Anat Cell Biol.  2020 Dec;53(4):481-492. 10.5115/acb.20.085.

A rice bran phytochemical, cyanidin 3-glucoside, inhibits the progression of PC3 prostate cancer cell

• Jongsomchai K
• Leardkamolkarn V
• Mahatheeranont S

• KMID: 2509694
• DOI: http://doi.org/10.5115/acb.20.085

Abstract

Prostate cancer is one of the high incidences and the most invasive cancer that is also highly resistant to chemotherapy. Currently, several natural products have been considering using as the supplements for anti-cancer therapy. This study aims to identify the potential active anti-cancer ingredients in the bran extracts of the native Thai rice (Luempua cultivar). Rice bran fraction enriched in anthocyanins was successively isolated and processed until the major purified compound obtained. The sub-fractions and the purified, rice bran, cyanidin 3-glucoside (RBC3G), were studied for biological effects (cell viability, migration, and invasion assays) on human prostatic cancer (PC3) cells using immunohistochemicalstaining and immuno-blotting approaches. The sub-fractions and the purified RBC3G inhibited epithelial mesenchymal transition (EMT) characteristics of PC3 cells by blocking the expression of several cytoskeletal associate proteins in a concentration dependent manner, leading to decreasing of the cancer cell motility. RBC3G reduced the expression of Smad/Snail signaling molecules but enhanced the expression of cell surface protein, E-cadherin, leading to a delay tumor transformation. The RBC3G also inhibited matrix metalloproteinase-9 and nuclear factor-kappa B expression levels and the enzymes activity in PC3 cells, leading to a slow cell migration/invasion process. The results suggested that RBC3G blunt and/ or delay the progressive cancer cell behaviors by inhibit EMT through Smad signaling pathway(s) mediating Snail/E-cadherin expression.

Keyword

Anthocyanins; Anticancer; β-catenin; Epithelial mesenchymal transition; Snail

Figure

• 

  Fig. 1 (A) Scheme of extraction, fractionation and purification of LP rice bran to yield the cyanidin-3-O-glucoside. (B) MTT assay showing the effect of rice bran methanol fractions (LBM1, LBM2, and LBM3), sub-fractions (LBM2.1, LBM2.2, and LBM2.3), semi-purified fractions (LBM2.2.1, LBM2.2.2) and the purified RBC3G on viability of PC3 cells in culture. The assay was performed after 24 and 48 hours treatments. LC-MS/MS, liquid chromatography/tandem mass spectrometry; LP, Luem Pua; RBC3G, rice bran, cyanidin 3-glucoside. Data was expressed as mean±standard error of the mean. *P<0.05, compared to control groups.

• 

  Fig. 2 (A–F) Confocal laser scanning micrographs of untreated PC3 cells (left row) and RBC3G treated PC3 cells (middle and right rows), cultured on FN-coated plate, showing the distribution patterns of proteins associated with EMT characteristics, cytoskeletal organization, and their signaling by double immuno-stained with antibodies to (A) β-catenin, probed with TRITC (red) and alpha-tubulin, probed with FITC (green). (B) β-catenin, probed with TRITC (red) and F-actin, probed with FITC (green). (C) pFAK-Alexa555 (red) and F-actin-Alexa488 (green). (D) Vimentin-conjugated Alexa555 (red). (E) Smad2/3-conjugated Alexa555 (red) and F-actin, probed with FITC (green). (F) Snail1/2-conjugated Alexa555 (red) and F-actin-Alexa488 (green). Nuclei were counter-stained with DAPI (blue), Scale bars=30 μm. (G–I) Western blot analysis of proteins associated with EMT characteristics and their signaling proteins involved in tumor transformation in the untreated and RBC3G treated PC3 cells, probed with antibodies to (G) β-catenin and pFAK, (H) E-cadherin and vimentin, (I) Smad2/3 and Snail1/2. The reactive bands density calculated in relative to β-actin. EMT, epithelial mesenchymal transition; FITC, fluorescein isothiocyanate; RBC3G, rice bran, cyanidin 3-glucoside; TRITC, tetramethylrhodamine isothiocyanate. The data presented as mean±standard error of the mean of triplicate independent experiments. *P<0.05 compared to control.

• 

  Fig. 3 (A) Western blot analysis of proteins associated with cell migration and invasion, MMP-9 and NF-κB, in PC3 cells after treatment with varied concentrations of RBC3G. The bands density of each protein calculated in relative to β-actin. The data presented as mean±standard error of the mean of triplicate independent experiments. (B) Zymography assay for MMP-9 enzyme released from PC3 cells during the treatment with RBC3G. The enzyme activity demonstrated from gelatinolytic band molecular weight (75 kDa). The bar graphs show relative bands intensity of the treated PC3 cells compared to untreated control. FN, fibronectin; MMP-9, matrix metalloproteinase-9; NF-κB, nuclear factor-kappa B; RBC3G, rice bran, cyanidin 3-glucoside. *P<0.05 compared to control. #P<0.05 compared to FN-coated plate control.

• 

  Fig. 4 (A) The phase contrast micrographs from scratch/wound healing assay shows the effect of RBC3G on migration of PC3 cells after 24 and 48 hours treatment. The bar graph shows the counting number of migrating cells into the scratched area in comparison with the controls (non-coated plate+PBS treatment and FN-coated plate+PBS treatment). (B) Cell invasion assay; through matrix gel-coated Transwell plate shows the inhibiting effect of RBC3G on PC3 cells. The bar graph shows the counting number of the invaded cells. FN, fibronectin; RBC3G, rice bran, cyanidin 3-glucoside. Data expressed as mean±standard error of the mean (n=3). ***P<0.001, compared to control groups. *P<0.05, compared to control groups.

Reference

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