1 activity. Furthermore, treatment with sirtinol (10 μmol/L) did not have any effect on apoptosis induced by resveratrol. These data provide evidence that resveratrol induces apoptosis via ROS, but independent of sirtuin1, in the human gastric cancer cell line SGC7901.
Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.