Background: Fucoidan is a sulfated polysaccharide found in brown algae; it has been shown to exhibit a number of biological effects, including anti-tumor effects. In this study, we evaluated the effects of fucoidan on apoptosis in HT-29 and HCT116 human colon cancer cells. Methods: HT-29 and HCT116 cells were cultured with various concentrations of fucoidan (0 - 20 μg/mL). Apoptosis was assayed via Hoechst staining and Annexin V staining followed by flow cytometric analysis. Western blot analyses and JC-1 staining were conducted to determine the levels of apoptosis-regulating proteins and mitochondrial membrane permeability, respectively. Results: Fucoidan induced substantial reductions in viable cell numbers and apoptosis of HT-29 and HCT116 cells in a dose-dependent manner. In HT-29 cells, fucoidan also increased the levels of cleaved caspases-8, -9, -7, and -3, and cleaved poly (ADP-ribose) polymerase (PARP) levels. The levels of the X-linked inhibitor of apoptosis protein and survivin were attenuated in the fucoidan-treated cells. Fucoidan was also shown to enhance mitochondrial membrane permeability, as well as the cytochrome c and Smac/Diablo release from the mitochondria. Fucoidan increased the levels of the Bak and truncated Bid proteins, but reduced the levels of Mcl-1. Additionally, fucoidan increased the levels of the tumor necrosis factor-related apoptosis-inducing ligand, Fas and death receptor 5 proteins. The caspase-8 and -9 inhibitors Z-IETD-FMK and Z-LEHD-FMK induced a reduction in fucoidan-mediated apoptosis. Caspase-8 inhibitor inhibited the fucoidan-induced cleavage of Bid, caspases-9 and -3, and PARP. Conclusion: The findings of this study indicate that fucoidan induces apoptosis in HT-29 and HCT116 human colon cancer cells, and that this phenomenon is mediated via both the death receptor-mediated and mitochondria-mediated apoptotic pathways. These results suggest that fucoidan may prove useful in the development of a colon cancer-preventive protocol.

Luteolin is 3′,4′,5,7-tetrahydroxyflavone found in celery, green pepper, and perilla leaf that inhibits tumorigenesis in animal models. We examined luteolin-mediated regulation of cell cycle progression and apoptosis in the HT-29 human colon cancer cell line. Luteolin decreased DNA synthesis and viable HT-29 cell numbers in a concentration-dependent manner. It inhibited cyclin-dependent kinase (CDK)4 and CDK2 activity, resulting in G₁ arrest with a concomitant decrease of phosphorylation of retinoblastoma protein. Activities of CDK4 and CDK2 decreased within 2 h after luteolin treatment, with a 38% decrease in CDK2 activity ( P < 0.05) observed in cells treated with 40 μmol/l luteolin. Luteolin inhibited CDK2 activity in a cell-free system, suggesting that it directly inhibits CDK2. Cyclin D₁ levels decreased after luteolin treatment, although no changes in expression of cyclin A, cyclin E, CDK4, or CDK2 were detected. Luteolin also promoted G₂/M arrest at 24 h posttreatment by downregulating cyclin B₁ expression and inhibiting cell division cycle (CDC)2 activity. Luteolin promoted apoptosis with increased activation of caspases 3, 7, and 9 and enhanced poly(ADP-ribose) polymerase cleavage and decreased expression of p21^CIP1/WAF1, survivin, Mcl-1, Bcl-x_L, and Mdm-2. Decreased expression of these key antiapoptotic proteins could contribute to the increase in p53-independent apoptosis that was observed in HT-29 cells. We demonstrate that luteolin promotes both cell cycle arrest and apoptosis in the HT-29 colon cancer cell line, providing insight about the mechanisms underlying its antitumorigenic activities.

