抗肿瘤新药S14161及其衍生化合物BENC-511抗前列腺癌的药理作用研究

发布时间：2018-03-31 10:51

本文选题：S14161　切入点：BENC-511　出处：《苏州大学》2014年硕士论文

【摘要】：[目的] PI3K/Akt信号通路是前列腺癌的一个重要治疗靶标,而S14161是本课题组前期筛选出的一个PI3K抑制剂,对恶性血液病等多种肿瘤细胞具有较强的凋亡诱导作用。为了进一步提高其抗肿瘤活性,我们对其进行了结构优化,得到一系列衍生物,其中BENC-511具有更强的抑制PI3K的活性。本课题中我们将比较S14161和BENC-511这两种化合物对雄激素非依赖型前列腺癌细胞株(PC3和DU145)的抑制作用及机制,并在裸鼠移植瘤模型中分析两种化合物的药效,从而为研发抗前列腺癌的新药提供一定的参考。 [方法] 1.观察S14161和BENC-511处理前列腺癌细胞(PC3和DU145)后细胞形态的变化,MTT法检测两种化合物对细胞增殖的抑制作用； 2.流式细胞技术检测两种化合物对细胞凋亡率的影响,并通过蛋白免疫印迹法检测凋亡相关蛋白表达水平的变化； 3.蛋白免疫印迹法检测两种化合物对PI3K/Akt/mTOR信号通路相关蛋白的表达及磷酸化的影响； 4.建立NOD/SCID裸鼠的皮下接种前列腺癌的移植瘤模型,在体内水平分析两种化合物对前列腺癌的抑制作用； 5.用转染技术使PTEN缺失的PC3细胞表达PTEN蛋白,检测PC3对化合物敏感程度的变化。 [结果] 1.S14161和BENC-511均能抑制多种实体瘤细胞株的存活,MTT实验表明两种化合物均能抑制前列腺癌细胞PC3和DU145的增殖,该抑制作用呈浓度的依赖性,但是PC3对BENC-511更为敏感； 2.S14161与BENC-511均能显著诱导前列腺癌细胞的凋亡,而BENC-511诱导凋亡的能力明显强于S14161；此外,PC3细胞株对这两种化合物的敏感程度显著高于DU145细胞株；这两种化合物均能诱导凋亡标志蛋白PARP和Caspase-3的激活,并呈现剂量依赖性关系； 3.S14161能在PC3细胞株中显著抑制PI3K信号通路的活化及信号传导,但是对DU145细胞株的抑制作用则不明显；而BENC-511不但能对PC3细胞株(PTEN缺失型)的PI3K通路产生更强的抑制作用,也能对DU145细胞株产生一定的抑制作用； 4.S14161和BENC-511均能在抑制裸鼠体内PC3及DU145的移植瘤生长,但是BENC-511对肿瘤的抑制作用要强于S14161；此外,这两种化合物均能抑制肿瘤组织中的Akt的磷酸化,并能促进PARP的剪切激活； 5.PC3是PTEN缺失型前列腺癌细胞,在PC3细胞中重新表达PTEN蛋白后,化合物诱导PARP的剪切程度下降,PC3对化合物BENC-511的敏感性下降。 [结论l S1416和BENC-511能显著诱导前列腺癌细胞株PC3和DU145的凋亡,抑制PI3K/Akt通路的激活及信号传导,并且BENC-511的作用强于其母体化合物S14161；PC3和DU145对这两种化合物的敏感性存在一定差异,原因可能是PC3是PTEN缺失型的细胞株,它的PI3K/Akt通路异常活化,更易被此通路的药物所抑制；在PC3细胞中成功表达PTEN蛋白后,PC3对化合物的敏感度下降,证明PC3比DU145具有的敏感度差异是PC3细胞的PTEN基因的缺失导致的；在体内水平,BENC-511也比S14161表现出更强的抗前列腺癌效应。因此,无论是在体外还是体内水平,BENC-511抗前列腺癌的作用均强于母体化合物S14161。
[Abstract]:[Objective]
PI3K/Akt signaling pathway is an important target for treatment of prostate cancer, and S14161 is the subject of a PI3K inhibitor group pre screened, with strong induction of apoptosis in malignant hematologic diseases and other tumor cells. In order to further improve its antitumor activity, we carried out the optimization of the structure, a series of derivatives. The inhibition of PI3K BENC-511 has stronger activity. In this paper we will compare the S14161 and BENC-511 of the two compounds on androgen independent prostate cancer cell lines (PC3 and DU145) the inhibitory effect and mechanism, and analyze the effects of two kinds of compounds in the nude mice model, to provide a reference to drug resistance for the development of prostate cancer.
[method]
1. the morphological changes of prostate cancer cells (PC3 and DU145) were observed by S14161 and BENC-511. The inhibitory effect of two compounds on cell proliferation was detected by MTT.
2. flow cytometry was used to detect the effect of two kinds of compounds on the apoptosis rate, and the expression level of apoptosis related proteins was detected by Western blot.
3. protein immunoblotting was used to detect the effect of two compounds on the expression of PI3K/Akt/mTOR signaling pathway related proteins and phosphorylation.
