Background

Visceral white adipose tissue (WAT) hypertrophy, adipokine production, inflammation and fibrosis are strongly associated with obesity, but the time-course of these changes in-vivo are not fully understood. Therefore, the aim of this study was to establish the time-course of changes in adipocyte morphology, adipokines and the global transcriptional landscape in visceral WAT during the development of diet-induced obesity.

Results

C57BL/6 J mice were fed a high-fat diet (HFD) or normal diet (ND) and sacrificed at 8 time-points over 24 weeks. Excessive fat accumulation was evident in visceral WAT depots (Epidydimal, Perirenal, Retroperitoneum, Mesentery) after 2–4 weeks. Fibrillar collagen accumulation was evident in epidydimal adipocytes at 24 weeks. Plasma adipokines, leptin, resistin and adipsin, increased early and time-dependently, while adiponectin decreased late after 20 weeks. Only plasma leptin and adiponectin levels were associated with their respective mRNA levels in visceral WAT. Time-course microarrays revealed early and sustained activation of the immune transcriptome in epididymal and mesenteric depots. Up-regulated inflammatory genes included pro-inflammatory cytokines, chemokines (Tnf, Il1rn, Saa3, Emr1, Adam8, Itgam, Ccl2, 3, 4, 6, 7 and 9) and their upstream signalling pathway genes (multiple Toll-like receptors, Irf5 and Cd14). Early changes also occurred in fibrosis, extracellular matrix, collagen and cathepsin related-genes, but histological fibrosis was only visible in the later stages.

Conclusions

In diet-induced obesity, early activation of TLR-mediated inflammatory signalling cascades by CD antigen genes, leads to increased expression of pro-inflammatory cytokines and chemokines, resulting in chronic low-grade inflammation. Early changes in collagen genes may trigger the accumulation of ECM components, promoting fibrosis in the later stages of diet-induced obesity. New therapeutic approaches targeting visceral adipose tissue genes altered early by HFD feeding may help ameliorate the deleterious effects of diet-induced obesity.

Background

Luteolin is a 3',4',5,7-tetrahydroxyflavone found in various fruits and vegetables. We have shown previously that luteolin reduces HT-29 cell growth by inducing apoptosis and cell cycle arrest. The objective of this study was to examine whether luteolin downregulates the insulin-like growth factor-I receptor (IGF-IR) signaling pathway in HT-29 cells.

Methods

In order to assess the effects of luteolin and/or IGF-I on the IGF-IR signaling pathway, cells were cultured with or without 60 μmol/L luteolin and/or 10 nmol/L IGF-I. Cell proliferation, DNA synthesis, and IGF-IR mRNA levels were evaluated by a cell viability assay, [3H]thymidine incorporation assays, and real-time polymerase chain reaction, respectively. Western blot analyses, immunoprecipitation, and in vitro kinase assays were conducted to evaluate the secretion of IGF-II, the protein expression and activation of IGF-IR, and the association of the p85 subunit of phophatidylinositol-3 kinase (PI3K) with IGF-IR, the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)1/2, and cell division cycle 25c (CDC25c), and PI3K activity.

Results

Luteolin (0 - 60 μmol/L) dose-dependently reduced the IGF-II secretion of HT-29 cells. IGF-I stimulated HT-29 cell growth but did not abrogate luteolin-induced growth inhibition. Luteolin reduced the levels of the IGF-IR precursor protein and IGF-IR transcripts. Luteolin reduced the IGF-I-induced tyrosine phosphorylation of IGF-IR and the association of p85 with IGF-IR. Additionally, luteolin inhibited the activity of PI3K activity as well as the phosphorylation of Akt, ERK1/2, and CDC25c in the presence and absence of IGF-I stimulation.

Conclusions

The present results demonstrate that luteolin downregulates the activation of the PI3K/Akt and ERK1/2 pathways via a reduction in IGF-IR signaling in HT-29 cells; this may be one of the mechanisms responsible for the observed luteolin-induced apoptosis and cell cycle arrest.

