Quercetin enhances 5-fluorouracil-induced apoptosis in MSI colorectal cancer cells through p53 modulation

Purpose: Colorectal tumors (CRC) with microsatellite instability (MSI) show resistance to chemotherapy with 5-fluorouracil (5-FU), the most widely used pharmacological drug for CRC treatment. The aims of this study were to test the ability of quercetin (Q) and luteolin (L) to increase sensitivity of MSI CRC cells to 5-FU and characterize the dependence of the effects on cells´ p53 status. Methods: Two MSI human CRC derived cell lines were used, CO115 wild-type (wt) for p53 and HCT15 that harbors a p53 mutation. Apoptosis induction in these cells by 5-FU, Q and L alone and in combinations were evaluated by TUNEL and western. The dependence on p53 of the effects was confirmed by small interference RNA (siRNA) in CO115 cells and in MSI HCT116 wt and p53 knockout cells. Results: CO115 p53-wt cells are more sensitive to 5-FU than the p53 mutated HCT15. The combination treatment of 5-FU with L and Q increased apoptosis with a significant effect for Q in CO115. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The significant apoptotic enhancement in CO115 incubated with Q plus 5-FU involved the activation of the apoptotic mitochondrial pathway. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence of the effect on p53 modulation by Q. Conclusion: This study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in MSI CRC therapy, especially Q in p53 wt tumors.CPRX was supported by the Foundation for Science and Technology (FCT), Portugal, through the grant SFRH/BD/27524/2006 and the work was supported by the FCT research grant PTDC/AGR-AAM/70418/2006

Angiopreventive Efficacy of Pure Flavonolignans from Milk Thistle Extract against Prostate Cancer: Targeting VEGF-VEGFR Signaling

The role of neo-angiogenesis in prostate cancer (PCA) growth and metastasis is well established, but the development of effective and non-toxic pharmacological inhibitors of angiogenesis remains an unaccomplished goal. In this regard, targeting aberrant angiogenesis through non-toxic phytochemicals could be an attractive angiopreventive strategy against PCA. The rationale of the present study was to compare the anti-angiogenic potential of four pure diastereoisomeric flavonolignans, namely silybin A, silybin B, isosilybin A and isosilybin B, which we established previously as biologically active constituents in Milk Thistle extract. Results showed that oral feeding of these flavonolignans (50 and 100 mg/kg body weight) effectively inhibit the growth of advanced human PCA DU145 xenografts. Immunohistochemical analyses revealed that these flavonolignans inhibit tumor angiogenesis biomarkers (CD31 and nestin) and signaling molecules regulating angiogenesis (VEGF, VEGFR1, VEGFR2, phospho-Akt and HIF-1α) without adversely affecting the vessel-count in normal tissues (liver, lung, and kidney) of tumor bearing mice. These flavonolignans also inhibited the microvessel sprouting from mouse dorsal aortas ex vivo, and the VEGF-induced cell proliferation, capillary-like tube formation and invasiveness of human umbilical vein endothelial cells (HUVEC) in vitro. Further studies in HUVEC showed that these diastereoisomers target cell cycle, apoptosis and VEGF-induced signaling cascade. Three dimensional growth assay as well as co-culture invasion and in vitro angiogenesis studies (with HUVEC and DU145 cells) suggested the differential effectiveness of the diastereoisomers toward PCA and endothelial cells. Overall, these studies elucidated the comparative anti-angiogenic efficacy of pure flavonolignans from Milk Thistle and suggest their usefulness in PCA angioprevention

Diastereoisomers exhibit anti-angiogenic effects through targeting signaling molecules in both prostate cancer cells and endothelial cells.

<p>Silybin A, silybin B, isosilybin A and isosilybin B target angiogenesis in prostate tumors through targeting signaling molecules in PCA cells as well as in endothelial cells, the important component of PCA microenvironment.</p

Flavonolignans inhibit VEGF-induced proliferation and invasion in HUVEC.

<p>(<b>A</b>) HUVEC were induced with VEGF and treated with each flavonolignan in 0.5% serum media, and total cell number was analyzed after 24 h. (<b>B</b>) HUVEC were plated in the upper chamber with DMSO or individual diastereoisomer, while VEGF was added in the lower chamber and HUVEC invasion was studied. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; *, p ≤ 0.001; #, p ≤ 0.01; $, p ≤ 0.05.</p

Flavonolignans decrease VEGFR1, HIF-1α, phosphorylated and total Akt levels in DU145 xenografts.

<p>DU145 xenograft tissues were analyzed for VEGFR1, HIF-1α, phosphorylated Akt^ser473 and total Akt levels by IHC. Quantitative analyses were performed using Zeiss Axioscope 2 microscope (Carl Zeiss, Germany) and photographs were originally captured (at 400x) with a Carl Zeiss AxioCam MrC5 camera with Axiovision Rel 4.5 software. The data shown in the bar diagrams is the mean ± SEM of 4–5 samples. Abbreviations: Sil A: Silybin A; Sil B: Silybin B; Iso A: Isosilybin A, Iso B: Isosilybin B; *, p ≤ 0.001.</p

Effect of flavonolignans on VEGF-induced signaling cascade in HUVEC.

<p>HUVEC were serum starved for 22 h, treated with diastereoisomers for 2 h and stimulated with VEGF (10 ng/ml) for 10 minutes. Total cell lysates were prepared and analyzed for mentioned signaling molecules. The densitometry values presented below the bands are ‘fold change’ compared to control after loading control (β-actin) normalization.</p