Anti-proliferative effect of Moringa oleifera Lam (Moringaceae) leaf extract on human colon cancer HCT116 cell line

Purpose: To investigate the in vitro anti-proliferative effect and mechanism of action of Moringa oleifera Lam. leaf extract on human colon carcinoma HCT116 cell line.Methods: M. oleifera leaves were extracted with methanol. It was fractionated by Sephadex LH-20 column chromatography. Several fractions were identified by thin layer chromatography (TLC), proton nuclear magnetic resonance (1H NMR) and mass spectrometry (MS). The growth inhibitory activity and mechanism of action of the extracts in HCT116 colon cancer cells were investigated by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blotting.Results: Successive fractions from M. oleifera leaf crude extracts by column  chromatography were combined into four pooled batches (MOL1 - MOL4) according to their absorbance at 260 nm and TLC pattern. MOL2 and MOL3 contain astragalin and isoquercetin, respectively. The results from MTT assay indicated that cell proliferation was significantly (p < 0.05) inhibited in a concentration-dependent fashion, especially by MOL2, MOL3 and MOL4. MOL2 and MOL3 exhibited a stronger cell growth  inhibition than their major ingredients. The anti-proliferative activity of MOL2 - MOL4 in HCT116 colon cancer cells was  mediated by downregulation of ERK1/2 phosphorylation.Conclusion: M. oleifera leaf extract has a strong anti-proliferative activity which is exerted by decreasing ERK1/2 phosphorylation. Thus, the extract has a potential for use in cancer chemoprevention.Keywords: Moringa oleifera, Anti-proliferation, Colon cancer, AKT, ERK1/2 phosphorylation, Chemopreventio

Effect of Curcumin on SMCT-1 Expression and Dichloroacetate Toxicity in HCT116 Colon Cancer Cells

Background: Colorectal cancer is a common cause of cancer-related deaths Epigenetic regulation of the influx sodium dependent monocarboxylate transporter-1 )SMCT1), a tumor suppressor, was recognized in colorectal cancer. In this study, effects of Curcumin (Cur), on SMCT1 gene expression was determined. A low SMCT1 expression, HCT116, cell line was used to test an in vitro effect of Cur on epigenetic regulation of SMCT1 expression via DNA methylation and its function. It was hypothesized that Cur can induce SMCT1 expression in the cells via hypomethylation effect. Measurement of increase in SMCT1 function was performed using dichloroacetate (DCA), a cytotoxic substrate of SMCT1. Methods: The effect of 5′Azacytidine (Aza), a hypomethylating agent, and Cur on SMCT1 expression and function was determined. Cells were treated with Aza and various concentrations of Cur for 72 h. After that SMCT1 expression was determined by real time PCR and Western blotting. To evaluate the SMCT1 function, DCA was used in MTT assay. Results: After treatment with 40 µM Cur, SMCT1 mRNA was significantly increased (p < 0.05). This was correlated with SMCT1 protein expression. Cells treated with 40 µM of Cur showed significant increase of cytotoxicity at DCA concentrations of 25 (p < 0.001) and 12.5 mM (p <0.01), respectively. Conclusion: Cur was shown to significantly induce the SMCT1 mRNA and protein expression in HCT116 cells. The induction of the SMCT1 protein increased DCA cytotoxicity, presumably through an increase of DCA transport into the cells. The mechanism underlying of SMCT1 induction by Cur may result from not only hypomethylation but other epigenetics

Chitosan-Mediated siRNA Delivery In Vitro: Effect of Polymer Molecular Weight, Concentration and Salt Forms

The aim of this study was to investigate chitosan/siRNA complexes formulated with various chitosan salts (CS) including chitosan aspartate (CS-Asp), chitosan glutamate (CS-Glu), chitosan acetate (CS-Ac), and chitosan hydrochloride (CS-HCl) for in vitro siRNA delivery into stable and constitutive enhanced green fluorescent protein (EGFP)-expressing HeLa cells. The CS/siRNA complexes were characterized by 2% agarose gel electrophoresis and investigated for their transfection efficiency in stable and constitutive EGFP-expressing HeLa cells. The cytotoxicity of the complexes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The formation of complexes CS/siRNA is mainly dependent on the weight ratio, whereas salt form and molecular weight has less effect. The particle sizes of the complete complexes were in the range of 270–373 nm except the complete complex of CS-Ac, with a slightly positive charge of less than 2 mV. The ability of CS to transfer functionally active siRNA into cell culture is mainly dependent on the weight ratio and molecular weight of CS whereas salt form of CS has less effect. The high gene-silencing efficiency was observed with low MW of CS (20 kDa) and high weight ratio of 32. Over 80% average cell viabilities were observed for CS/siRNA complexes in all weight ratios comparison to untreated cells. This study suggests CS salts have the potential to be used as safe siRNA delivery vectors