本文選題：S14161　切入點(diǎn)：BENC-511　出處：《蘇州大學(xué)》2014年碩士論文

【摘要】：[目的] PI3K/Akt信號(hào)通路是前列腺癌的一個(gè)重要治療靶標(biāo),而S14161是本課題組前期篩選出的一個(gè)PI3K抑制劑,對(duì)惡性血液病等多種腫瘤細(xì)胞具有較強(qiáng)的凋亡誘導(dǎo)作用。為了進(jìn)一步提高其抗腫瘤活性,我們對(duì)其進(jìn)行了結(jié)構(gòu)優(yōu)化,得到一系列衍生物,其中BENC-511具有更強(qiáng)的抑制PI3K的活性。本課題中我們將比較S14161和BENC-511這兩種化合物對(duì)雄激素非依賴型前列腺癌細(xì)胞株(PC3和DU145)的抑制作用及機(jī)制,并在裸鼠移植瘤模型中分析兩種化合物的藥效,從而為研發(fā)抗前列腺癌的新藥提供一定的參考。 [方法] 1.觀察S14161和BENC-511處理前列腺癌細(xì)胞(PC3和DU145)后細(xì)胞形態(tài)的變化,MTT法檢測(cè)兩種化合物對(duì)細(xì)胞增殖的抑制作用； 2.流式細(xì)胞技術(shù)檢測(cè)兩種化合物對(duì)細(xì)胞凋亡率的影響,并通過蛋白免疫印跡法檢測(cè)凋亡相關(guān)蛋白表達(dá)水平的變化； 3.蛋白免疫印跡法檢測(cè)兩種化合物對(duì)PI3K/Akt/mTOR信號(hào)通路相關(guān)蛋白的表達(dá)及磷酸化的影響； 4.建立NOD/SCID裸鼠的皮下接種前列腺癌的移植瘤模型,在體內(nèi)水平分析兩種化合物對(duì)前列腺癌的抑制作用； 5.用轉(zhuǎn)染技術(shù)使PTEN缺失的PC3細(xì)胞表達(dá)PTEN蛋白,檢測(cè)PC3對(duì)化合物敏感程度的變化。 [結(jié)果] 1.S14161和BENC-511均能抑制多種實(shí)體瘤細(xì)胞株的存活,MTT實(shí)驗(yàn)表明兩種化合物均能抑制前列腺癌細(xì)胞PC3和DU145的增殖,該抑制作用呈濃度的依賴性,但是PC3對(duì)BENC-511更為敏感； 2.S14161與BENC-511均能顯著誘導(dǎo)前列腺癌細(xì)胞的凋亡,而BENC-511誘導(dǎo)凋亡的能力明顯強(qiáng)于S14161；此外,PC3細(xì)胞株對(duì)這兩種化合物的敏感程度顯著高于DU145細(xì)胞株；這兩種化合物均能誘導(dǎo)凋亡標(biāo)志蛋白PARP和Caspase-3的激活,并呈現(xiàn)劑量依賴性關(guān)系； 3.S14161能在PC3細(xì)胞株中顯著抑制PI3K信號(hào)通路的活化及信號(hào)傳導(dǎo),但是對(duì)DU145細(xì)胞株的抑制作用則不明顯；而BENC-511不但能對(duì)PC3細(xì)胞株(PTEN缺失型)的PI3K通路產(chǎn)生更強(qiáng)的抑制作用,也能對(duì)DU145細(xì)胞株產(chǎn)生一定的抑制作用； 4.S14161和BENC-511均能在抑制裸鼠體內(nèi)PC3及DU145的移植瘤生長(zhǎng),但是BENC-511對(duì)腫瘤的抑制作用要強(qiáng)于S14161；此外,這兩種化合物均能抑制腫瘤組織中的Akt的磷酸化,并能促進(jìn)PARP的剪切激活； 5.PC3是PTEN缺失型前列腺癌細(xì)胞,在PC3細(xì)胞中重新表達(dá)PTEN蛋白后,化合物誘導(dǎo)PARP的剪切程度下降,PC3對(duì)化合物BENC-511的敏感性下降。 [結(jié)論l S1416和BENC-511能顯著誘導(dǎo)前列腺癌細(xì)胞株P(guān)C3和DU145的凋亡,抑制PI3K/Akt通路的激活及信號(hào)傳導(dǎo),并且BENC-511的作用強(qiáng)于其母體化合物S14161；PC3和DU145對(duì)這兩種化合物的敏感性存在一定差異,原因可能是PC3是PTEN缺失型的細(xì)胞株,它的PI3K/Akt通路異常活化,更易被此通路的藥物所抑制；在PC3細(xì)胞中成功表達(dá)PTEN蛋白后,PC3對(duì)化合物的敏感度下降,證明PC3比DU145具有的敏感度差異是PC3細(xì)胞的PTEN基因的缺失導(dǎo)致的；在體內(nèi)水平,BENC-511也比S14161表現(xiàn)出更強(qiáng)的抗前列腺癌效應(yīng)。因此,無論是在體外還是體內(nèi)水平,BENC-511抗前列腺癌的作用均強(qiáng)于母體化合物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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

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