Inhibitory Effects Of Flavones And Flavonols On Two 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,and are categorized,according to chemical structure,into flavones,flavonols, isoflavones,flavanols,flavanones,flavanonols,isoflavones and chalcones.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 are two main forms of esophageal cancer,each with distinct etiological and pathological characteristics:squamous cell carcinoma(＞90%of esophageal malignancies) and adenocareinoma (＜10%of esophageal malignancies).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 (luteolin,apigenin and chrysin) and flavonols(quercetin,kaempferol and myricetin) 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 luteolin＞quercetin＞chrysin＞kaempferol＞apigenin＞myricetin and quercetin＞luteolin＞chrysin＞kaempferol＞apigenin＞myricetin,respectively.These results suggested that the inhibitory effect of flavones and flavonols on proliferation of KYSE-510 and OE33 cells might be dependent on the presence of 3'-hydroxyl group and/or the absence of 4'-hydroxyl group and 3-hydroxyl group in these compounds,but independent on the number of hydroxyl groups.Flow cytometric analysis indicated that flavones and flavonols were able to induce cell cycle arrest in G₂/M phase in KYSE-510 and OE33.In agreement with the results of MTT assay,similar potency of these flavonoids on induction of G₂/M arrest in KYSE-510 and OE33 cells was also observed,respectively.It was suggested that inhibitory effects of flavones and flavonols might be mediated by G₂/M cell cycle arrest.Moreover,KYSE-510 and OE-33 cells treated with flavones and flavonols displayed typical characteristics of apoptosis by DNA fragmentation and acridine orange staining methods.The apoptosis induced by these flavonoids was quantified by flow cytometry after labeling the cells with FITC-annexin V/PI.The data showed that potency of these flavonoids on induction of apoptosis was also similar to that observed on growth inhibition and G₂/M cell cycle arrest.It was suggested that flavones and flavonols inhibited proliferation of KYSE-510 and OE33 ceils by causing cell cycle arrest and inducing apoptosis.To gain insight into the molecular mechanisms underlying inhibitory effects of flavones and flavonols on KYSE-510 and OE33 cells,the expression of genes was assessed by gene chip. Seventy-five genes showed＞1.5-fold change in KYSE-510 cells treated with luteolin.Among these genes,three genes(p21^wafl,PIG3 and egr-1) were up-regulated and five genes(cyclin B1, PSCA,vav3,WISP2 and ear-1) were down-regulated,which are related to cell proliferation,cell cycle progression and apoptosis.Fifty-two genes showed＞1.5-fold change in OE33 cells treated with quercetin.Among these genes,four genes(PIG3,GADD45β,p18 and 14-3-3s) were up-regulated and three genes(cyclin B1,PSCA and FBP1) were down-regulated.To verify the alterations of gene expression in two esophageal cancer cells at the mRNA level, which appeared in the microarray,we conducted real-time RT-PCR analysis for eight genes in KYSE-510 and seven genes in OE33 cells.Moreover,comparative effects of all six flavonoids on the regulation of these gene expressions were also assayed by real-time RT-PCR.The results of real-time RT-PCR analysis for these genes were in direct agreement with the microarray data.In KYSE-510 cells,alterations in expression levels of three genes(p21^wafl,PIG3 and cyclin B1) were corresponding to the potency of flavones and flavonols on induction of cell cycle arrest and apoptosis.In OE33 cells,alterations in expression levels of four genes(PIG3,GADD45β,14-3-3s and cyclin B1) were corresponding to the potency of flavones and flavonols on induction of cell cycle arrest and apoptosis.It was suggested that p21^wafl,PIG3 and cyclin B1 in KYSE-510 cells and PIG3,GADD45β,14-3-3s and cyclin B1 in OE33 cells were the target genes which mediated cell cycle arrest and apoptosis 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 caused G₂/M arrest through up-regulation of p21^wafl and down-regulation of cyclin B1 at the protein level in KYSE-510 cells, and caused G₂/M arrest through up-regulation of GADD45βand 14-3-3s and down-regulation of cyclin B1 at the protein level in OE33 cells.Flavones and flavonols induced p53-independent mitochondrial-mediated apoptosis through up-regulation of PIG3 and cleavage of caspase-9 and caspase-3 at the protein level in KYSE-510 cells and OE33 cells.The results of Western-blot analysis further showed that increases of p63 and p73 protein translation or stability might be responsible for the regulation of p21^wafl,GADD45g,14-3-3s,cyclin B1 and PIG3.