The contribution of dietary and plasma folate and cobalamin to levels of angiopoietin-1, angiopoietin-2 and Tie-2 receptors depend on vascular endothelial growth factor status of primary breast cancer patients

Figures

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  Figure 1. Dietary intakes of patients. Daily dietary intake of macronutrients including total intake levels of protein, carbohydrate, fat and fiber (graph A), dietary and total (dietary plus supplemented amounts) intake levels of folate and cobalamin and dietary folate equivalent (DFE: μg/d of food folate plus 1.7 times the μg/d of supplemented folic acid) (graph B) were compared with their dietary reference intake (DRI). Age- and gender-dependent reference amounts of intake based on DRI26 were as follow: carbohydrate ≥130 g/d, protein ≥46 g/d, fat ≥27.5 g/d (based on DRI 2002/2005), fiber ≥23 g/d, folate ≥400 µg/d and cobalamin ≥2.4 µg/d (based on DRI 1998). *p < 0.05. Mean values ± S.D. are presented. µg/d, microgram/day; ng/d, nanogram/day.

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  Figure 2. Plasma levels of angiogenic markers, folate and cobalamin were compared with the upper limit of the normal range measured in healthy individuals (a,b). Normal ranges were ascribed as follows: ANG1 0.600–6.000 ng/ml, ANG2 0.500–3.000 ng/ml, Tie2 10–92 ng/ml, VEGF-C 0.0–0.50 ng/ml, folate 2–20 ng/ml and cobalamin 200–900 pg/ml according to protocol of kits (folate: Cat N. 7525-300 and cobalamin: Cat N. 7526-300)47,52. *p < 0.05. Mean values ± S.D. are presented.

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  Figure 3. Scatter plots depicted to show the crude correlation of protein expression levels of ER (a–d) and PR (e-h) with plasma levels of studied angiogenic growth factors in primary breast cancer patients. The linear regression analysis has been done to obtain standardized beta (β). p < 0.05 considered statistically significant.

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  Figure 4. The proposed path diagram of the implied regression models fitted well in the parsimonious model (a) and just identified confirmatory multi-regression model (b) to show the contribution of folate and cobalamin with plasma Angiopoietins. Maximum likelihood estimation was performed to test the fitting function or estimation procedure of parameters in models. The plasma ANG-2 was weighted with interindividual levels of plasma VEGF-C. Rectangles were observed variables. Ellipses were latent (construct) variables. Values on the single-headed arrows (recursive) were standardized regression weights. Values on the double-headed arrows (nonrecursive) were beta (β) of intercorrelation between two variables. Each observed indicator included in models with measurement errors (e) and residual errors (top right corner of the rectangle) to predict the latent variable. Factor loading of a link between indicator and construct (latent) was also estimated. An asterisk (*) is shown to express critical ration (CR) >1.96 of estimated β, which means that the path (parameter) is significant at p < 0.05.

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  Figure 5. Microscope images (10 × ) of cologenic assays on MCF-7 cells at free folic acid medium as control (a) and folic acid concentration of 20 μM (b) after 5 days of treatments. Effects of folic acid treatment at different concentrations (0.1–200 μM) on the MCF-7 proportion of cell viability (c). The effects of different concentrations of folic acid were compared to control using ANOVA followed by Dunnet test (c). Calculated EC50 value to express the half-maximal viability of MCF-7 cells in response to folic acid treatment was 29.9 μM folic acid after 24 h. Data were expressed as the mean ± S.D. of three independent experiments.

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  Figure 6. The effects of folic acid treatments in MCF-7 cells on expression levels of tested genes (ANGPT1, ANGPT2, VEGF, and Tie-2) relative to HGPRT as an internal control. Mean value of relative expression at each concentration of folic acid were represented at mean ± S.D. and compared using ANOVA followed by Dunnett analysis. (*p < 0.05 considered statistically significant).