新型Ⅰ类组蛋白脱乙酰化酶（HDAC）抑制剂4CS-202抗结肠癌作用机制研究

发布时间：2019-05-30 03:57

【摘要】：目的：观察Ⅰ类组蛋白脱乙酰化酶(HDAC)抑制剂4SC-202体内外抗结肠癌细胞的作用,旨在为结肠癌的分子靶向治疗进行新的探索。方法：1)MTT法、台盼蓝染色法、细胞集落试验检测不同浓度的4SC-202处理不同作用时间后对结肠癌细胞存活、细胞死亡及细胞增殖的影响；2) Histone DNA ELISA法、Caspase-3/-9试剂盒方法、Annexin V-PI流式细胞仪法检测4SC-202对结肠癌细胞凋亡的作用；4SC-202处理后对其抗凋亡蛋白Bcl-2的表达进行检测,运用的方法为Western Blotting;3) PI流式细胞仪法观察4SC-202处理结肠癌细胞后,细胞周期的变化。4)用药理学和shRNA等手段抑制结肠癌细胞内AKT的活性,观察抑制后4SC-202体外抗结肠癌细胞活性的变化。5)MTT法、Histone DNA ELISA法等方法检测比较奥沙利铂组、4SC-202组以及奥沙利铂联合4SC-202组对结肠癌细胞的存活和凋亡的影响。6)建立裸鼠荷瘤模型,观察奥沙利铂和/或4SC-202在体内抗结肠癌细胞活性。结果：1)4SC-202显著抑制结肠癌细胞(原代人结肠癌细胞和HT-29,HCT-116,HT-15,DLD-1细胞株)的存活和增殖,其抑制作用呈时间和浓度依赖性。2)4SC-202诱导结肠癌细胞凋亡。4SC-202可以抑制抗凋亡蛋白Bcl-2的表达。3)当4SC-202浓度不断的增大时,结肠癌细胞的G1和S期所占比例逐渐减少,G2-M期比例增加。4)添加AKT选择性抑制剂perifosine、MK-2206或shRNA敲减AKT1/2显著增加4SC-202诱导的体外抗结肠癌细胞的活性。而导入持续活化的AKT1(CA-AKT1)则抑制4SC-202抗结肠癌细胞的作用。5)奥沙利铂诱导的体外抗结肠癌细胞的活性在有低浓度4SC-202存在时明显增强。两者协同给药诱导的抗结肠癌细胞的活性要显著强于单独给药。6)在裸鼠荷瘤模型中,4SC-202 (100mg/kg, Q2D)口服给药显著抑制HT-29移植瘤的在体生长。而同时腹腔注射奥沙利铂(5.0mg/kg, Q3D)则进一步增强4SC-202在体抗癌活性。结论：1)4SC-202浓度及时间依赖性的抑制人结肠癌细胞存活、增殖,并诱导癌细胞凋亡；2)4SC-202诱导结肠癌细胞G2-M周期停滞；3)抑制AKT能增强结肠癌细胞对4SC-202的敏感性；4)4SC-202增强奥沙利铂体外抗结肠癌细胞的作用；5)4SC-202和奥沙利铂协同抑制HT-29移植瘤的在体生长。
[Abstract]:Aim: to observe the effect of class I histone deacetylase (HDAC) inhibitor 4SC-202 on colon cancer cells in vitro and in vivo in order to explore the molecular targeting therapy of colon cancer. Methods: 1) MTT assay, trypan blue staining and cell colony test were used to detect the effects of different concentrations of 4SC-202 on the survival, cell death and cell proliferation of colon cancer cells. 2) Histone DNA ELISA assay, Caspase-3/-9 kit method and Annexin V-PI flow cytometry were used to detect the effect of 4SC-202 on apoptosis of colon cancer cells. After 4SC-202 treatment, the expression of anti-apoptosis protein Bcl-2 was detected by Western Blotting;. 3) PI flow cytometry was used to observe the changes of cell cycle in colon cancer cells treated with 4SC-202. 4) Pharmacology and shRNA were used to inhibit the activity of AKT in colon cancer cells. To observe the changes of anti-colon cancer cell activity of 4SC-202 in vitro after inhibition. 5) MTT, Histone DNA ELISA assay was used to detect and compare the activity of oxaliplatinum group. The effects of 4SC-202 group and oxaliplatin combined with 4SC-202 group on the survival and apoptosis of colon cancer cells. 6) the tumor-bearing model of nude mice was established to observe the anti-colon cancer cell activity of oxaliplatin and / or 4SC-202 in vivo. Results: 1) 4SC-202 significantly inhibited the survival and proliferation of colon cancer cells (primary human colon cancer cells and HT-29,HCT-116,HT-15,DLD-1 cell lines). The inhibitory effect was time-and concentration-dependent. 2) 4SC-202 induced apoptosis in colon cancer cells. 4SC-202 could inhibit the expression of anti-apoptosis protein Bcl-2. 3) when the concentration of 4SC-202 increased, The proportion of G _ 1 and S phase of colon cancer cells decreased gradually, while the proportion of G _ 2-M phase increased. 4) the addition of AKT selective inhibitor perifosine,MK-2206 or shRNA knockdown AKT1/2 significantly increased the activity of 4SC-202-induced anti-colon cancer cells in vitro. However, the introduction of continuously activated AKT1 (CA-AKT1) inhibited the anti-colon cancer cell effect of 4SC-202. 5) the activity of oxaliplatin induced anti-colon cancer cells in vitro was significantly enhanced in the presence of low concentration of 4SC-202. The activity of anti-colon cancer cells induced by co-administration was significantly stronger than that induced by single administration. 6) in nude mice tumor-bearing model, oral administration of 4SC-202 (100mg / kg, Q2D) significantly inhibited the growth of HT-29 transplantable tumor in vivo. At the same time, intraperitoneal injection of oxaliplatin (5.0 mg 路kg / kg, Q3D) further enhanced the anticancer activity of 4SC-202 in vivo. Conclusion: 1) 4SC-202 can inhibit the survival, proliferation and apoptosis of human colon cancer cells in a concentration-and time-dependent manner, 2) 4SC-202 can induce the arrest of G2 / M cycle of colon cancer cells. 3) inhibition of AKT could enhance the sensitivity of colon cancer cells to 4SC-202, 4) 4SC-202 enhanced the anti-colon cancer cell effect of oxaliplatin in vitro, and 5) 4SC-202 and oxaliplatin combined with oxaliplatin could inhibit the growth of HT-29 transplantation tumor in vivo.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

