Department of Biological Sciences, Michigan Technological University, Houghton, MI, USA.
School of Medicine, University of California, San Diego, CA, USA.
Phytomedicine. 2019 Apr;57:364-376. doi: 10.1016/j.phymed.2018.12.034. Epub 2018 Dec 31.
Rice 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/PURPOSE: The anti-inflammatory potential of RCSC and its bioactive fractions on normal colon epithelial cell lines, was investigated.
Three 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.
RCSC 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.
RCSC 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.
水稻愈伤组织悬浮培养（RCSC）已被证明在多种癌细胞系中具有很强的抗增殖活性。RCSC 及其生物活性化合物可以满足无副作用药物的需求。
假设/目的：研究了 RCSC 及其生物活性成分对正常结肠上皮细胞系的抗炎潜力。
用促炎细胞因子处理三种细胞系 InEpC、NCM356 和 CCD841-CoN，然后用 RCSC 处理。用荧光显微镜和流式细胞仪检测细胞质和核内 ROS。在 RCSC 处理的细胞系中进行免疫相关基因的表达分析。使用柱层析和 HPLC 对 RCSC 进行分段。使用合并的馏分 10-18 测试对结肠腺癌细胞系 SW620 的增殖活性和对 CCD841-CoN 的抗炎活性。对四个馏分进行质谱分析，以鉴定候选化合物。
RCSC 处理显示出不同的效果，NCM356 和 CCD841-CoN 中的细胞质 ROS 水平较高，而 InEpC 中的 ROS 水平较低。所有三种处理的细胞系中的核生成 ROS 水平均增加。碘化丙啶染色细胞的流式细胞术分析表明，在 RCSC 处理的 NCM356 和 CCD841-CoN 组中，炎症引起的细胞死亡得到缓解。用 RCSC 处理的 NCM356 和 CCD841-CoN 细胞系中，编码转录因子和细胞因子的基因表达水平不同，这为可能的途径提供了线索。用 HPLC 分析合并的馏分 10-18 鉴定出 8 个峰。用 SW620 进行的馏分 10-18 的细胞活力测定表明，活细胞数量大大减少，与 6X 和 33X RCSC 相似，对正常细胞几乎没有影响，与 1X RCSC 相似。RCSC 馏分与未处理和炎症对照相比，增加了核内和细胞质 ROS。用质谱分析四种馏分鉴定出 4-去甲根皮素、5'-甲氧基姜黄素、白藜芦醇苷和羽扇豆醇作为候选化合物，它们可能是 RCSC 具有增殖抑制、抗炎和免疫调节特性的原因。
RCSC 及其馏分对炎症性结肠上皮细胞具有抗炎活性。鉴定出可能介导 RCSC 作用的下游靶候选基因。RCSC 及其馏分的增殖抑制和抗炎活性的候选化合物为可能的药物靶点提供了依据。
