Inducing Differentiation Of Flavones And Flavonols On Human Esophageal Cancer Cells

Evidence has been accumulated indicating that dietary intake of plant-based diets,in particular those rich in vegetables and fruits,can reduce the risk of human cancers.Plant-derived foods generally are good sources of many bioactive compounds(phytochemicals) which are contributed to anticancer activity of these foods.Flavonoids are a group of polyphenols and are ubiquitously found in food plants.Compelling data from in vitro and in vivo laboratory studies,epidemiological investigations,and human clinical trials indicate that flavonoids have important effects on cancer chemoprevention and therapy.However,the molecular basis of the effect of flavonoids on cancer remains to be elucidated.Cancer of the esophagus has been reported as the ninth most common malignancy in the world. It also constitutes 7%of all gastrointestinal cancers and is one of the most lethal of all cancers. There is considerable epidemiological evidence suggesting that diets deficient in vegetables and fruits are important in the pathogenesis of esophageal cancer,apart from alcohol,tobacco,intake of carcinogens and thermal injuries.As a large number of bioactive ingredients in vegetables and fruits,dietary flavonoids may bring new hope to the prevention of esophageal cancer.In this study,inhibitory effects of structurally related flavones and flavonols on a human esophageal squamous cell carcinoma cell line(KYSE-510) and a human oesophageal adenocarcinoma cell line(OE33) were studied.The results of MTT assay showed that flavones (baicalein,chrysin) and flavonols(galangin,kaempferol) were all able to inhibit proliferation of KYSE-510 and OE33 cells in a dose- and time-dependent manner.The potency of these flavonoids on inhibition of KYSE-510 and OE33 cells was in the order of galangin＞chrysin＞baicalein＞kaempferol.These results suggested that flavones and flavonols had the inhibitory effect of on proliferation of KYSE-510 and OE33 cells.KYSE-510 and OE-33 cells treated with flavones and flavonols displayed typical differentiation phenotype by observation of phase-contrast microscopy.Cofocal laser scanning microscopy and flow cytometry analysis indicated that flavones and flavonols were able to induce differentiation marker(cytokeratin 8) in KYSE-510 cells and c-erbB-2 in OE33 cells.Moreover,flavones and flavonols were also able to down-regulate telomerase reverse transcriptase in KYSE-510 and OE33. The data showed all four flavnoids were able to induce differentiation in KYSE-510 and OE33 and the potency of these flavonoids on induction of differentiaion was also similar to that observed on growth inhibition.It was suggested that flavones and flavonols inhibited proliferation of KYSE-510 and OE33 cells by inducing differentiation.To verify the alterations of gene expression in two esophageal cancer cells at the mRNA level, we conducted real-time RT-PCR analysis for three genes in KYSE-510 and OE33 cells.Moreover, comparative effects of all four flavonoids on the regulation of these gene expressions were assayed by real-time RT-PCR.It was found that flavones and flavonols mighe be up-regulation of p21^wafl and down-regulation of cyclin B1 and cyclin D1 in KYSE-510 cells,and up-regulation of 14-3-3σand down-regulation of cyclin B1 and cyclin D1 in OE33 cells.It was suggested that p21^wafl,cyclin B1 and cyclin D1 in KYSE-510 cells and 14-3-3σ,cyclin B1 and cyclin D1 in OE33 cells were the target genes which mediated cell differentiation induced by flavones and flavonols.To verify whether the alternations in expression of these target genes at the mRNA level ultimately result in the alternations at the protein level we conducted a series of western-blot analyses for them.It was found that flavones and flavonols mighe be up-regulation of p21^wafl and down-regulation of cyclin B1 and cyclin D1 at the protein level in KYSE-510 cells,and up-regulation of 14-3-3σand down-regulation of cyclin B1 and cyclin D1 at the protein level in OE33 cells.It was speculated that P21^wafl,cyclin B1 and cyclin D1 in KYSE-510 cells 14-3-3σand cyclin B1 and cyclin D1 in OE33 cells might be target genes of flavones and flavonols in inducing differentiation.