【作者】 凌晖；

【导师】 苏琦；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 胃癌是我国最常见的恶性肿瘤,其发病率和死亡率仍居首位,每年约有40余万人患胃癌,而死于胃癌者多达30余万,大大高于欧美发达国家。目前,传统的手术治疗、放射治疗、化疗治疗等疗效较差、易复发、副反应严重,5年生存率仅为30%左右。因此,提高5年生存率,探索治疗胃癌的新方法,寻找新一代更有效、低毒和价廉的抗肿瘤药物,一直是亟待解决的课题。现在,逆转人胃癌细胞的研究已成为生命科学研究前沿领域。二烯丙基二硫（diallyl disulfide, DADS）是大蒜中的一种脂溶性的有效成分,对乳腺癌、肝癌、肺癌、膀胱癌、结肠癌、前列腺癌和白血病等多种肿瘤均有明显的抑制作用,是一种很有开发潜力的抗肿瘤候选药物。【DADS诱导人胃癌细胞增殖抑制、分化与周期阻滞的研究】本研究采用MTT法、电镜观察、Con A凝集实验、碱性磷酸酶（ALP）比活性测定、骨架蛋白细胞化学、划痕标记荧光染料示踪技术（SLDT）、免疫组化等方法,观察DADS对体外培养的人胃癌MGC803细胞的生长抑制与诱导分化效应。MTT比色实验结果显示,DADS浓度从20、25、30到35mg·L^-1处理人胃癌MGC803细胞,其吸光值比对照组低,而且随浓度增加逐渐降低（P<0.05）;相反,随着浓度增加,DADS对癌细胞增殖的抑制率从11.3%、35.2%、50.7%到60.6%逐渐增强（P<0.05）,表明DADS对人胃癌细胞具有明显的生长抑制作用,且呈显著的剂量-效应依赖关系。电镜观察发现30mg·L^-1DADS引起瘤细胞异型性降低,核质比下降,细胞表面微绒毛数目减少,胞浆内细胞器丰富,胞核及部分细胞器呈退行性变,并可见细胞间连接结构;DADS处理后瘤细胞对Con A的凝集率从79.10%降为26.98%（P<0.05）;ALP比活性由0.120 U/g下降至0.040 U/g（P<0.05）,下降率为66.12%;骨架蛋白细胞化学研究表明,细胞骨架蛋白合成增加与重组,划痕标记染料示踪技术（SLDT）显示细胞间缝隙连接通讯功能得以恢复;免疫组化发现,DADS处理后的MGC803细胞p21WAF1表达增强,p21ras、c-Myc、突变型p53表达减弱而pRb的表达没有改变。上述结果提示,DADS可诱导人胃癌MGC803细胞增殖抑制与分化。DADS对人胃癌BGC823细胞也具有一样的生长抑制效应,且呈显著的剂量-效应依赖关系（P<0.05）。流式细胞分析表明,DADS可将MGC803和BGC823细胞阻滞在G₂/M期。DADS作用12 h时,G₂/M期细胞分别增加到35.7%和30.1%;24 h时,分别增加到51.7和58.1%;36h时,分别达58.7%和50.2%（P<0.05）。Western Blot结果显示,20、25、30、35mg·L^-1DADS处理,呈浓度依赖性抑制人胃癌细胞中ERK的活化（P<0.05）;并且DADS +MEK抑制剂U0126能完全阻断ERK活化（P<0.05）;时间效应上,30mg·L^-1DADS处理后15-30min抑制作用最强,2h左右恢复正常（P<0.05）。提示DADS抑制ERK通路可能是诱导人胃癌细胞增殖抑制与分化的重要分子机制。结论:DADS具有诱导人胃癌细胞分化及抑制增殖的作用。DADS诱导人胃癌细胞增殖抑制和分化与细胞周期G₂/M期阻滞有关。ERK活性抑制是DADS诱导人胃癌细胞分化与抑制增殖的重要分子机制之一。【DADS对人胃癌MGC803和BGC823细胞G₂/M期检查点激酶通路的影响】肿瘤是多基因变化导致细胞周期紊乱的一类疾病,周期调控机制失活,特别是G1/S和G₂/M期检测点（checkpoint）的失活在细胞癌变过程中起着至关重要的作用。采用药物激活细胞周期调控机制,从而诱导肿瘤细胞凋亡和分化已成为肿瘤治疗的一个可行的新途径。前期工作证实,DADS对人胃癌MGC803细胞周期的作用可能与调节Cdc25C、cyclinB1蛋白的表达有关。本实验发现,DADS呈时间依赖性抑制人胃癌BGC823细胞周期Cdc25C磷酸酶的表达,cyclinB1表达在DADS处理24 h后开始下降,其改变与MGC803细胞中的一致。Cdc25C作用于cyclinB1/ CDK1复合物,而其磷酸酶活性受细胞周期检查点Chk1和/或Chk2激酶调节。本研究显示,30mg·L^-1DADS分别刺激1h和2h后,MGC803和BGC823细胞磷酸化Chk1表达明显增加,并呈时间依赖性（P<0.05）,提示DADS对两种细胞中的磷酸化Chk1的作用相仿,而人胃癌MGC803和BGC823细胞处理组与未处理组磷酸化Chk2表达无明显差异（P>0.05）;总Chk1和Chk2蛋白表达在两种细胞处理前后均无改变（P>0.05）。RT-PCR检测发现,Chkl和Chk2 mRNA水平在处理组与未处理组之间无明显差异（P>0.05）,表明DADS只对蛋白磷酸化有影响,而对其mRNA水平无影响。以上结果提示,DADS是通过激活Chk1而不是Chk2调节Cdc25C、cyclinB1的改变。Chk1是ATR的下游分子,我们进一步检测ATR的活性,结果显示,磷酸化ATR在MGC803和BGC823细胞中未处理组无表达,DADS处理15min后被激活,并呈时间依赖性（P<0.05）,而ATR总蛋白表达均无改变,这进一步说明DADS可能是通过ATR/Chk1/Cdc25C/cyclin B1引起细胞周期G₂/M阻滞。免疫共沉淀分析结果表明,MGC803和BGC823细胞中DADS能促进Chk1与Cdc25C结合,而对Chk2与Cdc25C结合无影响,证明DADS作用的周期信号通路是Chk1/Cdc25C,而不是Chk2/Cdc25C。结论:DADS可能是通过靶向检查点激酶Chk1引起G₂/M期阻滞。DADS阻滞人胃癌细胞于G₂/M期与调节ATR/ Chk1/ Cdc25C/ cyclin B1信号通路有关,可能成为治疗胃癌的新靶点。【Chk1和Chk2基因沉默对DADS G₂/M期阻滞作用及检查点激酶通路的影响】RNAi（RNA interference）是转录后基因沉默的一种调控机制,是机体固有的对抗外源侵袭基因及维持自身基因稳定性的一种自我保护机制。本研究采用RNAi技术分别在MGC803和BGC823细胞中将Chk1、Chk2基因沉默,进一步确定DADS的作用靶点。