Accumulating evidence from epidemiological studies indicates that chronic inflammation and oxidative stress play critical roles in neoplastic development. The aim of this study was to investigate the anti-inflammatory, anti-oxidative stress activities, and differential regulation of Nrf2-mediated genes by tea Chrysanthemum zawadskii (CZ) and licorice Glycyrrhiza uralensis (LE) extracts. The anti-inflammatory and anti-oxidative stress activities of hexane/ethanol extracts of CZ and LE were investigated using in vitro and in vivo approaches, including quantitative real-time PCR (qPCR) and microarray. Additionally, the role of the transcriptional factor Nrf2 (nuclear erythroid-related factor 2) signaling pathways was examined. Our results show that CZ and LE extracts exhibited potent anti-inflammatory activities by suppressing the mRNA and protein expression levels of pro-inflammatory biomarkers IL-1β, IL-6, COX-2 and iNOS in LPS-stimulated murine RAW 264.7 macrophage cells. CZ and LE also significantly suppressed the NO production of LPS-stimulated RAW 264.7 cells. Additionally, CZ and LE suppressed the NF-κB luciferase activity in human HT-29 colon cancer cells. Both extracts also showed strong Nrf2-mediated antioxidant/Phase II detoxifying enzymes induction. CZ and LE induced NQO1, Nrf2, and UGT and antioxidant response element (ARE)-luciferase activity in human hepatoma HepG2 C8 cells. Using Nrf2 knockout [Nrf2 (−/−)] and Nrf2 wild-type (+/+) mice, LE and CZ showed Nrf2-dependent transactivation of Nrf2-mediated antioxidant and phase II detoxifying genes. In summary, CZ and LE possess strong inhibitory effects against NF-κB-mediated inflammatory as well as strong activation of the Nrf2-ARE-anti-oxidative stress signaling pathways, which would contribute to their overall health promoting pharmacological effects against diseases including cancer.

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.

The anti-inflammatory activity of licorice (LE) and roated licorice (rLE) extracts determined in the murine phorbol ester-induced acute inflammation model and collagen-induced arthritis (CIA) model of human rheumatoid arthritis. rLE possessed greater activity than LE in inhibiting phorbol ester-induced ear edema. Oral administration of LE or rLE reduced clinical arthritis score, paw swelling, and histopathological changes in a murine CIA. LE and rLE decreased the levels of proinflammatory cytokines in serum and matrix metalloproteinase-3 expression in the joints. Cell proliferation and cytokine secretion in response to type II collagen or lipopolysaccharide stimulation were suppressed in spleen cells from LE or rLE-treated CIA mice. Furthermore, LE and rLE treatment prevented oxidative damages in liver and kidney tissues of CIA mice. Taken together, LE and rLE have benefits in protecting against both acute inflammation and chronic inflammatory conditions including rheumatoid arthritis. rLE may inhibit the acute inflammation more potently than LE.

Background

3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 – 30 μmol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells.

Methods

HT-29 cells were cultured with various concentrations of DIM (0 – 30 μmol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted.

Results

The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1.

Conclusion

Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

Introduction

Tumor-associated macrophages, which are derived from the infiltration of circulating bone marrow-derived monocytes, consist primarily of a polarized M2 macrophage (M2-Mϕ) population and are associated with poor prognosis in various cancers. In the present study, we attempted to assess whether M2-Mϕs derived from bone marrow stimulate the promotion and progression of mammary tumors.

Methods

4T1 murine mammary carcinoma cells were injected either alone or coupled with M2-Mϕs into the mammary fat pads of syngeneic female Balb/C mice. M2-Mϕs were prepared by treating monocytes isolated from female Balb/C mouse bone marrow with IL-4. Tumor cell growth was determined using an in vivo imaging system and the expression of cell proliferation-related, angiogenesis-related, and lymphangiogenesis-related proteins in tumor tissues was immunohistochemically analyzed. To evaluate the effects of the crosstalk between 4T1 cells and M2-Mϕs on the secretion and mRNA expression of cytokines and the migration of monocytes, 4T1 cells and M2-Mϕs were co-cultured and cytokine antibody array, real-time RT-PCR, and trans-well migration assays were conducted.

Results

The co-injection of M2-Mϕs into the mammary fat pads of mice increased solid tumor growth and lung metastasis of 4T1 cells as well as the infiltration of CD45+ leukocytes into tumor tissues. The proportions of Ki-67+ proliferating cells and the expression of hypoxia inducible factor-1α, vascular endothelial cell growth factor A, CD31, vascular endothelial cell growth factor C, and lymphatic vessel endothelial receptor-1 were increased significantly in the tumor tissues of mice co-injected with 4T1 cells and M2-Mϕs. The in vitro results revealed that the proliferation of 4T1 cells, the migration of monocytes, and the secretion of granulocyte colony-stimulating factor, IFNγ, IL-1α, IL-2, IL-16, IFNγ-induced protein-10, keratinocyte-derived chemokine, macrophage colony-stimulating factor, monocyte chemotactic protein-1, macrophage inflammatory protein-1α, and RANTES were increased when 4T1 cells were co-cultured with M2-Mϕs, as compared with when the 4T1 cells were cultured alone.

Conclusion

The crosstalk between 4T1 cells and M2-Mϕs increased the production of cytokines, which may have induced immune cell infiltration into tumor tissues, tumor cell proliferation, angiogenesis, and lymph angiogenesis, thereby increasing solid tumor growth and lung metastasis.