Dietary peptides from the non-digestible fraction of Phaseolus vulgaris L. decrease angiotensin II-dependent proliferation in HCT116 human colorectal cancer cells through the blockade of the renin-angiotensin system.

This study aimed to determine the ability of peptides present in the non-digestible fraction (NDF) of common beans to decrease angiotensin II (AngII) through the blockade of RAS and its effect on the proliferation of HCT116 human colorectal cancer cells. Pure synthesized peptides GLTSK and GEGSGA and the peptide fractions (PF) of cultivars Azufrado Higuera and Bayo Madero were used. The cells were pretreated with pure peptides, PF or AGT at their IC50 or IC25 values, in comparison with the simultaneous treatment of peptides and AGT. For western blot and microscopy analysis, 100 µM and 0.5 mg mL(-1) were used for pure peptides and PF treatments, respectively. According to the ELISA tests, GLTSK and GEGSGA decreased (p < 0.05) the conversion rate of AGT to angiotensin I (AngI) by 38 and 28%, respectively. All the peptides tested reduced (p < 0.05) the conversion rate of AngI to AngII from 38 to 50%. When the cells were pretreated with both pure peptides and PF before exposure to AGT, the effectiveness inhibiting cell proliferation was higher than the simultaneous treatment suggesting their preventive effects. GLTSK and GEGSGA interacted with the catalytic site of renin, the angiotensin-I converting enzyme, and the AngII receptor, mainly through hydrogen bonds, polar, hydrophobic and cation-π interactions according to molecular docking. Through confocal microscopy, it was determined that GLTSK and GEGSGA caused the decrease (p < 0.05) of AngII-dependent STAT3 nuclear activation in HCT116 cells by 66 and 23%, respectively. The results suggest that peptides present in the common bean NDF could potentially ameliorate the effects of RAS overexpression in colorectal cancer.

Simplified method for quantitation of angiotensin peptides in tissue.

A simple method for extraction, separation, identification and quantitation of angiotensin-like immunoactivity from tissue is described. Homogenized acetic acid extracts of tissue samples were lyophilized and reconstituted in mobile phase. Separation was performed by reversed-phase high-performance liquid chromatography on a phenyl silica gel column with an eluent consisting of 20% acetonitrile in 0.1 M aqueous ammonium phosphate buffer, pH 4.9. Elution of standard peptides under isocratic conditions revealed clear resolution of angiotensin I, II and III and the (1-7) and (3-8) peptides. Recoveries of labeled angiotensin peptide standards from the extraction step were > 90%. Radioimmunoassay of relevant peaks revealed detectable levels of angiotensin I-, II- and III-like immunoactivity in single rat hypothalami and brain stems.

Identification of angiotensin-(1-7) in rat brain. Evidence for differential processing of angiotensin peptides.

Tissue and plasma forms of angiotensin (Ang) peptides were characterized by reverse-phase high performance liquid chromatography and three specific radioimmunoassays. This method allowed resolution of 10 Ang peptides and revealed distinctive distributions for the three principal Ang peptides in the brain, adrenal gland, and plasma. In extracts from the rat hypothalamus, approximately equimolar amounts of Ang-(1-7), Ang-II, and Ang-I were detected (1.10, 1.18, and 1.45 pmol/g of tissue, respectively). A similar profile was observed in the medulla oblongata and amygdala, although the content of these three peptides was 40-70% less than that seen in the hypothalamus. In the adrenal gland, the predominant peptide was Ang-II (1.07 pmol/g); levels of Ang-(1-7) (0.19 pmol/g) and Ang-I (0.14 pmol/g) were approximately 20% that of Ang-II. In plasma, the major angiotensin was Ang-I (0.13 pmol/ml), with lower levels of Ang-(1-7) and Ang-II (0.01-0.02 pmol/ml). This study is the first demonstration of the endogenous presence of Ang-(1-7) in central and peripheral tissues of the rat. Moreover, the data suggest tissue-specific processing of angiotensins, with Ang-(1-7) being a predominant Ang peptide in the central nervous system. In light of the recent biological properties described for this peptide, Ang-(1-7) may represent an active member of Ang peptides in the brain.

Immunocytochemical and biochemical characterization of angiotensin I and II in cultured neuronal and glial cells from rat brain.

Neuronal and glial cells cultured from neonatal rat brains showed staining for both angiotensin I and II using the peroxidase-antiperoxidase method. In glial cell extracts of normotensive Wistar-Kyoto rats, the concentrations of angiotensin I and II were 12.47 +/- 2.71 (n = 4) and 66.73 +/- 13.28 fmol/mg protein (n = 4). Angiotensin I and II found in neuronal cell extracts of normotensive Wistar-Kyoto rats were 11.29 +/- 2.99 (n = 4) and 60.25 +/- 12.77 fmol/mg protein (n = 4). No significant difference was found in the concentration of angiotensin I and II in both cell types from the same rat strain. Angiotensin I concentrations of 16.83 +/- 3.43 fmol/mg protein (n = 5) determined in neuronal cell extracts derived from spontaneously hypertensive rats did not differ significantly from those found in neuronal cell extracts of Wistar-Kyoto rats. However, neuronal cell extracts from spontaneously hypertensive rats revealed values of 25.19 +/- 4.31 fmol angiotensin II/mg protein (n = 4). This was significantly different (p less than 0.05) and represented a 58% reduction in the angiotensin II levels in neuronal cells from spontaneously hypertensive rats compared to Wistar-Kyoto rat cultures. Angiotensin I and II measured in the growth medium containing 10% plasma-derived horse serum was below the detection limit of both radioimmunoassays. No difference in the angiotensin I and II levels was found in cells kept in serum-free medium. The angiotensin I and II immunoreactive material determined in the cell extracts could be characterized on reversed-phase high pressure liquid chromatography as (Ile5)-angiotensin I and II. (Ile5)-angiotensin III was not detectable.

The distribution of immunoreactive angiotensins I and II extracted from rat brain.

A method was developed to measure immunoreactive angiotensin I and II extracted from rat brain tissue. The angiotensin-immunoreactive materials extracted from brain tissue resembled angiotensins I and II in many of their physiochemical properties. The concentration of angiotensin-immunoreactive materials was highest in extracts of pituitary, hypothalamus and thalamus. The concentrations of angiotensin-immunoreactive materials were not decreased in extracts of brain tissue from bilaterally nephrectomized rats. The concentrations of immunoreactive materials in extracts of pituitary far exceed those which could be accounted for by the blood and suggests that these endogenous peptides may regulate the neurosecretion of antidiuretic hormone.