将Chk1、Chk2 RNAi分别转染MGC803和BGC823细胞后,Western blot和实时荧光定量PCR验证结果显示,MGC803和BGC823细胞Chk1 mRNA表达分别被抑制76.0%和84.7%,蛋白表达分别下降85.0%和90.2%;chk2 mRNA表达分别降低84.1%和69.0%,蛋白表达分别下调83.4%和93.5%。MTT结果发现,Chk1基因沉默后,对MGC803和BGC823细胞增殖有明显抑制作用,其抑制率分别为38%和48.1%（P<0.05）,而加以30mg·L^-1DADS后,其抑制率分别增加至56.3%和68.4%,与Chk1基因未沉默而用30mg·L^-1DADS处理组无差异（P>0.05）。相反,Chk2基因沉默后对细胞增殖无明显抑制作用（P>0.05）。流式细胞术分析Chk1和Chk2基因沉默后对BGC823细胞周期的影响,发现Chk1基因沉默后G₂期明显比未处理组减少12.5%（P<0.05）,Chk2基因沉默后G₂期下降8.1%（P>0.05）,Chk1和Chk2基因同时沉默G₂期下降13.6%（P<0.05）,与Chk1基因单独沉默的影响接近（P>0.05）。Chk1基因沉默后加入DADS后,在24h、48h,G₂期分别为10.4%、17.1%,与未处理组18.6%、20.4%比较,对细胞周期G₂期影响不大（P>0.05）,但较处理组58.1%、36.2%G₂期,分别下降47.7%、19.1%（P<0.05）,这表明DADS的作用靶点可能是Chk1。Chk1和Chk2下游分子的改变显示,Chk1基因沉默后Cdc25C和cyclinB1表达较未处理组明显增加,而加入DADS后,Cdc25C和cyclinB1表达与未处理组相比无差异;但Chk2基因沉默后对Cdc25C和cyclinB1表达无影响,对DADS的作用无影响,证实DADS的主要作用靶点是Chk1而不是Chk2。结论:Chk1基因沉默可明显抑制人胃癌细胞增殖。DADS阻滞G₂/M期是通过Chk1/Cdc25C/cyclinB1信号通路起作用,其主要作用靶点是Chk1。Chk2不参与DADS对胃癌G₂/M期的阻滞作用。更多还原

【Abstract】 Gastric cancer is the most common type of malignancy in China, and its morbidity and mortality rank first now. There are 400,000 people or so with gastric cancer, and 300,000 people succumb to it, which is much higher than that in the west developed countries. At present, therapeutic effect of orthodox surgical therapy, radiation therapy and chemotherapy is comparatively weak, easy to palindromia, furthermore , their side effect is serious and the 5-year survival rate of gastric cancer remains only about 30%. Therefore, it is an urgently solved topic to raise the 5-year survival rate, explore new method to treat gastric cancer, and look for new effective anticancer drugs with low toxicity and inexpensive price. Now human gastric cancer reversion research has become leading edge field in life science research. DADS inhibits the in vitro growth of colon, lung, breast and gastric cancer cell lines, and leukemia cell lines and it becomes a very potential candicate anticancer drug.【DADS inhibited growth of human gastric cancer MGC803 cells and induced them differentiation】 MTT assay, electron microscope,Con A-mediated cell agglutination, ALP specific activity detection and optics microscope, cytochemical staining, scrape-loading and dye transfer technology were employed to confirm the DADS-induced cell growth inhibition and differentiation in MGC803 cell line. Exposure of human gastric cancer MGC803 cells to DADS from 25 to 35mg·L^-1 for 96 h had less absorbance value than control,which exhibited a dose-dependent model （P<0.05）,whereas DADS induced-growth inhibition rate enhanced from 11.3%,35.2%,50.7% to 60.6% as assessed by the MTT test（P<0.05）.As exposed to electron microscope, MGC803 cells took on malignance declining as cellular heteromorphism