4. the tumor model of NOD/SCID nude mice was established by subcutaneous inoculation of prostate cancer, and the inhibitory effects of two compounds on prostate cancer were analyzed in vivo.
5. the transfection technique was used to express the PTEN protein in PC3 cells missing from PTEN and to detect the changes in the sensitivity of PC3 to the compounds.
[results]
Both 1.S14161 and BENC-511 could inhibit the survival of many kinds of solid tumor cell lines. MTT experiments showed that all two compounds could inhibit the proliferation of prostate cancer cells PC3 and DU145, and the inhibitory effect was concentration dependent, but PC3 was more sensitive to BENC-511.
The apoptosis of 2.S14161 and BENC-511 can significantly induce prostate cancer cells, and apoptosis induced by BENC-511 was stronger than S14161; in addition, the sensitivity of PC3 cells to the two kinds of compounds were significantly higher than that of DU145 cells; the two compounds could induce apoptosis marker of activated protein PARP and Caspase-3, in a dose-dependent manner the relationship between;
3.S14161 could significantly inhibit the PI3K signaling pathway in PC3 cells and the activation of signal transduction, but the inhibitory effect on DU145 cells was not obvious; while BENC-511 can not only on PC3 cell line (PTEN deletion) inhibition of PI3K pathway produced stronger, can also have a certain inhibitory effect on DU145 cell line;
Both 4.S14161 and BENC-511 can inhibit the growth of transplanted tumor of PC3 and DU145 in nude mice, but the inhibitory effect of BENC-511 on tumor is stronger than that of S14161. In addition, these two compounds can inhibit the phosphorylation of Akt and promote the shear activation of PARP.
5.PC3 is a PTEN deficient prostate cancer cell. After re expressing PTEN protein in PC3 cells, the degree of PARP induced by compounds decreases, and the sensitivity of PC3 to compound BENC-511 decreases.
[Conclusion L
S1416 and BENC-511 can significantly induce apoptosis of prostate cancer cell lines PC3 and DU145, and the activation of PI3K/Akt signaling pathway, and the effect of BENC-511 is stronger than its parent compound S14161; there are some differences in the sensitivity of PC3 and DU145 of the two compounds, may be the cause of PC3 is PTEN deficient cells, abnormal PI3K/Akt it's easier to access the activation of this pathway was inhibited by the drug; the successful expression of PTEN protein in PC3 cells after PC3 of compounds decreased sensitivity, sensitivity that difference of PC3 compared with DU145 is the lack of PTEN based PC3 cells due to lead; in vivo, BENC-511 than S14161 show the effect of stronger anti prostate cancer. Therefore, both in vitro and in vivo levels of BENC-511 against prostate cancer have stronger effects on the parent compounds S14161.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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