【作者】 张强；

【导师】 赵新淮；

【作者基本信息】 东北农业大学 ， 食品科学， 2008， 博士

【摘要】 大量证据表明,摄入植物性食物特别是蔬菜和水果,能够降低各种癌症的发病风险。植物性食物含有的多种生物活性物质（植物化学成分）,是其发挥抗癌活性的重要成分。类黄酮化合物是广泛存在于食用性植物中的多酚类化合物,根据化学结构可分为黄酮醇化合物、黄酮化合物、黄烷酮化合物、黄烷醇化合物、花色苷化合物、异黄酮化合物、二氢黄酮醇化合物以及查耳酮化合物等。许多体内外研究、流行病学调查以及某些临床应用结果表明,类黄酮化合物对于癌症的预防和治疗具有积极作用。但是,类黄酮化合物发挥抗癌活性的内在分子机制,仍有待进一步研究。世界范围内,食管癌在恶性肿瘤中排位第九,约占胃肠癌发病总数的7%,是所有肿瘤中最具致死性的癌症之一。食管癌主要包括两种类型,食管鳞癌（占食管癌总数的90%以上）和食管腺癌（占食管癌总数的10%以下）。大量流行病学研究结果表明,食管癌的发生不仅与饮酒、吸烟、致癌物的摄入以及热损伤直接相关,而且与食物中蔬菜和水果的摄入不足存在密切关系。膳食类黄酮化合物作为蔬菜和水果中的生物活性成分,很可能会为食管癌的预防带来新的希望。本课题研究化学结构相似的黄酮和黄酮醇化合物,对人食管鳞癌细胞系KYSE-510细胞和人食管腺癌细胞系OE33细胞的抑制作用。MTT分析结果表明,黄酮化合物（木犀草素、芹菜素和白杨素）和黄酮醇化合物（槲皮素、山奈酚和杨梅素）,均能以剂量-效应和时间-效应依赖方式,抑制KYSE-510和OE33细胞增殖。它们对KYSE-510和OE33细胞增殖的抑制活性强弱顺序分别为:木犀草素＞槲皮素＞白杨素＞山奈酚＞芹菜素＞杨梅素和槲皮素＞木犀草素＞白杨素＞山奈酚＞芹菜素＞杨梅素。上述结果表明,这些化合物分子中的3’-OH可能有助于黄酮和黄酮醇化合物对KYSE-510和OE33细胞的增殖抑制活性,而分子中的4’-OH和3-OH可能降低化合物抗癌活性的发挥。同时,黄酮化合物和黄酮醇化合物对KYSE-510和OE33细胞的增殖抑制活性,不依赖于分子中的-OH总数。流式细胞仪检测结果显示,黄酮化合物和黄酮醇化合物能够诱导KYSE-510和OE33细胞的周期循环停滞于G₂/M期,并且化合物对KYSE-510和OE33细胞G₂/M周期停滞的诱导活性强弱顺序与MTT检测结果一致,表明黄酮化合物和黄酮醇化合物可能通过诱导细胞周期停滞,抑制两株食管癌细胞增殖。另外,DNA片段化检测和吖啶橙荧光染色结果显示,KYSE-510和OE33细胞经黄酮化合物和黄酮醇化合物作用后,两株细胞均出现了细胞凋亡的典型特征,表明两类化合物能够诱导KYSE-510和OE33细胞发生细胞凋亡。采用FITC-annexin V和PI双荧光标记,通过流式细胞仪定量比较两类化合物对KYSE-510和OE33细胞的凋亡诱导活性,相应实验数据显示,黄酮化合物和黄酮醇化合物对两株食管癌细胞的凋亡诱导活性强弱顺序,与其G₂/M细胞周期停滞的诱导活性强弱顺序以及细胞增殖抑制活性强弱顺序完全一致,表明黄酮化合物和黄酮醇化合物可通过诱导细胞周期停滞和细胞凋亡,实现对两株人食管癌细胞增殖的有效抑制。为探明黄酮化合物和黄酮醇化合物抑制KYSE-510和OE33细胞的分子机制,采用基因芯片技术检测两株食管癌细胞中基因的表达改变。结果表明,KYSE-510细胞经木犀草素作用后,表达改变倍数大于1.5的基因共有75个,其中与细胞增殖、细胞周期循环和细胞凋亡相关的上调表达基因有3个(p21^waf1、PIG3和egr-1),下调表达基因有5个（cyclin B1、PSCA、vav3、WISP2和ear-1）;OE33细胞经槲皮素作用后,表达改变倍数大于1.5的基因共有52个,其中与细胞增殖、细胞周期循环和细胞凋亡相关的上调表达基因有4个（PIG3、GADD45β、p18和14-3-3s）,下调表达基因有3个（cyclin B1、PSCA和FBP1）。为验证两株食管癌细胞中基因表达的芯片检测结果,采用荧光定量RT-PCR对上述KYSE-510细胞中的8个基因以及OE33细胞中的7个基因的mRNA表达水平进行分析,并比较6种黄酮、黄酮醇化合物对各基因表达的调控能力。各基因表达的荧光定量RT-PCR分析结果,与相应基因芯片的分析结果相符,表明基因芯片分析结果较为准确可靠。荧光定量RT-PCR分析结果还显示,在KYSE-510细胞中,黄酮化合物和黄酮醇化合物对3个基因(p21^waf1、PIG3和cyclin B1)表达的调控活性强弱,对应于其G₂/M细胞周期停滞和细胞凋亡的诱导活性强弱;在OE33细胞中,黄酮化合物和黄酮醇化合物对4个基因（PIG3、GADD45B、14-3-3s和cyclin B1）表达的调控活性强弱,对应于其G₂/M细胞周期停滞和细胞凋亡的诱导活性强弱。上述结果说明,KYSE-510细胞中的p21^waf1、PIG3和cyclin B1以及OE33细胞中的PIG3、GADD45B、14-3-3s和cyclin B1为介导黄酮和黄酮醇化合物诱导细胞周期停滞和细胞凋亡的目标基因。为确定上述目标基因的mRNA表达水平改变是否最终导致相应蛋白的表达水平变化,采用Western-blot检测了上述目标基因的蛋白表达水平。检测结果表明,黄酮化合物和黄酮醇化合物通过增加p21^waf1的蛋白表达并抑制cyclin B1的蛋白表达,诱导KYSE-510细胞发生G₂/M周期停滞;通过增加GADD45B和14-3-3s的蛋白表达并抑制cyclin B1的蛋白表达,诱导OE33细胞发生G₂/M周期停滞。同时,黄酮化合物和黄酮醇化合物通过增加PIG3的蛋白表达,提高活体caspase-9和caspase-3的蛋白水平,诱导KYSE-510和OE33细胞发生非p53依赖的线粒体介导的细胞凋亡。Western-blot进一步分析表明,黄酮和黄酮醇化合物可能通过增加p63和p73蛋白翻译或蛋白稳定性,调控p21^waf1、GADD45β、14-3-3s、cyclin B1和PIG3等基因的表达。更多还原

【Abstract】 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.更多还原