Introduction: High-fat diets (HFDs) are known to cause obesity and are associated with breast cancer progression and metastasis. Because obesity is associated with breast cancer progression, it is important to determine whether dietary fat per se stimulates breast cancer progression in the absence of obesity. This study investigated whether an HFD increases breast cancer growth and metastasis, as well as mortality, in obesity-resistant BALB/c mice. Methods: The 4-week-old, female BALB/c mice were fed HFD (60% kcal fat) or control diet (CD, 10% kcal fat) for 16 weeks. Subsequently, 4T1 mammary carcinoma cells were injected into the inguinal mammary fat pads of mice fed continuously on their respective diets. Cell-cycle progression, angiogenesis, and immune cells in tumor tissues, proteases and adhesion molecules in the lungs, and serum cytokine levels were analyzed with immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). In vitro studies were also conducted to evaluate the effects of cytokines on 4T1 cell viability, migration, and adhesion. Results: Spleen and gonadal fat-pad weights, tumor weight, the number and volume of tumor nodules in the lung and liver, and tumor-associated mortality were increased in the HFD group, with only slight increases in energy intake and body weight. HF feeding increased macrophage infiltration into adipose tissues, the number of lipid vacuoles and the expression of cyclin-dependent kinase (CDK)2, cyclin D1, cyclin A, Ki67, CD31, CD45, and CD68 in the tumor tissues, and elevated serum levels of complement fragment 5a (C5a), interleukin (IL)-16, macrophage colony-stimulating factor (M-CSF), soluble intercellular adhesion molecule (sICAM)-1, tissue inhibitors of metalloproteinase (TIMP)-1, leptin, and triggering receptor expressed on myeloid cells (TREM)-1. Protein levels of the urokinase-type plasminogen activator, ICAM-1, and vascular cell adhesion molecule-1 were increased, but plasminogen activator inhibitor-1 levels were decreased in the lungs of the HFD group. In vitro assays using 4T1 cells showed that sICAM-1 increased viability; TREM-1, TIMP-1, M-CSF, and sICAM-1 increased migration; and C5a, sICAM-1, IL-16, M-CSF, TIMP-1, and TREM-1 increased adhesion. Conclusions: Dietary fat increases mammary tumor growth and metastasis, thereby increasing mortality in obesity-resistant mice.

A greater intake of vegetables and fruits has been linked to a reduced incidence of colon cancer. Flavonoids are polyphenolic compounds which are broadly distributed in fruits and vegetables and display a remarkable spectrum of physiological activities, including anti-carcinogenic effects. The objective of this study was to determine the mechanisms by which kaempferol, a flavonol present in tea, apples, strawberries, and beans, inhibits the growth of HT-29 human colon cancer cells. To examine the effects of kaempferol on cell cycle progression in HT-29 cells, cells were treated with various concentrations (0-60 μmol/L) of kaempferol. Cell proliferation and DNA synthesis were evaluated by MTT assay and [(3)H]thymidine incorporation assay, respectively. Fluorescence-activated cell sorting analyses were conducted to calculate cell cycle phase distribution. Western blot analyses and in vitro kinase assays were used to estimate the expression of proteins involved in the regulation of cell cycle progression and the activity of cyclin-dependent kinase (CDK)s, respectively. Kaempferol decreased viable cell numbers and [(3)H]thymidine incorporation into DNA of HT-29 cells in a dose-dependent manner. Kaempferol induced G1 cell cycle arrest within 6 h and G2/M arrest at 12 h. Kaempferol inhibited the activity of CDK2 and CDK4 as well as the protein expression of CDK2, CDK4, cyclins D1, cyclin E, and cyclin A, and suppressed the phosphorylation of retinoblastoma protein. Additionally, kaempferol decreased the levels of Cdc25C, Cdc2, and cyclin B1 proteins, as well as the activity of Cdc2. The present results indicate that kaempferol induces G1 and G2/M cell cycle arrest by inhibiting the activity of CDK2, CDK4, and Cdc2. The induction of cell cycle arrest may be one of the mechanisms by which kaempferol exerts anti-carcinogenic effects in colon cancer cells.

Fisetin, a natural flavonol present in edible vegetables, fruits, and wine, was reported to exert anticarcinogenic effects. The objective of the current study was to examine the effect of fisetin on the cell cycle progression of the human colon cancer cell line HT-29. HT-29 cells were cultured in serum-free medium with 0, 20, 40, or 60 μmol/L fisetin. Fisetin dose dependently inhibited both cell growth and DNA synthesis (P < 0.05), with a 79 ± 1% decrease in cell number observed 72 h after the addition of 60 μmol/L fisetin. Perturbed cell cycle progression from the G₁ to S phase was observed at 8 h with 60 μmol/L fisetin treatment, whereas a G₂/M phase arrest was observed after 24 h (P < 0.05). The phosphorylation state of the retinoblastoma proteins shifted from hyperphosphorylated to hypophosphorylated in cells treated with 40 μmol/L fisetin. (P < 0.05). Fisetin decreased the activities of cyclin-dependent kinases (CDK)2 and CDK4; these effects were likely attributable to decreases in the levels of cyclin E and D1 and an increase in p21^CIP1/WAF1 levels (P < 0.05). However, fisetin also inhibited CDk4 activity in a cell-free system (P < 0.05), indicating that it may directly inhibit CDk4 activity. The protein levels of cell division cycles (CDC)2 and CDC25C and the activity of CDC2 were also decreased in fisetin-treated cells (P < 0.05). These results indicate that inhibition of cell cycle progression in HT-29 cells after treatment with fisetin can be explained, at least in part, by modification of CDK activities.