Anti-inflammatory and immune-modulating effects of rice callus suspension culture (RCSC) and bioactive fractions in an in vitro inflammatory bowel disease model

Abstract

BackgroundRice callus suspension culture (RCSC) has been shown to exhibit potent antiproliferative activity in multiple cancer cell lines. RCSC and its bioactive compounds can fill the need for drugs with no side effects. Hypothesis/PurposeThe anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated. Study designThree cell lines, InEpC, NCM356 and CCD841-CoN were treated with proinflammatory cytokines followed by RCSC. Cytoplasmic and nuclear ROS were assayed with fluorescent microscopy and flow cytometer. Expression analysis of immune-related genes was performed in RCSC-treated cell lines. RCSC was fractionated using column chromatography and HPLC. Pooled fractions 10–18 was used to test for antiproliferative activity using colon adenocarcinoma cell line, SW620 and anti-inflammatory activity using CCD841-CoN. Mass spectrometric analysis was performed to identify candidate compounds in four fractions. ResultsRCSC treatment showed differential effects with higher cytoplasmic ROS levels in NCM356 and CCD841-CoN and lower ROS levels in InEpC. Nuclear generated ROS levels increased in all three treated cell lines. Flow cytometry analysis of propidium iodide stained cells indicated mitigation of cell death caused by inflammation in RCSC treated groups in both NCM356 and CCD841-CoN. Genes encoding transcription factors and cytokines were differentially regulated in NCM356 and CCD841-CoN cell lines treated with RCSC which provided insights into possible pathways. Analysis of pooled fractions 10–18 by HPLC identified 8 peaks. Cell viability assay with fractions 10–18 using SW620 showed that the number of viable cells were greatly reduced which was similar to 6X and 33X RCSC with very little effect on normal cells which similar to 1X RCSC. RCSC fractions increased nuclear and cytoplasmic ROS vs. both untreated and inflammatory control. Analysis of four fractions by mass spectrometry identified 4-deoxyphloridzin, 5′-methoxycurcumin, piceid and lupeol as candidate compounds which are likely to be responsible for the antiproliferative, anti-inflammatory and immune-regulating properties of RCSC. ConclusionRCSC and its fractions showed anti-inflammatory activity on inflamed colon epithelial cells. Downstream target candidate genes which are likely to mediate RCSC effects were identified. Candidate compounds responsible for the antiproliferative and anti-inflammatory activity of RCSC and its fractions provide possible drug targets.