diminished, nucleocytoplasmic proportion was reliable to reasonableness, the number of microvilli on cell surface decreased, cellular apparatus were abundant in plasm and intercellular conjunctional structure appeared after it being exposed to 30mg·L^-1 DADS; Con A-mediated cell agglutination ratio was 26.98% versus controls 79.10% （p<0.05） ; cell ALP specific activity decreased by 66.12% from 0.120 U/g to 0.040 U/g （p<0.05）. Cellular skeletin synthesized and reconstructured and recruitment of intercellular communication of gap junction were also observed with cytochemical technology; Gastric cancer cells showed negative of dye transfer,while positive transfer of LY dye from the load cells at the scraped-line to neighboring cells in gastric cancer cells induced by DADS. Immunihistichemical stain indicated that expression of p21WAF1 enhanced and that mutant p53,p21ras,c-myc downregulation expression and ther was no change in pRb. All these showed above suggested that human gastric cancer cells were induced differentiation into normal cells by DADS. Flow cytometry analysis revealed that treatment MGC803 cell with increasing quantities of DADS increased in the percentage of cells in the G₂/M phase. The proportion of cells in the G₂/M phase after treatment with 30mg·L^-1 DADS for 36 h was comparable （58.7%）, and more than four times that occurring in untreated cells （13.8%）. Exposure of human gastric cancer BGC823 cells to DADS from 5 to 20 mg·L^-1 for 96 h had less absorbance value than control, which exhibited a dose-dependent model （P<0.05）, whereas DADS induced-growth inhibition rate enhanced from 34.2、39.2、55.7、69.6% as assessed by the MTT test （P<0.05）.Which suggest that DADS inhibited growth of human gastric cancer BGC823 cells. After BGC823 cells were treated with 15mg·L^-1 DADS, flow cytometry analysis displayed that cell percentage in S phase of the DADS treated MGC803 cells Was decreased,whereas cell percentage in G₂ phase Was significantly increased（P<0.05）,and little change in Gl phase was found. Which is the same as that of MGC803 cells. Much attention has been focused on the ERK pathway as a possible target for newly designed anti-neoplastic drugs. Therefore, it is essential to establish the role that the ERK pathway plays in the process that leads to differentiation within a specific tissue or cell type. In this study, ERK activity is observed in the DADS-induced differentiation effect in human gastric cancer cell line MGC803. Western blot analysis revealed that although DADS did not influence the quantity of ERK1/2 protein expressed, it did decrease its phosphorylation in a concentration-dependent manner, compared with the controls （P<0.05）. At 30 mg·L^-1, DADS inhibited the activation of