ObjectiveOsteogenic repair in response to bone injury is characterized by activation and differentiation of mesenchymal stem cells (MSCs) to osteoblasts. This study determined whether activation of Sirt-1 (a NAD+-dependent histone deacetylase) by the phytoestrogen resveratrol affects osteogenic differentiation.MethodsMonolayer and high-density cultures of MSCs and pre-osteoblastic cells were treated with an osteogenic induction medium with/without the Sirt-1 inhibitor nicotinamide or/and resveratrol in a concentration dependent manner.ResultsMSCs and pre-osteoblastic cells differentiated to osteoblasts when exposed to osteogenic-induction medium. The osteogenic response was blocked by nicotinamide, resulting in adipogenic differentiation and expression of the adipose transcription regulator PPAR-γ (peroxisome proliferator-activated receptor). However, in nicotinamide-treated cultures, pre-treatment with resveratrol significantly enhanced osteogenesis by increasing expression of Runx2 (bone specific transcription factor) and decreasing expression of PPAR-γ. Activation of Sirt-1 by resveratrol in MSCs increased its binding to PPAR-γ and repressed PPAR-γ activity by involving its cofactor NCoR (nuclear receptor co-repressor). The modulatory effects of resveratrol on nicotinamide-induced expression of PPAR-γ and its cofactor NCoR were found to be mediated, at least in part, by Sirt-1/Runx2 association and deacetylation of Runx2.Finally, knockdown of Sirt-1 by using antisense oligonucleotides downregulated the expression of Sirt-1 protein and abolished the inhibitory effects of resveratrol, namely nicotinamide-induced Sirt-1 suppression and Runx2 acetylation, suggesting that the acetylated content of Runx2 is related to downregulated Sirt-1 expression.ConclusionThese data support a critical role for Runx2 acetylation/deacetylation during osteogenic differentiation in MSCs in vitro. (242 words in abstract)

ObjectiveDevelopment of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.MethodsWild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins.ResultsThe individual IC50 of curcumin and 5-FU were approximately 20 µM and 5 µM in HCT116 cells and 5 µM and 1 µM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC50 values for combination treatment were approximately 5 µM and 1 µM in HCT116 and 5 µM and 0.1 µM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-κB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IκBα kinase activation and IκBα phosphorylation.ConclusionsCombining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-κB/PI-3K/Src pathways and NF-κB regulated gene products.

ObjectiveInteraction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment.MethodsColon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU.ResultsMonolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment.ConclusionEnrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.

ObjectiveTreatment of colorectal cancer (CRC) remains a clinical challenge, as more than 15% of patients are resistant to 5-Fluorouracil (5-FU)-based chemotherapeutic regimens, and tumor recurrence rates can be as high as 50–60%. Cancer stem cells (CSC) are capable of surviving conventional chemotherapies that permits regeneration of original tumors. Therefore, we investigated the effectiveness of 5-FU and plant polyphenol (curcumin) in context of DNA mismatch repair (MMR) status and CSC activity in 3D cultures of CRC cells.MethodsHigh density 3D cultures of CRC cell lines HCT116, HCT116+ch3 (complemented with chromosome 3) and their corresponding isogenic 5-FU-chemo-resistant derivative clones (HCT116R, HCT116+ch3R) were treated with 5-FU either without or with curcumin in time- and dose-dependent assays.ResultsPre-treatment with curcumin significantly enhanced the effect of 5-FU on HCT116R and HCR116+ch3R cells, in contrast to 5-FU alone as evidenced by increased disintegration of colonospheres, enhanced apoptosis and by inhibiting their growth. Curcumin and/or 5-FU strongly affected MMR-deficient CRC cells in high density cultures, however MMR-proficient CRC cells were more sensitive. These effects of curcumin in enhancing chemosensitivity to 5-FU were further supported by its ability to effectively suppress CSC pools as evidenced by decreased number of CSC marker positive cells, highlighting the suitability of this 3D culture model for evaluating CSC marker expression in a close to vivo setting.ConclusionOur results illustrate novel and previously unrecognized effects of curcumin in enhancing chemosensitization to 5-FU-based chemotherapy on DNA MMR-deficient and their chemo-resistant counterparts by targeting the CSC sub-population. (246 words in abstract).

Background: Resveratrol has been proposed to have beneficial health effects due to its anti-inflammatory properties. Results: Resveratrol suppressed IL-1␤-induced activation of NF-B and PI3K in a dose-and time-dependent manner. Conclusion: Anti-inflammatory effects of resveratrol may be mediated at least in part through inhibition/deacetylation of PI3K and NF-B. Significance: Activated Sirt-1 plays an essential role in anti-inflammatory effects of resveratrol.