【相似文献】

相关期刊论文 前10条

1 胡平,琚立华,徐元鼎;结肠癌细胞核DNA含量测量及形态分析[J];复旦学报(医学版);2003年05期

2 王春晖;欧阳钦;唐承薇;黄明慧;李献;;选择性及非选择性COX-2抑制剂对结肠癌细胞生长的影响[J];四川大学学报(医学版);2006年04期

3 韩忠宝;李洪祥;杨翊研;;生长抑素联合阿霉素体外抑制结肠癌细胞的实验研究[J];吉林医学;2006年09期

4 张朝阳;徐克森;杨广运;王金申;高颖;刘炎峰;帅晶;王家勇;李少燕;寿楠海;牛军;;反义整合素β6基因对结肠癌细胞作用的研究[J];中华医学杂志;2007年37期

5 沈丹;邓长生;;过氧化物酶体增殖物激活受体γ在结肠癌中的表达及其激动剂对结肠癌细胞的生长抑制作用[J];临床内科杂志;2008年01期

6 田永刚;许军;刘昶;;人工气腹对结肠癌细胞生物学行为的影响[J];腹腔镜外科杂志;2009年10期

7 高喜廉，傅志民，，姜宗源;结肠癌细胞肠壁纵向浸润与临床意义[J];中国医科大学学报;1994年03期

8 郑平菊,黄威权,王瑞安,孙岚,高平;5-羟色胺及其受体在结肠癌细胞的免疫组织化学定位[J];科学通报;1995年21期

9 林洁;黄琼;来茂德;;5-氮-2′-脱氧胞苷对结肠癌细胞生物学行为的影响[J];临床与实验病理学杂志;2008年04期

10 王德力;齐东丽;;光镊拉曼光谱技术检测结肠癌细胞的研究[J];长春大学学报;2009年04期

相关会议论文 前10条

1 许峰;王杉;叶颖江;崔志荣;;肿瘤浸润淋巴细胞在结肠癌细胞杀伤中的作用及机制[A];中华医学会第七次全国消化病学术会议论文汇编（下册）[C];2007年

2 王贵玉;王锡山;;激光捕获微切割技术分离结肠癌细胞用于蛋白组学研究[A];中国蛋白质组学第三届学术大会论文摘要[C];2005年

3 沈丹;邓长生;;过氧化物酶体增殖物激活受体γ在结肠癌中的表达及其激动剂对结肠癌细胞的生长抑制作用[A];中华医学会第七次全国消化病学术会议论文汇编（下册）[C];2007年

4 郭俊明;赖依峰;肖丙秀;刘琼;;大豆异黄酮对体外培养的结肠癌细胞生长和细胞周期的影响[A];华东六省一市生物化学与分子生物学会2003年学术交流会论文摘要集[C];2003年

5 李本辉;杨贤子;张文杰;;人类结肠癌细胞缺陷型Stat6~(null)活化表型与癌细胞高凋亡率及低侵袭转移力相关[A];湖北省抗癌协会青年委员会成立大会暨第一届青年学术论坛资料汇编[C];2009年

6 王国强;方m锞

本文编号：2488502