ERK1/2 in 15-30 min （P<0.05）. These results suggested that the DADS-induced differentiation of MGC803 cells involved an alteration of the ERK1/2 signaling pathway .Conclusion: Differentiation and growth inhibition was induced by DADS. Cell Cycle G₂/M Arrest was involved in growth inhibition and differentiation induced by DADS in the Human Gastric Cancer Cells. ERK is involved in the differentiation and growth inhibition induced by diallyl disulfide in the human gastric cancer cells.【Checkpoint Kinase Signaling is Involved in Cell Cycle G₂/M Arrest Induced by Diallyl Disulfide in the Human Gastric Cancer Cell Line MGC803 and BGC823】 Anticancer insights derived from cell cycle research has given birth to the idea of cell cycle G₂ checkpoint abrogation as a cancer cell specific therapy, based on the discovery that many cancer cells have a defective G1 checkpoint resulting in a dependence on the G₂ checkpoint during cell replication. The most promising target to date seems to be Chk1 and there will be a growing number of selective inhibitors of Chk1 available in the future with a variety of activities that promise to have potential G₂ checkpoint abrogation qualities. Cell cycle has recently become more appealing as a new target of anti-carcinogenic agent.It is a new mechanism that the role of Chk was reduced by anti-carcinogenic drug in tumor cells. Furthermore, G₂/M cell cycle arrest is mainly related with the Chk2-Cdc25C-Cdc2/cyclin B1 pathway in cancer cells.It was found that Cdc25C and CyclinB1 expression are critical events in G₂/M arrest of MGC803 cells by DADS. DADS increased the expression of cyclinB1 by 20% after 12 h. DADS inhibited the expression of the cell cycle-associated phosphatase Cdc25C by 80% after 48 h. Western blot analysis showed that DADS treatment of BGC823 cells decreased the level of Cdc25C in a time-dependent model （P<0.05）. DADS increased the expression of cyclinB1 after 12h and then its expression decreased after 24h （P<0.05）, which tendency is similar to MGC803 cells by DADS. Cdc25C was thought to be the major effector of the G₂/M DNA damage checkpoint response. Cdc25C which triggers cyclinB1/CDK1 complex is controlled by Chk1 or/and Chk2. So Chk1 and Chk2 activity was observed by DADS on human gastric cancer cells. Western blot analysis showed that phosphorylation of Chk1 was decreased following treatment of MGC803 and BGC823 cells with DADS after 1hrs in a time-dependent model. At the same time,the total Chk1 abundance did not change.Expression of Phospho-Chk2 was weak in these two untreated cell lines,furthermore, its expression wasn’t changed by DADS （P>0.05）. Expression of Chk1 and Chk2 had no change after treated with DADS. The level of Chkl and Chk2 mRNA had no significant difference between the DADS treatment and contro1 by RT-PCR （P>0.05）. Which showed that the level of Chkl and Chk2 mRNA had no relationship with cell cycle arrest.ATR is capable of specifically phosphorylating Chkl. Western blot analysis showed that Phospho-ATR was inactivated by DADS from 15 or 30 to 90 min in MGC803cells or BGC823 cells respectively（P<0.05）. Expression of ATR had no change after treated with DADS. Coimmunoprecipitated Chk1 or Chk2 was detected by anti-Chk1 or anti-Chk2 immunoprecipitation （IP） followed by anti-Cdc25C immunoblotting （IB）, DADS enhanced binding activity of Chk1 kinase with Cdc25C in MGC803 and BGC823 cells,but didn’t influence binding activity of Chk2 kinase with Cdc25C. Which verified that DADS induced G₂/M arrest by Chk1- Cdc25C on MGC803cells and BGC823 cells.Conclusion: Checkpoint kinase Chk1 may be the target to arrest cell cycle by DADS. ATR/ Chk1/Cdc25C/ cyclin B1 was regulated by DADS to arrest Human Gastric Cancer Cells at G₂/M.【Checkpoint Kinase Signaling is regulated in Cell Cycle G₂/M Arrest Induced by Chk1 and Chk2 RNAi】To investigate the effect of Chk1 or Chk2 gene silence on the cell viability of human gastric cancer cell line MGC803 and BGC823, and explore the role of Chk1 and Chk2 in cell cycle arrest. The siRNA targeting at Chk1 or Chk2 gene was transfected into MGC803 and BGC823 cells. The mRNA and protein expression of Chk1 was detected by RT-PCR and Western-blot respectively. The Chk1 or Chk2 expression at mRNA and protein levels in Chk1 or Chk2 siRNA-transfected group was reduced markedly respectively as compared with that in negative control and empty vector-transfected group （P<0.05）.Cell viability was determined via MTT assay. Treated with DADS, cells-transfected viability by Chk1 SiRNA was decreased by 18.3% or 20.3% in MGC803 and BGC823 cells respectively （P<0.05）. Inhibition of the Chk1 expression in Chk1 siRNA transfected group in BGC823 cells significantly abrogated G₂/M arrest induced by DADS, and the proportion of the cells in G₂/M phase was more than two double lower than that in other control groups （P<0.05）.While inhibition of the Chk2 expression in Chk2 siRNA transfected group had little effect on G₂/M arrest.Chk1 gene silence increased expression of Cdc25C and cyclinB1,and inhibition of expression of Cdc25C and cyclinB1 by DADS was blocked （P<0.05）. On the contrary,Chk2 gene silence can not do so.Conclusion: Chk1 gene silence can inhibited and growth of Human Gastric Cancer Cells, and take synergistic effect with DADS. DADS arrestted Human Gastric Cancer Cells at G₂/M by Chk1/Cdc25C /CyclinB1, and its main target was Chk1. Chk2 didn’t participate G₂/M arrest by DADS in Human Gastric Cancer Cells.更多还原